Focus on Mascarenes Endemic Plants with Specific Phytochemical Composition, Potent Antioxidant and Antiproliferative Properties

Tropical forests constitute prolific sanctuary of unique floral diversity and potential medicinal sources, however, many of them remains unexplored. Herein, seven Mascarene endemic plants leaves were extracted and evaluated for their in vitro antioxidant properties and antiproliferative effects on a panel of cancer cell lines using MTT and clonogenic cell survival assay. Flow cytometry and comet assay were used to investigate the cell cycle and DNA damaging effects, respectively. Bioassay guided-fractionation coupled with LC-Mass spectrometry (MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopy analysis were used to identify the bioactive compounds. Among the seven plants tested, Terminalia bentzoë was comparatively the most potent antioxidant extract with significantly (p < 0.05) higher cytotoxic activities. T. bentzoë extract further selectively suppressed the growth of human hepatocellular carcinoma cells and significantly halted the cell cycle progression in G0/G1 phase, decreased the cells replicative potential and induced significant DNA damage. Ten phenolic compounds including punicalagin and ellagic acid were identified and likely contributed to the extract potent antioxidant and cytotoxic activities. These results established a promising basis for further in-depth investigations on the potential use of T. bentzoë as supportive therapy in cancer management.

Madagascar and its neighbouring islands in the Western Indian Ocean regions are known as biodiversity hotspots [8]. Undoubtedly, untapped endemic plant species from these niche areas broadened the structural variation of novel chemotypes [9,10]. The tropical forest of Madagascar was once acknowledged as a fertile source of economically valuable plants with pharmacologically active ingredients [11]. Indeed, the anticancer drugs vinblastine and vincristine were derived from the Madagascan endemic Catharanthus roseus (Apocynaceae) [12]. Certainly, the market value of vincristine alone was estimated to be 15 million USD per kilogram, in the year 2016 [13].
Phytogeography investigations revealed that the Mascarene endemic plants islands have their ancestral lineages traced back from Madagascar [14]. As such, the unique floral biodiversity of Mauritius is expected to possess similar medicinal and therapeutic prolificacy as the Madagascan rain forest. However, instead of conserving such valuable biodiversity, human activities are pushing endemic taxa towards an unprecedented extinction crisis. In less than 400 years of human settlement, Mauritius has witnessed the shrinking of its native forest to around 5 % of the original cover, leading to the permanent loss of 30 (10.9 %) of its endemic plant species and driving 81.7 % of the remnant endemic taxa on the brink of extinction [15][16][17]. Nevertheless, the remnant areas of the pristine forest are still home to a plethora of endemic flora rich in high genetic diversity representing interesting sources for complementary and alternative medicine, nutraceuticals as well as pharmaceutical leads [16].
With this in mind, the in vitro antioxidant propensities of leaf extracts from seven plants endemic to Mascarene islands were investigated. The plants under study have documented traditional uses against ailments ranging from dermatological conditions, asthma to infectious diseases ( Table 1). The cytotoxic effect of Terminalia bentzoë, on a panel of cancer cell lines, and its ability to impede the cell cycle progression in hepatocellular carcinoma (HepG2) cells were determined. The bioactive constituents in T. bentzoë leaf extract were characterised following bioassay-guided fractionation.

