The Apoptotic Potential Activity of Terminalia Brownii Extracts against Various Cancer Cell Lines

The present study aimed to investigate the potential inhibitory activities of various Terminalia brownie extracts against the proliferation of different cancer cell lines. Fractionation was carried out through the process of liquid-liquid extraction produced four fraction types using polarity-enhancing solvents such as, n-hexane, chloroform, ethyl acetate, and water. All extracts have been tested for their cytotoxicity against cancer cell lines using SRB assay and their inhibitory activity on cell cycle phases using flow cytometry. The chloroform extract exhibited a cytotoxic effect against all three types of cancer cell lines and especially the HepG2 cells at 100-µg concentration. Moreover, the most prominent plant extracts of chloroform, acetyl acetate, and hexane showed significant induction of cell death during the S phase cell cycle. The findings of this study suggest that compounds in the Terminalia brownie plant used in traditional Saudi medicine may have the potential to suppress the growth of cancer cell lines.


Introduction
Cancer is a disease with a high mortality risk that constitutes a major health burden in developed and developing countries alike. The transformation of normal cells into cancerous cells exhibits a wide range of biological alterations. At global level, cancer remains the second cause of human mortality, despite the availability of a multitude of treatments (Abdel-Magid, 2014). The progression of cancerous cells to different pathological and metabolic conditions is greatly aided by mutations (Kuppusamy et al., 2016). Normal cells are transformed to be cancerous when mutant genes are generated in greater amount due to the dysfunction of anti-apoptotic mechanisms and pathways by various transcriptional pathway disorders (Bradner et al., 2017). Furthermore, the regular functionality of neighboring cells may be disrupted due to growing of cancerous cells, irregular proliferation and differentiation. The expression of apoptotic genes inhibits chemical response, making cells unresponsive to signals or chemicals promoting programmed cell death (Newman et al., 2000). Among mechanisms that modulate the differentiation and proliferation of cancerous cells is the equilibrium of gene expression pathways respectively promoting and suppressing cancerous cells, as well as inhibition of anti-cancer genes and promotion of pro-cancer genes (Newman et al., 2000). A wide range of terrestrial and marine flora and fauna have supplied natural product medicines (Machumi et al., 2013). Indeed, ample Mountains of Saudi Arabia during the year 2017 where the identification process was conducted at the Herbarium of Biological Sciences, Biology Department, King Khalid University. Fresh plant branches were cut and the leaves were plucked and air-dried at ambient temperature (Ediriweera et al., 2016). After drying, the leaves were ground to a fine powder. A technique involving 80% aqueous methanol was employed to perform the extraction process (Ediriweera et al., 2016). Fractionation was carried out through the process of liquid-liquid extraction, which involved the production of four fractions through the use of four polarity-enhancing solvents, namely, n-hexane, chloroform, ethylacetate and water. The excess solvents were removed after every fraction through rotary evaporation.

Cell culture
Human breast cancer cells (MCF-7), human liver cancer cells (HEPG-2) and human colon cancer cells (HCT 116) were the cancer cell types that were employed for the purposes of this work. These cells were subjected to culturing with 10% foetal bovine serum and antibioticcontaining fresh growth medium (RBMI) in an incubator with 5% carbon dioxide at a temperature of 37°C for 24 hours. Following the period allowed for culturing, an inverted microscope was used to verify that the flask in which the culturing took place did not contain any fungi or bacteria, as well as to assess cell confluence. The trypsinization procedure was subsequently implemented to collect cells by employing 1 ml of trypsin/EDTA solution for a duration of 2 -5 minutes in the incubator. To ensure comprehensive cell coverage with trypsin, the tissue culture flask was rotated, while an inverted microscope facilitated the observation of cellular dislocation and rounding. The requisite quantity of cells was then transferred to distinctly labelled 96-well plates acquired from Sigma Aldrich, with each well accommodating 1,000 -2,000 cells. In anticipation of the sulforhodamine B (SRB) assay, the plates underwent a 24-hour incubation period (Keepers et al., 1991). The crude extract, along with its respective fractions, was employed at varying concentrations (0.01, 0.1, 1, 10, 100, and 1,000 μg/μl), with dimethyl sulfoxide (DMSO) serving as the positive control.

