biomphalaria glabrata of - hindawi publishing...

Research ArticleLarvicidal Activity against Aedes aegypti andMolluscicidal Activity against Biomphalaria glabrata ofBrazilian Marine Algae

Eliacuteca Amara Ceciacutelia Guedes12 Cenira M de Carvalho1

Karlos Antonio Lisboa Ribeiro Junior1 Thyago Fernando Lisboa Ribeiro1

Lurdiana Dayse de Barros1 Maria Raquel Ferreira de Lima1

Flaacutevia de Barros Prado Moura2 and Antocircnio Euzebio Goulart SantrsquoAna1

1 Laboratorio de Produtos Naturais Instituto de Quımica e Biotecnologia Universidade Federal de Alagoas Campus AC Simoes Avenida Lourival Melo Mota sn Tabuleiro do Martins 57072-970 Maceio AL Brazil

2 Universidade Federal de Alagoas Icbs Campus A C Simoes Avenida Lourival Melo Mota sn Tabuleiro do Martins57072-970 Maceio AL Brazil

Correspondence should be addressed to Elıca Amara Cecılia Guedes eacguedesgmailcom

Received 26 September 2013 Revised 23 December 2013 Accepted 23 December 2013 Published 13 February 2014

Academic Editor D S Lindsay

Copyright copy 2014 Elıca Amara Cecılia Guedes et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

This study investigated the biological activities of five benthicmarine algae collected fromNortheastern Region of BrazilThe testedactivities included larvicidal activity againstAedes aegypti molluscicidal activity against Biomphalaria glabrata and toxicity againstArtemia salina Extracts ofUlva lactuca (Chlorophyta) Padina gymnospora Sargassum vulgare (Phaeophyta)Hypnea musciformisand Digenea simplex (Rhodophyta) were prepared using different solvents of increasing polarity including dichloromethanemethanol ethanol andwater Of the extracts screened the dichloromethane extracts ofHmusciformis and P gymnospora exhibitedthe highest activities and were subjected to bioassay-guided fractionation in hexane and chloroform The chloroform fractions ofthe P gymnospora and H musciformis extracts showed molluscicidal activity at values below 40120583gsdotmLminus1 (111460120583gsdotmLminus1 and258689 120583gsdotmLminus1 resp) and the chloroform and hexane fractions of P gymnospora showed larvicidal activity at values below40120583gsdotmLminus1 (29018 120583gsdotmLminus1 and 17230 120583gsdotmLminus1 resp) The crude extracts were not toxic to A salina whereas the chloroformand hexane fractions of P gymnospora (788277 120583gsdotmLminus1 and 706990 120583gsdotmLminus1) showed moderate toxicity indicating that the toxiccompounds present in these algae are nonpolar

1 Introduction

Marine environments particularly in the tropics have greaterspecies richness than tropical forests These diverse speciesare capable of producing a wide variety of chemical com-pounds with unique structures and functions and manyof these compounds can be used for the development ofnovel drugs [1 2] As an abundant resource in Braziliancoastal waters marine algae exhibit a great potential sourceof novel compounds It is known that species of marine algae

synthesize bioactive compounds with diverse activities [2]Many studies aiming to identify representative genera ofmarine algae that produce bioactive substances have beenconducted In the last three decades the rate of discovery ofbiologically active metabolites produced by macroalgae hasincreased [3ndash5]

The secondary metabolites of marine algae are analogousto those of terrestrial plants with respect to the productionof terpenes aromatic compounds acetogenins amino acidderivatives and polyphenols [5] Many of these metabolites

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2014 Article ID 501328 6 pageshttpdxdoiorg1011552014501328

2 Journal of Parasitology Research

are known to possess antibacterial antifungal antiviral andantitumor activities [1]

The Brazilian Northeast Region is the poorest region ofBrazil and has the worst Human Development Indices inthe country [6] Most of the population in this region is atrisk of contracting neglected tropical diseases In particulardengue fever and schistosomiasis are major public healthconcerns in Brazil and other developing tropical countries[7 8] Natural products with different biocidal activitiescan help fight parasite vectors at the adult or larval stagesand are good alternatives to synthetic products due to theirrapid biodegradation and lower costs [9] thus contributingto improved public health conditions in tropical developingcountries