Materials and Methods
2.1. Plant material and preparation of total extracts. Healthy fresh leaves of seven Mascarene endemic plants were collected in Mauritius and deposited at the Mauritius herbarium, where plant species were authenticated by the botanist ( Table 1). The leaves were air-dried followed by exhaustive maceration with aqueous methanol (80 %, v/v) and freeze-dried as described previously [18]. The assay results were expressed in terms of the lyophilised weight of extracts.
2.2. Estimation of polyphenolic contents. The total phenolic, flavonoid, and proanthocyanidin level in the crude extracts were estimated using the Folin-Ciocalteu assay, aluminium chloride assay and HCl/Butan-1-ol assay as described [18].
2.3. In-vitro antioxidant capacities of extracts. The antioxidant potential of the extracts was investigated according to reported methods [18,19]. The final reaction volume of 3.4 mL of the ferric reducing antioxidant power assay contained 100 µL of extract and 300 µL of water followed by addition of 3 mL FRAP reagent. The FRAP reagent was prepared immediately before use by mixing 100 mL of 0.25 M acetate buffer (pH 3.6), 10 ml of 20 mM ferric chloride (source of Fe 3 +) and 10 mL of 10 mM 2,4,6-tripyridyl-s-triazine. After incubating the mixture for 4 minutes at ambient temperature, the absorbance was read at 593 nm against a blank. The DPPH assay protocol involved mixing varying concentrations of 100 µL of the methanolic extract with 200 µL of 100 µM methanolic DPPH and absorbance were read 30 minutes post-incubation at ambient temperature.
The reaction mixture for iron-chelating activity contained 40 µL of plant extract (concentrations ranging between 0 to 10 mg/ml), 10 µL of FeCl2⋅4H2O (0.5mM) and 150 µL of distilled deionized water. The mixture was incubated at ambient temperature for 5 minutes, before the addition of 10 µl of ferrozine (2.5mM) and the absorbance was read at 562 nm. The final 250 µL reaction volume for superoxide anion scavenging assay contained 25 µL of plant extract (0 to 300 µg/mL), 100 µL of 156 µM of nitroblue tetrazolium and 100 µL of 200 µM beta-nicotinamide adenine dinucleotide reduced disodium salt hydrate and 30 µL of phenazine methosulphate. The absorbance was read at 560 nm following 30 minutes incubation at 25 ˚C. The nitric oxide radical scavenging activity was conducted in a 96-well plate. 50 µL of aqueous extract (0 to 100 µg/ml) and 100 µL of 5 mM of sodium nitroprusside (in phosphate saline buffer, pH 7.4) was incubated at 25 ˚C for 150 minutes. After incubation, 125 µL of the reaction mixture was transferred to another 96-well plate, to which 100 µL of 0.33 % sulfanilic acid in 20 % glacial acetic acid was added. After 5 minutes, 100 µL of 0.1 % of N-1-napthyethylenediamine dihydrochloride was added and the pink coloration formed was read at 546 nm.
The deoxyribose degradation inhibitory assay protocol was optimised to a 24 well microtiter plate format. Each well contained 50 µL of aqueous extract, 50 µL of 1 mM EDTA, 100 µL of 500 µM FeCl3, 50 µL of 1 mM ascorbic acid, 50 µL of 10 mM hydrogen peroxide, 100 µL of 100 mM KH2PO4-KOH buffer (pH 7.4) and 100 µL of 15 mM 2-deoxyribose. The reaction mixture was incubated at 37 ˚C for 90 minutes. At the end of the incubation period, 500 µL of 10 % (w/v) trichloroacetic acid followed by 500 µL of 1% (w/v) thiobarbituric acid were added to each well and the solutions were heated in a water bath at 80 ˚C for 20 minutes to develop the pink chromogen. The absorbance of the reaction mixture was read both before and after incubation at 80 ˚C. Results were given in mg lyophilised extract/mL.
Extract vehicle and gallic acid (or otherwise stated) were used as negative and positive controls respectively. The percentage activity of the extracts was calculated relative to the negative control. GraphPad Prism 6 software (GraphPad Inc., USA) was used to plot the dose-response curves and to generate the half-maximal inhibitory concentration (IC50) values. All experiments were performed in triplicates in three independent assays. The results were expressed as mean ± SEM.

