Essential Oil Composition and Antimicrobial Activity of Diplotaenia damavandica

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1 Essentil Oil Composition nd Antimicrobil Activity of Diploteni dmvndic Fereshteh Eftekhr, *, Mortez Yousefzdi, Din Azizin, Ali Sonboli b, nd Peymn Slehi c Deprtment of Biology, Fculty of Sciences, Shhid Beheshti University, Evin, Tehrn, Irn. Fx: (+98-21) E-mil: f-eftekhr@cc.sbu.c.ir b Deprtment of Biology, Medicinl Plnts nd Drugs Reserch Institute, Shhid Beheshti University, Evin, Tehrn, Irn c Deprtment of Phytochemistry, Medicinl Plnts nd Drugs Reserch Institute, Shhid Beheshti University, Evin, Tehrn, Irn *Author for correspondence nd reprint requests Z. Nturforsch. 60 c, 821Ð825 (2005); received Mrch 21/April 27, 2005 Antimicrobil ctivity of the essentil oils obtined from leves, root nd the seeds of Diploteni dmvndic Mozffrin, Hedge & Lmond, n endemic plnt to Irn, ws determined ginst 10 microorgnisms using the disk susceptibility test s well s mesuring minimum inhibitory concentrtions. The results showed tht ll three oils hd ntibcteril ctivity ginst Bcillus subtilis, Stphylococcus ureus, Stphylococcus epidermidis nd Escherichi coli. The essentil oil from the leves hd the highest ntimicrobil ctivity ginst ll test microorgnisms including the fungl strins. The essentil oils compositions were nlyzed nd determined by GC nd GC-MS. The oils nlyses resulted in the identifiction of 16, 17 nd 20 compounds representing 94.2%, 96.4% nd 95.1% of the totl oils, respectively. The min components of the lef essentil oils were (Z)-β-ocimene (21.6%), α-phellndrene (21.3%) nd terpinolene (20%). Dill piol (30.1%) nd γ-terpinene (16.2%) were the min components of the root nd seed essentil oils, respectively. Key words: Antimicrobil Activity, Essentil Oil Compositions, Diploteni dmvndic Introduction Essentil oils re rich sources of biologiclly ctive compounds. Recently, there hs been profound interest in the ntimicrobil properties of the romtic plnts, prticulrly in their essentil oils (Lis-Blchin nd Dens, 1997; Pttnik et l., 1997; Knobloch et l., 1989). Members of the prsley fmily (Apicee) re well known with regrd to their diversity of essentil oils (Yss et l., 2003). This fmily is well represented in the Irnin flor with t lest 112 gener nd 316 species of which 75 re endemic (Hedge et l., 1987). Diploteni dmvndic Mozffrin, Hedge & Lmond is perennil wild herb which grows exclusively in centrl Alborz Mountins round Tr Lke, Dmvnd, Irn (Hedge et l., 1987). The plnt is loclly clled kozl nd upon contct with skin followed by exposure to sunlight cuses photosensitiztion (Aynechi et l., 1999). The extrcts obtined from the eril prts of the plnt hve been reported to hve ntifungul ctivity nd contin furnocoumrins (Aynechi et l., 1999; Srdri et l., 2000). As fr s our literture survey could scertin, composition of the essentil oils from D. dmvndic nd its ntimicrobil ctivity hve not been reported previously. Here, we report the ntimicrobil ctivity of the essentil oils isolted from leves, root nd the seeds, s well s their compositions. Mteril nd Methods Plnt mteril Leves, root nd the seeds of Diploteni dmvndic were collected from the Tr Lke re, Dmvnd, Irn, t n ltitude of 2200 m round JuneÐSeptember A voucher specimen (No ) is deposited t the Herbrium of Biology Deprtment, Shhid Beheshti University. Essentil oil isoltion The powdered plnt prts (250 g) of D. dmvndic were hydrodistilled using Clevenger type pprtus for 3 h. The resulting essentil oils 0939Ð5075/2005/1100Ð0821 $ Verlg der Zeitschrift für Nturforschung, Tübingen D

