Antioxidant, Antibacterial and Anti-Proliferative Activities of Opuntia Ficus-Indica L. Nano-Emulsion Against Breast Cancer: An In Vivo and In Vitro Study

INTRODUCTION
Breast cancer is the most common neoplastic malignancy in women worldwide and represents the leading cause of cancer-related deaths among women [1,2]. Although its global incidence continues to rise, prevalence rates vary significantly across different countries [3]. While developed nations report the highest frequency of breast cancer, studies indicate a sharp increase in prevalence in developing countries [4]. 
One of the primary drivers of cancer development is exposure to environmental factors, which can trigger mutations and genetic alterations, ultimately leading to malignancy [5]. Notably, research suggests that dietary choices play a crucial role in cancer prevention, as certain antioxidant-rich foods may help reduce cancer risk [6].The cactus (Opuntia ficus-indica L.) is widely used as both a food source and medicinal plant in the Americas, with at least 84 of the approximately 200 known Opuntia species found in Mexico [7,8]. The sweet edible fruit of this cactus, commonly called prickly pear (Cactaceae family) [9], has been traditionally used as a remedy [10] and contains an extensive array of bioactive compounds including sugars, ascorbic acid, betalains, fat-soluble pigments, polyphenolic compounds, and flavonoids [11]. Modern research demonstrates that cactus pear fruits serve as a rich source of phytonutrients with multiple health benefits, exhibiting antioxidant properties, neuroprotective and cardioprotective effects, anti-inflammatory activity, antidiabetic potential [12], as well as anti-chromosomal aberration [13] and anti-mutation effects [14]. Furthermore, the fruit demonstrates protective effects on red blood cell membranes [15] and against acute gastric lesions [16], while also improving platelet function [17] and showing cancer-preventive properties [8].Given the rising prevalence of breast cancer in Iraq, its increasing incidence over the past two decades, and the high mortality rate among patients—coupled with the accessibility, affordability, and widespread consumption of herbal products—this study aimed to evaluate the therapeutic potential of Opuntia spp. against breast cancer cells. The MCF-7 cell line, a well-established model for in vitro breast cancer research, was derived in 1970 from the breast tissue of a 69-year-old woman with breast cancer [18] and serves as an ideal system for such investigations.
This study focused on the antioxidant, antibacterial, and anti-proliferative properties of NE-OFI (nanoparticle-enhanced Opuntia ficus-indica extract) against the MCF-7 cell line, while also conducting a comparative analysis of its in vivo and in vitro efficacy in a breast cancer animal model.

MATERIALS AND METHODS
Herbal extraction protocol
Fresh Opuntia ficus-indica (OFI) fruits (500 g) were thoroughly washed with tap water to remove surface impurities. Following washing, the outer skin was partially removed, and the fruits were sliced into thin pieces. The samples were then dried at 60°C for 24 hours in a light-protected environment using a drying oven (Shimiazma, Iran). The dried material was ground into a fine powder, after which a hydroalcoholic extract was prepared by soaking 100 g of OFI powder in 500 mL of absolute ethanol for 72 hours at room temperature. The mixture was periodically agitated to enhance extraction efficiency. After 3 days, the extract was filtered through Whatman No. 1 filter paper to remove particulate matter, followed by concentration under reduced pressure using a rotary evaporator (40–50°C).

Preparation of Opunita Ficus-Indica extract Nanoemulsions
To prepare the nanoemulsion, 1% (w/w) herbal extract was combined with 1% (w/w) soybean oil and 40% (w/w) Tween 80 in deionized water. The aqueous phase was gradually added to the oil phase at a rate of 2 mL/min under homogenization (28 ± 2 °C) using a conventional ultrasonic device, maintaining a 45:55 (aqueous:oil) phase ratio. After complete addition, homogenization continued for an additional 15 minutes to ensure uniform dispersion and stable nanoemulsion formation.

Characterization of nanoparticles
Dynamic Light Scattering (DLS) 
The particle size distribution of NE-OFI was characterized using dynamic light scattering (DLS) with a Horiba SZ-100 nanoparticle analyzer (Kyoto, Japan).

