Optimization of extraction using response surface methodology and chromatographic characterization of phenolics and flavonoids from plants

Abstract

Empetrum nigrum is a dwarf evergreen shrub rich in phenolics. Among these compounds, batatasin-III is a topic on which comprehensive research is increasing due to its significant impact on the ecosystem. It can affect vegetation growth through a characteristic allelopathic properties. Although reindeer are known to live in the habitat of Empetrum nigrum, they generally avoid consuming this plant. However, DNA from Empetrum nigrum found in faecal samples has raised questions about the reindeer's diet and grazing practices. Furthermore, little is known about how phenolic and flavonoid compounds are presented and metabolized after digestion. Therefore, optimizing extraction conditions for accelerated solvent extraction (ASE) to recover phenolic and flavonoid compounds from Empetrum nigrum and reindeer faeces is studied.

This thesis aims to systematically optimize the extraction conditions for ASE. Additionally, gas chromatography-mass spectrometry (GC-MS) analysis was applied to discover the metabolic profile of different parts of Empetrum nigrum, including young and old shoots and litter, and reindeer faeces and to assess the presence and content of batatasin-III. The variability and interaction between the factors under study are expressed through response surface methodology (RSM). Extraction results were evaluated based on total phenolic content (TPC) and total flavonoid content (TFC). The matrix patterns were verified by extraction under optimized conditions. TPC and TFC were determined by UV-Vis spectroscopy, while results related to metabolite composition and batatasin-III content were obtained using GC-MS.

The optimization models gave good predictive results and were statistically verified. Young shoots contained the highest concentrations of TPC and TFC, followed by older shoots, litter, and reindeer faeces. GC-MS analysis also captured various compound groups present in Empetrum nigrum. Batatasin-III was also detected at approximately 10.7 mg/g DW in young and older shoots. These concentrations were significantly lower in litter and reindeer faeces, at 0.76 mg/g DW and 0.4 mg/g DW, respectively.

These findings demonstrate the potential of applying ASE methods to the recovery of organic compounds. Furthermore, batatasin-III persists after digestion by herbivores, which is evidence that reindeer may have accidentally consumed Empetrum leaves. However, more research is required. To our knowledge, this is the first study on batatasin-III in reindeer faeces, offering new insights into the metabolism of post-digestion bioactive compounds and the herbivorous-plant relationship in the northern region.