Phytochemical Analysis and Anticancer Properties of Drimia maritima Bulb Extracts on Colorectal Cancer Cells

Cancer is a worldwide health problem and is the second leading cause of death after heart disease. Due to the high cost and severe side effects associated with chemotherapy treatments, natural products with anticancer therapeutic potential may play a promising role in anticancer therapy. The purpose of this study was to investigate the cytotoxic and apoptotic characteristics of the aqueous Drimia maritima bulb extract on Caco-2 and COLO-205 colorectal cancer cells. In order to reach such a purpose, the chemical composition was examined using the GC-MS method, and the selective antiproliferative effect was determined in colon cancer cell lines in normal gingival fibroblasts. The intracellular ROS, mitochondrial membrane potential, and gene expression changes in selected genes (CASP8, TNF-α, and IL-6 genes) were assessed to determine the molecular mechanism of the antitumor effect of the extract. GC-MS results revealed the presence of fifty-seven compounds, and Proscillaridin A was the predominant secondary metabolite in the extract. The IC50 of D. maritima bulb extract on Caco-2, COLO-205, and the normal human gingival fibroblasts were obtained at 0.9 µg/mL, 2.3 µg/mL, and 13.1 µg/mL, respectively. The apoptotic effect assay indicated that the bulb extract induced apoptosis in both colon cancer cell lines. D. maritima bulb extract was only able to induce statistically significant ROS levels in COLO-205 cells in a dose-dependent manner. The mitochondrial membrane potential (MMP) revealed a significant decrease in the MMP of Caco-2 and COLO-205 to various concentrations of the bulb extract. At the molecular level, RT-qPCR was used to assess gene expression of CASP8, TNF-α, and IL-6 genes in Caco-2 and COLO-205 cancer cells. The results showed that the expression of pro-inflammatory genes TNF-α and IL-6 were upregulated. The apoptotic initiator gene CASP8 was also upregulated in the Caco-2 cell line and did not reach significance in COLO-205 cells. These results lead to the conclusion that D. maritima extract induced cell death in both cell lines and may have the potential to be used in CRC therapy in the future.


Introduction
Cancer can be defined as a non-communicable disease with progressive uncontrolled cell growth/division with a probability to invade nearby and/or distant tissues and could appear in any part of the body [1][2][3][4]. Colorectal cancer, "CRC", is the second most common cancer in both genders [5]. Natural products with anticancer therapeutic potential have earned an increasing interest in recent years. Pharmacological research of plant extracts as a source of secondary metabolites is probably the most important step in the identification of plant-derived compounds with therapeutic promise [6]. Plant extracts are a major source of phenolic compounds and other secondary metabolites, which have shown potential anticancer activities by regulating cell death pathways [7]. Furthermore, most of the anticancer drugs in the market are plant-derived such as taxanes (e.g., paclitaxel (Taxol) and docetaxel (Taxotere)) or synthetic compounds. Therefore, a need for basic and applied research to find novel cancer prevention and treatment options from plants as a source for safe and effective anticancer treatment [8].
To our knowledge, the cytotoxicity of the bulb extract of the medicinal plant D. maritima toward CRC cell lines was not previously investigated. Therefore, this research was initiated to study the therapeutic potential of D. maritima on CRC via assessment of the cytotoxic activity of D. maritima bulb extract on the colorectal Caco-2 and COLO-205 cancer cell lines. In this study, we prepared an aqueous extraction of the medicinal plant D. maritima bulb to assess the in vitro antiproliferative effects of D. maritima bulb extract against colorectal cancer cells. We also aimed to determine the mechanism of action of active D. maritima bulb extract against colorectal cancer cells. Finally, we aimed to examine the gene expression of some cell growth-related genes in bulb extract-treated colorectal cancer cells using quantitative RT-PCR.

