Antioxidant Activity and Phytochemicals of Locally Consumed Plant Foods from

In the Philippines, Baguio City – known as the “City of Pines” – holds the country’s major source of temperate climate vegetables. With increased dietary awareness, the consumption of plant foods rich in antioxidants has become relevant. Twenty-nine methanolic extracts from Baguio-produced plant foods were evaluated for antioxidant potential using DPPH, ferric reduction antioxidant power (FRAP), metal chelation, superoxide anion, nitric oxide, hydroxyl radical scavenging activities, MTT reduction, and phytochemical tests. Fagopyrum tataricum leaves, Vaccinium myrtoides fruit, and Morus alba fruit showed the most effective DPP radical, concentration-dependent reducing power, but low metal chelating activity. Solanum tuberosum tuber (22.86±63.26%) showed effective concentration-dependent chelating activity at 125 μg/ mL. Citrus aurantium fruit (26.77±9.24%) and Raphanus raphanistrum root (41.13±0.11%) demonstrated an effective scavenging activity against superoxide anions at 45.5 μg/mL. Significant nitric oxide scavenging activity was observed in some fruits. Brassica oleracea Cab leaves (54.36 ± 2.38%) showed the highest inhibitory activity against hydroxyl radicals at 166.7 μg/mL. Phytochemical analyses showed that most plant samples revealed the presence of glycosides, terpenes/terpenoids, and steroids/phytosterols, while few contained phenolic and tannin components. These phytochemicals may explain the dual behavior as an antioxidant or a prooxidant observed. Thus, determining food antioxidant component types and


INTRODUCTION
Reactive Oxygen Species (ROS) are normal oxidant by-products of aerobic metabolism 1 . These reactive intermediates, which include superoxide anion (O2•), the hydroxyl radical (OH•), and the singlet oxygen, are derived from the incomplete oneelectron reduction of molecular oxygen 2 . Prolonged exposure to high ROS concentrations may lead to non-specific damage to proteins, lipids, and nucleic acids, often inducing irreversible functional alterations or complete destruction 3 . Antioxidants have gained importance for years due to their ability to neutralize the pathological effects of increased oxidative concentrations 4,5 . Though our bodies are equipped with innate antioxidant machinery, such as antioxidant enzymes and vitamins, these may not be enough to protect against the detrimental effects of excess free radicals. Consumption of foods rich in phytochemicals with strong antioxidant properties may help prevent and repair cellular damage to our bodies 5 . Natural antioxidants, particularly in fruits and vegetables, have gained increasing interest among consumers and the scientific community. Fruit and vegetable juices are rich sources of numerous phytochemicals, polyphenols, carotenoids, fiber, vitamins, and minerals 6,7 . Complex mixtures of these phytochemicals present in fruits and vegetables provide additive and synergistic effects on health promotion 8 . Thus, dietary intake of this food as a source of antioxidants is recommended.
In the Philippines, Baguio City -known as the "City of Pines" -supplies the Philippines' daily requirements for highland vegetables 9 . Due to the temperate climate and wide land area, Baguio provided a conducive environment to cultivate quality fruits, vegetables, and other agricultural products. The Philippine Department of Agriculture stated that Baguio and neighboring provinces in Cordillera supply 80% of the highland vegetable requirement in major markets of Metro Manila and other lowland provinces 10 . Knowing the possible beneficial effects of plant foods cultivated from the country's primary agricultural source of prime vegetables, fruits, and crops is essential. Currently, limited studies are conducted on Baguio's commonly consumed plant foods. Thus, the study aims to determine the phytochemical constituents of some Baguioderived vegetables and evaluate their antioxidant activities in vitro.

