Mohammad Wahsha. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 450-456.
450
e- ISSN 0976 - 3651
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International Journal of Biological
&
Pharmaceutical Research
Journal homepage: www.ijbpr.com
IJBPR
PROTECTIVE ACTION OF FLAVONOIDS EXTRACTED FROM
DIFFERENT JORDANIAN PLANTS AGAINST OXIDATIVE STRESS
Mohammad Wahsha1*, Anas Al-Omari2, Mohammed Hassan2, Fuad A. Abuadas2,
Emad T.Ahmed2, Khodadad Mostafavi3, Mohsen Moradi4 and Mitra Ghotbi5
1
Department of Environmental Sciences, Ca' Foscari University of Venice, Italy.
2
College of Applied Medical Science, Taif University, KSA.
3
Department of Agronomy and Plant Breeding, Karaj Branch, Islamic Azad University, Iran.
4
Faculty of Agricultural Science, University of Mazandaran, Iran.
5
Department of Ecology and Natural Resources Management, Faculty of Agriculture, University of Bonn, Germany.
ABSTRACT
Oxidative stress represents a potential health risk. Prevention of such possible risks in human and animals is of great
importance. To date, however, there is no effective treatment of acute tissue injury induced by free radicals. Understanding the
mechanism of free radicals damage is beneficial and critical to plan new strategies in order to reduce its effects. In this study
three compounds from Jordanian plants were chosen as a source of flavonoids; Hesperidin, Naringin and Silymarin extracts
were identified and quantified. The radical scavenging activities of the selected flavonoids were determined using the
haemolysis of human Red Blood Cells mediated by Ferrous Sulfate. This work highlights the importance of Hesperidin,
Naringin and Silymarin protective action against oxidative stress as supported by biochemical results. Moreover the study
indicates that Silymarin has the highest protective capacity against free radicals damage followed by Naringin and Hesperidin
respectively. As a result of this work, combined with our previous studies, we now confirm that the protective effect of
Hesperidin, Naringin and Silymarin could provide a new insight into the potential therapeutic solution to free radicals induced
cell injury.
Keywords: Hesperidin, Naringin, Silymarin, Oxidative stress.
INTRODUCTION
Antioxidants are agents that inhibit or neutralize
potentially harmful elements known as free radicals (Galati
and O’brien, 2004; Zielinska el al., 2001). In our daily diet,
one of the most important sources of antioxidants is
flavonoids (Heim et al., 2002). Flavonoids are naturally
occurring polyphenolic compounds in plants that are
thought to have positive effects on human health (Wahsha
and Al-Jassabi, 2009). Flavonoids have attracted the
Corresponding Author
Mohammad Wahsha
Email:- dr.mohammadwahsha@yahoo.com
greatest attention and have been studied extensively,
because they are highly effective antioxidants with a lower
toxicity than synthetic antioxidants (Cai et al., 2010). Plant
flavonoids are emerging as potent therapeutic drugs
effective against a wide range of free radical mediated
diseases (Subash and Subramanian, 2010). The flavonoid
compounds can be regarded as C6-C3-C6 compounds, in
which each C6 moiety is a benzene ring. Multiple
combinations of hydroxyl groups, sugars, oxygens, and
methyl groups attached to these structures are the most
common basis of the classification of flavonoids (Bergman
et al., 2003; Heim et al., 2002). Several studies indicate
that flavonoids are potent antioxidants; they have the
Mohammad Wahsha. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 450-456.
451
ability of scavenging hydroxyl radicals, superoxide anions,
and lipid peroxy radicals (Wahsha et al., 2010; Edenharder
and Grünhage, 2003).
