In Vitro Anticancer Activity of Green-Synthesized Zinc Oxide Nanoparticles from Root Extract of Sida schimperiana against MDA-MB-231 Breast Cancer Cell Lines

Abstract

Objective:

This work addressed the fact that nano phytoformulations have emerged as promising, biocompatible alternatives to conventional cancer drugs, helping to overcome drug resistance in cancer and offering sustainable, biocompatible, and effective drug delivery. This study aimed to synthesize zinc oxide nanoparticles (SS-ZnO NPs) using an eco-friendly green synthesis method from Sida schimperiana aqueous root extract (SS-AQ), and to evaluate their selective cytotoxicity against MDA-MB-231 breast cancer cells.

Methods:

The SS-ZnO NPs were characterized using spectroscopic (FTIR, XRD, DLS), microscopic (SEM, TEM), and chemical (EDX) analytical techniques. The selective cytotoxicity of SS-ZnO NPs against breast cancer cell lines (MDA-MB-231) and normal cell lines (L929) was evaluated using the MTT assay.

Results:

XRD analysis confirmed that SS-ZnO NPs possess a crystalline, hexagonal wurtzite structure with an average size of 55.4 nm. DLS analysis indicated that the SS-ZnO NPs are monodispersed with a negative surface charge of –28.9 mV, suggesting high colloidal stability. SEM and TEM-EDX analyses revealed that the SS-ZnO NPs exhibit a pseudo-spherical, rough morphology with an average particle size of 22.65 nm. Strong absorption peaks at 1.01 keV and 0.52 keV were observed, corresponding to the characteristic signals of Zn and oxygen, respectively. The MTT assay demonstrated that SS-ZnO NPs exhibited significant, dose-dependent selective cytotoxicity against MDA-MB-231 breast cancer cell lines, with inhibition ranging from 10.14% to 62.44% at concentrations of 6.25–100 µg/mL, and an IC₅₀ value of 45.28 µg/mL (p ≤ 0.01). In comparison, SS-AQ exhibited 8.81% to 58.11% inhibition at the same concentration range, with an IC₅₀ of 50.16 µg/mL (p ≤ 0.01).

Conclusion:

The findings of the current study highlight that bio-inspired SS-ZnO NPs possess enhanced anticancer properties and can be considered a promising anticancer agent with potent, specific cytotoxic efficacy against MDA-MB-231 breast cancer cells, offering a potential alternative nanotherapeutic approach with reduced toxicity.

Introduction

Nano-oncology has emerged as a one of the most cutting-edge interdisciplinary fields of the twenty-first century, combining clinical oncology and advanced materials science, with applications in timely identification, efficient therapy, real-time monitoring, and targeted drug delivery to specific cancer sites [1]. Researchers around the world have invested a lot of capital, time, and expertise in the production and manipulation of nonmaterials at the nanoscale (1-100 nm) [2]. These nanomaterials have received prodigious attention due to their exceptional physicochemical, electrical, optical, magnetic, catalytic, and biological properties by tailoring features such as large surface area and quantum confinement [3]. For a long time, the scientific community has developed various physical and chemical methods to generate nanomaterials with high purity, scalability, controlled particle size, and a wide range of applications [4]. However, contempt of their use, these traditional methods frequently present significant challenges, such as high energy consumption, environmental pollution, high costs, and the use of toxic reducing agents [5]. To address these challenges, researchers have developed sustainable, environmentally friendly, and cost-effective approaches based on green chemistry principles, plant-mediated nanoparticle synthesis stands out as a promising alternative to traditional methods [6].

The phytosynthesis method successfully uses secondary metabolites such as polyphenols, flavonoids, terpenoids and tannins as reducing, stabilizing, and capping agents [7]. These secondary metabolites, with their diverse functional groups, not only act as active medical agents, but also helps in advancing cancer therapeutics including enhanced biocompatibility, improved bioavailability, controlled drug release, increased stability, multifunctionality, and precise dosing [8]. Among various metal oxide nanoparticles, zinc oxide nanoparticles (ZnONPs), are used for developing novel nano-drugs for breast cancer treatment [9], also, ZnONPs are believed to effectively combat breast cancer progression due to their unique physicochemical properties, that include high surface area, biocompatibility, and ease of functionalization, which enable targeted drug delivery, increased bioavailability, and potent anticancer activity in a short period of time [10]. Green-synthesized ZnONPs interact with cancer cells acidic microenvironment, releasing zinc ions as well as phyto-compounds that enable the mitochondria-dependent intrinsic apoptotic pathway [11].