Field Emission Scanning Electron Microscopy (FE-SEM) analyses
The size and morphology of NE-OFI were characterized using FE-SEM (TESCAN, Brno, Czech Republic). Prior to analysis, NE-OFI was centrifuged at 10,000 rpm for 15 min at 25°C. The pellet was washed three times with ultrapure water and air-dried overnight. For FE-SEM imaging, the sample was deposited onto a carbon-coated copper grid and sputter-coated with platinum to enhance conductivity.

Determination of the main bioactive compounds of Opunita Ficus-Indica extract
Bioactive compounds in the OFI extract were identified after clarification (12,000 × g, 15 min, room temperature). Total flavonoid content was quantified using a colorimetric method [19].

DPPH Radical Scavenging Assay
A 0.0789 mM DPPH solution was prepared by dissolving 7.89 mg DPPH in 100 mL of 99.5% ethanol, followed by 2-hour incubation in the dark. For the assay, 800 μL Tris-HCl buffer (pH 7.4) was mixed with 1,000 μL DPPH solution, and 200 μL NE-OFI was added. The mixture was incubated at 25°C for 30 min, and absorbance was measured at 517 nm using a spectrophotometer. A blank control (800 μL Tris-HCl + 1,200 μL ethanol) was used for baseline correction. Radical scavenging activity (%) was calculated as:
Inhibition ratio (%) = (A1 - A2) × 100∕A1 
where A1 shows the absorbance of blank sample and A2 shows the absorbance of NE-OFI solution.

MIC/MBC, Disk Diffusion
The antibacterial properties of NE-OFI were evaluated against Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) using disk diffusion and MIC/MBC assays. Bacterial suspensions were adjusted to 0.5 McFarland standard (10⁸ CFU/mL) and diluted 1:100. Then, 15 mL of Mueller-Hinton agar and 2 mL of bacterial suspension were plated per petri dish. After 5 minutes of incubation, excess liquid was removed, and 6 mm sterile filter paper discs (Whatman No. 1, Oxoid) impregnated with 15 μL of NE-OFI were placed on the inoculated plates. The plates were refrigerated at 4°C for 2 hours to allow diffusion, followed by incubation at 37°C for 24 hours. The diameter of inhibition zones was measured in millimeters.

Preparation and proliferation of MCF-7 (Human breast cancer cell line) 
The MCF-7 cell line (ATCC: HTB-22) was obtained from the Pasteur Institute of Iran Cell Bank. Cells were cultured in DMEM (Gibco™, Cat. No. 41965039) supplemented with 10% FBS (Gibco, Cat. No. 10082147, USA) and maintained in a humidified incubator at 37°C with 5% CO₂ until reaching 80-90% confluency. The medium was replaced every three days. For cell counting, trypsinized cells were centrifuged at 300 × g for 5 minutes, and the resulting pellet was resuspended in 1 mL of fresh DMEM. A 1:9 dilution was prepared by mixing 10 μL of cell suspension with 80 μL of DMEM. Subsequently, 10 μL of the diluted suspension was loaded into a hemocytometer, and viable cells were counted in four corner quadrants using a light microscope. Cell density (cells/mL) and viability were calculated based on these counts.

Cell Viability assay; MTT protocol 
MTT powder (Sigma, UK) was mixed at a concentration of 5 mg/ml in PBS (Merck, Germany) as a stock solution. The cells were seeded in each well of a 96-well plate and treated with NE-OFI (50, 100, 200, 400, 800 μg/ml) in triplicate, with approximately 10,000 cells per well. After treatment, the plate was placed in an incubator at 37°C for 72 hours to assess the toxicity of the different concentrations. 100 μl of MTT working solution with 0.5 mg/ml concentration was added to each well. After 4 hours incubation; formazan crystals were produced. Tetrazolium salts were disolved in 100 μL of DMSO (Merck, Germany) and changed to purpale color.  The optical density of the treated cells was assessed using a spectrophotometer at 570 nm wavelength.

