Usnic Acid in Cladonia Foliacea
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molecules Article Microwave-Assisted Extraction and HPLC-UV-CD Determination of (S)-usnic Acid in Cladonia foliacea Valeria Cavalloro 1,†, Giorgio Marrubini 2,† , Rita Stabile 2, Daniela Rossi 2 , Pasquale Linciano 2,* , Gabriele Gheza 3, Silvia Assini 1, Emanuela Martino 1,* and Simona Collina 2 1 Department of Earth and Environmental Sciences, University of Pavia, 27100 Pavia, Italy; [email protected] (V.C.); [email protected] (S.A.) 2 Department of Drug Sciences, University of Pavia, 27100 Pavia, Italy; [email protected] (G.M.); [email protected] (R.S.); [email protected] (D.R.); [email protected] (S.C.) 3 Department of Biological Geological and Environmental Sciences University of Bologna, 40126 Bologna, Italy; [email protected] * Correspondence: [email protected] (P.L.); [email protected] (E.M.) † Authors contributed equally to this work. Abstract: During the years, many usnic acid (UA) conjugates have been synthesized to obtain potent endowed with biological properties. Since (S)-UA is less abundant in nature than (R)-enantiomer, it is difficult to source, thus precluding a deeper investigation. Among the lichens producing UA, Cladonia foliacea is a valuable (S)-UA source. In the present work, we report on a rapid HPLC-UV/PAD- CD protocol suitable for the analysis and the identification of the main secondary metabolites present in C. foliacea extract. Best results were achieved using XBridge Phenyl column and acetonitrile and water, which were both added with formic acid as mobile phase in gradient elution. By combining analytical, spectroscopical, and chiroptical analysis, the most abundant analyte was unambiguously identified as (S)-UA. Accordingly, a versatile microwave-assisted extractive (MAE) protocol, assisted Citation: Cavalloro, V.; Marrubini, G.; Stabile, R.; Rossi, D.; Linciano, P.; by a design of experiment (DoE), to quantitatively recover (S)-UA was set up. The best result in ◦ Gheza, G.; Assini, S.; Martino, E.; terms of UA extraction yield was obtained using ethanol and heating at 80 C under microwave Collina, S. Microwave-Assisted irradiation for 5 min. Starting from 100 g of dried C. foliacea, 420 mg of (S)-UA were achieved. Thus, Extraction and HPLC-UV-CD our extraction method resulted in a suitable protocol to produce (S)-UA from C. foliacea for biological Determination of (S)-usnic Acid in and pharmaceutical investigation or commercial purposes. Cladonia foliacea. Molecules 2021, 26, 455. https://doi.org/10.3390/ Keywords: Cladonia foliacea; usnic acid; chiroptical properties; absolute configuration; HPLC- molecules26020455 UV/PAD-CD; DoE; MAE; lichens Academic Editor: Marcello Locatelli Received: 15 December 2020 Accepted: 13 January 2021 1. Introduction Published: 16 January 2021 Metabolites produced by lichens are still poorly investigated, although these organ- Publisher’s Note: MDPI stays neu- isms are commonly used in folk medicine to treat pathological conditions [1–9]. Among tral with regard to jurisdictional clai- the most investigated metabolites, usnic acid (UA) deserved attention due to its intrinsic ms in published maps and institutio- properties and its potential use as chiral synthon. UA is a dibenzofuran derivative, and nal affiliations. it is characterized by the presence of a stereogenic center (Figure1). Both enantiomers occur in nature, depending on the producing organism. The (R)-configured enantiomer of UA is the most abundant in nature, and Cladonia arbuscula, C. mitis, Ramalina boninen- sis, R. pacifica, R. roesleri, R. farinacea, Usnea diffracta, U. longissima, U. hirta, U. steineri, Copyright: © 2021 by the authors. Li- Flavoparmelia caperata, and Xanthoparmelia chlorochroa were identified as valuable source for censee MDPI, Basel, Switzerland. its extraction. Conversely, (S)-UA was isolated as an exclusive enantiomer in few species of This article is an open access article Cladonia (C. uncialis and C. foliacea) and Alectoria (A. lata and A. ochroleuca)[10]. In another distributed under the terms and con- few species of lichens, UA was detected as a mixture of (R)- and (S)-isomers in different ditions of the Creative Commons At- ratios [10]. tribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Molecules 2021, 26, 455. https://doi.org/10.3390/molecules26020455 https://www.mdpi.com/journal/molecules Molecules 2021, 26MoleculesMolecules, x 20212021,, 2626,, 455x 2 of 15 22 ofof 1515 Figure 1. ChemicalFigure structure 1. Chemical of usnic structure acid (UA) of usnic enantiomers. acid (UA) enantiomers. UA has demonstratedUA has has demonstrated demonstrated interesting bioactivity interesting interesting from bioactivity bioactivity a biological from from standpoint, a