Tocotrienols are members of the vitamin E family. In nature, twelve members of the vitamin E family are known, collectively they are called tocols. These are α-, β-, γ- and δ-tocopherols, α-, β-, γ- and δ-tocotrienols, desmethyltocotrienol, didesmethyltocotrienol and two isomers of α-tocomonoenol. Tocopherol has saturated phytyl side chain attached to the chroman ring whereas tocotrienol has three double bonds in the farnesyl side chain. Tocomonoenol has a single double bond in the hydrocarbon side chain. Besides the side chain, tocopherols, tocomonoenols and tocotrienols share the similar chemical structure of having a chroman ring.
α-Tocol refers to tocol with positions 5, 7 and 8 of the chroman ring substituted by methyl groups, whereas β-tocol refers to tocol with positions 5 and 8 of the chroman ring substituted by methyl groups, γ-tocol refers to tocol with positions 7 and 8 of the chroman ring substituted by methyl groups and δ-tocol refers to tocol with position 8 of the chroman ring substituted by a methyl group.
Unlike tocopherols, little attention was paid to tocotrienols until the last decade. This is not surprising as the sources of tocotrienols are very limited. However, recent research findings revealed that tocotrienols have good chemo-preventive properties that are not shared with tocopherols. Some of the research findings include                anti-angiogenic properties potentially for inhibiting growth and proliferation of cancer cells (Inokuchi et al, 2003, Biosci., Biotech. Biochem., 67, 1623-1627),        inducing apoptosis of human breast cancer cells (Guthrie et al, 1997, J. Nutr. 127, 544S-548S; Nesaretnam et al, 1998, Lipids, 33, 461-469; Yu et al, 1999, Nutr Cancer, 33, 26-32; Nesaretnam et al, 2000, Int. J. Food Sci. Nutr. 51 Suppl. S95-103; Chao et al, 2002, J. Nutr. Sci. Vitaminol. 48: 332-337;),        natriuretic (Saito et al, 2003, J. Lipid Res. 44, 1530-1535),        cholesterol lowering (Pearce et al, 1992, J. Med. Chem. 35, 526-541 & 3595-3606; Parker et al, 1993, J Biol, Chem. 268, 11230-11238; Qureshi et al, 1995, Lipids, 30, 1171-1177; Theriat et al, 1999, Clin. Biochem. 32, 309-319; Chao et al, 2002, Nutr. Sci. Vitaminol. 48, 332-337; Qureshi et al, 2002, Atherosclerosis, 161, 199-207),        anti-platelet aggregation (Qureshi et al, 1991, Am. J. Clin. Nutr., 53, 1021S-1026S),        regression of carotid stenosis (Kooyenga et al, 1997, Asia Pacific J. Clin. Nutr, 6, 72-75),        neuro-protection against glutamate induced toxicity (Sen et al, 2000, J. Biol. Chem. 275, 13049-13055; .Khanna et al, 2003, J. Biol. Chem. 278, 43508-43515).        
These research findings also implicated the potential of tocotrienols in substituting for established drugs like tamoxifen, aspirin and statins or use in combination with these drugs. The advantages of tocotrienols over the drugs include their multi-functional therapeutic properties, no known side effects and no known overdose toxicity was reported.
Unlike tocopherols, the sources of tocotrienols are scarce in nature. It is unlikely that significant amount of tocotrienols can be derived by normal food intake. Tocotrienols are found in low levels in palm oil, rice bran oil, barley, wheat germ, rye, coconut oil and palm kernel oil. There are three known commercial sources of tocotrienols—palm oil, rice bran oil and annatto bean.
Crude palm oil contains 600-1000 ppm of tocols and is the most reliable commercial source of tocotrienols. The current annual world production of crude palm oil exceeded twenty million tonnes and is growing steadily. Palm oil mainly consists of triacylglycerols. The other components include 1-5% free fatty acids, 4-7.5% diacylglycerols and minor components such as monoacylglycerols, sterols, glycolipids, phospholipids, squalene, carotenoids, other hydrocarbons and triterpene alcohols. Based on the current trade specifications, crude palm oil quality specifications comprising of three parameters, free fatty acid contents, moisture and impurities contents and a recently included locally developed parameter called deterioration of bleachability index (DOBI).
