Phytoconstituents from Piliostigma foveolatum (Dalzell) Thoth. leaves induce antiproliferative effect, apoptosis, and cell cycle arrest in Hop-62 cells

Abstract The study evaluated the therapeutic potential of ethanolic leaf extract of Piliostigma foveolatum (Dalzell) Thoth. (EEBF), its toluene, ethylacetate, methanol soluble fractions (viz. TFBF, EFBF, MFBF), and isolated phytoconstituents against lung cancer. Four compounds were isolated from MFBF by column chromatography and preparative HPLC. Structures were elucidated by IR, 13C-NMR, 1H-NMR, mass spectroscopy and identified as Quercetin, Kaempferol, Isorhamnetin, and ß-glucogallin. EEBF and its biofractions exhibited remarkable antiproliferative activity with GI50<85µg/mL, while isolated Quercetin, Kaempferol, Isorhamnetin, and ß-Glucogallin displayed GI50 values of 56.15 ± 1.16 μM, 68.41 ± 3.98 μM, 55.08 ± 0.57 μM and 58.99 ± 12.39 μM respectively. MFBF demonstrated significant apoptotic activity with 42.24 ± 0.57% cells in early and 4.61 ± 0.88% cells in late apoptosis comparable to standard Doxorubicin. Kaempferol exhibited 23.03 ± 0.37% cells in early and 2.11 ± 0.55% cells in late apoptosis, arresting Hop-62 cells in S-phase. In silico molecular docking, revealed that isolated constituents effectively bound to the same binding site of caspase-3 as Doxorubicin, highlighting their apoptotic mode of action. Graphical Abstract


Introduction
Lung cancer continues to be an alarming pandemic contributing to several co-morbidities, loss of millions of lives with 1.80 million deaths reported worldwide in 2020 (Thandra et al. 2021).Therapeutics from herbal sources are being continuously screened as prospective anticancer agents with an aim of improving the quality of life of the individual undergoing cancer treatment.Several plant species of the genus Bauhinia have been used in folk medicine to treat different kinds of pathologies and possess a variety of pharmacological activities viz.antioxidant, antimicrobial and cytotoxic effects (Negi et al. 2012;Rahman et al. 2016;Vijayan et al. 2019).Piliostigma foveolatum (Dalzell) Thoth.(Syn.Bauhinia foveolata Dalzell.)belonging to the family Fabaceae, is a tropical tree found along the lush semi-evergreen forests of the Indian Sahyadris (Bandyopadhyay et al. 2005).It has large porous leaves and is endemic to the Indian western ghats (Habbu et al. 2020).Limited research has been carried out on this species with key developments relating to antibacterial activity against Streptococcus pyogenes and antimalarial activity against Plasmodium falciparum by the acetone fraction and ethyl acetate extract respectively (Gamit et al. 2018), exploration of the cytotoxic principles of odoratin-7-glucoside and quercetin against HT-29 and HCT-15 human colon cancer cell lines (Habbu et al. 2020) and toluene fraction of the ethanolic leaf extract against MCF-7 human breast cancer cells with significant in vitro antioxidant activities (Mendonça et al. 2021).Based on the presence of substantial amounts of flavonoids and phenolics in Piliostigma foveolatum (Dalzell) Thoth, its antioxidant profile, and its ability to inhibit the proliferation of cancerous cells in breast, the current study was undertaken to evaluate and explore the anti-proliferative, apoptotic and cell cycle arrest potential of the ethanolic leaf extract of Piliostigma foveolatum (Dalzell) Thoth.(EEBF), its toluene (TFBF), ethyl acetate (EFBF) and methanol (MFBF) soluble biofractions along with the isolated phytoconstituents against lung cancer using Hop-62 cell lines.

