Antimicrobial Efficacy of Achillea ligustica All. (Asteraceae) Essential Oilsagainst Reference and Isolated Oral Microorganisms

by Cinzia Cecchinia), Stefania Silvia), Alberto Crescia), Andrea Piciottib), Giovanni Capriolic),Fabrizio Papac), Gianni Sagratinic), Sauro Vittoric), and Filippo Maggi*d)

a) School of Biosciences and Biotechnologies, University of Camerino, Via Gentile III da Varano,IT-62032, Camerino

b) Private Dentist, IT-62024 Matelicac) School of Pharmacy, University of Camerino, Via Sant�Agostino 1, IT-62032 Camerino

d) School of Pharmacy, University of Camerino, Via Pontoni 5, IT-62032 Camerino

The aim of this study was to verify the effectiveness of Achillea ligustica essential oils against severaloral microorganisms in comparison with a commercial essential oil-containing mouthrinse (Listerine�)and clove oil (containing 89% eugenol). The inhibition efficacy of A. ligustica essential oils alone and incombination with Listerine� was evaluated by the micro-dilution method. The most susceptiblemicroorganisms were Bacillus cereus, Streptococcus pyogenes, and Candida albicans. The efficacy wassimilar to that of the clove oil. The antiseptic mouthwash Listerine� did not exert a strong inhibition onmicrobial strains tested, whereas its effectiveness increased significantly when essential oil was added.The study provides additional evidence for the in vitro inhibitory activity of A. ligustica essential oils onseveral pathogens, suggesting their usefulness in mouthrinse formulations as an adjunct to mechanicaloral hygiene regimens. Essential oil-containing mouthrinses can be beneficial, safe components of dailyoral health routines, representing an efficient and without side effect alternative to prevent and controloral infections.

Introduction. – Dental caries is one of the most common chronic diseasesworldwide: 90% of people have had dental problems or toothache caused by caries, andin low-to-middle income countries most caries cases remain untreated [1]. Theeconomic impact of oral diseases is considerable with up to 10% of public-healthexpenditure, related to curative and restorative dental care, in developed countries [2].

In recent years, there has been an increasing interest in antibacterial mouthrinsesthat are effective in decreasing tooth surface plaque. In general, mouthrinses containfluorides, alcohols, and detergents, or antibacterial substances. Frequently usedantibacterial chemicals include povidone iodine products, chlorhexidine, and cetylpyr-idinium chloride. However, these chemicals can lead to side effects, such as staining ofteeth, alteration of taste, desquamation of oral epitelium, and oral cancer [3]. For thisreason, natural antibacterial substances have attracted attention over the last decades[4], notably essential oil-containing mouthrinses.

Essential oils are aromatic oily liquids derived from plants and industrially obtainedby means of steam distillation. Since antiquity, they have been used for their perfume,flavor, preservative, and pharmaceutical properties. The recent �green� consumerismhas led to an increased scientific interest in these substances, since they possessantimicrobial properties. These characteristics are possibly related to the function of

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)12

� 2012 Verlag Helvetica Chimica Acta AG, Z�rich

essential oils in plants. Various studies contributed to the evidence that essential oilsmay be suitable additives in products as dental root canal sealers [5], antiseptics [6],and products for the maintenance of oral hygiene or prevention of dental diseases [7].Several studies over the years have demonstrated not only the ability of essential oilmouthrinses to kill a broad spectrum of microorganisms, both in vitro and in vivo, butalso significant clinical outcomes associated with cidal activity, including the preventionand reduction of supragingival plaque and gingivitis, the reduction and control ofintrinsic oral malodor, the reduction of inflammation after periodontal surgery, and thesignificant reduction in viable bacteria contained in aerosols generated during dentalprocedures [8– 10]. Moreover, mouthrinses containing essential oils reach areas thatare highly vulnerable to plaque accumulation and difficult to access. Commercialmouthwashes containing essential oils are useful in the long-term control of plaque andmild-to-moderate gingivitis, and are preferred to those containing chlorhexidine forlong-term daily use [11]. Indeed, when used in combination, some essential oilssignificantly reduced the amount of chlorhexidine required to inhibit the growth of oralpathogens [12]. Thus, essential oil-containing mouthrinses should be recommended toat-risk patients, because they act in a non-mechanical way, offering additional andcomplementary advantages to brushing and interdental cleaning in the maintenance oforal health [13].

In our search for natural products that may be suitable additives in products usedfor the maintenance of oral hygiene or prevention of dental disease, we considered theessential oils from Achillea ligustica All. (Ligurian yarrow, Asteraceae). This is aperennial, herbaceous plant with a slightly aromatic scent, growing in Italy in theTyrrhenian region of Sicily and Sardinia [14]. In Italian folk medicine, it was used as anantihelmintic, and against rheumatism, and skin disorders and inflammation [15]. Here,we report the antimicrobial activity of the essential oils obtained from different parts ofA. ligustica against both reference and isolated cariogenic and periodonthopathicstrains. A comparison of the antimicrobial efficacy of the plant oils with that of its majorcomponents, as well as with efficacies of a commercial essential oil-containingmouthrinse (Listerine�) and one of the most used natural substances in dental care(clove oil; Syzygium aromaticum) was also carried out.

