Antioxidant activity of extract from elder flower (Sambucus nigra L.)

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Acta Alimentaria
DOI: 10.1556/AAlim.2014.0009
TOTAL PHENOLIC CONTENT, ANTIOXIDANT AND
ANTIMICROBIAL ACTIVITY OF HABERLEA RHODOPENSIS
EXTRACTS OBTAINED BY PRESSURIZED LIQUID EXTRACTION
D. Mihaylovaa*, a. lanteb and a. Krastanova
aDepartment of Biotechnology, University of Food Technologies, 26 Maritza Blvd., 4002, Plovdiv. Bulgaria
bDepartment of Agronomy, Food, Natural Resources, Animals and Environment, Università di Padova,
Viale dell`Universita 16, Agripolis, 35020 Legnaro, Padova. Italy
(Submitted: 08 April 2013; accepted: 26 July 2013)
The present study was designed to investigate the antioxidant and antimicrobial activities of pressurized liquid
extracts from Haberlea rhodopensis Friv. The total phenolic content was performed using the Folin-Ciocalteu
phenol reagent. To determine the antioxidant activities of the extracts, several complementary tests were used:
ABTS and DPPH radical scavenging activities, oxygen radical absorbance capacity, and ferric-reducing antioxidant
power assay. The phenolic concentration was 15.98±0.09 and 9.42±0.06 mg GAE g–1 DW for 70 and 85% ethanol
extracts, respectively. Of all the performed methods, the highest antioxidant activity values were measured by the
ORAC assay – 224.6±6.6 and 154.0±9.9 μM TE g–1 DW for 70 and 85% ethanol extracts, respectively. Results also
showed that both extracts exhibited very weak antimicrobial activity against the examined microorganisms.
However, the 70% ethanol extract possessed higher inhibition ability, which correlated with higher total phenolic
content and antioxidant activity.
Keywords: pressurized liquid extract, Haberlea rhodopensis, antioxidant activity, antimicrobial activity
Natural antioxidants play a very important role in the prevention of different diseases, such
as cancer, arteriosclerosis, and neurodegenerative diseases (Chu et al., 2012; FernánDez-Mar
et al., 2012). Therefore, there is an increasing interest in the antioxidant effects of natural
compounds from medicinal plants and pharmaceutical products for health (raMFul et al.,
2011; ventuani et al., 2011).
Recovery of antioxidant compounds from plant materials is typically accomplished
through different extraction techniques, taking into account their chemistry and uneven
distribution in the plant matrix. Solvent extraction is the most frequently used technique for
isolation of plant antioxidant compounds. However, the extract yields and resulting
antioxidant activities of the plant materials are strongly dependent on the nature of the
extracting solvent, due to the presence of different antioxidant compounds of varied chemical
characteristics and polarities that may or may not be soluble in a particular solvent (PesChel
et al., 2006).
Alternative novel extraction procedures are now being sought after that will reduce
extraction time and solvent consumption, increase sample throughput and improve analyte
recovery. Pressurized liquid extraction (PLE) operates at high pressures and temperature
above point of the boiling point of the organic solvent. The use of higher pressure facilitates
the extraction of the analytes from samples by improving the solvent accessibility to the
analytes that are trapped in the matrix pores. The use of PLE decreases signicantly the total
* To whom correspondence should be addressed.
Phone ++ 359 32 603 645; e-mail: dashamihaylova@yahoo.com

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time of treatment and in addition, this method can be more effective and selective by changing
some parameters (Choi et al., 2003; ong & len, 2003).
Haberlea rhodopensis is a resurrection species and glacial relic endemic in the mountains
of the Balkan Peninsula in southeastern Europe (Djilianov et al., 2011). It has been proven
that it can survive long periods of desiccation (for up to 2 years) and quickly resume normal
growth within hours of re-watering. Despite the fact that several reliable methods were
applied to study the antioxidant (BerKov et al., 2011; Mihaylova et al., 2011) and enzyme
activity (yahuByan et al., 2005) of its different extracts, this plant is still less explored. raDev
and co-workers (2009) and Mihaylova and co-workers (2011) studied different extracts of the
plant and reported inhibition against St. aureus and Ps. uorescens.
The aim of the present study was to obtain extracts from H. rhodopensis for the rst time
using alternative PLE technique and thus to extend knowledge of this less explored plant.
The potential antioxidant properties of the extracts were studied as well and the antimicrobial
activity was measured.
1. Materials and methods
1.1. Plant material
Haberlea rhodopensis Friv. was collected from its natural habitat (Permission
№201/07.05.2009-Bulgarian Ministry of Environment and Water) at Plovdiv region,
Bulgaria. The plant leaves were air-dried at room temperature (25-28 ºC), roughly grounded,
and stored in air-tight dark containers.
1.2. Extracts preparation
About 67 and 69 g of H. rhodopensis were randomly sampled from 200 g dry plant material.
The PLE was carried out for 1.45 h in an automatic equipment (NM LAB/M Deputex 88,
Limena, Padova, Italy) with 580 and 600 ml of ethanol/water solutions (70:30 and 85:15 v/v
acidied with 0.1 M HCl, pH 3), previously deoxygenated by ushing with nitrogen as
reported by Rossetto and co-workers (2005). The volume of each sample was measured to
calculate the extract concentration (g l–1). The extraction rate was 0.140 and 0.142, resp. The
extracts were subdivided in dark glass bottles without headspace and stored at –20 ºC.
1.3. Total phenolic content (TPC)
The TPC was analyzed using the method of Kujala and co-workers (2000) with some
modications. Each extract was mixed with Folin-Ciocalteu reagent and 7.5% Na2CO3. The
mixture was vortexed and left for 5 min at 50 ºС. After incubation, the absorbance was
measured at 765 nm by room temperature. The TPC was expressed as mg gallic acid
equivalents (GAE) per g dry weight (DW).
1.4. DPPH radical scavenging assay
The ability of the extracts to donate an electron and scavenge 2,2-diphenil-1-picrylhydrazyl
(DPPH) radical was determined by the slightly modied method of BranD-WilliaMs and co-
workers (1995). Freshly prepared 4×10–4 M methanolic solution of DPPH was mixed with the
samples in a ratio of 2:0.5 (v/v). After 30 min incubation at room temperature the light
absorption was measured at 517 nm. The DPPH radical scavenging activity was presented as
a function of the concentration of Trolox. The unit of Trolox equivalent antioxidant capacity

