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Journal of Agricultural and Food Chemistry
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Indicaxanthin from Opuntia Ficus Indica crosses the blood-brain barrier and modulates
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neuronal bioelectric activity in rat hippocampus at dietary-consistent amounts.
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Mario Allegra, Fabio Carletti, Giuditta Gambino, Marco Tutone, Alessandro Attanzio, Luisa
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Tesoriere, Giuseppe Ferraro, Pierangelo Sardo, Anna Maria Almerico, Maria Antonia Livrea.
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Palermo, Via M. Cipolla 74, 90121- Palermo - Italy.
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Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, University of
Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, Sezione di Fisiologia Umana
“G.Pagano”, University of Palermo, Corso Tukory 129, 90134 - Palermo - Italy
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Corresponding author: Maria A. Livrea, Dipartimento di Scienze e Tecnologie Biologiche,
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Chimiche e Farmaceutiche, University of Palermo, Via M. Cipolla 74, 90121- Palermo - Italy.
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Phone: +39 091 23896803, email address:
[email protected] 15 16
Acknowledgement: The present study has been supported by grants from the Ministero
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dell’Istruzione, Università e Ricerca (University of Palermo, Fondi di Ateneo ex 60 %).
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Abstract
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Indicaxanthin is a bioactive and bioavailable betalain pigment from the Opuntia ficus indica fruits.
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In this in vivo study, kinetic measurements showed that indicaxanthin is revealed in the rat brain
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within 1h from oral administration of 2 µmol/kg, an amount compatible with a dietary consumption
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of cactus pear fruits in humans. A peak (20 ± 2.4 ng indicaxanthin per whole brain) was measured
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after 2.5 h, thereafter the molecule disappeared with first order kinetics within 4 h. The potential of
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indicaxanthin to affect neural activities was in vivo investigated by a microiontophoretic approach.
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Indicaxanthin, administered in a range between 0.085 ng and 0.34 ng per neuron, dose-dependently
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modulated the rate of discharge of spontaneously active neurons of the hippocampus, with
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reduction of the discharge and related changes of latency and duration of the effect. Indicaxanthin
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(0.34 ng/neuron) showed inhibitory effects on glutamate-induced excitation, indicating activity at
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the level of glutamatergic synapses. A molecular target of indicaxanthin is suggested by in silico
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molecular modeling of indicaxanthin with N-methyl-D-aspartate receptor (NMDAR), the most
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represented of the glutamate receptor family in hippocampus. Therefore at nutritionally compatible
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amounts indicaxanthin i) crosses the rat BBB and accumulates in brain; ii) can affect the bioelectric
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activity of hippocampal neurons locally treated with amounts comparable with those measured in
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the brain and iii) modulates glutamate-induced neuronal excitation. The potential of dietary
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indicaxanthin as a natural neuromodulatory agent deserves further mechanistic and
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neurophysiologic investigation.
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Keywords: indicaxanthin, phytochemicals, BBB, electrophysiology, hippocampus,
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microiontophoresis, molecular modeling.
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Journal of Agricultural and Food Chemistry
Introduction A number of dietary phytochemicals are now accepted as important factors to maintain health and
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decrease risk of diseases. In this context, indicaxanthin ((2S)-2,3-dihydro-4-[2-[(2S)-2-
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carboxypyrrolidin-1-yl]ethenyl]pyridine-2,6-dicarboxylic acid, Fig.1), a betalain pigment from
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the fruit of cactus pear, has been the object of sound experimental work in recent years. As many
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phytochemicals indicaxanthin is a redox-active compound1 and has been shown to act as
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antioxidant in a number of in vitro studies in solution and in cells1-8. Interestingly, because of
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charged portions, ionizable groups and lipophilic moieties the molecule is amphiphilic at
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physiological pH9. This is critical to drive interaction with the cell surface10, 11, where a bioactive
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compound binds or interacts with lipids and/or membrane effectors to initiate cell signalling
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transduction or crosses the cell membrane to reach inner sites. Indicaxanthin indeed has appeared to
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exert signalling activity, being able to modulate specific redox-driven pathways involved in
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macrophage apoptosis, epithelial dysfunction and endothelial activation in vitro12-14. Remarkably,
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and in contrast with the majority of dietary phytochemicals, indicaxanthin is highly bioavailable.
