In vitro cytotoxicity and phytochemical analysis of the crude extract from Hypoxis hemerocallidea and its apoptosis-inducing effect on breast and skin can

Análise citotóxica e fitoquímica in vitro do extrato bruto de Hypoxis hemerocallidea e seu efeito indutor de apoptose em câncer de mama e de pele

Abstract

Hypoxis hemerocallidea, a medicinal plant traditionally used for its therapeutic properties, has demonstrated potential anticancer activity in vitro. This study evaluated the cytotoxic and pro-apoptotic effects of methanol (MeOH) and aqueous extracts of H. hemerocallidea on A375 melanoma, MCF-7 breast cancer, and HEK293 normal cell lines. MTT assays revealed concentration-dependent inhibition of cancer cell proliferation, with IC₅₀ values of 44.82–63.1 µg/mL for MeOH extract and 55.02–57.6 µg/mL for aqueous extract, while sparing normal HEK293 cells. Morphological analysis showed characteristic apoptotic changes, including cell shrinkage, rounding, membrane blebbing, and formation of apoptotic bodies. Flow cytometric analysis demonstrated increased caspase-3 activity (13–15% for extracts vs. 47.8% for Doxorubicin) and mitochondrial membrane depolarization (17.4% for aqueous extract, 48.4% for MeOH extract vs. 56.5% for Doxorubicin), indicating activation of intrinsic apoptotic pathways. Collectively, these findings suggest that H. hemerocallidea extracts selectively induce apoptosis in cancer cells through mitochondrial-dependent mechanisms, highlighting their potential as natural anticancer agents. Further studies are warranted to isolate active compounds and elucidate their molecular targets.

Keywords:
Hypoxis hemerocallidea; cytotoxicity; phytochemical composition; apoptosis; breast cancer; skin cancer

Resumo

Hypoxis hemerocallidea, uma planta medicinal tradicionalmente utilizada por suas propriedades terapêuticas, demonstrou potencial atividade anticancerígena in vitro. Este estudo avaliou os efeitos citotóxicos e pró-apoptóticos do metanol (MeOH) e dos extratos aquosos de H. hemerocallidea em linhagens celulares de melanoma A375, câncer de mama MCF-7 e células HEK293 normais. Os ensaios de MTT revelaram inibição da proliferação de células cancerígenas de forma dependente da concentração, com valores de IC50 de 44,82–63,1 µg/mL para o extrato de MeOH e 55,02–57,6 µg/mL para o extrato aquoso, poupando células HEK293 normais. A análise morfológica demonstrou alterações apoptóticas características, incluindo encolhimento celular, arredondamento, formação de bolhas na membrana e formação de corpos apoptóticos. A análise por citometria de fluxo demonstrou aumento da atividade da caspase-3 (13-15% para extratos vs. 47,8% para doxorrubicina) e despolarização da membrana mitocondrial (17,4% para extrato aquoso, 48,4% para extrato de MeOH vs. 56,5% para doxorrubicina), indicando ativação de vias apoptóticas intrínsecas. Coletivamente, esses achados sugerem que extratos de H. hemerocallidea induzem seletivamente apoptose em células cancerígenas por meio de mecanismos dependentes de mitocôndrias, destacando seu potencial como agentes anticancerígenos naturais. Estudos adicionais são necessários para isolar compostos ativos e elucidar seus alvos moleculares.

Palavras-chave:
Hypoxis hemerocallidea; citotoxicidade; composição fitoquímica; apoptose; câncer de mama; câncer de pele

1. Introduction

Cancer is a noncommunicable disease with a high incidence and fatality rate globally. In many parts of the world, it remains a life-threatening condition due to uncontrolled cell proliferation (Parikh et al., 2022; Correia et al., 2014).. It is distinguished by diverse etiologies and phases, driven by dysregulation of genetic and epigenetic programming triggered by microbial infections, chemical carcinogenesis, and hormonal imbalances (Supek and Lehner, 2015; Wynendaele et al., 2015). According to the World Health Organization (WHO), 9.7 million people died from cancer in 2022, with nearly 20 million new cases reported worldwide (WHO, 2024). The rising global incidence of cancer, with projections indicating a potential doubling of cases in the coming decades, highlights the urgent need to explore and optimize traditional sources for the development of potent anticancer agents that minimize systemic toxicity (Murugesan et al., 2021)