Human cell lines and culture conditions.
Human liposarcoma cells (SW872), Human lung carcinoma cells (A549) and Human hepatocellular carcinoma cells (HepG2), human ovarian carcinoma cell lines OVCAR-4 and OVCAR-8 were purchased from American Type Culture Collection (USA). Human ovarian epithelial (HOE) cells immortalized using SV40 large T antigen was obtained from Applied Biological Materials Inc (Canada). All cell lines, except the ovarian cell lines, were cultured in Dulbecco's Modified Eagle's Medium. Roswell Park Memorial Institute (RPMI) 1640 medium was used in the case of ovarian cell lines. Culture medium was supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 100 U/L streptomycin-penicillin. Cells were grown in a humidified atmosphere of 5 % carbon dioxide and 95 % humidity at 37 ˚C.

Cell-based assays.
The viability of the investigated cells treated with test samples was evaluated using the methyl thiazolyl diphenyl-tetrazolium bromide (MTT) cell viability assay. Following overnight acclimatisation of cells in 96-well plate, cells were treated with test samples for 48 hours and assayed for different parameters. For 96-well plate, the seeding densities for cancer cell lines and HOE cells were 5 x 10 3 cells and 2 x 10 3 cells per well, respectively. All experiments were performed in triplicates (unless otherwise specified) in three independent assays.

MTT viability assay
The MTT assay was performed as previously described [20]. The percentage cell viability relative to DMSO control (0.025 % v/v, final concentration) was calculated and the IC50 value determined using GraphPad Prism 6 software (GraphPad Inc., USA).

Clonogenic cell survival assay
The effect of T. bentzoë leaf extract on the cell reproductive death was assessed by clonogenic cell survival assay according to reported methods, with slight modifications (Al-Dabbagh et al., Franken et al., 2006). HepG2 cells were seeded in a 6-well plate (500/well) and allowed to attach overnight. Following 48 hours treatment period with extract/ control, the media was replaced with fresh complete culture media and cells were grown under standard recommended culture conditions for an additional 7 days to allow large colonies formation. Colonies were then fixed with 4 % paraformaldehyde for 30 minutes and stained with 0.5 % (w/v) crystal violet. The individual wells were imaged using a digital camera and the colonies counted using ImageJ software (the US, National Institute of Health). The cytotoxic effect was expressed as the percentage of surviving colonies relative to untreated control.

Single Cell Gel Electrophoresis
Comet assay was carried out according to the method described [21,22] with minor modifications. Briefly, 30 µL of pre-treated cells were mixed with 70 µL of 1 % (w/v) low melting agarose (LMA) and 40 µL of the cell-LMA mixture was placed on frosted microscope slides pre-coated with 1.5 % normal melting agarose. A coverslip was placed on top of the cell-LMA mix and allowed to solidify at 4°C for 1 hour in dark. Following solidification, the coverslip was gently slid off and the slides were immersed in pre-chilled lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris base, 1% v/v Triton X-100 (added 30 minutes before use), pH 10, 4 °C) for 1 hour in dark. Following this period, the excess lysis solution was drained and the slides were submerged in electrophoresis buffer (0.2 M NaOH, 1 mM EDTA, pH 13, 4 °C) for 30 minutes in dark, to allow the DNA to unwind.
Electrophoresis was conducted for 30 minutes at 30 volts and 350 mA. The gels were neutralized by immersing in pre-chilled neutralisation buffer (0.4 Tris-HCL, pH 7.5, 4 °C) for 10 minutes in dark. The slides were washed in distilled water, fixed with 4 % formalin solution for 20 minutes and allowed to air-dry overnight. The slides were stained with Hoechst 33342 (1 µg/ml), air-dried in dark and visualised at 200 X magnification in DAPI, using EVOS fluorescence microscope (Life Technologies). Damaged DNA was measured for 100 randomly selected cells (for each independent experiment) using the Comet Assay IV 4.3.1 (Perceptive instrument, UK).

Flow cytometric analysis
Apoptosis/necrosis analysis was performed on HepG2 cells after 48 hours treatment by flow cytometry (Beckman Coulter's CytoFLEX and Cytexpert software) using Annexin V-FITC and Propidium iodide (PI) double staining as described in a previous study [23]. Cell cycle analysis was performed using propidium iodide for DNA staining as described [24]. Percentage of cells in different phases (GO/G1, S and G2M phases) were quantified from propidium iodide fluorescence intensityarea (PI-A) histograms corresponding to the DNA content of HepG2 cells.

