Screening of Three Echinoderm Species as New Opportunity...

Research ArticleScreening of Three Echinoderm Species as NewOpportunity for Drug Discovery Their Bioactivities andAntimicrobial Properties

Loredana Stabili 12 Maria Immacolata Acquaviva2 Rosa Anna Cavallo2

Carmela Gerardi3 Marcella Narracci2 and Patrizia Pagliara1

1Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali Universita del Salento Via Provle Lecce-Monteroni Lecce Italy2Istituto per lrsquoAmbiente Marino Costiero UOS di Taranto CNR Via Roma 3 Taranto Italy3Istituto di Scienze delle Produzioni Alimentari UOS di Lecce Via Provle Lecce-Monteroni Lecce Italy

Correspondence should be addressed to Loredana Stabili loredanastabiliiamccnrit

Received 20 September 2017 Accepted 21 January 2018 Published 1 March 2018

Academic Editor Jae Youl Cho

Copyright copy 2018 Loredana Stabili et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Echinoderms are a renewable resource with an economic value due to their increasing demand as food andor source of bioactivemolecules exerting antitumor antiviral anticoagulant antioxidant and antimicrobial activities In this framework the presentstudy is aimed at investigating the antibacterial antioxidant and hemolytic activities in the three Echinoderm species Echinastersepositus Arbacia lixula and Sphaerechinus granularis The sea star E sepositus showed lysozyme-like activity (mean diameterof lysis of 134 plusmn 02mm) an antimicrobial activity against the human emerging pathogens Staphylococcus aureus Pseudomonasaeruginosa and Candida famata and a strong lytic activity (100plusmn005) towards the human red blood cells FurthermoreA lixulaand E sepositus had the highest antioxidant activity (179275 plusmn 2337 and 176565 plusmn 48458 nmolTEmL resp) From toxicologicalassays it was shown that E sepositus was not toxic towards HeLa cells and Vibrio fischeri encouraging the exploitation of thisspecies in the pharmaceutical field Therefore our findings have implications due to the ongoing explosion of antibiotic-resistantinfections because of the new opportunistic pathogens and the need to discover antibacterial agents with newmodes of action Alsothe recorded antioxidant activity taking into account the need to find natural antioxidants useful for human health is intriguing

1 Introduction

Echinodermata is a phylum containing approximately 7000living species and is a remarkable economic renewableresource holothurian and echinoid cultures indeed are animportant economic activity especially in Asia With theincreasing demand for sea urchin roe and trepang (a genericname for sea cucumbers) commercial culture venues havegrown in order to maintain the demands for these organisms[1ndash3] Moreover recently echinoderms have received greatattention as an unexploited source of new bioactivemoleculeswith important antimicrobial antiviral antiprotozoal anti-fungal and antihelminthic anticancer activities suggestingtheir potential applicability for drug discovery The peculiar-ities of these molecules are stability activity at low temper-ature and specificity of action Often these molecules are

part of their innate immune system As invertebrates lackingadaptive immunity echinoderms are an excellent model forstudying innate immunity Their defense mechanisms aremediated by cellular and humoral responses [4] Cellularresponses are carried out by several types of coelomocyteswhich are free roaming cells circulating in the coelomic cavityand able also to infiltrate tissues and organs [4] Differentmorphologically distinguishable cell types can be found in thecoelomic fluid phagocytic amebocytes colored and colorlessspherula cells vibratile cells hemocytes and crystal cells[5 6] Their action includes phagocytosis of cellular debristhe formation of a layer of cells at the site of injury cellclumping and the formation of capsules around ingestedparasite [7] Humoral immunity is mediated by a broad vari-ety of secreted molecules that can be found in the coelomicfluid These molecules are capable of recognizing foreign

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 7891748 8 pageshttpsdoiorg10115520187891748

2 Evidence-Based Complementary and Alternative Medicine

matter neutralizing or destroying pathogens inducing orenhancing cellular responses (opsonization) and helpingduring wound healing [4 5 8] These secreted immunemolecules include lectins hemolysins cytokines the comple-ment protein family and antimicrobials that have been thesubjects of extensive research even to the point of potentialmedical applications [9] As an example sea cucumber hasbeen valued in Chinese medicine for hundreds of years asa cure for a wide variety of ailments [9] More recentlycompounds exerting antimicrobial antiviral and antitumoractivity mainly from sea cucumbers and starfish have beenisolated leading to a growing interest for the discoveryof immunostimulatory activities useful for human health[10 11] In particular much attention has been addressedto antimicrobial peptides (AMPs) small proteins of theinnate immune response were evolutionary conserved [12]AMPs act on Gram-negative and Gram-positive bacteria andenvelop viruses fungi and even transformed or cancerouscells In echinoderms also lysozymes and fragments of largerproteins display antimicrobial activities [13] Lysozyme is aubiquitous enzyme widely distributed in the animal kingdomand induces cell lysis of the bacterial cell wall by the hydrolysisof 1205731ndash4 glycoside bond between NAM and NAG Thereare several lysozymes or lysozyme-like proteins identifiedfrom coelomic fluid coelomocytes and other tissues ofechinoderms [13]

The search for bioactive compounds with antioxidantactivity is also intriguing considering the positive associa-tion between nutraceutical or functional food and humanhealth In particular natural antioxidant compounds play animportant role as health-protecting factors from free radicalsand ROS (reactive oxygen species) effects [14] Recently aninterest in natural antioxidants has increased because theyare widely distributed and safer than synthetic antioxidantsThe antioxidants may play a major therapeutic role in humandisease causing also the regression of premalignant lesionsand inhibiting their development into cancer [15]

