Thonningia sanguinea Extract: Antioxidant and Cytotoxic Activities Supported by Chemical Composition and Molecular Docking Simulations

The current study was designed to investigate the antioxidant and cytotoxic activities of Thonningia sanguinea whole-plant extract. The total phenolic content was determined using Folin–Ciocalteu reagent and found to be 980.1 mg/g, calculated as gallic acid equivalents. The antioxidant capacity was estimated for the crude extract and the phenolic portion of T. sanguinea, whereupon both revealed a dose-dependent scavenging rate of DPPH• with EC50 values of 36.33 and 11.14 µg/mL, respectively. Chemical profiling of the plant extract was achieved by LC-ESI-TOF-MS/MS analysis, where 17 compounds were assigned, including ten compounds detected in the negative mode and seven detected in the positive mode. The phenolic portion exhibited promising cytotoxic activity against MCF-7 and HepG2 cells, with IC50 values of 16.67 and 13.51 μg/mL, respectively. Phenolic extract treatment caused apoptosis in MCF-7 cells, with total apoptotic cell death 18.45-fold higher compared to untreated controls, arresting the cell cycle at G2/M by increasing the G2 population by 39.7%, compared to 19.35% for the control. The apoptotic investigation was further validated by the upregulation of proapoptotic genes of P53, Bax, and caspases-3,8 9, and the downregulation of Bcl-2 as the anti-apoptotic gene. Bcl-2 inhibition was also virtualized by good binding interactions through a molecular docking study. Taken together, phenolic extract exhibited promising cytotoxic activity in MCF-7 cells through apoptosis induction and antioxidant activation, so further fractionation studies are recommended for the phenolic extract for specifying the most active compound to be developed as a novel anti-cancer agent.


Introduction
Cancer is a significant cause of death around the world [1,2]. Because of their independence from normal regulatory signals, cancer cells develop into tumor tissues, which can then move to other organs in a process known as metastasis, which is responsible for approximately 90% of cancer-related fatalities [3][4][5]. As a result, developing novel chemotherapeutic drugs capable of being used in cancer therapy is critical. As healthcare becomes less paternalistic and the demand for more candidate drugs grows more critical, plant-based health systems will continue to play an essential role in healthcare, and their use by various cultures will change and increase [6][7][8]. Medicinal plants have made a significant contribution to the rapidly expanding discipline of natural product chemistry. Natural products have also been accepted as a significant source in satisfying the healthcare demands of many societies, as a result of several discoveries [9]. Hence, natural products are part of current and effective tools for developing novel pharmaceuticals, as well as a bank of pharmacological templates [10][11][12][13]. Medicinal plants possess a significant impact in the following areas: antimicrobial [14], anti-inflammatory [15], anthelminthic [16,17], antidiabetic [18], and anticancer activity [19,20].
In a continuing effort to discover biologically active phytochemicals from medicinal plants [21][22][23][24][25], our group focused on the study of T. sanguinea, a locally well-known medicinal plant widely distributed throughout the forest zones of tropical Africa. T. sanguine is rich in phenolic compounds, such as brevifolin carboxylic acid and gallic acid [26]. In addition, dihydrochalcone glucoside derivatives, coniferin, (+)-pinoresinol, and lariciresinol were reported [27,28]. The compounds isolated from this plant are well known for their interesting biological activities. A previous survey was conducted and declared its use in folk medicine to treat coughs, urinary tract infections, and erectile dysfunction, and as an anthelmintic [29]. Additionally, it is also used for the treatment of sore throat, skin infections, abscesses, dental caries, gingivitis, fever, malaria, heart disease, and rheumatism [30,31]. Previous studies proved antimicrobial and antifungal effects, alongside the hepatoprotective activities of the T. sanguinea extracts [27,28]. Additionally, Ayim and Bayor et al. evaluated the cytotoxic activity of a selected number of Ghanaian plants. Among the investigated plants were T. sanguinea roots, where its activity was studied against three human cancer cell lines, DLD-1 (colon), MCF-7 (breast), and M14 (melanoma). The study revealed moderate IC 50 values of 40 ± 1.0, 55 ±1.2, and 43.2 ± 2.0, respectively [32]. These findings prompted us to investigate the whole plant in terms of chemical composition using LC-ESI-TOF-MS/MS analysis, the total phenolic contents, and the antioxidant activity of T. sanguinea. The plant extract was found to inhibit the proliferation of human hepatocellular carcinoma cells (HepG-2) through apoptosis induction. This was elucidated by cell cycle arrest in the sub-G1 cell population and DNA fragmentation [33]. Therefore, the current study was planned to screen the cytotoxic activity of crude and phenolic extract of T. sanguinea against another panel of cancer cell lines, and investigate the apoptosis induction in the tested cell line. Furthermore, the scope of this paper was extended for exploring the virtual mechanism of binding of the identified compounds through molecular docking towards the Bcl-2 protein.

