Apparatus and reagentsApparatus

The current study utilized a range of equipment, including a biosafety cabinet of grade (class 3), carbon dioxide incubator (level 3), water bath, and various culture flasks (25, 50, 75, 100, 125, 150, and 175 mL). Additionally, multi-well plates with 6, 12, 24, 48, and 96 wells were used. The study also employed pipettes, (single and multi) pipette channels, pipette tips of various volumes, filter units (500 mL and 1 L), 96-well plates, cryovial tubes, and falcon tubes (15, 30, and 50 mL). Furthermore, a centrifuge, − 80 °C freezer, nitrogen tank, automated cell counter, cell counter slides, micropipette, and a refrigerator maintained at 2–4 °C were all used during the experimental procedures for data generation in this study.

Reagents

The study used a variety of reagents including Hepes buffer, DMEM, RPMI, fetal bovine serum (FBS), penicillin–streptomycin solution, hemocytometer, trypsin, l-glutamine, and sodium bicarbonate. In addition, two prostate cancer cell lines (DU145 and LNCaP) were utilized. Furthermore, other key reagents included liquid nitrogen, carbon dioxide gas, phosphate-buffered saline (PBS), and the chemotherapy drug doxorubicin, dimethyl sulfoxide, distilled water, methanol, and from R. tridentata tuber extracts were all used.

Plant collection

The plant R. tridentata root tubers used in this experimental study were carefully collected from the slopes of Mount Elgon, a sub-region renowned for its rich biodiversity and unique ecosystem [23]. The plant was identified by a taxonomist at the Makerere University Herbarium laboratory and assigned a voucher number, KA051, which was deposited therein. These were specifically collected from the slopes of Mount Elgon in Sironko and Bulambuli districts. To ensure the integrity and authenticity of the root tubers, they were washed with running tap water, cut into small pieces, air-dried, and ground into a coarse powder using a hammer mill (Model BRONICA).

Serial extraction process and storage

This study employed a serial extraction protocol based on previous work by Ngouana et al. [24] with modifications. The serial extraction used water and methanol (1%) as solvents due to their differing polarities, allowing for the extraction of compounds with varying degrees of polarity [25]. The serial extraction consisted of the following steps: (1) Methanol extraction (1%): 500 g of powdered plant material underwent maceration in 1500 mL of methanol for 72 h at 25 °C. After filtration and concentration via rotary evaporation (50–60 °C), 44 g of extract were obtained, yielding 8.8%. (2) Aqueous extraction: The residue was air-dried and extracted with 2 L of distilled water (previously boiled and cooled) for 24 h. Following filtration and vacuum concentration (60–70 °C), 22.5 g of aqueous extract were obtained, yielding 4.5%. (3) Storage: The dried extracts were stored in a refrigerator at − 20 °C to preserve potency and integrity for later use.

Preparation of extract concentrations

To create the stock solution, aqueous and methanol extracts of 0.5 g each were dissolved in 5 mL of their respective solvents. The resulting solution was diluted by a factor of five to achieve a concentration of 0.1 g/mL, which is equivalent to 100,000 µg/mL (since 0.1 g/mL × 1,000,000 = 100,000 µg/mL). From this solution, subsequent concentrations of drugs used in the study interventions were prepared through serial dilution, based on the formula M1V1 = M2V2.

Spectrophotometric identification and quantificationPrinciple of spectrophotometry

Phytochemicals in the R. tridentata were identified using spectrophotometry, based on the method previously described by Laxmi et al. [26], with necessary modification. This technique measures the absorbance of light by phytochemicals at various wavelengths, allowing for the detection and quantification of specific compounds. This method facilitated the identification of a diverse range of phytochemicals, including alkaloids, flavonoids, phenols, and tannins, which are known for their potential anticancer properties and contribution to cytotoxic activity. Many of these compounds have been widely utilized in the development of anticancer drugs, highlighting their therapeutic significance.

Optimization of cell concentration for in vitro cell culture tests

To determine the optimal cell concentration, we tested varying concentrations in 96-well plates. Specifically, one plate was seeded at 8.8 × 10^5 cells/mL, while two additional plates were seeded at 4.4 × 10^5 cells/mL. Following incubation in a CO2 incubator under standard conditions, our results showed that 4.4 × 10^5 cells/mL was the optimal concentration for both LNCaP and DU145 cell lines was used.