Sida schimperiana is a medicinal plant from the Malvaceae family that distributed widely in Ethiopia, Kenya, Uganda, Tanzania, and Eritrea, and widely used as traditional medicine to treat many kinds of ailments, including wounds, respiratory infections, fever, intestinal disorders, malaria, joint pain, coughs, colds, asthma, stomach aches, and skin diseases [12].Phytochemical analysis of Sida species such as Sida acuta and Sida rhombifolia revealed the presence of secondary metabolites such as alkaloids, flavonoids, saponins, and phenolic compounds [13]. These compounds demonstrated for their significant biological properties such as antioxidant, anticancer, anti-inflammatory, antibacterial, and antifungal activities, highlighting the genus’ therapeutic potential [13-15].

According to current World Health Organization (WHO) reports, breast cancer will remain the leading cause of cancer-related deaths in women worldwide in 2023. Approximately 2.3 million new cases of breast cancer were diagnosed in 2022, about 685,000 deaths worldwide [16]. Globally, one in eight women is diagnosed with breast cancer at some point in her life, and one in any eleven dies from the disease [17]. Despite the clinical effectiveness of chemotherapy regimens as a primary treatment strategy for breast cancer, there are several major drawbacks, including insufficient drug absorption, systemic toxicity, a lack of specificity [18], and the rapid emergence of multidrug resistance in breast cancer tissues. These challenges point out the critical need for novel therapeutic approaches that improve treatment efficacy, minimize side effects, and improve patient outcomes [19]. Metal oxide nanoparticles, particularly ZnONPs synthesized using novel nano-phytoformulations have showed promising anticancer activity by activating intrinsic apoptotic pathway by inhibiting proliferation, and decreasing adverse effects.

Therefore, to the best of our knowledge, the present study was first attempt to green synthesize SS-ZnO NPs using an aqueous root extract of Sida schimperiana (S.schimperiana) as a reducing and capping agent. These obtained SS-ZnO NPs were characterized using spectroscopic, microscopic techniques furthermore; these SS-ZnO NPs were evaluated for their potential in vitro selective cytotoxicity against MDA-MB-231 human breast cancer cell lines.

Materials and Methods

Chemicals and reagents

Zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O) (99.9%) (Sigma-Aldrich; USA), Dimethyl sulfoxide (DMSO), (Sigma-Aldrich; USA), Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific; USA), fetal bovine serum (FBS),(Thermo Fisher Scientific; USA), Trypsin-EDTA solution (Thermo Fisher Scientific; USA)Penicillin/Streptomycin (Thermo Fisher Scientific; USA) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), (Thermo Fisher Scientific; USA). All solvents and chemicals used in the experiments were of analytical grade and were purchased from local suppliers.

Collection, Authentication and Preparation of Plant extract

Fresh Sida schimperiana (S. schimperiana) roots were collected in February 2021 from Axum City, Tigray region, Ethiopia, authenticated and assigned the voucher specimen number AH-001 by a botanist Mr. Melaku Wendaferash at the National Herbarium of Ethiopia, Addis Ababa University. The roots were washed with running tap water, followed by distilled water, and then shade-dried at room temperature for two weeks. According to the modified methodology of Chaithanya et al. [20] a Soxhlet extractor was used to extract approximately 100 g of powdered root material (1:5 w/v) over three cycles for 8 hours using 500 mL of distilled water. After extraction, the plant extract was filtered using Whatman filter paper No. 1. The filtrates were concentrated at 50–60°C using a rotary evaporator under reduced pressure [21]. The percentage yield of each extract was calculated using the following formula:

$\text{Percentage yield}=\frac{\text{Extract weight of the plant (g)}}{\text{Dry weight of the plant (g)}}\times 100$ (1)

Qualitative Phytochemical Screening

Preliminary phytochemical analysis was performed to detect various phytochemical constituents, including alkaloids, phenolic compounds, flavonoids, tannins, saponins, steroids, glycosides, terpenoids, and anthraquinones, in the aqueous root extract of S. schimperiana using standard methods described by Harborne and Williams [22].