Flavonoids are known to operate via direct
scavenging of Reactive Oxygen Species (ROS), chelation
of redox active transition metal ions, inhibition of enzymes
involved in ROS production, regeneration of endogenous
antioxidants such as α-tocopherol (Zielinska et al., 2001;
Fitzgeorge et al., 1994). Formation of ROS in cells is
associated with the development of many pathological
states (Wahsha and Al-Jassabi, 2009). This has contributed
to the creation of the oxidative stress concept; in this view,
ROS are unavoidable toxic products of O2 metabolism, and
aerobic organisms have evolved antioxidant defenses to
protect against this toxicity. Oxidative stress can increase
sharply in cells either due to the decrease in the activity of
the antioxidant defense systems or to the overproduction of
ROS (Wahsha et al., 2012). Dufour et al. (2007) explained
that the possible mechanisms of antioxidant activity have
been thoroughly studied from simple chemical models.
According to Mukherjee et al. (2007) the polyphenolics
including flavonoids, which are found in many herbal
extracts, have been shown to be strong ROS scavengers,
antioxidants and protectors of neurons from lethal damage
in both vivo and vitro. Epidemiological studies have
described the beneficial effects of dietary flavonoids on the
reduction of the risk of chronic diseases, including cancer
(Ramos, 2007). Tripoli et al. (2007) in their review
reported that dietary flavonoids may help to supplement
the body antioxidant defenses against free radicals. Prakash
et al. (2007) reported that flavonoids are promising antioxidants, and can reduce the excess of oxidants and other
deleterious molecules due to their ability to quench
oxygen-derived free radicals by donating hydrogen atom or
an electron, to chelate redox active metals and inhibit
lipooxygenases.
Naringin is the major flavonoid glycoside found is
found in the juice, flower, and rind of the grapefruit fruit
(responsible for its characteristic bitter flavor) and
constitutes up to 10% of the dry weight. The most
important beneficial effects of Naringin are its antioxidant
and the ability of lowering the amount of lipid in the blood,
since it is metabolized to the flavonone naringenin (Kaur
and Kapoor, 2001). It was found that Hesperidin has an
important antioxidant activity in humans, it enhances the
integrity of the blood vessels and it is found in great
quantity in citrus fruits (lemons and oranges) (Tripoli et
al., 2007). Silymarin is a polyphenolic flavonoid
antioxidant which is extracted from the fruits and seeds of
the milk thistle (Silybum marianum) (Vinh et al., 2002).
It is obvious that oxidative stress represent a
potential health risk. Prevention of such possible risks in
human and animals is of great importance. To date,
however, there is no effective treatment of acute tissue
injury induced by free radicals. Understanding the
mechanism of free radicals damage is beneficial and
critical to plan new strategies in order to reduce its effects.
Therefore, the main goal of this study was to investigate
the antioxidant power of some local Jordanian nutritional
antioxidants extracted from grapefruits (Naringin), oranges
(Hesperidin) and milk thistle seeds (Silymarin).
MATERIALS AND METHODS
Preliminary purification of flavonoids
Hesperidin
Identified oranges species (Moro blood,
Washington navel and Valencia) were obtained from a
farm in Shobak (south Jordan). According to the method
recommended by Lombardo et al. (2006), orange peels of
these species were dried using an oven at 50 oC for 12 h.
The dried peels were then mashed into a powder (150 g)
and a solution of petroleum ether (750 mL) was added to
the powder in a round bottom flask then heated at reflux
for one hour. The mixture was filtered through a Buchner
funnel while it was still hot, and allowed to dry at 25 oC in
water bath for 30 min then re-extracted with methanol (750
mL) in another round bottom flask and heated at reflux for
two hours. The hot solution was then filtered, and the
supernatant was concentrated to provide syrup. 25 ml of
the syrup was added to 7 ml of dimethylformamide, the
mixture was then stirred vigorously at 25 oC in water bath
for 20 min, and the solution was then filtered to remove
any insoluble material. The filtrate was added drop wise
with stirring to a boiling solution in a fume hood, the
boiling solution consisted of 20 ml d.H2O and 0.5 ml acetic
acid, then refrigerated overnight. The precipitated
Hesperidin was collected by suction filtration and washed
with cold water and stored at -20 oC.
