pharmaceuticals

Article Investigation of (Nees) Engl. Oil and Its Main Components for Antiviral Activity

Valentina Noemi Madia 1,† , Marta De Angelis 2,† , Daniela De Vita 3,*, Antonella Messore 1, Alessandro De Leo 1 , Davide Ialongo 1 , Valeria Tudino 1, Francesco Saccoliti 4, Giovanna De Chiara 5 , Stefania Garzoli 6 , Luigi Scipione 1 , Anna Teresa Palamara 2 , Roberto Di Santo 1 , Lucia Nencioni 2,† and Roberta Costi 1,†

1 Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” Università di Roma, p.le Aldo Moro 5, I-00185 Rome, Italy; [email protected] (V.N.M.); [email protected] (A.M.); [email protected] (A.D.L.); [email protected] (D.I.); [email protected] (V.T.); [email protected] (L.S.); [email protected] (R.D.S.); [email protected] (R.C.) 2 Laboratory Affiliated to Pasteur Italia-Fondazione Cenci Bolognetti, Department of Public Health and Infectious Diseases, “Sapienza” University of Rome, p.le Aldo Moro 5, I-00185 Rome, Italy; [email protected] (M.D.A.); [email protected] (A.T.P.); [email protected] (L.N.) 3 Department of Environmental Biology, “Sapienza” University of Rome, p.le Aldo Moro 5, I-00185 Rome, Italy 4 D3 PharmaChemistry, Italian Institute of Technology, Via Morego 30, I-16163 Genova, Italy; [email protected]  5 Institute of Translational Pharmacology, National Research Council (CNR), Via del Fosso del Cavaliere 100,  I-00133 Rome, Italy; [email protected] 6 Citation: Madia, V.N.; De Angelis, Department of Drug Chemistry and Technologies, Sapienza University of Rome, p.le Aldo Moro 5, M.; De Vita, D.; Messore, A.; De Leo, I-00185 Rome, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-06-4991-2788 A.; Ialongo, D.; Tudino, V.; Saccoliti, † These authors contributed equally. F.; De Chiara, G.; Garzoli, S.; et al. Investigation of Commiphora myrrha Abstract: The resinous exudate produced by Commiphora myrrha (Nees) Engl. is commonly known (Nees) Engl. Oil and Its Main as true and has been used since antiquity for several medicinal applications. Hundreds of Components for Antiviral Activity. Pharmaceuticals 2021, 14, 243. metabolites have been identified in the volatile component of myrrh so far, mainly sesquiterpenes. https://doi.org/10.3390/ph14030243 Although several efforts have been devoted to identifying these sesquiterpenes, the phytochemical analyses have been performed by gas-chromatography/mass spectrometry (GC–MS) where the high Academic Editor: temperature employed can promote degradation of the components. In this work, we report the Monica Notarbartolo extraction of C. myrrha by supercritical CO2, an extraction method known for the mild extraction conditions that allow avoiding undesired chemical reactions during the process. In addition, the Received: 5 February 2021 analyses of myrrh oil and of its metabolites were performed by HPLC and GC–MS. Moreover, we Accepted: 3 March 2021 evaluated the antiviral activity against influenza A virus of the myrrh extracts, that was possible Published: 9 March 2021 to appreciate after the addition of vitamin E acetate (α-tocopheryl acetate) to the extract. Further, the single main bioactive components of the oil of C. myrrha commercially available were tested. Publisher’s Note: MDPI stays neutral Interestingly, we found that both furanodienone and curzerene affect viral replication by acting on with regard to jurisdictional claims in different steps of the virus life cycle. published maps and institutional affil- iations. Keywords: Commiphora myrrha; sesquiterpenes; myrrh oil; vitamin E acetate; supercritical CO2 fluid extraction; HPLC; antiviral activity; influenza A H1N1 virus

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. 1. Introduction This article is an open access article distributed under the terms and Commiphora genus () includes more than 150 species especially occurring conditions of the Creative Commons in northeastern , southern Arabia and India [1]. The species Commiphora myrrha Attribution (CC BY) license (https:// (Nees) Engl. produces a resinous exudate known as true myrrh that has been used for creativecommons.org/licenses/by/ centuries as an embalming ointment, wound-healing remedy and for other medicinal ap- 4.0/). plications [2]. Myrrh consists of alcohol-soluble resins and volatile oil together with a gum

Pharmaceuticals 2021, 14, 243. https://doi.org/10.3390/ph14030243 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, x FOR PEER REVIEW 2 of 11

Pharmaceuticals 2021, 14, 243 2 of 11 centuries as an embalming ointment, wound-healing remedy and for other medicinal applications [2]. Myrrh consists of alcohol-soluble resins and volatile oil together with a gum soluble in water containing polysaccharides, proteins and long chain aliphatic derivatives. Thesoluble lipophilic in water part containing of myrrh is polysaccharides, composed of steroids, proteins sterols and long and chainterpenes aliphatic [3]. derivatives. To date, hundredsThe lipophilicof metabolites part have of myrrh been isidentified composed in the of steroids,volatile component sterols and of terpenes myrrh, [3]. To date, where sesquiterpeneshundreds are of the metabolites major components have been. identifiedNotably, furanosesquiterpenes in the volatile component suchof as myrrh, where furanoelemanes,sesquiterpenes furanoeudesmanes, are the major and components.furanogermacranes Notably, are furanosesquiterpenes the characteristic such as fura- constituents ofnoelemanes, myrrh oil [4]. furanoeudesmanes, As important co andmponents furanogermacranes of volatile oil, are sesquiterpenoids the characteristic constituents possess diverseof biological myrrh oil [activities,4]. As important including components antibacterial, of volatile antifungal, oil, sesquiterpenoids and antiparasitic possess diverse properties. It biologicalis also worthy activities, of note including that recent antibacterial, papers reported antifungal, the in and vitro antiparasitic and in vivo properties. It is analgesic andalso anticancer worthy ofactivities note that [5–8] recent of papersfour main reported components the in vitro of andthe involatile vivo analgesic oil, and anti- namely furanodienonecancer activities (1), furanoeudesma-1,3-diene [5–8] of four main components (2), curzerene of the ( volatile3), and β oil,-elemene namely (4 furanodienone) β (Figure 1). (1), furanoeudesma-1,3-diene (2), curzerene (3), and -elemene (4) (Figure1).

Figure 1. StructuresFigure 1. of Structures the main sesquiterpenes of the main sesquiterpen in myrrh oil:es furanodienonein myrrh oil: furanodienone (1), furanoeudesma-1,3-diene (1), furanoeudesma- (2), curzerene (3) and β-elemene1,3-diene (4). (2), curzerene (3) and β-elemene (4).