Cell culturing process and cryopreservation

The cell culturing was performed based on the protocol described by Lin et al. [27], with modifications to suit the current study objectives. The cells were procured from Cytion, a company based in Düsseldorf, Germany, and shipped directly to the Biomedical Research Laboratory (Immunology section) at Makerere University, where cryopreservation and subsequent experiments were performed. For long-term storage, the cells were cryopreserved in liquid nitrogen. To achieve this, the cryotube was first placed in a Mr. Frosty container, which gradually reduced the temperature by 1 °C per minute to − 80 °C, and then the cells were transferred to liquid nitrogen at − 196 °C. This controlled freezing process helped prevent ice crystal formation and preserved cell viability.

Culture media preparation

The RPMI cell culture medium was prepared by supplementing 870 mL of RPMI 1640 with 100 mL of Fetal Bovine Serum (FBS, 10% v/v), 10 mL of l-glutamine (1% v/v), and 10 mL of penicillin–streptomycin (1% v/v), resulting in final volume of 1 L. The medium was then sterilized by filtration using a 0.22 μm filter unit under vacuum pump.

Cell culturing and incubation

The cells were pipetted into a falcon tube washed and centrifuged for 10 min at 1200 rpm after adding 5 mL of the prepared cell culture media. The cell pellets were then resuspended and pipetted into a T25 cell culture flask to facilitate the initial cell signaling. The flask was placed in a carbon dioxide incubator and maintained at 37 °C, 95.5% relative humidity, and 5% carbon dioxide for three days. The cells were monitored daily for signs of contamination and proliferation until they reached 90% confluency.

Sub-culturing and passaging

Once the cells reached the 90% confluency, the old medium was serologically discarded, and the remaining cells were then washed with phosphate-buffered saline (PBS). Trypsin was added to the cells, which were then incubated for 2 min to facilitate detachment. The cells were subsequently observed under low power magnification (×10) for morphological changes as indicative of detachment. The cells were counted using a hemocytometer under a microscope or with an automated cell counter. The cells were again sub-cultured in T125 mL flasks, and the process continued until the required number of cells was attained.

Cell treatment and MTT (cytotoxicity) assay

The current investigation for this assay adopted the procedure previously established by Vajrabhaya and Korsuwannawong [28], with modification. Following a 24-h incubation, cells seeded at 1 × 10^4 cells per well were treated with R. tridentata extracts (0–800 μg/mL). Cytotoxicity was evaluated at 24, 48, and 72 h using the MTT assay. The cells were incubated with 20 μL of MTT solution (5 mg/mL) for 3–4 h, and formazan crystals were dissolved with DMSO (0.5%). The absorbance was measured at 590 nm using a microplate reader, and optical density readings were used to compute cytotoxicity.

Calculation of cytotoxicity and inhibitory concentration at 50 (IC 50)

The Inhibitory concentration 50 (IC50) was calculated using the formula described by Tunca et al. [29]. Following a 24-h incubation, cells seeded at 1 × 10^4 cells per well were treated with R. tridentata extracts (0–800 μg/mL). The Cytotoxicity was evaluated at 24, 48, and 72 h using the MTT assay. The cells were incubated with 20 μL of MTT solution (5 mg/mL) for 3–4 h, and formazan crystals were dissolved with DMSO. The absorbance was measured at 590 nm using a microplate reader, and optical density readings were used to compute cytotoxicity. The cytotoxicity of the test substance was calculated using the following formula: \(Cytotoxicity \left(\%\right)=OD Control-OD treated\times 100\%\) or \(Cytotoxcity \left(\%\right)=100-viability\). The IC50 was computed from the formula: IC50 = (50–b)/m, with b representing the y-intercept and m representing the slope of the linear regression line.

Data analysis & presentationData analysis

The data were analyzed using GraphPad Prism and Sigma plot14 to determine the IC50 value, which represents the concentration of the test substance required to inhibit 50% of cell growth. The IC50 value was used to evaluate the cytotoxic potential of the test substance.

Data presentation

The data were summarized as mean percentages ± standard deviation (SD) (Mean ± SD) in tables and figures to facilitate interpretation.