Anti-proliferative and photodynamic activities of Senna didymobotrya (Fresen.) leaf alkaloid-rich extracts against breast cancer cells

Abstract

Background

Amongst all neoplastic diseases, breast cancer represents a major cause of death among the female population in developed and developing countries. Since alkaloid drugs are commonly used in chemotherapy to manage this disease, this study investigated the anti-proliferative effectiveness of alkaloid-rich fractions of Senna didymobotrya leaves only and with laser irradiation against MCF-7 breast cancer cells.

Method and materials

A powdered sample of the plant leaves was extracted with 50% ethanol, filtered and their pH was adjusted with acid and base solution followed by partitioning with chloroform and ethyl acetate solvents. Cells were treated with 240 μg/mL of the respective extracts, while those in the photodynamic therapy groups, cells were exposed to laser (405 nm wavelength and 10 Jcm⁻²) irradiation 6 h post extracts' administration.

Results

Treatment with the S. didymobotrya leaves alkaloid extracts significantly decreased the ATP concentration and overall viability of the MCF-7 cells. Reactive oxygen species (ROS) levels in cell groups treated with the extracts and laser light were considerably higher than in experimental groups treated with only the extracts. Moreover, the molecular docking analysis revealed the involvement of only hydrophobic bonds in the interactions of the plant’s alkaloid-derived phytoconstituents’ with selected cancer protein biomarkers.

Conclusion

Although the in silico analysis suggests that the plant-identified alkaloid phytoconstituents inhibition of estrogen receptor-alpha, human epidermal growth factor receptor-2 and progesterone receptor proteins involved in breast cancer pathogenesis could explain a possible mechanism for the observed anticancer effect, more detailed in vitro molecular experiments are necessary to confirm these findings.

Introduction

Cancer is described as a neoplastic disease that remains a global health challenge with continually increasing reported incidence. According to Sung et al. [1] year 2020 report, cancer accounts for about 10 million deaths worldwide. Breast cancer is a major cancer type that constitutes a growing public health concern amongst the adult female population in Africa, even though other forms of this pathology affect people living on the continent. For instance, the data released by the South African National Cancer Registry, as reported by Swinny et al. [2], suggest that women in the country have a 1 in 27 chance of developing breast cancer.

Breast cancer is a collective terminology given to the various subtypes of breast cancer, which vary depending on their molecular characteristics, clinical staging, and gene expression [3]. Generally, breast cancer could be invasive when it originates in ductal or lobular tissues with inflammation that leads to the redness of the breast tissue, whereas it is non-invasive when it presents as lobular or ductal carcinoma in situ. Ultimately, these underlying mechanisms may result in blood-stained discharge, nipple retraction, scaling of the skin, lumping, and pain in the breast [4].

Various therapeutic interventions, including chemotherapy, radiotherapy, hormonal therapy, and tissue removal via surgery, exist for the management of breast cancer. While some patients may experience a resurgence of this disease after receiving some of these treatments, some studies have reported additional use of photodynamic therapy (PDT) to mitigate the risk of relapse [5]. PDT is a type of local cancer treatment where visible spectrum light is employed to initiate tissue damage after administering a photosensitizer (PS) compound to tumor cells [6]. Though it might be ineffective in combating metastasized or advanced cancer forms, PDT is often combined with other forms of treatment, such as chemotherapy, to increase effectiveness while reducing the incidence of drug resistance [7]. One of the PSs that displayed potency in unveiling the role of PDT in breast cancer treatment in vitro is sulfonated zinc phthalocyanine [8]. Despite the successful use of this compound in PDT, its self-aggregation with consequent reduction of singlet oxygen (¹O₂) production, as documented in some studies [9], has encouraged the search for alternative photoactive chemical compounds from plant sources.