Identification of Chemical Constituents of D. maritima Bulb Extract by GC-MS
Analysis of the bioactive constituents of the bulb extract of D. maritima by GC-MS demonstrated the presence of 57 compounds and their chromatogram, as shown in Figure 1 and Table 1. Major peaks determined in the GC-MS chromatogram and their corresponding components were recognized according to NIST 20 and Willy 19 Libraries. The bulb extract was found to include a variety of essential phytochemicals. The results of GC-MS analysis showed that D. maritima bulb extract was rich in the phytosterol "Campesterol" containing 14%, 2,3-Butanediol (glycol and secondary alcohol) and 5-Hydroxymethylfurfural (furans, an arenecarbaldehyde, and primary alcohol) (~10% each), and~8% for each of the fatty acid derivatives "Hexadecanoic acid ethyl ester" and "9-Octadecenamide (Z)-". Interestingly, the cardiac glycoside "Proscillaridin" proved to be one of the predominant secondary metabolites in the bulb extract of D. maritima with about 5% content which may play an effective role as a pharmaceutical anticancer agent (Table 1).

Drimia maritima Bulb Extract and ProA Can Selectively Inhibit the Proliferation of COLO-205 and Caco-2 Cancer Cells
The MTT assay was performed to evaluate the cell viability and proliferation of COLO-205 and Caco-2 cells upon treatment with different concentrations of D. maritima bulb extract, ProA, and Doxorubicin (positive control). The treatment of COLO-205 and Caco-2 with different concentrations of D. maritima bulb extract showed cytotoxic effects proportional to the concentration of D. maritima bulb extract used. The IC 50 of D. maritima bulb extract on COLO-205 cell lines was~2.3 µg/mL and for Caco-2 cells was~0.9 µg/mL ( Figure 2). In order to determine whether D. maritima bulb extract has cytotoxicity effects on normal human cells, normal HGF cells were treated with the plant extract using the same concentrations used in colon cancer cell lines and incubated for 48 h. The results showed that D. maritima bulb extract has minimal cytotoxic effects on normal human cells at low concentrations, and the IC 50 was achieved at a relatively high concentration of 13.1 µg/mL (Figure 2), indicating that D. maritima bulb extract potentially has selective cytotoxic effects on cancer cells but not on normal cells. From the reported IC50s, an estimation for the selectivity index can be found to be approximately 5.7 for the COLO-205 cells and 14.55 for Caco-2 cells.

Drimia maritima Bulb Extract and ProA Can Selectively Inhibit the Proliferation of COLO-205 and Caco-2 Cancer Cells
The MTT assay was performed to evaluate the cell viability and proliferation of COLO-205 and Caco-2 cells upon treatment with different concentrations of D. maritima bulb extract, ProA, and Doxorubicin (positive control). The treatment of COLO-205 and Caco-2 with different concentrations of D. maritima bulb extract showed cytotoxic effects proportional to the concentration of D. maritima bulb extract used. The IC50 of D. maritima bulb extract on COLO-205 cell lines was ~2.3 µg/mL and for Caco-2 cells was ~0.9 µg/mL ( Figure 2). In order to determine whether D. maritima bulb extract has cytotoxicity effects on normal human cells, normal HGF cells were treated with the plant extract using the same concentrations used in colon cancer cell lines and incubated for 48 h. The results showed that D. maritima bulb extract has minimal cytotoxic effects on normal human cells at low concentrations, and the IC50 was achieved at a relatively high concentration of 13.1 µg/mL (Figure 2), indicating that D. maritima bulb extract potentially has selective cytotoxic effects on cancer cells but not on normal cells. From the reported IC50s, an estimation for the selectivity index can be found to be approximately 5.7 for the COLO-205 cells and 14.55 for Caco-2 cells.
On the other hand, to evaluate the cytotoxic effect of ProA, which is one of the major active compounds of D. maritima (Table 1), COLO-205 and Caco-2 cells, as well as the normal HGF cells, were treated with different concentrations of ProA ranging from 0.0024 µg/mL to 1.32 µg/mL. The results, as indicated in Figure 2B, showed a significant increase in Caco-2 and COLO-205 cell death, and the IC50 was achieved at 0.0029 µg/mL and 0.012 µg/mL, respectively. Moreover, ProA showed a minimal cytotoxic effect on normal HGF, and the IC50 was achieved at 0.229 µg/mL.

