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Vol. 13(5), pp. 96-103, 10 March, 2019
DOI: 10.5897/JMPR2018.6724
Article Number: 213B77160480
ISSN 1996-0875
Copyright © 2019
Author(s) retain the copyright of this article
http://www.academicjournals.org/JMPR
Journal of Medicinal Plants Research
Full Length Research Paper
Edraianthus pumilio (Schult.) A. DC.: Phytochemical
and biological profile
Olivera Politeo1*, Stanislava Talic2 and Mirko Ruscic3
1Department of Biochemistry, Faculty of Chemistry and Technology, University of Split, 21000 Split, Croatia.
2Department of Chemistry, Faculty of Science and Education, University of Mostar, 88000 Mostar, Bosnia and
Herzegovina.
3Department of Biology, Faculty of Science, University of Split, 21000 Split, Croatia.
Received 12 December, 2018; Accepted 5 March, 2019
Edraianthus pumilio is stenoendemic plant native to Dalmatia, Croatia. This paper deals with its
phytochemical and biological profile. Phytochemical profile of volatile oil was performed by the gas
chromatography–mass spectrometry (GC/MS), while total phenolic content of its aqueous extract was
performed by Folin-Ciocalteu method. The phytochemical analysis showed that the main volatile oil
compounds were nonanal (21.2%) and myristicin (16.4%). This oil could be characterized as nonanal-
myristicin type. Total phenolic content of aqueous extract was 30.6 ± 1.1 mg GAE/g extract. Results of
testing antioxidant potential of E. pumilio volatile oil and aqueous extract showed low antioxidant
potentials as tested by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power
(FRAP) methods. Results also showed low acetylcholinesterase inhibition potential of volatile oil and
low to moderate potential of aqueous extract, as tested by Ellman method.
Key words: Edraianthus pumilio (Schult.) A. DC., volatile oil, aqueous extract, gas chromatography–mass
spectrometry (GC/MS), total phenolic content, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant
power (FRAP), Ellman.
INTRODUCTION
In terms of biological diversity Croatia is one of the
richest European countries. Croatian flora, with about
5000 species and subspecies is characterized by a
markedly high level of diversity per unit of surface (Nikolic
et al., 2015). Genus Edraianthus in flora Europe includes
9 species, 6 of which belong to the flora Croatica (Tutin
et. al., 1980: Nikolic, 2018). Edraianthus pumilio (Schult.)
A. DC. is stenoendemic species of flowering plant in the
family Campanulaceae, native to Dalmatia in Southern
Croatia. It is a strictly protected and almost endangered
species. It is widespread on Mount Biokovo, near
Makarska (Nikolic, 2018). The plant is highly heliophilic,
thermophilic and xerophilic. It grows on limestone-rock
cracks. This is a low-growing perennial plant that grows
in dense pads. Linear and silver leaves grow in tufts. The
underside of leaves is naked, while upper side of leaves
*Corresponding author. E-mail: olivera@ktf-split.hr. Tel: +38521329437. Fax: +38521329461.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution
License 4.0 International License
is hairy. The flowers are violet-blue bells, while fruits are
capsules. The plant belongs to alliance Seslerion
juncifoliae Horvat 1930 (Nikolic et al., 2015).
Previous Edraianthus studies mainly focused on
systematization of plants. The genus was the subject of
cytogenetic investigations (Siljak-Yakovlev et al., 2010),
molecular phylogenetic and phylogeographic studies
(Park et al., 2006; Lakušić et al., 2016) as well as finding
new species and determining their taxonomic status
(Boštjan et al., 2009).
According to molecular phylogeny analysis the E.
pumilio and E. dinaricus are two very closely related
species. Both species are closely restricted to the
mountains of Middle Dalmatia. These two species are
quite different from other Edraianthus species in terms of
morphological features such as leaf, inflorescence and
habits (Stefanović et al., 2008).
Chemical taxonomy at this genus is poorly researched.
The chemical structures of phenolics and terpenoids are
often specific and restricted to taxonomically related
organisms and hence useful in classification (Singh,
2016). Secondary metabolites and beneficial biological
effects of E. pumilio have not been investigated so far.