The present study investigates the biological activities offive marine algaeUlva lactuca Linnaeus Padina gymnospora(Kutzing) Sonder Sargassum vulgare C Agardh Hypneamusciformis (Wulfen in Jacque) JV Lamouroux andDigeneasimplex (Wulfen) C Agardhmdashall collected from the coastof Alagoas State The activities tested included larvicidalactivity against Aedes aegypti molluscicidal activity againstBiomphalaria glabrata and toxicity against Artemia salinaThis is the first report on the biological activity of marinealgae collected from the coast of Alagoas

2 Materials and Methods

21 Collection of Marine Algae Algae were manuallycollected from the Riacho Doce beach (9∘341015840010158401015840S and35∘391015840010158401015840W) during low tides between October 2007and July 2009 The collected samples were immediatelytransported to the Phycology Laboratory of the BiologicalSciences Institute (ICBS) Universidade Federal de Alagoas(UFAL) Five species of algae from three divisions wereused for this study ChlorophytamdashUlva lactuca LinnaeusMAC51238 PhaeophytamdashPadina gymnospora (Kutzing)Sonder MAC51235 and Sargassum vulgare C AgardhMAC51236 and RhodophytamdashHypnea musciformis (Wulfenin Jacque) JV Lamouroux MAC51234 and Digenea simplex(Wulfen) C Agardh MAC51231 A voucher specimen of eachalgae species was deposited in the MAC Herbarium at theEnvironmental Institute of the city of Maceio Alagoas (Br)as a reference material

22 Extract Preparation and Fractionation Algae werewashed with distilled water dried in air-circulating ovens(BLUE MOD-1401440SC USA) at 45∘C for three hoursand crushed using a MetVisa industrial blender (modeltype TA-2) The dried and crushed material from eachcollection and each alga was mixed in equal quantities Toobtain crude extracts 500 g of dried algae was suspendedin 1000mL of dichloromethane methanol ethanol or waterand macerated for 72 hours this process was repeated threetimes The organic extracts were filtered and rotoevaporated(MOD Rotoevaporator with a Buchi heating bath B-490Switzerland) at 25∘Cndash40∘C and the aqueous extract waslyophilized (Edwards Freeze dryer Mod E2 MB of Brazil)Extracts of the respective algal species were stored at 4∘C

The dichloromethane extracts of H musciformis and P gym-nospora were selected for fractionation because they gave thebest yields 1186 g (258) and 1396 g (304) respectivelyThe yields of the other extracts were less than 05 g (01) andwere therefore not sufficiently large for fractionation

For fractionation by liquid-liquid partitioning the crudeextracts ofH musciformis (273 g) and P gymnospora (190 g)were suspended in methanol water (3 1) and extracted withhexane and chloroform to yield chloroform and hexanefractions All solvents were purchased from VERTEC FineChemicals of Brazil

23 Mosquito Larvicidal Activity Bioassays were run with Aaegypti larvae at the Chemistry and Biotechnology Instituteof Federal University from Alagoas IQBUFAL laboratoryusing the WHO protocol [10] The bioassays used fourthinstar larvae with two repetitions Each experimental courseused 10 larvae placed in a 250mL cup containing 27mLof test solution at 100 120583gsdotmLminus1 Extracts that led to le50mortality were subjected to more precise tests In these testsfour replicates were run using 25 larvae in each replicateTheconcentrations used were defined based on the preliminaryresults

Larvae were considered dead when they failed to reachthe surface of the solution after the liquid was stirred Deadlarvae counts were performed at 24 hours and 48 hours afterthe start of the experiment and the samples were run inquadruplicate In parallel control tests were performed with1 aqueous dimethyl sulfoxide (DMSO) solution and thesynthetic larvicide Temephos at 3 120583gsdotmLminus1 The activity levelof extracts tested was calculated based on the average percentmortality of larvae at 48 hours (gt75 highly active 50ndash75 moderately active 25ndash50 weakly active and lt25inactive)

For A aegypti larvae treatments that result in at least50 mortality within 48 h were selected for subsequentbioassays using different concentrations to determine theconcentrations required to kill 10 (LC

