research article biological activities of aerial parts...

Research ArticleBiological Activities of Aerial Parts Extracts ofEuphorbia characias

Maria Barbara Pisano1 Sofia Cosentino1 Silvia Viale1 Delia Spanograve2 Angela Corona2

Francesca Esposito2 Enzo Tramontano2 Paola Montoro3

Carlo Ignazio Giovanni Tuberoso2 Rosaria Medda2 and Francesca Pintus2

1Department of Public Health Clinical and Molecular Medicine University of Cagliari Cittadella Universitaria09042 Monserrato Italy2Department of Sciences of Life and Environment University of Cagliari Cittadella Universitaria 09042 Monserrato Italy3Department of Pharmacy University of Salerno 84084 Fisciano Italy

Correspondence should be addressed to Francesca Pintus fpintusunicait

Received 22 January 2016 Revised 27 April 2016 Accepted 3 May 2016

Academic Editor Ibrahim M Banat

Copyright copy 2016 Maria Barbara Pisano et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of the present studywas to evaluate antioxidant antimicrobial anti-HIV and cholinesterase inhibitory activities of aqueousand alcoholic extracts from leaves stems and flowers of Euphorbia characias The extracts showed a high antioxidant activityand were a good source of total polyphenols and flavonoids Ethanolic extracts from leaves and flowers displayed the highestinhibitory activity against acetylcholinesterase and butyrylcholinesterase showing potential properties against Alzheimerrsquos diseaseAntimicrobial assay showed that leaves and flowers extracts were active against all Gram-positive bacteria tested The ethanolicleaves extract appeared to have the strongest antibacterial activity against Bacillus cereus with MIC value of 3125120583gmL followedby Listeria monocytogenes and Staphylococcus aureus that also exhibited good sensitivity withMIC values of 1250 120583gmLMoreoverall the extracts possessed anti-HIV activityThe ethanolic flower extract was themost potent inhibitor ofHIV-1 RTDNApolymeraseRNA-dependent and Ribonuclease H with IC

50values of 026 and 033 120583gmL respectivelyThe LC-DADmetabolic profile showed

that ethanolic leaves extract contains high levels of quercetin derivatives This study suggests that Euphorbia characias extractsrepresent a good source of natural bioactive compounds which could be useful for pharmaceutical application as well as in foodsystem for the prevention of the growth of food-borne bacteria and to extend the shelf-life of processed foods

1 Introduction

There has been a remarkable increment in scientific articlesdealing with research of antioxidant molecules because oftheir protective action from the damage induced by oxidativestress It causes serious cell and tissue damage leading it tobe the major cause of the pathogenesis of several diseaseprocesses like cancer diabetes aging and cardiovascularand neurodegenerative diseases Plant materials representa great source of antioxidant and bioactive compoundswhich are different in their composition and physical andchemical properties [1] Phenolics are broadly distributed inthe plant kingdom and are the most abundant secondarymetabolites of plants The identification and development

of phenolic compounds or extracts from different plantshas become a major area of health- and medical-relatedresearch Among these compounds flavonoids have beenespecially highlighted because they have been shown to haveprotective roles against many human diseases due to theirantioxidant capacity which depends mainly on the numberand position of hydroxyl groups within their structure andtheir anti-inflammatory anticancer and antiviral activities[2] Their activity as inhibitors of cholinesterase has alsobeen demonstrated and correlated with their structure andcould be useful for treatment of Alzheimerrsquos disease (AD)[3] AD results from a deficit of cholinergic functionsin the brain Hence one of the most promising approachesfor treating this disease is to restore the acetylcholine level

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 1538703 11 pageshttpdxdoiorg10115520161538703

2 BioMed Research International

by inhibiting cholinesterase activity Acetylcholinesterase(AChE) and butyrylcholinesterase (BChE) are two cholin-esterase enzymes that metabolize acetylcholine but differin substrate specificity enzyme kinetics and activity indifferent brain regions In a healthy brain AChE is the mostresponsible enzyme in regulating acetylcholine levels but inpatients with AD AChE activity gradually decreases with theconcomitant increase of BChE Thus both AChE and BChEare legitimate therapeutic targets for treatment of cholinergicdeficit characteristic of AD Effective therapeutic options forAD are limited up to now thus there is a demand for newnatural drugs without side effects

Moreover the demand for bioactive compounds fromnatural sources as an alternative to synthetic molecules iscontinuously increasing also because of the emerging prob-lem of the multidrug resistance of microorganisms relatedto antibiotics and their extensive use Thus several studiesrelated to plant antimicrobials have demonstrated their effi-cacy towards a large number of pathogens and food-borneagents causing disease [4ndash6] as well as viral infections [7ndash10] Although many plant-derived compounds are currentlybeing used for the treatment of infectious diseases and for thepreservation and extension of the shelf-life of foods severalmedicinal and aromatic plants present worldwide remain stillunexplored

Euphorbiaceae is a large flowering plant family (300genera and 8000 species) widely distributed all around theworld and composed of all sorts of plants (large woodytrees climbing lianas or simple weeds) with a wide varietyof chemical substances many of them with a medicinalapplication Some extracts from Euphorbiaceae plants havebeen characterized and patented as modern drugs [11]Among Euphorbiaceae the species Euphorbia characias anonsucculent shrub commonly occurring in vast areas of theMediterranean region has been analyzed and several biolog-ical active compounds were identified [12] The plant latexhas been the object of several researches and its screeninghas revealed the presence of natural rubber and numerousenzymes some of which interact in a common metabolism[13ndash22] On the other hand only little attention has been paidto the other parts of the plant [23ndash25]

Thus the objective of this research was to evaluateantioxidant antimicrobial and anticholinesterase propertiesof the aqueous and ethanolic extracts of leaves stems andflowers from E characias in order to find a novel potentialsource of bioactivemoleculesMoreover the antiviral efficacyof E characias extracts was also evaluated on the humanimmunodeficiency virus type 1 (HIV-1) reverse transcriptase-(RT-) associated RNA-dependent DNA polymerase (RDDP)and RibonucleaseH (RNaseH) activities Both RT-associatedfunctions are essential for viral replication and are validateddrug targets for which new drugs are still needed [26 27]

2 Materials and Methods

All chemicals were obtained as pure commercial productsand used without further purification Acetylcholinesterase(AChE) from Electrophorus electricus acetylthiocholine

Figure 1 The Mediterranean shrub Euphorbia characias subspcharacias

iodide 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) aluminum nitrate butyrylcholinesterase(BChE) from equine serum S-butyrylthiocholine chloride3-O-caffeoylquinic acid (chlorogenic acid) catechin 22-diphenyl-1-picrylhydrazyl radical (DPPH∙) 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) ellagic acid Folin-Ciocalteuphenol reagent ferric chloride galantamine hydrobromidegallic acid 4-hydroxybenzyl alcohol 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) myricetin246-tris(2-pyridyl)-135-triazine (TPTZ) and quercetinwere purchased from Sigma Aldrich (Milan Italy) LC-MSgrade acetonitrile and formic acid were purchased fromMerck (Darmstadt Germany) Standards of myricetin-3-O-glucoside quercetin-3-O-glucoside and acacetin werepurchased from Extrasynthese (Genay France) HPLCgrade water (18MΩsdotcm) was prepared by using a Millipore(Bedford MA USA) Milli-Q purification system

Spectrophotometric determinations were obtained withan Ultrospec 2100 spectrophotometer (Biochrom Ltd Cam-bridge England) using cells with a 1 cm path length

