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International Journal of Toxicological and Pharmacological Research 2015; 7(3); 140-146
ISSN: 0975-5160
Research Article
*Author for Correspondence
Free Radical Scavenging Activity of Leaf Extract of Rumex vesicarius
L. Obtained by Different Methods
Laouini S Eddine1*, Ladjel Segni2, Ouahrani M Redha1, Gherraf Noureddine3
1Laboratory of Valorisation and Technology of Saharian resources, Hamma Lakhdar University, El-Oued, Algeria
2Process engineering Laboratory, Kasdi Merbah University, Ouargla, Algeria
3Laboratory of Biomolecules and Plant Breeding, Larbi Ben M’hidi University, Algeria
Available Online 30th May, 2015
ABSTRACT
In this study, phenolic content, flavonoids and in vitro antioxidant activities of leaves extracts of Rumex vesicarius L
obtained by classical, ultrasonic assisted and Soxhlet method were investigated. The total phenolic content and
flavonoids were determined by a spectrophotometric method. Hydroxyl radical, superoxide radical scavenger, DPPH
radical-scavenging, Fe2+-chelating activity and hypochlorous acid scavenging methods were applied to test the
antioxidant activities. The results indicated that the extract obtained by the Soxhlet extraction showed the highest
antioxidant activities and contained higher total contents of phenolic and flavonoids than the extracts obtained by two
other extraction techniques (ultrasonic assisted and classical method). From the results that not only the more bioactive
components are obtained but also the extract has better free radical and reactive oxygen species scavenging activities
through Soxhlet extraction method. These findings further illustrate that Soxhlet extraction has a bright prospect for
extracting active ingredients from plant materials. From the above study it is concluded that the ethanolic leaves extract
of Rumex vesicarius L is a potential source of natural antioxidant.
Keywords: Rumex vesicarius L, antioxidant, DPPH, superoxide, hydroxyl, phenolic content
INTRODUCTION
Many diseases like diabetes, liver damage,
nephrotoxicity, inflammation, cancer, neurological,
cardiovascular disorders and aging are associated with
oxidative stress caused by free radicals both reactive
oxygen species (ROS) and reactive nitrogen species
(RNS) which contains one or more unpaired electrons and
can donate or receive electrons to become stable. It can
propagate and produce further radicals and also can be
made inactive by antioxidants1,2. Reactive oxygen species
(ROS), such as superoxide radical (O2•−), hydro-gen
peroxide (H2O2), hydroxyl radical (HO·), perhydroxyl
radical(HO2•) and singlet oxygen (1O2) are the most
prevalent free radicals in plants and living cells3,4. When
the ROS production is excessive, oxidative damage to
proteins, lipids and nucleic acids occurs. These alterations
are involved in various pathologies and/or complications5
such as obesity, atherosclerosis, diabetes, cancer,
neurodegenerative diseases, liver cirrhosis and the aging
process6. Antioxidants are usually reducing agents, such
as vitamins, carotenoids and polyphenols, which can
scavenge reactive oxygen species and inhibit the chain
reaction by donating an electron to the free radical. The
antioxidant defence system, supported by dietary
antioxidants, protects the body from free radicals7,8. Plant
based antioxidant compounds play a defensive role by
preventing the generation of free radicals and hence could
be beneficial in alleviating the diseases caused by
oxidative stress9. In this regard, plants have been a source
of medicinal agents for thousands of years, and an
impressive number of modern drugs have been isolated
from natural sources, many based on their use in
traditional medicine and there is an increasing interest in
the therapeutic use of natural products especially those
derived from plants10. Rumex vesicarius L is an annual,
glabrous herb of the Polygonaceae, It spreads throughout
desert and semi-desert areas of North Africa, Asia and
Australia11, and is distributed widely in the tropics as an
ornamental. It is also known as bladder dock, rosy dock,
blister sorrel or country sorrel and is mostly cultivated as
a leafy vegetable. In South Algeria, Rumex vesicarius L is
widely used as food, as a medicinal herb12, it is used in
treatment of liver diseases, digestive problems, toothache,
nausea, pain, anti-inflammatory, antitumor as well as
antischistosomal, and antimicrobial activities13,14. It was
also found to have aphrodisiac effect15,16, diseases of the
spleen, hiccough, flatulence, asthma, bronchitis,
dyspepsia, piles, scabies, leucoderma, toothache and
nausea. The plant also used as cooling, laxative,
stomachic, tonic, analgesic, appetizer, diuretic, astringent,
purgative, antispasmodic and antibacterial agents17. The
roasted seeds were eaten for cure of dysentery. Finally,
the plant can be used also to reduce biliary disorders and
control cholesterol levels18,19. Despite its importance,
only few studies have been conducted on Rumex
vesicarius L. Thus, the aim of the present work was to
Eddine et al. / Free Radical Scavenging Activity…
IJPTR, Volume 7, Issue 3, June 2015- July 2015 Page 141
quantify phenolic content, flavonoids of leaf extracts of
Rumex vesicarius L obtained by different methods
classical, ultrasonic assisted and Soxhlet, and to evaluate
the antioxidant activity.