Apoptotic assay by flow cytometry 
For apoptosis analysis by flow cytometry, approximately 100,000 cells were aliquoted into a microtube and resuspended in 500 μL of 1× Binding Buffer from the apoptosis detection kit. An unstained cell sample served as the negative control for instrument calibration. The cell suspension was then stained with 5 μL of Annexin V-FITC (BD Biosciences, USA) and incubated at 4°C in the dark for 15 minutes. After incubation, 1 mL of 1× Binding Buffer was added to each tube, followed by centrifugation at 1,500 rpm for 5 minutes. The supernatant was carefully aspirated, and the cell pellet was resuspended in 500 μL of fresh 1× Binding Buffer. Immediately prior to analysis, 3 μL of propidium iodide (PI) was added to each sample. Flow cytometry was performed using a BD FACSCalibur flow cytometer, and data were acquired using the manufacturer’s software. Data analysis was conducted using FlowJo software (version X.X, Tree Star) for graphical representation and Prism 5 (GraphPad Software) for statistical analysis.

Antioxidant enzymes assay and lipid peroxidation level analysis
Assessment of Lipid Peroxidation via Malondialdehyde (MDA) Quantification
Lipid peroxidation levels in NE-OFI-treated MCF-7 cells (IC50 dose) were quantified by measuring malondialdehyde (MDA) concentration using a ZellBio MDA assay kit (ZellBio GmbH, Germany). Briefly, 125 μL of cell homogenate was vigorously mixed with 50 μL of Tris-buffered saline (TBS) and 125 μL of trichloroacetic acid (TCA) containing 0.01% butylated hydroxytoluene (BHT) for protein precipitation. After centrifugation (1,000 × g, 10 min, 4°C), the supernatant was reacted with 40 μL of 0.6 M HCl and 160 μL of thiobarbituric acid (TBA) solution (prepared in Tris buffer) and heated at 80°C for 10 min. MDA-TBA adduct formation was quantified spectrophotometrically at 532 nm. A standard curve was generated using five MDA concentrations (0–20 μM). MDA concentrations in samples were calculated using a molar extinction coefficient of 156 mM⁻¹cm⁻¹ and expressed as nmol MDA per mg protein.

Analysis of Antioxidant Enzyme Activities
The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were analyzed using commercial assay kits (ZellBio GmbH, Germany). SOD activity was measured via the ZellBio SOD assay kit, while CAT activity was determined colorimetrically (ZellBio CAT assay kit) by monitoring H₂O₂ decomposition at 240 nm. GPx activity was quantified as μmol oxidized GSH/min/mg protein at 25°C using the ZellBio GPx assay kit. Total protein concentration in cell lysates was determined by the Lowry method [20] with bovine serum albumin (BSA) as the standard.

Gene expression analysis
Total RNA was isolated from NE-OFI-treated MCF-7 cells using TRIzol™ reagent (Thermo Fisher Scientific, MA, USA) and quantified using a NanoDrop spectrophotometer. RNA integrity was verified (A260/A280 ratio ≥1.8) prior to storage at −80°C. Complementary DNA (cDNA) was synthesized from 1 μg RNA using a reverse transcription kit (manufacturer’s protocol). Quantitative real-time PCR (qPCR) was performed on an ABI StepOnePlus™ system with SYBR® Green Master Mix (Takara Bio, USA) to analyze expression of apoptosis-related genes (Bax, Bcl2, Caspase3) normalized to Gapdh (housekeeping gene). Gene-specific primers (Table 1), designed using GeneRunner and validated by NCBI BLAST, were employed. The thermal profile consisted of: (1) initial denaturation (95°C, 5 min); (2) 40 cycles of denaturation (95°C, 30 s), primer annealing (58°C, 30 s), and extension (72°C, 10 s). All reactions were performed in quintuplicate. Relative gene expression was calculated via the 2−ΔΔCt method using REST software.

Animal keeping conditions
 All animal experiments were conducted under controlled environmental conditions (23 ± 3°C, 50 ± 10% humidity) with ad libitum access to food and water and a standardized 12-hour light/dark cycle. Twelve male BALB/c mice (20 ± 2 g body weight) obtained from the Pasteur Institute Laboratory Animal Center were randomly divided into six groups: (1) tumor model group receiving a subcutaneous injection of 5×10⁵ 4T1 breast cancer cells in 50 μL PBS; (2) NE-OFI low-dose treatment group (200 mg/kg/day, oral gavage, 4 weeks); (3) NE-OFI high-dose treatment group (400 mg/kg/day, oral gavage, 4 weeks); and (4) positive control group receiving doxorubicin (1.5 mg/kg, intraperitoneal injection). Animals was cared for in accordance with  the University of Babylon-College Science for Woman code of practice for the care and use of animals for scientific purpose (Approval No: IR-UU-AEC-1401-001).

