Abstract: This study was conducted to investigate the total phenolics content and the antioxidant activities from methanolic extracts of Vigna ungiculata, Bidens pilosa and Cleome gynandra. Antioxidant and free radical scavenging activity were studied using DPPH and reducing power assays, aβ-carotene linoleic acid model system and the inhibition of lipid peroxidation in rat brain. Phenolic compounds were also quantified using HPLC. Total phenolic compounds for Vigna ungiculata, Bidens pilosa and Cleome gynandra were 1136.603±3.869 g g-1, 1102.797±2.239 mg g-1 and 1327.333±1.658 mg g-1 dry mass, respectively. All three vegetable extracts contained vanillin, caffeic acid, p-coumaric acid and ferulic acid. There was an increase in the reducing power effects and inhibition of lipid peroxidation in all samples. All samples showed a time dependent decrease in radical scavenging of DPPH andβ-carotene.
INTRODUCTION
Indigenous vegetables could play a crucial role in overcoming malnutrition in Zimbabwe (Tredgold, 1986). Bidens pilosa (blackjack, mhuwu) is a weak annual herb usually a metre or less in height which has many applications including the treatment of coughs, conjunctivitis, dysentery and respiratory infections (Shava, 2000).
Vigna ungiculata (cowpea, munyemba) is a trailing vigorous-growing annual herb with large seeds (Shava, 2000). Hausa and Edo tribes use cowpeas medicinally to treat stubborn boils (Tredgold, 1986).
Cleome gynandra (spider plant, nyevhe) is an annual herb which can grow up to a size of 60 cm. The leaves and flower buds are washed and boiled in water with a little salt. A relatively long cooking time (2 h) is normally used to remove the bitter flavour (Tredgold, 1986).
There is now increasing interest in antioxidant activity of phytochemicals present in the diet (Ou et al., 2002). An antioxidant can be defined as any substance that when present at low concentrations compared to that of an oxidisable substrate, significantly delays or inhibits the oxidation of that substrate (Atoui et al., 2005). Antioxidants prevent damage to cellular components arising as a consequence of chemical reactions involving free radicals (Vertuani et al., 2002). Most antioxidants isolated from higher plants are polyphenols, which show biological activity as antibacterial, anti-carcinogenic, anti-inflammatory, antiviral and immune-stimulating effects.
The antioxidant effect of phenolics is mainly due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers (Shon et al., 2003). The objective of this research was to estimate the phenolic content, evaluate the antioxidant activity and determine the phenolic profile of the methanolic extracts of Bidens pilosa, Vigna ungiculata and Cleome gynandra.
MATERIALS AND METHODS
Sample Collection and Preparation
Bidens pilosa, Vigna ungiculata and Cleome gynandra
were obtained form Mbare green market, sundried and ground into a fine powder
and stored at room temperature until further analysis.
Chemicals
The chemical standards used were all of analytical grade. Catechin, vanillin,
caffeic acid, p-coumaric acid, protocatechuic acid, ferulic acid, p-hydroxy-benzoic
acid, p-hydroxybenzaldehyde, 1, 1-dipheny l-2 picrylhydrazyl radical (DPPH),
ascorbic acid, trichloroacetic acid (TCA) and potassium ferricyanide were obtained
from Sigma-Aldrich Chemie (Steinheim, Germany).Thiobarbituric acid (TBA) and β-carotene
were obtained from Boehringer, Manheim, Germany. Sodium carbonate, methanol
(HPLC grade), ascorbic acid, acetonitrile (super purity solvent) and acetic
acid were obtained locally.
Folin-Ciocalteu reagent (1 N), 20% sodium carbonate, standard gallic acid (0.5 mg mL-1), 50% methanol in distilled water (1:1 v/v) and ferric chloride were used for analysis.
Extraction and Determination of the Total Phenolics in the Vegetables
Total phenolic compounds were extracted from the vegetables as described
by Makkar (1999). The sample of vegetable (2 g) was extracted twice with cold
50% aqueous methanol (10 mL). The two extracts were combined, made up to 20
mL with 50% aqueous methanol, centrifuged at 3000 rpm for 10 min sand transferred
into small sample bottles for analysis. Total phenolic compounds were determined
following the method by Makkar (1999). To a sample (50μL), distilled water
(950μL) was added to make up to 1 mL followed by 1N Folin C. reagent (500μL)
and sodium carbonate (2.5 mL). After 40 min at room temperature absorbance was
read at 725 nm on a Spectronic 20®
GenesysTM spectrophotometer against
a blank that contained methanol instead of sample. Total phenolics were in terms
of gallic acid equivalent.
Evaluation of Antioxidant Activity of the Extracts
Antiradical Activity
The radical scavenging activity was determined following the method by Kuda
et al. (2005). Methanolic solutions of DPPH (1990μL, 1 mM) containing
10μL sample were monitored at 517 nm for 30 min on a Spectronic 20®
GenesysTM spectrophotometer. Ascorbic acid was used as a positive
control. The scavenging activity was calculated as:
Scavenging activity (%) = [1-(Abs sample/Abs control)] |
Where A0 is the initial absorbance at increasing time, t. The antiradical activity was expressed as k.