Total Phenolic Content
The total phenolic content was determined in the extract using Folin-Ciocalteu reagent by spectrophotometric technique, and was found to be 980.1 mg/g, calculated as gallic acid equivalents.

Biological Activities 2.2.1. Antioxidant Activity
The antioxidant activity was estimated for the crude extract and the phenolic portion of T. sanguinea using DPPH • reagent. As shown in Figure 1, both the crude extract and the phenolic portion revealed a dose-dependent scavenging rate of DPPH • . T. sanguinea crude extract revealed EC 50 values of 36.33 ± 1.02 µg/mL, and the phenolic portion exhibited EC 50 values of 11.14 ± 1.06 µg/mL compared with the EC 50 (24.42 ± 0.87 µg/mL) that was shown by the positive control, Trolox. The exhibited scavenging rate of the measured samples, as well as the used positive control, are illustrated in Figure 1. These results agree with the previously published work [23] that suggests T. sanguinea with radical scavenging action might ameliorate AFB1-induced liver toxicity. well as the used positive control, are illustrated in Figure 1. These results agree with the previously published work [23] that suggests T. sanguinea with radical scavenging action might ameliorate AFB1-induced liver toxicity.

Cytotoxic Activity
Previous investigation of T. sanguinea roots for its cytotoxic activity against three human cancer cell lines, DLD-1 (colon), MCF-7 (breast), and M14 (melanoma), revealed moderate IC50 values of 40, 55, and 43.2, respectively [10]. In another previous study on T. sanguinea subaerial extract, the isolation of ten dihydrochalcone glucoside derivatives was reported. Namely, they were: 3-hydroxyphloridzin, 2′-O- In addition, coniferin, (+)-pinoresinol, and lariciresinol were reported [27]. In our present study, both crude and phenolic extract of the whole-plant T. sanguinea were tested for their cytotoxic activity using an MTT assay. As seen in Table 1 and Figure  2, phenolic extract exhibited promising cytotoxic activity against MCF-7 and HepG2 cells with IC50 values of 16.67 and 13.51 µg/mL, respectively. These promising activities were compared to 5-FU of higher IC50 values of 26.98 and 15.8 µg/mL, respectively. At the same time, it did not have any detected activity with IC50 values higher than 50. This might indicate their selectivity of action. On the other hand, crude extract was less cytotoxic than the phenolic extract by exhibiting higher IC50 values than the 5-FU. 5-FU was chosen as a reference drug due to its potent chemotherapeutic activity for the treatment of solid cancers.