These compounds have shown to have antioxidant,
antimicrobial, antiinflammatory, anticarcinogenic,
antimutagenic and antiallergic properties (Roy et al.,
2017; Murugesan and Deviponnuswamy, 2014; Bharti et
al., 2012: Namita and Mukesh, 2012; Allesiani et al.,
2010).
Antioxidants are vital substances, which possess the
ability to protect the body from damages caused by free
radical-induced oxidative stress (Birben et al., 2012). A
large amount of evidence has demonstrated that
oxidative stress is intimately involved in age-related
neurodegenerative diseases. There have been a number
of studies which examined the positive benefits of
antioxidants to reduce or to block neuronal death
occurring in the pathophysiology of these disorders
(Loizzo, 2009; Ramassamy et al., 2006).
Acetylcholinesterase (AChE) is the enzyme involved in
the hydrolysis of acetylcholine neurotransmitter which
plays important role in memory and cognition. Low level
of AChE in brain is usually connected with
neurodegenerative disease, that is, Alzheimer’s disease
(AD). The most promising approaches in treating AD are
usually based on AChE inhibitors (Wszelaki et al., 2010;
Menichini et al., 2009).
Therefore, the aim of this work was to determine the
phytochemical composition of volatile oil, total phenolic
content of aqueous extract as well as antioxidant and
acetylcholinesterase inhibition potential of volatile oil and
aqueous extract isolated from E. pumilio. There are no
known records of this plant being used for medicinal
purposes but this or another research may contribute
otherwise. Also, the analysis of volatile components can
contribute to a better determination of the species E.
pumilio. To the best of our knowledge, this is the first
work that shows the phytochemical and biological profile
Politeo et al. 97
of this species.
MATERIALS AND METHODS
Plant material
Plant material (whole plants, without roots) was collected from its
natural habitat, via St. Ilija, Biokovo Mountain, Croatia (Central
Dalmatia, Makarska) during flowering in July, 2017, at 1500 m a.s.l.;
Gauss-Krüger coordinates: X=5663111, Y=4804120. The botanical
identity of the plant material was confirmed by a botanist PhD Mirko
Ruscic, associate professor, Department of Biology, Faculty of
Science, University of Split. Voucher specimens were deposited in
herbarium at Department of Biology, Faculty of Science, University
of Split, Croatia, under number EP_7/17.
Volatile oil isolation
Air-dried aerial parts of plants were hydro-distilled using Clavenger
apparatus for 3 h. Obtained essential oil was dried over anhydrous
Na2SO4 and stored in sealed vials, under - 20 oC before use (Nikbin
et al., 2014).
Preparation of aqueous extract
Air-dried aerial parts of plants and water were placed in an
Erlenmeyer flask and refluxed in an ultrasound bath for 2 h. The
mixture was then filtered through a filter paper and evaporated
under vacuum at 40°C and stored at -20°C in fridge before use.
Gas chromatography-mass spectrometry analysis
The analysis of the volatile oil was carried out using Shimadzu gas
chromatography–mass spectrometry (GC/MS), QP2010 system
equipped with an AOC 20i autosampler, using fused silica capillary
column Inert Cap (5% diphenyl, 95% dimethylpolysiloxane, 30 m ×
0.25 mm i.d., film thickness 0.25 μm). The operating conditions
were as follows: injection volume: 1.0 µl of volatile oil solution
(1:500 v/v in pentane); injection mode: splitless; injection
temperature: 260 oC; carrier gas: helium, 1.11 ml/min; the oven
temperature program: 50°C (5 min), 50 - 260°C (3°C/min); MS
conditions: ion source temperature: 200°C, ionization voltage: 70
eV, mass range: m/z 40 - 400 u. GCMSolution 2.5 (Shimadzu) was
used to handle data. Identification of volatile oil components was
based on (a) retention indices on non-polar column relative to a
homologous series of n-alkanes (C8 - C40), (b) on the comparison of
their mass spectra and retention indices with the NIST and Wiley
spectra library and with those reported in the literature (Adams,
2007; Linstrom and Mallard, 2014). GC/MS analysis was performed
in triplicate and results were averaged.
Total phenolic content
Total phenolic content was measured using Folin-Cocalteu
spectrophotometric method (Singleton and Rossi, 1965; Katalinic et
al., 2013) and gallic acid as a standard. 15 ml water and Folin-
Ciocalteu reagent (1.25 ml, diluted 1:2) were added to the sample
solution (0.25 ml, 1 mg/ml). The resulting solution was mixed. After
3 min, in solution were added Na2CO3 (3.75 ml, 20%) and water to
volume of 25 ml. The resulting mixture was then left for 2 h. The
absorbance of the resulting blue color was measured at 765 nm.