Analysis of cell survival via SRB assay
The SRB protein stain assay was conducted alongside another calorimetric assay. The in vitro SRB assay exhibited preferable linearity when the number of cells was assessed, while it exhibited heightened sensitivity during staining (Alshehri, 2016). All three types of cancer cells were used to determine the cytotoxic effect of the TB crude extract and associated fractions. Live and dead cells could be distinguished from one another through the SRB assay (Van Tonder et al., 2015).
The three types of cancer cells were subjected to culturing in labeled microtiter plates with 96 wells, with each well containing 1,000-2,000 cells, followed by 24-hour incubation (Gaidhani et al., 2009;Van Tonder et al., 2015). The next step was applying the TB crude extract and its fractions from a stock solution in different concentrations to the wells, followed by 72-hour incubation (Gaidhani et al., 2009;Van Tonder et al., 2015). 10% trichloroacetic acid (TCA) was used for fixation of the incubation medium for 60 minutes at 4°C. The medium was subsequently washed with tap water 3-5 times and air-dried for 60 minutes.0.4% SRB stain was afterwards used for staining the wells, followed by dissolution in 1% acetic acid for 10-30 minutes at 20-25°C. 1% acetic acid was then used to wash the stain and eliminate the unbound dye until the acetic acid became transparent. This was followed by 24-hour air-drying without UV exposure. The next step was solubilization of the dye with 10 Mm tris base with 7.4 pH (150µl/well) for 5 minutes (Liu et al., 2015). An ELISA microplate reader was employed for measurement of the optical density of the wells and the rate of cell survival at a wavelength of 454-540 nm (Van Tonder et al., 2015).

Evaluation of apoptosis
The AO/EB staining technique was utilized to assess apoptosis, focusing on quantifying live cells, early and late apoptotic cells, and necrotic cells, and examining aberrantly organized cells (Amna, 2019). AO penetration and staining of viable cells with a green color by employing a suitable filter is the basis of this method. On the other hand, EB can penetrate solely non-viable cells with compromised membrane, so the dead cells were noticeable by the orange color from EB staining.
The MCF-7, HEPG-2 and HCT 116 cancer cells were subjected to culturing in tissue culture plates with 96 wells, each well containing 1,000-2,000 cells, followed by 24-hour incubation. IC 50 of crude extract and its fractions was then applied to the cells before another round of incubation for 48 hours. The next step was transferring 2 μl of AO/EB mixture (100 μg/ml of AO and 100 μg/ml of EB in phosphate buffer saline) to the 96-well tissue culture plates. A fluorescence microscope (Nikon Eclipse E400) was then used to observe the dead cells, the necrotic cells and the live cells. AO-stained viable cells exhibited a bright green color and their structure was uncompromised, while AO-stained cells in early apoptosis exhibited a bright green region in the nucleus. By contrast, cells in late apoptosis subjected to both AO and EB staining were small in size and displayed a red-orange color, with dense orange regions indicating chromatin condensation (Elbehairi et al., 2020).

Flow cytometric analysis of cell cycle with propidium iodide DNA staining
Cell cycle analysis was conducted according to the manufacturer's protocol as follows: cells were harvested appropriately and washed in PBS; fixed in cold 70% ethanol, which was added dropwise to the pellet while vortexing to ensure fixation of all cells and minimize clumping; fixed for 30 minutes at 4°C; washed twice in PBS; centrifuged at 850 g, ensuring care was taken to avoid cell loss when discarding the supernatant, particularly after spinning out of ethanol; treated with ribonuclease by adding 50 µl of a 100 µg/ml stock of RNase, ensuring only DNA, not RNA, was stained; and finally, 200 µl of PI (from a 50 µg/ml stock solution) was added. extract of TB leaves (see Table 1). To name a few, the deprotonated molecular ion peak [M -H]+ for a component eluted at a retention time (Rt) 27.6 sec and observed at m/z 92.8 ( Figure 1A) can be assigned as phenolate anion. As shown in the mass spectrum of the compound eluted at Rt = 29.9 sec, the main peak was observed at m/z 110.8 could be ascribed to deprotonation {[M -H]+} of another phenolic compound,catechol. Besides, two additional peaks were observed at m/z 92.8 and 82.8 attributable for the loss of water [M -H 2 O]+. and carbonyl [M -CO]+. groups, respectively. The participation of carboxylic acids in the chemical composition of this extract was revealed from notice of peak at m/z 113.8 for the compound eluted at Rt = 46.1 ( Figure 1C) corresponding to [M -H] of the

Statistical analysis
All data were presented as the mean ± SE. Statistical analysis was performed using SPSS statistics software (Version 16.0) one-way ANOVA for all assays. However, statistical study of obesity induction follow-up parameters before starting the treatment was analyzed using SPSS statistics software (Version 16.0) Paired sample T-test. For all analyses, P-value < 0.05 was considered to be statistically significant.

