Olivero-Verbel et al.

Chemical Composition and Toxicity Against Artemia franciscana of t he Essential Oil of Callisternon speciosus (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 r y Grozrl), Deportinwrit of Ci~ernistnjUnizjersity of Cnrtclgrnn, Cnrnl)irs of Zc~rtr~ocilln, Crrrtngenn, Colornhicl, E-itu~il jo~izjcn>zj@ur~ic.c~rtt~gentl.rclu.co

Elena Stashenko,
Cilrotrlcitogr(lphy Lnhortltory, Re.seorclh Centre o$Erce:<?lleilce, CENIVAM. Irrtlustritll Uiiiocrsity (fSnritcln(br, Cm-rerti 27, Calle 9, Bucnrtlrrztingc~, Coloinhicr, E-rnoil: c,le~ia@ttlcnr~.uis.etlu.~o

Abstract
Tlie esseiiti:il oil horn tlie fiesli leaves of Calli.str?~r~on sl)rciostts (Siiris) DC. was isolated by Iiytlrotlistillatio~i. Tliirty-oiie volatilt, coiiipoiinds (relative anioiiiit > O . l % ) were separated aiid positively ideiitified by GC (FID) aiid gas cliroinatogr~ipliy-iiiass spectroinetry (GC/MS). Major constituerits were 1,H-cineole (55%), a-piiiene (18%), a-terpineol (4.1%), and a-ptiellaiidrerie (3.0%). Tlie oil preseiitt:<l a irieaii Ietlial coiicrntratiori agaiiist Arten~in f'runciscc~rici 56.7 pgL arid 38.8 pgnil, after 24 and 48 11 exposition, respectively, antl liad a milcl but sigiiificaiit of efkct ori tlie criistaceaii cloiigation.

Key Word lndex

Cnllisten~ot,s~>eciosus, Myrtaceae, esseritial oil coinposition, 1,8-ciiieole, a-piiiene, acute Ietlial coriceiitration, Arteinic~ fr<inc,iscur~a, toxicity.

lntroduction
Despite its worldwide distribution, there are few reports about tlie cheiiiical or biological properties of Cnllistei~lor~ sl)ecioais esseritial oil. Tlie tree beloiigs to the Myitaceae faiiiilyaiid is kiiou~ii tlie bottlebrusli tree. It reaclies a lieiglit as of 6 to 10 iii and a 10 to 20 ciii diaiiieter, witli red flowers of iiiteiise mlor Altliougli the tree ir originaiiy froin easteni mic1 soutlieasterii Aiotrdia, it d s o grows in tropical aiid siibtropical regioiis of Anierica. 111 Coloinbia, it is foiiiid at several altitudes in departiiierits sucli as Antioquia, Ciiridiiiaiiiarca, Cauca, and Bolivar. Aiiioiig tlie basic screening iiietliods tliat are available to exaiiiiiir tlie biological activity of plant extracts, the 1)riiie sliriiiilj assay iisiiig Arteir~in frcln,ciscclncr, coiniiiorily referred as A.scllintl, is coiisidered one of the iiiost reliable, siiiiple, iiiexpeiisive, and less tiiiie co~isuiiiing screeiiing bioassays for iiatural products (1,2). Tlie followiiig are tlie required coiiditioiis for tlie developirient of brine shriinp (hatcliiiig of tlie cysts aild growtli of the larvae): triiiperature 25-30C; saliiiity
'Address for correspondence

15-35 %O, pH 8; saturated dissolved oxygeri levels; aiid siif. ficieiit illiiiiriatioii (3,4). Duringtlie first 24 h ofiiicubatioii iii seawater, brine sliriiiip cysts bursts and the eiiibrios leave tlie shell as free-swiiniiiing iiaiipliis, reacliirig body lerigtlis betweeii 800-920 pin witliiii 72 11 (5, 6). Iriliibitioii of tlie ciustaceaiii growth aiid lethality evaluatioii after cIieinical exposilre, are two ideal tests for prrlilIiiliai) sssessliieiit of general toxicity screeliing (7,8,, T~~~~ ,yave tised in teratologr (9,10), toxicity evaluatioii (11,12), aiid in tlie assessineiit of the biological effects of iiiariiie natural proclucts (13), lieavy inetals (14), pesticides (15), dental iiiaterials (16), arid iiiycotoxins (17), aiiioiig others. Iii additioii, the Arteitzici letliality bioassay has sliown good correlatiori betu,een tlle in Cito alid ilz zjitrO tests (18),