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The molecule, that has been shown to cross unaltered an intestinal epithelial cell monolayer and is
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absorbed through paracellular junctions9, has been found in human plasma at a 7 µM peak
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concentration, 3h after the ingestion of four cactus pear fruits containing 28 mg7. Moreover, the oral
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administration of a dose of pure indicaxanthin (2 µmol/kg), equivalent to a dietary amount ingested
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with cactus pear fruits in humans7, generated in rats a plasma peak concentration of 0.2 µM, that
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appeared to be safe and exhibited a strong anti-inflammatory effect in a carrageenan-induced acute
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inflammation model15.
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While consumption of most phytochemicals has been associated to an extended life span16, the
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intake of quite a few has been reported to exert neuroprotective effects17, mainly due to the sealing
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nature of the blood brain barrier (BBB) that shields brain from the majority of xenobiotics18.
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Among all flavonoids, flavanols and anthocyanins are the only that have been reported to cross the
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BBB, accumulate within several regions of the brain, and modulate memory and learning ACS Paragon Plus Environment
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proceses17-19. However, because of limited bioavailability, these molecules are poorly absorbed in
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the brain, especially anthocyanins17. On the other hand many studies claimed accumulation of
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phytochemicals assayed at amounts much higher than those ingested with a normal diet20.
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Taking into account its bioavailability in rats15, this work first researched whether indicaxanthin
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crossed the BBB and accumulated in the rat brain. In addition, eventual action of the molecule on
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the neuronal function in the central nervous system (CNS), in particular the hippocampus, was
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investigated. This study was carried out exploiting recording techniques associated to
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microiontophoresis, a current-based approach for direct drug delivery, applied to locally investigate
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the neuromodulatory effects of molecular messengers on the electrophysiological properties of CNS
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neurons in vivo21. Indicaxanthin activity in the framework of glutamatergic synaptic system has
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been considered on the basis of inhibition studies in the presence of glutamate. The impact of
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indicaxanthin on the neuronal bioelectric activity in a structure involved in basic physiological
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processes is discussed.
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Materials and Methods
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Reagents
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Unless stated otherwise, all reagents were from Sigma-Aldrich (Milan, Italy) and of the highest
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grade commercially available.
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Extraction and purification of indicaxanthin from cactus pear fruits.
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Indicaxanthin was isolated from cactus pear (Opuntia ficus-indica) fruits (yellow cultivar). Briefly,
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the phytochemical was separated from a methanol extract of the pulp by liquid chromatography on
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Sephadex G-25 as previously reported1. Fractions containing the pigment were submitted to
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cryodesiccation, purified as described1, quantified by HPLC as below reported and suspended in
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PBS for the experiments.
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Animals and surgery.
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Adult male Wistar rats (Morini, Milan, Italy) weighing 220–280 g were used in all experiments.
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Animals were fed and had access to water ad libitum. The light cycle was automatically controlled
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(on at 07.00, off at 19.00) and the room temperature was thermostatically regulated at 22°±1°C.
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Before the experiments, rats were housed and acclimatized under these conditions for 3–4 d. In each
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experimental step, all efforts were made to minimize the number of animals used and their
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sufferance. All experiments were performed in strict accordance with the European Directive
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(2010/63/EU) on animal experimentation and with the National Institute of Health Guide for the
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Care and Use of Laboratory Animals (1986).
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As for pharmacokinetic studies, the animals were anaesthetized on the day of the experiment with
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urethane at the dose of 1.2 g/kg (i.p.). One group received indicaxanthin (2 µmol/kg) by oral
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gavage, while control rats receiving saline alone. Animals were sacrificed at 1, 2.5, 3, 4 and 5 h
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after indicaxanthin administration and brain samples were collected 15 and processed as described
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below. Each experiment was performed three times with groups of 10 rats. Data from separate
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experiments were pooled.