biological a biological with standpoint, standpoint,anti- with withanti- microbial, cytotoxic,antimicrobial,microbial, or cytotoxic,anti-inflammatory cytotoxic, or anti-inflammatory or anti-inflammatory properties [10]. properties Moreover, properties [10]. UA Moreover, [was10]. exploited Moreover, UA was as UAexploited was ex- as a useful intermediateploiteda useful asintermediate to a obtain useful potent intermediate to obtain analog potents to with obtain analog improved potents with analogsbiological improved with profiles biological improved [11–14]. profiles biological [11–14]. pro- The chemicalfilesThe modifications [chemical11–14]. The modifications introduced chemical modifications in introduced the main scaffold in introduced the main of UA inscaffold theare main resumed of scaffoldUA arein Figure ofresumed UA are in resumed Figure 2 [13,15–25]. Allin2 [13,15–25].Figure these 2chemical[13 All,15 these– 25modifications]. chemical All these modifications chemical preserve modifications the stereochemistrypreserve the preserve stereochemistry of thethe stereochemistryUA used of the UA of used the as starting material.UAas starting used as material. starting material. Figure 2. ChemicalFigure modifications2. ChemicalFigure modifications 2. of Chemical the main modificationsof core the scaffoldmain core of of thescaffold UA main apported of core UA scaffold inapported the derivatives of inUA the apported derivatives described in the de- in derivatives the litera- de- ture [13,15–25scribed]. in the literaturescribed in [13,15–25].the literature [13,15–25]. However, mostHowever, of the published most of thethe data publishedpublished concern datadata (R)-UA, concernconcern whereas ((RR)-UA,)-UA, only whereaswhereas a few studies onlyonly aa fewfew studiesstudies regarding (S)regarding-UA are described. ((S)S)-UA-UA are are Moreover, described. described. Moreover,only Moreover, a small only onlynumber a smalla sm ofall number reports number of refer reportsof reportsto (R)- refer refer to (R)- to and(R)- and (S)-UA activity(S)-UAand (S)-UA when activity activity examined when when examined in examinedcomp inarison. comparison. in compThe discrepancyarison. The discrepancyThe aboutdiscrepancy the about investiga- about the investigationsthe investiga- tions aroundaroundtions the ( Raround)- theand ( R (theS)-)-UA and (R)- results ( andS)-UA (S )-UAfrom results theresults from different from the different theaccessibility different accessibility andaccessibility availability and and availability of availability of the of the two enantiomers.twothe two enantiomers. enantiomers. Indeed, (R Indeed,)-UA Indeed, is (commerciallyR)-UA (R)-UA is commercially is commercially available due available available to the due high due to abundance theto the high high abundance abundance of of accessible accessibleofnatural accessible sources. natural natural In sources. contrast, sources. In ( contrast,SIn)-UA contrast, is (lessS)-UA ( Snaturally)-UA is less is less naturallyabundant. naturally abundant. It abundant.is not avail- It is It not is not available avail- able from thefromable leading from the leading chemicalthe leading chemical suppliers; chemical suppliers; otherwise, suppliers; otherwise, it otherwise, is sold it isat sold ait retailis at sold a retailcost at a30 cost retail times 30 cost times 30 higher times higher than thanhigherthe (R the)-enantiomer. than (R)-enantiomer. the (R)-enantiomer. Therefore, Therefore, the Therefore, prior the priorextraction the extraction prior of extraction (S)-UA of (S)-UA was of required(S)-UA was required was required before before proceedingproceedingbefore withproceeding withthe biological the with biological the or biological pharmacological or pharmacological or pharmacological investigation investigation investigation or the or thefurther further or the chemical further modification, thus hugely limiting the use and investigation of the (S)-UA so far. chemical modification,chemical modification, thus hugely limithusting hugely the use limi andting investigation the use and investigation of the (S)-UA of so the far. (S )-UA so far. As an alternative to the extractive procedure for the production of UA, only two As an alternativeAs an to alternative the extractive to the proced extractiveure for proced the productionure for the of production UA, only two of UA, syn- only two syn- synthetic strategies have been reported (Figure3)[26,27]. thetic strategiesthetic have strategies been reported have been (Figure reported 3) [26,27]. (Figure 3) [26,27]. Molecules 26 Molecules 2021, 26, x 2021, , 455 3 of 15 3 of 15 Figure 3. Chemical Figureand chemoenzymatic 3. Chemical and synthesis chemoenzymatic of UA. synthesis of UA. These processesThese suffer processes from