21 Plant Material E characias subsp characias (Figure 1)was identified by Professor Annalena Cogoni and a voucherspecimen has been deposited in the Department of Sci-ences of Life and Environment University of Cagliari Italy(number 121616 Herbarium CAG) The different parts of Echaracias were collected from February to June in southernSardinia (Dolianova CA Italy) The GPS coordinates were39∘24101584019010158401015840N and 9∘12101584057610158401015840E

Leaves stems and flowers were immediately frozenat minus80∘C and then lyophilized in intact condition Thelyophilized plant materials (1 g) were reduced in powderand 10mL of water (aqueous extract) or ethanol (ethanolextract) was added to the dried samples The extraction wascarried out in the dark at room temperature for 24 h undercontinuous stirring Ethanol extracts were diluted 10-foldwith water in order to freeze and then lyophilize the samples[24] Before use 1mg of dried powders was dissolved inwater or 10ethanol (1mL) for aqueous and ethanol extractsrespectively For antimicrobial and antiviral activity dried

BioMed Research International 3

powderswere dissolved inDMSO (100) as solventThe yield( ww) from all the dried extracts was calculated as followsyield () = (1198601 times 100)1198602 where 1198601 is the weight of thedried extract (after lyophilization) and1198602 is the weight of theplant powder

22 Antioxidant Assays In every extract total free radi-cal scavenging molecules were determined by ABTS∙+ andDPPH∙ methods using Trolox as antioxidant standard aspreviously reported [28 29] For both free radical meth-ods antioxidant activity was expressed as Trolox equivalentantioxidant capacity (TEAC mmolg dw) The FRAP (ferricreducing antioxidant power) assay was performed as previ-ously described [30] Quantitative analysis was performedaccording to the external standard method (FeSO

4 01ndash

2mmolL 119903 = 09997) and results were expressed as mmolFe2+g dw

23 Determination of the Total Polyphenols and FlavonoidsTotal content of polyphenols and flavonoids in the extractswas determined as previously reported [29] Polyphenolconcentration was calculated using gallic acid as a referredstandard and was expressed as mg of gallic acid (GAE) per 1 gof dry weight (dw) Flavonoid concentrationwas expressed asmg of quercetin equivalent (QE) per 1 g of dry extract

24 Acetylcholinesterase and Butyrylcholinesterase ActivityAcetylcholinesterase from Electrophorus electricus and equineserum butyrylcholinesterase were used for the inhibitoryassays AChE activity was measured using Ellmanrsquos reagentaccording to the method previously reported [31] Briefly thereaction mixture contained 01M phosphate buffer (pH 80)15mM 551015840-dithiobis-2-nitrobenzoate (DTNB) acetylthio-choline iodide (15mM) and extract at the desired con-centrations or solvent alone (control) in a final volume of1mL Finally enzyme was added to the reaction mixtureand the absorbance immediately monitored at 405 nm Forbutyrylcholinesterase assay the same procedure was followedexcept for the use of enzyme and substrate which wereBChE and S-butyrylthiocholine respectively Galantaminewas used as the standard cholinesterase inhibitor Resultswere expressed as IC

50values calculated as concentration of

extracts that produces 50 cholinesterase activity inhibition

25 Microbial Strains Culture Conditions and Antimicro-bial Activity Staphylococcus aureus ATCC 6538 Bacilluscereus ATCC 11178 Listeria monocytogenes ATCC 19115Escherichia coli ATCC 35150 (serotype O157H7) Salmonellatyphimurium ATCC 14028 Candida albicans ATCC 10231Saccharomyces cerevisiae ATCC 2601 Aspergillus flavusATCC 46283 (aflatoxin producer) and Penicillium chryso-genum ATCC 10135 were obtained from the American TypeCulture Collection (ATCC Rockville MD USA) and usedas indicators strains All bacterial strains were stored onnutrient broth (NB Microbiol Cagliari Italy) plus 20 (vv)glycerol at minus20∘C except yeasts and molds strains which weremaintained in potato dextrose broth (Microbiol) with 15

(vv) glycerol Before use they were subcultured twice inappropriate medium

Minimum inhibitory concentrations (MICs) and mini-mum bactericidalfungicidal concentrations (MBCsMFCs)of the E characias extracts were determined by a brothmicrodilution method [5] All tests were performed with NBfor bacteria and RPMI 1640 (Sigma Milan Italy) bufferedto pH 70 with morpholinepropanesulfonic acid (MOPSSigma) for yeasts and molds The extracts were dissolved inDMSO (5 vv) Serial doubling dilutions of each extractwere performed in a 96-well microtiter plate ranging from195 to 5000120583gmL Overnight broth cultures were preparedin NB or RPMI and adjusted so that the final concentra-tion in each well following inoculation was approximately50 times 105 cfumL The concentration of each inoculum wasconfirmed using viable counts on Tryptic Soy Agar (TSAMicrobiol) plates for bacteria Sabouraud Dextrose Agar(SDA Microbiol) for yeasts and potato dextrose agar (PDAMicrobiol) for molds The controls included sterility of NBand RPMI broths sterility of the extracts control culture(inoculum) and control DMSO to check the effect of solventon the growth of microorganisms Furthermore gentamicinketoconazole and amphotericin B were used as positivecontrols for bacteria yeasts and molds respectively

The MICs and MBCs were determined after 24 h incu-bation of the plates at 37∘C for bacteria and 30∘C for fungiMicrobial growth was indicated by the presence of turbidityand a ldquopelletrdquo on the well bottom MICs were determinedpresumptively as the first well in ascending order whichdid not produce a pellet To confirm MICs and to establishMBCs 10 120583L of broth was removed from each well andinoculated on TSA SDA or PDA plates After incubationunder the conditions described above the number of sur-viving microorganisms was determined The MIC was thelowest concentration which resulted in a significant decreasein inoculum viability (gt90) while the MBCMFC was theconcentration where 999 or more of the initial inoculumwas killed

All tests were conducted in triplicate and with threereplications and the modal MIC and MFC values wereselected

26 HIV-1 RT-Associated Functions Biochemical Assays HIV-1 RT gene subcloned into the p6HRT prot plasmid waskindly provided by Stuart LeGrice (NationalCancer InstituteFrederick USA) Protein expression and purification wasperformed in E coliM15 strain as described [32] The HIV-1RT-associated RDDP and RNase H activity were measured aspreviously described [33 34]

27 LC Detection and Quantitative Analysis ofPhenolic Compounds

271 (HR) LC-ESI-Orbitrap-MS and (HR) LC-ESI-Orbitrap-MSMS The electrospray ionisation (ESI) source of aThermo Scientific LTQ-Orbitrap XL (Thermo ScientificGermany) mass spectrometer was tuned in negative ionmode with a standard solution of kaempferol-3-O-glucoside


Table 1 Yield and antioxidant and antiradical properties of E characias extracts DPPH and ABTS values are expressed as mmol TEACgdw FRAP value is expressed as mmol Fe2+g dw