MATERIALS AND METHODS
Plant material
The Rumex vesicarius L were collected from southeast of
Algeria, state of El Oued on December 2013. The leaves
then separated from each other, washed and dried at room
temperature. All these organs were ground to a powder
with a basic electric grinder and stored in the dark at
room temperature before use. Then the powder was put in
a hot air oven at 60 °C until complete drying. Depending
on the physical characteristics of the samples, the time
ranged from 18 at 30 h.
Classical extraction (CE)
Fifty grams of powdered leaves (50 g) and ethanol (300
ml) were put in a series of the Erlenmeyer flasks (500
ml), no additional stirring was applied. The extraction
was performed at 30 °C for 60 min. At the end of the
extraction cycle the liquid extract was separated from the
solid residue by vacuum filtration. The solid residue was
washed twice with fresh solvent (60 ml). The filtrates
were collected and the solvent was evaporated in a rotary
vacuum evaporator at 45 °C. Extracts were stored at +4
°C in dark until use.
Ultrasonic-assisted extraction (UAE)
Ultrasonic apparatus from Branson (40 kHz, 1500W,
dimension: 49 cm×14 cm×15 cm) was used for
accelerated extraction. A beaker was partially submerged
in an isothermal water bath to maintain the extraction
temperature at 30 °C. Fifty grams (50 g ) leaves were then
extracted with ethanol (300 ml) for 60 min. The extract
was filtered through Whatman paper on a Büchner funnel
by vacuum; the solids were washed with an additional 60
ml of ethanol. The filtrate was rotary-evaporated under
vacuum at 40 ◦C to dryness. The crude extracts were
dried in a vacuum oven at 45 ◦C. Extracts were stored at
+4 °C in dark until use.
Soxhlet extraction (SE)
Fifty grams of powdered were mixed with 300 ml ethanol
and extracted in a Soxhlet apparatus for 6 h. The extracts
concentrated under vacuum at 40 °C by using a rotary
evaporator. To obtain ethanol extracts, air dried powdered
plants were boiled with 250 ml of ethanol for 30 min. The
ethanol extract were filtered and concentration using
rotary-evaporated under vacuum at 45 ◦C to dryness.
Extracts were stored at +4 °C in dark until use.
Total phenolic content
The total phenolic contents in all organs were determined
by the folin-Ciocalteu method20. Briefly, 100 µl of both
the sample and the standard (gallic acid) of known
concentrations were made up to 2.5 ml with water and
mixed with 0.25 mL of 1N Folin-ciocalteu reagent. After
5 min, 2.5 ml of sodium carbonate aqueous solution (2%,
w/v) was added to the mixture and was completed the
reaction for 30 minutes in darkness at room temperature.
The absorbance was read at 765 nm using a UV-visible
spectrophotometer (Shimadzu UV-1800, Japan). For the
blank the same protocol was used but the extract was
replaced by solvent. The concentration of total
polyphenols in the extracts was expressed as mg gallic
acid equivalent (GAE) per g of dry weight using UV-
Visible (Shimadzu UV-1800, Japan) and the equation of
calibration curve: Y= 0.00778x, R2 =0.991, x was the
absorbance and Y was the gallic acid equivalent. All
results presented are means (±SEM) and were analyzed in
three replications.
