Physicochemical analysis of Malaysian Sacha Inchi (Plukenetia volubilis L.) seed oil and its lipid-modulating effects with proteomic profiling

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a highly prevalent metabolic liver disorder characterized by excessive hepatic lipid accumulation in the context of cardiometabolic dysfunction, for which conventional treatments remain limited by modest efficacy, side effects, and poor adherence, thereby driving interest in plant-derived nutraceuticals as safer, multi-targeted therapeutic alternatives. Sacha Inchi (Plukenetia volubilis L.), native to the Amazon and increasingly cultivated in Malaysia, has gained attention due to its reported antioxidant, anti-inflammatory, hypoglycaemic, and anti-hyperlipidaemic effects. These effects are largely attributed to its ω3 rich fatty acid composition and antioxidant constituents. Previous in vitro and in vivo studies have reported improvements in lipid-related outcomes following Sacha Inchi oil (SIO) supplementation, supporting its relevance to metabolic disorders such as MASLD. Despite increasing cultivation and use, the mechanistic basis for SIO mediated hepatic lipid modulation remains insufficiently verified, limiting its scientific validation as a nutraceutical for MASLD. In particular, how SIO influences lipid-metabolizing pathways at the proteome level in hepatic models like HepG2 cells remains underexplored. This study evaluated the physicochemical properties and lipidmodulating effects of Malaysian SIO in oleic acid (OA)-induced HepG2 cells using physicochemical analysis, lipid accumulation assays, and LC-MS/MS based proteomics for protein identification processed in Proteome Discoverer, followed by pathway mapping and enrichment using DAVID (KEGG) and MetaboAnalyst. Three SIO types, including Peruvian commercial, Malaysian commercial, and lab-extracted oil, were investigated by Fatty Acid Methyl Ester analysis. Lab SIO had the highest ω3 (52.89%) and ω6 (36.23%), with an ω3/ω6 ratio of 1.46, compositionally similar to the Peruvian oil. In contrast, Malaysian commercial oil h ad l ow ω 3 a nd a n imbalanced ratio, reflecting compositional differences. Based on these findings, the Lab oil was selected for further bioactivity analysis due to its favorable fatty acid profile. Moreover, the physicochemical characterization of the Lab SIO confirmed its high quality, with a refractive index of 1.4739, an acid value of 1.10 mg KOH/g, and a peroxide value of 2.82 mEq/kg, indicating minimal oxidation and suitability for biological assays. In the lipid accumulation assays, Lab SIO reduced OA-induced lipid accumulation in a concentration-dependent manner based on Oil Red O staining. Proteomics and pathway analyses revealed that Lab SIO significantly modulated the expression of key differentially expressed proteins, including VDAC3, ALDH9A1, SOD2, GSTK1, and ACAT1, which were linked to four highly enriched lipid-related pathways. Fatty acid degradation exhibited the highest fold enrichment (48-fold, p<0.001), followed by peroxisome (24.85-fold), ferroptosis (24.56-fold), and fat digestion and absorption (23.99-fold), all showing statistical significance (p<0.05). These findings suggested that SIO modulated lipid metabolism through several key lipid-related pathways, highlighting its potential as a therapeutic agent for regulating metabolism and promoting liver health. Further in vivo and clinical studies are needed to validate its therapeutic potential. Keywords: Sacha Inchi; Plukenetia volubilis; HepG2 cells; proteomics; lipid metabolism; MASLD