Although current treatments such as surgery, radiation, and chemotherapy can be effective, they often lack specificity, inflict damage on healthy tissues, and lead to significant acute and chronic complications (Bouyahya et al., 2018; Mutazah et al., 2020; Mosel et al., 2011). Amidst these challenges, natural products continue to serve as foundational sources for anticancer drug development. Indeed, over 50% of small-molecule drugs approved between 1981 and 2014, and 68% of small-molecule anticancer agents, were derived from nature (Naeem et al., 2022). Similarly, an estimated 60% of present-day anticancer drugs are either direct derivatives or inspired by natural compounds (Asma et al., 2022). Plant secondary metabolites, such as alkaloids, terpenoids, and phenolics, exhibit notable anticancer properties and remain indispensable in ongoing drug discovery efforts (Fridlender et al., 2015; Panthi et al., 2023). These compounds offer a promising avenue to overcome systemic toxicity and resistance associated with conventional therapies.

Breast and skin cancers are among the most prevalent malignancies worldwide, characterized by dysregulation of molecular pathways governing proliferation, survival, and apoptosis. In breast cancer, amplification of the human epidermal growth factor receptor 2 (HER2) gene occurs in 25–30% of cases, driving aggressive tumor growth and poor prognosis. The phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mechanistic target of rapamycin (mTOR) pathway is frequently activated in triple-negative breast cancer (TNBC), contributing to tumor progression and therapy resistance (Zhang et al., 2024; Ryspayeva et al., 2025; Leyland-Jones, 2009). In skin cancers, particularly melanoma, activating mutations in the BRAF gene (e.g., V600E) or NRAS lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, promoting uncontrolled proliferation and survival (Lidsky et al., 2014). Importantly, evasion of apoptosis is a hallmark of both breast and skin cancers. Overexpression of anti-apoptotic proteins, such as B-cell lymphoma 2 (Bcl-2), and downregulation of pro-apoptotic mediators, like Bcl-2-associated X protein (Bax), disrupt the mitochondrial apoptotic balance (Hartman and Czyz, 2020). Furthermore, aberrant activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway enhances survival signaling and confers resistance to chemotherapy. These alterations highlight apoptosis restoration as a promising therapeutic strategy for these cancers (Kannan et al., 2025). Mitochondria serve as pivotal regulators of intrinsic apoptosis by controlling membrane permeability and the mitochondrial membrane potential (ΔΨm), which is often irreversibly disrupted during apoptotic progression (Chen et al., 2000). This perturbation facilitates the release of cytochrome c into the cytosol, where it associates with Apaf-1 to form the apoptosome, which activates caspase-9 and downstream executioner caspases, such as caspase-3 (Garrido et al., 2006; Eleftheriadis et al., 2016). Consequently, simultaneous measurement of ΔΨm disruption and caspase activation affords critical insight into the mitochondrial-mediated apoptotic cascade.

H. hemerocallidea, commonly known as the African potato, is a medicinal plant native to southern Africa that has gained research interest due to its traditional applications in managing chronic illnesses, including cancer. The tuberous corm has historically been used as a tonic in the treatment of tuberculosis, testicular tumors, prostate hypertrophy, and other cancers (Owira and Ojewole, 2009; Bassey et al., 2023). It is also used as an immune booster in individuals with cancer and HIV/AIDS. The major phytochemical hypoxoside, upon ingestion, is hydrolyzed to rooperol, a potent antioxidant with anticancer and anti-inflammatory properties (Owira and Ojewole, 2009). Additional compounds, such as β-sitosterol and sterol glycosides, commonly present in H. hemerocallidea, have been shown to exert significant immunomodulatory and therapeutic effects. β-Sitosterol and its glucoside have been demonstrated to stimulate lymphocyte proliferation and modulate key immune responses (e.g., cytokine production), while broader groups of steroidal glycosides are known for antitumor and immunostimulatory activities (Paniagua-Pérez et al., 2008; Fraile et al., 2012). Despite the well-documented traditional use of H. hemerocallidea, limited data exist on its ability to modulate these pathways in breast and skin cancer models. Addressing this gap, the present study evaluates the in vitro cytotoxicity, phytochemical composition, and apoptosis-inducing effects of H. hemerocallidea, with particular focus on mitochondrial integrity and caspase activation.