Ion mass spectrum (EPI-MS)
Several compounds were detected in the methanolic  deprotonated Levulinic acid form. Moreover, its peak at m/z 85.8 is assigned to [M-CH3OH] ( Figure 1C).

The activity of different extraction methods on cell viability
The extraction process was conducted to determine the plant extract efficiency and their activity on cell viability and potency of the extraction products against cancer cell development and proliferation. The chloroform and ethyl acetate-based extraction of TB leaves displayed the highest level of toxicity against HepG2 cells, with IC50 11.6 ± 0.7 µg/ml and 12.3 ± 0.86 µg/ml, respectively. The action of the water extract and crude whole extract showed moderate action of activity against the three types of cancer cells was moderate Furthermore; the greatest and lowest sensitivity to the various extracts was exhibited for HepG2 cells and MCF-7 cells, respectively ( Figure  2A). Additionally, TB ethyl acetate extract for MCF-7, HepG-2, and HCT 116 cancer cells at 100 µg showed a significant suppressing rate accompanied with reducing the viability, and their action was increased beyond this action as shown in Figure 2B.
The growth analysis was conducted 48 hours after treatment with the different extracts. As the extract concentration was elevated up to 100, growth was suppressed at a growing rate, reducing the viability; however, beyond the 100-µg concentration, there was a rise in viability, which might be indicative of drug interactions. The TB chloroform extract displayed great toxicity towards all three types of cells, with maximum toxicity at 1000 µg/ml, as shown in (Figure 2C). Similarly, Terminalia Brownii Activity against Cancer Cell Lines the TB hexane extract was most toxic at 1000 µg/ml concentration, while at a concentration of 100 µg/ml it displayed cytotoxicity against the MCF-7 and HCT 116 cells and more moderately against HepG2 cells ( Figure  2D). The TB water extract had a toxic effect solely at 1,000 µg/ml, whereas at lower concentrations it lacked toxicity as shown in figure 2E. The crude extract also had a toxic effect only at 1,000 µg/ml, whereas at lower concentrations it did not display any toxic effect ( Figure 2F). On the morphological basis, Figure 3 (A-G) illustrated the modifications in the morphology of MCF-7 cells exposed to different TB extracts and their various fractions at IC 50 concentration for 48 hours. Figure 3A shows the untreated cells exhibiting rounding and relative deterioration of the nuclear membrane. Figure 3B reveals that the majority of cells have chromatin regions of a greenish color and high density, while some other cells are in various stages of apoptosis with necrotic aspects, as reflected by their rounding and red color. Figure 3C shows that the majority of cells are green or yellowish, while other cells are red and had fragmented nucleases, suggesting they are in final apoptosis. Figure 3D illustrates that cells have rounded and normal nuclei, with a couple of distributed cells displaying a yellow color and high    chromatin density and small fragmented cells of red color. Figure 3E shows cells in early apoptosis and a couple of distributed cells in late apoptosis. Figure 3F reveals massively deteriorated cells of green color. Figure 3G shows the positive control, with early apoptotic green cells and late apoptotic red cells. Figure 4 (A-G) illustrates the modifications in morphology suffered by the HepG2 cells exposed to TB extract and its various fractions at IC50 concentration for 48 hours. Figure 4A shows that every cell is viable and normal, displaying a green color and rounded nucleus and nuclear membrane. Figure 4B shows that most cells have structural deterioration and fragmentation. Figure 4C reveals distributed fragmented cells of green and red color. Figure 4D illustrates that the majority of cells are viable and of green or yellowish color, with a couple of distributed fragmented cells of red color. Figure 4E shows that most cells have structural fragmentation and are of green and red color. Figure 4F shows distributed and viable cells of green and yellowish color. Figure 4G illustrates the positive control with viable green-and red-colored cells in early and late apoptosis, respectively. Figure 5 (A-G) illustrates the modifications in morphology suffered by the HCT 116 cells exposed to TB extract and its various fractions at IC 50 concentration for 48 hours. Figure 5A shows fully viable green cells with rounded and normal nuclei. Figure 5B shows viable cells of green color and high chromatin density. Figure 5C shows viable cells of green color and a couple of fragmented and late apoptotic cells of red color. Figure 5D illustrates a couple of distributed cells of greenish color  alongside rounded necrotic cells of red color. Figure 5E shows green viable cells with high chromatin density and a couple of interstitial necrotic cells of red color. Figure  5F illustrates partially viable cells of green color alongside a couple of necrotic cells of red color. Figure 5G shows the positive control, with green viable cells and red cells in early and late apoptosis, respectively.