Experimental
Plant material recollection and treatment: Tlie Callistt:rnon .~))ccio.stls leaves \vere liarvested iii Bogota, Departnieiit of Ciiiidinaiiiarca, Coloiiibia. Tliey were inaiiually picked
Received: December 2006 Revised: July 2007

1041-2905/08/0003-0272$14.00/04 2008 Allured Publishing Corp.

Accepted: July 2007

A. franciscana

early iii tlie iiioriiiiig, and only fresh undainaged leaves were used for extractioii. Plaiit inaterial was identified by botanist Jaime Aguirre Santoro froiii the Iilstitute of Natural Scierices at the National Uiiiversity of Coloiiibia (Bogota). A respective speciiiien voucher (Col No 512803) has been deposited at the Iiistitute's Herbariiiiii. After collectioii, saiiiples were stored iri the dark aiid traiisported to tlie laboratory, where they were geiitly washed aiid iised fr~sli. During this process the laboratory was coiiditioiid so that the sainple liad ininiinal liglit exposure. Essential oil isolation: Hydrodistillatiori (HD) was perforiiied in a 5 L-rourid Aask with 500 g of plant inaterial aiid 4 L ofwater, usiiig aii electric heater (boiling water) for 2 11, and after tliat, the oil was decanted froiri the condensate, previously saturated with NaCl, arid dried with aiihydrous sodiuin sulpliate, as described elsewhere (19). The oil isolatiori was repeated three tiiiies. The average oil yield was 0.2%. Essential oil analysis: Coiripound identificationwas based ori mass spectra (EI, 70 eV) obtained with a gas chroinatograph (Agilent Technologies 6890 Pliis, Palo Alto, CA, USA), equipped with a iriass selective detector (Agilent Tec!hiiologies 5973),split/splitless injector (1:50split ratio), andadatasystein (HP CheinStation 1.05),~vith WILEY 138K, NIST 2002 and QUADLIB 2004 inass spectralibraries. A fused-silica cqillary coluinn DB-5MS (J&W Scieiitific, Folsoin, CA, USA) of 60 in (L) x 0.25 iiiin x 0.25 Fin (df) was einployed. The GC overi teiilperature was prograiniried froin 45OC (15 iiiin) to 250C (15 inin) at 5OC/ iriiri. The teinperatures of t l i ~ ionisatioii chaiiiber arid ofthe traiisfer liiie were set at S30C aiid 285OC, respectively. Mass spectra aiid recoiistructed ion curreiits (chroinatograiirs) were obtaiiied by autoinatic scaiiiiiiigat 5.19 scan/s, in the iriass range iir/z 3 0 3 0 0 . Chroinatograpliic peaks were checked for tlieir hoinogerieity with the aid of tlie iiiass cliroinatograiiis for the cliaracteristic fragineiit ioiis. A gas chroinatogrqh (HP 5890 A Series 11), eqiiipped with flaine ioiiisation detectioii (FID), split/splitless iiij~ctor (1:50 split ratio), anda data systein (HP CheiiiStatioii IIP Rev. A.06.03 [509]) was used for GC/FID aiialysis of tlie oils and quaiititation of tlieir coinponents. Tlie detector aiid iiijector teinperatiire: were set at 250C. The saiiie capillaq coluiriri, as for the GC/MS a i i ~ ~ l ~was, used for GC/FID separatiori sis aiid detectioii. The oven teniperature was prograiiiiiied froiii 40C (15 iniri) to 250C (20 iiiiii) at 5C/iiiiii. Heliuiii was used. as carrier gas, with 152 kPa coluiiiii liead pressure arid 35.7 cm/s linear velocity. Hydrogeii aiid air at 30 and 300 iiiWiiiiii, respectively, were utilised iii the FID, with N, (30 inWiiiiii) as a inake-up gas. The various coinpourids were ideiitified by coiiiparison oftheir Kovits reteiition iiidices (SO),deteriniiied utiliziiig a linear scale on the DB-5MS (60 iii) coluinii, aiid of the inass spectra ofeach GC coiriponeiit with those ofstaiidard substances aiid reported data (21-23). Screening tozicity assays: The acute lethal concentration (LC50) (24) aiid tlie growtli-iiihibitory activity (25) of the frcrncisccrric~. oil were evaluated using tlie crustaceuin Arte~nic~ Both assays were coriducted by dissolviiig the oil iii DMSO (26,27) at a inaxiinuiii coiiceritration of 1.3% v/v. Tlie LC50 assay was started by puttiiig 10 larvae in 10 inL solutio~i for each tested coriceiitratioii (0.01-100 pg/inL) aiid vehicle control (DMSO). Couiitiiig of dead organisins was carried