MTT-guided fractionation and identification of bioactive molecules
The total extract of T. bentzoë was solubilised in distilled water and sequentially partitioned with ethyl acetate, followed by n-butanol. Each fraction was dried and their cytotoxicity evaluated against SW872, A549, HepG2 using the MTT cell viability assay. The butanol fraction being selective towards HepG2 cells was subjected to Sephadex LH-20 column chromatography (30 cm X 2.1 cm internal diameter) and eluted with water, water: methanol (3:1 v/v, 1:1 v/v and 1:3 v/v respectively), methanol and acetone. The flow rate was maintained at 1.5 mL/min. Guided by the cytotoxicity against HepG2 cells and purity profile, potent sub-fraction was further fractionated using semi-preparative HPLC column. The crude extract and thereof derived purified fractions were analysed by extensive spectroscopic methods including GC-MS, LC-MS, HPLC, 1 H-NMR and 13 C-NMR (Supporting Information). For analytical HPLC, the concentration of standards in the crude extract was determined from the linear regression of the analytical standards curve namely y = 12.317 x, R 2 = 0.9996: gallic acid and y = 16.066 x, R 2 = 0.9992: methyl gallate.

STATISTICAL ANALYSIS
Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Inc., San Diego, California). The mean values among extracts were compared using One-Way ANOVA. Student t-test and/or Tukey's multiple comparisons as Post Hoc test was used to determine significances in mean phytochemicals, antioxidants and cytotoxic activities among different species. All charts were generated using Microsoft Excel software (version 2010).

Estimation of polyphenols level in the investigated leaf extracts.
The phenolic content varied significantly among the seven leaf extracts under study (p < 0.05) with amounts ranging between 70.2 ± 4.72 mg and 385 ± 24.1 mg gallic acid equivalent/g. Total flavonoid levels ranged between 2.43 ± 0.06 mg and 12.9 ± 0.45 mg quercetin equivalent/g. Based on the spectrophotometric assay results, the estimated level of phenolics and flavonoids were significantly highest (p < 0.05) in T. bentzoë leaf extract as compared to the other investigated leaf extracts ( Table  2). While the proanthocyanidin content prevailed in E. sideroxyloides leaf, both S. lineata leaf and T. bentzoë leaf had a remarkably negligible amount of proanthocyanidin detected by the butanol/HCl assay.

In-vitro antioxidant activities of the investigated leaf extracts.
An array of five analytical models was used to benchmark the antioxidant potential of the investigated leaf extracts. The Mascarene endemic plant leaf extracts exhibited a varying degree of activities in the different antioxidant assays. All extracts showed a dose-dependent metal chelating and free radical scavenging activity ( Table 2). In terms of iron chelation, all the extracts were weak chelator compared to EDTA with an IC50 value of 0.01 ± 0.00 mg/mL (23.6 ± 0.22 µM) (p ≤ 0.05). As depicted in Table 2, among the seven accessions of plants, T. bentzoë showed the most effective antioxidant potential in all the five antioxidant assays. Thus, the free radical quenching activity of T. bentzoë was further evaluated in hydroxyl radical scavenging assay. T. bentzoë (IC50 = 0.25 ± 0.03 mg/mL) exhibited a significantly (p < 0.0001) greater degree of protection against Fenton-mediated oxidative damage to 2-deoxyribose sugar moiety as compared to gallic acid (IC50 = 1.65 ± 0.09 mg/mL). 8