The concentration of the total phenolic content was calculated by
using an equation obtained from gallic acid calibration curve and
98 J. Med. Plants Res.
expressed as mg gallic acid equivalent per gram of extract (mg
GAE/g extract). The determination of total phenolic content was
carried out in triplicate and the results were expressed as mean ±
standard deviations.
Antioxidant capacity
Antioxidant capacity of volatile oil and aqueous extract from E.
pumilio were tested using two different methods: 2,2-diphenyl-1-
picrylhydrazyl (DPPH) method and ferric reducing antioxidant power
(FRAP) method.
Capacity of DPPH radical scavenging by volatile oil and aqueous
extract was measured according to the method of Brand-Williams et
al. (1995) (Katalinic et al., 2010). DPPH method is based on the
reaction between free DPPH radicals and antioxidants. As a result,
a stable non-radical form of the DPPH is obtained, with
simultaneous change of the violet color to pale yellow. The
decrease in absorbance was measured at 517 nm. DPPH radical
solution was prepared by dissolving the stock solution (4 mg of
DPPH in 100 ml of ethanol). To optimize the conditions used to run
the DPPH assay in microplates, 10 µl of sample (1 mg/ml) was
placed in a well and 290 µI of DPPH solution was added. The
mixture was shaken vigorously and left to stand at room
temperature in the dark. The decrease in the absorbance was
measured after 1 h, with ethanol as blank. The DPPH radical
scavenging activity of the sample was calculated according to the
formula:
% inhibition = [(A0 – Asample)/A0] ×100
where A0 was absorbance of the DPPH ethanol solution measured
at the beginning and Asample was absorbance of the sample
measured after 60 min. The results were expressed as percentage
inhibition of DPPH. Butylated hydroxyanysole (BHA) was used as
positive control.
The reducing power of volatile oil and aqueous extract were also
performed using FRAP method (ferric reducing antioxidant power)
(Skroza et al., 2015; Benzie and Strain, 1996). FRAP method is
based on the reduction of ferric-tripyridyltriazine (Fe3+-TPTZ)
complex to ferrous (Fe2+) complex with an intense blue color and
maximum absorption at 593 nm. The method was performed in 96-
well microplates, with slight modifications. The FRAP solution was
freshly prepared by mixing 0.3 M acetate buffer (pH = 3.6) and 10
mM TPTZ in 40 mM HCl and 20 mM FeCl3 in a ratio of 10:1:1 (by
volume). The assay was carried out by placing 10 µl of the sample
(1 mg/ml) and 300 µl of FRAP reagent in a well. The absorbance
was measured after 4 min. The reducing power of sample was
calculated by comparing with the reaction signal given by solution
of Fe2+ ions in known concentration and expressed as µmol/I Fe2+.
BHA was used as positive control. The determination of antioxidant
capacity, performed by both methods was carried out in triplicate
and the results were expressed as mean ± standard deviations.
Tested stock solution concentration was 1 mg/ml.
Acetylcholinesterase inhibition potential
Acetylcholinesterase (AChE) inhibition potential of volatile oil and
aqueous extract were carried out by a slightly modified Ellman
assay (Politeo et al., 2018; Ellman, 1961). A typical run consisted of
180 I of phosphate buffer (0.1 M, pH 8), 10 µl of DTNB (at a final
concentration of 0.3 mM prepared in 0.1 M phosphate buffer pH 7
with 0.12 M sodium bicarbonate added for stability), 10 µl of sample
solution (dissolved in 80% EtOH), and 10 µl of AChE solution (with
final concentration 0.03 U/ml). Reactants were mixed in a 96-well
plate wells and reaction was initiated by adding 10 µl of
acetylthiocholine iodide (ATChI to reach a final concentration of 0.5
mM). As a negative control, 80% EtOH was used instead of sample
solution. Non-enzymatic hydrolysis was also monitored by
measurement of two blank runs for each run. All spectrophotometric
measurements were performed at 405 nm and at room temperature
for 6 min. The results are expressed as percentage inhibition of
enzyme activity. Eserine was used as positive control. The
determination of acetylcholinesterase inhibition potential was
carried out in triplicate and the results were expressed as mean ±
standard deviations. Tested stock solution concentration of E.
pumilio samples was 1 mg/mL, while stock solution concentration of
eserine was 0.1 mg/ml.