Cytotoxic Activity
Previous investigation of T. sanguinea roots for its cytotoxic activity against three human cancer cell lines, DLD-1 (colon), MCF-7 (breast), and M14 (melanoma), revealed moderate IC 50 values of 40, 55, and 43.2, respectively [10]. In another previous study on T. sanguinea subaerial extract, the isolation of ten dihydrochalcone glucoside derivatives was reported. Namely, they were: 3-hydroxyphloridzin, 2 -O- In addition, coniferin, (+)-pinoresinol, and lariciresinol were reported [27]. In our present study, both crude and phenolic extract of the whole-plant T. sanguinea were tested for their cytotoxic activity using an MTT assay. As seen in Table 1 and Figure 2, phenolic extract exhibited promising cytotoxic activity against MCF-7 and HepG2 cells with IC 50 values of 16.67 and 13.51 µg/mL, respectively. These promising activities were compared to 5-FU of higher IC 50 values of 26.98 and 15.8 µg/mL, respectively. At the same time, it did not have any detected activity with IC 50 values higher than 50. This might indicate their selectivity of action. On the other hand, crude extract was less cytotoxic than the phenolic extract by exhibiting higher IC 50 values than the 5-FU. 5-FU was chosen as a reference drug due to its potent chemotherapeutic activity for the treatment of solid cancers.  We suggest that these findings could be attributed to the high content of certain phenolic compounds such as thonningianin-A (145 mg were obtained from 1.5 g of T. sanguinea subaerial extract) [27], gallic acid (20 mg were obtained from 20 g of T. sanguinea flower extract), brevifolin carboxylic acid (17 mg were obtained from 20 g of T. sanguinea flower extract) [26], and (+)-epipinoresinol (100 mg was obtained from 423 g extract of T. sanguinea) [28]. To confirm this point of view, it was previously reported that thonningianin-A has a potent antihepatocarcinoma activity in a dose-dependent manner. It is an effective antitumor constituent capable of inhibiting the proliferation of HepG-2 cells by apoptosis induction through caspase activation [33,34]. Additionally, gallic acid showed a potential cytotoxic activity against MCF-7 human breast cancer cells through the activation of caspases, which are responsible for programmed cell death, suggesting its probable use as antitumor agents [35,36]. Similarly, brevifolin carboxylic acid had potent cytotoxicity against human lung adenocarcinoma cells (IC50 = 3.95 µg/mL), which was almost similar to the IC50 value (95 µg/mL) against mouse leukemia cell [37]. Moreover, (+)epipinoresinol showed significant inhibitory activity against human hepatoma (HepG2), human ovary serous adenocarcinoma (HO-8910), and human bladder cancer (T24) [38].

Apoptosis Investigation
Phenolic extract exhibited promising cytotoxic activities against both MCF-7 and HepG2 cancer cell lines, so apoptosis investigation with flow cytometric and RT-PCR analyses in MCF-7 cells treated with phenolic extract (16.67 µg/mL, 48 h) were investigated. We suggest that these findings could be attributed to the high content of certain phenolic compounds such as thonningianin-A (145 mg were obtained from 1.5 g of T. sanguinea subaerial extract) [27], gallic acid (20 mg were obtained from 20 g of T. sanguinea flower extract), brevifolin carboxylic acid (17 mg were obtained from 20 g of T. sanguinea flower extract) [26], and (+)-epipinoresinol (100 mg was obtained from 423 g extract of T. sanguinea) [28]. To confirm this point of view, it was previously reported that thonningianin-A has a potent antihepatocarcinoma activity in a dose-dependent manner. It is an effective antitumor constituent capable of inhibiting the proliferation of HepG-2 cells by apoptosis induction through caspase activation [33,34]. Additionally, gallic acid showed a potential cytotoxic activity against MCF-7 human breast cancer cells through the activation of caspases, which are responsible for programmed cell death, suggesting its probable use as antitumor agents [35,36]. Similarly, brevifolin carboxylic acid had potent cytotoxicity against human lung adenocarcinoma cells (IC 50 = 3.95 µg/mL), which was almost similar to the IC 50 value (95 µg/mL) against mouse leukemia cell [37]. Moreover, (+)-epipinoresinol showed significant inhibitory activity against human hepatoma (HepG2), human ovary serous adenocarcinoma (HO-8910), and human bladder cancer (T24) [38].

Apoptosis Investigation
Phenolic extract exhibited promising cytotoxic activities against both MCF-7 and HepG2 cancer cell lines, so apoptosis investigation with flow cytometric and RT-PCR analyses in MCF-7 cells treated with phenolic extract (16.67 µg/mL, 48 h) were investigated.