Total flavonoids
The determination of flavonoids was performed
according to the colorimetric assay21. Distilled water (4
ml) was added to 1 ml of leaf extract. Then, 5% sodium
nitrite solution (0.3 ml) was added, followed by 10%
aluminum chloride solution (0.3 ml). Test tubes were
incubated at ambient temperature for 5 min, and then 2
ml of 1 M NaOH were added to the mixture.
Immediately, the volume of reaction mixture was made to
10 ml with distilled water. The mixture was thoroughly
vortexed and the absorbance of the pink colour developed
was determined at 510 nm. A calibration curve was
prepared with catechin and the results were expressed as
mg catechin equivalents (CE)/g of dry weight.
Hydroxyl radical scavenging capacity22,23: A volume a
0.1 ml of EDTA (1 mM) was added to 0.01 ml of FeCl3
and 0.1 ml of H2O2. After, the precedent reaction mixed
with 0.36 ml of deoxyribose (10 mM) and 1.0 ml of
ethanolic extract (various concentrations) or standard
ascorbic acid, finally followed by the addition of 0.33 mL
of phosphate buffer (50 mM, pH-7.4) and 0.1 ml of
ascorbic acid in sequence. After incubation for1 h at 37°C
in stand, 1 ml of the incubated mixture was mixed with 1
ml of TCA and 1 ml of 0.5% TBA (in 0.025 M NaOH
containing 0.025% BHA) to develop the pink chromogen
which was measured at 532 nm, ascorbic acid used for
comparison. The hydroxyl radical scavenging activity of
the extract is reported as % inhibition of deoxyribose
degradation and is calculated using the equation .
Percentage inhibition = [(C-T)/C] x100
Where,
C : Absorbance at 532 nm of the control.
T : Absorbance at 532 nm of the test.
Scavenging activity of superoxide radicals
The superoxide anion scavenging of extracts was
estimated using the inhibition of NBT reduction24 by
photochemical generated O2‾. To the assay mixture
contained 2 µM of riboflavin, we added 6 µM EDTA, 50
µM NBT and 3 µg of sodium cyanide in 67 mM
phosphate buffer (pH= 7.8) in a final volume of 3 ml.
Initial absorbance was measured at 530 nm, the tubes
were illuminated uniformly with incandescent lamp at
530 nm. The sample extract was added to the reaction
mixture, in which O2- radicals are scavenged, thereby
inhibition the NBT reduction. Querecetin used as a
positive control and the percentage of scavenging
inhibition was calculated as: % inhibition = [(Acontrol –
Asample)/Acontrol] x 100, IC50 value is the concentration
(µg/ml) of the tested material that causes 50% loss of
superoxide radicals calculated by the linear regression
analysis.
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IJPTR, Volume 7, Issue 3, June 2015- July 2015 Page 142
DPPH radical scavenging activity
The radical scavenging activity using free-radical DPPH
assay was carried out using the spectrophotometric
method25. 1 ml aliquot of each extract was added to 0.5
ml of a DPPH ethanolic solution (7.8 mg DPPH in 100
mL ethanol 70 %). The mixture was vigorously shaken
and left to stand in the dark for 30 min at room
temperature. The antioxidant activity was then measured
by the decrease in absorption at 517 nm using UV-Visible
spectrophotometer and corresponds to the extract ability
to reduce the radical DPPH to the yellow-colored
Diphenylyldrazine. The Anti-radical activity was
expressed as IC50 (μg/ml). The Anti-radical percentage
inhibition calculated by the following equation:
DPPH Inhibition % =[ (Ao ̶ A1)/Ao] x 100, Where Ao is
the absorbance of control test after 30 min. A1 is the
absorbance of the sample extract after 30 min. All results
are means ±SD.
Metal chelating activity
The chelating activity of leaves extracts from Rumex
vesicarius L for ferrous ions Fe2+ was measured
according to the method described by Lucci et al26.
Briefly, to 0.5 ml of extract at different concentrations,
1.6 ml of deionized water and 0.05 ml of FeCl2 (2 mM)
was added. After, 0.1 ml ferrozine (5 mM) was added.
Ferrozine reacted with the divalent iron to form stable
magenta complex species that were very soluble in water.
