PHENOLIC ACID PROFILE ALTERATIONS IN AQUEOUS AND ETHANOLIC STEVIA EXTRACTS ACROSS SEQUENTIAL PURIFICATION STAGES

Abstract

Stevia extracts are valued for their health benefits, primarily due to the phenolic acids they contain, which contribute to the antioxidant properties of stevia. This study investigated the alterations in the phenolic acid composition of aqueous and ethanolic stevia extracts across several purification stages. Accordingly, dried stevia leaves were mixed with distilled water or ethanol, centrifuged, filtered, clarified at 50°C with aids, and then passed through stepwise membrane filtration using 30 kDa ultrafiltration and 5 kDa nanofiltration membranes. The phenolic acid composition was analyzed by extracting samples with a 75% methanol and formic acid solution, followed by centrifugation. Detection was performed using an LCMS/ MS device equipped with a C18 column. Chlorogenic acid was the most prevalent in both aqueous and ethanolic stevia extracts. However, the ethanolic extract exhibited a higher concentration (approx. 600 mg/kg) compared to the aqueous extract (approx. 100 mg/kg). After centrifugation and clarification, the phenolic acid concentrations in both extracts significantly decreased. The 30 kDa retentate phase was crucial for the enrichment of chlorogenic and caffeic acids. The aqueous extract reached approximately 120 mg/kg chlorogenic acid, while the ethanolic extract reached about 400 mg/kg. Nanofiltration phases (5 kDa permeate and retentate) significantly reduced the levels of these acids. Other phenolic acids, such as luteolin, apigenin, and rutin, remained at low levels throughout all phases. The solvent used had a significant impact on the phenolic profile, with ethanol being more effective in retaining chlorogenic acid. While the 30 kDa retentate phase concentrated phenolic acids, the type of solvent used played a key role in shaping the overall profile.