Plant material

Fresh tomato fruits (Solanum lycopersicum var. saladette L. Family Solanaceae) were collected from Mepaco-medifood pharmaceutical company’s medicinal farm in El Sharqiya, Egypt. Dr. Labib, T. Taxonomist of the Botanical Orman Garden, Egypt, generously authenticated them.

Lycopene extraction

Fresh tomatoes were cleaned with tap water, then mixed with a home blender and the homogenate of seeds and skins was strained. Sixty-five mL of methanol was added to fifty grams of tomato paste. To avoid hard lumps formation, the mixture was immediately agitated. The thick solution was filtered after 2 h, and the red residue was agitated for an extra 15 min with 70 mL of methanol and carbon tetrachloride mixture of equal parts before being separated by filtration. The carbon tetrachloride layer was moved to a separating funnel, followed by the addition of one volume of water and vigorous shaking, and then the carbon tetrachloride phase was separated and evaporated. Consequently, the residue was diluted with 2 mL of benzene then 1 mL of boiled methanol, and immediately, lycopene crystals were formed. Further purification on active acidic alumina column chromatography using toluene as mobile phase was done. The dark red zone was gathered, evaporated, and stored in a dark vial to avoid oxidation [24].

Lycopene identification by nuclear magnetic resonance (NMR) spectrometer

The identification of lycopene was done by nuclear magnetic resonance (NMR) spectrometer using Bruker Ascend 400/R (Bruker®, AVANCE III HD, 400 MHz, Switzerland)-Ain Shams University- Cairo, Egypt, Faculty of Pharmacy, Drug Discovery, Research and Development center [42,43,44,45].

Assessment of cytotoxic activity of lycopene using MTT assay

American Type Culture Collection (ATCC) provided the cell line MDA-MB-435. DMEM (Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone), 10 ug/mL of insulin (Sigma), and 1% penicillin–streptomycin was used to cultivate the cells. The remaining chemicals, components, and reagents were all purchased from Sigma Chemical Co. At 37 °C and 5% CO2, MDA-MB-435 cells were sub-cultured into a 96-well plate with 1 × 104 cells per well in medium before being exposed with or without several dilutions of test agents, each in triplicate for 24 h. PBS was used at the end of the incubation period to wash the harvested cells. To each well a concentration of 20 μL of MTT was poured and incubated for 2 h before adding the 200 μL of DMSO. At a wavelength of 570 nm, the absorbance was measured using an ELISA reader (Multiskan EX, Lab Systems) [46, 47].

DNA analysis using flow cytometry for cell cycle and apoptosis.

Before the cells were harvested, MDA-MB-435 cells (2 × 105 cells/well) in 12-well plates were exposed to various concentrations of the lycopene-testing compound for various amounts of time. The cells were suspended in PBS containing 40 g/mL protease inhibitor, 0.1 mg/mL RNase A (Sigma, USA), and 0.1% triton x-100 after being gently fixed in 70% ethanol (in PBS) on ice for an overnight period. The cells were examined by flow cytometry (Becton–Dickinson, San Jose, CA, USA) using an argon laser at 488 nm after 30 min at 37 °C in the dark. After that, the cell cycle and apoptosis were identified and analyzed [26, 48, 49].

In silico studies: molecular modeling studies

Utilizing the CDOCKER protocol and the Discovery Studio 4.1 Software, a molecular docking study was conducted. The isolated component underwent docking against the CDK1 active site. CDK1 X-ray crystallographic protein structure was obtained from PDB (ID: 4YC6) [50]. Hydrogens were added, unnecessary chains were cut out, the protein was cleaned, and heavy atoms were created. CHARMm forcefield along with MMFF94 as a partial charge were used for simulation. Fixed constraints and protein minimization were carried out. Complex ligand interaction site was used for the identification of the receptor binding site. Lycopene binding mode was examined to justify its biological effectiveness in addition to gain further perception into binding interactions and orientations, where the best pose out of ten was chosen.

Standard dynamic simulations

The dynamic simulation investigation were carried out with Discovery Studio 4.1 and applied to free protein and Lycopene docked against CDK1. Standard Dynamic Cascades was applied where the first minimization algorithm was set to steepest descent with maximum steps 2000 and RMS gradient 1.0. The second minimization algorithm was set to conjugate gradient with maximum steps 1000. The initial temperature was set to 50, and the target temp. 300 with a maximum velocity of 2000. On the other hand, the equilibration phase was set with a simulation time of 10 Ps and an interval of 2 Ps. The Implicit Solvent Model was set to Generalized Born with Simple Switching (GBSW).

Ramachandran plot

Ramachandran Plot was generated using Discovery Studio 4.1 for both free protein and lycopene to verify predicted torsion angles in protein during interaction with it.

ADMET/TOPKAT prediction

Discovery studio 4.1software was used to conduct the in-silico ADMET studies. Results were then compared to that obtained by the drug-likeness studies applying Swiss-ADME Boiled egg chart (https://www.swissadme.ch/index.php) [51]. These investigations were utilized to speculate on the pharmacokinetic features of the drug under consideration. The results also afforded the structure requirements for estimation of the probable antitumor activity. ADMET protocol was applied to prepare ligands of the extracted lycopene. Graph plots and numerical schedules were created and displayed. Similarly, the TOPKAT Toxicity protocol was applied to the same prepared lycopene after the criteria to be measured were determined, which are Ames Applicability, Prediction, Score, Probability, Skin Irritancy, Rat Oral LD50, and Rat Max. Tolerated Dose Feed Carcinogenic Potency TD50 for both Mice and Rats [52, 53].