The content of total polyphenols and antioxidant activity were evaluated in different types of leafy vegetables consumed by specific community of Bangladesh. We presumed that there exist a significant difference with respect to total polyphenol content and antioxidant activity, measured by DPPH method, in different indigenous leafy vegetable species.

Folin-Ciocaltu reagent and gallic acid were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). The analytical grade acetone, petroleum ether, and methanol were purchased from Merck (Darmstadt, Germany).

Muli-region sampling plan was employed for the vegetable sampling. In order to conform the representative sample principle – “what the mass people consume’ and from where they collect it”? (Greenfield and Southgate, 2003), the vegetables were collected from the retail markets located at wholesale markets where the vegetables are arrived from different geographical regions of the country and also from cultivation fields. It was, thus, ensured the representative sample.

A taxonomist who also accompanied the collection team, confirmed the sample identity. The identified samples undertaken in this study were Cajanus cajan, Stereospermum suaveolens, Calamus tenuis, Commelina benghalensis, Enhydra fluctuans, Albizia procera, Tamarindus indica, Hydrocotyle sibthorpiodes, Leucas aspera, and Bauhinia acuminate. Photographs of the studied vegetables are also of given in the Figure 1.

The samples were collected fresh, packed into autoseal poly bags with little water spray and brought to the laboratory for processing and analysis. Two to three samples (250 – 500 g) were collected for each of the items from every market and growing fields, which were then mixed to make three analytes or composite test samples. Samples were first gently washed with tap water to remove sand and other extraneous material before being washed with distilled water. Surface water was removed with tissue paper, air dried, cut into small pieces. The sample was freeze-dried at -48 °C (il Shin lab.Co. Ltd., Korea) and stored in refrigerator for phenolics analysis.

Approximately two grams of freeze-dried powdered sample was taken into a 250 mL conical flask and 42.5 mL methanol and 7.5 mL 1N hydrochloric acid were added. It was soaked for 24 hours at room temperature with intermittent shaking. Extracts were filtered through No. 1 Whitman filter paper and the filtrate was concentrated using a rotary evaporator at low temperature under reduced pressure. Methanol was added to make a final concentration of 1 mg.mL^-1 to be used as stock solution.

The total phenolics content was estimated by Folin-Ciocalteu colorimetric (Alam, Rana and Islam, 2016). In brief, 150 μL diluted sample extract was added to 225 μL of x2 diluted Folin-Ciocalteu reagent and was kept for 5 minutes at room temperature. Then 1.125 mL of 2% Na₂CO₃ solution was added, mixed well and kept for 15 minutes at room temperature and finally, the absorbance was measured at 750 nm by UV-VIS Spectrophotometer (UV-1800, Shimadzu, Japan). Total phenol content was calculated using a standard curve made by standard gallic acid. Results were expressed as mg gallic acid equivalent (GAE) per 100 g dry weight (DW).

The antioxidant activity of the plant extracts was evaluated by utilizing 1,1-diphenyl-2-pycrylhydrazyl (DPPH) free radical according to Piang-Siong et al. (2017) with slight modification. Briefly, DPPH stock solution was prepared by dissolving 6.5 mg of DPPH in 5 mL of 100% methanol and protected from light. 100 μL of varying concentrations (100 – 500 μg.mL^-1) of the plant sample extracts were taken and the volume was made up to 200 μL using methanol. Each of the samples was then further diluted with methanol up to 4 mL and to each 200 μL of DPPH stock solution was added. The mixture was then shaken vigorously and allowed to stand at room temperature for 30 min in the dark. After 30 min, the mixture was measured spectrophotometrically at 520 nm (UV-1800, Shimadzu, Kyoto, Japan). Absorbance of the control (200 μL DPPH in 4 mL methanol) and blank (methanol without DPPH) were also measured. The DPPH free radical inhibition capacity was calculated according to the following equation:

DPPH inhibition = ((1 -((Abs sample - Abs blank )/(Abs control - Abs blank )))×100

where, Abs_blank is the absorbance of the blank (containing only methanol), Abs_control is the absorbance of the control reaction (containing all reagents minus plant extracts), and Abs_sample is the absorbance of the plant extracts. The plant extracts concentration required for 50% inhibition of DPPH free radical (IC₅₀) was estimated from the dose-response graph plotted with percentage inhibition and concentrations of plant extract.

