Kinetics of Renin and Angiotensin Converting Enzyme Inhibition by African Giant Land Snail (<em>Archachatina marginata</em>) Protein Hydrolysate and Membrane Ultrafiltration Peptide Fractions

Research Article

Austin J Nutri Food Sci. 2015; 3(4): 1071.

Kinetics of Renin and Angiotensin Converting Enzyme Inhibition by African Giant Land Snail (Archachatina marginata) Protein Hydrolysate and Membrane Ultrafiltration Peptide Fractions

Girgih AT¹, Nwachukwu ID¹, Iwar MI², Fagbemi TN³ and Aluko RE¹*

¹Department of Human Nutritional Sciences, University of Manitoba, Canada

²Department of Wild Life and Range Management, Federal University of Agriculture, Makurdi, Nigeria

³Department of Food Science and Technology, Federal University of Technology, Akure, Nigeria

*Corresponding author: Rotimi E. Aluko, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB R3T 2N2, Canada

Received:August 27, 2015;Accepted:December 04, 2015;Published: December 11, 2015

Abstract

The aim of the study was to produce potential antihypertensive peptides with multi-enzyme inhibitory effects from defatted snail protein meal (SnPM). The SnPM was enzymatically hydrolysed by sequential pepsin and pancreatin addition to mimic gastrointestinal tract digestion. After the digestion, approximately 81% of the initial SnPM protein was converted into soluble peptides, which were collected as the snail protein hydrolysate (SnPH). The SnPH was then fractionated using ultrafiltration membranes to obtain peptide fractions with <1, 1-3, 3-5, 5-10 and >10 kDa molecular weight sizes. The SnPH and fractionated peptides were investigated for in vitro inhibitions of angiotensin I-converting enzyme (ACE) and renin activities followed by determination of enzyme inhibition kinetics parameters.The SnPH had 68 and 59% in vitro inhibition of ACE and renin activities, respectively in comparison to the membrane fractions with 46-78 and 41-66% values. The ACE-inhibitory IC50 values were 0.22-0.79 mg/mL when compared to 0.41-0.96 mg/mL for renin, which suggests that the peptides have higher potency against ACE. The SnPH and membrane fractions inhibited ACE activity through mainly a non-competitive mechanism whereas renin inhibition was of the mixed-type. The results suggest that the snail peptides bind mainly to ACE non-active sites but could bind to both the active and non-active sites of renin.

Keywords: Snail; Protein hydrolysate; Angiotensin converting enzyme; Renin; Enzyme inhibition kinetics; Membrane ultrafiltration; IC50 values

Introduction

Hypertension is defined as sustained increases in systolic blood pressure (SBP) of 140 mmHg or greater and/or diastolic blood pressure (DBP) >90 mmHg. Hypertension is classified into two types: primary or essential hypertension (no underlying cause) and secondary hypertension (hypertension due to other disorders like kidney disease, vascular or endocrine disorders) [1,2]. Hypertension in human beings is controlled mainly through the renin-angiotensin system (RAS), which has become a critical physiological target for the development of anti-hypertensive and other cardio-protective agents. The RAS plays vital roles in the progression of human cardiovascular and chronic kidney diseases [3,4]. The RAS pathway is associated with series of enzyme-catalyzed reactions that produce compounds capable of regulating human blood pressure. Renin and angiotensin I-converting enzyme (ACE) are the two principal enzymes involved in RAS control. The key reactions in the RAS pathway involve renin (an aspartyl protease), which catalyzes the first and rate-limiting step by converting angiotensinogen to angiotensin I (AT-I), a decapeptide [5]. This is followed by ACE (peptidyldipeptidase A) that converts AT-I to a potent vasoconstrictor octapeptide called angiotensin II (AT-II). ACE also catalyzes bradykinin (a vasodilator) degradation and inactivation, which contributes to increased vasoconstriction [5]. Several ACE inhibitors such as captopril, enalapril, lisinopril, quinapril, ramipril, perindopril, and benazepril in addition to the only commercially approved renin inhibitor (aliskiren) as well as angiotensin receptor blockers (ARB) have found relevant clinical applications as drugs in hypertension treatment. However, prolonged usage of these drugs has been reported to be associated with undesirable side effects in some patients, which undermines compliance with physician-prescribed dosage [6,7].

Therefore, it has become increasingly necessary to discover safer and cheaper therapeutic compounds from natural food sources for lowering human blood pressure during hypertension. Moreover, it has been postulated that the direct renin activity inhibition could provide a better control of elevated blood pressure than ACE only inhibition (mono-therapy). This is because renin inhibition will lead to a reduction in AT-I (ACE substrate) level, which would otherwise be converted to AT-II in some organs via an ACEindependent pathway catalyzed by the passive action of chymase [8]. It must be noted however, that renin inhibition does not prevent the progression of ACE-catalyzed bradykinin degradation. Thus even in the presence of renin inhibitors, ACE-catalyzed bradykinin degradation may continue to cause blood vessel stiffening, which could ultimately contribute to elevated blood pressure [9]. Therefore, there is growing and urgent need to develop antihypertensive agents that will exert multi-enzyme inhibitory effects, especially with the capacity to simultaneously inhibit ACE and renin activities. Such multi-enzyme inhibition could enable more effective controls of elevated blood pressure, which will contribute to a reduced risk of hypertension development. Previous works have reported food protein hydrolysates with in vitro inhibition of both ACE and renin activities [10-12]. The in vitro renin and/or ACE inhibitions were also shown to be associated with lowering of elevated blood pressure in spontaneously hypertensive rats [12-16] and in hypertensive humans [17,18].