10

) 50 (LC50

) and90 (LC

90

) of the larvae Analysis was performed accordingto the probit method [11]

24 Toxicity Test with Artemia salina (Nontarget ModelOrganism) Artemia salina toxicity (ie LC

50

) is largelyaccepted [12] as a parameter reflecting the general biologicaltoxicity of a compound in nontarget organisms Toxicitytests using Artemia salina Leach nauplii were carried outaccording to the method described by Bastos and colleagues[13] with some modifications Artemia cysts (approximately1 gsdotLminus1) were added to filtered seawater and maintained at27ndash30∘C with aeration and under continuous light Samplesthat showed ge30 mortality at 1000mgsdotmLminus1 in preliminarytests were subjected to quantitative assays After 24 hoursthe nauplii hatched were collected with a micropipette andwere concentrated in a Petri dish with seawater maintainedat the same temperature and luminosity for more than 24hours Fifteen grams of each crude extract (dichloromethanemethanol ethanol and water) was diluted in 5mL of distilled

Journal of Parasitology Research 3

Table 1 Lethal concentrations (LC50) of the chloroform and hexane fractions (in 120583gsdotmLminus1) of P gymnospora for Aedes aegypti

Algaefraction LC10 LC50 LC90

C6H14 (a) 3004 ge 767 le 12373 29018 le 39155 ge 54584 117977 ge 199791 le 572243

CHCL3 (b) 0992 ge 3009 le 5559 17230 le 25340 ge 37063 116650 ge 213374 le 633942

(a) Hexane fraction (b) chloroform fraction

water The control solution was prepared with seawater and01 dimethyl sulfoxide (DMSO)

Ten Artemia nauplii and 50 120583L algae extract were placedin each well Four different concentrations of each extractwere tested in six replicates A control solutionwas also testedin six replicates After 24 hours the number of dead larvaewas determined with the aid of a Zoom stereomicroscopeThe dead larvae counts also included larvae that remainedimmobile after stimulation for at least 10 seconds

The degree of toxicity of each plant extract fractionwas determined as recommended by Deciga-Campos et al[14] These authors consider extracts that have an LC

50

ge

1000 120583gsdotmLminus1 to be nontoxic an LC50

between 500 and1000 120583gsdotmLminus1 to be weakly toxic and LC

50

lt 500120583gsdotmLminus1to be highly toxicThe LC

50

values (concentrations that cause50 mortality) and their confidence intervals (90 IC and95 IC) were calculated using probit analysis [11]

25 Molluscicidal Activity Molluscicidal activity was testedin Biomphalaria glabrata (Say 1818) that were propagatedin the laboratory and were descendants of specimens notinfected by trematodes

Snails were kept in glass tanks at room temperature in adechlorinated water system for 8 hours with a mixture of soilsand and calcium carbonate During the assay period theywere fed fresh lettuce and fish foodMolluscicidal activity wasevaluated by immersing B glabrata in an aqueous solutionmixed with 01 dimethyl sulfoxide (DMSO) containing theextract under investigation at different concentrations (010to 100 120583gsdotmLminus1) [8]

The test solution for each algal extract was prepared at100 120583gsdotmLminus1 for the preliminary tests with the snails In thepreliminary stage five adult snails were used per concen-tration Two sets of controls were used to verify that thesnails hadnormal susceptibility to toxinsThepositive controlused was niclosamide at 3 120583gsdotmLminus1 and the negative controlwas dechlorinated water The organisms were exposed to theextracts for 24 hours and observed for 72 hours with readingsand water exchanges every 24 hours Dead specimens wereremoved at the time of the readings The death of thesnails was indicated by discoloration the absence of musclecontractions bleeding and the deterioration of body tissuesThe algal extract promoted approximately 30 mortalityat 100 120583gsdotmLminus1 In parallel control tests were performedwith aqueous DMSO solution (01) and the molluscicideniclosamide at 3120583gsdotmLminus1 Probit version 15 [11] was usedto calculate the lethal concentration values (LC