Retention Time (min)
Relative intensity (a.u.)*  Doxorubicin was also used to assess the efficacy and the potential of ProA and bulb extract as natural cancer therapeutics. Importantly, ProA showed a more powerful cytotoxic effect than D. maritima bulb extract on both Caco-2 and COLO-205 cell lines when compared to Doxorubicin, which was achieved at IC50 at 0.78 µg/mL for COLO-205 and 0.59 µg/mL for Caco-2 ( Figure 2C).

D. maritima Bulb Extract Can Induce Early and Late Apoptosis in Colon Cancer Cell Lines
In order to clarify whether D. maritima bulb extract can induce cell apoptosis in colon cancer cells, D. maritima-treated and -untreated Caco-2 and COLO-205 cells were stained with Annexin V and PI to analyze different apoptotic stages using flow cytometry. When compared to untreated cells (control cells), D. maritima bulb extract was able to significantly increase both the early and late cell apoptotic percentages in treated colon cancer cells in a concentration-dependent manner. In the COLO-205 cell culture, there was a statistically significant increase in the percentage of early and late apoptotic cells ( Figure 3A). These results clearly emphasize that D. maritima bulb extract is able to induce cell early and late apoptosis in Caco-2 and COLO-205 cell lines in a concentration-dependent manner ( Figure 3B). On the other hand, to evaluate the cytotoxic effect of ProA, which is one of the major active compounds of D. maritima (Table 1), COLO-205 and Caco-2 cells, as well as the normal HGF cells, were treated with different concentrations of ProA ranging from 0.0024 µg/mL to 1.32 µg/mL. The results, as indicated in Figure 2B, showed a significant increase in Caco-2 and COLO-205 cell death, and the IC 50 was achieved at 0.0029 µg/mL and 0.012 µg/mL, respectively. Moreover, ProA showed a minimal cytotoxic effect on normal HGF, and the IC 50 was achieved at 0.229 µg/mL.
Doxorubicin was also used to assess the efficacy and the potential of ProA and bulb extract as natural cancer therapeutics. Importantly, ProA showed a more powerful cytotoxic effect than D. maritima bulb extract on both Caco-2 and COLO-205 cell lines when compared to Doxorubicin, which was achieved at IC 50 at 0.78 µg/mL for COLO-205 and 0.59 µg/mL for Caco-2 ( Figure 2C).

D. maritima Bulb Extract Can Induce Early and Late Apoptosis in Colon Cancer Cell Lines
In order to clarify whether D. maritima bulb extract can induce cell apoptosis in colon cancer cells, D. maritima-treated and -untreated Caco-2 and COLO-205 cells were stained with Annexin V and PI to analyze different apoptotic stages using flow cytometry. When compared to untreated cells (control cells), D. maritima bulb extract was able to significantly increase both the early and late cell apoptotic percentages in treated colon cancer cells in a concentration-dependent manner. In the COLO-205 cell culture, there was a statistically significant increase in the percentage of early and late apoptotic cells ( Figure 3A). These results clearly emphasize that D. maritima bulb extract is able to induce cell early and late apoptosis in Caco-2 and COLO-205 cell lines in a concentration-dependent manner ( Figure 3B).

D. maritima Bulb Extract Induces the Production of ROS in Colon Cancer Cells
ROS generation was measured after treatment with D. maritima bulb extracts in colon cancer cells. D. maritima bulb extract was only able to induce statistically significant ROS levels in COLO-205 cells in a dose-dependent manner when treated with concentrations above 2 µg/mL (i.e., 3.5, 2.75, and 2 µg/mL; p-value > 0.001) (Figure 4). Although D. mari-

D. maritima Bulb Extract Induces the Production of ROS in Colon Cancer Cells
ROS generation was measured after treatment with D. maritima bulb extracts in colon cancer cells. D. maritima bulb extract was only able to induce statistically significant ROS levels in COLO-205 cells in a dose-dependent manner when treated with concentrations above 2 µg/mL (i.e., 3.5, 2.75, and 2 µg/mL; p-value > 0.001) (Figure 4). Although D. maritima bulb extract was able to induce ROS in Caco-2 cells at lower plant extract concentrations (0.5 and 1 µg/mL), these results were statistically non-significant ( Figure 4). Moreover, D. maritima bulb extract was not able to induce a statistically significant level of ROS in Caco-2 cells. Perhaps, this could be referred to the lower concentrations of D. maritima bulb extract used to treat Caco-2 cells (1.5, 1, and 0.75 µg/mL) when compared to that used to treat COLO-205.
Molecules 2023, 28, x FOR PEER REVIEW 8 of 18 tima bulb extract was able to induce ROS in Caco-2 cells at lower plant extract concentrations (0.5 and 1 µg/mL), these results were statistically non-significant ( Figure 4). Moreover, D. maritima bulb extract was not able to induce a statistically significant level of ROS in Caco-2 cells. Perhaps, this could be referred to the lower concentrations of D. maritima bulb extract used to treat Caco-2 cells (1.5, 1, and 0.75 µg/mL) when compared to that used to treat COLO-205.

