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Antibacterial potential of the essential oil from Sideritis montana L. (Lamiaceae)

Authors:
  • Faculty of Medicine, University of Niš, 18000 Niš, Serbia

Abstract

The antibacterial potential of essential oil from Serbian Sideritis montana L. (Lamiaceae) on different bacteria was investigated. The essential oil was obtained from air dried aerial parts of the plant with 0.03% (w/w) yield by hydrodistillation for 4 h using a Clevenger-type apparatus. The essential oil analyses were performed simultaneously by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) systems. Dominant compounds class is sesquiterpene hydrocarbons. The main constituents of oil were germacrene D (37,9%) and transgeraniol (26,1%). The antibacterial activity of the essential oil was tested towards 4 different bacteria, laboratory control strain obtained from the American Type Culture Collection (ATCC). Gram negative bacteria were represented by Escherichia coli ATCC 13706, Pseudomonas aeruginosa ATCC 27853, Salmonella enteritidis ATCC 13076 while researched Gram positive strain was Staphylococcus aureus ATCC 29213. Broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Essential oil has been found to have antibacterial activity against all tested bacteria, with a MIC values ranging from 0.03 to 0.06 mu l/ml and MBC values from 0.06 to 0.12 mu l/ml. Reference antibiotic tetracycline was active in concentrations between 0.025 to 0.1 mu g/ml. Essential oil from Serbian S. montana has high antibacterial potential which should be studied further.
541
Antibakterijski potencijal etarskog ulja
S
ideritis montana L. (Lamiaceae)
Dragoljub L. Miladinović1, Budimir S. Ilić1, Tatjana M. Mihajilov-Krstev2, Nikola D. Nikolić3,
Violeta N. Milosavljević4, Dejan M. Nikolić5
1Univerzitet u Nišu, Medicinski fakultet, Odsek za farmaciju, Niš, Srbija
2Univerzitet u Nišu, Prirodno–matematički fakultet, Departman za biologiju i ekologiju, Niš, Srbija
3Univerzitet u Nišu, Laboratorija za geohemiju, kosmohemiju i astrohemiju, Niš, Srbija
4Osnovna škola „Desanka Maksimović“, Čokot, Srbija
5Odsek za sanitarnu hemiju, Institut javnog zdravlja, Niš, Srbija
Izvod
U radu su prikazani rezultati proučavanja hemijskog sastava i antibakterijskog potencijala
etarskog ulja Sideritis montana L. (Lamiaceae) iz Srbije. Hemijska analiza etarskog ulja je
izvršena primenom GC i GC-MS metoda, dok je antibakterijska aktivnost ispitivana
mikrodilucionom metodom na četiri referentna bakterijska soja: Escherichia coli ATCC
13706, Pseudomonas aeruginosa ATCC 27853, Salmonella enteritidis ATCC 13076 i
Staphylococcus aureus ATCC 29213. Najzastupljenija klasa jedinjenja u ispitivanom uzorku
etarskog ulja su ugljovodonični seskviterpeni. Dominantne komponente etarskog ulja su
germakren D (37,9%) i trans-geraniol (26,1%). Etarsko ulje je delovalo na sve testirane
sojeve inhibitorno u intervalu od 0,03–0,06 μl/ml i baktericidno u intervalu od 0,06–0,12
μl/ml.
K
ljučne reč
i
: Sideritis montana, etarsko ulje, antibakterijska aktivnost.
NAUČNI RAD
UDK 547.913:543.544:615.281:58
Hem. Ind. 66 (4) 541–545 (2012)
doi: 10.2298/HEMIND111003001M
Dostupno na Internetu sa adrese časopisa: http://www.ache.org.rs/HI/
Lečenje infektivnih bolesti u svetu se poslednjih
decenija sve više komplikuje, zbog razvijene rezistencije
bakterija na sintetičke antibiotike [1]. Poznati su u isto-
riji slučajevi rezistetnosti Escherichia coli na treću gene-
raciju cefalosporina, zatim pojava vankomicin-rezistent-
nih Staphylococcus aureus i multirezistentne Pseudo-
monas aeruginosa [2]. Zbog toga postoji stalna potreba
za novim antibakterijskim agensima. Poznato je da ter-
penoidi, konstituenti etarskih ulja delimično ili potpuno
inhibiraju rast bakterija [3].