Effect of T. bentzoë leaf extract on cell survival.
To assess the antiproliferative properties of T. bentzoë leaf extract on cancer cells, first, the influence of the extracts on the cell viability of five cancer cell lines notably, SW872, A549, HepG2, Ovcar-4 and Ovcar-8 cells, were investigated using the MTT assay. T. bentzoë suppressed the growth of all cancer cell lines in a dose-dependent manner. However, the dose of extract required to reach the halfmaximal inhibitory concentration differed considerably among the various cancer cell types ( Table  3). The cytotoxicity of T. bentzoë was also evaluated against the non-malignant human ovarian epithelial (HOE) cells and the IC50 value obtained (55.5 ± 9.1 µg/mL). HepG2 cells were more sensitive to T. bentzoë treatment (selective index value 2.4 compared to HOE cells) (Table 3). At 48 hours of exposure, the highest concentration of T. bentzoë extract (100 µg/mL) reduced HepG2 cell viability to 20 % as compared to untreated control cells (Supplementary figure S1 A). Subsequently, the effect of T. bentzoë extract on the replicative ability of HepG2 cells was evaluated using the clonogenic cell survival assay. T. bentzoë treatment significantly (ρ < 0.0001) reduced the number of surviving HepG2 colonies as compared to untreated control cells (Figure 1). Data represent mean calculated IC50 values with a standard error of the mean (n=3). NA= Etoposide at 1 µg/ml. Inhibited above 80 % cancer cell growth, indicating a much lower concentration is required for determination of IC50 value. Asterisks represent significant differences between T. bentzoë and etoposide (positive control), **** p ≤ 0.0001.

Figure 1:
HepG2 cells were treated with indicated concentrations (µg/mL) of test extracts for 48 hours and subsequently allowed to grow into colonies for 14 days. After 14 days, the colonies were stained with 0.1 % crystal violet and the images of the wells were captured using a digital camera. The colonies were counted using Image J software. Each experiment was performed three times. Asterisks represent significant differences between extracts treatments and untreated control, **** P ≤ 0.0001.

Genotoxic effect of T. bentzoë extract in HepG2 cells.
DNA damage induced by T. bentzoë leaf extract was assessed in HepG2 cells by the alkaline comet assay. Treatment with 10 µg/mL T. bentzoë leaf extract for 24 hours resulted in the induction of significant (p ≤ 0.0001) DNA damage in HepG2 cells as compared to untreated control cells. The occurrence of DNA damaged was scored in terms of tail length, tail intensity and olive tail moment of cell cultures by comet assay software ( Table 4). The increased olive tail moment in response to T. bentzoë treatment was 4 fold higher than that of untreated control cells. Cells treated with 200 µM H2O2 for 30 minutes, was used as a positive control. Asterisks represent significant differences between extracts and untreated control, **** p ≤ 0.0001.

T. bentzoë induced cell death.
The

Effect of T. bentzoë on HepG2 cell viability
HepG2 cells were used as a model to fractionate and characterise the bioactive components present in T. bentzoë leaf extract. The first round of fractionation was achieved using liquid-liquid portioning with organics solvents of increasing polarities. T. bentzoë butanol fraction, being most active, was further fractionated on a Sephadex LH-20 column. A total of 11 sub-fractions was derived with cytotoxic activity being limited between fractions F4 to F10 only (Figure 3). Assessment of their chromatographic patterns revealed differences in the chemical compositions of the fractions, albeit multiple overlapping peaks highlighting the chemical complexity of the subfractions. Given the most potent cytotoxic activity of the subfraction F6 (IC50 = 15.2 ± 1.8 µg/mL), the further fractional separation of the latter was achieved using preparative HPLC to yield 11 HPLC subfractions (F6.1 -F6.11). Of these, HPLC subfraction F6.1 retained most of the cytotoxic activity, providing an IC50 value of 15.8 ± 0.3 µg/mL. By contrast, HPLC fractions F.2, F.7, F.9, F.10 and F.11 failed to effectively inhibit the growth of HepG2 cell cultures; hence no IC50 values were determined for these subfractions. A summary of the bioassay-guided fractionation employed is depicted in figure 3.