Extracts Yield (ww) ABTS DPPH FRAP

Leaves Aqueous 204 plusmn 15 252 plusmn 018b 18 plusmn 021b 270 plusmn 017c

Ethanolic 249 plusmn 26 468 plusmn 049a 673 plusmn 070a 457 plusmn 012a

Stems Aqueous 123 plusmn 14 010 plusmn 001d 017 plusmn 002c 089 plusmn 004f

Ethanolic 152 plusmn 18 085 plusmn 010c 088 plusmn 009c 136 plusmn 007e

Flowers Aqueous 176 plusmn 23 062 plusmn 005cd 053 plusmn 005c 195 plusmn 002d

Ethanolic 206 plusmn 19 091 plusmn 010c 058 plusmn 005c 349 plusmn 009b

Results are expressed as mean plusmn standard deviation of three independent experiments Means followed by distinct letters in the same column are significantlydifferent (119901 lt 005)

(1 120583gmL) infused at a flow rate of 5 120583Lmin with a syringepump In the FT experiment resolution of the Orbitrapmass analyzer was set at 30000 The mass spectrometricspectra were acquired by full range acquisition covering119898119911 120ndash1200 in LC-MS The data recorded were processedwith Xcalibur 20 software (Thermo Fisher Scientific) Initialcalibration of the instrument was performed using the stan-dard LTQ calibration mixture with caffeine and the peptideMRFA dissolved in 50 50 (vv) wateracetonitrile solutionLCESILIT-Orbitrap-MS was performed using a FinniganSurveyor HPLC (Thermo Finnigan San Jose CA USA)equipped with a Waters (Milford MA USA) Xselect CSHC18 35 120583m column (150mm times 21mm id) and coupled to ahybrid Linear Ion Trap- (LIT-) Orbitrap mass spectrometer(Thermo Scientific) Linear gradient elution with a mobilephase comprising water acidified with 01 formic acid(solvent A) and acetonitrile acidified with 01 formic acid(solvent B) starting from 95 A was converted in 65 A in45min from 65 to 0 (A) in 1min remaining 0 A for 4minutes and then from 0 to 95 (A) followed by 10minof maintenance The mobile phase was supplied at a flow rateof 200120583Lmin keeping the column at room temperature andthe effluent was injected directly into the ESI source Themass spectrometer was operated in negative ion mode ESIsource parameters were as follows capillary voltage minus12 Vtube lens voltage minus12147V capillary temperature 280∘Csheath and auxiliary gas flow (N

2) 30 and 5 sweep gas 0

and spray voltage 5V MS spectra were acquired by full rangeacquisition covering119898119911 120ndash1600 LC-ESI-LIT-MSMS datawere obtained by applying a data dependent scan experimentby directing to fragmentation the two highest peaks obtainedin LC-ESI-Orbitrap-MS trace Each parent ion was submittedto fragmentation with energy of 30 to produce an MSMSspectrum in the MS range specific relative to its mass 1mg of dried extract obtained from ethanolic extract from Echaracias leaves was dissolved in 10mL of a mixture of waterin acetonitrile and 10 120583L was injected in the LC-MS system

272 LC-DAD Detection and quantitative analysis of phe-nolic compounds were carried out using an HPLC-DADmethod [30] Chromatograms and spectra were elaboratedwith a ChromQuest V 251 data system (ThermoQuestRodano Milan Italy) Flavonols were detected and quan-tified at 360 nm and all the other compounds at 280 nm

Stock solutions were prepared at 1mgmL dissolving purestandards in methanol water (50 50 vv) The calibrationcurves for each compound were calculated by regressionanalysis by plotting the peak area obtained after standardsinjection (3 replicates at each concentration) against theknown standard concentrations The stock solutions werediluted with methanol in order to obtain work solutions andthe correlation values were 09992ndash09998 E characias leavesextract was dissolved and injected in the LC-DAD systemwith the same condition of the LC-MS analysis

28 Statistical Analysis Data are reported as mean plusmn stan-dard deviation of three independent experiments The datawere analyzed using one-way analysis of variance (one-wayANOVA) and Tukeyrsquos posttest Statistical analysis was per-formed with GraphPad Prism 7 software (GraphPad Soft-ware San Diego California USA) A difference was consid-ered statistically significant at 119901 lt 005

3 Results and Discussion

31 Antioxidant Activity Polyphenol and Flavonoids Contentand Cholinesterase Activity Inhibition Total free radical scav-enging capacities determined with ABTS and DPPH assaysare reported in Table 1 Comparable TEAC values of eachextract were detected using the two methods Leaves extractsexhibited significantly higher free radical scavenging activity(119901 lt 005) than other extracts with ethanolic extract showingthe highest activity Antioxidant activity was also examinedwith FRAP assay confirming that ethanolic extract of leavespossessed the significantly highest antioxidant activity (119901 lt005) followed by flower ethanolic extract

The polyphenol and flavonoid content of the extractsexpressed as mg of gallic acid or quercetin equivalentrespectively are reported in Figure 2 Total phenolic con-tent varied widely among analyzed parts of the plant andthe highest value was found in leaves in both aqueousand ethanolic extracts In fact the amount of phenoliccompounds in leaves aqueous extract (680mgGAEg dw)was about 6- and 23-fold higher than the same extractfrom stems (1021mgGAEg dw) and flowers (2892mgGAEg dw) respectively (Figure 2(a)) Moreover these resultsshowed that ethanolic extract of leaves is rich in polyphenolscontent (8157mgGAEg dw) with a value about 15-fold


Leaves Flowers

GA

E (m

gg

dw)

Et Aq Et Aq EtAqStems

0

200

400

600

800

1000

(a)

QE

(mg

g dw

)

Leaves FlowersEt Aq Et Aq EtAq

Stems

0

50

100

150

200

250

(b)

Figure 2 Polyphenol and flavonoid content in aqueous (Aq) and ethanolic (Et) leaves stems and flowers extracts from E characias (a)Polyphenol amount is expressed as mg of gallic acid equivalent (GAE) per g of dry weight (dw) (b) the amount of flavonoids is expressed asmg of quercetin equivalent (QE) per g of dry weight (dw) All data are expressed as mean of three measurements plusmn standard deviation

minus2

0

2

4

6

ABT

S TE

AC (m

mol

g d

w)

200 400 600 8000GAE (mgg dw)

r = 08448

y = 00055x minus 11362

(a)

minus2

0

2

4

6

8

DPP

H T

EAC

(mm

olg

dw

)

200 400 600 8000GAE (mgg dw)

r = 07346

y = 00070x minus 16991

(b)

Figure 3 Correlation between phenolic content and antioxidant capacity of E characias extracts (a) ABTS assay (b) DPPH assay TEACTrolox equivalent antioxidant capacity GAE gallic acid equivalents

higher than the same extract from stems and flowers (about5446mgGAEg dw for each extract) Polyphenols have beenreported to have antioxidant activity mainly based on theirredox properties which have a key role in scavenging freeradicals and chelating oxidant metal ions High phenoliccontent in all E characias extracts resulted in high TEACvalues determined by ABTS or DPPH methods which couldindicate that phenolic compounds were capable of function-ing as free radical scavengers Good correlation was foundbetween TEAC values for both ABTS and DPPH (119903 = 08448and 119903 = 07346 resp) of the different plant extracts and theirphenolic contents (Figure 3) confirming a strong relationshipbetween antioxidant capacity and phenolic content

Among phenolic compounds flavonoids are ubiquitousplant compounds with an important role as attractants to

pollinators as sunscreens to protect against UV irradia-tion and as antimicrobial and antiherbivory factors [37]Content of flavonoids is shown in Figure 2(b) Flavonoidcontent of flowers ethanolic extract was about 2-fold higher(2417mgQEg dw) than the correspondent extract from theother part of the plant (sim62mgQEg dw) This extract alsoshowed the highest ratio of total flavonoids (TFC) andphenolic content (TPC) being 044 indicating that flavonoidswere almost 40 of the total phenolic content Howeveraqueous extracts of both leaves and stems exhibited the lowestamounts of total flavonoids (332mg and 181mgQEg dwresp) with aqueous extract from leaves showing the lowestTFCTPC ratio of 005

