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The invention consists in an original procedure to purify dirty manna, an under-utilized waste matter from the lymph poured from fraxinus plants, to obtain a product comparable with the pure edible “manna”, and enriched in bioactive components with respect to pure manna. Methods to treat the so obtained purified manna are also part of the invention.

Autophagy is a highly conserved catabolic process that degrades and recycles intracellular components through the lysosomes [1]. The role of this process in tumorigenesis and tumor progression is controversial: in the early stages, it can block tumor growth and conversely it can promote its progression in the later stages [2]. The tumor suppressor BECN1 gene, encodes the protein Beclin 1, a marker of autophagy down-regulated in several types of cancer, such as colorectal cancer [3]. There are a lot of both genetic and environmental risk factors for colorectal cancer, including diet: for this reason, in accordance with epidemiological studies, consumption of foods rich in phytochemicals is widely promoted. The betalain indicaxantin (Ind) is a phytochemical from the Opuntia Ficus-Indica fruit having several biological activities, such as antioxidant, anti-inflammatory. It showed antiproliferative and proapoptotic effects in colorectal adenocarcinoma (Caco2) cells where was able to regulate gene expression through modulation of methylation state of DNA at CpG islands [4]. For the first time, using Methylation-Sensitive Restriction Endonuclease PCR (MSRE-PCR), we report that Ind (50 e 100 µM) decreases the methylation of BECN1 promoter in Caco2 cells, to the same extent as 5-azacytidine (Zcyd, positive control). Interestingly, colorimetric detection of DNA Methyltransferases activity, indicates that Ind reduced the activity of these enzymes, like Zcyd did. These preliminary data, indicating that Ind is able to decrease the methylation of BECN1 gene, allow us to propose an epigenetic hypothesis of autophagy regulation in Caco2 cells.

Intestinal inflammation is a natural process crucial to maintain gut integrity, but its deregulation is involved in the pathogenesis of severe intestinal disorders[1]. Intestinal epithelial cells play a crucial role in the inflammatory response, modulating the immune cell exposure to antigens and by their ability to secrete many inflammatory mediators. IL-1β represents a pivotal player: secreted by infiltrated leucocytes, it induces the expression of several pro-inflammatory genes. Also the anti-inflammatory IL-10, whose function is to terminate the inflammatory process, modulates the intestinal physiology[2]. Recent clinical reports showed that patients with ulcerative colitis in remission phase have significantly higher IL10 gene expression in mucosa compared with active patients and controls[3]. Moreover, in the latest years aberrant epigenetic mechanisms were put in binomial relationship with chronic inflammatory diseases[4]. Previously, we described a demethylation of pro-inflammatory IL6 and IL8 genes in human colonic Caco-2 cells differentiated into an enterocyte-like phenotype and exposed to the inflammatory action of IL-1β[5]. In the present study we evaluate whether the IL-1β treatment affected the methylation status of the anti-inflammatory IL10 gene, in the same in vitro model. Our results showed that IL-1β treatment did not change the hypermethylation status of the IL10 promoter. Moreover, in cell lysates from IL-1β-treated Caco-2 cells, we observed a dose-dependent increase of DNMTs activity and, surprisingly, a decrease of DNMT3b expression. These findings put in evidence the complexity of relationship between IL-1β and DNMTs, and may suggest a potential role of IL-1β as pleiotropic modulator of DNA methylation in Caco-2 cell line.

Inflammation is a complex physiological response that requires the activity of a sophisticated regulatory network involving the activation of specific genes for defense, tissue repair and remodeling. Although transcriptional activation has been shown to be critical in the regulation of inflammatory genes (1) the role of epigenetic phenomena in the modulation of the inflammatory response is now emerging (2). Specifically, it has been recently reported that proinflammatory stimuli induce DNA demethylation in the interleukin IL-1 promoter of human articular chondrocytes (3). IL-1 cytokine, among several proinflammatory agents, represents an essential player in the inflammatory conditions of the gut (4): functioning as the strongest signal transduction to NF-kB, IL-1 increases in intestinal paracellular permeability and over-expression of proinflammatory genes (5). In this tissue, moreover, inflammatory response is crucial to maintain its structural integrity and function, thus, alteration and deregulation of inflammatory pathways contribute to tissue damage and ulceration, and are pivotal factors in the pathogenesis of several inflammatory gut diseases. In the present study we evaluate both wide-ranging and gene-specific epigenetic changes in the inflammatory response of Caco-2 cells differentiated into intestinal epithelial cells and exposed to the inflammatory actions of IL-1β. Our results clearly show that IL-1induces changes in the DNA methylation either at genome and gene level and that the local methylation changes are induced in two pro-inflammatory genes that are IL-1-regulated. In particular, we show that a cell exposure to IL-1β for 24 h induces, in a dose-dependent manner, hypomethylation of genomic DNA in respect to untreated cells. We also observe a reduced DNA methyltransferases activity of cell lysates obtained from IL-1 treated cells. Finally our data show that IL-1 is able to induce hypomethylation of specific CpG sites in IL-6 and IL-8 genes. These preliminary results suggest that IL-1 in intestinal epithelial cells is able to act as an epigenetic modulator towards the entire genome and specific genes. Modulation of epigenetic changes in inflammation may provide a new “reading frame” of the inflammatory diseases molecular basis. Since epigenetic modifications are potentially reversible, a thorough understanding of these changes during inflammatory response opens opportunities to develop efficient agents for specific targets.

