Enhanced physicochemical stability and efficacy of angiotensin I-converting enzyme (ACE) - inhibitory biopeptides by chitosan nanoparticles optimized using Box-Behnken design

Abstract

Bromelain-generated biopeptides from stone fish protein exhibit strong inhibitory effect against ACE and can potentially serve as designer food (DF) with blood pressure lowering effect. Contextually, the DF refer to the biopeptides specifically produced to act as ACE-inhibitors other than their primary role in nutrition and can be used in the management of hypertension. However, the biopeptides are unstable under gastrointestinal tract (GIT) digestion and need to be stabilized for effective oral administration. In the present study, the stone fish biopeptides (SBs) were stabilized by their encapsulation in sodium tripolyphosphate (TPP) cross-linked chitosan nanoparticles produced by ionotropic gelation method. The nanoparticles formulation was then optimized via Box-Behnken experimental design to achieve smaller particle size (162.70 nm) and high encapsulation efficiency (75.36%) under the optimum condition of SBs:Chitosan mass ratio (0.35), homogenization speed (8000 rpm) and homogenization time (30 min). The SBs-loaded nanoparticles were characterized for morphology by transmission electron microscopy (TEM), physicochemical stability and efficacy. The nanoparticles were then lyophilized and analyzed using Fourier transform infra-red spectroscopy (FTIR) and X-ray diffraction (XRD). The results obtained indicated a sustained in vitro release and enhanced physicochemical stability of the SBs-loaded nanoparticles with smaller particle size and high encapsulation efficiency following long period of storage. Moreover, the efficacy study revealed improved inhibitory effect of the encapsulated SBs against ACE following simulated GIT digestion.

Figure 1

Response optimization for the formulation of SBs loaded Chitosan:TPP NPs, composite and individual desirabilities (D, d₁ and d₂) of the predicted outcomes R₁ = particle size (nm), R₂ = Encapsulation efficiency (%), A; (SBs:Chitosan mass ratio), B; (Homogenization speed and C; (Homogenization time).

Figure 2

Three dimensional response surface plots for the effect of process conditions on; (1). Particle size (nm): (a) SBs:Chitosan mass ratio (A) and Homogenization speed (B); (b) SBs:Chitosan mass ratio (A) and Homogenization time (C); (c) Homogenization speed (B) and Homogenization time (C), (2). Encapsulation efficiency (%): (d) SBs:Chitosan mass ratio (A) and Homogenization speed (B); (e) SBs:Chitosan mass ratio (A) and Homogenization time (C); (f) Homogenization speed (B) and Homogenization time (C).

Figure 3

Changes in the physicochemical properties of the optimized Chitosan:TPP NPs containing SBs during 12 weeks of storage at 4 °C (a) Particle size and polydispersity index (pdi) (b) zeta potential (ζ) and (c) Encapsulation efficiency.

Figure 4

Physicochemical characterization of Empty Chitosan:TPP NPs and SBs loaded Chitosan:TPP NPs: (a) FTIR spectra of the Empty Chitosan:TPP NPs and SBs loaded Chitosan:TPP NPs; (b) X ray diffractogram of the Empty Chitosan:TPP NPs and SBs loaded Chitosan:TPP NPs; (c) In vitro cumulative release profile of the encapsulated SBs from the SBs loaded Chitosan:TPP NPs following 12 h of incubation in phosphate buffered saline, pH 7.4; (d) ACE-inhibitory activity of free SBs and SBs loaded Chitosan:TPP NPs before and after simulated GIT digestion, different letters indicate significant difference (p <0.05).

Figure 5

TEM images of the SBs loaded Chitosan:TPP NPs.