Reducing Power Assay
Reducing power effects were determined following the method by Kuda et al. (2005). Up to 80μL sample or ascorbic acid control solution was
mixed with phosphate buffer (0.2 mL, 0.2 M pH 7.2) and 1% potassium ferricyanide
(0.2 mL). The mixture was incubated at 50°C for 20 min after which TCA (0.2
mL, 10%) was added. After transferring an aliquot of the mixture (0.125 mL)
into microtitre plate, distilled water (0.125 mL) and FeCl3 (0.02
mL, 0.1%) was added. The absorbance at 655 nm was measured on a Spectra MAX
340 (USA, Sunnyvale, California) microtitre plate spectrophotometer.
Inhibition of Phospholipid Peroxidation
Homogenization of rat brain (2 g) was done with a chloroform:methanol mixture
(2:1, v/v) followed by centrifugation at 3000xg for 5 min. The supernatant obtained
was used as the phospholipids source.
The blank contained the phospholipid solution (50μL) mixed with distilled water (0.5 mL) and 50% methanol (0.2 mL). The test run contained the phospholipids solution (50μL), the vegetable extract (0.5 mL), 50% methanol (0.2 mL), FeSO4 (0.5 mL), TBA (0.5 mL) and TCA (4 mL). Ascorbic acid (5%) was used as the positive control. Incubation at 37°C was followed by the addition of TBA and TCA and the solution was then heated in a boiling water bath for 15 min. After cooling on ice, absorbance was read at 532 nm.
The β-carotene-linoleic Acid Model System
The inhibition of lipid oxidation was assayed using the method of Amin and
Tan (2002). β-Carotene 0.2 mg mL-1 (1 mL) solution in chloroform
was placed into 100 mL round bottomed flask. The mixture was evaporated for
10 min using a rotary vapor to remove the chloroform. Linoleic acid (40μL)
and Tween 80 (400μL) and the mixture was diluted immediately with distilled
water to 100 mL and agitated vigorously to form an emulsion. Emulsion (4.8 mL)
was placed in test tubes and sample extracts (20μL) were added to each
test tube. The test mixture was shaken and incubated at 50°C for 2 h. The
absorbance was read at 470 nm at 15 min interval. A control was also set with
50% methanol being added instead of sample. The antioxidant activity was calculated
as the degree of inhibition of lipid oxidation using the formula:
Antioxidant activity = 100 x {1-[(Ao-At)
/Aoo-Aot)]} |
where Ao and A°o are the absorbance values measured at the initial incubation time for the samples and control, respectively while At and A°t are the absorbance values measured in the samples or standards and control at t = 120 min.
HPLC Analysis for Phenolic Acids
A Shimadzu HPLC system with a SCL-6B Shimadzu system controller, C-R AX
Shimadzu Chromatopac, Shimadzu SPD-10 AV UV-V is detector equipped with a Dynamax
60 A C18 column was used for analysis of phenolic compounds. Five microlitres
of sample were injected and the flow rate set at 1 mL min-1. All
samples in duplicate were filtered through a 0.22μm filter unit (Millex®-GV,
Molsheim, France) before injection and the solvents were filtered through a
0.45μm filter (Whatman, Maidstone, England). Two mobile phases, A, which
contained water: acetic acid (98:2 v/v) and B, which contained water-acetonitrile-acetic
acid (78: 20: 2 v/v/v), were used. The gradient profile used had 0% Solvent
B at the start, rising to 80% within 55 min, remaining at 80% up to 70 min and
falling back to 0% at time 80 min. Detection was carried out by measuring absorbance
at 280 nm according to Pena-Neira et al. (2000). After each run, the
system was reconditioned for 15 min before analysis of the next sample.
Statistical Analysis
Samples were analysed in duplicate and results are given as averages ±standard
deviations. One-way ANOVA and the Student's t-test, both packaged in the
Statistical Package for Social Sciences (SPSS) for Windows Standard Version
8.0.0 were used for the statistical evaluation with p<0.05 considered statistical
significant.
RESULTS AND DISCUSSION
Among all the vegetable extracts, Cleome gynandra had the highest phenolic content (1327.333±1.658 mg g-1) followed by Vigna ungiculata (1136.603±3.869 mg g-1) and Bidens pilosa (1102.797±2.239 mg g-1) (Fig. 1).
The vegetable extracts showed a time dependent scavenging of DPPH, which may be attributed to its hydrogen-donating ability (Fig. 2). When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced (Kuda et al., 2005). The bleaching (changes in colour from deep-violet to light yellow) that occur at 515 nm consequently result in the decrease in scavenging effect of all vegetable extracts.
