Identification of guanine, guanosine, and inosine for α-amylase inhibitors in the extracts of the earthworm Eisenia fetida and characterization of their inhibitory activities against porcine pancreatic α-amylase

Abstract

Earthworms are known as a source of a traditional medicine, and bioactive components have been reported. We have reported that a fraction (U3EE) with molecular mass under 3 kDa from the water extract of Eisenia fetida inhibits porcine pancreatic α-amylase (PPA) activity with a half-maximal inhibitory concentration (IC50) of 73.7 ± 4.0 mg/mL. Here we purified PPA-inhibitory components from U3EE by sequential procedures of 85 %-ethanol (EtOH) extraction, solid-phase extraction (SPE), and RP-HPLC. The water eluate from SPE of the 85 %-EtOH extract was a major inhibitory fraction, from which three components were separated by 2nd RP-HPLC and identified with MS, TLC, and UV spectroscopy as guanine (Gua), inosine (Ino), and guanosine (Guo). Kinetic analysis showed that Gua and Guo were non-competitive inhibitors and Ino a mixed-type one, suggesting a key role of the purine ring in inhibition. The inhibitor constants (Ki) of Gua and Guo were 0.28 ± 0.07 and 1.64 ± 0.14 mM, respectively, and Ki and Ki’ of Ino in the EI and ESI complexes were 5.8 ± 1.1 and 59 ± 12 mM, respectively. U3EE might be useful for food supplements to prevent obesity and diabetes.