1Department of Botany, Sant Gadge Baba Amravati University, Amravati, Maharashtra State, India

2Department of Botany, Anjuman Islam Janjira, Degree College of Science, Murud-Janjira, Raigad MS India

Corresponding author email: kamlakarmore@sgbau.ac.in, dramanullak@gmail.com

Article Publishing History

Received: 10/04/2026

Accepted After Revision: 27/06/2026

ABSTRACT:

The current study based on phenolic composition and antioxidant potential of Mucuna nivea (Roxb.) seed extract using a multi-assay approach. Methanolic extraction was employed to obtain bioactive constituents, followed by estimations of total phenolic content (TPC) with estimation of total flavonoid content (TFC) with using Folin Ciocalteu and aluminium chloride-based methods, respectively. The plant extract is used for antioxidant activity and testes with the help of DPPH, ABTS, and CUPRAC assays to assess radical scavenging and reducing capacity.The results revealed that the extract contains appreciable levels these metabolites, indicating its strong redox potential. Among different solvents, acetone exhibited the highest phenolic content, while methanol showed superior flavonoid extraction efficiency, highlighting the influence of solvent polarity on phytochemical recovery. The DPPH assay demonstrated a radical scavenging activity of 68.30%, whereas the ABTS assay showed comparatively higher activity (73.97%), suggesting enhanced effectiveness against hydrophilic radicals. The CUPRAC assay further confirmed the antioxidant potential, with the extract exhibiting a Trolox equivalent value of 76 µM, indicating moderate reducing power. The record the antioxidants activity can be based on the synergistic action of secondary metabolites, particularly phenolics and flavonoids, which play a crucial role in neutralizing reactive oxygen species. These findings are consistent with the traditional medicinal relevance of Mucuna species and especially Mucuna nivea as a promising source of natural antioxidants. The study provides a scientific basis for the utilization of Mucuna nivea in pharmaceutical and nutraceutical applications and suggests the need for further investigation into its bioactive compounds and therapeutic properties.

KEYWORDS:

Antioxidant Activity, Cuprac Assay, Dpph And Abts Assays, Flavonoids, Medicinal Plants, Mucuna Nivea And Phenolic Compounds.

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More K, Pande D, Kale V, Ekapure K, Gawande P. A, Khan A. Evaluation of Phenolic-Flavonoid Content and Antioxidant Activity of Mucuna nivea Seeds Using DPPH, ABTS and CUPRAC Assays. Biosc.Biotech.Res.Comm. 2026;19(2).

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More K, Pande D, Kale V, Ekapure K, Gawande P. A, Khan A. Evaluation of Phenolic-Flavonoid Content and Antioxidant Activity of
Mucuna nivea Seeds Using DPPH, ABTS and CUPRAC Assays. Biosc.Biotech.Res.Comm. 2026;19(2). Available from: <ahref=”https://shorturl.at/MyKIG“>https://shorturl.at/MyKIG</a>

INTRODUCTION

Approximately 80% of the global population continues to rely on traditional medicine for primary healthcare needs, with plant-based remedies forming the cornerstone of such practices (WHO, 1993, Gerland et al., 2014; Tinitana et al., 2016, Divya et al 2024). A substantial proportion of traditional therapeutic systems involves the use of plant extracts and their bioactive constituents (Domingo-Fernández et al., 2023). Medicinal, aromatic, and herbal plants constitute an important component of global biodiversity and serve as a vital source of raw materials for pharmaceutical, cosmetic, and fragrance industries (Anju & Kumar, 2024; Patil & Khan, 2017; Zamani et al., 2025).

Their longstanding utilization is largely attributed to their diverse therapeutic properties and accessibility (El-Saadony et al., 2025). These plants are extensively employed in the treatment of various ailments and are recognized for their significant contribution to preventive and curative healthcare systems ( Sujarwo et al 2015, Misganaw et al., 2025).

In the context of increasing global trade and the rising prevalence of lifestyle-related disorders, oxidative stress has emerged as a critical factor underlying numerous pathological conditions (Krishnaiah et al., 2007; Pizzino et al., 2017). This has intensified scientific interest in identifying natural antioxidants from plant sources. Notably, a considerable number of modern pharmacological agents have been derived directly or indirectly from medicinal plants, underscoring their importance as reservoirs of novel bioactive compounds, ( Latif & Nawaz, 2026; McClatchey et al., 2009 El Saadony, 2025 ).