The pharmacological activities associated with plant products have been attributed to their endogenous chemical compounds. Interestingly, reports have indicated that some phytochemicals possess photoactive properties that could make them pharmacologically relevant third-generation PSs for improving cancer treatments [10]. Amongst the group of plant secondary metabolites that have been described to be photoactive are nitrogen-containing alkaloids such as cinchonamine and quinine, which have demonstrated impressive anticancer activities [11, 12]. The mechanism of action of these alkaloids in cancer pathology has been described to include modulation of Bax, Bcl-2, Bcl-xL, NF-κB and other caspase proteins, regulation of cyclic-dependent kinase, as well as interference with cell cycle at the G1 or G2/M phases [13].

Senna plants are deciduous shrubs of the family Fabaceae or Leguminosae. Biological activities associated with species of this plant have been attributed to alkaloids identified in various parts of the plants. According to Pereira et al. [14], Senna spectabilis alkaloid-related plant chemicals impeded cell cycle processes in G1/S transition in liver cancer cells. Senna auriculata leaf extract phytochemicals similarly caused nuclear fragmentation and condensation associated with DNA laddering in MCF-7 and Hep-2 cell lines [15]. Besides their reported anticancer potentials, alkaloids identified in Senna species have been reported to display cholinergic, purinergic and antidiabetic activities [16,17,18]. Although the studies by Oliveira et al. [19] demonstrated the photodynamic therapy activities of Senna alata, Senna splendida and Senna macranthera against antibiotic resistance bacteria strains, other findings that showcase similar potentials of other Senna species, especially in cancer disease treatment are scarce. Therefore, our study aims to investigate the photodynamic therapeutic potential of Senna didymobotrya leaves alkaloid-rich extract against MCF-7 breast cancer cells for the first time. Moreover, the effects of the plant alkaloid-derived phytoconstituents on breast cancer etiology biomarkers were also determined in silico.

Materials and methods

Plant material and alkaloid-rich sample preparation

Leaves of Senna didymobotrya plant were obtained from its shrub found within the Durban Botanical Garden, Durban, South Africa, in September 2022. The aerial sample of the plant with an intact flower was taken to the University of KwaZulu-Natal ward herbarium for identification before a specimen voucher (K. Olofinsan 8) of the sample was deposited. The raw leaves of the plant were washed with running water and then dried under the shade. The dried leaves samples were powdered with a Wiley industrial miller (Philadelphia, USA) before being transferred into air-tight plastic bags. Then, alkaloid-rich extracts of the leaves were prepared using the modified method of Xie et al. [20]. About 60 g of the pulverized leaves were extracted exhaustively in 50% ethanol. The pH of the recovered filtrate was adjusted to 10 with a 1 M solution of sodium hydroxide and then partitioned with ethyl acetate. The upper layer was separated as the ethyl acetate alkaloid-rich extract, while the lower layer was transferred into a beaker, and the pH was adjusted to 2 with a 1 M hydrochloric acid solution. The solution was subsequently fractionated with chloroform, and the lower layer was obtained as the chloroform alkaloid-rich extract. The two extracts were concentrated under reduced pressure at 40⁰C in a rotatory evaporator and then air-dried for in vitro analysis. The stock solution of the dried chloroform or the ethyl acetate extract used for the experimental assays was each prepared by first dissolving a dried mass of the extract in a small volume of DMSO before adding distilled water to a final concentration of 0.5% DMSO.

Senna didymobotrya extracts’ phytochemical characterization

The bioactive metabolites in the leaf alkaloid-rich samples were characterized using a AOC-20i model Shimadzu gas chromatograph attached to a QP2010 SE Mass spectrophotometer. 1 µl of the sample solution injected into the column was transferred through the separation chamber with ultra-pure helium gas. The flow rate was kept at 1.03 mL.min⁻¹ and 37 cm.s⁻¹ linear velocity. While the injector and the oven temperatures were maintained at 60⁰C and 250⁰C respectively, other operating parameters, including electron ionization mode and the electron multiplier voltage of the equipment, were set as described by Olofinsan et al. [21]. Subsequent identification of the S. didymobotrya chemical compounds was done by comparing the mass spectral data generated from the analysis with those in the National Institute of Science and Technology library,

Culturing of breast cancer cells

The breast cancer cell line (MCF-7) obtained from the American Type Culture Collection (ATCC) used for this experiment was cultured in 15 mL of a complete media containing 1% of penicillin–streptomycin, 10% Fetal Bovine Serum, 1% amphotericin B and Dulbecco's Modified Eagle's Medium. The cells were allowed to grow at 37 °C temperature, 85% humidity, and 5% CO₂ in an incubator until they reached around 90% confluency.