Compounds like flavonoids alkaloids terpenoids andcoumarins are known to have the capacity to inhibit


Table 2 Inhibition of AChE and BChE by E characias extracts

Extracts IC50values (mgmL)

AChE BChE

Leaves Aqueous 42 plusmn 025c mdashEthanolic 06 plusmn 0056d 039 plusmn 004c

Stems Aqueous 69 plusmn 071a mdashEthanolic 58 plusmn 043b mdash

Flowers Aqueous 525 plusmn 035b 42 plusmn 039a

Ethanolic 06 plusmn 0045d 122 plusmn 008b

Galantamine 027 plusmn 007 120583gmL 812 plusmn 061 120583gmLResults are expressed as mean plusmn standard deviation of three independentexperiments Means followed by distinct letters in the same column aresignificantly different (119901 lt 005)

cholinesterase types key enzymes in the cholinergic nervoussystem [38] This inhibitory activity is usually the first of anumber of requirements for the development of medicinesfor treating some neurological disorders such as Alzheimerrsquosdisease

Table 2 shows the acetylcholinesterase and butyrylcholin-esterase inhibitory activities of the E characias extractscompared with those of standard inhibitor galantamine Asurvey on IC

50values revealed that all the extracts exhibited

acetylcholinesterase inhibitory activity while only few ofthem inhibited butyrylcholinesterase This is not surprisingbecause AChE and BChE enzymes display distinct substrateand inhibitor specificities Most of the other IC

50values

were about three orders of magnitude higher compared tothe results obtained with galantamine However ethanolicleaves extract showed the significantly highest (119901 lt 005)butyrylcholinesterase inhibition that is about 50 times lessthan the effect of galantamine This is a good result since thestandard inhibitor is a singlemolecule whereas plant extractsare a mixture of numerous compounds and might containonly few active components

32 Antimicrobial and Anti-HIV Activities Tables 3(a) and3(b) report the antagonistic activity of E characias extractsagainst a panel of microorganisms including Gram-negativeGram-positive and spore-forming bacteria yeast and moldspecies As can be observed the leaves extracts appeared tohave the strongest antibacterial activity followed by flow-ers and stems extracts Gram-negative bacteria yeasts andmolds were the least sensitive being resistant to all extractsat the maximum concentration tested (5000120583gmL) Leavesextracts exhibited antibacterial activity towards all Gram-positive bacteria tested B cereus ATCC 11778 was the mostsusceptible strain being totally inhibited by the ethanolicleaves extract at the concentration of 3125 120583gmL (MICequivalent to MBC) L monocytogenes ATCC 19115 and Saureus ATCC 6538 also exhibited a good sensitivity to theethanolic leaves extract as they both showed MIC valuesof 1250 120583gmL Higher MIC values were observed for theaqueous leaves extracts towards these bacterial strains Asregards the flowers extracts all Gram-positive strains testedwere moderately inhibited by both aqueous and ethanolic

extracts withMIC values equal to or greater than 2500120583gmLwith the exception of B cereus strain which was moresensitive to the ethanolic extract (MIC 1250 120583gmL) Stemsextracts showed the lowest activity with ethanolic extractslightly inhibiting the abovementioned bacterial strains (MIC5000 120583gmL) Our findings are in agreement with thosereported by Lin et al for Euphorbia macrorrhiza species[39] who observed inhibitory activity against S aureus butno effect towards E coli and C albicans strains tested Incontrast Perumal et al [40] observed antimicrobial effectfor Euphorbia hirta ethanolic extracts towards both Gram-positive and Gram-negative bacteria with MIC values lowerthan those observed in this work On the other hand theantagonistic effect of plants extracts even within the samespecies is variable and depends on several factors such asthe concentration of active components due to differenttissue composition variation in the extraction protocol andtechnique used to detect antimicrobial activity and resistanceof the test microorganisms [41ndash43]

Finally E characias extracts were also tested for theirability to inhibit the HIV-1 RT-associated RDDP and RNaseH functions using the known nonnucleoside RT inhibitorefavirenz [27] and the diketo acid derivative RDS1759 [33] ascontrol for RDDPandRNaseH inhibition respectively Inter-estingly all extracts were able to inhibit both RT functions(Table 4) In all cases ethanolic extractsweremore active thanaqueous extracts and the flower extracts were themost potenton both enzyme activities

33 LC-ESI-Orbitrap-MS LC-ESI-Orbitrap-MSMS and LC-DAD Analysis of E characias Ethanolic Leaves Extract Con-sidering all the antioxidants and biological activities of theextracts ethanolic extract from leaves appeared to be themost active extractThus this extract was selected for furtherstudy by liquid chromatography (LC) It was analyzed byan analytical method developed in LC-ESI-Orbitrap-MS andLC-ESI-Orbitrap-MSMS in negative ionmodeThenegativeLC-MS profile highlighted the presence of a large group ofcompounds corresponding to the deprotonated molecularions of different flavonoids and ellagitannin derivatives (Fig-ure 4) Individual components were identified by comparisonof their 119898119911 values in the Total Ion Current (TIC) profilewith those of the selected compounds described in literature(Table 5) Additional LC-ESI-Orbitrap-MSMS experimentswere carried out in order to select and submit these ionsto fragmentation experiments using the parameters previ-ously chosen by ESI-MS and ESI-MSMS direct infusionexperiments Bymatching experimental MSMS spectra withthose reported in literature andor with those reported ina public repository of mass spectral data called MassBank[35] compounds 1ndash16 were identified with the exception ofcompounds 3 5 and 8 (unknown compounds)

Table 5 reports identification of compounds based onhigh resolution mass spectrometric data chemical for-mula derived by accurate mass maturation retention timesMSMS results and references used for identification Com-pounds 1 and 2 were identified by the diagnostic [M minusH]minus ion shown in HR ESI-MS analysis compared with


Table 3 Antimicrobial activity of E characias aqueous (a) and ethanol (b) extracts MIC and MBCMFC values are expressed in 120583gmL

(a)

Target microorganisms Leaves Stems FlowersMIC MBCMFC MIC MBCMFC MIC MBCMFC

E coli gt5000 mdash gt5000 mdash gt5000 mdashS typhimurium gt5000 mdash gt5000 mdash gt5000 mdashS aureus 5000 gt5000 gt5000 mdash 5000 gt5000B cereus 1250 1250 5000 gt5000 2500 5000L monocytogenes 2500 2500 gt5000 mdash 2500 gt5000C albicans gt5000 mdash gt5000 mdash gt5000 mdashS cerevisiae gt5000 mdash gt5000 mdash gt5000 mdashA flavus gt5000 gt5000 gt5000 mdash gt5000 gt5000P chrysogenum gt5000 gt5000 gt5000 mdash gt5000 gt5000

(b)


