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Ol ivero -Verbe l e t al.

Chemical Composition and Toxicity Against Artemiafranciscanaof t he Essent al Oil of Callisternonspeciosus (Sims) DC. Collected in Bogota

(Colombia).Jorge Gette-Fernndez, Jess Olivero-Verbel, lndira O'Byrne-Hoyos and Beatriz Jaramillo,Envirortrtwrltcd crnd Coir~putntiorrnlC i~e~n i r t ryrozrl), Deportinwrit of Ci~ernistnjUnizjersity of Cnrtclgrnn, Cnrnl)irs f

Zc~rtr~ocilln,rrrtngenn, Colornhicl, E-itu~il o~izjcn>zj@ur~ic.c~rtt~gentl.rclu.coElena Stashenko,

Cilrotrlcitogr(lphy Lnhortltory, Re.seorclh Centre o$Erce:

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A. franciscana

ii i tlie iiioriiiiig, and only fresh undainaged leaves werePlaiit inaterial was identified by botanist

irre Santoro froiii the Iilstitute of Natural Sciericesve speciiiien voucher (Col No 512803) has been depositedthe Iiistitute's Herbariiiiii . After collectioii, saiiiples were

were geiitly washed aiid iised fr~s li .During this processry was coiid itioiid so that the sainple liad ininiinallit exposure.Essential oil isolation: Hydrodistillatiori (HD)was per-

usiiig aii electric hea ter (boiling water) for 2 11, andwas decanted froiri the condensate, previ-

iate, as described elsewhere (19). The oil isolatiori washree tiiiies. The average oil yield was 0.2%.

Essential oil analysis:Coiripound identification was basedi mass spectra (EI, 70 eV) obtained with a gas chroinato-

(Agilent Technologies 6890 Pliis, Palo Alto, CA, USA),ed with a iriass selective detector (Agilent Tec!hiiologiessplit/splitless injector (1:50split ratio), andadatasystein

P CheinStation 1.05),~vithWILEY 138K, NIST 2002 andass spectralibraries. Afused-silica cqil lary(J&W Scieiitific, Folsoin, CA, USA) of 60

x 0.25 iiiin x 0.25 Fin (df) was einployed. The GC overierature was prograiniried froin 45OC (15 iiiin) to 250C

inin) at 5OC/ iriiri. The teinperatures of t l i ~onisatioiith e traiisfer liiie were set at S30C aiid 285OC,

Mass spectra aiid recoiistructed ion curreiitswere obtaiiied by autoinatic scaiiiiiiig at 5.19

, in the iriass range iir/z 30 300 . Chroinatograpliic peakschecked for tlieir hoinogerieity with the aid of tlie iiiass

A gas chroinato grq h (H P 5890 A Series 11), eqiiippedne ioiiisation detectioii (F ID), split/splitless iii j~ ct or

split ratio), and a data systein ( HPCheiiiStatioii IIP Rev.) was used for GC/ FID aiialysis of tlie oils and

tlieir coinponents. Tlie detector aiid iiijectorwere set at 250C. The saiiie capillaq coluiriri,

for the GC/MS ai i~ ~l ~s is ,as used for GC/FID separatiorihe oven tenipera ture was prograiiiiiied froiii

15 iniri) to 250C (20 iiiiii) at 5C/iiiiii. Heliuiii was used.Hydrogeii aiid air at 30 and 300 iiiWiiiiii,

were utilised iii the F ID , with N, (30 inWiiiiii)The various coinpourids were ideiitified by

arison of thei r Kovits reteiition iiidices (SO),deteriniiiedziiig a linear scale on the DB-5MS (60 i i i ) coluinii, aiid ofinass spectra ofeach G C coiriponeiit with those ofstaiidard

nces aiid reported data (21-23).Screening tozic ity assays: The acu te lethal concentra-(LC50) (24) aiid tlie growtli-iiihibitory activity (25)of the

were evaluated using tlie crustaceuin Arte~nic~rcrncisccrric~.,27) at a inaxiinuiii coiiceritration of 1.3% v/v. Tlie LC50

ay was sta rted by puttiiig 10 larvae in 10 inL solutio~i orDMS O). Couiitiiig of dead organisins was carried

out after 24 aiid 48 11. Al1 tlie experiirieiits were carried oiitfour tiines aiid LCj, ,vdu esas ~ 1 ~ 1 1s 95%) onfideiice iiiten~alswere calculated by the probit iiietliod (28).

