Influence of the extraction solvent on antioxidant activity of Althaea officinalis L. root extracts

Abstract

Althaea officinalis (Malvaceae) is a well-known plant that is widely distributed throughout the world. Aqueous and hydroalcoholic extracts from A. officinalis root are used mainly because of their antitussive and expectorant activity. It is well known that these activities are based on the polysaccharide composition, but little is known about the possible antioxidant activity of root extract. The present study evaluated antioxidant activity of root extracts prepared with different extraction solvents applying ABTS·+ (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), hypochlorous acid scavenging assay and iron-induced lipid peroxidation. The results showed that the extract prepared with water as extraction solvent did not possess antioxidant activity, whereas the extracts obtained using ethanol:water as extraction agent showed well pronounced antioxidant activity. In particular, the extracts obtained at low concentration of ethanol in the mixed solvent (50:50 and 70:30, v/v) showed higher scavenging activity for ABTS·+ radicals and hypochlorite ions than the extract obtained with the higher ethanol concentration (90:10, v/v). These results correlated very well with phenolic and flavonoid content of the extracts. The extracts did not show cytotoxic effect on human BV-173 leukemic cells but may have immunomodulating effects due to their antioxidant properties.

Full text

Central European Journal of Biology
* E-mail: krassi.yoncheva@gmail.com
Research Article
1Department of Pharmacognosy,
Medical University of Soa, 1000 Soa, Bulgaria
2Department of Pharmaceutical Technology,
Medical University of Soa, 1000 Soa, Bulgaria
3Department of Physics and Biophysics, Medical Faculty,
Medical University of Soa, 1431 Soa, Bulgaria
4Department of Pharmacology, Faculty of Pharmacy,
Medical University of Soa, 1000 Soa, Bulgaria
Niko Benbassat1, Krassimira Yoncheva2*, Vera Hadjimitova3, Nadia Hristova3,
Spiro Konstantinov4, Nikolai Lambov2
Inuence of the extraction solvent on antioxidant
activity of
Althaea ofcinalis
L. root extracts
1. Introduction
Althaea ofcinalis L. (genus Althaea) belongs to the
botanical family Malvaceae and is widely distributed
throughout the world. It is a perennial plant well-known
for its healing properties since ancient time. The activity
of various root extracts of A. ofcinalis against cough,
throat irritation and inammation of gastrointestinal
mucosa has been described [1,2]. Syrups prepared by
maceration of root (usually diluted with sucrose syrup)
are used in many countries for the treatment of cough
and inammation of the mouth and pharynx. Other
preparations of the plant include decoction and tea
preparation [3]. Decoction is mainly administered for
constipation [4], whereas infusion is used for bronchial
catarrh [5]. Infusions have been reported to be applied
orally for treatment of asthma and as expectorant [6,7].
Comminuted herbal substance for tea preparation
is applied to alleviate abdominal aches of digestive
origin. Antibacterial activity of root extract and its
incorporation in mouthwash for topical periodontal
prophylactics has been also reported [8]. Other studies
mentioned the potential use of root extract in topical
formulation due to its wound healing effects [7]. The
capacity of the A. ofcinalis extracts to soothe chapped
skin and to reduce the inammation is well established.
Some studies have evaluated antioxidant activity
of extracts prepared from dried plant and its owers
[9,10]. Although the above-ground parts displayed a
mild antioxidant activity [9,10], the antioxidant activity
Cent. Eur. J. Biol. • 9(2) • 2014 • 182-188
DOI: 10.2478/s11535-013-0245-2
182
Received 15 February 2013; Accepted 18 July 2013
Keywords: Althaea ofcinalis • ABTS•+ • Hypochlorous acid scavenging assay • Lipid peroxidation • Total phenolic content • Total avonoids
Abstract: Althaea ofcinalis (Malvaceae) is a well-known plant that is widely distributed throughout the world. Aqueous and hydroalcoholic extracts
from A. ofcinalis root are used mainly because of their antitussive and expectorant activity. It is well known that these activities are based
on the polysaccharide composition, but little is known about the possible antioxidant activity of root extract. The present study evaluated
antioxidant activity of root extracts prepared with different extraction solvents applying ABTS•+ (2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulphonic acid), hypochlorous acid scavenging assay and iron-induced lipid peroxidation. The results showed that the extract prepared
with water as extraction solvent did not possess antioxidant activity, whereas the extracts obtained using ethanol:water as extraction
agent showed well pronounced antioxidant activity. In particular, the extracts obtained at low concentration of ethanol in the mixed solvent
(50:50 and 70:30, v/v) showed higher scavenging activity for ABTS•+ radicals and hypochlorite ions than the extract obtained with the
higher ethanol concentration (90:10, v/v). These results correlated very well with phenolic and avonoid content of the extracts. The extracts
did not show cytotoxic effect on human BV-173 leukemic cells but may have immunomodulating effects due to their antioxidant properties.
© Versita Sp. z o.o.