Some works [9,10]Some have works also [9 ,been10] have devoted also beento the devoted identification to the identificationor characterization or characterization of of these aforementionedthese aforementioned sesquiterpenes sesquiterpenes but most of the but phytochemical most of the phytochemical analyses have analyses been have been performed byperformed gas chromatography by gas chromatography where the high where temperature the high employed temperature can employed promote can promote degradation ofdegradation the components, of the thus components, influencing thus the influencing qualitative–quantitative the qualitative–quantitative composition composition of the mixture.of For the instance, mixture. Forthe rearrangem instance, theent rearrangement of furanodiene of to furanodiene curzerene during to curzerene the during the essential oil extractionessential process oil extraction and/or process during and/or conventional during gas conventional chromatographic gas chromatographic analysis, analysis, ◦ in which temperaturesin which temperaturesover 200 °C are over employed, 200 C are is reported employed, [11]. is reported [11]. Here we describeHere the we extraction describe the of extractionC. myrrha ofbyC. supercritical myrrha by supercriticalCO2, an extraction CO2, an extraction method that ismethod well known that is for well its knownmany advantages for its many in advantagesobtaining volatile in obtaining extracts volatile or aroma extracts or aroma substances and,substances more generally, and, more to extract generally, lipophilic to extract compounds lipophilic from compounds a wide variety from a wideof variety of matrices. Indeed,matrices. in addition Indeed, to inbeing addition non-toxic to being and non-toxic non-flammable, and non-flammable, the mild extraction the mild extraction conditions allowconditions avoiding allow undesired avoiding chemical undesired reactions chemical during reactions the process. during This the process.leads to This leads to shorter extractionshorter times extraction without times withoutdecomp decomposingosing compounds compounds vulnerable vulnerable to to high temperatures temperatures differentlydifferently from hydro- or steam- steam-distilleddistilled oils oils [12]. [12]. In In the the present present work, work, the the analysis of analysis of myrrhmyrrh oil oil and and the the quantification quantification of of its its metabolites metabolites were were performed performed by HPLC,HPLC, so as to avoid so as to avoidhigh high temperatures temperatures also also during during the analyticalthe analytical process. process. Our interest Our interest in substances in endowed substances endowedwith antiviral with antiviral activity [activity13] and [13] in the and field in the of biologicallyfield of biologically active natural active products [14] natural productsprompted [14] prompted us to evaluate us to theevalua antiviralte the activity antiviral against activity influenza against A influenza virus of bothA the volatile myrrh extracts and of the single sesquiterpenes furanodienone (1), furanoeudesma-1,3- virus of both the volatile myrrh extracts and of the single sesquiterpenes furanodienone diene (2), curzerene (3) and β-elemene (4). In particular, we tested the myrrh extracts (1), furanoeudesma-1,3-diene (2), curzerene (3) and β-elemene (4). In particular, we tested both alone and in combination with vitamin E acetate (α-tocopheryl acetate) in order to the myrrh extracts both alone and in combination with vitamin E acetate (α-tocopheryl reduce the cytotoxic effects of the volatile oil. We decided also to investigate the activity of acetate) in order to reduce the cytotoxic effects of the volatile oil. We decided also to curzerene, furanodienone, furanoeudesma-1,3-diene and β-elemene 1–4, being the main investigate the activity of curzerene, furanodienone, furanoeudesma-1,3-diene and β- bioactive components of the volatile oil of C. myrrha [2] commercially available. elemene 1–4, being the main bioactive components of the volatile oil of C. myrrha [2] commercially 2.available. Results and Discussion To determine the amount of the major sesquiterpenoids (1–4) in our extract, an HPLC 2. Results and Discussion analysis was carried out. The percentage of each sesquiterpene is reported in Table1 . It To determineis possible the amount to notice of the that major furanoeudesma-1,3-diene sesquiterpenoids (1–4) in is our the extract, most abundant an HPLC while no fura- analysis was carriednodienone out. wasThe percentage detected in of our each extracts. sesquiterpene Our results is reported are in goodin Table agreement 1. It is with those possible to noticefound bythat Marongiu furanoeudesma-1, et al. [12] who3-diene obtained is the supercritical most abundant fluid extraction while (SCFE)no myrrh oil furanodienonemade was detected up of the in following our extracts. main Our components: results are furanoeudesma-1,3-diene in good agreement with those (26.2%), curzerene (9.7%), and β-elemene (3.0%) without detecting any amount of furanodienone. A run at

Pharmaceuticals 2021, 14, 243 3 of 11

low temperature was used for the HPLC analysis to isolate compound 3 given that it could not be separated from other compounds in the mixture at higher temperatures.

Table 1. Percentage of compounds analyzed in the oil of C. myrrha extracted by supercritical fluid

extraction (SCFE)-CO2 technique.

Compound % (w/w) LOQ LOD 1 nd 1 3.62 1.19 2 10.48 1.61 0.53 3 2.35 3.81 1.26 4 1.04 2.59 0.86 1 Not detected; LOQ = limit of quantification (µg/mL): 10 × (SE/S); LOD = limit of detection (µg/mL): 3.3 × (SE/S); SE = standard deviation of regression of y intercept; S: slope of the linear calibration line.

To investigate the volatile composition of C. myrrha oil, gas-chromatography/mass spectrometry (GC/MS) technique was used. Twenty-two volatile compounds were iden- tified representing an average of 99.9% of the oil composition and they are listed in Table2 . The most abundant component was furanoeudesma-1,3-dione (31.1%) followed by curzerene (23.1%), germacra-1(10),7,11-trien-15-oic acid, 8,12-epoxy-6-hydroxy-gamma- lactone (14.4%), lindestrene (11.9%) and other minor compounds.

Table 2. Chemical volatile composition (%) of myrrh oil.

