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ANTIMUTAGENIC EFFECT OF ESSENTIAL OIL OF SAGE(SALVIA OFFICINALIS L.)

AND ITS FRACTIONS AGAINST UV-INDUCED MUTATIONS

IN BACTERIAL AND YEAST CELLS

Arch. Biol. Sci., Belgrade, 57 (3), 163-172, 2005.

JELENA KNEEVI-VUKEVI1, BRANKA VUKOVI-GAI, TATJANA STEVI1, JASNA STANOJEVI,BILJANA NIKOLI, andDRAGA SIMI

Faculty of Biology, University of Belgrade, 11000 Belgrade,Serbia and Montenegro1Dr. Josif Pani Institute for Medicinal Plant Research, 11000 Belgrade, Serbia and Montenegro

Abstract-The inhibition of spontaneous and UV-indued mutations by essential oil (EO) of sage (Salvia ofcinalis L.) andits frations F1-F5 ontaining different proportions of mono- and sesquiterpenes was studied with the Salmonella/miro-some,E. coliK12, and S. cerevisiaeD7 reversion assays. The EO, F1, and F2 exhibited antimutageni potential againstUV-indued mutations in all tests. Frations F3 and F4 produed a toxi, mutageni, or antimutageni response, depend-ing on the test organism used. Redution of spontaneous and UV-indued mutations by F5 was deteted only in permeablestrains ofE. coli. The obtained results demonstrate antimutageni ativity of volatile sage terpenes and reommend themfor further antimutagenesis and antiarinogenesis studies.

UDc 635.74:665.5:577.21

INTRODUcTION

Mutations are impliated in many important human dis-eases, inluding atheroslerosis, autoimmune and neuro-degenerative diseases, some types of diabetes and aner.There is inreasing evidene that many plant extrats andtheir omponents an at as inhibitors of mutagenesis andarinogenesis (cr a i g , 1999; We i s b u r g e r , 2001).chemoprevention of mutation-related diseases and aneris an important researh eld, and the dietary use of an -timutagens and antiarinogens has been proposed as the

most promising approah to protetion of human health(W a t e n b e r g , 1985; H a y a t s u et al.1988; Fe r g u -s o n , 1994).

For many years our researh efforts have been fo-used on detetion of antimutageni properties of medii-nal and aromati plants of our region. For that purposewe designed and validated anEscherichia coliK12 assaysystem and used it along with standard mutageniity testsreommended by OEcD, the Salmonella/mirosome mu-tation assay and the Saccharomyceserevisiae D7 muta-tion assay. Results of sreening indiated that terpenoidfrations of wild and ultivated sage (Salvia ofcinalis

L.) possess antimutageni potential against mutations in-dued by UV-c and ethidium bromide in E. coliand S.typhimurium (Vukov i -Ga i and S imi , 1993 ;S i m i et al.1994,1997; M i t i et al.2001). Interest-ingly, between differently prepared extrats, only the fra-tions from ultivated sage with high ontent of low-mo-leular-weight terpenes inhibited UV-indued mutations.The antimutageni effet was lost when the same plantwas steam distilled before extration and frationation( S i m i et al.1994, 1997, 1998). The obtained data ledus to hypothesize that volatile terpenes from ultivated

sage possess antimutageni potential. An inhibitory ef-fet of sage essential oil on hromosome aberrations in-dued by mitomyin c in mie was reently reported byV u j o e v i a nd B l a g o j e v i (2004).

To verify further the proposed antimutageni poten-tial of volatile terpenes from sage we investigated the in-hibitory potential of essential oil of ultivated sage and itsfrations ontaining different proportions of mono- andsesquiterpenes on UV-indued mutations. The study wasonduted with two standard mutageniity tests, namelythe Salmonella/mirosome and S. cerevisiaeD7 reversionassays, as well as with our E. coliK12 reversion assay,

163

Key words: Sage, essential oil, UV-irradiation, antimutagenesis, Salmonella/mirosome,E. coli,S.cerevisiae


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whih was used in a previous study of ours on sage.