Green Synthesis of SS- ZnONPs

A green synthesis of SS-ZnO NPs was carried out using the aqueous root extract of S.schimperiana, by following a modified protocol of Wafaeyet al. [23]. In brief, 10 mL of sterile aqueous root extract of S. schimperiana was added drop wise with continuous stirring to 90 mL of a 1 mol/L solution of zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O), at a temperature of 60–80°C. The mixture was stirred magnetically at 300 rpm for 15 minutes. The resulting solution was centrifuged at 3,500 rpm for 10 minutes to remove unreacted plant extract. The green-synthesized nanoparticles were then collected by centrifugation at 12,000 rpm for 20 minutes. The synthesized SS-ZnO NPs, were allowed to dry in a watch glass at 50–60°C followed by further purification through continuous washing with sterile distilled water to obtain pure SS-ZnO NPs in pellet form. The colour change of the reaction mixtures was the initial indication of SS-ZnO NP formation. Dimethyl sulfoxide (2% DMSO) was used to dissolve the S. schimperiana root aqueous extract (SS-AQ) and SS-ZnO NPs. Stock solutions were prepared in DMEM culture medium at a concentration of 1 mg/mL. The final DMSO concentration never exceeded 0.1% after filtration of the prepared samples using 0.2 µm porous membrane filters.

Characterization of SS- ZnONPs

According to Gholami Shabani et al. [24] and Joudeh and Linke [25], the obtained SS-ZnO NPs were validated using spectroscopic and microscopic techniques. A noticeable colour change was observed, indicating the formation of nanoparticles. UV-Vis spectroscopy (Shimadzu) was performed in the range of 200–800 nm to confirm the surface plasmon resonance (SPR) peaks of SS-ZnO NPs. FT-IR (IRTracer-100, Shimadzu) analysis was conducted in the range of 400–4000 cm⁻¹ to identify functional groups present in the aqueous root extract of S. schimperiana and the surface chemistry of SS-ZnO NPs, which act as reducing and capping agents. X-ray diffraction (XRD-6100, Shimadzu) analysis was performed in the range of 5° to 80° to determine the crystalline structure. Dynamic Light Scattering (DLS) and zeta potential analyzers (Zetasizer Nano ZS) were used to measure the size distribution and zeta potential of the nanoparticles. Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X-ray Spectroscopy (EDS) (Shimadzu) was used to analyze the surface morphology and elemental composition of SS-ZnO NPs. Transmission Electron Microscopy (TEM) (Zeiss EM10 C, 100 kV) was used to analyze the internal morphology and size of the nanoparticles.

Cell Cultures and Maintenance

MDA-MB-231 (breast carcinoma) and normal mouse fibroblast cell line (L929) were purchased from the American Type Culture Collection (ATCC). These stock cell lines were cultured in 24-well plates with RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 µg/mL), penicillin (100 Units/mL), and 2 mM L-glutamine, maintained at 37°C in a 90% humidified 5% CO₂ atmosphere incubator until they reached 80% confluence.

In vitro Cytotoxicity Activity

The selective in vitro cytotoxic effect of the UV-sterilized aqueous crude root extract of S. schimperiana (SS-AQ) and its green-synthesized SS-ZnO NPs against MDA-MB-231 (breast carcinoma) and normal mouse fibroblast L929 cell lines were determined by the MTT colorimetric reduction assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye) according to the modified methods of Mfengwana and Bertrand [26].These cell lines were seeded at a density of 1×10⁴ cells/well in a flat-bottom 96-well plate in complete DMEM and incubated for 24 hours at 37°C in a humidified atmosphere of 95% air with 5% CO₂. After 24 hours, the cells were subsequently treated with five different concentrations of SS-AQ and SS-ZnO NPs (6.25, 12.5, 25, 50, and 100 µg/mL). Cisplatin (2.5, 5, 10, 20, and 40 µg/mL) was used as a positive control. These treatments were added to each well in triplicate, except for the basal control wells, where only nutrient medium (DMEM) was added. The plates were incubated for 24 hours in a 5% CO₂ incubator at 37°C. Following the incubation period, 20 µl of MTT (5 mg/ml) reagent was added to each well and incubated for 4 hours in the dark in a 5% CO₂ incubator at 37°C. After incubation, the MTT reagent was aspirated, and 100 µl of 10% Sodium Dodecyl Sulfate (SDS) was added, leading to the formation of purple-coloured formazan products. The absorbance was measured spectrophotometrically using a micro-ELISA plate reader at 570 nm. Cytotoxicity was determined using the following formula, and IC₅₀ values were calculated:

Cytotoxicity (%) = (OD of control-OD of test)/ (OD of control) ×100 (2)

To determine the selective cytotoxicity of SS-AQ, SS-AuNPs, and SS-ZnO NPs against the L929 and MDA-MB-231 cell lines, the selective index (SI) was calculated using the given equation.

Selective Index (SI)= (IC₅₀ of normal cell lines)/(IC 50 of cancer cell lines) ×100 (3)

Statistical analysis

Statistical differences between the Mean ± SD values of the anticancer activity (% cell viability) between the control (without plant extract) and experimental group was assessed by using unpaired Student’s t-test. Statistical analysis were performed using Microsoft Excel 2007 and Graph Pad Prism version 9.0, with statistical significance indicated as *p < 0.05, **p < 0.01, and ***p < 0.001 versus control.

Results

Percentage of yield

As shown in Table 1, the percentage yield of the aqueous root extract of S. schimperiana was 10.5%, extract appeared a dark brown colour with a slightly viscous nature. These results are consistent with Donkor et al. [27], who reported that the aqueous leaf extract of Sida acuta had highest percentage yield of 9.3%.

Table 1.

Colour, Consistency and Percentage of Yield (w/w %) of Aqueous Root Extract of S.schimperiana

Extract	Colour	Consistency	Percentage of yield
Aqueous extract of root of S.schimperiana	Dark brown	Slightly viscous	10.50%

Qualitative phytochemical analysis

The preliminary phytochemical analysis of the aqueous root extract of S. schimperiana revealed the moderate presence of alkaloids, phenolic compounds, tannins, and flavonoids, and the minimal presence of steroids and saponins (Table 4), which are responsible for the bioreduction of Zn²⁺ ions to Zn⁰ during the synthesis of SS-ZnO NPs. Additionally, these compounds also act as stabilizing and capping agents, preventing agglomeration of SS-ZnO NPs. Our results align with the previous phytochemistry Sida species reported by Membe et al. [13], who indentified simalr secondary metabolites alkaloids, flavonoids, tannins, phenolic compounds, sterols, and triterpenes in hydroethanolic extracts of Sida acuta (whole plant) and Sida rhombifolia (aerial parts) (Table 2).

Table 4.

Comparative Selective Cytotoxic Effect and IC₅₀ Values of SS-AQ, and SS-ZnO NPs on MDA–MB-231cell Lines and Normal L929 Fibroblast Cells

Extracts	Concentration	% Cytotoxicity		IC50 (µg/mL)		Selective Index (SI)
/Standard	(µg/mL)	MDA-MB 231	L929	MDA-MB 231	L929
SS-AQ	6.25	8.11 ±044	5.22±0.11	50.16	>100
	12.5	19.19±0.11	8.22±0.01			1.99
	25	26.11±0.63	12.10±0.96
	50	44.09±0.96	14.11±0.85
	100	58.11±0.93	15.96±0.05
SS-ZnO NPs	6.25	10.14±0.10	5.55±0.16
	12.5	21.34±0.38	10.54±0.14	45.28		2.2
	25	31.22±0.18	13.81±0.53		>100
	50	48.34±0.40	15.22±0.12
	100	62.44±0.26	18.31±0.03
Cisplatin	2.5	18.15±38	ND	8.34	ND	ND
	5	39.74±29	ND
	10	56.69±0.43	ND
	20	77.09±0.19	ND
	40	91.74±0.76	ND

Table 2.

