Beneficial Effects of Tamarind Trypsin Inhibitor in Chitosan-Whey Protein Nanoparticles on Hepatic Injury Induced High Glycemic Index Diet: A Preclinical Study

Abstract

Several studies have sought new therapies for obesity and liver diseases. This study investigated the effect of the trypsin inhibitor isolated from tamarind seeds (TTI), nanoencapsulated in chitosan and whey protein isolate (ECW), on the liver health status of the Wistar rats fed with a high glycemic index (HGLI) diet. The nanoformulations without TTI (CW) and ECW were obtained by nanoprecipitation technique, physically and chemically characterized, and then administered to the animals. The adult male Wistar rats (n = 20) were allocated to four groups: HGLI diet + water; standard diet + water; HGLI diet + ECW (12.5 mg/kg); and HGLI diet + CW (10.0 mg/kg), 1 mL per gagave, for ten days. They were evaluated using biochemical and hematological parameters, Fibrosis-4 Index for Liver Fibrosis (FIB-4), AST to Platelet Ratio Index (APRI) scores, and liver morphology. Both nanoparticles presented spherical shape, smooth surface, and nanometric size [120.7 nm (ECW) and 136.4 nm (CW)]. In animals, ECW reduced (p < 0.05) blood glucose (17%), glutamic oxalacetic transaminase (39%), and alkaline phosphatase (24%). Besides, ECW reduced (p < 0.05) APRI and FIB-4 scores and presented a better aspect of hepatic morphology. ECW promoted benefits over a liver injury caused by the HGLI diet.

Keywords: hyperglycemia; nanoencapsulation; protease inhibitor.

Figure 1

Isolation of TTI and characterization of nanoformulations (ECW and CW). (A) Chromatographic profile of F2 (which showed the highest antitrypsin activity) of tamarind seeds evaluated by spectrophotometry (Ultrospec™ 2100 pro UV/Visible Spectrophotometer, GE Heathcare Bio-Sciences Corp., Piscataway, NJ) at 280 nm. F2 = protein fraction 2. (B) Electrophoresis in denaturing polyacrylamide gel (SDS-PAGE) at 12.5% stained with silver nitrate. M = Marker; CE = Crude Extract; F1 = Protein fraction 1 (saturation with 0–30% ammonium sulfate); F2 = Protein fraction 2 (saturation with 30–60% ammonium sulfate); TTI = Trypsin inhibitor isolated from tamarind seeds after Trypsin-Sepharose affinity chromatography. (C) Scanning Electron Microscopy (SEM) of the ECW with a magnitude of 30 kX. (D) Scanning Electron Microscopy (SEM) of the CW with a magnification of 50 kX. (E) Laser diffraction obtained for ECW by dispersing nanoparticles in water and previous crosslinking using formaldehyde. (F) Laser diffraction obtained by CW by dispersing nanoparticles in water and previous crosslinking using formaldehyde. (G) ECW Fourier Transform Infrared Spectrum (FTIR): a. ECW; b. Tween 80; c. purified chitosan; d. whey protein isolate; e. TTI. (H) CW Fourier Transform Infrared Spectrum (FTIR): a. CW; b. Tween 80; c. purified chitosan; d. whey protein isolate. ECW trypsin inhibitor isolated from tamarind seeds encapsulated with whey protein isolate and chitosan (1:2:2 w/w/w); CW: chitosan–whey protein isolate nanoparticles (2:2 w/w).

