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FARMACIA, 2017, Vol. 65, 4
624
ORIGINAL ARTICLE
INVESTIGATION OF ANTIOXIDANT AND ANTIMICROBIAL
POTENTIAL OF SOME EXTRACTS FROM HEDERA HELIX L.
CARMEN ELENA POP1#, MARCEL PÂRVU2#, ANDREEA LETIȚIA ARSENE3#*, ALINA
ELENA PÂRVU4#, DAN CRISTIAN VODNAR5#, MONICA TARCEA6#, ANCA MARIA TOIU7#,
LAURIAN VLASE8#
1“Iuliu Haţieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Drug Industry and
Pharmaceutical Biotechnology, 8 Victor Babeș Street, Cluj-Napoca, Romania
2“Babeș - Bolyai” University, Faculty of Biology and Geology, Department of Biology, 42 Republicii Street, Cluj-Napoca,
Romania
3“Carol Davila” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of General and Pharmaceutical
Microbiology, 6 Traian Vuia Street, District 2, Bucharest, Romania
4“Iuliu Hațieganu” University of Medicine and Pharmacy, Faculty of Medicine, Department of Pathophysiology, 3 Victor
Babeș Street, Cluj-Napoca, Romania
5University of Agricultural Sciences and Veterinary Medicine, Faculty of Food Science and Technology, Department of Food
Science, 3-5 Mănăștur Street, Cluj-Napoca, Romania
6University of Medicine and Pharmacy, Faculty of Medicine, Department of Community Nutrition and Food Safety, 38
Gheorghe Marinescu Street, Târgu Mureș, Romania
7“Iuliu Hațieganu” University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacognosy, 8 Victor
Babeș Street, Cluj-Napoca, Romania
8“Iuliu Hațieganu” University of Medicine and Pharmacy, Department of Pharmaceutical Technology and
Biopharmaceutics, 8 Victor Babeș Street, Cluj-Napoca, Romania
*corresponding author: andreeanitulescu@hotmail.com
#All authors have equal contribution to this paper.
Manuscript received: September 2016
Abstract
The aim of this study was the investigation of the antioxidant and the antimicrobial potential of some ethanolic extracts from
Hedera helix (leaves, flowers, immature and ripe fruits) and the correlation with the profile of polyphenols, using an LC/MS
method. In all samples, the chlorogenic acid, isoquercitrin, rutin and quercetin have been identified. The DPPH (2,2-
diphenyl-1-picrylhydrazyl) assay, Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) were used to evaluate
the antioxidant effects and the results showed a better antioxidant activity for H. helix ripe fruits ethanolic extract and a
positive correlation between antioxidant effects, polyphenolic and flavonoid contents. The antimicrobial effect was tested
using six bacterial strains and the microdilution method. The immature fruits extract showed a significant antibacterial
activity against Staphylococcus aureus, while both immature fruits and flowers extracts possess a good antibacterial activity
against Listeria monocytogenes.
Rezumat
Scopul acestui studiu a constat în testarea potențialului antioxidant și antimicrobian ale unor extracte etanolice din frunze,
flori, fructe verzi și coapte de Hedera helix, precum și corelarea rezultatelor cu profilul polifenolilor determinat prin LC/MS.
În toate probele au fost identificați: acidul clorogenic, isoquercitrina, rutozida și quercetolul. Pentru evaluarea efectului
antioxidant s-a utilizat metoda DPPH (2,2-difenil-1-picrilhidrazil) și s-a evaluat conținutul total în polifenoli și flavonoide
totale. Rezultatele au evidențiat o acțiune antioxidantă mai bună pentru extractul din fructe coapte de H. helix, ceea ce a fost
în concordanță cu datele experimentale obținute la calculul conținutului de compuși polifenolici și de flavonoide. Efectul
antimicrobian a fost testat pe șase tulpini bacteriene, prin tehnica microdiluțiilor. Extractul obținut din fructe imature a avut o
activitate antibacteriană semnificativă pe Staphyloccocus aureus. De asemenea, extractul din fructe imature și cel din flori au
prezentat o activitate antibacteriană bună pe Listeria monocytogenes.
