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Evaluation of the antiproliferative, cytotoxic and phytochemical properties of Zimbabwean medicinal plants used in cancer treatment

BMC Complementary Medicine and Therapies volume 25, Article number: 156 (2025) Cite this article

Abstract

Background

Cancer cases have been on the rise globally and several treatment strategies have been developed but mortality rates remain high. Zimbabwe, like many other countries, has also experienced a surge in cancer cases. In Zimbabwe, medicinal plants have been widely used to treat cancer for centuries. However, there has been limited research on the effectiveness, safety, and chemical composition of these plants. The current study assessed antiproliferative, cytotoxic and phytochemical properties of selected Zimbabwean medicinal plants.

Method

Cytotoxic activity of Agelenthus pungu, Carissa edulis, Dombeya rotundifolia, Flacourtia indica, Lannea discolor, Leonotis ocymifolia, Leucas martinicensis, Plicosepalus kalachariensis, Pseudolachnostylis maproneifolia, Solanum incanum, Strychnos cocculoides, Strychnos spinosa and Viscum verrucosum extracts were evaluated on normal murine peritoneal cells and sheep erythrocytes while antiproliferative activity was assessed on Jurkat T and HL60 cell lines. Cell viability was determined using the trypan blue exclusion and sulforhodamine B assay. Additionally, the effect of reduced glutathione on cytotoxic extracts was examined. The phytochemicals of the methanolic extracts were qualitatively determined using standard methods.

Results

Agelenthus pungu, Carissa edulis, Flacourtia indica, Strychnos cocculoides, Strychnos spinosa and Viscum verrucosum were cytotoxic to normal murine peritoneal cells. Flacourtia indica and Viscum verruscosum caused haemolysis of sheep erythrocytes at a concentration of 250 µg/mL for both plant extracts and 125 µg/mL for Viscum verrucosum. Cell viability increased on addition of 25 µg/mL of reduced glutathione to the extracts considered the most cytotoxic extracts, Agelenthus pungu and Viscum verrucosum. Agelenthus pungu, Carissa edulis, Leonotis ocymifolia, Leucas martinicensis and Viscum verrucosum significantly inhibited Jurkat T and HL60 cell proliferation. Viscum verrucosum was the most potent with the lowest half-maximum inhibitory concentration (IC50) values of 33 and 34 µg/mL on Jurkat T and HL60 cell lines respectively. The most dominant phytochemical classes were alkaloids, flavonoids and saponins.

Conclusion

This study demonstrates that Agelenthus pungu, Carissa edulis, Leonotis ocymifolia, Leucas martinicensis and Viscum verrucosum have antiproliferative activity against Jurkat T and HL60 cell lines. Viscum verrucosum was the most potent. These findings emphasise the importance of medicinal plants as well as their potential use as sources of novel compounds in anticancer drug discovery.

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Introduction

Cancer is a leading cause of death in the world, with the cancer burden being greater in developing countries [1]. It is estimated that 70% of cancer deaths occur in developing countries [2]. This is largely due to poverty, limited healthcare access and inadequate cancer treatment resources [3]. In 2020, Zimbabwe had 16,083 cancer cases and 10,676 cancer deaths, according to a World Health Organization (WHO) report [4]. Cancers such as breast cancer, cervical cancer, lung cancer, Kaposi sarcoma, prostate cancer, and leukaemia contribute to the cancer burden in Zimbabwe [5].

Leukaemia is the most common childhood cancer, accounting for 30% of all cancer cases diagnosed in children under the age of fifteen [6]. It is a type of cancer that affects white blood cells. It starts when the bone marrow produces abnormal white blood cells that fail to function properly leading to the body's inability to fight infections. Chemotherapy typically serves as the primary treatment option for leukaemia. It involves administering drugs aimed at eradicating or impeding the proliferation of cancer cells [7].

Despite the effectiveness of chemotherapy in destroying cancer cells, this method may have some drawbacks, as some cancer cells may become resistant to the drugs. Furthermore, most chemotherapeutic drugs damage both normal and cancerous cells, leading to other health problems [8]. To alleviate the drawbacks of chemotherapy, patients resort to herbal remedies or antioxidant supplements. These complementary treatments have shown promising activity in enhancing the effectiveness of chemotherapy and minimising damage to normal cells [9]. Alternative treatment options, such as radiation, targeted therapy, stem cell transplant, immunotherapy, and gene therapy are often considered for leukaemia treatment [10]. However, in Zimbabwe, despite advancements in treatment, accessibility and affordability remain notable challenges.

In Zimbabwe, over 75% of the population relies on medicinal plants for primary healthcare [11]. For centuries, medicinal plants have been used to treat various ailments and their use in cancer treatment has recently gained global attention. It is estimated that 60% of cancer drugs are plant-based [12]. These plant-based cancer drugs belong to various phytochemical classes, such as alkaloids, flavonoids, terpenoids, and polyphenols. Each class exhibits unique properties and can reduce cancer cell proliferation via several mechanisms of action, making them valuable assets in the fight against cancer [13]. As a result, research continues to explore the potential of phytochemicals in developing effective cancer therapies.