Annexin-V-FITC/PI Staining and Cell Cycle Analysis
Untreated and treated MCF-7 cells with phenolic extract were investigated for apoptotic activity. As shown in Figure 3, phenolic extract treatment induced total apoptosis in MCF-7 cells by 21.04 %, compared to 1.14 % in the control. It induced early apoptosis by 7.55 %, and late apoptosis by 13.49 %, while phenolic extract induced necrotic cell death by 6.66 %, compared to 0.99% in the control. These results illustrate apoptosis induction rather than necrosis as the mechanism of cytotoxic activity in MCF-7 cells.
In this study, MCF-7 cells untreated and treated with the phenolic extract were subjected to the DNA flow to determine the cell population in each cell phase and to determine at which cell cycle cells were arrested. As seen in Figure 4, phenolic extract treatment significantly increased the G2 population by 39.7 %, compared to 19.35 % for the control, while it significantly decreased the G1 population by 19.71%, compared to 53.35 %. Hence, phenolic extract treatment may induce apoptotic cell death in the G2/M phase. These data, regarding apoptotic investigation in MCF-7 cells upon treatment, agree with previous studies [39,40], which exhibited a higher ratio of apoptosis over necrosis and illustrated G2/M as the phase at which cell cycle was arrested. Annexin-V-FITC/PI Staining and Cell Cycle Analysis Untreated and treated MCF-7 cells with phenolic extract were investigated for apoptotic activity. As shown in Figure 3, phenolic extract treatment induced total apoptosis in MCF-7 cells by 21.04 %, compared to 1.14 % in the control. It induced early apoptosis by 7.55 %, and late apoptosis by 13.49 %, while phenolic extract induced necrotic cell death by 6.66 %, compared to 0.99% in the control. These results illustrate apoptosis induction rather than necrosis as the mechanism of cytotoxic activity in MCF-7 cells. In this study, MCF-7 cells untreated and treated with the phenolic extract were subjected to the DNA flow to determine the cell population in each cell phase and to determine at which cell cycle cells were arrested. As seen in Figure 4, phenolic extract treatment significantly increased the G2 population by 39.7 %, compared to 19.35 % for the control, while it significantly decreased the G1 population by 19.71%, compared to 53.35 %. Hence, phenolic extract treatment may induce apoptotic cell death in the G2/M phase. These data, regarding apoptotic investigation in MCF-7 cells upon treatment, agree with previous studies [39,40], which exhibited a higher ratio of apoptosis over necrosis and illustrated G2/M as the phase at which cell cycle was arrested.

RT-PCR
To investigate the apoptotic pathway of the phenolic extract, 16.67 µg/mL of the sample was added to MCF-7 cells and left for 48 h; after RNA extraction, cDNA was produced. Then, the mRNA expression of caspases 3, 8, and 9, and proapoptotic (P53 and Bax) as well as anti-apoptotic genes (Bcl-2) in the treated cells, was traced by RT-PCR analysis. As demonstrated in Figure 5, the expression of the P53 gene was noticeably elevated by the phenolics extract (≈6.54-fold), with concomitant activation of the Bax and caspases 3, 8, and 9, by 8.2-fold, 7.44-fold, 3.76-fold, and 6.65-fold, respectively. Meanwhile, it markedly suppressed the expression of Bcl-2 (the anti-apoptotic gene) by 0.27-fold. These findings are in harmony with the apoptotic mechanism suggested for anti-cancer activity.

RT-PCR
To investigate the apoptotic pathway of the phenolic extract, 16.67 µg/mL of the sample was added to MCF-7 cells and left for 48 h; after RNA extraction, cDNA was produced. Then, the mRNA expression of caspases 3, 8, and 9, and proapoptotic (P53 and Bax) as well as anti-apoptotic genes (Bcl-2) in the treated cells, was traced by RT-PCR analysis. As demonstrated in Figure 5, the expression of the P53 gene was noticeably elevated by the phenolics extract (≈6.54-fold), with concomitant activation of the Bax and caspases 3, 8, and 9, by 8.2-fold, 7.44-fold, 3.76-fold, and 6.65-fold, respectively. Meanwhile, it markedly suppressed the expression of Bcl-2 (the anti-apoptotic gene) by 0.27-fold. These findings are in harmony with the apoptotic mechanism suggested for anti-cancer activity.
In agreement with the apoptotic investigation of the flow cytometric results, the RT-PCR results exhibit upregulation of the proapoptotic proteins P53, Bax, and caspase-3, 8, and 9, and downregulation of anti-apoptotic proteins (Bcl-2) in the MCF-7 cells untreated and treated with phenolic extract. Caspases are key components in apoptosis in various cells and their activation is a vital episode in the progression of apoptosis, while the mitochondrial cytochrome c and caspase activation are controlled by Bcl-2 proteins.