Although most efforts have been devoted to terrestrialplants and microorganisms little attention has yet been paidto marine invertebrates as potential sources of antioxidants[16] In this framework with the aim of discovering newbioactivemolecules we focused on three echinoderm speciesinvestigating their antibacterial antioxidant and hemolyticactivities In particular the antibacterial activity against somenew emerging human pathogens was investigatedThe searchof novel molecules as alternatives to conventional antibi-otics is needed taking into account the ongoing explosionof antibiotic-resistant infections due to new opportunisticpathogens that continue to plague global health care

2 Materials and Methods

21 Echinoderm Collection Adult specimens of the sea starEchinaster sepositus and of the sea urchins Sphaerechinusgranularis andArbacia lixulawere collected by using SCUBAequipment in a coastal area of theNorthern Ionian Sea (PortoCesareo Lecce Italy) at a depth of 5ndash10mAfter the samplingthe animals were kept in circulating seawater transported tothe laboratory and utilized for the sequent assays

22 Samples Preparation The coelomic fluid (CF) of Esepositus was obtained by a transverse cut of the sea stararms From the two sea urchins coelomic fluid was harvestedby bleeding through the peristomial membrane For eachspecies the CF samples from 20 individuals were collectedand then divided into two aliquots The first aliquot wasemployed for the evaluation of cell vitality and light micro-scope observations The second aliquot was immediatelycentrifuged at 400timesg for 10min at 4∘CThe supernatant (CF)of the different individuals was pooled and employed forthe evaluation of the lysozyme-like activity and antibacterialantioxidant and hemolytic activities The pelleted coelo-mocytes were resuspended in distilled water pooled andsonicated on ice with an ultrasound probe (Sonifer sonicatorModel 250240 Brain Ultrasonic Corporation) for 4minat 50 duty cycles Sonicated samples were checked underthe microscope to ensure cell breakage and centrifuged at12000timesg for 30min The supernatants reported as coelomo-cyte lysates (CL) were used for the biological assays

23 Cell Vitality and Microscopic Observations To evaluatecell vitality an aliquot of 1mL of each individual CF fromeach species was rapidly poured into 1mL of ice-cold filtered(022 120583m) seawater (FSW) with 30mM EDTA Cell viabilityhas been evaluated using the Trypan blue exclusion methodFor this 10 120583L of 08 Trypan blue in FSW-EDTA was addedto 90 120583L of CF loaded into a hemacytometer and observedunder a light microscope (Nikon Eclipse 50i) Bright cellswere counted as live and blue cells were considered dead Atthe same time the presence and the normal morphology ofeach cell type were monitored

24 Lysozyme-Like Activity To detect lysozyme-like activityinoculated Petri dishes were used as standard assay [17]Briefly 700120583L of 5mgmL of driedMicrococcus lysodeikticuscell walls (Sigma) was diluted in 7mL of 005M PB-agarose(12) (pH 52) and then spread on a Petri dish Whenthe agarose gel has solidified 63mm diameter wells weresunk and filled with 30 120583L of sample (CF and CL of eachspecies) The diameter of the cleared zone due to the lysisof bacterial cell walls of five replicates for each sample wasrecorded after overnight incubation at 37∘C The diameter ofthe cleared zone was then compared with those of a referencesample represented by hen egg-white lysozyme (Merck) usedat a concentration ranging from 02mgmL to 15mgmLand producing diameters of lysis comprised between 15 and105mm

25 Bacteria and Fungi To test the antimicrobial activityof the examined species the following emerging humanpathogenic bacteria which are indicator organisms com-monly used in programs to monitor antibiotic resistance andkindly furnished by theMicrobiology Laboratory ofOspedaleldquoVito Fazzirdquo Lecce (Italy) were employed Pseudomonasaeruginosa Enterococcus sp and Staphylococcus aureus Inaddition the antimicrobial assay was carried out on the clini-cal isolates of human pathogenic fungi Candida albicans andCandida famata Strains were routinely maintained at +4∘Cas already described by Stabili et al [18] and subcultured on

Evidence-Based Complementary and Alternative Medicine 3

a fresh appropriate agar plates 24 h prior to any antimicrobialtest

26 Disc Diffusion Assay for Antimicrobial Activity To mea-sure the antimicrobial activity of the examined species on theselected microbial strains the same methodology describedby Stabili et al [18] was used Briefly sterile paper discs wereimpregnated with test samples (CF or CL of each species atthe same proteic concentration of 02mgmL) while discsimpregnated with an equivalent volume of FSW were usedas negative controls For each assay agar plates were seededusing a sterile swab to give a uniform covering with 100120583L oftest bacteria or fungi suspension adjusted to 108 CFU (colonyforming unit)mL and impregnated discs and controls werelaid onto the surface (indirect cell counting method withabsorbance value at 540 nm Perkin Elmer model Lambda 3Bspectrophotometer) After incubation for 24 hours at 37∘Cthe clear zone around each disc due to microbial growthinhibition was observed and the diameter of the clear zonewas measured The diameter of microbial growth inhibitionwas then calculated as total diameter of clear zone

27 Hemolytic Activity Hemolytic activity was assayed aspreviously described by Pagliara and Canicattı [19] Brieflysuspensions of human red blood cells (HRBC) (25 inTris buffer Tris HCl 005M 015MNaCl pH 8) were usedto test the ability of CF and CL samples to exert a lyticactivity The test was performed in triplicate by adding onehundred microliters of each sample (protein concentration02mgmL) to an equal volume of HRBC suspension in glasstubes with U bottom The test tubes were incubated for60min at 37∘C and then centrifuged for 5min at 1500timesgOne milliliter of Tris buffer was added to the supernatantin order to obtain an adequate amount of volume forspectrophotometric evaluation (541 nm) of the hemoglobincontent As controls 100120583L of HRBC suspension was mixedwith an equal volume of Tris buffer The degree of hemolysiswas calculated by the formula [(absorbance of sample minusabsorbance of control)absorbance of total hemolysis] times 100Total hemolysis (100) was achieved by adding 100 120583L ofdistilled water to the same volume of the red cell suspension