Antioxidant potential of T. bentzoë fractions
Both the ethyl acetate (22 ± 0.29 mmol Fe 2+ ) and butanol (21.01 ± 0.56 mmol Fe 2+ ) fractions were significantly (p ≤ 0.05) higher compared to the aqueous residual fraction. Furthermore, the FRAP value of the organic fractions was greater as compared to the total extract. The butanol fraction of T. bentzoë was a more effective scavenger of DPPH• radical as opposed to ethyl acetate and aqueous residual fractions ( Table 5). As far as the butanol subfractions were concerned, notably F6, F7, and F8 had better antioxidant activity in FRAP and superoxide radical scavenging assays ( Table 5).

Characterisation of the cytotoxic components of T. bentzoë.
In an effort to identify the bioactive constituents in T. bentzoë, bioassay-guided fractionation was carried out. LC-MS analysis in conjunction with NMR spectroscopy of T. bentzoë butanol fraction 6 and its semi-prep HPLC sub-fractions allowed the identification of 8 phenolic compounds including punicalagin (1), isoterchebulin (2) S7 and Table  S1), with a ratio of 5:2 based on the 1H NMR integration of single hydrogen in each compound was identified from the non-active subfraction F6.9. 2"-O-galloylvitexin (7), and ellagic acid (8) were detected only by LC-MS from the butanol fraction 6 (Supplementary Figures S4). Additionally, two simple phenolics, gallic acid and methyl gallate were detected in both the ethyl acetate and butanol fractions and identified by GC-MS analysis (Supplementary figure S8). They were likely present in other less bioactive fractions. The levels of gallic and methyl gallate were quantified as 15.6 ± 1.1 and 26.2 ± 8.1 µg/mg total extract) (n=3) by HPLC, respectively.