Keywords: polyphenols, Hedera helix, antioxidant, antimicrobial, LC/MS
Introduction
The structural class of polyphenols comprises more
than 8000 compounds, generally identified in higher
plants. Throughout the past ten years, researchers
have become very interested in plants containing
polyphenols, so the literature abounds in scientific
publications emphasizing the positive effects associated
to these compounds.
Polyphenols are naturally occurring biomolecules,
secondary metabolites biosynthesized in plants. Their
FARMACIA, 2017, Vol. 65, 4
625
role is to defend plants against ultraviolet radiation
and aggression of pathogen microorganisms. It is
scientifically proved that plants with high content in
polyphenols are efficient for the prevention of some
diseases associated with oxidative stress such as
cancer, neurodegenerative and cardio-vascular diseases
[4, 6]. Additionally, the polyphenols also have the
capacity to reduce the number of tumours and their
growth in some types of cancer and may possess an
antidiabetic effect. The polyphenols modulate glycaemia
through a complex mechanism that can be reduced
to the inhibition of reabsorption of glucose in the
gut [4, 6, 11].
Hedera helix L. (ivy or English ivy) is a member of
Araliaceae family, well known as an ornamental
plant, but also for its harmful effects such as contact
dermatitis, gastrointestinal irritation, bloody diarrhoea
and even death produced by the fresh fruits and
leaves [17]. Yet, in the folk medicine it is used to treat
the benign warts and for its antioxidant, antispasmodic
and antiallergic properties. Various authors have
reported the positive effect of dry extracts on
respiratory functions of children with chronic bronchial
asthma and other therapeutic effects such as: anti-
bacterial, antihelmintic, leishmanicidic and antifungal
properties [12, 13].
There is a direct correlation between the significant
number of therapeutic properties and the chemical
composition of ivy. Thus, multiple studies have
indicated a complex chemical composition for ivy
leaves: phenolic acids (caffeic, neochlorogenic,
chlorogenic), flavonoids (quercetin, kaempferol, iso-
quercitrin), phytosterols (stigmasterol, sitosterol),
polyacetylenes (falcarinose, falcarinol), hederagenin,
oleanolic acid, hederasaponins, etc. [3, 5, 12].
Concerning the chemical composition of ivy fruits and
flowers, there is little published data. Determination of
fruits composition revealed the presence of triterpene
saponins, fatty acids, β-lectins and polyacetylenes
[3, 12].
The major goal of the present study is the
investigation of LC/MS profile of polyphenols,
antioxidant and antimicrobial potentials of H. helix
ethanolic extracts obtained from leaves, flowers,
immature and ripe fruits.
Materials and Methods
Plant material and extraction
Ivy (H. helix L.) was collected from the “Alexandru
Borza” Botanical Garden of Cluj-Napoca, Romania
(46°45′36″N and 23°35′13″E). The material was
identified by Dr. M. Pârvu, “Babeș - Bolyai”
University of Cluj-Napoca, Romania. A voucher
specimen (CL 664210) is deposited at the
Herbarium of “Babeș - Bolyai” University, Cluj-
Napoca, Romania. The plant material was harvested
in April 2014 (ripe fruit), in September 2014 (leaves
and flowers) and in November 2014 (immature
fruits).
Small fragments (0.5 - 1 cm) of fresh H. helix L.
(ivy) were extracted with 70% ethanol (Merck,
Germany) by cold repercolation method [6, 16], at
room temperature, for 3 days [16]. The content of
ivy extract (w/v; g/mL) was: 1/1.5 (for leaf extract),
1/1 (for immature fruit extract and ripe fruit extract)
and 1/1.1 (for flower extract).
Determination of total polyphenols and flavonoid
contents
The total phenolic content (TPC) of the extracts
was measured using the Folin-Ciocâlteu method,
with some modifications [18]. The absorbance was
measured at 760 nm, using a JASCO UV-VIS
spectrophotometer. Standard curve was prepared
using different concentrations of gallic acid. TPC
was expressed as mg gallic acid/g dry material
plant (mg GAE/g plant material).
