OBJECTIVES: GST inhibition activity and characterization of Kanji and methanol extract of D. carota roots, and oral absorption pattern of ferulic acid from Kanji in rats.
MATERIALS AND METHODS: GST inhibition activity of Kanji and methanol extract of D. carota roots in concentration range 0.001-100.00 mg/mL was determined using Sprague Dawley rat liver cytosolic fraction. Methanol extract upon column chromatography gave ferulic acid, which was used to characterize Kanji and determine its oral absorption pattern in Wistar rats.
RESULTS: The GST inhibition activity of Kanji (100.00 μg/mL), methanol extract of D. carota roots (100.00 μg/mL) and tannic acid (10.00 μg/mL, positive control) was found to be 0.162 ± 0.016, 0.106 ± 0.013 and 0.073 ± 0.004 μM/min/mg, respectively. Different Kanji samples and methanol extract contained ferulic acid (0.222-0.316 mg/g) and 0.77 mg/g, respectively. Ferulic acid did not appear in plasma after oral administration of Kanji.
DISCUSSION: Kanji having solid contents 80.0 μg/mL, equivalent to 0.0025 μg/mL ferulic acid, does not inhibit the activity of GST. The oral administration of Kanji, in human equivalent dose (528 mg/kg, 16.67 μg ferulic acid), to rats indicated poor absorption of ferulic acid.
CONCLUSION: Kanji having solid contents 14-36 mg/mL does not inhibit GST activity, hence may not interfere with drugs that are the substrates of GST, if taken concomitantly.
METHODS: ACE inhibitory proteins were isolated from P. cystidiosus based on the bioassay guided purification steps, i.e. ammonium sulphate precipitation, reverse phase high performance liquid chromatography and size exclusion chromatography. Active fraction was then analysed by LC-MS/MS and potential ACE inhibitory peptides identified were chemically synthesized. Effect of in vitro gastrointestinal digestions on the ACE inhibitory activity of the peptides and their inhibition patterns were evaluated.
RESULTS: Two potential ACE inhibitory peptides, AHEPVK and GPSMR were identified from P. cystidiosus with molecular masses of 679.53 and 546.36 Da, respectively. Both peptides exhibited potentially high ACE inhibitory activity with IC50 values of 62.8 and 277.5 μM, respectively. SEC chromatograms and BIOPEP analysis of these peptides revealed that the peptide sequence of the hexapeptide, AHEPVK, was stable throughout gastrointestinal digestion. The pentapeptide, GPSMR, was hydrolysed after digestion and it was predicted to release a dipeptide ACE inhibitor, GP, from its precursor. The Lineweaver-Burk plot of AHEPVK showed that this potent and stable ACE inhibitor has a competitive inhibitory effect against ACE.
CONCLUSION: The present study indicated that the peptides from P. cystidiosus could be potential ACE inhibitors. Although these peptides had lower ACE inhibitory activity compared to commercial antihypertensive drugs, they are derived from mushroom which could be easily obtained and should have no side effects. Further in vivo studies can be carried out to reveal the clear mechanism of ACE inhibition by these peptides.