Barzegar et al., Int. J. Rev. Life. Sci.

, 5(9), 2015, 1709-1715

ISSN 2231-2935
Research Article
www.ijrls.pharmascope.org

Ultrasonic Extraction of Antioxidants from Different Species of Wild Hawthorn

Leaves
Hanieh Barzegar 1, Reihaneh Ahmadzadeh Ghavidel *1, Sodabeh Einafshar 2
1
Department of Food Science and Technology, Quchan Branch, Islamic Azad University, Quchan, Iran
2
Department of Agricultural Engineering Institute, Khorasan Razavi Agricultural and Natural Resources Research
and Education Center, AREEO, Mashhad, Iran

ABSTRACT

The hawthorn is a fruit with antioxidant properties that make this fruit useful for the treatment of many diseases.
In this study eight hawthorn species were assayed for their antioxidant capacities evaluating total phenolic com-
pounds and ferric reducing antioxidant power. In addition, these extraction methods (cold solvent, cold solvent
plus sonication for 5 min and cold solvent plus sonication for 10 min) were investigated. The results from this
study thus indicate that there is a significant difference between hawthorn species in terms of antioxidant activity,
total phenolic compounds and ferric reducing antioxidant power. The assadii2 and pontica species were known as
the best species in terms of antioxidant capacities. In addition, the results demonstrated that sonication for 5 min
could enhance the level of antioxidant compounds in hawthorn leaves extracts.
Keywords Antioxidants; Hawthorn; Phenolic Compounds; Ultrasonic.
INTRODUCTION thocyanidins, catechins, phenolic acids, essential oils and
terpenoids (Bahorum et al., 1994 and García-Mateos et al.,
Crataegus commonly called hawthorn, is a large genus of
2012) explain their use as natural therapy for the treatment
shrubs and trees in the family Rosaceae, native to temperate
of neurodegenerative diseases, in some types of cancer, in
regions of the Northern Hemisphere in Europe, Asia and
the affectation of the immunological system and cardiovascu-
North America. There are descriptions of 150-200 species of
lar disorders (Craig, 1999 and Chang et al., 2002 and Cui et
thisgenus in the world (García-Mateos et al., 2013). It is a
al., 2006). Hawthorn is well known in phytotherapy for the
small tree growing to 8 m tall, with a dense crown. The leaves
treatment of many cardiovascular diseases; it regulates blood
are 2–6 cm long and 2–5 cm broad, with 2–3 shallow, for-
pressure, increases the strength of heart muscle, and is used
ward-pointing lobes on each side of the leaf.
against arteriosclerosis and angina pectoris. There are a
The hermaphrodite flowers are produced in corymbs of 6– number of medicinally active phytochemicals such as terpe-
12, each flower with five white or pale pink petals and two or noids, polyphenols and flavonoids (Shao-Jiang et al., 2011
three styles, and are pollinated by midges. The fruit is a dark and Calişkan et al., 2012 and Edwards et al., 2012). That have
red or yellow pome 6–10 mm diameter, slightly broader than been isolated from hawthorn plants with most of the data
long, containing 2–3 nutlets. generated in studies of those species that are native to Eu-
rope and Asia. Besides, hawthorn has a soothing effect on
The pathophysiology of many diseases is associated with an the nervous system, and is also used as a mild diuretic (Kostić
increase of free radicals derived from reactive oxygen species et al., 2012). Apart from phytotherapy, hawthorn is used in
(ROS) in the cells (Singh et al., 2008). A higher production of the food industry for the production of jam and various bev-
ROS can cause oxidative damage in DNA (Spencer et al., erages including wine, juice, compote and herbal tea (WHO,
2012). Antioxidants play an important role in protecting cells 2002).
against ROS (Jayakumar, 2012).
The wide diversity that exists in the Iranian hawthorn species
Some fruits have protective properties and help prevent the demands the characterization of its fruits and the determina-
complications of chronic degenerative diseases, such as ath- tion of its antioxidant properties to be recommended as a
erosclerosis, chronic inflammation, diabetes mellitus, cata- food. The objective of the present study was to evaluate the
racts, coronary diseases and certain types of cancer content of phenolic compounds and the antioxidant proper-
(Havsteen, 2002). These characteristics have been attributed ties in leaves of eight species of Iranian hawthorn.
to the antioxidant activity of many secondary metabolites in
fruits. Many types of fruits also have nutraceutical properties MATERIALS AND METHODS
(Andlauer, 2002) and a nutritional capacity to prevent chron-
Plant materials
ic degenerative diseases (Willis and Wians, 2003) .The high
contents of phenolic compounds such as flavonoids, proan- In order to evaluate phenolic compounds and the antioxidant
properties of eight hawthorns species (C. pentagyna Sub sp.
* Corresponding Author pentagyna1, C. pseudoheterophylla Sub sp. turkestanica, C.
Reihaneh Ahmadzadeh Ghavidel assadii1, C. azarolus var. aronia, C. pentagyna Sub sp. pen-
reahmadzadeh@yahoo.com tagyna2, C. kurdistanica, C. azarolus var. pontica and C. assa-
dii2), leaves samples were collected from KhorasanRazavi
©JK Welfare & Pharmascope Foundation | International Journal of Review in Life Sciences 1709

