Biogenic metal-oxide nanoparticles synthesized from Morus nigra leaf extract: A dual therapeutic approach for antioxidant and antidiabetic applications

2.1 Chemicals and sample collection

Zinc acetate dihydrate and D-glucose (anhydrous) were obtained from Qualigens (Thermo Fisher Scientific India Pvt. Ltd., Mumbai, India). Fresh mulberry leaves were collected from the garden of Isabella Thoburn College, located at 26°52′18″N 80°56′32″E, Lucknow, Uttar Pradesh, India.

2.2 Preparation of Morus nigra aqueous leaf extract

The leaves were washed, air-dried, and powdered. 15 g of leaf powder was boiled with 200 mL of Milli-Q water at 60°C for approximately 30 min. After cooling and overnight incubation, the extract was filtered and stored at 4°C.

2.3 Biogenic synthesis of G-ZNPs

The literature indicates that ZnO nanoparticles have been prepared from the leaf extract of Morus nigra(Tang et al., 2020). Unlike previous studies that rely on NaOH to stabilize the reaction pH, no external pH-adjusting agent was used in the present work. This preliminary study focuses on the growth and subsequent nucleation of ZnO nanoparticles, influenced by the alkaline range of phytochemical constituents of the leaf extract of Morus nigra. The present work preserves the biogenic nature of the synthesis process and ensures that nanoparticle formation reflects the natural reducing and stabilizing capacity of the plant extract. The synthesis was carried out using the native pH and ionic composition of the Morus nigra leaf extract. In this study, 0.5 M zinc acetate dihydrate was dissolved in 100 mL of Milli-Q water. 25 mL of leaf extract was added dropwise under magnetic stirring at 70°C. After an hour, 2.2 g of glucose was gradually added and stirred for 2-3 h. The mixture was centrifuged, washed, dried at 70°C, and calcined at 400°C, followed by grinding. The synthesis protocol was partially adapted from previously reported studies on leaf extract-mediated ZnO nanoparticle synthesis.

3.1 UV-visible spectroscopy

A min quantity of nano-powder was dispersed in a solvent mixture of 30μL of Dimethyl sulfoxide (DMSO) and 970μL of Milli-Q water. This solution was diluted six-fold, and the resultant solution was subjected to UV-visible scanning between 200 to 800 nm using a Shimadzu 1601 spectrophotometer (India) to assess the absorbance.

3.2 Field emission scanning electron microscopy (FE-SEM) analysis

FE-SEM analysis coupled with Energy-dispersive X-ray spectroscopy (EDX) was performed to determine the shape and surface morphology of G-ZNPs. The morphology was evaluated using a JEOL JSM 7610f instrument (Tokyo, Japan).

3.3 Dynamic light scattering/zeta potential analysis

The Malvern Zeta Sizer (United Kingdom) examined the size distribution and ζ-potential of G-ZNPs. The biosynthesized nano-powder was dispersed in water, and the average particle size was measured as the hydrodynamic diameter and zeta potential.

3.4 FT-IR spectroscopy

A Bruker Alpha II 210966 (USA) FTIR Spectrometer (3200-400 cm^-1) analyzed the functional groups in the plant extract responsible for stabilizing and facilitating the formation of zinc oxide nanoparticles.

3.5 X-Ray diffraction (XRD) analysis

The structural properties of G-ZNPs were analyzed using X-ray diffraction. The pattern was obtained within a range of 5̊ to 80̊ using Cu-Kα radiation with a wavelength of 1.54059 Å.

3.6 α-Amylase inhibitory activity of G-ZNPs

The anti-diabetic properties of glucose-capped ZnO nanoparticles were evaluated using the DNSA method (Robkhob et al., 2020). A stock solution of 1mg/mL G-ZNPs was prepared by dissolving 10 mg of nanoparticles in 10 mL of DMSO. Different volumes of G-ZNPs (40 μg/mL, 60 μg/mL, 80 μg/mL, 100 μg/mL, and 120 μg/mL) were diluted with PBS (pH 6.9) to make 1 mL of solution and incubated with 100 μL of α-amylase solution at 37°C for 30 min. A further 100 μL of 1% starch solution was added to the test tubes and incubated at 37°C for 30 min. The reaction was quenched by adding 200 μL of 3,5-dinitrosalicylic acid (DNSA) and kept in a water bath at 100°C for 5 min (Faisal et al., 2021). The absorbance of the enzymatic reaction mixture was determined at 540 nm using a UV-spectrophotometer. The same procedure was followed for metformin.