Results and discussion
The yield of the ethanolic leaf extract of Piliostigma foveolatum (Dalzell) Thoth.was reported to be 151.29 g while that of its biofractions TFBF, EFBF and MFBF were 27.99 g, 13.79 g and 28.68 g respectively.The phytochemical analysis reported by Mendonça et al. 2021, revealed that the leaf extract and its methanolic biofraction contained considerable amounts of flavonoids, alkaloids, glycosides, tannins, phenolics etc.Since MFBF exhibited a remarkable ability to scavenge the DPPH, nitric oxide, and hydrogen peroxide free radicals, it was further subjected to liquid chromatography-mass spectroscopy.
Column chromatography resulted in the isolation of column fraction LMF, which upon further purification by preparative HPLC led to the isolation of four compounds LMF1, LMF2, LMF3 and LMF4 (Supplementary material, Figure S1), the yields of which were 21.1 mg, 242.6 mg, 21.7 mg, and 19.1 mg respectively.The structures of the isolated compounds were elucidated by IR, 1 H-NMR, 13 C-NMR and mass spectroscopic methods and identified as Quercetin (LMF1), Kaempferol (LMF2), Isorhamnetin (LMF3) and β-glucogallin (Syn.1-o-galloyl-β-D-glucose) (LMF4) respectively.The spectral data of the isolated compounds has been listed in the supplementary material.
In vitro anticancer activity of the leaf extract, its biofractions, and isolated phytoconstituents was evaluated using the Sulforhodamine B antiproliferative assay and subsequently investigated for apoptosis and cell cycle arrest using flow cytometry after staining with Annexin V-FITC and/or Propidium Iodide.
Amongst the isolated phytoconstituents investigated, all four compounds viz.LMF1 (Quercetin), LMF2 (Kaempferol), LMF3 (Isorhamnetin) and LMF4 (β-glucogallin) displayed significant Hop-62 cell antiproliferative activity with GI 50 values of 56.15 ± 1.16 µM, 68.41 ± 3.98 µM, 55.08 ± 0.57 µM and 58.99 ± 12.39 µM respectively as against the standard doxorubicin with GI 50 value of 19.49 ± 1.70 µM.Apoptosis study of the isolated compounds at their GI 50 concentrations showed that all compounds induced substantial apoptotic activity.LMF2 (Kaempferol) exhibited considerable apoptotic activity with 23.03 ± 0.37% cells experiencing early apoptosis, 2.11 ± 0.55% cells in late apoptosis and 0.51 ± 0.37% cells in necrotic stage (Figures 2 and S21), as compared to other compounds tested.In cell cycle analysis, all the compounds showed an increase in the number of cells in the sub-G 1 phase, while a decrease in the number of cells in the G 1 -G 0 phase as compared to the control, thus indicating induction of apoptosis-mediated cell death.The percentage of cells in the S-phase had increased from 2.57 ± 0.52% to 4.45 ± 0.33%, 6.55 ± 0.57%, 5.60 ± 0.34% and 5.54 ± 0.40% with quercetin, kaempferol, isorhamnetin and β-glucogallin respectively, with a decrease in cellular percentage in G 2 -M phase (Figures 2 and S22).It can hence be inferred that the isolated constituents contribute to the cell cycle arrest at the S-phase checkpoint.Thus, the phytoconstituents may have induced activation of caspase-7, caspase-6, caspase-9, or caused up-regulation of pro-apoptotic genes like Bax, p53 etc. or down-regulation of expression of anti-apoptotic genes like Bcl-2, cyclinD1, which could facilitate apoptosis and/or trigger cell cycle arrest (Goda et al. 2021).
In silico molecular docking studies were performed to investigate the probable mechanism of action and intermolecular interactions of the isolated compounds with the crystal structure of human caspase-3 enzyme (PDB ID 1QX3, 1.90 Å, X-ray Diffraction), using the surflex-dock programme of sybyl-X 2.0 software.Surflex-Dock studies revealed that all four isolated compounds showed the same type of interaction with amino acid residues (PHE250, SER209, ARG207, TYR204) of the active site of the human caspase-3 enzyme, as that of the standard drug doxorubicin (Figure S23).The detailed intermolecular interactions of all isolated compounds and standard drug doxorubicin has been elaborately explained in the supplementary data, while the predicted binding energies of the compounds are listed in Table S9.All the compounds exhibited a significant docking score against the enzyme, with a consensus score in the range 4.38 − 2.66, indicating the summary of all forces of interaction between compounds and the enzyme.Quercetin demonstrated a consensus score of 3.92, while kaempferol, isorhamnetin and ß-glucogallin exhibited a consensus score of 2.66, 4.04 and 4.38 respectively as against doxorubicin, that displayed a consensus score of 6.64 (Table S9) indicating their apoptotic mode of action via the caspase pathway (Eom et al. 2005).
Since apoptosis causes cellular death by non-inflammatory processes, the enriched bioactive components of Piliostigma foveolatum (Dalzell) Thoth.leaves may be regarded as safe and effective therapeutics for lung cancer therapy (Tilekar et al. 2020).

Experimental
The materials and methods adopted for the study have been described in the Supplementary material.

Conclusion
The extract, biofractions and phytoconstituents quercetin, kaempferol, isorhamnetin and β-glucogallin isolated from MFBF have shown to possess significant anti-proliferative effects against Hop-62 cells via apoptotic and cell cycle arrest pathways, accrediting these as potential therapeutic candidates in management of lung cancer.