Results and Discussion. – 1. Chemical Analysis of Essential Oils. The composition ofessential oils obtained from different parts of A. ligustica is compiled in Table 1. In theessential oils from whole flowering aerial parts (AP), inflorescences (FL), and leaves(LV), 101, 111, and 117 volatile components were identified, respectively, for a total of124 volatiles. The components identified amounted to 91.0– 96.6% of the total oils.

Hydrodistillation gave a yield of 0.6% for whole flowering aerial parts, 3.0% forinflorescences, and 1.5% for leaves. In particular, the yields from inflorescences andleaves were higher than those obtained in our previous investigation (0.91% and 0.18%for inflorescences and vegetative parts, resp.) [16]. This can be due to the use of freshmaterial instead of dry one. In literature, the following values were reported: 0.4 and0.13– 0.90% for population from Corsica [17] [18], respectively, 0.43 – 0.88% forpopulation from Sardinia [19], and 0.38 – 0.48% for population from Sicily [20].

Hydrodistilled oils showed a strong blue color (darker for inflorescences and meanfor leaves) due to the presence of chamazulene and other compounds of azulenic

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 13

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)14

Table 1. Chemical Composition of Achillea ligustica Essential oils

Componenta) RI HP-5b) RI Lit.c) Essential oil fromd) IDe)

ADAMS NIST 08 FL LV AP

Oct-1-ene 792 791 792 tr. f) tr. MS, RIHexanal 802 801 802 tr. tr. 0.1 Std7-Methyloct-1-ene 853 853 tr. tr. 0.1 MS, RI(3Z)-Hex-3-en-1-ol 864 859 864 tr. 0.1 MS, RIHexan-1-ol 879 870 880 tr. tr. MS, RINon-1-ene 892 892 0.1 0.1 0.1 MS, RIHeptanal 902 902 902 tr. tr. MS, RIIsobutyl isobutyrate 919 911 913 tr. MS, RITricyclene 923 926 923 tr. tr. MS, RIa-Thujene 928 930 928 0.1 0.2 0.1 MS, RIa-Pinene 934 939 935 0.8 1.8 1.0 Std1-(1-Methylethyl)-4-methylidene-bicyclo[3.1.0]hex-2-ene

946 946 tr. tr. tr. MS, RI

Camphene 949 954 949 0.1 0.1 0.1 StdBenzaldehyde 960 960 960 tr. tr. MS, RISabinene 974 975 974 1.1 1.8 1.0 MS, RIb-Pinene 977 979 977 7.0 16.5 11.7 Stdtrans-Dehydrolinalool oxide 993 991 tr. MS, RIMyrcene 995 990 995 0.1 0.1 tr. StdDehydro-1,8-cineole 995 988 0.1 0.1 tr. MS, RIOctanal 998 998 999 tr. Stda-Phellandrene 1007 1002 1007 tr. tr. tr. StdIsobutyl 2-methylbutanoate 1009 1009 0.1 tr. MS, RIIsobutyl isovalerate 1013 1013 0.1 tr. MS, RIa-Terpinene 1019 1017 1019 2.6 2.5 1.5 Stdp-Cymene 1029 1022 1029 0.7 0.6 Stdb-Phellandrene 1032 1029 1031 0.2 0.5 0.4 MS, RI1,8-Cineole 1034 1031 1033 6.8 7.0 5.4 Std(E)-b-Ocimene 1046 1050 1046 0.2 0.1 MS, RIIsobutyl angelate 1061 1051 tr. tr. MS, RIg-Terpinene 1063 1059 1063 2.6 4.6 3.1 StdOctan-1-ol 1068 1068 1068 tr. MS, RIcis-Sabinene hydrate 1072 1070 1072 0.3 3.1 0.2 MS, RITerpinolene 1090 1088 1090 0.5 1.0 0.7 MS, RIIsobutyl tiglate 1090 1090 1090 tr. tr. MS, RItrans-Sabinene hydrate 1098 1098 0.5 1.2 0.2 MS, RILinalool 1106 1096 1106 25.9 0.1 7.7 StdNonanal 1110 1100 1110 0.6 0.1 0.2 MS, RI2-Methylbutyl isovalerate 1115 1104 1113 0.4 0.1 0.1 MS, RIcis-p-Menth-2-en-1-ol 1125 1121 0.4 0.8 0.3 MS, RIa-Campholenal 1131 1126 1130 0.1 0.1 0.1 MS, RI4-Acetyl-1-methylcyclohexene 1137 1136 0.3 0.6 0.5 MS, RItrans-Pinocarveol 1141 1139 1141 1.4 2.1 1.9 MS, RItrans-p-Menth-2-en-1-ol 1144 1140 0.1 0.3 0.2 MS, RICamphor 1146 1146 1146 0.5 0.3 0.1 StdIsoamyl tiglate 1153 1153 0.1 tr. MS, RIPinocarvone 1164 1164 1164 2.0 1.5 1.2 MS, RIBorneol 1168 1169 1168 1.4 0.6 0.3 Std

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 15

Table 1 (cont.)

