Antioxidant Activity of Phenolic Rich Fraction Obtained from Convolvulus arvensis L. Leaves Grown in Egypt
Acidic ethyl acetate fraction was prepared from the leaves of Convolvulus arvensis L. contents of total phenolics and total flavonoids, antioxidant activity and reducing power were evaluated in this fraction. Total phenolics and total flavonoids were measured as 244.6±2.9 and 174.4±0.4 mg gallic acid and rutin equivalents per gram extract, respectively. The fraction exhibited strong antioxidant activity measured by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method and its IC50 value was 66.9±0.1 μg mL-1. Further it possessed strong reducing power and inhibited the oxidation of β-carotene. GC-MS and HPLC analyses indicated that, this fraction contained a variety of phenolic compounds including p-hydroxybenzoic acid, syringic acid, vanillin, benzoic acid and ferulic acid. This study revealed that acidic ethyl acetate fraction of C. arvensis L. leaves has strong antioxidant activity which is correlated with its high content of phenolic compounds and it may serve as a phenolic rich fraction in food industry.
Received: July 22, 2011;
Accepted: September 28, 2011;
Published: December 31, 2011
Reactive Oxygen Species (ROS) have been considered to cause oxidative injury
to living organisms and thus play an important role in many human diseases such
as arthritis, atherosclerosis, emphysema, aging process, diabetes mellitus and
cancer (Elzaawely et al., 2005; Gupta
et al., 2007; Juan and Chou, 2010). Synthetic
antioxidants such as Tert-butyl Hydroxyanisole (BHA) and Tert-butyl Hydroxytoluene
(BHT) are used to protect foods against oxidative damage caused by ROS (Liu
et al., 2011). However, these synthetic antioxidants have been proved
to have undesirable side effects; therefore, there has been an increasing interest
in the substitution of synthetic antioxidants by natural ones (Al-Soqeer,
2011). Phenolic compounds are natural plant-derived substances that have
positive antioxidant, antimutagenic, anticarcinogenic, anti-inflammatory and
antimicrobial activities (Su et al., 2009; Caliskan
and Polat, 2011; Anago et al., 2011).
Convolvulus arvensis (Family: Convolvulaceae) is a wild plant native
to Europe and Asia and widely distributed in Egypt. It is a climbing or creeping
herbaceous perennial plant growing to 0.5-2 m high. It showed an allelopathic
activity (Om et al., 2002; Hegab
and Ghareib, 2010), antibacterial activity (Sener et
al., 1998) and antioxidant activity (Thakral
et al., 2010). Previous phytochemical studies revealed that C.
arvensis contained tropane alkaloids (Molyneux et
al., 1993; Todd et al., 1995) and phenolic
compounds (Hegab and Ghareib, 2010).
This study was designed to prepare a phenolic rich fraction from the leaves of C. arvensis grown in Egypt and to investigate its antioxidant capacity as well as its contents of phenolic compounds.
MATERIALS AND METHODS
Chemicals: Standard phenolic compounds (p-hydroxybenzoic acid, syringic acid, vanillin, benzoic acid and ferulic acid), Folin- Ciocalteus reagent, aluminum chloride, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Tert-butyl Hydroxytoluene (BHT), β-carotene, linoleic acid, polyoxyethylene sorbitan monopalmitate (Tween-40), potassium ferricyanide, trichloroacetic acid, ferric chloride and all solvents used were of analytical grade and purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Plant material: C. arvensis plants were collected from Tanta province, Egypt. The leaves were air dried for one week and the dried materials were powdered and kept in the refrigerator till use.
Preparation of the acidic fraction: Five grams dried powdered leaves of C. arvensis were extracted with aqueous ethanol 80% (3x200 mL) for 24 h at room temperature. The extracts were collected, filtered and the volume was reduced under vacuum at 40°C. After the pH of the solution adjusted to 11.0 by NaOH 4 N, it was extracted with chloroform (3x150 mL) to remove basic material that mainly contains alkaloids. The remaining aqueous solution was made acidic to pH 2.0 with HCl 6 N and then extracted with ethyl acetate (5x150 mL). The ethyl acetate fraction was collected, filtered and dried under vacuum at 40°C to yield 0.17 g of crude acidic fraction. The fraction was hydrolyzed with 50 mL NaOH 4 N at 50°C with stirring for 4 h to release phenolic acids. After the pH was adjusted to 2.0 by HCl 6 N, it was extracted with ethyl acetate (2x150 mL). The acidic ethyl acetate fraction was filtered and dried under vacuum to yield 0.13 g of phenolic-rich ethyl acetate fraction of C. arvensis leaves.