out after 24 aiid 48 11. Al1 tlie experiirieiits were carried oiit four tiines aiid LCj,,vduesas ~ 1 ~ as 95%)confideiice iiiten~als 1 1 were calculated by the probit iiietliod (28). The irihibitory effect of tlie oil on the growth (elongitioii) of Atteniicl fr(it~ci.scat~n ineasured 24, 48 aild 72 11 was after cyst expositioii to differeiit coriceritratioris. Briefly, 20 ing of cysts were placed oii iridi\idual Petri dishes coiitaiiiiiig tlie vehicle control (DMSO) or tlie saiiie oil conceiitratioiis used in the lethality assay. Lerigths of 60 larvile were ineasured for each conce~itratioii, usiiig aii optical iiiicroscope aiid a Neiibauer camera. A statistically sigiiificarit differerice in lerigtli averages obtained for the differeiit coriceiitratioil treatinents was taken as indicative of growth disruptioii. Al1 experiinents were carried out by tetraplicates. No dead aiiiiiials or cliaiiges iii larvae growth were observed iii the control group. Statistical analysis: Al1 the results were prese~ited the as irieaii f standard deviation (XkSD). Sigiiificaiit differeiices between irieans for differeiit oil coiiceiitratioiis were assessed by ANOVA, using the Dunnet test as a post test. Iii al1 cases,

Table l. Percentage composition of the oil of Callisternon speciosus. Compounds RI' GC area, % Mean ISD"'
isoamyl acetate isobutyric acid a-thujene a-pinene" camphene sabinene P-pinene" myrcene" a-pheliandrene isoamyl isobutyrate limonene" 1,8-cineole" y-terpinene terpinolene linaloolff trans-sabinene hydrate trans-p-rnentha-2,8-dien. cis-p-mentha-2-en-1-01 trans-pinocarveol pinocarvone terpinen-4-01" n-terpineol geranial" eugenol" geranyl acetate p-caryophyllene allearomadendrene bicyclogermacrene spatulenol globuiol P-eudesmol

'Experimentally determined retention indices (DB-5MS Column, 60 m); "For tdentification. standard compounds were also used; "'Mean of three HD extractions (n =3, ISD).

Olivero-Verbel et al.

Table II. LC-. of the Callisternon s~eciosus aaainst Artemia franciscana. oil

Oil

Exposition time (h)

LC,, (~glmL)

24-h LC,J48-h LC,,

Callisternon speciosus

24

'95% Confidence Intervals.

900

-24h

8h

72 h

800

700

600

0,001

DMSO

0,l

10

100

1000

Concentration of

Callisternon speciosus

(~9/m L)

Figure 1. Effect o the Callistemon speciosus oil on the Artemia franciscana body length. f

iioriiialdistnbiitio~i nndeqiiality betweeiivariaiiceswereevduetedusingKoliiiogorov-Siiiiniov Barlett tests, respectively. aiid Iii abseiice of iioriiiality, log trniisforinatioii of the data were carried out or coinparisoiis doiie by tlie Kruskal IVallys test. For al1 cases, the sigiiificaiice leve1 was set at p<0.05.

represeiited 81.1% of the C. speciosus esseiitial oil.