90

LC50

andLC10

) TheWorld Health Organization (WHO) suggests that

crude extracts of plants with LC50

values lt40 120583gsdotmLminus1 (ie004 or 04mgsdotmLminus1) have some potential as molluscicidalor larvicidal compounds [10]

The results shown for themolluscicidal tests represent theaverageplusmn standard deviation of three replicates (119899 = 3)Theseresults were submitted to ANOVA followed by the Tukeytest (119875 le 005) All tests were performed using the MicrocalOrigin version 80 and Graph Pad Prism version 50

3 Results and Discussion

The crude dichloromethane methanol ethanol and waterextracts from different algal species (Ulva lactuca (Chloro-phyta) Padina gymnospora Sargassum vulgare (Phaeophyta)Hypnea musciformis and Digenea simplex (Rhodophyta))and the chloroform and hexane fractions of extracts from Hmusciformis and P gymnospora were screened for biologicalactivity Different concentrations of extracts were testedaccording to organism susceptibility The initial concentra-tions used were 1000120583gsdotmLminus1 for the Artemia salina toxicitytest 500120583gsdotmLminus1 for the Aedes aegypti larvicidal test and100 120583gsdotmLminus1 for theBiomphalaria glabratamolluscicidal test

The A aegypti larvicidal tests revealed that only thehexane and chloroform fractions of P gymnospora exhibitedlarvicidal activity with LC

50

values of 2901120583gsdotmLminus1 and1723 120583gsdotmLminus1 respectively (Table 1) the other extracts exhib-ited lower activities

Selvin and Lipton [9] evaluated the biocidal potentialof Ulva fasciata and H musciformis isolated in India andverified their larvicidal activity against the mosquito Culexsp at the second and fourth instars These authors foundthat larvae at second instar exhibited 100 mortality whenexposed to 10mgsdotmLminus1 methanol dichloromethane extracts(1 1) Although the mosquito species used in our study wasnot the same as that used by those authors both Culex spand A aegypti are vectors of viral diseases and extractsfrom H musciformis showed no lethality against A aegyptiAn analysis of the larvicidal activities of methanol extractsof 20 species of seaweed collected off the coast of Indiaagainst the second instar larvae of A aegypti [2] revealedthat Lobophora variegata (Phaeophyta-Dictyotaceae) had arelatively high LC

50

(7038 120583gsdotmLminus1) In our work only thehexane and chloroform fractions of P gymnospora (Phaeo-phyta) had higher larvicidal potentials (29018120583gsdotmLminus1 and17230 120583gsdotmLminus1 resp)

The chloroform fractions from H musciformis andP gymnospora showed molluscicidal activity against B


Table 2 Lethal concentration (LC50) of the hexane (C6H14) and chloroform (CHCL3) fractions of extracts frommarine algae species collectedon the beach of Riacho Doce AL against B glabrata

Algaeextract LC10 (120583gsdotmLminus1) LC50 (120583gsdotmLminus1) LC90 (120583gsdotmLminus1)(95 CI) (95 CI) (95 CI)

H musciformisCHCL3 27831 (22906ndash32838) 111460 (101599ndash12161) 446382 (395253ndash513482)P gymnosporaCHCL3 5963 (48189ndash71286) 258689 (234123ndash284624) 1122189 (968318ndash1334954H musciformislowastC6H14 mdash mdash mdashP gymnosporalowastC6H14 mdash mdash mdashlowastThe LC could not be calculatedlowastlowastValues in parentheses correspond to the confidence intervals

Table 3 Accurate toxicity test with crude extracts of marine algae collected on the beach Riacho Doce AL against A salina (CH2Cl2dichloromethane MeOH methanol EtOH ethanol H2O water)