Descriptive statistics were performed and values were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) was employed to evaluate the differences among varieties for total polyphenol content and antioxidant activity and was declared significant when p <0.05 at 5% level of significance. SPSS (version 20.0 SPSS Inc, IL, USA) was used to analyze the data.

The TPC of selected wild vegetables was analyzed using the Folin–Ciocalteu method, and also presented in Table 1. Generally, these vegetables had high TPC. Bauhinia acuminata had the highest phenolic content (710.42 ±4.32 mg GAE.100g^-1 DW), followed by Commelina benghalensis L. (701.33 ±5.01 mg GAE.100g^-1 DW), Hydrocotyle sibthorpiodes L. (633.45 ±3.73 mg GAE.100g^-1 DW), Albizia procera (550.89 ±1.58 mg GAE.100g^-1 DW), Cajanus cajan Millsp. (491.51 ±2.71 mg GAE.100g^-1 DW). The lowest TPC was found in Leucas aspera (102.20 ±1.10 mg GAE.100g^-1 DW). The TPC in the wild leafy vegetables of the present study ranged from 102.20 to 710.42 mg GAE.100g^-1 DW. Previous study reported that the TPC of some common Indian leafy vegetables was in the range of 5 – 69.5 mg of tannic acid.g^-1 of extract (Shyamala et al., 2005). Compared to other studies, the wild vegetables under investigation had higher TPC than some frequently consumed leafy and non-leafy vegetables (Uusiku et al., 2010; Kaur and Kapoor, 2002; Gupta et al., 2005; Salvatore et al., 2005; Gupta and Prakash, 2009; Mohankumar, Uthira and Maheswari, 2018).

Secondary plant metabolites, such as aromatic phenolic compounds, are widely distributed throughout the plant kingdom and related with colour, sensory qualities and nutritional and antioxidant attributes of food. The antioxidant activity of phenolic compounds is mainly due to redox properties, which allow them to act as reducing agents, hydrogen donors, singlet oxygen quenchers, heavy metal chelators and hydroxy radical quenchers (Kaur and Kapoor, 2002). Phenolic compounds such as flavonoids, phenolic acid and tannins exerts varied biological activities such as anti-inflammatory, anti-carcinogenic and anti-atherosclerotic activities and these effects could be attributed to their antioxidant activity (Podsędek, 2007; Mertz et al., 2009).

However, it has been reported that the TPC of vegetables varies widely depending on the variety of vegetable, climatic conditions, stage of maturation, soil features, extraction methods, especially the extraction solvent, time and pH (Kamffer, Bindon and Oberholster, 2010) and thus makes it difficult for effective comparison, as different standard compounds have been used for their analysis.

Several studies have reported an inverse relation between flavonoid intake and risk of lung cancer, cardiovascular diseases (Wang et al., 2011; Bystrická et al., 2017; Lenková et al., 2017; Brindza et al., 2019) and biomarkers of inflammation (Shaik et al., 2006). Hence, the consumption of such wild vegetables, rich in phenolic compound, could ameliorate or prevent the generation of chronic diseases.

The DPPH free radical assay is an easy, reliable and quick way to evaluate antioxidant potential of various extracts (Piang-Siong et al., 2017). This method is simple and do not call for a special reaction and device. In this method, the antioxidant activity is assessed on the basis of the ability of the antioxidant to donate hydrogen or electron to DPPH· to produce a stable DPPH-H (diphenylhydrazine) molecule. Changes in the color occur as DPPH radicals in the environment decreases and this (Piang-Siong et al., 2017). Hence the more potent antioxidant, more decrease in absorbance is seen and accordingly the IC₅₀ value will be minimum.