28 Antioxidant Activity The antioxidant activity wasassayed as previously described by Stabili et al [20] by theOxygen Radical Absorbance Capacity (ORAC) and theTrolox Equivalent Antioxidant Capacity (TEAC) assaysIn particular for ORAC the method [21] samples werediluted with 75mM phosphate buffer (pH 74) The assaywas carried out in black-walled 96-well plates (Greiner-BioOne) and each well contained a final volume of 200120583L Toeach well 20120583L of sample and 120120583L of fluorescein (FL70 nM final concentration) were added and the plate wasincubated at 37∘C for 15min The AAPH (60 120583l 12mM finalconcentration) was added to each well and fluorescenceintensity was estimated using an Infinite 200 Pro plate reader(Tecan Mannedorf Switzerland) every minute for a totalof 80min using an excitation wavelength of 4859 nm andan emission wavelength of 53520 nm A standard curve was

constructed using 6-hydroxy-2578-tetramethylchroman-2-carboxylic acid (Trolox Sigma-Aldrich 15ndash105 120583M) Ablank (fluorescein + AAPH) using phosphate buffer insteadof the antioxidant solution was carried out in each assayResults were calculated usingMagellan v 72 software (TecanSwitzerland) on the basis of the difference in area under thecurve between the control and the sample and expressed as120583moles of Trolox equivalents (TE) per ml of CF or CL Allthe reaction mixtures were prepared in triplicate and at leastthree independent assays were performed for each sample

The TEAC assay was performed by the classical method[22]modified withminormodifications Briefly 221015840-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt(ABTS Sigma-Aldrich) radical cations were prepared bymixing an aqueous solution of potassium persulfate 245mM(final concentration) and an aqueous solution of ABTS 7mM(final concentration) and were allowed to stand in the darkat room temperature for 12ndash16 hours before use The ABTSradical cation solution was diluted in PBS (pH 74) to anabsorbance of 040 at 734 nm Trolox was used as antioxidantstandard and a standard calibration curve was constructedfor Trolox (0ndash16120583M) After addition of 200120583L of dilutedABTS to 10 120583L of Trolox standard or extracts diluted inPBS in each well of a 96 well-plate (Costar) the absorbancereading at 734 nm was taken 6min after initial mixingusing an Infinite 200 Pro plate reader (Tecan MannedorfSwitzerland) Appropriate solvent blank was run in eachplate The CF and CL of each species were assayed at leastat three separate dilutions and in triplicate The percentageinhibition of absorbance at 734 nm is calculated and plottedas a function of concentration of Trolox and the TEAC valueexpressed as Trolox equivalent (in 120583molar) per ml of CF andCL using Magellan v72 software

29 Protein Content Protein concentration of samples wasdetermined by the method of Bradford [23] with bovineserum albumin (Bio-Rad) as standard All the data werenormalized at the same protein concentration

210 Toxicity Assays

2101 HeLa Cells Culture Conditions and Treatments andMTT Assay HeLa cells were cultured in sterile conditionsin 25 or 75 cm2 plastic flasks with D-MEM supplementedwith 10 (vv) FBS 2mM L-glutamine 100 120583gmL peni-cillinstreptomycin and maintained in 5 CO

2at 37∘C

(Thermo Forma Scientific incubator) For propagation cellswere detached and harvested with a 03 (vv) trypsinsolution and then transferred to new flasks every 2-3 days(70ndash90 confluence) The culture medium was changedevery 2 days All experiments were performed betweenpassage 3 and passage 10 propagation For assays CF and CLsamples were added to the cell culture medium Control cellswere incubated with fresh medium

The MTT assay was used to evaluate the effects of Esepositus CF and CL on mitochondrial activity and cel-lular viability Cells were seeded in 96-well plates (20 times103 cellswell) and incubated for 24 h at 37∘C After incu-bation the medium was removed and replaced with a


(a) (b)

Figure 1 (a)Microscopic images of freshly isolated coelomocytes from S granularis and (b) from E sepositus arrow amebocytes arrowheadcolorless spherula cells double arrowhead red spherula cells asterisk vibratile cells Scale bar corresponds to 10120583m

medium containing CF and CL samples After treatmentMTT solution (5mgmL in sterile filtered PBS pH 74)was added to each well to reach a final concentration of05mgMTTmL and plates were incubated at 37∘C for 3 hThe dark-blue formazan crystals were then solubilized by celllysis with 200120583lwell 2-propanolHCl 4N and absorbancewas measured at 550 nm with a microplate reader Data werereported as of control and were the mean (plusmnSEM) of8 samples replicates of each treatment Three independentexperiments were performed

2102 Microtox Bioassay The Microtox Basic Test (BT)was performed according to standard operating procedure[24]The bacteria (Vibrio fischeri)were obtained fromAZUREnvironmental as freeze-lyophilized cells In the BT theCF and CL samples of E sepositus were diluted 1 10 usingthe diluent reagent (Microtox Diluent) The light emissionof the bacteria was measured after 5 15 and 30min andcompared to an aqueous standard control The tests wereperformed at 15∘Cand pH 80plusmn05with control and replicatedthree times for each sample All the measurements were per-formed by using theM500 luminometerThe instrument wasinterfaced with PC operating with the Microtox Omni 116software for Windows 98 for acquisition and data handling

211 Statistical Analysis All data were analyzed by using theStatSoft STATISTICA v 60 [25] (2001)

3 Results

31 Cell Vitality and Microscopic Observations The Trypanblue exclusion test revealed a coelomocytes vitality of 914 plusmn261 in E sepositus 883 plusmn 353 in A lixula and 902 plusmn 233 inS granularis