E coli gt5000 mdash gt5000 mdash gt5000 mdashS typhimurium gt5000 mdash gt5000 mdash gt5000 mdashS aureus 1250 2500 5000 gt5000 5000 gt5000B cereus 3125 3125 5000 gt5000 1250 2500L monocytogenes 1250 1250 5000 gt5000 5000 gt5000C albicans gt5000 mdash gt5000 mdash gt5000 mdashS cerevisiae gt5000 mdash gt5000 mdash gt5000 mdashA flavus gt5000 mdash gt5000 mdash gt5000 gt5000P chrysogenum gt5000 mdash gt5000 mdash gt5000 gt5000Positive controls ampicillin MICs (S aureus ATCC 6538 25120583gmL B cereus ATCC 11778 10120583gmL and L monocytogenes ATCC 19115 25120583gmL)gentamicinMICs (E coliATCC 35150 10120583gmL S typhimuriumATCC 14028 10 120583gmL) ketoconazoleMICs (C albicansATCC 10231 25120583gmL S cerevisiaeATCC 2601 25 120583gmL) amphotericin B MICs (A flavus ATCC 46283 and P chrysogenum ATCC 10135 5 120583gmL)

Table 4 Effects of E characias extracts on HIV-1 RT-associatedfunctions

ExtractsIC50(120583gmL)lowast

HIV-1 HIV-1RDDP RNase H

Leaves Aqueous 0785 plusmn 0003c 195 plusmn 103ab

Ethanolic 075 plusmn 0028c 0685 plusmn 0155bc

Stems Aqueous 687 plusmn 081a 2235 plusmn 0245a

Ethanolic 305 plusmn 02b 1615 plusmn 0035abc

Flowers Aqueous 103 plusmn 00c 151 plusmn 053abc

Ethanolic 026 plusmn 008c 033 plusmn 01c

Efavirenz 00016 plusmn 00003lowastlowast NDlowastlowastlowast

RDS1759 ND 71 plusmn 05lowastlowastlowastExtracts concentration required to inhibit HIV-1 RT-associated functionsby 50lowastlowastValues expressed in 120583M concentrationlowastlowastlowastNot doneMeans followed by distinct letters in the same column are significantlydifferent (119901 lt 005)

standards and literature and from the MSMS data obtainedworking in LC-ESI-Orbitrap-MSMS in Product Ion Scanin negative ion mode and the compounds were identified

by MassBank as gallic acid and catechin and confirmedby standard analysis Compounds 7 9 10 11 and 15 wereidentified by the diagnostic [M minusH]minus ions shown in HR ESI-MS analysis their fragmentation profiles obtained in LC-ESI-(Orbitrap)-MSMS in Product Ion Scan in negative ionmodecompared with literature data and resulting compoundspreviously reported in E characias leaves [25] They arederivatives of quercetin Compounds 4 and 6were tentativelyidentified by the diagnostic [M minus H]minus ions shown in HRESI-MS analysis their fragmentation profiles obtained in LC-ESI-Orbitrap-MSMS in Product Ion Scan in negative ionmode compared with MassBank data They were proposedas myricetin derivatives The identity of compound 12 washypothesized from the MSMS data obtained working inLC-ESI-Orbitrap-MSMS in Product Ion Scan in negativeion mode and the compound was tentatively identified byMassBank as dicaffeoylquinic acid Compounds 13 and 14were tentatively identified by the diagnostic [M minus H]minus ionshown in HR ESI-MS analysis and from the MSMS dataobtained working in LC-ESI-Orbitrap-MSMS in ProductIon Scan in negative ion mode compared with literatureThese compounds were not described in E characias but inanother plant of the same genus Euphorbia pekinensis [36]

From a quantitative point of view the most significantpolyphenolic compounds were flavonoids mainly quercetin


SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

Figure 4 Identification of polyphenolic compounds in E characias leaves using LC-ESI-Orbitrap-MSMS in negative ion modeChromatographic conditions are described in the text A list of compounds is reported in Table 5

derivatives (Table 5) Quercetin-3-(2-O-acetyl)-arabinoside(15) and quercetin-3-O-rhamnoside (11) were themost abun-dant compounds (3999 plusmn 252 and 3188 plusmn 275 gL resp)followed by quercetin-3-O-arabinoside (10) and quercetin-3-O-xyloside (9) These findings are in agreement with theresults of previous investigations on the aerial part of Echaracias [25] For the first time other compounds such asgallic acid (1) catechin (2) myricetin derivatives and ellagicacid derivativeswere quantified inE characias leaves extractsSome of these compounds such as gallic acid catechin andquercetin-3-O-rhamnoside were isolated from other plantsources and displayed cholinesterase inhibitory activity [44ndash46] Several compounds are derivatives of myricetin (4 and6) and quercetin (7 9ndash11 and 15) two flavonoids whichshowed anti-HIV-1 activity [8 47] Finally previous studiesindicated that quercetin derivatives possess antimicrobialproperties and the mechanisms of these compounds wereattributed to their ability to form complexeswith extracellularand soluble proteins and bacterial cell walls [48] This mightexplain the lack of activity or the minor susceptibility ofthe Gram-negative bacteria and the greater inhibition ofE characias extract against Gram-positive bacteria In factGram-negative bacteria possess an effective permeability bar-rier represented by the outer lipidic membrane which could

restrict the penetration of plant extractsThe next step will bethe isolation of pure compounds of the extract and the evalua-tion and characterization of their biological activities in orderto find new bioactive compounds from a natural source

4 Conclusions

The results of this study indicate that E characias aerial partsare a good source of antioxidant antimicrobial antiviraland anti-ChE compounds Leaves extracts exhibited thestrongest antioxidant activities and the highest amount ofpolyphenols and cholinesterase inhibitors molecules whileflowers extracts seemed to be the best sources of flavonoidsand exerted the best antiviral activity Extracts also exhibitedacetyl- and butyrylcholinesterase inhibition and these arepromising results since ChE inhibitors represent the standardtherapeutic approach to the treatment of Alzheimerrsquos disease

Analyzing all results extraction with ethanol was moreefficient than use of plain water In fact ethanol extractswere found to provide the highest antioxidant and anti-ChE activity and phenolic and flavonoids contents comparedwith the correspondent aqueous extract The same result wasfound for antimicrobial assays since ethanol extracts werefound to bemore effective towards the food-borne pathogens


Table 5 Identification of polyphenolic compounds in E characias leaves ethanolic extract using LC-ESI-Orbitrap-MSMS in negative ionmode and quantification by LC-DAD

Putative identification RT (min) gL (mean plusmn SD) MW [M minusH]minus Molecularformula MSMS References

1 Gallic acida 717 094 plusmn 023 1700215 1691195 C7H6O5

12502 [35]

2 Catechina 1475 065 plusmn 004 2902680 2890715 C15H13O6

245052050412502

[35]

3 Unknown 2039 NQ 9510734 93270 mdash4 Myricetin-hexoseb 2316 002 plusmn 000 4800904 4790824 C

21H19O13

31706 [35]5 Unknown 2456 NQ 960789 91301 mdash6 Myricetin-deoxyhexoseb 2649 001 plusmn 000 4640954 4630873 C

21H19O12

31706 [35]7 Quercetin-3-O-glucosidea 2752 194 plusmn 009 4640954 4630873 C

21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12

39307 mdash9 Quercetin-3-O-xylosidec 2920 225 plusmn 016 4340849 4330771 C

20H17O11

30107 [25]10 Quercetin-3-O-arabinosidec 3018 954 plusmn 036 4340849 4330771 C

20H17O11

30107 [25]11 Quercetin-3-O-rhamnosidea 3102 3188 plusmn 275 4481005 4470924 C

21H19O11

30125 [25]

12 Di-O-caffeoylquinic acidd 3107 002 plusmn 000 5160962 5150800 C17H23O18

3531019102 [35]