Naringin
Identified grapefruits species (Kara, King and
Fortune) were obtained from a farm Al-Shuna Al-Janubiya
(Southwest Jordan). Naringin was extracted according to
the method recommended by Markham (1982) with some
modifications, as follows: the dried peels of grapefruit (2.5
Kg) were extracted with 2 L of 95 % ethanol at 25 oC in a
water bath for 1 week. The extract was concentrated by
lyophilization, the residue was then dissolved and
suspended in 1 L of H2O and partitioned with 0.75 L
hexane. The H2O layer was then extracted successively
with 0.55 L ethyl acetate and 0.5 L n-butanol. The nbutanol extract was lyophilized to give 40-50 g of residue.
This lyophilized extract was dissolved in ethyl acetate:
methanol: d.H2O (13: 0.5: 0.5 ml) and subjected to
Sephadex LH-20 column. EtOAc-MeOH-d.H2O was used
as eluent with increasing methanol and water content
(12:1:1, 11:1:1, l0:l:l, 8:1:1,6: 1 : 1,2: 1 :0.5), 5 mL
fractions
were
collected,
and
tested
using
Mohammad Wahsha. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 450-456.
452
spectrophotometer. Fractions of crude Naringin were
lyophilized and kept in -20 oC.
Silymarin
Milk thistle seeds were collected from local areas
Ajloon and Irbid (North Jordan), and according to Duan et
al. (2004), seeds were crushed using a Braun KSM 2B
coffee grinder. 900 g of ground seeds were divided into 3
parts (300 g each); 250 ml of n-hexane was added to each
part. The suspension obtained was heated at 70 °C with
stirring using a mechanical mixer and heated to reflux for
30 min. The refluxing suspension was stirred for an
additional 3 h. After cooling, the herbal material was
filtered using a Buchner funnel leaving pale yellow oil
residues which was heated at 70 °C in vacuum for 2 h to
remove any remaining traces of hexane. Then the herbal
material was placed into a 250 ml necked flask, 150 ml of
acetone was added, and the obtained suspension was
stirred with a mechanical mixer at 20 oC in a water bath for
72 h, after that, the product was lyophilized. 100 g of the
lyophilized seeds were packed into the extraction container
containing 25 ml methanol. The loaded extraction
container was then placed on a heater; d.H2O was added
through the cell at a constant flow rate (5 ml/min). When
the desired temperature was reached (100 – 120 oC),
continuous sample collection began using for each sample
tube. After collection, fractions were lyophilized and
stored at -20 oC.
Antioxidant Power Assay
The radical scavenging activities of flavonoids
were determined using the haemolysis of human Red
Blood Cells (RBCs) mediated by Ferrous Sulfate (FeSo4)
according to the method described by Edenharder and
Grünhage (2003). The underlying principle is radicalmediated oxidation of lipids in cell membrane and thereby
induction of haemolysis. 50 ml of blood containing
heparin, obtained from a healthy non-smoker donor was
centrifuged at 1500 g for 20 min, the plasma was discarded
and the precipitate was re-suspended in 250 ml phosphate
buffer saline (PBS) pH 7.2 and centrifuged at 1500 g for 15
min. These steps were repeated once more, and then the
erythrocyte sediment was suspended in PBS at a dilution of
1:10. 2 ml of the suspension was mixed with 2 ml of 0.01
mM FeSO4 in PBS solution containing 1 mg extracted
flavonoids. The reaction mixture was incubated at 37 oC
for 2.5 hrs on a rotary shaker. After that, 3.5 ml of PBS
then was added and the diluted reaction mixture was
centrifuged for 10 min at 1500 g. All measurements were
performed in duplicates. The absorbance of the supernatant
was determined at 540 nm and the percentage of
membrane damage inhibition was calculated using Trolox
as a standard.
RESULTS AND DISSCUSSION
A number of studies have shown that antioxidants
from plant sources can effectively inhibit oxidative agents.
In this study three compounds from Jordanian plants were
chosen as a source of flavonoids. Hesperidin, Naringin and
Silymarin extracts were identified and quantified by
spectrophotometer (Fig. 1) according to Bushra et al.