LC-ESI-TOF-MS/MS Analysis
T. sanguinea extract was previously investigated for its chemical constituents. As a result, 25 compounds were isolated and identified [27,28]. The structures of the previously isolated compounds are illustrated in Supplementary Materials Figure S1. In the current study, LC-ESI-TOF-MS/MS analysis was applied to the crude methanolic extract of the plant to investigate the additional chemical constituents ( Figure 6). As a result, 17 com- and treated with phenolic extract. Caspases are key components in apoptosis in various cells and their activation is a vital episode in the progression of apoptosis, while the mitochondrial cytochrome c and caspase activation are controlled by Bcl-2 proteins.

LC-ESI-TOF-MS/MS Analysis
T. sanguinea extract was previously investigated for its chemical constituents. As a result, 25 compounds were isolated and identified [27,28]. The structures of the previously isolated compounds are illustrated in Supplementary Materials Figure S1. In the current study, LC-ESI-TOF-MS/MS analysis was applied to the crude methanolic extract of the plant to investigate the additional chemical constituents ( Figure 6). As a result, 17 compounds were revealed, including ten compounds detected in the negative ion mode ( Table 2) and seven compounds detected in the positive ion mode (Table 3).

LC-ESI-TOF-MS/MS Analysis
T. sanguinea extract was previously investigated for its chemical constituents. As a result, 25 compounds were isolated and identified [27,28]. The structures of the previously isolated compounds are illustrated in Supplementary Materials Figure S1. In the current study, LC-ESI-TOF-MS/MS analysis was applied to the crude methanolic extract of the plant to investigate the additional chemical constituents ( Figure 6). As a result, 17 compounds were revealed, including ten compounds detected in the negative ion mode ( Table   2) and seven compounds detected in the positive ion mode (Table 3).              * Mass accuracy was calculated as [measured mass-expected mass/expected mass] × 10 6 and is expressed in parts per million (ppm) error [49]. * Mass accuracy was calculated as [measured mass-expected mass/expected mass] × 10 6 and is expressed in parts per million (ppm) error [49].  Daidzein [48] * Mass accuracy was calculated as [measured mass-expected mass/expected mass] × 10 6 and is expressed in parts per million (ppm) error [49].

Molecular Docking Studies
In this present study, T. sanguinea phenolic extract exhibited antiproliferative activity against breast cancer cells via apoptosis. Hence, all compounds, whether identified by the present LC-ESI-TOF-MS/MS analysis or previously isolated from the plant, were chosen for a simulated molecular docking investigation to gain insights into the possible molecular targets for the cytotoxic and apoptosis-inducing activities. As seen in Table 4 and Table S3, most of the identified compounds revealed good binding interactions with the key amino acids inside the "B-cell lymphoma 2 (Bcl-2), PDB ID: 4IEH", while there were some minor compounds that did not show any binding activity towards the studied target. Interestingly, compounds thonningianin-A and -B were promising compounds, possessing strong interactions with the key amino acids (mainly Arg 66) of the binding site, as shown in Table 4 with 2D and 3D disposition images. Accordingly, our docking experiment proposed their mechanism of action as Bcl-2 suppressors, congruent with the flow cytometric and RT-PCR analyses, illustrating their apoptosis-inducing activity. It is worth mentioning that thonningianin-A is a major active constituent of T. sanguinea, as it was Daphnetin [56] * Mass accuracy was calculated as [measured mass-expected mass/expected mass] × 10 6 and is expressed in parts per million (ppm) error [49].