2 822 F. Eftekhr et l. Oil Composition nd Antimicrobil Activity of Diploteni dmvndic were dried over nhydrous sodium sulfte nd stored t Ð4 C until nlyzed nd tested. Essentil oil nlysis GC nlyses of the essentil oils were conducted using Thermoquest gs chromtogrph instrument equipped with fused silic cpillry DB-1 column (30 m 0.32 mm i.d., film thickness, 0.25 µm). Nitrogen ws used s the crrier gs t the constnt flow rte of 1.1 ml/min. The oven temperture ws held t 60 C for 10 min, then progrmmed to 250 C trte of 4 C/min, nd then held for 10 min. The injector nd detector (FID) tempertures were kept t 250 C nd 280 C, respectively. GC-MS nlysis ws crried out on Thermoquest-Finnign Trce GC-MS instrument equipped with fused silic cpillry DB-1 column (60 m 0.25 mm i.d., film thickness 0.25 µm). The oven temperture ws rised from 60 C to 250 C t rte of 5 C/min, held t 250 C for 10 min; trnsfer line temperture ws 250 C. Helium ws used s the crrier gs t flow rte of 1.1 ml/min; split rtio ws 1/50. The qudruple mss spectrometer ws scnned over the 45Ð465 mu with n ionizing voltge of 70 ev nd n ioniztion current of 150 µa. The constituents of the oils were identified by clcultion of their retention indices under progrmmed temperture conditions for n-lknes (C 6 ÐC 24 ) nd the oil on DB-1 column under the sme conditions. Identifiction of individul compounds ws mde by comprison of their mss spectr with those of the internl reference mss spectr librry or with uthentic compounds nd confirmed by comprison of their retention indices with uthentic compounds or with those reported in the literture (Adms, 1995; Dvies, 1987; Shibmoto, 1987). Quntittive dt ws obtined from FID re percentges without the use of correction fctors. Microbil strins Ten microbil reference strins were used which included Bcillus subtilis (ATCC 465), Enterococcus feclis (ATCC 29737), Stphylococcus ureus (ATCC 25923), Stphylococcus epidermidis (ATCC 12228), Escherichi coli (ATCC 25922), Klebsiell pneumonie (ATCC 10031), Pseudomons eruginos (ATCC 85327), Aspergillus niger (ATCC 16404), Cndid lbicns (ATCC 10231) nd Scchromyces cerevisie (ATCC 9763). Antimicrobil screening by the disk diffusion method The ntimicrobil ctivities of essentil oils were determined by the disk diffusion method (NCCLS, 1997). Briefly, 0.1 ml of suspension of the test microorgnism (10 8 cells/ml) ws spred on Mueller-Hinton gr pltes for bcteri nd Sbourud gr for the fungi. Sterile 6 mm disks, ech contining 20 µl of essentil oil, were plced on the microbil lwns. Disks contining dill piol, ocimene, terpinolene, γ-terpinene nd p-cymene (Fluk Chemicls, Tufkirchen, Germny) were used to study the ntimicrobil ctivity of the mjor oils components t 10 µg/disk for bcteri nd 30 µg/disk for the fungl strins. The pltes were incubted t 37 C for 24 h for bcteri nd 30 C for 48 h for fungi. The dimeters of the zones of inhibition were mesured nd reported in mm. Triplicte tests were crried out for ech oil. Minimum inhibitory concentrtions (MIC) MIC vlues were determined by the broth microdilution ssy recommended by the NCCLS (1999). Seril two-fold dilutions of the essentil oils were mde in Mueller-Hinton broth