D. maritima Bulb Extract Affects Mitochondrial Membrane Potential (ΔΨm) in Colon Cancer Cells
The effect of D. maritima bulb extract on mitochondrial membrane potential (ΔΨm) was assessed in colorectal cancer cells using TMRE cell-permeant red-orange dye to label

D. maritima Bulb Extract Affects Mitochondrial Membrane Potential (∆Ψm) in Colon Cancer Cells
The effect of D. maritima bulb extract on mitochondrial membrane potential (∆Ψm) was assessed in colorectal cancer cells using TMRE cell-permeant red-orange dye to label active mitochondria. FCCP was used as a positive control, which can significantly diminish mitochondrial membrane potential. The results showed that treatment of COLO-205 cells concentrations of D. maritima bulb extracts above 2 µg/mL (2.75 and 3.5 µg/mL) resulted in a statistically significant (p < 0.001) decrease in ∆Ψm of COLO-205 cells (4.569 ± 0.726, and 0.666 ± 0.72, respectively) when compared to control cells ( Figure 5). Although the effect of D. maritima bulb extracts on ∆Ψm in COLO-205 cells at 2 µg/mL concentration was minor, it was statistically significant (15.38 ± 0.72; p < 0.05). However, treatment of COLO-205 with 1 µg/mL D. maritima bulb extract showed no significant reduction in their ∆Ψm. active mitochondria. FCCP was used as a positive control, which can significantly diminish mitochondrial membrane potential. The results showed that treatment of COLO-205 cells concentrations of D. maritima bulb extracts above 2 µg/mL (2.75 and 3.5 µg/mL) resulted in a statistically significant (p < 0.001) decrease in ΔΨm of COLO-205 cells (4.569 ± 0.726, and .666 ± 0.72, respectively) when compared to control cells ( Figure 5). Although the effect of D. maritima bulb extracts on ΔΨm in COLO-205 cells at 2 µg/mL concentration was minor, it was statistically significant (15.38 ± 0.72; p < 0.05). However, treatment of COLO-205 with 1 µg/mL D. maritima bulb extract showed no significant reduction in their ΔΨm.
On the other hand, treatment of Caco-2 cells with different concentrations of D. maritima bulb extract of 0.5, 0.75, 1, 1.5, and 2 µg/mL resulted in a statistically significant (p < 0.001) reduction in their ΔΨm (260 ± 194.4, 250.3 ± 194.4, 290.3 ± 194.4, 326.0 ± 194.4, and 287.7 ± 194.4, respectively) when compared to the control cells ( Figure 5). These results confirmed that D. maritima bulb extract could significantly reduce ΔΨm in COLO-205 cells in a dose-dependent manner and also in the Caco-2 cell line.  Figure 5). These results confirmed that D. maritima bulb extract could significantly reduce ∆Ψm in COLO-205 cells in a dose-dependent manner and also in the Caco-2 cell line.