2. Materials and Methods

2.1. Plant collection and extraction

Hypoxis hemerocallidea roots were collected from Reservoir Hills, Durban, washed thoroughly with distilled water, air-dried, and ground into a fine powder. Extraction was performed using methanol and water as solvents. For the methanolic extract, 50 g of powdered root material was shaken with 400 mL of 80% methanol for 48 h, filtered, and concentrated under reduced pressure using a rotary evaporator. For the aqueous extract, 50 g of powdered root material was shaken with 400 mL of distilled water for 48 h, filtered, and lyophilized. The resulting extracts were freeze-dried to obtain crude dry material, which was stored at −20 °C until further use. For cytotoxicity assays, the extracts were reconstituted in 10% DMSO (v/v) to prepare working solutions (Nayim et al., 2021).

2.2. Preliminary phytochemical analysis

Phytochemical Analysis of H. hemerocallidea was conducted to identify the main classes of secondary metabolites, including alkaloids, steroids, terpenoids, flavonoids, phenols, saponins, glycosides, and tannins, using established techniques previously described (Nandagoapalan et al., 2016).

2.3. Determination of total phenolic

The total phenolic content of H. hemerocallidea extracts was determined using the Folin–Ciocalteu method, with gallic acid as the reference standard, following a modified procedure from Saeed et al. (2012). Briefly, 1 mL of extract solution (1 mg/mL) was mixed with 9 mL of distilled water in a volumetric flask, followed by the addition of 2.5 mL of 10-fold diluted Folin–Ciocalteu reagent and 10 mL of 7.5% sodium carbonate (Na₂CO₃) solution. The mixture was adjusted to volume with distilled water and incubated in the dark at room temperature for 90 min. Standard gallic acid solutions (100–200 μg/mL) were prepared using the same procedure. Absorbance was measured at 760 nm using a UV–Visible spectrophotometer, and total phenolic content was expressed as milligrams of gallic acid equivalent (GAE) per gram of extract.

2.4. Determination of flavonoid contents

The total flavonoid content of H. hemerocallidea extracts was determined using the aluminum chloride colorimetric method, as described by John et al. (2014). with slight modifications. Briefly, 1 mL of extract solution (1 mg/mL) was mixed with 4 mL of distilled water in a volumetric flask. After 5 min, 0.30 mL of 5% sodium nitrite solution and 0.30 mL of 10% aluminum chloride (AlCl₃·6H₂O) solution were added, followed by 2 mL of 1.0 M sodium hydroxide (NaOH). The mixture was then made up to volume with distilled water and incubated at room temperature. Standard quercetin solutions (20–100 μg/mL) were prepared in the same manner. Absorbance of both extracts and standards was measured at 510 nm using a UV–Visible spectrophotometer. Total flavonoid content was calculated from the quercetin calibration curve and expressed as milligrams of quercetin equivalent (QE) per gram of extract.

2.5. Cell lines and cell culture

Human embryonic kidney cells (HEK293), human melanoma cells (A375), and human breast cancer cells (MCF-7) were obtained from Highveld Biological (South Africa). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂ in growth medium (Gibco™, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. Cell growth, medium color, density, and morphology were monitored daily using a Zeiss inverted light microscope.

