Studies On Purification, Structure And Biological Activities Of Angiotensin Converting Enzyme Inhibitors From Tricholoma Matsutake And Leucopaxillus Tricolor

Hypertension, as a silent and an "invisible killer", is a worldwide problem of epidemic proportions. The study of prevention and treatment of hypertension is one of the arduous tasks for the world’s medical community. Angiotensin converting enzyme (ACE) plays a key role in the regulation of peripheral blood pressure mainly through the renin-angiotensin (RAS) and kallikrain-kinin systems (KKS). Hence, it is a big challenge to develop novel, safe and natural angiotensin converting enzyme inhibitors (ACEIs) from food-derived antihypertensive peptides as alternatives to synthetic drugs. Mushrooms are popular not only because of their delicious taste, but also due to their proven valuable nutritional and medicinal attributes. So the research on ACEIs from edible mushrooms has a tremendous potential in prevention and treatment of hypertension-associated diseases. In this study, an ACE inhibitory peptide TMP and an ACE inhibitory protein LTP were purified from the mushrooms Tricholoma matsutake and Leucopaxillus tricolor, respectively. The amino acid sequences, characterization and biological activities of these ACE inhibitors were investigated.To explore the hypertensive effect of the highly prized mushroom Tricholoma matsutake, a series of purification methods including extraction, heating, ultrafiltration using a membrane with the molecular weight cutoff of 5 kDa, ion exchange chromatography on Q-Sepharose and Mono-Q, and fast protein liquid chromatography on Superdex peptide were performed. An ACE inhibitory peptide with an IC50 of 0.40μM was purified from the extract and designated as TMP. Its amino acid sequence was elucidated to be WALKGYK through LC-MS/MS analysis and its molecular mass was 865.04 Da. TMP exhibited remarkable stability over a wide range of temperatures from 40℃ to 90℃. After incubation at 90℃ for 2 hours, TMP retained 80% ACE inhibitory activity. TMP also displayed good stability from pH 2.0 to pH 11.0. When TMP was exposed to pH 2.0 for 2 hours,40% ACE inhibitory activity was retained. The Lineweaver-Burk plot suggested that TMP was a non-competitive inhibitor of ACE. It also demonstrated 2,2-diphenyl-l-picrylhydrazyl (DPPH) radical scavenging activity and Fe2+ chelating activity. At the concentration of 10 mg/mL,50% of the DPPH radicals were scavenged. TMP showed 24% Fe2+ chelating activity at the dosage of 20 mg/mL. A short-term assay of antihypertensive activity demonstrated that both TMP and the water extract of T. matsutakei fruiting bodies exerted a clear antihypertensive action by lowering the systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs). The biggest drop in SBP of SHRs were 18 mmHg for TMP (25 mg-kg-1 body weight) and 36 mmHg for water extract of T. matsutakei fruiting bodies (400 mg-kg-1 body weight), respectively. At a higher dosage of 50 mg-kg-1 body weight, the biggest decrement in SBP produced by TMP was 36 mmHg. TMP and the water extract of T. matsutakei fruiting bodies had no effect of the heart rate of SHRs. Hence T. matsutake can be used as a functional food to help prevent hypertension-associated diseases.An 86-kDa homodimeric ACE inhibitory protein designated as LTP was isolated from the fruiting bodies of the mushroom Leucopaxillus tricolor with 12.7-fold purification. The purification protocol entailed ultrafiltration through a membrane with a molecular weight cutoff of 10-kDa, ion exchange chromatography on Q-Sepharose, and finally fast protein liquid chromatography-gel filtration on Superdex 75. LTP exhibited an IC50 value of 1.64 mg/mL for its ACE inhibitory activity and the yield was 0.9%. The unique N-terminal amino acid sequence of LTP was disclosed by Edman degradation to be DGPTMHRQAVADFKQ. The percentage of hydrophobicity of the N-terminal amino acids of LTP was 40%. In addition, seven internal sequences containing more hydrophobic amino acid residues of LTP were elucidated by LC-MS/MS analysis. The percentage of hydrophobicity of the seven internal sequences was 60% at least. Results of the Lineweaver-Burk plot suggested that LTP competitively inhibited ACE. About 60% of the ACE inhibitory activity of LTP remained after incubation at pH 2.0 for 2 hours while only 30% ACE inhibitory activity was retained after treatment at pH 9.0 for 2 hours. A decline in ACE inhibitory activity occurred after incubation for 2 hours at temperatures ranging from 30 to 90℃, indicating that LTP had poor thermostability. Both LTP (100 mg-kg-1 body weight) and the water extract of L. tricolor (200 mg-kg-1 body weight) exhibited a clear antihypertensive effect on SHRs and the biggest decrements in SBP were 34 and 43 mmHg, respectively. LTP and the water extract of L. tricolor had no effect of the heart rate of SHRs. This may provide experimental evidence for L. tricolor being a functional food.