Chemical Composition, Antimicrobial and Cytotoxic Activity of Heracleum verticillatum PANCIC and H. ternatum VELEN. (Apiaceae) Essential Oils

Abstract

In this work, the chemical composition, antimicrobial and cytotoxic activity of Heracleum verticillatum Pančić and H. ternatum Velen. root, leaf and fruit essential oils were investigated. The composition was analyzed by GC and GC/MS. Heracleum verticillatum and H. ternatum root oils were dominated by monoterpenes, mostly β-pinene (23.5 and 47.3%, respectively). Heracleum verticillatum leaf oil was characterized by monoterpenes, mainly limonene (20.3%), and sesquiterpenes, mostly (E)-caryophyllene (19.1%), while H. ternatum leaf oil by the high percentage of phenylpropanoids, with (Z)-isoelemicin (35.1%) being dominant constituent. Both fruit oils contained the majority of aliphatic esters, mostly octyl acetate (42.3% in H. verticillatum oil and 49.0% in H. ternatum oil). The antimicrobial activity of the oils was determined by microdilution method against eight bacterial and eight fungal strains. The strongest effect was exhibited by H. verticillatum root oil, particularly against Staphylococcus aureus, Salmonella typhimurium (MICs=0.14 mg/ml, MBCs=0.28 mg/ml) and Trichoderma viride (MIC=0.05 mg/ml, MFC=0.11 mg/ml). Cytotoxic effect was determined by MTT test against malignant HeLa, LS174 and A549 cells (IC50 =5.9-146.0 μg/ml), and against normal MRC-5 cells (IC50 >120.1 μg/ml). The best effect was exhibited by H. verticillatum root oil on A549 cells (IC50 =5.9 μg/ml), and H. ternatum root oil against LS174 cells (IC50 =6.7 μg/ml). This article is protected by copyright. All rights reserved.

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FULL PAPER
Chemical Composition, Antimicrobial and Cytotoxic Activity of Heracleum
verticillatum PAN

CI

Cand H.ternatum VELEN. (Apiaceae) Essential Oils
by Ljubo
sJ.U

sjak
a
), Silvana D. Petrovi
c*
a
), Milica M. Drobac
a
), Marina D. Sokovi
c
b
), Tatjana P. Stanojkovi
c
c
),
Ana D.

Ciri
c
b
), Na da Ð. Grozdani
c
c
), and Marjan S. Niketi
c
d
)
a
) Department of Pharmacognosy, University of Belgrade - Faculty of Pharmacy, Vojvode Stepe 450, RS-11221 Belgrade
(phone: +381-11-3951322; fax: +381-11-3972840; e-mail: silvana.petrovic@pharmacy.bg.ac.rs)
b
) Institute for Biological Research “Sini
sa Stankovi
c”, University of Belgrade, Bulevar Despota Stefana 142, RS-11000
Belgrade
c
) Institute of Oncology and Radiology of Serbia, Pasterova 14, RS-11000 Belgrade
d
) Natural History Museum, Njego
seva 51, RS-11000 Belgrade
In this work, the chemical composition, antimicrobial and cytotoxic activity of Heracleum verticillatum PAN

CI

Cand
H.ternatum VELEN. root, leaf, and fruit essential oils were investigated. The composition was analyzed by GC and GC/MS.
Heracleum verticillatum and H. ternatum root oils were dominated by monoterpenes, mostly b-pinene (23.5% and 47.3%,
respectively). Heracleum verticillatum leaf oil was characterized by monoterpenes, mainly limonene (20.3%), and
sesquiterpenes, mostly (E)-caryophyllene (19.1%), while H.ternatum leaf oil by the high percentage of phenylpropanoids,
with (Z)-isoelemicin (35.1%) being dominant constituent. Both fruit oils contained the majority of aliphatic esters, mostly
octyl acetate (42.3% in H.verticillatum oil and 49.0% in H.ternatum oil). The antimicrobial activity of the oils was
determined by microdilution method against eight bacterial and eight fungal strains. The strongest effect was exhibited by
H.verticillatum root oil, particularly against Staphylococcus aureus,Salmonella typhimurium (MICs=0.14 mg/ml,
MBCs=0.28 mg/ml), and Trichoderma viride (MIC =0.05 mg/ml, MFC =0.11 mg/ml). Cytotoxic effect was determined by
MTT test against malignant HeLa, LS174, and A549 cells (IC
50
=5.9 –146.0 lg/ml), and against normal MRC-5 cells
(IC
50
>120.1 lg/ml). The best effect was exhibited by H.verticillatum root oil on A549 cells (IC
50
=5.9 lg/ml), and
H.ternatum root oil against LS174 cells (IC
50
=6.7 lg/ml).
Keywords: Heracleum verticillatum,Heracleum ternatum, Essential-oil composition, Antimicrobial activity, Cytotoxic activity.
Introduction
The genus Heracleum L. (Apiaceae) comprises about 125
biennial or perennial plants predominantly distributed in
the temperate Northern Hemisphere [1]. It is named after
ancient Greek hero Heracles, because of the size and
healing properties of these plants [2]. The widely dis-
tributed H.sphondylium L. s.l. is traditionally used for
the treatment of epilepsy, digestive disorders, such as
diarrhea and dysentery, hypertension, respiratory ailments
including infections, sexual weakness, skin diseases, and
infected wounds. All plant parts, particularly young leaves
and stems, are consumed as food [3–5]. Heracleum verti-
cillatum PAN

CI

C(a Central Balkan endemic) and H.terna-
tum VELEN. [2][6][7] were the subject of present
investigation. They belong to a widely circumscribed
H.sphondylium group [8], although some authors con-
sider them as subspecies of H.sphondylium [9].
Heracleum verticillatum is a biennial herbaceous plant
with whorled upper branches and a softly pubescent to
villous young branches. Leaves are divided into 5 –7 seg-
ments, flowers are white, and the outer ones are zygomor-
phic. This species is restricted to siliceous mountain
massifs in Central Balkan (eastern Serbia, western and
southern Bulgaria, north-eastern Greece, and western
FYR Macedonia) and its natural populations inhabit wet
places along the springs in subalpine region [2][6][9–11].
Heracleum ternatum is a biennial herbaceous plant,
branched at the top, with leaves divided into three seg-
ments (ternate leaves) and greenish, actinomorphic flow-
ers. It inhabits forests, thickets, and road edges in the
mountainous areas of the Balkan Peninsula, and northern
and central Anatolia, as well as in the northern and cen-
tral Apennine Mountains [2][6][9][12].
Previously, several furanocoumarins were identified in
the roots and in the fruits of H.verticillatum, and the
anticonvulsant activity of the combined root fura-
nocoumarins was demonstrated [13]. The chemical com-
position, and cytotoxic, antioxidant and/or antimicrobial
activity of the fruit essential oils of H.ternatum from two
©2016 Verlag Helvetica Chimica Acta AG, Z€
urich DOI: 10.1002/cbdv.201500151
466 Chem. Biodiversity 2016,13, 466 – 476