The health promoting benefits of food protein-derived peptides have remained a subject of interest to many food and nutrition scientists who are continually exploring new and underutilized food protein sources. This is especially true for the non-conventional protein sources that can be used for isolation of bioactive compounds to serve as therapeutic agents in hypertension.The African giant land snail (Archachatina marginata) is a soft-bodied type of mollusk that is composed basically of a head with a flattened foot inside a protective calcified shell. The African giant land snail is a non-conventional wildlife dietary meat with high protein and iron contents that is particularly relished by the African consumers as a delicacy meal [19]. Proximate composition studies of the most popular species of edible African giant land snails have shown them to have 17-21% crude protein contents [20,21]. The snail protein compares well with other conventional livestock, meats like mutton, duck and chicken, which have crude protein values of 16.9%, 18.6% and 20.5%, respectively. In addition, snail proteins possess an excellent amino acid profile that could enhance isolation of peptides with the required amino acids for antihypertensive activity. Snails are cheap to rear both at subsistent and commercial levels with high returns on low input and could serve as a valuable source of raw materials for nutritional products [21]. However, to date, there is no information on the potential enzymatic release of bioactive peptides from snail muscle proteins. Therefore, the objective of this work was to determine the in vitro ACE and renin-inhibitory activities as well as the enzyme inhibition kinetics of African giant land snail protein-derived peptides.

Materials and Methods

Materials

Dried (<12% moisture content) ground African giant land snail protein meal (SnPM) was obtained from the Department of Wild Life and Range Management, University of Agriculture, Makurdi, Nigeria. Renin inhibitor screening assay kit was purchased from Cayman Chemicals (Ann Arbor, MI). Rabbit lung ACE, N-[3-(2-Furyl) acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG), pepsin (porcine gastric mucosa), and pancreatin (porcine pancrease) were purchased from Sigma-Aldrich (St. Louis, MO). Other analytical-grade reagents and ultrafiltration membranes (1, 3, 5, and 10 kDa molecular weight cut-off) were obtained from Fisher Scientific (Oakville, ON, Canada).

Preparation of snail protein hydrolysate and ultrafiltration membrane fractions

Prior to enzymatic hydrolysis, the ground SnPM was first defatted using acetone (1:10, w:v); the mixture was then stirred in the fume hood for 3 h and decanted followed by a second and a third consecutive extractions of the residue. The defatted SnPM was air-dried overnight in the fume hood and used for further studies. The defatted SnPM was enzymatically hydrolyzed according to a previous method [22] as follows. Briefly, a 5% (w/v, protein basis) defatted SnPM slurry was heated to 37 °C and adjusted to pH 2.0 using 2 M HCl. Protein hydrolysis was initiated by pepsin (4% w/v, protein basis) addition and the mixture stirred for 2 h. After peptic hydrolysis, the reaction mixture was adjusted to pH 7.5 with 2 M NaOH, pancreatin (4% w/v, protein basis) was then added and the mixture incubated at 37 °C for 4 h. The enzymatic reaction was terminated by adjusting the mixture to pH 4.0 with 2 M HCl followed by heating to 95 °C for 15 min to ensure a complete denaturation of residual enzymes. The mixture was centrifuged (7000g at 4 °C) for 30 min and the resulting supernatant was labeled snail protein hydrolysate (SnPH). The SnPH was sequentially passed through ultrafiltration membranes with molecular weight cut-off (MWCO) of 1, 3, 5, and 10 kDa in an Amicon stirred ultrafiltration cell. The retentate from 1 kDa was passed through the 3 kDa membrane whose retentate was passed through the 5 kDa with the retentate passed through the 10 kDa membrane; the permeate from each membrane was collected to obtain <1, 1-3, 3-5, 5-10 kDa peptide fractions, respectively. Permeates and the 10 kDa membrane retentate (peptides >10 kDa) were collected, lyophilized and stored at -20 °C. The above digestion and ultrafiltration membrane fractionation were performed in triplicate and the freeze-dried products combined and used for in vitro screening of renin and ACE-inhibitory properties.

Determination of protein content and yield of snail peptides

The percent protein contents of the SnPM, SnPH and its ultrafiltration membrane fractions were determined by modified Lowry method [23] while the yield for the SnPH and its membrane peptide fractions were determined according previously described procedures [22]. Briefly, SnPH yield was determined as the ratio of peptide weight of lyophilized SnPH to the protein weight of unhydrolyzed SnPM. Similarly, the percent yields of the ultrafiltration membrane fractions were calculated as the ratio of the respective weights of the lyophilized peptide permeates to the SnPH peptide weight.