MATERIAL AND METHODS

Bacterial strains

Salmonella typhimurium TA102 (hisG428/pAQ1rfa/pKM101) and Escherichia coliK12 SY252 (argE3)and IB103 (argE3mutS215::Tn10) were used in prokary-oti mutageniity and antimutageniity assays (M a r o nand Ames, 1983; Simi et al. 1997). Permeable E.

coli strains IB112 and IB113 were onstruted for thiswork by seleting spontaneous mutants of SY252 andIB103 resistant to oliphage T7 (M c o y et al.1985).Suseptibility to large moleules was asertained by thedemonstration of the inreased sensitivity to rystal violet(Ma r o n and A m e s , 1983). The S. cerevisiaeD7 dip-loid strain ade2-40/119 trp5-12/27 ilv1-92/92 was usedin eukaryoti mutageniity and antimutageniity assays( Z i m m e r m a n n et al.1975). Fresh overnight ulturesof all tester strains, to whih glyerol was added as a ryo-

protetive agent, were stored at -20c. The strains were

JELENA KNEEVI-VUKEVI et al.164

Table 1. composition of essential oil of sage and its frations


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routinely heked to onrm geneti features.

Media and growth conditions

The bateria were grown in LB medium (5 g Nacl,10 g bato tryptone, 5 g yeast extrat, 1 L distilled water)at 37oc with aeration. Saccharomyces. cerevisiaeD7 wasgrown in YPD medium (10 g yeast extrat, 20 g bato-

peptone, 20 g D-gluose, 1 L distilled water) at 30oc withaeration. All media for the S. typhimuriumreversion as-say were as desribed by M a r o n and A m e s (1983).The Semi-enrihed minimal medium (SEM) for the E.coli K12 reversion assay was a minimal agar medium

supplemented with 3% (v/v) NB (N i k o l i et al.2004).The seletive c medium (4 g yeast nitrogen base withoutamino aids, 10 mg tryptophan, 20 g D-gluose, 15 g agar,1 L distilled water) was used in the S. cerevisiaereversionassay.

The S9 fration was isolated from the liver of albinoWister male rats (170-180 g) indued with pheno-barbi-tal/-naphtho avone (O n g et al.,1980). The S9 mix-ture ontained 4% (v/v) S9 fration, 33 mM Kcl, 8 mMMgcl

2, 5 mM gluose-6-phosphate, and 4 mM NADP in

0.1 M phosphate buffer (pH 7.4).

Preparation of essential oil of sage (Salvia ofcinalis L.)

and its fractions

Sage (Salvia ofcinalis L.) was ultivated by theDr. Josif Pani Institute for Mediinal Plant Researhin Panevo. The essential oil was prepared aording toPh. Jug. IV by distillation of the dried aerial part (Salviaeherba) in a 2 - m3 steam distiller (Hromil) for 2 hours ata pressure of 3-4 bars and temperature of 135-145oc. Theessential oil was frationated by vauum retiation on alow-resolution olumn to yield ve frations, designatedF1-F5 (B r k i et al.1999).

The omposition of essential oil and frationswas determined using analytial Gc/FID and Gc/MStehniques and the Wiley/NBS library of mass spetra( M a r i n k o v i et al.2002). The quality of essential oilmeets the standards of Ph. Jug. IV and ISO9909. Essentialoil and frations were stored at 4c and dissolved in 98%ethanol (1/10) just before use.

Ultraviolet irradiation

Ultraviolet irradiation was arried out with a ger-

miidal lamp (camag) having maximum output at 254nm. Dose rates were measured with the Latarjet dosim-eter (L a t a r j e t et al.1953). cell suspensions in 0.01 MMgSO

4were irradiated in glass Petri dishes at a thikness

of less than 1 mm. cell suspensions were kept in the darkto prevent photoreativation.