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7-Ketocholesterol (7-KC)-induced apoptosis of macrophages is considered a key event in the development of human atheromas. In the present study, the effect of indicaxanthin (Ind), a bioactive pigment from cactus pear fruit, on 7-KC-induced apoptosis of human monocyte/macrophage THP-1 cells was investigated. A pathophysiological condition was simulated by using amounts of 7-KC that can be reached in human atheromatous plaque. Ind was assayed within a micromolar concentration range, consistent with its plasma level after dietary supplementation with cactus pear fruit. Pro-apoptotic effects of 7-KC were assessed by cell cycle arrest, exposure of phosphatidylserine at the plasma membrane, variation of nuclear morphology, decrease of mitochondrial trans-membrane potential, activation of Bcl-2 antagonist of cell death and poly(ADP-ribose) polymerase-1 cleavage. Kinetic measurements within 24 h showed early formation of intracellular reactive oxygen species over basal levels, preceding NADPH oxidase-4 (NOX-4) over-expression and elevation of cytosolic Ca2þ, with progressive depletion of total thiols. 7-KC-dependent activation of the redox-sensitive NF-kB was observed. Co-incubation of 2·5mM of Ind completely prevented 7-KC-induced pro-apoptotic events. The effects of Ind may be ascribed to inhibition of NOX-4 basal activity and over-expression, inhibition of NF-kB activation, maintaining cell redox balance and Ca homeostasis, with prevention of mitochondrial damage and consequently apoptosis. The findings suggest that Ind, a highly bioavailable dietary phytochemical, may exert protective effects against atherogenetic toxicity of 7-KC at a concentration of nutritional interest.

Phytochemicals play prominent roles in human diet and nutrition as protective redo-active substances in prevention of several disorders and chronic diseases in humans. Today, their function as potent modulators of the mammalian epigenome-regulated gene expression is rapidly emerging. In the present study antiproliferative effects of Indicaxanthin (Ind) from the fruits of Opuntia ficus-indica (1), and potential influence on DNA methylation has been investigated on Caco-2 cells, a human cell line of colorectal carcinoma. Ind caused a clear dose- and time-dependent decrease of the cell proliferation (IC(50) 50 M) associated to apoptosis as demonstrated by phosphatidylserine externalization and depolarization of mithocondrial membrane. Ind decreased the Go-G1phase whereas increased S and G2-M phases of the cell cycle. The phytochemical did not altered the intracellular ROS levels but decreased the [Ca2+]i. Investigation on DNA methylation using MESAP-PCR (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction) (2), showed that 100 M Ind induced a slight global demethylation after a 48 h treatment. Analysis of epigenetic changes in the DNA methylation pattern at CpG promoter of p16 (INK4a), using MSRE (Methylation-Sensitive Restriction Endonucleases Multiplex-Polymerase Chain Reaction), showed that Ind caused CpG demethylation. Western blotting analysis carried out with p16 monoclonal antibody, confirmed the reactivation of the protein expression. Present data, suggesting that a long-term exposure to indicaxanthin in diet might potentially affect epigenetic machines of the intestinal cells, preventing or repairing initial derangements/disorders, encourage studies on the mechanism involved.

In the present study antiproliferative effects of Indicaxanthin (Ind), a highly bioavailable pigment from the fruits of Opuntia ficus-indica (1), were investigated on a number of human cancer cell lines including hepatocarcinoma cells (HepG2, Ha22T, HUH 7), breast cancer cells (MCF7), cervix epithelial carcinoma (HeLa), and colorectal carcinoma cells (Caco-2). Cytotoxicity of Ind, in a concentration range between 25 to 100 M, was evaluated by Trypan blue exclusion method and MTT assay. Ind caused a clear dose- and time-dependent decrease in the proliferation of Caco-2 and Ha 22T cells with an IC(50) of about 50 M, with minor effect on the other cell lines. Flow cytometric analysis after Annexin V-FITC and propidium iodure double staining, at 24, 48 and 72 h of treatment with 100 M Ind, showed a pro-apoptotic effect of the pigment at 48 and 72 h. Effect of Ind on DNA methylation investigated on DNA from Ha22T cells line and Caco2 cells line at 48 h of treatment with 10 M Ind, using MESAP-PCR (Methylation-Sensitive Arbitrarily-Primed Polymerase Chain Reaction) (3) showed that Ind induces a slight global demethylation. While antiproliferative effects of indicaxanthin add further value to the nutritional characteristics of the fruits of O. ficus-indica (2), our results also are consistent with the emerging role of dietary phytochemicals on the epigenetic regulation of gene expression.