localities in Italy and from two localities in Turkey were
reported [5][14][15]. Additionally, the tocopherols and
phospholipids of H.ternatum fruit fatty oil were analyzed
[16].
The aim of this work was to investigate the chemical
composition, antimicrobial and cytotoxic activity of
H.verticillatum and H.ternatum root, leaf, and fruit
essential oils.
Results and Discussion
Chemical Composition of the Essential Oils
Heracleum verticillatum roots and leaves yielded 0.32%
and 0.10% of the yellow essential oils, respectively, while
the fruits afforded 1.11% of the colorless oil. The total of
58 components were identified by GC and GC/MS in the
root oil, 66 in the leaf oil and 38 in the fruit oil (repre-
senting 93.1%, 95.3%, and 95.5% of the total oils, respec-
tively). Heracleum ternatum roots, leaves, and fruits
afforded 0.19%, 0.25%, and 0.85% of the yellow oils,
respectively. The GC and GC/MS analysis revealed 70
components in the root oil, 65 in the leaf oil, and 59 in
the fruit oil (representing 92.1%, 96.4%, and 89.8% of
the total oils, respectively). The results of the chemical
analysis of H.verticillatum and H.ternatum oils are given
in Table 1.
Heracleum verticillatum root essential oil was domi-
nated by monoterpenes (67.5%), mostly b-pinene (23.5%)
and limonene (19.2%), followed by sesquiterpenes
(20.3%), mainly intermedeol (10.9%). Heracleum terna-
tum root oil was also characterized by monoterpenes
(77.3%), with b-pinene (47.3%) being the most abundant,
followed by (Z)-b-ocimene (15.6%). Similarly, monoter-
penes prevailed in the root oils of nine previously investi-
gated Heracleum species grown at experimental station in
Leningrad Oblast’ (Russia), and b-pinene was the main
compound in seven of them (17.6 –39.0%). The highest
amounts of b-pinene were determined in the root oils of
H. wilhelmsii FISCH. & C. A. MEY. and H. ponticum (LIP-
SKY)SCHISCHK.exGROSSH. Monoterpene hydrocarbon
limonene (19.4 –20.0%) was the second most abundant
constituent, after octyl acetate (33.0 –35.0%), in the
essential oils of H. stevenii MANDEN. roots, collected dur-
ing budding and at the end of vegetation [17].
Monoterpenes (31.0%) and sesquiterpenes (34.2%)
characterized the essential oil of H.verticillatum leaves.
Its main constituents were limonene (20.3%) and (E)-car-
yophyllene (19.1%). Limonene was also the principal
component in H. mantegazzianum SOMMIER &LEVIER and
H.wilhelmsii leaf oils (50.4% and 60.2%, respectively)
[18]. Leaf oil sampled by microdissection from the com-
panion canals of the type subspecies H.sphondylium
subsp. sphondylium L. mainly contained (E)-caryophyl-
lene (28.0%), while limonene (4.0%) was the most promi-
nent among monoterpenes [19]. Heracleum ternatum leaf
oil had similar amount of sesquiterpenes (34.4%) as
H.verticillatum leaf oil, with germacrene D (9.4%) and
(E)-caryophyllene (8.5%) being dominant. However,
H.ternatum leaf oil differed significantly in the high per-
centage of phenylpropanoids (58.4%), mostly (Z)-isoele-
micin (35.1%), elemicin (12.6%), and methyl eugenol
(10.7%). The majority of phenylpropanoids was also
found in oils of H. transcaucasicum MANDEN. and
H. rechingeri MANDEN. aerial parts, with elemicin being
the most abundant among them (41.1% and 23.1%,
respectively) [20][21].
Regarding chemical composition, tested Heracleum
fruit essential oils remarkably differed from the investi-
gated root and leaf oils. Both H.verticillatum and H.ter-
natum fruit oils were predominantly composed of
aliphatic esters (88.0% and 72.7%, respectively), mostly
octyl acetate (42.3% and 49.0%, respectively). It was fol-
lowed by octyl 2-methyl butanoate (22.3%) and octyl
isobutanoate (15.2%) in H.verticillatum fruit oil, and
octyl hexanoate (11.3%) in H.ternatum fruit oil. The fruit
essential oils of H.ternatum from two localities in Turkey
(Elmayanı village in Denizli and Kirikkale road in
Ankara) and two localities in Italy (Montelago in Appen-
nino Umbro-Marchigiano and Pian Grande in Sibillini
Mountains) were previously analyzed, and all of them
also contained significant amounts of aliphatic esters [5]
[14][15]. The most prominent constituent in Italian H.ter-
natum fruit oils was also octyl acetate (54.9 –60.2%), fol-
lowed by octyl butanoate (10.1 –13.4%) [5]. The fruit oil
of H.ternatum from Ankara was dominated by octyl
butanoate (37.7%), followed by octyl acetate (31.6%),
and from Denizli by n-octanol (50.3%), followed by octyl
butanoate (24.6%) and octyl acetate (7.3%) [14][15].
n-Octanol (9.6%) and octyl butanoate (2.4%) were also
identified in H.ternatum fruit oil in the present study.
Similarly, the fruit oils of six Heracleum species from
Iran were dominated by aliphatic esters. Three of them,
H.rawianum C. C. TOWNS., H.pastinacifolium C. KOCH
and H.anisactis BOISS.&HOHEN., contained high
amounts of octyl acetate (48.71 –75.36%), while H. per-
sicum DESF., H.gorganicum RECH. f. and H.rechingeri
had similar content of octyl acetate (13.84 –20.48%) and
hexyl butanoate (17.73 –38.36%) [22]. Results obtained
in our study agree with previous finding that aliphatic
esters can be considered as marker compounds of
Heracleum fruit oils [23].
Antimicrobial Activity
The antibacterial activity of H.verticillatum and H.terna-
tum root, leaf, and fruit essential oils is shown in Table 2.
The root essential oils, particularly the one of H.verticilla-
tum, exhibited significantly better antibacterial activity than
the leaf and fruit oils. Heracleum verticillatum root oil was
more active than ampicillin against Staphylococcus aureus
(MIC =0.14 mg/ml, MBC =0.28 mg/ml) and Pseu-
domonas aeruginosa (MIC =0.28 mg/ml, MBC =0.55 mg/
ml), while the activity of this oil against Salmonella
Chem. Biodiversity 2016,13, 466 – 476 467
©2016 Verlag Helvetica Chimica Acta AG, Z€
urich www.cb.wiley.com

