**No. Component Cas # Content (%)** <sup>α</sup>-D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl 597-12-6 0.843 <sup>α</sup>-D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl 597-12-6 0.482 3-Methoxybenzyl alcohol 6971-51-3 0.538 5-Acetoxymethyl-2-furaldehyde 10551-58-3 0.449 5-Hydroxymethylfurfural 67-47-0 0.335 Glutaric acid, 2-naphthyl tridecyl ester 998725-55-8 0.56 8-Oxabicyclo[5.1.0]oct-5-en-2-ol, 1,4,4-trimethyl 58795-43-0 0.14 Cycloheptasiloxane, tetradecamethyl 107-50-6 0.16 Ethyl hydrogen succinate 1070-34-4 1.679 Phenol, 2,4-bis(1,1-dimethylethyl) 96-76-4 1.029 β-D-Glucopyranose, 1,6-anhydro 498-07-7 1.072 <sup>α</sup>-D-Glucopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl 597-12-6 0.217 6Methoxy-2-amido-5,6-dihydrothiazolo[2,3-c]-1,2,4-triazole 997204-67-5 3.361 Myristic acid 544-63-8 0.155 Ethyl hydrogen succinate 1070-34-4 0.339 Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester 761-35-3 0.067 Palmitoleic acid 373-49-9 0.063 n-Hexadecanoic acid 57-10-3 0.105 Hexadecanoic acid, ethyl ester 628-97-7 8.057 Heptadecanoic acid 506-12-7 0.159 Octadecanoic acid 57-11-4 0.417 Octadecenoic acid (Z) 9 112-80-1 1.578 Octadecanoic acid 57-11-4 0.73 Hexadecanamide 629-54-9 2.309 Stearic acid, 2-hydroxy-1-methylpropyl ester 14251-39-9 1.497 9-Octadecenamide, (Z) 301-02-0 8.142 Octadecanamide 124-26-5 1.513 (Z)-(S)-Octadec-9-en-11-olide 997490-85-5 3.409 n-Propyl 9-octadecenoate 997641-34-3 0.439 Stearic acid, 2-hydroxy-1-methylpropyl ester 14251-39-9 1.569 Bis(2-ethylhexyl) phthalate 117-81-7 0.499 Elaidamide 301-02-0 0.475 Ethyl iso-allocholate 112-84-5 1.244 13-Docosenamide, (Z) 112-84-5 0.545 Campesterol 474-62-4 13.874

#### **Table 1.** *Cont*.

5,13-dione 997713-65-8 0.723

6,12-dimethoxy-8-methyl-5,8,9,13-tetrahydro-7H-cyclohept[b]anthracene-


**Figure 2.** *Cont*.

**Figure 2.** Cell viability MTT assay of Caco-2 and COLO-205 colon cancer cells and hGFs after treatment with the following: (**A**) increasing concentration of aqueous *Drimia maritima* bulb extract (0.122–31.25 μg/mL); (**B**) Proscillaridin (0.0024–1.32 μg/mL); (**C**) Doxorubicin (from 50 to 0.195 μg/mL). The IC50 values were calculated using log-probit analysis. Each value is presented as the mean ± SD of an average of three independent experiments.

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.

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).

#### *2.3. 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).

**Figure 3.** Flow cytometry analysis of D. maritima (DM) bulb extract effect on COLO-205 and Caco-2 cells. (**A**) Representative flow cytometer plots are presented for the untreated group (control) and DM-treated groups (4 μg/Ml, 3.5 μg/mL, 2.75 μg/mL, 2 μg/mL, 1 μg/mL and 0.5 μg/mL) in COLO205 cells. (**B**) The bar graph represents the percentage of early and late apoptotic cells detected by flow cytometer from three separate experiments (mean ± SD, n = 3). (**C**) Representative flow cytometery plots are presented for the control group (Untreated) and DM treated groups (2 μg/mL, 1 μg/mL, 0.75 μg/mL, and 0.5 μg/mL) in Caco-2 cells. (**D**) The bar graph represents the percentage of early and late apoptotic cells detected by flow cytometer from three different individual experiments (mean ± SD, n = 3). \*\* Significant differences were observed between the DM-treated (2 μg/mL and 1 μg/mL) and untreated control group (*p*-values \*\*\* < 0.001, \*\* < 0.01 and \* < 0.05).

#### *2.4. 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.

**Figure 4.** Production of reactive oxygen species (ROS) in COLO-205 and Caco-2 cells incubated for 48 h with *D. maritima* (DM) bulb extract: (**A**,**B**) Histograms and bar graphs of ROS production in COLO-205 cells were obtained by flow cytometer in the FITC channel in different groups. The bar graph shows a remarkable increase in the level of intracellular ROS in the treated group. (**C**,**D**) Representative histograms and bar graphs from Caco-2 cells treated with indicated concentrations of *D. maritima* extract on intracellular ROS levels production in comparison to control (untreated cells) detected by flow cytometer from three separate experiments (mean ± SD, n = 3). *p*-values \*\*\* < 0.001, \*\* < 0.01.

#### *2.5. D. maritima Bulb Extract Affects Mitochondrial Membrane Potential (*ΔΨ*m) 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 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 0.666 ± 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.

**Figure 5.** Assessment of the mitochondrial membrane potential (ΔΨm) in COLO-205 and Caco-2 cells after treatment with *D. maritima* (DM) bulb extract for 48 h: (**A**,**B**) Histograms and bar graphs show changes in COLO-205 cells ΔΨm response to various concentrations of DM extract (3.5 μg/mL, 2.75 μg/mL, 2 μg/mL, 1 μg/mL, and 0.5 μg/mL) in comparison to untreated cells. (**C**,**D**) Histogram and bar graphs show loss of ΔΨm following exposure to different concentrations of DM extract (2 μg/mL, 1.5 μg/mL, 1 μg/mL, 0.75 μg/mL, and 0.5 μg/mL) compared to control (untreated cells). All data are expressed as mean ± SD of three separate experiments. All data are expressed as mean ± SD of three separate experiments. (*p*-values \*\*\* < 0.001 \*\* < 0.01, and \* < 0.05).

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.

#### *2.6. 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*.

**Figure 6.** Gene expression analysis of apoptotic initiator gene *CASP8* and two inflammatory cytokine genes *TNF-α* and *IL-6* in COLO-205 and Caco-2 cells after treatment with various concentrations of *D. maritima* (DM) bulb extract for 48 h as examined by quantitative RT–PCR: (**A**,**D**) The graphs reveal fold changes in the expression of *CASP8* in COLO-205 and Caco-2 cells, respectively. (**B**,**E**) *TNF-α* gene in COLO-205 and Caco-2 cells at various extract concentrations (2.0, 1.5, 1.0, 0.75, and 0.5 μg/mL). (**C**,**F**) *IL-6* in COLO-205 and Caco-2 cells treated with different extract concentrations (1.5, 1.0, 0.75, and 0.5 μg/mL). Fold change was calculated using the ΔΔCt method. Untreated cells were used as the control; therefore, the fold change for untreated cells is 1 in all plots. *GAPDH* gene served as an internal reference gene. The error bars indicate the standard deviation from triplicate experiments (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001).

#### **3. 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 [10–14]. 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 IC50 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 IC50 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 (IC50 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 IC50 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, ΔΨm, is of significant value in this context. In particular, ΔΨm has considered one of the key markers of apoptosis [23]. Accordingly, the changes in the ΔΨm 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 ΔΨm 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 ΔΨm, 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 ΔΨm

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*.
