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    US8586109B2

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    (2) oy os) ow ay @ 6s) (3) () os) «2 8) United States Patent Tan et al. ANNATTO EXTRACT COMPOSITIONS, INCLUDING TOCOTRIENOLS AND TOCOPHEROLS AND METHODS OF USE Inventors: Barrie Tan, Amherst, MA (US) Jose LLlobrera, Beleheston, MA (US) Assignee: American River Nutition, Ine Hadley, MA (US) Notice: Subject to any disclaimer, the team ofthis, patent is extended or adjusted under 35 USC. 1S4(b) by 817 days. Appl. Now 12/168819 Piled: Su, 7, 2008 Prior Publication Data US 200910041870.A1 Feb. 12,2009 Related Application Data Continuation-in-part of application No, 100%23,083, filed on Apr. 12,2004, now abandoned Provisional application No. 604488,310, 18, 2003, provisional application No. 60/461,932, filed on Apr. 10,2003, Int. Cl AGIK 36/185 (2006.01) Us.Cl usp 424727, 4240725; 424/764; 4241776: 424/750, 424/757, ld of Class None ‘See pplication file for complete search history. ation Search References Cited US. PATENT DOCUMENTS 6350453 Bi? 22002 Tan ecal 076 ‘US008586109B2 (10) Patent No. 4s) Date of Patent: US 8,586,109 B2 Noy. 19, 2013 FOREIGN PATENT DOCUMENTS wo oxone7s AL 22003 (OTHER PUBLICATIONS Frew ¢al, Metication and Estimation of Tocowienols inthe Amato Liquid Fraction by Gas Chyomstography-Mass Spectrum, 1998, J4OCS, 75: 1723-1727" Murty, 2003, ps wo ifeexteasionitamins conde? eevie imi Pearectal, Hypocholeteolmic Activity of Syahetic and Natural Tocotienol, 1992, Med Chem, 35:3895-3606." Office Acton, dated May 24 2011, Canadian Inllectal Property Otte Morrison, Erol ¥ St A; Wes Maney E, The eect of Bia ore Tn annatio) on bod supa levels in the anaesthetized dog, West Indian med (1) 8-42. Mar 1988 WM Wan Nizaimoon and BAK Khalid, Toctrenlsrich dit Assreses nant pysoeyation end-pret in nonin as tnd improves ghyemne contol in sropenzooci-indoed diabetic fats, vo 24 No.2 Dee 2002, 728 Canaan Oifice Aston dated Aug 20,2013 correspon o Appl ‘ation No.2.52,020, * cited by examiner Primary Examiner — Terry McKelvey Catheryne Chen Hogan Lovells Assistant Examiner (74) Attornes, Agent, oF Firm ABSTRACT Compositions and methods of use of anatt extracts [350- 450 Dalton molecular weight fraction] including ocotrienols and tocopherols with an appropriate spectrum. This spectrum Jncludes but not limited to Ho alpha tocopheeo, high delta ‘and gamma-tocols and mixtures with other extracts [350-450 Dalton moteculae weight feaction] lke palm and rice andlor 18 Claims, 3 Drawing Sheets U.S. Patent Nov. 19, 2013 Sheet 1 of 3 US 8,586,109 B2 100 BB Annatto Rice Annatto Tocotrienols 15 25 Percentage of Tocos (T1 + T3) ort yf BT a3 yrs 813 Tocopherol Tocotrienol FIGURE 1 U.S, Patent Nov. 19,2013 Sheet 2 of 3 TOCOPHEROL US 8,586,109 B2 Methyl Group Position(s) Tocopherol 5,78 - Trimethy! alpha-T1 5,8 - Dimethyl beta-Tl 7,8 - Dimethyl gamma-T1 8 - Monomethyl delta-TI FIGURE 2 Tocotrienol alpha-T3 beta-T3 gamma-T3 delta-T3 U.S. Patent Nov. 19, 2013 Sheet 3 of 3 US 8,586,109 B2 TCNDL (Female) TCDL (Mate) ore Cardiovascular Risk Index 0 1 2 3 ‘Treatment Period (month) FIGURE 