Biljne vrste roda Sideritis su u upotrebi u mnogim
zemljama kao fitopreparati [4]. Vekovima se koriste
zbog antiinflamatornih [5], antimikrobnih [6] i gastro-
protektivnih svojstava [7]. Najnovija istraživanja uka-
zuju da neka jedinjenja izolovana iz vrsta roda Sideritis
ispoljavaju anti-HIV i antiproliferativne aktivnosti [8].
Rod Sideritis (Lamiaceae) obuhvata 140 vrsta, koje
su rasprostranjene uglavnom na Mediteranu i nekim
regionima Azije. U flori Srbije je opisana samo vrsta
Sideritis montana [9].
Pregledom dostupnih literaturnih podataka, nismo
našli potvrdu da je do sada ispitivana antibakterijska
aktivnost etarskog ulja S. montana. Stoga je etarsko
ulje ove vrste ušlo u fokus našeg naučnog intereso-
vanja.
Prepiska: D.L. Miladinović, Odsek za farmaciju, Medicinski fakultet,
Univerzitet u Nišu, Bulevar dr Zorana Đinđića 81, 18000 Niš, Srbija.
E-pošta: dragoljubm@gmail.com
Rad primljen: 3. oktobar, 2011
Rad prihvaćen: 16. januar, 2012
EKSPERIMENTALNI DEO
Biljni materijal
Nadzemni delovi S. montana sakupljeni su u junu
2011. godine u fazi cvetanja u selu Kravlje, jugoistočna
Srbija. Taksonomsku pripadnost populacije je determi-
nisao prof. dr Novica Ranđelović sa Odseka za biologiju
i ekologiju, Prirodno–matematičkog fakulteta u Nišu,
gde je herbarijumski uzorak deponovan.
Izolovanje etarskog ulja
Sprašena herba podvrgnuta je procesu hidrodestila-
cije na Clavenger aparatu u trajanju od 4 sata. Izo-
lovano etarsko ulje je osušeno anhidrovanim natrijum-
sulfatom i čuvano na temperaturi od 4 °C.
Gasna hromatografija (GC)
GC analiza etarskog ulja je urađena na gasnom hro-
matografu HP-5890 Series II, opremljenim split-splitless
injektorom, kapilarnom kolonom sa HP-5MS stacionar-
nom fazom (30 m×0,25 mm; debljina filma 0,25 µm),
helijumom kao nosećim gasom (1 ml/min) i plameno-
-jonizujućim detektorom (FID). Temperatura injektora
iznosila je 250, a detektora 280 °C, dok je temperatura
kolone linearno povećavana od 50 do 250 °C (3 °C/min).
Gasna hromatografija/masena spektrometrija (GC–MS)
GC–MS analiza je urađena na Agilent Technologies
aparatu, model GS 6890N pri 70 eV, sa detektorom tipa
MSD 5975C, pod istim gasno–hromatografskim uslovima.
D.L. MILADINOVIĆ i sar.: ETARSKA ULJA
Sideritis montana
L. Hem. ind. 66 (4) 541–545 (2012)
542
Identifikacija komponenti
Identifikacija komponenti ulja vršena je maseno-
spektrometrijski, poređenjem retencionih vremena i
masenih spektara komponenti sa referentnim supstan-
cama i/ili jedinjenjima iz raspoložive baze podataka
(Wiley 275, NIST/NBS). Eksperimentalno dobivene vre-
dnosti Kovačevih indeksa, IK, primenom programa
AMDIS (ver. 2.64), u saglasnosti su sa literaturnm po-
dacima [10].