Discussion
Medicinal plants are known to be the epicentre of numerous well established ethnomedicinal systems across the globe [4]. However, more than 84 % of these medicinal plants have been poorly studied in regards to their phytochemical compositions, clinical efficacy as well as their safety and toxicological profiles [4]. Moreover, forest cover is being uprooted across the world, at an unprecedented rate, threatening the survival of at least 15000 medicinal plant species [25]. Plant secondary metabolites have contributed enormously to the modern-day pharmaceutical industry by providing the chemical backbone for almost 25 [58] approved by the US Federal Drugs Administration between 1981 and 2014 [1,2,26]. As such it is of utmost importance to evaluate the unexplored terrestrial flora for their therapeutic potential, as the dwindling medicinal plant's species continues to stand as a rich repository to probe for novel chemotypes in the drug developmental process.
The current findings highlighted the richness of the different subclasses of polyphenolics, notably phenolics, flavonoids and proanthocyanidins, the distribution of which differed significantly (p < 0.05) among the investigated accessions ( Table 2). The biosynthesis of flavonoids, in particular flavonols, are known to be upregulated in response to ultra-violet radiation [27]. The accumulation of flavonoids in the endemic plant leaves collected from Mauritius may be attributed to the high sunlight conditions and UV radiation which are characteristic to tropical islands like the Mascarene [28]. Given the ubiquitous involvement of oxidative damage in carcinogenesis, antioxidant-rich secondary metabolites, notably, polyphenolics have attracted much interest in the search of novel and alternative treatment modalities for cancer [29,30]. In this vein, the in vitro antioxidant activities correlated strongly with the growth inhibitory activity against cancer cell lines [31,32]. The antioxidant mechanism of action of polyphenols, especially flavonoids are multifaceted, thus a panel of in vitro assay models allowed to gauge the antioxidant potential of the evaluated extracts [33]. T. bentzoë having the highest abundance of TPC as well as TFC also exhibited the most potent antioxidant activity in all six in vitro assays and was further evaluated for its cytotoxicity against cancer cell lines.
The cytotoxicity of different Terminalia species against multiple cancerous cell lines is documented. Investigation of the cytotoxic activity of methanolic leaf extracts of T. arjuna against human chronic myelogenous leukaemia cells led to the isolation of the bioactive ursolic acid (triterpenoid) [34]. Leaf extracts of T. chebula suppressed the growth of human breast and lung cancer cell lines [35]. Along a similar line, the leaf extract from T. catappa significantly (p < 0.05) suppressed the proliferation of human colorectal (SW480) cell line in a dose-dependent manner by downregulating the level of Bcell lymphoma 2 (BCL-2) gene expression while upregulating the level of Caspase 9 and Caspase 3, indicative of the mitochondrial pathway of apoptosis in SW480 cells [36].
The foremost aim of oncologic agents is to precisely target cancerous cells with minimal effect on non-malignant cells. T. bentzoë crude extract had a selective index value of approximately 2.5, indicating that the extract had some degree of selectivity towards HepG2 cells as opposed to nonmalignant human ovarian epithelial cells. Nevertheless, it is crucial to also assess the cytotoxicity of the extract against a panel of normal cells of different tissue origins, to provide greater insight regarding its toxicity window and safety profile.
In line with the United States National Cancer Institute cytotoxicity guidelines [37,38], T. bentzoë crude extract with an IC50 value of 22.8 ± 1.3 µg/mL can be considered as a potent candidate for further investigation with regards to its anticancer potential. It is known that, following exposure to toxicants, a subpopulation of cells enter a dormant state. These dormant viable cells further retained their ability to replicate into stem-like progeny cells which might lead to the development of drugresistance and cancer relapse [39,40]. Thus, while investigating the potential anticancer effect of extracts, it is warranted to also evaluate their long-term effect on the replicative ability of the cancer cell line. The colony formation assay is a simple and useful in vitro model, considered as the gold standard to predict the long-term sensitivity of cancer cell response to therapeutics treatment [39,41]. In this vein, the current study evidenced the ability of T. bentzoë to impede the replicative potential of HepG2 cells (Figure 1). Numerous anticancer agents, including polyphenols, are known to abrogate the limitless replicative ability of different cancer cell variants [42,43]. Catechin, catechin-3-O-gallate, 7-O-galloyl catechin, and methyl gallate purified from Acasis hydaspica is reported to suppress the long-term clonal proliferation of prostate cancer (PC-3) cells [44].
This study also attempted to delineate the mode of T. bentzoë induced HepG2 cell death. Flow cytometric analysis of annexin V-FITC and PI dual stained HepG2 cells treated with 40 µg/mL, revealed a significant (p < 0.05) increased in both Annexin V-FITC and PI fluorescence level, compared to the untreated control, thus indicating the putative activation of both apoptotic and necrotic pathways in HepG2 cells. Cells grown as monoculture are known to initiate apoptosis which is terminated by necrosis like events, also termed as secondary necrosis, due to the absence of phagocytic scavenger cells [45,46]. Numerous studies evidenced this type of cancer cell death following treatment with cytotoxic agents. Ellagic acid was reported to induce both apoptotic and necrotic cell death mechanism in human pancreatic cancer cell cultures [47]. A similar effect of Lepidium sativum and Vitis vinifera extract were highlighted in human breast and skin cancer cells, respectively [48,49].
Plant extracts and/or thereby derived phytochemicals are known to provoke cancer cell death by causing oxidative damage to genetic material mediated cell cycle arrest and subsequent cell death [50,51]. With this in mind, the DNA damage to HepG2 cellular DNA following T. bentzoë crude extracts treatment was investigated using the alkaline comet assay and scored in terms of tail length, tail intensity, and olive tail moment. The comet assay is a well-established and highly sensitive method for the detection of DNA damage and fragmentation pattern [52]. T. bentzoë induced 4 folds significantly (p < 0.001) higher DNA damaged to HepG2 cells compared to the untreated control, as reflected by the olive tail moments ( Table 4). Consistent with the DNA damaging ability, T. bentzoë halted the cell cycle progression significantly (p < 0.01, versus control) in the G0/G1 phase (Figure 2). A similar observation was reported in lung cancer cells, where casuarinin, a tannin purified from T. arjuna L. bark, provoked apoptotic mechanism via DNA fragmentation and G0/G1 phase cell cycle arrest [53].
It is noteworthy that, the cytotoxicity of the extract may arise from the complex interplay of the cocktails of secondary metabolites present, which may act either synergistically or antagonistically to produce the overall results. Also, crude extracts often comprised a pool of inactive phytoconstituents that dilute the efficacy of the active components [54,55]. It is therefore of paramount importance to purify and identify the principal bioactive components to conduct further mechanistic studies to establish their molecular mode of action [54,56]. Moreover, isolating the lead compounds also allows for the potential structural modification in an attempt to enhance their selectivity and potency [57]. As such, the MTT-guided fractionation revealed that only six out of the eleven preparative HPLC subfractions retained the potent cytotoxicity of the crude extract (Figure 3). The HPLC subfraction F6.1 was 1.4 folds more potent as compared to the crude extract.
LC-MS analysis in conjunction with NMR spectroscopy allowed the identification of 8 phenolic compounds from the most bioactive subfraction F6. Punicalagin, isoterchebulin and ellagic acid have been previously reported from the bark extract of the same species collected in Réunion Island [58]. Although, the other identified compounds are being reported for the first time, to the best of our current knowledge, in T. bentzoë leaf, some were reported in the leaf extracts of other Terminalia species. For instance, 2"-O-galloylvitexin and gallic acid were identified from T. brachystemma Welw. ex Hiern and T. mollis M. Laws leaves, respectively [59]. Likewise, terflavin A was found in T. catappa L. leaf [60].
Punicalagin and ellagic acid were reported to induce S Phase arrest and G0/G1 phase arrest in HepG2 cells, respectively [61]. Ellagic acid administration in prostate cancer patients was associated with decreased prostate-specific antigen as well as reduced chemotherapy-induced myelotoxicity [62]. It may be strongly proposed that the overall cytotoxicity of T. bentzoë leaf extract may be a synergistic effect of the identified compounds. Gallic acid was reported to induce S phase arrest in HepG2 cells [63] and be cytotoxic to ovarian cancer cells [64]. Furthermore, gallic acid was also shown to impair centrosomal clustering in Hela cells, thus causing a mitotic catastrophe and ultimate cell G2/M phase cell cycle arrest.