Consequently, the study of plants as medicine acts as good source of new therapeutics and becomes increasingly relevant (Domingo-Fernández et al., 2023).Plant-derived phytochemicals are broadly classified into primary forms of metabolites and secondary forms of metabolites based on their functional roles ( Agidew, 2022; Altemimi et al., 2017; Aly et al., 2022; Patil & Khan, 2017). Primary metabolites are essential for plant growth and development and serve as fundamental nutritional components for humans (Aly et al., 2022; Salam et al., 2023 Afakhar et al., 2025 ).

In contrast, secondary metabolites like phenolics, alkaloids, tannins, saponins, etc., play a pivotal role in plant defense like abiotic and biotic types of stresses and are widely exploited for their medicinal, aromatic, and pharmacological properties (Duke, 1994; El-Saadony et al., 2025; Khan & Hafiz, 2023; Salam et al., 2023, 2023).These compounds are known to exhibit strong antioxidant activities by normalising reactive oxygen species and protecting from oxidative damage (Naqbi et al., 2022; Pizzino et al., 2017).

Wild plant species possess enhanced resilience to environmental stresses compared to cultivated crops and are often rich sources of bioactive compounds (Altemimi et al., 2017). The oxidative stress conditions encountered by plants stimulate the biosynthesis of antioxidant molecules as part of their defence strategy (Pizzino et al., 2017). Conversely, in biological systems, an imbalance in antioxidant mechanisms can lead to the onset of various diseases (Pham-Huy et al., 2008). Therefore, understanding the antioxidant potential of plant-derived metabolites is crucial for their therapeutic application.

Mucuna nivea (Roxb.), commonly known as Kuyari and belonging to the family Fabaceae (subfamily Papilionoideae), is traditionally used in herbal medicine for the management of neurological disorders such as Parkinson’s disease and for enhancing testosterone levels (Figure 1.) (Khan, 2026; Lampariello et al., 2012; Tayade et al., 2020). Despite its ethnomedicinal importance, there is limited scientific data available regarding its phytochemical composition and antioxidant potential, particularly in seed extracts (Tayade et al., 2020). With this importance, the current work aims to evaluate the plant phyto-profile and antioxidant activity of M. nivea seeds by quantifying phenolic and flavonoid content and assessing high radical-neutralising capacity using multiple in vitro assays, including DPPH, ABTS, and CUPRAC. This multi-assay approach provides a clear assessment of the antioxidant potential of the plant sample and supports its possible application in pharmaceutical and nutraceutical development.

Figure 1. :Mucuna nivea (Roxb.)

MATERIALS AND METHODS

Materials and Extracts Preparation: The mature dry pods of M. nivea, were collected from the vicinity of Kasod, India (Location: Lat. 21.1927º Log. 77.011378º), Shivpur, Akola, Maharashtra. The seeds were manually separated from the pods and subsequently crushed using a mortar and pestle to make fine powder to ensure uniform particle size and homogeneity. 5 g of the seed powder was subjected to solvent extraction using 50 mL of methanol (Figure 1.) (Razmjou et al., 2025).

Using a rotary evaporator, the extract was concentrated by evaporating the solvents under reduced pressure. The concentrated extract was further subjected to sonication at a frequency range of 20-25 kHz for 15-30 minutes to enhance the extraction ability of phytochemicals (Bin Mokaizh et al., 2024; Bitwell et al., 2023). Following sonication, the extract was filtered extract was using Whatman’s filter paper No. 42 to remove particulate matter. The filtrate was collected and stored under appropriate conditions for subsequent analysis (Altemimi et al., 2017; Bitwell et al., 2023).

Phenolic Estimation: TPC was determined by the Folin Ciocalteu colourimetric method in methanolic seed extract as reported by Jagdish et al. (2009), with suitable changes (Jagdish et al., 2009). Catechol was employed as the reference standard for calibration (Siddiqui et al., 2017). A volume of 200 µL of the crude extracts (1 mg/mL) was mixed with 3 mL of distilled water (DW) in a capped test tube. To establish alkaline conditions necessary for the redox reaction, 1.5 mL sodium carbonate solution (20%) was added to the mixture. Subsequently, 0.5 mL of Folin Ciocalteu reagent was mixed, and incubated in the dark until complete colour development was achieved (Eshwarappa et al., 2014; Omer Abuelgassim, 2020). Absorbance of blue complex colour was recorded at 565 nm using a spectrophotometer. All analyses were conducted in triplicate to ensure reproducibility. TPC was recorded using a catechol standard curve and expressed as µg catechol equivalents per mL of extract (Lukiati et al., 2020).