The Impact of D. maritima Bulb Extract on Gene Expression in Colon Cancer Cell Lines
In order to figure out changes in colon cancer cell lines at the molecular level after the observed growth-inhibitory effect of D. maritima bulb extract, RT-qPCR was applied to assess gene expression of multiple genes: Casp8, TNF-α, and IL-6 in COLO-205 and Caco-2 cells pre-treated with different concentrations of D. maritima bulb extract.
In COLO-205 cells, there was a non-significant upregulation in Casp8 gene by when the cells were treated with the extract at a concentration of 3.5 µg/mL ( Figure 6A). Moreover, treatment with the extract for 48 h at 2.75 and 3.5 µg/mL resulted in statistically significant elevations in the target gene TNF-α (5.59-fold and 4.8-fold, respectively). Although there was an elevation in the expression of the TNF-α gene when cells were treated with the extract at 2 µg/mL by 1.65-fold, this elevation was statistically non-significant ( Figure 6B). Furthermore, there was a non-significant elevation in the expression of the pro-inflammatory cytokine gene IL-6 when COLO-205 was treated at extract concentrations 2, 2.75, and 3.5 µg/mL (1.2-, 1.6-, and 2.1-fold, respectively) ( Figure 6C).
In comparison to the control group (untreated cells), the pro-inflammatory TNF-α expression levels in Caco-2 cells were significantly increased by 6.5-and 6.4-fold following treatment with 2 and 1.5 µg/mL of D. maritima bulb extracted for 48 h, respectively (p < 0.0001), and 5.2-fold when cells were treated with 1 µg/mL (p < 0.0007) ( Figure 6D). Although the expression levels of TNF-α elevated by 3.3-and 3.6-fold when Caco-2 cells were treated with 0.75 and 0.5 µg/mL, respectively, these elevations were not significant. The expression of the Casp8 gene was significantly upregulated (1.5-fold) following 48 h treatment with 1.5 µg/mL (p < 0.001) in Caco-2 cells. Moreover, the expression still elevated significantly by 1.31-and 1.3-fold when decreasing the concentration to 1 and 0.75 µg/mL (p < 0.041 and <0.014), respectively ( Figure 6E). An elevation also occurred when Caco-2 cells were treated with 0.5 µg/mL, yet, this elevation was statistically nonsignificant. Furthermore, results showed that D. maritima bulb extract could induce the activation of the effector Casp8 gene in Caco-2 cells ( Figure 6E). With respect to the expression of pro-inflammatory cytokine gene IL-6, results showed that there was a statistically significant upregulation (6.5-fold increase) when Caco-2 cells were treated with 1.5 µg/mL (p < 0.0244) when compared to control cells ( Figure 6F). Although there was an increase in the expression of gene IL-6 (3.4-, 3.2-, and 5.5-fold) upon treatment of Caco-2 cells with 0.5, 0.75, and 1 µg/mL of D. maritima bulb extract, respectively, these elevations were statistically non-significant.
In general, the results showed that D. maritima bulb extract could affect the expression of cell proliferation-related genes that were examined in this study in both cell lines, i.e., it can upregulate the expression of inflammatory cytokine genes TNF-α and IL-6 and the apoptotic initiator gene Casp8.

Discussion
Plants are an interesting and cost-effective natural source of potential therapeutic candidates, and their secondary metabolites, phenols, and extracts have been extensively investigated for a mode of action on cancer cell death and their anticarcinogenic properties [9]. In the current study, the anticancer potential of a novel medicinal plant common in Mediterranean regions, namely D. maritima was investigated. D. maritima has long been used for the management and treatment of many pathological conditions [10][11][12][13][14]. In cancer settings, several preceding in vitro studies have reported potent anticancerous and selective cytotoxic effects of D. maritima on different cancer types, including breast, lung,

