Strategy for the Enzymatic Acylation of the Apple Flavonoid Phloretin Based on Prior α-Glucosylation

Abstract

The acylation of flavonoids serves as a means to alter their physicochemical properties, enhance their stability, and improve their bioactivity. Compared with natural flavonoid glycosides, the acylation of nonglycosylated flavonoids presents greater challenges since they contain fewer reactive sites. In this work, we propose an efficient strategy to solve this problem based on a first α-glucosylation step catalyzed by a sucrose phosphorylase, followed by acylation using a lipase. The method was applied to phloretin, a bioactive dihydrochalcone mainly present in apples. Phloretin underwent initial glucosylation at the 4'-OH position, followed by subsequent (and quantitative) acylation with C8, C12, and C16 acyl chains employing an immobilized lipase from Thermomyces lanuginosus. Electrospray ionization-mass spectrometry (ESI-MS) and two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) confirmed that the acylation took place at 6-OH of glucose. The water solubility of C8 acyl glucoside closely resembled that of aglycone, but for C12 and C16 derivatives, it was approximately 3 times lower. Compared with phloretin, the radical scavenging capacity of the new derivatives slightly decreased with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and was similar to 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+). Interestingly, C12 acyl-α-glucoside displayed an enhanced (3-fold) transdermal absorption (using pig skin biopsies) compared to phloretin and its α-glucoside.

Keywords: acylation; antioxidants; dihydrochalcones; flavonoids; hydrophile–lipophile balance (HLB).

Figure 1

Reaction pathway proposed to obtain acylated derivatives of polyphenols. (A) The polyphenol is α-glucosylated using sucrose as the glucosyl donor catalyzed by the sucrose phosphorylase mutant R134A from T. thermosaccharolyticum. (B) The α-glucoside is acylated with vinyl esters (C8–C16) catalyzed by the lipase from T. lanuginosus, with high regioselectivity at 6-OH of the glucose, yielding the corresponding acyl α-glucosides.

Figure 2

HPLC chromatograms (at 0, 1, and 6 h) showing the acylation of phloretin α-glucoside. Reaction conditions: phloretin 4′-O-α-glucopyranoside (0.016 mmol), vinyl ester (0.32 mmol), Lipozyme TL IM (7 mg), tert-butanol (1 mL), 60 °C. Acyl donors: (A) vinyl octanoate, (B) vinyl laurate, and (C) vinyl palmitate.

Figure 3

Progress of phloretin monoglucoside acylations. Reaction conditions: phloretin 4′-O-α-glucopyranoside (7 mg, 0.016 mmol), vinyl ester (0.32 mmol), Lipozyme TL IM (7 mg), tert-butanol (1 mL), 60 °C. Acyl donors: (A) vinyl octanoate, (B) vinyl laurate, and (C) vinyl palmitate.

Figure 4

Molecular structure of the synthesized compounds: (1a) phloretin 4′-O-(6-O-octanoyl)-α-d-glucopyranoside; (1b) phloretin 4′-O-(6-O-lauroyl)-α-d-glucopyranoside; and (1c) phloretin 4′-O-(6-O-palmitoyl)-α-d-glucopyranoside.

Figure 5

Antioxidant activity on ABTS^•+ (A) and DPPH (B) of phloretin, its 4′-O-α-glucoside, and the corresponding acylated α-glucosides. Data are expressed as TEAC value ± SD (n = 3, ^#p < 0.01 vs phloretin; ^●p < 0.02 vs Glc-phloretin).

Figure 6

In vitro percutaneous absorption of acylated derivatives of phloretin and their precursors (phloretin and its 4′-O-α-glucoside) within the different skin layers. W is the surface excess, SC is the stratum corneum, E is the epidermis, D is the dermis, FR is the fluid receptor, and Perc. Abs. is the percutaneous absorption. The results are expressed as μg/cm² of the active pharmaceutical ingredient (API). Mean values ± standard deviations, n = 3; *p < 0.05 vs phloretin; ^#p < 0.05 vs α-Glc-phloretin.