Abstract: This study was designed to investigate the chemical composition of fourteen vegetables popularly consumed in the South Western part of Nigeria and also to evaluate some in vitro antioxidant activities of the selected vegetables. Ethanolic extracts of the vegetables were injected into GC-MS to investigate the presence of some chemical compounds and were also screened for their lipid peroxidation inhibitory potentials and free radical scavenging activities by two complementary. GC-MS studies showed that caffeic acid was present in all the vegetable samples with the exception of Celocia argentia and Talinum triangulare in addition to other compounds identified in some of the vegetables (phenylacetic acid, vanillic acid, genticic acid, protocatechuic acid, syringic acid, p-coumaric acid, garlic acid, ferulic acid, epicatechin and catechin. Lipid peroxidation inhibition studies revealed that lipid peroxide inhibition ranges from 3.59-69.47 (%) while conjugated diene inhibition ranges from 84.33-97.31 (%). Scavenging ability of the vegetables towards hydrogen peroxide ranges from 4.89±0.55-60.26±1.23 (%) while superoxide scavenging activities ranges from 75.62±1.42-97.01±1.32 (%). Total flavonoid mg mL-1 and total antioxidant activity (mg mL-1) ranges from 0.12 to 3.08 and 0.13 to1.60, respectively. Results from conjugated diene formation and superoxide radical scavenging activity show that the evaluated vegetables exhibited a relatively high lipid peroxidation inhibitory potentials and free radical scavenging activity which could possibly be as a result of caffeic acid and other compounds present in the vegetables.
Introduction
The role of oxygen and free radicals in tissue damage related to aging and other disease conditions are becoming increasingly recognized. Free radicals, having one or more unpaired electrons in the outer orbit, include superoxide anion (O·2 ), hydroxyl (HO·), peroxyl (ROO·), alkoxyl (RO· ) and nitric oxide, which are oxygen-centered free radicals sometimes known as Reactive Oxygen Species (ROS) (Squacrito and Peyer, 1998).
Modern theories of ROS have revealed that the oxygen centered free radicals play a dual role in organisms. ROS are not only strongly associated with lipid peroxidation, which lead to the deterioration of food but also involved in the development of a variety of diseases including cellular aging, mutagenesis, carcinogenesis, coronary heart disease, diabetes and neuro-degenerative disorders (Harman, 1980; Sasaki et al., 1996; Moskovitz et al., 2002). The efficiency of phenolic compounds as antioxidant is diverse and depends on many factors, such as the number of hydroxyl groups bonded to the aromatic ring, the site of bonding and mutual position of hydroxyls in the aromatic rings. The cell possesses a natural antioxidant defense mechanism that enables it to take care of these free radicals. However, when the free radicals outweigh the defense mechanism, the resulting effect is oxidative stress.
Recently, various phytochemicals and their effect on health, especially the suppression of free radicals by natural antioxidants have been studied (Ho et al., 1994). Increased intakes of dietary antioxidants may help to maintain an adequate antioxidant defense status, defined as the balance between oxidant and antioxidants in living organisms (Pulido et al., 2000; Halliwell et al., 1995). Food rich in antioxidants plays an essential role in the prevention of cardiovascular diseases and cancers (Gerber et al., 2002; Kris-Ethertn et al., 2002; Serafini et al., 2002), neurodegenerative diseases, as well as inflammation and problems caused by cell and cutaneous aging (Ames, 1983).
Many vegetables and fruits are potentially useful for decreasing the risks of several chronic diseases, such as coronary heart diseases and some cancers (Block et al., 1992; Hertog et al., 1995; Lampe, 1999). These protective effects have been particularly attributed to various antioxidant compounds such as vitamins C and E, β-carotene and polyphenolics (Diplock et al., 1998).
Recent investigation on the phytochemical screening and antioxidant indices of the selected tropical vegetables indicates that they could be potentially useful as natural antioxidants (Akindahunsi and Salawu, 2005a, b). However, detailed chemical composition and in vitro antioxidant studies is necessitated by lack of quantitative information to compare antioxidant activities of the vegetables. Therefore, this study was aimed to correlate the chemical composition of some Nigerian vegetables, mostly phenolic compounds with the in vitro antioxidant activities of the selected vegetables to provide useful information on their antioxidant potentials.
