Evaluation of Free Radical Scavenging Activity and Antioxidant Potential of a Few Popular Green Leafy Vegetables of Malaysia

Abstract

In this study, phenolic contents and antioxidant activity of five popular green leafy vegetables of Malaysia—Kangkung, Kucai, Di Huang Miao, Ubi Keledek, and Yaw Mak Choy—were evaluated. Methanol, acetone, and distilled water were the solvent systems used for extraction. The antioxidant capacity of the extracts was evaluated using ferric reducing antioxidant potential assay and the free radical scavenging capacity was evaluated using the DPPH· radical-scavenging assay. The total flavonoids, flavonols, and tannins were also quantified. The efficacy of the three solvents used for extraction differed significantly for both the levels of antioxidants and free radical scavenging activity. In general, methanol was the best of the three solvents for extraction of antioxidants. The results of this study will be useful for health conscious consumers, and they will provide a basis for future exploration of these green leafy vegetables as potential sources of natural antioxidants for food and nutraceutical applications.

Keywords:

• Antioxidants
• Leafy vegetables
• Solvent extraction
• Phenolic compounds
• Flavonoids
• Flavonols

INTRODUCTION

The increasing gap between population growth and food supply, especially in developing and underdeveloped regions of the world, has created renewed interest in finding reliable and cheap sources of plant resources with potential nutraceutical and health promoting properties.[1] Providing detailed scientific evidence about the therapeutic value of plant-based food resources that are used traditionally can serve as incentive for their greater consumption among local populations. Green leafy vegetables (GLVs) constitute a vital part of a balanced diet and are rich in bioactive compounds (polyphenols, carotenoids, flavonoids, flavones, isoflavones, catechins, vitamins (vitamins A and C), and minerals such as iron and selenium) and their consumption can reduce the risk of developing several degenerative diseases, such as cancer and cardiovascular diseases.[2, 3] Malaysia, being a tropical country, has an abundance of plant species that grow naturally, and many of them are part of the traditional Malaysian diet.

The five GLVs analyzed in this study have varied growth habitats and are eaten either cooked or raw as a part of a salad by inhabitants of Malaysia. Kang Kung and Di Huang Miao are also consumed in neighboring countries, such as Thailand and Indonesia. Kangkung is a semi-aquatic plant with arrowhead-shaped leaves. This vegetable is widely popular among the local Malays and Chinese communities. Usually the leaves are eaten fresh or are lightly cooked (like spinach) or are stir fried. In some households, the leaves are cooked along with cuttlefish, shrimp, or with mussels. Kucai is a garlic-flavored green leafy vegetable that generally is used for salads and seasoning. It is either stir fried or boiled with other ingredients to make a soup. The leaves are also used for preparing omelets and fish dishes. Traditionally, the leaves are used to treat urinary problems and impotence and for recovering from fatigue. Di Huang Miao is a tender, fast-growing, climbing vine with thick heart-shaped leaves with a red to purplish stem. The leaves have succulent mucilage and are used for preparing salads (usually mixed with yogurt and salt), steamed with tofu and ginger, or used as a thickener in soups. Ubi Keledek leaves are long, finger-like and star shaped, and they usually are boiled until fully cooked before being consumed. Sometimes they are used to thicken gravy. These leaves are highly popular among inhabitants of Malaysia and Indonesia. Yaw Mak Choy has long and slender leaves with a unique aromatic flavor. It is one of the most popular GLVs in Malaysia, and it is generally stir fried like spinach. The leaves also are eaten raw, used to prepare soup, or mixed with meat or seafood.

The main objectives of this study were to: (i) determine the antioxidant activities of several GLVs locally consumed in Malaysia by measuring percent DPPH· inhibition, total antioxidant capacity, ferric reducing antioxidant potential (FRAP), and concentrations of flavonoid, flavonol, total phenolic compounds, tannins, and vitamin C; and (ii) to evaluate the effectiveness of different solvents (methanol, acetone, distilled water) on the extractability of various antioxidants. The results of this study will provide information about the availability of health promoting factors (in the form of antioxidants) in some popular and commonly consumed GLVs in the normal diet in Malaysia and other neighboring countries.

MATERIALS AND METHODS

Plant Material

Five popular and commonly used GLVs of Malaysia were analyzed included: Kangkung (Ipomoea aquatica Forsk; swamp cabbage or water spinach); Kucai (Allium tuberosum Rottler ex Sprengel; Chinese leek, Chinese chive); Di Huang Miao (Basella alba L.; Indian spinach, Malabar spinach, emperor vegetable); Ubi Keledek (Ipomoea batatas L. Lam.; sweet potato leaves); and Yaw Mak Choy (Lactuca sativa var longifolia; Romaine lettuce). These GLVs were purchased from the local supermarket (TESCO Extra, Penang, Malaysia).

