Polyphenol ellagic acid (EA) possesses antioxidant, anti-inflammatory, anti-carcinogenic, anti-diabetic and cardio protection activities, making it an interesting multi-targeting profile. EA also controls the central nervous system (CNS), since it was proven to reduce the immobility time of mice in both the forced swimming and the tail-suspension tests, with an efficiency comparable to that of classic antidepressants. The proposed mechanism revealed that EA mimics clonidine at the presynaptic release-regulating α2 autoreceptors in hippocampal noradrenergic nerve endings.
pomegranate tannins;ellagic acid;molecular modelling;α2-adrenoreceptors;α2-ARs;molecular dynamic simulations;antioxidant;natural compounds;antidepressant activity;food chemistry
Pomegranate (Punica granatum
L.) is a very ancient edible fruit originating in the Middle East and North Africa, used from the dawn of history as a healing and health-promoting fruit in traditional medicine 
. In the past decades, this rustic crop has obtained high popularity as a nutraceuticals source, becoming a high-value crop. Moreover, it has demonstrated increased importance due to its adaptability to different climatic conditions, its resilience and longevity, and its high drought and salinity resistance 
Today, pomegranate cultivation is widely spread in many tropical and subtropical regions, and about two million tons of fruits are produced annually worldwide 
. Particularly, India, Iran, China, Turkey, the United States, Spain, South Africa, Peru, Chile, and Argentina represent the major producers and exporters of this fruit 
Pomegranate fruit is of great economic and nutritional interest, and it is in high demand due to its wide range of industrial uses, especially for direct consumption, for juice production, and oil extraction from its seeds 
. The nutritional value of pomegranate is linked to its naturally high content of phenolic compounds with antioxidant properties 
. Many studies, mainly in vitro, demonstrated the health benefits and the functional properties of this fruit in the prevention of several peripheral disorders such as cancer, cardiovascular diseases, chronic inflammatory diseases, and metabolic disorders (i.e., diabetes, obesity) 
. Moreover, the beneficial effects of pomegranate phenolic compounds on central neuroinflammatory and neurodegenerative pathologies, including multiple sclerosis, Alzheimer’s disease, Parkinson’s diseases, epilepsy, and depression, have been highlighted too [7,8,9]
The main industrial product from pomegranate is its juice, obtained by aril squeezing, which represents the edible portion of the fruit, but recently there has been an increased focus on pomegranate by-products as a source of nutraceuticals. It has been determined that by-products, especially the peels, have higher levels of bioactive compounds and antioxidant activity than juice, opening new possibilities for pomegranate manufacturers to recover and exploit these by-products in a zero-waste economy perspective 
. A major class of compounds in pomegranate fruit is the hydrolysable tannins, including ellagic acid, punicalagin, and gallic acid 
Ellagic acid (EA) is a chromene-dione derivative (3,7,8-tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione), which is derived from the spontaneously lactonization of hexahydroxydyphenic acid (HHDP) 
. In its free form, or as constituent of ellagitannins (ETs), or conjugates with different monosaccharides (EA-glycosides), EA is considered the main phenolic compound responsible for the numerous health properties of pomegranate 
. ETs are esters of gallic acid (GA) and hexahydroxydiphenic acid (HHDP) units, connected with mainly β-d
-glucose as sugar residue. Punicalagin (2,3-(S)-hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-d
-glucose), a large molecule consisting of ellagic acid and gallagic acid linked via a glucose unit, is the most abundant ET in pomegranate and it is specific to the Punica
. Data from preclinical studies in the literature support the healthy properties of both EA and ETs. These include the ability to interfere with tumor cell proliferation, the cell cycle, invasion and angiogenesis by making it a multi-target candidate for various cancer treatments 
. It also places a particular emphasis on the role of EA in central inflammatory and (auto)-immunological diseases, but also for depression, anxiety and aged-related neurological impairments [7,14,15,16]
Figure 1. The polypharmacological effects regulated by ellagic acid contained within the pomegranate.
As far as the mood disorders are concerned, EA was proven to reduce the immobility time of mice in both the forced swimming and the tail-suspension tests, with an efficiency comparable to that of classic antidepressants 
. Interestingly, the anti-depressant-like activity was almost nulled by the concomitant administration of selective antagonists of the noradrenergic receptors (namely the α1
, the α2
and the β receptors) and by modulators of the serotonergic systems as well (including receptor antagonists and synthesis inhibitors) [17
suggesting the involvement of these cellular targets in the central effects elicited by EA and its derivatives.