The total flavonoid content (TFC) was determined
and expressed as rutin equivalents (mg RE/g plant
material), using the method described in the Romanian
Pharmacopoeia (Xth Edition) [20]. The absorbance
was measured at 430 nm.
LC/MS analysis of polyphenolic compounds
For the qualitative and quantitative determination
of polyphenols we used an HPLC-MS method. The
experiment was carried out using an Agilent 1100
Series HPLC system (Agilent, USA) consisting of a
G1322A degasser, G1311A binary gradient pump
and a G1313A autosampler and a UV detector. The
chromatographic separation was achieved using a
reversed-phase analytical column (Zorbax SB-C18
100 mm x 3.0 mm i.d., 3.5 µm particle) maintained
at 48°C. The mobile phase consisted of a binary
gradient: methanol and acetic acid 0.1% (v/v). The
mobile phase was delivered with a flow rate of
1mL/min and the injection volume was 5 µL. The
detection of polyphenols was performed on UV
(330 nm and 370 nm) and MS mode. The MS
system operated using an ion trap mass
spectrometer with electrospray negative ionization.
The chromatographic data were processed using
Chemstation and Data Analysis software from
Agilent, USA. Also, the calibration curves in the
0.5 - 5 µg/mL range showed good linearity (R2 < 0.999)
for a five point plot [7, 8, 10].
DPPH assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay
was used to evaluate the radical scavenging activity,
by bleaching of purple methanolic solution of the
stable radical. The antioxidant effect implies the
disappearance of the DPPH absorption through the
action of antioxidants. 20 µL of diluted extracts
were added to 980 µL DPPH solution (100 µM).
After 30 min incubation period, the decrease in
absorbance was measured at 517 nm, using a UV-
VIS JASCO V-530 spectrophotometer. Both hydrophilic
FARMACIA, 2017, Vol. 65, 4
626
and lipophilic synthetic antioxidants, quercetin and
butylated hydroxytoluene (BHT) were used as
standards. The percentage inhibition of the DPPH
radical after adding individual samples was calculated
using the following equation: I = 100 (Ac - As)/Ac,
where: I – DPPH inhibition (%), Ac - absorbance of
the control sample, As - absorbance of the tested
sample. The antioxidant activity was also expressed
as inhibitory concentration IC50, defined as the
concentration of the sample required to cause a
50% decrease in initial DPPH radical absorbance.
IC50 values in DPPH assay were calculated graphically.
All experiments were performed in triplicate [1, 19].
Antibacterial activity
Microorganisms and culture conditions. For the
bioassay, six bacterial strains were used: three Gram
positive bacteria: Staphylococcus aureus (ATCC
49444), Bacillus cereus (ATCC 11778), Listeria
monocytogenes (ATCC 19114) and three Gram
negative bacteria: Pseudomonas aeruginosa (ATCC
27853), Salmonella typhimurium (ATCC 14028)
and Escherichia coli (ATCC 25922). All of the
tested microorganisms were obtained from Food
Biotechnology Laboratory, Life Sciences Institute,
University of Agricultural Sciences and Veterinary
Medicine Cluj-Napoca, Romania. The bacteria
were cultured on Müller-Hinton agar. Cultures were
stored at 4°C and sub-cultured once a month. For
the antimicrobial activity evaluation, the obtained
extract was evaporated to dryness under reduced
pressure at 30°C and re-suspended in 1 mL of bi-
distilled water.