Cancer cells’ treatment

Confluent cells were washed with Hank's Balanced Salt Solution (HBSS) and detached from the culture flask with colourless TrypLE™ solution. The cells were seeded (300,000 cells/mL) into 6 groups, namely control (untreated cells), laser-only (untreated cells exposed to laser irradiation only), DE (cells treated with S. didymobotrya ethyl acetate alkaloid-extract only), LDE (cells treated with S. didymobotrya ethyl acetate alkaloid-extract and laser irradiation), DC (cells treated with S. didymobotrya chloroform alkaloid-extract only) LDE (cells treated with S. didymobotrya chloroform alkaloid-extract and laser irradiation). After seeding and incubation for 24 h, cells in the plant material groups were incubated with media containing 240 μg/mL of the respective extracts for 6 h before those in the laser irradiation groups were further exposed to laser light under the experimental conditions described in Table 1. In Oliveira et al. [19] previous experiment the absorption spectra of three different Senna species were observed to be in the 400 nm blue light spectrum.

Table 1 Laser light parameters utilized for the irradiation of Senna didymobotrya leaves alkaloid-extracts’ treated MCF-7 cells

Morphological assessment

Cells in the various experimental groups were washed with phenol red-free HBSS and subjected to morphological observation under a Wirsam Olympus inverted light microscope (CKX 41) with an Olympus C5060-ADUS digital camera. The camera's user interface was accessed on a computer with the imaging analysis software CellSens (V1.16), which was employed to capture possible cellular alteration images of the various experimental groups at 400 X magnification. Moreover, other assays carried out on the treated cells are detailed below.

3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) colorimetric assay

In this assay, dehydrogenase enzymes in the mitochondria of viable cells hydrolysis the tetrazolium ring of MTT (3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide) to give a coloured insoluble formazan product. Consequently, about 10,000 cells from the treated and the untreated experimental groups in suspension were incubated for 4–5 h at 37 °C with 0.1 mL of 10% Sigma MTT Cell Proliferation (kit I) assay solution prepared in serum-free media. Then, 100 μL of lysis buffer solution supplied with the kit was added to each well before further incubation overnight for complete cell lysis. The absorbance of the coloured solution produced was measured at 540 nm in a PerkinElmer VICTOR NivoTM multi-plate reader.

Trypan blue exclusion assay

Trypan blue is a negatively charged azo dye which can only interact with membrane components of compromised cells. In the assay, cell suspensions obtained from the various experimental groups were diluted first to lower their cell population. Then, 20 μL aliquot of cell suspension and 0.4% trypan blue solution (Invitrogen) were mixed with a pipette in a 500 μL Eppendorf tube. After that, 10 μL of the dye-cell suspension was transferred to each side on a countess slide before the number of live and dead cell populations was estimated automatically with the Invitrogen (counting II FL) cell counter.

Lactate dehydrogenase (LDH) release assay

This assay determines the amount of LDH leaked into the culture media and was carried out using a BioVision LDH-cytotoxicity colorimetric assay kit. Briefly, the culture medium containing the experimental cell samples will be aspirated and centrifuged at 4000 rpm for 5 min. Then, 0.1 mL of the supernatant was transferred to a 96-well plate before incubation with 100 μL of the reaction mixture supplied with the kit for 30 min in the dark at 25 °C. After terminating the reaction with the kit stop solution, the absorbance of the coloured solution produced was measured in a Victor Nivo Multimode plate reader at 450 nm. Subsequently, the LDH level in the experimental samples was calculated per the kit instructions.

