An Endemic Plant of the Mediterranean Area: Phytochemical Characterization of Strawberry Tree (Arbutus unedo L.) Fruits Extracts at Different Ripening Stages

Abstract

This work focused on the extraction, quantification, and characterization of bioactive compounds of Arbutus unedo L. fruits, comparing the results obtained from the different ripening states. Extractions were performed by different methods (such as maceration extraction and ultrasonic extraction) and food grade solvents (aqueous and hydroalcoholic solvents) in each of the all ripening states (four states considered, associated with four different colors, i.e., green, yellow, orange, and red). The presence of (poly)phenols was quantified and characterized, and scavenging activity was determined by the Folin-Ciocâlteu reagent and the DPPH method, respectively. The content of bioactive compounds was characterized by LC-MS/MS, such as multiple reaction monitoring (MRM) mass spectrometry. The results showed that ultrasound-assisted extraction (UAE) performed better than maceration extraction; ethanol-water mixture extracts showed a more positive effect than the use of aqueous extracts regarding the content of total phenolic compounds. Overall, the total phenolic compounds in the EtOH:H₂O mixture at a ratio of 7:3 (v:v) were higher than that of the other solvents for both extraction methods. Some bioactive molecules were characterized for the first time in the extracts of A. unedo. The chemical profile of the strawberry tree extracts depended on the degree of fruit ripeness. The results suggest that A. unedo fruits may be of great interest for food and nutraceutical applications.

Keywords: Arbutus unedo L.; MRM mass spectrometry; radical scavenging activities; ripening process; total phenolic compounds.

FIGURE 1

Graphical representation with sample flow chart: V, green fruit; G, yellow fruit; A, orange fruit; R, red fruit; H1, extraction with 100% H₂O; E7, extraction with 70% EtOH in hydroalcoholic solution; E3, extraction with 30% EtOH in hydroalcoholic solution; ME, maceration extraction; UAE, ultrasound-assisted extraction; DPPH, 2,2-diphenyl-1-picrylhydrazyl radical.

FIGURE 2

MRM chromatograms of glutamic acid (A) and malvidin-3-O-glucoside (B) standard molecules. The best transitions 148 m/z→130 m/z, 148 m/z →102 m/z and 148 m/z →84 m/z and 493 m/z →331 m/z and 493 m/z →270 m/z were monitored for glutamic acid and malvidin-3-O-glucoside, respectively. Analytical parameters calculated for the standards (LOD, LOQ), angular coefficient (m), y-intercept (q), and R² are reported.

FIGURE 3

Two-dimensional principal component analysis (PCA) biplot showing associations between experimental samples (A. unedo fruit) and compounds abundance (clr-transformed data). Original data are summarized in Table 6 and in Supplementary Table 1. Compounds extracted in H1 are represented. Samples are shown according to the ripening stages (green, yellow, orange, and red) and compounds are shown according to polyphenol categories.

FIGURE 4

Two-dimensional principal component analysis (PCA) biplot showing associations between experimental samples (A. unedo fruit) and compounds abundance (clr-transformed data). Original data are summarized in Table 6 and in Supplementary Table 1. Compounds extracted in E3 are represented. Samples are shown according to the ripening stages (green, yellow, orange, and red) and compounds are shown according to polyphenol categories.

FIGURE 5

Two-dimensional principal component analysis (PCA) biplot showing associations between experimental samples (A. unedo fruit) and compounds abundance (clr-transformed data). Original data are summarized in Table 6 and in Supplementary Table 1. Compounds extracted in E7 are represented. Samples are shown according to the ripening stages (green, yellow, orange, and red) and compounds are shown according to polyphenol categories.

FIGURE 6

Summary of MRM/MS E7, E3, and H1 analysis of butanol extracts of A. unedo fruit samples in different ripening stages (green, yellow, orange, and red) (A1) free amino acid distribution. (B1) Pie charts pointing the percentage composition of A. unedo polyphenolic fraction extracted with the mixture E7. Four different compound classes were grouped: anthocyanins, phenolic acids, catechins, and other flavonoids. (A2) free amino acid distribution. (B2) Pie charts pointing the percentage composition of A. unedo polyphenolic fraction extracted with the mixture E3. A total of four different compound classes were grouped: anthocyanins, phenolic acids, catechins, and other flavonoids. (A3) Free amino acid distribution. (B3) Pie charts pointing the percentage composition of A. unedo polyphenolic fraction extracted with the mixture H1. A total of four different compound classes were grouped: anthocyanins, phenolic acids, catechins, and other flavonoids.