Development and optimization of nanoparticles loaded with erucin, a dietary isothiocyanate isolated from Eruca sativa: Antioxidant and antiproliferative activities in ehrlich-ascites carcinoma cell line

Abstract

The study on Erucin (ER) has gained interest of nutraceutical and pharmaceutical industries because of its anti-cancer properties. Erucin is an isothiocyanate obtained from the seeds of Eruca sativa which possess certain drawbacks such as poor aqueous solubility and bioavailability. Therefore, the present study aimed at developing ER-cubosomes (CUB) by solvent evaporation technique followed by applying Central Composite Design to optimize ER loaded cubosomes. For this purpose, independent variables selected were Monoolein (MO) as lipid and Pluronic-84 (P-84) as a stabilizer whereas dependent variables were particle size, percentage of ER loading and percentage of its entrapment efficiency. The cubosomal nanocarriers exhibited particle size in the range of 26 nm, entrapment efficiency of 99.12 ± 0.04% and drug loading of 3.96 ± 0.0001%. Furthermore, to investigate the antioxidant potential, we checked the effect of ER and ER-CUB by DNA nicking assay, DDPH assay and Phosphomolybdate assay, and results showed significant improvement in antioxidant potential for ER-CUB than ER. Similarly, ER-CUB showed enhanced anticancer activity with a marked reduction in IC50 value than ER in MTT assay. These results suggested that ER-CUB produced notable escalation in antioxidant potential and enhanced anticancer activity than ER.

Keywords: antioxidant assay; central composite design; colon cancer; cubosomes; ehrlich ascites carcinoma; erucin.

FIGURE 1

Schematic representation of preparation of ER loaded Cubosomes (ER-CUB) and in vitro cytotoxitity studies.

FIGURE 2

Partition co-efficient of ER using Octanol/water.

FIGURE 3

3D images of Central composite design of experiment showing (A) Size of ER-CUB (B) Entrapment efficiency (C) Drug loading of ER.

FIGURE 4

FTIR spectra of ER, MO, P84, Physical mixture, BLK-CUB and ER-CUB.

FIGURE 5

TEM images of (A) BLK-CUB and (B) ER-CUB (C) Size of BLK-CUB and (D) ER-CUB.

FIGURE 6

In vitro release of pure ER and ER-CUB using dialysis bags method. The release study was performed at 37°C at 100 rpm in 0.1N HCl, PBS (pH 6.8 and 7.4), values are mean±SD (n=3). The release ER fabricated formulation exhibited controlled release compared to pure ER (p < 0.05) at different time intervals.

FIGURE 7

Cell viability was determined by using MTT assay. Cells were treated with varying concentrations of the ER and ER-CUB, EAC cancer cells for 24 h. Data represented as mean ± SE at the level of significance p ≤ 0.05.

FIGURE 8

In vitro cytotoxicity of ER, and ER-CUB in EAC cancer cells after 24 h of incubation by MTT assay. Values are mean ± SD (n = 3), *p < 0.05, **p < 0.01 versus ER treatment at same dose. EAC cancer cells stained with Hoechst IC30, IC50 and IC70 images of acridine orange and ethidium bromide staining of EAC cancer cell line.

FIGURE 9

Total antioxidant capacity (TAC) of (A) ER-CUB and (B) ER (Pure) were determined using plasmid DNA on the protection of supercoiled pBR322 DNA against hydroxyl radical generated by Fenton’s reagent. CN, Negative control (pBR322 DNA + distilled water); FR, Positive control (pBR322 DNA + FR); Rutin, pBR322 DNA + FR + 100 µg/ml rutin; Conc. pBR322 DNA + FR + 1, 2, 4, 8, 16µg/ml respectively.

FIGURE 10

Antioxidant potential of different concentrations of ER and ER-CUB.

FIGURE 11

Showing the reducing potential of ER and ER-CUB in ferric reducing power assay.