Chemical profiling of the secondary metabolites of the ethyl alcohol extract of L. nudicaulis together with hexane, CH2Cl2, ethyl acetate, and n-butanol fractions was carried out using LC-HRMS which resulted in dereplication of 15 compounds belonging to different chemical classes (Supplementary data; Figure S1a-S1d); three alkaloids were identified, as aspidofractinin, N-(2-methylpropylamide)-2E,4E-dodecadienamide, and ruscopine; three coumarins were annotated as umbelliferone, aesculetin, and 3,4-dihydroscopoletin; three flavonoid glycosides were dereplicated as luteolin 7-(6″-malonylneohesperidoside), kaempferol-3-O-[6″-malonyl-β-d-apiofuranosyl-(1 → 2)-β-d-glucopyranoside], and luteolin-7-O-β-d-glucopyranoside. Other identified compounds were luteolin, caffeic acid, 1-O-caffeoylgalactoside, and chicoric acid methyl ether. Finally, the sphingolipid nidicaulin B and the triterpene lupeol (Supplementary data; Table S1, Figure S2) completed the metabolomics analysis of the different extracts with annotation for each dereplicated compound.

Depending on the physicochemical properties and spectroscopic data (1H, and DEPT-Q NMR) (Supplementary data: Figure S4-S15) as well as comparison with the reported data and the results of metabolomic analysis, six compounds L1-6 (Supplementary data: Figure S3) were isolated and identified as caffeic acid (L1) (Atoui et al. 2005), luteolin (L2) (Elwekeel et al. 2022a, b), luteolin-7-O glucoside (L3) (Lin et al. 2015), β-sitosterol (L4) (El-Hawary et al. 2016), lupeol (L5) (Silva et al. 2018), and palmitic acid (L6) (Di Pietro et al. 2020).

The cytotoxic activity was evaluated for hexane, CH2Cl2, EtOAc, and n-butanol extracts against three human cancer cell lines, i.e., HL-60, HT-29, and MCF-7 using MTT assay; the results was expressed as IC50 (Table 1). The results revealed that the DCM extract displayed a significant cytotoxic activity toward HL-60 and HT-29 cells with IC50 values of 5.8 and 8.2 µg/ml, respectively, followed by the n-butanol extract with IC50 values of 11.6 and 9.6 µg/ml, respectively. Also, the EtOAc extract showed a good activity against HT-29 with IC50 value 10.7 µg/ml and moderate activity against HL-60 with IC50 value 12.40 µg/ml. CH2Cl2, n-butanol, and EtOAc extracts showed a moderate activity against MCF-7 with IC50 values 15.1, 18.2, and 19.5 µg/ml, respectively, while the hexane extract exhibited a weak cytotoxic activity against the tested cell lines. Previous study on the anticancer activity of the ethanol extract of L. fragilis and L. nudicaulis against six cell lines using sulforhodamine B (SRB) assay revealed that the ethanol extract of L. nudicaulis exhibited cytotoxic activity against lung carcinoma cell line (H1299) (El-Darier et al. 2021). LC/MS analysis of different extracts disclosed that the activity of the CH2Cl2-soluble fraction could be attributed, in part, to the alkaloid content, since aspidofractinine alkaloid showed potent cytotoxic activity against BGC-823 cells (human gastric carcinoma), HepG2 cells (Human hepatocellular carcinoma), MCF-7 cells (human breast cancer), SGC-7901 cells (human gastric adenocarcinoma), SK-MEL-2 (human skin cancer), and with SK-OV-3 (ovarian) compared with doxorubicin (Wang et al. 2017). The polar extracts EtOAc and n-butanol showed a rich content of phenolic acids, such as caffeic acid, chicoric methyl ether (caffeic acid derivative), and 1-O-caffeoylgalactose previous studies revealed that caffeic acid was active as anticancer against colon cancer cell lines (Hashim et al. 2008), while chicoric acid was active against human gastric cancer progress (Sun et al. 2019). Flavonoids as luteolin and luteolin-7-O-glucoside, which were detected in the plant extract, have been described as cytotoxic agents against many cancer cell lines (Seelinger et al. 2008). So, the cytotoxic effect of EtOAc and n-butanol extracts could be attributed to the presence of phenolic constituents, active redox agents, and fully recognized as antioxidants or scavengers.