Sample collection
Four weeks following NE-OFI treatment, mice were humanely euthanized by cervical dislocation. Tumor tissues were promptly excised, with portions fixed in 10% neutral buffered formaldehyde for histopathological analysis and remaining aliquots flash-frozen at -80°C for molecular studies.

Histopathological examination
Fixed tumor tissues underwent standard dehydration through a graded ethanol series (70%, 80%, 90%, and 100%) with 50-minute immersions at each concentration. Tissues were then cleared in xylene (Merck, Cat. #108298) through two changes (50 minutes each) and embedded in paraffin using two successive baths (50 minutes each). Following processing, 5 μm sections were cut using a rotary microtome, mounted on glass slides, and prepared for hematoxylin and eosin (H&E) staining and subsequent microscopic evaluation. 

Statistical analysis
This randomized controlled trial was conducted with three independent experimental replicates. All statistical analyses were performed using GraphPad Prism (version 8.4.3). Normality of data distribution was verified using the Shapiro-Wilk test. One-way ANOVA followed by LSD post hoc testing was employed for group comparisons, with P-values < 0.05 considered statistically significant.

RESULTS AND DISCUSSION
Phytochemical contents analysis of OFI fruit extract 
The results of the phytochemical analysis of the hydroalcoholic extract of OFI fruit confirmed the presence of various bioactive compounds, including phenolic compounds, sugar derivatives, flavonoids, proteins, fiber, and fats (Fig. 1). As shown in Fig. 1, the extract contained several antioxidant compounds, such as quercetin, gallic acid, and vanillic acid. Additionally, other phenolic acids, including p-coumaric acid, caffeic acid, and cinnamic acid, were identified as components of the extract. Benzoic acid and ferulic acid were also detected during the phytochemical analysis of OFI. The remaining constituents of the OFI extract included chlorogenic acid and m-coumaric acid. Trace amounts of sinapic acid and syringic acid were also identified in the extract.

DPPH assay
The antioxidant potential of the OFI fruit extract was assessed using the DPPH radical scavenging assay (Fig. 2). The results demonstrated that the extract exhibited significant antioxidant activity, with an inhibition rate of 92.68 ± 1.62% in vitro. This finding aligns closely with the observations of Roghelia and Panchal [21], who reported comparable free radical scavenging activity in OFI extracts.

Characteristics of Ginger Nano-emulsion
The characterization results of the optimized nano-emulsion formulation (NE-OFI) are presented in Fig. 3. All NE-OFI formulations met the required organoleptic standards for nano-emulsions, including color, odor, clarity, and viscosity. Furthermore, the particle size of all NE-OFI formulations fell within the specified range of 90–100 nm, confirming the formation of a nano-emulsion with uniform particle size and a narrow size distribution (Fig. 3).

Antimicrobial effect of NE-OFI on Escherichia coli
As illustrated in Fig. 4 and Fig. 5, the minimum inhibitory concentration (MIC) of NE-OFI against E. coli and S. aureus was determined to be 80 µg/mL and 160 µg/mL, respectively. The largest zone of inhibition was observed for E. coli, while the smallest inhibition zone corresponded to the S. aureus strain (Table 2).
Anticancer effect of NE-OFI on MCF7
Evaluation of cell viability using the MTT assay revealed that NE-OFI exhibited a dose- and time-dependent effect on MCF-7 cell proliferation. As demonstrated in Fig. 6, lower concentrations (50–100 µg/mL) had minimal impact on cell survival, whereas higher doses (200–800 µg/mL) significantly reduced viability, inducing cell death. The half-maximal inhibitory concentration (IC50) was determined to be 230 µg/mL after 72 hours of treatment.