Molecular Docking Studies
In this present study, T. sanguinea phenolic extract exhibited antiproliferative activity against breast cancer cells via apoptosis. Hence, all compounds, whether identified by the present LC-ESI-TOF-MS/MS analysis or previously isolated from the plant, were chosen for a simulated molecular docking investigation to gain insights into the possible molecular targets for the cytotoxic and apoptosis-inducing activities. As seen in Table 4 and Table S3, most of the identified compounds revealed good binding interactions with the key amino acids inside the "B-cell lymphoma 2 (Bcl-2), PDB ID: 4IEH", while there were some minor compounds that did not show any binding activity towards the studied target. Interestingly, compounds thonningianin-A and -B were promising compounds, possessing strong interactions with the key amino acids (mainly Arg 66) of the binding site, as shown in Table 4 with 2D and 3D disposition images. Accordingly, our docking experiment proposed their mechanism of action as Bcl-2 suppressors, congruent with the flow cytometric and RT-PCR analyses, illustrating their apoptosis-inducing activity. It is worth mentioning that thonningianin-A is a major active constituent of T. sanguinea, as it was Daphnetin [56] * Mass accuracy was calculated as [measured mass-expected mass/expected mass] × 10 6 and is expressed in parts per million (ppm) error [49].

Molecular Docking Studies
In this present study, T. sanguinea phenolic extract exhibited antiproliferative activity against breast cancer cells via apoptosis. Hence, all compounds, whether identified by the present LC-ESI-TOF-MS/MS analysis or previously isolated from the plant, were chosen for a simulated molecular docking investigation to gain insights into the possible molecular targets for the cytotoxic and apoptosis-inducing activities. As seen in Table 4 and Table S3, most of the identified compounds revealed good binding interactions with the key amino acids inside the "B-cell lymphoma 2 (Bcl-2), PDB ID: 4IEH", while there were some minor compounds that did not show any binding activity towards the studied target. Interestingly, compounds thonningianin-A and -B were promising compounds, possessing strong interactions with the key amino acids (mainly Arg 66) of the binding site, as shown in Table 4 with 2D and 3D disposition images. Accordingly, our docking experiment proposed their mechanism of action as Bcl-2 suppressors, congruent with the flow cytometric and RT-PCR analyses, illustrating their apoptosis-inducing activity. It is worth mentioning that thonningianin-A is a major active constituent of T. sanguinea, as it was

Molecular Docking Studies
In this present study, T. sanguinea phenolic extract exhibited antiproliferative activity against breast cancer cells via apoptosis. Hence, all compounds, whether identified by the present LC-ESI-TOF-MS/MS analysis or previously isolated from the plant, were chosen for a simulated molecular docking investigation to gain insights into the possible molecular targets for the cytotoxic and apoptosis-inducing activities. As seen in Table 4 and Table S3, most of the identified compounds revealed good binding interactions with the key amino acids inside the "B-cell lymphoma 2 (Bcl-2), PDB ID: 4IEH", while there were some minor compounds that did not show any binding activity towards the studied target. Interestingly, compounds thonningianin-A and -B were promising compounds, possessing strong interactions with the key amino acids (mainly Arg 66) of the binding site, as shown in Table 4 with 2D and 3D disposition images. Accordingly, our docking experiment proposed their mechanism of action as Bcl-2 suppressors, congruent with the flow cytometric and RT-PCR analyses, illustrating their apoptosis-inducing activity. It is worth mentioning that thonningianin-A is a major active constituent of T. sanguinea, as it was isolated in an amount of 145 mg from 1.5 g of the plant extract [27]. Our rationalization for targeting Bcl-2 as the molecular target for the phenolic extract agreed with the study of Verma et al. [57], which developed some natural polyphenolics as inhibitors against the anti-apoptotic Bcl-2 gene. Table 4. Summary of the ligand-receptor interactions of the promising docked compounds inside the active site of Bcl-2 (PDB: 4IEH).

Ligand-Receptor Interactions (HB and Van Der Waals Interactions)
Thonningianin-A Arg 66 Ala 59 Gly 104 Tyr 161 Asn 102 1 HB-Acceptor 1 HB-donor 1 HB-Acceptor 1 HB-Acceptor 1 HB-Acceptor isolated in an amount of 145 mg from 1.5 g of the plant extract [27]. Our rationalization for targeting Bcl-2 as the molecular target for the phenolic extract agreed with the study of Verma et al. [57], which developed some natural polyphenolics as inhibitors against the anti-apoptotic Bcl-2 gene. Table 4. Summary of the ligand-receptor interactions of the promising docked compounds inside the active site of Bcl-2 (PDB: 4IEH).

