Aspalathin and Other Rooibos Flavonoids Trapped α-Dicarbonyls and Inhibited Formation of Advanced Glycation End Products In Vitro

Abstract

The excessive dietary intake of simple sugars and abnormal metabolism in certain diseases contribute to the increased production of α-dicarbonyls (α-DCs), such as methylglyoxal (MGO) and glyoxal (GO), the main precursors of the formation of advanced glycation end products (AGEs). AGEs play a vital role, for example, in the development of cardiovascular diseases and diabetes. Aspalathus linearis (Burman f.) R. Dahlgren (known as rooibos tea) exhibits a wide range of activities beneficial for cardio-metabolic health. Thus, the present study aims to investigate unfermented and fermented rooibos extracts and their constituents for the ability to trap MGO and GO. The individual compounds identified in extracts were tested for the capability to inhibit AGEs (with MGO or GO as a glycation agent). Ultra-high-performance liquid chromatography coupled with an electrospray ionization mass spectrometer (UHPLC-ESI-MS) was used to investigate α-DCs' trapping capacities. To evaluate the antiglycation activity, fluorescence measurement was used. The extract from the unfermented rooibos showed a higher ability to capture MGO/GO and inhibit AGE formation than did the extract from fermented rooibos, and this effect was attributed to a higher content of dihydrochalcones. The compounds detected in the extracts, such as aspalathin, nothofagin, vitexin, isovitexin, and eriodictyol, as well as structurally related phloretin and phloroglucinol (formed by the biotransformation of certain flavonoids), trapped MGO, and some also trapped GO. AGE formation was inhibited the most by isovitexin. However, it was the high content of aspalathin and its higher efficiency than that of metformin that determined the antiglycation and trapping properties of green rooibos. Therefore, A. linearis, in addition to other health benefits, could potentially be used as an α-DC trapping agent and AGE inhibitor.

Keywords: AGEs; Aspalathus linearis; MGO; advanced glycation end products; dihydrochalcones; flavonoids; glyoxal; methylglyoxal; rooibos; trapping of dicarbonyls.

Figure 1

Antiglycation activity after seven days of incubation of bovine serum albumin with glycation agents (0.5 mM) and tested compound (1.5 mM) or hydroethanolic extracts (500 µL, DER 1:100) expressed as % inhibition of: (a) MGO mediated-AGE formation, (b) GO-mediated AGE formation. Results are representative of three experiments performed in triplicate ± SD. Values not sharing a common letter are significantly different at p < 0.05 according to Tukey’s multiple comparisons test. Abbreviations: AG, aminoguanidine; MET, metformin; GRE, green rooibos extract; RRE, red rooibos extract; ASP, aspalathin; PLT, phloretin; PLG, phloroglucinol; VT, vitexin; IVT, isovitexin; ER, eriodictyol.

Figure 2

Mass spectra of phloroglucinol and its methylglyoxal/glyoxal adducts after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), phloroglucinol; (B), mono-MGO-phloroglucinol; (C), di-MGO-phloroglucinol; (D), tri-MGO-phloroglucinol; (E), mono-GO-phloroglucinol; (F), di-GO-phloroglucinol. Other isomers are also possible.

Figure 3

Proposals for chemical structures of adducts formed in the reaction of phloroglucinol with methylglyoxal/glyoxal after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), phloroglucinol; (B1), hemiacetal form of mono-MGO-phloroglucinol; (B2), hemiketal form of mono-MGO-phloroglucinol; (C1), hemiacetal form of di-MGO-phloroglucinol; (C2), hemiketal form of di-MGO-phloroglucinol; (D1), hemiacetal form of tri-MGO-phloroglucinol; (D2), hemiketal form of tri-MGO-phloroglucinol; (E), mono-GO-phloroglucinol; (F), di-GO-phloroglucinol isomer a; (G), di-GO-phloroglucinol isomer b. Other isomers are also possible.

Figure 4

Mass spectra of phloretin and its methylglyoxal/glyoxal adducts after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), phloretin; (B), mono-MGO-phloretin; (C), di-MGO-phloretin; (D), mono-GO-phloretin. Other isomers are also possible.

Figure 5

Representative LC chromatograms of aspalathin and its methylglyoxal (A)/glyoxal (B) adducts after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), mono-MGO-aspalathin; (B) mono-GO-aspalathin; a, mono-MGO-aspalathin a; b, mono-MGO-aspalathin b; c, mono-MGO-aspalathin c; d, mono-MGO-aspalathin d; a’, mono-GO-aspalathin a; b’, mono-GO-aspalathin b; c’, mono-GO-aspalathin c; asp, aspalathin.

Figure 6

Mass spectra of aspalathin and its methylglyoxal/glyoxal adducts after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), aspalathin; (B), mono-MGO-aspalathin; (C), mono-GO-aspalathin.

Figure 7

Mass spectra of nothofagin and its methylglyoxal adduct after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), nothofagin; (B), mono-MGO-nothofagin.

Figure 8

Proposals for chemical structures of adducts formed in the reaction of aspalathin/nothofagin with methylglyoxal/glyoxal after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), aspalathin/nothofagin; (B1,B2), hemiacetal forms of mono-MGO-aspalathin/nothofagin; (C1,C2), hemiketal forms of mono-MGO-aspalathin/nothofagin; (D1,D2), mono-GO-aspalathin isomers. Other isomers are also possible.

Figure 9

Mass spectra of vitexin and its methylglyoxal/glyoxal adducts after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), vitexin; (B), mono-MGO-vitexin; (C), mono-GO-vitexin.

Figure 10

Mass spectra of isovitexin and its methylglyoxal adduct after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), isovitexin; (B), mono-MGO-isovitexin.

Figure 11

Proposals for chemical structures of adducts formed in the reaction of vitexin with methylglyoxal/glyoxal after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), vitexin; (B1,B2), hemiacetal forms of mono-MGO-vitexin; (C1,C2), hemiketal forms of mono-MGO-vitexin; (D1,D2), mono-GO-vitexin isomers. Other isomers are also possible.

Figure 12

Proposals for chemical structures of adducts formed in the reaction of isovitexin with methylglyoxal after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), isovitexin; (B), hemiacetal form of mono-MGO-isovitexin; (C) hemiketal form of mono-MGO-isovitexin. Other isomers are also possible.

Figure 13

Mass spectra of eriodictyol and its methylglyoxal/glyoxal adducts after 1 h of incubation in pH 7.4 phosphate buffer solution at 37 °C; (A), eriodictyol; (B), mono-MGO-eriodictyol; (C), mono-GO-eriodictyol.