Table 1. Chemical composition of Heracleum root, leaf, and fruit essential oils
RI exp
a
)RI lit
b
) Compound
c
) Content [%]
d
)
H.verticillatum H.ternatum
Root Leaf Fruit Root Leaf Fruit
890 880 Isopropyl 2-methylbutanoate –––––0.7
896 –Isopropyl isovalerate –––––0.5
901 900 n-Nonane ––0.1 0.6 –tr
902 –4-Nonene+3-Nonene 1.3 0.5 ––––
902 901 Heptanal tr 0.2 tr 0.6 tr tr
914 908 Isobutyl isobutanoate ––0.1 ––0.2
916 –2-Nonene 0.5 tr ––––
933 924 a-Thujene –––tr tr –
940 932 a-Pinene 0.7 0.2 –5.5 0.8 0.6
956 946 Camphene –––0.4 0.1 –
966 952 Benzaldehyde –––tr tr –
977 969 Sabinene tr 0.4 –tr tr tr
985 974 b-Pinene 23.5 5.7 tr 47.3 0.3 0.1
989 981 6-Methyl-5-hepten-2-one ––––tr –
992 988 Myrcene 0.9 0.6 –2.8 0.6 tr
1005 998 n-Octanal 0.5 0.5 1.0 1.5 tr 2.1
1008 –Isobutyl isovalerate –0.1 0.1 tr –0.3
1016 1007 Isoamyl isobutanoate –––––0.1
1019 –2-Methylbutyl isobutanoate tr tr 0.1 ––0.5
1030 1020 p-Cymene tr tr –tr 0.1 –
1036 1024 Limonene 19.2 20.3 tr 2.3 0.4 0.2
1042 1032 (Z)-b-Ocimene 1.1 1.5 –15.6 0.4 0.1
1046 –Butyl 2-methylbutanoate –––––0.3
1050 –Butyl isovalerate –––––0.2
1053 1044 (E)-b-Ocimene 1.2 1.7 –0.3 0.2 –
1058 –(4Z)-2-Methyl-4-decene –––0.2 ––
1063 1054 c-Terpinene –––tr 0.2 –
1067 1047 (3Z)-Octen-1-ol –––––0.7
1067 –2-Methyldecane –––0.3 ––
1074 1063 n-Octanol –tr 1.6 ––9.6
1082 –Isobutyl 3-methyl-2-butenoate tr tr 0.2 tr –0.1
1093 1086 Terpinolene 9.0 0.4 –1.1 tr –
1095 –(E)-4-Undecene –––0.3 ––
1101 1100 n-Undecane –––tr ––
1102 1100 Isopentyl 2-methylbutanoate –––––0.1
1104 1100 n-Nonanal –0.5 –0.3 ––
1106 1100 2-Methylbutyl 2-methylbutanoate tr –tr –tr 0.9
1108 1102 Isopentylisovalerate –––tr ––
1110 1103 2-Methylbutyl isovalerate 0.4 1.1 tr tr tr 0.3
1129 –Cyclooctanone 0.2 –– – ––
1131 1122 a-Campholenal –––tr tr tr
1133 1128 allo-Ocimene –––tr ––
1136 1128 (Z)-Epoxyocimene –––tr ––
1141 1137 trans-Limonene oxide –0.1 ––––
1143 1135 Nopinone 0.2 –– tr ––
1145 1137 trans-Sabinol 0.7 –– 0.6 ––
1151 –4,8-epoxy-p-Menth-1-ene 1.7 tr ––––
1152 1147 Hexyl isobutanoate ––0.4 ––0.6
1163 1157 (2E)-Nonen-1-al tr tr ––––
1168 1160 Pinocarvone tr tr –0.2 tr –
1176 1168 2-Methoxy-3-(1-methylpropyl)pyrazine 0.3 tr ––––
1181 1174 Terpinen-4-ol –––tr tr –
1181 –1,8-Menthadien-4-ol 0.4 tr –– ––
1185 –1-Methylbutyl 3-methyl-2-butenoate 0.5 0.4 0.2 tr tr 0.2
1191 1167 Octanoic acid –––––0.1
1191 1179 p-Cymen-8-ol 7.4 –– tr tr –
1195 1186 a-Terpineol 0.6 tr –0.5 tr –
1196 1193 (4Z)-Decenal ––1.2 ––0.5
468 Chem. Biodiversity 2016,13, 466 – 476
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urich

Table 1. (cont.)
RI exp
a
)RI lit
b
) Compound
c
) Content [%]
d
)
H.verticillatum H.ternatum
Root Leaf Fruit Root Leaf Fruit
1200 1195 Myrtenal 0.4 tr –0.2 ––
1200 1195 Chavicol methyl ester –––tr ––
1208 1201 n-Decanal ––0.7 ––1.2
1221 1211 Octyl acetate –tr 42.3 –tr 49.0
1241 1233 Hexyl 2-methylbutanoate ––0.4 ––0.7
1246 1241 Hexyl isovalerate ––0.1 tr tr 0.3
1249 1239 Carvone 0.2 tr ––––
1261 1255 (4Z)-Decen-1-ol –––––tr
1265 1260 (2E)-Decenal –––0.7 ––
1288 1284 Bornyl acetate –––tr tr 0.1
1295 1292 (2E,4Z)-Decadienal –––tr ––
1299 1300 n-Tridecane –––tr ––
1305 –Octyl propaonate ––1.4 ––0.1
1309 1305 Undecanal –––––0.1
1318 1315 (2E,4E)-Decadienal –––0.3 tr tr
1324 –Hexyl 3-methyl-2-butenoate ––0.1 –––
1342 1339 trans-Carvyl acetate 0.5 –– – ––
1352 –Octyl isobutanoate ––15.2 ––0.2
1353 1346 a-Terpinyl acetate –––0.4 ––
1362 1356 Eugenol ––––tr 0.1
1372 1369 Cyclosativene 0.3 –– – ––
1376 1373 a-Ylangene 0.6 tr ––––
1379 1374 a-Copaene ––––0.1 –
1383 1380 Daucene 0.3 tr ––––
1388 1387 b-Bourbonene ––––0.4 tr
1391 –Octyl butanoate –––––2.4
1392 1389 b-Cubebene ––––0.1 –
1394 1389 b-Elemene ––––1.0 –
1395 –1-Butenylidenecyclohexane ––0.5 ––0.6
1398 1397 (Z)-Trimenal ––1.5 ––0.2
1405 1403 Methyl eugenol –––0.5 10.7 tr
1412 1407 Decyl acetate ––0.4 ––0.6
1412 1408 Dodecanal –tr 0.1 ––0.6
1418 –Bornyl isobutanoate –––tr tr tr
1426 1417 (E)-Caryophyllene –19.1 ––8.5 –
1429 1421 (E)-Trimenal ––0.7 –––
1433 1430 b-Copaene ––––tr –
1439 1432 a-trans-Bergamotene 0.2 tr –tr ––
1442 –Octyl 2-methylbutanoate ––22.3 ––0.1
1444 –Octyl isovalerate ––2.6 ––0.1
1456 1452 a-Humulene tr 1.4 ––1.5 tr
1460 1454 (E)-b-Farnesene tr –– tr 0.8 tr
1464 1464 a-Acoradiene ––––tr –
1471 1465 c-Decalactone –––tr –0.1
1483 1479 ar-Curcumene 0.4 0.3 –– ––
1487 1487 (E)-b-Ionone –0.2 ––––
1488 1484 Germacrene D ––––9.4 –
1489 1486 Phenylethyl 2-methylbutanoate ––––tr –
1491 1499 4-epi-cis-Dihydroagarofuran –––0.5 ––
1493 1489 b-Selinene 0.6 tr tr –tr –
1493 1490 Phenylethyl isovalerate ––––tr –
1495 1495 2-Tridecanone –0.2 ––––
1496 1493 a-Zingiberene ––––tr –
1497 1496 Valencene 0.8 –tr –––
1497 1500 Bicyclogermacrene –0.8 –1.3 1.3 –
1502 1500 Isodaucene 0.2 –– tr 1.4 –
1511 1505 b-Bisabolene 0.5 0.2 –tr 1.5 –
Chem. Biodiversity 2016,13, 466 – 476 469
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urich www.cb.wiley.com