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With regards to electrophysiological experiments, after having been anaesthetized as above
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reported, rats were positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA). A
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3-mm burr hole was drilled in the skull, 5 mm anterior to the interaural line and 2.4 mm lateral to
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the midline. Physiological parameters were monitored throughout all experimental sessions.
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Quantification of indicaxanthin in brain samples
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The amount of indicaxanthin within brain samples was evaluated at selected time intervals after a
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single oral administration of the pigment, as reported below. Briefly, animals were perfused with
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normal saline to remove any compounds still circulating in the blood. Brains (10 per time point)
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were then isolated, carefully washed with saline, pooled and homogenized in PBS. Indicaxanthin
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was extracted from samples with chloroform:methanol, 2:1, by vol (1g tissue with 3 volumes of ACS Paragon Plus Environment
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extraction mixture). The methanol phase from all samples at each time point was dried under
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nitrogen, resuspended in 1% acetic acid in water, analyzed on a Varian Microsorb C-18 column (4.6
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x 250 mm; Varian, Palo Alto, CA) and eluted with a 20-min linear gradient elution from solvent A
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(1% acetic acid in water) to 20% solvent B (1% acetic acid in acetonitrile) with a flow rate of 1.5
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mL/min. Spectrophotometric revelation was at 482 nm. Under these conditions, indicaxanthin
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eluted after 8.15 min and was quantified by reference to standard curves constructed with 5–100 ng
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purified compounds and by relating its amount to the peak area.
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Electrophysiological recordings
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A seven-barrel glass microelectrode was directed stereotaxically to the hippocampus for both
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recording and drug ejection (4.8–5.8 mm anterior to the interaural line, 1–3 mm lateral to the
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midline, 2.4–4 mm ventral to the cortical surface)22. The system was set as previously reported23.
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The center recording barrel (1.1-2.0 MΩ resistance) was filled with 2 M NaCl with 1% Fast Green
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(Sigma), one side barrel was filled with 2 M NaCl 0.9% buffered saline solution (for automatic
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current balancing) and the others with 12 mM, 6 mM and 3 mM indicaxanthin (pH 7.4) and 100
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mM L-glutamic acid monosodium salt (GLU, pH 8).
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Retaining currents of 8–10 nA (positive for GLU and negative for the indicaxanthin solutions) were
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applied to drug barrels (20–70 MΩ) (Neurophore BH-2 System, Harvard Apparatus, Hamden, CT).
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At the beginning of each track (0.5 mm ventral to the cortical surface), maximal ejection currents
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were applied to each barrel for 30 min, to concentrate the drug at the pipette tip. Electrical signals
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were amplified, filtered, acquired and stored on a computer together with their temporal markers
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(time precision 100 s) for off-line analysis as previously described24. A ratemeter histogram of
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neuronal activity was continuously displayed and updated every 5 s on the computer screen with a
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counter window, to detect variations of neuronal firing rate on-line. All computer operations were
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performed using the SciWorks package, version 5.0 (Datawave Technologies, Loveland, CO).
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Neuronal units were not added to the database if marked changes in amplitude or configuration of ACS Paragon Plus Environment
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the spike were observed, or if there was early death of the cell in the course of recording. Baseline
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activity of each neuron was recorded for 3-5 min before acute vehicle or drug administration.