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(TEAC) was dened the concentration of Trolox having equivalent antioxidant activity
expressed as the μM per g DW (μM TE g–1 DW).
1.5. ABTS radical cation decolourization assay
The radical scavenging activity of the extracts against 2,2´-azino-bis(3-ethylbenzothiazoline-
6-sulfonic acid) (ABTS•+) was estimated according to re and co-workers (1999). The results
were expressed as TEAC value (μM TE g–1 DW).
1.6. Ferric-reducing antioxidant power (FRAP) assay
The FRAP assay was carried out according to the procedure of Benzie & strain (1996) with
slight modication. The FRAP reagent was prepared fresh daily and was warmed to 37 °C
prior to use. 150 µl of plant extracts were allowed to react with 2850 µl of the FRAP reagent
for 4 min at 37 °C and the absorbance was recorded at 593 nm. The results were expressed as
μM TE g–1 DW.
1.7. Oxygen radical absorbance capacity (ORAC) assay
The ORAC assay measures the antioxidant scavenging function against peroxyl radical
induced by AAPH at 37 °C. The loss of uorescence of uorescein is an indication of the
extent of damage from its reaction with the peroxyl radical (ou et al., 2001; huang et al.,
2002; goMes et al., 2005). ORAC values were expressed as µM TE g–1 DW.
1.8. Determination of antimicrobial activity (AMA)
Escherichia coli ATCC 25922, Salmonella enterica subsp. enterica ATCC BAA-2162,
Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 25093 were
purchased from the National Bank of Industrial Microorganisms and Cell Cultures (Soa,
Bulgaria). Strains Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus niger and
Rhizopus sp. were provided by the Culture collection at the Department of Microbiology,
University of Food Technologies, Klebsiella pneumoniae and Listeria monocytogenes were
isolated from clinical samples.
The AMA of the examined extracts was analyzed by the agar-well diffusion method. To
prepare suspensions, the tested microorganisms (TM) were cultured on Luria-Bertani (Sigma-
Aldrich) agar and incubated at 37 °C overnight. Appropriate dilutions of each microbial
suspension were used to inoculate the molten plate count agar (PCA) (Sigma-Aldrich),
equilibrated in water bath, to obtain the concentrations given in Table 2. A. niger was grown
on Wort agar (Sigma- Aldrich) at 30 °C for 48 hours and a suspension was prepared to
inoculate the molten medium. The wells (6 mm in diameter) were cut from the agar and 60 µl
of the tested extracts were delivered into them. Equal volumes of the solvents were used as
controls. PCA plates were incubated at 37 ºC for 24 hours and Wort agar plates at 30 °C for
48 hours. All plates were examined for any zones of growth inhibition, and the diameters
were measured (mm).
1.9. Statistical analysis
All measurements (accept AMA) were carried out in triplicates. The results were expressed
as mean±SD and statistically analyzed using MS-Excel software.