Componenta) RI HP-5b) RI Lit.c) Essential oil fromd) IDe)

ADAMS NIST 08 FL LV AP

d-Terpineol 1172 1166 1172 0.2 0.1 0.2 MS, RIcis-Pinocamphone 1176 1175 0.2 0.3 0.2 MS, RITerpinen-4-ol 1180 1177 1180 7.6 13.8 8.0 Stda-Terpineol 1192 1188 1192 2.1 2.0 1.6 StdMyrtenal 1196 1195 1196 0.3 0.7 0.3 StdMyrtenol 1197 1195 1196 0.7 0.9 0.5 MS, RItrans-Piperitol 1211 1208 1211 0.1 0.3 0.1 MS, RIcis-Carveol 1226 1229 1226 0.1 Stdcis-Myrtanol 1257 1253 0.1 0.1 0.1 MS, RIcis-Chrysanthenyl acetate 1266 1265 0.4 0.2 0.2 MS, RIPerilla aldehyde 1279 1271 1279 tr. tr. tr. MS, RIBornyl acetate 1287 1288 1287 0.9 0.6 0.4 MS, RItrans-Pinocarvyl acetate 1301 1298 0.4 0.5 0.6 MS, RIMyrtenyl acetate 1330 1324 1328 0.1 0.1 0.1 MS, RId-Elemene 1337 1338 1337 tr. tr. MS, RItrans-Carvyl acetate 1345 1342 tr. tr. MS, RIEugenol 1364 1363 1366 tr. 0.1 0.4 StdCyclosativene 1365 1364 1363 0.3 0.1 MS, RIcis-Carvyl acetate 1368 1365 1364 0.4 0.2 0.2 MS, RIa-Copaene 1374 1376 1374 0.1 0.2 0.3 Stdb-Bourbonene 1382 1390 1382 tr. MS, RItrans-Myrtanol acetate 1386 1386 0.1 0.1 0.1 MS, RI7-Episesquithujene 1390 1390 tr. tr. MS, RIb-Elemene 1391 1390 1391 0.1 0.1 0.2 MS, RIBenzyl valerate 1398 1396 0.1 MS, RIa-Gurjunene 1407 1409 1407 0.1 tr. 0.1 Std(E)-Caryophyllene 1415 1419 1414 0.8 1.2 2.2 Stdb-Copaene 1427 1432 tr. 0.1 0.1 MS, RIAromadendrene 1444 1441 1441 tr. 0.2 Std(Z)-b-Farnesene 1446 1442 1447 tr. 0.1 0.1 MS, RIa-Humulene 1451 1454 1451 0.1 0.1 0.2 StdAmorpha-4,11-diene 1451 1451 tr. MS, RIallo-Aromadendrene 1458 1460 1458 0.4 0.4 0.6 Std(E)-b-Farnesene 1464 1456 1462 0.4 tr. 0.1 Stdcis-Cadina-1(6),4-diene 1462 1463 tr. 0.4 0.9 MS, RIa-Acoradiene 1466 1466 1462 tr. tr. tr. MS, RI10-epi-b-Acoradiene 1475 1475 0.4 MS, RIg-Muurolene 1476 1479 1476 0.1 MS, RIGermacrene D 1479 1485 1479 2.1 6.0 7.0 MS, RIg-Curcumene 1482 1482 0.2 0.1 0.4 MS, RIar-Curcumene 1484 1480 1484 0.3 0.6 MS, RIViridiflorene 1493 1496 1493 0.1 0.1 MS, RIBicyclogermacrene 1493 1500 1494 0.1 0.3 0.7 MS, RIa-Zingiberene 1496 1493 1495 0.2 0.2 MS, RIa-Muurolene 1498 1500 tr. 0.1 0.2 MS, RI(Z,E)-a-Farnesene 1498 1496 1496 tr. 0.1 0.2 MS, RI(E,E)-a-Farnesene 1511 1505 1511 0.6 1.3 2.3 MS, RId-Cadinene 1524 1523 1524 0.6 0.4 2.1 MS, RI

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)16

Table 1 (cont.)

Componenta) RI HP-5b) RI Lit.c) Essential oil fromd) IDe)