Bakterijski sojevi
Antibakterijska aktivnost etarskog ulja je ispitivana
na četiri laboratorijska referentna soja iz American
Type Culture Collection (ATCC). Iz grupe Gram-nega-
tivnih bakterija: Escherichia coli ATCC 13706, Pseudo-
monas aeruginosa ATCC 27853 i Salmonella enteritidis
ATCC 13076. Iz grupe Gram-pozitivnih bakterija: Sta-
phylococcus aureus ATCC 29213. Od prekonoćnih kul-
tura bakterijskih sojeva su pripremljene suspenzije tur-
biditeta 0.5 McFarlanda (gustine 107–108 CFU/ml),
zavisno od soja – propisano od strane Clinical and Labo-
ratory Standards Institute, CLSI [11].
Mikrodiluciona metoda
Upotrebom mikrodilucione metode određena je mi-
nimalna inhibitorna koncentracija (MIC) i minimalna
baktericidna koncentracija etarskog ulja (MBC) [11]. U
etanolu (70%) napravljene su serije duplih razblaženja
ulja i zatim je unešeno u mikrotitar ploče sa 96 udu-
bljenja, po 10 μl u 90 μl inokulisane tečne podloge
(Mueller–Hinton bujon). Finalna zapremina u svakom
udubljenju je bila 100 μl, finalna gustina bakterijskih će-
lija 106 CFU/ml, a koncentracije proučavanog ulja su
bile u opsegu 0,025–50,0 μl/ml. Mikrotitar ploče su
inkubirane 24 h na 37 °C. Svi eksperimeni su urađeni u
tri ponavljanja. Korišćene su dve kontrole, hranljivi me-
dijum sa rastvaračem etanolom (negativna kontrola) i
antibiotik tetraciklin (pozitivna kontrola). Bakterijski
rast je detektovan dodavanjem po 20 μl 0,5% vodenog
rastvora trifenil-tetrazolium-hlorida (TTC). MIC je defi-
nisana kao najniža koncentracija etarskog ulja pri kojoj
nema vidljivog rasta bakterija, crveno obojenih kolonija
na dnu udubljenja mikrotitar ploče nakon dodavanja
TTC-a. Da bi se odredila MBC, sadržaj udubljenja u ko-
jima nije bilo vidljivog rasta je prenešen na petri ploče
sa Mueller–Hinton agarom (MHA) i inkubiran 24 h na
37 °C, nakon čega su brojane porasle kolonije. MBC je
ona koncentracija ulja pri kojoj je ubijeno 99,9% od
početnog broja bakterijskih ćelija.
Statistička obrada podataka
Rezultati su statistički obrađeni analizom varijanse
(ANOVA) sa sigurnošću od 95% (p0,05), primenom
programa SPSS Statistics.
REZULTATI I DISKUSIJA
Prinos etarskog ulja S. montana iznosio je 0,03%
(w/w), što odgavara podacima u literaturi za biljnu
vrstu S. montana podvrsta montana iz Turske [12]. Na
osnovu GC i GC–MS analize etarskog ulja identifikovano
je 8 komponenata, koje predstavljaju 100% ukupno
detektovanih komponenata (tabela 1).