2.6. In vitro cytotoxicity Hypoxis hemerocallidea

To evaluate the anticancer potential of H. hemerocallidea, two cancer cell lines were selected: MCF-7 (breast cancer) and A375 (skin melanoma). MCF-7 cells are a well-established model for estrogen receptor-positive breast cancer, commonly used in cytotoxicity and apoptosis studies, while A375 cells represent malignant melanoma, an aggressive and therapy-resistant cancer. Including these distinct cancer types allows assessment of the extracts' potential broad-spectrum anticancer activity. Human embryonic kidney cells (HEK293) were included as a normal cell line to assess selectivity and minimize potential cytotoxic effects on non-cancerous cells.

The cytotoxicity of the extracts was evaluated using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as previously described (Kasumbwe et al., 2017). This method is based on the conversion of the yellow tetrazolium salt (MTT) into purple formazan crystals by metabolically active cells, which can be quantified colorimetrically. Cancer cell lines (A375 and MCF-7) and a noncancerous cell line (HEK293) were used to assess both cytotoxicity and selectivity. Cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and allowed to adhere overnight. They were then treated with varying concentrations of the extracts (6.25–250 µg/mL) for 48 h. After treatment, 20 µL of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h. The supernatant was carefully removed, and the resulting formazan crystals were solubilized with 100 µL of dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm using a Multiscan Go microplate reader (Thermo Fisher Scientific). Untreated cells served as the negative control, Doxorubicin (5 µg/mL) as the positive control, and a vehicle control (0.1% DMSO) was included. The percentage of cytotoxicity was calculated using the following Formula 1:

$$\%Cytotoxicity=\left(A_c-A_s\right)/A_{c}x100$$ (1)

Where: Ac is the absorbance of untreated control cells

As is the absorbance of sample-treated cells

2.7. Statistical analysis

The half-maximal inhibitory concentration (IC₅₀) values of the extracts were determined using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). All experiments were performed in triplicate, and IC₅₀ values were expressed as mean ± standard deviation (SD).

2.8. Assessment of apoptosis-inducing properties in Mcf-7

2.8.1. Measurement of mitochondrial membrane potential (ΔΨ) Assay

The mitochondrial membrane potential (ΔΨm) was assessed using the BD MitoScreen JC-1 assay kit (BD Biosciences) according to the manufacturer's instructions. Cells were harvested, washed twice with cold PBS (4 °C), and centrifuged at 1200 rpm for 3 minutes. Pellets were resuspended in 500 µL of JC-1 working solution, gently vortexed, and incubated at 37 °C in a humidified 5% CO₂ incubator for 15 minutes. After incubation, the cells were washed twice with JC-1 assay buffer (centrifuged at 1450 rpm for 5 minutes each) and then resuspended in 250 µL of JC-1 assay buffer. Samples were transferred to 5 mL round-bottom tubes for flow cytometry analysis. During flow cytometry, JC-1 monomers (green fluorescence, ~530 nm) and aggregates (red fluorescence, ~590 nm) were detected. The mitochondrial membrane potential was evaluated based on the red/green fluorescence ratio: a decrease in red fluorescence, accompanied by a corresponding increase in green fluorescence, indicated mitochondrial depolarization, consistent with apoptosis. Untreated cells served as negative controls, while Doxorubicin-treated cells were used as positive controls.

2.8.2. Caspase activation assay

Caspase-3 activity was assessed using the PE Active Caspase-3 Apoptosis Kit (BD Biosciences) according to the manufacturer's instructions. Briefly, cells were harvested, washed twice with cold PBS (4°C), and then centrifuged at 1200 rpm for 3 minutes. The pellets were resuspended in 500 μL of BD Cytofix/Cytoperm solution and incubated on ice for 20 minutes to fix and permeabilize the cells. Following fixation, cells were washed twice with BD Perm/Wash buffer, then incubated at room temperature for 30 minutes with 120 μL of PE-conjugated anti-active caspase-3 antibody diluted in BD Perm/Wash buffer. After staining, cells were pelleted, resuspended in 250 μL of BD Perm/Wash buffer, and transferred into 5 mL round-bottom tubes for flow cytometry analysis. Data were acquired using a flow cytometer, and analysis was performed by gating viable cells and measuring the percentage of PE-positive cells. The level of caspase-3 activation was expressed as the percentage of caspase-3–positive cells relative to untreated negative controls. Doxorubicin-treated cells were used as positive controls for apoptosis induction.