ADAMS NIST 08 FL LV AP

b-Sesquiphellandrene 1524 1522 1524 0.3 0.2 0.6 MS, RIa-Calacorene 1544 1545 0.2 0.1 0.1 MS, RIb-Calacorene 1565 1565 tr. tr. tr. MS, RIPalustrol 1566 1568 1561 0.3 0.3 MS, RISpathulenol 1578 1578 1578 0.1 tr. 0.5 MS, RICaryophyllene oxide 1580 1583 1580 0.5 0.8 0.8 Stdar-Turmerol 1583 1583 0.2 0.2 0.2 MS, RIallo-Cedrol 1589 1589 0.5 0.3 0.5 MS, RIGlobulol 1590 1590 1592 0.5 0.7 0.5 StdViridiflorol 1592 1592 1592 8.6 8.7 9.8 MS, RIGuaiol 1601 1600 1601 0.5 0.7 0.8 Std1-Epicubenol 1629 1628 0.3 0.5 0.8 MS, RIMuurola-4,10(14)-dien-1b-ol 1632 1631 0.3 0.5 0.8 MS, RICaryophylla-4(12),8(13)-dien-5-olg) 1636 1640 1.3 0.7 0.6 MS, RIb-Acorenol 1636 1637 0.4 MS, RIa-Cadinol 1657 1654 1658 0.4 0.4 0.7 MS, RIcis-Calamenen-10-ol 1662 1661 0.2 tr. 0.2 MS, RItrans-Calamenen-10-ol 1667 1668 0.5 0.4 0.7 MS, RIa-Bisabolol 1685 1685 1685 0.5 0.7 0.8 StdGermacra-4(15),5,10(14)-trien-1-a-ol 1686 1686 tr. tr. tr. MS, RI(Z)-a-trans-Bergamotol 1688 1690 0.2 tr. 0.2 MS, RI10-Norcalamenen-10-one 1697 1702 tr. tr. MS, RI1,2,3,5,6,7,8,8a-Octahydro-6-iso-propenyl-4,8a-dimethylnaphthalen-2-ol

1717 1714 0.2 MS, RI

Chamazulene 1728 1731 1732 0.2 tr. 0.3 MS, RIMint sulfide 1734 1741 MS, RI(6S,7R)-Bisabolone 1750 1749 0.1 0.1 0.3 MS, RIHexadecan-1-ol 1875 1875 1876 tr. 0.2 0.2 MS, RIOctadecan-1-ol 2080 2077 2081 0.1 MS, RIPhytol 2120 2112 2122 0.6 0.2 Std

Compound groups [%]Monoterpene hydrocarbons 14.6 29.9 20.4Oxygenated monoterpenes 53.9 37.6 30.5Sesquiterpene hydrocarbons 7.0 12.2 19.7Oxygenated sesquiterpenes 15.5 15.7 18.5Aromatics 0.1 0.1 0.4Aliphatics 1.8 0.6 1.0

Oil yield [% (w/w)] 3.0 1.5 0.6Identified compounds 111 117 101Total identified [%] 92.5 96.6 91.0

a) Compounds in order of their elution from a HP-5 column. b) Retention index (RI) on HP-5 column,experimentally determined using homologous series of C8 –C30 alkanes. c) Relative RIs taken fromADAMS [21] and/or NIST08 [22] for DB-5 and HP-5 capillary column, respectively. d) FL:Inflorescences; LV: leaves; AP: whole aerial parts. e) Identification methods: MS, by comparison ofthe mass spectra with those in the computer mass libraries Wiley, ADAMS, and NIST08 ; RI, bycomparison of RI values with those reported in [21] [22]; Std, by comparison of tR and MS data with thoseof available authentic standard. f) tr., Traces (mean value below 0.1%). g) Correct isomer not identified.

nature; this observation suggests their use also as natural colorant for food, cosmetics,and oral hygiene products.

In the whole flowering aerial parts, the major volatiles (> 5%) were b-pinene(11.7%), viridiflorol (9.8%), terpinen-4-ol (8.0%), linalool (7.7%), germacrene D(7.0%), and 1,8-cineole (5.4%). Monoterpenes constituted the main fraction (50.9%)of the oil, with oxygenated compounds being more prevalent (30.5%). Sesquiterpenesaccounted for 38.2%, with equal amounts of hydrocarbons (19.7%) and oxygen-containing compounds (18.5%). Among O-containing compounds, alcohols were themost abundant.

The major components of the oil from inflorescences were linalool (25.9%),viridiflorol (8.6%), b-pinene (7.0%), terpinen-4-ol (7.6%), and 1,8-cineole (6.8%).This oil was dominated by O-containing compounds, i.e., monoterpenoids andsesquiterpenoids accounting for 53.9% and 15.5%, respectively, followed by mono-terpene hydrocarbons (14.6%).

In leaves, the main components were b-pinene (16.5%), terpinen-4-ol (13.8%),viridiflorol (8.7%), 1,8-cineole (7.0%), germacrene D (6.0%), and g-terpinene (4.6%),whilst linalool was present only in scant amounts (0.1%). Hydrocarbons and O-containing compounds afforded the following contributions: 29.9% for monoterpenehydrocarbons, 37.6% for oxygenated monoterpenes, 12.2% for sesquiterpene hydro-carbons, and 15.7% for oxygenated sesquiterpenes.

These findings were consistent with those obtained in our previous investigations[16], resulting in a different composition of the oils, qualitatively richer with respect tothose obtained from other regions [18 – 20]. This confirms that A. ligustica presentsseveral chemotypes, reflected in the strong variability in the essential oil compositionsfrom different areas, and supports the hypothesis that the chemical compositions ofAchillea species are complex and susceptible to variation [23].

2. Antimicrobial Activity. Among the seven solvents and emulsifiers tested with APoil and assayed against the test strain Streptococcus mutans, PEG 400 assured the bestresults in terms of inhibition (MIC of 38 mg/ml). The inhibition values for all the otherswere: acetone, 78 mg/ml; DMSO, 155 mg/ml, PBSþTween 80, 310 mg/ml; PBS, 1250 mg/ml; acetone þ Tween 80, 1250 mg/ml; and Tween 80, 2500 mg/ml.