RESULTS AND DISCUSSION
Phytochemical composition of E. pumilio volatile oil
E. pumilio volatile oil yield was 0.31% (w/w). The analysis
revealed forty two compounds separated into five
classes: nonterpene compounds, phenyl propanes,
terpene compounds, norisoprenoids, and other
compounds (Table 1). Nonterpene compounds (54.6%)
were predominated compound class in E. pumilio volatile
oil. Among them nonterpene aldehydes (41.8%) were the
most common class of compounds with nonanal (21.2%)
as a major one. (E,E)-2,4-Decadienal (3.9%), tridecanal
(3.4%), decanal (2.5%), octanal (2.5%), (E,Z)-2,4-
decadienal (2.1%) and others nonterpene aldehydes
were identified in lower quantity. Nonterpene
hydrocarbons (7.4%), ketones (4.0%), alcohols (1.1%)
and esters (0.3%) were also identified in lower quantity.
Among these compounds, the most common compound
was 4-methyldecane (2.9%), while other compounds
were identified in quantity lower than 2%. Second one
quantitatively important compound class, phenyl
propanes (17.3%), was presented with two compounds:
myristicin (16.4%) as predominant compound and anisole
(0.9%). Terpene compounds (15.1%) were mainly
presented with monoterpene compounds. Among them
the main ones were hexahydrofarnesyl acetone (5.1%),
prenol (2.2%) and ß-myrcene (2.0%). Other monoterpene
compounds were identified in quantity lower than 2%.
Sesquiterpene compounds were present only with one
compound, ß-caryophyllene (0.4%). Norisoprenoids
(0.4%) were presented with two compounds: ß-ionone
(0.1%) and ß-ionone epoxide (0.3%). 4-Vinyl phenol
(1.6%), benzothiazole (0.7%) and indole (0.5%) were
presented as other compounds (2.8%).
Total phenolic content of E. pumilio aqueous extract
The total phenolic content of E. pumilio aqueous extract
(the extraction yields was 14.8%, w/w) was determined
using Folin-Ciocalteu reagent and external calibration
with gallic acid, according to the method previously
described by Singleton and Rossi (Katalinic et al., 2013;
Singleton and Rossi, 1965). The total phenolic content
was 30.6 ± 1.1 mg GAE/g extract (Table 2).
Politeo et al. 99
Table 1. E. pumilio: Volatile oil constituents.