N. Benbassat
et al.
of extracts from the root of A. ofcinalis remains to be
determined.
The aim of the present study was to evaluate the
inuence of the extraction process on antioxidant
activity of the extracts prepared from A. ofcinalis root.
Extraction solvent, the ratio of the quantity of the herbal
substance to the quantity of the resulting extract and the
physical state of the herbal substance inuenced the
properties of the nal herbal preparation [11-14]. Since
the extraction solvent has a great inuence on the nal
phytochemical composition, this factor was evaluated in
the present study by using different extraction solvent.
2. Experimental Procedures
2.1 Plant material and chemicals
A. ofcinalis L. was cultivated and two years old
roots were obtained from Botanical Garden of Soa
University “St. Kl. Ohridski” (Soa, Bulgaria), where
voucher specimen is deposited. ABTS (2,2’-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid), Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid), pyrogallol and quercetin were obtained from
Sigma-Aldrich. BV-173 cell line was obtained from the
German Collection of Microorganisms and Cell Cultures
(DSMZ GmbH, Braunschweig, Germany).
2.2 Preparation of the extracts
The extracts were prepared using different solvents
as extraction media, in particular E1 (puried water),
E2 (50:50, v/v ethanol/water), E3 (70:30, v/v ethanol/
water) and E4 (90:10 v/v ethanol/water). The liquid
extracts were prepared at a ratio 1:10 giving a nal
concentration of 100 mg mL-1. The extraction process
included ultrasonic treatment of the soaked herbal drug
into the selected medium for 2 h. The extraction was
carried out at ultrasonic power of 300 W, frequency
of 45 kHz and temperature of 25oC (Sonica 4300,
Milano, Italy). The obtained extract was ltered trough
the cellulose membrane (3.5 kDa Roth, Karlsruhe,
Germany) and kept in dark glass bottles.
2.3 ABTS radical cation scavenging assay
The ABTS•+ scavenging test was used to determine
the antioxidant activity. ABTS•+ radical was obtained
by reaction between ABTS and potassium persulfate
[15]. Blank sample was prepared from the daily solution
by adding 1 mL PBS, which gives an absorbance of
0.7 ± 0.01 at 723 nm. The radical scavenging activity
was assessed by mixing 2 ml of ABTS•+ solution with
1 mL PBS solutions of the investigated plant extracts
with different concentrations. The reactive mixture was
allowed to stand at room temperature for 10 min and
the absorbance was recorded at 734 nm. The value of
IC-50 was dened as the concentration of substrate
that causes 50% loss of the ABTS•+ activity and was
calculated as mg L-1.
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid) was used as a reference compound.
The Trolox equivalent antioxidant capacity (TEAC) was
expressed as mol Trolox per g of sample.
2.4 Determination of total phenolic content
Phenolic content in the liquid extracts was studied using
the Folin-Ciocalteu method with some modications
[16]. The extracts (0.5 mL) were mixed with 0.5 mL Folin-
Ciocalteu reagent and incubated for 5 min. Then, 1 mL
20% solution of sodium carbonate was added and nally
the volume was diluted to 10 ml with puried water. The
mixtures were incubated for 2 h and the absorbance
was measured at 765 nm (Hewllet Packard 8452A).
Pyrogallol (2-15 µg mL-1) was used as a standard to
produce the calibration curve (r=0.9970). The mean of
three readings was used and the total phenolic content
was expressed as µg of pyrogallol equivalents (PE) per
ml of extracts.
2.5 Determination of total avonoid content
The total avonoid content was determined using the
method adapted from Arvouet-Grand et al. [17]. In
the present study, 5 mL of 2% solution of AlCl3•6H2O
in ethanol was mixed with the same volume of the
extracts. The samples were incubated for 15 min and the
absorbance was measured at a wavelength of 410 nm
(Hewllet Packard 8452A). The total avonoid content
was calculated using a standard curve of quercetin as
a standard (0–50 mg L-1, r=0.9986). The total avonoid
content was expressed as μg of quercetin equivalents
(QE)/ml of extracts.
2.6 Hypochlorous acid scavenging assay
The samples for the assay were prepared in 2 mL
PBS (pH=7.4) and each contained 15 mmol L-1 taurine,
20 mmol L-1 potassium iodide (KI), 0.06 mmol L-1
sodium hypochlorite and the investigated extracts in
different concentrations. Control sample did not contain
extract. The samples were incubated for 30 min at room
temperature and the absorbance was measured at
350 nm. The results were presented using antioxidant
activity (AOA) that was calculated applying the equation:
AOA = (Ao – A)/Ao, where Ao was the absorbance of the
control and A was the absorbance of the extract samples.
The values of IC-50 were dened as the concentration
of the extract that caused 50% loss of the AOA and were
calculated in mg mL-1.
183

