In Vitro Inhibition of Enzymes and Antioxidant and Chemical Fingerprinting Characteristics of Azara serrata Ruiz & Pav. Fruits, an Endemic Plant of the Valdivian Forest of Chile

Abstract

The World Health Organization has emphasized the importance of consuming small fruits for the prevention of chronic health problems, including diabetes, cardiovascular diseases, cancer, and obesity, which are named chronic non-communicable diseases (NCDs). Azara serrata Ruiz & Pav., commonly called "aroma de Castilla", is a shrub endemic to Chile from the Salicaceae family that produces an underutilized blue-grey berry that grows wild in southern Chile. The species is widely used as a medicinal plant by the Andean communities of southern Chile. In this work, a high-resolution mass spectrometric analysis of the methanolic extract revealed several phenolic compounds for the first time in the edible berry of this endemic species. Furthermore, several glycosylated anthocyanins were detected and quantified using UHPLC coupled with UV/Vis detection and trapped ion mobility mass spectrometry (UHPLC-DAD-TIMS-TOF) for the anthocyanin-rich extract, which was prepared using an optimized anthocyanin extraction protocol. The extract proved to be active in the inhibition of several enzymes linked to NCDs, such as acetylcholinesterase, tyrosinase, amylase, lipase, and glucosidase (IC₅₀ = 3.92 ± 0.23, 12.24 ± 0.03, 11.12 ± 0.10, 32.43 ± 0.0, and 371.6 ± 0.0 μg/mL, respectively). Furthermore, the extract concentrated in anthocyanins showed good antioxidant activity evidenced by the bleaching of the radicals DPPH and ABTS, ferric-reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC). The results show that these neglected endemic small berries can be a source of healthy phytochemicals. These Chilean berries can be used as functional food and their extracts are candidates for use as functional ingredients in naturally healthy products.

Keywords: TIMS-TOF; analytical membrane chromatography; anthocyanins; antioxidants; collision cross-section; corcolen; endemic berries; enzyme inhibition; pyrano anthocyanins; salicaceae.

Figure 1

Azara serrata Ruiz & Pav. fruits and its leaves, collected in February 2022 from the Botanical Garden, UACh, Valdivia.

Figure 2

UHPLC-DAD-TIMS-TOF-MS analysis of the methanolic extract of the berries from Azara serrata Ruiz & Pav., with base peak all MS ne—gative (I), DAD signal at 280 nm (II), and DAD signal at 520 nm (III).

Figure 3

Chemical structures of some salicinoids in the berries of Azara serrata Ruiz & Pav.

Figure 4

Proposed mechanism of the formation of the pyranoanthocyanin species in the berries of Azara serrata Ruiz & Pav.

Figure 5

Chromatogram of the UHPLC-DAD analysis of the anthocyanin-rich extract of the berries of Azara serrata Ruiz & Pav. at 520 nm.

Figure 6

Structures of a selection of salicinoids in the berries of A. serrata used for the docking calculations.

Figure 7

Predicted intermolecular interactions of selected compounds in A. serrata methanolic extract and the residues of the Torpedo Californica acetylcholinesterase (TcAChE) catalytic site. Yellow dotted lines indicate hydrogen bond interactions, cyan dotted lines represent π-π interactions, and magenta dotted lines represent T-shaped interactions. (A). Idescarpin in the catalytic site. (B). Coumaroyl idescarpin in the catalytic site. (C). Coumaroyl idesin in the catalytic site.

Figure 8

Predicted intermolecular interactions of selected compounds in A. serrata methanolic extract and the residues of the human butyrylcholinesterase (hBuChE) catalytic site. Yellow dotted lines indicate hydrogen bond interactions and cyan dotted lines represent π-π interactions. (A). Idescarpin in the catalytic site. (B). Coumaroyl idescarpin in the catalytic site. (C). Coumaroyl idesin in the catalytic site.

Figure 9

Predicted intermolecular interactions of selected compounds in the A. serrata methanolic extract and the residues of the human Agaricus bisporus mushroom tyrosinase catalytic site. Yellow dotted lines indicate hydrogen bond interactions and cyan dotted lines represent π-π interactions. (A). Idescarpin in the catalytic site. (B). Coumaroyl idescarpin in the catalytic site. (C). Coumaroyl idesin in the catalytic site.

Figure 10

Predicted intermolecular interactions of selected compounds in the A. serrata methanolic extract and the residues of the human bile salt activated lipase catalytic site. Yellow dotted lines indicate hydrogen bond interactions, cyan dotted lines represent π-π interactions, and red lines represent the π-cation. (A). Idescarpin in the catalytic site. (B). Coumaroyl idescarpin in the catalytic site. (C). Coumaroyl idesin in the catalytic site.

Figure 11

Predicted intermolecular interactions of selected compounds in the A. serrata methanolic extract and the residues of the human glucosidase catalytic site. Yellow dotted lines indicate hydrogen bond interactions, cyan dotted lines represent π-π interactions, and magenta dotted lines represent T-shaped interactions. (A). Idescarpin in the catalytic site. (B). Coumaroyl idescarpin in the catalytic site. (C). Coumaroyl idesin in the catalytic site.

Figure 12

Predicted intermolecular interactions of selected compounds in the A. serrata methanolic extract and the residues of the human pancreatic alpha-amylase catalytic site. Yellow dotted lines indicate hydrogen bond interactions and magenta dotted lines represent T-shaped interactions. (A). Idescarpin in the catalytic site. (B). Coumaroyl idescarpin in the catalytic site. (C). Coumaroyl idesin in the catalytic site.