N◦ COMPONENT 1 LRI 2 LRI 3 MS 4 (%) 5 1 δ-elemene 1345 1347 + 0.8 2 copaene 1377 1379 + 0.4 3 β-elemene 1381 1387 + 3.9 4 γ-elemene 1443 1445 + 2.3 5 humulene 1470 1473 + 0.2 6 curzerene 1491 1495 + 23.1 7 germacrene D 1498 1500 + 0.3 8 δ-guajene 1501 1508 + 1.0 9 δ-cadinene 1505 1509 + 0.5 10 γ-cadinene 1533 1536 + 0.6 11 elemol 1539 1543 + 0.4 12 caryophyllene oxide 1585 1583 + 0.5 13 spathulenol 1603 1601 + 0.5 14 γ-eudesmol 1631 1630 + 0.3 15 isoserecenin 1790 1799 * + 0.5 16 furanodienone 1870 § + a 1.7 17 furanoeudesma-1,3-dione 2155 § + a 31.1 18 lindestrene 2162 § + 11.9 germacra-1(10),7,11-trien-15-oic acid, 19 2170 § + 14.4 8,12-epoxy-6-hydroxy-gamma-lactone 20 1-(2,4-diemthylphenyl)-2-(2-furyl) cyclopropane 2180 § + 2.6 21 cycloisolongifolene, 8,9-dehydro-9-formyl- 2200 § + 2.4 8-hydroxy-3,8a-dimethyl-5-methylene-2- 22 oxododecahydronaphto[2,3-b]furan-4-yl 2210 § + 0.5 acetate TOTAL (%) 99.9 1 The components are reported according their elution order on an apolar column; 2 linear retention indices measured on an apolar column; 3 linear retention indices from the literature; §: linear retention indices (LRI) not available for apolar column; *: Kovats Index RI; 4 MS: identification by comparison of the mass spectra with those present in the libraries; a: identification confirmed by co-injection of an authentic sample; 5 percentage mean values of C. myrrha oil components (%).

The extract obtained by supercritical fluid extraction (SCFE) using supercritical CO2 as solvent has been evaluated in vitro for its antiviral activity against influenza A Puerto Rico 8/34/H1N1 virus (PR8). First, we evaluated the eventual toxicity of compounds on Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 11

comparison of the mass spectra with those present in the libraries; a: identification confirmed by co-injection of an authentic sample; 5 percentage mean values of C. myrrha oil components (%).

The extract obtained by supercritical fluid extraction (SCFE) using supercritical CO2 Pharmaceuticals 2021, 14, 243 4 of 11 as solvent has been evaluated in vitro for its antiviral activity against influenza A Puerto Rico 8/34/H1N1 virus (PR8). First, we evaluated the eventual toxicity of compounds on the cell monolayers. Therefore, intact monolayers of A549 cells were treated with different concentrationsthe (3–100 cell monolayers. µg/mL) of myrrh Therefore, oil. intactDMSO monolayers was used as of control. A549 cells The were integrity treated of with different cell monolayersconcentrations was evaluated (3–100 by usingµg/mL) a vital of dye myrrh (Cell oil. Tag) DMSO of cells was and used revealed as control. by In- The integrity Cell Westernof assay cell monolayers(ICW). Myrrh was oil evaluated alone was by toxic using at a the vital highest dye (Cell concentrations: Tag) of cells andthe revealed by integrity of cellIn-Cell monolayer Western was assay damaged (ICW). Myrrhof about oil 30, alone 71 wasand toxic76% compared at the highest to DMSO concentrations: the alone at 25, 50integrity and 100 of µg/mL, cell monolayer respectively was (data damaged not shown). of about Due 30, to 71 the and low 76% stability compared of to DMSO myrrh oil andalone its components at 25, 50 and in 100 theµ air-oxygeng/mL, respectively environment, (data notwe hypothesized shown). Due tothat the the low stability of cytotoxicity ofmyrrh myrrh oil could and itsbe componentsascribed to the in thedecomposition air-oxygen environment, of the components we hypothesized of this that the oil. Thus, wecytotoxicity decided to ofadd myrrh a compound could be ascribedendowed to with the decomposition antioxidant properties of the components to the of this oil. extract. To thisThus, end, we α-tocopheryl decided to addacetate a compound and a mixture endowed myrrh with oil antioxidant(3–100 µg/mL) properties plus α- to the extract. tocopheryl acetateTo this (1% end, wα/v-tocopheryl) were tested acetate for their and atoxicity. mixture We myrrh found oil (3–100that theµ g/mL)addition plus of α-tocopheryl 1% vitamin Eacetate acetate (1% to myrrhw/v) wereoil protected tested for the their cell toxicity. monolayers We found starting that from the addition50 µg/mL. of 1% vitamin Indeed, the percentageE acetate to of myrrh relative oil protectedfluorescence the cellintensity monolayers (RFI) of starting Cell Tag from indicated 50 µg/mL. a Indeed, the recovery of cellpercentage integrity, ofreaching relative the fluorescence same percentage intensity of not (RFI) toxic of Cellconcentrations Tag indicated (Figure a recovery of cell 2). integrity, reaching the same percentage of not toxic concentrations (Figure2).

Figure 2. TheFigure mixture 2. myrrhThe mixture oil and myrrh vitamin oil E and (Vit. vitamin E) acetate E (Vit. increased E) acetate cell viability.increased A549 cell viability. cells were A549 treated cells with different concentrationswere (3–100 treatedµg/mL) with myrrh different oil, concentrations Vit. E acetate (0.03–1 (3–100µ µg/mL)g/mL) andmyrrh a mixture oil, Vit. of E myrrhacetate oil (0.03–1 (3–100 µg/mL)µg/mL) with Vit. E acetate (0.03–1andµ ag/mL). mixture Cells of myrrh treated oil with (3–100 DMSO µg/mL) were with used Vit. as control. E acetate After (0.03–1 24 h, µg/mL). cell monolayers Cells treated were with fixed and stained with Cell Tag.DMSO The effect were of used compounds as control. on After the integrity 24 h, cell of mono cell monolayerslayers were wasfixed analyzed and stained byIn-Cell with Cell Western Tag. The assay (ICW), using LI-COReffect Image of Studiocompounds Software. on the Images integrity are of representative cell monolayers of one was experiment analyzed by of In-Cell two replicates, Western eachassay performed in duplicate. The(ICW), graph using indicates LI-COR the percentage Image Studio of relative Software. fluorescence Images are intensity representative (RFI) of of Cell one Tag experiment expressed of on two myrrh oil and myrrh oil + Vit.replicates, E acetate each compared performed to Vit. in Eduplicate. acetate is The considered graph indicates as 100%. the Values percentage are the of mean relative± S.D. of two replicates of fluorescence intensity (RFI) of Cell Tag expressed on myrrh oil and myrrh oil + Vit. E acetate one experiment performed in duplicate (n = 2); ** p < 0.01 vs. Vit. E acetate. compared to Vit. E acetate is considered as 100%. Values are the mean ± S.D. of two replicates of one experiment performedTherefore, in duplicate since the (n addition= 2); ** p < of 0.01α-tocopheryl vs. Vit. E acetate. acetate (1% w/v) increased the viability of cells treated with the extract, in these conditions we could appreciate the antiviral activity Therefore,of thesince mixture the addition myrrh of oil α +-tocopherylα-tocopheryl acetate acetate (1% compared w/v) increased to DMSO the viability treated–infected cells of cells treated(control) with the measured extract, by in means these ofconditions hemagglutination we could assay appreciate (HAU/mL). the antiviral The mixture strongly activity of thereduced mixture viral myrrh replication oil + α-tocopheryl in a dose-dependent acetate compared manner (seeto DMSO Table3 ).treated– The viral titer was infected cellsdetermined (control) measured by evaluating by means the presence of hemagglutination of hemagglutinating assay (HAU/mL). activity in the The supernatant of mixture stronglyinfected reduced cells. viral When replication the cells in were a dose-dependent treated with myrrh manner oil (see alone Table or in 3). combination The with viral titer wasvitamin determined E (Vit. by E), evaluating a decrease the in pr viralesence titer of expressed hemagglutinating as HAU/mL activity values in the was measured, supernatant ofwith infected no observed cells. When viral titer the at cell concentrations were treated of 50 withµg/mL. myrrh The oil Vit. alone E acetate or in alone did not combination withinterfere vitamin with E viral (Vit. replication:E), a decrease indeed, in viral at titer concentrations expressed as ranging HAU/mL from values 0.03 to 1 µg/mL, was measured,the with values no observed of viral titer viral were titer equal at concentration to 8 HAU/mL, of 50 as µg/mL. for the The control Vit. E (DMSO). acetate alone did not interfereTo confirm with viral the replication: antiviral activity indeed, of at the concentrations mixture, cells ranging were treated from 0.03 with the highest to 1 µg/mL, theconcentrations values of viral (25, titer 50 were and 100equalµg/mL) to 8 HAU/mL, of myrrh as oil for alone, the control myrrh (DMSO). oil + Vit. E (1%) and Vit. E (1%). The expression of viral nucleoprotein (NP) was analyzed by ICW, a method that allows the evaluation of the expression of viral proteins directly on an infected mono- layer [15]. As shown in Figure3, the expression of NP was significantly reduced using myrrh oil at 25 and 50 µg/mL in mixture with Vit. E acetate, compared to Vit. E treated– infected cells (considered as 100%). In particular, the percentage of RFI was 89.7 ± 3% Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 11

