1 Introduction

Basella alba L. (B. alba) also known as Malabar spinach or Ceylon spinach, is an annual herbaceous plant of Basellaceae [1]. Presence of minerals, protein, oil, carbohydrates, fiber, carotenoids, and organic acids, makes it a key herb for good health [2]. It is commonly used in salads, dips, and soups to reduce sticky texture. Betalains impart a red-violet color to leaves, stalks, petioles, and fruits [3, 4]. Additionally, this plant possesses considerable economic, nutritional, and medicinal value. It serves as a promising source of safe and effective skin-moisturizing polysaccharides, with the potential for industrial-scale adaptation [5]. Itis rich in vitamins A and C, folate, and manganese, with moderate levels of vitamin B and several dietary minerals [6, 7].

Species of Basella contains carotenoids, betacyanin, bioflavonoids, β-sitosterol and lupeol [8], which are reported to have antioxidant, antimicrobial, antiproliferative, anti-inflammatory, anticancer, anti-ulcer, antiviral, anti-cholesterol, wound healing, anti-hypoglycemic, androgenic properties [9,10,11] and used to treat constipation and act as a diuretic, detoxifying agent [12, 13].

Green synthesis, also known as biogenic synthesis, is considered an environmentally benign, economical, and sustainable method for producing nanomaterials. It enables the development of novel methods to replace, minimise, or even eliminate the production of hazardous chemical products that have a negative impact on both the environment and human health. The technique of producing nanomaterials using plant extracts, microbes, and biological components such as enzymes and whole cells is known as “green synthesis of nanoparticles” [14]. They have several benefits, including being naturally occurring, easy to obtain, free of adverse effects, generally economical, and using phytochemical substances as stabilisers or reducing agents throughout the synthesis process.

Amongst diverse types of metal oxide NPs, manganese (IV) oxide or MnO2 has gained interest since this metal occupies the twelfth most abundant transition metal element belonging to group 7 in nature. Also, it exhibits low toxicity and is reported to be stable, cost-effective, and eco-friendly [15]. Furthermore, manganese has the capability to form manifold bonds with oxygen through spontaneous oxidation to a range of stoichiometric hydroxides/oxides/oxyhydroxides that are collectively termed “manganese oxides” (MnO2) [16]. Hence, the distinct potential of MnO2-NPs from biogenic methods has been reported for various purposes, depending on the mode of action and application [17]. Strong antibacterial and antibiofilm properties were shown by Mn-O₂-NPs made with Viola betonicifolia leaf extract [18]. Additionally, biogenic MnO₂ nanoparticles have demonstrated little toxicity towards normal cells and promising lethal effects against cancer cell lines [19]. Seed germination and seedling growth in chickpea (Cicer arietinum) were improved seed priming with aqueous root extract of Kigelia africanabased Mn-O₂-NPs [20]. According to these results, green-synthesized MnO₂ nanoparticles have a variety of biological functions that make them appealing for use in agriculture and biomedicine.

The present study aimed at the biogenic synthesis of Ba-MnO2-NPs using an aqueous extract of B. alba and characterized by various techniques such as UV-Visible, FT-IR, SEM, EDX, p-XRD, and a particle size analyzer. Further, antibacterial, antioxidant, and anticancer activities were evaluated.

2 Materials and methods

2.1 Collection and identification of plant material

Basella alba L. leaves (fresh, healthy and disease free ) were harvested from matured, mass cultivated plants grown in our University medicinal plant garden. The plant material was collected during the study period from May 2024 to December 2025 and plant specimens were identified by Dr. Haleshi C, Assistant Professor and plant taxonomist, Department of studies in Botany, Davangere University, Davangere, Karnataka, India. The specimen voucher was prepared for the collected plant, allotted voucher number HDUD583 and maintained at Davangere University herbaria, Department of Botany, Davangere University, Davangere, Karnataka, India for future reference.

About 500 g of fresh leaves of B. alba sample was washed under running tap water, shade dried, and then homogenized to coarse powder with the grinder and stored in an airtight bottle at -20 °C at department of studies in Biotechnology, Davangere University, Davangere, Karnataka, India for further use.