Table 1. (cont.)
RI exp
a
)RI lit
b
) Compound
c
) Content [%]
d
)
H.verticillatum H.ternatum
Root Leaf Fruit Root Leaf Fruit
1518 1513 c-Cadinene 0.4 –– – ––
1519 –Bornyl isovalerate –––0.3 tr tr
1522 1520 7-epi-a-Selinene 0.7 –– – ––
1522 –Octyl 3-methyl-2-butenoate ––0.7 –––
1527 1521 b-Sesquiphellandrene –––tr 2.0 tr
1532 1529 Kessane –––2.4 ––
1545 1545 Selina-3,7(11)-diene ––––0.2 –
1548 1544 a-Calacorene tr –– – ––
1561 1555 Elemicin tr tr –tr 12.6 tr
1566 1561 (E)-Nerolidol –––0.4 ––
1578 1568 (Z)-Isoelemicin –––tr 35.1 0.1
1583 1577 Spathulenol 3.7 1.8 –1.2 ––
1585 –Octyl hexanoate ––1.2 ––11.3
1587 1582 Caryophyllene oxide –5.4 –– 3.7 –
1599 1594 Salvial-4(14)-en-1-one ––––0.1 –
1599 1600 Guaiol –––0.4 ––
1611 1608 Humulene epoxide II –0.4 ––0.7 –
1619 1618 1,10-di-epi-Cubenol 0.4 –– – ––
1635 1641 Caryophylla-4(14),8(15)-dien-5b-ol –0.8 ––––
1640 1641 Caryophylla-4(14),8(15)-dien-5a-ol –1.8 ––––
1641 1631 (E)-Sesquilavandulol –––0.4 ––
1644 –Isospathulenol 0.3 –– – ––
1667 1665 Intermedeol 10.9 1.1 0.1 0.5 ––
1670 1670 Bulnesol –––0.7 ––
1673 1670 14-Hydroxy-9-epi-(E)-Caryophyllene –0.8 ––––
1684 1685 a-Bisabolol tr 0.4 –– ––
1691 1685 Germacra-4(15),5,10(14)-trien-1-a-ol ––––1.7 –
1779 –Octyl octanoate ––0.2 ––3.2
1838 –Neophytadiene –2.7 ––0.3 –
1844 –Hexahydrofarnesyl acetone –0.9 –– 0.1 –
1878 1874 n-Hexadecanol –0.6 ––––
1966 1959 Hexadecanoic acid –1.6 –0.6 ––
2028 2033 Isobergapten tr tr tr tr ––
2038 2035 (Z)-Falcarinol 0.9 5.1 –0.8 ––
2058 2056 Bergapten tr tr tr tr –tr
2076 –(Z)-9-Octadecen-1-ol tr 0.8 –– ––
2107 –c-Palmitolactone –8.9 ––tr –
2112 –Phytol isomer –2.1 –– 0.1 –
2126 –Pimpinellin 0.5 0.6 tr 0.3 ––
2140 2140 Osthole tr 1.6 ––––
2233 2237 Isopimpinellin tr –– tr ––
2296 2300 n-Tricosane ––––tr –
2493 2500 n-Pentacosane –0.2 ––tr –
2693 2700 n-Heptacosane –0.9 ––tr –
2892 2900 n-Nonacosane –0.5 ––tr –
Monoterpene hydrocarbons 55.6 30.9 tr 75.1 3.1 1.1
Oxygenated monoterpenes 11.9 0.1 –2.2 tr 0.1
Sesquiterpene hydrocarbons 5.0 21.7 tr 1.3 28.2 tr
Oxygenated sesquiterpenes 15.3 12.5 0.1 6.5 6.2 –
Phenylpropanoids tr tr –0.5 58.4 0.2
Aliphatic esters 0.9 1.6 88.0 tr tr 72.7
Others 4.4 28.4 7.4 6.5 0.5 15.7
Total identified 93.1 95.3 95.5 92.1 96.4 89.8
–, not detected; tr, trace (<0.1%);
a
)RI exp, retention indices on HP-5MS column relative to C
8
–C
40
n-alkanes;
b
)RI lit, retention indices
obtained from the literature [36];
c
) Constituents listed in the order of elution on HP-5MS column;
d
) [%], relative area percentage of the
compounds obtained from FID area percent data.
470 Chem. Biodiversity 2016,13, 466 – 476
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typhimurium (MIC =0.14 mg/ml, MBC =0.28 mg/ml) was
better than the activity of both ampicillin and streptomycin.
The strongest effect of H. ternatum root oil, which was
comparable with the effect of streptomycin and better than
the effect of ampicillin, was exhibited against P.aeruginosa
(MIC =0.12 mg/ml, MBC =0.52 mg/ml).
The significance of obtained results is reflected in the
fact that these microorganisms cause various diseases. Sta-
phylococcus aureus infection is a major cause of skin, soft
tissue, respiratory, bone, joint, and endovascular disorders
[24]. Infection by the enteric pathogen S.typhimurium
generally results in severe abdominal cramping and diar-
rhea [25]. Pseudomonas aeruginosa is a significant source
of hospital-acquired infections, such as urinary tract infec-
tions in catheterized patients, pneumonia in patients on
respirators, and infections of patients with burns or cystic
fibrosis [26].
Heracleum ternatum fruit oils from both localities in
Italy and from Denizli in Turkey, which chemical compo-
sition was discussed earlier, were also tested for antibacte-
rial activity [5][14]. The oil from Denizli, dominated by
n-octanol (50.3%), was the most active. It inhibited the
growth of Bacillus cereus,Listeria monocytogenes,S. au-
reus,S. typhimurium,Escherichia coli, and P. aeruginosa
(MICs=0.125 –1.0 mg/ml). Its antimicrobial activity was
explained by the presence of n-octanol, which showed the
