Solvent extraction optimisation and validation of the HPLC-DAD method for quantification of 5-hydroxyindolin-2-one in Protaetia brevitarsis seulensis larvae extracts

Abstract

Recent studies on the composition of edible insects have placed more emphasis on the quantity and quality of macro- and micronutrients and less emphasis on their bioactive components. This study aimed to assess the effect of 0-100% ethanol solvent ratio considering 5-hydroxyindolin-2-one (5-HI) content on the extraction efficiency in Protaetia brevitarsis seulensis larvae (PBSL) extract and to investigate the effect of PBSL on the induction of cell adhesion molecules (CAMs) in tumour necrosis factor (TNF)-α-activated human umbilical vein endothelial cells (HUVECs). A reversed-phase high-performance liquid chromatography method coupled to a diode array detector (HPLC-DAD) was validated for the quantification of 5-HI. The HPLC-DAD method was validated for its specificity, linearity, precision, accuracy, limit of detection, and limits of quantitation. The calibration curve of 5-HI showed significant linearity (R2>1). The limits of detection and quantitation were 0.016 μg/ml and 0.048 μg/ml, respectively. The relative standard deviation of the precision and intraday tests was less than 0.5%. In the recovery test, the accuracy ranged from 94.48-105.5% and the relative standard deviation value was less than 0.13%. The method validation parameters indicate the applicability of the HPLC method for quality control of food or pharmaceutical formulations containing PBSL. Comparative analysis of the 5-HI content by extraction solvent revealed that it was the highest at 357.7 μg/ml in 70% ethanol. Additionally, the analysis result of 5-HI content according to the growth conditions was the highest at 439 μg/ml at 31 °C for 30 days. Furthermore, in the condition where PBSL was at 31 °C for 30 days inhibition of the TNF-α-induced upregulation of protein expression of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and endothelial-leukocyte adhesion molecule-1 (E-selectin) was observed. Therefore, PBSL may interfere with early atherosclerotic events involving endothelial CAM induction.