3. Result and Discussion

3.1. Analysis of phytochemicals; determination of total phenolic and flavonoid levels

The medicinal properties of herbal medicines are largely attributed to their bioactive compounds, or secondary metabolites, produced during plant metabolism (Thomford et al., 2018). These compounds play important roles in disease prevention and treatment, including cancer (Xiong et al., 2015). In this study, methanolic and aqueous extracts were subjected to preliminary phytochemical screening to identify bioactive constituents (Table 1). The methanol extract contained steroids, saponins, phenols, flavonoids, alkaloids, and glycosides, whereas the aqueous extract contained terpenoids, saponins, phenols, and flavonoids. Tannins were absent in both extracts, and glycosides were only present in the methanol extract, indicating potential differences in pharmacological effects. Saponins have been reported to inhibit cancer cell growth by inducing apoptosis and cell cycle arrest (Kuznetsova et al., 1982; Mimaki et al., 1998; Hanausek et al., 2001). Phenols and flavonoids possess antioxidant, anti-inflammatory, anticancer, and cardiovascular protective properties, scavenging free radicals and chelating metal ions (Han et al., 2007; Middleton et al., 2000; López et al., 2003). The presence of these bioactive compounds in the studied extracts underscores their potential therapeutic value, particularly in cancer prevention and treatment.

Table 1
Phytochemical analysis conducted on various solvent extracts of Hypoxis hemerocallidea.

Test	Methanol Extract	Aqueous Extract
Steroids	+	-
Terpenoids	+	+
Tannins	+	-
Saponnins	+	+
Phenols	+	+
Glycosides	-	-
Flavonoids	+	+
Alkaloids	+	-

The quantitative analysis of total phenolic and flavonoid contents is presented in Table 2. The methanol extract contained higher levels of phenolics (148.6±0.7 mg GAE/g dwt) and flavonoids (41.2±1.2 mg QE/g dwt) than the aqueous extract, which had phenolics (27.5±0.3 mg GAE/g dwt) and flavonoids (20.3±0.8 mg QE/g dwt). Polyphenols are well-known for their potential to inhibit cancer cell growth, tumor development, angiogenesis, inflammation, and metastasis, making them promising agents for cancer therapy (Bhosale et al., 2020). Flavonoids, abundant in edible plants, exhibit antioxidant, anti-inflammatory, antimicrobial, enzyme-inhibitory, and antitumor properties, while generally showing low toxicity (Cushnie and Lamb, 2005). In line with these findings, flavonoids were detected in all active plant extracts, with some exhibiting minimal toxicity towards the HEK293 cell line. Specific flavonols, such as quercetin and kaempferol, commonly present in glycoside derivatives in the aerial parts of A. tinctoria and A. sylvestris, demonstrate multiple anticancer activities. Quercetin, for example, scavenges free radicals, protects cells from oxidative stress, inhibits cell proliferation, and induces apoptosis in cancer cells (Niedzwiecki et al., 2016). Moreover, certain polyphenols, including apigenin and quercetin, modulate key signaling pathways involved in cancer initiation, progression, and metastasis (Cháirez-Ramírez et al., 2021).

Table 2
Total phenolic and flavonoids content of methanol and aqueous extracts of Hypoxis hemerocallidea .

	Fraction	Total Phenolic Content (mg GAE/g of d. wt.)	Flavonoid Content (mg QE/g of d. wt.)
1.	Methanol fraction	148.6+0.7	41.2+1.2
2.	Aqueous fraction	27.5+0.3	20.3+0.8

3.2. In vitro cytotoxicity of Hypoxis hemerocallidea

Despite advancements in modern therapeutic strategies, including gene therapies and genome editing, the treatment of cancer remains challenging due to issues such as drug resistance, off-target effects, and toxicity (Yao et al., 2015; Stone et al., 2016). Herbal medicines and secondary metabolites have long been recognized for their therapeutic potential, particularly in the prevention and management of chronic diseases and cancer (Ben‐Arye et al., 2016). Natural plant-derived compounds offer a low-risk, accessible alternative that can complement conventional treatments while potentially enhancing efficacy (Lin et al., 2019). In particular, polyphenolic-rich herbal extracts have been associated with reduced cancer incidence and possess bioactive properties that make them valuable candidates for anticancer drug development (Syed et al., 2007; Zaki et al., 2017).