By observation at light microscopy we pointed out thatin each sample diverse cell types with their characteristicheterogeneous size and morphology could be found Inparticular in the two sea urchins A lixula and S granularis(Figure 1(a)) the coelomic fluid was characterized by thepresence of red spherula cells with their peculiar amoeboid

CL

E sepositus S granularis A lixula

CF

16

12

8

4

0

mm

of l

ysis

diam

eter

Figure 2 Lysozyme-like activity in coelomocytes lysate (CL) andcell free coelomic fluid (CF) of the three examined echinoderms

movement determining rapid changes in shape Colorlessspherula cells and amebocytes were also present moreoverin very freshly samples vibratile cells rapidly swimmingthrough the fluid by a long flagellum were observed In Esepositus (Figure 1(b)) most of the same cell types have beenobserved with a prevalence of amebocytes and spherula cellsVibratile cells have also been detected

32 Lysozyme Activity In Figure 2 the lysozyme-like activityof the CL and CF in the examined species is reported Asregards CL the highest activity was recorded for E seposituswith amean diameter of lysis of 134plusmn02mm correspondingto 182mgmL of hen egg-white lysozyme followed by Sgranularis (diameter of lysis = 94 plusmn 05mm) and A lixula(diameter of lysis of 77 plusmn 05mm) For CF a valuable activitywas recorded only in the case of E sepositus producing adiameter of lysis of 90 plusmn 11mm

33 Antimicrobial Activity towards Emerging Pathogens Allthe three echinoderm species used in this studywere screenedfor antibacterial activity towards some human emerging


Table 1 Antimicrobial activity of CL and CF from the examined echinoderm species

Microbial strainDiameter of growth inhibition (mm)

E sepositus A lixula S granularisCL CF CL CF CL CF

Enterococcus sp 0 0 0 0 0 0Pseudomonas aeruginosa 75 plusmn 02 0 0 0 0 0Staphylococcus aureus 14 plusmn 05 0 0 0 0 0Candida albicans 0 0 0 0 0 0Candida famata 8 plusmn 03 0 0 0 0 0

Table 2 Antioxidant activity of CL and CF of A lixula S granularis and E sepositus

Sample TEAC ORAC(nmolTEmL sample) (nmolTEmL sample)

CLA lixula 1079725 plusmn 9440 179275 plusmn 233699E sepositus 832475 plusmn 28956 196565 plusmn 48458S granularis 435757 plusmn 27595 6712 plusmn 140290

CFA lixula 88385 plusmn 1770 140500 plusmn 4767E sepositus 85270 plusmn 1440 140345 plusmn 3444S granularis 6443 plusmn 0978 4605 plusmn 1768

Data are the mean plusmn SD (119899 = 3) TE = Trolox equivalent

pathogens In particular tests were performed against Ente-rococcus sp P aeruginosa and S aureus and on the clinicalisolates of human pathogenic fungiC albicans andC famataThe diameters of inhibition zones were used as a measure ofthe degree of the antimicrobial activity on each strain TheCL of E sepositus exerted an antimicrobial activity againstS aureus (diameter of growth inhibition = 14 plusmn 05mm)(Figure 3) P aeruginosa (75 plusmn 02mm) and C famata (8 plusmn03mm) (Table 1) None of the samples tested demonstratedinhibition against the other bacterial and fungal strains

34 Hemolytic Activity The hemolytic activity of E sepositusCL was very high inducing 100plusmn 005 of HRBC hemolysiswhile the CF did not possess this activity An opposite resultwas obtained for S granularis in this species the CF possessa strong lytic activity (86 plusmn 003) while only 11 plusmn 001 ofhemolysis was exerted by CLArbacia lixula did not show anyactivity neither in the CF nor in the CL

35 Antioxidant Activity The antioxidant activity of the CLand CF of the three examined species tested by TEAC andORAC assays is reported in Table 2 Antioxidant capac-ity measured by TEAC assay was higher than that mea-sured by ORAC assay for almost all the samples In par-ticular the highest activity was recorded by TEAC assayfor E sepositus and A lixula CL (196565 plusmn 48458 and179275 plusmn 2337 nmolTEmL sample resp) Also the activityobserved in the S granularis CL was remarkable The resultsobtained by TEAC assay in the CF samples were around140 nmolTEmL sample except for S granularis that showed

Figure 3 Disc diffusion assay CL of E sepositus against Staphylo-coccus aureus (A = disc impregnated with CL B = negative control)

the lowest antioxidant activity by both ORAC and TEACassay

36 Toxicity Assays Since E sepositusCL showed the highestvalues for the screened activities in the light of its potentialemployment as natural source of bioactive compounds weverified the nontoxicity of the samples by the classical MTTtest and the specificMicrotox test againstVibrio fischeriMTTtest revealed that the sea star cell lysate proved to be nontoxictowards HeLa cells This result was also corroborated byMicrotox data revealing no acute toxicity


4 Discussion

In the present study we screened three echinoderm specieswidespread in the Mediterranean area such as Echinastersepositus Sphaerechinus granularis and Arbacia lixula aspotential source of antibacterial and antioxidant compoundsFrom the obtained results some interesting issues can beinferred

A strong lysozyme-like activity was evidenced inE sepositus Lysozymes are enzymes widely distributedthroughout the animal kingdom able to damage the bacterialcell wall catalyzing the hydrolysis of the 1205731ndash4 glycoside bondbetween NAM and NAG In this way they act as nonspecificinnate immunity molecules against the invasion of bacterialpathogens [26]There are several lysozymes or lysozyme-likeproteins identified from coelomic fluid and coelomocytesof echinoderms [13] The lysozyme-like activity evidencedin E sepositus is noteworthy as potential novel antibacterialcompound at the time when multidrug-resistant pathogensare on the rise This emergence leads to the need for newantibacterial agents with fundamentally different modes ofaction than those of traditional antibiotics [27 28] Bacterialcell wall hydrolases (BCWH) are among the most promisingcandidates and lysozyme was recently chosen as a modelprotein [29 30] thus lysozyme from E sepositus couldrepresent a great opportunity in drug systems as a newantimicrobial agent