A significant contribution made by Mlilo and Sibanda [14] was the cataloguing of plants traditionally used for the treatment and management of cancer in Matebeleland, Zimbabwe. Based on the results and recommendations of this study, we evaluated the effectiveness of these documented medicinal plants as potential in vitro leukaemia remedies. By assessing the plants’ ability to inhibit cancer cell proliferation, toxicity to normal cells, and analysing for the presence of different classes of bioactive compounds. The study aimed to identify plants with potential for advancement in cancer therapy research and development.

Materials and Methods

Chemicals

Analytical-grade chemicals and drugs were purchased from Sigma-Aldrich (Steinheim, Germany) for the study. This included methanol, Foetal Bovine Serum (FBS), Roswell Park Memorial Institute 1640 media (RPMI), reduced-glutathione (GSH), penicillin and streptomycin solution (PNS), Hanks Buffered Saline Solution (HBSS), foetal calf serum (FCS), trichloroacetic acid (TCA), sulforhodamine B (SRB), trizma base, sodium hypochlorite, dimethyl sulfoxide (DMSO), trypan blue dye, chlorambucil, camptothecin and phytochemical screening reagents.

Collection and authentication of plant material

The plant species were collected from five districts of the Matebeleland region (Bulawayo, Gwanda, Hwange, Matobo and Tsholotsho) between September and December 2018. Approval to collect plant samples was sought from the relevant Rural District Councils (RDC) and Community Leaders. The samples were collected using sustainable harvesting methods to minimise the impact on biodiversity. Plant specimens were deposited at the Zimbabwe National Herbarium and Botanical Garden in Harare, Zimbabwe, for identification and authentication. The plants were identified by Anthony Mapaura and deposited specimens were filed by Christopher Chapano. Table 1 provides a comprehensive list of collected species along with the plant parts used and their corresponding specimen numbers.

Table 1 List of medicinal plants used traditionally in Zimbabwe for the treatment of cancer
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Extraction of plant material

Fresh leaves, stem barks, fruits, tubers, or roots were collected from September to December 2018 using traditional sampling methods. These plants were first washed with distilled water to remove soil and dust particles. The plant materials were air-dried at room temperature (25 – 30℃) in the shade, pulverised into a fine powder with a blender (MRC Laboratory Blender model 800-G, Waring Commercial) and kept in sealed containers in the dark until required for analysis. Maceration was employed as a method of extraction using methanol solvent. Known weights (200 g) of powdered material were loaded onto a fritted glass column. The material was allowed to stand in 1000 mL of methanol for 24 h to allow the soluble phytochemicals to dissolve. The extract was filtered after 24 h and fresh solvent was added and this procedure was repeated twice. The solvent was removed using a rotary evaporator at 37℃. The dry samples were placed in airtight sample tubes and kept in the fridge at -4℃ until needed.

The cytotoxic effects of crude methanolic plant extracts on normal animal cells

Animal cells were used in the experimental work and researchers prioritised animal welfare and ensured responsible experimental designs were followed. All tissue culture experiments were carried out following relevant ethical regulations. Experimental protocols used in this study were approved by the National University of Science and Technology Ethics Committee (NUST/IRB/2022/48).

The cytotoxic effect of methanolic plant extracts on normal murine peritoneal cells

Mice used in this study were obtained from the University of Zimbabwe Animal House. The normal murine peritoneal cells were isolated from 6–8 week-old mice using a method previously described by Ray and Dittel [15]. To increase peritoneal cell yield, 1 mL of starch solution (20%) was injected intraperitoneally into male Balb/C mice (31 ± 3 g) and left for 24 h. The mouse was euthanised by cervical dislocation and this was performed by trained personnel to ensure compliance with ethical standards. The mouse was then sprayed with 70% ethanol and mounted on a styrofoam board on its back. The outer peritoneum skin was cut using scissors and forceps and gently pulled, exposing the inner peritoneal skin. Cold PBS with 3% FBS (5 mL) was injected into the peritoneal cavity without puncturing any organs. The peritoneum was then massaged to remove any attached cells to the PBS. A 25 g needle was inserted, bevelled up, and attached to a 10 mL syringe in the peritoneum. This was done to extract as much fluid as possible. The collected fluid was put into tubes kept on ice. An incision was made in the inner skin to collect the remaining peritoneal fluid from the cavity. Samples with visible blood contamination were discarded. The collected suspension was centrifuged for 10 min at 1500 rpm in a Hettich Rotofix 32 centrifuge (Tuttlingen, Germany). The supernatant was discarded, and the cells were resuspended in RPMI and incubated overnight at 37℃ in a 5% CO2 Shel lab incubator (CO2 series Sheldon Mfg. Inc, Cornelius, USA). Cells were exposed to 0.4% trypan blue and manually counted with a haemocytometer counting chamber under a Celestron digital light microscope (Celestron, Los Angeles, USA) using a × 10 objective lens. The crude methanolic extracts of A. pungu, C. edulis, D. rotundifolia, F. indica, L. discolor, L. ocymifolia, L. martinicensis, P. kalachariensis, P. maproneifolia, S. incanum, S. cocculoides, S. spinosa and V. verrucosum were prepared in triplicates with the highest concentration being 250 µg/mL. This concentration was chosen due to the insolubility observed in some extracts at higher concentrations. To ensure consistency, this concentration was applied uniformly across all extracts in this study. The cells (1 × 104 per well) and test solutions were incubated for 72 h at 37℃ in a 5% CO2 Shel Lab incubator (CO2 series Sheldon Mfg. Inc, Cornelius, USA). This was followed by staining the cells with 0.4% trypan blue dye and counting using a haemocytometer counting chamber under a Celestron digital light microscope. The extracts that were found to be cytotoxic were subjected to a concentration-dependent assay in triplicates at the following concentrations: 0, 32, 63, 125, and 250 μg/mL.