3 US 8,586, 1 ANNATTO EXTRACT COMPOSITIONS INCLUDING TOCOTRIENOLS AND ‘TOCOPHEROLS AND METHODS OF USE, RELATED APPLICATIONS, s ‘This application is a Continuation-in-Pant of US, Non provisional application Se. No. 10'823.083 filed on Apr. 12, 200$ (pending), which clsims priority upon U.S. provisional ‘application Ser No. 60/461,932 filed on Apr. 10, 2008 and ‘claims priority upon U.S. provisional application Ser. No. {60/488 510 fled on Jul. 18,2003, thecontentsof which reall herein incorporated by tis reference in ther entireties. ‘All publications, patents, patent applications, databases and other references cited inthis application, al related appl cations referenced herein, and all references cited therein, are Jncomporated by reference in their entirety as ifrestated herein {ul andasif each individual publication, patent, patent appli cation, database or other reference were specifically and indi vidal indicated to be incorporated by reference (OTHER REFERENCE Al, K. S., ef al. 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Gu, J, et al (1997). “Combined Fffects of Sesamin with ‘Alpha TI or T3s oa Lipid and Immune Indices in Brow ‘Norway Rats” Nate, Res, 17: 339-38. Guille-Deniau, Let al. (2003), “Glucose induees de nove fatty acid synthesis in rat skeletal muscle through a SREBP-Ie dependent pathway.” Diabetes $2(Suppl. 1: extended abstract, 1024P. Guthrie, NA. Gapor, et al. (1997). “Inhibition of Prlifert tion of Estrogen Receptor-negative MDA-MB-435 and “positive MCF Human Breast Cancer Cells by Pala Oil ‘Tocotrienols and Tamoxifen, Alone and in Combination, J. Nut,127(3): 448-5488. Hayes, K., A. Pronezak, etal. (1993), “Differences in the ‘plasma iraasport ad Hssve concentrations of locopherols And tocolrienols: abservations in humans and hamsters” roe Soe Exp Biol Med. 202(3): 353-359, Igarashi, O., otal. (2003). “Diureties containing gamma- tocotrienol, US Patent Application Pub No: US 2003/ (0130467 Al, Pub, Date: J. 24, 2003. Ikeda, ST. Tohyama, etal. 2003). “Dietary alpha-toco- ‘hero decreases alpha-tocotrienal bt ot gamma-oc ‘enol concentration in rats J, Nutz. 133(2) 428-44, Ima-Nirwana, S. et.al. (2000). “Palm vitamin F prevents ‘osteopomss in orchideetomized yrowing male rats.” Nat rod. Sei 6: 155-160. Jel, A. etal (2003), "Identfcston of amador! modified proteins by western blot and mass spectrometry in plasma of type-2 diabetes patients.” Diabetes S2(Suppl. 1): AIST extended abstract, 6752. Jenkins, A. J. and T. J. Lyons (2000), “Preventing Vascular Disease in Diabetes” Practical Diabetology 19: 19-34, Siang, Q..S. Christen, etal. 2001), "Gamms- Tocopherol, the ‘Major Fon of Vitamin Fin the US Diet, Deserves More Atiention’" Am. J. Clin, Nutr, 74: 714-722. Kaku, S.,S. Yunoki, etal. (1999). “Effet of dietary ano ‘dantsonserum lipid contentsand immunoglobulin produc tivity oflymphocytesin Sprague-Dawley rats" Biosei Bio techool Biochem. 