13 331015840-Dimethyl ellagic acid pentosee 3153 001 plusmn 000 4620798 4610877 C22H19O12

32902 [36]14 331015840-Dimethyl ellagic acid deoxyhexosee 3570 001 plusmn 000 4760954 4750877 C

22H19O12

32902 [36]15 Quercetin-3-(2-O-acetyl)-arabinosidea 3574 3999 plusmn 252 4760954 4750877 C

22H19O12

30008 [25]16 Acacetin glucuronidef 3984 050 plusmn 002 4601005 4590923 C

22H19O11

28327 [35]aQuantified using corresponding authentic standard bquantified as equivalent of myricetin-3-O-glucoside cquantified as equivalent of quercetin-3-O-glucoside dquantified as equivalent of chlorogenic acid equantified as equivalent of ellagic acid fquantified as equivalent of acacetin NQ not quantified

L monocytogenes S aureus and B cereus HPLC-DAD andLC-ESI-MS chromatogram revealed that leaves ethanolicextract showed the presence of several phenolic and flavonoidcompounds known for their antioxidant activity and thatcould be responsible for the biological activity of the extractFurther studies will be performed for the isolation andcharacterization of single active compounds which could beused for pharmaceutical application as well as in food systemfor the prevention of the growth of food-borne bacteria

Abbreviations

AChE AcetylcholinesteraseAD Alzheimerrsquos diseaseABTS 221015840-Azinobis-(3-ethylbenzothiazoline-6-

sulfonic acid)BChE ButyrylcholinesteraseDPPH∙ 22-Diphenyl-1-picrylhydrazyl radicalFRAP Ferric reducing antioxidant powerGAE Gallic acid equivalentMBCsMFCs Minimum bactericidalfungicidal

concentrationsMICs Minimum inhibitory concentrationsQE Quercetin equivalentRDDP RNA-dependent DNA polymerase

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

The authors thank Professor Annalena Cogoni (Departmentof Sciences of Life and Environment Section of Botany andBotanical Garden University of Cagliari Italy) for plantspecies identification This work was partially supported bygrants from Universita di Cagliari (Contributo di Ateneo perla Ricerca CAR 2013-14)

References

[1] M Garcıa-Alonso S De Pascual-Teresa C Santos-Buelga andJ C Rivas-Gonzalo ldquoEvaluation of the antioxidant propertiesof fruitsrdquo Food Chemistry vol 84 no 1 pp 13ndash18 2004

[2] S Kumar andA K Pandey ldquoChemistry and biological activitiesof flavonoids an overviewrdquo The Scientific World Journal vol2013 Article ID 162750 16 pages 2013

[3] Y Xie W Yang X Chen and J Xiao ldquoInhibition of flavonoidson acetylcholine esterase binding and structurendashactivity rela-tionshiprdquo Food amp Function vol 5 no 10 pp 2582ndash2589 2014

[4] S Cosentino C I G Tuberoso B Pisano et al ldquoIn-vitroantimicrobial activity and chemical composition of SardinianThymus essential oilsrdquo Letters in Applied Microbiology vol 29no 2 pp 130ndash135 1999

[5] S Cosentino A Barra B Pisano M Cabizza F M Pirisiand F Palmas ldquoComposition and antimicrobial properties ofSardinian Juniperus essential oils against foodborne pathogensand spoilage microorganismsrdquo Journal of Food Protection vol66 no 7 pp 1288ndash1291 2003

[6] A Upadhyay I Upadhyaya A Kollanoor-Johny and K Venki-tanarayanan ldquoCombating pathogenic microorganisms using


plant-derived antimicrobials a minireview of the mechanisticbasisrdquo BioMed Research International vol 2014 Article ID761741 18 pages 2014

[7] F Esposito L Zinzula A Maxia E Tramontano and C SannaldquoInhibition of HIV-1 reverse transcriptase associated activitiesby the hydroalcoholic extract ofCasimiroa edulis seedsrdquoNaturalProduct Research vol 25 no 11 pp 1067ndash1073 2011

[8] F Esposito C Sanna C Del Vecchio et al ldquoHypericumhircinum L components as new single-molecule inhibitors ofboth HIV-1 reverse transcriptase-associated DNA polymeraseand ribonuclease H activitiesrdquo Pathogens and Disease vol 68no 3 pp 116ndash124 2013

[9] B-S Min N Nakamura H Miyashiro Y-H Kim and MHattori ldquoInhibition of human immunodeficiency virus type1 reverse transcriptase and ribonuclease H activities by con-stituents of Juglans mandshuricardquo Chemical amp PharmaceuticalBulletin vol 48 no 2 pp 194ndash200 2000

[10] L Xu N Grandi C Del Vecchio et al ldquoFrom the traditionalChinese medicine plant Schisandra chinensis new scaffoldseffective on HIV-1 reverse transcriptase resistant to non-nucleoside inhibitorsrdquo Journal ofMicrobiology vol 53 no 4 pp288ndash293 2015

[11] J T Mwine and P Van Damme ldquoWhy do Euphorbiaceae tickas medicinal plants A review of Euphorbiaceae family and itsmedicinal featuresrdquo Journal of Medicinal Plants Research vol 5no 5 pp 652ndash662 2011

[12] Q-W Shi X-H Su and H Kiyota ldquoChemical and pharma-cological research of the plants in genus Euphorbiardquo ChemicalReviews vol 108 no 10 pp 4295ndash4327 2008

[13] D Spano F Pintus C Mascia et al ldquoExtraction and charac-terization of a natural rubber from Euphorbia characias latexrdquoBiopolymers vol 97 no 8 pp 589ndash594 2012

[14] D Spano F Pintus F Esposito D Loche G Floris and RMedda ldquoEuphorbia characias latex micromorphology of rub-ber particles and rubber transferase activityrdquo Plant Physiologyand Biochemistry vol 87 pp 26ndash34 2015

[15] E Dainese A Sabatucci F Pintus et al ldquoDomain mobilityas probed by small-angle X-ray scattering may account forsubstrate access to the active site of two copper-dependentamine oxidasesrdquo Acta Crystallographica Section D BiologicalCrystallography vol 70 no 8 pp 2101ndash2110 2014

[16] F Pintus D Spano A Bellelli F Angelucci G Floris and RMedda ldquoNucleotide pyrophosphatasephosphodiesterase fromEuphorbia characias latex purification and characterizationrdquoPlant Science vol 177 no 6 pp 636ndash642 2009

[17] F Pintus D Spano A Bellelli et al ldquoEuphorbia peroxidasecatalyzes thiocyanate oxidation in two different ways the distalcalcium ion playing an essential rolerdquo Biochemistry vol 49 no40 pp 8739ndash8747 2010

[18] F Pintus R Medda A C Rinaldi D Spano and G FlorisldquoEuphorbia latex biochemistry complex interactions in a com-plex environmentrdquo Plant Biosystems vol 144 no 2 pp 381ndash3912010

[19] F Pintus D Spano S Corongiu G Floris and R MeddaldquoPurification primary structure and properties of Euphorbiacharacias latex purple acid phosphataserdquo Biochemistry vol 76no 6 pp 694ndash701 2011

[20] D Spano K Pospiskova I Safarik et al ldquoChitinase III inEuphorbia characias latex purification and characterizationrdquoProtein Expression and Purification vol 116 pp 152ndash158 2015