Materials and Methods
Collection of Plant Materials
Some popularly consumed green leafy vegetables in Nigeria namely: Telfairia
occidentalis (Ugu), Hibiscus esculentus (Ewe Ila), Crassocephalum
crepidioides (Worowo), Occimum graticimum (Efinrin), Xanthosoma
maffafa (Ewe Koko), Vernonia amygdalina (Ewuro), Solanum macrocarpon
(Igbagba), Structium Sparejanophora (Ewuro-Odo), Celocia argentia
(Soko), Talinium triangulare (Gbure), Corchorus olitorus (Ewedu),
Amaranthus hybridus (Arowojeja), Amaranthus caudatus (Tete) and Manihot
utilisima (Ewe Ege) were collected from a local market in Akure. The vegetables
were rinsed with water and the edible portions were separated from the inedible
portions. The edible portions were chopped into smaller pieces and air-dried,
for further analysis.
Preparation of Samples for GC-MS Studies
Fifty milliliter of 96% ethanol was added to 5 g of the powdered vegetable
materials in a pyrex tube and 500 μg g-1 (powdered vegetables
in o-coumaric acid as internal standard) was added. The samples were extracted
by sonication at room temperature for 2 h. The samples were then filtered using
a Whatman No.1 filter paper. One milliliter each of filtered samples was evaporated
by nitrogen flow and re-dissolved in 100 μL of ethyl acetate. The samples
were added to 100 μL of BSTFA + TMCS (Supelco) and heated at 80°C for
20 min. One millilitre of derivatized samples were then injected into GC-MS.
Preparation of Methanol Extracts for Invitro Antioxidant Assay
The air-dried and finely ground vegetable samples (25 g) were extracted
with 150 mL of methanol at 30°C for 5 h and mixed by using a magnetic stirrer.
Each extract was filtered through Whatman No. 4 filter paper and re-extracted
with the same solvent for the extraction of antioxidant fractions. All extracts
were pooled and concentrated under vacuum at 4°C and kept at +4°C prior
to analysis.
Lipid Peroxidation Assay
Lipid peroxidation assay was carried out by measuring lipid peroxide content
and the conjugated diene formation.
Lipid Peroxide Formation
A modified thiobarbituric acid reactive species (TBARS) assay (Okhawa et
al., 1979) was used to measure the lipid peroxide formed using egg yolk
homogenates as lipid rich media. Egg yolk homogenate was prepared according
to a standard method (Wang et al., 1997). Malondialdehyde (MDA), a secondary
end product of the oxidation of polyunsaturated fatty acids, reacts with two
molecules of thiobarbituric acid (TBA) yielding a pinkish red chromogen with
absorbance maximum at 532 nm (Janero, 1990). Egg homogenate (0.5 mL of 10% v/v)
and 0.1 mL of each extract were added to a test tube and made up to 1 mL with
distilled water. 0.05 mL of FeSO4 (0.07 M) was added to induce lipid
peroxidation and incubated for 30 min. Then 1.5 mL of 20% acetic acid (pH adjusted
to 3.5 with NaOH) and 1.5 mL of 0.8% (w/v) TBA in 1.1% sodium deodecyl sulphate
and 20% TCA were added and the resulting mixtures were vortexed and then heated
at 95°C for 60 min. After cooling, 5.0 m of butan-1-ol was added to each
tube and centrifuged at 3000 rpm for 10 min. The absorbance of the organic layer
was measured at 532 nm. Inhibition of lipid peroxide formation (%) by the extract
was calculated according to [(1-E/C) x 100] where C is the absorbance value
of the fully oxidized control and E is (Abs 532+TBA-Abs532-TBA).
Conjugated Diene Formation
1.8 mL of asolectin liposome (500 μM final concentration of phospotidylcholine
equivalent) was incubated in a water bath at 37°C with 0.05 mL of the extract.
