Isolation, purification and characterization of naturally derived Crocetin beta-d-glucosyl ester from Crocus sativus L. against breast cancer and its binding chemistry with ER-alpha/HDAC2

Abstract

Saffron plant (Crocus sativus L.) is being used as a source of saffron spice and medicine to cure or prevent different types of diseases including cancers. We report the isolation, characterization of bioactive small molecule ([crocetin (β-d-glucosyl) ester] from the leaf biowastes of saffron plant of Kashmir, India. MTTC assay and Bio-autography aided approach were used to assess anti-oxidant activity and anti-cancer properties of crocin (s) against DPPH free radical and breast cancer cell line respectively. Crocetin beta-d-glucosyl ester restrained proliferation of human breast adeno-carcinoma cell model (MCF-7) without significantly affecting normal cell line (L-6). Further studies involving molecular mechanics generalized born surface area and molecular docking showed that crocetin beta-d-glucosyl ester exhibits strong affinity for estrogen receptor alpha and histone deacetylase 2 (crucial receptors involved in breast cancer signalling) as evidenced by the negative docking score and binding free energy (BFE) values. Therefore, crocetin beta-d-glucosyl ester from Crocus sativus biowastes showed antiproliferative effect possibly by inhibiting estrogen receptor alpha and HDAC2 mediated signalling cascade.

Keywords: Antioxidant; Breast cancer; Crocetin beta-d-glucosyl ester; DMEM, Dulbecco’s Modified Eagle’s Medium; DPPH, 2,2-diphenyl-1-picrylhydrazyl; FBS, Fetal Bovine serum; FTIR, Fourier-transform infrared spectroscopy; Floral biowastes; LC-MS/MS, Liquid chromatography–mass spectrometry; MTT, 3-(4,5–dimethyl thiazol–2–yl)–5–diphenyltetrazolium bromide; Molecular docking; NMR, Nuclear magneticresonance; Saffron; TLC, Thin layer chromatography; UV, Ultra violet.

Fig. 1

(A) TLC separation of petroleum ether leaf extract of Crocus sativus using mobile phase Toluene: acetone: water: acetic acid (16:2:2:2), (B) Antioxidant TLC Bio autography assay of crocetin beta-d-glucosyl ester using DPPH as free radical.

Fig. 2

A Spectrums of crocetin beta-d-glucosyl ester based on hyphenated spectroscopic techniques (A) UV–Vis (B) FT-IR (C) ¹H NMR (D) ¹³C NMR (E) Mass spectrum (ESI⁺). The interpretation of the obtained data along with its comparison with literature data has characterized the compound as [crocetin (β-d-glucosyl) ester] (Molecular formula = C₂₆H₃₄O₉).

Fig. 3

Structure of crocetin (β-d-glucosyl) ester retrieved from Pubchem (CID-10368299).

Fig. 4

Cytotoxicity effect of [crocetin (β-d-glucosyl) ester] on cells from MCF-7 cell line. The cells were seeded in 96-well plate (10,000 cells/well) for 24 h, then exposed to 0.1% DMSO based extract of [crocetin (β-d-glucosyl) ester] 1 for 72 h at different concentrations (31.25, 62.5, 125, 250, 500, 1000 µg/ml). The statistically significant concentration dependent cytotoxicity (P value = 0.0019) was observed with IC₅₀ as 628.36 µg/ml. Data are representative of three independent experiments (mean ± SD). Representative slides of MTT assay for MCF-7 cell line upon treatment with [crocetin (β-d-glucosyl) ester]. The prominent morphological changes are seen in treated cells at concentration gradient of 250, 500, 1000 µg/ml, representing apoptosis. L-6 cell line has not demonstrated any significant levels of cytotoxicity upon treatment by [crocetin (β-d-glucosyl) ester] at 500, 1000 µg/ml concentrations with IC₅₀ > 1000 µg/ml. Untreated MCF-7 and L-6 cells act as negative control without any morphological variations. Data are representative of three independent experiments (mean ± SD).

Fig. 5

Relationship between dose of [crocetin (β-d-glucosyl) ester] and % cytotoxicity against MCF and L-6 cell lines at different concentrations (31.25, 62.5, 125, 250, 500, 1000 µg/ml). The statistically significant concentration dependent cytotoxicity (P value = 0.0019) was observed with IC₅₀ as 628.36 µg/ml. Non-significant levels of cytotoxicity was observed in L-6 cell line.

Fig. 6

Pose validation by self-docking. The native ligand of crystal structure was redocked to its host receptor using extra precision flexible docking protocol. The RMSD between native (pink) and redocked (yellow) pose of ligand was found to be 1.9341 Å clearly suggesting that docking algorithm is working correctly.

Fig. 7

Residues of estrogen alpha receptor targeted by 4-Hydroxytamoxifen. This inhibitor targets Arg 394, Glu 353 and Asp 351 by forming hydrogen bonding interactions with them.

Fig. 8

Residues targeted by Crocetin beta-d-glucosyl ester in estrogen receptor alpha. [crocetin (β-d-glucosyl) ester]-1 forms hydrogen bonding interaction with residues Glu 380, Leu 536 and Arg 394. Moreover, the defined inhibitor forms a salt bridge with Arg 394.

Fig. 9

Crocetin beta-d-glucosyl ester targets HDAC2 residue Pro 211 by forming hydrogen bonding interaction with it and Arg 39 by forming salt bridge.