Qualitative Phytochemical Screening of Aqueous Root Extract of S. schimperiana

Chemical constituents	Test	Result
Alkaloid	Mayer’s test	++
Phenolic compound	Ferric chloride test	++
Tannins	Ferric chloride test	++
Flavonoids	Alkaline reagent test	++
Steroid	Liebermann–Buchard’s test	+
Saponins	Foam test	+

+, Minor presence; ++, Moderate presence

Characterization of SS-ZnO NPs

Visual Colour Change Observation

As depicted in Figure 1, the formation of ZnONPs from the aqueous root extract of S. schimperiana was initially confirmed by the observed colour changes from colourless to light brown. This colour change revealed the bio-reduction of Zn²⁺ions to Zn⁰, leading to the synthesis of biogenic SS-ZnO nanoparticles. The characteristic colour change is due to specific optical properties of SS-ZnO nanoparticles, including quantum confinement, localized surface plasmon resonance (LSPR) [28].

Figure 1.

The Colour Changes during Green Synthesis of SS-ZnO NPs with Aqueous Root Extract of S. schimperiana (a) Zinc(II) acetate dehydrate solution; (b) Aqueous root extract of S. schimperiana ; (c) SS-ZnO NPs

Our results align with previous reports of Chai et al. [29], observed similar colour changes from colourless to light brown, confirming the formation of zinc oxide nanoparticles (ZnO NPs) from Hibiscus rosa-sinensis flower extract through bioreduction of Zn²⁺ ions to Zn⁰.

UV-Vis Spectroscopy of SS-ZnO NPs

The optical properties of SS-ZnO NPs in solutions were measured using UV-Vis spectroscopy in the wavelength range of 200–800 nm. As shown in Figure 2, the UV-Vis spectra of SS-ZnO NPs showed a wide absorption range between 300 to 400 nm, with a distinct peak at 330 nm, which corresponds to the oscillation of free conduction band electrons in zinc oxide nanoparticles. The results align with findings of Mongy and Shalaby [30], who reported that ZnO NPs prepared from Rhuscoriaria fruit aqueous extract exhibited maximum absorbance at approximately 359 nm.

Figure 2.

UV-Vis Analysis of SS-ZnO NPs Prepared from Aqueous Root Extract of S.schimperiana

FT-IR Analysis

The FT-IR spectrum provides information about the functional groups present in the aqueous root extract of S.schimperiana responsible for the bio-reduction, stabilization, and synthesis of SS-ZnO NPs (Table 3).

Table 3.

FT-IR Chemical Finger Printing and Vibrations of SS-ZnO NPs from Aqueous Root Extract of S.schimperiana

Wave number (cm⁻¹)	Functional Group / Bond	Vibration Mode
3653	O-H (free hydroxyl group)	Stretching (sharp, strong)
3481	N-H (amines)	Stretching (broad for O-H)
2494	S-H (thiols)	Stretching (medium to weak)
2181	C≡C (alkynes) / C≡N (nitriles)	Stretching (sharp)
2032	C≡C (conjugated alkynes)	Stretching (medium, sharp)
991	=C-H (alkenes)	Out-of-plane bending (medium)
445	Zn-O(Zinc-Oxygen bond)	Stretching (lattice vibrations)

Figure 3, show the FTIR spectrum of SS-ZnO NPs, indicating the presence of characteristic stretching frequency peaks at 3653, 3481, 2494, 2181, 2032, 991, and 445 cm⁻¹, corresponding to their respective functional groups: O-H (hydroxyl group), N-H (amines or amides), S-H (thiols), C≡C (alkynes) / C≡N (nitriles), C≡C (conjugated alkynes), C=O (carbonyl in conjugation) / C=C (aromatic or alkenes), =C-H (alkenes), and Zn-O (zinc-oxygen bond). These functional groups on the surface of SS-ZnO NPs may be responsible for the bio-reduction and stabilization of Zn²⁺ ions to Zn⁰ and help prevent the agglomeration of SS-ZnO NPs.Similar results were reported by Mongy and Shalaby [30] for synthesis of R. coriaria fruit extract-mediated ZnO NPs. Our findings also supported by previous reports of Ifeanyichukwu et al. [31].