TPC was calculated in mg/g of plant extract, where C: Gallic acid concentration obtained by standard curve (mg/L), V: Extract volume (mL), m: Mass of extract in g, and DF: Dilution factor.

Flavonoid Estimation: The TFC of seed extract was tested by the aluminium chloride colorimetric method, with standard as quercetin (Chaves et al., 2020; Sulastri et al., 2018). Briefly, a 5% (w/v) solution  of sodium nitrite, sodium hydroxide (1 M) and aluminium chloride solution (10% w/v) were prepared according to standard protocols (Ahmad et al., 2025; Sulastri et al., 2018).

Quercetin (100 µg/mL) as a stock solution was prepared in 80% ethanol. An aliquot of 100 µL (1 mg/mL) was diluted with 1 mL methanolic solvent, and then the process was further followed by the addition of 1 mL DW. After 5 min incubation, 0.3 mL of sodium nitrite (5%) was added, followed by 0.3 mL aluminum chloride (10%). The mixture was react for 6 min, after 2 mL of sodium hydroxide (1 M) was added, final volume adjusted to 10 mL with DW and incubated for 15 min at room temperature to allow complete change of color (Dureshahwar et al., 2017).

Spectroscopic value recorded at 450 nm in triplicate for all tests. A calibration curve was constructed using quercetin standard solutions of varying concentrations, treated under identical conditions, with potassium acetate as a stabilizing agent. A reagent blank was prepared by replacing aluminum chloride with DW (Dilkalal et al., 2024; Sulastri et al., 2018). TFC were recorded as µg quercetin equivalents (QE) per mL extracts.

 Determination of antioxidant activity assessment: The seed extract was evaluated using three complementary in vitro assays: DPPH, ABTS, and CUPRAC, following standardized protocols (Apak et al., 2007; Knez et al., 2025; Rumpf et al., 2023; Shahidi & Samarasinghe, 2025). Experiment repeated in triplicate, and the data are reordered to nullify the errors.

DPPH Assay: A 0.0024% (w/v) DPPH solutions were prepared by using 80% methanol. Ascorbic acid was taken as standard antioxidant.  The test mixture, consisting of 100 µL of extracts (1 mg/mL) and 3 mL of DPPH solution, was incubated in dark at for 30 minutes and then taken absorbance at 517 nm against a reagent blank.

The radical’s scavenging activity is calculated with:

ABTS Assay: It was tested to find out proton radical-scavenging activity from plant extract. The ABTS solution and potassium persulfate were prepared separately by dissolving 3.4mg 2, 2’-azino-bis (3 ethylbenzothiazoline-6 sulfonic acid and 6.4 mg  in 10 ml of DW. Both solutions were taken 5ml and incubated in the dark for 12 hours. The ABTS solution was later diluted with 10ml of DW. The extract was mixed with ABTS solution and incubated for 30 min in dark. The antioxidant activity of the extract was determined by recording absorbance at 734 nm. The following formula were used to calculation of activity.

CUPRAC Assay: Antioxidant capacity was tested using CUPRAC method (HiMedia EZAssay™ kit). Trolox was used as the standard. Serial dilutions of Trolox were prepared for calibration. The reaction mixture contained sample (100 µL), DW, and CUPRAC reagents (copper solution, chromogenic reagent, and ammonium acetate buffer). All tubes were incubated at dark for 10 minutes in room temperature and recorded absorption at 450 nm. Results represented as µM Trolox equivalents (TE) using its calibrations curve:

RESULTS AND DISCUSSION

Phenolic Estimation: The calibration curve exhibited a strong and linear relationship between concentration and absorbance in M. nivea (Roxb.) seeds extract, indicating the reliability of the assay (Table 1).

Table 1. Catechol standard calibration data.

Catechol (µg/mL) Absorbance (650 nm) 200 0.886 400 1.687 600 2.491 800 3.256 1000 4.121

The increase in absorbance with concentration confirms the proportional relationship between phenolic compounds and reducing capacity. The phenolics content of the plant extract calculated using the regression equations derived from this standard curve Figure 2 (a).

