Decoding Variation among the Phenolic Composition and Antioxidant Properties of Some Finger Millet [Eleusine coracana (L.) Gaertn.] Landraces Grown in Maharashtra, India

Finger millet [Eleusine coracana (L.) Gaertn.] has a considerable historical, cultural and nutritional significance, notably in Asia and Africa (Pokharia et al., 2014). More than 25 countries on the African and Asian continents farm it, making up 12% of the world’s total millet area (Vetriventhan et al., 2016). In Maharashtra, locally known as ragi, nachani and nagli, are mostly cultivated in hilly areas of Western Ghats. Potentially climate-resistant and nourishing, finger millet has strong nutraceutical and antioxidant qualities (Kumar et al., 2016). It supplies a steady supply of food, making it an essential crop in certain environments. The crop yields food grain as well as straw, which is an important source of animal feed (Rurinda et al., 2014). In addition to being gluten-free, finger millet grain is high in calcium, fibre and iron, as well as having great malting capabilities and a low glycemic index (GI). As a result of these characteristics, finger millet grain is a preferred diet for diabetics (Padhan et al., 2010). 
       
Additionally, it has gained importance because of a higher amount of calcium (344 mg/100 gm), an excellent source of natural iron 3.9 mg (Gopalan et al., 2009), phenolic compounds (Geetha et al., 1990) and its functional components, such as slowly digestible starch, protein (5-8%), carbohydrates (65-75%), dietary fibre (15-20%) and minerals (2.5-3.5%) (Chethan and Malleshi, 2007). However, the millet also contains phytates (0.48%), polyphenols, tannins (0.61%), trypsin inhibitory factors and dietary fibre, which were once considered “anti-nutrients” due to their metal chelating and enzyme inhibition activities (Thompson, 1993). Apart from nutritional studies, several therapeutic properties of finger millet including antibacterial, antifungal, antidiabetic, antioxidant activity, anti-inflammatory, anti-carcinogenic and anti-cardiovascular activities have been reported (Devi et al., 2014).
       
Finger millet exhibits great genetic diversity as it grows in a wide range of environmental conditions. Native landraces, on the other hand, are more often associated with traditional and subsistence agriculture (Joshi et al., 2021; Ceasar et al., 2023). Local farmers select seeds based on empirical criteria such as the high yield, flavour and sustainability of the local environment. Native varieties may be capable of providing currently undiscovered benefits, making germplasm banks necessary to preserve diversity. Research into unknown characteristics of local landraces may produce findings that provide economic incentives for farmers to continue using native landraces. For example, if a local landrace proves to have high antioxidant properties, its commercial viability would surely improve (Syedd-León et al., 2020). Different landraces of finger millet are grown in different regions of Maharashtra based on the suitability of the local environment and its use. Previously, we investigated the morpho-agronomic diversity, nutritional value, polyphenol content and DPPH radical scavenging assay of finger millet landraces grown in various regions (Auti et al., 2017, Kazi et al., 2021; Kazi et al., 2022). Following our investigation into finger millet landraces, we present the results of antioxidant activity and LC-MS phenolic compound characterization, thereby adding value to this crop as a nutraceutical source and generating data that could be used in future breeding programmes.

Chemicals
 
The standards including ascorbic acid, quercetin, catechol and tannic acid were obtained from Sigma, Mumbai. The solvents employed, Folin-Ciocalteu reagent, Phosphomoly bdenum reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Ferrozine, hydrogen peroxide and aluminium chloride were of analytical grade and purchased from Hi Media, Mumbai. The LC-MS grade methanol, acetonitrile, water and formic acid for analysis were obtained from Sigma, Mumbai. 
 
Collection of landraces
 
Ten finger millet (Eleusine coracana) landraces were collected in November 2018 from different locations in the Nashik and Dhule districts of Maharashtra, India and accession numbers were given (T1-T29). In addition to that, a known variety (Dapoli-1) rose by Balasaheb Konkan Krishi Vidyapeeth Dapoli, was considered as a control (Table 1). The whole finger millet grains harvested from the plants were cleaned to remove soil and other particles and ground using mortar and pestle to obtain a fine powder. The obtained powder was placed in plastic containers and stored at room temperature (20°C) for further analysis.
 

 
Preparation of the extracts
 
The whole seed samples of landraces (0.5 g) were homogenized in 10 ml of 1% HCl-methanol. The homogenate was centrifuged at 10,000 rpm for 20 minutes. The extraction was repeated three more times with 1% HCl-methanol. The supernatants were filtered using filter paper (Whatman 1 Sigma, Mumbai) and collected in a round flask. The obtained polyphenol-rich extracts were then used for the subsequent analysis.
 
In vitro antioxidant activity
 
Hydrogen peroxide scavenging activity
 
The ability of finger millet to scavenge H₂O₂ was determined as per the method suggested by Halliwell and Whiteman (2004). Samples were extracted as mentioned in the DPPH assay. Hydrogen peroxide solution (20 mM) was prepared in phosphate-buffered saline (PBS) (pH 7.4). 0.2 ml of extract was added to 2.0 ml of H₂O₂ solution in PBS. The absorbance of H₂O₂ was measured at 230 nm, after 10 minutes against a blank solution that contained PBS without H₂O₂. Ascorbic acid was used standard as a compound.
 