Adenosine triphosphate (ATP) assay

This assay was done using the CellTiter-Glo® Luminescent ATP kit. Briefly, a mixture of the treated or the control cells (50 µL) suspension was incubated in the dark with 50 µL of ATP reagent and 50 µL phosphate buffered solution (1X). After shaking for 20 min at 25⁰C, the reaction mixture was transferred to the wells of an opaque white plate. Then, the ATP luminescence of the live cells was measured with the earlier mentioned multi-plate reader.

Reactive oxygen species (ROS) production

This experiment was carried out using the dichlorodihydrofluorescin diacetate (DCFH-DA) dye via a method modified from an earlier protocol [22]. Briefly, breast cancer cells were seeded at about 10,000 cells/well into black 96-well culture plates. After washing the cells with HBSS, they were made to take up the dye by incubation (37⁰C and 5% CO₂) for 1 h 30 min with 100 μL of complete media containing 100 μM of the DCFH-DA. Then, the cells were exposed to the previously described treatments (Control, laser-only, DE, LDE, DC, and LDC). After removing the DCFH-DA dye-media mixture in each well, the cells were quickly washed in the dark with phenol red-free HBSS solution before adding 100 μL of a lysis buffer containing 2.5 mM sodium chloride, 0.1 M ethylenediaminetetraacetic acid, 10 mM Tris (pH 10.00) and 1% Triton X-100. Subsequently, the fluorescence generated from each well was measured at 485 nm excitation and 535 nm emission wavelengths in the multimode spectrophotometer.

Molecular docking

The possible modulatory effects of the S. didymobotrya leaf alkaloid-phytoconstituents was determined on three proteins linked with breast cancer pathogenesis. The 3D structure of these proteins, which include estrogen receptor-alpha (ER-alpha), human epidermal growth factor receptor-2 (hEGFR-2) and progesterone receptor (PR) with access code 3ERT, 3RCD and 1SQN, respectively, were retrieved from the protein data bank. Each of the protein targets was prepared separately with the protein preparation tool of Schrodinger Maestro software (V11.5). Ligand and water molecules co-crystalized with the protein were removed before amino acid residues were missing from the protein crystallographic structure or the side chains were added before further hydrogen atoms were added. Non-polar charges were added to the carbon atoms, while Gasteiger charges were added to the polar hydrogen atoms. Then, the processed protein molecules were subjected to energy minimization. The 3D-chemical structures of the phyto-compounds identified in the extracts (Table 2) were downloaded from the PubChem database in SDF format. After processing and optimizing the compounds to their global minimal structures, they were subjected to molecular docking at the active site of the protein molecules. Subsequently, the best configuration of the ligand–protein complex with the lowest binding energy was saved in PDB format, and their 2D-molecular interaction was visualized with Discovery Studio software.

Table 2 Chemical structure of alkaloid-derived phytocompounds identified from Senna didymobotrya leaves

Data analysis

All the experiments were performed in triplicates (n = 3), and their data were expressed in mean ± standard errors. The statistical significance between experimental treatment groups was established at p < 0.05 with IBM SPSS version 25. While this software was used for one-way analysis of variance calculation, data presentation was done with GraphPad Prism for Windows (Version 6.01).

Results

The images captured after MCF-7 cells were subjected to various treatments, including the extracts and laser irradiation, are presented in Fig. 1. No pronounced changes were observed between the untreated cells’ morphology and those in the laser laser-only group. Among the alkaloid extracts-only groups, cells in the DE experimental group showed a higher level of cellular vacuolation than those in the DC group. Moreover, a similar trend was noticed for cells in the LDE group, which appeared to show more significant shrinkage than those in the LDC group.