After 10 min at room temperature, the absorbance of the
Fe2+-ferrozine complex was measured at 562 nm. EDTA
was used as positive control. The metal chelation activity
was calculated using the following equation: Metal
chelating activity (%) =[(Acontrol – Asample) /Acontrol] x 100.
where AControl is the absorbance of control devoid of
sample and ASample is the absorbance of sample in the
presence of the chelator. The extract concentration
providing 50% metal chelating activity was calculated
from the graph of Fe2+ chelating effects percentage
against extract concentration.
Figure 1. Rumex vesicarius L
Figure 2. Total phenolic content (mg GAE/g DW) and
total flavonoids (mg CE/g DW) of leaves extracts from
Rumex vesicarius L obtained by different methods.
Figure 3. Hydroxyl radical scavenging activity of leaves
extracts from Rumex vesicarius L obtained by different
methods and the standard ascorbic acid
Figure 4. The HOCl scavenging activity of leaves extracts
from Rumex vesicarius L obtained by SE, UAE, CE and
standard ascorbic acid. The data represent the percentage
of HOCl inhibition.
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IJPTR, Volume 7, Issue 3, June 2015- July 2015 Page 143
Hypochlorous acid scavenging
Hypochlorous acid (HOCl) was freshly prepared
adjusting the pH of a 10% (v/v) solution of NaOCl to 6.2
with 0.6 M H2SO4, and the concentration of HOCl was
determined by measuring the absorbance at 235 nm using
the molar extinction coefficient of 100 M−1 cm−1. The
reaction mixture contained, 1.5 mM of HOCl and
different concentration of the extract or standard ascorbic
acid and incubated for 1 h at 37°C. After that taurine (30
mM) was added and incubate again 30 min at 37°C
followed by the addition of thionitro benzoic acid
(TNB)27. Absorbance was measured at 412 nm against
blank and % scavenging was calculated according to the
standard formula28.
Statistical analyses
The data obtained in this study were expressed as the
mean of three replicate determinations plus or minus the
standard deviation (SD). Statistical comparisons were
made with Student’s test. P values <0.05 were considered
to be significant
RESULTS
Total phenolic contents and flavonoids
Ethanolic leaves extracts of Rumex vesicarius L obtained
by classical, ultrasonic assisted and Soxhlet method were
found to be rich in total phenolics and flavonoids
contents. The total phenolic content is given in figure 2.
SE was found to have the highest value 38.94.15 ± 1.2
mg GAE/g DW, following by the UAE 30.27.45 ± 0.9 mg
GAE/g DW and the lowest value in CE 25.76 ± 0.7 mg
GAE/g DW.
Similar results were observed in quantification of total
flavonoids, the content of total flavonoids was also found
to vary significantly (p <0.05) and content ranged from
9.43±0.2 mg CE/g DW to 16.88± 0.5 mg CE/g DW. The
Total flavonoids in increasing order was: SE > UAE >
CE.
OH˙ scavenging capacity
Figure 3 shows the percentage of hydroxyl radical
scavenging activity of the three extracts obtained CE,
UAE and SE. Hydroxyl radical has been implicated as
capable of damaging almost every molecule found in
living cells28. The extracts have shown a dosage-
dependent increase in inhibition of OH˙ radicals. The
results show that SE presented the highest antioxidant
activity behavior, with values of 181.52±6.5 μg/ml,
followed by UAE with results of 212.36±7.82 μg/ml and
the lowest value in CE 230.42±8.5 μg/ml.
Superoxide anion radical method
The results for the selected extracts are presented in Table
1 and compared with the standard querecetin value of the
superoxyde scavenger capacity. The results for Table 1
are expressed in terms of IC50 (mean±standard deviation).
The SE show the best results in antioxidant potential,
with values of IC50=208.56±7.5 μg/ml, higher than the
querecetin standard (IC50= 287.95 ± 8.25 μg/ml). the
extract obtained by ultrasonic assisted method shows a
moderate antioxidant potential (IC50=264.56±8.75 μg/ml),
while the extract obtained by classical method resulted in
IC50 = 278.32±8.52 μg/ml.
Antioxidant activity by DPPH method
The DPPH method is recommended as a simple and rapid
screening method for obtaining basic information about
the antioxidant activity of the extracts. It is a commonly
and widely used method despite some disadvantages29.