Among the noradrenergic receptors, we focussed on the α2
-ARs) that, in the central nervous system (CNS), act as presynaptic inhibitory autoreceptors in noradrenergic nerve endings/varicosities [19,20]
. The α2
-ARs are indirectly tuned by antidepressants acting as noradrenaline (NA) re-uptake inhibitors (NRI) 
. By increasing NA bioavailability in the synaptic cleft, these drugs cause the continuous stimulation of the presynaptic α2
-Ars, leading to their down regulation. A comparable outcome also can be triggered by the continuous direct activation of the presynaptic α2
-ARs with agonists. In both cases, the final outcome is the silencing of the presynaptic mechanism of autocontrol of the release of the biogenic amine and, consequently, the reinforcement of the noradrenergic transmission [22,23,24]
. Notably, the α2
-ARs also exist in astrocytes, where they control the phenotype of the glial cells favouring the non-inflammatory one 
; whether and how these receptors desensitize was so far scarcely investigated.
-ARs are G protein coupled receptors (GPCRs) negatively associated to the adenylyl cyclase (AC) that reduce the gating of the voltage-operated calcium channels (VOCCs), concomitantly favouring the opening of the K+
-channels, to inhibit cellular functions. There are three characterized α2
-AR subtypes, the α2A
-AR and α2C
, which are well conserved across mammals and are differently distributed in postganglionic sympathetic neurons and CNS noradrenergic neurons.
-ARs and the α2C
-ARs are mainly expressed in noradrenergic neuronal projections from the Locus coeruleus to other central regions, with higher expression on the varicosities in dendritic and axonal processes, as well as in nerve terminals 
. The α2A
-ARs and the α2C
-ARs are also present peripherally, on postganglionic sympathetic neurons, where again they act as inhibitory autoreceptors. Differently, the α2B
-ARs are preferentially expressed in the periphery, their presence in the CNS still representing a matter of debate 
2. Computational Studies
In silico studies were carried out to explore the binding modes of EA into α2A
-ARs and α2C
-AR catalytic pockets. In addition to this, these specific docking studies were carried out to investigate the possible differences and similarities of the interactions established between a known agonist (clonidine) or antagonist (yohimbine) with the receptors. For the current analysis, the crystal structures of the human α2A
-AR in complex with the indole derivative, a partial agonist (PDB code: 6KUY) 
-AR in complex with the naphthyridine derivative, an antagonist (PDB code: 6KUX) 
, and, α2C
-AR in complex with the naphthyridine derivative, an antagonist (PDB code: 6KUW 
, were used.
2.1. Molecular Docking Studies
For each structure, the docking protocol was validated by docking the co-crystallized ligand into the binding site (Figure 32
a,b). Root mean square deviation (RMSD) values between the native pose of α2A
ligands and the related best re-docked conformations were found to be 0.19 Å (Figure 32
c), 0.89 Å (Figure 32
d), and 0.39 Å (Figure 32
e), respectively, thus revealing the reliability of docking protocol (Figure 32
Figure 32. (a) Top view: Ribbon representation of overlaid binding orientation of co-crystallized ligands into the binding site of α2A-AR6KUY (orange), α2A-AR6KUX (green) and α2C-AR6KUW (magenta) structures; (b) Front view of the superposed α2A-C-ARSs. Three-dimensional superimposition between the re-docked pose (blue carbon ball and sticks) and the conformation of the native ligand; (c) Indole derivative into the binding site of 6KUY; (d) Naphthyridine derivative into both 6KUX (green) and (e) 6KUW (magenta) X-ray structures.
The three ligands were docked to the minimized structures of the α2
-ARs subtypes A and C, respectively (Table 1
). Regarding yohimbine in complex to α2A
, it was observed that it was able to better recognize the orthosteric pocket of the receptor, compared to EA and clonidine. In detail, the positively charged amine group in the pyrido[1,2-b]isoquinoline displays a hydrogen bond with D113 (1.81 Å) and a π-cation interaction with F412. The hydroxyl and the carboxylic group create a hydrogen bond with Y98 side chain and I190 backbone, respectively. Furthermore, the indole moiety enhances the binding by two π-π interactions with F390 and F391 (Figure 43
Figure 43. Key contacting elements inside (a–c) the α2A-AR6KUX/clonidine, (d–f) the α2A-AR6KUX/EA, and (g–i) the α2A-AR6KUX/yohimbine best docked pose. Panels (b,e,h) show all side chains involved in H-bonds (violet), π-π interactions (cyan) and π-cation interactions (red) in stick representation. Panels (a,d,g) show the surface area of α2A-AR6KUX complexed to clonidine, EA and yohimbine, respectively. The surface area of the receptor is shown in solid orange solid. 2D representation of the key interactions of (c) clonidine, (f) EA and (i) yohimbine into the α2A-AR6KUY structure.
Table 1. Glide score, calculated in kcal/mol, of the best yohimbine, EA and clonidine docked pose towards α2A-AR6KUX, α2A-AR6KUY and α2C-AR6KUW structures.
|Glide SP Score *
||Glide SP Score *
||Glide SP Score *