same MICs as the oil against these microorganisms [14].
The oils of H.ternatum fruits from Italy, dominated by
octyl acetate, exhibited weaker antibacterial activity
(MICs more than 20 mg/ml), which authors explained by
the lower content of n-octanol (0.9 –4.0%) [5][15].
Results obtained in the present investigation agree with
these findings. Tested H.ternatum fruit oil, which con-
tained 9.6% of n-octanol, exhibited weaker activity
(MICs=1.88 –3.75 mg/ml) than the oil from Turkey and
better activity than the oil from Italy.
The antifungal activity of H.verticillatum and H.terna-
tum root, leaf, and fruit essential oils is presented in
Table 3.Heracleum verticillatum root oil was again the
most active. The effect of this oil against Trichoderma vir-
ide (MIC =0.05 mg/ml, MFC =0.11 mg/ml) was better
than the effects of bifonazole and ketoconazole. The
activity against Aspergillus fumigatus,A.versicolor
(MICs=0.11 mg/ml, MFCs=0.22 mg/ml), and A.ochraceus
(MIC =0.22 mg/ml, MFC =0.44 mg/ml) was comparable
with the activity of bifonazole and better than the activity of
ketoconazole. The effects against A.niger,Penicillium
funiculosum, and P.verrucosum var. cyclopium were
slightly weaker than the effects of both antibiotics.
The obtained results are interesting because the
members of Aspergillus and Penicillium genera are com-
mon food contaminants and producers of some carcino-
genic mycotoxins [27]. Besides being a food contaminant,
A.fumigatus is an airborne fungal pathogen that can
usually cause a fatal invasive aspergillosis in immuno-
compromised patients [28]. Trichoderma species rarely
infect humans, but some members of this genus can
Table 2. Antibacterial activity of Heracleum essential oils and antibiotics expressed as minimum inhibitory and minimum bactericidal concentrations, MICs and MBCs [mg/ml]
Bacteria H.verticillatum H.ternatum Streptomycin Ampicillin
Root Leaf Fruit Root Leaf Fruit
MIC
a
)
b
)
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
Staphylococcus aureus 0.14 0.03
b
0.28 0.00
b
1.10 0.00
d
3.30 0.01
d
4.30 0.01
f
8.60 0.03
g
0.52 0.00
c
1.04 0.03
c
2.40 0.00
e
4.80 0.10
f
1.88 0.06
de
3.75 0.06
e
0.04 0.00
a
0.09 0.00
a
0.25 0.02
bc
0.37 0.01
b
Bacillus cereus 0.55 0.01
c
1.10 0.03
c
0.55 0.03
c
1.10 0.06
c
4.30 0.03
f
8.60 0.10
g
1.04 0.06
d
2.09 0.06
d
2.40 0.06
e
4.80 0.06
f
1.88 0.03
de
3.75 0.01
e
0.09 0.30
a
0.17 0.07
a
0.25 0.00
b
0.37 0.01
b
Micrococcus flavus 0.55 0.06
c
2.20 0.00
c
3.30 0.01
f
4.40 0.03
e
6.30 0.10
g
8.60 0.10
f
1.49 0.06
d
2.09 0.03
c
0.60 0.03
c
1.20 0.03
b
2.53 0.00
e
3.75 0.06
d
0.17 0.01
a
0.34 0.00
a
0.25 0.02
b
0.37 0.00
b
Listeria monocytogenes 1.10 0.06
c
2.20 0.06
c
2.20 0.02
d
4.40 0.00
d
4.30 0.06
f
8.60 0.06
f
2.09 0.03
d
4.15 0.06
d
4.80 0.06
f
9.60 0.10
g
3.75 0.06
e
7.50 0.03
e
0.17 0.01
a
0.34 0.00
a
0.37 0.00
b
0.49 0.03
b
Pseudomonas aeruginosa 0.28 0.00
ab
0.55 0.01
b
4.40 0.03
e
6.60 0.00
e
6.30 0.03
f
8.60 0.06
f
0.12 0.00
a
0.52 0.01
b
2.40 0.06
d
4.80 0.00
d
1.88 0.01
c
3.75 0.01
c
0.17 0.01
a
0.34 0.00
a
0.74 0.03
b
1.24 0.00
c
Salmonella typhimurium 0.14 0.02
a
0.28 0.03
a
3.30 0.06
e
4.40 0.06
d
8.60 0.00
f
17.20 1.20
e
2.09 0.03
d
4.15 0.06
cd
2.40 0.03
d
4.80 0.06
d
1.88 0.06
c
3.75 0.06
c
0.17 0.01
a
0.34 0.00
ab
0.37 0.01
b
0.49 0.03
b
Escherichia coli 0.41 0.01
c
1.10 0.01
c
0.55 0.06
cd
1.10 0.00
c
8.60 0.10
g
17.20 1.20
f
0.75 0.00
d
1.04 0.03
c
4.80 0.06
f
9.60 0.00
e
2.53 0.06
e
3.75 0.00
d
0.17 0.00
a
0.34 0.03
a
0.25 0.02
b
0.49 0.01
b
Enterobacter cloacae 1.10 0.01
c
2.20 0.00
c
2.20 0.03
d
4.40 0.06
e
12.90 1.20
e
17.20 1.20
f
2.09 0.06
d
4.15 0.06
e
2.40 0.03
d
4.80 0.01
e
1.88 0.03
cd
3.75 0.00
d
0.26 0.00
a
0.52 0.01
a
0.37 0.01
b
0.74 0.01
b
a
)MICs and MBCs are expressed as the mean SD determined from the results obtained in three independent experiments.
b
) Different letters in superscript indicate significant
differences between the mean values of MICsorMBCs(P<0.05).
Chem. Biodiversity 2016,13, 466 – 476 471
©2016 Verlag Helvetica Chimica Acta AG, Z€
urich www.cb.wiley.com

