Arch. Biol. Sci., Belgrade, 66 (4), 1539-1545, 2014 DOI:10.

2298/ABS1404539S

ANTIFUNGAL ACTIVITY OF HELICHRYSUM ITALICUM (ROTH) G. DON (ASTERACEAE)

ESSENTIAL OIL AGAINST FUNGI ISOLATED FROM CULTURAL HERITAGE OBJECTS

MILOŠ STUPAR1,*, MILICA LJALJEVIĆ GRBIĆ1, ANA DŽAMIĆ1, NIKOLA UNKOVIĆ1,

MIHAILO RISTIĆ2 and JELENA VUKOJEVIĆ1

1
University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden „Jevremovac“, Studentski trg 16,
11000 Belgrade, Serbia
2
Institute for Medicinal Plant Research “Dr Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia

*Corresponding author: smilos@bio.bg.ac.rs

Abstract - There is considerable interest in the use of essential oils as alternative methods to control micromycetes from cul-
tural heritage objects. We investigated the chemical composition and antifungal activity of the essential oil of Helichrysum
italicum. The main components of the oil were γ-curcumene (22.45%), α-pinene (15.91 %) and neryl acetate (7.85 %). H.
italicum essential oil showed moderate antifungal activity against fungi isolated from cultural heritage objects. The most
susceptible fungi to oil treatment were Epicoccum nigrum and Penicillium sp., while the most resistant was Trichoderma
viride. The H. italicum essential oil showed demelanizing activity against Aspergillus niger.

Key words: antifungal activity; demelanization; essential oils; Helichrysum italicum (Roth) G. Don

INTRODUCTION used in folk medicine. In the Greek-Roman system

of medicine, H. italicum was used as an anti-inflam-
The genus Helichrysum (family Asteraceae, tribe In- matory and anti-infective plant (Ballero and Maxia,
uleae) consists of approximately 600 species wide- 2006). Also, dried flowers of H. italicum had a great
spread all over the world and 25 species native to the reputation in traditional medicine as a choleretic,
Mediterranean area (Morone Fortunato et al., 2010). diuretic and expectorant (Chinou et al., 1996, 1997).
The name of the genus is derived from the Greek The biological activities of the many metabolites of
words “helios” (sun) and “chryos” (gold) and relates to H. italicum, especially volatile components of the es-
the typical long-lasting bright yellow inflorescences, sential oils, produced in the glandular hairs present
known as gold-everlasting or eternal flowers (Pign- on the leaves and flower heads of the plant (Morone
atti, 1982). The best investigated species of this genus Fortunato et al., 2010), have been confirmed in many
is Helichrysum italicum (Roth) G. Don (immortelle recent studies. Previous studies reported many dif-
on Italian), a woody dwarf shrub with yellow flow- ferent activities: anti-inflammatory (Sala et al. 2002),
ers growing on dry cliffs and sandy soil widespread antioxidant (Rosa et al., 2007), anti-allergic (Chinou
along the coast and on the islands of the Adriatic Sea et al., 1997), antibacterial (Nostro et al., 2001) and
(Mastelić et al., 2008). H. italicum has been widely antiviral (Appendino et al., 2007).

1539
1540 MILOŠ STUPAR ET AL.

