Preparation, separation, purification, characterization and human cell line anti-cancer evaluation of rice bran peptides.

摘要

In this research rice bran was used for preparing peptides with an intention of providing benefits in controlling cancer cell growth. The protein was directly hydrolyzed using food grade enzyme to prepare peptides. These were treated with simulated gastro-intestinal juice before fractionation and collection according to their molecular sizes using ultra-filtration technique. The different sized peptides were tested for bio-activity by cell culture techniques to assess their ability to control cancer cell proliferation.;Identifying and characterizing rice bran peptides that can arrest human cancer cell proliferation formed the broad objective of this study. As a summary heat stabilized defatted rice bran was treated with endoprotease, alcalase after optimizing conditions for enzymatic hydrolysis using response surface design. Degree of hydrolysis (DH) was considered as the dictating response variable for enzymatic hydrolysis, which was set at nearly 25% to avoid both excessive hydrolysis (over 40% DH) and limited hydrolysis (under 15%). The resulting hydrolysates were treated with simulated gastric and intestinal juices to obtain gastrointestinal (GI) resistant peptides. The peptides were fractionated into molecular size ranges of >50, 10-50, 5-10, and 5 kDa using ultrafiltration. They were tested for their abilities to reduce cell viability and cause cytotoxicity to human cancer cells in vitro employing the trypan blue dye exclusion assay as well as a cell titer assay that uses a tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner sa (MTS)] and the electron coupling reagent, phenazine methosulfate or [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT).;In order to provide a comprehensive picture for cancer cell proliferation inhibitory effects of the peptide fractions, and also due to the specificity of each cancer cell type, cancer cells derived from multiple sites representing colon, breast, lung and liver were chosen. The idea was to evaluate rice bran peptide(s) for multiple site activity against cancers. Two colon cancer cell lines including a tumorogenic cell line, two breast cancer cell lines including a tumorogenic cell line, a lung and a liver cancer cell line were selected for the study. The idea of including tumorogenic cell lines representing colon and breast was to decipher any probable anti-tumorigenic activity of the bran peptide fractions because colon and breast organs are prone to involve tumors as part of cancer pathology more commonly than other cancer types chosen for the study. Both GI and non-GI resistant fractions were initially screened with each cancer cell line for evaluating cell viability patterns of the fractions. Based on the findings the 5kDa fraction exhibited better anti-cancer activities followed by the 5-10 kDa fraction compared to other fractions, controls as well as non-GI resistant fractions. The 5 kDa fraction was selected for further confirmatory anti-cancer properties using relatively more specific assays. Dosage and anti-tumorigenic assays were also performed on the 5kDa fraction that initially showed better anti-cancer properties. This fraction was selected for characterization purposes.;The highlight of the research has been the demonstration of GI-resistant 5 and 5-10kDa bran peptide hydrolysates to inhibit growth of certain cancer cell types more effectively compared to controls, and nonresistant fractions. GI resistant fractions had better anti-cancer activities than non-GI resistant fractions of which the 5 and 5-10kDa fractions served as prime candidates for possessing anti-cancer activities based on initial screening. Specifically, the 5kDa fraction showed 70-75% cytotoxicity to colon (Caco-2, HCT-116), 70-80% cytotoxicity to breast (MCF-7, HTB-22) and 80% cytotoxicity to liver (HepG2) cells respectively. The 5-10kDa fraction caused 80% cytotoxicity to liver (HepG2) cancer cells alone. This implies that 5kDa fraction is able to arrest cancer cell proliferation and could also serve as anti-tumorogenic agent. The 5kDa fraction was characterized to obtain a pure peptide that when tested for cancer cell proliferation showed nearly 80% inhibition to colon, breast and liver cancer cells. Amino acid analysis of the peptide revealed the presence of Arginine, Proline and Glutamic acid residues. Full characterization of the peptide by proteomic tools coupled to mass spectrometry enabled determination of the amino acid sequence, Gln-Glu-Arg-Pro-Arg. Thus a penta-peptide from rice bran has been isolated and characterized for multiple-site activity against cancers. This could mean that a food-derived peptide could act as a cheaper natural alternative over synthetic anti-cancer drugs for treatment against cancers originating from multiple sites. (Abstract shortened by UMI.)