cause pulmonary infections or peritoneal dialysis-asso-
ciated peritonitis in patients with impaired immune sys-
tem [29].
Significant numbers of essential oils and their compo-
nents have been screened for antimicrobial activity
against a wide range of microorganisms over the years.
The antimicrobial action of essential oils and their con-
stituents is usually attributed to their lipophilic nature,
which allows them to disrupt bacterial membrane struc-
ture and to penetrate through membrane to the interior
of the cell, causing, for example, loss of ions and reduc-
tion in membrane potential, collapse of the proton pump
and depletion of the ATP pool, impairment of bacterial
enzyme systems, coagulation of cytoplasm, leakage of
macromolecules, and lysis. The antimicrobial activity of
an essential oil is often attributed to its major compo-
nents, but the synergistic or antagonistic effects of com-
pounds which are present in minor quantities has to be
considered [30]. Previously, some of the microorganisms
used in present research showed susceptibility to several
H.verticillatum and H.ternatum oils components, which
are present both in major or minor quantities (b-pinene,
limonene, (E)-caryophyllene, methyl eugenol, n-octanol,
germacrene D, terpinolene, a-pinene, caryophyllene
oxide, (Z)-falcarinol, and a-humulene) [14][31].
Cytotoxic Effect
All the tested essential oils, except H.verticillatum leaf
oil, exhibited significant activity (IC
50
=5.9 –25.3 lg/ml)
on human malignant cervix adenocarcinoma HeLa, colon
carcinoma LS174, and nonsmall cell lung carcinoma A549
cell lines. Their effect satisfied the criterion of the
National Cancer Institute (NCI) for cytotoxicity
(IC
50
<30.0 lg/ml) [32]. The results are shown in Table 4.
Among H.ternatum oils, the most active was the root oil
against LS174 cells (IC
50
=6.7 lg/ml), followed by the
leaf oil against A549 cells (IC
50
=7.4 lg/ml). Regarding
H.verticillatum oils, the strongest effect was exhibited by
the root oil, especially on A549 cell line (IC
50
=5.9 lg/
ml). This effect was comparable with the effect of the
positive control, cisplatin on A549 cell line (IC
50
=4.2 lg/
ml). Cisplatin showed stronger cytotoxic activity than
the tested oils against malignant cell lines (IC
50
=
0.84 –4.2 lg/ml), but also much higher cytotoxicity
against human normal fetal lung fibroblast MRC-5 cell
line (IC
50
=15.2 lg/ml). IC
50
values of the tested oils on
MRC-5 cell line were higher than 120.1 lg/ml.
The mechanisms of the cytotoxic activity of essential
oils and their components on cancer cell lines are similar to
the mechanisms of their antimicrobial activity [30]. The
cytotoxicity of some major as well as minor compounds of
investigated oils (e.g. b-pinene, limonene, (E)-caryophyl-
lene, elemicin, n-octanol, caryophyllene oxide, (Z)-falcari-
nol, and a-humulene), has been previously demonstrated
against some of the cell lines used in the present study [33].
Moreover, limonene exhibited chemopreventive and
Table 3. Antifungal activity of Heracleum essential oils and antibiotics expressed as minimum inhibitory and minimum fungicidal concentrations, MICs and MFCs [mg/ml]
Fungi H. verticillatum H. ternatum Bifonazole Ketoconazole
Root Leaf Fruit Root Leaf Fruit
MIC
a
)
b
)
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
Aspergillus fumigatus 0.11 0.00
a
0.22 0.01
a
0.80 0.00
c
1.10 0.00
c
2.15 0.00
e
4.30 0.03
e
4.15 0.03
f
8.30 0.03
f
0.60 0.01
c
1.20 0.00
c
1.88 0.03
d
3.75 0.02
d
0.15 0.00
ab
0.20 0.01
a
0.20 0.01
b
0.50 0.00
b
Aspergillus versicolor 0.11 0.03
a
0.22 0.02
a
1.10 0.01
c
4.40 0.03
e
1.08 0.01
c
2.15 0.06
d
2.09 0.03
d
4.15 0.03
e
0.30 0.01
b
0.60 0.03
b
0.48 0.03
b
0.95 0.06
c
0.10 0.01
a
0.20 0.02
a
0.20 0.00
ab
0.50 0.02
b
Aspergillus ochraceus 0.22 0.06
a
0.44 0.06
b
1.60 0.06
c
4.40 0.06
d
1.08 0.03
bc
2.15 0.03
c
4.15 0.02
d
8.30 0.01
e
4.80 0.03
e
9.60 0.06
f
0.95 0.00
b
1.88 0.06
bc
0.15 0.02
a
0.20 0.03
a
1.50 0.07
c
2.00 0.10
c
Aspergillus niger 0.33 0.03
c
0.44 0.00
b
3.30 0.03
f
4.40 0.01
d
2.15 0.06
e
8.60 0.03
e
4.15 0.00
g
8.30 0.03
e
4.80 0.03
h
9.60 0.01
f
0.95 0.04
d
1.88 0.02
c
0.15 0.00
a
0.20 0.02
a
0.20 0.01
ab
0.50 0.02
b
Trichoderma viride 0.05 0.03
a
0.11 0.06
a
2.20 0.00
f
4.40 0.03
f
1.08 0.03
e
2.15 0.01
e
2.09 0.03
f
4.15 0.06
f
0.60 0.00
d
1.20 0.03
d
0.48 0.06
c
0.95 0.00
c
0.15 0.01
b
0.20 0.02
b
1.00 0.01
e
1.00 0.00
c
Penicillium funiculosum 0.22 0.01
a
0.88 0.03
c
1.10 0.01
c
4.40 0.02
e
1.61 0.02
cd
2.15 0.00
d
2.09 0.03
e
4.15 0.03
e
1.20 0.03
c
2.40 0.06
d
0.48 0.06
b
0.95 0.06
c
0.20 0.02
a
0.25 0.02
a
0.20 0.00
a
0.50 0.02
ab
Penicillium ochrochloron 0.44 0.06
b
0.88 0.06
b
1.10 0.06
c
2.20 0.06
d
1.08 0.03
c
2.15 0.03
d
4.15 0.02
e
8.30 0.01
g
2.40 0.03
d
4.80 0.00
f
0.95 0.06
c
1.88 0.00
c
0.20 0.00
a
0.25 0.00
a
2.50 0.07
d
3.50 0.03
e
Penicillium verrucosum 0.22 0.00
a
0.44 0.01
b
2.20 0.03
d
4.40 0.06
e
1.08 0.06
bc
2.15 0.03
d
4.15 0.06
e
8.30 0.06
f
1.20 0.02
c
2.40 0.00
d
0.95 0.03
b
1.88 0.00
c
0.10 0.00
a
0.20 0.01
a
0.20 0.01
a
0.30 0.00
ab
a
)MICs and MFCs are expressed as the mean SD determined from the results obtained in three independent experiments.
b
) Different letters in superscript indicate significant
differences between the mean values of MICsorMFCs(P<0.05).
472 Chem. Biodiversity 2016,13, 466 – 476
www.cb.wiley.com ©2016 Verlag Helvetica Chimica Acta AG, Z€
urich