contining 0.5% Tween 80 for bcteri nd Sbourud dextrose broth with 0.5% Tween 80 for fungi within the rnge of 28.6 to 0.9 mg/ml in 96-well microtiter pltes. Fresh microbil suspensions prepred from overnight grown cultures in the sme medi were dded to give finl concentrtion of orgnisms/ml. Controls of medium with microorgnisms or the essentil oil lone were included. The micropltes were incubted t 37 C for 24 h for bcteri nd 30 C for 48 h for fungi. The first dilution with no microbil growth ws recorded s MIC. Results nd Discussion Essentil oils composition The constituents of the essentil oils obtined from leves, root nd the seeds of D. dmvndic re presented in Tble I, where the compounds re listed in order of their elution from DB-1 column. The essentil oils nlyses resulted in identifiction of 16, 17 nd 20 compounds representing

3 F. Eftekhr et l. Oil Composition nd Antimicrobil Activity of Diploteni dmvndic 823 Tble I. Composition of essentil oils from leves, root nd seeds of Diploteni dmvndic. Compound RI %ofthe oils Lef Root Seed α-pinene Sbinene Myrcene α-phellndrene Crene p-cymene β-phellndrene (Z)-β-Ocimene (E)-β-Ocimene γ-terpinene Cmphenone Terpinolene llo-ocimene p-cymene-8-ol Estrgol Cmphenyl cette Sbinyl cette α-terpinyl cette Gernyl cette β-cryophyllene γ-elemene γ-curcumene Germcrene-D Elemicin Kessne Elemol Germcrene-B Spthulenol Dill piol Monoterpene hydrocrbons Oxygented monoterpenes Sesquiterpene hydrocrbons Oxygented sesquiterpenes Totl Compounds listed in order of their elution from DB-1 column. RI, retention index reltive to n-lknes (C 6 ÐC 24 ). 94.2%, 96.4% nd 95.1% of the totl oils, respectively. Ten compounds were common in ll three oils, of which (Z)-β-ocimene (21.6%), α-phellndrene (21.3%) nd terpinolene (20.0%) hd higher quntities in the lef oil compred to the root nd seed oils. Dill piol (30.1%) ws the mjor component of the root oil nd p-cymene (15.9%), γ-terpinene (16.2%) nd kessne (8.5%) were mostly found in the seed oil. β-phellndrene (11.2%) nd elemicin (2.8%) were two constituents found only in the lef oil wheres 6-cmphenone (9.2%) nd 6-cmphenyl cette (7.3%) were found exclusively in the root oil. The ltter two compounds were lso reported to be present in the root oil of D. cchrydifoli (Hrkis nd Slehy-Surmghy, 1987). Spthulenol (4.7%) ws one of the five mjor components found specificlly in the seed oil. Overll, monoterpene hydrocrbons were found to be the mjor compounds with 84.1%, 52.6% nd 66.5% in lef, root nd seed oils, respectively. Oxygented sesquiterpenes were found minly in root oil (40.0%) with dill piol (30.1%) s the min component. Sesquiterpene hydrocrbons (17.6%) were mostly found in seed oil with kessne (8.5%) s the second mjor compound. Biologicl ctivity All essentil oils showed ntibcteril ctivity by the disk diffusion ssy. However, the best results were obtined with the lef oil which ws ctive not only ginst the bcteril strins but lso produced good zones of inhibition ginst the fungl test orgnisms (Tble II). The most susceptible bcteri were: Bcillus subtilis nd Stphylococcus ureus (MIC vlues of 3.6 mg/ml) followed by Stphylococcus epidermidis nd Escherichi coli (MIC 7.2 mg/ml), nd Enterococcus feclis (MIC 14.3 mg/ml). Klebsiell pneumonie showed little susceptibility nd Pseudomons eruginos ws resistnt to ll essentil oils. There ws correltion between the MIC results with