The irihibitory effect of tlie oil on the growth (elon-gitioii ) of Atteniicl fr(it~ci.scat~nas ineasured 24, 48 aild 72 11after cyst expositioii to differeiit coriceritratioris. Briefly, 20 ingof cysts were placed oii iridi\idual Petri dishes coiitaiiiiiig tlievehicle control ( DM SO) or tlie saiiie oil conceiitratioiis usedin the lethality assay. Lerigths of 60 larvile were ineasured foreach conce~itratioii, siiig aii optical iiiicroscope aiid a Neii-bauer camera. A statistically sigiiificarit differerice in lerigtliaverages obtained for the differeiit coriceiitratioil treatinentswas taken as indicative of growth disruptioii. Al1 experiinentswere carried out by tetraplicates. No dead aiiiiiials or cliaiigesiii larvae growth were observed i i i the control group.

Statistical analysis: Al1 the results were p rese~i ted s theirieaii f tandard deviation (XkSD). Sigiiificaiit differeiicesbetween irieans for differeiit oil coiiceiitratioiis were assessedby ANOVA, using the Dunnet test as a post test. Iii al1 cases,

Table l. Percentage composition of the oil of Callisternonspeciosus.

Compounds RI' GC area, %MeanI D"'

isoamyl acetateisobutyric acida-thujenea-pinene"camphenesabineneP-pinene"myrcene"a-pheliandreneisoamyl isobutyratelimonene"1,8-cineole"y-terpineneterpinolenelinaloolfftrans-sabinene hydratetrans-p-rnentha-2,8-dien.cis-p-mentha-2-en-1-01trans-pinocarveolpinocarvoneterpinen-4-01"n-terpineolgeranial"eugenol"geranyl acetatep-caryophylleneallearomadendrenebicyclogermacrenespatulenolglobuiolP-eudesmol

'Experimentally determined retention ndices (DB-5MS Column, 60 m); "Fortdentification. standard compounds were also used; "'Mean of three HDextractions (n=3,I D).

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Olivero-Verbel et al.

Table II . LC-. of the Callisternon s~ecios us il aaainst Artemia franciscana.

Oil Exposition time (h) LC,, (~glmL) 24-h LC,J48-h LC,,

Callisternon spec iosus 24

'95% Confidence Intervals.

- 2 4 h 4 8900 - 72 h800 -700 -600 -

0,001 DMSO 0,l 1 10 100 1000

Concentration of Callisternon speciosus ( ~ 9 / m)Figure 1. Effect of the Callistemon speciosusoil on the Artemia franciscanabody length.

iioriiialdistnbiitio~i ndeqiiality betweeiivariaiiceswereevdu- represeiited 81.1% of the C. speciosus esseiitial oil.etedusingKoliiiogorov-Siiiiniov iid Barlett tests, respectively. Acute toxicity against Artemia franciscana: Th e LC,,,Iii abseiice of iioriiiality, log trniisforinatioii of the data were of tlie C. sy~eciosi~sil agaiiist Artenzirr .frnnci.rcart.a after 24carried out or coinparisoiis doiie by tlie Kruskal IVallys test . arid 48 Ii exposiire, are presented iri Table 11. Based o11 botliFor al1 cases, the sigiiificaiice leve1 was set at pO . 1%)were separated aiid ideiiti-fied as seeii iii Table 1. The iiiost abiiiiclaiit coiiipouiid iii tlieoil was 1,8-ciiieole, witli aii averageIestaiidard error rrlativeabuiidaiice ofSSI3.3%. This coiiipouiiclhas also beeii reporte tlasairiajorcoiripoiierit forthe geiiiisCa11istenu)n 29,30). ,8-ciii-eole together witli a-piiieiie, a-terpiiieol, aiid a- ph el la ~i dr e~ ie

froiii 24 to 48 11 did iiot lead to a significaiit iiicrease iii toxicity(LC,,,s of 56.7 and 39.8 pghiiI,, respectively), siiggestiiig tIiatriciupliar siisceptibility to this oil did not chaiige tliroughoiitdevelopiiieiit.