(2009) and Liu and Zhu (2007).
Table 1 shows the yield product of Hesperidin,
Naringin and Silymarin extracted from local fruits. A high
yield of 3 mg of Hesperidin / g dried peels was obtained by
this method while for Naringin, the yield was 0.9 mg
Naringin / g dried peels. Regarding Silymarin, it was 4
folds of that of Hesperidin and 13 folds of Naringin.
Total antioxidant power
This method measures the relative ability of
antioxidant substances to scavenge the radical in an
aqueous phase as compared to standard amount of the
antioxidant Trolox. It was important to be able to
quantitatively measure the total antioxidant power within
local fruit and seeds sources. The activity of tested
compounds was expressed as Trolox equivalent.
The millimolar concentration of a Trolox solution
shows an antioxidant capacity equivalent to a 1.0 mM
solution of the substance under investigation. As shown in
(Table 2) the 1.8 Trolox equivalent antioxidant capacity of
Silymarin is higher than the capacity of Naringin (1.6
Trolox equivalent), and Naringin antioxidant capacity is
higher than that of Hesperidin (1.5 Trolox equivalent).
Various studies using compounds that directly or
indirectly cause cell damage, have been performed to
demonstrate the antioxidant action of Naringin, Hesperidin
and Silymarin. In our study, these three antioxidants were
investigated to measure their antioxidant power against the
formation of peroxyl radical initiator induced by FeSO4
using human red blood cells lysis as an indicator for ROS
damage. ROS initiate cell damage which can lead to loss of
membrane integrity and cell death. Therefore, it is
sufficient to use a reducing agent like FeSO4 to estimate
the antioxidant power in preventing the damaging
mechanism. Our results using a Trolox as a standard
indicated that Silymarin was the strongest scavenger and it
could prevent the total disruption of RBCs membrane;
Naringin ranked second, and Hesperidin was the weakest.
Naringin, Hesperidin and Silymarin are polyphenolic
compounds which play an important role as antioxidants;
they can directly quench free radicals, inhibit the enzymes
of oxygen reduction pathways and also prevent the
sequestration of transient metal actions (Berker et al.,
2007; Chatterjee et al., 1999). Our results showed that
these three antioxidants can prevent RBCs massive injury
caused by FeSO4. Moreover
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453
Previous studies have shown that certain
antioxidants are capable of reducing cell damage if
administered prior to the toxin dose (Wahsha et al., 2010).
The best described property of almost every group of
flavonoids is their capacity to act as antioxidants
(Andersen & Markham, 2006). Some studies which have
illustrated the antioxidant activities of flavonoids indicate
them as hydrogen donors and free radical scavengers,
besides having the ability to inhibit the oxidation of lipids
(Turkoglu et al., 2007). Furthermore, other studies have
indicated that flavonoids can inhibit free radical formation
and the propagation of free-radical reactions by chelating
of the transition metal ions (Berker et al., 2007).
Antioxidants were proved to be beneficial in reversing
oxidative stress produced by FeSO4.
We have also calculated the descriptive statistic
for the selected antioxidants; our observations on the
extraction yield results, combined with antioxidant
capacity assay proved that the selected nutrition
antioxidants can be subjected to our daily nutrients diet.
The descriptive statistic test boxplot for Hesperidin,
Naringin and Silymarin graphs the percentiles and the
median of column data. The ends of the boxes define the
25th and 75th percentiles, with a line at the median and
error bars defining the 10th and 90th percentiles (Fig. 2).
Silymarin exhibit 41.64 % of protection / yield and it was
the best antioxidant among the studied flavonoids.
Naringin was extracted form 3 different sources as well as
Hesperidin but the percentage of protection / yield was less
than Silymarin. On the other hand it better than Hesperidin
with a 35.44 % of protection / yield.
Kaur et al. (2006) reported that Hesperidin
attenuates lipopolysa-ccharide induced hepatotoxicity
possibly by preventing cytotoxic effects of ROS. Kim et al.