Microdilution method. The modified microdilution
technique was used to evaluate the antimicrobial
activity. Anaerobic bacteria were cultured overnight
at 37°C on Tryptic Soy Broth (TSB) medium. The
bacterial cell suspensions were adjusted with sterile
saline to a concentration of approximately 2 × 105
colony-forming unit (CFU)/mL in a final volume of
100 µL per well. The inoculum was stored at 4°C
for further use. Dilutions of the inoculums were
cultured on solid Müller-Hinton (MH) for bacteria
to verify the absence of contamination and to check
the validity of the inoculums. Determinations of the
minimum inhibitory concentrations (MICs) were
performed by a serial dilution technique using 96 -
well microtitre plates. Different dilutions of the
ethanolic extracts were carried out over the wells
containing 100 µL of Müller-Hinton (MH) broth
and afterwards, 10 µL of inoculum was added to all
the wells. The microplates were incubated for 24 -
48 h at 37°C. The MIC of the samples was detected
following the addition of 20 µL (0.2 mg/mL) of
resazurin solution to each well and the plates were
incubated 2 h at 37°C. A change from blue to pink
indicates reduction of resazurin and therefore
bacterial growth. The MIC was defined as the
lowest drug concentration that prevented this colour
change. The minimum bactericidal concentrations
(MBCs) were determined by serial sub-cultivation
of a 2 µL into microtitre plates containing 100 µL
of broth per well and further incubation for 48 h at
37°C. The lowest concentration with no visible
growth was defined as MBC, indicating 99.5%
killing of the original inoculum. Streptomycin (Sigma
P 7794, Santa Clara, CA, USA) (0.05 - 3 mg/mL)
was used as positive control for the bacterial
growth. A 10% solution of ethanol in water was
used as negative control [7, 8].
Results and Discussion
Polyphenols analysis
The total phenolic content and total flavonoid content
of the extracts varied considering the natural product,
with higher amounts in ripe fruits extract (2.17 mg
GAE/g and 1.75 mg RE/g, respectively), followed by
flowers extract (1.64 mg GAE/g and 1.33 mg RE/g,
respectively), immature fruits extract (1.28 mg GAE/g
and 1.02 mg RE/g, respectively), and leaves extract
(1.28 mg GAE/g and 1.02 mg RE/g, respectively).
The concentrations of the total polyphenols (1.03 -
2.17 mg GAE/g) and flavonoids (0.87 - 1.75 mg RE/g)
are presented in Table I.
Table I
TPC and TFC in H. helix extracts (± standard
deviation (SD))
Extract
TPC
(mg GAE/g)
TFC
(mg RE/g)
H. helix leaves
1.03 ± 0.09
0.87 ± 0.01
H. helix flowers
1.64 ± 0.11
1.33 ± 0.1
H. helix immature fruits
1.28 ± 0.08
1.02 ± 0.07
H. helix ripe fruits extract
2.17 ± 0.14
1.75 ± 0.12
Previous work on H. helix has shown that aqueous
methanol extract of ivy stems is related with the
presence of triterpenes, flavonoids, tannins and
saponins [14]. Other phytochemical investigations
of our research group on H. helix harvested from
Romania showed a high correlation between all anti-
oxidant methods used. The extract obtained from
the flowers presented a higher content in the total
poly-phenols then those from the fruits and leaves,
which is in accordance with the results of our study [9].
The polyphenolic profile obtained in this work was
similar to that presented by Bahafar et al. on other
Hedera species, H. pastuchovii [2]. TPC and TFC
of the berry extract (213.5 and 54.2, respectively)
were approximately two times higher than those
found in the leaf extract (121.4 and 39.3, respectively).
Their results were expressed in mg GAE/g of dry
extract, which can explain the difference in amounts
compared with our study.
In order to determine the LC/MS polyphenolic
profile, 18 polyphenolic compounds were used as
standards: caffeic, chlorogenic, caftaric, gentisic,
FARMACIA, 2017, Vol. 65, 4
627
ferulic, sinapic, p-coumaric acids, quercitrin, iso-
quercitrin, quercetin, rutin, myricetin, hyperoside,
fisetin, patuletin, luteolin, kaempferol and apigenin.
The results are summarized in Table II. The
concentrations of the identified polyphenols are
organized in the order of their retention times. MS
data and retention times were compared to those of
the reference standards.
Table II
The polyphenolic compounds content in the analysed H. helix extracts (µg/mL)
Polyphenolic
compound
m/z
RT ± SD
(min)
Leaves extract
Flower extract
Fruits extract
Immature
Ripe
Chlorogenic acid
353
5.62 ± 0.05
126.400
530.894
118.775
59.740
p-Coumaric acid
163
9.48 ± 0.08
NF
3.571
NF
NF
Ferulic acid
193
12.8 ± 0.10
NF
0.759
2.680
0.810
Hyperoside
463
19.32 ± 0.12
NF
NF
NF
1.440
Isoquercitrin
463
19.60 ± 0.10
1.583
50.898
5.590
33.021
Rutin
609
20.20 ± 0.15
124.663
278.011
48.954
183.746
Quercitrin
447
23.64 ± 0.13
NF
8.404
0.365
NF
Quercetin
301
26.80 ± 0.15
0.284
1.220
0.394
3.862
Kaempferol
285
32.48 ± 0.17
NF
2.937
0.283
0.947
Note: NF-not found, below limit of detection. Values are the mean ± SD (n = 3)
Figure 1.