Table 3. Antiviral activity of C. myrrha SCFE-CO2 extract alone and in combination with Vit. E Pharmaceuticals 2021, 14, 243 5 of 11 acetate.

Viral Titer (HAU/mL) 1 Myrrh oil 8 4 4 4 1.5 2 0 0 (*Myrrhp < 0.05 oil +vs. Vit. VitE acetate E), 58.3 (1% ±w/v5%) (**8p < 0.014 vs.4 Vit.4E) and4 23.4 0± 5%0 (** p < 0.01 vs. Vit. E) at 25, 50 andDMSO 100 µ g/mL, respectively.8 8 8 8 8 8 8 Myrrh oil concentration (µg/mL) 0 3 6 12.5 25 50 100 1 Viral titer was measured by hemagglutination assay (HAU/mL) in the supernatant of infected cellsTable treated 3. Antiviralor not with the activity compounds of C. alone myrrha or in combinationSCFE-CO2 withextract Vit. E alone(1%), DMSO and was in combination used with Vit. E asacetate. control. Viral titer for Vit. E acetate was equal to 8 HAU/mL at all the tested concentrations (0.03–1 µg/mL); 2 SD ± 0.5. Viral Titer (HAU/mL) 1 To confirm the antiviral activity of the mixture, cells were treated with the highest concentrations (25,Myrrh 50 and oil 100 µg/mL) of myrrh 8 oil alone, 4 myrrh oil 4 + Vit. E (1%) 4 and1.5 Vit.2 0 0 E (1%).Myrrh The oil expression + Vit. E acetateof viral (1%nucleoproteinw/v) (NP)8 was analyzed 4 by 4 ICW, a method 4 that 4 0 0 allows the evaluationDMSO of the expression of viral proteins 8 directly 8 on an 8 infected 8 monolayer 8 8 8 [15]. As shown in Figure 3, the expression of NP was significantly reduced using myrrh oil atMyrrh 25 and oil 50 concentration µg/mL in mixture (µg/mL) with Vit. E acetate, 0 compared 3 to Vit. 6 E treated–infected 12.5 25 50 100 cells1 Viral (considered titer was measuredas 100%). In by particular, hemagglutination the percentage assay(HAU/mL) of RFI was 89.7 in the ± 3% supernatant (* p < 0.05 ofvs. infected cells treated or Vitnot E), with 58.3 the ± 5% compounds (** p < 0.01 alone vs. Vit. or inE)combination and 23.4 ± 5% with (** p Vit. < 0.01 E (1%), vs. Vit. DMSO E) at was 25, 50 used and as 100 control. Viral titer for Vit. µg/mL,E acetate respectively. was equal to 8 HAU/mL at all the tested concentrations (0.03–1 µg/mL); 2 SD ± 0.5.