Discussion
Plants are an interesting and cost-effective natural source of potential therapeutic candidates, and their secondary metabolites, phenols, and extracts have been extensively investigated for a mode of action on cancer cell death and their anticarcinogenic properties [9]. In the current study, the anticancer potential of a novel medicinal plant common in Mediterranean regions, namely D. maritima was investigated. D. maritima has long been used for the management and treatment of many pathological conditions [10][11][12][13][14]. In cancer settings, several preceding in vitro studies have reported potent anticancerous and selective cytotoxic effects of D. maritima on different cancer types, including breast, lung, lymphoma, and prostate cancer [10,11,13,15]. However, [16] the cytotoxic effect of D. calcarata bulb extracts against p53 mutant HT-29 and p53 wild-type Caco-2 colorectal cancer cells has recently been demonstrated. Herein, to our knowledge, the current study is the first to investigate the anticancerous effect of D. maritima bulb extracts on COLO-205 and Caco-2 colorectal cancer cell lines.
In order to evaluate the potential antitumor effect mediated by D. maritima bulb extract on the cell proliferation of colon COLO-205 and Caco-2 cancer cell lines, an MTT assay was used to measure mitochondrial activity in viable cells. Interestingly, D. maritima bulb extract showed a dose-dependent antiproliferative effect against both colon cancer cell lines (Caco-2 and COLO-205). D. maritima bulb extract was able to achieve IC 50 at 0.92 and 2.3 µg/mL in Caco-2 and COLO-205, respectively. In this study, ProA, which accounts for about 5% of the D. maritima bulb extract, was one of the major active phytochemical compounds with potent anticancer activity on different cancer types, including, but not limited to, glioblastoma, lung, and prostate cancers [17,18]. Accordingly, it was assumed that the cytotoxic activity of D. maritima bulb extracts on colon COLO-205 and Caco-2 cell lines could be achieved, at least in part, through ProA as an active compound. Interestingly, ProA was highly effective in inhibiting COLO-205 and Caco-2 cell lines and achieved IC 50 at concentrations of 0.0029 µg/mL and 0.012µg/mL, respectively. Additionally, both D. maritima bulb extract and ProA express minimal cytotoxic effects against normal HGF when compared to cancer cells (IC 50 equals 13.1 µg/mL and 0.229 µg/mL, respectively), indicating that their cytotoxic activities are selective to colon cancer cells, with a very attractive selectivity index of 14.6 for Caco-2 and 5.6 for COLO-205. Interestingly, the obtained IC50 values in this study are relatively lower than the previously reported IC50 against different breast cancer cell lines. For example, Hamzeloo-Moghadam reported that the IC50 values for the non-hemolytic ethanol extract of U. maritima against breast cancer MCF7 cells were 11.01 µg/mL. Additionally, IC50 values for the non-hemolytic acetone extract of U. maritima against breast cancer MCF7 cells was 6.01 µg/mL, while the Doxorubicin drug (positive control) exhibited a cytotoxic effect with a higher IC50 value (52.35 µg/mL) [19]. Another example is the study conducted by Obeidat and Sharan, which found that the effective doses of D. maritima that inhibited 50% of growth (IC50) of MCF-7 and MDA-MB-468 cells were 20.48 ± 1.17 µg/mL and 25.74 ± 2.05 µg/mL, respectively. Interestingly, Obeidat and Sharab's study revealed that D. maritima displays significantly lower cytotoxicity against AGO1522, a normal human fibroblast cell, with IC 50 values of 43.5 ± 1.73 µg/mL [13].
Under normal physiological conditions, apoptosis "programmed cell death" is considered a mechanism to eliminate aged or abnormal cells; however, in cancer settings, cancer cells are, in general, resistant to apoptosis and induce apoptosis "therapeutically", which is considered an effective way to eliminate cancer cells [20]. The findings of this study showed that when compared to untreated cells (control), D. maritima bulb extract was able to induce early and late cell apoptosis in a concentration-dependent manner.
D. maritima bulb extract was able to significantly (p < 0.0001) induce ROS production in both Caco-2 and COLO-205 cancer cell lines after 48 h of treatment in a dose-dependent manner. As aforementioned, intracellular ROS generation in large amounts is considered a mechanism to trigger cell apoptosis, in part, through triggering the endoplasmic reticulum stress [21], and thus, colon cancer cells apoptosis investigated in this study could be a result of excessive intracellular ROS generation mediated by D. maritima bulb extract. On the other hand, excessive ROS generation can cause cell cycle arrest as described previously [22], but still, our results did not confirm the finding; at the same time, this possibility cannot be excluded.
In order to further understand the mechanisms that promoted apoptosis in colon cancer cells upon treatment with D. maritima bulb extract, studying the mitochondrial membrane potential, ∆Ψm, is of significant value in this context. In particular, ∆Ψm has considered one of the key markers of apoptosis [23]. Accordingly, the changes in the ∆Ψm were evaluated by tracking TMRE fluorescent signal in colon cancer cells (Caco-2 and COLO-205 cell lines) after treatment with different concentrations of D. maritima bulb extract for 48 h. Importantly, when compared to untreated cells (control), a significant reduction in ∆Ψm was observed in colon cancer cells (p < 0.0001), and this effect was dose-dependent. Notably, the Colo-205 represents a metastatic colorectal cancer, as this cell was isolated from the ascitic fluid of a 70-year-old Caucasian male with carcinoma of the colon. The patient had been treated with 5-fluorouracil for 4-6 weeks before the removal of the fluid specimen. Additionally, Caco-2 cells represent relatively sensitive cells toward the 5-fluorouracil. Interestingly, the extract used in this study shows a relatively higher activity toward the colorectal Caco-2 cells. Interestingly, the apoptosis assay has also shown less apoptosis percentage than those seen in the case of the Caco-2 cells. These results inversely correlate with the ROS results, indicating that there is a negative correlation between ROS generation and ∆Ψm, which is consistent with the results of [24], and showed that curcumin is able to induce apoptosis in melanoma cancer cells by increasing the production of ROS and reducing ∆Ψm Finally, in order to track the molecular pathway through which D. maritima bulb extract was able to induce apoptosis in colon cancer cell lines, RT-qPCR was used to quantify the following target genes: genes encoding for pro-inflammatory cytokines, including TNF-α and IL-6, as well as the gene encoding for caspase-8. TNF-α is an important pleiotropic pro-inflammatory cytokine that, once produced at the site of inflammation, can mediate a wide range of cellular responses that include but are not limited to stimulating the production of pro-inflammatory cytokines, affecting the survival and proliferation of cells and/or inducing cell death under certain circumstances [25]. In fact, TNF-α can positively regulate the survival and proliferation of cells if it activates certain NF-kB-dependent genes involved in cell survival and proliferation [26,27]. The latter can be achieved through the activation of distinct caspase-8 activation pathways [27]. Furthermore, it has been reported that upregulating the gene expression of TNF-α and IL-6 can participate in inducing Cacocell death [28]. This explains why the above-mentioned three gene targets are examined in this study. Importantly, the results showed that upon treatment of both colon cancer cell lines with the D. maritima bulb extract, there was a statistically significant increase in the expression of all three gene targets tested in this study. This may indicate that D. maritima bulb extract induces apoptosis in colon cancer cells through the TNF-α, IL-6, and caspase-8 activation pathways [29]. The results of this study are in agreement with [13], which showed that the expression levels of TNF-α and IL-6 gene were induced in MCF7 cells treated with fruit extracts of D. maritima.

