RESUMO
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 3â¯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⯱â¯4.0â¯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⯱â¯0.07 and 1.64⯱â¯0.14â¯mM, respectively, and Ki and Ki' of Ino in the EI and ESI complexes were 5.8⯱â¯1.1 and 59⯱â¯12â¯mM, respectively. U3EE might be useful for food supplements to prevent obesity and diabetes.
Assuntos
Oligoquetos , Animais , Guanina , Guanosina , Inosina , Cinética , alfa-Amilases Pancreáticas , Suínos , alfa-AmilasesRESUMO
We have reported previously that the water extract of the earthworm Eisenia fetida has inhibitory effect on human dipeptidyl-peptidase IV (DPP IV) in vitro. Here we studied to identify DPP IV inhibitors in a low-molecular mass extract (designated U3EE) under 3 kDa prepared from the water extract. U3EE showed 50 % inhibition (IC50) at the concentration of 5.3 ± 0.3 mg/mL. An inhibitory active fraction obtained by solid-phase extraction of U3EE was separated into three parts by reversed-phase HPLC. These parts were shown by GC/MS to be composed of ten (Ala, Gly, Thr, Ser, Asn, Asp, Lys, His, Orn, and cystine), two (Leu and Ile), and one (Met) amino acids, respectively. Among them, Met, Leu, and His showed strong inhibition with IC50 values of 3.4 ± 0.3, 6.1 ± 0.3 and 14.7 ± 1.2 mM, respectively; Ala, Lys, Orn, and Ile showed rather weaker inhibition than those, while the others showed no inhibition. Met, Leu, and Ile were competitive inhibitors and His was a mixed-type one. DPP IV inhibition by U3EE might be due to additive and/or synergistic effects of the inhibitory amino acids, suggesting that it could be useful as pharmaceutical and supplement for diabetes prevention.