Phytochemistry xxx (2017) 1e20

Contents lists available at ScienceDirect

Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Review

Separation of phytochemicals from Helichrysum italicum: An analysis

of different isolation techniques and biological activity of prepared
extracts
Svetolik Maksimovic a, *, Vanja Tadic b, Dejan Skala a, Irena Zizovic a
a
University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, 11120 Belgrade, Serbia
b
Institute for Medical Plant Research Dr Josif Pancic, Tadeusa Koscuska 1, 11000 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Helichrysum italicum presents a valuable source of natural bioactive compounds. In this work, a literature
Received 29 August 2016 review of terpenes, phenolic compounds, and other less common phytochemicals from H. italicum with
Received in revised form regard to application of different separation methods is presented. Data including extraction/separation
29 December 2016
methods and experimental conditions applied, obtained yields, number of identied compounds, con-
Accepted 4 January 2017
Available online xxx
tent of different compound groups, and analytical techniques applied are shown as corresponding tables.
Numerous biological activities of both isolates and individual compounds are emphasized. In addition,
the data reported are discussed, and the directions for further investigations are proposed.
Keywords:
Helichrysum italicum
2017 Elsevier Ltd. All rights reserved.
Asteraceae
Terpenes
Phenolic compounds
Distillation
Supercritical CO2 extraction
Extraction by organic solvents
Biological activity

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Terpenes of H. italicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1. Distillation of H. italicum aerial parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1.1. Hydrodistillation of H. italicum aerial parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1.2. Steam distillation of H. italicum aerial parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Supercritical CO2 extraction from H. italicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.3. Isolation of terpenes from H. italicum using organic solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Phenolic compounds of H. italicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.1. Extraction of H. italicum by acetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.2. Extraction of H. italicum by methanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.3. Extraction of H. italicum by ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3.4. Extraction of H. italicum by other solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
4. Other phytochemicals of H. italicum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
5. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

* Corresponding author.
E-mail addresses: smaksimovic@tmf.bg.ac.rs (S. Maksimovic), vtadic@mocbilja.
rs (V. Tadic), skala@tmf.bg.ac.rs (D. Skala), zizovic@tmf.bg.ac.rs (I. Zizovic).

http://dx.doi.org/10.1016/j.phytochem.2017.01.001
0031-9422/ 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
2 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
1. Introduction
The genus Helichrysum consists of an estimated 600 species. Its
name is derived from the Greek words helios (sun) and chrysos
(gold). H. italicum (Roth) G. Don l. (syn. H. angustifolium subsp.
italicum (Roth) Briq. & Cavill.) from the family Asteraceae is
commonly known as the curry plant or the everlasting plant. It
grows on dry, rocky, or sandy ground around the Mediterranean.
The stems are woody at the base and can reach 30e70 cm in height
(Viegas et al., 2014). It is a small aromatic shrub with yellow
owers. H. italicum can be further divided into six subspecies
distributed in different regions of the Mediterranean basin:
H. italicum (Roth) G. Don subsp. italicum, native to the Mediterra-
nean basin; H. italicum subsp. microphyllum (Willd.) Nyman, char-
acteristic for Balearic Islands (Majorca and Dragonera), Sardinia,
Corsica, Crete, and Cyprus; H. italicum subsp. picardii Franco, found
in the ora of France, Italy, Portugal, and Spain; H. italicum subsp.
pseudolitoreum (Fiori) Bacch, related to Argentario, Gargano, and
Monut Conero; H. italicum subsp. serotinum (Boiss.) P. Fourn., found
in Iberian Peninsula; and H. italicum subsp. siculum (Jord. & Fourr.)
Galbany, found in Sicily (Viegas et al., 2014). The relationships be-
tween the location and the genetic data among some subspecies
have already been reported (Galbany-Casalas et al., 2011).
Isolates of H. italicum consist of many different compound
classes, among which the most common are terpenes and phenolic
compounds. Because of the variety of specialized metabolites,
H. italicum has been reported to possess a wide range of biological
activities. Viegas et al. (2014) in their review article compared re-
ports on the traditional use of H. italicum with those on the activity
of single isolated compounds stating the abundance of literature
data. Traditional use of this plant includes the application for the
treatment of allergies, colds, cough, skin, liver and gallbladder
disorders, inammation, infections, and sleeplessness. It was one of
the main topics of Lourens et al.'s (2008) review article in which
biological activity and phytochemistry of South African Helichrysum
species were also investigated. The results presented by Guinoiseau
et al. (2013) referred to the classication of the main biological
activities of H. italicum essential oils and extracts into ve groups:
antimicrobial, anti-inammatory, anti-viral, antioxidant, and anti-
larvicidal activities. The emphasis was given to the correlation be-
tween the most common individual compounds and their reported
activities.
This review article is an attempt to classify and critically
examine the literature data on biologically active compounds iso-
lated from H. italicum with regard to the particular experimental
procedure of isolation. According to the results of application of
distinct isolation procedures presented in literature, special atten-
tion was given to terpenes and phenolic compounds, followed by
other less signicant phytochemicals. The analyzed isolation pro-
cedures were hydro- and steam distillation and supercritical CO2
extraction, as the favorable procedures for the separation of ter-
penes, and extraction with organic solvents, which is mainly used
for isolation of phenolic compounds. The reported results based on
the isolation procedures are presented as tables. In addition, the
biological activities of extracts and pure compounds are also given
but to the extent that ensures nonrepetition of the presented data
in the abovementioned review articles (Guinoiseau et al., 2013;
Lourens et al., 2008; Viegas et al., 2014).
2. Terpenes of H. italicum
Terpenes, one of the most signicant specialized metabolites
present in H. italicum, possess numerous biological activities, both
individually or as a part of plant isolates. Among them, the greatest
contribution to the composition and biological activity of
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
H. italicum isolates belongs to mono- and sesquiterpenes. As
mentioned, the majority of reported results indicated that hydro-
distillation, including steam distillation, followed by supercritical
CO2 extraction and, in a few cases, extraction with organic solvents
were used for the separation of terpenes from H. italicum. There-
fore, this section mainly focuses on terpene constituents of
H. italicum essential oils, obtained by distillation processes, and
supercritical extracts, emphasizing the data of isolation/extraction
procedures and biological activities of extracts/pure compounds. In
addition, special attention was given to the most signicant rep-
resentatives of H. italicum terpenes.
2.1. Distillation of H. italicum aerial parts
Distillation of H. italicum aerial parts is the most commonly used
technique for obtaining the essential oils, according to the large
amount of literature data. The majority of literature data regarding
H. italicum essential oils obtained by distillation referred to the two
main subspecies: H. italicum (Roth) G. Don subsp. italicum and
H. italicum subsp. microphyllum (Willd.) Nyman. Two distinct types
of distillation are presented in the literature: hydrodistillation
performed in a Clevenger-type apparatus and steam distillation
performed in a spring-type apparatus.
2.1.1. Hydrodistillation of H. italicum aerial parts
Studies reporting hydrodistillation of H. italicum essential oils in
a Clevenger-type apparatus are numerous. According to the liter-
ature data, the applied time for distillation varied from 1 to 5 h, and
the obtained yields were in the range of 0.02e0.78%. The most
common methods for analysis and identication of essential oil
components were GC-FID and GC-MS. Reported experimental
conditions and results in terms of number and overall percentage of
the identied compounds and the main components of the com-
pounds are given in Table 1. Some authors reported the contents of
different compound groups in essential oils, and these data are
presented in Table 2.
Activities of essential oils obtained by hydrodistillation are
diverse. Leonardi et al. (2013) investigated the composition of 21
essential oil samples isolated from H. italicum (Roth) G. Don subsp.
italicum collected at 7 locations of Elba Island (Tuscany, Italy),
characterized by different soil types during three different periods
(January, May, and October 2010). The results of applied statistical
analysis showed a difference in the composition of the essential oils
mainly because of the environment where the plant grew, and, in
particular, to the soil type. Essential oils isolated from samples
collected in locations characterized by intrusive igneous rocks were
rich in a-humulene, g-muurolene, b-caryophyllene, cis-a-berga-
motene, eudesm-5-en-11-ol, eudesmol isomers, and neryl acetate.
Essential oils of sandstone soil samples were characterized by the
presence of a- and b-pinene, camphene, g-terpinene, 1,8-cineole, a-
terpineol, borneol, nerol, and linalool. Furthermore, essential oils of
samples growing on quaternary deposits were rich in eudesm-5-
en-11-ol, viridiorol, nerol and its acetic acid a propanoic acid es-
ters, d-selinene, ar-curcumene, and limonene. Finally, essential oils
of samples collected from serpentine soils were characterized by
the presence of neryl acetate, neryl propanoate, ar-curcumene,
italicene, and limonene. Similar to Leonardi et al. (2013), Paolini
et al. (2006) studied essential oils of 11 samples of H. italicum
(Roth) G. Don subsp. italicum collected from six Tuscan archipelago
islands that showed similarities in the main composition with that
of Corsican, Sardinian, and North American H. italicum subsp.
mycrophyllum essential oils. Satta et al. (1999) studied the chemical
composition of essential oils of H. italicum subp. microphyllum
(Willd.) Nyman collected at different locations in Sardinia, at alti-
tudes between 100 and 900 m. Essential oils obtained at altitudes
 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20 3

Table 1
Literature results of Clevenger-type distillation of H. italicum.

Distillation Yield, Number of Overall percentage of Number Main components Identication References
time, h % isolated identied compounds, of methods
compounds % samples

2 0.1 115 96.8e99.9 21 nerol, neryl acetate, eudesm-5-en-11-ol, g-curcumene, a-pinene, GC-FID, GC- Leonardi
e0.6 limonene, 1,8-cineole, neryl propanoate MS et al. (2013)
e 0.27 49 83.6e97.7 11 nerol, b-diketones, eudesm-5-en-11-ol, ar- and g-curcumene, a- GC-FID, GC- Paolini et al.
e0.78 pinene, b-selinene, neryl acetate MS, 13C NMR (2006)
1.5 0.09 33 e 8 linalool, nerol, neryal acetate, neryl propanoate, g-curcumene GC-MS Satta et al.
e0.21 (1999)
e 0.02 97 47.3e66.7 4 trans-a-bergamotene, b-acoradiene, neryl acetate, rosifoliol GC-MS Zeljkovic
e0.12 et al. (2015)
1 0.31 34 e 146 limonene, linalool, neryl acetate, neryl propanoate, g- and ar- GC-MS Melito et al.
e0.45 curcumene, b-eudesmol, eudesmen-7-(11)-en-4-ol (2016)
3 0.11 57 89.7 1 neryl acetate, d- and g-cadinene, rosifoliol, neryl propanoate GC-FID, GC- Ornano et al.
MS (2014)
1 0.03 e ~90 10 limonene, linalool, a-terpineol, nerol, neryl acetate, neryl GC-MS Angioni et al.
e0.18 propanoate, ar- and g-curcumene, guaiol, rosifoliol (2003)
1 e 35 e 50 caryophyllene, eudesm-5-en-11-ol, neryl propanoate, nerolidol, GC-MS Melito et al.
neryl acetate, cis-b-guaiene, trans-b-guaiene, a-terpinene, g- (2013)
cadinene
3 0.02 44 90.0 1 isoitalicene epoxide, 8-cedren-13-ol, (Z)-a-trans-bergamotol, b- GC-FID, GC- Mancini
costol MS et al. (2011)
4 0.07 48 83.0e96.5 48 nerol, neryl acetate, eudesm-5-en-11-ol GC-FID, GC- Usai et al.
e0.22 MS (2010)
1 0.67 16 90.63 1 neryl acetate, a-pinene, a- and b-eudesmol, geranyl propionate, GC-FID, GC- Charles and
limonene, camphene MS Simon
(1991)
5 0.11 43 82.9e93.6 9 neryl acetate, g-curcumene GC-FID, GC- Bianchini
e0.42 MS et al. (2001)
4 0.25 45 84.5e94.2 8 neryl acetate, nerol, neryl propanoate, b-diketones, a-pinene, a- and GC-FID, GC- Bianchini
e0.41 b-selinene, g-curcumene, eudesm-5-en-11-ol MS, 13C NMR et al. (2003)
5 0.02 28 67.8e94.9 48 limonene, 4,6-dimethyloctane-3,5-dione, nerol, neryl acetate, neryl GC-FID, GC- Bianchini
e0.44 propanoate, g-curcumene, eudesm-5-en-11-ol MS, 13C NMR et al. (2009)
3 0.12 52 90.6 1 a-pinene, a-cedrene, aromadendrene, b-caryophyllene, limonene, GC-MS Mastelic
neryl acetate, geranyl acetate et al. (2005)
3 0.21 44 98.0 1 a-pinene, neryl acetate, 2-methyl-cyclohexyl pentanoate, 1,7-di- GC, GC-MS Mastelic
epi-a-cedrene et al. (2008)
e e 18 91.8 1 neryl acetate, a-pinene, limonene, g-curcumene, neryl propanoate, GC-FID, GC- Conti et al.
nerol MS (2010)
e e e e 1 neryl acetate, neryl propanoate, 2,4,6,9-tetramethyldec-8-en-3,5- e Drapeau
dione et al. (2009)
e 0.3 e e 1 e GC-FID, GC- Moretti et al.
MS (2002)
e e 18 97 1 a-pinene, limonene, nerol, neryl acetate, neryl propanoate, g- GC-FID, GC- Bertoli et al.
curcumene MS (2012)
e e 7 61.9 1 limonene, neryl acetate, neryl propanoate, a-curcumene GC-FID, GC- Idaomar
MS et al. (2002)
4 e 19 84.05 1 a-pinene, neryl butyrate, g-curcumene, trans-b-caryophyllene GC-FID Rossi et al.
(2007)
4 0.23 46 76.8 1 neryl acetate, trans-b-caryophyllene, g-curcumene, a- and b- GC-FID, GC- Ivanovic
selinene MS et al. (2011)
4 0.19 67 e 1 a-pinene, neryl acetate, italicene, trans-b-caryophyllene GC-FID, GC- Maksimovic
MS et al. (2013)
e 0.47 16 75.48 1 neryl acetate, trans-caryophyllene, g-curcumene, a- and b-selinene GC, GC-MS Micic et al.
(2009)
4 0.7 74 94.3 1 neryl acetate, cis-dihydro-occidentalol, nerol, neryl propanoate, ar- GC-MS Marongiu
and g-curcumene, a-cadinol et al. (2003)
3 0.6 30 91.7 1 a-pinene, italicene, g- and ar-curcumene, epi-b-bisabolol GC-FID, GC- Costa et al.
MS (2015)
e 0.15 27 96.1 1 neryl acetate, neryl propanoate, ar- and g-curcumene GC-MS Kladar et al.
(2015)
3 0.11 100 93.65e95.58 2 a-pinene, b-selinene, g-curcumene GC-MS Roussis et al.
e0.68 (2000)
3 e 68 86.1 e guaiol, nerol, b-caryophyllene GC-FID, GC- Tsoukatou
MS et al. (1999)
3 0.14 34e54 95.2e97.3 2 a-pinene, b-caryophyllene, a-selinene, eremophilene, a-muurolol, GC-FID, GC- Maggio et al.
e0.44 b-eudesmol MS (2016)
e 0.04 29e35 63.09e91.19 2 g-curcumene, neryl acetate, neryl propanoate, nerol, linalool, GC-MS Tucker et al.
e0.07 limonene (1997)
e e 40e48 e 10 a-pinene, limonene, b-caryophyllene, aromadendrene, g- GC-FID, GC- Schipilliti
curcumene, a-selinene, neryl acetate, ledol, rosifoliol MS, GC-C- et al. (2016)
IRMS
e e 14 80.47 1 neryl acetate, limonene, g-curcumene, b-diketones GC-MS Cui et al.
(2015)
(continued on next page)

Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
4 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20

Table 1 (continued )

Distillation Yield, Number of Overall percentage of Number Main components Identication References
time, h % isolated identied compounds, of methods
compounds % samples

e e 12 81.09 1 neryl acetate, limonene, g-curcumene, b-diketones GC-MS Cui et al.

(2016)
e e 60 98.47 1 a-pinene, b-selinene, g-curcumene, neryl acetate GC-FID, GC- Stupar et al.
MS (2014)
e e 9 75.91 1 italicene, b-diketones, neryl acetate, neryl propanoate GC Bakkali et al.
(2005)
e e 37 92.8 1 a-pinene, limonene, a-copaene, caryophyllene, a-cedrene, neryl GC-MS Politeo et al.
acetate, b-bisabolene, ar-curcumene, 2-methylcyclohexyl octanoate (2006)

between 100 and 200 m were rich in linalool and g-curcumene,
while those obtained at altitudes between 500 and 900 m were rich
in nerol, neryl acetate, neryl propionate, and g-curcumene.
Zeljkovic et al. (2015) tested four samples of H. italicum essential oil
collected from different parts of Dalmatia and indicated similarities
in qualitative but differences in quantitative composition of
essential oils; this was because the plants were collected at
different locations at different altitudes and sun exposure levels.
Lower altitude and solar exposure inuenced high total amount of
oxygen-containing compounds and low content of monoterpene
hydrocarbons in the samples. Melito et al. (2016) explored the
chemical proles of 146 H. italicum subsp. microphyllum genotypes
from two contrasting habitats in Sardinia: seaside (0e60 m above
sea level) and mountains (600e1250 m above sea level). Applied
statistical evaluation and multivariate analysis strongly suggested
that altitude level and climatic conditions affected the differences
in essential oil proles of this species. It was also shown that in
terms of relationship between meteorological variables and
chemical compounds, nerolidol was mostly positively correlated
with mean winter temperature, while italicene, bergamotene,
nerol, and curcumene were positively inuenced by spring and
summer precipitation. Ornano et al. (2014) tested essential oils of
H. italicum subsp. microphyllum (Willd.) Nyman for cytotoxicity on
three human tumor cell lines: MDA-MB 231 (human breast
adenocarcinoma), HCT116 (human colon carcinoma), and A375
(human malignant melanoma) cells by 3-(4,5-dimethylthiozol-2-
yl) 2e5 diphenyltetrazolium bromide (MTT) assay. Cytotoxicity
was expressed as the concentration of the compound inhibiting cell
growth by 50% (IC50). Essential oils of H. italicum showed a strong
inhibitory activity on A375 cells (IC50 of 16 mg/ml). In addition, the
oil was assessed for antioxidant activity by 2,20 -diphenyl-1-
picrylhydrazyl (DPPH), 2,20 -azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid) (ABTS) and ferric reducing antioxidant power
(FRAP) assay. Antioxidant activity of the essential oils, expressed as
IC50 values and in micromoles of Trolox equivalents (TE)/g, was
weak, probably due to the lack of components endowed with
hydrogen- or electron-donor functional groups. Some authors
proved the presence of different chemotypes in essential oils of
H. italicum using appropriate genetic assays (Angioni et al., 2003;
Melito et al., 2013). Mancini et al. (2011) evaluated essential oils
of H. italicum (Roth) G. Don subsp. italicum for its potential in vitro
phytotoxic activity against germination and early radicle elongation
of radish and garden cress. The essential oil in watereacetone
mixture (99.5:0.5) was assayed in different doses. The radicle
elongation of radish was signicantly inhibited at the highest doses
tested (0.25e2.5 mg/ml). The phytotoxic activity was probably due
to the presence of bioactive sesquiterpenes (Table 1); this nding
can suggest helpful models for lead compounds in the development
of new herbicides. Usai et al. (2010) isolated essential oils from 48
different H. italicum samples collected from vegetative to post-
blooming period. They concluded that July was the best period to
harvest plants to obtain the best essential oil composition, i.e.,
having the highest percentage of terpenes. Charles and Simon
(1991) investigated the essential oil of H. italicum (Roth) G. Don
subsp. italicum, whereby 16 compounds were dened and played
signicant roles in the specic, curry-like aroma of the plant.
Among them, the most common were neryl acetate, a-pinene, a-
and b-eudesmol, geranyl propionate, limonene, and camphene.
Bianchini et al. (2001, 2003, 2009) published three articles in
which essential oil of H. italicum (Roth) G. Don subsp. italicum of
Corsica was investigated in terms of differences in sampling time
and compared with those obtained from the plant collected from
Tuscany and Sardinia (Italy). In terms of chemical variability ac-
cording to the vegetation cycle, environment, and geographic ori-
gins around Corsica, the obtained data were analyzed. These
studies showed that oils produced from plants harvested in the
stage of owering had high content of neryl acetate (Bianchini et al.,
2001). The authors also showed that the variation in the amount of
neryl acetate essentially correlated, on the one hand, with the
relatively weak soil acidity and low percentages of clay, ne sand,
and coarse silt, and on the other hand, with high percentages of
coarse sand and ne silt (Bianchini et al., 2009).
Mastelic et al. (2005) investigated the essential oil of H. italicum
(Roth) G. Don subsp. italicum and conrmed strong antimicrobial
activity against Staphylococcus aureus and Candida albicans mainly
because of its terpenoid fraction. The essential oil and its fractions
were investigated for antimicrobial activity by the disc diffusion
method, including measuring the diameter of inhibition zone, and
by the agar method to determine the minimum inhibitory con-
centration (MIC). The authors also showed that the essential oil of
H. italicum possessed signicant amount of ester-bonded acids,
obtained by the hydrolysis of ester-containing fractions after the
essential oil neutralization (Mastelic et al., 2008).
Some authors compared particular activities of essential oil of
H. italicum and those of other plants from different families. Conti
et al. (2010) tested essential oils of six Mediterranean plants
against mosquitoes Aedes albopictus by exposing three groups of 20
fourth-instar larvae to different test dosages of the essential oils in
mineral water with 0.1% of Tween 80. The mortality was recorded at
the end of the test, during which no food was given to the larvae.
Lethal concentrations (LC50 and LC90) were also calculated. Among
the investigated plants, only essential oil of H. italicum caused the
highest mortality of these insects (100% at 300 ppm dosage).
Drapeau et al. (2009) investigated the repellent effect of H. italicum
essential oil together with 18 essential oils of plants from other
families against A. aegyptii. The results indicated a capability of the
H. italicum essential oil to reduce the human nger attractivity for
yellow fever mosquitoes in a Y-tube olfactometer. In addition, two
tests on the olfactory perception of these 19 oils were performed,
involving 25 female and 25 male human volunteers. The aspects
studied were the hedonic dimension of these oils and their
acceptance as a nal fragrance for a repellent formulation. Con-
cerning both the aspects, the essential oil of H. italicum did not
show signicant results. H. italicum essential oil also showed

Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different

Table 2
Percentage of different compound groups isolated by Clevenger-type distillation of H. italicum.a

Percentage of different compound groups, % References

b
MH OM MA ME M SH OS SA S D TH TE LK NTK b-DK OX H A E ET P HAKE AL AR O

2.3 38.6 e e e 5.1e20.1 3.1e25.4 e e e e e e e e e e e e e e e e e 2.3 Leonardi et al. (2013)

e41.9 e62.7 e11.6
0.7 27.5 e e e 9.1e19.7 e e e e e e e 0 3.7e42 e e e e e e e e e 2.1e11 Paolini et al. (2006)

S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20

e22.8 e57.9 e0.6
1.0 14.7 e e e 15.2 6.7e22.6 e e e e e e e e e e e e e e e 1.3 0.2 e Zeljkovic et al. (2015)
e11.6 e22.1 e21.2 e3.4 e9.7
e e e e 0 e e e 0 e e e e e e e e 0 0 0 e e e e e Melito et al. (2016)
e96.6 e78 e60 e91.9 e11.5
e 1.3 e 24.7 e 33.1 28.3 e e e e e e e e e e e e e e e e e 2.2 Angioni et al. (2003)
1.1 e e e e 0.7 73.6 e e e e e e e e e e e e e e e e e 13.7 Mancini et al. (2011)
6.1 1.5e52.7 e e e 13.7 3.3e11.9 e e e e e e 0 0.4e9.4 e e e e e e e e e e Bianchini et al. (2003)
e60.8 e38.7 e3.0
0.5 e 3.3 8.3 e 2e18.3 e 0 e e e e 0.9 e 3.2 0.3 e e e e e e e e e Bianchini et al. (2009)
e31.3 e21.1 e60 e20.1 e20.6 e22.7 e8.8
e e e e e e e e e e 46.9 43.7 e e e e e e e e e e e e e Mastelic et al. (2005)
e e e e e e e e e e e e e e e 1 24 18 59 e 1 e e e e Moretti et al. (2002)
e e e e 10.38 e e e 61.3 4.22 e e e e e e e e e e e 1.03 e e e Ivanovic et al. (2011)
54.5 0.59 e e e 34.79 1.78 e e e e e e e e e e e e e e e e e e Costa et al. (2015)
0.4 45.9 e e e 42.2 7.6 e e e e e e e e e e e e e e e e e e Kladar et al. (2015)
e 20.2 e e e 25.4 9.8 e e e e e e e e e e e e e e e e e 30.7 Tsoukatou et al. (1999)
4.5e9 0.-1.2 e e e 37.9 45.4 e e e e e e e e e 0 e e e e e e e 0.3e1 Maggio et al. (2016)
e39.3 e51.8 e1.8
19.83 4.49 e e e 60.6 3.73 e e e e e e e e e e e e e e e e e 9.76 Stupar et al. (2014)
a
The authors tried to keep the authenticity of the data taken from literature in terms of the nomenclature below, keeping in mind their heterogeneity and the possible uncertainties that may occur among the readers,
considering the possible overlap between the mentioned compound groups.
b
MH e monoterpene hydrocarbons, OM e oxygenated (oxygen-containing) monoterpenes, MA e monoterpene alcohols, ME e monoterpene esters, M e monoterpenes, SH e sesquiterpene hydrocarbons, OS e oxygenated
(oxygen-containing) sesquiterpenes, SA e sesquiterpene alcohols, S e sesquiterpenes, D e diterpenes, TH e terpene hydrocarbons, TE e terpenoids, LK e linear ketones, NTK e non-terpenic ketones, b-DK e b-diketones, OX e
oxides, H e hydrocarbons, A e alcohols, E  esters, ET e ethers, P e phenols, HAKE e hydrocarbons, aldehydes, ketones and esters, AL e aliphatic compounds, AR e aromatic compounds, O e other.