Figure 3.

FT-IR Spectrum of Green Synthesized SS-ZnO NPs from Aqueous Root Extract of S.schimperiana by potassium bromide (KBr) pellet method and, scanned wave number range of 400- 4000 cm⁻¹

XRD Analysis

The XRD patterns of SS-ZnO NPs are shown in Figure 4, showed characteristic peaks at 2θ values of 31.7º, 34.4 º, 36.2º, 47.5 º, 56.6 º, 62.8º, 67.9º and 72.5º corresponding to the lattice planes (100), (002), (101), (102), (110), (103), (112), and (201) respectively. These diffraction peaks align with hexagonal wurtzite crystalline structure of zinc oxide (ZnO), as confirmed by the JCPDS card No. 36-1451. The calculated average crystallite size, based on the Debye-Scherer equation, yielded an average value of 55.4 nm, indicating a relatively small crystallite size for SS-ZnO NPs.

Figure 4.

XRD Spectra of SS-ZnO NPs from Aqueous Root Extract of S. schimperiana Recorded in the 2θ Range of 10°–80° Using Cu Kα Radiation (λ = 1.5406 Å).

Furthermore, the XRD pattern of SS-ZnO NPs agrees with Faisal et al.,[32], who reported that zinc oxide nanoparticles synthesized from aqueous fruit extracts of Myristica fragrans also exhibited peaks corresponding to the (100), (002), (101), (102), (110), (103), and (112) planes. This comparison further confirms the hexagonal wurtzite structure of ZnO nanoparticles.

Dynamic Light Scattering (DLS) analysis

The particle size distribution of SS-ZnO NPs was determined using a dynamic light scattering (DLS) by measuring light interference based on the Brownian motion of NPs dispersed in a liquid medium. This method measures the size distribution based on the polydispersity index of NPs, which ranges from 0 to 1.

The dynamic light scattering (DLS) analysis of SS-ZnO NPs as shown in Figures 5, revealed a distinct size distribution. SS-ZnO NPs had an average size of 396.2 nm within the same range, with a higher polydispersity index (PDI) of 0.865, suggesting greater size variability. Our findings similar with Faisal et al. [32] reported that ZnO NPs from the aqueous extract of Myristica fragrans was (45.3 nm) with a PDI of 0.5, indicating a more uniform distribution.

Figure 5.

Particle Size Distribution of SS-ZnO NPs from Aqueous Root Extract of S. schimperiana by DLS Analysis was Performed at 25°C Using a 633 nm

Zeta potential analysis

As shown in Supplementary Figure 1, SS-ZnO NPs showed a zeta potential of –28.9 mV, indicating high colloidal stability. The negative zeta potential helps prevent agglomeration, by electrostatic repulsion. However, with a value close to the stability threshold range of -20 mV to +20 mV, the colloidal stability may decrease over time. Our findings are consistent with Kaliyaperumal et al. [33], who reported a zeta potential of -27.1 mV for ZnO NPs from Phyllanthus emblia extract, equally indicating stable dispersions.

EDAX Analysis

As shown in Supplementary Figure 2, the EDX spectrum of SS-ZnO NPs, showed characteristic peak for Zn at 1.01 keV, while the oxygen peak at 0.52 keV, confirm the presence of Zn-O bonding and the formation of ZnO nanoparticles.

Our results were similar with previous reports of Jaishi et al. [34],who reported that ZnO NPs synthesized using the aqueous bark extract of A. nepalensis D. Don displayed a strong EDX peak for zinc at approximately 1 keV, verifying the ZnO composition.

SEM Analysis

As shown in Supplementary Figure 3, the SEM images of SS-ZnO NPs, revealed a pseudo-spherical morphology with a rough and rocky surface appearance. The nanoparticles exhibited irregularities and flake-like structures, indicative of partial aggregation during green synthesis. The rough and flaky morphology may be attributed to the ZnO nuclei fusing under the influence of bioactive compounds from the extract. Ahmad and Kalra [35] reported similar results for ZnO nanoparticles prepared with using the leaf extract of Euphorbia hirta, which also showed a pseudo-spherical shape with a few aggregates.