Reaction was initiated with the addition of 200 μL of azo-initiator (5
mM AAPH final concentrations). Aliquots of liposome (60 μL) were dissolved
with 940 μL of methanol directly in 1 cm quartz cell (Goncalves et al.,
1998). The formation of conjugated diene was followed by comparing oxidation
of liposome by AAPH with oxidation of AAPH-liposome-extract system.
Superoxide Radical (O·2 ) Scavenging Activity
The assay was based on the capacity of the extract to inhibit the photochemical
reduction of nitroblue tetrazolium (NBT) in the presence of riboflavin-light-NBT
system (Beauchamp and Fridovich, 1971). Superoxide dismutase was followed after
modification of a standard method (Martinez et al., 2001). Each 3 mL
reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 mM methionine,
2 μM riboflavin, 100 μM EDTA, NBT (75 μM) and 0.1 mL of the sample
solution. The production of blue formazan was followed by monitoring the increase
in absorbance at 560 nm after 10 min illumination. The entire reaction assembly
was enclosed in a box lined with aluminium foil. Identical tubes with reaction
mixture were kept in the dark and served as blanks. The percentage inhibition
of superoxide generation was measured by comparing the absorbance value of the
control and those of the reaction mixture containing sample solution.
Hydrogen Peroxide Scavenging Activity
The ability of extracts to scavenge hydrogen peroxide was determined according
to a standard method (Ruch et al., 1989). Briefly, a solution of (40
mM) H2O2 was prepared in phosphate buffer (0.1 M, pH 7.4).
0.1 mL of the methanolic extracts of each vegetable was dissolved in 4 mL phosphate
buffer and mixed with 600 μl of 40 mM hydrogen peroxide. Hydrogen peroxide
concentration was determined spectrophotometrically at 230 nm in the presence
and absence of the extract. For each extract, a separate blank sample solution
containing the extract in buffer solution without hydrogen peroxide was used.
Percentage hydrogen peroxide scavenging activity was calculated as A0-A1/A0
where A0 is the absorbance of the control (Abs230.H2O2)
and A1 is the absorbance in the presence of sample (Abs230.
H2O2+ sample).
Total Flavonoid
Total flavonoid content of the extracts was determined according to a colorimetric
method with some modifications (Bao et al., 2005). One milliliter of
methanolic extract were transferred into an Eppendorf tube containing 1 mL of
distilled water and mixed with 75 μL of 5% NaNO2. After 5 min,
75 μL of 10% AlCl3 solution was added. The mixture was allowed
to stand for another 5 min and then 0.5 mL of 1 M NaOH was added. The reaction
solution was mixed and kept for 15 min. The increase in absorbance was measured
at 510 nm. Total flavonoid content was calculated using a standard quercetin
calibration curve. The results were expressed as milligram per ml of quercetin
equivalent.
Total Antioxidant Activity
The assay was based on the reduction of Mo (VI)-Mo (V) by the extracts and
the subsequent formation of a green phosphate/Mo (V) complex at acidic pH (Prieto
et al., 1999). 0.1 mL of the extracts was combined with 3 mL of reagent
solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate).
The tubes were incubated at 95°C for 90 min. After which the mixture had
cooled to room temperature, the absorbance of the solution was measured at 695
nm against blank. The total antioxidant activity was expressed as gallic acid
equivalent.
Results and Discussion
There are numerous antioxidant methods with modifications for the evaluation of antioxidant capacities of plant materials. Of these, total antioxidant activity, reducing power, DPPH assay, active oxygen species such as H2O2, O·−2, OH. quenching assay and lipid peroxidation are most commonly used for the determination of antioxidant activities of plant extracts (Amarowicz et al., 2000; Mitsuda et al., 1996).