5
6 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
considerable results (LC50 of 0.42 ml/g) in the treatment of Limantria
dispar larvae (Moretti et al., 2002). The mortality at 1.0% of active
principle was statistically signicant after 48 and 72 h of exposure.
Bertoli et al. (2012) tested the bioactivity of six essential oils against
the stored food insect Sitophylus zeamais. Bioassays included con-
tact toxicity test in topical applications on insects using a Burkard
microapplicator and repellency test using half paper lter discs
treated with essential oil solution for exposure to insects. Essential
oil of H. italicum had relatively high insecticidal and repellent ac-
tivities. This essential oil when mixed with that of Ledum groen-
landicum L., Ericaceae and Ravensara aromatica L., Lauraceae showed
antimutagenic effect against urethane (Idaomar et al., 2002). After
performing the somatic mutations and recombination test (SMART)
in the wings of Drospohila melanogaster, it was shown that the
antimutagenic effect of these essential oils could be explained by
the interaction of their constituents with cytochrome P-450 acti-
vation system leading to a reduction in the formation of the active
metabolite. Rossi et al. (2007) investigated the essential oils from
28 plants of Corsica and evaluated their antimicrobial activities
against a large panel of human pathogenic bacteria, including
gram-positive (S. aureus) and gram-negative bacteria (Escherichia
coli, Pseudomonas aeruginosa, Enterobacter aerogenes, and
Campylobacter jejuni). The agar diffusion method was used for the
determination of the MIC values. The essential oil of H. italicum was
reported to possess the highest antimicrobial activity.
In few articles, hydrodistillation of H. italicum was applied with a
goal of essential oil and supercritical extract comparison (Costa
et al., 2015; Ivanovic et al., 2011; Maksimovic et al., 2013;
Marongiu et al., 2003; Micic et al., 2009). Furthermore, Marongiu
et al. (2003) compared the antibacterial activity of H. italicum
essential oil and supercritical extract against Bacillus cereus,
wherein the essential oil showed stronger activity. The agar dilu-
tion method was used for the comparison.
Kladar et al. (2015) performed hydrodistillation of H. italicum to
compare antioxidant activity of essential oil with that of ethanolic
extract by the DPPH method. Essential oil exhibited relatively weak
DPPH-scavenging potential but signicantly higher than that of
ethanolic extract.
Some authors made comparisons within Helichrysum genus or
H. italicum species. Roussis et al. (2000) in their work investigated
essential oil obtained from samples of H. italicum subsp. mycro-
phyllum before and during anthesis. Essential oil obtained from the
samples collected during anthesis showed strong bacteriostatic
activity against S. aureus, S. epidermidis, Klebsiella pneumoniae, P.
aeruginosa, E. coli, and E. cloacae and exhibited the strongest anti-
bacterial activity among the four assayed plant species (H. italicum,
H. orientale, H. heldreichii, and H. doereri). The in vitro antibacterial
studies were carried out by the paper disc diffusion assay.
Tsoukatou et al. (1999) studied essential oils from H. italicum subsp.
serotinum (Boiss) and H. stoechas subsp. stoechas (Moench) to
compare their chemical compositions and activities against two
gram-positive (S. aureus and S. epidermidis) and four gram-negative
bacteria (E. coli, E. cloacae, K. pneumoniae and P. aeruginosa). The
in vitro antibacterial studies were carried out using the paper disc
diffusion test. The most characteristic difference in the composition
of the oils was the dominance of monoterpene hydrocarbons in the
oil of H. stoechas in contrast to the dominance of oxygenated
monoterpenes in the oil of H. italicum. However, both the oils
exhibited signicant levels of activity against S. aureus and
S. epidermidis, whereas the essential oil of H. italicum was partially
active against gram-negative bacteria. It was active against
K. pneumoniae and P. aeruginosa and inactive against E. coli and
E. cloacae. Maggio et al. (2016) analyzed the chemical proles of the
essential oils in 10 populations of the genus Helichrysum in Sicily.
The obtained results conrmed that the principal component
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
analysis (PCA) of specialized metabolites could be used to nger-
print wild populations of Helichrysum. According to the chemical
proles of the essential oils, investigated populations were classi-
ed into three main clusters referring to the aggregates of
H. stoechas, H. rupestre, and H. italicum. Tucker et al. (1997) per-
formed distillation of two main H. italicum subspecies: H. italicum
(Roth) G. Don subsp. italicum and H. italicum subsp. microphyllum
(Willd.) Nyman. It was shown that the essential oil of the former
was rich in g-curcumene, neryl acetate, neryl propanoate, and
nerol, whereas the essential oil of the latter possessed signicant
amount of limonene and linalool, beside neryl acetate and g-cur-
cumene. Schipilliti et al. (2016) analyzed the chemical compositions
of the essential oils of H. italicum collected in Sicily and Corsica.
Furthermore, they successfully introduced the Carbon Isotope Ratio
Mass Spectrometry (GC-C-IRMS), with the use of the international
standards (i-std), to differentiate the genotypes of H. italicum (Roth)
G. Don subsp. italicum plants of different geographic origin. It was
also shown that the Sicilian oils were rich in a- and b-selinene,
rosifoliol, and aromadendrene, whereas the Corsican oils were rich
in neryl acetate, a-pinene, and g-curcumene.
Few articles referring to the chemical composition of purchased
samples of H. italicum provided valuable information on essential
oils' composition, and the results are presented in Table 1.
Furthermore, different biological activities of the samples were
tested. Cui et al. (2015) evaluated the antibacterial activity of
H. italicum essential oil in vitro on vegetables. The antibacterial
activity of H. italicum essential oil was evaluated using the plate
count method. Furthermore, the antibacterial mechanism of the
essential oil of H. italicum was observed by screening the integrity
of the cell membrane, loss of 260-nm absorbing material, quanti-
cation of DNA in bacterial cells, and measurement of cellular ATP
concentrations. The lowest concentration showing no visible
growth was regarded as the MIC. The minimum bactericidal con-
centration (MBC) was determined as the lowest concentration at
which no growth occurred on the plates. Strong activity was shown
for E. coli and S. aureus, which were exposed to oil both in vitro and
on vegetables and for B. subtilis, B. pumilus, Salmonella typhimurium,
K. pneumoniae, and P. aeruginosa, which were exposed to oil only
in vitro. Furthermore, Cui et al. (2016) investigated the combined
treatments of H. italicum essential oil and cold nitrogen plasma
against S. aureus biolm on different food-contact surfaces.
Compared to the results of independent cold nitrogen plasma and
H. italicum essential oil treatment, the combined treatment
Fig. 1. Chemical structures of the most common monoterpenes of H. italicum essential
oil: 1 e a-pinene, 2 e limonene, 3 e nerol, 4 e neryl acetate, 5 e neryl propanoate.
 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
indicated better dispersion effect against S. aureus biolms on the
surfaces of stainless steel, glass sheet, and plastic. Stupar et al.
(2014) conrmed the antifungal activity of H. italicum essential oil
against Epicoccum nigrum and Penicillium sp. and the demelanizing
activity against Aspergillus niger. Antifungal activity was tested us-
ing the microatmosphere method. Bakkali et al. (2005) reported the
least cytotoxicity of H. italicum essential oil in the yeast Saccharo-
myces cerevisiae in comparison to those of four other plants by
testing the capacity of the essential oils to induce nuclear DNA
damage-responsive genes, using suitable Lac-Z fusion strains for
RNR3 and RAD51, which are the genes involved in DNA metabolism
and DNA repair, respectively. Finally, Politeo et al. (2006) analyzed
the chemical composition and related total antioxidant capacities
of the essential oils of 12 spices. Antioxidant effectiveness was
examined by four different methods: the DPPH radical scavenging
method, determination of FRAP, determination of antioxidant ac-
tivity with thiobarbituric acid reactive species (TBARS), and auto-
matic determination of the oxidative stability of fat (RANCIMAT).
Essential oil of H. italicum was shown to have high antioxidant
activity.
Different hydrodistillation times and the impossibility to dene
orderliness among values of distillation yields suggested that
distillation yield was not proportional to experimental time as
shown in Table 1. It can be assumed that the most of the yields were
obtained after 2e3 h of hydrodistillation. By analyzing data pre-
sented in Table 2, it could be concluded that the essential oil of
H. italicum consisted mainly of mono- and sesquiterpenes, followed
by other components, among which the most signicant were b-
diketones. It was shown that the differences in the number of
isolated and identied compounds and content of compound
groups depended mainly on the geographic region, soil type, and
harvest time. Among these compounds, the most common indi-
vidual components of H. italicum essential oil obtained by hydro-
distillation in a Clevenger-type apparatus were monoterpenes - a-
pinene, limonene, nerol, neryl acetate, and neryl propanoate (Fig. 1)
and sesquiterpenes (Fig. 2) - a- and b-selinene, g-curcumene, trans-
b-caryophyllene, and eudesm-5-en-11-ol, which is a stereoisomer
of rosifoliol (Bianchini et al., 2004). Thus, these compounds might
be considered as the principal carriers of the biological activity of
H. italicum essential oil. However, considering all the presented
data, it might be interesting to direct further investigation in
establishing which constituents from the identied compounds
Fig. 2. Chemical structures of the most common sesquiterpenes of H. italicum essential oil: 1 e a-selinene, 2 e b-selinene, 3 e g-curcumene, 4 e trans-b-caryophyllene, 5 e
eudesm-5-en-11-ol.
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
7
present in essential oils might have the largest effect on dened
activity.
At the end of this section, few articles that dealt with com-
mercial samples of H. italicum essential oil are also presented. Amer
and Mehlhorn (2006a, 2006b, 2006c) tested the larvicidal effect
and repellency of a large number of essential oils against mosqui-
toes. For this purpose, both in vitro and in vivo tests were per-
formed. In vitro tests included investigations of the activities of
different concentrations of essential oils dissolved in tap water
using 2 ml of 100% acetone against the third-instar larvae aiming to
calculate the LC50 of the selected oils. In vivo tests included the
evaluation of essential oils against mosquitoes using the skin of
human volunteers to determine the protection time and repellency.
The essential oil of H. italicum showed signicant positive results
for both the experimental types. Chao et al. (2008) also used the
essential oil of H. italicum for the analysis of inhibitory activity
against methicillin-resistant S. aureus by using the disc diffusion
assay method. Among 91 single and 54 blended essential oils, this
essential oil of H. italicum had relatively high level of inhibition. Foti
et al. (2013) reported the rst case of allergic contact dermatitis
caused by an emollient cream in which H. italicum essential oil
obtained from owering tops was incorporated. Wang et al. (2008)
in their work employed 45 kinds of commonly used essential oils to
investigate free-radical scavenging ability and showed that the
essential oil of H. italicum achieved an average value. The antioxi-
dant activity was tested by the DPPH free-radical scavenging assay,
including the calculation of the scavenging effect of a DPPH free
radical (%). Finally, Plant (2016) investigated the anti-aging effects
of 31 essential oils on human K562 cells in the presence or absence
of hydrogen peroxide, aiming to evaluate the effects of essential oils
on telomere length. Changes in telomere length were measured
using the quantitative PCR-based procedure. The essential oil of
H. italicum did not show the telomere-protective effect.
2.1.2. Steam distillation of H. italicum aerial parts
After detailed search of the available literature, only two articles
(Morone-Fortunato et al., 2010; Perrini et al., 2009) regarding
H. italicum essential oil obtained by steam distillation in a spring-
type apparatus were found. Experimental data and reported re-
sults are summarized in Table 3.
Perrini et al. (2009) investigated two genotypes of H. italicum
(Roth) G. Don subsp. microphyllum (Willd.) Nyman, collected from
8 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
Table 3
Literature results of steam distillation of H. italicum in spring-type apparatus.
Distillation Yield, Number of Overall Number Percentage of different
time, h % isolated percentage of the of compound groups, %
compounds identied samples
Ma S b-DK O
compounds, %
2.5 0.5 30 99.7e99.8 2 70.3 15.7 1.2
e0.54 e81.4 e27.19 e1.5
2.5 0.1 62 80.8e100.0 20 26.4 61.6
e0.5 e53.6 e91.3
a
M e monoterpenes, S e sesquiterpenes, b-DK e b-diketones, O e other.
two different locations in Corsica, planted under the same condi-
tions and then analyzed their morphological, histological, and
chemical characteristics using isolated essential oils. Moreover, one
genotype was used to establish an efcient micropropagation
protocol. Morone-Fortunato et al. (2010) described the morpho-
logical, histological, and chemical characteristics of 20 native
H. italicum (Roth) G. Don subsp. italicum genotypes collected from
different locations in Italy and Corsica and planted under similar
conditions. The chemical analysis of their essential oils recognized
at least three different chemotypes on the basis of their major
constituents, which are listed in Table 3. These authors used the
same apparatus for their experiments (Albrigi, Italy).
Essential oils of investigated H. italicum subspecies consisted
mainly of mono- and sesquiterpenes, with signicant content of b-
diketones in the second case (Table 3). Differences in distillation
yields and number and percentage of the identied isolated com-
pounds could be a consequence of different geographic region and
planting conditions. However, similar to hydrodistillation, the main
compounds of the obtained essential oils were monoterpenes
nerol, neryl acetate, and neryl propanoate and sesquiterpenes ar-
and g-curcumene.
Considering individual analytical results of hydro- and steam
distillation of H. italicum essential oil, the possible assumption
might be that these two procedures resulted in essential oils with
similar compositions.
The dominance of terpenes among volatile organic compounds
(VOCs) in H. italicum was conrmed by a novel method performed
by Giuliani et al. (2016). These authors introduced headspace solid
phase microextraction coupled with GC-MS (HS-SPME-GC-MS) to
screen the annual leaf shoots of Helichrysum populations in Tuscany
for the emissions of VOCs. Nine selected Helichrysum populations,
including H. italicum complex, H. litoreum, and H. stoechas, were
investigated. The VOC composition analysis revealed the produc-
tion of 386 different compounds, with terpenes being the most
represented compound class. Considering H. italicum, the most
common terpenes were a-pinene, limonene, p-cymene, g-terpi-
nene, (Z)-geraniol, and neryl acetate. Statistical data conrmed the
results of some previously published articles related to the effects
of the geographical provenance area on determining the volatile
proles.
2.2. Supercritical CO2 extraction from H. italicum
Supercritical uid extraction from plant material has become a
very popular process because of its advantages over traditional
extraction techniques. It is a exible process because of the possi-
bility of continuous modulation of solvent power/selectivity, by
changing uid's density when pressure and temperature are
changed. This process avoids the application of polluting organic
solvents and expensive postprocessing of the extract for solvent
elimination (Reverchon and De Marco, 2006). In addition, possible
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
Main components Identication References
methods
0.9 nerol, neryl acetate, neryl propanoate, ar- and g- GC-FID, GC- Perrini
e1.2 curcumene MS et al.
(2009)
11.2 e nerol, neryl acetate, neryl propanoate, a- and b- GC-FID, GC- Morone-
e24.2 selinene, ar- and g-curcumene, b-diketones, b- MS Fortunato
caryophyllene, carvacrol, eudesm-5-en-11-ol et al.
(2010)
thermal degradation of particular compounds during the isolation
procedure, like in case of distillation, can be avoided using super-
critical uid extraction at mild temperature conditions (40e50  C).
At the end of the process, supercritical uid is simply removed from
the system by pressure reduction.
The most popular solvent used for supercritical uid extraction
is carbon dioxide. It is safe, is readily available, and has a low cost. It
enables supercritical extraction at relatively low pressures and
near-room temperatures (Reverchon and De Marco, 2006). When
polar components are extracted, a polar modier (cosolvent) may
be mixed with supercritical CO2 to enhance the solubility (Oman
et al., 2013).
However, there are only few reports on supercritical uid
extraction of H. italicum in the literature. In the reported studies,
extraction pressure and temperature were varied in the range of
80e350 bar and 35.86e64.14  C, respectively, with extraction times
of 1.5e4 h. Obtained yields were in the range of 0.35e6.31%. The
results of the reports are listed in Table 4.
Ivanovic et al. (2011) analyzed the kinetics and selectivity of
supercritical CO2 extraction of H. italicum owers of different
moisture contents at pressures in the range of 100e200 bar and
temperatures of 40 and 60  C. Increased moisture content of
H. italicum owers resulted in enhanced solubility of extractable
substances enabling decrease in supercritical CO2 consumption.
The analysis of chemical composition of the extracts showed that
the most abundant group of terpenes was sesquiterpenes: ar-
curcumene, a- and b-selinene, caryophyllene oxide, and eudesm-
7(11)-en-4-ol (Table 4).
Research performed by Maksimovic et al. (2013) was aimed to
determine the yield and composition of H. italicum extract obtained
by supercritical CO2 extraction at 150 bar and 40  C and explore the
possible process intensication. The main idea was to investigate
the possible effects of specialized metabolites of Salvia ofcinalis on
the kinetics of extraction from H. italicum. In nature, these two
plants often grow very close to each other. Supercritical extraction
was performed using a mixture of these two plants: a mixture of
H. italicum owers and essential oil of S. ofcinalis. It was shown
that the average selectivity of monoterpenes and some individual
sesquiterpenes and diterpenes (manool and cembrenol) present in
pure S. ofcinalis extract or pure H. italicum extract were signi-
cantly changed when a mixture of these plants was used for the
extraction. The cosolvent effect of S. ofcinalis essential oil in su-
percritical extraction of H. italicum owers was demonstrated. By
acting as the cosolvent, the essential oil of S. ofcinalis changed the
solubility in supercritical CO2 of different compounds present in
H. italicum. Increased extraction of heavier compounds from
H. italicum owers was recorded. Literature data on supercritical
extraction of pure H. italicum owers are presented in Table 4.
Micic et al. (2009) investigated the effects of pressure in the
range of 80e350 bar on the yield of supercritical CO2 extraction of
H. italicum aerial parts at 40  C. It was shown that the extraction
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
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Table 4
Literature results of supercritical CO2 extraction of H. italicum.