Fig. 1

Morphological comparison of MCF-7 cells exposed to different treatments. Control = Untreated cells, Laser only = Laser light, DE = Cells + SD leaves ethyl acetate alkaloid fraction, DC = SD leaves chloroform alkaloid fraction, LDE = Cells + SD leaves ethyl acetate alkaloid fraction + laser light, LDC = Cells + SD leaves chloroform alkaloid fraction + laser light. Magnification × 400

Data in Fig. 2 display cell viability 24 h after S. didymobotrya extracts and laser light treatment. In the MTT assay presented in Fig. 2A, the viability in the DE group (45.3%) was statistically lower than the 68.5% viability estimated in the DC group. Although viability in LDE and LDC were 17.5% and 23.4%, respectively, these treatment groups had no significant differences (p < 0.05). While viability in the plant leaves chloroform alkaloid extract group (DC) was 74.5% in Fig. 2B trypan blue assay, further treatment with laser light decreased the viability below this value in the LDC group (51.1%). With the cells in the LDE group having the lowest cell viability (35%), estimated values in their similar group not exposed to laser irradiation (DE) were not significantly different from those in the LDC experimental group.

Fig. 2

Effect of Senna didymobotrya leaves alkaloid extracts and laser light irradiation on MCF-7 (A) MTT and (B) Trypan blue dye cell viability assays. Data are presented as mean ± SD triplicate experiments. Bars with different letters a-d are statistically different at p < 0.05

The result of the relative concentration of lactate dehydrogenase released from cells in each experimental group after treatment is shown in Fig. 3. Although the amount of the enzyme in the Control, DE, DC, and laser-only groups were 13.6%, 20.4%, 22.2% and 14.6%, respectively, statistically, these treatments are not significantly different from each other. However, the concentration of LDH produced from cells in the LDE group was significantly (12.8%) higher than those in the LDC treatment group.

Fig. 3

LDH cellular toxicity assay of MCF-7 cells treated with Senna didymobotrya leaves alkaloid extracts and laser irradiation. Data are presented as mean ± SD triplicate experiments. Bars with different letters a-c are statistically different at p < 0.05

Data in Fig. 4 compares the result of the relative amount of ATP generated in live cells in the different treatment groups. ATP in the laser-only group was significantly lower compared with those of untreated cells, representing the control. Within the alkaloid extract-only groups, energy equivalent concentration in the DE group was about 62.9% lower than in the DC group. However, similar energy production parameters measured in the LDE and LDC alkaloid and laser light-treated groups are not significantly different.

Fig. 4

ATP production assay of MCF-7 cells treated with Senna didymobotrya leaves alkaloid extracts and laser light. Data are presented as mean ± SD triplicate experiments. Bars with different letters a-e are statistically different at p < 0.05

The extent of ROS production in cells of the various experimental treatments is presented in Fig. 5. These chemical species estimate in the DC, DE and laser-only groups were not statistically different from each other despite being higher than the 23.9% relative concentration recorded in the untreated cell group. Furthermore, the result showed that the MCF-7 cells in the LDE treatment had the highest concentration of ROS, followed by those in the LDC group.

Fig. 5

Intracellular ROS production assay of MCF-7 cells treated with Senna didymobotrya leaves alkaloid extracts and laser light. Data are presented as mean ± SD triplicate experiments. Bars with different letters a-d are statistically different at p < 0.05

Table 2 shows the chemical information about the different alkaloid-related compounds detected via characterization analysis in the S. didymobotrya leaves chloroform and ethyl acetate alkaloid extracts. These compounds include 1,3,6,8-tetratert-butyl-9H-carbazole, 4-(4-methylphenyl) piperidine, 4-Acetyl-2-ethoxy-6-methoxyquinoline and 2,3-bis(1-methylallyl) pyrrolidine alkaloid derivatives. Other compounds of the same phytochemical classification also found in the extracts were N-acetyl-4-(4-methoxyphenyl)-6-phenyl-2(1H)-pyrimidinone and 9-Aza-D-homogona-13(14)-en-6-one.