[21] A Mura F Pintus A Fais et al ldquoTyramine oxidation bycopperTPQ amine oxidase and peroxidase from Euphorbia

characias latexrdquo Archives of Biochemistry and Biophysics vol475 no 1 pp 18ndash24 2008

[22] F Pintus D Spano G Floris and R Medda ldquoEuphorbia chara-cias latex amine oxidase and peroxidase interacting enzymesrdquoProtein Journal vol 32 no 6 pp 435ndash441 2013

[23] M Palomino-Schatzlein P V Escrig H Boira J PrimoA Pineda-Lucena and N Cabedo ldquoEvaluation of nonpolarmetabolites in plant extracts by 13CNMRspectroscopyrdquo Journalof Agricultural and Food Chemistry vol 59 no 21 pp 11407ndash11416 2011

[24] F Pintus D Spano A Corona and R Medda ldquoAntityrosinaseactivity of Euphorbia characias extractsrdquo PeerJ vol 3 articlee1305 2015

[25] P V Escrig D J Iglesias A Corma J Primo E Primo-Milloand N Cabedo ldquoEuphorbia characias as bioenergy crop astudy of variations in energy value components according tophenology and water statusrdquo Journal of Agricultural and FoodChemistry vol 61 no 42 pp 10096ndash10109 2013

[26] A Corona T Masaoka G Tocco E Tramontano and S F J LeGrice ldquoActive site and allosteric inhibitors of the ribonucleaseH activity of HIV reverse transcriptaserdquo Future MedicinalChemistry vol 5 no 18 pp 2127ndash2139 2013

[27] F Esposito A Corona and E Tramontano ldquoHIV-1 reversetranscriptase still remains a new drug target structure func-tion classical inhibitors and new inhibitors with innovativemechanisms of actionsrdquo Molecular Biology International vol2012 Article ID 586401 23 pages 2012

[28] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[29] F Pintus D Spano C Mascia A Macone G Floris andR Medda ldquoAcetylcholinesterase inhibitory and antioxidantproperties of Euphorbia characias latexrdquo Records of NaturalProducts vol 7 no 2 pp 147ndash151 2013

[30] C I G Tuberoso M Boban E Bifulco D Budimir and FM Pirisi ldquoAntioxidant capacity and vasodilatory properties ofMediterranean food the case of Cannonauwine myrtle berriesliqueur and strawberry-tree honeyrdquo Food Chemistry vol 140no 4 pp 686ndash691 2013

[31] G L Delogu M J Matos M Fanti et al ldquo2-Phenylbenzofuranderivatives as butyrylcholinesterase inhibitors synthesis bio-logical activity and molecular modelingrdquo Bioorganic amp Medici-nal Chemistry Letters vol 26 no 9 pp 2308ndash2313 2016

[32] F Esposito A Corona L Zinzula T Kharlamova and ETramontano ldquoNew anthraquinone derivatives as inhibitorsof the HIV-1 reverse transcriptase-associated ribonuclease Hfunctionrdquo Chemotherapy vol 58 no 4 pp 299ndash307 2012

[33] A Corona F S Di Leva S Thierry et al ldquoIdentificationof highly conserved residues involved in inhibition of HIV-1 RNase H function by diketo acid derivativesrdquo AntimicrobialAgents and Chemotherapy vol 58 no 10 pp 6101ndash6110 2014

[34] A Corona A Schneider K Schweimer P Rosch B MWohrl and E Tramontano ldquoInhibition of foamy virus reversetranscriptase by human immunodeficiency virus type 1 RNaseH inhibitorsrdquo Antimicrobial Agents and Chemotherapy vol 58no 7 pp 4086ndash4093 2014

[35] H Horai M Arita S Kanaya et al ldquoMassBank a publicrepository for sharing mass spectral data for life sciencesrdquoJournal of Mass Spectrometry vol 45 no 7 pp 703ndash714 2010

[36] P Hou Y Zeng B Ma et al ldquoA fast sensitive and high-throughput method for the simultaneous quantitation of three


ellagitannins from Euphorbiae pekinensis Radix in rat plasmaby ultra-HPLC-MSMSrdquo Journal of Separation Science vol 36no 15 pp 2544ndash2551 2013

[37] R A Dixon and GM Pasinetti ldquoFlavonoids and isoflavonoidsfrom plant biology to agriculture and neurosciencerdquo PlantPhysiology vol 154 no 2 pp 453ndash457 2010

[38] P J Houghton Y Ren and M-J Howes ldquoAcetylcholinesteraseinhibitors from plants and fungirdquo Natural Product Reports vol23 no 2 pp 181ndash199 2006

[39] J Lin J Dou J Xu and H A Aisa ldquoChemical compositionantimicrobial and antitumor activities of the essential oils andcrude extracts of Euphorbia macrorrhizardquoMolecules vol 17 no5 pp 5030ndash5039 2012

[40] S Perumal RMahmud S PillaiW C Lee and S RamanathanldquoAntimicrobial activity and cytotoxicity evaluation ofEuphorbiahirta (L) extracts fromMalaysiardquo APCBEE Procedia vol 2 pp80ndash85 2012

[41] Y Deng G Yang J Yue et al ldquoInfluences of ripening stages andextracting solvents on the polyphenolic compounds antimicro-bial and antioxidant activities of blueberry leaf extractsrdquo FoodControl vol 38 no 1 pp 184ndash191 2014

[42] H Ikigai T Nakae Y Hara and T Shimamura ldquoBactericidalcatechins damage the lipid bilayerrdquo Biochimica et BiophysicaActa (BBA)mdashBiomembranes vol 1147 no 1 pp 132ndash136 1993

[43] A M Janssen J J C Scheffer and A Baerheim SvendsenldquoAntimicrobial activity of essential oils a 1976ndash1986 literaturereview Aspects of the test methodsrdquo Planta Medica vol 53 no5 pp 395ndash398 1987

[44] T Kulisic-Bilusic V Katalinic V Dragovic-Uzelac et alldquoAntioxidant and AChE inhibiting activity of teardquo Food Tech-nology and Biotechnology vol 46 pp 368ndash375 2008

[45] N Suganthy and K Pandima Devi ldquoIn vitro antioxidant andanti-cholinesterase activities of Rhizophora mucronatardquo Phar-maceutical Biology vol 54 no 1 pp 118ndash129 2016

[46] M A Aderogba A R Ndhlala K R R Rengasamy and J VanStaden ldquoAntimicrobial and selected in vitro enzyme inhibitoryeffects of leaf extracts flavonols and indole alkaloids isolatedfrom Croton menyharthiirdquoMolecules vol 18 no 10 pp 12633ndash12644 2013

[47] S Pasetto V Pardi and R M Murata ldquoAnti-HIV-1 activity offlavonoid myricetin on HIV-1 infection in a dual-chamber invitro modelrdquo PLoS ONE vol 9 no 12 Article ID e115323 18pages 2014

[48] M M Senthamilselvi D Kesavan and N Sulochana ldquoAnanti-inflammatory and anti-microbial flavone glycoside fromflowers of Cleome viscosardquo Organic and Medicinal ChemistryLetters vol 2 article 19 pp 1ndash5 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


by inhibiting cholinesterase activity Acetylcholinesterase(AChE) and butyrylcholinesterase (BChE) are two cholin-esterase enzymes that metabolize acetylcholine but differin substrate specificity enzyme kinetics and activity indifferent brain regions In a healthy brain AChE is the mostresponsible enzyme in regulating acetylcholine levels but inpatients with AD AChE activity gradually decreases with theconcomitant increase of BChE Thus both AChE and BChEare legitimate therapeutic targets for treatment of cholinergicdeficit characteristic of AD Effective therapeutic options forAD are limited up to now thus there is a demand for newnatural drugs without side effects