Detection of mutagenic and antimutagenic potential in

S. typhimurium

The overnight ulture of S. typhimurium TA102strain was washed by entrifugation, resuspended in thesame volume of 0.01 M MgSO

4, and UV-irradiated. The

UV dose was 24 J/m2. Samples (0.1 mL) of un-irradiatedand UV-irradiated ells were added to 2 mL of molten topagar with and without the S9 mixture (0.3 mL), mixed,and poured in dupliates onto minimal gluose agar plateswith different onentrations of essential oil or frations.Ethanol was used as a negative ontrol. After inubationat 37c for 48 h, the number of His+revertants was deter-mined and the presene of the baterial bakground lawnon all plates was inspeted.

Detection of mutagenic and antimutagenic potential in

E. coli and S. cerevisiae

Overnight ultures of E. coli strains SY252 andIB112 (wild type) and IB103 and IB113 (mutS) werewashed by entrifugation and resuspended in the samevolume of 0.01 M MgSO

4. cell suspensions of SY252

and IB112 strains were UV-irradiated with a dose 28 J/m2. Samples (0.1 mL) of un-irradiated and UV-irradiatedells, appropriately diluted for determination of ell sur-vival and undiluted for determination of Arg+revertants,were spread in dupliates onto 3% SEM plates with dif-ferent onentrations of essential oil or frations and inu-

bated at 37c for 48 h. Mutagenesis and antimutagenesisassay with S. cerevisiaeD7 was performed in the sameway, exept that an exponential ulture ontaining about3 x 107ells/mL was used. The UV dose was 130 J/m2.cell survival was determined on YPD plates. Soring ofIlv+revertants were sored on seletive c medium. Plateswere inubated at 30c for 72 h. In all experiments etha-nol was used as a negative ontrol.

Statistical analysis

The Student t-test was employed for statistial anal-ysis. Signiane was tested at the P< 0.05 level. Ex-

periments were repeated twie. The results presented in

ANTIMUTAGENIc EFFEcT OF ESSENTIAL OIL OF SAGE 165


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gures are the means of two dupliates obtained in repre-sentative experiments.

RESULTS AND DIScUSSION

The effet of EO and frations on spontaneous andUV-indued mutations was ompared in S. typhimuriumTA102 and E. coli K12 SY252 and IB103 strains. Allstrains used ontain point mutations leading to auxotro-

phy and an revert to prototrophy by base substitutions.The S. typhimurium TA102 strain is repair-proientand ontains an ohre hisG428mutation, loated on themultiopy plasmid pAQ1; the hromosomal opy of thehisG gene has been deleted. It also ontains an rfamuta-tion onferring inreased permeability to large moleulesand arries the mutator plasmid pKM101 (M a r o n andA m e s , 1983). The repair proientE. colistrain SY252and its mutS ounterpart IB103 are from ourE. coliK12assay system and arry the ohre hromosomal mutationargE3(S i m i et al.1997). We have used these strainsin the past for detetion of antimutageni ativities ofmodel antimutagens and different plant extrats and forassessing the mehanisms of antimutagenesis (S i m i etal. 1997, 1998; N i k o l i et al.2004).Escherichia coliIB112 and IB113 strains with inreased permeability to

large moleules are derivatives of SY252 and IB103, re-spetively, onstruted to math the permeability of Sal-monella strains(B e r i and B j e d o v, 2003).

Ultraviolet c radiation (254 nm) is one of the mostextensively studied mutagens, shown to indue mutationsin both prokaryoti and eukaryoti test systems. We usedUV-c as a model mutagen beause many hemial muta-gens/arinogens indue mutations by the same meha-nism as UV-c (Friedberg et al. 1995). Sine UV-cis not known to produe reative oxygen speies in addi-tion to pyrimidine dimers (Yo u n g et al.2003), possiblehemial interation between mutagen and antimutagenis prevented.