therapeutic effects against mammary tumors in rats and
metastasis of human gastric cancer [34].
Conclusions
This study contributes to the investigations of the chemi-
cal composition of the essential oils of the genus Hera-
cleum, providing the comparison of the qualitative and
quantitative similarities of H.verticillatum and H.terna-
tum essential oils with previously investigated Heracleum
oils. It can be concluded that tested essential oils isolated
from different plant parts, particularly those from the
roots, exhibited promising antimicrobial and cytotoxic
activity, and that they are good candidates for further
bioactivity and mode of action testing.
This work was financially supported by the Ministry of
Education, Science, and Technological Development of the
Republic of Serbia (Project Nos. 173021, 173032, and
175011).
Experimental Part
General
Dimethylsulfoxide (DMSO) was purchased from Merck
KGaA (Darmstadt, Germany); Tryptic Soy broth (TSB)
and malt agar from Institute of Immunology and Virology
Torlak (Belgrade, Serbia); streptomycin, Tween 80 and p-
iodonitrotetrazolium violet (INT) from Sigma–Aldrich
(St. Louis, MO, USA); ampicillin from Panfarma (Bel-
grade, Serbia); bifonazole from Srbolek (Belgrade, Serbia);
ketoconazole from Zorka Pharma (

Sabac, Serbia); 2-[4-(2-
hydroxyethyl)piperazinyl]ethanesulfonic acid (HEPES),
RPMI-1640 nutrient medium, fetal bovine serum (FBS), 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), and L-glutamine from Sigma Chemical Co. (St.
Louis, MO, USA); homologue series of n-alkanes
(C
8
–C
40
) from Fluka (Buchs, Switzerland).
Gas chromatography (GC) analysis of the essential
oils was carried out using an Agilent 6890N gas
chromatograph (Agilent Technologies, Palo Alto, CA,
U.S.A.), while gas chromatography/mass spectrometry
(GC/MS) analysis was performed on an Agilent 6890-5975
GC/MS system (Agilent Technologies).
Plant Material
Heracleum verticillatum roots and fruits were collected in
August 2012, and the leaves in July 2014 on Mt. Stara
Planina in eastern Serbia. Heracleum ternatum roots,
leaves, and fruits were collected in August 2013 on Mt.
Durmitor in the northwestern part of Montenegro. Vou-
cher specimens are deposited in the Herbarium of the
Natural History Museum, Belgrade (BEO) under acces-
sion numbers 20120803/04 for H.verticillatum and
20130807/14 for H.ternatum, respectively. The plants
were identified by Dr. Marjan Niketi
c, curator/botanist of
the BEO.
Isolation of the Essential Oils
Air-dried plant material was powdered (roots and fruits)
or crashed (leaves) and hydrodistilled using Clevenger-
type apparatus, according to the procedure of the Euro-
pean Pharmacopoeia 7.0 [35]. Collecting solvent was
n-hexane. Plant material was hydrodistilled for 2.5 h. The
oils were dried over anhydrous Na
2
SO
4
and kept at 4 °C
until analysis.
Essential Oils Analysis
GC Analysis.Agilent 6890N gas chromatograph was
equipped with a split/splitless injector (200 °C), attached
to a HP-5MS capillary column (Agilent Technologies;
30 m 90.25 mm; film thickness 0.25 lm) and connected
to a flame-ionization detector (FID). The FID and trans-
fer line temperatures were set at 300 and 250 °C, respec-
tively. Split ratio was 1:10 and the injected volume was
1ll of 3% solution of oil in 99.9% (v/v) EtOH. The car-
rier gas was He (1.0 ml/min). The thermal program was
set from 60 to 280 °C at a rate of 3 °C/min.
Table 4. Cytotoxic activity of Heracleum essential oils and cisplatin expressed as IC
50
[lg/ml]
Essential oils IC
50a
)[lg/ml]
Malignant cells Normal cells
HeLa LS174 A549 MRC-5
H. verticillatum root 8.3 0.5 13.9 1.1 5.9 0.2 >200
H. verticillatum leaf 66.8 5.2 146.0 1.2 135.5 1.3 120.1 1.2
H. verticillatum fruit 11.3 1.2 14.1 0.9 10.8 0.9 >200
H. ternatum root 17.7 0.9 6.7 0.9 12.0 0.6 >200
H. ternatum leaf 14.4 1.0 25.3 1.3 7.4 0.5 >200
H. ternatum fruit 10.5 0.7 23.5 1.8 12.0 1.2 >200
Cisplatin 0.84 0.11 2.8 0.1 4.2 0.7 15.2 0.1
a
)IC
50
values are expressed as the mean SD determined from the results of MTT assay in three independent experiments.
Chem. Biodiversity 2016,13, 466 – 476 473
©2016 Verlag Helvetica Chimica Acta AG, Z€
urich www.cb.wiley.com