Flow cytometry and cell cycle
The cell cycle results showed that the S phase was the most prominent in the phases through the chloroform extracts for the TB among treated MCF-7 cells, followed by ethyl acetate extracts for HepG2, and finally, Hexane extracts for HCT11 as shown in Table 2, Figure 6.

Discussion
Cancer continues to be associated with a high mortality rate throughout the world, despite the various available treatments and preventive measures, including surgical intervention, which alleviates the tumor burden, chemotherapy, which eradicates residual and micrometastatic tumors systematically and in a surgical setting, and radiation, which eradicates residual tumor cells within a surgical setting. However, radiation and chemotherapy lack tumor specificity, so they can affect healthy cells with fast proliferation just like tumors, such as the cells of the bone marrow, hair follicles and gastrointestinal tract lining. Another limitation of these interventions is that they are severely invasive and occasionally their effect is only a palliative one. Therefore, new interventions of greater efficiency are urgently needed (Elbehairi et al., 2020). Terminalia Brownii Activity against Cancer Cell Lines Although, radiotherapy and chemotherapy lack tumor specificity, they affect healthy cells with fast proliferation just like tumors, such as the cells of the bone marrow, hair follicles, and gastrointestinal tract lining. Additionally, another limitation of these interventions is that they are severely invasive and occasionally have a palliative action. Therefore, new interventions of greater efficiency are urgently needed (Haupt et al., 2002). On this background, increasing attention is being paid to immunological strategies for cancer treatment as more and more insight is gained into the immune system and its regulatory mechanisms (Haupt et al., 2002). The immune system displays exceptional specificity, so cancer cells could be targeted whilst healthy cells are left unaffected.
Tumor development is highly dependent on excessive expression of oncogenes or mutated tumor inhibiting genes, which could therefore be targeted in treatment strategies. Cancer is typically associated with mutations in the tumor inhibiting genes p53 (Nigro et al., 1989;Chiba et al., 1990;Berzofsky et al., 2004) and HER-2/neu, which represents an extra oncogene exhibiting excessive expression rather than mutation in several carcinomas (Yanuck et al., 1993). Exposure to chemotherapy or radiation causes the death of cancer cells primarily through the mechanism of apoptosis. By evolving mechanisms that confer resistance to apoptosis, cancer cells become less susceptible to clinical interventions (Slamon et al., 1989).
The phytochemistry of Terminalia laxiflora and Terminalia brownie has been the focus of several studies. It has been discovered that the roots of both species and the stem bark of TB contain medium-polar-to-polar phenolic compounds, including gallic acid, ellagic acid, and its derivatives, gallotannins and the ellagitannins punicalagin, terchebulin, methyl-(S)-flavogallonate, and its isomer (Hanahan and Weinberg, 2011;Schrader et al., 2016;Yamauchi et al., 2016;Salih et al., 2017). Furthermore, the stem wood of T. laxiflora has been found to contain terchebulin, flavogallonic acid dilactone, and ellagic acid and its derivatives (Muddathir and Mitsunaga, 2013). The stem bark of both species and the root bark of T. laxiflora have also been reported to contain sterols (β-sitosterol and stigmasterol), triterpenoids (betulinic acid, arjungenin, terminolic acid and monogynol A), a diterpenoid (laxiflorin), and a chromone (terminalianone) (Rashed et al., 2016).
The premise postulated by the present study is that the ratios of the different polyphenolic compounds, particularly those of ellagitannins, may be the reason that explains the activity of extracts from various parts of T. laxiflora and TB differ in their effectiveness as anti-cancer drug. The compounds contained in the roots might have the ideal ratio since extracts from those parts were observed to suppress growth most efficiently. Indeed, by comparison to other plant parts, the roots have been reported to have a greater proportion of compounds with antimicrobial effect (Balmer and Mauch-Mani, 2013). This might be explained by the occurrence of synergistic interaction between certain compounds such as ellagitannins and other phenolic compounds in the roots of T. laxiflora and TB (Balmer and Mauch-Mani, 2013). A correlation between the antioxidant action and the antimicrobial effect of phenolic compounds has been proposed, meaning that the pathogenic activity of microorganisms is diminished by phenols through reduction of the generation of stressassociated defense compounds in those microorganisms (de Freitas Araújo et al., 2012). Thus, inflammatory events caused by microbial infections could be managed with plant extracts containing antioxidant compounds (Courtney et al., 2015).