FigureFigure 3. The 3. The mixture mixture myrrh myrrhoil and Vit. oil E and acetate Vit. inhibi E acetatets influenza inhibits A virus influenza replication. A A549 virus cells replication. A549 cells werewere infected infected with with PR8 and, PR8 after and, viral after adsorption viral adsorption,, treated with treated different with concentrations different myrrh concentrations oil myrrh oil (25, (25, 50 and 100µ µg/mL), Vit. E acetate (0.25–1 µg/mL)µ and myrrh oil (25, 50 and 100 µg/mL) in µ mixture50 and with 100 Vit.g/mL), E acetate Vit. (0.25–1 E acetate µg/mL). (0.25–1Cells infectedg/mL) with PR8 and and myrrh treated oil with (25, DMSO 50 and were 100 g/mL) in mixture usedwith as Vit.control. E acetate After 24 (0.25–1h, cell monolayersµg/mL). were Cells fixed infected and the expression with PR8 of andviral treatednucleoprotein with DMSO were used as (NP)control. was analyzed After 24 by h,ICW, cell usin monolayersg LI-COR Image were Studio fixed Software. and the Images expression are representative of viral of nucleoprotein (NP) was one experiment of two replicates, each performed in duplicate. analyzed by ICW, using LI-COR Image Studio Software. Images are representative of one experiment of twoTo further replicates, investigate each performed the antiviral in activity duplicate. of myrrh oil components, we decided to test also furanodienone (1), furanoeudesma-1,3-diene (2), curzerene (3), and β-elemene (4) on A549To cells further infected investigate with PR8 virus the. Indeed, antiviral these activity secondary of myrrhmetabolites oil are components, reported we decided to totest be the also main furanodienone bioactive components (1), furanoeudesma-1,3-diene of C. myrrha oil commercially available. (2), curzerene (3), and β-elemene (4) All the compounds were first tested for their potential toxicity on cell monolayer at differenton A549 concentrations: cells infected furanodienone with PR8 virus.(1), curzerene Indeed, (3) and these β-elemene secondary (4) at metabolitesa range of are reported to 30–500be the µg/mL; main furanoeudesma-1,3-diene bioactive components (2)of at C.2.5–30 myrrha µg/mLoil (due commercially to the availability available. of a low amountAll the of compoundsthe standard compound). were first We tested found for that their compounds potential 1 and toxicity 3 showed on cell monolayer at toxicitydifferent at higher concentrations: doses (startingfuranodienone from 100 µg/mL), while (1), curzerene compound 4 ( 3was) and highlyβ-elemene toxic at (4) at a range of all30–500 the concentrationsµg/mL; furanoeudesma-1,3-diene used. For this reason, the latter ( 2compound) at 2.5–30 wasµ g/mLexcluded (due for the to the availability of following experiments. a lowCompounds amount were of theadded standard at different compound). concentrations We(30, found60 and 100 that µg/mL) compounds after the 1 and 3 showed infectiontoxicity and at maintained higher doses for the (starting following from24 h. Viral 100 replicationµg/mL), was while measured compound by means4 was highly toxic ofat HAU/mL. all the concentrations As shown in Table used. 4, compounds For this furanodienone reason, the ( latter1) and curzerene compound (3) were was excluded for the following experiments. Compounds were added at different concentrations (30, 60 and 100 µg/mL) after the infection and maintained for the following 24 h. Viral replication was measured by means of HAU/mL. As shown in Table4, compounds furanodienone ( 1) and curzerene (3) were effective at 60 and 100 µg/mL, even though the latter concentration was quite toxic for both compounds. In particular, for compound 1, the viral titer was of 6 and 2 HAU/mL, at 60 and 100 µg/mL, respectively; for compound 3, the viral titer was of 3 at 60 µg/mL, while no viral titer was observed at a concentration of 100 µg/mL. With regard to 2, no inhibition was observed at any concentrations used (data not shown). Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 11

Pharmaceuticals 2021, 14, 243 6 of 11

effective at 60 and 100 µg/mL, even though the latter concentration was quite toxic for both compounds. In particular, for compound 1, the viral titer was of 6 and 2 HAU/mL, at 60 andTable 100 µg/mL, 4. Antiviral respectively; activity offor1 compoundand 3. 3, the viral titer was of 3 at 60 µg/mL, while no viral titer was observed at a concentration of 100 µg/mL. With regard to 2, no inhibition was observedCompound at any concentrations used (data notViral shown). Titer (HAU/mL) 1 1 8 8 6 2 2 Table 4. Antiviral activity of 1 and 3. 3 8 8 3 3 0 1 CompoundConcentration (µg/mL) 0 Viral Titer (HAU/mL) 30 60 100 1 8 8 6 2 2 1 Viral titer was measured by hemagglutination assay (HAU/mL) in the supernatant of infected cells treated or 3 not with3 the compounds; 2 SD8 ± 2; 3 SD ± 01.8 3 0 Concentration (µg/mL) 0 30 60 100 1 Viral titer was measured by hemagglutination assay (HAU/mL) in the supernatant of infected Thus, the antiviral activity of the effective compounds 1 and 3 was analyzed by cells treated or not with the compounds; 2 SD ± 2; 3 SD ± 01. measuring the RFI of HA protein by ICW assay. However, the highest concentration of Thus,100 theµ antiviralg/mL was activity quite of toxicthe effective for both compounds compounds, 1 and therefore, 3 was analyzed we chose by meas- a lower concentration uring theto RFI exclude of HA thatprotein the by antiviral ICW assay. activity However, may be the in highest part due concentration to the cytotoxicity of 100 of compounds. µg/mL wasTherefore, quite toxic to for deepen both compounds, the mechanism therefore, of we action chose of a furanodienonelower concentration (1) to and curzerene (3), exclude that80 µtheg/mL antiviral was activity added may to cells be in at part different due to stepsthe cytotoxicity of infection: of compounds. only before infection (2 h, Therefore,PRE), to deepen only the during mechanism infection of action (1 h, DUR),of furanodienone immediately (1) and after curzerene infection (3),and 80 maintained for µg/mL wasthe added following to cells 24 at h different (POST). steps Compounds of infection: were only also before maintained infection for(2 h, all PRE), the time of infection only during(before, infection during (1 h, and DUR), after) immediately but in this after condition, infection they and both maintained became for toxic the forfol- the cells, and data lowing 24were h (POST). not considered Compounds valid. were Asalso shown maintained in Figure for all4 ,the both time compounds of infection were(before, effective at specific during and after) but in this condition, they both became toxic for the cells, and data were phases: compound 1 was mainly effective when added PRE or POST (** p < 0.001); for not considered valid. As shown in Figure 4, both compounds were effective at specific phases: compoundcompound 1 3,wasalthough mainly effective quite effective when added at all thePRE phases, or POST the (** most p < 0.001); inhibition for (46% compared compoundto 3, untreated although quite cells) effective was reached at all the during phases, viral the challengemost inhibition (DUR). (46% On compared the basis of these results, to untreatedwe cells) hypothesize was reached that during compound viral ch1allengemight (DUR). act before On the infection basis of bythese interfering results, with some cell we hypothesizefactors that on thecompound plasma 1 membranemight act before or, due infection to the by antioxidant interfering activitywith some of cell furanodienone [16], factors onit the may plasma maintain membrane reduced or, due conditions to the antioxidant into the activity cells making of furanodienone them more [16], resistant to virus it may maintaininfection reduced [17–19 conditions]. With regard into the to compound cells making3 ,them since more it mainly resistant acts to during virus viral adsorption, infection it[17–19]. might With interfere regard with to compound the viral 3 attachment, since it mainly or entry acts during into the viral cells. adsorption, it might interfere with the viral attachment or entry into the cells.