The aim of this study was to estimate the anti- tor, attached to a HP-5 column (25 m × 0.32 mm,
fungal potential of H. italicum essential oil against 0.52-µm film thickness) and fitted to FID. Carrier
selected fungal species isolated from wooden and gas flow rate (H2) was 1 ml/min, split ratio 1:30,
stone cultural heritage objects. Literature reports injector temperature 250°C, detector temperature
regarding the antifungal properties of H. italicum 300°C, while the column temperature was linearly
essential oil are scarce. However, Angioni et al. programmed from 40-240°C (at the rate of 4°/min).
(2003) reported strong antifungal activity of H. The same analytical conditions were employed for
italicum essential oil against Globisporangium ulti- GC-MS analysis, where a HP G 1800C Series II
mum (Trow) Uzuhashi, Tojo & Kakish, Athelia rolf- GCD system, equipped with a HP-5MS column (30
sii (Curzi) C.C. Tu & Kimbr and moderate activity m × 0.25 mm, 0.25 µm film thickness) was used.
against Phytophthora capsici Leonian and Zymosep- The transfer line was heated to 260°C. The mass
toria tritici (Desm.) Quaedvl. & Crous. Mastelić spectra were acquired in EI mode (70 eV), in m/z
et al. (2005) reported that H. italicum essential oil range 40-400. Identification of individual EO com-
could inhibit growth of Candida albicans (C.P. Rob- ponents was accomplished by comparison of reten-
in) Berkhout. tion times with standard substances and by match-
ing mass spectral data with those held in the Wiley
MATERIALS AND METHODS 275 library of mass spectra. Confirmation was per-
formed using AMDIS software and literature (Ad-
Essential oil ams, 2007). Area percentages obtained by FID were
used as a base for the purpose of quantitative analy-
The essential oil used in this study was a commercial sis.
sample H. italicum (Herba d.o.o, Belgrade, Serbia)
analyzed by the Institute for Medicinal Plant Re- Micro-atmosphere method
search “Dr Josif Pančić”, Belgrade.
The following method allows study of the effect of
Tested fungi the volatile fractions of the EO. The test was per-
formed in sterile Petri plates (85 mm Ø) containing
The fungal isolates used in this research were As- 20 ml of MEA (Maruzzella and Sicurella, 1960). Af-
pergillus niger Tiegh (wood, w), Aspergillus ochraceus ter the inoculation of the tested fungi at the center
G. Wilh (w), Bipolaris spicifera (Bainier) Subram of the MEA, the Petri plates were overturned. Steri-
(stone, s), Epicoccum nigrum Link (s), Penicillium lized filter paper disc was placed in the center of the
Link sp. (w) and Trichoderma viride Pers (w). The Petri plate lid soaked with various amount of EO
molds were deposited with the Mycotheca of the De- in final concentrations of 10, 25, 50, 75 and 100 μL
partment for Algology, Mycology and Lichenology, mL-1. The Petri plates were incubated at 28 ± 1˚C.
Institute of Botany, Faculty of Biology, University of The growth of the tested fungi was measured after
Belgrade. Isolates were maintained on malt extract 21 days and percent of inhibition was computed af-
agar (MEA), potato dextrose agar (PDA), stored at ter comparison with the control. Fungistatic effect
4˚C and subcultured once a month. was expressed in terms of mycelia growth inhibi-
tion (%) and calculated by the formula of Pandey
Gas chromatography (GC) and GC-mass et al. (1982):
spectrometry (GC/MS)
Mycelial growth inhibition (%) = 100 (dc – dt)/dc
Qualitative and quantitative analyses of the EOs
were performed using GC and GC-MS. The GC where dc = average diameter of fungal colony in con-
analysis of the oil was carried out on a GC HP-5890 trol and dt = average diameter of fungal colony in
II apparatus, equipped with a split-splitless injec- treatment.
 ANTIFUNGAL ACTIVITY OF HELICHRYSUM ITALICUM (ROTH) G. DON (ASTERACEAE) ESSENTIAL OIL AGAINST FUNGI 1541

Fig. 1. Morpho-physiological changes documented in Aspergillus niger colony grown in microatmosphere conditions with Helichrysum
italicum essential oil. a. Control; b. Albino colony grown at concentration of 100 μL mL-1.