After transporting the samples to the laboratory, the edible leafy portion of each plant was separated and washed under running water followed by three washes with distilled water. The samples were drained completely and dried on filter paper. Any leaf samples with apparent physical or insect damage were discarded. The leaves were oven dried at 40°C (up to 24 h) and the dried leaves were ground into a fine powder using a kitchen mixer to a mesh size of 30 mm. The powdered samples were used for analysis.

Preparation of Extract

Powdered samples of leaves were extracted using three different solvent systems (methanol, acetone, and distilled water) at a ratio of 1:10 (w/v). Exhaustive extraction was conducted for ∼24 h in a shaker at 37°C with gentle shaking. Further, it was filtered (Whatman no. 1) and was used for analyses. All spectrophotometric measurements were made using a UV-visible spectrophotometer (Shimadzu UV-160A, Kyoto, Japan).

Antioxidant Compounds

Determination of total phenolics, tannin, flavonoid, and flavonol contents

The total phenolic contents of the GLV extracts obtained using the three different extraction solvents was determined using the Folin–Ciocalteu method,[4] while to determine the tannin content, the vanillin-HCl method was used with some modifications.[5] The colorimetric method described by Sakanaka et al.[6] was used to determine the total flavonoid content. The total flavonol content of the extracts was estimated using the Miliauskas et al.[7] method.

Vitamin C

The vitamin C content in the extracts was determined using the 2,6-dichloroindophenol titrimetric method of AOAC.[8]

Determination of Antioxidant Activity

Free radical scavenging ability by DPPH· assay

Determination of the DPPH· (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the extracts was performed spectrophotometrically based on the method described by Blois.[9] The DPPH· radical scavenging activity was calculated using the following formula:

where A₀ is the absorbance of the control and A ₁ is the absorbance of the extracts. Data are presented as percent inhibition.

Phosphomolybdenum and Ferric Reducing Antioxidant Potential (FRAP) Assays

The total antioxidant capacity of the extracts was determined using the phosphomolybdenum method described by Prieto et al.[10] The ability of extracts to reduce ferric ions was evaluated using the modified method described by Benzie and Strain.[11]

Statistical Analysis

All experiments were conducted in triplicate and results are given as mean ± standard deviation (SD). All data analyses were performed using SPSS for Windows Version 12.0 (SPSS Inc., Chicago, IL, USA). Differences between means were assessed using a one-way analysis of variance (ANOVA) with a post-hoc determination using Tukey's test. The level of significance was set at P < 0.05. All the results are expressed on dry weight basis.

RESULTS AND DISCUSSION

Total Phenolics and Tannin Contents

Polyphenols present in plants consist of a wide variety of compounds that include flavonoids (anthocyanins, flavanols, flavones, flavonols, and others) and pro-anthocyanidins.[12] These phenolic compounds are aromatic secondary metabolites that are associated with imparting color and sensory and nutritional qualities along with health benefits. Generally, the polyphenolic compounds present in plants are antioxidants that act as reducing agents, hydrogen donors, heavy metal chelators, hydroxyl radical quenchers, and lipid peroxidation stabilizers.[13] Consumption of plant products rich in polyphenols is correlated with reduced risk of heart and cardiovascular diseases.[14, 15] Folin–Ciocalteu (FC) method measures the reduction of FC reagent by phenolic compounds (present in the samples) by the formation of a blue complex, which can be measured at 760 nm with a suitable standard, such as gallic acid. In the present study, significant differences were observed in the amount of total phenolics found in the extracts obtained using different solvents (Table 1). Among the various solvents used, methanol showed higher levels of phenolics in Ubi Keledek followed by Kang Kung, Di Huang Miao, and Yaw Mak Choy (679.77 > 65.67 > 50.38 > 34.07 mg GAE/g extract, respectively). However, irrespective of the extraction solvents used, overall, Ubi Keledek showed the highest phenolics content (34.68–680.01 mg GAE/g extract) compared to other GLVs (Table 1).