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2. Results and discussion
2.1. Determination of TPC
The TPC in 70 and 85% ethanol extracts from H. rhodopensis were 15.98±0.09 and 9.42±0.06
mg GAE g–1 DW, respectively. Phenols and polyphenolic compounds, such as avonoids, are
widely found in many food products derived from plant sources, and they have been shown
to possess signicant AOAs (van aCKer et al., 1996). Furthermore, phenols have been
determined to play an important role in the survival of the plants under extreme conditions
(Műller et al., 1997). Consequently the presence of those compounds in the extracts
suggested their important role in the plant. Several studies have successfully correlated the
phenolic content with AOA (tePe et al., 2006; Mihaylova et al., 2011), which provoked our
interest.
2.2. Determination of antioxidant activity (AOA) of the extracts
Various methodologies are widely used for evaluation of the antioxidant potential and, in this
work, we determined the free radical scavenging capacity of H. rhodopensis extracts using
ABTS and DPPH methods, ORAC assay and their reducing capacity by the FRAP method.
The use of several methods to measure AOA may seem a redundancy, but since different
authors used various methods, comparison of properties becomes easier if a large set of data
is available.
2.3. DPPH, ABTS, FRAP and ORAC assays
In order to investigate the AOA, experiments with two stable radicals DPPH• and ABTS•+
were conducted, TEACDPPH• values were 72.42±0.18 and 30.44±0.25 μM TE g–1 DW and the
TEACABTS•+ values were 72.98±0.77 and 37.99±2.84 μM TE g–1 DW for the 70 and 85%
ethanol extracts, respectively (Table 1). Higher TEAC value indicates that a sample has
stronger AOA. The FRAP values for the investigated extracts of H. rhodopensis were as
follows: 120.54±3.57 and 58.57±1.42 μM TE g–1 DW. Using the ORAC assay the established
results were 224.6±6.60 and 154.0±9.90 μM TE g–1 DW.
The results of the total antioxidant capacity assays (Table 1.) showed that the investigated
extracts possessed AOA, which for the 70% ethanol extract was approximately two times
higher than the capacity of the 85% one. This conrmed the results obtained from the TPC
assay. Interestingly, the highest AOA values were measured by the ORAC assay. A slight
difference among the results obtained by the DPPH, ABTS, ORAC, and FRAP assays was
observed. This might be explained by the unique mechanism and the unequal sensitivity of
each method applied. The authors therefore strongly suggested that, when analyzing the AOA
of samples, it is better to use at least two methods due to the differences between the test
systems (ou et al., 2002). From the results obtained it can be concluded that the 70% ethanol
is more efcient as solvent in order to obtain extract with higher content of biologically
active substances in terms of AOA. This is in agreement with previously conducted studies
that established the effectiveness of 70% ethanol as solvent (Mihaylova et al., 2011).
2.4. Evaluation of antimicrobial activity of the extracts
The AMA was evaluated by measuring the inhibition zone against the TMs after correction
with the diameter of wells and the zones of appropriate controls. According to the results of

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antimicrobial screening given in Table 2, neither extracts showed any signicant activity
against the TMs. However, higher antimicrobial activities were obtained for 70% ethanol
extract. The 85% ethanol extract has been shown to possess antimicrobial effect only against
A. niger among the studied TMs, which corresponds well with the results of TPC and AOA
assays. The 70% ethanol extract showed weak activity against Kl. pneumoniae, A. niger,
B. subtilis, and Rh. sp., while neither of the extracts showed inhibitory activity toward
L. monocytogenes. In comparison raDev and co-workers (2009) reported effect of
H. rhodopensis against S. aureus.
Table 1. Total phenol content and antioxidant activity of extracts from H. rhodopensis
Sample
/Assay
TPC, mg
GAE g–1 DW
TEACDPPH•,
μM TE g–1 DW
TEACABTS•+,
μM TE g–1 DW
ORAC,
μM TE g–1 DW
FRAP,
μM TE g–1 DW
70% ethanol 15.98±0.09 72.42±0.18 72.98±0.77 224.6±6.60 120.54±3.57
85% ethanol 9.42±0.06 30.44±0.25 37.99±2.84 154.0±9.90 58.57±1.42
Table 2. Antimicrobial activity of extracts from H. rhodopensis
Test microorganism Inhibition zone diameter, mm Concentration
of TM, CFU а ml–1 in media
70% ethanol 85% ethanol
E. coli n.d.bn.d.b1.0*1010
Kl. pneumoniae 1;0cn.d.b1.9*1011
S. enterica subsp. enterica n.d.bn.d.b1.7*109
Ps. aeruginosa n.d.bn.d.b1.5*106
L. monocytogenes n.d.bn.d.b2.5*106
St. aureus n.d.bn.d.b1.4*1011
B. subtilis 1;1 n.d.b1.0*105
S. cerevisiae n.d.bn.d.b1.0*105
A. niger 1;0c3;3c1.0*105
Rhizopus sp. 1;1cn.d.b1.0*105
a:CFU: colony-forming units; b: n.d.–not detected; c: the results of two parallel determinations
3. Conclusions
In summary, due to the insufcient information on H. rhodopensis, we applied the pressurized
liquid extraction method for the rst time in order to obtain new extracts and to reveal the
characteristics of this resurrection plant. The extraction technique was applied with two
different solvents – 70 and 85% ethanol and the antioxidant and the antimicrobial activities
were evaluated. Based on the conducted experiments the 70% ethanol extract was found to
possess higher inhibition, which correlated with the higher total phenolic content and
antioxidant activity.
*

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This is a collaborative project and has been partially supported by the „Science and Business” Program at the
Ministry of Education and Science, Bulgaria, BG051PO001/3.3-05, co-nanced by the European Social Fund.
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