Plant Collection, Classification, and Extraction
The plant D. maritima was collected in March 2021 from Princess Tasneem bint Ghazi Technological Research Station in Al-Salt (Jordan), with coordinates: 32 • 05 N 35 • 40 E. The collected plant was identified by a taxonomist specialist Mr. Refad Khawaldeh at Jordan Royal Botanical Garden, where a voucher specimen (073/2021) is conserved for future reference. Then, a powder of D. maritima bulbs was prepared after the bulbs were cleaned with water. The grinding process was achieved manually by mortar and pestle. In the next step, the crude powdered plant (30 g) was macerated in 100 mL water for two weeks with continuous shaking at 150 rpm at RT. At the end of this period, the mixture was filtered using a clean white canvas and filter papers to yield the aqueous D. maritima bulb extract. Then, the aqueous part of the solution was dried out (evaporated) using a lyophilizer to obtain the desired D. maritima bulbs to extract in a lyophilized form. Finally, 100 mg of the D. maritima bulb extract was dissolved in 1 mL of DMSO to make a 100 mg/mL stock solution for the treatment experiments.

Identification and Characterization of D. maritima Bulb Extract by Gas Chromatography-Mass Spectrometry (GC-MS)
Gas chromatography-mass spectrometry (GC-MS) analysis of the extracted sample was performed using an Agilent 5977B GC/MSD (Agilent, Santa Clara, CA, USA). Gas Chromatograph was equipped and coupled to a mass detector Turbo Mass Gold, PerkinElmer Turbo Mass 5.1 spectrometer with an Elite-1 (100% dimethylpolysiloxane), DB-5MS, 30 m × 0.25 mm i.d., 0.25 µm film thickness of capillary column. The instrument was set to an initial temperature of 80 • C and maintained at this temperature for 1 min. At the end of this duration, the oven temperature was raised to 300 • C, at the rate of an increase of 15 • C/min, and maintained for 9 min. The injection port temperature was ensured at 290 • C, and the helium flow rate was one mL/min. The ionization voltage was 70 eV. Samples were injected in split mode as 10:1. Mass spectral scan range was set at 30-600 (m/z) [30].

