Indicaxanthin, a multi-target natural compound from Opuntia ficus-indica fruit: From its poly-pharmacological effects to biochemical mechanisms and molecular modelling studies.

Over the latest years phytochemical consumption has been associated to a decreased risk of both the onset and the development of a number of pathological conditions. In this context indicaxanthin, a betalain pigment from Opuntia ficus-indica fruit, has been the object of sound research. Explored, at first, for its mere antioxidant potential, Indicaxanthin is now regarded as a redox-active compound able to exert significant poly-pharmacological effects against several targets in a number of experimental conditions both in vivo and in vitro. This paper aims to provide an overview on the therapeutical effects of indicaxanthin, ranging from the anti-inflammatory to the neuro-modulatory and anti-tumoral ones and favored by its high bioavailability. Moreover, biochemical and molecular modelling investigations are aimed to identify the pharmacological targets the compound is able to interact with and to address the challenging development in the future research.

Indicaxanthin from Opuntia Ficus Indica (L. Mill) impairs melanoma cell proliferation, invasiveness, and tumor progression.

BACKGROUND: A strong, reciprocal crosstalk between inflammation and melanoma has rigorously been demonstrated in recent years, showing how crucial is a pro-inflammatory microenvironment to drive therapy resistance and metastasis. PURPOSE: We investigated on the effects of Indicaxanthin, a novel, anti-inflammatory and bioavailable phytochemical from Opuntia Ficus Indica fruits, against human melanoma both in vitro and in vivo. STUDY DESIGN AND METHODS: The effects of indicaxanthin were evaluated against the proliferation of A375 human melanoma cell line and in a mice model of cutaneous melanoma. Cell proliferation was assessed by MTT assay, apoptosis by Annexin V-Fluorescein Isothiocyanate/Propidium Iodide staining, protein expression by western blotting, melanoma lesions were subcutaneously injected in mice with B16/F10 cells, chemokine release was quantified by ELISA. RESULTS: Data herein presented demonstrate that indicaxanthin effectively inhibits the proliferation of the highly metastatic and invasive A375 cells as shown by growth inhibition, apoptosis induction and cell invasiveness reduction. More interestingly, in vitro data were paralleled by those in vivo showing that indicaxanthin significantly reduced tumor development when orally administered to mice. The results of our study also clarify the molecular mechanisms underlying the antiproliferative effect of indicaxanthin, individuating the inhibition of NF-κB pathway as predominant. CONCLUSION: In conclusion, we demonstrated that indicaxanthin represents a novel phytochemical able to significantly inhibit human melanoma cell proliferation in vitro and to impair tumor progression in vivo. When considering the resistance of melanoma to the current therapeutical approach and the very limited number of phytochemicals able to partially counteract it, our findings may be of interest to explore indicaxanthin potential in further and more complex melanoma studies in combo therapy, i.e. where different check points of melanoma development are targeted.

Betaxanthins and antioxidant capacity in Stenocereus pruinosus: Stability and use in food.

Betalains are important pigments for the food, pharmaceutical, and cosmetics industry. In the yellow Stenocereus pruinosus fruits (pitayas), total betalain concentration, Folin-Ciocalteu reduction capacity, and antiradical capacity per dry weight were 2345.9µgg-1, 7.3mg gallic acid equivalentsg-1, and 48.8µmol Trolox equivalentg-1, respectively. The stability of betaxanthins, which represent 89% of total betalains in yellow pitayas, was evaluated over a range of pH, temperature, as well as in the presence of food additives. Maximum stability was observed at pH6.6, and addition of ascorbic acid increased the half-life 1.8 times. Thermal stability at pH6.48±0.05 was also evaluated from 50°C to 80°C, over which the activation energy for betaxanthin degradation was determined to be 66.2kJmol-1. Model gelatin gummies and beverages were then prepared with pitaya juice or pulp, and pigment retention and color parameters were investigated during storage under various conditions. To match the yellow color of commercial products, gummies were supplemented with 4.6% w/w juice or pulp, and beverages were supplemented with 5% w/v juice, achieving H° values of 69.0-86.2° and 64.6-87.1°, respectively. Results indicate that betaxanthins were more stable in gummies than in beverages, and that pigment retention increased when products were stored in the dark or at low temperatures. Also, different changes in color during storage were observed between gummies and beverages.