Antioxidant activity of
Althaea ofcinalis
root extracts
2.7 Thiobarbituric acid reactive substances
(TBARS) assay
The TBARS of lipid peroxidation was measured in
liposomal suspension obtained from phospholipids
extracted from egg yolk. The samples were prepared in
PBS and contained 1 mg lipid mL-1 and diluted extracts.
Control sample did not contain extract. After addition
of 0.1 mmol L-1 FeCl2 the samples were incubated at
37ºC for 30 min. Then, 0.5 mL of 2.8% trichloroacetic
acid and 0.5 ml of 0.5% TBA were added. The samples
were heated at 100ºC for 20 min and the absorbance
was measured at 532 nm. The results were presented
using antioxidant property (AOP) that was calculated
as AOP=(A/Ao)x100, where Ao was the absorbance of
the control and A was the absorbance of the samples
containing the extracts.
2.8 In vitro cytotoxicity
The cell viability after exposure to the extracts was
examined applying the MTT-dye reduction assay,
based on the biotransformation of the yellow dye
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide to a violet formazan product. Exponentially
growing cells were seeded in 96-well at-bottomed
microplates (100 μL/well) at a density of 1×105 cells per
mL and after a 24 h incubation at 37°C they were treated
with graded concentrations of the extracts. The incubation
after the treatment was for 72 h. After the exposure
period 10 μL MTT solution (10 mg mL-1 in PBS) aliquots
were added to each well. The microplates were further
incubated for 4 h at 37°C and the MTT-formazan crystals
formed were dissolved through addition of 100 μL/well
5% formic acid in 2-propanol. The MTT formazan
absorption was determined using a microprocessor
controlled microplate reader (Labexim LMR-1) at 580 nm.
3. Results and Discussion
Liquid formulations of A. ofcinalis (syrups, extracts,
infusions) are widely used mainly due to their antitussive
and anti-inammatory activity. It is well known that these
activities are based on the polysaccharide composition
[18,19]. The polysaccharides possessing bioadhesive
properties form a coating layer on the oral and pharyngeal
mucosa that reduce the irritation associated with the
dry cough [20,21]. This study evaluates the antioxidant
activity of A. ofcinalis root extracts and examines the
inuence of the extraction solvent on this activity.
3.1 ABTS radical cation scavenging assay
The study showed that the aqueous extract of
A. ofcinalis did not possess antioxidant activity. The
absence of antioxidant properties of aqueous extract
could be due to the extraction mainly of polysaccharides.
Some polysaccharides extracted from the leaves of
A. ofcinalis, in particular glucuronoxylans, showed high
antioxidant activity evaluated by their ability to inhibit
peroxidation of soyabean lecithin liposomes by OH•
radicals [22]. Polysaccharides extracted from the root of
A. ofcinalis are mainly D-glucans, rhamnogalacturonan
and arabinans [23,24]. However, a previous study
reported the weak antioxidant activity of glucans [25].
Thus, the absence of antioxidant activity of the root
aqueous extract in the present study corresponded with
the weak activity of glucans.
The extracts obtained using ethanol:water
as the extracting agent showed well pronounced
antioxidant activity (AOA). The antioxidant properties
of the ethanol:water extracts were investigated by
concentration dependence of AOA (Figure 1). The slope
of the curve is proportional to antioxidant properties of
the extracts. IC-50 was calculated by using the data
from Figure 1. The values of IC-50 for E2, E3 and E4
extracts were 59.6 ± 0.8 mg L-1, 69.5 ± 3.9 mg L-1 and
313.3 ± 43.8 mg L-1, respectively. The extracts obtained
by low concentration ethanol solution manifested higher
antioxidant activity than the extract obtained with the
highest ethanol concentration (Figure 2). These results
led to the suggestion that the antioxidant components
were extracted more efcient at low concentration of
ethanol.
Total phenolic and total avonoid contents were
determined to investigate their relation with the different
antioxidant activity of the three extracts. According to
literature, the root extract contains phenolic acids, in
particular caffeic, p-coumaric, ferulic, p-hydroxybenzoic,
salicylic, syringic, p-hydroxyphenylacetic and vanillic
Figure 1.
Relationship between concentration of the extracts
(E2, E3 and E4) and their antioxidant activity (AOA). Data
expressed as the mean ± SD, n=3.
184