Effect of NE-OFI on apoptosis in MCF-7 cell
Flow cytometric analysis using the Annexin V-FITC/propidium iodide (PI) assay revealed a significant alteration in the cellular profile of MCF-7 cells following treatment with 230 μg/mL NE-OFI for 72 hours (Fig. 7). Both NE-OFI and doxorubicin (Dox) at their IC50 concentrations induced a statistically significant increase (P < 0.05) in total apoptosis compared to the control group. Notably, treatment with the IC50 concentration of NE-OFI resulted in 68% of cells undergoing apoptosis (Fig. 7).

Determination of antioxidant activity and lipid peroxidation of NE-OFI
Fig. 8 presents the comparative levels of antioxidant enzymes (CAT, SOD, and GPx) among the control group and cell groups treated with NE-OFI (230 μg/mL) and doxorubicin (5 μM). The untreated control group demonstrated significantly higher (p ≤ 0.05) CAT, SOD, and GPx activities compared to treated groups (Fig. 8A-C). Conversely, lipid peroxidation analysis revealed a significant increase (p ≤ 0.05) in MDA levels in MCF-7 cells following 72-hour treatment with either NE-OFI (230 μg/mL) or doxorubicin (5 μM) (Fig. 8D).

Bax, Caspase3 and Bcl2 mRNA expression in NE-OFI treated MCF7 cell lines 
Quantitative PCR analysis demonstrated that NE-OFI treatment significantly modulated apoptosis-related gene expression in MCF-7 cells, with a 5.56 ± 0.74-fold upregulation (p < 0.05) of pro-apoptotic Bax mRNA and a 2.34 ± 0.06-fold increase (p < 0.05) in Caspase 3 mRNA compared to untreated controls, while simultaneously causing significant downregulation of anti-apoptotic Bcl-2 expression (Fig. 9A-C), collectively indicating NE-OFI’s potent activation of apoptotic pathways.

Tumor growth inhibition after 28 days treatment with NE-OFI treatment
Longitudinal monitoring of tumor dimensions at 7-day intervals revealed that both NE-OFI (400 mg/kg) and doxorubicin (DOX) monotherapy induced maximal tumor growth inhibition (TGI) of 33%, while combination treatment demonstrated significantly enhanced efficacy (40% TGI, p < 0.05 versus control) (Figs. 10-11).

