Comparative Study on Assisted Solvent Extraction Techniques for the Extraction of Biologically Active Compounds from Sideritis raeseri and Sideritis scardica

Abstract

The plants in the Sideritis genus are postulated to exhibit several important medicinal properties due to their unique chemical composition. To isolate the targeted phytochemical compounds, the selection of a suitable extraction method is of primary importance. In this work, a comparative study on the phytochemical profiles of various Sideritis raeseri and Sideritis scardica extracts has been carried out. An untargeted metabolomics approach based on ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry was applied to investigate the metabolic differences between extracts obtained by conventional extraction and extractions assisted by microwaves, ultrasounds and high pressure. Additionally, the influence of extraction solvents on HPLC antioxidant profiles obtained following the derivatization of analytes with ABTS reagent was evaluated. A total of 102 metabolites have been putatively identified. The major secondary metabolites groups were classified as flavonoids, terpenoids, phenylethanoid glycosides and phenolic acids. The main antioxidants in the extracts were isoscutellarein and hypolaetin derivatives as well as verbascoside and chlorogenic acid. The results showed that 70% ethanol was the most effective extractant for different classes of phytochemicals including antioxidants. In addition, extraction supported with microwaves, ultrasounds or high pressure improved the overall recovery of metabolites by about 3 times compared to the conventional extraction method.

Keywords: HPE; MAE; Sideritis raeseri; Sideritis scardica; UPLC-HRMS; USAE; antioxidants; extraction.

Figure 1

Total ion chromatograms obtained by LC-Q-Orbitrap in negative mode (black) combined with chromatograms registered by UV-Vis detector at 270 nm (orange) and antioxidant profiles registered at 734 nm after post-column derivatization with ABTS (grey) (A), assembled with heat maps representing the mean MS peak area value of the identified compounds in four different Sideritis raeseri extracts: SRH₂O—water extract; SR30—30% ethanol extract; SR70—70% ethanol extract; SR100—ethanol extract (B). For identity of peaks, see Table 1.

Figure 2

Total ion chromatograms obtained by LC-Q-Orbitrap in negative mode (black) combined with chromatograms registered by UV-Vis detector at 270 nm (orange) and antioxidant profiles registered at 734 nm after post-column derivatization with ABTS (grey) (A), assembled with heat maps representing the mean MS peak area value of the identified compounds in four different Sideritis scardica extracts: SSH₂O—water extract; SS30—30% ethanol extract; SS70—70% ethanol extract; SS100—ethanol extract (B). For identity of peaks, see Table 1.

Figure 3

Extraction efficiency of major classes of bioactive phytochemicals from two Sideritis species with four different solvents based on sum of peak areas of compounds assigned to the appropriate classes. SS—Sideritis scardica; SR—Sideritis raeseri; H₂O—water extract; 30—30% ethanol extract, 70—70% ethanol extract; 100—ethanol extract. Asterisks representing p-value classification refer to: (*) p ≤ 0.05; (**) p ≤ 0.01; (***) p ≤ 0.001; (****) p ≤ 0.0001.

Figure 4

Volcano plot combining the results of fold change (FC) analysis and t-tests comparing Sideritis raeseri (SR) and Sideritis scardica (SS) extracts prepared with 70% ethanol (A) juxtaposed with a bar graph showing the relative peak area of the 10 characteristic metabolites differentiating the SS and SR varieties (the contents of each higher one converted to 1.0) (B). For identity of peaks, see Table 1.

Figure 5

Venn diagrams showing the number and relations between the significantly better-extracted metabolites, in terms of peaks areas obtained by using alternative solvent extraction (ASE) compared to the extracts obtained with conventional solvent extraction method. SR—Sideritis raeseri; SS—Sideritis scardica; CSE—conventional solvent extraction; USAE—ultrasound-assisted extraction; MAE—microwave-assisted extraction; HPE—high-pressure extraction (S—20 min of extraction; L—18 h of extraction).

Figure 6

Comparison of the extraction methods in terms of bioactive compound recovery. Bar graphs represent total peak areas of compounds isolated with different extraction methods and grouped into classes characteristic for Sideritis species (A). The heatmaps show the relative peak areas of main identified bioactive metabolites obtained by different extraction methods. The results are presented in relation to the conventional solvent extraction areas, which have been assumed to equal 0.0 (B). Red indicates an improvement in bioactive extraction, blue means deterioration. The abbreviations refer to: SR—Sideritis raeseri; SS—Sideritis scardica; CSE—conventional solvent extraction; USAE—ultrasound-assisted extraction; MAE—microwave-assisted extraction; HPE—high-pressure extraction (S—20 min of extraction; L—18 h of extraction). Asterisks representing p-value classification are as follows: (*) p ≤ 0.05; (**) p ≤ 0.01; (***) p ≤ 0.001. For identity of peaks, see Table 1.