The findings of this study are consistent with those of Courtney et al., (2015) which discovered that ellagitannincontaining Terminalia ferdinandiana extracts displayed satisfactory antimicrobial and antioxidant actions, despite the lack of correlation between the two properties. A particularly potent antioxidant effect has been demonstrated by corilagin, with 53 dg/ml IC 50 (Anokwuru et al., 2015). Different phytochemicals are present in the methanolic bark extracts of TB and some of them may underpin its effect against inflammation. Flavonoids also have an anti-inflammatory effect because they suppress the action of synthetase, a prostaglandin enzyme (Tapas et al., 2008;Chatterjee et al., 2015). Furthermore, inflammatory events are alleviated by steroids through suppression of phospholipase A2, which causes prostanoids and leukotrienes to form by hydrolyzing arachidonic acid from the membrane phospholipids (Mencarelli et al., 2009). Additional compounds with anti-inflammatory effects are triterpenoids, which block not only prostaglandin secretion but also macrophage and neutrophil function (Salminen et al., 2008). Hence, the anti-inflammatory effect observed in the present study might have been generated by the individual or synergistic action of the flavonoids, steroids, and terpenoids in the TB extract.
The current study primarily sought to assess the suppressing effect of diluted TB leaf extracts in different concentrations on three types of cancer cells. Employing a range of extraction techniques, the cytotoxicity of the extracts and their disruption of cancer cell viability were demonstrated. The findings were validated via SRB cell survival assay and dual staining techniques for examination of cell morphology. Similarly, Wani et al., (2016) stated that the homeopathic preparations of Terminalia chebula were shown to hinder the proliferation of the breast cancer cell line MCF-7, whilst leaving normal cells unaffected. Likewise, at 10 µg/ml LC 50 T. chebula affected leukemia cell line K562, which is equivalent to prostate cancer cell line.
On one side, the anti-cancer potential activity of T. chebula was attributed to the highly effective bioactive secondary metabolites gallic acid, ellagic acid, chebulic acid, and tannin (Bupesh et al., 2016). On the other side, it was shown that T. bellerica extracts were more efficient at suppressing the viability of MCF-7 cells in high dosage (Diab et al., 2015). Furthermore, n-hexane extract from other types of plants was reported to make HepG2 cell line significantly less viable, while the common fruit extract malvidin has been proven to suppress the migration of HCT 116 cells. Additionally, the chloroform, ethyl acetate, and Hexane extract revealed significant changes in the replication of the cancer cell for MCF-7, HepG2, and HCT116, respectively through the S phase cell cycle (Al Groshi et al., 2018). That may trigger the activity of the TB treatment with their different extractions that enhance DNA damage that occurs in the S phase due to replication forks, nucleotide excision/repair process, or as intermediates of DSB resolution, the intra S phase checkpoint is activated to prevent further replication (Errico et al., 2012). Additionally, Peasland et al., (2011) stated drugs targeting the S and G2/M checkpoint are considered potent promising pathways that may be developed against breast and ovarian carcinoma cell lines.
The findings of this study suggest that compounds in the TB plant used in traditional Saudi medicine may have the potential to suppress the growth of cancer cells. The extracts obtained with ethyl acetate and chloroform displayed particularly high cytotoxicity against the different types of cancer cells that were examined. More specifically, the chloroform extract exhibited a cytotoxic effect against all three types of cancer cells and especially the HepG2 cells at 100 µM concentration. By contrast, the crude extract demonstrated cytotoxicity solely against the HepG2 cancer cells. Given that the IC50 value of the TB plant is less than 20, further research is required to determine how extracts of this plant affect healthy cells. At the same time, it is also worth conducting in vivo studies to find out how its suppressing effects are underpinned by molecular and cellular mechanisms. Moreover, the chloroform, ethyl acetate, and Hexan extracts of the TB plant are considered good inhibitors that can sensitize cancer cells to DNA damaging drugs forcing the cells with DNA damage to bypass the S and G2/M arrest and enter mitosis, leading to cell death by mitotic inhibition.

Author Contribution Statement
All authors contributed equally in this study.