Evaluation of antiproliferative activity, apoptotic induction and LC-HRMS/MS analyses of the VLC fractions of L. numidicum

Abstract In this study, six vacuum liquid chromatography (VLC) fractions (F1–F6) of the n-BuOH extract of L. numidicum Murb. (BELN) were examined for their anticancer capacity. The composition of secondary metabolites was analyzed by LC-HRMS/MS. The antiproliferative effect against PC3 and MDA-MB-231 lines was evaluated by MTT assay. Apoptosis of PC3 cells was detected by annexin V-FITC/PI staining using a flow cytometer. The results showed that only fractions 1 and 6 inhibited PC3 and MDA-MB 231 cell proliferation in a dose-dependent manner and induced dose-dependent apoptosis of PC3 cells, evidenced by the accumulation of early and late apoptotic cells, and by the decrease in viable cells. LC-HRMS/MS profiling of fractions 1 and 6 revealed the presence of known compounds that may be responsible for the observed anticancer activity. F1 and F6 may be an excellent source of active phytochemicals for cancer treatment. Graphical Abstract


Introduction
According to the World Health Organization (WHO) 2020, Cancer is the main cause of death in the world with nearly 10 million deaths, the highest incidences in the world are observed for breast and prostate cancers.
Linum genus (Linaceae) includes more than 200 species distributed worldwide (Rogers 1982). Several studies have reported that Linum species could inhibit the growth of various types of cancer cell lines through cell cycle arrest and induction of apoptosis (Asl et al. 2018;Alejandre-Garc ıa et al. 2015).
The aim of this study was to investigate the ability of vacuum liquid chromatography (VLC) fractions (F1-F6) of the n-BuOH extract of the endemic species L. numidicum Murb. (BELN) to inhibit cancer cell proliferation and induce apoptosis. In addition, the secondary metabolites composition of the most active fractions (F1 and F6) was analyzed by LC-HRMS/MS to determine the relationship between their anticancer activity and their chemical composition.

Induction of apoptosis by F1 and F6 in PC3 cells
To explore the mechanism by which F1 and F6 inhibit cell proliferation, a flow cytometer analysis of apoptosis using annexin V-FITC/PI double staining was performed. F1 and F6 induced dose-dependent apoptosis of PC3 cells, evidenced by the accumulation of early and late apoptotic cells, and the decrease in viable cells, with a greater effect at 125 and 250 lg/mL ( ÃÃÃÃ p < 0.0001) compared with the negative control ( Figure S3(a,b) and S4 (a,b)).
The apoptotic effect induced by F1 and F6 can be attributed to its main bioactive compounds and other compounds present in the extract through a direct and also synergistic effect (Table S2).

LC-HRMS/MS analyses of F1 and F6
The evaluation of the antiproliferative and apoptotic activity of the fractions obtained indicated that the inhibitory activity was localized in two fractions F1 and F6. It was therefore necessary to perform chemical analyses to reveal the molecules responsible for this activity. Therefore, LC-HRMS/MS analyses were necessary to study the structure-activity relationship. LC-HRMS/MS analyses of F1 and F6 revealed the presence of 72 and 58 compounds, respectively (Table S2 and Figure S5(a,b)). Flavonoids (32 compounds were detected in F1 and F6) and phenolic acids (15 compounds in F1 and 8 in F6) are the most important classes.
The anticancer activity induced by F1 and F6 could be due to the major compounds detected, namely vicenin-2, malvidin 3-O-galactoside, vitexin and isovitexin, genkwanin, oleamide, orientin, and luteolin-7-O-glucoside. Indeed, vicenin-2 has been reported to possess numerous pharmacological properties, including antioxidant, antiinflammatory, and anticancer effects (Yang et al. 2018). Studies conducted by Nagaprashantha et al. (2011) showed that vicenin-2 induces antiangiogenic, pro-apoptotic, and antiproliferative activity in prostate cancer cells. It has also been reported that malvidin-3-galactoside inhibits proliferation and induces apoptosis in hepatocellular carcinoma (HCC) cell lines HepG2 and Huh-7 (Wang et al. 2019;Lin et al. 2020). Vitexin and isovitexin are flavones, anticancer effects on various cancer models were also observed for these two compounds, inhibit cell growth and induce apoptosis (Ganesan and Xu 2017). Genkwanin also showed strong antiproliferative activity on HeLa cell line (Awouafack et al. 2016) and MDA-MB-468 (Androutsopoulos et al. 2009). In addition, oleamide exerts antiproliferative effects on RG2 glioblastoma cells through cell cycle arrest in the S phase and induction of apoptosis (Torres-Rom an et al. 2020). Studies have reported that orientin exerts a potent antiproliferative effect against EC109 and MCF-7 cancer cells (Thangaraj and Vaiyapuri 2017) and colorectal carcinoma cells (HT29) by inducing apoptosis and blocking the cell cycle in G0/G1 (Thangaraj et al. 2019). Luteolin-7-O-glucoside inhibits the proliferation of various COLO 320 DM, AGS, MCF-7, and A549 lines. The ability to induce apoptosis in in COLO 320 cells was determined by its antiradical activity by scavenging ROS and regulating b-catenin expression (Baskar et al. 2011).

Experimental
The experimental section is available online in the supplementary material.

Conclusion
Our results demonstrated for the first time that the VLC fractions F1 and F6 of the n-BuOH extract of L. numidicum (BELN) exert anticancer activity by inhibiting cell proliferation and inducing apoptosis. This could be attributed to its major bioactive compounds such as vicenin-2, malvidin 3-O-galactoside, vitexin and isovitexin, genkwanin, oleamide, orientin, and luteolin-7-glucoside. F1 and F6 can be used as an excellent source of phytochemicals to treat cancer. Future studies should aim to explore the anticancer activity of each compound, its detailed mechanism, and its synergistic effects.