S/N
Compounds
%
RIa
Mode of identification
Nonterpene compounds
54.6
Nonterpene aldehydes
41.8
1
Benzaldehyde
1.4
961
RI, MS
2
Octanal
2.5
1001
RI, MS
3
Phenyl acetaldehyde
1.0
1044
RI, MS
4
Nonanal
21.2
1104
RI, MS
5
(E)-2-Nonenal*
1.1
1161
RI, MS
6
Decanal
2.5
1204
RI, MS
7
(E)-2-Decenal*
0.8
1263
RI, MS
8
(E,Z)-2,4-Decadienal*
2.1
1299
RI, MS
9
Undecanal
1.0
1305
RI, MS
10
(E,E)-2,4-Decadienal*
3.9
1314
RI, MS
11
(E)-2-Undecenal*
0.2
1360
RI, MS
12
Tridecanal
3.4
1504
RI, MS
13
Pentadecenal
0.7
b
-, MS
Nonterpene hydrocarbons
7.4
14
3,5,5-Trimethyl-2-hexene
0.8
986
RI, MS
15
4-Methyldecane
2.9
1060
RI, MS
16
2- Methyldecane
0.4
1072
RI, MS
17
1-Dodecene
1.1
1193
RI, MS
18
1-Tetradecene
1.8
1390
RI, MS
19
Pentadecane
0.4
1500
RI, MS
Nonterpene ketones
4.0
20
(E,E)-3,5-Octadien-2-one*
0.8
1093
RI, MS
21
(E)-2-Methyl-2-nonen-4-one*
1.7
1216
RI, MS
22
(Z)-3-Nonen-2-one*
0.6
1332
RI, MS
23
6,10-Dimethyl-undecan-2-one
0.9
1403
RI, MS
Nonterpene alcohols
1.1
24
3,5-Octadien-2-ol
1.1
1037
RI, MS
Nonterpene esters
0.3
25
cis-3- Hexenyl tiglate
0.3
1319
RI, MS
Phenyl propanes
17.3
26
Anisole
0.9
1235
RI, MS
27
Myristicin
16.4
1513
RI, MS
Terpene compounds
15.1
Monoterpene ketones
5.5
28
Neryl acetone
0.4
1443
RI, MS
29
Hexahydrofarnesyl acetone
5.1
b
-, MS
Monoterpene hydrocarbons
5.1
30
α-Thujene
1.3
925
RI, MS
31
β-Myrcene
2.0
991
RI, MS
32
p-Cymene
0.7
1022
RI, MS
33
Limonene
1.1
1028
RI, MS
100 J. Med. Plants Res.
Table 1. Cont.
Monoterpene alcohols
4.1
34
Prenol
2.2
778
RI, MS
35
Linalool
1.6
1101
RI, MS
36
cis-Sabinol
0.3
1142
RI, MS
Sesquiterpenes
0.4
37
ß-Caryophyllene
0.4
1410
RI, MS
Norisoprenoids
0.4
38
β-Ionone
0.1
1475
RI, MS
39
(E)-5,6-Epoxy-ß-ionone*
0.3
1479
RI, MS
Other compounds
2.8
40
p-Vinylphenol
1.6
1222
RI, MS
41
Benzothiazole
0.7
1224
RI, MS
42
Indole
0.5
1292
RI, MS
Total
90.2
* Correct isomers were not identified; RI = Kovats index determined on a Inert Cap column using the homologous series of n-
hydrocarbons C8-C40; MS = mass spectra; b = The RI was outside of the RI interval of series of n-alkanes.
Table 2. Total phenolic content, antioxidant and anticholinesterase inhibition capacity of volatile oil and water extract
from E. pumilio
Variable
Total phenolic
content mg
GAE/g extract
Antioxidant capacity
AChE inhibition
%
DPPH
inhibition %
FRAP
µmol/L Fe2+
Ep-volatile oila
-
ni
15.3 ± 0.7
26.6 ± 2.1
Ep-aqueous extracta
30.6 ± 1.1
10.7 ± 0.7
118.7 ± 7.2
46.9 ± 4.7
BHAa
-
91.9 ± 2.9
5586.3 ± 72.6
-
eserineb
-
-
-
95.9 ± 1.9
Ep = Edraianthus pumilio; astock solution concentration was 1 mg/ml (0.048 mg/mL for DPPH, 0.029 mg/ml for FRAP and
0.045 mg/ml for AChE in reaction system); bstock solution concentration was 0.1 mg/mL (4.5 µg/ml for AChE in reaction
system); ni = inhibition not identified.
Antioxidant capacity of volatile oil and aqueous
extract from E. pumilio
Antioxidant potential of volatile oil and aqueous extract
from E. pumilio (Ep) are presented in Table 2 and Figure
1. Results obtained were compared with those of well-
known antioxidant BHA. Result showed no antioxidant
potential of Ep-volatile oil and low antioxidant potential of
Ep-aqueous extract (10.7±0.7%) tested by DPPH method
as well as low antioxidant capacity of Ep-volatile oil
(15.3±0.7 ek. µmol/L Fe2+) and Ep-aqueous extract
(118.7±7.2 ek. µmol/L Fe2+) tested by FRAP method for
tested stock solution concentration of 1 mg/ml (0.048
mg/ml for DPPH and 0.029 mg/ml for FRAP in reaction
system). In comparison, well-known antioxidant
compound BHA inhibited DPPH with 91.9±2.9%, while
antioxidant capacity tested by FRAP method was eq..