The cytotoxic effect of methanolic plant extracts on sheep erythrocytes

Sheep erythrocytes were used in this assay and were obtained from the University of Zimbabwe Animal House. The haemolysis assay was conducted according to the procedure previously documented by Mapfunde et al. [16]. The assay was performed on the following concentrations of the methanolic plant extracts: 0, 32, 63, 125, and 250 μg/mL. A volume of 50 mL of sheep blood was collected under the guidance of veterinary experts. The blood was placed in a flask and an equal volume of Alsever solution was immediately added. Blood was centrifuged at 3000 rpm for 10 min. The supernatant was discarded, and the residue was washed three times with 1:5 volume of PBS by centrifuging at 4000 rpm for 5 min in a Rotofix 32 centrifuge. The supernatant was discarded. Cells were diluted four folds with PBS and the resulting suspension was used to determine haemolysis. A volume of 500 µL was incubated with 500 µL of the test sample extract in PBS for 90 min at 37℃. After incubation, the tubes were spun in a microcentrifuge at 3000 rpm for 1 min. The resulting supernatant 200 µL was added to 3 mL of Drabkin’s reagent. The positive control consisted of a 500 µL uncentrifuged mixture of erythrocyte suspension and 500 µL of PBS from which 400 µL was added to 3 mL Drabkin’s reagent to obtain 100% haemolysis. The negative control was used to measure the level of spontaneous haemolysis and thus was made by mixing 500 µL of erythrocyte suspension and 500 µL of buffer, centrifuging at 3000 rpm for 60 s then adding 200 µL of supernatant to 3 mL of Drabkin’s reagent. Aliquots of 200 µL of the supernatant in Drabkin’s reagent were placed in round-bottomed 96-well plates to determine the amount of haemoglobin released, the absorbance was read at 590 nm. The percentage of haemolysis was calculated using Eq. 1.

$$Percentage\;Haemolysis=\frac{\left(Sample\;absorbance-Negative\;control\;absorbance\right)}{(Positive\;control\;absorbance-Negative\;control\;absorbance)}x10$$
(1)

Effect of reduced-glutathione (GSH) on crude plant extracts

The effect of GSH on the activity of the most cytotoxic methanolic extracts on normal murine peritoneal cells was investigated. To a 12-well plate, the following samples were added in triplicates: cells only; cells + GSH; cells + extract; cells + extract + GSH. The GSH concentration utilised was 25 µg/mL, while the plant extracts were tested at their highest concentration of 250 µg/mL. The plates were incubated at 37℃ with 5% CO₂ for 72 h. The Trypan blue dye exclusion method was used to count the number of viable cells.

The antiproliferative effect of methanolic plant extracts on cancer cell lines

The effect of crude methanolic plant extracts on Jurkat T cell lines

The in vitro antiproliferative activity was assessed on Jurkat E6-1 human leukaemic T lymphoblastoid cell lines (Hudson Alpha/Caltech ENODE). The cells were grown in RPMI-1640 media supplemented with 10% FBS and 1% PNS solution at 37℃ with 5% CO2 in an incubator (Shel Lab CO2 Series, Sheldon Mfg. Inc., Cornelius, USA). The antiproliferative effect of the thirteen extracts was evaluated using the Trypan blue exclusion assay as previously described by Machingauta et al. [17]. The extracts were first tested at the highest concentration of 250 μg/mL and 10 µg/mL chlorambucil was used as a positive control. Wells with cells only were used as negative controls to ensure that any observed antiproliferative effect was not due to factors other than the tested compounds. To determine half-maximal inhibitory concentration (IC50) the extracts that were considered antiproliferative were further subjected to the following concentrations: 0, 32, 63, 125, and 250 μg/mL, with all experiments conducted in triplets.

The effect of crude methanolic plant extracts on HL60 cell lines

The antiproliferative effect of extracts was assessed on HL60 cell lines (European Collection of Authenticated Cell Cultures; (ECACC), U K). A colourimetric SRB assay was used to assess growth inhibition in a manner previously described by Skehan et al., with minor modifications [18]. All thirteen extracts were first tested at the highest concentration of 250 µg/mL, and 10 µg/mL of camptothecin served as a positive control. Wells containing cells only served as a negative control. A concentration-dependent assay (0, 32, 63, 125, 250 µg/mL) was conducted on extracts that showed significant antiproliferative effects and IC50 values were determined. All experiments were done in triplicates.

Selectivity index

Selectivity index (SI) was calculated for the most cytotoxic extracts, this was done to assess the safety of the extracts [19, 20]. It was calculated using the following Eq. (2):

$$SI=\frac{{IC}_{50}\;of\;Extracts\;on\;Normal\;Murine\;Peritoneal\;Cells}{{IC}_{50}\;of\;Extracts\;on\;Cancer\;Cells}$$
(2)

Phytochemical screening

The methanolic plant extracts were screened for various phytoconstituents. Screening was done for the following phytoconstituents: alkaloids, saponins, phenolic compounds, steroids, phytosteroids, anthraquinones, terpenoids, quinones, tannins, and coumarins. The tests were conducted according to standard methods by Guevara; and Harbone [21, 22]. The qualitative results are expressed as ( +) for the presence and ( −) for the absence of phytochemicals.