The binding energies obtained after molecular docking of the identified S. didymobotrya alkaloid derivatives with three key proteins linked with cancer pathogenesis in the breast are displayed in Table 3. 9-Aza-D-homogona-13(14)-en-6-one produced the highest binding energy (-9.1 kcal mol^–1) with progesterone receptor protein. This calculated value is followed by 8.0 kcal mol^–1 for 4-(4-Methoxyphenyl)-6-phenyl-2(1H)-pyrimidinone and the same protein. Although all the 3D chemical structures of all these plant-derived alkaloids have negative binding energies that suggest good interactions with ER-alpha and human EGFR-2 proteins, with -8.5 kcal mol^–1 and -8.8 kcal mol^–1 estimated binding energies, 9-Aza-D-homogona-13(14)-en-6-one amongst all the other compounds had the strongest affinities with the proteins respectively.

Table 3 Binding energies computed from the docking of Senna didymobotrya leaves derived alkaloids with breast cancer-linked protein targets

Since 9-Aza-D-homogona-13(14)-en-6-one had the lowest binding energies with the selected protein targets, the 2D images showing the molecular interactions between the compound and the active site amino acids of the selected breast cancer linked markers are present with their respective 3D pictures in Fig. 6. From the in silico analysis results, the stability of the compound at the active site pockets of the protein is mainly due to alkyl and pi-alkyl hydrophobic interactions. The progesterone receptor amino acids involved in the bonding complex with this compound include LEU80, LEU114, LEU203, LEU38, CYS207, MET76, PHE95 and TYR206. For the human EGFR-2 protein, MET116, MET38, LEU220, LEU41, LEU86, LEU44, ALA45 and HIS219 residues were associated with the protein–ligand complex formations, whereas in estrogen receptor-α protein, only LEU17, LEU139, ALA42 and PHE266 moieties were linked with the molecular bonding.

Fig. 6.

2D and 3D images of the binding interactions of 9-Aza-D-homogona-13(14)-en-6-one with active site amino acids of (A) Progesterone receptor (B) Epidermal growth factor receptor-2 and (C) Estrogen receptor-α

Discussion

Among the different cancer pathologies affecting people globally, breast cancer continues to represent a significant cause of mortality amongst the women population, especially in developing countries [23]. Chemical compounds like alkaloids derived from plants have been improved to produce drugs, including vincristine and vinblastine, as chemotherapeutic agents to manage this female-related malignancy [24]. The fact that the use of these drugs was associated with various unwanted adverse reactions has resulted in the search for alternative anticancer chemical molecules of natural origin with similar pharmacological bioactivity but with reduced side effects. With previous findings documenting the presence of various alkaloids in Senna plant species [14, 16, 25], the current study investigated the potential anti-proliferative effects of S. didymobotrya leaves alkaloid-rich extracts as a single treatment or combined with phototherapy in MCF-7 breast cancer cells.

The mitochondria is a unique organelle found in all eukaryotic cells, that generate energy in the form of ATP, allowing cell survival in an infested host. This process of energy generation is this cellular structure is mediated or regulated by different enzymes. One of these enzymes, which constitute one of the components of the enzyme complexes of the respiratory chain and plays a role in the Krebs cycle, is succinate dehydrogenase. Although this reductase physiologically converts succinate to fumarate, it has been evidenced that it could also use MTT as a substrate to produce an insoluble coloured formazan product [26]. Consequently, the ability of this enzyme to generate formazan molecules in living cells via viability estimates can be employed to determine the effectiveness of chemical entities targeted to destroy cancerous cells. The significant reduction in the MCF-7 cell population of the plant treatment groups compared with the untreated controls, as observed in the viability result in Fig. 2A, is supported by the decrease in total ATP concentration estimated in the living cells, as presented in Fig. 4. Since ATP is only generated in the live cells, this observation may be linked with the anticancer effect of the Senna plant extract on cell survival.