2.2 Solvents and reagents

All the solvents, reagents, and chemicals used in this study were analytical grade (AR), and they were procured from Hi-Media, India.s.

2.3 Preparation of plant extract

About 25 g of powdered B. alba, was added with 250 mL of double-distilled water in an Erlenmeyer flask using a cold extraction method and kept at room temperature for 24 h under dark conditions. The extract was filtered using Whatman filter paper and stored in an airtight bottle at 4 °C until use. The aqueous extract was used as a reducing and stabilizing agent for the synthesis of Ba-MnO2-NPs.

2.4 Phytochemical analysis

The crude aqueous extract of B. alba was qualitatively tested for the presence of different phytochemical constituents, namely alkaloids, flavonoids, glycosides, phenols, lignins, saponins, sterols, tannins, anthraquinones, and reducing sugar [21,22,23].

2.5 Biogenic synthesis of Ba-MnO2-NPs using Basella alba

An aliquot of 10 mL of B. alba aqueous extract was added dropwise into the 200 mL of 10 mM potassium permanganate (KMnO4) at a 1:20 (v/v) ratio. The phytochemicals present in Basella alba leaf extract, such as phenolics, flavonoids, alkaloids, proteins, and carbohydrates, contain functional groups capable of donating electrons. These biomolecules reduce KMnO₄ (permanganate ions) into MnO₂ nanoparticles through redox reactions. In addition, the same phytoconstituents adsorb onto the nanoparticle surface and act as capping/stabilizing agents, preventing agglomeration and improving the stability of the nanoparticle. This step was performed under dark conditions at room temperature. After the completion of the incubation period, the pink-colored reaction mixture changed to a dark brown color indicating the oxidation or reduction reaction [24]. After the desired reaction period, the aqueous mixture containing a precipitate of Ba-MnO2-NPs was centrifuged at 10,000 rpm for 10 min and washed with double-distilled water, air dried for 24 h, and followed by drying at 80 °C for 15 h to obtain Ba-MnO2-NPs in powder form. Similar drying temperatures (65–80 °C) have been widely employed in the green synthesis of metal oxide nanoparticles to improve particle stability and facilitate subsequent physicochemical characterization [25].

2.6 Measurements and characterization

The synthesized Ba-MnO2-NPs were subjected to various characterization techniques for analysis. The UV-visible analysis was carried out using the UV-visible spectrophotometer, in the wavelength range of 200–800 nm, Model: HITACHI 2J1-0004. The functional groups are identified using the FT-IR instrument (KBr Pellet) in the spectral range of 4000–400 cm⁻¹, Model: NICOLET 6700. The surface morphology of Ba-MnO2-NPs was determined by SEM. Also, the zeta potential analysis was performed in the Zetasizer Nano ZS instrument (HORIBA SZ-100) in the 50–800 nm range. The crystalline nature, phase purity, and crystal structure of the synthesized Ba-MnO2-NPs were analyzed by p-XRD, Rigaku MiniFlex 600 X-ray diffractometer (Rigaku Corporation, Japan) recorded over a 2θ range of 10–80°. The reduced IO-NPs were dried on a carbon-coated copper grid, which was subjected to EDX, and the elemental composition was detected.

2.7 Antibacterial activity

The antibacterial potential of synthesized Ba-MnO2-NPs was evaluated against bacterial strains K. pneumonia and S. aureus. Various concentrations of Ba-MnO2-NPs (30, 60, and 90 µg) dispersing the nanoparticles in 1% dimethyl sulfoxide (DMSO) were subjected to the antibacterial study using the agar well diffusion method [26]. The results were compared with the standard drug Ampicillin (25 µg). The results were represented as a zone of inhibition, which was measured after 24 h of incubation at 37 °C.