63(3) 575-576. Kamat J,etal. (1997). “Tocotrienols from Palm Oils Filee- tive Inhibitors of Protein Oxidation and Lipid Poroxidation in Rat Liver Microsomes” Molecular aad Cellular Bio- chemist 170: 131-138. ‘Kamat, J. and T. Devasagayam (1995). “Tocotrienols from ‘ali ol as potent inhibitors of ip peroxidation and pro- {ein oxidation in at brain mitoehondea.” Neurosci Lett 195(3) 179-182. Khor, H. and . Ng (2000). “fteets of Administration of a-Tocopherol and Tocotrinols on Serum lipids and Liver HIMG Coa Reductase Activity” Int. J. of Food Sei. aud Nate SI: S381 Kooyenga, D, T. Watkins, etal, (2001). Antioxidants Mod Tate the Course of Carotid Atherosclerosis: Four-year Study. Micronutrients and Health, Molecular Biological ‘Mechanisms. K. Nesarctnam and [., Packer, AOCS Pres: 366.375, US 8,586,109 B2 3 Kraegen, E, (1998), “Physiologie manifestations of PPAR- ‘gamma gctivation: preclinical studies” Clinical Courier 16(88): 5-7. Lohmann, J. (1981). “Comparative Sensitivitios of Toco- pheroiievels of Platelets, Red Blood Cells, and Plasma for Estimating Vitamin E Ntritional Status inthe Rat.” Ama J lin, Note 34: 2108-2110, Lino, JK, (1998), “Endothelium and Acute Coronary Sya- sdromes Clin Chem. 44: 1799-1808. Liu, M., R. Wallin, etal. 2002)."Mixed Tocopherols Have a ‘Songer Inhibitory Effect on Lipid Peroxidation Than ‘Tocopherol Alone” J. Cardiovase. Phurmacel. 395): 714-721 Melntyre, B.,K. Briski tal. 2000). “Antiproiferative and Apoptotic Effects of Tocopherols and Tocotrenols on Pre ‘neoplastic and Neoplastic Mouse Mammary Epithelial Cells" PSEBM 224: 292-301, McLatghlin, tal. (2003), "Prediction of IR with plasma 1G or TGINDL ratio Diabetes S2¢Suppl. 1): A224 extended abstract, 9622. Meigs, J, F Hu, et al- 2003). “Endothelial! Dysfunction Predicts Development of Type 2 Diabetes.” Diabetes '52(Suppl. 1): ASK extended abstract. 249-OR. Mensink, R., A. Houwelingen, eta. (1999). “A Vitamin E ‘Concentrate Rich in Tocotrienols Had No Effect on Serum Lipids, Lipoproteins, or Platelet Funetion in Men With Mildly Elevated Serum Lipid! Concentrations.” Amer. J Clin, Nue. 692): 213-219, Mezey, FA. Parmslee, eta. (2003), “OF splice and men: ‘what does the distribution of KAP mRNA in he eat tel 1s bout the pathogenesis of familial dysoutonomia?” Brain Res. 98%(1-2) 200-214. Mustad, VC. Smith, etal (2002). "Supplementation With 3 Compositionally Different Tocotrenol Supplements Does [Not lmprove Cardiovascular Disease Risk Factors in Men and Women With Hypercholesterolemia” Am. J. Clin. [Nate 76(6): 1287-1243, Nazaimoon, W. and B. Khalid (2002). “Tocotrenol-tich diet ‘decreases AGE in non-diabetic rats and improves alycemic ‘onto in streptozotoein-indueed diabetic rats” Malay J. Pathol. 24: 77-82 Newaz, M, and N. Nawal (1999), “Effeet of gmma-tocot- ‘enol on blood pressure, lipid peroxidation and total ant ‘oxidant status in spontaneously hypertensive rats” Chia, Exper. Hypertension 21: 1297-1313, Nowa, M., Z. Yousefipaur, etal (2008). “Nitric oxide syn- ‘hate aetvity in blood vessels of spontaneously hyper sve rats: antioxidant protection by gamma-tocotrieno.” J Physiol Pharmacol. $43): 319-327 Norazlina, M., et al. (2002). “Tocotrienols are Needed for "Normal Hone Caleification of Grossing Female Rats.” Asis Pacific Clin, Nute: 194-199, Norazlina, M., etal. (2007). “Fitees of vitamin E