The inhibition values of A. ligustica oils, expressed as MICs, are collected in Table 2.No significant differences in activity against oral pathogens were observed among theoils obtained from different parts of the plant. With regard to microbial strains tested,the most susceptible microorganisms were Bacillus cereus, Streptococous mutans, S.pyogenes, and Candida albicans with MIC ranges of 78 –310, 38– 155, 78– 155, and 78–155 mg/ml, respectively. FL Oil was more active on B. cereus and S. pyogenes, whereasLV oil had a more pronounced inhibition on C. albicans.

Among all the standard compounds, representatives of the major constituents of theoils, linalool and b-pinene, exhibited highest activity on the microbial strains (lowestMIC of 155 mg/ml on C. albicans). However, they did not equal the inhibition activity ofthe Ligurian yarrow (A. ligustica) oils, indicating that the action of the majorcomponent is modulated by other minor compounds occurring in the oils.

Comparing the MIC values of essential oils with that of the standard drugchlorhexidine, it was concluded that the latter has a potent anti-oral-pathogen activityexcept for Lactobacillus acidophilus and C. albicans on which the activity of Ligurian

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 17

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)18

Tabl

e2.

Ant

imic

robi

alA

ctiv

ityas

Min

imal

Inhi

bito

ryC

once

ntra

tion

(MIC

[mg/

ml]

)of

Ess

entia

lO

ilsfr

omA

chill

ealig

usti

caag

ains

tO

ral

Pat

hoge

nSt

rain

s

MIC

[mg/

ml]

a )

B.c

ereu

sIM

C10

2E

.avi

umIM

C10

3L

.aci

doph

ilus

IMC

101

Stap

h.au

reus

AT

CC

2592

3St

rep.

dysg

alac

tiae

IMC

106

Stre

p.m

utan

sD

SM20

523

Stre

p.py

ogen

esIM

C10

5St

rep.

saliv

ariu

sIM

C10

4C

.alb

ican

sA

TC

C14

053

Oil

sam

ples

b)

FL

7850

0031

031

062

515

578

1250

155

LV31

025

0025

0062

512

5015

515

512

5078

AP

155

2500

1250

625

1250

3878

625

155

Maj

orco

mpo

nent

sb

-Pin

ene

1250

1250

2500

2500

1250

1250

310

625

155

1,8-

Cin

eole

2500

2500

5000

2500

2500

2500

1250

1250

2500

g-T

erpi

nene

2500

2500

5000

2500

1250

2500

625

625

2500

Terp

inen

-4ol

1250

2500

5000

1250

2500

1250

1250

625

310

Lin

aloo

l31

062

550

0025

0012

5062

562

562

515

5V

irid

iflo

rol

n.t.

c )n.

t.n.

t.n.

t.n.

t.25

0062

562

5n.

t.

Posi

tive

cont

rols

Chl

orhe

xidi

ne38

1931

019

50.

60.

319

155

Lis

teri

ne25

0010

000

5000

5000

2500

2500

1250

5000

1250

Lis

teri

neþ

AP

oil

625

2500

1250

1250

625

1250

7812

5062

5C

love

oil

155

625

2500

155

1250

155

310

310

155

Neg

ativ

eco

ntro

lP

EG

400

2500

5000

5000

nad)

5000

2500

1250

1250

na

a )M

ean

ofth

ree

dete

rmin

atio

ns.

b)

FL

:Inf

lore

scen

ces;

LV:l

eave

s;A

P:w

hole

aeri

alpa

rts.

c )n.

t.:N

otte

sted

.d)

n.a.

:Not

acti

ve.

yarrow oils was equal to that of the positive control (MIC values of 310 and 155 mg/ml,resp.).

Instead, there were no significant differences in inhibition against the mostsusceptible strains exhibited by Ligurian yarrow oils with respect to clove oil (S.aromaticum, 89% eugenol) that is one of the most frequently used natural substances indental care, very effective against oral bacteria associated with dental caries andperiodontal disease [24]. Clove oil contains mainly eugenol, which has been used indentistry for numerous years, and is added to dental products and medications,including mouth washes and tooth pastes. Dentists also mix clove oil with zinc oxideand prepare a white filling material as a temporary alternative to root canal.

Noteworthy was that the antiseptic mouthwash Listerine� did not exert a stronginhibition on microbial strains tested (Fig.). When A. ligustica oil was added, theeffectiveness of the mixture increased significantly (p<0.01), especially on Strepto-coccus pyogenes (MIC from 1250 to 78 mg/ml), highlighting an additive effect of the oilto that of the mouthwash.

Frequently, oral pathogens develop drug resistance leading to a resurgence in theuse of natural alternative therapies instead of synthetic pharmaceuticals, often withmany side effects. The antibacterial properties of essential oils are well-known, andactivity against bacteria found in the oral cavity, including pathogens, has beendocumented [25]. Therefore, mouthrinses with essential oils can be a beneficial, safecomponent of daily oral health routines [13].