Tabela 1. Hemijski sastav etarskog ulja biljne vrste S. montana;
RT – retenciono vreme, min; IK – Kovačev indeks (eksperi-
mentalno utvrđeni retencioni indeksi koiniciranjem sa homo-
logom serijom n-alkana)
Table 1. Essential oil composition of S. montana
Komponenta RT / min IK Sadržaj, %
1-Okten-3-ol 9,997 969 2,8
trans-Geraniol 19,748 1228 26,1
Timol 21,108 1270 10,3
trans-Geranil acetat 23,998 1363 7,6
β
-Kariofilen 25,339 1414 8,1
(E)-
β
-Farnezen 26,366 1440 3,0
Germakren D 27,312 1472 37,9
Biciklogermakren 27,712 1497 4,2
Ukupno 100
Seskviterpeni su dominantna klasa jedinjenja etar-
skog ulja sa glavnom komponentom germakrenom D
(37,9%). Sledeća klasa jedinjenja po zastupljenosti su
oksidovani monoterpeni, sa geraniolom (26,1%), kao
dominantnom komponentom. Timol (10,3%), predstav-
nik fenolnih jedinjenja, treće je jedinjenje po zastup-
ljenosti. Ovakav sastav etarskog ulja S. montane iz Sr-
bije je sličan ulju iste biljne vrste iz Bugarske [13], dok u
etarskom ulju S. montana iz Irana dominantna kompo-
nenta je geraniol [14]. Na osnovu višegodišnjih prouča-
vanja hemijskog sastava etarskog ulja roda Sideritis u
Turskoj, Kirimer i sar. [12] klasifikovali su ulja u šest
glavnih grupa: ulja bogata ugljovodoničnim monoter-
penima, ulja bogata oksidovanim monoterpenima, ulja
bogata ugljovodoničnim seskviterpenima, ulja bogata
oksidovanim seskviterpenima, ulja bogata diterpenima
i ostala ulja. Od ukupnog broja vrsta roda Sideritis u
Turskoj, 57% pripada grupi ulja bogatih ugljovodo-
ničnim monoterpenima. Dve podvrste S. montana se
svrstavaju u grupu ulja bogatih ugljovodoničnim seskvi-
terpenima, u koju se može uvrstiti i etarsko ulje S. mon-
tana iz Srbije.
U tabeli 2 prikazani su rezultati antibakterijske ak-
tivnosti etarskog ulja. Rezultati pokazuju izraženu anti-
bakterijsku aktivnost ulja na sve proučavane bakterijske
sojeve. Etarsko ulje je delovalo na sve testirane sojeve
inhibitorno u intervalu 0,03–0,06 μl/ml i baktericidno u
opsegu 0,06–0,12 μl/ml. Referentni antibiotik tetraci-
klin je delovao inhibitorno u intervalu 0,025–0,10 μl/ml
i baktericidno u opsegu 0,025–0,1 μl/ml.
D.L. MILADINOVIĆ i sar.: ETARSKA ULJA
Sideritis montana
L. Hem. ind. 66 (4) 541–545 (2012)
543
U literaturi ne postoje podaci o antimikrobnom de-
lovanju etarskog ulja S. montana. Iscan i sar. [15] mik-
rodilucionom metodom su proučavali antibakterijsko
delovanje etarskih ulja biljnih vrsta Sideritis cilicica i Si-
deritis bilgerana na sojevima Gram-negativnih bakterija
(Salmonella typhimurium i Escherichia coli) i Gram-po-
zitivnih bakterija (Staphylococcus aureus, Bacillus ce-
reus i Staphylococcus epidermidis). Zabeležene su MIC
vrednosti u opsegu 0,125–0,50 μg/ml. Pretpostavljeno
je da su nosioci ove aktivnosti
α
-pinen i
β
-pinen, domi-
nantni konstituenti etarskih ulja obe biljne vrste. U stu-
diji pročavanja antibakterijskog delovanja etarskog ulja
Sideritis italica utvrđene su MBC vrednosti u intervalu
62,6–125 μg/ml. Takođe je zabeležena veća antibak-
terijska aktivnost na Gram-negativne bakterije [7].