Moreover the demand for bioactive compounds fromnatural sources as an alternative to synthetic molecules iscontinuously increasing also because of the emerging prob-lem of the multidrug resistance of microorganisms relatedto antibiotics and their extensive use Thus several studiesrelated to plant antimicrobials have demonstrated their effi-cacy towards a large number of pathogens and food-borneagents causing disease [4ndash6] as well as viral infections [7ndash10] Although many plant-derived compounds are currentlybeing used for the treatment of infectious diseases and for thepreservation and extension of the shelf-life of foods severalmedicinal and aromatic plants present worldwide remain stillunexplored

Euphorbiaceae is a large flowering plant family (300genera and 8000 species) widely distributed all around theworld and composed of all sorts of plants (large woodytrees climbing lianas or simple weeds) with a wide varietyof chemical substances many of them with a medicinalapplication Some extracts from Euphorbiaceae plants havebeen characterized and patented as modern drugs [11]Among Euphorbiaceae the species Euphorbia characias anonsucculent shrub commonly occurring in vast areas of theMediterranean region has been analyzed and several biolog-ical active compounds were identified [12] The plant latexhas been the object of several researches and its screeninghas revealed the presence of natural rubber and numerousenzymes some of which interact in a common metabolism[13ndash22] On the other hand only little attention has been paidto the other parts of the plant [23ndash25]

Thus the objective of this research was to evaluateantioxidant antimicrobial and anticholinesterase propertiesof the aqueous and ethanolic extracts of leaves stems andflowers from E characias in order to find a novel potentialsource of bioactivemoleculesMoreover the antiviral efficacyof E characias extracts was also evaluated on the humanimmunodeficiency virus type 1 (HIV-1) reverse transcriptase-(RT-) associated RNA-dependent DNA polymerase (RDDP)and RibonucleaseH (RNaseH) activities Both RT-associatedfunctions are essential for viral replication and are validateddrug targets for which new drugs are still needed [26 27]

2 Materials and Methods

All chemicals were obtained as pure commercial productsand used without further purification Acetylcholinesterase(AChE) from Electrophorus electricus acetylthiocholine

Figure 1 The Mediterranean shrub Euphorbia characias subspcharacias

iodide 221015840-azinobis-(3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) aluminum nitrate butyrylcholinesterase(BChE) from equine serum S-butyrylthiocholine chloride3-O-caffeoylquinic acid (chlorogenic acid) catechin 22-diphenyl-1-picrylhydrazyl radical (DPPH∙) 551015840-dithiobis(2-nitrobenzoic acid) (DTNB) ellagic acid Folin-Ciocalteuphenol reagent ferric chloride galantamine hydrobromidegallic acid 4-hydroxybenzyl alcohol 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox) myricetin246-tris(2-pyridyl)-135-triazine (TPTZ) and quercetinwere purchased from Sigma Aldrich (Milan Italy) LC-MSgrade acetonitrile and formic acid were purchased fromMerck (Darmstadt Germany) Standards of myricetin-3-O-glucoside quercetin-3-O-glucoside and acacetin werepurchased from Extrasynthese (Genay France) HPLCgrade water (18MΩsdotcm) was prepared by using a Millipore(Bedford MA USA) Milli-Q purification system

Spectrophotometric determinations were obtained withan Ultrospec 2100 spectrophotometer (Biochrom Ltd Cam-bridge England) using cells with a 1 cm path length

21 Plant Material E characias subsp characias (Figure 1)was identified by Professor Annalena Cogoni and a voucherspecimen has been deposited in the Department of Sci-ences of Life and Environment University of Cagliari Italy(number 121616 Herbarium CAG) The different parts of Echaracias were collected from February to June in southernSardinia (Dolianova CA Italy) The GPS coordinates were39∘24101584019010158401015840N and 9∘12101584057610158401015840E

Leaves stems and flowers were immediately frozenat minus80∘C and then lyophilized in intact condition Thelyophilized plant materials (1 g) were reduced in powderand 10mL of water (aqueous extract) or ethanol (ethanolextract) was added to the dried samples The extraction wascarried out in the dark at room temperature for 24 h undercontinuous stirring Ethanol extracts were diluted 10-foldwith water in order to freeze and then lyophilize the samples[24] Before use 1mg of dried powders was dissolved inwater or 10ethanol (1mL) for aqueous and ethanol extractsrespectively For antimicrobial and antiviral activity dried




4 01ndash


































Leaves Flowers

GA

E (m

gg

dw)


0

200

400

600

800

1000

(a)

QE

(mg

g dw

)


Stems

0

50

100

150

200

250

(b)


minus2

0

2

4

6

ABT

S TE

AC (m

mol

g d

w)

200 400 600 8000GAE (mgg dw)

r = 08448

y = 00055x minus 11362

(a)

minus2

0

2

4

6

8

DPP

H T

EAC

(mm

olg

dw

)

200 400 600 8000GAE (mgg dw)

r = 07346

y = 00070x minus 16991

(b)









AChE BChE










50values









(a)



(b)


















SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance




4 Conclusions







12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




4 01ndash


































Leaves Flowers

GA

E (m

gg

dw)


0

200

400

600

800

1000

(a)

QE

(mg

g dw

)


Stems

0

50

100

150

200

250

(b)


minus2

0

2

4

6

ABT

S TE

AC (m

mol

g d

w)

200 400 600 8000GAE (mgg dw)

r = 08448

y = 00055x minus 11362

(a)

minus2

0

2

4

6

8

DPP

H T

EAC

(mm

olg

dw

)

200 400 600 8000GAE (mgg dw)

r = 07346

y = 00070x minus 16991

(b)









AChE BChE










50values









(a)



(b)


















SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance




4 Conclusions







12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





















Leaves Flowers

GA

E (m

gg

dw)


0

200

400

600

800

1000

(a)

QE

(mg

g dw

)


Stems

0

50

100

150

200

250

(b)


minus2

0

2

4

6

ABT

S TE

AC (m

mol

g d

w)

200 400 600 8000GAE (mgg dw)

r = 08448

y = 00055x minus 11362

(a)

minus2

0

2

4

6

8

DPP

H T

EAC

(mm

olg

dw

)

200 400 600 8000GAE (mgg dw)

r = 07346

y = 00070x minus 16991

(b)









AChE BChE










50values









(a)



(b)


















SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance




4 Conclusions







12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




AChE BChE










50values









(a)



(b)


















SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance




4 Conclusions







12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)



(b)


















SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance




4 Conclusions







12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


SM 11G3565

3107

3018230

2457 2929 4972492639792039 4241 46281909351 717 988 14752 3

5

4

6

7

8 9

10

11

12

13

14

15

161

RT 000ndash5207

5 10 15 20 25 30 35 40 45 500Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance




4 Conclusions







12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





12502 [35]


245052050412502

[35]


21H19O13


21H19O12


21H19O12

30107 [25]8 Unknown 2897 NQ 4690516 C

22H13O12


20H17O11


20H17O11


21H19O11

30125 [25]


3531019102 [35]



22H19O12


22H19O12


22H19O11



Abbreviations




Competing Interests


Acknowledgments


References





























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article biological activities of aerial parts...

Documents