The SE exhibited the strongest antioxidant activities
against DPPH radicals IC50= 116.54± 3.72 μg/ml,
followed by UAE IC50= 157.28± 5.75 μg/ml and the
lowest value in CE IC50= 177.58± 6.85 μg/ml.
Chelating ability of ferrous ion
The metal ion chelating activity extracts were analyzed
and shown in Table 1. The decolorization of red color of
the reaction mixture depends upon the reduction of
ferrous ions by the plant extracts. The results were
expressed as mg EDTA equivalents/ g DW. The metal
chelating activity of ethanolic leaf extract by classical
method, Soxhlet and ultrasonic assisted extract was
45.5±1.5, 61.35± 1.45 and 39.62±1.14 mg EDTA
equivalents/ g DW, respectively. Among the different
methods extraction, the Soxhlet ethanolic extract
exhibited better metal ion chelating property.
Hypochlorous acid scavenging
Figure 4 shows the dose-dependent hypochlorous acid
scavenging activity of CE, UAE and SE of leaf from
Rumex vesicarius L compared to that of ascorbic acid.
The results indicate that the extracts scavenged
hypochlorous acid more efficiently (IC50 ranges from
157.42 ± 2.56 to 185.34 ± 3.15 μg/ml) than ascorbic acid
(IC50 = 143.39 ± 1.75 μg/ml) . Similar results were
observed for the metal ion chelating activity, the highest
inhibition founded in SE IC50=157.42 ± 2.56 μg/ml
followed by the UAE IC50=176.91 ± 2.83 μg/ml and the
CE IC50=185.34 ± 3.15 μg/ml.
DISCUSSION
Table 1. Scavenging activity of superoxide radicals, metal chelating activity and DPPH of SE, UAE and CE of leaf of
Rumex vesicarius L and standards (qurerecetin). Antioxidant activity was expressed as % inhibition IC50 values
(μg/ml) for superoxyde radicals and DPPH
Extraction technique
superoxide radicals IC50=
(μg/ml)
DPPH
IC50= (μg/ml)
Metal chelating activity (mg
EDTA/ g DW)
SE
278.32±8.52
177.58 ±6.85
61.35±1.45
UAE
264.56±8.75
157.28±5.75
45.53±1.54
CE
208.56±7.50
116.54±3.72
39.62±1.14
Querecetin
287.95±8.25
-
Data are expressed as means ± standard deviation of triplicate samples. Values with different row are significantly (P
< 0.05).
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IJPTR, Volume 7, Issue 3, June 2015- July 2015 Page 144
In the present study, three extraction methods were used
to evaluate the total phenolic contents, total flavonoids
and antioxidant activity of leaf extracts from Rumex
vesicarius L. Among the different solvent extraction
methods, successive Soxhlet extraction could provide
comparable or even better results than the maceration and
ultrasonic assisted for extracting polyphenolic
compounds and showed a significant antioxidant activity
over the other two methods. Nowadays, several novel
extraction techniques such as sonication and microwave-
assisted have been developed and employed along with
conventional extraction techniques for the extraction of
bioactive compounds and nutraceuticals from plants30.
However, it is important to define clearly which type of
capacity is being measured by each one. Moreover, the
different actions measured by each method could produce
some discrepancies between the results obtained31. As a
simple assay that provides all the antioxidant information
does not exist, it is necessary to apply different methods
to obtain the more complete antioxidant profile of the
extracts32. The preferred extraction method should be
simple, fast, economical and able to retain the
phytoconstituents of therapeutical and nutraceuticals
value. Free radicals are produced in higher amounts in a
lot of pathological conditions and involved in the
development of the most common chronic degenerative
diseases, such as cardiovascul- ar disease and cancer33,
degenerative diseases and also lysis of the cells and
tissues. The antioxidant activity of phenolics is mainly
due to their redox properties, which allow them to act as
reducing agents, hydrogen donors and singlet oxygen
quenchers. Many synthetic drugs protect against
oxidative damage but they have adverse side effects. An
alternative solution to the problem is to consume natural
antioxidants from food supplement as traditional
medicines.