Table 1 Total phenolics, tannins, flavonoid, flavonol, and vitamin C in green leafy vegetables extracted in different solvents (on dry weight basis)

Sample	Solvent	Total phenolics (mg GAE/g extract)^A	Tannins (mg CAE/g extract)^B	Flavonoids (mg CAE/g extract)^B	Flavonols (mg QE/g extract)^C	Vitamin C (mg AA/100 g extract)^D
Kang Kung	Methanol	65.67 ± 1.1^f	0.01 ± 0.0^a	64.68 ± 0.2^e	3.41 ± 0.6^a	317.83 ± 1.3^a
	Acetone	32.20 ± 0.8^c	0.02 ± 0.0^a	31.62 ± 0.2^c	4.23 ± 0.1^b	392.37 ± 0.1^c
	Water	41.99 ± 0.1^d	0.12 ± 0.0^b	41.55 ± 1.0^d	1.16 ± 0.1^a	669.59 ± 0.1^e
Kucai	Methanol	23.11 ± 0.2^b	0.50 ± 0.0^c	30.72 ± 1.5^c	6.10 ± 0.2^b	601.34 ± 0.8^e
	Acetone	26.46 ± 0.2^b	0.44 ± 0.0^c	55.94 ± 1.4^d	8.52 ± 0.4^c	390.95 ± 2.5^c
	Water	29.72 ± 0.1^b	0.07 ± 0.0^a	10.18 ± 0.1^a	2.01 ± 0.1^a	468.72 ± 0.0^d
Di Huang Miao	Methanol	50.38 ± 0.2^e	0.40 ± 0.0^c	25.03 ± 0.8^c	5.8 ± 0.11^b	707.8 ± 0.3^f
	Acetone	36.55 ± 0.3^c	0.43 ± 0.0^c	28.22 ± 1.0^c	5.93 ± 0.2^b	346.6 ± 3.4^b
	Water	40.02 ± 0.3^d	0.03 ± 0.0^a	16.36 ± 0.3^b	2.92 ± 0.8^a	468.7 ± 0.0^d
Ubi Keledek	Methanol	679.77 ± 0.2^g	0.08 ± 0.0^a	164.5 ± 0.5^g	8.36 ± 0.1^c	708.0 ± 0.0^f
	Acetone	680.01 ± 0.6^g	0.44 ± 0.0^c	128.2 ± 1.0^f	8.29 ± 0.2^c	348.1 ± 1.3^b
	Water	34.68 ± 0.2^c	0.02 ± 0.0^a	231.8 ± 1. 4^h	5.92 ± 0.1^b	736.3 ± 0.4^f
Yaw Mak Choy	Methanol	34.07 ± 0.2^c	0.21 ± 0.0^b	47.41 ± 2.2^d	19.53 ± 0.1^d	830.8 ± 1.0^g
	Acetone	29.99 ± 0.1^b	0.51 ± 0.0^c	135.5 ± 3.0^f	8.98 ± 0.2^c	348.8 ± 0.0^b
	Water	17.37 ± 0.1^a	0.01 ± 0.0^a	24.8 ± 0.2^c	8.26 ± 0.1^c	602.1 ± 1.0^e
Results are presented as the mean of triplicate measurements (n = 3 ± SD). Values followed by the same letter (a–h) within the same column are not significantly different from each other (P > 0.05).
^AGAE: gallic acid equivalents; ^BCAE: catechin equivalents; ^CQE: quercetin equivalents; ^DAA: ascorbic acid.

Tannins are high molecular weight phenolic compounds known to quench free radicals in a biological system. According to Hagerman et al.,[16] the bioactivity of tannins depends on their molecular weight, the number of aromatic rings present, and the nature of hydroxyl group substitution, rather than on the specific functional groups. In the authors' study, the methanolic extract of Kucai yielded the highest tannin content (0.50 mg CAE/g extract) among the five GLVs studied. Although tannins were present in low amounts in the GLVs analyzed (Table 1), their presence even at threshold levels might still be beneficial, as they are known free radical scavengers and may be one of the potent nutraceuticals that can prevent free radical-mediated diseases, especially those of the gastrointestinal tract.[17]

Total Flavonoids and Flavonols Contents

Flavonoids and flavonols possess a wide spectrum of chemical and biological activities, including free radical scavenging properties. Previously, epidemiological studies have reported a positive correlation between increased consumption of flavonoids and flavonols and reduced risk of cardiovascular diseases and cancer.[18] Flavonoids exist widely in the plant kingdom and are common in leaves, flowering tissues, and pollens.[19] Among the five GLVs analyzed in the present study, the total flavonoids content in the extracts ranged from 10.18 (in aqueous extract of Kucai) up to 231.8 mg CAE/g extract (in aqueous extract of Ubi Keledek) (Table 1). High flavonoids content was recorded in Ubi Keledek extracts, which ranged from 128.16 to 231.76 mg CAE/g extract, whereas extracts of Yaw Mak Choy had flavonols content ranging from 8.26 to 19.53 mg QE/g extract (Table 1). The presence of flavonols in adequate amounts in leaves is beneficial, as flavonols are potent antioxidants with health-promoting activities.[20] These results re-confirm the fact that flavonoids and flavonols to be the main representative group of phenolic compounds in plants and their products.[21]