The UV chromatogram of H. helix flowers extract. Identified compounds: 1, Chlorogenic acid; 2, p-Coumaric
acid; 3, Ferulic acid; 4, Isoquercitrin; 5, Rutin; 6, Quercitrin; 7, Quercetin; 8, Kaempferol.
The only phenolic acid identified and quantified in the
ethanolic extract of H. helix leaves was chlorogenic
acid (126 µg/mL). Two flavonoid glycosides, iso-
quercitrin and rutin were identified in the same
extract, while the class of flavonoid aglycons was
represented by quercetin. These results are not in
accordance with those obtained by our research
group by analysing H. helix leaves harvested on
September 2011. The amount of rutin was much
smaller (34 µg/mL) compared to the amount quantified
in the plant material collected in September 2014
(124.663 µg/mL) [9].
The flowers of H. helix are the richest in phenolic
acids. Thus, among these substances, there were
quantified chlorogenic, p-coumaric and ferulic acids,
with the first being the most abundant (530.894
µg/mL). The dominant flavonoid was rutin (278.011
µg/mL) followed by isoquercitrin. Also, we identified
one flavonol, quercetin, but in a minor amount
(1.220 µg/mL). For the plant material collected in
September 2011, the chlorogenic acid was absent,
while the quantity of rutin was inferior (130 µg/mL).
Yet, the amount of quercetin and kaempferol was
superior, 7.11 µg/mL and 7.90 µg/mL respectively [9],
compared to 1.22 µg/mL and 2.937 µg/mL respectively,
quantified in H. helix flowers harvested in 2014. It
is difficult to find an explanation for all these
differences, considering that we used the same protocol
for extracts preparation, but in the same time we
have to take into consideration that environmental
factors have a major effect on the polyphenol
contents. These factors may be pedoclimatic (ground
type, sun exposure, rain fall). Apparently, exposure to
light has a considerable effect on most flavonoids [4].
The analysis of ivy fruits extracts revealed interesting
results. In both fruits, immature and ripe, were
quantified two phenolic acids, chlorogenic and ferulic
acids, but the concentrations of these compounds were
FARMACIA, 2017, Vol. 65, 4
628
higher in immature fruits. Concerning the flavonoid
glycosides, isoquercitrin and rutin, the ripe fruits
are richer than the immature fruits, while hyperoside
was quantified only in ripe fruits. These results
support the observations published by Manach et al.
2004 [4]. According to them, the degree of ripeness
strongly affects the concentration and proportion of
polyphenolic compounds. It has been observed that
phenolic acid content decreases during ripening and
the degree of ripeness at the time of harvest, other
environmental factors and also processing and storage
of plant material have a major effect on polyphenols
concentration.
Antioxidant activity assay
In order to evaluate the ability of ivy extracts and
synthetic antioxidants quercetin and BHT to donate
the hydrogen atom, the stable free radical DPPH
was used. All extracts obtained from H. helix were
able to reduce DPPH radical with different degrees of
scavenging activity. A lower IC50 value represents a
higher bleaching effect, thus a better antioxidant
activity.
The results obtained for the evaluation of the anti-
oxidant activity using the DPPH bleaching assay
are presented in Table III.
The strongest antioxidant was the positive control
quercetin, with IC50 value of 5.59 µg/mL. All analysed
extracts showed lower DPPH scavenging activity
compared to both reference compounds, quercetin
and BHT. The highest radical scavenging activity
was determined for H. helix ripe fruits extract
(68.55 ± 4.21 µg/mL), with positive correlation
between the scavenging activity on DPPH, the total
phenolic content and the total flavonoid content.