In this study, the cytotoxic effects of methanol (MeOH) and aqueous extracts of H. hemerocallidea were evaluated in vitro against two cancer cell lines (MCF-7 breast cancer and A375 melanoma) and one normal cell line (HEK293) using the MTT assay. The assay measures cell metabolic activity based on the reduction of MTT tetrazolium dye to purple formazan by viable cells. Both extracts exhibited concentration-dependent cytotoxicity against cancer cells, while showing minimal effects on normal HEK293 cells. At the highest tested concentration (250 µg/mL), the aqueous extract demonstrated cytotoxicity of 79.01% in MCF-7 (Figure 1) and 72.1% in A375 cells (Figure 2), with only 19.2% inhibition in HEK293 cells (Figure 3). Similarly, the MeOH extract inhibited 78.1% of MCF-7 and 71.8% of A375 cells at the same concentration, while having a lesser effect on HEK293 cells (29.6%).

Figure 1
Cytotoxic activity of aqueous (HyhAq) and methanol (HyhMeOH) extracts of Hypoxis hemerocallidea, and Doxorubicin against MCF-7 cancer cells. Different lowercase letters (a, b, c) indicate statistically significant differences between groups (p < 0.05, one-way ANOVA followed by Tukey's post hoc test).

Figure 2
Cytotoxic activity of aqueous (HyhAq) and methanol (HyhMeOH) extracts of Hypoxis hemerocallidea, and Doxorubicin against A375 cancer cells. Different lowercase letters (a, b, c) indicate statistically significant differences between groups (p < 0.05, one-way ANOVA followed by Tukey's post hoc test).

Figure 3
Cytotoxic activity of aqueous (HyhAq) and methanol (HyhMeOH) extracts of Hypoxis hemerocallidea, and Doxorubicin against HEK293 cells. Different lowercase letters (a, b, c) indicate statistically significant differences between groups (p < 0.05, one-way ANOVA followed by Tukey's post hoc test).

The IC₅₀ values further confirmed these observations. The aqueous extract had IC₅₀ values of 57.6 µg/mL (MCF-7), 55.02 µg/mL (A375), and >250 µg/mL (HEK293), while the MeOH extract showed IC₅₀ values of 63.1 µg/mL (MCF-7), 44.82 µg/mL (A375), and >250 µg/mL (HEK293) (Table 3). In comparison, the positive control Doxorubicin displayed much lower IC₅₀ values across all cell lines (11.64 µg/mL for MCF-7, 17.34 µg/mL for A375, and 2.678 µg/mL for HEK293), reflecting its potent but non-selective cytotoxicity. These results suggest that H. hemerocallidea extracts preferentially inhibit cancer cell proliferation while sparing normal cells, indicating potential therapeutic selectivity.

Table 3
IC₅₀ values of various solvent extracts of Hypoxis hemerocallidea on different cell lines.

IC50 (µg/mL)
Samples	MCF-7	A375	HEK293
HyhAq	63.1 ± 1.8	44.82 ± 1.39	˃250
HyhMeOH	57.6 ± 1.76	55.02 ± 1.74	˃250
Doxorubicn	11.64 ± 1.07	17.34 ± 1.24	2.678 ± 2.49

