Molecular Studies of Purple Rice Extracts on Carcinogens Induced Liver and Colon Carcinogenesis

Abstract

Oryza sativa L. var. indica cv. Kum Doisaket is a pigmented rice variety cultivated in northern Thailand. In the present study, purple rice seed and hull were extracted by various solvents, including hexane, dichloromethane, methanol, acidified methanol, ethanol, and water. Purple rice hull extracts contained higher total phenolic acids than the seed extracts, while the seed extracts showed higher flavonoid contents than the hull extracts. The most abundant phenolic acids containing in the rice hull extracts were vanillic acid, p-coumaric acid, and protocatechuic acid, while rice seed contained the highest amount of protocatechuic acid and vanillic acid, respectively. The extraction by acidified methanol yielded higher anthocyanin contents than the other extractions. The major anthocyanins containing in purple rice extracts were cyanidin-3-glucoside and peonidin-3-glucoside. Alpha-tocopherol was the major vitamin E containing in both part of purple rice seed and hull. The mutagenic and antimutagenic properties of purple rice seed and hull extracts were determined using Salmonella mutation assay. The purple rice seed and hull extracts, with the exception of acidified methanol extract of rice hull, showed no mutagenicity in strains TA98 and TA100 in both presence and absence of metabolic activation. All purple rice seed and hull crude extracts inhibited the number of mutant colonies induced by aflatoxin B1 and 2-amino-3, 4-dimethylimidazo[4,5-f]quinolone. None of the extracts inhibited the mutagenicity of 2-(2-furyl-3-(5-nitro-2-furyl) acrylamide or sodium azide in the absence of metabolic activation. All purple rice extract, except water extract, enhanced the number of revertant colonies induced by benzo(a)pyrene. The acidified methanol extract of rice hull was further analyzed using solvent partitioning with n-hexane, chloroform, ethyl acetate, butanol and water. All of the solvent-partitioned fractions, with the exception of aqueous-partitioned fraction, displayed antimutagenicity against aflatoxin B1. However, mutagenicity was observed in the tests with the n-hexane-, chloroform- and ethyl acetate-partitioned fractions. To determine whether acidified methanol rice hull extract and its solvent partitioned fractions present mutagenicity in mammalian cell, the rpsL mutation assay was used in this study. The results found that the extracts, at the maximum concentration, 100 µg/ml, did not show mutagenic activity in the mutation assay using the rpsL gene of Escherichia coli as a reporter gene. Due to the highest antimutagenicity of methanol seed extract and acidified methanol extract of purple rice hull in Salmonella mutation assay, the anticarcinogenicity of these extracts were determined using dual organ carcinogenicity test in rats. To establish the dual organ carcinogenicity test, triple injection of diethylnitrosamine (DEN) and double injection of 1, 2-dimethylhydrazine (DMH) were administrated to male Wistar rats under various protocols. The glutathione-S-transferase placental form (GST-P) positive foci and aberrant crypt foci (ACF) were used as the end-point markers of the early stages of liver and colon carcinogenesis, respectively. The protocol in which DENs (Days 0, 4, 9) were treated during DMH (Days 0, 7) administrations showed the highest number of hepatic GST-P positive foci and colonic ACF at week 8 of the experiment. The serum AST and ALT activities were increased in combined treatment of DEN and DMH group. The expression of hepatic phase I metabolizing enzymes were decreased, while the detoxifying enzyme activity was increased when compared to carcinogen alone groups. In addition, combined treatment of DEN and DMH also increased hepatic and colonic DNA adducts when compared with control group. This model can be used as an alternative model for determining chemical carcinogenicity and anticarcinogenicity in rat liver and colon. Oral administration of 500 mg/kg bw the purple rice seed and hull extracts did not induce both preneoplastic lesions in liver and colon tissues. Interestingly, they significantly decreased the number of GST-P positive foci in rat liver but could not inhibit the number of ACF in colon. The inhibitory mechanisms of purple rice extracts were involved in the reduction of cell proliferation and induction of cell apoptosis. In conclusion, the seed and hull extracts of purple rice cv. Kum Doisaket showed no mutagenicity in bacterial model or carcinogenicity in laboratory rats. However, they presented antimutagenicity in Salmonella mutation assay and chemopreventive activity against diethylnitrosamine-induced rat hepatocarcinogenesis. The knowledge on this research provides health promoting effect of Thai purple rice for the use as a candidate of cancer chemoprevention. The identification of anticarcinogenic compounds should be further investigated.