Table 1 shows the GC-MS analysis of some phenolic compounds in the selected vegetables (Telfairia occidentalis, Hibiscus esculentus, Crassocephalum,Occimum graticimum, Xanthosoma maffafa, Vernonia amygdalina, Solanum macrocarpon, Structium sparejanophora, Celocia argentia, Talinium triangulare, Corchorus olitorus, Amaranthus caudatus, Amaranthus hybridus and Manihot utilisima).
| Table 1: | GC-MS analysis of some compounds (μg g-1) in the selected tropical vegetables |
| PHA = Phenylacetic acid, VA = Vanillic Acid, GEA = Gentisic acid, PRA = Protocatechiuc acid, SA = Siringic Acid, p-CA = p-Coumaric acid, GA = Gallic acid, FA = Ferulic Acid, CA = Caffeic Acid, t-RES = trans-resveratrol, (-)-EPI = (-)-Epicatechin, (+)-CAT = (+)-Catechin, QUE = Quercetin, ND = Not Detected, Tr = Trace | |
The results revealed that caffeic acid was the main phenolic compound present in all vegetables with the exception of Celocia argentia and Talinum triangulare. Caffeic acid is a phenolic compound widely present in the plant kingdom (Duke, 1992). It has been studied extensively and known to share a spectrum of physiological activities including anti-inflammatory (Taguchi et al., 1993; Moreira et al., 2000), anti-allergic (Murota and Koshihara, 1985; Kimata et al., 2000) and anti-tumour (Li, 1999; Hudson et al., 2000; Soleas et al., 2002). Other compounds includes; phenylacetic acid (Manihot utilisim, Xanthosoma maffafa, Crassocephalum, Occimum graticimum, Amaranthus hybridus) vanillic acid (Manihot utilisima, Hibiscus esculentus, Xanthosoma maffafa, Crassocephalum crepidiodes, Occimum graticimum, Amaranthus caudatus, Amaranthus hybridus), gentisic acid (Manihot utilisima, Vernonia amygdalina, Telfairia occidentalis and Xanthosoma maffafa), protocatechuic acid (Manihot utilisima, Vernonia amygdalina, Telfairia occidentalis, Solanum macrocarpon, Structium sparejanophora, Amaranthus caudatus, Amaranthus hybridus and Occimum graticimum), syringic acid (Manihot utilisima and Crassocephalum crepidiodes), p-coumaric acid (Manihot utilisima, Vernonia amygdalina, Telfairia occidentalis, Structium sparejanophora, Xanthosoma maffafa, Crassocephalum crepidiodes, Amaranthus caudatus, Amaranthus hybridus and Occimum graticimum), garlic acid (Manihot utilisima, Xanthosoma maffafa, Amaranthus hybridus and Occimum graticimum), ferulic acid (Manihot utilisima, Solanum macrocarpon, Xanthosoma maffafa, Crassocephalum crepidiodes, Amaranthus caudatus, Amaranthus hybridus and Occimum graticimum) epicatechin (Manihot utilisima, Amaranthus caudatus and Amaranthus hybridus) and catechin (Manihot utilisima, Amaranthus caudatus and Amaranthus hybridus) most of which shared some physiological properties.
The peroxidation inhibitory capacities of the selected vegetables were shown on Table 2 by measuring the lipid peroxide content and conjugated diene formation. The results of the investigation indicates that all the evaluated vegetables were able to inhibit lipid peroxidation but at different rates. The inhibition of lipid peroxide formation ranges from 4.70-69.46 with Vernonia amygdalina having the highest inhibitory tendency while the least was recorded for Crasscocphalum crepidiodes. Lipid peroxides are likely involved in numerous pathological events, including inflammations, metabolic disorders and cellular aging (Ames, 1983; Wiseman and Halliwell, 1996). The result suggests that the consumption of the vegetables, most especially Vernonia amygdalina and Corchorous olitorus may afford a better cytoprotective effects. The inhibition of conjugated diene formation as presented on Table 2 indicates that all the evaluated vegetables possess a relatively high inhibitory activity (84.33-99.73%). This implies that most of the evaluated vegetables could protect lipids in the presence of extracts by decreasing the induction rates of oxidation. Thereby, predicting the high potency of the selected vegetables as good natural antioxidants.
| Table 2: | Lipid peroxidation inhibitory activity of the selected vegetables |
Structium sparejanophora was reported to exhibit the highest ability to inhibit conjugated diene formation while Celocia argentia has the least inhibitory activity.