It is worth noting that an antibacterial activity of theE sepositus coelomocytes lysate against some new emerginghuman pathogens was observed for the first time In particu-lar the antimicrobial activity was exerted against S aureusP aeruginosa and C famata These microorganisms maybecome dangerous in particular conditions (body debilitatedetc) and assume different characteristics of pathogenicitycompared to those known These nosocomial isolates showincreasing resistance towards antibiotics representing a greatchallenge for the management of hospital-acquired infec-tions and the modern pharmacology Staphylococcus aureusis indeed notorious for its ability to become resistant toantibiotics P aeruginosa develops antimicrobial resistancerapidly which complicates medical treatment of infections[31] and C famata belongs to the nonalbicans Candida(NAC) species Most of these exhibit primary resistance orreduced susceptibility towards antifungals [32] Previouslyseveral studies demonstrated that echinoderm coelomocyteslysates and coelomic fluid possess activity against poten-tial pathogenic bacteria fungi and even tumor cells Inparticular coelomic fluid from Echinus esculentus possessesbactericidal activity against Pseudomonas sp [33] In the seaurchin Paracentrotus lividus coelomocytes induced growthinhibition against several Vibrio sp including V alginolyti-cus [34 35] presented activity against both Gram-positiveand Gram-negative bacteria and fungi [36] and showed acytotoxic activity against rabbit erythrocytes and the K562tumor cell line [37] Thus our findings about antimicrobialsfrom echinoderms may be a valuable therapeutic supportopening new perspectives for the utilization of new andstill unexploited sources of drugs Moreover E sepositusCL exerted an high lytic effect on human red blood cells

This is an interesting observation since hemolytic activityappears to be common in other echinoderm extracts thatshow high antibacterial activity [38] On account of theseobservations the same effector was supposed to be respon-sible for the two observed activities as a consequence ofthe transmembrane channels formation [39] Lytic activityindeed is usually assayed on vertebrate red cells howeverother kinds of cells including bacteria are lysed Thusalso bacteria represent useful targets to demonstrate killingproperties of the lytic system as already suggested by Cani-cattı [39] In addition as the lytic activity is known to bealso directed against mammalian malignant cells includinghuman leukemia [13] further studies will be also undertakento explore the potential antitumor activity of the studiedechinoderm species

In addition in the coelomic fluid and circulating cellsof all the examined species an antioxidant activity wasfirst recorded The highest values were in the cell lysateof E sepositus and A lixula In echinoderms an antiox-idant activity was already evidenced in the viscera of theAtlantic sea cucumber Cucumaria frondosa and in diges-tive tract and noncommercial grade gonads of green seaurchin Strongylocentrotus droebachiensis [40] Moreover inthe crude ethanol extract of the starfish Luidia maculataa good antioxidant activity was observed by Suguna et al[41] In all these cases the activity was assayed in tissueextracts and there are few studies only on sea cucumbersdemonstrating the coelomic fluid as a good source of antiox-idants [42ndash44] Due to differences in sample preparationmethod data here obtained are not comparable with theantioxidant activity observed in the other investigated echin-oderms Up to now available studies on aquatic animalshave reported data with significant antioxidant ability onlyfor protein hydrolysates [45] For this reason the hereinhighest recorded values (about 1970 nmolTEmL sample) arecomparable only to the well-known natural antioxidant com-pounds such as the orange juice (1130ndash3840 nmolTEmL)and the red wine (500ndash6000 nmolTEmL) [46 47] It isknown from the literature that antioxidants play an impor-tant role in the protection of human body against damageby ROS [48] These molecules indeed induce oxidativedamage of DNA and other cellular components leading tocancer degenerative and cardiovascular related mutationsConsequently the intake of natural antioxidant has beenassociated with reduced risk of cancer and other diseasesassociated with oxidative damage [49] In this scenariothe here evidenced activity indicates that the examinedechinoderms and in particular E sepositus could repre-sent a new source of natural antioxidants of marine ori-gin

In conclusion this study points out the potential of Esepositus cell lysate as promising source of antimicrobial andantioxidant compounds Obviously before its exploitationseveral toxicity assays will be undertakenThe first performedtoxicological tests revealed that E sepositus samples arenot toxic towards HeLa cells and Vibrio fischeri encour-aging looking at this echinoderm species as a new toolfor biotechnological applications in the pharmaceutical andnutraceutical field


Conflicts of Interest

The authors declare that there are no conflicts of interest

Authorsrsquo Contributions

Loredana Stabili and Patrizia Pagliara conceived and de-signed the experiments Loredana Stabili Maria ImmacolataAcquaviva Rosa Anna Cavallo Carmela Gerardi MarcellaNarracci and Patrizia Pagliara performed the experimentsand analyzed the data Loredana Stabili and Patrizia Pagliaracontributed reagentsmaterialsanalysis tools and wrote thepaper All authors read and approved the manuscript

References

[1] C Conand ldquoCommercial sea cucumbers and trepang marketsrdquoin Fisheries and Aquaculture vol II pp 221ndash250 Safran EOLSSPublishers Co Ltd Oxford UK 2009

[2] P A Pantazis ldquoThe culture potential of Paracentrotus lividus(Lamarck 1816) in Greece A preliminary reportrdquo AquacultureInternational vol 17 no 6 pp 545ndash552 2009

[3] R Furesi F A Madau P Pulina R Sai M G Pinna and APais ldquoProfitability and sustainability of edible sea urchin fisheryin Sardinia (Italy)rdquo Journal of Coastal Conservation vol 20 no4 pp 299ndash306 2016