Pressure, Temperature, Extraction Yield, % Number of Overall Number Percentage of different compound groups, % Main components Identication References
o
bar C time, h isolated percentage of methods
MHb OM SH OS M S HAE W O
compounds of samples
the
identied
compounds,
%

S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20

100e200 40e60 1.5 1.37 59e65 75.75e76.92 7 e e e e 3.48 20.56 1.15 23.07 18.74 a-pinene, neryl acetate, GC-FID, GC-MS Ivanovic et al.
e4.1 e5.48 e26.42 e3.61 e28.0 e22.46 ar-curcumene, a- and (2011)
b-selinene,
caryophyllene oxide,
eudesm-7(11)-en-4-ol,
waxes
150 40 1.7 5.7 64 e 1 e e e e e e e e e a-pinene, epi-b- GC-FID, GC-MS Maksimovic
bisabolol, canellal, et al. (2013)
cembrenol, a-
santonine, waxes
90 50 2e4 0.4e1 59e77 88.9e96.3a 2 e e e e e e e e e neryl acetate, nerol, GC-MS Marongiu et al.
neryl propanoate, (2003)
linalool, g-curcumene
80e350 40 3 0.35 18e20 75.78e94.62 2 e e e e e e e e e trans-caryophyllene, g- GC, GC-MS Micic et al.
e5.71 curcumene, waxes (2009)
90e120 40 e 0.36 26e30 86.99e97.76 4 7.37 0.56 48.48 0.57 e e e e e a-pinene, italicene, 9- GC-FID, GC-MS Costa
e0.60 e47.84 e0.90 e79.18 e2.94 epi-(E)-caryophyllene, et al. (2015)
g- and ar-curcumene
260 50 3 3.9e4.9 e e 4 e e e e e e e e e e e Poli
et al. (2003)
79.3 35.86e64.14 1.5 0.43 e e 13 e e e e e e e e e scopoletin RP-HPLC, UV-VIS Jokic
e6.31 et al. (2016)
e220.7
a
Without waxes.
b
MH e monoterpene hydrocarbons, OM e oxygen-containing monoterpenes, SH e sesquiterpene hydrocarbons, OS e oxygen-containing sesquiterpenes, M e monoterpenes, S e sesquiterpenes, HAE e hydrocarbons, acids
and esters, W e waxes, O e other.

9
10 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20

yield increased with the increase in pressure. Chemical analysis of extracts of H. italicum was investigated. All the extracts showed
volatile fraction showed signicant content of sesquiterpenes in the signicant protective action against the superoxide radical.
obtained extracts. Chemical characterization of the obtained extracts was not per-
Marongiu et al. (2003) performed supercritical CO2 extraction of formed in the same study.
H. italicum (Roth) Don subsp. microphyllum (Willd.) Nyman leaves According to Table 4, the applied identication methods (mostly
and owers at 90 bar and 50  C by a fractional separation technique. GC-FID and GC-MS) showed that the most common compounds
It was shown that the ower extract was richer in heavier sesqui- present in volatile fractions of supercritical extracts of H. italicum
terpenes than the leaf extract. Moreover, the extracts were were similar to those obtained by hydro- and steam distillation: a-
screened for antimicrobial activity against S. aureus, Enterococcus pinene, neryl acetate, and g-curcumene, whereby supercritical
faecalis, B. cereus, Listeria monocytogenes, E. coli, C. albicans, extracts contained signicant content of waxes also. However, it is
C. tropicalis, and Cryptococcus neoformans by the agar dilution also possible to isolate heavier sesquiterpenes and diterpenes by
method. Only moderate extracts' activity against B. cereus was using supercritical CO2 extraction. They were followed by nerol,
reported. neryl propanoate, selinene, and caryophyllene isomers (Ivanovic
Costa et al. (2015) extracted volatiles from H. italicum subsp. et al., 2011; Maksimovic et al., 2013). Except one recent article
picardii by using supercritical CO2 at 40  C and under different (Jokic et al., 2016) presented in Section 3, there are no data reported
conditions of pressure (90 and 120 bar) and particle size (<4 mm on HPLC analysis of H. italicum supercritical extracts. In addition,
and 4 cm) to analyze their effects on the extraction yield, chemical besides specialized metabolites of S. ofcinalis (Maksimovic et al.,
prole, organoleptic properties, and antioxidant activity. The 2013), no other substances were tested as cosolvents in supercrit-
increment of extraction pressure had a signicant positive effect on ical extraction of H. italicum.
the yield of the extracts with the largest particle size. Moreover, the
particle size affected the yield of the extracts where the smallest
particle size generated the highest extraction yield. Furthermore, 2.3. Isolation of terpenes from H. italicum using organic solvents
only the supercritical extract isolated at 120 bar could reduce the
DPPH radical. Few articles dealt with the investigation of terpenes isolated by
Poli et al. (2003) tested the antioxidant activity of supercritical
CO2 extracts obtained at 260 bar and 50  C from H. italicum dried
ower heads derived from the commercial drug and the plants
grown in different areas of North-East Italy. Characterization of the
antioxidant activity was performed by the DPPH- and b-carotene
bleaching test methods. The DPPH free-radical scavenging activity
of H. italicum supercritical extracts was expressed as decoloration
percentage (%), and the antioxidant activity in b-carotene bleaching
test was calculated using the relative antioxidant activity (RAA)
coefcient. All the extracts showed considerable antioxidant ac-
tivity according to all the methods performed. Furthermore, inhi-
bition of the production of superoxide radicals (O 2 ) generated by
the xanthine-xanthine oxidase enzyme system by supercritical
Fig. 3. Chemical structure of arzanol.

Table 5
Literature results of acetone extraction of H. italicum.