Drugs Preparation
Proscillaridin A (Sigma-Aldrich, St. Louis, MO, USA) and Doxorubicin (EBEWE Pharma, Rome, Italy) were completely dissolved in DMSO and then prepared in 20 mM and 100 mM stock solutions, respectively. Then serial dilutions for both were prepared in DMEM medium and stored at −20 • C until use.

Cell Culture
Two human colorectal carcinoma cell lines from the American Type Culture Collection (ATCC), namely COLO-205 (ATCC ® CCL-222 ™ ) and Caco-2 (ATCC ® HTB-37™), were used in this study. Additionally, normal human gingival fibroblasts (hGFs) samples prepared at the Cell Therapy Center (CTC) were used to assess the cytotoxic effects of the plant extract and drugs/treatments. All cells were cultured under the same conditions in 75 cm 2 tissue culture flasks and maintained in cell culture media (CCM) consisting of DMEM (Euroclone, Pero, Italy) high-glucose supplemented with 10% (v/v) heat-inactivated FBS, 1X penicillin/streptomycin, 1X non-essential amino acid, sodium pyruvate, and 1X L-Glutamine and incubated in a humidified incubator at 37 • C and 5% CO 2 . When the confluence of cells reached 70-80 percent, cells were passaged with 1X trypsin/EDTA (Euroclone, Italy) and centrifuged at 1400 rpm, 25 • C for five min. Different characteristics for the used cell line is listed in Table 2.

Cell Viability and Proliferation Assay
The antiproliferative effect and the median inhibitory concentration (IC 50 ) of D. maritima bulb extract and ProA was assessed on COLO-205, Caco-2, and hGF cells using an MTT assay. In brief, cells were seeded in 96-well plates at a concentration of 8 × 10 3 cells/well and incubated for 24 h at 37 • C. The next day, cells were treated with D. maritima bulb extract and ProA at concentrations ranging from 0.122 µg to 500 to /mL and 2.46 to 5307 µg/mL, respectively, in a quadruplicate manner. The plates were incubated at 37 • C for 48 h. COLO-205, Caco-2, and hGF cells were also treated with a 2-fold serial dilution of Doxorubicin at concentrations ranging from 0.024 to 50 µg/mL under the same conditions. of 95 • C for 15 s (denaturation), "58 • C" for 60 s (annealing), and "72 • C" for 30 s (extension). The primers sequences are listed in Table 3. The PCR data analysis was performed using the ∆∆Ct method (delta delta cycle threshold); the analysis was performed automatically according to CFX Maestro Software of Bio-Rad Company (Hercules, CA, USA). The data were normalized, across all plates, to the housekeeping gene GAPDH. Table 3. List of primers for qPCR amplification.

Gene
Forward Primer Sequence (

Statistical Analysis
All statistical analyses were performed using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, CA, USA). The comparison between different groups of numerical variables was performed using one-way or two-way ANOVA. Homogeneity of variances was tested using Dunnett's and Bonferroni's multiple comparisons tests, and a p-value less than 0.05 (p < 0.05) was considered statistically significant.
The IC 50 values were determined by using log-probit analysis; log (concentration) plotted on the x-axis and inhibition percentage plotted on the y-axis; percent of inhibition was calculated, after correction of the absorbance (A) measurements for the background (blank) absorbance, according to the equation; Inhibition% = ((A control − A treatment )/A control ) × 100%.

Conclusions
In summary, D. maritima bulb extract and ProA showed a selective inhibitory activity on the proliferation of colorectal cancer cell lines. Such a finding was supported by the results of the apoptosis, intracellular ROS, and mitochondrial membrane potential assays. On the molecular level, the expression of pro-inflammatory genes TNF-α and IL-6 and the apoptotic initiator gene CASP8 was also confirmative for the antiproliferative effect of the D. maritima bulb extract. Taken together, the findings of this study elect D. maritima for further investigation and validation on CRC animal cancer xenograft models.