Algaeextract LC10 (120583gsdotmLminus1) LC50 (120583gsdotmLminus1) LC90 (120583gsdotmLminus1)H musciformisCH2CL2 54054 ( 41067ndash62021) 107090 (93020ndash148002) 213046 (150039ndash478061)P gymnospora CH2CL2 44056 (25032ndash55061) 116001 (94048ndash213066) 295041 (177011ndash1633047)H musciformisMeOH 59073 (470908ndash670167) 113002 (97018ndash157013) 213085 (154077ndash468026)P gymnosporaMeOH 64042 (56031ndash700235) 100078 (90174ndash117073) 157069 (130087ndash225043)P gymnosporaEtOH 83084 (72039ndash97064) 143059 (115001ndash307008) 245094 (163069ndash1077090)H musciformisH2O 55025 (31095ndash65074) 145061 (108068ndash47640) 383069 (200035ndash636094)Values in parentheses correspond to the confidence intervals

glabrata and their LC50

values were determined to be111460 and 258689120583gsdotmLminus1 respectively The hexane frac-tions of these two algal species did not show mollusci-cidal activity sufficient to calculate a lethal concentration(Table 2)

Given the molluscicidal activity of the chloroform frac-tions of H musciformis and P gymnospora and the factthat the WHO [10] recommends that plant extracts withLC50

lt 40 120583gsdotmLminus1 be used directly against B glabrata thesefractions might be promising sources of new compoundswith molluscicidal activity Patel et al [15] evaluated the mol-luscicidal potential of methanolic extracts from 60 marinealgae and found that only extracts from Fucus serratusF vesiculosus Pelvetia canaliculata Ascophyllum nodosumHalidrys siliquosa Bifurcaria bifurcata Dictyota dichotomaand Halopithys incurve exhibited activity against B glabrataat 500 120583gsdotmLminus1

A salina toxicity upon exposure to crudedichloromethane methanol ethanol and water extractsand to the chloroform and hexane fractions of algae Hmusciformis and P gymnospora was analyzed No mortalitywas observed at any concentration tested for any samplesexcept the chloroform and hexane fractions of algae Hmusciformis and P gymnospora thus the LC values couldnot be calculated

The LC50

value reflects the concentration necessary tocause the death of 50 of the A salina individuals in asample Therefore lower LC

50

values reflect greater toxicity[16] The results of the A salina assay indicated that crudeextracts of P gymnospora prepared with dichloromethane

methanol and ethanol and crude extracts of H musciformisprepared with dichloromethane methanol and water hadlow toxicities (Table 3) The chloroform and hexane fractionsof the dichloromethane extract of P gymnospora showed lowtoxicity againstA salina (lower than 1000 120583gsdotmL-1) (Table 4)For the other marine algae extracts tested no LC could becalculated

Ara et al [17] investigated the toxic activity of ethanolextracts of 22 algal species from the Karachi coast againstA salina and found that five brown algae showed valuesbelow 1000 120583gsdotmLminus1 (Spatoglossum asperum Stokeyiaindica (507120583gsdotmLminus1) Stoechospermum marginatum(612 120583gsdotmLminus1) Sargassum swartzii (928120583gsdotmLminus1) andS binderi (735 120583gsdotmLminus1)) whereas only one green algaCaulerpa racemosa (929 120583gsdotmLminus1) was toxic In the samestudy these authors found that the hexane fractions of Smarginatum (349 120583gsdotmLminus1) and S swartzii (61 120583gsdotmLminus1) werealso toxic In our study the chloroform and hexane fractionsof P gymnospora (788277120583gsdotmLminus1 and 706990 120583gsdotmLminus1resp) showed low activity indicating the polar nature of thetoxic compounds of this alga

Lhuillier et al [18] screened 26 benthic macroalgae inthe state of Santa Catarina and assayed their lethality usingA salina larvae the samples included ethanol extracts fromred (Rhodophyta) brown (Phaeophyta) and green (Chloro-phyta) algae These authors found that 25 species had sig-nificant toxicity especiallyAcanthophora spicifera Galaxauramarginata Gracilaria dominguensis Pterocladia capillaceaPadina gymnospora Ectocarpus breviarticulatus Chaeto-morpha antennina and Codium decorticatum (50 120583gsdotmLminus1)


Table 4 More precise toxicity test of the hexane (C6H14) and chloroform (CHCl3) fractions of the extracts of algal species collected on thebeach Riacho Doce AL against A salina