Since the isolated compounds L1–L6 have been reported previously to have cytotoxic properties against various cell lines (Baskar et al. 2011; Rajavel et al. 2017) with some of them identified as inhibitors of topoisomerase I (topo I) and/or topoisomerase II (topo II) (Webb and Ebeler 2004), molecular docking study of both isolated and detected metabolites in L. nudicaulis extracts has been accomplished to investigate binding modes and interactions of the compounds with topoisomerases. Analysis of binding free energies of the compounds docked to topo I (1T8I) (Supplementary data: Table S2) demonstrated that the detected metabolite luteolin-7-(6″-malonylneohesperidoside) (1) exhibited the lowest binding energy (− 11.341 kcal/mol) (Fig. 1a). This flavonoid intercalated with DNA where the planar aromatic flavonoid nucleus is stacked between the diphosphate cytosine (DC112) and diphosphate adenosine (DA113); binding mode of this compound with topo I depicted three hydrogen bonds with side chains of Arg364 and Lys436 and backbone of Tyr426, in addition to an ionic interaction between malonyl group and Lys436. Similarly, kaempferol-3-O-[6″-malonyl-β-d-apiofuranosyl-(1 → 2)-β-d-glucopyranoside] (2) (− 10.796 kcal/mol) exhibited hydrogen bond interaction between the apiose moiety and Tyr426 in addition to stabilization through π-π sacking of the flavonoid nucleus with four DNA bases (Supplementary data: Figure S16a). Among the isolated compounds, luteolin-7-O-β-d-glucopyranoside (3) showed the highest binding affinity (− 10.367 kcal/mol) with four π-π sacking interactions with DNA and two hydrogen bonds formed between both 2″-OH and 6″-OH of the glucose unit and Asp533 (Supplementary data: Figure S16b). Docking simulation of the compounds to topo IIα (PDB: 5gwk) and topo IIβ (PDB: 3QX3) revealed again luteolin-7-(6″-malonylneohesperidoside) (1) with the lowest energy of binding (− 10.866 and − 10.111 kcal/mol, respectively) where the docking interaction with topo IIα exhibited stabilization of the compound in the binding site through formation of hydrogen bonding with DNA base in addition to two hydrogen bondings with the enzyme residues Glu461 and His759 and two ionic contacts with Mg ion that further contributed to the high binding affinity (Fig. 1b) while its proposed binding mode to topo IIβ showed the planar aromatic nucleus stacked with diphosphate guanosine (DG13) and stabilized by additional formation of hydrogen bonding, in addition to two hydrogen bonds with the amino acids Glu477 and Lys505 (Fig. 1c). Another noticeable compound is kaempferol-3-O-[6″-malonyl-β-d-apiofuranosyl-(1 → 2)-β-d-glucopyranoside] (2) with topo IIα and topo IIβ binding affinity values of − 10.102 and − 9.916 kcal/mol, respectively. Its interactions with topo IIα (Supplementary data: Figure S17a) revealed the presence of both π-staking interaction and hydrogen bonding with DNA base pairs, along with two hydrogen bonds with Glu461 and Lys489 and two contacts with Mg ion, while binding mode to topoisomerase IIβ depicted three hydrogen bonds with Glu477, Asp557, and Gly776 along with three ionic interactions with Mg ion (Supplementary data: Figure S17b). The higher binding affinity of luteolin-7-O-β-d-glucopyranoside (3) to topoisomerase I (− 10.367 kcal/mol) compared to topoisomerases IIα and IIβ (− 7.048 and − 6.923 kcal/mol, respectively) suggests that presence of more than one sugar moiety substituted into the flavonoid nucleus may have a role in stabilization of the inhibitor binding to topoisomerases IIα and IIβ. Altogether, the provided docking results endorse the studied compounds as candidates for further investigations to develop anticancer therapies.

2D diagram of the binding interactions of luteolin-7-(6″-malonylneohesperidoside) with the active site residues of the enzymes a topoisomerase I (1T8I), b topoisomerase IIα (5GWK), and c topoisomerase IIβ (3QX3). Pink circles with red and blue borders indicate polar acidic and basic amino acids, respectively, green circles indicate nonpolar amino acids, and light blue circles indicate DNA nucleotides