(2004) suggested that Hesperidin is a powerful
peroxynitrite scavenger and promotes cellular defense
activity in the protection against peroxynitrite involved
diseases. In agreement of our findings, various other
studies show that Hesperidin reduces oxidative stress; the
protective effect of Hesperidin can be correlated directly to
its strong, iron chelators as well as ROS scavenger's
properties (Kim et al., 2004).
Our results showed that Naringin exhibited
antioxidant activity, stopped the generation of cellular ROS
production induced by FeSO4. It has been found that
Naringin can play a role as a direct scavenger of H2O2, and
O2- and so it consequently blocks the cytotoxicity and
apoptosis caused by ROS (Andersen & Markham, 2006).
In two different studies, the first by Dixit et al.
(2007) and the other by Lakshmana et al. (2004), the
authors explain that Silymarin can offer a strong protection
against ROS in mice. Silymarin has been shown to prevent
the formation of lipid peroxidation after the exposure to the
hepatotoxin Microcystin-LR.
In a review by Moon et al. 2006 reported the
effects of Naringin, Hesperidin and Silymarin on
cytochrome P450 enzymes involved in activation of
inhibitors of phase II enzymes. These represent an
important character for those antioxidants and their role in
the protection of liver injury.
The radical scavenging power of flavonoids is
thought to be related to their structure. Flavonoids in
general scavenge oxidizing radicals preferentially via their
B-ring catechol; in particular the ortho-dihydroxy structure
in the B ring gives a higher stability during the formation
of aroxyl radicals and participation in electron dislocation:
this character is found in these three extracted antioxidants.
The presence of the 3' and 5' OH functions together give a
maximum radical scavenging potential: this property is
found in both Silymarin and Hesperidin (Andersen &
Markham, 2006; Joshi et al., 2005; Markham, 1982). From
this point of view this means that Naringin seems to be the
weakest among them, but this could not be proved during
our work. It might be that the structure is not the only way
by which the power of the antioxidants can be predicted
from, because radical scavenging activities depend also on
the nature of the radical and its specific reaction
mechanism. These systems will be influenced by the
presence of glycosidic moieties, the position of
glycosylation, and the number and positions of hydroxy
and methoxy groups. Thus, the final outcome of flavonoid
antioxidant properties will be effectively determined by
analyzing all their structural elements (Edenharder and
Grünhage, 2003).
Table 1. The yield of Hesperidin, Naringin and Silymarin extracted from local fruits
Group type
Hesperidin
Naringin
Silymarin
Yield of antioxidant / g dried fruit
3 ± 0.05
0.9 ± 0.02
12 ± 0.1
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454
Table 2. Measurement of Trolox equivalent antioxidant capacity for antioxidants used in this study, Hesperidin,
Naringin and Silymarin. Each value is the mean ± S.E.M of duplicate measurements
Group
Silymarin
Naringin
Hesperidin
Trolox
OD at 450 nm
0.265
0.378
0.533
1.000
% of RBCs membrane damage inhibition
0.8675
0.8110
0.7335
0.5000
Trolox equivalent
1.8 ± 0.1
1.6 ± 0.1
1.5 ± 0.1
1.0 ± 0.1
Figure 1. The mass spectrum of Naringin (N), Hesperidin (H) and Silymarin (S)
Figure 2. Vertical boxplot of the combined results of Hesperidin, Naringin and Silymarin
Mohammad Wahsha. et al. / International Journal of Biological & Pharmaceutical Research. 2012; 3(3): 450-456.
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CONCLUSION
From the results obtained in the current study we
can suggests that natural antioxidant flavonoids
Hesperidin, Naringin and Silymarin extracted from local
Jordanians oranges, grapefruits and Milk thistles seeds,
could effectively prevent the action of free radicals induced
acute red blood cells membrane damage.
ACKNOWLEDGEMENT
The authors are grateful to the reviewers and the
Editor in chief for their interest and support. Special thanks
go to Eng. Abeer Wahsha for her valuable help in this
work.
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