Data analysis

GraphPad Prism 8 for Windows (GraphPad Software Inc., San Diego, California, USA) version 8.02 was used to analyse the results using a one-way analysis of variance test (ANOVA) with Dunnett’s Multiple Comparison Test. Values with a P-value of 0.05 or less were considered statistically significant.

Results

Extraction of plant material

A total of thirteen medicinal plant species were selected from a previous ethnobotanical survey conducted by Mlilo and Sibanda [14] for antiproliferative, cytotoxic and phytochemical screening. The selection was based on the availability of plant samples, which ensured that enough material was available for screening purposes. In addition, literature searches were performed on plant species to ensure all plants with therapeutic potential were included in the analysis. Table 1 lists the plants screened, (botanical, family and local isiNdebele names are included as additional information) and percentage yields which were obtained following extraction with methanol.

The cytotoxic effects of methanolic plant extracts on normal cells

The effects of extracts on normal murine peritoneal cells

The cytotoxic effects of thirteen plant methanolic extracts on normal murine peritoneal cells are shown in Fig. 1. The plant extracts (A. pungu, C. edulis, F. indica, S. cocculoides, S. spinosa and V. verrucosum) were cytotoxic on normal murine peritoneal cells at a concentration of 250 µg/mL. The results showed a significant difference between the untreated cells and those subjected to treatment, with a P-value < 0.0001. Four plant extracts, including A. pungu, F. indica, S. spinosa and V. verrucosum which demonstrated a reduction in cell viability exceeding 40%, underwent a concentration-dependent assay as illustrated in Fig. 2. The resulting IC50 values are shown in Table 2.

Fig. 1
figure 1

The effect of crude methanolic plant extracts on normal murine peritoneal cells. Crude methanolic plant extracts were tested on normal murine peritoneal cells for their cytotoxic activity at a concentration of 250 µg/mL and chlorambucil was used as a positive control at a concentration of 10 μg/mL. The negative control contained cells and RPMI media only. The cells were incubated with test solutions and cell viability was determined after 72 h. The values are for mean ± SD for n = 3. All treatments were compared to cells only to determine the significant difference. The level of significance was denoted as follows: ****P < 0.0001. Methanolic plant extracts (A. pungu, C. edulis, F. indica, S. cocculoides, S. spinosa and V. verrucosum) significantly reduced cell viability and they were considered cytotoxic

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Fig. 2
figure 2

Concentration-dependant effect of plant methanolic extracts on normal murine peritoneal cells. The cells were incubated with test solutions at a varying concentration from 32 to 250 μg/mL and cell viability was determined after 72 h. Chlorambucil was used as a positive control at a concentration from 1.25 to 10 μg/mL. The negative control contained cells and RPMI media only. The cell viability decreased with an increase in the concentration of the test extract

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Table 2 Half-maximum inhibitory concentration (IC50) values (μg/mL) and selectivity index of the potent methanolic plant extracts
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The effect of methanolic plant extracts on sheep erythrocytes

Haemolysis assay was conducted on the four plant extracts A. pungu, F. indica, S. spinosa and V. verrucosum and their haemolytic activities are shown in Fig. 3. Extracts were considered to be haemolytic if the percentage of haemolysis was greater than 30%. The A. pungu and S. spinosa extracts exhibited low haemolytic activity of less than 30% at all concentrations. F. indica exhibited haemolytic activity above 30% at the highest concentration 250 µg/mL, while V. verrucosum showed haemolytic activity above 30% at the highest concentration at both 125 µg/mL and 250 µg/mL.

Fig. 3
figure 3

The haemolytic effect of crude methanolic plant extracts on sheep erythrocytes. The erythrocyte suspension was incubated with an equal volume of test samples dissolved in PBS at a concentration range of 32 to 250 μg/mL at 37℃. Methanolic plant extracts had greater haemolytic activity at higher concentrations. F. indica and V. verrucosum methanolic extracts caused haemolysis at the highest concentration of 250 μg/mL, with V. verrucosum also exhibiting haemolytic activity at 125 μg/mL

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The effect of reduced-glutathione (GSH) on the activity of cytotoxic extracts on normal murine peritoneal cells

The effects of GSH were investigated on the extracts which reduced cell viability of normal murine peritoneal cells by more than 50%, specifically A. pungu and V. verrucosum. The effects of GSH were investigated at the highest extract concentration of 250 μg/mL. As depicted in Fig. 4, the addition of GSH to the extracts of A. pungu and V. verrucosum to a noteworthy enhancement in cell viability.