The percentage of H₂O₂ scavenging by the plant extracts was calculated as,
 
Where,
A₀- Absorbance of control.            
A₁- Absorbance in the presence of plant extract.
 
Phosphomolybdenum reduction
 
The total antioxidant activity of the finger millet grains was estimated using the phosphor-molybdenum assay (Prieto et al., 1999). The powdered samples were extracted in 1% HCl-methanol and 0.2 ml was used. To that 3.0 ml of Phosphomolybdenum reagent was added and the final volume was adjusted using DW 3 ml of Phosphomoly bdenum reagent alone serves as blank. The reaction mixture was incubated at 97^oC for 90 minutes. After cooling the absorbance was measured at 695 nm using a UV/Vis spectrophotometer against the blank. The antioxidant capacity was expressed as Ascorbic acid equivalent (AAE) by using the standard ascorbic acid.
 
Ferric reducing antioxidant potential (FRAP)
 
The ability of finger millet extracts to chelate ferrous ions was measured according to a prescribed method (Dinis et al., 1994). Briefly, 0.4 ml of extracts in distilled water was added to a solution of 2 mM FeCl₂ (0.05 ml). The reaction was initiated by adding 5 mM Ferrozine (0.2 ml) and the total volume was adjusted to 4 ml with distilled water. The mixture was vigorously shaken and left at room temperature for 10 min. The absorbance of the reaction mixture was measured at 562 nm. For the control distilled water was used instead of the extract. The antioxidant power of the extract was compared with standard antioxidant Ascorbic acid. The results were expressed as micromoles of ascorbic acid equivalents per gram of sample.
 
Identification of phenolic compounds by LC-ESI-QTOF/MS
 
Phenolic compounds are characterized using the prescribed method (Gu et al., 2019) and were performed by the Agilent LC1200 series (Agilent Technologies, USA) equipped with an Agilent 6540 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, CA, USA). Reverse-phase HPLC column Zorbax Eclipse plus C18 with dimensions of 2.1*50 mm and pore size of 1.8 µM was used for the study. The column was maintained at a temperature of 40°C. The mobile phase consisted of 0.1% formic acid in acetonitrile. The flow rate was maintained at 0.35 ml/min. The total run for separation of samples was 15 min. 1 µl of the sample was injected into the column. Gradient elution was done to the identification of various components in the solution. Agilent 6540UHD Q-TOF-MS was used for the analysis of various phenolic compounds in the sample. The instrument had a mass range of 50-2000. Electrospray ionization with positive and negative ion modes was used for the identification of the compounds. The gas flow was maintained at 8l/min and the gas temperature at 295°C. The data analysis was done using Mass hunter workstation software V. B.05.01. The database used for analysis was METLIN.
 
Statistical analysis
 
All antioxidant assays were performed in triplicate and the data were analysed for one-way ANOVA by using Graph Pad Prism software version 7. All the values were expressed as Mean ± S.D. Pearson’s correlation coefficient was done by bivariate correlation analysis. Three correlation levels were defined as strong (r-value = 0.600–1.000), moderate (r-value = 0.400-0.599) and weak (r-value = 0.000-0.399).

Antioxidant studies
 
Numerous in-vitro assays can be used to assess the antioxidant potential of various plant extracts. Each of these tests focuses on a different aspect of antioxidant activity, such as the ability to scavenge free radicals or inhibit lipid peroxidation. However, due to their complex composition, a single method is not recommended for evaluating the antioxidant activities of various plant products. To obtain relevant data, the antioxidant effects of plant products must be evaluated by combining two or more different in vitro assays (Arfa et al., 2015; Khyade et al., 2017). In this regard, three different assays were used to investigate the antioxidant potential of landraces.
       
Fig 1 shows the concentration-dependent hydrogen peroxide decomposition activity of methanolic extracts. Hydrogen peroxide is a weak oxidising agent that can directly inactivate a few enzymes by oxidising essential thiol (-SH) groups. Hydrogen peroxide can rapidly cross cell membranes; once inside the cell, H₂O₂ is likely to react with Fe²⁺ and possibly Cu²⁺ ions to form hydroxyl radicals, which may be the source of many of its toxic effects (Gupta and Sharma, 2010: Halliwell and Gutteridge, 2015; Khyade et al., 2017). As a result, it is biologically advantageous for cells to control the amount of hydrogen peroxide that accumulates. From the results, it appeared that H₂O₂ scavenging activity is remarkably higher as compared to DPPH scavenging assay (Kazi et al., 2022). The highest H₂O₂ scavenging activity was shown by T1, T2, T3, T5, T8, T10 and T29 (90.12%, 82%, 79.8%, 82.9%, 79% and 85.78% respectively); however, the control sample T had significantly lower activity (55.87%), indicating selected finger millet landraces possess good radical scavenging activity over control, (Fig 1). Because of their high phenolic and flavonoid content, the above landraces have the highest H₂O₂ scavenging activity (Kazi et al., 2021). Additinally, the H₂O₂ radical scavenging potential of the methanolic extracts of finger millet landraces indicates quite similar results as compared to those in DPPH reactions (Kazi et al., 2021).
 