the disk sensitivity profile s shown in Tble II. With inhibition zones of 19Ð21 mm, the MIC vlues were 3.6 mg/ml. Inhibition zones of 15Ð19 mm corresponded with MIC vlues of 7.2 mg/ml, nd zones of 14 mm or less yielded higher MIC vlues (14.3Ð 28.6 mg/ml). The MIC for the lef oil ginst Scchromyces cerevisie with n inhibition zone of 25 mm ws 1.8 mg/ml. Overll, the lef oil hd the highest ctivity ginst ll microorgnisms used. We believe tht this is the first report on the ntimicrobil ctivity of D. dmvndic. Tble III shows the ntimicrobil ctivity of the five commercil oils tested. Among these, terpinolene hd the highest ntimicrobil ctivity ginst ll test orgnisms. The MIC vlues obtined for terpinolene were generlly lower thn the results obtined for the lef oil but were comprble for Bcillus subtilis, Stphylococcus ureus, Enterococcus feclis nd Escherichi coli. Antimicrobil ctivity of the other commercil oils vried from wek to cceptble. Terpinolene is monoterpene hydrocrbone which ws shown to hve ntifungl, ntibcteril nd insecticidl properties

4 824 F. Eftekhr et l. Oil Composition nd Antimicrobil Activity of Diploteni dmvndic Tble II. Antimicrobil ctivity of essentil oils from Diploteni dmvndic. Microorgnism Lef oil Root oil Seed oil Stndrd ntibiotics DD MIC b DD MIC DD MIC Ampicillin c Nysttine d Bcillus subtilis 20 ð ð ð ð ð ð ð 0.4 nt Enterococcus feclis 13 ð ð ð ð 0.6 Ð nt 11 ð 0.3 nt Stphylococcus ureus 19 ð ð ð ð ð ð ð 0.3 nt Stphylococcus epidermidis 17 ð ð ð ð ð ð ð 0.5 nt Escherichi coli 17 ð ð 0.2 Ð nt 12 ð ð ð 0.2 nt Klebsiell pneumonie 11 ð ð 0.4 Ð nt Ð nt Ð nt Pseudomons eruginos Ð nt Ð nt Ð nt 9.7 ð 0.2 nt Aspergillus niger 19 ð ð 0.4 Ð nt Ð nt nt 16 ð 0.4 Cndid lbicns 21 ð ð 0.3 Ð nt Ð nt nt 18 ð 0.5 Scchromyces cerevisie 25 ð ð 0.4 Ð nt Ð nt nt 18 ð 0.2 Vlues given s men ð stndrd devition of triplicte tests. Zone of inhibition includes dimeter of disc (6 mm). b Minimum inhibitory concentrtion vlues in mg/ml. c Tested t 10 µg/disc. d Tested t 30 µg/disc. Inctive (Ð); modertely ctive (7Ð13); ctive (> 14); nt, not tested. Tble III. Antimicrobil ctivity of the min compounds of the essentil oils from Diploteni dmvndic. Microorgnism Dill piol Ocimene Terpinolene γ-terpinene p-cymene DD MIC b DD MIC DD MIC DD MIC DD MIC B. subtilis 15 ð (32.4) ð ð (55.0) ð ð (26.4) ð ð (27.5) ð ð (27.9) ð 0.4 E. feclis 9 ð 0.1 >14.3 (> 64.3) ð ð 0.1 >15 (110.1) ð ð (52.8) ð ð (55.1) ð 0.2 Ð nt S. ureus 12 ð (64.3) ð ð (110.1) ð ð (26.4) ð ð 0.1 >15 (110.1) ð ð (55.9) ð 0.3 S. epidermidis 14 ð (32.4) ð ð (55.0) ð ð (13.2) ð ð (55.1) ð ð (55.9) ð 0.2 E. coli 10 ð 0.1 > 14.3 (> 64.3) ð ð 0.1 > 15 (> 110.1) ð ð (52.8) ð ð (55.1) ð ð (55.9) ð 0.4 K. pneumonie Ð nt Ð nt 17 ð (52.8) ð 0.4 Ð nt 11 ð 0.2 > 15 (> 55.9) ð 0.3 P. eruginos Ð nt Ð nt Ð nt Ð nt Ð nt A. niger 11ð 0.1 > 14.3 (> 64.3) ð 0.6 Ð nt 25 ð (13.2) ð ð 0.3 > 15 (> 110.1) ð 0.4 Ð nt C. lbicns 12 ð (64.3) ð 0.5 Ð nt 32 ð (6.6) ð ð (55.1) ð ð 0.2 > 15 (> 55.9) ð 0.3 S. cerevisie 10 ð 0.1 > 14.3 (> 64.3) ð 0.4 Ð nt 30 ð (6.6) ð ð 0.4 > 15 (> 110.1) ð ð 0.1 > 15 ( > 15 (55.9) ð 0.4 Vlues given s men ð stndrd devition of triplicte tests. Zone of inhibition includes dimeter of disc (6 mm). b Minimum inhibitory concentrtion vlues in mg/ml (millimolr). Min compounds tested t 10 µl/disc on bcteri nd 30 µl/disc on fungi. Inctive (Ð); modertely ctive (7Ð13); ctive (> 14); nt, not tested.