Statistical analyses increase of H. italicum essential oil concentrations, a

higher percentage of mycelial growth inhibition was
One-way ANOVA was performed for mycelia growth recorded (Table 2).
assay using Microsoft office Excel 2007. A P value less
than 0.05 was considered statistically significant. In addition to inhibited growth, colonies of A.
niger formed in the presence of H. italicum essential
RESULTS oil exhibited distinct morphological variations when
compared to the control, such as visible loss of co-
The H. italicum essential oil predominantly con- nidia melanization and a significantly lower number
tained sesquiterpene hydrocarbons (60.66%), fol- of conidial heads. Colonies with demelanized conidia
lowed by monoterpene hydrocarbons (19.83%). were regarded as albino (Fig 1). After the reinocula-
The oxygenated monoterpenes comprised 4.49% tion of albino colonies on sterile MEA, colonies of A.
and the sesquiterpenes class 3.73% of the total oil. niger with typical species morphology were formed,
Results of the essential oil analysis showed a total of suggesting that morphophysiological changes in-
60 components (98.47% of oil) (Table 1). The main duced with oil were reversible. Demelanizing activity
component of the oil was γ-curcumene (22.45%). was documented toward A. niger only.
Other components present in significant percentage
were α-pinene (15.91%), neryl acetate (7.85%), and DISCUSSION
β-selinene (6.94%).
With regard to the chemical composition of H. itali-
Fungi tested in the micro-atmosphere method cum essential oils, Satta et al. (1999) suggested the
showed different susceptibility to H. italicum es- presence of two different chemotypes, one rich in
sential oil. The most resistant species was T. viride. nerol and esters (chemotype A), and the other abun-
The mycelial growth of this fungus was not inhib- dant with rosifoliol (chemotype B). However, Roussis
ited with any of the oil concentrations used in the et al. (2000) reported the presence of another, third,
experiment (Table 2). E. nigrum and Penicillium sp. chemotype of H. italicum (chemotype C1) with es-
were the most sensitive fungal species (P<0.05). For sential oils rich in β-selinene, γ-curcumene and
these fungi, inhibition of mycelial growth was docu- α-pinene. The oil examined in this research was dom-
mented at the concentration of 25 μL mL-1. With an inated by γ-curcumene (22.45%) and a significant
1542 MILOŠ STUPAR ET AL.

Table 1. Chemical composition of Helichrysum italicum essential oil

Component KIE1 KIL2 %
α-pinene 926.4 932 15.91
α-fenchene 939.2 945 0.39
camphene 949.1 946 0.20
β-pinene 969.0 969 0.31
p-cymene 1019.1 1020 0.06
limonene 1022.4 1024 2.52
1,8-cineole 1025.4 1026 0.30
isobutyl angelate n/a* 1045 0.21
γ-terpinene 1052.9 1054 0.25
α-terpinolene 1082.3 1086 0.19
linalool 1096.5 1095 0.51
isoamyl 2-methyl butyrate 1100.1 1100 0.15
endo-fenchol 1108 1114 0.08
trans-pinocarveol 1133.3 1135 0.08
isoamyl tiglate 1149.6 1148 0.77
nerol oxide 1150.4 1154 0.11
borneol 1160.3 1165 0.10
cis-pinocamphone 1179.1 1172 0.53
terpinen-4-ol 1172.2 1174 0.55
α-terpineol 1185.8 1186 0.26
decanal 1201.8 1201 0.06
nerol 1224.7 1227 0.78
hexyl 2-methyl butanoate 1232.7 1233 0.16
hexyl 3-methyl-2-butenoate 1281.8 n/a* 0.30
2-undecanone 1291.4 1293 0.06
neryl acetate 1362.5 1359 7.85
α-ylangene 1364 1373 0.42
α-copaene 1368.7 1374 3.52
italicene 1395.8 1405 5.42
cis-α-bergamotene 1408.1 1411 1.44
trans-caryophyllene 1411.6 1417 4.74
trans-α-bergamotene 1428.6 1432 3.24
neryl propanoate 1449.4 1452 1.39
allo-aromadendrene 1464.4 1458 0.28
α-acoradiene 1471.0 1464 0.12
β-acoradiene 1473.8 1469 0.64
selina-4,11-diene 1468.2 1475 1.13
γ-curcumene 1474.3 1481 22.45
ar-curcumene 1477.0 1479 1.90
β-selinene 1479.1 1489 6.94
α-selinene 1488.1 1498 4.78
α-muurolene 1493.2 1500 1.39
β-curcumene 1505.0 1514 0.60
δ-cadinene 1516.0 1522 1.52
italicene ether 1526.6 1536 0.54
α-calacorene 1535.4 1544 0.13
trans-nerolidol 1557.1 1561 0.06
caryolan-8-ol 1561.5 1571 0.05
geranyl 2-methylbutyrate 1570.2 1574 0.57
neryl isovalerate 1573.7 1582 0.47
caryophyllene oxide 1585.5 1582 0.08
 ANTIFUNGAL ACTIVITY OF HELICHRYSUM ITALICUM (ROTH) G. DON (ASTERACEAE) ESSENTIAL OIL AGAINST FUNGI 1543