The differential cytotoxicity observed between cell lines may reflect variations in cell metabolism, membrane permeability, or susceptibility to specific bioactive compounds present in the extracts. H. hemerocallidea contains several bioactive phytochemicals, including hypoxoside, which have been reported to induce apoptosis and inhibit cancer cell growth through oxidative stress modulation, mitochondrial dysfunction, and cell cycle arrest (Hirano et al., 1995; Faried et al., 2007). The selective cytotoxicity toward cancer cells observed in this study is consistent with these mechanisms. The observed cytotoxicity also highlights the potential synergistic effects of multiple bioactive compounds in the extracts, which may simultaneously target several cellular pathways, reduce adverse effects, and enhance therapeutic efficacy. Such multi-target activity is particularly valuable in cancer treatment, where redundancy in survival pathways often leads to chemoresistance. Thus, the results of this study demonstrate that both methanol and aqueous extracts of H. hemerocallidea possess significant anticancer activity, particularly against A375 and MCF-7 cells, while showing limited toxicity to normal HEK293 cells.

3.3. Morphological observations

To confirm the cytotoxic effects of methanol and aqueous extracts on A375 cancer cells, the cells were examined for morphological changes after exposure to IC₅₀ concentrations of 44.8 and 63.1 μg/mL of methanol and aqueous extracts, respectively (Figure 4). Untreated cells exhibited normal morphology, with high cell density and intact cellular structure, whereas treated cells showed a significant reduction in cell numbers and the appearance of intracellular spaces (Figures 4A and 4B). Distinct apoptotic features, including cell shrinkage, rounding, membrane blebbing, and formation of apoptotic bodies, were observed in treated cells, indicating induction of apoptosis. Apoptotic morphological changes result from specific molecular and biochemical processes, such as activation of proteolytic enzymes, leading to DNA fragmentation and cleavage of structural proteins that maintain cytoplasmic and organelle integrity (Saraste and Pulkki, 2000). Previous studies have highlighted that cell rounding and membrane blebbing are hallmark features of apoptosis (Archana et al., 2013). Early-stage apoptotic cells typically exhibit shrinkage and a rounded or oval appearance under light microscopy (Elmore, 2007). These findings suggest that methanol and aqueous extracts of H. hemerocallidea exert cytotoxic effects on A375 cells by triggering apoptotic cell death, consistent with the results of the MTT assay.

Figure 4
shows the morphological changes in A375 cells treated with H. hemerocallidea extracts at IC₅₀ concentrations of 44.8 and 63.1 μg/mL after 48 hours. The images depict untreated cells (A), DMSO (0.2%) controls (B), MeOH extract (C), and aqueous extracts (D).

3.4. Apoptosis detection using the caspase-3 kit

Caspases are critical proteins that mediate apoptosis, a genetically controlled form of programmed cell death characterized by biochemical and morphological changes, including caspase activation, phosphatidylserine externalization, membrane blebbing, chromatin condensation, and the formation of apoptotic bodies (Fulda and Debatin, 2006; Sinha et al., 2013). Both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways converge on caspase-3, which executes apoptosis by cleaving key cellular proteins and DNA (Dho et al., 2025; Mustafa et al., 2024).

Flow cytometry was employed to assess caspase-3 activity in A375 cells treated with methanol (MeOH) and aqueous extracts of H. hemerocallidea, compared to untreated cells and cells treated with Doxorubicin, a known inducer of apoptosis (Adan et al., 2017). Doxorubicin treatment significantly increased caspase-3 activity by 47.8% relative to the untreated control (Figure 5B), confirming its potent apoptotic effect. Both the MeOH and aqueous extracts elevated caspase-3 activity, but to a lesser extent, with increases of 14.6% and 13.4%, respectively (Figures 5C and 5D). These results suggest that the extracts have the potential to induce apoptosis in A375 cells, consistent with the morphological changes and cytotoxicity observed in previous assays. Although their effect is not as pronounced as that of Doxorubicin, the increase in caspase-3 activity indicates activation of the apoptotic pathway. Caspase-3 activity is widely recognized as a reliable marker of apoptosis (Abd El-Karim et al., 2019), highlighting the relevance of these findings in evaluating the anticancer potential of H. hemerocallidea extracts.