[4] F J Ramırez-Gomez and J E Garcıa-Arraras ldquoEchinodermimmunityrdquo Invertebrate Survival Journal vol 7 no 2 pp 211ndash220 2010

[5] L C Smith J Ghosh K M Buckley et al ldquoEchinodermImmunityrdquo in Invertebrate Immunity vol 708 of Advances inExperimental Medicine and Biology pp 260ndash301 Springer NewYork NY USA 2010

[6] V Matranga ldquoMolecular Aspects of Immune Reactions inEchinodermatardquo in Invertebrate Immunology vol 15 of Progressin Molecular and Subcellular Biology pp 235ndash247 SpringerBerlin Germany 1996

[7] Z Glinski and J Jarosz ldquoImmune phenomena in echinodermsrdquoArchivum Immunologiae etTherapia Experimentalis vol 48 no3 pp 189ndash193 2000

[8] P S Gross W Z Al Sharif L A Clow and L C SmithldquoEchinoderm immunity and the evolution of the complementsystemrdquoDevelopmentalampComparative Immunology vol 23 no4-5 pp 429ndash442 1999

[9] M S Kelly ldquoEchinoderms their culture and bioactive com-poundsrdquo Progress in Molecular and Subcellular Biology vol 39pp 139ndash165 2005

[10] R J Layson M Criselda A Rodil E E Mojica and C CDeocaris ldquoPotential Anti-cancer and Anti-bacterial Activitiesof Philippine Echinoderm Extractsrdquo Journal of Tropical LifeScience vol 4 no 3 pp 175ndash181 2014

[11] F Farshadpour S Gharibi M Taherzadeh et al ldquoAntiviralactivity of Holothuria sp a sea cucumber against herpessimplex virus type 1 (HSV-1)rdquo European Review for Medical andPharmacological Sciences vol 18 no 3 pp 333ndash337 2014

[12] N Mookherjee and R E W Hancock ldquoCationic host defencepeptides Innate immune regulatory peptides as a novelapproach for treating infectionsrdquo Cellular and Molecular LifeSciences vol 64 no 7-8 pp 922ndash933 2007

[13] C Li T Haug and K Stensvag ldquoAntimicrobial peptides inechinodermsrdquo Invertebrate Survival Journal vol 7 no 1 pp 132ndash140 2010

[14] D P Gelain G A Behr R B de Oliveira and M TrujilloldquoAntioxidant therapies for neurodegenerative diseases mecha-nisms current trends and perspectivesrdquo in Oxidative MedicineandCellular Longevity D PGelainGA Behr R B deOliveiraand E Trujillo Eds Hindawi Publishing Corporation 2012

[15] A Pachiayappan A Muthuvel and G Sadhasivam ldquoIn vitroantioxidant activity of different gastropods bivalves and echin-oderm by solvent extraction methodrdquo International Journal ofPharmaceutical Sciences and Research vol 5 pp 2539ndash25452014

[16] F ShahidiMaximizing the Value of Marine By-Products Wood-head Publishing Ltd Cambridge UK 2007

[17] L Stabili AMMiglietta andG Belmonte ldquoLysozyme-like andtrypsin-like activities in the cysts of Artemia franciscanaKellog1906 Is there a passive immunity in a resting stagerdquo Journalof Experimental Marine Biology and Ecology vol 237 no 2 pp291ndash303 1999

[18] L Stabili M I Acquaviva F Biandolino et al ldquoThe lipidicextract of the seaweed Gracilariopsis longissima (RhodophytaGracilariales) A potential resource for biotechnological pur-posesrdquo New Biotechnology vol 29 no 3 pp 443ndash450 2012

[19] P Pagliara and C Canicattı ldquoIsolation of cytolytic granules insea urchin amebocytesrdquo European Journal of Cell Biology vol60 pp 179ndash184 1993

[20] L Stabili S Fraschetti M I Acquaviva et al ldquoThe PotentialExploitation of the Mediterranean Invasive Alga Caulerpacylindracea Can the Invasion Be Transformed into a GainrdquoMarine Drugs vol 14 no 11 pp 210ndash223 2016

[21] A Davalos C Gomez-Cordoves and B Bartolome ldquoCommer-cial dietary antioxidant supplements assayed for their antioxi-dant activity by differentmethodologiesrdquo Journal of Agriculturaland Food Chemistry vol 51 no 9 pp 2512ndash2519 2003

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

[23] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquo Analytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[24] Azur Environmental Microtox M500 Manual A ToxicityTesting Handbook Carlsbad Calif USA 1994

[25] Statsoft Inc Electronic Statistics Textbook Statsoft Tulsa OKUSA 2001

[26] P Jolles and J Jolles ldquoWhatrsquos new in lysozyme research -Alwaysa model system today as yesterdayrdquo Molecular and CellularBiochemistry vol 63 no 2 pp 165ndash189 1984

[27] A Parisien B Allain J Zhang R Mandeville and C Q LanldquoNovel alternatives to antibiotics Bacteriophages bacterial cellwall hydrolases and antimicrobial peptidesrdquo Journal of AppliedMicrobiology vol 104 no 1 pp 1ndash13 2008

[28] H W Boucher G H Talbot D K Benjamin et al ldquo10 timesrsquo20 progress - Development of new drugs active against gram-negative bacilli An update from the infectious diseases societyofAmericardquoClinical InfectiousDiseases vol 56 no 12 pp 1685ndash1694 2013

[29] H R Ibrahim T Aoki and A Pellegrini ldquoStrategies for newantimicrobial proteins and peptides Lysozyme and aprotinin asmodel moleculesrdquo Current Pharmaceutical Design vol 8 no 9pp 671ndash693 2002