The omposition of essential oil of sage (EO) and itsfrations is shown in Table 1. Among 44 different terpenesidentied in EO, the oxygen-ontaining monoterpenes -thujone, 1,8-ineole, and amphor were dominant. Fra-tions F1 and F2 ontain exlusively monoterpenes. Themost abundant monoterpenes in F1 and F2 are 1,8-ineoleand -thujone, respetively. Frations F3 and F4 ontaina small proportion of sesquiterpenes in addition to mono-terpenes. They both ontain a very high proportion of -thujone and amphor. Fration F5 ontains about 40% of

sesquiterpenes, the most abundant being -humulene.Salmonella typhimurium mutagenicity and

antimutagenicity assays

The effet of EO and frations on spontaneous andUV-indued mutagenesis in S. typhimuriumTA102 wastested in a range of onentrations (1-10 L/plate), bothwith and without metaboli ativation by S9 enzymes.Toxiity was determined by observing alteration of the

baterial bakground lawn and spontaneous revertantounts signiantly dereased in omparison with the sol-vent ontrol. Sine similar results were obtained in the

presene of S9 enzymes, only the results with S9 are pre-sented.

No mutageni or o-mutageni potential of EO andfrations was deteted in the range of onentrations ap-

plied; there was no inrease in the number of spontaneousor UV-indued revertants ompared with orrespondingsolvent ontrols. Moreover, the number of spontaneousand UV-indued revertants dereased in a onentrationdependent manner on plates with EO and frations. Theindiation for an antimutageni effet, i.e., signiantderease of UV-indued revertants without any effet on

spontaneous revertants, is seen with EO and F1-F3 (Fig.1). The inhibition was 35-60% and dereased in the orderof EO>F1>F2>F3. With F4 and F5, there was alterationof the baterial bakground lawn and signiant redutionof spontaneous revertants at all onentrations applied, in-diating toxiity (data not shown).

Escherichia coli mutagenicity assay

The effet of EO and frations on survival and spon-taneous mutagenesis inE. coliSY252 and its permeableounterpart IB112 was tested. The range of onentrationsto be used was determined by monitoring ell ounts on

plates with EO or frations. The SY252 strain was about10 times less sensitive than TA102 and IB112, owing toinreased permeability of the latter strains.

There was no evidene of mutageniity of EO andfrations F1, F2, and F5 at any of the onentrations test-ed (data not shown). However, on plates with F3 and F4there was about two-fold inrease in the spontaneous re-vertant ounts, in both the non-permeable SY252 and per-meable IB112 strain (Fig. 2), indiating a mild mutagenieffet of these frations. Frations F3 and F4 ontain ahigh proportion of thujone, whih has been reported to



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have antioxidative properties (P e r r y et al.2001). It isknown that antioxidants may at as oxidants and indue

DNA damage (L a b i e n i e et al.2003).It has been shown reently that mismath repair or-

Fig. 1. Effet of EO and F1-F3 of sage on spontaneous and UV-indued mutagenesis in the TA102 Salmonella/mirosome assay. UV-dose 24 J/m2. Number of revertants:spontaneous 28711; UV-indued 122420. *p


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reported data (S i m i et al.1994, 1997, 1998; M i t i et al.2001; Vu j o e v i et al.2004), reommend sageterpenes for further antimutagenesis/antiarinogenesisstudies.

Acknowledgements: This researh was supported by the Ministry of Siene andEnvironmental Protetion of the Republi of Serbia (Projet No. 1502). The skilfulassistane of Ljubia Lali is gratefully aknowledged.

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A (SALVIA OFFICINALIS L.)

UV-

JE -, -, 1, ,

, , 11000 , ,1 . , 11000 ,

UV- () (Salvia ofci-nalis L.) F1-F5, - , Sal-monella/, E. coli K12 S. cerevisiae D7

. , F1 F2 UV- .

, F3 F4 , . F5

UV- E. coli K12. .


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