Vitamin C Content

Generally, fresh green leaves are rich sources of ascorbic acid. In the present study, almost all of the GLVs contained high amounts of vitamin C (317.83–830.80 mg AA/100 g extract). The highest content was found in the methanolic extract of Yaw Mak Choy (830.80 mg AA/100 g) (Table 1). Vitamin C, a potential antioxidant with known health benefits, is a sensitive bioactive compound that can be easily degraded in the presence of oxygen by oxidative processes stimulated by light, oxygen, and enzymes, such as ascorbate oxidase and peroxidase.[22] In addition, the vitamin C content of GLVs might vary significantly based on the age of the leaves and the irradiance that reaches the leaf surface.[23]

Free Radical Scavenging Activity

The free radical scavenging activity of the antioxidants present in plants or their extracts are routinely evaluated (at room temperatures) using synthetic radicals like that of DPPH· in the presence of polar organic solvents. DPPH· is a stable free radical, which is capable of accepting an electron or a hydrogen radical to become a stable diamagnetic molecule. Generally, the scavenging of DPPH· radicals is used to evaluate chain-breaking activity in the propagation phase of lipid (and protein) oxidation. The mechanism of action involves the reaction of specific compounds or plant extracts with DPPH· in methanol solution. The disappearance of DPPH· radical absorption at a characteristic wavelength (517 nm) is monitored by measuring the decrease in optical density.[24] In the present study, the percent inhibition of the DPPH· radical in the GLVs varied significantly among the extracts and ranged between 4.5 and 84.8% (in Kucai and Ubi Keledek, respectively) (Table 2). The significant differences observed indicate that irrespective of the solvent used, GLV extracts are capable of scavenging free radicals via electron- or hydrogen-donating mechanisms. Hence, they are capable of preventing the initiation of deleterious free radical-mediated chain reactions, indicating their potentiality to be used for therapeutic purposes.

Table 2 DPPH· radical scavenging assay, antioxidant capacity, and FRAP values for green leafy vegetables extracted in different solvents (on dry weight basis)

Sample	Solvent	DPPH assay (% inhibition)	FRAP value (μmol Fe(II)/g)	Antioxidant capacity (mg ascorbic acid equivalent/g extract)
Kang Kung	Methanol	26.1 ± 0.1^d	50.02 ± 0.9^c	12.45 ± 0.3^a
	Acetone	9.0 ± 0.5^b	20.65 ± 1.4^a	8.85 ± 0.1^a
	Water	20.3 ± 1.4^d	72.57 ± 1.0^e	9.65 ± 0.2^a
Kucai	Methanol	4.5 ± 0.1^a	27.67 ± 0.5^b	22.48 ± 0.1^c
	Acetone	12.0 ± 0.3^c	35.01 ±0.2^b	25.47 ± 0.1^c
	Water	5.7 ± 0.6^a	26.98 ± 0.1^b	13.59 ± 0.6^b
Di Huang Miao	Methanol	5.6 ± 0.1^a	18.63 ± 1.0^a	15.28 ± 1.2^b
	Acetone	6.6 ± 0.2^a	18.31 ± 0.5^a	21.20 ± 1.0^c
	Water	9.4 ± 0.4^b	54.69 ± 0.1^d	18.96 ± 0.3^b
Ubi Keledek	Methanol	84.8 ± 2.2^f	173.69 ± 1.1^f	18.72 ± 0.5^b
	Acetone	63.1 ± 0.2^e	180.08 ± 1.7^f	24.95 ± 1.5^c
	Water	60.1 ± 0.2^e	26.56 ± 0.44^b	25.25 ± 1.1^c
Yaw Mak Choy	Methanol	12.2 ± 0.3^c	40.23 ± 0.2^c	15.99 ± 0.2^b
	Acetone	9.5 ± 0.6^b	40.12 ± 0.4^c	24.88 ± 0.6^c
	Water	14.1 ± 1.0^c	47.96 ± 0.2^c	8.63 ± 0.4^a
Results are presented as the mean of triplicate measurements (n = 3 ± SD). Values followed by the same letter (a–f) within the same column are not significantly different from each other (P > 0.05).