Eflect of the oil on elongation ofArtemiafranciscana:Tlie eFfects of tlie oil oir tlie elongatioii of A. ~fr(~nci.scnnare

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ii Figure 1. Tlie average size of the iiaiipliis ofA.finri-for the coiitrol groups (DM SO)duriiig 24 ,48 aiid 72 11

e of 525.4, 733.5 and 835.0 piri, respec-ely. These results were coinparable with those reported by

(S), arid Olsoii (6) , who observed that dunng tlie11 after cyst wettiiig, average iiauplii body length was

h it had cha~igedo 800-920 prn. However,trr and Scliaeffer (26), reported that the body size of tlie

0 f A . j - n n c i s c r r n n duriiig the first 24 and 48 11 were 430piii, respectively. Exposure dunng 24, 48 and 72 11

C. spec iosu .~ il did not produce significaiit effects oii thef the criistacean at concentratioiis below 10 pg/rriL.coiicentrations, the oil iriducrd a sigiiificaiit decrease

iuin iirar 12% after 72exposure to 250 pg/iiiL. At 24 ,48 and 72 11 of exposure, the

a lowest observed adverse effect leve1 (LOAEL) values ofaiid 50 pg/mL, respectively.Ekeii togetlier, these resiilts suggest that the oil of C.s ari iinportant source of 1,8-cineole, aiid that in

preliiiiinary screeiiing, it had a low toxicity iii a crustacea~iassay, siiggesting an i~nportaiit otential as a tlierapeutical

Fiticri ic icrl si lpport ~ I - O I I ~olcienci(r.s. Bogot(r, Coloinhi(r (Grcrnt432-2004) , is g i -cr t~f i r l l~ jri:knowledgetl. \[.e thank ]ose L ~ l i s

zr~tl bxcriuler O'B yni efo r tl i r col/cction ofthr plant

J.M. McLaughlin, Crown gall tumours on potato discs and brine shrimplethality: wo~simple ioassays trhigherplant screening andfractlonation.In:AssaysforBioactivify,Edit., K. Hostettmann,pp.2-32, Academic Press,San Diego, CA (1991).P. Vanhaecke, G. Persoone, C. Claus, and P Sorgeloos, Proposal for ashort-term toxicify test with Artemia nauplii. Ecotoxicol. Envi ron. Saf., 5,382-387 (1981 .S. Persoone, P Sorgeloops, O. Roels, and E. Jaspers, The brine shrimp,Artemia. Universa Press Ediciones, 3, 11-13 (1980).G. Persoone and P.G. Wells, Artemia in aquatic toxicology: a review. In:Artemia research and its applications: 1 . Morphology. genetics, straincharacterization, toxicology Edits., P Sorgeloos, et a l. pp.259-275 (1987).Proceedings of the Second lnternational Symposium on the Brine ShrimpArtemia. (1987).D.T. Anderson, Larva1 development and segment formation in thebranchiopod crustaceans Limnadia stanleyana King (Conchostraca) andArtemia salina. (L.) (Anostraca). Aust. J. Zool., 15,47-91 (1967).C.S. Olson, Timing of developmental events in Artemia salina (L)(Anostraca). Crustaceana, 36, 303-308 (1979).A.S. Michael, C.G.Thompson and M. Abramovitz. Artemia salina asa testorganism for a bioassay. Science, 123,464 (1956).W.Tarpley, Studies on the use of the brine shrimp Artemia salina (Leach)as a test organism for bioassay. J. Econ. Entomol.. 51, 780-783 (1958).