Algaeextract LC10 (120583gsdotmLminus1) LC50 (120583gsdotmLminus1) LC90 (120583gsdotmLminus1)P gymnospora C6H14 330474 (139004ndash437601) 788277 (687856ndash998205) 1880271 (1307848ndash5926118)lowastH musciformis C6H14 mdash mdash mdashP gymnospora CHCL3 42349 (322234ndash489074) 706990 (648747ndash776152) 1180111 (1009600ndash1593490)lowastH musciformis CHCL3 mdash mdash mdashlowastThe LC could not be calculated values in parentheses correspond to the confidence intervals

In the present study the ethanol extract of H musciformiswas not sufficiently lethal to allow the calculation of the LC

50

andP gymnosporawas not lethal Guedes et al [19] found thatfractions ofHmusciformis andP gymnospora have a selectivecytotoxic activity against cancerous cells

The generally low toxicity of algal extracts against Asalina as reported by these authors is consistent with thefindings of this studyThese results indicate that these extractshave some potential for the development of products tocontrol vectors of neglected diseases without any importantimpacts on nontarget organisms that is organisms that couldbe inadvertently affected by toxic substances as a result ofsharing an environment or resources with the target species

4 Conclusions

The biological activity of two marine algae collected from thecoast of Alagoas State showed larvicidal activity againstAedesaegypti (based on Aedes aegypti larvae in the fourth instar)and molluscicidal activity against Biomphalaria glabrata(based on the snail) The chloroform fractions of H musci-formis and P gymnosporamight be promising sources of newcompounds with molluscicidal activity and the hexane andchloroform fractions of P gymnospora could be sources ofnovel larvicides

The primary advantage of using these extracts would betheir low cost of production as the algae from which theywere obtained are an abundant resource on the Braziliancoast These algae are easy to cultivate and their crudeextracts have low off-target toxicity as demonstrated in theArtemia nauplii

Therefore we suggest the extension of studies to establishthe potential of these extracts for use in natural environmentsconsidering the potential low impact on nontarget organisms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] T F Molinski D S Dalisay S L Lievens and J P SaludesldquoDrug development from marine natural productsrdquo NatureReviews Drug Discovery vol 8 no 1 pp 69ndash85 2009

[2] A Manilal N Thajuddin J Selvin A Idhayadhulla R SKumar and S Sujith ldquoIn vitro mosquito larvicidal activity of

marine algae against the human vectorsCulex quinquefasciatus(say) andAedes aegypti (linnaeus) (diptera Culicidae)rdquo Interna-tional Journal of Zoological Research vol 7 no 3 pp 272ndash2782011

[3] M T Cabrita C Vale and A P Rauter ldquoHalogenated com-pounds from marine algaerdquo Marine Drugs vol 8 no 8 pp2301ndash2317 2010

[4] P A Miyasato T Kawano J C Freitas R G S Berlinck ENakano and L F Tallarico ldquoMolluscicidal activity of somemarine substances against the snail Biomphalaria glabrata(Mollusca Planorbidae)rdquo Parasitology Research vol 110 pp1873ndash1879 2012

[5] D J Faulkner ldquoMarine natural productsrdquo Natural ProductReports vol 19 no 1 pp 1ndash48 2002

[6] Programa das Nacoes Unidas para o Desenvolvimento ldquoAVerdadeira Riqueza das Nacoes Vias para o DesenvolvimentoHumanordquo 2010 httpwwwpnudorgbrrdh

[7] P Hotez ldquoA new voice for the poorrdquo PLoS Neglected TropicalDiseases vol 1 no 1 article e77 2007

[8] E A D Santos C M D Carvalho A L S Costa A SConceicao F D B P Moura and A E G Santana ldquoBioac-tivity evaluation of plant extracts used in indigenous medicineagainst the snail Biomphalaria glabrata and the larvae ofAedes aegyptirdquo Evidence-Based Complementary and AlternativeMedicine vol 2012 Article ID 846583 9 pages 2012

[9] J Selvin and A P Lipton ldquoBiopotentials of Ulva fasciata andHypnea musciformis collected from the peninsular coast ofIndiardquo Journal of Marine Science and Technology vol 12 no 1pp 1ndash6 2004

[10] World Health Organization (WHO) ldquoInstruction for deter-mining the susceptibility or resistance of mosquito larvae toinsecticidesrdquo WHOVBC75583 1975