Table 1. Continued

Component KIE1 KIL2 %

globulol 1589.8 1590 0.17
viridiflorol 1595.8 1592 0.40
rosifoliol 1599.1 1600 0.39
humulane-1,6-dien-3-ol 1604.0 1619 0.14
γ-eudesmol 1623.0 1630 0.17
β-eudesmol 1625.8 1649 0.06
selin-11-en-4-a-ol 1646.3 1658 0.40
epi-β-bisabolol 1661.7 1670 0.09
α-bisabolol 1663.2 1674 0.14
Grouped constituents
Monoterpene hydrocarbons 19.83
Oxygenated monoterpenes 4.49
Sesquiterpene hydrocarbons 60.66
Oxygenated sesquiterpenes
3.73
Others 9.76
Total: 98.47
1
Kovats retention index, experimental data
2
Kovats retention index (Adams, 2007)
*
not available

Table 2. Antifungal activity of Helichrysum italicum essential oil against selected fungi
Oil Mycelial growth inhibition (mean ± SE)*(%)
concentration A.n A.o B.s E.n P. T.v
(μL mL-1)
10 0 0 0 0 0 0
25 0 0 0 24.31±2.16 7.26±1.40 0
50 0 0 0 59.66±2.03 12.50±1.66 0
75 2.67±1.4 9.1±0.81 18.67±1.33 65.33±3.72 31.68±8.23 0
100 29±1.4 11.5±1.22 42.33±6.17 76.93±4.05 59.56±4.07 0
*mean of three replication (P < 0.05); A.n - Aspergillus niger; A.o - Aspergillus ochraceus; B.s - Bipolaris spicifera; E.n - Epicoccum nigrum;
P. – Penicillium sp. T.v - Trichoderma viride.

presence of α-pinene (15.91 %) and β-selinene (6.94 during anthesis and rich in β-selinene, α-pinene and
%), which places this oil in the chemotype group C1. γ-curcumene (chemotype C1) have strong antibacte-
The ability of H. italicum essential oil to inhibit myc- rial activity. The tested fungi showed different suscep-
elial growth of tested fungi was monitored using the tibility to oil treatment. T. viride appeared to be the
micro-atmosphere method that allows estimation of most resistant, while E. nigrum and Penicillium sp.
the growth inhibition of mycelia exposed to oil vapor were the most sensitive. The resistance of T. viride can
components. According to Angioni et al. (2003), the be explained by a variety of enzymes produced and
antimicrobial activity of H. italicum essential oils can secreted by mycelia that can detoxify oil components
be considered as moderate. Chinou et al. (1996) sug- into inactive forms (Farooq et al., 2002). Although
gested that the antimicrobial activity of H. italicum oil 100% of mycelia growth inhibition was not accom-
was due to its richness in nerol and ester components plished, even with the highest concentration of oil
(chemotype A). However, Roussis et al. (2000) point- used in the experiment (100 μL mL-1), the morpho-
ed out that the essential oil of H. italicum synthesized physiological variations documented in the A. niger
1544 MILOŠ STUPAR ET AL.

