International Journal of Molecular Sciences Article Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L. Virginia Brighenti 1, Ramona Iseppi 1 , Luca Pinzi 1, Annamaria Mincuzzi 2, Antonio Ippolito 2 , Patrizia Messi 1 , Simona Marianna Sanzani 3, Giulio Rastelli 1,* and Federica Pellati 1,* 1 Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 103/287, 41125 Modena, Italy; [email protected] (V.B.); [email protected] (R.I.); [email protected] (L.P.); [email protected] (P.M.) 2 Department of Soil, Plant and Food Sciences, University of Bari Aldo Moro, Via Amendola 165/A, 70126 Bari, Italy; [email protected] (A.M.); [email protected] (A.I.) 3 CIHEAM-Bari, Via Ceglie 9, 70010 Valenzano, Italy; [email protected] * Correspondence: [email protected] (G.R.); [email protected] (F.P.); Tel.: +39-059-2058564 (G.R.); +39-059-2058565 (F.P.) Abstract: Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target Citation: Brighenti, V.; Iseppi, R.; of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics Pinzi, L.; Mincuzzi, A.; Ippolito, A.; was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration Messi, P.; Sanzani, S.M.; Rastelli, G.; (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as Pellati, F. Antifungal Activity and potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition DNA Topoisomerase Inhibition of assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the Hydrolysable Tannins from Punica Alternaria granatum L.. Int. J. Mol. Sci. 2021, 22, fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for 4175. https://doi.org/10.3390/ alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth ijms22084175 control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC Academic Editor: coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able Marie-Laure Fauconnier to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I Received: 12 March 2021 and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate Accepted: 10 April 2021 to be further exploited as an antifungal agent in particular against human fungal infections. Published: 17 April 2021 Keywords: Punica granatum L.; pomegranate; punicalagin; antifungal activity; Candida albicans; Publisher’s Note: MDPI stays neutral topoisomerase with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction It is well-known that natural compounds derived from plants represents a rich source of privileged chemical entities for drug discovery, since they are characterized by favorable Copyright: © 2021 by the authors. chemical properties, a high structural diversity, and positive effects on human health [1,2]. Licensee MDPI, Basel, Switzerland. This article is an open access article A recent literature review has identified nearly a hundred novel natural products having distributed under the terms and promising antifungal activity against human pathogens [2]. These compounds originate conditions of the Creative Commons from a variety of organisms, comprising bacteria, algae, fungi, sponges, and plants [2]. Attribution (CC BY) license (https:// Focusing on natural antifungal compounds derived from plants, previous studies have creativecommons.org/licenses/by/ demonstrated that Punica granatum L. (pomegranate) extracts rich in punicalagin present 4.0/). Int. J. Mol. Sci. 2021, 22, 4175. https://doi.org/10.3390/ijms22084175 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 20 Int. J. Mol. Sci. 2021, 22, 4175 2 of 18 ditions of the Creative Commons At- studies have demonstrated that Punica granatum L. (pomegranate) extracts rich in puni- tribution (CC BY) license (http://crea- calagin present compelling antifungal activity [3,4], even though the mechanism/s of ac- tivecommons.org/licenses/by/4.0/). tion behindcompelling this activity antifungal has never activity been [3,4 fully], even elucidated. though the mechanism/s of action behind this Inactivity a recent has study, never pomegranate been fully elucidated. cultivars with different susceptibility to the fungal etiological Inagent a recent of fruit study, dry rot pomegranate Coniella granati cultivars, also known with different as Pilidiella susceptibility granati, were to tested the fungal for theiretiological phenolic agent profile. of fruit The dry HPLC rot Coniella analyses granati of the, also extracts known indicated as Pilidiella the granati presence, were of tested characteristicfor their peaks phenolic in profile.the fungal The inoculated HPLC analyses samples, of the corresponding extracts indicated to phenolic the presence com- of char- poundsacteristic derived peaks from inthe the degradation fungal inoculated of punicalagin, samples, which corresponding is the main to ellagitannin phenolic compounds pre- sent inderived this fruit from [5]. the Since degradation C. granati ofis punicalagin,fairly able to whichenzymatically is the main lysate ellagitannin this classpresent of com- in this pounds,fruit it is [5 ].reasonable Since C. granati that theis phenolic fairly able prod to enzymaticallyuction is a response lysate thisto the class pathogen of compounds, attack, it is i.e., thereasonable fungus hydrolyses that the phenolic them in production a positive feedback is a response mechanism to the pathogen that ends attack, up in i.e., a minor the fungus susceptibilityhydrolyses of some them invarieties a positive [5]. feedbackThis finding mechanism supports that also ends the up hypothesis in a minor that susceptibility pome- of granatesome polyphenols varieties [ 5might]. This play finding a role supports in the different also the hypothesis susceptibility that of pomegranate pomegranate polyphenols cul- tivars mightto C. granati play a, role their in content the different being susceptibility a putative marker of pomegranate in selecting cultivars programs to C. of granati new, their resistantcontent germplasm being a [5]. putative marker in selecting programs of new resistant germplasm [5]. Punicalagin,Punicalagin, highly highly present present in pomegranate in pomegranate rind, rind, is the is theputative putative precursor precursor of ofpuni- punicalin calin (Figure(Figure 11))[ [6].6]. Gallagic Gallagic acid acid is is an an intermediate intermediate molecule, molecule, which which in in turn turn is is generated generated after after punicalinpunicalin degradation, degradation, and and is is the the precursor precursor of of ellagic ellagic acid, acid, which which is the is the last last product product of of ellegitanninellegitannin biodegra biodegradationdation (Figure (Figure 1) [6].1)[ 6]. Figure 1. BiodegradationFigure 1. pathway Biodegradation of punicalagin. pathway of punicalagin. The surgeThe surgein resistance in resistance to fungicides to fungicides among among pathogenic pathogenic populations populations is an emergent is an emergent issue inissue both in agriculture both agriculture and medicine and medicine [7], an [d7], a andclear a example clear example of the ofimportance the importance of a One of a One HealthHealth approach. approach. As for As human for human pathogenic pathogenic fungi,fungi, several several classes classes of antifungal of antifungal drugsdrugs are are in clinicalin clinical use, but use, drug-resistant but drug-resistant CandidaCandida albicans albicans and toxicity-relatedand toxicity-related reactions reactions to existing to existing compoundscompounds strongly strongly suggest suggest that new that therapeu new therapeutictic agents agents are required are required [7]. The [7]. emergence The emergence of fungicide-resistantof fungicide-resistant isolates isolates of human of human pathogens pathogens has been has related been related to the to exposure the exposure to to triazoletriazole fungicides fungicides used in used agro-ecosystems in agro-ecosystems [7]. The [7]. low The number low number of antifungal of antifungal agents agents in in clinicalclinical use, as use, opposed as opposed to the tolarge the number large number of agricultural of agricultural fungicides fungicides with similar with similar mode mode of action,of action, is considered is considered as a risk as afactor risk factorthat limits that limitsthe success the success of the of therapeutic the therapeutic use of use of existing antifungal