Pro-oxidant activity of indicaxanthin from Opuntia ficus indica modulates arachidonate metabolism and prostaglandin synthesis through lipid peroxide production in LPS-stimulated RAW 264.7 macrophages.

Macrophages come across active prostaglandin (PG) metabolism during inflammation, shunting early production of pro-inflammatory towards anti-inflammatory mediators terminating the process. This work for the first time provides evidence that a phytochemical may modulate the arachidonate (AA) metabolism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, promoting the ultimate formation of anti-inflammatory cyclopentenone 15deoxy-PGJ2. Added 1 h before LPS, indicaxanthin from Opuntia Ficus Indica prevented activation of nuclear factor-κB (NF-κB) and over-expression of PGE2 synthase-1 (mPGES-1), but up-regulated cyclo-oxygenase-2 (COX-2) and PGD2 synthase (H-PGDS), with final production of the anti-inflammatory cyclopentenone. The effects were positively related with concentration between 50 and 100 µM. Indicaxanthin did not have any effect in the absence of LPS. A kinetic study investigating the redox status of LPS-stimulated macrophages between 0.5 and 12 h, either in the absence or in the presence of 50-100 µM indicaxanthin, revealed a differential control of ROS production, with early (0.5-3 h) modest inhibition, followed by a progressive (3-12 h) concentration-dependent enhancement over the level induced by LPS alone. In addition, indicaxanthin caused early (0.5-3 h) concentration-dependent elevation of conjugated diene lipid hydroperoxides, and production of hydroxynonenal-protein adducts, over the amount induced by LPS. In LPS-stimulated macrophages indicaxanthin did not affect PG metabolism when co-incubated with either an inhibitor of NADPH oxidase or vitamin E. It is concluded that LPS-induced pro-oxidant activity of indicaxanthin at the membrane level allows formation of signaling intermediates whose accumulation modulates PG biosynthetic pathway in inflamed macrophages.

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16(INK4a) gene in human colorectal carcinoma (Caco-2) cells.

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25 mg fresh pulp equivalents/mL, and 115±15 µM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16(INK4a) gene promoter, reactivation of the silenced mRNA expression and accumulation of p16(INK4a), a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells.

Indicaxanthin inhibits NADPH oxidase (NOX)-1 activation and NF-κB-dependent release of inflammatory mediators and prevents the increase of epithelial permeability in IL-1ß-exposed Caco-2 cells.

Dietary redox-active/antioxidant phytochemicals may help control or mitigate the inflammatory response in chronic inflammatory bowel disease (IBD). In the present study, the anti-inflammatory activity of indicaxanthin (Ind), a pigment from the edible fruit of cactus pear (Opuntia ficus-indica, L.), was shown in an IBD model consisting of a human intestinal epithelial cell line (Caco-2 cells) stimulated by IL-1ß, a cytokine known to play a major role in the initiation and amplification of inflammatory activity in IBD. The exposure of Caco-2 cells to IL-1ß brought about the activation of NADPH oxidase (NOX-1) and the generation of reactive oxygen species (ROS) to activate intracellular signalling leading to the activation of NF-κB, with the over-expression of inflammatory enzymes and release of pro-inflammatory mediators. The co-incubation of the cells with Ind, at a nutritionally relevant concentration (5-25 µM), and IL-1ß prevented the release of the pro-inflammatory cytokines IL-6 and IL-8, PGE2 and NO, the formation of ROS and the loss of thiols in a dose-dependent manner. The co-incubation of the cells with Ind and IL-1ß also prevented the IL-1ß-induced increase of epithelial permeability. It was also shown that the activation of NOX-1 and NF-κB was prevented by Ind and the expression of COX-2 and inducible NO synthase was reduced. The uptake of Ind in Caco-2 cell monolayers appeared to be unaffected by the inflamed state of the cells. In conclusion, our findings suggest that the dietary pigment Ind may have the potential to modulate inflammatory processes at the intestinal level.

