HPTLC QUANTIFICATION OF 4, 4’-METHYLENE BIS (2,6-DI-TERT-BUTYL PHENOL) IN FLAX MICROGREEN EXTRACTS AND ITS ANTICANCER POTENTIAL AGAINST PROSTATE CANCER

MUDASSIR LAWAL; NEETA RAJ SHARMA; AWADHESH KUMAR VERMA; IBRAHIM HAMZA KANKIA; VETRISELVAN SUBRAMANIYAN; GURMEEN RAKHRA

doi:10.22159/ijap.2025v17i4.53939

Authors

MUDASSIR LAWAL Department of Biochemistry, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India. Department of Biochemistry and Molecular Biology, Federal University Dutsin-Ma, Katsina-Nigeria https://orcid.org/0000-0002-5899-084X
NEETA RAJ SHARMA Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India
AWADHESH KUMAR VERMA Department of Bioinformatics, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India https://orcid.org/0000-0002-1497-6210
IBRAHIM HAMZA KANKIA Department of Biochemistry, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University, Katsina-Nigeria https://orcid.org/0000-0001-7474-2347
VETRISELVAN SUBRAMANIYAN Department of Medical Sciences, School of Medical and Life Sciences Sunway University, No. 5, Jalan Universiti, Bandar Sunway, 47500 Selangor Darul Ehsan Malaysia
GURMEEN RAKHRA Department of Biochemistry, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, Punjab, India

DOI:

https://doi.org/10.22159/ijap.2025v17i4.53939

Keywords:

ADMET, Antioxidants, Anti-prostate cancer, Flax microgreens, HPTLC, Molecular docking

Abstract

Objective: This research aimed to evaluate the antioxidant activity of methanolic extract of flax microgreens (MEFM), to identify and quantify 4,4’-Methylenebis (2,6-Di-tert-butylphenol) [4,4’-M(2,6-DTBP)] using GC-MS and HPTLC, and to assess its inhibitory activity against prostate cancer.

Methods: In vitro antioxidant activity was determined by 2,2-Diphenyl-2-picryl-hydrazyl (DPPH) scavenging activity. 4,4’-M(2,6-DTBP) was identified and quantified by Gas Chromatography-Mass Spectrometry (GC-MS) and High Performance Thin Layer Chromatography (HPTLC) analysis. The docking simulation had been carried out in PyRx 0.8 software. Toxicity studies were performed using ADMETlab 3.0 and ProTox 3.0 prediction tools, respectively. The cytotoxic effects and induction of apoptotic cell death by MEFM and 4,4’-M(2,6-DTBP) on PC-3 cell lines were assessed by MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Annexin V apoptosis assays, respectively.

Results: The HPTLC fingerprint confirmed the presence of 4,4’-M(2,6-DTBP) in the MEFM and indicated its existence in high content. 4,4’-M(2,6-DTBP) exhibited the highest binding energies (−17.1 kcal/mol) and favorable interactions against prostate cancer target proteins. The Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) prediction studies revealed that this 4,4’-M(2,6-DTBP) compound had low toxicity and distinct metabolic properties. The MEFM showed strong growth inhibition against PC-3 (IC₅₀: 377.5 μg/ml), whereas 4,4’-M(2,6-DTBP) exhibited weak growth inhibition (IC₅₀: 2324.78 μg/ml). The Annexin V assay revealed that the MEFM and 4,4’-M(2,6-DTBP) significantly increased total apoptosis to 41.03% and 22.86%, respectively. In early apoptotic cells, the MEFM and 4,4’-M(2,6-DTBP) caused 40.9% and 19.5% cell death, while in late apoptotic cells, cell death was found to be 0.13% and 3.36%, respectively.

Conclusion: The extract and its bioactive compound demonstrate anticancer potential, but in vivo studies are required to further evaluate efficacy, metabolism, and toxicity in a living system.

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