Development and validation of a high-performance thin-layer chromatography‒densitometric method and mass spectroscopy profiling for the determination of bioactive phytosterol from Manilkara zapota L. P. Royen leaves and correlating its antioxidant and antiinflammatory potential

Abstract

A simple, reliable high-performance thin-layer chromatography (HPTLC)‒densitometry method was developed and validated for the quantification of β-sitosterol, a significant bioactive phytosterol, in Manilkara zapota L. P. Royen leaves. This method is combined with mass spectroscopy (MS) for relevant structural identification, responsible for their antioxidant and antiinflammatory therapeutic potential. The pet ether (PE), chloroform (CH), ethyl acetate (EA), and hydro-ethanolic (HE) leaf extracts were prepared using the cold maceration technique. These extracts were screened for preliminary qualitative tests, and based on the screening results, secondary metabolites such as flavonoids, phenols, steroids, terpenoids, and tannins were further quantified. Antioxidant potential was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), ferric reducing antioxidant power (FRAP), and nitric oxide (NO) free radical scavenging assays. antiinflammatory activity was evaluated through protein denaturation and human red blood cell (HRBC) membrane stability methods. Additionally, an HPTLC method was developed and validated for β-sitosterol quantification. Among all the extracts, the ethyl acetate leaf extract exhibited the highest steroidal content [50% inhibitory concentration (IC₅₀) 2.92 ± 0.21 mg β-sitosterol equivalent (BSE)/g dry extract (DE)], flavonoid content [IC₅₀ 22.9 ± 0.25 mg quercetin equivalent (QE)/g DE], and phenolic content [IC₅₀ 103.8 ± 0.23 mg gallic acid equivalent (GAE)/g DE]. The ethyl acetate extract also demonstrated the highest efficacy against DPPH (IC₅₀ 16.35 ± 1.49), ABTS (IC₅₀ 17.52 ± 2.36), FRAP [41.32 ± 0.02 mg ascorbic acid (AA)/g crude extract], NO (IC₅₀ 17.13 ± 2.06), protein denaturation (IC₅₀ 31.75 ± 2.1), and HRBC membrane stability (IC₅₀ 24.8 ± 2.44). Correlation analysis further supported these findings. Furthermore, following the International Council for Harmonisation guidelines, an HPTLC method was developed, and 434.4 ng of β-sitosterol was quantified, with the highest content found in the ethyl acetate extract. Therefore, M. zapota leaf extract exhibits beneficial antioxidant and antiinflammatory effects due to its richness in phytosterols. This extract has the potential to be further explored as a therapeutic agent against inflammatory diseases.