The study reported here provides additional evidence for the in vitro inhibitoryactivity of A. ligustica essential oils on several bacteria associated with the oral cavityand also against the oral opportunistic fungal pathogen C. albicans, suggesting their use

Figure. Growth inhibition, expressed as MICs, of Bacillus cereus IMC 102, Streptococcus pyogenes IMC105, S. mutans DSM 20523, and Candida albicans ATCC 14053 with different A. ligustica oils, clove,Listerine�, and Listerine�þAP oil. FL: inflorescences; LV: leaves; AP: aerial parts. *: Statisticallysignificant (p<0.01) compared to Listerine� alone. Concentrations tested ranged from 10 to

0.019 mg/ml.

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 19

in mouthrinse formulations as an adjunct to mechanical oral hygiene regimens and/oras natural colorant in oral care products due to their strong blue color.

In general, essential oils and many of their components have limited solubility inaqueous media, and this property may prejudice their performances in standardsusceptibility test methods. In addition, often they form a turbid suspension in anaqueous broth medium, which makes the detection of growth difficult. Variousmodifications to methods, including the addition of solvents and/or emulsifiers, for theadequate dispersion of the essential oils on in vitro antimicrobial tests and to stabilizethem in water-based culture media, have been used previously to overcome thesedifficulties. Additionally, detergents possess negligible antimicrobial activity of theirown, and the possibility that they may act synergistically with the oil components orenhance the growth of test organisms cannot be neglected.

In the present study, we showed that PEG 400 provided the best results in terms ofinhibitory activity of yarrow with respect to the other solvents and emulsifiersinvestigated, indicating that the choice of the proper dispersion agent is of greatimportance when antimicrobial activity of essential oils is considered. Moreover, thisprocedure should be always performed, since the dispersion properties of an essentialoil, that influence the results of this kind of experiments, depends mostly on itscomposition that is quite variable with the geographic origin of samples.

In numerous studies, active compounds or plant extracts have been compared withcurrently used antibacterial compounds employed in dentistry, such as chlorhexidineand triclosan, to determine relative effectiveness [26]. Using the above criteria, extractswith MIC values around or below 100 mg/ml and isolated phytochemicals with MICvalues below 20 mg/ml may be considered useful for the development as products forapplication against oral infections [27]. Instead, plant derivatives with MIC valuesabove 100 mg/ml are unlikely to be useful chemotherapeutic agents, since such highconcentrations are almost impossible to achieve in vivo and could in many cases betoxic [28].

The inhibition activity of A. ligustica oils can be explained by the wide spectrum ofantimicrobial compounds that they contain, most of them being O-containingcompounds, notably alcohols. Terpinen-4-ol, ranging from 7.6 to 13.8% in A. ligusticaoils, is the main component of the �Tea Tree Oil� (TTO) that is employed largely for itsantimicrobial properties [29] and, for this reason, incorporated as the active ingredientin many topical formulations used to treat cutaneous infections. Additional compo-nents of A. ligustica oils with MIC values similar to or lower than those of TTO includea-pinene, b-pinene, and linalool [30]. Concerning the latter, it has been shown that alinalool-rich essential oil inhibited the growth of reference samples of Candida albicans,Streptococcus sobrinus, and S. mutans, all of them associated with oral cavity disease[31]. The O-containing viridiflorol (8.6– 9.8% in yarrow oils) was reported to be themajor sesquiterpenoid in niaouli essential oils obtained from M. quinquenervia [32].Niaouli oils are known to have biological activity, notably antimicrobial, and they havebeen used since old times in traditional medicines and more recently as activeingredients in modern formulations [33] [34].

Therefore, the inhibition activity of A. ligustica oils can be expected to be related tothe chemical configuration of its components, the proportions in which they arepresent, and the interactions between them. In the present study, the activities of A.

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ligustica oils against most susceptible oral strains have been found higher than those ofthe major compounds tested alone. Some studies have led to the conclusion that wholeessential oils have a greater antibacterial activity than the major components mixed[35] [36], suggesting that the minor components are critical to the activity, and theymay have a synergistic effect or enhancing influence. The complexity of the oil of A.ligustica, with more than 100 components, increases the likelihood that synergisticinteractions are occurring between components. In fact, although some molecules maynot belong to one of the primary antimicrobial components, they may permeabilizebacterial membranes and facilitate the entry of other, more active components.

Conclusions. – Of the major components occurring in A. ligustica oils, it seems thatmost possess at least some degree of antimicrobial activity, and this is considered tocorrelate with the presence of functional groups, such as alcohols, and the solubility ofthe component in biological membranes [37]. The study reported here providesadditional evidence for the in vitro inhibitory activity of A. ligustica essential oils onseveral bacteria associated with the oral cavity and also against the oral opportunisticfungal pathogen C. albicans, suggesting their use in mouthrinse formulations as anadjunct to mechanical oral hygiene regimens and/or as natural colorant in oral careproducts due to their strong blue color. These findings tend to reinforce the use of plantextracts by inhabitants of financially unprivileged regions, representing an efficient andunrestricted alternative to prevent and control oral infections. However, beforeconsidering the essential oil of A. ligustica as a real promising agent to be used in rinse,toothpastes, or gel preparations produced by pharmaceutical industries, additionaltests, including experimental models, and pharmacological and safety applicability, arerequired.