Results and Discussion

Thirty-oiie coiiipo~iiids 1%)were separated aiid ideiiti(>O. fied as seeii iii Table 1. The iiiost abiiiiclaiit coiiipouiid iii tlie oil was 1,8-ciiieole, witli aii averageIestaiidard error rrlative abuiidaiice ofSSI3.3%. This coiiipouiiclhas also beeii reportetl asairiajorcoiripoiierit forthe geiiiisCa11istenu)n(29,30). l,8-ciiieole together witli a-piiieiie, a-terpiiieol, aiid a-phella~idre~ie
2741Journal of Essential Oil Research

Acute toxicity against Artemia franciscana: The LC,,, of tlie C. sy~eciosi~s agaiiist Artenzirr .frnnci.rcart.a after 24 oil arid 48 Ii exposiire, are presented iri Table 11. Based o11botli 24 aiid 48 11 LC?,,rc-sults, iiauplii were uiiifori~il~ seiisitive to tlie oil. Tlie ratio of tlie estiiiiated LC,,,s (24t1-LC,~,,/4811-LC,,, was 1.4. Tliis iridicated that aii increase iii tlie exposiire tiiiie 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 tIiat riciupliar siisceptibility to this oil did not chaiige tliroughoiit developiiieiit. Eflect of the oil on elongation ofArtemia franciscana:
Tlie eFfects of tlie oil oir tlie elongatioii of A. ~fr(~nci.scnna are
Vol. 20, MayIJune 2008

sliow~i Figure 1. Tlie average size of the iiaiipliis ofA.finriiii c i s c n n o for the coiitrol groups (DMSO) duriiig 24,48 aiid 72 11 after cyst wetting were of 525.4, 733.5 and 835.0 piri, respectively. These results were coinparable with those reported by Aiidersoii (S), arid Olsoii (6), who observed that dunng tlie first 24 11 after cyst wettiiig, average iiauplii body length was lower tliaii 600 piii, within the secoiid 24 h it was 725-740 piii, and after 72 h it had cha~iged 800-920 prn. However, to Kerstrr and Scliaeffer (26), reported that the body size of tlie iiauplii 0 f A . j - n n c i s c r r n n duriiig the first 24 and 48 11 were 430 and 660 piii, respectively. Exposure dunng 24, 48 and 72 11 to C. s p e c i o s u . ~ did not produce significaiit effects oii the oil growtli of the criistacean at concentratioiis below 10 pg/rriL. At higlier coiicentrations, the oil iriducrd a sigiiificaiit decrease in nauplii elongation, reacliing a inaxiiiiuin iirar 12% after 72 11 exposure to 250 pg/iiiL. At 24,48 and 72 11 of exposure, the oil presented a non-observed adverse effect Ievrl (NOAEL) and a lowest observed adverse effect leve1 (LOAEL) values of 10 aiid 50 pg/mL, respectively. Ekeii togetlier, these resiilts suggest that the oil of C. s / ~ e c i o s u sis ari iinportant source of 1,8-cineole, aiid that in a preliiiiinary screeiiing, it had a low toxicity iii a crustacea~i bioassay, siiggesting an i~nportaiit potential as a tlierapeutical natural prodiict.
Acknowledgments
Fiticriicicrl silpport ~ I - O IColcienci(r.s. Bogot(r, Coloinhi(r (Grcrnt I~ R C 432-2004), is g i - c r t ~ f i r l l ~ j cri:knowledgetl. \[.'e thank ]ose L ~ l i s Montemirczr~tln czr~tl Abxcriuler O ' B y n i e f o r t l i r col/cction ofthr plant rrurtrrinl.

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Journal of Essential Oil Researchl275