Determination of total phenolic content: The amount of total phenolics
was determined according to the Folin-Ciocalteu procedure described by Kahkonen
et al. (1999). Briefly, 1.0 mL Folin-Ciocalteus reagent (50%)
and 0.8 mL 7.5% (w/v) Na2CO3 were added to 0.2 mL (500
ppm) of methanolic solution of EtOAc fraction. After shaking, the mixture was
incubated at room temperature for 30 min. Absorption was measured at 765 nm
using a Shimadzu UV-160A spectrometer, Kyoto (Japan). Total phenolic content
was expressed as Gallic Acid Equivalents (GAE) in milligrams per gram extract.
Determination of total flavonoids: The amount of total flavonoids was
determined according to the method described by Djeridane
et al. (2006). Briefly, 1.0 mL of methanolic solution of EtOAc fraction
(1000 ppm) was mixed with 1 mL aluminum chloride (2% in methanol). After shaking,
the mixture was incubated at room temperature for 15 min and then the absorption
was measured at 430 nm using a Shimadzu UV-160A spectrometer, Kyoto (Japan).
Total flavonoids content was expressed as Rutin Equivalents (RE) in milligrams
per gram extract.
Antioxidant activity by DPPH method: The radical scavenging activity
was evaluated as described previously by Abe et al.
(1998). Two millileter of the methanol solution of the sample (25, 50 and
100 ppm) were mixed with 1 mL of 0.5 mM DPPH methanol solution and 2 mL of 0.1
M sodium acetate buffer (pH 5.5). After shaking, the mixture was incubated at
room temperature in the dark for 30 min and then the absorbance was measured
at 517 nm using a Shimadzu UV-160A spectrometer, Kyoto (Japan). BHT (10 ppm)
was used as positive reference while methanol was used as negative one. The
IC50 value was determined as the concentration required to give 50%
DPPH radical scavenging activity.
Antioxidant activity by β-carotene bleaching method: Antioxidant
activity was evaluated according to the β-carotene bleaching method as
reported by Siddhuraju and Becker (2003). β-Carotene
(2.0 mg) was dissolved in 10 mL chloroform. One milliliter of the chloroform
solution was mixed with 20 μL linoleic acid and 200 mg Tween-40. The chloroform
was evaporated under vacuum at 45°C, then 50 mL oxygenated water was added
and the mixture was vigorously shaken. The emulsion obtained was freshly prepared
before each experiment. An aliquot (250 μL) of the β-carotene-linoleic
acid emulsion was distributed in each of the 96-wells of the microtitre plates.
Methanolic solutions (30 μL) of the sample and BHT at 1000 ppm were added.
An equal amount of methanol was used for control. The microtitre plates were
incubated at 50°C and the absorbance was measured using a model MTP-32 microplate
reader (Corona Electric, Ibaraki, Japan) at 492 nm. Readings of all samples
were performed immediately at zero time and every 15 min up to 180 min.
Reducing power: Reducing power was determined as described previously
by Yildirim et al. (2003). One milliliter of
the ethyl acetate fraction of C. arvensis leaves and BHT at different
concentrations in methanol was mixed with 2.5 mL phosphate buffer (0.2 M, pH
6.6) and 2.5 mL potassium ferricyanide [K3Fe(CN)6] (10
g L-1), then the mixture was incubated at 50°C for 30 min. Afterwards,
2.5 mL trichloroacetic acid (100 g L-1) was added to the mixture
which was subsequently centrifuged at 4000 rpm for 10 min. Finally, 2.5 mL of
the supernatant solution was mixed with 2.5 mL distilled water and 0.5 mL FeCl3
(1 g L-1) and the absorbance was measured at 700 nm. Increased absorbance
of the reaction mixture indicated increased reducing power.
GC-MS analysis: A 1 μL aliquot of the acetone solution of the ethyl acetate fraction was injected into the GC-MS (QP-2010, Shimadzu Co., Kyoto, Japan). The DB-5MS column was 30 m in length, 0.25 mm id and 0.25 μm in thickness (Agilent Technologies, J and W Scientific Products, Folsom, CA, USA). The carrier gas was helium. The GC oven temperature program was as follows: 50°C hold for 6 min, raised at 5°C min-1 to 280°C and hold for 5 min. The injector and detector temperatures were set at 250 and 280°C, respectively. The mass range was scanned from 20 to 900 amu. The control of the GC-MS system and the data peak processing were carried out by means of Shimadzus GC-MS solution software, version 2.4.