Agricultural Research Center, Mashhad, Iran. The leaves
samples were dried at 30̊ C and the dry leaves samples were
pulverized into powdered form using a grinder machine.
Extraction
The powdered samples were mixed with 96% methanol (1:
20 w: w), extracted using magnetic blender at room tempera-
ture for 15 min. Then samples were subjected to ultrasonic
waves (Heilscher, Germany – UP400S, 24 KHz, 400W) for 5
and 10 min. Solid part was separated from blend by passing
through Whatman #1 filter paper by using Buchner funnel
with vacuum. Sediment was extracted again as above. Extrac-
tion solvent was evaporated at room temperature under
laboratory hood. The dried samples were put in dark-glass
containers and stored at cold and dry place. Control samples
were not subjected to ultrasound waves.
Antioxidant capacity (IC50)
Antioxidant capacity was determined by the DPPH (1, 1-
diphenyl-2-picrylhydrazyl) assay according to the method
reported by (Ersus, S., and Yurdagel, U 2007). First, 0.006%
DPPH solution was prepared by dissolving DPPH in methanol.
Then 1 ml DPPH solution was added into methanol extracted
samples. Samples were mixed well and left to stand for 1 h in
the dark. The absorbance was then measured at 512 nm.
Standard carve was plotted using different concentration of
iron sulphate II. The DPPH scavenging activity was calculated
as follows.
Equation 5.
Ac  As
A(%) 100
Ac
Where A: scavenging activity, Ac: absorbance of control and
as: absorbance of sample. The values were plotted onto
graph and IC50 was defined as the concentration in which
antioxidant is able to inhibit 50% of free radicals.
Total of total phenolic compounds (FOLIN)
The content of total phenolic was determined by using folin-
ciocalteu method (Huang and Ling, 2011). The sample (5 mg)
was extracted in 10 ml methanol and then 2.5 ml folin-
ciocalteu agent (folin-ciocalteu was dissolved in water 1:10
v/v) was added into a falcon tubes. Samples were left on the
bench to start reaction. Afterwards, 5 ml of 7.5% Na2CO¬3
solution was added into aqueous phase and after 1 min the
samples were diluted to 50 ml with distilled water. Each
sample was allowed to stand for 24 h in dark place at room
temperature and absorbance was measured at 765 nm.
Standard carve was plotted using different concentration of
gallic acid. Total phenolic was calculated using following
equation and expressed as mg ml-1.
Equation 6.
Y terms of antioxidant activity. Several studies suggest that the
P 100
W
Where P: Total phenolic compounds, W: sample weight
Ferric reducing antioxidant power (FRAP)
Briefly, 100 mg sample was dissolved in 10 ml ethanol. Then
30 µl of the solution was mixed into 900 µl FRAP solution and
90 µl distilled water in test tube. Test tubes were vortexed
and placed into benmary until 37̊ C. At this time absorbance
©JK Welfare & Pharmascope Foundation | International Journal of Review in Life Sciences
Barzegar et al., Int. J. Rev. Life. Sci., 5(9), 2015, 1709-1715
was recorded at 595 nm (Benzie and Strain, 1996). The Fe II
content was calculated using following equation.
Equation 4.
Y= 1782X-9.211
Where Y: Fe II (µmol l-1), X: absorbance at 595 nm
STATISTICAL ANALYSIS
The experimental design was a completely randomized de-
sign arranged in factorial with three replications. The first
factor was eight species of hawthorn and the second factor
was sonication time. Statistical analysis was conducted using
MSTAT-C. Significant differences between means were de-
termined by LSD test. Differences were considered to be
significant at P< 0.05.
RESULTS AND DISCUSSION
Extraction method
The effect of extraction methods on IC50, Frap and phenolic
compounds are illustrated in (table 1). As can be seen, there
are significant difference between cold solvent and 5 and 10
min sonication treatments. However, 5 min sonication signif-
icantly increased Frap and phenolic compounds and de-
creased IC50 in hawthorn leaves extract. These results indi-
cate that ultrasound application has positive effect on antiox-
idant activates and 5 min ultrasonic was more effective than
the other treatment.
Increase in sonication time up to 10 min, significantly in-
creased IC50 and decreased FOLIN and FRAP values (Table 2).
These results suggest a higher extraction of antioxidants
compounds due to 5 min sonication, 10 min sonication and
cold solvent extraction, respectively. In other words, the
highest and the lowest extraction efficiency were related to 5
min then 10 min sonication. Increase in antioxidants com-
pounds extraction could be due to the decomposition of the
bioactive constituents by sonication or due to the initial rins-
ing effect of sonication, which facilitated the release of most
of the active constituents inside the cells to the solvent dur-
ing the first 5 minutes (Annegowda et al., 2010). Further-
more, prolonged sonication lead to a decrease diffusion area,
diffusion rate, and increased diffusion distance at a pro-
longed interval of sonication. A similar finding was also re-
ported by (Lu, 2008).
Antioxidant capacity (IC50)
The results indicated that the highest antioxidant activity was
related to CA2 and CAP species with the lowest IC50 value
(9.50 and 15.53 µg ml-1 respectively) (Figure 1). So CA2 and
CAP can be applied as natural and available species providing
antioxidants in food and drug industries. On the other hand,
the CPP1 showed the highest IC50 value (403.40 µg ml-1) and
subsequently the lowest antioxidant activity (Figure 1). There
was no significant difference between CAA, CK and CPP2 in
antioxidant activity of fruits is influenced bygenotype, habitat
conditions and ripeness ofthe fruits (Orhan, 2007). Other
factors, such as altitude,light, temperature, and content of
nutritivematter in the soil, can influenceantioxidant activity
(Dixon and Paiva, 1995).
Interaction between Howthorn species and extraction meth-
ods on IC50 is shown on figure 2. As can be seen in figure2
CPP1 and 5 min sonicated treatments had the lowest IC50
and the most antioxidative activity.
1710

