Singlet Oxygen Produced by Aspalathin and Ascorbic Acid Leads to Fragmentation of Dihydrochalcones and Adduct Formation

Abstract

Singlet oxygen-mediated fragmentation of various dihydrochalcones and chalcones was reported. (Dihydro)cinnamic acids formed in the fragmentation showed a B-ring substitution pattern of the precursor (dihydro)chalcone. For the first time, the intrinsic generation of singlet oxygen by aspalathin and ascorbic acid under mild aqueous conditions (37 °C, pH 7.0) and exclusion of light was verified using HPLC-(+)-APCI-MS² experiments. If a 4 molar excess of aspalathin or ascorbic acid was used, fragmentation of dihydrochalcones with monohydroxy and o-hydroxymethoxy B-ring substitution was induced up to 2 mol %, respectively. Incubations of the dihydrochalcone phloretin with ascorbic acid not only led to p-dihydrocoumaric acid but also to a novel ascorbyl adduct, which was isolated and identified as 2,4,6-trihydroxy-5-[3-(4-hydroxyphenyl)propanoyl]-2-[(1R, 2S)-1,2,3-trihydroxypropyl]-1-benzofuran-3(2H)-one. The impact of different structural elements on adduct formation was evaluated and verified to be a phloroglucinol structure linked to an acyl moiety. Formation of the ascorbyl adduct was shown to occur in apple puree when both ascorbic acid and phloretin were present at the same time.

Keywords: apple; ascorbic acid; aspalathin; chalcone; dihydrochalcone; oxidative fragmentation; phloretin; singlet oxygen.

Figure 1

Formation of dihydrocinnamic acids from dihydrochalcones triggered by singlet oxygen via oxidative rearrangement-fragmentation.

Figure 2

Incubation of 0.5 mM phloridzin with 2 mM DMN-EP at 37 °C and pH 7 under aeration. Verification of p-dihydrocoumaric acid as the trimethylsilyl ether by GC-MS, (A) authentic reference standard; (B) incubation workup.

Figure 3

Stability of DMN-EP (●) and DPA-EP (○) at 37 °C and pH 7 under aeration.

Figure 4

Formation of specific singlet oxygen endoperoxids in aspalathin and ascorbic acid incubations (37 °C, pH 7, aeration). Verification of DPA-EP by collision induced dissociation (CID) of m/z 363 (M + H)⁺ via HPLC(+)-APCI-MS², (A) authentic reference standard; (B) aspalathin incubation; (C) ascorbic acid incubation.

Figure 5

Incubation of 0.5 mM phloretin in the presence of 2.0 mM ascorbic acid (37 °C, pH 7, aeration): phloretin (○), ascorbic acid (●), p-dihydrocoumaric acid (Δ), and phloretin ascorbyl adduct (▼).

Figure 6

Proposed mechanism for the formation of the novel phloretin ascorbyl adduct.

Figure 7

Incubation of commercial pasteurized apple puree spiked with 0.5 mM phloretin (24 h, 37 °C, aeration). Verification of the phloretin ascorbyl adduct by collision induced dissociation (CID) of m/z 419 (M – H)⁻ via LC(−)-ESI-MS², (A) authentic reference standard; (B) apple puree workup.