Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer

Huang D, Ou B, Hampsch-Woodill M, Flanagan JA, Deemer EK.

J Agric Food Chem. 2002 Mar 27;50(7):1815-21.

We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using fluorescein (FL) as the fluorescent probe. The current ORAC(FL) assay is limited in hydrophilic antioxidant due to the aqueous environment of the assay. Lipophilic antioxidants mainly include the vitamin E family and carotenoids, which play a critical role in biological defense systems. In this paper, we expanded the current ORAC(FL) assay to lipophilic antioxidants. Randomly methylated beta-cyclodextrin (RMCD) was introduced as the water solubility enhancer for lipophilic antioxidants. Seven percent RMCD (w/v) in a 50% acetone-H(2)O mixture was found to sufficiently solubilize vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). This newly developed ORAC assay (abbbreviated ORAC(FL-LIPO)) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORAC(FL-LIPO) assay is reliable and robust. For the first time, by using 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid as a standard (1.0), the ORAC values of alpha-tocopherol, (+)-gamma-tocopherol, (+)-delta-tocopherol, alpha-tocopherol acetate, tocotrienols, 2,6-di-tert-butyl-4-methylphenol, and gamma-oryzanol were determined to be 0.5 +/- 0.02, 0.74 +/- 0.03, 1.36 +/- 0.14, 0.00, 0.91 +/- 0.04, 0.16 +/- 0.01, and 3.00 +/- 0.26, respectively. The structural information of oxidized alpha-tocopherol obtained by liquid chromatography/mass spectrometry reveals that the mechanism for the reaction between the vitamin E and the peroxyl radical follows the hydrogen atom transfer mechanism, which is in agreement with the notion that vitamin E is the chain-breaking antioxidant.

The effects of various commercial hydrothermal processes (steaming, autoclaving, and drum drying) on levels of selected oat antioxidants were investigated. Steaming and flaking of dehulled oat groats resulted in moderate losses of tocotrienols, caffeic acid, and the avenanthramide Bp (N-(4′-hydroxy)-(E)-cinnamoyl-5-hydroxy-anthranilic acid), while ferulic acid and vanillin increased. The tocopherols and the avenanthramides Bc (N-(3′,4′-dihydroxy-(E)-cinnamoyl-5-hydroxy-anthranilic acid) and Bf (N-(4′-hydroxy-3′-methoxy)-(E)-cinnamoyl-5-hydroxy-anthranilic acid) were not affected by steaming. Autoclaving of grains (including the hulls) caused increased levels of all tocopherols and tocotrienols analyzed except beta-tocotrienol, which was not affected. Vanillin and ferulic and p-coumaric acids also increased, whereas the avenanthramides decreased, and caffeic acid was almost completely eliminated. Drum drying of steamed rolled oats resulted in an almost complete loss of tocopherols and tocotrienols, as well as a large decrease in total cinnamic acids and avenanthramides. The same process applied to wholemeal made from groats from autoclaved grains resulted in less pronounced losses, especially for the avenanthramides which were not significantly affected.

Palm oil is an excellent choice for food manufacturers because of its nutritional benefits and versatility. The oil is highly structured to contain predominantly oleic acid at the sn2-position in the major triacylglycerols to account for the beneficial effects described in numerous nutritional studies. Oil quality and nutritional benefits have been assured for the variety of foods that can be manufactured from the oil directly or from blends with other oils while remaining trans-free. The oxidative stability coupled with the cost-effectiveness is unparalleled among cholesterol-free oils, and these values can be extended to blends of polyunsaturated oils to provide long shelf-life. Presently the supply of genetic-modification-free palm oil is assured at economic prices, since the oil palm is a perennial crop with unparalleled productivity. Numerous studies have confirmed the nutritional value of palm oil as a result of the high monounsaturation at the crucial 2-position of the oil’s triacylglycerols, making the oil as healthful as olive oil. It is now recognized that the contribution of dietary fats to blood lipids and cholesterol modulation is a consequence of the digestion, absorption, and metabolism of the fats. Lipolytic hydrolysis of palm oil glycerides containing predominantly oleic acid at the 2 position and palmitic and stearic acids at the 1 and 3 positions allows for the ready absorption of the 2-monoacrylglycerols while the saturated free fatty acids remain poorly absorbed. Dietary palm oil in balanced diets generally reduced blood cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides while raising the high-density lipoprotein (HDL) cholesterol. Improved lipoprotein(a) and apo-A1 levels were also demonstrated from palm oil diets; an important benefits also comes from the lowering of blood triglycerides (or reduced fat storage) as compared with those from polyunsaturated fat diets. Virgin palm oil also provides carotenes apart from tocotrienols and tocopherols that have been shown to be powerful antioxidants and potential mediators of cellular functions. These compounds can be antithrombotic, cause an increase of the prostacyclin/thromboxane ratio, reduce restenosis, and inhibit HMG-CoA-reductase (thus reducing) cholesterol biosynthesis). Red palm oil is a rich source of beta-carotene as well as of alpha-tocopherol andtocotrienols.

Antioxidants in dietary oils: Their potential role in breast cancer prevention

Sylvester PW Dr, Shah S.

Malays J Nutr. 2002 Mar;8(1):1-11.

Edible oils contain variable amounts of natural antioxidants such as vitamin E. Antioxidants act not only to prevent lipid peroxidation and free-radical production, but also display potent anticancer activity. The vitamin E family of compounds is divided into two subgroups called tocopherols and tocotrienols, but only tocotrienols display potent anticancer activity at treatment doses that have little or no effect on normal cell growth or viability. Palm oil contains the highest concentrations of natural tocotrienols. Tocotrienols induced apoptosis or programmed cell death in breast cancer cells. Morphological and biochemical characteristics of apoptosis, such as nuclear and cytoplasmic condensation and DNA fragmentation, are mediated by the activation of cysteine proteases called caspases. Apoptosis is triggered by the activation of initiator caspases (caspase-8 or 9) that subsequently activate effector caspases (caspase-3, 6, and 7). Studies were conducted using the highly malignant +SA mouse mammary epithelial cell line to determine if tocotrienol-induced programmed cell death is mediated through the caspase-8 or caspase-9 pathway. Treatment with cytotoxic doses of tocotrienol resulted in a large increase in caspase-8 and caspase-3, but not caspase-9 activity. Combined treatment of tocotrienol with selective caspase-8 or caspase-3 inhibitors completely blocked tocotrieno-linduced apoptosis and activation of caspase-8 and caspase-3, respectively. These findings demonstrate that tocotrienol-induced apoptosis in highly malignant mammary epithelial cells is mediated through caspase-8 activation, and may provide essential information necessary for understanding the potential health benefits of these compounds in preventing and/or reducing the risk of breast cancer in women.

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