Date of Graduation

5-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor

Navam S. Hettiarachchy

Committee Member

Michael G. Johnson

Second Committee Member

Dan Davis

Third Committee Member

Timothy Kral

Fourth Committee Member

Narayan Rath

Keywords

Biological sciences; Bioactive compounds; Cancer; Gastrointestinal; Peptides; Rice bran

Abstract

Bioactive compounds are revolutionizing the nutritional and medicinal world with their inherent disease-fighting properties. A wide range of functional groups fall under the category of imparting health benefits. Compounds from both animal and plant origins have been generated as bioactive agents that have opened up new vistas for alternative medicine and natural healing. For example, in a debilitating disease like cancer, these compounds can act to suppress or delay the underlying pathology over and above the conventional treatment strategies involving drugs or chemotherapy. In other words conventional and invasive therapy, although still considered appropriate at certain stages of cancer, their low success rates have necessitated and ushered in an era of nutraceuticals or naturally available bioactive compounds that can through diet reduce the progression of cancers thereby providing a replacement or supplement to pharmacologic treatments. As a result many, including Americans are supplementing traditional health care by turning to nutraceuticals and functional foods.

Animal and plant derived bioactive compounds have been shown to render positive health benefits. However with the outbreaks of a few meat-related illnesses, vegetarianism is on the rise. Compounds derived from plants including fruits and vegetables are being researched for the presence of bioactive compounds. Cereal grains are also being identified to be valuable sources of bioactive proteins and peptides that help arrest disease progression. The increasing knowledge of peptides with anti-disease properties like antihypertensive, antimutagenic, and anticancer has created commercial interest in using them as functional ingredients in food products. Bioactive peptide research had focused primarily on animal, fish, and dairy proteins but owing to disease propensities and religious reasons many are turning to study and recommend plant derived products. Protein isolates and fractions from food sources have been shown to exert anti-mitogenic, anti-hypertensive, anti-cancerous, and anti-microbial effects, particularly peptides that are broken down from proteins available in foods have been documented to possess anti-disease characteristics. Hence exploring and testing for bioactivities, in compounds originating from plant sources has gained interest. Cereal proteins particularly have been utilized to obtain hydrolysates and peptides for testing against disease attributes like anti-hypertensive or anti-cancer etc.

The following research has been based on grounds of utilizing cereal grains' by-product and hopes to provide scope for reducing cancerous states with the use of natural, inexpensive and readily available value-added byproducts obtained during processing of certain cereal grains.

Rice bran is one such byproduct obtained during milling of rice. It contains 90% of the nutrients and nutraceuticals, and forms 8-10% of the total weight. The rice bran components include high quality protein, phenolics, dietary fiber, source of vitamins and minerals. Rice bran is primarily used as animal feed and to extract oil which is used in cooking. It has nearly 20% protein and could be a source of bioactive peptides and hence a functional food for health benefits. The State of Arkansas is ranked number one in the production of rice and, rice bran is a cheap co-product of rough rice milling. Rice bran production rate in the United States has reached 800,000 tons annually. Although rice bran production is so high, it is mostly under-utilized.

In spite of numerous potential health benefits little work has been done with rice bran to produce functional bioactive compounds. Rice bran protein has the potential for creating anticancer peptides. The high quality protein can be broken down to generate peptides. There are no systematic studies reported on the production of anti-cancer peptides from commercially available heat stabilized defatted rice bran by proteolytic enzyme hydrolysis and, particularly their resistance to gastrointestinal environment. Therefore, a systematic study on the production of gastrointestinal resistant peptides from rice bran by enzymatic hydrolysis, and testing for anticancer activity was needed.

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 ultrafiltration 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 salt; (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 pentapeptide 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.

The significance of the findings observed in this study lies in the potential capacity by which a natural nutraceutical agent like rice bran could bear positive impact on human health through its protein components providing potentials for advancement in medicinal application from an agricultural standpoint, while it is considered as a co– product of a larger process. Successful utilization of data from this research will revolutionize the concept of preparing peptides with bioactivity against human cancers and promote further study of value added products from the cereal grains like rice bran.

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