Plant Extract Isolated compounds Elements of isolation procedure Identication References

material mass, g methods
Fractionation Chromatography and other Elution agents
mass, g
agents techniques

1000 51 arzanol, u-oleoyloxylinalool, petroleum vacuum chromatography on petroleum ethereethyl acetate IR, 1H NMR, Appendino
13
helipyrone, micropyrone, ether, ethyl silica gel, gravity column (95:5, 8:2, 5:5, 4:6, 3:7) C NMR, et al. (2007)
tremetones acetate, chromatography on silica gel HREIMS
acetone
1
540 12 arzanol, methylarzanol, ethyl acetate, recrystallization from toluene n-hexaneeethyl acetate (6:4, 9:1, H NMR, 13C Rosa et al.
helipyrone, micropyrone, n-hexane eacetone mixture, gravity 7:3, 6:4) NMR, UV, IR, (2007)
rosifoliol, 10-hydroxytremetone, column chromatography on HR-MS
acetoxytremetone, silica gel
acetoxyhydroxytremetone
5000 202 ursolic acid, neryl acetate, u- petroleum recrystallization from methanol, methanol-water (93:7), petroleum 1H NMR, 13C Tagliatela-
oleoyloxylinalool, coumarates, ether, crystallization from ether, ethereethyl acetate (9:1, 7:3), n- NMR, ESIMS, Scafati et al.
helipyrone, oleoylbitalin A, aqueous ltration over neutral alumina, hexaneeethyl acetate (8:2, 9:1, HRESIMS (2013)
nonanoylbitalin A, santinols, methanol gravity column chromatography 85:15, 55:45)
micropyrone, propanoylbitalin A, (1:9), acetone on silica gel, HPLC on normal
isocaproylbitalin A, arzanol phase
e e galangin, galangin-3-methyl ether, methanol gravity column chromatography methanol, toluene, methylethyl UV-VIS, 1H Wollenweber
8-hydroxygalangin, 8- on Sephadex LH-20, silica gel, ketone, PE100-140etoluene NMR, 13C et al. (2005)
hydroxygalangin-3-methyl ether, polyamide SC-6 and acetylated emethylethyl ketoneemethanol NMR, APCI-
gnaphaliin, kaempferol-3- polyamide, TLC on polyamide (12:6:1:1, 6:12:2:1), toluene MS/MS
methyleter, herbacetin-3- and silica gel edioxaneemethanol (8:1:1),
methylether, quercetin-3- tolueneemethylethyl ketone
methylether, gossypetin-3- emethanol (12:5:3), toluene
methylether, gossypetin-3,8- emethylethyl ketone (9:1), toluene
dimethylether, pinocembrin edioxaneeacetic acid (18:5:1)

Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
extraction using organic solvents. Extraction from H. italicum using
acetone resulted in isolation of triterpene ursolic acid (Tagliatela-
Scafati et al., 2013) and sesquiterpenes - u-oleoyloxylinalool
(Appendino et al., 2007; Tagliatela-Scafati et al., 2013), rosifoliol
(Rosa et al., 2007), and neryl acetate (Tagliatela-Scafati et al., 2013).
Rosifoliol showed signicant protective effect against linoleic acid
oxidation (Rosa et al., 2007). Methanol was used to isolate ursolic
acid (Sala et al., 2001, 2003b). Mari et al. (2014) identied one novel
drimane sesquiterpene 6,11-diacetyl-9,11-dihydroxydrimane in
H. italicum methanolic extract. Terpenes were also identied in
H. italicum diethyl ether extract (Nostro et al., 2000). The experi-
mental conditions, results, and biological activities of the extracts
rich in the abovementioned terpenes are described in detail in the
next section.
Tundis et al. (2005) analyzed the methanol extracts of
H. italicum aerial parts collected from Calabria and Sardinia (Italy)
for their antioxidant, antibacterial, and antifungal activities. Anti-
oxidant activity, evaluated using DPPH- and b-carotene bleaching
test, showed that the Calabrian samples were more active than
those from Sardinia. Antibacterial activity of all the extracts was
determined against gram-positive (S. aureus, E. faecalis, and
Micrococcus luteus) and gram-negative (Proteus mirabilis, P. vulgaris,
and E. coli) bacteria. The best activity was detected against M. luteus.
Antifungal activity was evaluated on phytopathogen Pythium ulti-
mum and dermatophyte Trichophyton mentagrophytes. Contrary to
previous comparison, in this case, the extracts from Sardinia were
more active than those from Calabria. This species was character-
ized through detection, isolation, and quantitative evaluation of the
chemical markers a-terpinolene, trans-caryophyllene, and neryl
acetate.
3. Phenolic compounds of H. italicum
Phenolic compounds are the second great phytochemical class
found in H. italicum isolates. Unlike distillation of H. italicum,
whereby mostly terpenes are obtained, the use of solvent extrac-
tion enables to isolate other specialized plant metabolites of
H. italicum. Among them, phenolic compounds are the most
signicant.
In this section, the class of phenolic compounds is considered in
terms of few distinctive classes, which are the main ingredients of
H. italicum extracts obtained by extraction using the organic sol-
vents: avonoids, acetophenones, phloroglucinols, tremetones,
coumarins and coumarates, phenolic acids and esters, with their
derivatives. These compounds, individually or as a part of
H. italicum extract, showed many biological activities. Therefore,
this section focuses on both individual phenolic compounds and
H. italicum extracts obtained using different organic solvents, with
respect to experimental procedures and obtained results.
After Clevenger-type hydrodistillation, solvent extraction is the
most reported method in literature for isolation of active com-
pounds from H. italicum. There are various reports on different
organic solvents applied for extraction from H. italicum. Never-
theless, the experimental procedures applied were similar. At rst,
the plant material is soaked in a particular solvent and kept for a
particular time at the room temperature. After that, the obtained
extract is further fractionated by other solvents. Subsequently,
fractions are subjected to different separation processes (mostly
thin layer and column chromatography) to isolate particular com-
pounds using an appropriate elution agent. Individual compounds
are identied by different spectroscopic methods. Because of the
complex separation of individual compounds, this isolation method
requires high amounts of raw plant material.
The obtained extraction yields are signicantly higher than
those obtained by the distillation and supercritical CO2 extraction,
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
11
with yield even up to 19.77% (Kladar et al., 2015).
According to the information reported in the literature, the
following organic solvents were the most frequently used for the
extraction of H. italicum: acetone, methanol, ethanol, chloroform,
petroleum ether, dichloromethane, and diethyl ether. For acetone,
methanol, and ethanol, the survey of reported experimental con-
ditions and the results in terms of listed isolated compounds and
other key elements of the applied isolation procedures are dis-
cussed in details.
3.1. Extraction of H. italicum by acetone
Literature data presented in Table 5 show that acetone used as a
solvent for the extraction of H. italicum is the most effective for the
isolation of arzanol (the prenylated heterodimeric pholoroglucinyl
a-pyrone) known as 3-(3-acetyl-2,4,6-trihidroxy-5-(3-methylbut-
2-en-1-yl)benzyl)-6-ethyl-4-hydroxy-5-methyl-2H-pyran-2-one
(Fig. 3) (Kothavade et al., 2013). Arzanol possesses various phar-
macological activities (Appendino et al., 2007; Bauer et al., 2011;
Rosa et al., 2007, 2011; Tagliatela-Scafati et al., 2013).
Appendino et al. (2007) performed extraction of H. italicum
leaves and owers by acetone to obtain arzanol and tremetones. It
was shown that arzanol inhibits nuclear factor-kappa B (NF-kB)
activation by luciferase and the release of proinammatory medi-
ators (interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis
factor (TNF)-a, and prostaglandin E2 (PGE2)) in lipopolysaccharide
(LPS)-stimulated monocytes isolated from the blood of healthy
human donors, thus qualifying as a novel plant-derived anti-in-
ammatory and antiviral chemotype. The levels of the proin-
ammatory mediatorsIL-6, IL-1, TNF-a, and PGE2 were measured by
ELISA or EIA according to the manufacturer's instruction. In addi-
tion, arzanol inhibited HIV-1 replication in Jurkat T cells infected
with the VSV-pseudotyped-pNL4-3.Luc.3-4-.
Bauer et al. (2011) used the same method for arzanol isolation
from H. italicum. Arzanol was tested against biosynthesis of
proinammatory eicosanoids. It inhibited 5-lipoxygenase activity
and related leukotriene formation in neutrophils as well as
inhibited the activity of cyclooxygenase-1 (COX-1) and the forma-
tion of cyclooxygenase-2 (COX-2)-derived PGE2 in vitro. Determi-
nation of COX-1 activity was performed in platelet homogenates
and that of COX-2 activity was performed in monocyte homoge-
nates after the stimulation of monocytes with LPS. Arzanol also
inhibited microsomal PGE2 synthase in human blood. PGE2 syn-
thase was isolated after preparation of crude microsomal prosta-
glandin E synthase (mPGES)-1 in microsomes of adenocarcinomic
human alveolar basic epithelial (A549) cells. Arzanol potently
suppressed the inammatory response of the carrageenan-induced
pleurisy in rats. It was injected 30 min before carrageenan
administration into the rats' pleural cavity. In this in vivo model,
arzanol showed its anti-inammatory properties of lowering PGE2
by inhibition of mPEGS-1.
Rosa et al. (2007) investigated various phenolics from
H. italicum, extracted by acetone, for their capacity to inhibit
nonenzymatic lipid peroxidation. Arzanol showed antioxidant ac-
tivity and could protect linoleic acid against free-radical attack in
assays of autoxidation and ethylendiaminetetraacetic acid dipo-
tassium salt (EDTA)-mediated oxidation in dry state. Methylarzanol
showed signicant protective effect against linoleic acid oxidation.
Arzanol also showed antioxidant activity against autoxidation of
cholesterol in dry state and strong inhibition of tert-butyl hydro-
peroxide (TBH)-induced stress in VERO cells derived from monkey
kidney. Furthermore, the cytotoxic effect of arzanol was assessed by
the MTT method and lactate dehydrogenase (LDH) release was
measured in VERO cells. Arzanol was not toxic at all the tested
concentrations.
12 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20

Table 6
Literature results of methanol extraction of H. italicum.

Extraction Plant Extract Isolated compounds Elements of isolation procedure Identication References
time, h material mass, g methods
Fractionation Chromatography and other Elution agents
mass, g
agents techniques

e 400 50 ursolic acid, gnaphaliin, n-hexane, precipitation, gel ltration methanol, dichloromethane UV, IR, 1H NMR, Sala et al.
13
pinocembrin, tiliroside, 4- dichloromethane, over Sephadex LH-20, VLC on emethanol, C NMR, EIMS, (2001)
hydroxy-3-(3-methyl-2- ethyl acetate, n- silica gel, gravity column dichloromethaneeethyl FABMS,
butenyl)acetophenone, 4- butanol chromatography on silica gel, acetate (9:1, 95:5), methanol HREIMS
hydroxy-3-(2-hydoxy-3- purication on a Lobar ewater (4:6, 2:8)
isopentenyl)acetophenone, LiChroprep RP-18 (Merck)
12-hydroxytremetone, 3-(3- column
methyl-2-butenyl)
acetophenone-4-O-b-D-
glucopyranoside, 12-
hydroxytremetone-12-O-b-
D-glucopyranoside, 3-(2-
hydroxyethyl)acetophenone-
4-O-b-D-glucopyranoside,
maltol-b-D-glucopyranoside
e 400 55 ursolic acid, 4-hydroxy-3- n-hexane, e e TLC, HPLC-DAD Sala et al.
(3-methyl-2-butenyl) dichloromethane, (2003b)
acetophenone, gnaphaliin, ethyl acetate, n-
avonoids, acetophenone butanol
derivatives, lipids, sitosterols
e 400 55 gnaphaliin, tiliroside water, gravity column dichloromethane-ethyl e Schinella
chromatography on silica gel, acetate, 40% methanol
dichloromethane, et al.
low-pressure column
methanol, ethyl (2007)
acetate chromatography on reverse
phase (RP-18)
48 500 e a-terpinolene, neryl acetate, methanol e water gravity column n-hexaneeacetone (98:2) GC-FID, GC-MS Tundis
trans-caryophyllene (9:1), n-hexane chromatography on silica gel et al.
(2005)
3 1e5 e phenolic acids e e e UPLC-MS/MS De la
(gallic acid, caffeic acid, Garza et al.
chlorogenic acid, chlorogenic (2013) a
acid-3-O-glucoside),
avonoids (avanones e
naringenin, naringenin-7-O-
glucoside, naringenin
diglycoside, narirutin,
naringenin-4-glucoside-7-
rutinoside, hesperidin;
avonols e kaempferol,
kaempferol-3-O-glucoside,
kaempferol rutinoside,
myricetin glucoside; avones
methoxyluteolin; avanols -
epigallocatechin-3-O-gallate)
1 e 38.7 styrylpyrones water, ethyl chromatography water, methanol, methanol UV, 1H NMR, D'Abrosca
13
isobenzofuranone glycosides, acetate, methanol on Amberlite XAD-4 and ewater (1:3), chloroform C NMR, Q-TOF et al.
3- Sephadex LH-20, TLC on SiO2, emethanolewater (13:7:2, HRMS, COSY, (2013) b
hydroxydihydrobenzofuran RP-18 column 13:7:3), n-hexane TOCSY, HSQC,
glycosides, neolignans, chromatography echloroformemethanol CIGAR-HMBC,
oxyneolignans and low- (1:1:1), acetonitrile H2BC, HSQC-
molecular weight phenol emethanolewater (1:1:8, TOCSY
glucosides 3:3:14)
1
e 500 e caffeic acid, chlorogenic acid, e size exclusion methanolewater (38:62), H NMR, 13C Mari et al.
quercetin-3-O-glucoside, 5- chromatography on 0.5% acetic acid, 0.5% acetic NMR, HRESIMS, (2014) c
O-caffeoyl-4-methylquinic Sephadex LH-20, HPLC on acideacetonitrile DQF-COSY,
acid, kaempferol 3-O- Phenomenex C18 Synergi- HSQC, HMBC,
glucoside, isorhamnetin 3-O- Hydro-RP column, Reverse ROESY, LC-
glucoside, 4,5- Phase-HPLC-IR, RP-HPLC-UV, ESI(IT)MSMS
dicaffeoylquinic acid, 3,4-
dicaffeoylquinic acid,
quercetin-30 -O-glucoside,
6,11-diacetyl-9,11-
dihydroxydrimane, 3,5
dicaffeoylquinic acid methyl
ester, tiliroside, 12-
hydroxytremetone,
gnaphaliol
a
Extraction solvent was methanol - water (3:1).
b
Extraction was ultrasound assisted.
c
Petroleum ether and chloroform were plant pretreatment agents.

Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20 13

Fig. 4. Chemical structures of 1 e gnaphaliin, 2 e pinocembrin, 3 e tiliroside.

Rosa et al. (2011) reported arzanol's protective effect on oxida-
tive modication of lipid components induced by Cu2 ions in
human low density lipoprotein (LDL) and the reduction of poly-
unsaturated fatty acids and cholesterol levels, inhibiting the in-
crease in oxidative products. Arzanol showed a protective effect on
TBH-induced oxidative damage in a cell line of broblasts derived
from VERO cells and human intestinal epithelial (Caco-2) cells. In
addition, the cytotoxic activity of arzanol was evaluated in VERO
cells by the MTT assay and in Caco-2 cells by the alamarblue test.
Arzanol did not exert a signicant reduction in cell viability under
50 mM concentration in both the cell lines.
Taglialatela-Scafati et al. (2013) reported isolation of arzanol
from H. italicum acetonic extract, together with bitalin A derivatives
(tremetones) and coumarates. Antibacterial activity of arzanol,
coumarates, and heterodimeric phloroglucinols was evaluated by
the broth dilution method and showed that only heterodimers had
potent antibacterial action against multidrug-resistant S. aureus.
Wollenweber et al. (2005) performed acetone extraction of
H. italicum aiming to isolate avonoids by using an appropriate
separation method as shown in Table 5. The major avonoid of
H. italicum extract was 8-hydroxygalangin-3-methyl ether accom-
panied by lesser amounts of galangin, galangin-3-methyl ether,
gnaphaliin, and only a trace of 8-hydroxygalangin. Other isolated
avonoids were 3-methyl ether derivatives of kaempferol, herba-
cetin, quercetin and gossypetin, gossypetin-3,8-dimetyhl ether, and
pinocembrin.
3.2. Extraction of H. italicum by methanol
Methanol used as a solvent for the isolation of different com-
pounds from H. italicum yielded extracts that mainly contained
avonoids and acetophenones and their glycoside and glucoside
derivatives. Classied experimental data, elements of isolation
procedures, and applied identication methods for the isolated
compounds are shown in Table 6.
Sala et al. (2001) performed methanol extraction of H. italicum
aerial parts and isolated three previously undescribed acetophe-
none glucosides and three known aglycons. All the compounds
tested showed anti-inammatory activity in a 12-O-tetradeca-
noylphorbol 13-acetate (TPA)-induced mouse ear edema test, and
among them, 4-hydroxy-3-(2-hydoxy-3-isopentenyl)acetophe-
none was the most active. Six isolated acetophenone compounds
were tested for their ability to inhibit enzymatic and nonenzymatic
lipid peroxidation (Sala et al., 2003a). Presence of these compounds
showed stable DPPH free-radical, superoxide scavenging, and
arachidonic acid metabolism. In addition, they were studied in
different experimental models such as the chronic inammation
induced by TPA, the phospholipase A2-induced mouse paw edema
test, the carageenan-induced mouse paw edema test, and writhing
induced by acetic acid in the mouse. Only 4-hydroxy-3-(3-methyl-
2-butenyl)acetophenone showed signicant improvement (Sala
et al., 2003a). On the other side, three avonoids gnaphaliin,
pinocembrin, and tiliroside (Fig. 4) were studied in vitro for their
antioxidant and/or scavenger properties and in vivo in different
models of inammation. The most active substance was tiliroside,
while pinocembrin was the only avonoid that exhibited anti-
inammatory activity in the sheep red blood cell-induced
delayed-type hypersensitivity reaction (Sala et al., 2003b).
Sala et al. (2002) reported the anti-inammatory and antioxi-
dant activities of methanolic extracts of H. italicum aerial parts that
had been established in various in vivo and in vitro experimental
models. Their ndings suggested that the anti-inammatory ac-
tivity was because of the effects of compounds expressed through a
corticoid-like mechanism. In addition, the antioxidant activity
seems to be implicated in the anti-inammatory activity, as the
former inhibits enzymatic and nonenzymatic lipid peroxidation
and has free-radical scavenger properties. The main compounds in
extracts were 4'-hydroxy-3'-(3-methyl-2-butenyl)acetophenone

Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
14 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20

Table 7
Literature results of ethanol extraction of H. italicum.

Extraction Plant Extract Isolated compounds Elements of isolation procedure Identication References
time, h material mass, g methods
Fractionation Chromatography and Elution agents
mass, g
agents other techniques

e e e apigenin, glycosyl-apigenin, e e e UV-VIS, EI-MS, Facino et al.

luteolin, gnafaliin, naringenin, FD-MS (1988)
glycosyl-naringenin, glycosyl-
chalcone
e e 20 4,20 ,40 ,60 -tetrahydroxychalcone- water TLC ethyl acetateeformic acid UV-VIS, FD-MS, Facino et al.
20 -glucoside, kaempferol-3- eacetic acidewater 1
H NMR, 13C (1990)
glucoside and naringenin- (100:11:11:27) NMR, HPLC
glycoside
e 6 0.5 naringenin-40 -glucoside, methanol, HPLC, TLC 2-propanolewater (20:80), HPLC, UV-VIS Pietta et al.
kaempferol-3-glucoside, water ethyl acetateeformic acid (1991)
4,20 ,40 ,60 -tetrahydroxychalcone- eacetic acidewater
20 -glucoside (100:11:11:27)
e 6 0.5 luteolin-7-glucoside, methanol, HPLC, TLC 2-propanolewater (20:80), -DAD, Pietta et al.
isoquercitrin, kaempferol-3- water ethyl acetateeformic acid HPLC-DAD, UV- (1992)
glucoside, apigenin-7-glucoside, eacetic acid-water VIS
hyperosid, quercitrin, 4,20 ,40 ,60 - (100:11:11:27)
tetrahydroxychalcone-20 -
glucoside, naringenin-40 -
glucoside
e e e p-coumaric acid, caffeic acid, e e UV-VIS, IR, PMR, Zapesochnaya
ferulic acid, chlorogenic acid, 7- MS et al. (1990)
hydroxy-5-methoxyphtalide,
bitalin A (12-hydroxytremetone)
e e e 3-caffeoylquinic acid, 4- e HPLC, TLC on Silufol watereacetonitrile HPLC, 1 NMR Zapesochnaya
caffeoylquinic acid, 5- 254, gravity column etetrahydrofuraneacetic acid et al. (1992)
caffeoylquinic acid caffeic acid, chromatography on (85:10:2:3), chloroform
bitaloside, 1,3-dicaffeoylquinic silica gel L 40/100 emethanolewater (26:14:3),
acid, p-coumaric acid, chloroformemethanol (from
helichrysin, bitalosidin, 100:0 to 60:40)
isoquercitrin, 3,5-
dicaffeoylquinic acid, 1,5-
dicaffeoylquinic acid, 1,4-
dicaffeoylquinic acid, 4,5-
dicaffeoylquinic acid
24 e e gallic acid and quercetin water e e UV-VIS Kladar et al.
equivalents (2015)
1
12 535 18 12-acetoxytremetone, [2,3- 10% aqueous gravity column chloroform-methanol (from UV, IR, H NMR, Rigano et al.
13
dihydro-2-[1-(hydroxymethyl) methanol, n- chromatography on 100:0 to 0:100 gradient), C NMR, COSY, (2013)
ethenyl]-5-benzofuranyl]- hexane, silica gel, TLC e spray petrol-ethyl acetate (1:1) HSQC, HMBC,
ethanone chloroform, reagent Ce(SO4)2 in ESIMS
n-butanol H2SO4, normal phase
HPLC on a silica column
12 235 18 12-acetoxytremetone, [2,3- 10% aqueous gravity column chloroform-methanol (from UV, IR, 1H NMR, Rigano et al.
13
dihydro-2-[1-(hydroxymethyl) methanol, n- chromatography on 100:0 to 0:100 gradient), C NMR, ESI/MS, (2014)
ethenyl]-5-benzofuranyl]- hexane, silica gel, TLC e spray petrol-ethyl acetate (1:1), HR-MALDITOF-
ethanone, 13-(2- chloroform, reagent Ce(SO4)2 in chloroform-methanol (90:10, MS, DQF-COSY,
methylpropanoyloxy)toxol, n-butanol H2SO4, normal phase 85:15), n-hexane-ethyl HSQC, HMBC,
gnaphaliol 9-O-propanoate, 1- HPLC on a silica column acetate (70:30, 50:50) TOCSY, ROESY
[2-[1-[(acetyloxy) methyl]
ethenyl]-2,3-dihydro-3-
hydroxy-5-benzofuranyl]-
ethanone, gnaphaliol

and gnaphaliin.
Schinella et al. (2007) isolated tiliroside and gnaphaliin from the
methanolic extract of H. italicum aerial parts. The compounds were
studied in vitro for their capacity to inhibit Cu2-induced human
LDL and diluted plasma oxidation, monitored by conjugated diene,
TBARS formation, and electrophoretic mobility on an agarose gel. In
the rst case, antioxidant activities of these two compounds were
in the same order of magnitude. In the second case, tiliroside
showed higher activity than gnaphaliin.
Schinella et al. (2002a, 2002b) also tested methanolic extracts of
H. italicum and other plant extracts and conrmed its anti-
inammatory activity in different experimental models. Using
free-radical-generating systems, the H. italicum extract was
analyzed for enzymatic and nonenzymatic lipid peroxidation in
model membranes, and this extract showed scavenging property
on the superoxide radical (Schinella et al., 2002a). In addition, the
prepared extract was tested against epimastigote Trypanosoma
cruzi and showed relatively low activity (Schinella et al., 2002b).
De la Garza et al. (2013) isolated phenolic compounds from two
compound groups e phenolic acids and avonoids (avanones,
avonols, avones, and avanols) e using a methanolewater (3:1
by volume) extract of H. italicum. All isolated individual compounds
are listed in Table 6. This study also showed that H. italicum extract
prepared by such a method inhibited in vitro enzyme activities,
reduced maltose digestion in noneverted intestinal sacs, and
inhibited sodium-glucose linked transporter 1 (SGLT1)-mediated
methylglucoside uptake in Caco-2 cells in the presence of Na.
In vivo studies demonstrated that the methanolewater H. italicum