Experimental Part

Plant Material. A population naturalized after cultivation at Botanical Garden in Camerino (centralItaly, 600 m above sea level, GPS coordinates: N 43808’02’’ E 13804’06’’) was considered in this study.Inflorescences and leaves were separately collected in June 2009 during the blooming period, whilstwhole flowering aerial parts were collected in June 2008. The plant was botanically confirmed by F. M.using available literature [38]. A voucher specimen was deposited with the Herbarium Camerinensis(included in the online edition of Index Herbariorum: http://sweetgum.nybg.org/ih/) [39] of School ofEnvironmental Sciences (University of Camerino, Italy) under the accession code CAME 13420; it isalso available at the website: http://erbariitaliani.unipg.it.

Essential Oil Extraction. Essential oils were obtained by hydrodistillation (3 h) in a Clevenger-typeapparatus of fresh inflorescences (FL), leaves (LV), and whole aerial parts (AP) of A. ligustica, until thecondensing oils could no more be seen. Oils were separated from the aq. soln., dried (Na2SO4),transferred into an amber glass flask, and kept at �208 until used. Three oils were obtained from eachsample by hydrodistillation and subsequently analyzed by GC/FID and GC/MS. The oil yield wasestimated on a dry-weight basis, by calculating the H2O content prior to the distillation by leaving plantmaterial in a stove at 1108 for 24 h. Clove oil was obtained by the same procedure from the buds ofSyzygium aromaticum (L.) Merrill & Perry purchased from a local market.

Chemicals. Standard compounds used for identification of essential oil constituents were purchasedfrom Sigma�Aldrich (I-Milan). For retention index (RI) determination, a mixture of hydrocarbonsranging from octane (C8) to triacontane (C30) (Supelco, USA) was used and run under the exper.conditions reported below. All compounds were of anal. grade. Anal. grade hexane was purchased fromCarlo Erba (I-Milan); it was successively distilled by a Vigreux column before use. Na2SO4 was of anal.grade from J. T. Baker (NL-Deventer).

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GC/FID and GC/MS Analysis. For GG separations, an Agilent 4890D instrument coupled to anionization flame detector (FID) was used. Volatile components were separated on a HP-5 cap. column(5% phenyl methylpolysiloxane, 25 m, 0.32 mm i.d.; 0.17 mm film thickness; J & W Scientific, Folsom,CA), with the following temp. program: 5 min at 608, subsequently 48/min up to 2208, then 118/min up to2808, held for 15 min, for a total run of 65 min. Injector and transfer line temps. were 2808. He was used asthe carrier gas, at a flow rate of 1.4 ml/min; injection volume, 1 ml ; split ratio, 1 : 34. A mixture of aliphatichydrocarbons (C8�C30; Sigma, I-Milan) in hexane was directly injected into the GC injector according tothe above temp. program, in order to calculate RI (as Kovats index) of each compound. All samples werediluted 1 : 100 in hexane for GC analysis, and the volume injected was 1 ml. The relative amounts ofvolatile components, expressed as %, were obtained by FID peak-area internal normalization, bycalculating the response factor (RF) of the FID for different classes of volatiles in the essential oils [40].They were means of three determinations. Data were collected by using HP3398A GC Chemstationsoftware (Hewlett Packard, Rev. A.01.01). GC/MS Analysis was performed on an Agilent 6890N gaschromatograph coupled to a 5973N mass spectrometer using a HP-5 MS (5% phenyl methylpolysiloxane,30 m, 0.25 mm i.d., 0.1 mm film thickness; J & W Scientific, Folsom), with the following temp. program:5 min at 608, then at 48/min to 2208, then 118/min up to 3008, held for 15 min, for a total run of 72 min.Injector and transfer line temps. were 2808. He was used as the carrier gas, at a flow rate of 1 ml/min.Samples were diluted 1 : 100 in hexane, and the volume injected was 2 ml ; split ratio, 1 :50; acquisitionmass range, 29–400 m/z. All mass spectra were acquired in electron-impact (EI) mode with a ionizationvoltage of 70 eV. Whenever possible, volatile components were identified by comparing the tR, RI, andMS of the chromatographic peak with those of standard (see chemicals) analyzed under the sameconditions. Otherwise, the peak assignment was based on computer matching with the WILEY275,NIST08, ADAMS, and a home-made (based on the analyses of reference oils and commercially availablestandards) libraries, taking into account the coherence of the RIs of the analyzed compounds with thosereported by ADAMS [21] and NIST08 [22] library. This is in accordance with the standard of theInternational Organization of the Flavor Industry (IOFI ; http://www.iofi.org/) statement. Data wereanalyzed by using MSD ChemStation software (Agilent, version G1701DA D.01.00).