DPPH ⋅ + A-H = DPPH-H + A ⋅

In this study, the extracts of the undertaken vegetable samples were assessed for the antioxidant potential by utilizing the above principle of DPPH radical scavenging method. Table 2 represents the DPPH· radical scavenging abilities of the wild vegetables used in this study. At 500 μg.mL^-1, Bauhinia acuminata (Shetokanchan) showed the highest DPPH inhibition (93.10 ±1.23%), followed by Commelina benghalensis (Bat baittashak) (91.97 ±1.31%), Hydrocotyle sibthorpiodes L. (Sakumubakla) (91.83 ±2.13%), Albizia procera (Shada Koroi) (91.21 ±4.90%), Cajanus cajan (Orohorpata) (90.79 ±5.81%). The lowest DPPH radical inhibition among the studied samples was observed in Leucas aspera (Shetodhron) (68.1 ±2.65%) compared to other samples studied. In this study, all extracts prepared from the vegetable leaves contain varying degrees of DPPH scavenging activity. At a higher concentration, these extracts exhibit more significant DPPH free radical scavenging activity. From Table 2, we can also observe that there is a dose response relationship in inhibiting DPPH· free radical.

Fabaceae species are one of the several indigenous vegetables that have been reported to be rich in antioxidant compounds (Gođevac et al., 2008). As stated, this was the first study to evaluate the antioxidant capacities of the undertaken wild vegetables, no reference data was available to compare. Thus, only few studies could be found examining the other members of the same species. In a study by Gođevac et al., (2008) using nine different Fabaceae species, they found that the Fabaceae species possess good antiradical activity. Similar results was also reported by Srinivasan et al. (2016). Similar to our study, few other studies have also reported that the Tamarindus indica, Commelina nudiflora L., Bauhinia variegata, Leucas aspera exhibited potent DPPH free radical scavengers (Rajani and Ashok, 2009; Das et al., 2011; Chew et al., 2012; Kuppusamy et al., 2015; Mbaye et al., 2017). It is speculated that the antioxidant activity of the extract is attributed to the presence of phenolic components (Rajani and Ashok, 2009; Das et al., 2011; Chew, Jessica and Sasidharan, 2012; Kuppusamy et al., 2015; Mbaye et al., 2017).

The DPPH radical scavenging activity was further expressed as the effective concentration (IC₅₀) at which antioxidant activity was 50% (Figure 2). The lowest IC₅₀ values for the vegetables were recorded of Bauhinia acuminata (11.64 μg.mL^-1), Hydrocotyle sibthorpiodes L. (12.78 μg.mL^-1), Enhydra fluctuans (81.19 μg.mL^-1), Cajanus cajan (94.28 μg.mL^-1), whereas Leucas aspera (313.79 μg.mL^-1) exhibited the highest IC₅₀ value in all the studied samples.

The IC₅₀ values observed in this study were varying from other reports (Gođevac et al., 2008; Rajani and Ashok, 2009; Das et al., 2011; Chew, Jessica and Sasidharan, 2012; Kuppusamy et al., 2015; Srinivasan et al., 2016; Mbaye et al., 2017). Ethanolic extract of L. aspera root exhibited a significant DPPH radical scavenging activity having an IC₅₀ value of 7.5 μg.mL^-1 (Rahman, Sadhu and Hasan, 2007) whereas we found 313.79 μg.mL^-1 in methanolic extract of the leaves. Moreover, the ethanolic extract of whole L. aspera plant showed an IC₅₀ value of 176.46 mg.mL^-1 (Das et al., 2011). This difference in scavenging activity could be due to the different solvent extract system, growing conditions, fractions of the plant and some other variables (Rahman Sadhu and Hasan, 2007; Chew, Jessica and Sasidharan, 2012).