Aerobic life is characterised by a steady formation of pro-oxidants, which is approximately balanced by antioxidant defense systems. When cell’s capacity to protect itself fails, oxidative stress occurs. Because of bioactive components vegetables are now considered helpful in preventing chronic pathologies in which oxidative damage is an important etiologic factor. Cactus pear (Opuntia ficus-indica) fruit contains substantial amounts of vitamin C, biothiols, and taurine, and redox-active betalain pigments. It has been shown that a short-term supplementation (500 g fruit pulp daily, 2 wk) positively affected the body’s redox balance, decreased lipid oxidation, and improved antioxidant status of healthy humans. Supplementation with vitamin C at a comparable dosage enhanced overall antioxidant defence but did not affect body oxidative stress. Oral administration of a water-soluble extract from the fresh fruit pulp resulted in a remarkable anti-inflammatory effect in rats. Betanin and indicaxanthin may be considered as contributors to the fruit effects. Both compounds behave as scavengers of biologically relevant radicals, and antioxidants in various experimental sets in vitro and ex vivo, or may affect redox-sensitive cell transduction pathways in cultured cells. Moreover both are bioavailable. Kinetic studies in healthy humans showed a plasma peak of the micromolar order at 3 h, after a single ingestion of 500 g cactus pear fruit pulp, providing 28 mg and 16 mg indicaxanthin and betanin, respectively. The urinary disposal of indicaxanthin and betanin over 12 h represented 76 % and 3.7 %, respectively, of the ingested compounds. Circulating low density lipoproteins and red blood cells incorporated both betalains time-dependently, in parallel with their plasma concentration, and exhibited enhanced resistance to an ex-vivo-induced oxidation, the higher the amount of betalains the higher the resistance. Because its bioactive phytochemicals, cactus pear may have an important role in health promotion.

Consumption of fruit and vegetables is now associated with protection against various pathologies, including cancer and cardiovascular and cerebrovascular diseases, in which oxidative damage is an important etiologic factor. Various compounds from plant food, acting independently of known nutrients and micronutrients, have appeared very important in protecting human health. Most of these components can act as antioxidants or affect redox-dependent cell activities involved both in physiological and pathological events. In this scenery fruits of cactus pear (Opuntia ficus-indica), and the characteristic betalain pigments have recently attracted the attention of researchers. While suggesting major benefits from diets including cactus pear fruits, to reduce the risk of age-related and degenerative diseases in which destruction of finely tuned redox-regulated transduction pathways may play a pathogenic role, our findings open to new studies directed to possible applications of purified fruit components either as such, or as lead compounds for novel therapeutics.

Pistacia vera L. is the only species of Pistacia genus producing edible nuts. This paper investigates the antioxidant potential of a Sicilian variety of pistachio nut by chemical as well as biological assays and measured antioxidant vitamins and a number of antioxidant polyphenols in either the hydrophilic and/or the lipophilic nut extract. In accordance with the majority of foods, the total antioxidant activity, measured as a TAA test, was much higher (50-fold) in the hydrophilic than in the lipophilic extract. Substantial amounts of total phenols were measured. The hydrophilic extract inhibited dose- dependently both the metal-dependent and -independent lipid oxidation of bovine liver microsomes, and the Cu+2-induced oxidation of human low-density lipoprotein (LDL). Peroxyl radical-scavenging as well as chelating activity of nut components may be suggested to explain the observed inhibition patterns. Among tocopherols, γ-tocopherol was the only vitamin E isomer found in the lipophilic extract that did not contain any carotenoid. Vitamin C was found only in a modest amount. The hydrophilic extract was a source of polyphenol compounds among which trans-resveratrol, proanthocyanidins, and a remarkable amount of the isoflavones daidzein and genistein, 3.68 and 3.40 mg per 100 g of edible nut, respectively, were evaluated. With the exception of isoflavones that appeared unmodified, the amounts of other bioactive molecules were remarkably reduced in the pistachio nut after roasting, and the total antioxidant activity decreased by about 60%. Collectively, our findings provide evidence that the Sicilian pistachio nut may be considered for its bioactive components and can effectively contribute to a healthy status.

Cactus pear fruits have recently been the object of scientific research aimed at revealing bioactive components. This short review summarizes results obtained by our group. The two characteristic betalain pigments, betanin and indicaxanthin, appear promising molecules to be considered nutraceutical compounds.