Materials
Twenty-six plant samples were collected from local markets of Baguio (shown in Table I). Edible plant parts commonly consumed by locals were utilized in the study. All samples gathered were authenticated by the Institute of Biology Herbarium -University of the Philippines, Diliman. 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitro blue tetrazolium (NBT), phenazine methosulfate (PMS), sodium nitroprusside (SNP), sulfanilamide, naphthyl ethylenediamine dihydrochloride (NED), potassium ferricyanide [K3Fe(CN)6], quercetin, butylated hydroxytoluene (BHT), ethylene diaminetetraacetic acid (EDTA), ascorbic acid, and reduced nicotinamide adenine dinucleotide (NADH) were purchased from Sigma-Aldrich Co. St. Louis, Germany. All other chemicals used were of analytical grade. Metal chelating activity: In a 96-well microplate, different concentrations of the 20 µL methanol extracts were loaded, and 100 µL of 0.2 mmol/L FeCl2 was added to each well. Afterward, 40 µL of 5 mmol/L ferrozine was added. The reaction mixture was incubated at room temperature for 10 minutes. The absorbance was measured at 562 nm 15 .
Superoxide scavenging activity: As much as 10 µL of standard and test compounds at different concentrations were loaded into a 96-well microplate. Then, 100 µL 468 µM NADH, 100 µL 156 µM NBT, and 50 µL 60 µM PMS were added into each well. Five-minute incubation was done at room temperature. The absorbance was measured at 560 nm 16 .
Nitric oxide scavenging assay: Sodium nitroprusside (10 mM, 2 mL) in phosphate buffer saline was incubated with test compounds in different concentrations at room temperature for 150 minutes. After 30 minutes, 0.5 mL of the incubated solution was added with 1 mL of Griess reagent (0.33% sulfanilamide in 20% glacial acetic acid, 0.5 mL, and 0.1% NED, 1 mL) and was incubated for 30 minutes at room temperature. The absorbance was measured at 546 nm 14 .
MTT assay: Stock solutions of test compounds and extracts were prepared in DMSO (250-1000 μg/mL). The MTT (1 mg/mL) was dissolved in water. An aliquot of 190 µL of MTT solution in water and 10 µL of test compounds or extracts in DMSO were vortexed in a capped glass vial (2 mL) for 1 minute. As much as 200 µL DMSO was added, and the solution was vortexed again. The reaction mixture was then incubated at 37°C for six hours, 200 µL of the reaction mixture was pipetted to a 96-well cell culture plate, and the absorbance was measured at 570 nm 17 . Test samples were kept at 37°C for an hour. The free radical damage imposed on the substrate, deoxyribose, was measured using the thiobarbituric acid test. As much as 1 mL of 1% thiobarbituric acid (TBA) and 1 mL of 2.8% trichloroacetic acid (TCA) was added to the test samples and incubated at 100°C for 20 minutes. After cooling, the absorbance was measured at 532 nm against a blank containing deoxyribose and buffer 18 .

Data analysis
Results are expressed as mean ± SD. The statistical analysis was performed using one-way ANOVA. The differences were considered statistically significant at p <0.05.

DPPH scavenging activity
One mechanism by which antioxidants inhibit oxidation is by quenching reactive species through hydrogen or electron donation 19 . The DPPH assay is one of the most popular and frequently employed methods among antioxidant assays. DPPH is a stable free radical with a deep purple color and a strong absorption around 517 nm 20 Figure 1.

FRAP assay
The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity. In this assay, antioxidant capacity measures the reduction of the ferric ion (Fe 3+ ) to the ferrous ion (Fe 2+ ) by donor electrons in the sample 29 . The activity is monitored by measuring the formation of Perls' Prussian blue complex at 700 nm 30

Metal chelating activity
Metals, such as Fe and Cu, and ultra-trace elements, Co and Ni, act as cofactors that assist enzymes in catalyzing biochemical reactions efficiently 36 . Transition metals become toxic at elevated tissue concentrations. Excess transition metals can initiate hydroxyl radical production through the Fenton reaction (H2O2 + Fe 2+ → Fe 3+ + OH -+ OH•) 37 . This process hastens the rate of lipid peroxidation by the continuous generation of peroxy radicals (LOO•) to form lipid hydroperoxides (LOOH) -the main primary products of lipid peroxidation 38 . Chelation therapy is the preferred medical treatment for reducing the toxic effects of metals 39 . The use of metal chelators can improve the symptoms of metal overload. Synthetic chelators have posed severe side effects; hence, using natural plant foods with chelating ability has been getting more attention 40 .
In metal chelation, a decrease in the absorbance of the red-violet Fe 2+ /ferrozine complex is monitored 41  The overall results are presented in Figure 3.