The cell membrane act as barrier that separates cell intracellular components such as enzymes that are vital for life process from chemicals found in the extracellular environment, that could negatively affect their functions. Lactate dehydrogenase, an enzyme confined normally to the cell cytoplasm produces reducing equivalents (NADH) needed for energy production during reduction of lactic acid to pyruvate. Interestingly, previous studies have revealed that the activity of this enzyme increased considerable in cancer cells to meet their energy demands [27]. Then, when cell dies, their cell components such as this protein could leak into the extracellular medium and thus their utilization as cell death biomarker. Beside the realise cytoplasmic content, dead cells due to their inability to maintain the membrane integrity allow extracellular chemicals like trypan blue to penetrate into their internal where they interact with cellular macromolecules [28]. The reduced cell viability in the S. didymobotrya alkaloid extracts group as observed in the trypan blue essay in Fig. 2 could further support the anti-proliferative efficacies of the plant mediated by its constituent alkaloid phytochemicals presented in Table 2. Interestingly, the role of plant derived alkaloids and their derivatives in cancer therapies has been well documented [13]. Moreover, in support of this study, alkaloid compounds from other Senna species have been reported to show anticancer activities [14]

Photodynamic therapy represents one of the clinically approved procedures for managing cancer. The less invasive cancer treatment method involves the chemical interplay of molecular oxygen, a photosensitizer and light with wavelength in the visible region [29]. The mechanism behind this therapeutic procedure involved the production of reactive oxygen species after electrons are released from light-excited photosensitizer molecules. Thus, The ROS generated triggers a reaction cascade that abstracts electrons from intracellular biomolecules, ultimately leading to cell death [30]. With the pharmacokinetic features, including solubility representing one of the challenges currently hampering the development of some existing synthetic photosensitizer drugs in PDT, there is a growing demand to seek nature as an alternative source of such chemical compounds. Interesting alkaloids such as berberine have been established to possess photosensitivity effects for cancer cell targeting [31]. The significant increase in ROS concentration in the laser light and S. didymobotrya extracts treated groups (LDE & LDC) in Fig. 5 relative to the extract-only treatments (DE & DC) could suggest photoactive properties of some phytoconstituents of this plant, as supported by the morphological images in Fig. 1. Interestingly, Oliveira et al. [19] reported the photodynamic activity of Senna macranthera, Senna alata and Senna splendida on some bacteria of medical significance.

Some of the drugs that have been directed toward breast cancer management function by modulating the activities of some proteins implicated in the disease pathogenesis. The molecular investigations carried out on some of these proteins, namely estrogen receptor-alpha, progesterone receptor and human epidermal growth factor receptor-2, had assisted researchers in understanding the possible roles of these proteins and their mechanism involved in the onset and progression of the pathology. Therefore, to save time and avoid the unnecessary sacrifice of animals during ex vivo and in vivo scientific experiments, several studies have employed molecular docking analysis to screen and predict the possible mechanisms involved in the pharmacological activities of lead chemical molecules against these proteins [32,33,34]. Consequently, the negative binding energies of the alkaloid derivatives identified in the S. didymobotrya extracts in this study (Tables 2 and 3) could suggest the inhibitory effects of these phytoconstituents on the functionality of target proteins in the MC7-cells. This observation may also be linked to the decreased cell viability of the cell groups treated with only the extract as compared with those in the normal untreated control (Fig. 2). While 9-Aza-D-homogona-13(14)-en-6-one amongst the compounds found in our studied plant showed high affinities for the active site amino acids of the breast cancer-associated biomarkers (Table 3), a chemical with the same quinolizidine scaffold as this alkaloid has been derivatized into other molecules that portrayed pronounced cytotoxic properties against triple-negative breast cancer cell lines [35].

Conclusion

This study, for the first time, revealed the anticancer potentials of Senna didymobotrya leaves alkaloid extracts as a single treatment and their photodynamic therapy effect in the presence of light in the visible region against MCF-7 cells. Although the in silico analysis carried out in this investigation suggests the possible inhibition of selected biomarkers linked to this onset and progression of breast cancer, an in-depth molecular study of these proteins is proposed for further research to provide a better understanding of the mechanism employed by the plant phytoconstituents in bringing about the observed findings in this study. Unravelling this mode of action could cause the discovery of lead anticancer drugs from this plant with efficacies similar to existing commercially available synthetic chemicals but relatively affordable to diseased individuals amongst the populations in developing countries.