[30] F Niyonsaba andH Ogawa ldquoProtective roles of the skin againstinfection implication of naturally occurring human antimi-crobial agents 120573-defensins cathelicidin LL-37 and lysozymerdquoJournal of Dermatological Science vol 40 no 3 pp 157ndash1682005

[31] M S Lee Ventola ldquoThe antibiotic resistance crisismdashpart 1causes and threatsrdquo Pharmacy and Therapeutics vol 40 no 4pp 277ndash283 2015

[32] N Papon V Courdavault M Clastre and R J BennettldquoEmerging and emerged pathogenic Candida species beyondthe Candida albicans paradigmrdquo PLoS Pathogens vol 9 no 9Article ID e1003550 2013

[33] A C Wardlaw and S E Unkles ldquoBactericidal activity ofcoelomic fluid from the sea urchin Echinus Esculentusrdquo Journalof Invertebrate Pathology vol 32 no 1 pp 25ndash34 1978

[34] P Gerardi M Lassegues and C Canicatti ldquoCellular distribu-tion of sea urchin antibacterial activityrdquo Biology of the Cell vol70 no C pp 153ndash157 1990

[35] L Stabili P Pagliara and P Roch ldquoAntibacterial activity inthe coelomocytes of the sea urchin Paracentrotus lividusrdquoComparative Biochemistry and Physiology - B Biochemistry andMolecular Biology vol 113 no 3 pp 639ndash644 1996

[36] D Schillaci V Arizza N Parrinello et al ldquoAntimicrobialand antistaphylococcal biofilm activity from the sea urchinParacentrotus lividusrdquo Journal of Applied Microbiology vol 108no 1 pp 17ndash24 2010

[37] V Arizza F T Giaramita D Parrinello M Cammarata and NParrinello ldquoCell cooperation in coelomocyte cytotoxic activityof Paracentrotus lividus coelomocytesrdquo Comparative Biochem-istry and Physiology - A Molecular and Integrative Physiologyvol 147 no 2 pp 389ndash394 2007

[38] T Haug A K Kjuul O B Styrvold E Sandsdalen Oslash MOlsen and K Stensvag ldquoAntibacterial activity in Strongylo-centrotus droebachiensis (Echinoidea) Cucumaria frondosa(Holothuroidea) and Asterias rubens (Asteroidea)rdquo Journal ofInvertebrate Pathology vol 81 no 2 pp 94ndash102 2002

[39] C Canicattı ldquoThe echinoderm lytic systemrdquo Bollettino diZoologia vol 59 no 2 pp 159ndash166 1992

[40] J Mamelona R Saint-Louis and E Pelletier ldquoNutritionalcomposition and antioxidant properties of protein hydrolysatesprepared from echinoderm byproductsrdquo International Journalof Food Science amp Technology vol 45 no 1 pp 147ndash154 2010

[41] A Suguna S Bragadeeswaran E Natarajan and M MohanrajldquoStudies on antioxidant properties of starfish Luidia maculata(Muller amp Troschel 1842) off Parangipettai Southeast coast ofIndiardquo Journal of Coastal LifeMedicine vol 2 pp 694ndash698 2014

[42] I Hawa M Zulaikah M Jamaludin A A Zainal Abidin M AKaswandi and B H Ridzwan ldquoThe potential of the coelomicfluid of sea cucumber as an antioxidantrdquo Malaysian Journal ofNutrition vol 5 pp 55ndash59 1999

[43] L S Dolmatova M G Eliseikina and V V RomashinaldquoAntioxidant enzymatic activity of coelomocytes of the Far EastSea Cucumber Eupentacta fraudatrixrdquo Journal of EvolutionaryBiochemistry and Physiology vol 40 no 2 pp 126ndash135 2004

[44] S Bordbar F Anwar and N Saari ldquoHigh-value componentsand bioactives from sea cucumbers for functional foodsmdashareviewrdquoMarine Drugs vol 9 no 10 pp 1761ndash1805 2011

[45] X He W Cao Z Zhao and C Zhang ldquoAnalysis of proteincomposition and antioxidant activity of hydrolysates fromPaphia undulaterdquo Journal of Food and Nutrition Research vol1 pp 30ndash36 2013

[46] P Rapisarda A Tomaino R Lo Cascio F Bonina A DePasquale and A Saija ldquoAntioxidant effectiveness as influencedby phenolic content of fresh orange juicesrdquo Journal of Agricul-tural and Food Chemistry vol 47 no 11 pp 4718ndash4723 1999

[47] M S Fernandez-Pachon D Villano M C Garcıa-Parrilla andAM Troncoso ldquoAntioxidant activity of wines and relationwiththeir polyphenolic compositionrdquo Analytica Chimica Acta vol513 no 1 pp 113ndash118 2004

[48] M Valko M Izakovic M Mazur C J Rhodes and J TelserldquoRole of oxygen radicals inDNAdamage and cancer incidencerdquoMolecular and Cellular Biochemistry vol 266 no 1-2 pp 37ndash562004

[49] A Rietveld and S Wiseman ldquoAntioxidant Effects of TeaEvidence fromHuman Clinical Trialsrdquo Journal of Nutrition vol133 no 10 pp 3285Sndash3292S 2003