Further, the MIC of Ba-MnO2-NPs (10–100 µg/mL) was determined using a broth micro-dilution assay. Briefly, microbial culture containing different concentrations of Ba-MnO2-NPs for each bacterial strain (1.5 × 105 CFU/mL) was loaded in flat-bottom micro-titer plate wells. The wells containing microbial inoculum served as a control. After 24 h incubation at 37 °C, absorbance was measured with a microplate reader for bacterial growth (Blue/purple), and Ba-MnO2-NPs were calculated. The MIC value was considered as at lowest concentration of Ba-MnO2-NPs that showed neither visible bacterial growth nor turbidity after incubation period indicating the inhibition of bacterial growth (color remained in blue).

2.8 Antioxidant activity

A 100 µL aliquot of diverse concentrations of Ba-MnO2-NPs (20, 40, 60, 80, and 100 µg/mL) was mixed with 3 mL of (0.609 mM) 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution prepared by dissolving 24 mg of DPPH granules in 100 mL of methanol. Each test sample was kept in the dark for 30 min at room temperature. After the incubation time, the optical density was measured at 517 nm, using a UV-visible spectrophotometer. A mixture of 100 µL of methanol and 3 mL of methanolic DPPH solution served as standard or blank, and ascorbic acid as a reference compound to compare the antioxidant capacity of the Ba-MnO2-NPs. The DPPH free radical scavenging activity of the prepared sample was expressed as a percentage of free radical inhibition using the following formula.

$$Inhibition\,(\%)=Ac-At/Ac\times 100$$

Where ‘At’ is the absorbance of the test sample and ‘Ac’ is the absorbance of the control reaction (100 µL of ethanol plus 100 µL of DPPH solution).

The experiment was carried out in triplicate and average values are taken. Further, for each sample, the IC50 value was computed. The lower absorbance of the reaction mixture was indicative of higher amounts of free radical activity.

2.9 Anti-cancer activity

The effect of Ba-MnO2-NPs at different concentrations (20 to 100 µg/mL) on the viability of lung cancer (A549) cells was determined by MTT assay using 3-(4, 5-dimethylthiazol- 2-yl)-2, 5-dimethyl tetrazolium bromide dye (Sigma, St. Louis, MO, USA), according to the reported method [27]. A549 cells are treated with different concentrations of Ba-MnO₂-NPs and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO₂. Cisplatin was used as positive control, while untreated cells served as the negative control of this assay is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a purple formazan product [28]. The percentage growth inhibition was calculated using the following formula and the concentration of test drug needed to inhibit cell growth by 50% (IC50) values is generated from the dose-response curves for each cell line.

$$Inhibition\,(\%)=Ac-At/Ac \times 100$$

Where ‘At’ is the absorbance of the test sample and ‘Ac’ is the absorbance of the control reaction.

3 Results and discussion

3.1 Phytochemical analysis

In the present study, the dried leaf sample of Basella alba was subjected to aqueous extraction using the cold method and screened for secondary metabolites. The phytochemical analysis revealed the presence of alkaloids, phenols, and terpenoids as major phytoconstituents.

Arya et al. [29]. reported the presence of alkaloids, flavonoids, phenols, tannins, saponins, glycosides, terpenoids, steroids, carbohydrates, and proteins. Phenolic compounds and flavonoids in leaf extract of B. alba.

3.2 Characterization

Synthesized Ba-MnO2-NPs exhibited a sharp absorption peak at 357 nm in the UV–Visible spectrum, confirming their formation (Fig. 1A). Additionally, the FTIR analysis revealed functional groups present in the samples the broad band at 3421 cm⁻¹ corresponding to the O-H stretching vibrations of phenolic compounds and alcohols, indicating the presence of hydroxyl groups that acted as reducing agents during nanoparticle synthesis. While peaks at 2920–2850 cm⁻¹ were attributed to aliphatic C–H stretching vibrations, while the band at 1632 cm⁻¹ was assigned to C = O stretching (amide/carbonyl groups), suggesting the involvement of proteins, flavonoids, and other phytochemicals in surface stabilization of the nanoparticles. The absorption bands between 1500 and 1400 cm⁻¹ were associated with C–H and N–H bending vibrations, further supporting the presence of biomolecules acting as capping agents. Strong peaks in the 1200–1000 cm⁻¹ region confirmed the presence of C–O and C–O–C groups, characteristic of carbohydrates and polysaccharides (Fig. 1B).