Angioni, A., Barra, A., Arlorio, M., Coisson, J.D., Russo, M.T.,
colonies suggested that the oil components interfered Pirisi, F.M., Satta, M. and P. Cabras (2003). Chemical
with fungal metabolism. It can be concluded that H. composition, plant genetic differences and antifungal ac-
italicum essential oil can prevent A. niger from com- tivity of Helichrysum italicum G. Don ssp. microphyllum
pleting its life cycle, which was demonstrated with (Willd) Nym. J. Agric. Food. Chem. 51, 1030-1034.
the depigmentation and scarce sporulation, leading Appendino, G., Ottino, M., Marquez, N., Bianchi, F., Giana, A.,
to the formation of albino colonies. Conidia of some Ballero, M., Sterner, O., Fiebich, B.L. and E. Munoz (2007).
Aspergillus and Penicillium species contain pigments Arzanol, an anti-inflammatory and anti-HIV-1 phloroglu-
belonging to melanins: a green-colored chromopro- cinol alpha-Pyrone from Helichrysum italicum ssp. micro-
tein and a black insoluble pigment (Eismann and Ca- phyllum. J. Nat. Prod. 70, 608-612.
sadevall, 2012). Abundant sporulation of these fungi Ballero, M. and A. Maxia (2006). Erbosteria Domani, 300, 52-
causes the formation of colonies in different shades 56.
of yellow, green, ochre, blue and black, etc. Altera- Butler, J.M., Day, A.W., Henson, J.M. and N.P. Money (2001).
tions of A. niger colonies induced by H. italicum es- Pathogenic properties of fungal melanins. Mycologia 93,
sential oil may be related to the interference of the oil 1-8.
components in melanin biosynthesis. Other essential Chinou, I.B., Roussis, V., Perdetzolou, D. and A. Loukis (1996).
oils can display demelanizing activity against differ- Chemical and biological studies on two Helichrysum spe-
ent fungi. Sharma and Tripathi (2008) reported the cies of Greek origin. Planta Med. 62, 339-377.
visible pigmentation loss of A. niger colonies grown
Chinou, I.B., Roussis, V., Perdetzolou, D., Tzakou, O. and A.
with essential oil isolated from Citrus sinensis (L.) Loukis (1997). Chemical and antibacterial studies of two
epicarp. Conidia of different Aspergillus species (A. Helichrysum species of Greek origin. Planta Med. 63, 181-
flavus Link, A. parasiticus Speare, A. ochraceus, A. 183.
fumigatus Fresenius and A. niger) lost their pigmen- Eismann, H.C. and A. Casadevall (2012). Synthesis and assem-
tation when treated with Hyptis suaveolens (L.) Poit bly of fungal melanin. Appl. Microbiol. Biotechnol. 93,
essential oil (Pessoa Moreira et al., 2010). Although 931–940.
there are reports that B. spicifera poroconidia can
Farooq, A., Choudhary, M.I., Rahman, A. and S.Tahara (2002).
be demelanized when treated with Nepeta rtanjensis Detoxification of terpinolene by plant pathogenic fungus
Diklić & Milojević essential oil (Ljaljević Grbić et al., Botrytis cinerea. Z. Naturforsch. 57, 863-866.
2011), the tested essential oil of H. italicum did not
Laljević Grbić, M., Stupar, M., Vukojević, J. and D. Grubišić
display any such activity, suggesting different target (2011). In vitro antifungal and demelanizing activity of
mechanisms of these oils. However, melanin produc- Nepeta rtanjensis essential oil against human pathogen Bi-
tion by certain fungi contributes to the virulence of polaris spicifera. Arch. Biol. Sci. 63, 897-905.
human, animal and plant pathogenic fungi (Butler
Maruzella, J.C. and N.A. Sicurella (1960). Antibacterial activity
et al., 2001), and therefore the demelanization effect of essential oil vapors. JAPhA. 49, 692-694.
caused by interaction with essential oils, as antifungal
agents, is significant. Mastelić, J., Politeo, O. and I. Jerković (2008). Contribution to the
analysis of the essential oil of Helichrysum italicum (Roth)
G. Don. − determination of ester bonded acids and phe-
Acknowledgments - This research was carried out as part of nols. Molecules. 13, 795-803.
the project No.173032, financially supported by the Ministry
Mastelić, J., Politeo, O., Jerković, I. and N. Radošević (2005). Com-
of Education, Science and Technological Development of the
position and antimicrobial activity of Helichrysum itali-
Republic of Serbia. cum essential oil and its terpene and terpenoid fractions.
Chem. Nat. Compd. 41, 35-40.
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