       
The ferric reducing antioxidant power (FRAP) is a commonly used metric to assess an antioxidant’s ability to donate an electron. The presence of a reductant (antioxidant) in the extract causes the Fe³⁺/ferricyanide complex to be reduced to the ferrous form (Megdiche-Ksouri et al., 2015; Khyade et al., 2020). In the different finger millet landraces evaluated, the FRAP activity ranged from 1.02-1.76 mM of Fe (II)/gm. The highest reducing power was shown by T2, T15 and T8 as 1.98, 1.94 and 1.89 mM of Fe (II)/gm respectively; while lower values were recorded in T19 (0.89 mM of Fe (II)/gm) and T1 (1.02 mM of Fe (II)/gm), (Fig 2).
 

       
This assay is based on the extract reducing Mo (VI) to Mo (V) and the subsequent formation of the green phosphate complex at acid pH (Behera, 2018). The total antioxidant activity of the methanolic extract was calculated as ascorbic acid equivalents (AAE) per gram. Based on the results, T2, T5, T8 and T10 all had a high reducing potential (158.71, 143.2, 149.5 and 152.65 equivalents of ascorbic acid in g/gm of a sample, respectively). Extracts with reducing capacities ranging from 73.86 to 158.71 equivalents of ascorbic acid µg/gm of extract. The control sample, on the other hand, had a much lower reducing capacity of 47.69 equivalents of ascorbic acid in µg/gm of extract of sampling (Fig 3).
 

 
Identification of the major phenolic compounds in ten landraces
 
The LC-ESI-QTOF/MS has been proved to be an effective tool for tentatively identifying and characterizing phenolic compounds in several plants (Hossain et al., 2010; Subbiah et al., 2020; Zhu et al., 2022). The phenolic compounds were identified and characterized based on their m/z value from MS spectra in both positive and negative ionization modes. As shown in Table 2, identified compounds were listed along with their molecular formula, retention times, ionization modes, molecular weight and mass error. Seventeen different phenolic compounds were characterized in all the ten selected landraces of finger millet, including 10 phenolic acids and 7 flavonoids (Table 2).
 

 
Flavonoids
 
Flavonoids are a diverse group of phenolic compounds that are present in many dietary plant foods. It has attracted interest due to their antioxidant, anti-inflammatory effects and their ability to modulate certain enzymatic functions (Panche et al., 2016; Al-Khayri et al., 2022). In this experiment, seven flavonoids were identified from the selected landraces quercetin, catechin, Iso-orientin, Iso-vitexin, lucenin, tricin and vitexin.
 
Phenolic acids
 
Phenolic acids are a subclass of phenolic compounds with a carboxyl group. Phenolic acids mainly comprise hydroxybenzoic acids and hydroxycinnamic acids and they have been extensively studied for their antioxidant, antimicrobial and anti-inflammatory effects (Kumar and Goel, 2019). In this study, two subclasses of phenolic acid were identified, which include hydroxybenzoic acids (5) and hydroxycinnamic acids (5).
       
Among the different types of hydroxybenzoic acid, hydroxycinnamic acid and flavonoid, the least frequent one is gallic acid; while p-hydroxybenzoic acid and catechin were identified in all the accessions. Ferulic acid, a common hydroxycinnamic acid derivative, was the predominant phenolic compound found in all finger millet landraces except T15. Obtained results revealed that T1, T19 and T29 have the highest fractionations over T2, T3 and T14. The landraces T3 and T14 do not show the presence of any flavonoid constituents. Among flavonoids, quercetin and catechin were the most predominant followed by iso-orientin. T2 and T8 landraces reveal the difference in the phenolic compounds; T2 and T8 exhibit 12 and 14 phenolic compounds respectively. The landraces T2 is specific for vanillic acid, trans-cinnamic, isovitexin and lucenin, while T8 is for genistic acid, syringic acid, sinapinic acid and tricin (Fig 1 and 2). Obtained results revealed that T1, T2, T5, T8, T10, T19 and T29 exhibit maximum types of phenolic compounds.

This study analysed the phenolic composition and antioxidant profile of ten finger millet landraces grown and consumed in Maharashtra, India. Phytochemicals and antioxidant activity were significantly higher in T1, T2, T5, T8, T10 and T19 finger millet landraces. The most abundant phenolic component was ferulic acid, a common hydroxycinnamic acid derivative found in all finger millet landraces except T15. In contrast, the phenolic compounds of T2 and T8 landraces differ. Taking this into account, T2 and T8 contain 12 and 14 phenolic compounds, respectively. The studied landraces can be considered health-promoting tools for local populations and their consumption should be encouraged to combat nutrient deficiencies and noncommunicable diseases. They also appear to be a valuable source of various phenolic compounds, with potential as a functional food as well as for nutraceutical applications. Furthermore, based on antiradical activities, T2, T5, T8, T10 and T29 could be potential cultivars as a climate resilient crop for sustainable agriculture.

All authors declare that they have no conflicts of interest.