Figure 2. Multi-panel representation of phytochemical and antioxidant analysis of M. nivea seed extract (a) Phenolic standard calibration curve (catechol), (b) Flavonoid standard calibration curve (quercetin), (c) Comparative bar graph of phenolic and flavonoid content in different solvent extracts, (d) Comparative antioxidant activity (DPPH vs ABTS assays), (e) CUPRAC standard calibration curve (Trolox), and (f) Comparative CUPRAC antioxidant capacity of Trolox and seed extract.

Flavonoid Estimation: The aluminium chloride colorimetric method revealed a linear increase in absorbance with increasing quercetin concentration table 2. This confirms the presence of flavonoid in the sample, which are known contributors to antioxidant activity Figure 2(b).

Table 2. Quercetin standard calibration data.

Quercetin (µg/mL) Absorbance (450 nm) 200 0.415 400 0.724 600 1.033 800 1.443 1000 1.912

The extraction efficiency of phytochemicals is highly dependent on the polarity of solvents. In the present study, four different solvents methanol, toluene, petroleum ether, and acetone were evaluated for their ability to extract phenolic and flavonoid compounds from M. nivea seeds (Table 3).

Table 3. Phenolic and flavonoid in different solvent extract

Solvent Phenolic Content (mg/g) Flavonoid Content (mg/g) Methanol 23.8 33.44 Toluene 14.0 22.09 Petroleum Ether 16.3 11.56 Acetone 56.42 33.08

Among the solvents tested, acetone extract exhibited the highest phenolic content (56.42 mg/g), indicating its superior efficiency in extracting phenolic compounds. This may be attributed to its intermediate polarity, which enhances the solubility of a wide range of phenolics. In contrast, methanol extract recorded the high flavonoid contents (33.44 mg/g), closely related by acetone-based extract (33.08 mg/g), suggesting that polar solvents are more effective for flavonoid extraction (Figure 2(c)). Non-polar solvents such as petroleum ether and toluene exhibited comparatively lower extraction efficiency, particularly for flavonoids, which are generally polar in nature. These results records are consistent with previous studies indicating that solvent polarity significantly influences the extraction of phytochemicals (Dai & Mumper, 2010).

Determination of antioxidant activity assessment: The antioxidant potential of M. nivea seed extract was evaluated using three complementary in vitro assays, namely DPPH, ABTS, and CUPRAC. These assays collectively assess the hydrogen-donating ability, reducing power of bioactive compounds and radical scavenging efficiency.

DPPH Radical Scavenging Activity: The 2,2-diphenyl-1-picrylhydrazyl (DPPH) analysis represent the reductions of the stable free radical DPPH (deep purple) to a yellow colored di-phenyl-picryl-hydrazine upon interaction with antioxidants present in sample. The absorbance decline at 517 nm reflects the radical scavenging activity of the extract. The seed extract of M. nivea exhibits 68.30% DPPH radical scavenging activity, indicating substantial antioxidant potential. In comparison, the standard ascorbic acid showed higher activity (82.63%), as expected for a pure antioxidant compound (Table 4). The observed activity is comparable with previously reported plant extracts such as virgin coconut oil (53.52%) (Syafitri et al., 2023), indicating moderate-to-high antioxidant efficiency as in Figure 2(d) as above.

ABTS Radical Scavenging Activity:  The ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay represent the antioxidants that neutralise ABTS radical cations, resulting in decolourization measurable at 734 nm. The methanolic extract demonstrated a higher ABTS scavenging activity (73.97%) compared to DPPH, suggesting a stronger ability to quench hydrophilic radicals. The standard ascorbic acid showed 90.84% inhibition, confirming assay validity. The higher ABTS activity compared to DPPH indicates that the extract contains a broad spectrum of antioxidant compounds with both hydrogen atom transfer and electron transfer mechanisms.

Table 4. Comparative radical scavenging activity of seed extract

Assay Control

(Mean ± SD)

Sample

(Mean ± SD)

% Inhibition (Extract) % Inhibition (Standard) DPPH 0.874 ± 0.190 0.277 ± 0.033 68.30% 82.63% ABTS 2.331 ± 1.125 0.257 ± 0.103 73.97% 90.84%

CUPRAC Assay: Cupric Ion Reducing Antioxidant Capacity assay (CUPRAC Assay) measures the reducing capacity of antioxidants through the conversion of Cu²⁺ to Cu⁺, developing a coloured complex measurable at 450 nm. This method is highly sensitive and applicable to a wide range of antioxidant compounds.