Phenolic compounds, tannins and flavonoids have been
reported to have multiple biological effects, including
antioxidant and anti-inflammatory properties34. Recent
evidences suggest that diets rich in polyphenolic
compounds play a significant role against oxidative stress
related disorders because of their antioxidant activities35.
The hydroxyl radical in the cells can easily cross cell
membranes at specific sites, react with most biomolecules
and is a highly reactive free radical species and capable of
damaging almost every molecule found in living cells36.
This radical has the capacity to join nucleotides in DNA
and cause strand breakage37, which contributes to
carcinogenesis, mutagenesis and cytotoxicity. In addition,
this species is considered to be one of the quick initiators
of lipid peroxidation process, abstracting hydrogen atoms
from unsaturated fatty acids38. Superoxide radical is
highly toxic species, which is generated by numerous
biological and phytochemical reactions. Flavonoids are
effective antioxidant mainly because they scavenge
superoxide anions39. The results suggest that
concentration-dependent increasing of superoxide radical
scavenging activity. The DPPH free radical is a stable
free radical, which has been widely used for estimating
the free radical-scavenging activities of plant
extracts/antioxidants36. The results of the present
investigation demonstrate that a successive Soxhlet
ethanolic extract can significantly decrease in vitro
DPPH˙ concentration, thus suggesting that plant extract
contains secondary metabolites with strong antioxidant
activity. Phenolic compounds of the ethanolic leaf extract
from Rumex vesicarius L are possibly involved in their
free radical reactions by reducing the stable DPPH radical
to a yellowish colored diphenylpicrylhydrazine
derivative.
Transition metal chelating activity depends on the ability
of samples to chelate transition metals (Fe2+ or Cu+). The
ability plays a vital role in antioxidant mechanism since it
reduces the concentration of the catalyzing transition
metal in lipid peroxidation mechanism. In addition,
liposome peroxidation and oxidative damage of protein
model systems induced by a Fenton reaction in which
ferrous ions catalyze the conversion of hydrogen peroxide
to hydroxyl radical with the production of ferric ion.
Although metal chelating agents are not antioxidants,
they play a vital role in the stabilization of fatty acids
against rancidity.
The results obtained revealed that Soxhlet extracts
demonstrate a marked capacity for iron binding,
suggesting that their action as a peroxidation protector
may be related to their iron binding capacity. Because
ferrous ions are commonly found in foods, the high
ferrous ion chelating ability of the extracts from Rumex
vesicarius L would be beneficial. HOCl, or its anion
hypochlorite (‾OCl), is produced in vivo by activated
phagocytes in myeloperoxidase–hydrogen peroxide-
chloride ion system which is the major strong oxidant
generated by the phagocytes cells. HOCl causes fast and
important changes in cell shape, electrical resistance, and
protein permeability41. Moreover, it is capable of
deactivating antioxidant enzymes. For example,
glutathione peroxidase is very quickly inactivated by low
concentration of HOCl. Inactivation of catalase is also
rapid, but requires higher HOCl concentrations and the
haem appears to be degraded. Hypochlorite is a strong
oxidant and reacts quickly with thiols and amines, thus
modifying proteins and enzymes42. Our results suggest
that leaf extract from Rumex vesicarius L may ameliorate
diseases associated with excessive production of HOCl.
CONCLUSION
In this study, in vitro antioxidant activities, phenolic
content and flavonoids of leaf extracts from Rumex
vesicarius L obtained by classical, Soxhlet and ultrasonic
assisted methods have been evaluated. The results
indicated that Soxhlet extract exhibited strongest
antioxidant activities. The contents of polyphenols and
flavonoids Soxhlet extract were significantly higher than
those by other extraction methods, which were possibly
responsible for higher antioxidant activities of Soxhlet
extract. From the results we can draw the conclusion that
not only the more bioactive components are obtained but
also the extract has better free radical and reactive oxygen
species scavenging activities through Soxhlet method.
These findings further illustrate that Soxhlet has a bright
Eddine et al. / Free Radical Scavenging Activity…
IJPTR, Volume 7, Issue 3, June 2015- July 2015 Page 145
prospect for extracting active ingredients from plant
materials than the classical and ultrasonic extraction
methods.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest
related to the publication of this manuscript.
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