Ligand-Receptor Interactions (HB and Van Der Waals Interactions)
Thonningianin-A isolated in an amount of 145 mg from 1.5 g of the plant extract [27]. Our rationalization for targeting Bcl-2 as the molecular target for the phenolic extract agreed with the study of Verma et al. [57], which developed some natural polyphenolics as inhibitors against the anti-apoptotic Bcl-2 gene.

Compound Ligand-Receptor Interactions (HB and Van Der Waals Interactions)
Thonningianin-A

Plant Material
T. sanguinea whole plant was collected from the eastern region of Ghana in January 2015. The plant was authenticated by the curator of the herbarium. A voucher specimen was deposited, labeled with the code CSRPM no. 140.

Preparation of Plant Extract
The whole plant of T. sanguinea (300 g) was shade dried for seven days and pulverized. The dried powdered plant material was extracted by cold maceration with MeOH (3 x 1 L for 3 days), followed by MeOH:CHCl3 (1:1; 3 × 1 L for 3 days) at room temperature. The filtrates were pooled together and concentrated in vacuo using the rotary evaporator (BÜ CHI Labortechnik AG, Flawil, Switzerland) to produce 20 g of dry residue.

Plant Material
T. sanguinea whole plant was collected from the eastern region of Ghana in January 2015. The plant was authenticated by the curator of the herbarium. A voucher specimen was deposited, labeled with the code CSRPM no. 140.

Preparation of Plant Extract
The whole plant of T. sanguinea (300 g) was shade dried for seven days and pulverized. The dried powdered plant material was extracted by cold maceration with MeOH (3 x 1 L for 3 days), followed by MeOH:CHCl3 (1:1; 3 × 1 L for 3 days) at room temperature. The filtrates were pooled together and concentrated in vacuo using the rotary evaporator (BÜ CHI Labortechnik AG, Flawil, Switzerland) to produce 20 g of dry residue.

Plant Material
T. sanguinea whole plant was collected from the eastern region of Ghana in January 2015. The plant was authenticated by the curator of the herbarium. A voucher specimen was deposited, labeled with the code CSRPM no. 140.

Preparation of Plant Extract
The whole plant of T. sanguinea (300 g) was shade dried for seven days and pulverized. The dried powdered plant material was extracted by cold maceration with MeOH (3 × 1 L for 3 days), followed by MeOH:CHCl 3 (1:1; 3 × 1 L for 3 days) at room temperature. The filtrates were pooled together and concentrated in vacuo using the rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) to produce 20 g of dry residue.

Determination of Total Phenolic Content
The Folin-Ciocalteu method [58] was used to determine total phenolic content. Briefly, sample solution (0.5 mL) was mixed with 2.5 mL of Folin-Ciocalteu reagent, and 2 mL of sodium carbonate was then added. After incubation at room temperature for 2 h, the absorbance of the reaction mixture was measured at 760 nm against a blank using a Spectrophotometer UV/Vis specor D 250 plus (Analytik Jena GmbH, Jena, Germany). Gallic acid was used as the standard, and the total phenolic content was expressed as mg/g, calculated as gallic acid equivalents

Preparation of Phenolic Extract
T. sanguinea phenolic compounds were extracted using 200 g of the powdered plant by treatment with 5% Na 2 CO 3 aqueous solution, which was left for one hour. Then, the mixture was filtered and washed with distilled water. The filtrate was diluted with distilled water, neutralized using HCl, then partitioned between chloroform, ethyl acetate, and nbutanol. The obtained extracts were combined, then concentrated under reduced pressure using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) [59].

Initial Screening Step
Solutions of the provided samples were prepared in final concentrations of 1000 and 100 µg/mL in MeOH, to identify the range within which the inhibitory concentration 50 (IC 50 ) lies.

Determination of IC 50
Extracts that exceeded 50% inhibition in any of the initial screening step concentrations were serially diluted to provide five concentrations.

DPPH • Assay
A DPPH • (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free radical assay was carried out according to the previously described method [60]. Briefly, 100 µL of freshly prepared DPPH reagent (0.1% in methanol) was added to 100 µL of the sample in a 96-well plate (n = 6), and the reaction was incubated at room temperature for 30 min in dark. At the end of incubation time, the resulting reduction in DPPH • color intensity was measured at 540 nm. The scavenging of the DPPH • radical (PI) was determined, and the data are represented as means ± SD according to the following equation: The results were recorded using the microplate reader FluoStar Omega.