Effect of NE-OFI on tumor tissue cell death by histopathological assessment
Histopathological analysis revealed that tumors from mice treated with NE-OFI (400 mg/kg) showed reduced inflammatory cell infiltration and decreased necrotic areas compared to the control group, which exhibited increased infiltration of granulocytes (neutrophils) (Figs. 12-13). Additionally, the destructive nuclear fragmentation process, known as karyorrhexis—a hallmark of late apoptosis, senescence, or cell death—was observed following NE-OFI (400 mg/kg) and DOX treatment. Microscopic examination also revealed the presence of vacuoles and nuclear pigments, as well as altered mitotic properties, in the NE-OFI-treated group. Furthermore, both NE-OFI and DOX significantly reduced cell proliferation potential relative to other groups (Figs. 12-13).
Breast cancer remains one of the most prevalent malignancies affecting women worldwide [22]. Given the established link between tumor progression, inflammation, and oxidative stress, compounds with anti-inflammatory or antioxidant properties may exhibit anticarcinogenic effects through pharmacological and biochemical mechanisms. These compounds—particularly those with antioxidant and anti-inflammatory activities—appear to play a role in anticarcinogenic and anti-mutagenic processes [23]. Numerous studies have demonstrated that bioactive components derived from OFI extract can inhibit cancer cell proliferation and induce apoptosis [24].
Research on various parts of Opuntia—including prickly pear fruits, seeds, peels, stems, cladodes, and roots—has shown that these plant components promote cell death in malignant cell lines [16]. For instance, Antunes-Ricardo et al. (2014) demonstrated the cytotoxic effects of purified isorhamnetin glycosides and cladode flour extracts from O. ficus-indica (var. Jalpa), which induced apoptosis in human colon cancer cells (Caco-2 and HT-29). Notably, these extracts exhibited selective anti-apoptotic effects against drug-resistant cancer cell lines while showing no toxicity toward normal fibroblast cells (NIH 3T3).These researchers demonstrated that the glycosylation pattern of purified isorhamnetin glycosides and cladode flour extract exhibited greater cytotoxic effects on Caco-2 and HT-29 cells compared to normal controls. The induction of apoptosis via Bax and Caspase-3 gene activation plays a critical role in the cytotoxic effects of O. ficus-indica on cancer cells [25] examined the impact of O. ficus-indica fruit aqueous extract on the proliferation of Caco-2 human colon cancer cells, revealing its epigenomic effect on the p16INK4A tumor suppressor through promoter demethylation and subsequent p16INK4A upregulation. Similarly, Sreekanth et al. [26] reported that betanin from O. ficus-indica fruits suppresses K562 cell proliferation by activating the intrinsic apoptotic pathway.
This study represents the first in vitro and in vivo evaluation of NE-OFI for anticancer activity. MCF-7 cells treated with NE-OFI for 72 hours exhibited dose-dependent anti-proliferative effects, with the highest phenolic and kaempferol content correlating with maximal efficacy. Notably, organic acids derived from O. ficus-indica—including malic, quinic, and aconitic acids—are predominantly concentrated in peels and cladodes. Further research is needed to compare phenolic levels across flowers and seeds.
Quinic acid (C7H12O6), a cyclohexanecarboxylic acid derivative, is a key metabolite in the biosynthesis of aromatic compounds (e.g., flavonoids, phenolic acids) in plants and mammals [27]. Beyond antibacterial effects, D-(-)-quinic acid exhibits antioxidant, immunomodulatory, and cytotoxic properties [28]. Phenolic compounds have recently gained significant attention as potent anticancer agents due to their ability to modulate apoptosis, suppress cell proliferation, and influence critical carcinogenesis processes such as angiogenesis, differentiation, and cell invasion [29]. These metabolites include hydroxybenzoic acids (derived from benzoic acid) and hydroxycinnamic acids (characterized by aromatic ring substitutions), both of which are subclasses of phenolic acids [30]. Among these, hydroxycinnamic acid derivatives—such as cinnamic acid and p-coumaric acid—are notable polyphenolic constituents. OFI also contains simple esters of quinic acid or glucose, with cinnamic, chlorogenic, coumaric, and ferulic acids being the most abundant hydroxycinnamic acids. Minor constituents include caffeic, sinapic, and dimethoxycinnamic acids [27].
Nanomedicines, defined as nanoscale drug carriers, enable targeted delivery of therapeutic agents to specific tissues while facilitating controlled release over extended periods [31]. These systems enhance drug solubility and bioavailability. Nanoemulsions, thermodynamically stable and homogeneous, consist of aqueous phases, surfactants, and oil phases [32]. Typically, 20–200 nm in size, they are classified as oil-in-water (o/w) or water-in-oil (w/o) systems, with particle size dependent on synthesis and homogenization methods.
The present study demonstrated that NE-OFI exhibits antibacterial activity against E. coli and Pseudomonas aeruginosa, along with antiproliferative and pro-apoptotic effects on MCF-7 breast cancer cells. Notably, NE-OFI showed comparable efficacy to Dox in inhibiting tumor growth after 28 days of oral administration. These effects may be attributed to bioactive phenolics in OFI, which are increasingly recognized for their anticancer potential. Future studies should evaluate NE-OFI against other cancer cell lines and in vivo models to further assess its tumor growth inhibition potential. 

CONCLUSION
This pioneering study demonstrates the anticancer potential of NE-OFI through both in vitro and in vivo investigations. At 800 µg/mL, NE-OFI exhibited 92.68 ± 1.62% antioxidant activity, while its minimum inhibitory concentrations were determined to be 80 µg/mL for E. coli and 160 µg/mL for Pseudomonas aeruginosa. Treatment with 230 µg/mL NE-OFI significantly upregulated pro-apoptotic genes (Bax and Caspase-3) while downregulating BCL-2 expression in MCF-7 cells compared to untreated controls. Furthermore, this concentration induced oxidative stress characterized by increased reactive oxygen species and decreased antioxidant enzymes. Most notably, NE-OFI promoted significant apoptotic cell death at both 230 µg/mL (in vitro) and 400 mg/kg (in vivo), confirming its dose-dependent antitumor efficacy against breast cancer cells.

ACKNOWLEDGMENTS
The authors are also thankful to the Institute of Histogenotech Company for providing the technical support.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.