Tabela 2. Antibakterijska aktivnost (μl/ml) etarskog ulja biljne
vrste S. montana i tetraciklina (μg/ml)
Table 2. Antibacterial activity (μl/ml) of S. montana essential
oil and tetracycline (μg/ml)
Bakterijski soj S. montana L. Tetraciklin
MIC MBC MIC MBC
E. coli ATCC 13706 0,060 0,120 0,100 0,100
P. aeruginosa ATCC 27853 0,030 0,120 0,025 0,025
S. enteritidis ATCC 13076 0,030 0,060 0,025 0,025
S. aureus ATCC 29213 0,060 0,060 0,025 0,025
Mehanizam delovanja terpenoida se intenzivno pro-
učava, ali još uvek u potpunosti nije objašnjena veza
između njihove antibiotske aktivnosti i hemijske struk-
ture. Mnoga hemijska jedinjenja u sastavu etarskih ulja
reaguju sa proteinima ćelijske membrane. Dokazano je
da ciklični ugljovodonici reaguju sa ATPazom, enzimom
smeštenim na ćelijskoj membrani koji je okružen mole-
kulima lipida. Takođe ugljovodonici mogu uticati na dis-
torziju proteina u lipidnoj membrani što može dovesti
do direktne interakcije lipofilne komponente sa hidro-
fobnim delom proteina [16]. Najjače inhibitorno dejs-
tvo na rast bakterija ispoljavaju fenoli i alkoholi, zatim
slede aldehidi i ketoni, dok je inhibitorna aktivnost
ugljovodoničnih monoterpena najslabija. Slobodna hid-
roksilna grupa fenola i alkohola je uzrok njihove aktiv-
nosti. Takođe je potvrđeno da terpenoidi imaju različitu
antiseptičku moć, zavisno od njihove rastvorljivosti u
vodi. Međutim, uočene su i neke anomalije u vezi ti-
mola i karvakrola koji i pored slabe rastvorljivosti u vodi
ispoljavaju izrazito antiseptičko dejstvo [3]. Mehanizam
delovanja timola se zasniva na povećanju permeabil-
nosti ćelijske membrane. Molekul timola može da ra-
zori spoljašnju membranu Gram-negativnih bakterija,
oslobađajući lipopolisaharide i povećavajući propust-
ljivost ćelijske membrane [17].
Bez obzira na najveću zastupljenost ugljovodoničnih
seskviterpena, geraniol i timol, sa zastupljenošću od
36,4% u etarskom ulju S. montana su nosioci antibak-
terijske aktivnosti. U studiji koja se bavila proučava-
njem odnosa hemijskog sastava i antibakterijske aktiv-
nosti etarskog ulja Sideritis sipylea izveden je sličan
zaključak [18]. Eksperimentalno utvrđene vrednosti
MIC i MBC za sve testirane bakterije bile su niže u
odnosu na literaturne podatke. Za razliku od Gram-po-
zitivnih bakterija, Gram-negativne bakterije se odlikuju
većom rezistentnošću prema antisepticima i antibio-
ticima upravo zbog spoljašnje membrane koja deluje
kao barijera za ulazak antibakterijskih agenasa [19]. U
našem istraživanju odabrane Gram-negativne bakterije
nisu ispoljile značajniju rezistentnost. Ovo je naročito
značajno kada se radi o soju P. aeruginosa, zbog hidro-
filnog karaktera površine njegovih ćelija, većina etar-
skih ulja pokazuje slabiju aktivnost. Evidentna je visoka
osetljivost Escherichia coli, za koju je zabeležena znatno
niža MIC vrednost etarskog ulja u poređenju sa tetra-
ciklinom (tabela 2).
ZAKLJUČAK
Antibiotska rezistencija humanih patogena na broj-
ne komercijalne antibiotike je realnost. Stoga je neop-
hodno razviti prirodan i bezbedan način za kontrolu in-
fektivnih bolesti, uzrokovanih bakterijama. Cilj ovog
rada bio je ispitivanje hemijskog sastava etarskog ulja S.
montana i njegovog antibakterijskog potencijala. Domi-
nantna klasa jedinjenja etarskog ulja su ugljovodonični
seskviterpeni sa glavnom komponentom germakrenom
D (37,9%). Geraniol i timol, nosioci anbakterijske aktiv-
nosti, zastupljeni su 36,4%. Relativno niske koncen-
tracije etarskog ulja su ispoljile antimikrobnu aktivnost
prema svim ispitanim bakterijama. Rast Escherichia coli
je značajnije inhibiran etarskim uljem u poređenju sa
referentnim antibiotikom. Na osnovu iznetog se može
zaključiti da etarsko ulje S. montana poseduje visok
antibakterijski potencijal i treba ga dalje proučavati.