Data Analysis
The data were analyzed using Microsoft Excel ® , and the IC 50 value was calculated using GraphPad Prism 5 ® by converting the concentrations to their logarithmic value and selecting the nonlinear inhibitor regression equation (log (inhibitor) vs. normalized response-variable slope equation) [61].

LC-ESI-TOF-MS/MS Analysis
The mobile phase was prepared from DI water:methanol:acetonitrile in a ratio of (2:1:1), then 50 mg of dry methanolic extract was mixed with 1 mL of the mobile phase. The mixture was ultra-sonicated for 10 min and centrifuged for 10 min at 11,200× g. Finally, the injected concentration was adjusted to be 1 µg/µL. The injected volume was 25 µL in both positive and negative mode; in addition, 25 µL of the mobile phase was injected as a blank sample. MasterView was used for peaks extraction based on the following criteria: features should have a signal-to-noise ratio greater than 5, and the feature intensities of the sample-to-blank ratio should be greater than 5. A cutoff score of 70% was used to finely select the identified metabolites [23]. LC-ESI-TOF-MS/MS parameters and conditions are listed in the Supplementary Materials, Table S2. 3.7. Biological Evaluation 3.7.1. Cytotoxic Activity Cytotoxic activities of crude extract and phenolic extract were screened against breast cancers of MCF-7 (ATCC ® HTB-22™ Homo sapiens) and MDA-MB-231 (ATCC ® HTB-26™ Homo sapiens); liver HepG2 (ATCC ® HB-8065™ Homo sapiens) and lung A549 (ATCC ® CCL-185™ Homo sapiens) cancer cell lines. Each cell line was cultured in a proper complete medium, according to the standard cell culture work. Following routine MTT assay, the treatment of cells with crude extract and phenolic extract for 48 h incubation was carried out, and standard fluorouracil (5-FU) was used. Cell viability percentages versus the log values of serial concentrations were calculated. Then, IC 50 values in µg/mL were calculated using GraphPad Prism 7 [62].

Apoptotic Investigation Flow Cytometric Analysis
Flow cytometric analyses: "FITC/Annexin-V-FITC/PI, and DNA content cell cycle" were performed following previously described methods. The detailed methodology is provided in the Supplementary Materials. In brief, MCF-7 cells were treated with phenolic extract (16.54 µg/mL, 48 h), and 10 5 cells were harvested by trypsinization and washed two times with ice-cold PBS (pH = 7.4). This was followed by the addition of 500 µL Annexin V-FITC/PI solution to the cells. Then, cells were injected into the flow cytometer. Finally, ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA) was employed for quadrant analysis of the produced data, to estimate the percentage of apoptotic cells, and to determine at which phase cell cycle would be arrested [39].

RT-PCR
Phenolic extract (16.54 µg/mL, 48 h) was treated for the MCF-7 cells. Cells were collected at the end of the treatments, and total RNA was extracted with the RNeasy ® Mini Kit (cat.no. 74104, Hilden, Germany), as directed by the factory manufacturer. The cDNA synthetic kit (Bio-Rad, Hercules, CA, USA) was performed with 500 ng of RNA by i-Script cDNA synthesis, as instructed by the manufacturer. All reactions were performed for 35 cycles using this temperature profile: 95 • C for 5 m (initial denaturation); 95 • C for 15 min (denaturation), 55 • C for 30 min (annealing), and 72 • C for 30 min (extension). The cycle threshold values (Ct) and the relative folds of change between all samples were collected [40]. The primer sequences are listed in Table S2.

Molecular Docking
The molecular docking study was performed on the identified compounds (phenolic extract) towards the apoptosis inhibitor, Bcl-2 (PDB: 4IEH), to elucidate their cytotoxic and apoptotic activity profiles. The molecular docking studies were carried out using the MOE 2008-10 (Chemical Computing Group, Canada) as the computational software. The protein structure complexed with the co-crystallized ligand was easily accessible from the Protein Data Bank. Routine work concerning receptor and ligand preparation and molecular docking was carried out as previously published [63,64].