Microorganisms. Representative microorganisms of the oral microbiota were included in this study.The reference sample representing caries-associated microorganism was Streptococcus mutans DSM20523 (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, D-Braunschweig). Othercommonly tested oral microorganisms used as reference strains were Staphylococcus aureus ATCC 25923(American Type Culture Collection, Rockville, MD, USA) and Candida albicans ATCC 14053 [41].

Human isolates, obtained from the caries and periodontal lesions of patients, included Lactobacillusacidophilus IMC101, Bacillus cereus IMC102, Enterococcus avium IMC103, Streptococcus salivariusIMC104, S. pyogenes IMC105, and S. dysgalactiae IMC106.

Isolation and Identification of Microorganisms. The human isolates were obtained from patients withcaries during a dental surgery. The specimens from five different people were collected using swabs andinoculating them in Tryptone Soya Broth (OXOID, Unipath Ltd., Basingstoke, Hampshire, England)supplemented with 5% sheep blood and inserted in a jar under anaerobic conditions obtained byinserting an AnaeroGen sachet (OXOID) for transportation. The specimens were incubated for 24 h at378, then streaked on different agar plates, i.e., Tryptone Soya Broth (OXOID) supplemented with 5%sheep blood for the isolation of Streptococci, Mac Conkey Agar (OXOID) for Gram-negative bacteria,Sabouraud Agar (OXOID) for Candida strains, and Mannitol Salt Agar (OXOID) for Staphylococci. Theplates were incubated at 378 for 48–72 h both aerobically and in an anaerobic cabinet (Concept 400,Ruskinn Technology Limited, Leeds, West Yorkshire, UK). The colonies obtained were analyzed formorphological, cultural, and physiological characteristics. The identification was performed by using API20 STREP kit for Streptococci identification, API 20 NE kit for non-entorobacteria Gram-negativestrains, API 50 CHB medium kit for Bacilli, API STAPH kit for Staphylococci, and API 50 CHL mediumkit for Lactobacilli (bioMerieux, F-Marcy-l�Etoile).

Screening of Solvent and Emulsifying Agents. Several solvents and emulsifiers were tested in order toselect the most effective one to enhance the anti-oral pathogen efficacy of the essential oils. A MinimalInnhibitory Concentration (MIC) test against Streptococcus mutans (DSM 20523; Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH, D-Braunschweig) as test strain for its involvement in

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caries diseases was applied using essential oil from aerial parts (AP) of A. ligustica diluted (20 mg/ml)with acetone, acetone þ polysorbate 80 (Tween 80 ; 1 : 1), polysorbate 80, dimethyl sulfoxide (DMSO),phosphate buffered saline (PBS), polyethylene glycol 400 (PEG 400), polysorbate 80 (0.1%). Theprocedure applied to evaluate the antimicrobial activity was the same as for the antimicrobial assay (seebelow).

Antimicrobial Assay. Antimicrobial activity of A. ligustica essential oils (FL, LV, and AP) wasevaluated using the broth microdilution method with a 96-well microtiter plate [42]. Bacterial strainswere cultivated on M�ller�Hinton Broth (OXOID), while Candida strain was cultivated on Sabouraudliquid medium (OXOID). Cell cultures were incubated at 378 for 24 h and then used. The cellsuspensions were adjusted with sterile saline soln. to obtain a turbidity comparable to that of McFarlandn. 0.5 standard (1.5�108 cells ml). One series of twofold dilutions of each oil sample in PEG400 (rangingfrom 10 to 0.019 mg/ml) for each microbial strain was prepared using the same media as above. Eachseries was inoculated with 0.8 ml of each microbial strain (1.5�108 cells/ml). MIC Determination, as thelowest concentration of essential oil that inhibited the visible growth of the microorganisms after 24 hincubation at 378, was carried out evaluating the microbial growth in the wells using a stereomicroscope(GSZ2, Ascania, Germany). Chlorhexidine (0.12%, oral soln., Curasept, Curadent Healthcare, I-Saronno) and an essential oil-containing mouthrinse (Listerine� Antiseptic Mouthwash, Pfizer ConsumerHealthcare, Morris Plains, NJ, USA) were used as positive controls, as well as clove bud oil (S.aromaticum; purchased from a local market), containing 89% of eugenol (determined at GC/FID), anatural analgaesic and antiseptic used primarily in dentistry. Moreover, to verify a possible synergisticeffect, AP oil was mixed with Listerine� at concentration of 20 mg/ml and tested. A negative control,inoculating the PEG 400 without oils, was also included. Available standards of b-pinene, 1,8-cineole,myrcene, g-terpinene, terpinen-4-ol, linalool (all from Sigma–Aldrich, I-Milan), and viridiflorol (kindlyprovided by Michael Russell, Department of Primary Industries, Industry and Investment NSW,Wollongbar, NSW, Australia), representatives of the major volatile components, were also tested underidentical conditions to compare their activities with those of the investigated oils.

Statistical Analysis. The antimicrobial activity of essential oils was evaluated by the One-WayANOVA. Values of p<0.05 were considered as statistically significant. All statistical evaluations werecarried out using the SPSS 13.0 software package for Windows (SPSS Inc., Chicago, Illinois, USA).

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Received July 17, 2011

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