SEM analysis of Ba-MnO₂-NPs revealed predominantly spherical, slightly agglomerated particles with sizes ranging from approximately 200–400 nm (Fig. 1C). Based on their morphology, the synthesized particles are classified as zero-dimensional (0D) nanostructures because they possess approximately spherical geometry rather than elongated (1D) or sheet-like (2D) structures. The observed agglomeration is attributed to intermolecular interactions among phytochemicals from Basella alba leaf extract during the green synthesis process and sample drying. Elemental composition characterized by EDX analysis confirmed the presence of manganese and oxygen, with the corresponding weight and atomic percentages presented in Table 1; Fig. 1D.

The crystalline nature and phase purity of the synthesized Ba-MnO2-NPs were determined using the p-XRD. The p-XRD pattern demonstrates four distinguish peaks at 2θ = 23°, 31°, 43° and 65°, indexed to the crystal plane (Table 2 and Fig. 1E).

Particle size analysis (PSA) revealed an average hydrodynamic diameter of 159.6 nm (Fig. 1F). The larger particle size obtained by PSA compared with the conventional nanoparticle size range is attributed to the hydrodynamic diameter, which includes the nanoparticle core, the surrounding hydration layer, and phytochemicals adsorbed on the particle surface. Furthermore, aggregation of primary nanoparticles during drying and sample preparation contributes to the larger particle size observed by SEM. Therefore, although the measured particle size exceeds the conventional 10–100 nm range, the synthesized MnO₂ particles retain nanoscale characteristics and are classified as zero-dimensional (0D) nanostructures based on their morphology. Similar observations have been reported for green-synthesized metal oxide nanoparticles due to phytochemical capping and particle aggregation.

A similar kind of work carried out by Manjula et al. [30], the synthesized MnO2 nanoparticles from G. resinifera leaf extract showed an absorption peak at 362 nm in the UV–visible spectrum confirming nanoparticle formation. The P-XRD showed main diffraction peaks corresponding to 200, 121, 211 and 161 planes indicating the crystalline nature of MnO₂-NPs. SEM and EDX showed MnO₂-NPs were predominantly spherical in shape with particle sizes ranging from 50 to 100, while EDX analysis further confirmed the elemental composition of the nanoparticles, showing characteristic peaks corresponding to manganese and oxygen, thereby verifying the successful formation and purity of MnO₂-NPs.

Table 1 Elemental composition of Ba-MnO2-NPs
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Table 2 p-XRD peak details of Ba-MnO2-NPs
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Fig. 1
Fig. 1
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Characterization of Ba-MnO2-NPs (A) UV-visible absorption spectrum; (B) FT-IR spectrum; (C) SEM images; (D) EDX analysis; (E) p-XRD analysis; (F) Particle size analysis

3.3 Antibacterial activity

Different concentrations of Ba-MnO2-NPs were subjected to antibacterial activity against two pathogens responsible for infectious diseases such as K. pneumoniae (Gram-negative) and S. aureus (Gram-positive), along with the standard drug Ampicillin (25 µg). For K. pneumoniae, the zone of inhibition was found to be 4 mm, 9 mm, and 16 mm at 30 µg, 60 µg and 90 µg concentrations, respectively and Ampicillinshowed9 mm. The zone of inhibition was found to be 5 mm, 8 mm and 23 mm at 30 µg, 60 µg and 90 µg concentrations, respectively, and the standard drug exhibited 8 mm zone of inhibition with S. aureus (Fig. 2).

Further, MIC results of Ba–MnO₂-NPs indicated that K. pneumoniae was inhibited at 20 µg/mL, whereas S. aureus required a higher concentration of 40 µg/mL for complete growth inhibition among the tested concentration range of 10 µg to 100 µg (Fig. 3).

In a similar kind of study, Saod et al. [31] evaluated the antimicrobial activity of manganese oxide nanoparticles prepared by green tea extract against E. coli, K. pneumoniae and P. aeruginosa. The result revealed that the nanoparticles showed zones of inhibition of 12 mm against E. coli, 14 mm against K. pneumoniae, and 18 mm against P. aeruginosa. The MIC of the MnO2-NPs was determined to be 12.5 U/mL using the resazurin microtiter assay, indicating strong antibacterial efficacy at low concentrations.