Figure 4. CompoundsFigure 1 and 4. Compounds 3 affect different 1 and 3 stepsaffect ofdifferent virus lifecycle.steps of vi A549rus lifecycle. cells were A549 infected cells were with infected PR8 with and treated or not with furanodienonePR8 (1) and or curzerenetreated or not (3) atwith different furanodienone phases ( of1) theor curzerene virus life (3 cycle:) at different the compounds phases of the were virus added life for 2 h before cycle: the compounds were added for 2 h before (PRE); during viral adsorption for 1 h (DUR); (PRE); during viral adsorption for 1 h (DUR); immediately after viral adsorption and maintained for 24 h (POST). After 24 h infection, supernatants were recovered and used to infect a fresh monolayer of MDCK cells. The expression of viral Hemagglutinin (HA) was analyzed by ICW assay, using LI-COR Image Studio Software, as described in Methods. The percentage of RFI was calculated in comparison to Infected (I) cells (considered 100%). Values are the mean ± S.D. of three replicates (n = 3) of one out two experiments performed. Statistical significance of the data vs. I was defined as * p < 0.01 and ** p < 0.001. Pharmaceuticals 2021, 14, 243 7 of 11

3. Materials and Methods 3.1. Materials Oleo-gum-resin of C. mhyrra (origin ) was purchased by Farmalabor (Italy). Before utilization, the material was ground with a Malavasi mill (Pinerolo, Italy) while care was taken to avoid overheating. The particles size was in the range 250–850 µm. CO2 (purity 99%) was supplied by SIO (Societa Italiana Ossigeno, Cagliari, Italy). Certified references furanoeudesma-1,3-diene and vitamin E acetate were purchased from Sigma- Aldrich (Milan, Italy), β-elemene, furanodienone and curzerene were purchased from Angene Chemicals (Nanjing, China). Acetonitrile HPLC grade was purchased from Sigma- Aldrich (Milan, Italy), and used without further purification. Water was purified before use by a Milli-Q plus 185 system (Milford, MA, USA).

3.2. Extraction The powdered vegetable matter (1.30 kg) underwent the extraction process with an industrial-scale supercritical fluid extraction system SCFE-10 (Exenia Group, Pinerolo, TO, Italy). The supercritical CO2 extractions were performed as previously reported [20]. Briefly, in an apparatus equipped with 4200 mL extraction basket (with an internal diameter of 10 cm and an effective height of 42 cm) about 1.30 kg of myrrh was loaded at each run. Two filters of 80 µm were placed at both ends of the extraction basket and then placed in the 5 kg extractor vessel. The extraction parameters were set up as follows: temperature ◦ 65 C, pressure 37 MPa and CO2 flow rate 18 kg CO2/h. The oil was collected in a glass container, previously autoclaved and weighted. An amount of 1.30 kg of powdered myrrh ◦ yielded 45.5 g of a brown oily extract at temperature 65 C, pressure 37 MPa and CO2 flow rate 18 kg CO2/h.

3.3. Analysis Method development for analysis of the extract was carried out with an HPLC system consisting of a Shimadzu LC-10AD pump, a SIL-10AD autosampler using a 200 µL sample loop, a CTO-10AC column oven, a SPD-10A detector. Data analyses were performed using the LC solutions software. The HPLC analyses were performed using an analytical column Waters Symmetry C18 (150 mm × 4.6 mm, 3.5 µm) by isocratic or in gradient elution. A concentrated stock solution of standards was prepared dissolving in acetonitrile an exactly weighted amount of the standards in a 5 mL volumetric flask; serial dilutions were performed obtaining solutions used to construct the calibration curves. A volume of 5 µL of each solution was injected. Peak area of each compounds was plotted vs. actual concentrations (mg/mL). Linearity was assessed through evaluation of the coefficient of determination (R2). Method details for each standard are reported below: − Furanodienone (compound 1): flow 0.5 mL/min, temperature 30 ◦C; gradient elution: 0–10 min (50% acetonitrile), 10–40 min (100% acetonitrile), 222 nm, tR = 14.10 min. − Furanoeudesma-1,3-diene (compound 2): flow 0.5 mL/min, temperature 30 ◦C; gra- dient elution: 0–15 min (80% acetonitrile), 15–30 min (100% acetonitrile), 222 nm, tR = 13.00 min. − Curzerene (compound 3): flow 0.5 mL/min, 18 ◦C, isocratic elution acetonitrile 70/water 30, 222 nm, tR = 30.02 min. − β-elemene (compound 4): flow 1 mL/min, 18 ◦C, isocratic elution acetonitrile 80/wa- ter 20, 205 nm, tR = 17.65 min. An exactly weighted amount of myrrh extract was solubilized in acetonitrile (concen- tration ranging from 4 to 8 mg/mL) and 5 µL of this solution was injected for each HPLC analysis.

3.4. Analysis Gas-Chromatography–Mass Spectrometry (GC–MS) Analysis The analysis of C. myhrra oil was carried out by a gas chromatograph equipped with flame ionization detector (FID) and a split–splitless injector and directly coupled to a mass spectrometer (MS) Perkin Elmer Clarus 500 model (Waltham, MA, USA). A Varian Pharmaceuticals 2021, 14, 243 8 of 11

FactorFour VF-1 fused-silica capillary column (length 60 m × 0.32 mm ID × 1.0 µm film thickness) was used with helium as carrier gas (1.0 mL/min). A volume of 1 µL of EO oil was diluted in 1 mL of acetonitrile and the injection volume was 1µL. The chromatograph was equipped with a split/split less injector used in the split-less mode. The oven GC temperature program was as follows: from 70 to 250 ◦C at a rate of 5 ◦C/min, and held for 10 min; the injector temperature was 280 ◦C. The electron-impact ionization mass spectrometer was operated as follows: ionization voltage, 70 eV; ion source temperature, 200 ◦C; scan mode, 40.0–450.0 mass range. GC–FID analyses were performed under the same operative conditions as described for the GC–MS measurements. The FID temperature was 280 ◦C. The identification of the components was assigned by matching their mass spectra with that reported on MS library search (Wiley and Nist). Furthermore, linear retention indices (LRIs) were calculated using a mixture of aliphatic hydrocarbons (n-alkanes C8-C30, Ultrasci) injected directly into GC injector at the same temperature program described above and compared with those reported in reference libraries. Two components were also identified by co-injection with an authentic sample. The semi-quantitative analysis was performed by normalizing the peak area generated in FID (%) without using corrections factors (RRFs). The analysis was repeated three times.

3.5. Cell Cultures and Viral Production A549 (ATCC catalogue no. CCL-185), and MDCK (ATCC catalogue no. CCL-34) were used to determine the antiviral activity of myrrh oil and its metabolites. Cell lines were maintained in high glucose Dulbecco’s Modified Eagle’s Medium with sodium pyruvate and L-glutamine (DMEM; Euroclone, Milan, Italy), Minimum Essential Medium Eagle (EMEM; Euroclone, Milan, Italy) or Roswell Park Memorial Institute medium (RPMI-1640, Sigma, Milan, Italy) supplemented with 10% Fetal Bovine Serum (FBS; Euroclone, Milan, Italy) and 1% Penicillin/Streptomycin (Pen/Strep, Euroclone, Milan, Italy). Allantoic cavities of 11-day-old embryonated chicken eggs were used to growth in- fluenza virus A/Puerto Rico/8/34 H1N1 (PR8 virus). Viral suspension was inoculated in the allantoic cavity and incubated for 48 h at 37 ◦C, then infected eggs were maintained overnight at 4 ◦C. Subsequently, the allantoic fluid was collected and clarified by centrifu- gation (2500× g for 30 min). The recovered virus was used for the infection of MDCK and A549 cells. Allantoic fluid from uninfected eggs was used as reference for mock infection.