The ferric reducing power assay which measures antioxidant capacity by the
reduction of the ferric tripyridyltriazine complex to the blue ferrous complex
was done (Lee et al., 2005). For all samples, there was a general increase
in absorbance with the increase in volume of the vegetable extracts as shown
in Fig. 3. Increased absorbance at 655 nm of the reaction
mixture indicates an increased reducing power (Ou et al., 2002).
| Fig. 1: | Total phenolic contents |
| Fig. 2: | DPPH radical scavenging assay |
| Fig. 3: | Reducing power effect |
| Fig. 4: | Inhibition of lipid peroxidation in rat brain |
The capacity to inhibit lipid peroxidation in rat brain by the vegetable extracts
was performed. Cleome gynandra exhibited the highest inhibitory effect
on rat brain peroxidation whilst Vigna ungiculata exhibited the lowest
inhibitory effect on peroxidation as shown in Fig. 4. Biological
specimens such as rat brain contain a mixture of thiobarbituric acid reactive
substances (TBARS), including lipid hydroperoxides and aldehydes, which increase
as a result of oxidative stress (Capecka et al., 2005). TBARS return
to normal levels over time, depending upon the presence of antioxidants (Wangensteen
et al., 2004).The total antioxidant activity, which reflected the ability
of the vegetable extracts to inhibit the bleaching of the β-carotene, was
measured and compared with that of the control. All vegetable extracts showed
similar trends with a significant decrease (p<0.05) of the absorbance values.
This indicates that the samples acted as effective antioxidants in the β-carotene
linoleate system, which inhibited the oxidation activity of β-carotene (Fig.
5). There was a decrease in absorbance values of β-carotene in the absence
of vegetable extracts due to the oxidation of β-carotene and linoleic acid.
The high absorbance values indicated that vegetable extracts possessed antioxidant
activity. Antioxidant activity of vegetable extracts depend on the type and
polarity of the extracting solvent, the isolation procedures and purity of active
compounds as well as the assay techniques and substrates used (Chun et al.,
2005).
| Fig. 5: | Beta-carotene linoleic acid model system |
| Fig. 6: | HPLC separation of dried, Vigna ungiculata. (1) Gallic
acid, (2) p-Hydroxybenzoic acid, (3) Vanillin, (4) Caffeic acid and (5)
Ferulic acid |
Qualitative analysis of the vegetable extracts was done using HPLC and the
chromatograms obtained are shown in Fig. 6-8.
All three vegetable extracts contained vanillin, caffeic acid, p-coumaric acid
and ferullic acid. However, protochatechuic acid and catechin were detected
only in Cleome gynandra.There is a wide degree of variation between
different phenolic compounds in their effectiveness as antioxidant (Ismail et
al., 2004). Ferulic acid and other hydroxycinnamic acids (caffeic and p-coumaric
acid derivatives) have been found to have good antioxidant activities. The presence
of the CH = CH-COOH group in the hydroxycinnamic acids is considered to be key
for the significantly higher antioxidative efficiency than the COOH in the hydroxybenzoicacids
(Kim et al., 2006). The presence of flavonoids in Cleome gynandra
could explain the high antioxidant activity observed in all the assays performed.
Flavonoids generally have more hydroxyl groups if compared to ferulic acids.
| Fig. 8: | HPLC separation of Bidens pilosa. (1) p-Hydroxybenzaldehyde,
(2) Vanillin, (3) Caffeic acid, (4) p-Coumaric acid and (5) Ferulic acid |
| Fig. 8: | HPLC separation of Cleome gynandra. (1) Gallic acid,
(2) Protocatechuic acid, (3) p-Hydroxybenzoic acid, (4) p-Hydroxybenzaldehyde,
(5) Catechin, (6) Vanillin, (7) Caffeic acid, (8) p-Coumaric acid and (9)
Ferulic acid |
Besides, orthosubstitution with electron-donating alkyl or methoxy groups of flavonoids increases the stability of the free radical and hence its antioxidant potential (Ismail et al., 2004).
Several studies have reported on the relationships between phenolic content and antioxidant activity. Some authors have found a correlation between the phenolic content and the antioxidant activity, while others found no such relationship. Velioglu et al. (1998) reported a strong relationship between total phenolic content and the antioxidant activity in selected fruits, vegetables and grain products. No correlation between antioxidant activity and phenolic content was found in the study by Kahkonen et al. (1999) on some plants extracts containing phenolic compounds (Ismail et al., 2004).
In this study, the findings do not show a significant relationship between antioxidant activity and total phenolic contents. For example, Cleome gynandra had the lowest antioxidant activity (61%) at 10 min in the DPPH radical scavenging assay and yet its total phenolic content (TPH) was higher than that of Bidens pilosa and Vigna ungiculata. The correlation between total phenolic contents and the antioxidant activity such as the DPPH radical scavenging activity of the vegetable extracts where R2 = 0.58 for TPH/DPPH was not highly significant according to the Pearson's test. It is known that the antioxidant properties of single compounds within a group vary remarkably so that the same levels of phenolics do not necessarily correspond to the same antioxidant responses. Moreover, the response of phenolics in the Folin-C assay also depends on their chemical structure and thus the radical scavenging capacity of an extract cannot be predicted on the basis of its total phenolic content (Atoui et al., 2005).