Superoxide scavenging activity
Superoxide (O2• -) is a free radical with a short biological lifespan produced by the one-electron reduction of molecular oxygen 44 . Superoxide anion initiates free radical formation of other reactive oxygen species in living systems 45 . It can also react with nitric oxide and form peroxynitrite 46 . Thus, knowing the right food which will provide good superoxide scavenging properties may help lower the risk of many degenerative diseases. The overall results are presented in Figure 4. In this assay, a decrease in absorbance at 560 nm indicates the scavenging of superoxide radicals 47  With these reported results demonstrated the dual action, as an antioxidant and as a prooxidant, of some plant foods studied. Therefore, natural antioxidants have been shown to act as prooxidants under certain conditions 5 .

Nitric oxide scavenging activity
Nitric oxide (NO) is an important cell signaling molecule. At lower concentrations, NO aids in angiogenesis 49 . Nitric oxide plays vital roles in humans, which include dilating blood vessels, raising blood supply and lowering blood pressure, regulating platelet aggregation, signaling molecules between neurons, and killing bacteria 50,51 . However, NO also possesses a dual role. As a free radical, nitric oxide readily reacts with other radical species and the metal centers of metalloproteins 52 . Moreover, when nitric oxide is near superoxide, peroxynitrite (ONOO -) may spontaneously form 44 . In this assay, the Griess reagent can estimate nitric oxide formed 14 . Nitric oxide scavengers compete with oxygen, producing reduced nitric oxide.

Hydroxyl radical scavenging assay
Hydroxyl radicals are highly reactive and short-lived molecules. These molecules can react with lipids, polypeptides, proteins, and DNA, especially thiamine and guanosine bases 53 Figure 7.

Phytochemical analysis
All  Table II. Many phytochemicals support the body's innate antioxidant machinery. Phytochemicals: polyphenols, flavonoids, anthocyanins, and carotenoids are the major contributors to their antioxidant properties 56 . Polyphenols, which include flavonoids, stilbenes, lignans, and phenolic acids, are chemical substances characterized by aromatic rings with one or more hydroxyl groups. These compounds react with free radicals, resulting in the delocalization of the gained electron and stabilization of the aromatic nucleus through resonance. This, in turn, stops the free radical chain reaction 57 . Carotenoids, which exhibit a characteristic, symmetrical tetraterpene skeleton found in colored pigments of plant foods, are effective "radical-trapping antioxidants" and one of the most efficient singlet oxygen quenchers 58 . Other phytochemicals such as terpenes and terpenoids, alkaloids, and saponin compounds were also reported for antioxidant activities 59-61 . However, several studies reported that phenolic antioxidants can also act as prooxidants under certain conditions, like high concentrations of transition metal ions, alkali pH, and the presence of oxygen molecules 62-64 . Large molecular weight phenolics, such as hydrolysable and condensed tannins, have little or no prooxidant properties compared to simple phenol. Polyphenols with low oxidation potentials (Epa) exhibit antioxidant activity, while those with high Epa values act as prooxidants 5 . This characteristic could describe a dual action of phenolic compounds, where high-Epa polyphenols exist in some extracts that simultaneously exhibit antioxidant and prooxidant activities.
In summary, the study revealed that phytochemicals from these plant foods exhibited various degrees and types of antioxidant components and activities. For example, some plant foods, F. tataricum, V. myrtoides, and M. alba, possess higher or equal antioxidant activity than known standards. The various composition of phytochemicals in plant foods exerts different "strength" against oxidants. Reports have shown that exogenous antioxidants may show prooxidant activities, especially when administered at high doses. Despite these drawbacks, food-based secondary metabolites hold promising avenues for health benefits. Further work may be done to elucidate these results in vivo and identify compounds responsible for these activities. Moreover, continuous studies on phytochemical mechanisms and interactions may be done to establish the most significant impact of antioxidant systems on alleviating chronic diseases.

CONCLUSION
In conclusion, the study revealed that Baguio plant foods contained various phytochemicals and showed promising antioxidant capacities. The findings of this study may provide information that consuming Baguio-cultivated plant foods are beneficial and may be applied in the management of various free radical-linked diseases.