$\%inhibition=\frac{AbsorbanceofControl-AbsorbanceofSample}{AbsorbanceofControl}\times 100$

3.7 In vitro antioxidant activity of G-ZNPs

The free radical scavenging activity of Morus nigra leaf extract and G-ZNPs was assessed using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Dhandapani et al., 2020). 1 mL (0.1 mM) of DPPH solution was added to varying concentrations (20, 40, 60, 80, and 100 μg/mL) of the plant extract and G-ZNPs. The solutions were kept in the dark for 30 min, and the absorbance was recorded at 517 nm by a UV-visible spectrophotometer. Ascorbic acid was used as the standard. The percentage of free radical scavenging activity was determined by the following formula:

$\begin{array}{c}\%ScavengingActivity\\ =\frac{AbsorbanceofControl-AbsorbanceofSample}{AbsorbanceofControl}\times 100\end{array}$

3.8 In vivo antioxidant activity of G-ZNPs

Ten-month-old zebrafish (Danio rerio) were bought from a local fish market in Lucknow, India. The experiments were performed at the Department of Zoology, University of Lucknow, Lucknow. 24 adult zebrafish were used, and the average adult zebrafish weight was 2.5±0.5 g, and 3.5±0.5 cm in length, and they spent two weeks being domesticated in dechlorinated tap water in a laboratory. To promote optimal health and adaptability, the fish were kept at an average temperature of 25°C±2 under natural photoperiod conditions during the acclimation period. They were also fed a commercial fish feed. After two weeks of acclimatization, non-contagious fish were randomly chosen for the experiment and divided into four groups (n=3 per group): control, 10 ng/L, 20 ng/L, and 40 ng/L G-ZNPs treatments. On the seventh, fourteenth, twenty-first, and twenty-eighth days, samples were taken for analysis. 0.05 mL of fish tissue homogenate was combined with reaction buffer, trailed by the addition of 0.1 mL of NADH (2.34 mM) to assess the superoxide dismutase (SOD) activity. After incubation, the absorbance was measured at 560 nm for 3.5 min (Aebi, 1984). The activity of catalase (CAT) was calculated using the methods described by Ellman (1959); Misra and Fridovich (1972). This procedure relies on the findings that dichromate is transformed into chromic acetate in acetic acid upon heating with H₂O₂, with perchromic acid emerging as an unstable intermediate. Colorimetric measurements of chromic acetate are made at 620 nm.

3.9 Statistical analysis

Duncan’s multiple range test was used to assess the difference between individual means, and data were analyzed using SPSS 26 window version for in vivo antioxidant activity. Statistical comparisons were made between the controls and the G-ZNPs-treated groups, and t-tests and one-way ANOVA were used to analyze the results. All experimental data are presented as mean ± 95% confidence limits (CL), and a significance threshold of p < 0.05 was adopted for all comparisons. The data for α-amylase inhibition and in vitro antioxidant activity were analyzed using Origin Pro Software.

4.1 UV-visible spectroscopy

UV-Vis analysis reported a sharp peak at 229 nm, indicating excitonic absorption and the formation of ZnO nanoparticles (Fig. 2a) (Talam et al., 2012; Upadhyay et al., 2020). The observed peak is sharp, which suggests a monodisperse distribution of nanoparticles (Upadhyay et al., 2020). The optical band gap of the capped nanoparticles was determined using the Tauc relation. The plot of (αhυ)^1/n vs hυ calculates the band gap of ZnO nanostructures, where α is the absorption coefficient, hυ is the energy of the incident photons, and the value of n depends on the type of transition. The band gap was found to be 5.34 eV (Fig. 2b), which may be due to the quantum confinement effect (Anbuvannan et al., 2015). The reduction in the size of capped nanoparticles increases the energy gap in the electronic energy states of semiconductors. The Coulombic interactions between the holes and electrons alter both the conduction and valence bands. This was more significant when the size of the synthesized glucose-capped nanoparticles was smaller than the Bohr radius. As a result, the band gap widens with decreasing particle size (Aldalbahi et al., 2020; Anbuvannan et al., 2015).

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Fig. 2. (a) UV Spectra of G-ZNPs from leaf extract of Morus nigra. (b) Tauc plot of synthesized G-ZNPs

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4.2 Particle size distribution/zeta potential

Dynamic light scattering (DLS) was performed to record the size distribution and zeta potential of the biosynthesized G-ZNPs. The zeta potential was recorded as -16.9 mV (Fig. 3a), confirming the negative surface charge and moderate colloidal stability, which is more likely attributed to the steric contributions from the phytochemical corona and coating of glucose, reducing particle-particle interactions and subsequently agglomeration (Barzinjy and Azeez, 2020). The hydrodynamic diameter measured by DLS was 401.5 nm, larger than the SEM-observed size (∼ 52 nm), likely due to solvation effects (Barzinjy and Azeez, 2020; Sattari et al., 2020) (Fig. 3b). The polydispersity index was 0.426, indicating moderate polydispersity, with a relatively narrow size distribution (Danaei et al., 2018). Therefore, a strong correlation existed between the metabolites adsorbed on the nanoparticles’ surface and the zeta potential.