N. Buu-hoi and P. Chanh, Effect of various fypes of carcinogens onthe hatching of Artemia salina eggs. J. Nat. Cancer Inst., 44. 795-799(1970).R.B. Sleet and K. Brendel, Homogeneous populations of Artemia naupliiand their potential use for in vitro testing in developmental toxicology.Teratog. Carcinog. Mutagen., 5, 41-54 (1985).T. Alves, A. Fonseca, M. Brando, T. Mesquita, E. Smania. A. Smania,and C. Zani, Biological screening of brazilian medicinalplants. Mem. Inst.Oswaldo Cruz. 95 , 367-373 (2000).R. Padmaja, P.C. Arun, D. Prashanth, M. Deepak, A. Amit, and M. Anjana,Brine shrimp lethalily bioassay of selected lnd~anmedicinal plants.Fitoterapia, 73 , 508-510 (2002).J. Carballo, Z. Hernndez-lnda, P. Perez, and M. Garcia-Grvalos, Acomparison between two brine shrimp assays to detect n vitro cytotoxicilyin marine naturalproducts. BMC Biotechnology, 2, 17-21 (2002).J. Bagshaw, P. Raflee, C . Matthews, and T. MacRae, Cadmium and zincreversibly arrest development of Artemia lawae. Bull. Environ. Contam.Toxicol., 37, 289-296 (1986).S.Sanchez-Fortun andV. Barahona, Theuseofcarbamates,atropine, and2-pyridinealdoximemethoiodideinheprotection ofArtemiasalina againstpoisoning by carbophenothion. Envi ron. Toxicol. Chem., 20, 200E2013(2001).M. Pelka, C. Danzl, W. Distler, and A. Petschelt, A new screening testtoxicify testing of dental materials. J. Dent., 28, 341 345 (2000).K.Tanaka, M. Manabeand S. Matsuura,Biologicaltestusing he brineshrimp(Artemia salina). The toxicities of metal compounds and the mycotoxinson the brine shrimp. Rep. Nat. Food Res. Inst., 39, 58-63 (1982).A. Logarto, R.Silva. I.Guerraand L. glesias,Comparativestudyoftheassayof Artemia salina and the estimate of the medium letal dose (LD,, value)in mice, to determine oral acute toxicify of plant extracts. Phytomedicine,8, 395-400 (2001).E. Stashenko, B. Jaramillo and J. Martinez, Comparison of differentextraction methods for the analysis of volatile secondary metabolites ofLippia alba (Mill.) N.E. Brown, grown in Colombia, and evaluation of its invitro antioxidant activity. J. Chromatogr. A,, 1025. 93-103 (2004).E. Kovhts and Marcel Dekker, The Retention lndex System. In: Advan.Chromatogr., Edits., J.C. Giddings and R.A. Keller, pp. 229 Marcel Dekker247, New York, NY (1965).W.Jennings and T. Shibamoto, Oualitative analysis of flawrandfragrancevolatiles by glass capillary gas chromatography, Academic Press, NewYork, NY (1980).R.P. Adams, ldentification of essential o il components by gaschromatography/mass spectroscopy, Allured Publishing Corporation,Carol Stream, IL (1995).D. Joulain and W.A. Konig, The atlas of specral data of sesquiterpenehydrocaaons, E.B.-Verlag, Hamburg, Germany (1998).P.D3Souza,M.Deepak, P.Rani, S.Kadamboor,A.Mathew,A.Chandrashekarand A. Agarwal, Brineshrimp lethalilyassayofBacopa rnonnieri.PhytotherRes., 16, 197 Marcel Dekker 198 (2002).R.B.Sleet and K. Brendel,Brineshrimp, Artemia salina:apotentialscreeningorganism for initial teratology screening. Proc. West Pharmacol. Soc., 26,16s-170 (1983).H.W. Kerster and D.J. Schaeffer, Brine shrimp (Artemia salina) nauplii asa teratogen test system. Ecotoxical. Environ. Saf., 7 , 342-349 (1983).A. Massele and C. Nshimo, Brine shrimp bioassay forbiological activity ofmedicinalplants used in traditional medicines in Tanzania. East Afr. Med.J., 72, 661 463 (1995).D. Finney, Probit analysis, 3Id ed. pp. 76-80. Cambridge Univ. Press,Cambridge, UK. London,. (1971).J. Brophy, P.Forster,R.Goldsack,D. Hibbertand A. Punruckvong, Variationincalli stemon viminalis (Myrtaceae):Newevidence from volatileoils.Aust.Syst. Bot., 10, 1 PP. 76-8013 (1997).J.Brophy,R.Goldsack,P.Forster, L.Cravenand B.Lepschi, Theleafessentialoils of the Australian mernbers of the genus Callistemon (Myrtaceae). J.Essent. Oil Res., 10, 595 406 (1998).

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