TEM Analysis

As shown in Supplementary Figure 4, TEM analysis of SS-ZnO NPs showed a pseudo-spherical morphology, consistent with negligible aggregation, with size ranging from 13 nm–38.6 nm and an average size of 22.65 nm. These findings demonstrate the ability of S. schimperiana root extract to produce nanoparticles with precise morphologies and sizes, demonstrating its potential for green nanotechnology applications.

Jamdagni et al. [36] reported that ZnO NPs synthesized using Nyctanthes arbor-tristis flower extract had a size range of 12–32 nm.

Cytotoxicity Analysis of SS-ZnO NPs on MDA MB-231 Cell Lines

Preliminary MTT assay results showed that SS-ZnO NPs and the aqueous root extract of Sida schimperiana (SS-AQ) exhibited dose-dependent selective cytotoxicity against MDA-MB-231 breast cancer cells and normal L929 fibroblast cells over 24 hours. As shown in Supplementary Figure 5, SS-ZnO NPs showed significantly higher selective anticancer activity against MDA-MB-231 cells (p < 0.01) compared to SS-AQ. The highest inhibition of 62.44% and 58.11% were observed respectively at a dosage of 100 µg/mL with significance of (P < 0.01) with IC₅₀ values of 45.28µg/mL and 50.16µg/mL against MDA-MB-231.

Cisplatin used as a standard showed cytotoxicity between 18.15% to 91.74% at doses of 2.5–40 µg/mL with an IC₅₀ value of8.34 µg/mL. Both SS-ZnO NPs and SS-AQ showed minimal cytotoxicity of in the range of 5.22% –15.96% and 5.55%-18.31%respectivelyat concentrations of 6.25 – 100µg/mL with IC₅₀of >100indicating their biocompatibility with non-cancerous cells. The selective cytotoxic activity of SS-ZnO NPs and SS-AQ against MDA-MB-231 cells followed this order:

SS-ZnO NPs> SS-AQ

The selective index (SI) values of SS-ZnO NPs, and SS-AQ against MDA–MB-231 cells, compared to L929 cells, were 2.22, and 1.99, respectively (Table 4). SS-ZnO NPs showed a cytotoxic concentration (CC50) >100 µg/mL in L929 cells and an IC₅₀ of 45.28 µg/mL in MDA–MB-231 cells, resulting in an SI of 2.2 This indicates that SS-ZnO NPs exhibited preferential cytotoxicity toward MDA–MB-231 cells, sparing normal cells. The high SI of SS-ZnO NPs confirms their anticancer potential against aggressive triple-negative breast cancer, likely due to their optimal size, shape, charge, and secondary metabolites like phenolic compounds, tannins, and flavonoids from Sida schimperiana. The factors such as enhanced cellular uptake, oxidative stress, ROS generation, mitochondrial damage, contribute apoptosis in MDA–MB-231 cells [37].

Effect of SS-ZnO NPs on Cells Morphology

Morphological analysis of MDA-MB-231 cells treated with SS-ZnONPs revealed significant apoptosis-related changes in a dose-dependent manner. Cells were exposed to ½ IC₅₀ (20 µg/mL), IC₅₀ (40 µg/mL), and 2× IC₅₀ (80 µg/mL) for 24 hours. As shown in Supplementary Figure 6, cellular shrinkage, membrane blebbing, detachment, and apoptotic body formation were prominent at IC₅₀ and 2× IC₅₀, confirming apoptosis and SS-ZnONPs’ cytotoxicity.

Discussion

Medicinal plants have extended played important role in traditional medicinal system for treatment of various cancers. Earlier research studies demonstrated that the isolated bioactive compounds Vinblastine, Capsaicin, Resveratrol and Lycopene from various medicinal plants showed potential anticancer activity by targeting and modulating different enzymatic, molecular mechanism and signal transduction pathways [38]. Hence in the present study roots of S.schimperiana were selected based on traditional use in Ethiopia medicinal system and earlier phytochemical studies demonstrated that presence of various secondary metabolites are capable of exhibiting various pharmacological activities, such as modulating cancer-related pathways in a line with significant ethno botanical evidence from Gedeo Zone and Amhara region, Ethiopia [39].