5586.3 ± 72.6 µmol/L Fe2+ for the same tested
concentration. Low antioxidant capacity of Ep-volatile oil
and slightly better, but still low, antioxidant capacity of
Ep-aqueous extract could be connected to chemical
composition of these extracts. Namely, tested Ep-volatile
oil does not contain components responsible for
antioxidant potential such as phenolic or monoterpenoid
compounds (eugenol, carvacrol, thymol, menthol)
(Brewer, 2011; Bakkali et al., 2008), while low antioxidant
capacity of Ep-aqueous extract is in correlation with low
total phenolic content of tested extract (30.6±1.1 mg
GAE/g extract). Therefore, Ep-aqueous extract probably
contains low quantity of phenolic components with good
antioxidant capacity such as phenolic acids (gallic,
protochatechuic, caffeic and rosmarinic acids), phenolic
Politeo et al. 101
Figure 1. Antioxidant capacity of volatile oil and aqueous extract from E. pumilio (tested concentrations were 1 mg/ml).
Figure 2. Acetylcholinesterase inhibition of volatile oil and aqueous
extract from E. pumilio (tested concentrations were 1 mg/ml and 0.1
mg/ml for eserine).
diterpenes (carnosol and carnosic acid) and flavonoids
(quercetin and catechin) (Brewer, 2011) but these
compounds cannot significantly contribute to antioxidant
capacity of Ep-aqueous extract.
Acetylcholinesterase inhibition potential of volatile
oil and aqueous extract from E. pumilio
Acetylcholinesterase inhibition potential of Ep-volatile oil
and Ep-aqueous extract is presented in Table 2 and
Figure 2. Results obtained were compared with those of
well-known AChE inhibition agent, eserine. Results
showed low AChE inhibition potential of Ep-volatile oil
(26.6±2.1%) and low to moderate AChE inhibition
potential for Ep-aqueous extract (46.9±4.7%) in tested
concentration of 1 mg/ml (0.045 mg/ml in reaction
system). In comparison, well-known AChE inhibitor
eserine showed 95.9±1.9% AChE inhibition in tested
concentration of 0.1 mg/ml (4.5 µg/ml in reaction system).
Low AChE inhibition potential of Ep-volatile oil and low to
moderate AChE inhibition potential for Ep-aqueous
extract could also be connected to chemical composition
of tested extracts. Namely, tested volatile oil probably
does not contain compounds responsible for AChE
inhibition potential, such as α-pinene, δ-3-carene, 1,8-
cineole, α- and β-asarone (Burcul et al., 2018), while Ep-
aqueous extract probably contains low quantity of
compounds responsible for AChE inhibition, such as
compounds with catechol moiety in their structure (Ji and
Zhang, 2006), polymers of resveratrol (Jang et al., 2007),
stilbene oligomers (Sung et al., 2002) or others
(Szwajgier, 2014; Suganthy et al., 2009; Mukherjee,
2007).
Conclusions
The phytochemical analysis of E. pumilio volatile oil
revealed forty two compounds separated into five
102 J. Med. Plants Res.
classes: nonterpene compounds, phenyl propanes,
terpene compounds, norisoprenoids, and other
compounds. Among identified compounds, the main E.
pumilio volatile oil compounds were nonanal (21.2%) and
myristicin (16.4%) and this oil could be characterized as
nonanal-myristicin type. The total phenolic content of E.
pumilio aqueous extract was 30.6 ± 1.1 mg GAE/g
extract.
Results of testing antioxidant potential of E. pumilio
volatile oil and aqueous extract showed no antioxidant
potential of volatile oil and low antioxidant potential of
aqueous extract tested by DPPH method as well as low
antioxidant capacities of volatile oil and aqueous extract
tested by FRAP method, in comparison with BHA.
Results of acetylcholinesterase inhibition potential test
showed low potential of volatile oil and low to moderate
potential of aqueous extract in comparison with eserine.
The volatile oils rich in nonanal as well as nonanal
isolated from volatile oil may have other useful biological
properties such as antidiarrhoeal (Zavala-Sanchez et al.,
2002). Myristicin (a phenylpropanoid) was reported to
have cytotoxic effects (Lee et al., 2005), anti-cholinergic,
antibacterial, hepatoprotective effects and anti-
inflammatory properties (Lee and Park, 2011) and also
can inhibit tumorigenosis in mice (Zheng et al., 1992).
Future studies of phytochemical compound should
focus on other possible biological effects such as anti-
inflammatory, antidiarrheal, and anticancer.
CONFLICT OF INTERESTS
The authors have not declared any conflict of interests.
ACKNOWLEDGMENTS
This work was supported by the Croatian Science
Foundation; project IP-2014-09-6897.
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