Unravelling the Chemical Landscape of Neolitsea pallens Essential Oil: A Comparative Study of Geographical Chemotypes Using t-SNE and Hierarchical Clustering

Neolitsea pallens essential oil (NPEO) is known for its diverse chemical composition, which can vary significantly depending on geographical origin. In this study, we aimed to explore the chemical variability of NPEOs across different geographical regions using advanced multivariate analysis techniques. The methodology involved the collection of Neolitsea pallens leaves from 13 different locations of Himachal Pradesh, followed by hydrodistillation using a Clevenger-type apparatus. Comprehensive qualitative and quantitative chemical profiling was conducted using gas chromatography–mass spectrometry (GC–MS) and gas chromatography with flame ionisation detection (GC-FID) analysis. The GC–MS analysis identified 66 distinct chemical compounds, dominated by sesquiterpenoids, ranging from 10.33% to 59.21%, respectively. Caryophyllene oxide emerged as the major compound, with concentrations ranging from 3.32% ± 0.18% to 33.8% ± 2.43%, with the highest concentration observed in the Shankar dehra location. To elucidate the underlying chemical patterns, differentiate between geographical chemotypes and explore altitudinal correlations between locations, we employed both t-distributed stochastic neighbour embedding (t-SNE), hierarchical clustering analysis (HCA) and Pearson's correlation matrix. The chemotypes were identified as caryophyllene oxide, β-caryophyllene, β-sabinene and α-muurolene-14-ol. This study highlights for the very first time the significant influence of geographic location on the chemical composition of NPEOs. These variations highlight the need for considering geographical factors when utilising NPEOs for industrial or therapeutic applications. The integration of t-SNE and hierarchical clustering offers a robust framework for chemotype differentiation, providing deeper insights into the chemical diversity of this species.