Stem Cells International

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Volume 2018

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Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


matter neutralizing or destroying pathogens inducing orenhancing cellular responses (opsonization) and helpingduring wound healing [4 5 8] These secreted immunemolecules include lectins hemolysins cytokines the comple-ment protein family and antimicrobials that have been thesubjects of extensive research even to the point of potentialmedical applications [9] As an example sea cucumber hasbeen valued in Chinese medicine for hundreds of years asa cure for a wide variety of ailments [9] More recentlycompounds exerting antimicrobial antiviral and antitumoractivity mainly from sea cucumbers and starfish have beenisolated leading to a growing interest for the discoveryof immunostimulatory activities useful for human health[10 11] In particular much attention has been addressedto antimicrobial peptides (AMPs) small proteins of theinnate immune response were evolutionary conserved [12]AMPs act on Gram-negative and Gram-positive bacteria andenvelop viruses fungi and even transformed or cancerouscells In echinoderms also lysozymes and fragments of largerproteins display antimicrobial activities [13] Lysozyme is aubiquitous enzyme widely distributed in the animal kingdomand induces cell lysis of the bacterial cell wall by the hydrolysisof 1205731ndash4 glycoside bond between NAM and NAG Thereare several lysozymes or lysozyme-like proteins identifiedfrom coelomic fluid coelomocytes and other tissues ofechinoderms [13]

The search for bioactive compounds with antioxidantactivity is also intriguing considering the positive associa-tion between nutraceutical or functional food and humanhealth In particular natural antioxidant compounds play animportant role as health-protecting factors from free radicalsand ROS (reactive oxygen species) effects [14] Recently aninterest in natural antioxidants has increased because theyare widely distributed and safer than synthetic antioxidantsThe antioxidants may play a major therapeutic role in humandisease causing also the regression of premalignant lesionsand inhibiting their development into cancer [15]

Although most efforts have been devoted to terrestrialplants and microorganisms little attention has yet been paidto marine invertebrates as potential sources of antioxidants[16] In this framework with the aim of discovering newbioactivemolecules we focused on three echinoderm speciesinvestigating their antibacterial antioxidant and hemolyticactivities In particular the antibacterial activity against somenew emerging human pathogens was investigatedThe searchof novel molecules as alternatives to conventional antibi-otics is needed taking into account the ongoing explosionof antibiotic-resistant infections due to new opportunisticpathogens that continue to plague global health care

2 Materials and Methods

21 Echinoderm Collection Adult specimens of the sea starEchinaster sepositus and of the sea urchins Sphaerechinusgranularis andArbacia lixulawere collected by using SCUBAequipment in a coastal area of theNorthern Ionian Sea (PortoCesareo Lecce Italy) at a depth of 5ndash10mAfter the samplingthe animals were kept in circulating seawater transported tothe laboratory and utilized for the sequent assays

22 Samples Preparation The coelomic fluid (CF) of Esepositus was obtained by a transverse cut of the sea stararms From the two sea urchins coelomic fluid was harvestedby bleeding through the peristomial membrane For eachspecies the CF samples from 20 individuals were collectedand then divided into two aliquots The first aliquot wasemployed for the evaluation of cell vitality and light micro-scope observations The second aliquot was immediatelycentrifuged at 400timesg for 10min at 4∘CThe supernatant (CF)of the different individuals was pooled and employed forthe evaluation of the lysozyme-like activity and antibacterialantioxidant and hemolytic activities The pelleted coelo-mocytes were resuspended in distilled water pooled andsonicated on ice with an ultrasound probe (Sonifer sonicatorModel 250240 Brain Ultrasonic Corporation) for 4minat 50 duty cycles Sonicated samples were checked underthe microscope to ensure cell breakage and centrifuged at12000timesg for 30min The supernatants reported as coelomo-cyte lysates (CL) were used for the biological assays

23 Cell Vitality and Microscopic Observations To evaluatecell vitality an aliquot of 1mL of each individual CF fromeach species was rapidly poured into 1mL of ice-cold filtered(022 120583m) seawater (FSW) with 30mM EDTA Cell viabilityhas been evaluated using the Trypan blue exclusion methodFor this 10 120583L of 08 Trypan blue in FSW-EDTA was addedto 90 120583L of CF loaded into a hemacytometer and observedunder a light microscope (Nikon Eclipse 50i) Bright cellswere counted as live and blue cells were considered dead Atthe same time the presence and the normal morphology ofeach cell type were monitored

24 Lysozyme-Like Activity To detect lysozyme-like activityinoculated Petri dishes were used as standard assay [17]Briefly 700120583L of 5mgmL of driedMicrococcus lysodeikticuscell walls (Sigma) was diluted in 7mL of 005M PB-agarose(12) (pH 52) and then spread on a Petri dish Whenthe agarose gel has solidified 63mm diameter wells weresunk and filled with 30 120583L of sample (CF and CL of eachspecies) The diameter of the cleared zone due to the lysisof bacterial cell walls of five replicates for each sample wasrecorded after overnight incubation at 37∘C The diameter ofthe cleared zone was then compared with those of a referencesample represented by hen egg-white lysozyme (Merck) usedat a concentration ranging from 02mgmL to 15mgmLand producing diameters of lysis comprised between 15 and105mm

25 Bacteria and Fungi To test the antimicrobial activityof the examined species the following emerging humanpathogenic bacteria which are indicator organisms com-monly used in programs to monitor antibiotic resistance andkindly furnished by theMicrobiology Laboratory ofOspedaleldquoVito Fazzirdquo Lecce (Italy) were employed Pseudomonasaeruginosa Enterococcus sp and Staphylococcus aureus Inaddition the antimicrobial assay was carried out on the clini-cal isolates of human pathogenic fungi Candida albicans andCandida famata Strains were routinely maintained at +4∘Cas already described by Stabili et al [18] and subcultured on









210 Toxicity Assays


2at 37∘C




(a) (b)





3 Results



CL


CF

16

12

8

4

0

mm

of l

ysis

diam

eter






















4 Discussion












References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























210 Toxicity Assays


2at 37∘C




(a) (b)





3 Results



CL


CF

16

12

8

4

0

mm

of l

ysis

diam

eter






















4 Discussion












References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b)





3 Results



CL


CF

16

12

8

4

0

mm

of l

ysis

diam

eter






















4 Discussion












References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




































4 Discussion












References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























References























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of












































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Screening of Three Echinoderm Species as New Opportunity...

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