The current world production of rice bran oil is estimated to be less than one million tonnes and the bulk being of industrial grade. The annual production of annatto beans was about ten thousand tonnes for the year 1992.
The tocols composition in palm oil has advantages over that of rice bran oil. Almost half of the tocols from rice bran oil is tocopherols whereas tocopherol content in palm oil constituted about 22%. In addition, rice bran oil practically does not contain δ-tocotrienol. δ-Tocotrienol was reported to have the highest potency amongst all the tocols in anti-angiogenesis, inducing apoptosis and in prevention of cardio-vascular diseases. δ-tocotrienol constituted about 12% in the tocols derived from palm oil. Although tocotrienol from annatto beans is rich in δ-tocotrienol, however it does not contain α-tocotrienol. α-Tocotrienol was reported to have the highest neuro-protection activity.
One main concern on tocotrienols is the poor absorption in the blood and/or lymphatic systems and their bioavailability. It is known that the absorption of tocotrienols is poor without the presence of dietary fat to stimulate the secretion of bile and lipases. U.S. Pat. No. 6,200,602 described formulation using monoacylglycerol and diacylglycerol of medium chain fatty acids and a dispersing agent to enhance the uptake of polar drugs from the colon whereas U.S. Pat. No. 6,596,306 described formulations using surfactants (labrasol™ and Tween 80™), palm olein and soybean oil to enhance the tocotrienol delivery system.
As tocotrienols are present in minute quantity in oils and fats, the triacylglycerols have to be separated or removed in order to increase the concentration of tocotrienols. This can be achieved by transesterification or solvent extraction. Other minor components have to be removed in order to enrich further on the tocotrienol concentration.
There are patents describing the production of tocotrienols from vegetable oils and fats. Most of these patents involved transesterification of the oil or fat prior to recovery of tocotrienols, followed by vacuum distillation and post-distillation treatment such as using adsorbents. These include U.S. Pat. Nos. 5,157,132, 6,072,092, 5,190,618, European Patent No. 0333472A2, U.K. Patent Nos. GB2218989A, GB2160874A and GB1515238.
There were many examples of using solvent extraction to remove impurities or undesired components from oil seeds and solvent oil miscella. Examples of the applications include U.S. Pat. No. 4,359,417 described a process using aqueous methanol for removing aflatoxin and/or gossypol from the residue meal of oil seed. IL58842 described a process involving extraction of hydrocarbon solvent oil miscella with aqueous methanol or ethanol. These inventions were not aimed at the recovery of components of interest of this invention.
There were patents describing the production of tocotrienols from vegetable oils and fats by an alcohol extraction followed by short path distillation. These include U.K. Patent No. GB2387390 and U.S. Pat. No. 6,649,781. The main disadvantages of these patents include low tocotrienol concentration obtained and relatively large volume of solvent was used during the extraction. Fatty acids and diacylglycerols were the main components in the alcohol extract and were removed by vacuum distillation.
Urea is a weak base. Solubility of urea in methanol is 35 g per 100 mL at 40° C. Urea can react with free fatty acids to form salts but this reaction is very slow and not significant under the conditions of this invention. These characteristics enabled urea solution in methanol to be used for liquid-liquid extraction; although strictly speaking, palm oil is a semi-solid, not a true liquid, but effective extraction can still be achieved by dispersing the semi-solid palm oil into fine droplets. In addition, urea forms urea inclusion compounds with hydrocarbons, fatty acids, fatty acid methyl esters and monoacylglycerols. Urea inclusion compound formation is a useful technique for concentrating certain fatty acids. The selective urea inclusion compound is based on the principle that certain categories of fatty acids formed urea inclusion compound in preference to other fatty acids, while branched chain and fatty acids with cyclic ring do not form urea inclusion complex.
USPTO Backgrounds
USPTO Backgrounds