We employed a completely randomized design (CRD) with a posttest-only control group to minimize selection bias by ensuring equal treatment assignment probability for all subjects. Healthy and energetic female mice (Mus musculus), aged 8 to 10 weeks and weighing 18 to 25 g, were included in the study.

Mice that refused food/water, died during the study, or were found to be pregnant at any point were excluded from the analysis. The dropout criteria included mice that died prior to the conclusion of the study. The mice were sourced from the Healthy Animal Laboratory in Malang, Indonesia.

Eleutherine bulbosa, a medicinal plant indigenous to Kalimantan, was harvested in amounts totaling 40 kg from the Kalimantan area. The reddish-purple tubers were separated, cleaned, sliced, and dried at 40 °C for 1 week at the Women’s Health Laboratory of Hasanuddin University. The dried tubers were pulverized and macerated with 96% ethanol over 3 periods of 24 hours at the Phytopharmaceutical Laboratory. The filtrate was subjected to filtration, evaporation, and subsequent drying to yield a concentrated dark crimson extract for further pharmacological evaluation.

This study adhered to ethical animal procedures. Breast cancer was induced in 36 BALB/c mice using the carcinogenic substance DMBA. Each mouse weighing 20 g was administered a dose of 1 mg of DMBA per day for 42 days, resulting in a total DMBA dose of 42 mg per mouse (1 mg/d × 42 days = 42 mg). Before administration, DMBA was dissolved in sesame oil to form a 1% solution, and each mouse received 0.1 mL of this solution per day via an oral tube. This procedure aimed to induce breast cancer in vivo as an experimental model and was performed at Satwa Sehat, Malang. The breast cancer model in mice was validated by examining histopathological changes (cell and tissue structure) and using immunohistochemistry (IHC) techniques. In addition, the presence of lumps and tumor masses physically detected in DMBA-induced mice was also an indication of the success of the model.

Experimental groups. This study employed a laboratory experiment utilizing a posttest-only control group design within a completely randomized design framework. This approach was implemented to mitigate selection bias and ensure each subject had an equal probability of being assigned to any treatment group. The study included 36 female BALB/c mice (Mus musculus), aged 8 to 10 weeks and weighing approximately 18 to 25 g. Female BALB/c strain mice that satisfied the inclusion and exclusion criteria were allocated to 2 control groups and 4 intervention groups, with 6 mice in each group. These mice were randomly allocated to groups. The negative control group consisted of healthy mice that were only fed and given water without any treatment. The positive control group consisted of breast cancer model mice that did not receive Eleutherine bulbosa ethanol extract or tamoxifen. Intervention group 1 was given Eleutherine bulbosa ethanol extract at 180 mg/kg daily for 14 days,18 while intervention group 2 was given tamoxifen at 10 mg/kg every 2 days for 7 doses. Intervention group 3 received a combination of Eleutherine bulbosa extract and tamoxifen simultaneously, and intervention group 4 received a combination of tamoxifen followed by Eleutherine bulbosa extract sequentially for 14 days.

Fourier transform infrared spectroscopy analysis. Fourier transform infrared (FTIR) spectroscopy analysis was performed at the Analytical Chemistry and Food Control Laboratory, Faculty of Agriculture, Hasanuddin University, Makassar, to nondestructively identify the functional groups of active chemicals in the concentrated E bulbosa ethanol extract. A droplet of the extract was deposited onto the surface of an attenuated total reflectance (ATR) crystal, and the instrument was scanned across the range of 4000 to 400 cm⁻¹. The resultant spectrum was examined to identify the –OH, –C=O, and –C–O functional groups, which are typically present in flavonoids and phenolic compounds. This technique is rapid, precise, and suitable for phytochemical analysis of plant extracts (Figure 1).

Histopathological examination was performed at the Satwa Sehat Laboratory in Malang using hematoxylin-eosin staining as the conventional method for breast cancer diagnosis. Tissue was acquired via biopsy, preserved in 10% formalin, embedded in paraffin blocks, and finely sectioned using a microtome. Hematoxylin staining highlights cell nuclei, whereas eosin imparts color to the cytoplasm. The data were analyzed microscopically to evaluate cell differentiation, mitotic activity, and invasion patterns, and to ascertain the malignancy grade using the Nottingham approach to inform the treatment plan.