Fig. 4
figure 4

The effect of reduced—glutathione (GSH) on the cytotoxic activity of A.pungu and V. verrucosum. The effect of combining GSH and crude methanolic plant extracts of A. pungu and V. verrucosum was evaluated on normal murine peritoneal cells. GSH and crude methanolic plant extracts were used at concentration of 25 and 250 µg/mL respectively. The cells were incubated with the test solution and cell viability was determined after 72 h. To determine significance (cells + extracts) was compared with (cells + extracts + GSH). ****P < 0.0001 and *** P 0.0001. The addition of GSH to A. pungu and V. verrucosum increased the percentage of viable normal murine peritoneal cells

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The antiproliferative effect of methanolic plant extracts on cancer cell lines

The effect of plant extracts on Jurkat T cell lines

A total of thirteen crude methanolic plant extracts were subjected to the antiproliferation assay on Jurkat T cell lines at a concentration of 250 µg/mL, the results are shown in Fig. 5. Eight methanolic plant extracts (A. pungu, C. edulis, F. indica, L. ocymifolia, L. martinicensis, P. kalachariensis, S. spinosa and V. verrucosum) were considered to have antiproliferative effects as there was a significant difference between the treated cells and the cells only with P-value < 0.0001. The extracts deemed effective included A. pungu, C. edulis, L. martinicensis, L. ocymifolia, L. martinicensis and V. verrucosum as they exhibited a reduction in cell viability by more than 50%. A concentration-dependant assay was conducted for the four methanolic plant extracts and the results are shown in Fig. 6. The IC50 values are shown in Table 2.

Fig. 5
figure 5

The effects of crude methanolic plant extracts on Jurkat T cell lines. Crude methanolic plant extracts were tested for their antiproliferative effect on Jurkat T cell line at a concentration of 250 µg/mL. Chlorambucil was used as a positive control at a concentration of 10 μg/mL. The negative control contained cells and RPMI media only. The cells were incubated with test solutions and cell viability was determined after 72 h. The values are for the mean ± SD for n = 3. All treatments were compared to cells only to determine the level of significance. The level of significance is denoted as follows: **** P < 0.0001. A. pungu, C. edulis, F. indica, L. ocymifolia, L.martinicensis, P. kalachariensis, S. spinosa and V. verrucosum, significantly inhibited the proliferation of Jurkat T cell line and V. verrucosum had the greatest potency

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Fig. 6
figure 6

Concentration-dependent effect of crude methanolic plant extracts on Jurkat T cell lines. The concentration dependent effect of crude methanolic plant extracts of A. pungu, C. edulis, L. ocymifolia, L. martinicensis and V. verrucosum were evaluated on Jurkat T cell lines. The cells were incubated with crude methanolic plant extracts at varying concentrations ranging from 0 to 250 μg/mL and cell viability was determined after 72 h. Chlorambucil was used as a positive control at a concentration of 1.25 to 10 μg/mL. The negative control contained cells and RPMI media only. The cell viability decreased with an increase in the concentration of the extract

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The effect of plant extracts on HL60 cell lines

The antiproliferative effect of thirteen crude methanolic extracts was also assessed on HL60 cell lines at a concentration of 250 µg/mL. The results are shown in Fig. 7 and it was noted that nine plant extracts ( A. pungu, C. edulis, F. indica, L. ocymifolia, L. martinicensis, P. kalachariensis, S. cocculoides, S. spinosa and V. verrucosum) were considered potent as there was a significant difference between the treated cells and the negative control with P-value < 0.0001. The extracts that reduced cell viability by 50% or more (A. pungu, C. edulis, L. ocymifolia, L. martinicensis and V. verucosum) were subjected to a concentration-dependent assay and the results are shown in Fig. 8 and the IC50 values were calculated and shown in Table 2.

Fig. 7
figure 7

The effects of crude methanolic plant extracts on HL60 cell lines. Crude methanolic plant extracts were tested for their antiproliferative effect on HL60 cell line at a concentration of 250 µg/mL. Camptothecin was used as a positive control at a concentration of 10 μg/mL. The negative control contained cells and RPMI media only. The cells were incubated with test solutions and cell viability was determined after 72 h. The values are for the mean ± SD for n = 3. All treatments were compared to cells only to determine the level of significance. The level of significance is denoted as follows: **** P < 0.0001. Plant extracts A. pungu, C. edulis, F. indica, L. ocymifolia, L. martinicensis, P. kalachariensis, S. cocculoides, S. spinosa and V. verrucosum significantly inhibited the proliferation of HL60 cells and V. verrucosum was the most active

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Fig. 8
figure 8

Concentration-dependent effect of crude methanolic plant extracts on HL60 cell lines. The concentration-dependent effect of crude methanolic plant extracts of A. pungu, C. edulis, L. ocymifolia, L. martinicensis and V. verrucosum were evaluated on HL60 cell lines. The cells were incubated with extracts at varying concentrations ranging from 0 to 250 μg/mL. Chlorambucil was used as a positive control at a concentration range of 1.25 to 10 µg/mL. The negative control contained cells and RPMI media only and cell viability was determined after 72 h. The cell viability decreased with an increase in the concentration of the extract

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Selectivity Index (SI) of potent methanolic plant extracts

The SI values for Jurkat T and HL60 cell lines versus normal murine peritoneal cells were determined for the most potent plant extracts (A. pungu and V. verrucosum) and are shown in Table 2.

Phytochemical screening of methanolic plant extracts

Thirteen plant extracts were screened for different classes of phytoconstituents and the results are shown in Table 3 and the frequency of phytochemical constituents are shown in Fig. 9.