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Fig. 3. (a) Zeta potential of G-ZNPs. (b) Particle size distribution of G-ZNPs synthesized from Morus nigra leaves.

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4.3 SEM and EDX analysis

The morphology of the bio-capped ZnO nanoparticles is analyzed using scanning electron microscopy. The SEM particle size histogram of G-ZNPs indicated an average size of 52 nm, with sizes ranging from 38 to 79 nm (Fig. 4). Images at different magnifications revealed agglomeration. This might be because smaller particles combine to form larger ones owing to van der Waals interactions between nanocrystallites to reduce their surface area and achieve thermodynamic stability (Khorsand Zak et al., 2013).

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Fig. 4. SEM images of G-ZNPs from Morus nigra leaf extract. (a) 30,000 X (b) 50,000 X (c) Nanoparticle Size Histogram (avg 52 nm).

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However, a discrepancy was reported in the size of nanoparticles observed by SEM and DLS analyses. SEM images reveal the size of the core of inorganic nanoparticles and indicate their physical dimension. However, DLS studies measure the Z-average diameter, which is influenced by the solvation shell and the capping agents adsorbed on the surface of nanoparticles, thereby enlarging the effective diameter of ZnO nanoparticles (Filippov et al., 2023).

Energy-dispersive X-ray Spectroscopy (EDX) analysis was used to determine the elemental composition of the G-ZNPs. The EDX spectra revealed peaks between 1 and 10 keV, with a prominent peak at 1 keV, indicating the presence of zinc (Fig. 5). The Elemental analysis showed 19% oxygen and 80% zinc, which was in agreement with the previous studies (Jamdagni et al., 2018). The presence of another peak could be due to bioactive entities from the plant extract.

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Fig. 5. Energy-dispersive XRD spectra of G-ZNPs.

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4.4 FT-IR analysis

Fourier transform infrared (FTIR) spectrophotometry was performed for the identification of chemical bonds or functional groups. FTIR results provide supportive evidence of surface functionalization by different organic moieties during green synthesis of nanoparticles; however, they cannot be interpreted as definitive chemical identification. The FTIR absorption spectrum of the dried mulberry leaf was reported in the ranges of 3375-3260 cm^-1, 1600-1300 cm^-1, 1450-1050 cm^-1, 1080-620 cm^-1, and 935-710 cm^-1 (Wongsa et al., 2022) (Fig. 6a). However, a shift in the absorption band was observed for the mulberry leaf extract with zinc salt. The peak between 400 and 600 cm^-1 is attributed to the stretching of the Zn-O bond (Rajiv et al., 2013) (Fig. 6b). The FTIR spectrum of capped green synthesized zinc oxide nanoparticles ranges from 3200 to 400 cm^-1. The IR bands within the range of 3100–3400 cm^-1 typically indicate the presence of hydroxyl groups and intermolecular hydrogen bonds. The broad peak at 3200 cm^-1 typically indicates the presence of hydroxyl groups and intermolecular hydrogen bonds between alcohols and polyphenols (Jagajjanani Rao and Paria, 2013; Sana et al., 2020). This slight change in the peak is likely due to the formation of chemical bonds between OH and metal ions. The presence of a peak at 2359 cm^-1 may be attributed to C-H stretching vibrations of the alkane groups found within the extract. The absorption band at 1504 cm^-1 corresponds to the stretching frequency of C=C in aromatic rings as well as the in-plane bending of the hydroxyl groups of quercetin. The band at 1415 cm^-1 (1475-1400) cm^-1 is associated with the C=C bond of the aromatic or heteroaromatic ring of flavonols and anthocyanin groups (Krysa et al., 2022). The 1054 cm^-1 band represents the bending vibration of the C-OH bond, stretching frequency of =C-O-C, or stretching frequency of C-C of flavonoids, steroids, or compounds that have a primary alcohol functional group, which may be attributed to the C-O bond vibration found in glucose (Adiana and Mazura, 2011; Nybacka, 2016)

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Fig. 6. FT-IR Spectrum (a) Leaf Extract of Morus nigra (b) G-ZNPs.