3.6. Cytotoxicity Assay The cytotoxicity assay was evaluated on cell lines used for influenza virus infection by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, cells were seeded in 96-well plates at a density of 2 × 104 cells/well in 100 µL of complete RPMI medium without phenol red for 24 h at 37 ◦C. Subsequently, cell monolayers were treated or not with increasing concentrations (2.5–500 µg/mL) of compounds for 24 h at 37 ◦C. After 24 h, 10 µL of MTT solution (5 mg/mL) was added to each well for 4 h at 37 ◦C. Afterwards, each sample was acidified by adding 0.1 N HCl in isopropanol (100 µL/well) for 30 min in slow agitation to ensure that all formazan crystals were dissolved. Absorbance of samples was read at 570 nm, using an automatic plate reader (Multiskan EX, Ascent Software, Thermo Fisher Scientific, Milan, Italy). Untreated cells were used as control.

3.7. Hemagglutination (HAU) Assay Virus production was evaluated in the supernatants of infected cells recovered 24 h after infection, by measuring the hemagglutinin units (HAU), using human type 0 Rh+ erythrocytes [19]. Stock solutions of compounds dissolved in DMSO were diluted in RPMI medium to final concentrations of 2.5–500 µg/mL. Compounds were added after the adsorption period and maintained in the culture media until the end of the experiments. The highest DMSO concentration present in the culture medium was 0.2%. Control cells were treated with DMSO alone at the same concentration present in the test substance being evaluated. Pharmaceuticals 2021, 14, 243 9 of 11

3.8. In Cell Western Assay The ICW assay was performed using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA) [19]. Briefly, A549 cells grown in 96-well plates (2 × 104 cells/well), either infected or mock-infected (Ctr) with PR8, were fixed with 4% formaldehyde, washed, permeabilized with 0.1% Triton X-100 and incubated with PBS containing Odyssey Blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). The cells were stained at 4 ◦C overnight with primary antibodies against influenza Nucleoprotein (NP) or Hemagglutinin (HA). After incubation, three washes with PBS plus 0.1% Tween 20 were performed and then the cells were stained with a mixture of fluorochrome-conjugated secondary antibodies (fluorescence emission at 800 nm), properly diluted in Odyssey blocking buffer and fluorochrome-conjugated Cell Tag (fluorescence emission at 700 nm), for 1 h at room temperature. Cell Tag was used as control of the integrity of cell monolayer. Subsequently, three washes with PBS plus 0.1% Tween 20 were performed and plates were analyzed by the Odyssey infrared imaging system (LI-COR). Integrated intensities of fluorescence were determined by the LI-COR Image Studio soft- ware and the relative fluorescence intensity (RFI) was expressed as percentage compared to untreated infected cells (100%).

3.9. Time-of-Addition Assay A549 cells were seeded in 12-well plates at a density of 2.5 × 105 cells/mL for 24 h at 37 ◦C, infected with PR8 and treated or not with furanodienone (1) or curzerene (3) (80 µg/mL) at different phases of the virus lifecycle, as indicated in the Results section. Un- treated infected cells were used as control. Cell supernatants were recovered to infect fresh monolayer and to analyze viral protein expression by ICW assay. Statistical significance of the data was analyzed by means of a Student t test and p values < 0.05 were considered significant. Data were expressed as mean ± the standard deviation (SD).

4. Conclusions

In conclusion, we reported the extraction of C. myrrha by supercritical CO2, an extrac- tion method that allowed avoiding undesired chemical reactions during the process. In addition, the analysis of the obtained myrrh oil and the quantification of its metabolites were performed by HPLC, so as to avoid high temperatures also during the analytical process. Interestingly, our study demonstrated that myrrh oil shows antiviral activity against influenza A Puerto Rico 8/34/H1N1 virus. Due the toxicity exerted by myrrh oil alone on the cell lines used for viral infection, we decided to add Vit. E acetate (1% w/v) that led to an increase in the viability of cells treated with the extract. It was also possible to appreciate the antiviral activity of myrrh oil plus Vit. E acetate. In particular, in the hemagglutination assay, the mixture of myrrh oil and the antioxidant compound showed a reduction in viral replication in a dose-dependent manner. Moreover, the expression of viral nucleoprotein was reduced using myrrh oil at 25 and 50 µg/mL in mixture with Vit. E acetate. Lastly, to investigate the antiviral activity of the main myrrh oil components, the single sesquiterpenes commercially available were tested. At non-toxic concentrations, the most active compounds, curzerene and furanodienone, proved to be effective at different steps of the virus lifecycle: furanodienone was mainly effective before and immediately after infection while curzerene was mainly active during viral adsorption. According to our results, myrrh and its metabolites may be useful to develop new antiviral agents active against influenza virus.

Author Contributions: Conceptualization, D.D.V., L.S., R.C., R.D.S. and L.N.; methodology, V.N.M., D.D.V., M.D.A., S.G. and G.D.C.; validation, A.M., A.D.L., V.T., D.I., M.D.A. and G.D.C.; formal analysis, M.D.A. and G.D.C.; investigation, D.D.V., V.N.M., A.M., A.D.L., V.T., D.I., M.D.A., S.G. and G.D.C.; resources and project administration, L.S., R.C., R.D.S. and L.N.; writing—original draft preparation, V.N.M., D.D.V., and L.N.; writing—review and editing, V.N.M., D.D.V., A.M., F.S., S.G., R.C. and L.N.; visualization, V.N.M., D.D.V., and L.N.; supervision, D.D.V., L.S., R.C., R.D.S., A.T.P. Pharmaceuticals 2021, 14, 243 10 of 11