In this study first time we successfully green-synthesized high stable, pseudo-spherical shape, with average size of 22.65 nm from the aqueous crude root extract of S.schimperiana (SS-AQ). The results evident that SS-ZnONPs’ demonstrated significant dose-depended selective cytotoxicity against MDA-MB-231 cell lines with promising IC₅₀ of 45.28 µg/mL while aqueous crude root extract of S.schimperiana (SS-AQ) also showed comparable strong selective cytotoxicity against MDA-MB-231 cell lines with IC₅₀ of 50.16 µg/mL. Hence introducing green, synthesized ZnO NPs from green sources like S.schimperiana roots rich in secondary metabolites will have great impact on nano medicine discovery, increased bioavailability, cost effective and less adverse effect compared to currently available chemotherapeutic agents.

ZnO NPs posses exceptional physicochemical properties such as high surface area, abundant surface defects, mechanical stability and enhanced electron mobility that enable integrated in bio medical system, for target drug delivery, carries medicine and facilitate the controlled release of nanomedicine at precise sites [40]. To support our results, different latest publication of green synthesis of ZnO NPs evident that ZnO NPs can exhibit their biological applications as antibacterial, antifungal, antidiabetic, anti-oxidant, anti-inflammatory and anticancer agents. Sishu and Selvaraj, green synthesized ZnO nanoparticle from root aqueous extract Cichorium intybus L produced average size of 26.66 nm with quasi-spherical particles that showed, significant DPPH radical scavenging (60.63 μg/mL), anti-diabetic by inhibiting α-amylase (41.74 μg/mL), showing strong anticancer activity on anticancer A549 lung cancer cell lines (26.56 μg/mL) and biocompatible properties by low hemolysis supporting their biomedical potential [41]. Akhras et al. [42], similarly reported that ZnO NPs prepared from aqueous extract of Artemisia absinthium L. exhibited strong antibacterial and antifungal activities against tested Staphylococcus aureus, Escherichia coli, and Candida albicans.

The present study aligns with existing reports by Mongy and Shalaby [30] who reported that zinc oxide nanoparticles (ZnO NPs) produced from Rhus coriaria fruit extract, showed dose-dependent anticancer activity with IC₅₀ values ranging from 35.04–44.86 μg/mL against MCF-7 cells and 55.54–63.71 μg/mL for MDA-MB-231 cells.In line with these findings, Ahamed et al. [37], reported that ZnO NPs synthesized from Phoenix dactylifera fruit extract showed enhanced anticancer activity against MCF-7 cells compared to pure ZnO NPs, highlighting the potential of plant-derived nanoparticles in cancer therapies, especially for triple-negative breast cancer. This study employed an eco-friendly, phyto-mediated approach to synthesize SS-ZnO NPs using the aqueous root extract of Sida schimperiana (SS-AQ) as a reducing and capping agent. The successful formation of SS-ZnO NPs was confirmed by distinct colour changes in the reaction medium. Phytochemical and FTIR analyses revealed that bioactive compounds such as phenolics, tannins, and flavonoids played a crucial role in reducing Zn²⁺ to Zn⁰, and stabilizing the nanoparticles via surface functionalization. Characterization studies demonstrated that SS-ZnO NPs were crystalline, predominantly pseudo-spherical, with average sizes of 22.65 nm.

In conclusion, the findings of current study confirmed that SS-ZnO NPsexhibited superior selective anticancer activity against MDA-MB-231 cells compared to SS-AQ extract without

effecting normal cell lines highlighting the potential of green-synthesized SS-ZnO NPs as promising biogenic nanotherapeutic agents and drug delivery tool against aggressive breast cancer therapies.

Author Contribution Statement

Asmelash Hagos, Zenebe Hagos and Kamalakararao K conducted the experimental workand acquisition of all the data in Department of Biology and Chemistry. Berihu Tekluu, Tesfay Welderfaeland Naveen Kumar A.Dinvolved in collection of review of literature, draftingmanuscript and editing. Krishna Chaithanya K performedstatistical analysis and organized manuscriptaccording to the submission guidelines of Journal.