ACE inhibition assay

The ability of snail peptide samples to inhibit ACE in vitro activity was measured according to a previously described spectrophotometric method that uses FAPGG as a substrate [24]. Briefly, 1 mL of 0.5 mM FAPGG (dissolved in 50 mM Tris-HCl buffer containing 0.3 M NaCl, pH 7.5) was mixed with 20 μL of ACE (1 U/mL, final activity of 20 mU) and 200 μL of sample dissolved in the same buffer as the FAPGG. The rate of decrease in absorbance at 345 nm was recorded for 2 min at room temperature. In the blank experiment, the buffer was used instead of peptide solution. ACE activity was expressed as rate of reaction (ΔA/min) and inhibitory activity was calculated as:

ACE inhibition (%) = [1 –ΔA.min-1 (sample)/ ΔA.min-1 (blank)] × 100

Where ΔA.min-1 (sample) and ΔA.min-1 (blank) are ACE activity in the presence and absence of inhibitory peptides, respectively.

Renin inhibition assay

In vitro assay of human recombinant renin activity was conducted using the Renin Inhibitor Screening Assay Kit according to a previously described method [25]. Briefly, snail peptide samples were diluted in Tris-HCl buffer (50 mM, pH 8.0, containing 100 mM NaCl), and pre-warmed to 37 °C. Before the reaction was started, (1) 20 μL substrate, 160 μL assay buffer, and 10 μL Double Distilled Water (DDW) were added to the background wells; (2) 20 μL substrate, 150 μL assay buffer, and 10 μL DDW were added to the control wells; and (3) 20 μL substrate, 150 μL assay buffer, and 10 μL sample were added to the inhibitor (sample) wells. The reaction was initiated by adding 10 μL renin to the control and sample wells. The microplate was shaken for 10 s for proper mixing and incubated at 37 °C for 15 min; fluorescence intensity (FI) was then recorded at excitation wavelength of 340 nm and emission wavelength of 490 nm using a fluorometric microplate reader (Spectra MAX Gemini, Molecular Devices, Sunnyvale, CA). The percentage renin inhibition was calculated as follows:

Renin inhibition (%) = [1 –ΔFIU.min-1 (sample)/ ΔFIU.min-1 (blank)] x 100

Where ΔFIU.min-1 (sample) and ΔFIU.min-1 (blank) are renin activity in the presence and absence of inhibitory peptides, respectively.

Kinetics studies of ACE and renin inhibition

The concentration of snail peptide that inhibited ACE activity by 50% (IC50) was calculated by non-linear regression from a plot of percentage ACE inhibition versus four peptide concentrations (0.125, 0.25, 0.5, and 1.0 mg/mL). The kinetics of ACE inhibition was studied with 0.0625, 0.125, 0.25 and 0.5 mM substrate (FAPGG) concentrations. The mode of ACE inhibition was determined from the Lineweaver-Burk plots while kinetic parameters (Vmax and Km) were estimated from non-linear regression fit of the data to the Michaelis-Menten equation using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). Inhibition constant (Ki) was calculated as the x-axis intercept from a plot of the slope of the Lineweaver-Burk lines against sample concentration while the catalytic efficiency (CE) was calculated fromVmaxx/Km ratio. Similarly for renin kinetics, the IC50 was calculated using the non-linear regression from a plot of percentage renin inhibition versus snail peptide concentrations (0.125, 0.25, 0.5, and 1.0 mg/mL). The renin inhibition kinetics was conducted using five (0.625, 1.25, 2.5, 5 and 10 μM) substrate concentrations in the absence and presence of samples while kinetic parameters were calculated as described above for ACE.

Statistical analysis

All assays were conducted in triplicate and analyzed by one-way analysis of variance (ANOVA). The means were compared using Duncan’s multiple range test and significant differences accepted at p<0.05.

Results and Discussion

Protein content and yield of SnPH and its ultrafiltration membrane peptide fractions

Enzymatic hydrolysis of the African giant land SnPM was carried out to mimic human gastrointestinal tract (GIT) digestion using a combination of pepsin and pancreatin proteases to produce SnPH. Protein contents were determined to be 18, 81, 68, 71, 73, 75 and 76% for SnPM, SnPH, <1 kDa, 1-3 kDa, 3-5 kDa, 5-10 kDa and >10 kDa samples, respectively (Table 1). The results indicate that enzymatic hydrolysis and the isolation methods enhanced the separation of peptides from non-protein materials, hence the significantly higher protein contents of the hydrolyzed products. The results confirm susceptibility of the snail proteins to enzyme-induced release of peptides, which could enhance value-added utilization of the snail meat. The comparatively lower 68% protein content of the initial peptide permeate (<1 kDa peptides) may be attributed to the presence of impurities, especially salt (NaCl) since the membrane was used first. Most of the salts are formed during the protein hydrolysis steps when the addition of NaOH is necessary to maintain reaction pH. The results are similar to a previous report on hemp seed peptides, which obtained a lower protein content for the <1 kDa peptides when compared to >1 kDa peptide fractions [22].