This might be related to the presence of higher
amounts of phenolic compounds in berries and
indicates that these compounds contribute to the
antioxidant effects of the natural product. The
different antioxidant activities between these extracts
may be due to the variability of composition and
content in various active compounds, and also to
the synergy between the natural substances.
Table III
Antioxidant activity for H. helix extracts
Sample
IC50 (µg/mL)
H. helix leaves extract
122.47 ± 9.31
H. helix flowers extract
82.59 ± 6.44
H. helix immature fruits extract
94.72 ± 7.68
H. helix ripe fruits extract
68.55 ± 4.21
Quercetin
5.59 ± 0.13
BHT
15.88 ± 1.06
Note: Values are the mean ± SD (n = 3).
Considering the obtained results, the following order
regarding the antioxidant activities was established:
H. helix leaves extract < H. helix immature fruits
extract < H. helix flowers extract < H. helix ripe
fruits extract < BHT < quercetin. According to this
method, the ethanolic extract of H. helix ripe berries
showed a good antioxidant activity (IC50 = 68.55
µg/mL).
These findings are in agreement with Bahafar et al.
[2], who reported IC50 values of 87.10 µg/mL and
139.02 µg/mL for berry and respectively leaf extracts
obtained from H. pastuchovii.
Antimicrobial activity assays
The in vitro antibacterial potential of H. helix extracts
against both Gram-positive and Gram-negative
bacteria is presented in Table IV.
Table IV
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of H. helix extracts
and streptomycin against bacterial strains tested with the microdilution method
Bacterial strain
Standard antibiotic
Minimum Inhibitory Concentration (mg/mL)
Minimum Bactericidal Concentration (mg/mL)
Streptomycin
Leaves
Flowers
Immature fruits
Ripe fruits
Staphylococcus aureus
0.03
1.25
0.15
0.078
1.25
0.06
2.5
0.3
0.15
2.5
Bacillus cereus
0.015
1.25
1.25
1.25
1.25
0.03
2.5
2.5
2.5
2.5
Listeria monocytogenes
0.015
0.62
0.15
0.15
0.62
0.03
1.25
0.3
0.3
1.25
Pseudomonas
aeruginosa
0.06
5
2.5
2.5
1.25
0.12
10
5
5
2.5
Salmonella
typhimurium
0.06
2.5
2.5
0.62
0.62
0.12
5
5
1.25
1.25
Escherichia coli
0.12
2.5
1.25
1.25
1.25
0.24
5
2.5
2.5
2.5
Note: Each value is the mean ± SD of three independent measurements.
As shown in Table IV, different plant parts exhibited
antibacterial activity, but to varying extents (MIC
values 0.078 - 5 mg/mL). The results indicate that
the bacterial strain Staphylococcus aureus was the
most sensitive to both H. helix immature fruits and
flower extracts, with MIC values of 0.078 mg/mL
and 0.15 mg/mL, respectively and MBC values of
0.15 mg/mL and 0.3 mg/mL, respectively. Also, these
FARMACIA, 2017, Vol. 65, 4
629
two extracts present a good activity against Listeria
monocytogenes with the same MIC value of 0.15
mg/mL, and the identical MBC value of 0.3 mg/mL.
According to Salvat et al. [15], if MIC value of the
plant extracts is less than/or around 0.5 mg/mL, the
antibacterial activity is appropriate, so we conclude
that H. helix immature fruits and flower extracts
exhibited good antimicrobial activity against S. aureus
and L. monocytogenes, while the growth inhibitory
effect of all the other plant parts tested showed low
activity against all evaluated bacterial strains.
Conclusions
This study revealed the antioxidant and antimicrobial
activities of some ethanolic extracts from leaves,
flowers, immature and ripe fruits of H. helix. The
highest amount of phenolic and flavonoid compounds
was identified in the ripe fruits extract; also, the
same extract exhibited the best antioxidant activity.
Regarding the results of the antibacterial test, the
immature fruits extract showed a significant activity
against S. aureus, followed by the flower extract
with a good growth inhibitory effect against the
same bacterial strain. Both immature fruits and
flowers extracts possess appropiate antibacterial
capacity against L. monocytogenes.
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