Hypoxis hemerocallidea herbarium specimen number Toona 01, H. caespititium Toona 02 and D. anomala 03 corms were collected from Bojanala District in Rustenburg Municipality, Mamerotse (25°25’03.0”S, 27°20’28.0”E) and Mogajane (25.4870°S, 27.3618°E) villages, Bafokeng area, on April 2024. The plant habitat was along the plain roadside, fully exposed to the sun, and the plants were moderately to abundantly distributed. In addition, the DU145 (ATCC^® HBT-81™) prostate cancer cell line used was purchased from Highveld Biological, Cellonex, South Africa.

The direct maceration extraction method was used, as adapted from Fonmboh et al. (2020). Five grams of dried H. hemerocallidea, H. caespititium and D. anomala, respectively, were mixed with 50 mL of methanol (MeOH) and 50 mL of aqueous (deionised water [ddH2O]) as solvents. The mixtures were placed on a constant mechanical shaker for 40 h at room temperature (20°C – 24°C); extracts were then filtered, and the supernatant was concentrated under reduced pressure using a vacuum rotary evaporator. The concentrated extracts were stored in small glass bottles at 4°C until further use (Ghomari et al. 2019). The residue was used for experiments.

Prostate cancer cell line DU145 was cultured in RPMI 1640, low-glucose cell culture medium, and foetal bovine serum (FBS) was purchased from GE Healthcare Life Sciences (Logan, UT, United States). Cultures of cells were seeded into 96-well microtitre plates at a density of 5000 cells/well using a volume of 100 µL aliquots in each well. The microtitre plates with cells were incubated at 37°C, with 5% CO2 for 24 h prior to the addition of the extracts compound to allow for the attachment of cells. Extracts were solubilised in dimethyl sulfoxide and diluted to the desired concentrations of 15, 31, 62.5, 125, 250 and 500 µg/mL in the medium. They were added to wells with exponentially growing cells, and all experiments were performed in triplicate.

The treatment medium was removed after 48 h and replaced with 100 µL DPBS-Dulbecco’s Phosphate Buffered Saline with Ca²⁺ and Mg²⁺ containing Hoechst 33342 at a final concentration of 5 µg/mL and incubated for 30 min at room temperature (20°C – 24°C). For cell death detailing effects, propidium iodide (PI) was added at a final concentration of 100 µg/mL using 20 µL per well of a 1 mL stock just before image acquisition, which was carried out using an ImageXpress Micro XLS fluorescent microscope and evaluated with MetaXpress software. Thus, the concentration to inhibit 50% of the cell population was determined.

In comparison to the untreated cells and melphalan control, a reduction in viability of the DU145 cell line was seen in D. anomala aqueous extracts at concentrations of 15 µg/mL and 62.5 µg/mL. Conversely, the H. hemerocallidea and H. caespititium aqueous extracts did not result in significant DU145 cell growth inhibition. Therefore, H. hemerocallidea and H. caespititium aqueous extracts did not exhibit much cytotoxicity even at the maximum extract concentration of 500 µg/mL, although at this concentration, D. anomala aqueous extracts still proved to be potent (Figure 1).

Treatment of DU145 prostate cancer cells with methanolic extracts from H. caespititium and D. anomala resulted in a concentration-dependent inhibition of cell proliferation. Specifically, concentrations of 15 µL/mg and 31 µL/mg of these extracts significantly arrested the DU145 cell line, an effect comparable to the positive control, melphalan, and a notable reduction compared with the untreated control (Figure 2). In contrast, the methanolic extract of H. hemerocallidea showed minimal inhibition of cell growth.

Further analysis revealed that methanolic extracts were more effective than their aqueous counterparts at the same concentrations (15 µL/mg and 31 µL/mg) in reducing the viability of DU145 cell line (Figures 1 and 2). The distinct cytotoxic effects were observed with the methanolic extracts of H. caespititium and D. anomala. DU145 cell proliferation rates were found to be 54% and 14% after treatment with 31 µL/mg of the methanolic extracts of H. caespititium and D. anomala, respectively.

These findings suggest that the methanolic extract of D. anomala is cytotoxic to the DU145 prostate cancer cell line, demonstrating a more potent antiproliferative effect than the other tested extracts (Figures 1 and 2).

In comparison with aqueous extracts of H. hemerocallidea, H. caespititium and D. anomala showed minimal effects on the DU145 cell line, similar to the methanolic extracts of H. hemerocallidea and H. caespititium. However, DU145 cell line treated with methanolic D. anomala showed a more cytotoxic effect. Therefore, the inhibition concentration (IC) of D. anomala methanol extracts proved to be cytotoxic against the DU145 cell line, with an IC value of 18.98 µg/mL (R² = 94%; Figure 3).

This study investigated the cytotoxic effects of extracts from H. hemerocallidea, H. caespititium and D. anomala on the DU145 prostate cancer cell line. Melphalan and untreated cells were used as positive and negative controls, respectively.

Analysis of the aqueous extracts revealed that H. hemerocallidea and H. caespititium did not inhibit DU145 cell proliferation, even at a high concentration of 500 µg/mL (Figure 1). In contrast, the aqueous extracts of D. anomala demonstrated notable cytotoxic activity at this same concentration. This finding aligns with the research of Steenkamp and Gouws (2006), who observed significant cytotoxicity from both H. hemerocallidea and D. capensis aqueous extracts on the same cell line. However, it contradicts the findings of Mamabolo et al. (2018), who reported low toxicity for H. caespititium aqueous extracts in rat hepatocytes, suggesting that the cytotoxicity of these extracts is cell type-dependent.

Furthermore, methanolic extracts of H. caespititium and D. anomala showed greater cytotoxic activity than the aqueous extracts. Helichrysum caespititium and D. anomala significantly inhibited DU145 cell proliferation at concentrations of 15 µg/mL and 31 µg/mL, an effect comparable to the positive control, melphalan (Figure 2). However, the methanolic extract of H. hemerocallidea showed only a minimal effect. Notably, the methanolic extract of D. anomala was the most potent, as it effectively arrested the DU145 cell line at both 15 µg/mL and 30 µg/mL (Figure 3). These results are consistent with the broader anticancer properties of D. anomala extracts observed by Chota et al. (2020) in the study performed on other cancers such as breast and lung cancer.

The study’s most significant and unexpected finding was that among the three plants traditionally used together, only the methanolic extract of D. anomala exhibited strong cytotoxic activity against the DU145 cell line. This outcome suggests that the active anticancer compounds are highly dependent on the extraction method and the specific plant species, which contrasts with the traditional practice of using aqueous preparations of all three plants.

This aligns with previous studies showing that variations in extraction procedures and the natural variability of plants can affect their therapeutic properties (Steenkamp & Gouws 2006). While some research has shown that H. hemerocallidea can inhibit the growth of other cancer cell lines, such as MCF-7 (Steenkamp & Gouws 2006), the results of this study suggest that aqueous extracts may have a limited effect on specific carcinoma cell lines (Chota et al. 2020).

Further investigations are needed to fully understand the anticancer mechanism of action of D. anomala and H. caespititium. Future investigations should focus on demonstrating whether these extracts induce apoptosis (programmed cell death) with minimal necrosis (cell injury), which is a crucial characteristic of a promising anticancer agent. This study encourages in-depth research to characterise the specific compounds responsible for the cytotoxic effects of these plant extracts, particularly D. anomala, on DU145 prostate cancer cell proliferation.