Table 3 Phytochemical screening of thirteen medicinal plants used traditionally for the treatment of cancer in Zimbabwe
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Fig. 9
figure 9

Frequency of phytochemical constituents of the crude methanolic plant extracts. Among the 13 methanolic plant extracts screened, 10 contained alkaloids, 9 contained flavonoids, and 9 had saponins. Alkaloids, flavonoids and saponins were the most common phytochemical classes present in the plant extracts

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Discussion

Plants are a rich source of natural compounds and the importance of such compounds has been proven since time immemorial [23]. Cancer has become a cause of morbidity and mortality across the globe, this is because there has been an increase in incidences of drug-resistant cancer and some cancer treatments have severe side effects [8, 24, 25], hence, there is a need to search for new anti-cancer drugs with little or no side effects. An ideal anti-cancer drug should be able to destroy cancer cells while leaving the normal cells undamaged [26]. Phytochemical compounds have offered promising alternatives to synthetic drugs and these significantly contributed to cancer treatment [27]. Hence, the current study evaluated the antiproliferative and cytotoxic effects of medicinal plants used traditionally in Zimbabwe for the treatment and management of cancer.

The cytotoxic effects of methanolic plant extracts on normal cells

The effects of plant extracts on normal murine peritoneal cells

Cytotoxicity is an important factor in the search for novel anticancer agents from plants. It is the toxic effect of a compound that leads to cell death [28]. By evaluating cytotoxicity on normal murine peritoneal cells, the current study aimed at assessing the potentially toxic effects of the extracts.

Mistletoes A. pungu and V. verrucosum methanolic extracts were the most cytotoxic on normal murine peritoneal cells. Currently, there is no available literature on the cytotoxicity of these mistletoes on normal cell lines. However, mistletoes in the same families (Santaliaceae and Loranthaceae) have been studied and found to contain several cytotoxic compounds [29,30,31]. It has been found that compounds from Viscum coloratum (Kom.) Nakai and Viscum album L. were cytotoxic to normal cell lines [32, 33]. Alkaloids and phenolic compounds were identified as primary biological active components in these mistletoe species [34,35,36]. Phytochemical screening of A. pungu and V. verrucosum in this study confirmed that both mistletoes contain alkaloids and phenolic compounds. This suggested that A. pungu and V. verrucosum might have similar compounds to those of mistletoes in the same family that have been studied. However, further research is required to determine the compounds responsible for the cytotoxic activity in A. pungu and V. verrucosum.

The effect of plant extracts on sheep erythrocytes

Haemolysis assay on sheep erythrocytes was used in this study to evaluate the effect of cytotoxic extracts on membrane integrity [37]. Haemolysis is the destruction of red blood cells resulting from the lysis of the membrane lipid bilayer [38]. Plant extracts may destabilise the membrane of red blood cells and this in turn can cause haemolysis [39]. The results show that haemolytic activity was concentration-dependent, wherein higher concentrations of plant extract correlated with increased haemolytic activity. The results are in agreement with previous studies by Zejli et al. [40] suggesting that haemolytic activity increases with extract concentration, as postulated by Fick’s Law which states that diffusion flux from a membrane is proportional to the concentration difference of both sides [41].

Plant extracts were considered to be haemolytic if the percentage of haemolysis was greater than 30% as a study by Khursheed et al. [42]. A. pungu and S. spinosa extracts exhibited low haemolytic activity of less than 30% at all concentrations. F. indica and V. verrucosum had haemolytic activity above 30% at the highest concentration (250 µg/mL), with V. verrucosum additionally exhibiting haemolytic effects at 125 µg/mL. Studies by Kim et al. and Podalak et al., have shown that the haemolytic activity of plant extracts can be attributed to the presence of phytochemicals such as saponins [43, 44]. In the current study, phytochemical screening revealed the presence of saponins in F. indica and V. verrucosum. As a result, saponins are likely to be responsible for the haemolytic activity observed. Several studies indicate that saponins can attach to erythrocyte membranes and destabilise them, resulting in haemolysis [39, 45]. As the concentration of saponins increases, there is a concurrent increase in haemolytic activity [45]. This could provide additional rationale for the higher degree of haemolysis observed at the highest concentration of 250 μg/mL as compared to lower concentrations.

The effect of reduced-glutathione (GSH) on the activity of cytotoxic extracts on normal murine peritoneal cells

It is common practice for cancer patients to use dietary supplements with antioxidants during or after conventional cancer treatments to enhance the benefits of the treatment and prevent side effects [9, 46, 47]. The use of antioxidants during chemotherapy has been condemned as it is believed to interfere with the efficacy of the drug [9]. However, some practitioners use antioxidant supplements so that patients can tolerate higher effective doses of chemotherapy [48]. Antioxidants are viewed as a safe means of protecting cells and tissues from damage caused by free radicals thus preventing the onset of cancer and side effects of chemotherapy [48, 49].

Reduced-glutathione (GSH) is a naturally occurring antioxidant that plays a critical role in defence, cellular process regulation, and nutrient metabolism [50]. The effect of GSH was investigated on A. pungu and V. verrucosum extracts which reduced cell viability of normal murine peritoneal cells by more than 50%. Based on the classification by López Villarreal et al., [51] these extracts were categorised as moderately toxic. The addition of GSH to the extracts of A. pungu and V. verrucosum led to an increase in cell viability. This finding aligns with previous by Singh et al., studies suggesting antioxidants mitigate drug-induced cytotoxic effects [48]. Furthermore, research has demonstrated a heightened therapeutic response to drugs when accompanied by antioxidant supplementation [48, 52]. However, since these were in vitro studies, further studies are necessary to confirm these findings and evaluate their potential therapeutic implications.