Figure 5
Flow cytometer analysis data from the PE active caspase 3 assay conducted on A375 cells to assess caspase 3 activity is depicted in histogram plots. The plots show cell number versus PE active caspase 3 fluorescence, with PE-A- and PE-A+ bi-sector gates indicating the percentage of caspase 3 negative and positive cells, respectively. The data is presented for untreated cells (A), Doxorubicin-treated cells (B), MeOH extract (C) and aqueous extract (D).

3.5. Assessment of mitochondrial membrane potential (ΔΨ)

Mitochondria are essential for energy production and metabolic processes that support cell survival (Chen and Wong, 2009). Mitochondrial membrane potential (MMP) is a key indicator of mitochondrial health, and its disruption is a hallmark of apoptosis. In this study, MMP was assessed using the BD MitoScreen assay, which measures JC-1 fluorescence. Polarized mitochondria display red aggregates (FL-2), while depolarized mitochondria show green monomers (FL-1). Untreated A375 cells exhibited a baseline depolarization of 7.35% (Figure 6A), reflecting normal mitochondrial function. Doxorubicin treatment markedly increased depolarization to 56.5% (Figure 6B), consistent with its well-known pro-apoptotic activity through DNA damage and topoisomerase I inhibition. The MeOH extract induced a depolarization of 48.4%, indicating substantial mitochondrial dysfunction, whereas the aqueous extract caused a milder depolarization of 17.4%. These results suggest that both extracts can impair mitochondrial integrity, with the MeOH extract having a more substantial effect.

Figure 6
Flow cytometer analysis of ΔΨ status conducted on A375 cells: (A) Untreated, (B) Doxorubicin, (C) MeOH extract, (D) Aqueous extract.

Mitochondrial depolarization increases membrane permeability, leading to the release of cytochrome c and the activation of the caspase cascade, ultimately resulting in apoptosis (Elmore, 2007). Our findings align with previous studies, which show that disruption of the mitochondrial outer membrane leads to loss of membrane potential and apoptotic cell death (Gottlieb et al., 2003). These results complement our MTT and caspase-3 assays, indicating that H. hemerocallidea extracts induce apoptosis via mitochondrial pathways.

3.6. Phytochemical contributions to apoptosis

The cytotoxic and pro-apoptotic effects of the MeOH and aqueous extracts of H. hemerocallidea may be partly attributed to their phytochemical composition. Flavonoids, saponins, and other polyphenolic compounds present in the extracts are known to modulate apoptotic pathways by promoting mitochondrial depolarization, activating caspases, and inducing DNA fragmentation (Abotaleb et al., 2018; Bhadra, 2022; Wani et al., 2023). These compounds may act synergistically to selectively target cancer cells while sparing normal cells (Devaraji and Thanikachalam, 2025). This mechanistic insight complements the observed morphological changes, caspase-3 activation, and mitochondrial membrane depolarization, providing a plausible biochemical basis for the selective anticancer activity of the extracts (Kicinska and Jarmuszkiewicz, 2020).

4. Conclusion

The present study demonstrates that methanol and aqueous extracts of Hypoxis hemerocallidea exhibit selective cytotoxicity against A375 melanoma and MCF-7 breast cancer cells while showing minimal effects on normal HEK293 cells. The extracts induce hallmark features of apoptosis, including morphological alterations, activation of caspase-3, and mitochondrial membrane depolarization, indicating that their anticancer effects involve mitochondrial-dependent apoptotic pathways. Among the two extracts, the methanol extract consistently showed stronger cytotoxic and pro-apoptotic effects. These findings highlight the potential of H. hemerocallidea as a source of natural anticancer agents. Future research should focus on isolating the active phytochemicals, elucidating their precise molecular mechanisms of action, and validating their efficacy and safety in in vivo models to advance their potential therapeutic application.

Data Availability Statement

All data supporting the findings of this study are contained within the manuscript.

Acknowledgements

The authors thank the Durban University of Technology for its support.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  27 Oct 2025
• Date of issue
  2025

History

• Received
  20 July 2025
• Accepted
  04 Sept 2025