Figure 2

Assessment of liver damage scores. (A) AST/ALT ratio. (B) FIB-4. (C) APRI. Experimental groups: (1) HGLI diet + water; (2) Standard diet + water; (3) HGLI diet + ECW; (4) HGLI diet + CW. Values were expressed as mean and standard deviation. The Kolmogorov-Smirnov test was used to evaluate the normality of the data. AST/ALT ratio presented parametric distribution, so the ANOVA test with Tukey’s post-hoc test was used to determine the significant differences.FIB-4 and APRI showed non-parametric distribution, so the Kruskal-Wallis test and Dunn’s post-test were used to detect significant differences. * Values were significantly different for APRI score, according to Kruskal-Wallis and Dunn’s post-hoc test (p < 0.05). ** Values were significantly different for FIB 4 score, according to Kruskal-Wallis and Dunn’s post-hoc test (p < 0.001). *** Values were significantly different for FIB 4 score, according to Kruskal-Wallis and Dunn’s post-hoc test (p < 0.0001). Mean and standard deviation. HGLI: high glycemic index and glycemic load diet; AST: oxalacetic glutamic transaminase; ALT: alanine transaminase; FIB-4: Fibrosis-4 Index for Liver Fibrosis; APRI: AST to Platelet Ratio Index; ECW trypsin inhibitor isolated from tamarind seeds encapsulated with whey protein isolate and chitosan (1:2:2 w/w/w); CW: chitosan–whey protein isolate nanoparticles (2:2 w/w).

Figure 3

Histopathological analysis Wistar rats livers stained with hematoxylin and eosin (H,E). (A,B): HGLI diet + water (n = 5); (C–E): standard diet + water (n = 5); (F–J): HGLI diet + ECW (n = 5); (K–O): HGLI diet + CW. (A): Panoramic evidence of the liver, with the presence of dilated blood vessels due to hyperemia (black arrow), in addition to an area of ballooning degeneration and hepatocytes suggestive of necrosis (black rectangle) in the peripheral organ portion (H,E-10× objective). (B): Presence of hepatocytes suggestive of necrosis (black arrows); hepatocytes showing ballooning degeneration (black circles); evidence of sinusoidal capillaries dilatation (blue arrowhead); hyperemia in the centrilobular vein (black arrowhead) (H,E-40× objective). (C): extensive area of hepatocytes suggestive of necrosis (black circle); evidence of pseudolobule (black arrow); bile ductal proliferation (black arrowhead); intense hyperemia (blue arrowhead) (H,E-10× objective). (D): an external band of ballooning degeneration (black arrowheads) (H,E-10× objective). (E): the presence of binucleated hepatocytes (black circle) and microvesicular steatosis (blue arrowhead) (H,E-40× objective). (F): liver panoramic view, showing few areas of necrosis (black circles) (H,E-10× objective). (G): evidence of a steatosis focal area, showing microvesicular steatosis (blue arrowhead) and macrovesicular steatosis (black arrowhead) (40× H,E-Objective). (H): area of ballooning hepatocytes degeneration (black arrow) (H,E-40× objective). (I): focal area of mononuclear inflammatory infiltrate (black circle) (H,E-10× objective). (J): hyperemia and dilation of sinusoidal capillaries (black arrow) (H,E-10× objective). (K): panoramic view of the liver showing the predominant maintenance of the normal architecture (H,E-10× objective). (L): evidence of hepatic cord morphology maintenance, without evidence of hyperemia (H,E-40× objective). (M): the presence of bile duct proliferation (black arrows) (H,E-40× objective). (N): scarce hepatocyte necrosis (black arrowhead) (H,E-40× objective). (O): scarce hepatocyte ballooning degeneration (black arrowhead) (H,E-40× objective). H,E: hematoxylin and eosin. HGLI: diet with a high glycemic index and high glycemic load. ECW trypsin inhibitor isolated from tamarind seeds encapsulated with whey protein isolate and chitosan (1:2:2 w/w/w); CW: chitosan–whey protein isolate nanoparticles (2:2 w/w).

Figure 4

Experimental design summary. HGLI: high glycemic index and glycemic load diet; ECW trypsin inhibitor isolated from tamarind seeds encapsulated with whey protein isolate and chitosan (1:2:2 w/w/w); CW: chitosan–whey protein isolate nanoparticles (2:2 w/w).