Investigation of Angiotensin-1 converting enzyme-inhibitory activity of extracts obtained from Tympanatonus fuscatus var radula (Linne) and Pachymelania aurita (Muller)

Paul, Abraham Abbey (2019)

xx,162 pages

Thesis

This study extracted aqueous protein fraction from Tympanotonus fuscatus radula and Pachymelania aurita; performed enzymatic hydrolysis of the extracts; fractionated the hydrolysates (peptides) of T. fuscatus and P. aurita previously obtained; and investigated the antioxidant potentials and angiotensin-1 converting enzyme-inhibitory activity of the peptides. These were with a view to providing information on the angiotensin -1 converting enzyme (ACE) inhibitory and antioxidant potentials of the extracts of T. fuscatus and P. aurita and their potential health benefits. The molluscs were collected from Oron beach market, Oron, Akwa Ibom State, Nigeria. The flesh and the hemolymph of the molluscs were homogenized and protein was extracted twice in phosphate-buffered saline. The proximate composition of the whole organisms was determined. The lyophilized aqueous extracts of the two samples were reconstituted in 0.01 N HCl (25 mg/ml) and subjected to proteolytic treatments using trypsin, chymotrypsin and pepsin individually for 16 hrs. Aliquots were collected at intervals, the proteolysis terminated, and the degree of hydrolysis determined. The antioxidant activities of the unhydrolysed samples, 2 and 16 hrs hydrolysates were evaluated. To assess the stability of the peptides to gastric digestion, simulated gastrointestinal digestion (SGID) was carried out by a combination of pepsin, trypsin and chymotrypsin. The hydrolysates of SGID were fractionated (using 3 kDa ultracentrifugal filter) into two fractions. The ACE inhibition and antioxidant activity of the crude hydrolysate and fractions were determined. The peptide distribution of the hydrolysate was determined by SDS-PAGE. The low molecular weight fraction of the two samples were subjected to size-exclusion chromatography (Sephadex G-50), the elution monitored by absorbance at 215, 225 and 280 nm and the fraction showing absorbance at these wavelengths were assayed for antioxidant activity. The results of the proximate analysis showed the crude protein content of the two samples to be 11.74 ± 0.08 and 9.23 ± 0.08 % in P. aurita and T. fuscatus respectively. ACE inhibition and antioxidant activity increased with progressive hydrolysis. Two hours of hydrolysis was sufficient for the production of bioactive peptides as further hydrolysis did not produce significant (P < 0.05) change in the biological (antioxidant) activity tested. Based on the elution profiles from Sephadex G-50 column, P. aurita were pooled into 5 fractions while T. fuscatus were pooled into 4 fractions. These pooled fractions showed DPPH radical scavenging activity, specifically two fractions of T. fuscatus exhibiting about 70 % inhibition with IC50 values of 1.08 ± 0.21 and 4.79 ± 1.07 μg/ml respectively compared with the ascorbic acid standard with IC50 value of 5.3 μg/ml. The ACE inhibitory potential (IC50) of the ultrafiltration fractions of P. aurita were respectively, 65.16 ± 6.41 μg/ml (≤ 3 kDa) and 301.9 μg/ml (> 3 kDa) and that of T. fuscatus (IC50) were 54.94 ± 2.83 μg/ml (≤ 3 kDa) and 291.67 μg/ml (> 3 kDa). The study suggested the potential health benefit of consuming Tympanotonus fuscatus var radula and Pachymelania aurita for the maintenance of general health and management of diseases such as hypertension.