Radical scavenging activities (H2O2, O·−2 scavenging activity) of the selected vegetables were presented on Table 3. The H2O2 scavenging activities (%) ranges from 4.89±0.55- 60.91±1.11, while superoxide scavenging activity (%) ranges from 75.62±1.42-95.28±1.33. The results of the investigation show that Vernonia amygdalina has the highest hydrogen peroxide scavenging activity while the least was recorded for Xanthosoma maffafa. The H2O2 scavenging activity of an extract may be attributed to the structural features of their active components, which determines their electron donating ability (Wettasingbe and Shahidi, 2000).
Results from the investigation revealed that all the evaluated vegetables possess a relatively high superoxide scavenging activity (75.62±1.42-97.01±1.32) with Manihot utilisima having as high as 97.01±1.32 scavenging ability while the least, though, a relatively high value was recorded for Talinium triangulare. Superoxide radicals (O·−2) also known as superoxide anion is produced in the body during aerobic respiration, enzymatic reactions and drug metabolism (Fridovich, 1972; Halliwell and Gutteridge, 1989). Although a superoxide radical itself is not so reactive to biomolecules, it helps in the generation of more powerful hydroxyl radicals through the Haber-Weiss reaction (Halliwell and Gutteridge, 1989 ). The toxicity of superoxide radicals and its role in deleterious processes in biology are well established. Hence the ability of plant food stuff to inhibit the formation of such toxic species is a good index for measuring antioxidant activities.
Total antioxidant activity is a measure of the capacity of substances extracted from the food matrix to delay oxidation process in a controlled system, (Cao et al., 1996; Fogliano et al., 1999; Miller and Rice-Evans, 1997; Pellegrini et al., 2000). Rather than determining the concentration of each antioxidant molecule individually, evaluation of total antioxidant activity, using different model assay systems has become increasingly important.
Total antioxidant capacity of the tropical vegetables is expressed as the number of equivalents of gallic acid (Table 4). The assay is based on the reduction of Mo (VI)-Mo (V) by the extract and subsequent formation of green phosphate/Mo (V) complex at acid pH. The result of the study shows that total antioxidant activity was in the following order: Solanum macrocarpon>Telfairia occidentalis>Xanthosoma maffafa>Manihot utilisima>Corchorus olitorus> Occimum graticimum>Amaranthus hyhbridus>Amaranthus caudatus>Structium sparejanophora> Crassocephalum>Talinium triangulare>Hibiscus esculentus>Celocia argentia> Vernonia amygdalina. Table 4 equally shows the total flavonoid content (mg mL-1 quercetin equivalent) of the selected vegetables. Flavonoids are one of the most powerful antioxidants found in plants. Typically, they possess one or more of the elements that are considered important to the antioxidant potential of plant materials.
| Table 3: | Radical scavenging activities of the selected vegetables |
| Table 4: | Total flavonoid and total antioxidant activity of the selected vegetables |
Solanum macrocarpon have the highest flavonoid content (3.08) while the least content was recorded for Amaranthus hybridus. The result is in agreement with the result of an investigation (Takeoka et al., 2001), antioxidant activity in methanolic fraction is due to the presence of phenolic compounds, such as cafeic acid and chlorogenic acid as this could be the reason why solanum macrocarpon with the highest total flavonoid content is equally having the highest total antioxidant activity.
Based on the data obtained from this study, the selected vegetables exhibit free radical and lipid peroxidation inhibitory or scavenging activity which may limit free radical damage occurring in the human body. However, this reveals that consumption of the selected vegetables may supply substantial antioxidant by synergy and may provide health promoting and disease preventing effect. This study could be considered as a new report and could be a starting point for further investigations.
Acknowledgments
The financial support of ICTP/IAEA received through the Ph.D Sandwich Training Educational Program (STEP) fellowship of ICTP is greatly acknowledged and appreciated. I also wish to acknowledge the useful contribution and suggestions of Dr. Luca Quaroni, Synchrotrone, Elettra, Italy and also the Management of Synchrotrone, Elettra, Trieste, Italy for allowing me to use their facilities for the present study.