The antiproliferative effect of plant extracts on cancer cell lines

Antiproliferative assays are widely used to assess the effectiveness of potential anticancer agents [53], by measuring their ability to inhibit cancer cell growth. These assays provide valuable insights into the therapeutic potential of test compounds, which can help slow or even halt disease progression [54]. In this study, antiproliferation assays were conducted on leukaemia cell lines specifically Jurkat T and HL60 cell lines to evaluate the effectiveness of methanolic plant extracts. Leukaemia was chosen as a target due to the high rate of leukaemia cases in Zimbabwe [55] and the fact that it is the leading cause of death for all haematological malignancies worldwide [56].

The effect of plant extracts on Jurkat T and HL60 cell lines

The plant species A. pungu, C. edulis, F. indica, L. ocymifolia, L. martinicensis, P. kalachariensis, S. cocculoides S. spinosa and V. verrucosum significantly inhibited the proliferation of both leukaemia (HL60 and Jurkat T) cell lines, as confirmed by statistical analysis (p < 0.0001). The extract of S. cocculoides extracts had a significant antiproliferative effect on the HL60 cell line, as supported by statistical analysis (p < 0.0001). Strychnos species (Loganiaceae) are considered among the most renowned plants for their health-promoting properties [57]. However, to the best of our knowledge, the antiproliferative effects of S. cocculoides have never been reported before.

It was noted that S. cocculoides had significant antiproliferative activity on HL60 than on Jurkart T cell lines. The difference in the antiproliferative effects of S. cocculoides on HL60 and Jurkat T cell lines could be attributed to variations in the genetic makeup and susceptibility of the cells to the compounds present in the plant [58]. Different cell lines can exhibit diverse responses to the same treatment due to variations in their molecular characteristics and underlying mechanisms of proliferation [59]. Additionally, the use of different methods to determine cell viability, such as the SRB assay for HL60 cell lines and the trypan blue exclusion method for Jurkat T cell lines, could have contributed to the observed difference in antiproliferative effects. One method may be more sensitive than the other, leading to variations in the reported percentage viability between the two cell lines.

The results revealed that the activity of methanolic plant extracts increased with an increase in the concentration of extract, which is consistent with previous studies by Gul et al. and Pieme et al. [60, 61]. This suggests that higher concentrations of these extracts may have a stronger antiproliferative effect on both cell lines, HL60 and Jurkat T. Extracts of C. edulis, and L. martinicensis have been used traditionally for the treatment of cancer [62, 63]. Research on C. edulis has proved that it has antitumor activity [62, 64] and its characterisation led to the isolation of lupeol [65], a compound with proven anti-cancer properties [66, 67]. Preliminary studies by Matata et al., show that L. martinicensis extract might have some anti-cancer properties [68] and this is in line with the results that were obtained in this study. However, to our knowledge, no studies have reported the antiproliferative effects of A. pungu, L. ocymifolia and V. verrucosum on HL60 and Jurkat T cell lines.

In the present study V. verrucosum was the most potent extract with IC50 values of 33 and 34 µg/mL on Jurkat T and HL 60 cell lines respectively. Based on the IC50 values, this extract shows potential for further development as a source of chemotherapeutic compounds. This potential aligns with the criteria set by the American Cancer Institute [69]. Viscum verrucosum is a type of mistletoe and traditionally a wide range of mistletoe species have been used in the treatment of several ailments, including cancer [70]. Mistletoes, including V. album from the Santalaceae family, have been identified as promising alternative therapies for treating various types of cancer [31]. Numerous studies on V. album have demonstrated that its compounds possess antiproliferative effects [71, 72], and it is likely that V. verrucosum, also from the same family, may exhibit similar bioactive compounds.

Selectivity Index (SI) of potent plant extract

Chemotherapy is the most widely used treatment for leukaemia, involving the use of drugs to slow the growth or destroy cancerous cells [8]. Despite its wide use, chemotherapy is known to have side effects since it damages both normal and cancerous cells, thus limiting its clinical application [8]. In the search for novel chemotherapeutic compounds from plants, it is important to evaluate the selectivity of plant extracts as this will indicate compound toxicity to normal cells [73]. In the present study, the effect of methanolic plant extracts was evaluated on leukaemia cell lines (Jurkat T and HL60) and normal animal cells.

To identify extracts that are both active on cancer cell lines and safe for normal cells, Selectivity Index (SI) values were calculated for potent extracts. An SI value of less than 1 meant that the plant extract was non-selective (toxic), between 1 and 10 the extract was weakly selective and greater than 10 meant the plant was safe (non-toxic), leading to better outcomes and fewer side effects for patients [20]. The SI values of A. pungu and V. verrucosum ranged between 1 and 10, indicating that these plant species are weakly selective, meaning they are slightly toxic to normal cells. Therefore, further research is needed to determine their safety profile.

Phytochemical Screening of plant extracts

Phytochemicals are responsible for the biological activities of plant extracts [74]. Results show that plant methanolic extracts have several phytochemical classes with alkaloids, flavonoids and saponins being the most abundant. Alkaloids are the most common phytochemicals and they exhibit a wide range of pharmacological activities including anti-cancer properties [75]. Hence, they have been used in the development of anticancer drugs such as vincristine, vinblastine and taxol [76]. Therefore, the results obtained are in line with literature findings that alkaloids are the most abundant phytochemicals in plant extracts [75].