5 F. Eftekhr et l. Oil Composition nd Antimicrobil Activity of Diploteni dmvndic 825 (Froog et l., 2002; Msten, 1999). The ntifungl ctivity hs been observed ginst vrious pthogens s well s the spores of Diplodi pine. We lso found tht terpinolene hd good ntifungl ctivity (Tble III). Antibcteril properties of terpinolene hve been reported including ctivity ginst Prpionibcterium which cuses cne. Wek ntibcteril ctivity of α-pinene, sbinene nd p- cymene hs lso been reported (Sokmen et l., 2003; Dormn nd Dens, 2000; Bougtsos et l., 2003). The ntimicrobil ctivity of the essentil oils from D. dmvndic my well be due to the presence of synergy between terpinolene nd other constituents of the oil with vrious degrees of ntimicrobil ctivity. Considering the fct tht the lef oil contined 20% terpinolene, the results obtined my be quite significnt. Adms R. P. (1995), Identifiction of essentil oils com- Lis-Blchin M. nd Dens S. G. (1997), Bioctivity of ponents by gs chromtogrphy/qudruple mss selected plnt essentil oils ginst Listeri monocytospectroscopy. Allured Publishing Co., Crol Strem, genese. J. Appl. Bcteriol. 82, 759Ð762. Illinois, USA. Msten S. (1999), Terpinolene. Review of Toxicologicl Aynechi A., Amin G., Slehy Surmghy M. H., nd J- Literture. Reserch Tringle Prk, Durhm, North ryny F. (1999), Furnocoumrins of Diploteni Crolin, USA. dmvndic. Phrmceut. Biol. 137, 161Ð162. Ntionl Committee for Clinicl Lbortory Stndrds Bougtsos C., Meyer J. J., Mgitis P., Vgis C., nd (NCCLS) (1997), Performnce Stndrds for Antimi- Chinou I. B. (2003), Composition nd ntimicrobil crobil Disk Susceptibility Test (6 th ed.). Approved ctivity of the essentil oils of Helichysum krussii Stndrd. M100-A6. Wyne, Pennsylvni, USA. Sch. Biop. nd H. rugulosum Less. from South Afric. Ntionl Committee for Clinicl Lbortory Stndrd Flvour Frgr. J. 18, 48Ð51. (NCCLS) (1999), Performnce Stndrds for Antimi- Dvies N. N. (1987), Gs chromtogrphic retention in- crobil Susceptibility Testing. 9 th Interntionl Supdices of monoterpenes nd sesquiterpenes on methyl plement. M100-S9. Wyne, Pennsylvni, USA. silicone nd DB-wx 20M phses. J. Chromtogr. 503, Pttnik S., Subrmnym V. R., Bpji M., nd Kole 1Ð24. C. R. (1997), Antibcteril nd ntifungl ctivity of Dormn H. J. D. nd Dens S. G. (2000), Antimicrobil romtic constituents of essentil oils. Microbios 89, gents from plnts: Antibcteril ctivity of plnt vol- 39Ð46. tile oils. J. Appl. Microbiol. 88, 308Ð316. Srdri S., Amin G., Sekhon A., Micetich R. G., nd Froog A., Chouhry M. I., Rhmn A., Thr S., Bser Dneshtlb M. (2000), Antifungl ctivity of Diplo- K. H. C., nd Demirci F. (2002), Detoxifiction of ter- teni dmvndic. Phrm. Phrmcol. Commun. 6, pinolene by plnt pthogenic fungus Botrytis ciner. 455Ð458. Z. Nturforsch. 57c, 863Ð866. Shibmoto T. (1987), Retention indices in essentil oil Hrkis K. J. nd Slehy-Surmghy M. H. (1987), Vol- nlysis. In: Cpillry Gs Chromtogrphy in Essentiles from the root of Diploteni cchrydifoli, the til Oil Anlysis (Sndr P. nd Bicchi C., eds). first nturl source of 6-cmphenone. J. Nt. Prod. Huethig Verlg, New York. (Lloydi) 50, 991Ð994. Sokmen A., Vrder-Unlu G., Polissiou M., Dferer D., Hedge I. C., Lmond J. M., nd Rechinger K. H. (1987), Sokmen M., nd Donmez E. (2003), Antimicrobil c- Umbellifere. In: Flor Irnic, Vol. 162 (Rechinger tivity of essentil oil nd methnol extrcts of Achille K. H., ed.). Akdemische Druck- u. Verlgsnstlt sintenisii (Astercee). Phytother. Res. 17, 1005Ð Grz, Austri Knobloch K., Puli A., Iberi B., Wegnd H., nd Weis Yss N., Akhni H., Aghmdi M., nd Slimin M. N. (1989), Antibcteril nd ntifungl properties of (2003), Essentil oil from two endemic species of essentil oil components. J. Essent. Oil Res. 1, 119Ð Apicee from Irn. Z. Nturforsch. 58c, 459Ð