Optimization of ultrasonic-assisted extraction (UAE) for phenolics and antioxidant activity from cocoa (Theobroma cacao) leaves and phytochemical profiling using GC-MS and LC-HRMS

Abstract

Cocoa (Theobroma cacao) leaves, often discarded as agricultural by-products, are a valuable source of phenolic compounds with strong antioxidant properties. This study aims to optimize the ultrasound-assisted extraction (UAE) process to maximize the total phenolic content (TPC) and antioxidant activity (AA) of cocoa leaves using response surface methodology (RSM) with a central composite design (CCD). Three variables—ethanol concentration (70–90%), ultrasonic irradiation time (30–60 min), and solvent-to-solid ratio (5–15 mL/g)—were evaluated for their effects on TPC and AA. The RSM quadratic model determined the optimum extraction conditions as 89.34% ethanol, 45.87 min ultrasonic irradiation, and 10.03 mL/g solvent-to-solid ratio, with predicting TPC and AA values of 129.67 mg GAE/g extract and 44.52 %, respectively. Experimental validation yielded TPC of 118.84 ± 0.83 mg GAE/g and antioxidant activity of 42.01 ± 1.14 %, aligning with predictions within a 5% error margin. Furthermore, GC-MS and LC-HRMS analyses identified key phenolics, including (+)-procyanidin B2, (−)-epicatechin, and D-(+)-catechin, alongside other compounds such as squalene. The findings demonstrate that UAE was a highly efficient method for extracting bioactive compounds from cocoa leaves, facilitating the repurposing of agricultural by-products and potentially enhancing the functional value of cocoa leaf-derived products.