GC/MS Analysis. Agilent 6890-5975 GC/MS system
operating in the electron ionization (EI) mode at 70 eV,
was equipped with a split/splitless injector (200 °C) and
attached to a HP-5MS capillary column (Agilent
Technologies;30m90.25 mm; film thickness 0.25 lm).
The analytical conditions were the same as that used for
the GC analysis.
Compound Identification. The identification of the
compounds was based on the comparison of their
retention indices (RI), retention times (Rt), and mass
spectra to those from the NIST/NBS 05, Wiley libraries
8th edition, and the literature [36]. The linear RIs were
determined in relation to homologue series of n-alkanes
(C
8
–C
40
) ran under the same operating conditions.
Relative percentages of the compounds were calculated
based on the peak areas from the FID data.
Antimicrobial Activity. Microbial Strains. The Gram-
positive bacteria Staphylococcus aureus (ATCC 6538),
Bacillus cereus (clinical isolate), Listeria monocytogenes
(NCTC 7973), and Micrococcus flavus (ATCC 10240), and
the Gram-negative bacteria Pseudomonas aeruginosa
(ATCC 27853), Escherichia coli (ATCC 35210), Salmonella
typhimurium (ATCC 13311), and Enterobacter cloacae
(human isolate) were used. The fungi Aspergillus fumigatus
(human isolate), A. versicolor (ATCC 11730),
A. ochraceus (ATCC 12066), A. niger (ATCC 6275),
Trichoderma viride (IAM 5061), Penicillium funiculosum
(ATCC 36839), P. ochrochloron (ATCC 9112) and
P. verrucosum var. cyclopium (food isolate) were tested.
The micromycetes were maintained on malt agar, the
cultures stored at 4 °C, and subcultured once a month.
Antibacterial Activity. Minimum inhibitory and
minimum bactericidal concentrations (MICs and MBCs)
were determined by the microdilution method in 96-well
microtiter plates [37][38]. Bacterial suspensions were
adjusted with sterile saline to a concentration of
1.00 910
5
CFU/ml. The oils were dissolved in 5%
DMSO solution that contained 0.10% Tween 80 (v/v)
(10 mg/ml) and added to TSB medium (100 ll) with
bacterial inoculum (1.00 910
4
CFU per well), to achieve
concentrations from 0.12 to 17.20 mg/ml. The MICs were
defined as the lowest concentrations without visible
bacterial growth (determined at binocular microscope).
Additionally, the MICs were determined by the
colorimetric microbial viability assay that is based on the
reduction of INT color. Results were compared to the
positive control [37][38]. The MBCs were determined by
serial subcultivations of 2 ll of tested oils (dissolved in
medium and inoculated for 24 h) into microtiter plates
that contained 100 ll of broth per well, after further
incubation for 24 h. The lowest concentration without
visible bacterial growth was defined as the MBC,
indicating that 99.5% of the original inoculum was killed.
The optical density of each well was measured by
microplate manager 4.0 (Bio-Rad Laboratories, Hercules,
CA, U.S.A.) at the wavelength of 655 nm and compared
to the blank and positive control. Streptomycin
(0.04 –0.52 mg/ml) and ampicillin (0.25 –1.24 mg/ml)
were used as the positive controls. DMSO (5%) was used
as the negative control.
Antifungal Activity. In order to investigate the
antifungal activity of the oils, modified microdilution
technique was used [39]. Fungal spores were washed off
from the surface of agar plates with 0.85% sterile saline
that contained 0.10% Tween 80 (v/v). Spore suspensions
were adjusted with sterile saline to a concentration of
1.00 910
5
in the final volume of 100 ll per well. The oils
were dissolved in 5% DMSO solution that contained
0.10% Tween 80 (v/v) (10 mg/ml) and added to broth
malt medium with the inoculum (to achieve
concentrations 0.05 –9.60 mg/ml). The lowest
concentrations without visible growth (at the binocular
microscope) were defined as MICs. The minimum
fungicidal concentrations (MFCs) were determined by
serial subcultivations of a 2 ll of the tested oils (dissolved
in medium and inoculated for 72 h) into microtiter plates
that contained 100 ll of broth per well, after further
incubation for 72 h at 28 °C. The MFC was defined as
the lowest concentration without visible growth,
indicating that 99.5% of the original inoculum was killed.
Commercial fungicides bifonazole (0.10 –0.25 mg/ml)
and ketoconazole (0.20 –3.50 mg/ml) were used as the
positive controls. A 5% DMSO was used as the negative
control.
Statistical Analysis. All of the tests were carried out in
triplicate. The results were expressed as mean
values standard deviation (SD), and analyzed by one-way
analysis of variance (ANOVA), followed by Tukey’s HSD
test with a=0.05, to determine whether there is a
statistically significant difference between them. The
analysis was carried out by Statistical Package for the Social
Sciences (SPSS) version 18.0 (IBM, Armonk, NY, USA).
Cytotoxic Activity. Cell Cultures. Cervix adenocarci-
noma HeLa, human colon carcinoma LS174, nonsmall cell
lung carcinoma A549, and human normal fetal lung
fibroblast MRC-5 cell lines (ATCC) were cultured as a
monolayer in the RPMI 1640 nutrient medium, supple-
mented with heat inactivated (at 56 °C) 10% FBS,
3 mmol/l of L-glutamine, and antibiotics, at 37 °C, in a
humidified air atmosphere with 5% CO
2
.
Treatment of Cell Lines. In vitro assay for the cytotoxic
activity of the oils was performed when the cells reached
70 –80% of confluence. The stock solution (100 mg/ml)
of each oil was dissolved in RPMI 1640 medium to obtain
required concentrations. Neoplastic HeLa (2000 cells per
474 Chem. Biodiversity 2016,13, 466 – 476
www.cb.wiley.com ©2016 Verlag Helvetica Chimica Acta AG, Z€
urich

well), LS174 (7000 cells per well), A549 (5000 cells per
well), and normal MRC-5 cells (5000 cells per well) were
seeded into 96-well microtiter plates and 24 h later, after
the cell adhesion, five different, double diluted
concentrations of the oils were added to the wells. The
final concentrations of the oils were 12.5, 25, 50, 100, and
200 lg/ml. Control wells contained only nutrient medium
that was made of RPMI 1640 medium, supplemented with
3 mmol/l L-glutamine, 100 mg/ml streptomycin, 100 IU/ml
penicillin, 10% heat inactivated (56 °C) FBS, and
25 mmol/l HEPES. The pH of the medium was adjusted
to 7.2 with bicarbonate solution. The cultures were
incubated for 72 h.
Determination of Cell Survival (MTT Test). The
effect of the oils on cancer cell survival was determined
by the MTT test (microculture tetrazolium test),
according to Mosmann [40], with modification by Ohno
and Abe [41], 72 h after the addition of the oils. Briefly,
20 ll of MTT solution (5 mg/ml phosphate-buffered
saline, PBS) was added to each well. The samples were
incubated for further 4 h, at 37 °C, in 5% CO
2
humidified
air atmosphere. During this period MTT dye was
converted to insoluble product, formazan, by viable cells.
This precipitate was then dissolved by adding 100 llof
10% sodium dodecylsulfate (SDS). The number of viable
cells in each well was proportional to the intensity of the
light absorbance (A) that was measured 24 h later by an
ELISA plate reader (Multiskan FC Microplate reader,
Thermo Fisher Scientific Inc., Australia Pty Ltd., Scoresby,
Australia) at 570 nm. To calculate cell survival [%], the
Aof a sample with cells grown in the presence of various
concentrations of the oils were divided with control
optical density (the Aof control cells grown only in
nutrient medium) and multiplied by 100. The Aof the
blank was always subtracted from the Aof the
corresponding sample with target cells. The IC
50
value was
defined as the concentration of an agent that inhibits the
survival of 50% cells, compared to the vehicle treated
control. Cisplatin was used as the positive control. The IC
50
values were expressed as mean values SD that were
determined on the basis of the results of three independent
experiments.
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Received May 11, 2015
Accepted September 4, 2015
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