and L.N.; funding acquisition, R.C., A.T.P. and L.N. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MIUR-PRIN 2017, 2017BMK8JR006 (project “ORIGINALE CHEMIAE in Antiviral Strategy—Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad Spectrum Antiviral Strategy”) (LN), MIUR-PONARS 01_00597_OR4 (ATP) grants. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We wish to thank the Exenia Group (Pinerolo, TO, Italy) for providing the SCFE extracts. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Suleiman, M.H.A. Prenylated flavonoids from the stem wood of Commiphora opobalsamum (L.) Engl.(Burseraceae). J. King Saud Univ. Sci. 2015, 27, 71–75. [CrossRef] 2. Latha, S.; Selvamani, P.; Prabha, T. Pharmacological uses of the belonging to the genus Commiphora. Cardiovasc. Hematol. Agents Med. Chem. 2020.[CrossRef][PubMed] 3. Hanuš, L.O.; Rˇezanka,T.; Dembitsky, V.M.; Moussaieff, A. Myrrh-Commiphora chemistry. Biomed. Pap. 2005, 149, 3–28. [CrossRef] [PubMed] 4. Morteza-Semnani, K.; Saeedi, M. Constituents of the essential oil of Commiphora myrrha (Nees) Engl. var. molmol. J. Essent. Oil Res. 2003, 15, 50–51. [CrossRef] 5. Shalaby, M.A.; Hammouda, A.A. Analgesic, anti-inflammatory and anti-hyperlipidemic activities of Commiphora molmol extract (Myrrh). J. Intercult. Ethnopharmacol. 2014, 3, 56–62. [CrossRef][PubMed] 6. Chen, Y.; Zhou, C.; Ge, Z.; Liu, Y.; Liu, Y.; Feng, W.; Li, S.; Chen, G.; Wei, T. Composition and potential anticancer activities of essential oils obtained from myrrh and frankincense. Oncol. Lett. 2013, 6, 1140–1146. [CrossRef][PubMed] 7. Zhai, B.; Zhang, N.; Han, X.; Li, Q.; Zhang, M.; Chen, X.; Li, G.; Zhang, R.; Chen, P.; Wang, W.; et al. Molecular targets of β-elemene, a herbal extract used in traditional Chinese medicine, and its potential role in cancer therapy: A review. Biomed. Pharmacother. 2019, 114, 108812. [CrossRef][PubMed] 8. Messina, F.; Gigliarelli, G.; Palmier, A.; Marcotullio, M.C. Furanodienone: An emerging bioactive furanosesquiterpenoid. Curr. Org. Chem. 2017, 21, 305–310. [CrossRef] 9. Zhu, N.; Kikuzaki, H.; Sheng, S.; Sang, S.; Rafi, M.M.; Wang, M.; Nakatani, N.; Di Paola, R.S.; Rosen, R.T.; Ho, C.-T. Fura- nosesquiterpenoids of Commiphora myrrha. J. Nat. Prod. 2001, 64, 1460–1462. [CrossRef][PubMed] 10. El Ashry, E.S.H.; Rashed, N.; Salama, O.M.; Saleh, A. Components, therapeutic value and uses of myrrh. Pharmazie 2003, 58, 163–168. [PubMed] 11. Chang, R.; de Morais, S.A.L.; Napolitano, D.R.; Duarte, K.C.; Guzman, V.B.; Nascimento, E.A. A new approach for quantifying furanodiene and curzerene: A case study on the essential oils of Eugenia uniflora L., Myrtaceae (pitangueira) leaves. Rev. Bras. Farmacogn. 2011, 21, 392–396. [CrossRef] 12. Marongiu, B.; Piras, A.; Porcedda, S.; Scorciapino, A. Chemical composition of the essential oil and supercritical CO2 extract of Commiphora myrrha (Nees) Engl. and of Acorus calamus L. J. Agric. Food Chem. 2005, 53, 7939–7943. [CrossRef][PubMed] 13. Pescatori, L.; Metifiot, M.; Chung, S.; Masoaka, T.; Cuzzucoli Crucitti, G.; Messore, A.; Pupo, G.; Madia, V.N.; Saccoliti, F.; Scipione, L.; et al. N-Substituted quinolinonyl diketo acid derivatives as HIV integrase strand transfer inhibitors and their activity against RNase H function of reverse transcriptase. J. Med. Chem. 2015, 58, 4610–4623. [CrossRef][PubMed] 14. Costi, R.; Di Santo, R.; Artico, M.; Miele, G.; Valentini, P.; Novellino, E.; Cereseto, A. Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase. J. Med. Chem. 2007, 50, 1973–1977. [CrossRef][PubMed] 15. Bizzarri, B.M.; Fanelli, A.; Piccinino, D.; De Angelis, M.; Dolfa, C.; Palamara, A.T.; Nencioni, L.; Zippilli, C.; Crucianelli, M.; Saladino, R. Synthesis of stilbene and chalcone inhibitors of influenza A virus by SBA-15 supported Hoveyda-Grubbs metathesis. Catalysts 2019, 9, 983. [CrossRef] 16. Joshi, S.C.; Mathela, C.S. Antioxidant and antibacterial activities of the leaf essential oil and its constituents furanodienone and curzerenone from Lindera pulcherrima (Nees.) Benth. ex hook. f. Pharmacogn. Res. 2012, 4, 80–84. [CrossRef] 17. Celestino, I.; Checconi, P.; Amatore, D.; De Angelis, M.; Coluccio, P.; Dattilo, R.; Alunni Fegatelli, D.; Clemente, A.M.; Matarrese, P.; Torcia, M.G.; et al. Differential redox state contributes to sex disparities in the response to influenza virus infection in male and female mice. Front. Immunol. 2018, 9, 1747. [CrossRef][PubMed] Pharmaceuticals 2021, 14, 243 11 of 11

18. Di Sotto, A.; Checconi, P.; Celestino, I.; Locatelli, M.; Carissimi, S.; De Angelis, M.; Rossi, V.; Limongi, D.; Toniolo, C.; Martinoli, L.; et al. Antiviral and antioxidant activity of a hydroalcoholic extract from Humulus lupulus L. Oxidat. Med. Cell. Long. 2018, 2018, e5919237. [CrossRef][PubMed] 19. Fioravanti, R.; Celestino, I.; Costi, R.; Cuzzucoli Crucitti, G.; Pescatori, L.; Mattiello, L.; Novellino, E.; Checconi, P.; Palamara, A.T.; Nencioni, L.; et al. Effects of polyphenol compounds on influenza A virus replication and definition of their mechanism of action. Bioorg. Med. Chem. 2012, 20, 5046–5052. [CrossRef][PubMed] 20. De Vita, D.; Messore, A.; Toniolo, C.; Frezza, C.; Scipione, L.; Bertea, C.M.; Micera, M.; Di Sarno, V.; Madia, V.N.; Pindinello, I.; et al. Towards a new application of amaranth seed oil as an agent against Candida albicans. Nat. Prod. Res. 2019, 1–6. [CrossRef] [PubMed]