Total phenolic
The highest (118.30 mg ml-1) and the lowest (6.47 mg ml-1)
phenolic compounds (FOLIN) were observed in CPP1 and
CPP2, respectively (Figure 3). Without any significant differ-
ence CPT, CPP2, CK and CAP varieties had the lowest phenolic
compounds and the middle amounts of phenolic compounds
were recorded from CA1 and CAA varieties (Figure 3). The
increase in phenolic compounds extraction might be due to
increase in mass transfer due to collapse of cavitation bub-
bles near the cell walls, which results in better contact be-
tween solvent and plant material. In addition, during the
collapse of cavitation bubbles, ultrasonic waves act as micro-
pomp for solvent and facilitate solvent penetration into the
plants cells (Albu, 2004). With increasing sample to solvent
ratio, during the short contact time, phenolic compounds
would increase slightly, whereas during the long contact
time, phenolic compounds would decrease. This is probably
due to this fact that during the long contact time, solvent
dissolves impure components (Chan, 2007). The results indi-
cated that total phenolic compounds are influenced by the
genotype of Crataegus. Phenolic compounds can protect
against free radicals in aerobic cell metabolic processes. The
antiradical activity of the polyphenols is attributed to the
interaction of structural parts catalyzing metal chelation, the
activation of certain antioxidant enzymes and the inhibition
of oxidase enzymes (Ji-Hyun and Kwang-Deog, 2011) point
out a high content of polyphenols in the fruits of Cratae-
guspinnatifida due to the presence of procyanidins of phe-
nols and of flavonoids (Svedström, 2002). Describe the pres-
ence of oligomericprocyanidins in leaves, in flowers and the
highest concentration detected was in fruits of Cratae-
guslaevigata.
The interaction between species and extraction treatments
on FOLIN is given in figure 4. Regarding FOLIN, the highest
value (152.50 mg ml-1) was obtained from CAPsonicated for
10 (figure 4). However, there was no significant difference
between this treatment and CA1 at both sonicated treat-
ments, CPT and CPP1 at cold extraction. The CPP2 cold ex-
traction showed the lowest FOLIN value (6.47 mg ml-1) (fig-
ure 4). The wide diversity and genotypic variability that exists
in hawthorn species demands the characterization of its
fruits and the determination of its antioxidant properties to
Table 1. 8 Hawthorn leaves species
Hawthorn leaves species
C. pentagynaSub sp. pentagyna1
C. pseudoheterophyllaSub sp. turkestanica
C. assadii1
C. azarolusvar. aronia
C. pentagynaSub sp. pentagyna2
C. kurdistanica
C. azarolusvar. pontica
C. assadii2
Table 2. Individual effect of extraction methods on IC50, FRAP and FOLIN
IC50
Extraction methods
(µg.ml-1)
Cold solvent 355.6a
Sonication for 5 minutes 9.508c
Sonication for 10 minutes 163.9b
©JK Welfare & Pharmascope Foundation | International Journal of Review in Life Sciences
Barzegar et al., Int. J. Rev. Life. Sci., 5(9), 2015, 1709-1715
be recommended as a food with high nutritional value. The
results affirmed that the extracted phenolic compounds of
hawthorn is not only associated with genetic background, but
also is attributed to extraction method. Similar results have
been found by (Saadatian, 2014).