Separation of polar betalain pigments from cacti fruits of Hylocereus polyrhizus by ion-pair high-speed countercurrent chromatography.

Polar betacyanin pigments together with betaxanthins from ripe cactus fruits of Hylocereus polyrhizus (Cactaceae) were fractionated by means of preparative ion-pair high-speed countercurrent chromatography (IP-HSCCC) also using the elution-extrusion (EE) approach for a complete pigment recovery. HSCCC separations were operated in the classical 'head-to-tail' mode with an aqueous mobile phase. Different CCC solvent systems were evaluated in respect of influence and effectiveness of fractionation capabilities to separate the occurring pigment profile of H. polyrhizus. For that reason, the additions of two different volatile ion-pair forming perfluorinated carboxylic acids (PFCA) were investigated. For a direct comparison, five samples of Hylocereus pigment extract were run on preparative scale (900 mg) in 1-butanol-acetonitrile-aqueous TFA 0.7% (5:1:6, v/v/v) and the modified systems tert.-butyl methyl ether-1-butanol-acetonitrile-aqueous PFCA (2:2:1:5, v/v/v/v) using 0.7% and 1.0% trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) in the aqueous phase, respectively. The chemical affinity to the organic stationary CCC solvent phases and in consequence the retention of these highly polar betalain pigments was significantly increased by the use of the more lipophilic fluorinated ion-pair reagent HFBA instead of TFA. The HFBA additions separated more effectively the typical cacti pigments phyllocactin and hylocerenin from betanin as well as their iso-forms. Unfortunately, similar K(D) ratios and selectivity factors alpha around 1.0-1.1 in all tested solvent systems proved that the corresponding diastereomers, 15S-type pigments cannot be resolved from the 15R-epimers (iso-forms). Surprisingly, additions of the stronger ion-pair reagent (HFBA) resulted in a partial separation of hylocerenin from phyllocactin which were not resolved in the other solvent systems. The pigments were detected by means of HPLC-DAD and HPLC-electrospray ionization-MS using also authentic reference materials.

Development of a protocol for the semi-synthesis and purification of betaxanthins.

A method for the analytical and semi-preparative chromatographic purification of betaxanthins is described together with an improved procedure for the semi-synthesis of these compounds from betalamic acid. Standard conditions for obtaining preparative amounts of betaxanthins free of the precursor amino acids are provided. Following this procedure, 14 pure betaxanthins were obtained with yields of up to 100%. A simple reversed-phase HPLC protocol for pigment identification and quantification is also provided. Calibration for betaxanthins is reported for the first time using the synthesised and purified pigments as standards. Structures were confirmed by UV-vis spectroscopy, HPLC retention times and electrospray ionization mass spectrometry. Betaxanthins can be obtained pure, and in sufficient amounts for further studies, which opens up new perspectives in the research and applications of these pigments.

Betaxanthins as pigments responsible for visible fluorescence in flowers.

Betalains are water-soluble nitrogen-containing pigments present in flowers and fruits of plants of the order Caryophyllales, where they replace anthocyanins. This article describes how flowers containing yellow betaxanthins are fluorescent. Betaxanthins exhibit spectra with excitation maxima between 463 nm and 474 nm and emission maxima between 509 nm and 512 nm. Thus, betaxanthins are able to absorb blue light and emit green light. Relations between fluorescence and the structural properties of the pigments are discussed. For the first time, pictures of flowers naturally emitting light are presented. Yellow flowers of the ornamental plant Portulaca grandiflora were chosen as a model for the studies in fluorescence due to the existence of the white phenotype, which was used as a control. Studies were also performed in Lampranthus productus flowers, which contain dopaxanthin as a single pigment. The visible fluorescence of betaxanthins inside the petal cells was detected in a confocal microscope after laser excitation.