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isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
extract decreased blood glucose levels and reduced postprandial
glucose levels. This extract also improved hyperinsulinemia and
homeostatic model assessment (HOMA) index in a dietary model of
insulin resistance in rats. Moreover, the activity of the H. italicum
extract was compared with that of Citrus X paradise.
D'Abrosca et al. (2013) performed ultrasound-assisted methanol
extraction of H. italicum dried leaves. Among 16 known aromatic
metabolites classied in several groups, isobenzofuranone glyco-
sides, 3-hydroxydihydrobenzofuran glycosides, and low-
molecular-weight phenol glucosides were isolated and detected
on the basis of different spectroscopic methods (Table 6). 3-
Hydroxydihydrobenzofuran glycosides, chlorogenic acid, and 3,5-
dicaffeoylquinic acid showed signicant activity against a strain
of biolm-forming P. aeruginosa by the broth dilution method.
Biolm formation was assayed by measuring the ability of bacterial
cells to adhere to a sterile 96-well polystyrene at-bottom micro-
titer plate.
Mari et al. (2014) investigated metabolite prole of methanolic
extract of H. italicum owers using LC-ESI(IT)MSMS. Extraction
with pretreatment of plant material by petroleum ether and chlo-
roform resulted in the identication of 14 compounds belonging
mainly to avonoids (Table 6). In all the analyzed samples, a high
amount of quinic acid derivatives was found.
Nostro et al. (2000) compared the antimicrobial activity of
H. italicum extracts obtained using different organic solvents
against gram-positive bacteria, gram-negative bacteria, and fungal
organisms with those obtained from few other plant species. The
disc diffusion test, agar dilution method, and bioautography were
used for testing purpose. The methanolic extract of H. italicum did
not show signicant antimicrobial activity.
3.3. Extraction of H. italicum by ethanol
Ethanol is the most commonly used organic solvent for extrac-
tion from H. italicum according to the literature data. Utilization of
this solvent and application of appropriate techniques resulted in
the isolation of mainly avonoids and their glycoside derivatives.
Reported literature data on this topic are presented in Table 7.
Facino et al. (1988) isolated the avonoid fraction from the
ethanolic extract of H. italicum with an aim to test its potential anti-
erythematous activity on the sunburned skin. Experiments carried
out in guinea pigs with the avonoid fraction isolated from the
crude H. italicum extract indicated that the photoprotective activity
against UVB-induced erythema was largely because of the action of
this avonoid complex consisting of apigenin, glycosyl-apigenin,
luteolin, gnafaliin, naringenin, glycosyl-naringenin, and glycosyl-
chalcone. Subsequent clinical studies including experiments with
UVB radiation conrmed that this complex is a potent anti-
erythematous agent. Spectrometric studies demonstrated that
this fraction also possesses strong UVA ltering capacity.
Fig. 5. Chemical structure of 12-acetoxytremetone
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isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
15
Furthermore, the same authors (Facino et al., 1990) in their second
article tested free-radical scavenger properties of the single
glycosyl-avonoids, isolated by ethanolic extraction of H. italicum
owering tops, and in toto glycosidic fraction with in vitro systems
where different reactive oxygen species were generated and on
lipid peroxidation induced by adenosine diphosphate (ADP)/Fe2
and nicotinamide adenine dinucleotide phosphate (NADPH) or
carbon tetrachloride (CCl4) in rat liver microsomes. It was shown
that in toto fraction inhibited superoxide ions and hydroxyl radicals
to a lesser extent than the lipid peroxidation in microsomes. On the
other side, single avonoids act with an additive mechanism, thus
resulting in a synergistic effect in diminishing lipid peroxidation.
Moreover, the action of the glycosidic fraction on the release of
thromboxane B2 (TXB2) and 12-hydroxyeicosatetraenoic acid (12-
HETE) in human platelets after collagen stimulation was also
evaluated, and no effect of the fraction was observed. In both the
articles, extractions were performed with 70% ethanol.
Pietta et al. (1991) developed an isocratic reversed-phase HPLC
method for the separation of the main avonoid glucosides from
the ethanolic extract naringenin-40 -glucoside, kaempferol-3-
glucoside, and 4,20 ,40 ,60 -tetrahydroxychalcone-20 -glucoside of
H. italicum. The applied procedure provided further support for the
validity of using eluents based on C3 alcohols. The same authors
(Pietta et al., 1992) in their second article improved the separation
method for the abovementioned avonoids with few additional
compounds from the same type, including micellar electrokinetic
capillary chromatography (MECC) and ultraviolet diode array
detection (DAD). As in the previous two articles, the extractions
were performed with 70% ethanol.
Zapesnochnaya et al. (1990, 1992) performed the extraction of
H. italicum owers using 80% ethanol and isolated hydroxycin-
namic, chlorgenic (Zapesochnaya et al., 1990), and dicaffeoylquinic
acids (Zapesochnaya et al., 1992). Hydroxycinnamic and chlorgenic
acids were detected by spectrometric methods, whereas the au-
thors proposed HPLC as the most promising method for the qual-
itative and quantitative study of dicaffeoylquinic acids.
Kladar et al. (2015) performed the extraction of H. italicum
owers using 45% ethanol and achieved the highest extraction yield
(19.77%) compared to that of other solvent extractions reported in
this article. Extraction was performed to estimate the total phenolic
and the total avonoid content expressed as gallic acid and quer-
cetin equivalents, respectively, using the FolineCiocalteau reagent
and to test the antioxidant activity of extract. It was shown that the
ability of the H. italicum ethanolic extract to scavenge DPPH free
radical was signicantly stronger than that of the extracts of some
other Helichrysum species reported in literature. This scavenging
potential was comparable to those obtained for quercetin dehy-
drate and propyl gallate. On the other side, the ethanolic extract of
H. italicum demonstrated notable potential to inhibit OH free
radicals generated during the Fenton reaction.
Extraction from H. italicum using ethanol was performed and
described in detail in two articles of Rigano et al. (2013, 2014). In
the rst article, the authors performed extraction of H. italicum
owers by ethanol to investigate the intestinal antispasmodic ef-
fects of the extract (Rigano et al., 2013). Contractility in vitro was
evaluated by stimulating the isolated ileum, in an organ bath, with
acetylcholine and barium chloride. Motility in vivo was evaluated
by measuring the upper gastrointestinal transit, both in the control
mice and in the mice with experimental intestinal inammation
induced by croton oil. It was shown that this extract elicited anti-
spasmodic actions in the isolated mouse ileum and inhibited transit
preferentially in the inamed gut. These actions were achieved by
the synergistic activity of the active ingredients of the extract 12-
acetoxytremetone and [2,3-dihydro-2-[1-(hydroxymethyl)
ethenyl]-5-benzofuranyl]-ethanone (Rigano et al., 2014). These two
16 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
compounds, along with a few others listed in Table 7, were isolated
from the ethanolic extract of H. italicum owers (Rigano et al.,
2014). The structures of all the isolated compounds were eluci-
dated by extensive spectroscopic methods as shown in Table 7. The
compounds were also investigated for their cytotoxicity, anti-
inammatory, and antioxidant properties. Cell viability was
assessed by the neutral red assay, anti-inammatory activity was
assessed by measuring the nitrite production induced by LPS
treatment in macrophages, and antioxidant activity was assessed
by detection of reactive oxygen species in human corneal epithelial
cells (HCEC). Tested compounds did not show negative effect on the
cell survival and anti-inammatory properties, while biological
assays on HCEC showed that 12-acetoxytremetone (Fig. 5)
possessed antioxidant effects specically by reducing the reactive
oxygen species.
Nostro et al. (2004) investigated the antibacterial activity of
H. italicum ethanolic extract against oral streptococci (Streptococcus
mutans, S. salivarius, S. sanguis) by the broth dilution method and
the effects on cell-surface hydrophobicity by the microbial adhe-
sion to hydrocarbon (MATH) test, in vitro sucrose-dependent
adherence to glass surface, and cellular aggregation of S. mutans
in the presence of dextran T2000. Prepared extract exerted inhib-
itory effect on the growth and enzymatic activities of S. mutans.
3.4. Extraction of H. italicum by other solvents
Besides hydrodistillation, Roussis et al. (2000) performed
extraction from small quantities of owers and leaves of four Hel-
ichrysum species (H. italicum, H. orientale, H. heldreichii, and
H. doereri) using a 1:1 mixture of pentane and dichloromethane to
investigate activity against S. aureus, S. epidermidis, K. pneumoniae,
P. aeruginosa, E. coli, and E. cloacae by the paper disc diffusion test.
Antibacterial activity of H. italicum extract was insignicant.
Nostro et al. (2000, 2001, 2002) used different organic solvents
and methods (A and B) for obtain the extract of H. italicum. In
method A, a known amount of powdered drug was sequentially
extracted at room temperature using petroleum ether, dichloro-
methane, dichloromethane-methanol (9:1), and methanol. In
method B, the powdered drug was pretreated with HCl and NaOH
and then extracted using diethyl ether (Nostro et al., 2000). Results
of the disc diffusion test, agar dilution method, and bioautography
indicated that the petroleum ether extract was active against gram-
positive bacteria, while dichloromethane and dichloromethane-
methanol (9:1) extracts were found to have low antimicrobial ac-
tivities. The diethyl ether extract showed considerable antimicro-
bial activity against gram-positive and gram-negative bacteria and
yeasts. Phytochemical analysis of diethyl ether extract showed the
presence of coumarins and avonoids. Compound identication
was performed by TLC and UV using chloroformemethanolewater
(80:18:2) as the elution agent (Nostro et al., 2000).
Diethyl ether extract of H. italicum was further investigated for
its effect on growth of S. aureus and the effect of sub-MICs on some
enzymes, which are considered as the virulence factors (Nostro
et al., 2001). The results indicated that the H. italicum extract had
Fig. 6. Chemical structures of 1 e helypyrone, 2 e micropyrone.
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
inhibitory effect on S. aureus strains reducing both their growth and
some of the enzymes such as coagulase, DNAse, thermonuclease,
and lipase. In addition, it was conrmed that this extract interfered
with the growth and production of some enterotoxins by S. aureus.
The production of enterotoxins in the presence or absence of
H. italicum diethyl extract was estimated in microtiter plates using a
reversed passive latex agglutination (SET-RPLA) kit (Nostro et al.,
2002).
Tomas-Barberan et al. (1990) performed extraction from
H. italicum owers using chloroform and isolated the main anti-
fungal compound identied as 4-hydroxy-3(isopenten-2-yl)aceto-
phenone. The compound was isolated using the column
chromatography on silica gel G-100 with n-hexane-ethyl acetate
(5:1) as the elution agent and identied by UV, IR, EI-MS, 1H NMR
(COSY), and 13C NMR (DEPT) techniques. Antifungal activity of this
acetophenone was investigated applying the liquid lm method
whereby the substance showed antifungal activity against Penicil-
lium sp., Cladosporium herbarum, and Phytophora capsicci. Results of
the agar dilution method showed that 4-hydroxy-3(isopenten-2-yl)
acetophenone was also active against gram-negative bacteria
E. coli.
Karasartov et al. (1992) applied chromatography on silica gel
columns with chloroformemethanol and chloroformepetroleum
ether as elution agents for the isolation of the weakly polar com-
pounds from chloroform extract of H. italicum owers. Three cou-
marins (esculetin, scopoletin, and isoscopoletin) and three
avonoids (kaempferol, 3,5,7-trihydroxy-8-methoxyavone, and
3,5-dihydroxy-6,7,8-trimethoxyavone) were isolated and identi-
ed by UV-VIS and 1 NMR.
Although isolation of phenolic compounds from H. italicum is
related mainly to extraction using organic solvents, as it was shown
in this section, recent work of Jokic et al. (2016) proved that this
could be possible also by supercritical CO2 extraction. Jokic et al.
(2016) performed supercritical extraction of scopoletin from
H. italicum owers under different conditions of pressure and
temperature determined by central composite rotatable design
(CCRD). The main objective of their study was to optimize the
extraction process using response surface methodology (RSM) in
terms of obtaining the highest extraction yield and yield of
extracted scopoletin (Table 4). Scopoletin was determined using
reversed-phase HPLC with UV detection. It was shown that the
highest extraction yield and the highest yield of scopoletin were
obtained under the extraction conditions of 200 bar and 40  C.
Furthermore, supercritical extracts of H. italicum showed good
antioxidant activity determined by the DPPH method and inu-
enced by variation in temperature and pressure.
Finally, it should also be noted that phenolic compounds were
found in aqueous extracts of H. italicum. Kazazic et al. (2016) per-
formed extraction from leaves and owers of 11 medicinal plants.
Ground plant material was used for the extraction using hot water
under stirring. Aqueous extracts were further investigated for their
antioxidant properties using ABTS- and DPPH radical scavenging
capacity assay and FRAP assay. Total phenolic content was deter-
mined by the Folin-Ciocalteau method. Among the tested plant
extracts, H. italicum aqueous extract was the one with high total
phenolic content and signicant antioxidant activity.
The FolineCiocalteau reagent method was performed to deter-
mine the total phenolic content of 45 essential oils investigated in
the work of Wang et al. (2008). Among the tested essential oils, that
of H. italicum was the one with the signicant total phenolic
content.
4. Other phytochemicals of H. italicum
As mentioned, reported data on distillation of H. italicum
 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
essential oils in few cases indicate that the most signicant com-
ponents in essential oils after mono- and sesquiterpenes are b-
diketones, which are also known as italidiones. Bianchini et al.
(2001) showed that the essential oils produced from plants in
early shoots possessed high amounts of ketones and b-diketones.
Furthermore, high content of sesquiterpenes in H. italicum su-
percritical extracts is followed by signicant content of waxes.
Marongiu et al. (2003) showed that the leaf supercritical extract
was particularly rich in waxes.
As in distillation and supercritical CO2 extraction, the main
components isolated by extraction using organic solvents were also
followed by other, less signicant phytochemical classes.
After phenolic compounds, the largest phytochemical class
detected in H. italicum extract was pyrones. Appendino et al. (2007)
performing the extraction of H. italicum using acetone obtained
helypyrone and micropyrone (Fig. 6). Helypyrone had an effect on
the inhibition of NF-kB activation. Furthermore, helypyrone, as a
part of H. italicum acetonic extract, showed antioxidant activity and
could protect linoleic acid against free-radical attack in assays of
autoxidation and EDTA-mediated oxidation (Rosa et al., 2007).
Helipyrone and micropyrone were also isolated by Taglialatela-
Scafati et al. (2013) performing extraction from H. italicum using
acetone. Methanol was used to isolate benzo-g-pyrone glucoside,
which showed anti-inammatory activity in a TPA-induced mouse
ear edema test (Sala et al., 2001). D'Abrosca et al. (2013) isolated
two previously undescribed styrylpyrones by ultrasound-assisted