Zahvalnica
Ovo istraživanje je realizovano u okviru projekta
Ministarstva za prosvetu i nauku Republike Srbije (br.
171025).
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D.L. MILADINOVIĆ i sar.: ETARSKA ULJA
Sideritis montana
L. Hem. ind. 66 (4) 541–545 (2012)
545
SUMMARY
ANTIBACTERIAL POTENTIAL OF THE ESSENTIAL OIL FROM Sideritis montana L. (Lamiaceae)
Dragoljub L. Miladinović1, Budimir S. Ilić1, Tatjana M. Mihajilov-Krstev2, Nikola D. Nikolić3, Violeta N. Milosavljević4,
Dejan M. Nikolić5
1University of Niš, Faculty of Medicine, Department of Pharmacy, Niš, Serbia
2University of Niš, Faculty of Science and Mathematics, Department of Biology and Ecology, Niš, Serbia
3University of Niš, Laboratory for Geochemistry, Cosmochemistry and Astrochemistry, Niš, Serbia
4Elementary school "Desanka Maksimović", Čokot, Serbia
5Department of Sanitary Chemistry, Institute of Public Health, Niš, Serbia
(Scientific paper)
The antibacterial potential of essential oil from Serbian Sideritis montana L.
(Lamiaceae) on different bacteria was investigated. The essential oil was obtained
from air dried aerial parts of the plant with 0.03% (w/w) yield by hydrodistillation
for 4 h using a Clevenger-type apparatus. The essential oil analyses were per-
formed simultaneously by gas chromatography (GC) and gas chromatography-
mass spectrometry (GC-MS) systems. Dominant compounds class is sesquiterpene
hydrocarbons. The main constituents of oil were germacrene D (37,9%) and trans-
geraniol (26,1%). The antibacterial activity of the essential oil was tested towards
4 different bacteria, laboratory control strain obtained from the American Type
Culture Collection (ATCC). Gram negative bacteria were represented by Escheri-
chia coli ATCC 13706, Pseudomonas aeruginosa ATCC 27853, Salmonella enteri-
tidis ATCC 13076 while researched Gram positive strain was Staphylococcus au-
reus ATCC 29213. Broth microdilution method was used to determine the mini-
mum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC). Essential oil has been found to have antibacterial activity against all tested
bacteria, with a MIC values ranging from 0.03 to 0.06 μl/ml and MBC values from
0.06 to 0.12 μl/ml. Reference antibiotic tetracycline was active in concentrations
between 0.025 to 0.1 μg/ml. Essential oil from Serbian S. montana has high anti-
bacterial potential which should be studied further.
Keywords: Sideritis montana Essential
oil Antibacterial activity
... Considering that a significantly lower amount of bicyclogermacrene was detected in the same sample compared to S. montana from Mosor (S. montana M), ledene may have been formed during the drying and storage period from the aforementioned bicyclogermacrene. Our results considering the major VOCs of investigated populations of S. montana are in accordance with the results of previous studies from Bulgaria [22], Turkey [18], Serbia [25] and Italy [23], in which germacrene D predominated in the chemical composition of EOs isolated from aerial parts by hydrodistillation, usually being followed by bicyclogermacrene. Different major compounds were reported for S. montana from Iran, in which geraniol particularly predominated in the EO obtained by hydrodistillation from the flowering spikes of the plant [26], while it was not detected in our samples of S. montana aerial parts, as well as for S. montana subsp. ...
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... Therefore, a quest for new and sustainable antibiotics or other treatment therapy is a necessity. Many researches have proved substantial information on antibacterial activity of natural products on resistant microbial strains (Bakkali et al., 2008;Miladinović et al., 2012). Tremendous increase in antibiotic resistance due to ESBL production in gram negative bacteria created a problem (Duman, et al., 2010;Duman, et al., 2014;Toner, et al., 2016). ...
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