In vitro and in vivo studies of nanoparticles of chitosan- Pandanus tectorius fruit extract as new alternative treatment for hypercholesterolemia via Scavenger Receptor Class B type 1 pathway

Abstract

Pandanus tectorius fruit, a natural product rich in tangeretin and ethyl caffeate, has been reported to have potential as anti-hypercholesterolemia agent via Scavenger Receptor Class B type 1 (SR-B1) pathway. However, due to its semi-polar properties, P. tectorius extract exhibits poor solubility when used as a medical remedy. The extract's solubility can potentially be improved through a synthesis of nanoparticles of chitosan-P. tectorius fruit extract. This can also increase the extract's SR-B1 gene expression activity. To date, no studies of nanoparticles of chitosan-P. tectorius fruit extract and its pathway via SR-B1 have been published anywhere. In this study, cytotoxicity properties against HepG2 were explored by MTT. Then luciferase assay was used to detect their effectiveness in increasing SR-B1 activity. An in vivo study using Sprague dawley was carried out to observe the extract nanoparticles' effectiveness in reducing the cholesterol levels and the toxicity property in rat's liver. As the results showed, the extract nanoparticles had no cytotoxic activity against HepG2 cells and exhibited higher SR-B1 gene expression activity than the non-nanoparticle form. As the in vivo study proved, nanoparticle treatment can reduce the levels of TC (197%), LDL (360%), and TG (109%), as well as increase the level of HDL cholesterol by 150%, in comparison to those for the untreated high-cholesterol diet group. From the toxicity study, it was found that there was non-toxicity in the liver. It can be concluded that nanoparticles of chitosan-P. tectorius fruit extract successfully increased P. tectorius fruit extract's effectiveness in reducing hypercholesterolemia via SR-B1 pathway. Hence, it can be suggested that nanoparticles of chitosan-P. tectorius fruit extract is safe and suitable as an alternative treatment for controlling hypercholesterolemia via SR-B1 pathway.

Keywords: Chitosan nanoparticles; Hypercholesterolemia; Pandanus tectorius; SR-B1.

Fig. 1

Effect of different concentration of acetic acid toward the particle size using probe sonication.

Fig. 2

Size distribution by intensity of nanoparticles of chitosan-P. tectorius fruit extracts with formulation 1a (a) and 2c (b).

Fig. 3

Infrared spectra of chitosan (red line) and nanoparticles of chitosan-P. tectorius fruit extract (blue line).

Fig. 4

Surface morphology of pure chitosan (a), chitosan nanoparticles (b) and nanoparticles of chitosan of P. tectorius fruit extract (c) using SEM.

Fig. 5

Cytotoxicity property of nanoparticles chitosan loaded by PHE, PEE, and PME against HepG2 Cell Line. The values are presented as mean ± SD.

Fig. 6

Luciferase activities among nanoparticles of chitosan-PHE, PEE, and PME in increasing SR-B1expression. Data are presented as mean ± SD (n = 3). PHE = hexane extracts of P. tectorius fruit, PEE = ethyl acetate extracts of P. tectorius fruit, PME = methanol extracts of P. tectorius fruit, NPs = nanoparticles.

Fig. 7

Comparison of luciferase activity value of SR-B1 promoter given by nanoparticles of chitosan-PME and pure PME. The values of data are presented as mean ± SD. Note: PME = methanol extracts of P. tectorius fruit.

Fig. 8

Body weight during 42-day treatment of normal group (A), cholesterol (B), simvastatin drug (10 mg/kgBW) (C) and nanoparticles of chitosan-P. tectorius fruit extract (500 mg/kgBW) (D).

Fig. 9

Food consumption during 42-day treatment by normal group (A), cholesterol (B), simvastatin drug (10 mg/kgBW) (C), and nanoparticles of chitosan-P. tectorius fruit extract (500 mg/kgBW) (D).

Fig. 10

Effect on total cholesterol (TC), triglyceride (TG), high density Lipoprotein (HDL) and low density lipoprotein (LDL) levels for normal group (A), cholesterol (B), simvastatin (10 mg/kgBW) (C), and nanoparticles of chitosan-P. tectorius fruit extract (500 mg/kgBW) (D) at day 14 of treatment. Data are presented as mean ± SD, with the number of rats per group n = 8. *p < 0.05 compared to normal group (A) using the Duncan test.

Fig. 11

Effect of total cholesterol (TC), triglyceride (TG), high density Lipoprotein (HDL) and low density lipoprotein (LDL) levels for normal group (A), cholesterol (B), simvastatin (10 mg/kgBW) (C), and nanoparticles of chitosan-P. tectorius fruit extract (500 mg/kgBW) (D) at D-28 of treatment toward the administration of nanoparticles of chitosan-P. tectorius fruit extracts. Data are presented as mean ± SD, with the number of rats per group n = 8. *p < 0.05 compared to normal group (A) using the Duncan test.

Fig. 12

Histology of liver tissue of group A, B, C, and D at day 28 and day 42 of treatment with magnification 20x. CV = Central Vein, PV = Portal vein, F = fatty liver degeneration.

Fig. 13

Effect of nanoparticles of chitosan-P. tectorius fruit extract in reducing hypercholesterolemia through increasing of SR-B1 gene expression (Modified from Andriani et al., 2015b). HDL = High-density Lipoprotein, LDL = Low-density Lipoprotein, SR-B1 = Scaverager Receptor Class B, LCAT = Lecithin Cholesterol Acyltransferase.