Conclusion
The findings evidenced the selective long-term cytotoxicity of the antioxidant-rich T. bentzoë leaf extract against HepG2 cells. This plant which is also used traditionally in the mitigation of asthma, haemorrhages, diarrhoea, and sexually transmissible diseases, amongst others, has also shown some in-vitro anticancer activities against HepG2 cells. The cytotoxic nature of T. bentzoë leaf extract against cancerous cells indicated that T. bentzoë leaf has the potential to be repurposed in the mitigation of cancer as part of traditional medicine. The results so far generated, supports the hypothesis that T. bentzoë extract induced apoptosis/necrosis cell death in HepG2 cells via the degradation of cellular genetic material and subsequent arrest of the cell cycle progression in G0/G1 phase. Overall, the MTTguided fractionation of the crude extract allowed the characterisation of 10 phenolic compounds including gallic acid, methyl gallate, punicalagin, and ellagic acid that are known to have established in vitro and in vivo anticancer activities. However, the contribution of the other non-identified phytoconstituents cannot be excluded and thus further evaluation of purified identified components is warranted alongside the crude extract. The metabolite profiling of the leaf extract may be envisaged in future studies. This will allow to precisely ascribe the bioactive molecules present in T. bentzoë leaf.
Taking into consideration the guidelines of the US national cancer institute as well as the selectivity index observed in this study, T. bentzoë leaf extract revealed as a promising candidate that can be exploited in the search for novel anticancer agents, Further mechanistic investigation, directed towards delineating the molecular mechanisms via which the purified bioactive entities target of the aberrant signalling pathways involved in carcinogenesis, is needed to fuel in-vivo studies.

Supplementary Materials
Figure S1: Cell viability profile of HepG2 cells treated with T. bentzoë extract and fractions.