[11] D J Finney Probit Analysis Cambridge University PressCambridge UK 3rd edition 1971

[12] J L Carballo Z L Hernandez-Inda P Perez andM D Garcıa-Gravalos ldquoA comparison between two brine shrimp assays todetect in vitro cytotoxicity in marine natural productsrdquo BMCBiotechnology vol 2 article 17 2002

[13] M L A BastosM R F Lima LM Conserva V S Andrade EM M Rocha and R P L Lemos ldquoStudies on the antimicrobialactivity and brine shrimp toxicity ofZeyheria tuberculosa (Vell)Bur (Bignoniaceae) extracts and their main constituentsrdquoAnnals of ClinicalMicrobiology andAntimicrobials vol 8 article16 2009

[14] M Deciga-Campos I Rivero-Cruz M Arriaga-Alba et alldquoAcute toxicity and mutagenic activity of Mexican plants usedin traditional medicinerdquo Journal of Ethnopharmacology vol 110no 2 pp 334ndash342 2007

[15] A V Patel D C Wright M A Romero G Blunden and MD Guiry ldquoMolluscicidal polyphenols from species of fucaceaerdquo


Natural Product Communications vol 3 no 2 pp 245ndash2492008

[16] M M Milhem A S Al-Hiyasat and H Darmani ldquoToxicitytesting of restorative dental materials using brine shrimp larvae(Artemia salina)rdquo Journal of Applied Oral Science vol 16 no 4pp 297ndash301 2008

[17] J Ara V Sultana S Ehteshamul-Haque R Qasim and V UAhmad ldquoCytotoxic activity of marine macro-algae on Artemiasalina (Brine Shrimp)rdquo Phytotherapy Research vol 13 pp 304ndash307 1999

[18] C Lhullier P A Horta and M Falkenberg ldquoAvaliacao deextratos demacroalgas benticas do litoral catarinense utilizandoo teste de letalidade para Artemia salinardquo Brazilian Journal ofPharmacognosy vol 16 no 2 pp 158ndash163 2006

[19] E A C Guedes T G da Silva J S Aguiar L D de BarrosL M Pinotti and A E G SantrsquoAna ldquoCytotoxic activity ofmarine algae against cancerous cellsrdquo Revista Brasileira deFarmacognosia vol 23 no 4 pp 668ndash673 2013

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are known to possess antibacterial antifungal antiviral andantitumor activities [1]

The Brazilian Northeast Region is the poorest region ofBrazil and has the worst Human Development Indices inthe country [6] Most of the population in this region is atrisk of contracting neglected tropical diseases In particulardengue fever and schistosomiasis are major public healthconcerns in Brazil and other developing tropical countries[7 8] Natural products with different biocidal activitiescan help fight parasite vectors at the adult or larval stagesand are good alternatives to synthetic products due to theirrapid biodegradation and lower costs [9] thus contributingto improved public health conditions in tropical developingcountries

The present study investigates the biological activities offive marine algaeUlva lactuca Linnaeus Padina gymnospora(Kutzing) Sonder Sargassum vulgare C Agardh Hypneamusciformis (Wulfen in Jacque) JV Lamouroux andDigeneasimplex (Wulfen) C Agardhmdashall collected from the coastof Alagoas State The activities tested included larvicidalactivity against Aedes aegypti molluscicidal activity againstBiomphalaria glabrata and toxicity against Artemia salinaThis is the first report on the biological activity of marinealgae collected from the coast of Alagoas

2 Materials and Methods

21 Collection of Marine Algae Algae were manuallycollected from the Riacho Doce beach (9∘341015840010158401015840S and35∘391015840010158401015840W) during low tides between October 2007and July 2009 The collected samples were immediatelytransported to the Phycology Laboratory of the BiologicalSciences Institute (ICBS) Universidade Federal de Alagoas(UFAL) Five species of algae from three divisions wereused for this study ChlorophytamdashUlva lactuca LinnaeusMAC51238 PhaeophytamdashPadina gymnospora (Kutzing)Sonder MAC51235 and Sargassum vulgare C AgardhMAC51236 and RhodophytamdashHypnea musciformis (Wulfenin Jacque) JV Lamouroux MAC51234 and Digenea simplex(Wulfen) C Agardh MAC51231 A voucher specimen of eachalgae species was deposited in the MAC Herbarium at theEnvironmental Institute of the city of Maceio Alagoas (Br)as a reference material