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4.5 XRD Pattern Analysis

The main diffraction peaks for G-ZNPs were observed at diffraction angles (2θ) and lattice planes 31.80 (100), 34.50 (002), 36.24 (101), 47.58 (102), 56.61 (110), 62.86 (110), 68.08 (103) (Fig. 7) have been identified as the hexagonal phase of ZnO, suggesting crystalline nature of the nanoparticles, as referenced in Joint Committee on Powder Diffraction Standards (JCPDS) card number 36-1451(Sedefoglu, 2023). This confirmed the hexagonal phase of the crystalline wurtzite structure of the ZnO nanoparticles (Jayachandran et al., 2021). The strong and narrow diffraction peaks observed suggested an increased level of crystallinity in the nanoparticles. However, the relatively broad diffraction peaks indicate lattice strain owing to surface functionalization and defect-rich structures, which are typically associated with green synthesis of ZnO nanoparticles. The lattice parameters, a and c, were determined by least-squares refinement of the relation

$\frac{1}{d^2}=\frac{4}{3}\frac{h^2+hk+k^2}{a^2}+\frac{l^2}{c^2}$

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Fig. 7. XRD pattern of G-ZNPs.

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using the indexed reflections. The obtained values, a ≈ 3.25 Å and c ≈ 5.20 Å (c/a ≈ 1.60), closely match those of standard bulk ZnO.

The crystallite size of these structures was calculated using the Debye- Scherrer formula D=kλ/β cos θ, where D is the average crystalline particle size, k is Scherrer’s constant or shape factor (0.9), λ is wavelength of the X-ray (1.5406 Å), β is the XRD peak full width at half maximum (FWHM), and θ is Bragg’s diffraction angle (Sharma et al., 2022) (Table 1). The XRD analysis detected the crystallite size of the obtained G-ZNPs as 10.5 nm, corresponding to the most intense peak located at 36.24 (101).

4.6 α-Amylase inhibitory activity

The antidiabetic properties of Morus nigra leaves and ZnO nanoparticles have been previously reported in the literature (Hago et al., 2021; Shaban et al., 2022). Based on the reported studies, G-ZNPs were screened for their antidiabetic properties against metformin. α-Amylase inhibition showed concentration-dependent activity. G-ZNPs revealed stronger inhibition (IC₅₀=88 ± 0.02 µg/mL) compared to metformin (IC₅₀=121± 0.03 µg/mL) (Fig. 8). This confirms the inhibitory potential of G-ZNPs, which has the potential to control hyperglycemia by inhibiting α-amylase. The polyphenols found in the leaves may have been responsible for their inhibitory activities by reducing starch breakdown. Therefore, G-ZNPs decreased the level of α-amylase and reduced hyperglycemia (Nagarajan and Arumugam Kuppusamy, 2013).

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Fig. 8. α-Amylase inhibition assay of G-ZNPs and metformin.

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4.7 In vitro antioxidant activity

DPPH assay demonstrated significant percentage inhibition of both G-ZNPs IC₅₀=54±0.2 µg/mL) and plant extract (IC₅₀=53±0.17µg/mL), with ascorbic acid showing IC₅₀=58±0.5µg/mL (Fig. 9). This suggests the effective radical scavenging ability of the nanoparticles, attributed to their propensity to generate reactive oxygen species (ROS) (Daré and Lautenschlager, 2025).

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Fig. 9. In vitro antioxidant activity of leaf extract, G-ZNPs, and ascorbic acid.

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4.8 In vivo antioxidant activity

In vivo antioxidant activity revealed significant increases (p ˂ 0.05) in SOD and CAT activities in all treated groups, with the highest increase observed in the 40 ng/L group. The SOD activity in all groups exposed to G-ZNPs was in the range 8.2 to 8.5 U/mg in group 2, 9.3 to 9.6 U/mg in group 3, and 10.4 to 10.8 U/mg in group 4, respectively, which was significantly increased in comparison to the control group, 6.2 U/mg. The CAT activity in group 2 ranged from 157.75 to 159.33 U/mg, 164.85 to 173.46 U/mg in group 3, and 170.83 to 184.71 U/mg in group 4 (Figs. 10a, b). These indicated enhanced antioxidant defense, possibly due to ZnO-induced upregulation of enzyme expression.

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Fig. 10. In vivo antioxidant activity of G-ZNPs (a) SOD activity (b) CAT activity.

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The concentration used in the in vivo antioxidant assay was in the ng/L range to determine the lowest effective concentration required to induce detectable physiological effects. This confirms the enhanced bioavailability of capped ZnO nanoparticles and provides a basis for broader dose-response assessments in future studies.

The observed biological activities of G-ZNPs are consistent with the known intrinsic antioxidant, α-amylase inhibitory, and enzyme-modulating properties of Morus nigra leaf extract, containing flavonoids, phenolic acids, anthocyanins, and tannins. Therefore, the results obtained in this study are consistent with previously reported phytochemical bioactivities, supported by the nanoparticles’ enhanced surface area.