Quantification by HPLC: Phenolic compounds were measured at 280 nm using a Shimadzu HPLC (SCL-10 A vp, Shimadzu Co., Kyoto, Japan) coupled with a UV-vis detector (SPD-20A, Shimadzu). Separations were achieved on a RP-18 ZORBAX ODS column (Agilent Technologies, USA) (25x0.46 cm i.d.; 5 μm particle size). The mobile phase was water with 1% acetic acid (v/v) (solvent A) and methanol: acetonitrile:acetic acid (75:24:1, v/v/v) (solvent B) at a flow rate of 0.8 mL min-1. The gradient elution was performed as follows: 0-2 min, 5% B isocratic; 2-10 min, linear gradient 5-25% B; 10-20 min, linear gradient 25-40% B; 20-30 min, linear gradient 40-50% B; 30-40 min, linear gradient 50-100% B; 40-45 min, 100% B isocratic and 45-55 min, linear gradient 100-5% B. A 5 μL Methanolic solution of the ethyl acetate fraction at 10,000 ppm was used and the identification of the compounds was carried out by comparing their retention times to those of standards. The quantification of each compound was determined based on peak area measurements which were reported to calibration curves of the corresponding standards.
Statistical analysis: Data were analyzed using SAS version 6.12 using ANOVA with the Least Significant Difference (LSD) at the 0.05 probability level.
RESULTS AND DISCUSSION
Total phenolic and total flavonoids: Contents of total phenolic compounds
were determined using Folin-ciocalteau method and total flavonoids determined
using the aluminum chloride colorimetric assay of the acidic ethyl acetate fraction
prepared from C. arvensis leaves are shown in Fig.
1. Results show that, this fraction contained high amounts of total phenolics
and total flavonoids and they were measured as 244.6±2.9 and 174.4±0.4
mg gallic acid and rutin equivalents per g extract, respectively. It has been
recorded that phenolic compounds including flavonoids are associated with strong
antioxidant activity (Liu et al., 2011) and they
possess healthy benefits (Shih and Diagle, 2003).
Antioxidant activity: The DPPH radical scavenging activity of acidic
ethyl acetate fraction of C. arvensis leaves and BHT as positive
control are presented in Fig. 2. The IC50 defined
as the concentration of the sample needed to scavenge 50% of DPPH present in
the test solution. Lower IC50 value reflects higher DPPH radical
||Total phenolics (mg GAE g-1 extract) and total
flavonoids (mg RE g-1 extract) in acidic ethyl acetate fraction
of C. arvensis leaves
||DPPH radical scavenging activity of acidic ethyl acetate fraction
of C. arvensis leaves and BHT
||Antioxidant activity of acidic ethyl acetate fraction of C.
arvensis leaves and BHT measured by β-carotene bleaching method
DPPH is a stable radical showing a maximum absorbance at 515 nm. It can be
reduced by an antioxidant molecule to uncolored solution (Erkan
et al., 2008). It is a convenient, accurate and easy method and therefore,
it is widely used to measure the antioxidant activity of plant extracts (Blois,
1958; Ali et al., 2011). Under the assay
conditions employed here, the acidic ethyl acetate fraction of C. arvensis
leaves showed strong DPPH activity with the IC50 value of 66.9±0.1
μg mL-1; while the IC50 of BHT as standard was 12.2
μg mL-1. This high activity may be due to the high amounts of
the corresponding phenolic compounds found in this fraction. Positive correlation
has been demonstrated between antioxidant activity and phenolic content of plant
extracts (Su et al., 2008; Biglari
et al., 2008; Socha et al., 2009;
Juan and Chou, 2010).
Effect of the acidic ethyl acetate fraction of C. arvensis leaves
as well as BHT on oxidation of β-carotene/linolic acid is shown in Fig.
3. The linoleic acid free radical, formed upon the abstraction of a hydrogen
atom from one of its diallylic methylene groups, attacks the highly unsaturated
β-carotene molecules. As β-carotene molecules lose their double bonds
by oxidation, the compound loses its orange color. The presence of extract/fraction
that has antioxidant activity can hinder the extent of β-carotene-bleaching
by neutralizing the free radicals formed in the system and thus, the degradation
rate of β-carotene depends on the antioxidant activity of the extract/fraction
(Jayaprakasha et al., 2001). In present study,
the acidic ethyl acetate fraction of C. arvensis leaves reduced
the oxidation of β-carotene to some extent and its effect was less than
that of BHT.