N. Benbassat
et al.
acid [26]. Our results revealed that the extract received
with the lowest ethanol concentration (E2) contained
the maximum phenolic content. Thus, the higher
phenolic content correlated with the higher antioxidant
activity of the extracts (Figure 3, r=0.9903). These
results could be considered in agreement with previous
studies that reported antioxidant activity of phenolic
acids, in particular caffeic, ferulic and p-cumaric acid
[27].
Content of some avonoids like hypolaetin-8-
glucoside, isoquercitrin, kaempferol, quercetin and
naringenin in the roots of A. ofcinalis has been
reported [26,28,29]. The phytochemical evaluation of
the extracts in the present study showed higher total
avonoid content for the extracts obtained by extraction
with lower ethanol concentration in the mixed solvent
(E2 and E3) (Figure 4). These results are in agreement
with a study dedicated on the extraction of quercetin
from onion. The authors have reported that the quercetin
extracted with 60% and 90% ethanol was 298 µg L-1 and
207 µg L-1, respectively [30]. It was considered that the
reason could be the presence of quercetin in the form
of glycoside having higher polarity than aglycone. In our
study similar phenomenon was observed, in particular
the lowest avonoid content was found for the extract
obtained with the highest ethanol concentration (E4).
Further, avonoid content in the extracts obtained by
extraction with the different concentrations of ethanol
correlated with the antioxidant capacity (Figure 4). As
shown, the higher the avonoid content, the higher
antioxidant activity.
3.2 Hypochlorous acid scavenging assay
It is well known that activation of phagocytizing cells
during inammatory processes is associated with
production of signicant amounts of ROS and the
neutrophils activation leads to release of hypochlorous
acid in the system. For this reason, the ability of
the three extracts to scavenge the hypochlorite
ions was investigated. Figures 5 and 6 represent
scavenging activity and the respective IC-50 values
of the extracts. All extracts possessed the ability to
react with hypochlorous acid. The highest effect
demonstrated the extract E2 (ethanol:water - 50:50),
since the value of IC-50 for this extract was the lowest
– 0.84 mg mL-1. IC-50 of E3 (ethanol:water - 70:30)
was slightly higher than the IC-50 of E2 (1.05 mg mL-1),
whereas IC-50 of E4 (ethanol:water - 90:10) was
signicantly higher than those of E2 and E3 extracts
(2.18 mg mL-1). Thus, the results suggested that
stronger anti-inammatory activity would be achieved
with A. ofcinalis extract prepared with ethanol:water
solvent in ratio 50:50.
Figure 2.
The antioxidant activity of the extracts (E2, E3 and E4)
presented by values of IC-50 (mmol TE g-1 extract).
Figure 3. Relationship between total phenolic content of the
extracts and antioxidant activity presented by IC-50
Trolox equivalent TE g-1 of the extracts. Data expressed
as the mean ± SD, n=3.
Figure 4. Relationship between total avonoid content of extracts
and antioxidant activity presented by IC-50 Trolox
equivalent TE g-1 of the extracts. Data expressed as the
mean ± SD, n=3.
185