22 Extract Preparation and Fractionation Algae werewashed with distilled water dried in air-circulating ovens(BLUE MOD-1401440SC USA) at 45∘C for three hoursand crushed using a MetVisa industrial blender (modeltype TA-2) The dried and crushed material from eachcollection and each alga was mixed in equal quantities Toobtain crude extracts 500 g of dried algae was suspendedin 1000mL of dichloromethane methanol ethanol or waterand macerated for 72 hours this process was repeated threetimes The organic extracts were filtered and rotoevaporated(MOD Rotoevaporator with a Buchi heating bath B-490Switzerland) at 25∘Cndash40∘C and the aqueous extract waslyophilized (Edwards Freeze dryer Mod E2 MB of Brazil)Extracts of the respective algal species were stored at 4∘C

The dichloromethane extracts of H musciformis and P gym-nospora were selected for fractionation because they gave thebest yields 1186 g (258) and 1396 g (304) respectivelyThe yields of the other extracts were less than 05 g (01) andwere therefore not sufficiently large for fractionation

For fractionation by liquid-liquid partitioning the crudeextracts ofH musciformis (273 g) and P gymnospora (190 g)were suspended in methanol water (3 1) and extracted withhexane and chloroform to yield chloroform and hexanefractions All solvents were purchased from VERTEC FineChemicals of Brazil

23 Mosquito Larvicidal Activity Bioassays were run with Aaegypti larvae at the Chemistry and Biotechnology Instituteof Federal University from Alagoas IQBUFAL laboratoryusing the WHO protocol [10] The bioassays used fourthinstar larvae with two repetitions Each experimental courseused 10 larvae placed in a 250mL cup containing 27mLof test solution at 100 120583gsdotmLminus1 Extracts that led to le50mortality were subjected to more precise tests In these testsfour replicates were run using 25 larvae in each replicateTheconcentrations used were defined based on the preliminaryresults

Larvae were considered dead when they failed to reachthe surface of the solution after the liquid was stirred Deadlarvae counts were performed at 24 hours and 48 hours afterthe start of the experiment and the samples were run inquadruplicate In parallel control tests were performed with1 aqueous dimethyl sulfoxide (DMSO) solution and thesynthetic larvicide Temephos at 3 120583gsdotmLminus1 The activity levelof extracts tested was calculated based on the average percentmortality of larvae at 48 hours (gt75 highly active 50ndash75 moderately active 25ndash50 weakly active and lt25inactive)

For A aegypti larvae treatments that result in at least50 mortality within 48 h were selected for subsequentbioassays using different concentrations to determine theconcentrations required to kill 10 (LC

10

) 50 (LC50

) and90 (LC

90

) of the larvae Analysis was performed accordingto the probit method [11]

24 Toxicity Test with Artemia salina (Nontarget ModelOrganism) Artemia salina toxicity (ie LC

50

) is largelyaccepted [12] as a parameter reflecting the general biologicaltoxicity of a compound in nontarget organisms Toxicitytests using Artemia salina Leach nauplii were carried outaccording to the method described by Bastos and colleagues[13] with some modifications Artemia cysts (approximately1 gsdotLminus1) were added to filtered seawater and maintained at27ndash30∘C with aeration and under continuous light Samplesthat showed ge30 mortality at 1000mgsdotmLminus1 in preliminarytests were subjected to quantitative assays After 24 hoursthe nauplii hatched were collected with a micropipette andwere concentrated in a Petri dish with seawater maintainedat the same temperature and luminosity for more than 24hours Fifteen grams of each crude extract (dichloromethanemethanol ethanol and water) was diluted in 5mL of distilled










50

ge



50


50





90

LC50

andLC10








50



50














The LC50


50









50



4 Conclusions






References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










50

ge



50


50





90

LC50

andLC10








50



50














The LC50


50









50



4 Conclusions






References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology












The LC50


50









50



4 Conclusions






References






























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





50



4 Conclusions






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

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