Flavonoids have recently gained importance due to their anticancer properties [77]; they have shown substantial potential as cytotoxic agents [36]. In the current study, a large number of methanolic plant extracts contained flavonoids and in the literature, this class of compounds has been reported to have beneficial effects on treating various diseases including cancer [78]. Plant-derived flavonoids like apigenin have proven anticancer properties with low toxicity to normal cells making them ideal anti-cancer drug candidates [79].

Saponins have shown potential in cancer treatment due to their ability to inhibit tumour cell growth, induce apoptosis, and suppress angiogenesis [44, 80, 81]. Their anticancer properties have been linked to structural diversity, making them promising candidates for novel anticancer therapies. Saponin-based anticancer drugs are currently unavailable, despite extensive research and significant anticancer effects [80]. This could be attributed to their inherent toxicities and complex chemical structures, which make it difficult to synthesise and purify them [82, 83].

Conclusion

The evaluation of antiproliferative, cytotoxic and phytochemical properties of Zimbabwean medicinal plants used in cancer treatment has provided valuable insight into their therapeutic effect. Plants that showed potential antiproliferative effects included A. pungu, C. edulis, L. ocymifolia, L. martinicensis and V. verrucosum on Jurkat T and HL60 cell lines. Mistletoe V. verrucosum was found to be the most potent with the lowest IC50 values of 33 and 34 µg/mL on Jurkat T and HL60 respectively. However, V. verrucosum displayed weak selectivity, suggesting that it is mildly toxic to normal cells as it had an IC50 of 134 µg/mL on normal murine peritoneal cells. Additionally the V. verrucosum extract caused destabilisation of the erythrocytes sheep membrane at 125 and 250 µg/mL. The addition of GSH appeared to have a protective effect on cells, potentially counteracting the cytotoxic effect of plant extracts on normal murine peritoneal cells. The study confirmed the presence of medicinally significant phytochemical classes such as alkaloids, flavonoids, terpenoids, quinones and saponins in plant extracts. Therefore, these findings emphasize the need for further research to evaluate the mechanism of action and the activity of extracts on other cancer cell lines. Additionally, investigating the phytochemical composition of potent methanolic plant extracts is essential.

Data availability

All data are included within the manuscript.

Abbreviations

FBS:

Foetal Bovine Serum

RPMI:

Roswell Park Memorial Institute

GSH:

Reduced—Glutathione

PNS:

Penicillin Streptomycin Neomycin

HBSS:

Hanks’ Balanced Salt Solution

DMSO:

Dimethyl sulfoxide

FCS:

Foetal Calf serum

TCA:

Trichloroacetic Acid

SRB:

Sulforhodamine B

SI:

Selectivity Index

IC50 :

Half-maximal inhibitory concentration

WHO:

World Health Organisation

RDC:

Rural District Council

ECACC:

European Collection of Authenticated Cell Culture

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Acknowledgements

The authors are grateful to the National University of Science and Technology (NUST) Research Board for funding the research, relevant Rural District Councils where plant samples were collected and the Biomolecular Interaction Analysis Laboratory at the University of Zimbabwe for allowing us to carry out tissue culture experiments.

Funding

This research work was funded by the National University of Science and Technology (NUST)

Research Board grant numbers RB/56/16, 57/16, and 19/17. The funders had no role in the design of the study design, data collection, analysis, interpretation of data, and writing of the manuscript.

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Authors and Affiliations

  1. Department of Applied Chemistry, National University of Science and Technology (NUST), P.O.Box AC 939, Ascot, Bulawayo, Zimbabwe

    Sigcono Mlilo & Samson Sibanda

  2. Department of Soil Science and Productivity, Sciences and Technology (MUAST), Marondera University of Agricultural, P.O Box 35, Marondera, Zimbabwe

    Simbarashe Sithole

  3. African Institute of Biomedical Science and Technology 911 Boronia Farm, Beatrice, Zimbabwe

    Stanley Mukanganyama

  4. Environmental Science, National University of Science and Technology (NUST), Bulawayo, Zimbabwe

    Yogehkumar S. Naik

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  3. Simbarashe Sithole
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  4. Stanley Mukanganyama
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  5. Yogehkumar S. Naik
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Contributions

S.M.1 conducted all the in vitro experimental studies with the assistance of S.S.2 and S.M.2. All authors read and approved the final manuscript. S.S.1 and Y.N directed the study. S.M.1 wrote the first draft. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Sigcono Mlilo.

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Ethics approval and consent to participate

The study was approved by the Faculty Higher Degrees Committee during its 318th meeting in November 2017 at the National University of Science and Technology (NUST), Zimbabwe. This approval was granted in the form of a written project proposal discussed in the board meeting. The NUST Institutional Review Board also approved the study, assigning it approval number NUST/IRB/2022/48. Permission to collect plant species was obtained from the relevant Rural District Councils and Community Leaders.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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Mlilo, S., Sibanda, S., Sithole, S. et al. Evaluation of the antiproliferative, cytotoxic and phytochemical properties of Zimbabwean medicinal plants used in cancer treatment. BMC Complement Med Ther 25, 156 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04883-1

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04883-1

Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

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