Ferric reducing-antioxidant power (FRAP) test the highest
FRAP was observed in CA1 (3400 µmol l-1) and CPP1(3250
µmol.l-1) (Figure 4). By contrast, the lowest FRAP was ob-
tained from CPT2 and CPP2 (100 and 90 µmol l-1 respective-
ly) (Figure 4). There was no significant difference between
CK, CAP and CA2 in terms of FRAP (Figure 5). Nonetheless the
highest FRAP was related to CA2, CK and then CAP.
As can be seen from the results, CPP1 showed the highest
IC50, phenolic compounds and FRAP, indicating its antioxi-
dant activity is related to its power of chelating matals not to
scavenging radical's power compared with other species.
Similar results have been found by (Lotito and Frei, 2004). in
apple, they have reported that there is significant differences
in FRAP values and antioxidant power in different apple spe-
cies.
The interaction between species and extraction treatments
on FRAP is given in (figure 6). From the results, the highest
value (5739 µmol l-1) was observed in CPT species sonicated
for 10 min, while the lowest values (79.20 µg ml-1) was ob-
served in CPP2 species extracted with cold solvent (figure 6).
There was no significant difference between CA1 sonicated
for 5 and 10 min, CAA for 5 and 10, and CPP1 extracted with
cold solvent (figure 6). Similar impact of sonication time on
FRAP and other antioxidants yield from various plants and
fruits were observed by (Chavan and Singhal, 2013 and Khan
et al., 2012 and Le et al., 2012 and Porto et al., 2013 and
Rumbaoa et al., 2009).
CONCLUSION
Comparison of IC50, FRAP and FOLIN in leaves of eight haw-
thorn species showed that CA1 and CAP species are the best
species in terms of antioxidant activity. In addition, the re-
sults clearly indicated that sonication treatment for 5 min is
more effective in enhancing the antioxidant compounds in
hawthorn leaves extracts.
abbreviation
CPP1
CPT
CA 1
CAA
CPP2
CK
CAP
CA2
FRAP FOLIN
(µmol.l-1) (mg.ml-1)
79.2c 6.474c
2962a 75.77a
1366b 31.8b
1711
 Barzegar et al., Int. J. Rev. Life. Sci., 5(9), 2015, 1709-1715

Values within the each column and followed by the same letter are not different
at P < 0.05 by an ANOVA protected LSD Test.

Figure 1. The effect of different Hawthorn leaves species on IC50. Values within the each column and followed
by the same letter are not different at P < 0.05 by an ANOVA protected LSD Test.

Figure 2. Interaction between species and extraction methods on IC50.

Figure 3. The effect of different species on FOLIN. Values within the each column and followed by the same
letter are not different at P < 0.05 by an ANOVA protected LSD Test.

©JK Welfare & Pharmascope Foundation | International Journal of Review in Life Sciences 1712
 Barzegar et al., Int. J. Rev. Life. Sci., 5(9), 2015, 1709-1715

Figure 4. Interaction between species and extraction methods on hawthorn leaves phenolic compounds.

Figure 5. The effect of different species on FRAP. Values within the each column and followed by the same let-
ter are not different at P < 0.05 by an ANOVA protected LSD test.

Figure 6. Interaction between hawthorn leaves species and extraction methods on FRAP.
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©JK Welfare & Pharmascope Foundation | International Journal of Review in Life Sciences 1715