The IHC procedure on the blood plasma of breast cancer mice was performed by collecting blood into an EDTA tube and centrifuging it at 1500g for 10 minutes at 4 °C to obtain plasma. A total of 100 μL of plasma was placed on a glass slide using the cytospin method (600 rpm, 4 minutes) and fixed with 4% paraformaldehyde for 10 minutes. After rinsing with phosphate-buffered saline (PBS), the sample was treated with 0.1% Triton X-100 for 10 minutes for permeabilization and blocked with 5% normal serum for 45 minutes. Next, the slides were incubated with p53 primary antibody overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)–labeled secondary antibody for 1 hour. Staining was performed with DAB substrate until a brown color appeared, followed by staining with hematoxylin contrast dye, gradual dehydration, and permanent mounting. Positive and negative controls, as well as 4 intervention groups, were used to ensure specific results were obtained.

This test was conducted by the Healthy Animal Laboratory in Malang using the enzyme-linked immunosorbent assay (ELISA) method, a quantitative immunological technique used to measure IL-10 levels in biological samples such as serum, plasma, or tissue homogenates. The ELISA method is a sandwich technique, wherein the IL-10 antigen in the sample binds to specific antibodies coated on a microtiter plate, followed by the addition of biotinylated antibodies and streptavidin-HRP enzyme. The enzymatic reaction produces a color change proportional to the concentration of IL-10, which was read using a spectrophotometer at 450 nm (Figure 2). The findings indicated a reduction in IL-10 levels, signifying an increase in the inflammatory response.

This result corresponds with findings in the journal Biomedical Research and Therapy, highlighting the significant function of IL-10 as an anti-inflammatory cytokine in the pathology of chronic illnesses.

In the data analysis using SPSS version 25, the normality test was performed first, as indicated by the results of the Kolmogorov-Smirnov and Shapiro-Wilk tests (P > 0.05), indicating that the data were normally distributed. Next, a homogeneity test was performed, and because the results were not homogeneous, a Brown-Forsythe test was performed to determine whether there were significant differences between the intervention groups. Subsequently, to identify the specific intervention groups that had significant differences, a post hoc Games-Howell test was used, which showed significant differences between the intervention groups.

FTIR analysis of the extract revealed the presence of several functional groups that significantly contributed to its biological activity (Table 1).

The absorption spectra at approximately 3348.85 cm⁻¹ signify the presence of hydroxyl (O-H) groups in phenolic chemicals or alcohols, which are known for their antioxidant properties. The absorption peak at 2927.28 cm⁻¹ signifies aliphatic C-H groups from alkyl chains, whereas the peak at 1649.65 cm⁻¹ denotes carbonyl groups (C=O), typically present in flavonoids, aldehydes, or ketones, which possess significant anti-inflammatory and anticancer properties. The detection of aromatic C=C bonds at 1451.43 cm⁻¹ corroborates the existence of aromatic chemicals such as flavonoids, and the peak at 1055.00 cm⁻¹ signifies C-O groups from alcohols, esters, or glycosides. These findings suggest that the extract contains bioactive chemicals that can be developed as pharmacological agents for cancer therapy.

Histopathological analysis of breast tissue from a murine model of breast cancer using hematoxylin-eosin staining revealed distinct cellular morphological variations among the different treatment groups. The positive control group exhibited predominant and dense proliferation of cancer cells, which displayed distinct malignant characteristics, such as hyperchromatic nuclei, pleomorphism, and an elevated nucleus-to-cytoplasm ratio. The tissue architecture was disordered, exhibiting stromal invasion with few signs of apoptosis. Concurrently, the cohorts administered tamoxifen or Eleutherine bulbosa ethanol extract exhibited a reduction in tumor cell density, expansion of intercellular gaps, and indications of apoptosis, including pyknosis, karyorrhexis, and cytoplasmic vacuolization. The combination of tamoxifen and Eleutherine bulbosa extract exhibited the most significant therapeutic effect, characterized by an extensive area of focal necrosis and a marked decrease in the number of active cancer cells, suggesting the synergistic potential of these agents in suppressing proliferation and inducing tumor cell apoptosis (Figure 3).

Immunohistochemical examination of breast tissue in experimental animals was performed to evaluate the expression of markers associated with breast cancer growth and responses. The tissue examination results showed differences in the responses of each group. In the control group (Figure 4A), there were no signs of cancer; therefore, the staining of cancer markers was almost invisible. In contrast, in the cancer-induced group (Figure 4B), cancer markers were clearly visible, indicating rapid cancer cell growth. After treatment, Eleutherine bulbosa (Figure 4C), tamoxifen (Figure 4D), and Eleutherine bulbosa and tamoxifen (Figure 4E) suppressed cancer growth, although tamoxifen showed a stronger reduction. The best results were observed in the group treated with a combination of tamoxifen and Eleutherine bulbosa (Figure 4F), where cancer marker expression decreased drastically, approaching normal conditions. This indicates that the combination of both is far more effective in combating cancer than when they are used separately.

Results from the ELISA analysis of IL-10 concentrations are presented below.