Fig. 2
Fig. 2
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Antibacterial activity results of Ba-MnO2-NPs (A) S. aureus (B) K. pneumonia (C) and (D) Standard Ampicillin and control Distilled water

Fig. 3
Fig. 3
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Minimum inhibition concentration of Ba-MnO2-NPs against bacterial strains

3.4 Antioxidant activity

The ability of green synthesized Ba-MnO2-NPs in neutralizing free radicals at its varying concentrations was assessed using DPPH. Different concentrations of (50,100,150, 200, 250 µg/mL) of MnO2−NPs synthesized from Basella alba leaf extracts exhibited inhibition of radical scavengers by dose dependent way (Table 3 and Fig. 4). At the highest concentration (250 µg/mL), Ba-MnO2-NPs showed the potent DPPH radical scavenging of 67.04%, while standard ascorbic acid showed 89.19% antioxidant activity. Further, the IC50 values of Ba-MnO2-NPs and standard ascorbic acid were 88.02 µg/mL and 186.24 µg/mL respectively.

Biosynthesized MnO₂ nanoparticles showed concentration-dependent DPPH radical-scavenging activity, with a maximum inhibition of 50.03% at 466 µg/mL. These results demonstrate the effective antioxidant potential of MnO₂-NPs through free radical neutralization [32].

Table 3 Antioxidant assay of Ba-MnO2-NPs
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Fig. 4
Fig. 4
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Antioxidant activity showing present inhibition using DPPH assay

3.5 Anticancer activity

The green synthesized Ba-MnO2-NPs were subjected to the anticancer studies using MTT cell viability assay on lung cancer A549 cell line along with the cisplatin as a positive group and untreated cells as a negative group. The results revealed that the synthesized Ba-MnO2-NPs have shown good results with a decrease in the percentage of cell viability with the increase in the concentration of the test sample. At 20 µg the percentage of viability was seen to be 84.65 ± 0.0127 and it was decreased to 25.81 ± 0.0091 at 100 µg concentration. Whereas, the standard drug cisplatin has shown around 10.46 ± 0.0084% of cell viability.

The IC50 value of the test sample was calculated to be 70.55 µg (Table 4 and Fig. 5). Morphological studies also correlate with the MTT results as the concentration increased there were changes in the cells’ appearance (Fig. 6) with a decrease in the cell count and appearance of abnormalities such as cell shrinkage, cell turgidity, cell elongation, and apoptotic bodies.

Saraca asoca mediated Mno2−NPs showed dose dependent cytotoxicity activity against MCF-7 and MDA-MB-231 cells with IC50 values above 20 µg/mL at 24 h [33].

Table 4 Anticancer activity of Ba-MnO2-NPs on lung cancer cell line A549
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Fig. 5
Fig. 5
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% Cell viability of Ba-MnO2-NPs against lung cancer cell line A549

Fig. 6
Fig. 6
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Morphological effects of Ba-MnO2-NPs against lung cancer A549 cell line

4 Conclusion

This study effectively demonstrated the green synthesis of manganese dioxide nanoparticles utilizing B. alba leaf extract (Ba-MnO₂-NPs). Aqueous extract of B. alba was evaluated for phytochemical constituents. The formation of Ba-MnO₂-NPs was confirmed through UV–Vis, FT-IR, SEM, EDX, p-XRD, and particle size analyses, revealing spherical, crystalline nanoparticles with an average particle size of 159.6 nm. The synthesized Ba-MnO₂-NPs exhibited significant biological activities. Antibacterial studies effectively inhibited both K. pneumoniae and S. aureus. In addition, Ba-MnO₂-NPs showed considerable antioxidant activity indicating their ability to scavenge free radicals. Furthermore, the Ba-MnO2-NPs displayed dose-dependent cytotoxicity against A549 lung cancer cells, suggesting promising anticancer potential.