Phosphomolybdenum Assay and FRAP

The determination of antioxidant capacity in sample extracts by the phosphomolybdenum method is quantitative, as the total activity is expressed as the number of equivalents of ascorbic acid. This assay is based on the reduction of the molybdenum complex [Mo(VI) to Mo(V)] by the sample analyte and the subsequent formation of the green phosphate/Mo(V) complex at acidic pH.[10] The methanol and acetone extracts of Kucai had the highest antioxidant capacities (22.48 and 25.47 mg ascorbic acid equivalents/g of extract, respectively) among the GLVs analyzed, followed by Ubi Keledek extracts (18.72 and 25.25 mg ascorbic acid equivalents/g of extract, respectively) (Table 2). Some researchers suggest that the antioxidant capacity of plant extracts depends directly on the amount of polyphenols present in fresh produce.[25] Hence, fresh plant products with high phenol content naturally can exhibit high antioxidant capacities, which was true in the present investigations also. However, antioxidant capacity is also directly based on the electron transfer reactions. Additionally, it should be noted that antioxidant compounds (or bioactive compounds) present in a plant species are also capable of reacting differently in various antioxidant assays, the results of which are directly related to the extraction solvents used.

The reducing ability of antioxidants present in the GLV extracts was measured using the FRAP assay. With this method, antioxidants present in the sample extract readily react with the ferric tripyridyl triazine complex (Fe³⁺-TPTZ) to form the colored ferrous tripyridyl triazine complex (Fe²⁺-TPTZ), which corresponds to the antioxidants or reductants present in a sample.[11] The results indicated significant differences among the extracts evaluated. Methanol and acetone extracts of Ubi Keledek exhibited the highest FRAP values (173.69 and 180.08 μmol Fe (II)/g, respectively) followed by the methanol and aqueous extracts of Kang Kung (50.02 and 72.57 μmol Fe (II)/g, respectively) (Table 2).

Effects of Extraction Solvents

Extraction of bioactive compounds from a plant material using various solvent systems is a vital step in accurately quantifying their levels. Solvents, such as methanol, ethanol, acetone, distilled water, chloroform, ethyl acetate, and petroleum ether, are routinely used for extraction of polyphenols and other bioactive compounds from plant sources.[26] Previous reports describe methanol and acetone as yielding maximum extraction of phenolics and other bioactive compounds compared to other solvents,[27, 28] and therefore these two solvents were used in the present study. Because a range of solvents with different polarity have been used, it is difficult to compare among studies and to draw conclusions about the actual amount of the antioxidant compounds present in a given plant sample. Among the different factors that contribute to varied results for polyphenols and antioxidant of a single plant source, the chemical nature of the compounds, the extraction solvents used, and the assay employed are the major ones.[29] Additionally, recovery of polyphenols and other antioxidant compounds from plant materials depends greatly on the compounds solubility in a particular solvent, the polarity of the solvents, viscosity, and vapor pressure.[29] Thus, solvents, such as methanol or acetone, can easily diffuse into the pores of the plant materials to leach out the active constituents. In addition, antagonistic and synergistic interactions might occur between phenols and other compounds, such as carbohydrate or proteins (soluble in water or other solvents), that might be responsible for the distinct antioxidant activities in the same plant material.[30] Based on these facts, the variations observed in the results of phenolics and other antioxidant compounds in the GLVs analyzed in the present study can be attributed directly to the type of solvent used and its polarity.

CONCLUSIONS

All five GLVs from Malaysia studied herein possess high antioxidant potential, and it is recommended that they be incorporated into the normal diet of the general population. The authors' results should enhance interest in evaluating various GLVs to be used in the development of new nutraceutical and pharmacological products. The consumption of these GLVs may play a vital role in preventing human diseases that involve free radicals, such as aging, cancer, and cardiovascular diseases. However, isolation and characterization of individual components, their in vivo antioxidant activities, and the various antioxidant mechanisms require further study. Because great variability was found in the quantity of antioxidant compounds present in different green leafy vegetables, it would be worthwhile to study the influence of factors, such as stage of maturity and harvesting time on the content of antioxidant compounds. As GLVs are consumed raw or cooked or stir fried, further investigations need to be performed to assess the changes that occur in the antioxidants and other bioactive compounds after cooking.

ACKNOWLEDGMENTS

This work was supported by the USM research grants (Incentive grants, dt.11.09.2009, RCMO, USM, Malaysia) sanctioned to Rajeev Bhat. The authors would like to thank the anonymous referees and the editor for constructive suggestions, which were useful in upgrading the quality of this manuscript.