Antioxidant activity of
Althaea ofcinalis
root extracts
3.3 Iron-induced lipid peroxidation
The antioxidant properties of the extracts in model
system of lipid peroxidation in vitro were investigated
(Figure 7). The values of IC-50 for all tested extracts
were similar and got into the concentration interval
5 - 6.5 mg mL-1. The observed values were 3 to 10
times higher than the values obtained by the other
methods (ABTS and hypochlorite ions). Unlike the
strong correlation between the radicals scavenging
capacity and the avonoid and phenolic content, the
lipid peroxidation did not show such relationship. This
observation could be due to the fact that in this model
system the activity depended on the variety of chemical
reactions including the participation of intermediate and
nal products.
Figure 5.
Relationship between concentration of the extracts
(E2, E3 and E4) and their antioxidant activity detected in
hypochlorite ions generating system. Data expressed as
the mean ± SD, n=3.
Figure 6. Antioxidant activity of the extracts in hypochlorite ions
generating system presented by values of IC-50. Data
expressed as the mean ± SD, n=3.
Figure 7. Antioxidant properties of Althaea ofcinalis extracts in
iron-induced lipid peroxidation system. Data expressed
as the mean ± SD, n=3.
Figure 8. Cytotoxicity of the different Althaea ofcinalis extracts
on leukemic BV-173 cells. Data expressed as the mean
± SD, n=3.
3.4 In vitro cytotoxicity study
Cytotoxicity of the different extracts of Althaea ofcinalis
was investigated on leukemic BV-173 cells (Figure 8).
This line was selected because of its lymphoid origin
and drug sensitivity despite of the expression of
Bcr-Abl fusion gene which is associated with resistance
toward apoptotic signaling. Previous reports found
that water extract of roots (10%) was inactive on
HeLa tumor cells [31]. Another study reported weak
antineoplastic effect (LC50>5 mg mL-1) of the root extract
obtained in ethanol [32]. This activity was estimated
on murine neuroblastoma cells, originally derived from
spontaneous malignant tumor. Water extract from
A. ofcinalis root had no effect on primary dermal human
broblasts (pNHF) but showed stimulating effect on
186

N. Benbassat
et al.
cell viability and proliferation of epithelial KB cells [33].
The results in the present study showed that the liquid
extracts at the concentration of 50 mg mL-1 exerted no
signicant cytotoxicity on human BV-173 leukemic cells.
4. Conclusions
The present study revealed that the hydroalcoholic
extract from A. ofcinalis root possesses antioxidant
properties. The concentration of the ethanol in the
extraction mixture inuenced the antioxidant activity
due to the different phytochemical composition of
the extracts. The lowest ethanol concentration in the
mixture led to higher content of phenolic and avonoid
content in the resulting extracts. The highest antioxidant
activity correlated with the highest phenolic as well
as the highest avonoid content of the extracts. The
optimal extraction regarding antioxidant activity of the
nal extract seemed to be with ethanol:water mixed
solvent at a 1:1 ratio. Taken together our experimental
ndings indicate that the plant extracts do not have any
antitumor properties but may have immunomodulating
effects due to their antioxidant features.
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