methanol extraction of H. italicum. Styrylpyrones showed signi-
cant activity against P. aeruginosa biolms.
D'Abrosca et al. (2013) also isolated neolignans and oxy-
neolignans from the methanolic extract of H. italicum.
Taglialatela-Scafati et al. (2013) characterized an unusual class of
lipids named santinols isolated from H. italicum acetonic extract.
Lipids followed by sitosterols were detected in H. italicum acetonic
extract by Sala et al. (2003b), whereas steroids were identied in
H. italicum diethyl ether extract by Nostro et al. (2000).
5. Discussion
Articles presented in this review showed a wide variety of bio-
logical properties of H. italicum extracts and some individual
compounds, divided into three phytochemical classes and obtained
by three distinctive isolation methods.
Distillation by water or steam resulted in different quantities of
essential oils, mainly rich in terpenes followed by b-diketones.
Hydrodistillation in a Clevenger-type apparatus was the most
common process and the reported literature data conrmed many
activities of H. italicum essential oil: antibacterial, antifungal, anti-
oxidant, anti-carcinogenic, antimutagenic, insecticidal, phytotoxic,
geno- and antigenotoxic, and cytotoxic. The dependence of essen-
tial oil chemical composition on planting conditions, geographic
region, soil type, and harvest time was reported as well. Chro-
matographic methods and mass spectrometry, in majority of the
articles, conrmed the presence of particular mono- and sesqui-
terpenes, previously listed, as the main phytochemicals and prob-
ably the carriers of biological activities of H. italicum essential oil.
However, although hydro- and steam distillation are simple pro-
cesses, which do not require additional purication of the product,
they were characterized by low yields (up to 0.78%).
Supercritical CO2 extraction, although being a very interesting
isolation procedure because of its advantages in terms of mild
operating conditions and complete and easy removal of solvent, has
not yet been studied enough in case of H. italicum to create a clearer
picture about the possible uses of the extracts obtained. Although it
is possible to obtain signicantly higher yields than that by distil-
lation process (up to 6.31%), only seven articles in the literature
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isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
17
have reported experimental data related to this topic. The presence
of terpenes (mostly heavier sesquiterpenes) and waxes in super-
critical extracts was conrmed by gas chromatography and mass
spectrometry. The presence of similar main compounds in three
articles conrmed the antimicrobial and antioxidant activities of
the supercritical extracts isolated under relatively low pressures
that leads to an assumption that the biological activities of the
supercritical extracts obtained under lower pressures and essential
oils obtained by distillation should be similar as well. However,
supercritical CO2 extraction at higher pressures certainly resulted
in the co-extraction of higher molecular weight compounds,
whereby the use of polar cosolvents could enhance the extraction
of polar compounds. Yet, with exception of one recent article (Jokic
et al., 2016), there are no data reported on HPLC analysis of extracts
obtained at higher pressures or cosolvent effects of polar
compounds.
Solvent extraction of H. italicum, as the second most popular
isolation method in the literature, revealed possibilities to separate
distinctive groups of phenolic compounds (avonoids, acetophe-
nones, phloroglucinols, tremetones, coumarins and coumarates,
phenolic acids and esters, with their derivatives), followed by
pyrones, lipids, and steroids. Thus far, the most common extraction
solvents are ethanol and methanol, followed by acetone. Literature
studies conrmed different activities of the obtained extracts: anti-
inammatory, antiviral, antioxidant, antibacterial, antifungal, in-
testinal antispasmodic, and cytotoxic. Additionally, various activ-
ities were reported for both cumulative extracts and particular
separated compounds. Among them, the most signicant activity
was found for arzanol, followed by avonoids gnaphaliin, pino-
cembrin, and tiliroside, as well as 12-acetoxytremetone. Because of
the low selectivity of the mentioned organic solvents, high
extraction yields were reported (up to 19.77%).
Ultrasound-assisted solvent extraction and Soxhlet extraction
are well-known effective methods for the extraction of valuable
compounds from plant material. In particular, extraction by the
Soxhlet method could result in high yields. However, no data on
extraction from H. italicum by the Soxhlet method have been re-
ported to date, and there is only one report on ultrasound-assisted
extraction of H. italicum by methanol (D'Abrosca et al., 2013).
An advantage of the supercritical CO2 extraction is its capability
for the isolation of both essential oils and phenolic compounds
from plant material, according to the literature. It was shown that
the supercritical CO2 extraction of essential oils could be optimally
performed at mild pressures (90e100 bar) and temperatures
(40e50  C) (Reverchon and De Marco, 2006). The recommended
temperature range is the consequence of terpenes' thermolability,
while the recommended pressure range is optimal from an equi-
librium point of view and based on the general rule: the higher is
the pressure, the larger is the solvent power and the smaller is the
extraction selectivity (Fornari et al., 2012). Furthermore, introduc-
tion of higher pressures (up to 600 bar) and temperatures (up to
120  C) changes the supercritical CO2 selectivity and leads to the
extraction of different phenolic compounds (Marostica Junior et al.,
2010). Because of the high polarity of phenolic compounds, espe-
cially avonoids, the addition of small amounts of organic cosol-
vents such as ethanol and methanol to change the solvent polarity
and increase the solvating power toward the desired compounds is
required (Marostica Junior et al., 2010; Pereira and Meireles, 2010).
Supercritical CO2 (without cosolvent) could be used for the
extraction of nonpolar and some moderately polar compounds
such as phenolic acids and their esters (Marostica Junior et al.,
2010). Hence, it could be also used for the extraction of avonoid
aglycones, because they are usually extracted using less polar sol-
vents such as benzene, chloroform, and diethyl ether, whereas
avonoid glycosides are commonly extracted using more polar
18 S. Maksimovic et al. / Phytochemistry xxx (2017) 1e20
solvents such as acetone, butanol, methanol, and ethanol (Tan et al.,
2014). Literature data on the supercritical CO2 extraction of avo-
noid aglycones without the addition of cosolvent are presented in
the review article of Oman et al. (2013). For most phenolic com-
pounds, a good recovery rate can be achieved by changing the
abovementioned parameters and the extraction time. Because of
the rich chemical composition of H. italicum, more R&D might be
expected in the future related to the comparison of yields and the
chemical composition of extracts obtained by supercritical CO2
extraction at high pressures and temperatures and by solvent
extraction, including appropriate identication methods, such as
HPLC.
Supercritical CO2 extraction can be used as the rst step in
fractional extraction also. For example, it can be used to isolate
valuable components and pretreat the herbaceous matrix prior to
solvent extraction. After the decompression step in the high pres-
sure processing, the plant material undergoes swelling, which en-
ables easier release of active principles in the posterior solvent
extraction.
Finally, the various biological activities of different isolates of
H. italicum and those of particular individual compounds listed
could be the reference for the future R&D of medical, pharmaceu-
tical, and cosmetic formulations based on H. italicum isolates or
compounds as the main ingredients.
6. Conclusion
Because of the various bioactive constituents, H. italicum has
been intensively studied in the last three decades. The aim of this
review article was to present and classify literature data on phy-
tochemicals from H. italicum terpenes, phenolic compounds, and
other phytochemicals with regard to their biological activities and
the isolation technique. According to the literature data, the phy-
tochemicals from H. italicum have been isolated by three distinctive
methods. Distillation and supercritical CO2 extraction provided
extracts rich in terpenes, while organic solvent extraction was
found to be the main method for isolation of phenolic compounds.
Phytochemicals were identied mostly by different chromato-
graphic techniques followed by spectroscopic methods. Classied
experimental conditions and the results obtained for each separa-
tion technique are given in tables.
According to the literature data, distillation of H. italicum aerial
parts, the most common procedure for isolation of phytochemicals,
resulted in obtaining the essential oils with different chemical
proles depending on the plant's geographic origin and planting
conditions. Reported chemical proles of essential oil indicated the
dominance of monoterpenes a-pinene, limonene, nerol, neryl ac-
etate, and neryl propanoate and sesquiterpenes aselinene, b-
selinene, g-curcumene, trans-b-caryophyllene, and eudesm-5-en-
11-ol. In few cases, b-diketones were detected in H. italicum
essential oils as the second main compound class. Biological assays
indicated that the essential oil of H. italicum possesses various
biological activities ascribed mainly to sesquiterpenes. Among
them, antimicrobial, antioxidant, insecticidal, and cytotoxic activ-
ities were the most tested and proven. Nevertheless, except few
cases related to insecticidal activity, these activities were tested
in vitro and left scope for their demonstration in vivo.
Regardless of its well-known advantages over the conventional
isolation procedures and the possibility to obtain signicantly
higher yields than that by the distillation process, supercritical CO2
extraction of H. italicum was presented in terms of relatively poor
information taken from the seven articles. Literature data indicated
the dominance of sesquiterpenes, followed by waxes, in chemical
proles of supercritical extracts. Moreover, there is an evident lack
of information on the biological activities of supercritical extracts,
Please cite this article in press as: Maksimovic, S., et al., Separation of phytochemicals from Helichrysum italicum: An analysis of different
isolation techniques and biological activity of prepared extracts, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.01.001
which is limited to antimicrobial and antioxidant activities tested
in vitro and presented in three articles. It was assumed that the
supercritical extracts obtained at conditions of lower pressure (up
to 200 bar) and essential oils obtained by distillation had no sig-
nicant difference in their biological activities because of their
quite similar chemical composition.
Data on solvent extraction of H. italicum, among which acetone,
methanol, and ethanol were the most commonly applied solvents,
showed diverse biological properties of both total extracts and in-
dividual separated phenolic compounds, followed by pyrones,
lipids, and steroids tested both in vivo and in vitro. The most
important compounds in H. italicum extracts obtained by organic
solvents were found to be arzanol, a prenylated phloroglucinyl a-
pyrone, avonoids gnaphaliin, pinocembrin and tiliroside, and 12-
acetoxytremetone. Arzanol was proven to be a very important
compound because of its strong anti-inammatory activity, tested
both in vitro and in vivo, in human intestinal epithelial cells.
Furthermore, extracts of H. italicum obtained using organic solvents
were proven to possess anti-inammatory activities in topical and
intestinal applications, followed by antioxidant, antimicrobial, and
cytotoxic as the main activities, tested both in vitro and in vivo,
mostly in experiments with rats and mice. These studies could be
the reference for introducing more in vivo and clinical in-
vestigations of biological activities of H. italicum isolates and their
individual compounds.
Finally, the review indicated that the ultrasound-assisted
extraction method was applied in one study only, and the data on
extraction of H. italicum by the Soxhlet method have not yet been
published. Isolation of phenolic compounds by supercritical CO2
with the addition of a cosolvent at higher pressures should be more
explored in the future.
Acknowledgments
Financial support of the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grant Nos
45001 and 45017) is gratefully acknowledged.
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Svetolik Maksimovic is a PhD student at the Faculty of
Technology and Metallurgy, University of Belgrade. He
obtained his degree in Chemical Engineering in 2010 and
the Master in Chemical Engineering in 2011 at the Faculty
of Technology and Metallurgy. His research interests are
related to natural products, including supercritical CO2
extraction from plant material and supercritical impreg-
nation of different solid carriers with plant extracts, with
an aim to produce bioactive materials for possible appli-
cations in medicine and pharmacy.
Vanja Tadic graduated at the Faculty of Pharmacy, Uni-
versity of Belgrade, and she obtained her PhD in Science -
Chemistry of natural products at Chemistry Faculty, Uni-
versity of Belgrade. She did her post-doc at Mayo Clinic,
Rochester, USA, at Department of Biochemistry and Mo-
lecular Biology. Presently, she works as Science Advisor
at the Department of Pharmaceutical Research and Devel-
opment, Institute for Medicinal Plant Research Dr Josif
Pancic, Belgrade. She has been participating as one of
the principal investigators in several research projects,
and was leader of the project whose patents won the
Gold Medal and Diploma for the best innovation of the
WIPO 2010. She is author of more than a hundred scienti-
c publications. Her main research interests are chemistry
of natural products (extraction, identication, fraction-
ation and isolation of chemical compounds from natural
matrices, especially medicinal plants) and their biological
activities. She has experience in the formulation of high
value products in pharmaceuticals, nutraceuticals and
food sectors. She has supervised several PhD and master
students. Dr Vanja Tadic is a reviewer in a number of
prominent international journals.
Dejan Skala is professor at University of Belgrade, who
retired in 2013, but he is still actively working on research
in different elds of chemical engineering. He has auth-
ored about 180 different publications, and a substantial
number of them focused on extraction of different com-
pounds from plants using supercritical or other commonly
used techniques such as hydrodistillation, steam distilla-
tion, extraction with common organic solvents or their
mixture with water, etc. His research activity of super-
critical extraction dated from 1987 and, in many cases, the
activities were focused on isolation of specic compounds
from medical and spicy herbs, their characterization, and
analysis of potential application of various extracts as
antioxidants or systems with acceptable antimicrobial
activity.
Irena Zizovic is Full Professor in Chemical Engineering at
the Faculty of Technology and Metallurgy, University of
Belgrade. She holds PhD in Chemical Engineering (2006,
University of Belgrade), MSc in Chemical Engineering
(1996, University of Belgrade) and BSc in Chemical Engi-
neering (1991, University of Belgrade). Her research ac-
tivities are in the eld of applications of supercritical
uids. Her contributions to the eld are in mathematical
modeling and optimization of supercritical uid extrac-
tion processes from plant materials as well as in the
development of added value materials using supercritical
uids. She leads a multidisciplinary research group that
consists of scientists in Chemical Engineering, Microbi-
ology, and Pharmacy. Prof. Irena Zizovic was the principal
investigator in four international and two national
research projects.