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Indicaxanthin treatment
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Microiontophoretic preliminary procedures were previously described24. Drug-test recordings were
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carried out at increasing ejection currents to both identify the kinetics of phytochemical action and
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the minimal current exerting an effect on all responsive neurons. For this purpose, 90 s pulses at 40,
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60 and 80 nA ejection currents were tested, with a 90 s inter-pulse interval between pulses to
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prevent any influence of the previous pulse on the following one. After this step, all recorded
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neurons underwent the iontophoretic administration of indicaxanthin (in the concentration range
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reported above) for 5 min at the current of 60 nA in order to outline a correlation between the
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modifications of neuronal response and the concentration of the drug. Once the application of
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indicaxanthin was terminated, recording continued until the spontaneous firing rate recovered to
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baseline. Control tests, applying the same ejection currents used for the treatments, were performed
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with vehicle solutions; no changes in the neuronal firing were recorded. It is worth to highlight that
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ejection times and currents in our microiontophoretic approach were set to result in an actual
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amount of indicaxanthin interacting with single neurons in a ng range compatible with the amount
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measured in the rat brain after the ingestion of the pigment. Indeed, indicaxanthin interacting with a
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single neuron was approximately 0.34 ng (for 12 mM), 0.17 ng (for 6 mM) and 0.085 ng (for 3
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mM).
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When required, the group of neurons that had received 0.34 ng indicaxanthin per neuron were
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tested in co-administration with GLU; namely, at the recovering of baseline, GLU was ejected (40
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nA ejection current)25 before and during subsequent administration of the phytochemical and
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responses were assessed. For all groups, only data from cells exhibiting recovery at the end of
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treatments were included in statistical analysis.
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At the end of each electrophysiological experiment, the recording sites were marked with Fast
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Green through the electrode by using a 50 A ejection current for 15 min. Coronal frozen sections
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were cut at 50 m and stained with cresyl violet for histological verification and reconstruction of
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recording sites.
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Preparation of protein and ligand
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The crystal structure of amino terminal domains of the N-methyl-D-aspartate (NMDA) glutamate
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receptor dimeric GluN1/GluN2B subunit, in complex with antagonist Ifenprodil (Pdb:3QEL)26, and
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dimeric GluN1/GluN2A subunit in complex with either D-(-)-2-amino-5-phosphonopentanoic acid
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(D-AP5) (Pdb:4NF5), or 1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA)
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(Pdb:4NF6)27, were retrieved from the RCSB Protein Data Bank. The starting coordinates of the
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crystal complexes were subjected to protein preparation using the protein preparation wizard of
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Glide software 5.9 (Schrodinger, LLC, New York, NY). The proteins were minimized by applying
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the OPLS-2005 force field. Water molecules and other non-ligand molecules were deleted, and the
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structures refined by means the optimization of H-bond assignment, and the minimization until the
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average root mean square deviation of the non-hydrogen atoms reached 0.3 Å.
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The structures of the ligands were then extracted from the X-ray complexes, and appropriate bond
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order was assigned using the LigPrep software 2.6 (Schrodinger, LLC, New York, NY). The same
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treatment was carried out on indicaxanthin, generating all the possible states and tautomers at pH =
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7.0 ± 2.0
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Docking experiments
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The ligands were extracted and docked by the Glide XP high performance docking procedure, to
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evaluate if the docking algorithm fits the crystallized conformation28-31.
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Prime/MM-GBSA software 3.2 (Schrodinger, LLC, New York, NY) was used to calculate the free
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energy of binding between the receptor subunits and either the ligands or indicaxanthin, according
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to the default parameters for in water estimation.
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Computational mutagenesis
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Alanine scanning studies were carried out using the Mutate Residue script from Maestro
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(Schrodinger, LLC, New York, NY). Computational mutagenesis is an approach used to find out
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the contribution of specific residues to the protein function by mutating the original residues to
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alanine32 to recognize the structural and energetic characteristics of the hotspots. The mutated
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systems were not minimized, and it was assumed that no local rearrangements occur after the
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mutation. The interacting residues with Indicaxanthin were mutated to alanine, and the binding free
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energy re-calculated.
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Statistical Analysis
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Data from electrophysiological study were analyzed using 1-factor ANOVA, followed by a
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Bonferroni’s test. Neuronal firing rate was off-line analysed before, during and after drug
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administration for each recorded unit. Individual rate-meter histograms (5 s bin width) were
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analysed by means of a non-parametric Mann–Whitney U test to detect any statistically significant
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treatment-related change in neuronal firing. To analyse the effects induced by treatments, neurons
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were considered responsive if changes were significant (probability level P