Reducing power is considered to be a strong indicator of the antioxidant activity
and was determined using a modified iron (III) to iron (II) reduction assay.
The presence of the reductants (antioxidants) in the solution can reduce the
Fe3+/ferricyanide complex to the ferrous form by donating an electron
(Ali et al., 2011; Liu et
al., 2011). The reducing power of the acidic ethyl acetate fraction
of C. arvensis leaves as well as standard BHT is presented in
Fig. 4. The reducing power of both samples increased with
its concentrations. The ethyl acetate fraction had high ability to reduce Fe3+
to Fe2+ and the effect was less than that of BHT. The reducing ability
is generally associated with the presence of reductants which exert antioxidant
action through breaking the free radical chain by donating a hydrogen atom or
preventing peroxide formation (Kumaran and Karunakaran,
2006). The results presented in Fig. 4 indicated that
the reducing activity of the ethyl acetate fraction attend to be due to the
presence of phenolic compounds that may act as reductants by donating the electrons
and reacting with free radicals to convert them to more stable products and
terminate radical chain reaction (Loganayaki et al.,
|| Reducing powers of acidic ethyl acetate fraction of C.
arvensis leaves and BHT
||Contents of phenolic compounds (μg g-1 DW)
in acidic ethyl acetate fraction of C. arvensis leaves LSD
(p<0.05) = 62.656. Values are means of 3 replications±SE. Means
followed by different letters are significantly different at p<0.
||HPLC chromatogram of phenolic compounds from acidic ethyl
acetate fraction of C. arvensis leaves. 1: p-Hydroxybenzoic,
2: Syringic, 3: Vanillin, 4: Benzoic and 5: Ferulic
Identification and quantification of phenolic compounds: Since phenolic
acids are often found in plants in bound complexes such as esters and glycosides
(Germano et al., 2006), it needs to be released
fro m its bound form by using alkaline, acidic or enzymatic hydrolysis (Madhujith
and Shahidi, 2009). In this study, alkaline hydrolysis with 4 M NaOH at
50°C was carried out to release conjugated phenolics and subsequently five
phenolic compounds were detected in the acidic ethyl acetate fraction of C.
arvensis by GC-MS and HPLC. These compounds are p-hydroxybenzoic acid,
syringic acid, vanillin, benzoic acid and ferulic acid. Figure
5 displays quantitative results of identified phenolic compounds in the
acidic ethyl acetate fraction of C. arvensis by HPLC and Fig.
6 shows the representative HPLC chromatogram of this fraction monitored
at 280 nm. The quantification of the compounds using HPLC was carried out by
comparing their retention times to those of standards. Furthermore, the identification
of the compounds was confirmed using the GC-MS by comparing their retention
times and spectral characteristics of their peaks with those of standards. Statistically
significant variation was found in the quantities of phenolic compounds in this
fraction. Benzoic acid was the predominant phenolics (350.6 μg-1
DW) while syringic acid was the lowest one (30.5 μg g-1 DW).
Previous study reported that the methanol extract and its ethyl acetate fraction
prepared from the aerial parts of C. arvensis grown in India showed
strong antioxidant activity; however, its chemical constituents were not identified
(Thakral et al., 2010). It was previously reported
that phenolic compounds have strong antioxidant activity (Elzaawely
et al., 2007; Zhang et al., 2009;
Mariod et al., 2010) and therefore, the phenolic
compounds in the phenolic rich fraction of C. arvensis may be
responsible for its antioxidant activity.
This investigation shows that, the acidic ethyl acetate fraction prepared from
C. arvensis leaves contains high amounts of total phenolics and
total flavonoids and it exhibited strong reducing power and antioxidant activity
measured by DPPH and β-carotene bleaching methods. GC-MS and HPLC revealed
that this fraction contains five phenolic compounds viz., p-hydroxybenzoic acid,
syringic acid, vanillin, benzoic acid and ferulic acid that are responsible
for its strong antioxidant activity. Further studies in isolation of individual
phenolic compounds particularly flavonoids in this fraction and its effect on
antioxidant status in animal models are needed to evaluate their potential benefits.
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