Table 5. CUPRAC assay and Trolox equivalent values

Reagent Volume (µL) Absorbance (450 nm) Trolox Equivalent (µM) Trolox-1 200 0.172 191 Trolox-2 400 0.217 416 Trolox-3 600 0.259 626 Trolox-4 800 0.273 696 Trolox-5 1000 0.313 896 Trolox-6 1200 0.383 1246 Seed extract 100 µL 0.149 76

The seed extract exhibited a total antioxidant capacity of 76 µM Trolox equivalents, indicating moderate reducing power. The increase in absorbance with increasing Trolox concentration confirms the linearity and reliability of the assay as in Table 5 and Figure 2(e-f). The present investigation provides a systematic and integrative assessment of the phytochemical composition and antioxidant potential of M. nivea seed extract, revealing its significant bioactivity mediated by its secondary metabolites like phenolic and flavonoid constituents.

The elevated total phenolic and flavonoid contents observed in this study underscore the extract’s strong redox properties, as these compounds function as effective hydrogen and electron donors, thereby stabilizing reactive oxygen species. This is consistent with established reports that correlate phenolic abundance with enhanced antioxidant efficacy in wild-medicinal plants (Rumpf et al., 2023). The flavonoids record further strengthens this biochemical framework, given their well-documented roles in radical scavenging, metal ion chelation’s, and modulation in oxidative enzymatic pathways (Stachelska et al., 2025).

The antioxidant assays employed in this study collectively demonstrate the functional efficiency of the extract through distinct mechanistic pathways. The DPPH assay indicated substantial radical scavenging activity, reflecting the hydrogen-donating capacity of phytoconstituents, while the comparatively higher ABTS activity suggests superior efficacy against hydrophilic radical systems. This differential response highlights the complexity of antioxidant interactions and supports the presence of a diverse pool of bioactive molecules capable of both electron transfer and hydrogen atom transfer mechanisms (Floegel et al., 2011). Furthermore, the CUPRAC assay validated the reducing power of the extract, confirming its ability to participate in redox cycling through Cu²⁺ reduction, thereby reinforcing its antioxidant potential across multiple chemical environments (Chaves et al., 2020; Özyürek et al., 2011)

Importantly, antioxidant property reports the synergistic interplay of secondary metabolites, including phenolics, flavonoids, and potentially alkaloids, which collectively contribute to cellular protection against oxidative stress (Kasote et al., 2015). These biological activity become relevance, to oxidative stress and a key etiological parameter for the development and persistence of chronic disease like cancer, diabetes, and neurodegenerative problems (Castelli et al., 2025). The findings also align with the traditional medicinal relevance of Mucuna species, especially in neurological conditions like Parkinson’s disease, thereby providing scientific validation to ethnopharmacological knowledge (Rai et al., 2026).  Notably, while Mucuna pruriens has been extensively investigated, the present study highlights Mucuna nivea as a comparatively underexplored yet promising candidate with considerable antioxidant potential (Lampariello et al., 2012; ; Tayade et al., 2020, Rai et al., 2026).

The present study establishes that Mucuna nivea (Roxb.) seed extract is a potent source of bioactive phytochemicals, particularly phenolics and flavonoids, which significantly contribute to its antioxidant efficacy. The extract demonstrated consistent and appreciable activity across multiple in vitro assays, including DPPH, ABTS, and CUPRAC, indicating its broad-spectrum radical scavenging and reducing capabilities. These findings substantiate its potential application as a natural antioxidant in pharmaceutical, nutraceutical, and functional food systems.From a broader perspective, the study opens avenues for advanced phytochemical investigations, including compound isolation and structural characterization using chromatographic and spectrometric techniques. Future research should focus on in vivo validation, mechanistic studies, and formulation development to translate these findings into therapeutic applications. The comparative studies within the Mucuna genus and exploration of its neuroprotective and metabolic regulatory roles could further enhance its scientific and industrial relevance.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the Department of Botany, Sant Gadge Baba Amravati University, Amravati, Maharashtra, for providing the necessary laboratory facilities and academic support to carry out this research work. The authors also express their sincere gratitude to the Department of Botany, A I J D College of Science, Murud-Janjira, Raigad, for continuous encouragement and institutional support.

Conflict of Interest: The authors declare that there is no conflict of interest regarding the publication of this manuscript.

Data availability: All data are available with the corresponding author on a reasonable request

Funding Agency-Nil

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