supple- ‘mentation on bone metabolism in nicotne-teated rs" Singapore Med. J. 48(3): 195-198, Packer, [., 8. Weber, etal. (2001) "Molecular aspects of ‘alpha-tocotrienol antioxidant ation and cell sigalg." J Nate: 131(2): 369-738. Pearce. B., R: Patker, et al. (1992), “ypocholesterolemic activity of synthetic and natural ocotrienols” J Med Chem. 35(20) 3595-3606. Prescot, S. M. TM. Meintyre etal 2001). “Events at the ‘Vascular Wall: The Molecular Basis of Inflammation” J Invest. Med. 49: 104-111 Qureshi, A. etal. 2001). “Novel Teeotrenols of Rice Bran Inhibit Atherosclerotic Lesions in CS7ALIG ApoF=defi= jee” J. Nu 13: 1-1, 4 Qureshi. A..etal. (2002), “EfTees of Stabilized Rice Bran, Is ‘soluble and Fiber Practions on Blood Gihucose Levels and ‘Serum Lipid Parameters i Humans with Diabetes Mellitus ‘Type [and I:" 1. Nutritional Biochemistry 13: 175-187 5. Qureshi, A. F, Bradlow, etal. (1997), "Novel Tocotrienols of ‘Rice Bran Modulate Cardiovascular Disease Risk Param- ters of Hypercholesterolemie Humans.” . Nutritional Biochemistry 8: 290-208. Qureshi, A. B: Peace, eal. (1996). “Dietary Tocopherol “Attenuates the Impacet of g-Tocotrienal on Hepatic 3-Hy- roxy 3-Methylglutary| Coenzyme A Reductase Activity in Chickens J. Nut, 126: 389-394, Qureshi, A. and D, Peterson (2001), “The combined Effects ‘of Novel Tocotsienols and Lovastatin oa Lipid Metaboli Jn Chickens" Atherosclerosis 156: 39-17 Qureshi, A.B. Bradlow, et al. (1995). “Response of Hyper cholesterolemic Subjects To Administration of Tocat- ‘enols” Lipids 30: 171-1177, Rekeneie, NR. Pei, etl (2008). "Inflammation, Insulin, “Glucose Ia Non Diabetie Older Persons. (Epidemiology).” Diabetes 52(Supp. 1: A218 extended abstract. 937P. Ridker, PJ, Buring, etal. (2003), “C-Reaetive Protein, the ‘Metabolic Syndrome, and Risk of Incident Cardiovascular Events: An §-Yeur Follow-Up of 14,719 Initially Healthy American Women" Cireuation 107(3): 391-397, Robbesyn, FV: Garcia, eal. 2003). “HDL Counterbalan the Proinlammatory Eflect of Oxidized LDL By Inhib ing Intracellular Reactive Oxygen Species Rise, Protes- some Activation, and Subsequent NF-Kappab Activation in Smooth Muscle Cells.” FASEB J. 17(6) 743-745, Saldeen, T,D.Li,etal. (1999), "Differential effects of alpha- ‘and gamma-focopherol on low-density lipoprotein oxida tion, superoxide activity platelet aggregation and arterial thrombogenesis" J Am Coll Cardiol, 34(3): 1208-1215. Schalkwiik. C, etal, 2008), “Incressed accumulation ofthe alyoxidation product N (carboxymethy?)Iysine in bears tiabeti patients” Dinbetes $2(Suppl. 1): A16S extended abstract, 709P. Sea, C., et al. (2000). “Tocotrieno! Poteatly_ Inhibits Gutamate-induced pp 60e-Ste Kinase Activation and Death of HT4 Neuronal Cells” J. Biological Chemistry 275: 13049-13085. Serbinova, EV. Kagan, etal (1991). “Pree Radical Recy- ling andl Intramembrne Mobility in the Antioxidant Properties of Alpha-Tocopherol and Alpha-Tocotrienol.” Free Rad. Biol. Mod, 10: 263-275 Sheppard, A.J. J. Pennington, etal (1993). Analysis and Distbution of Vitamin F in Vegetable Oils and Foods, ‘Vitansin Ein Heath ad Disease. L. Packer and J, Puebs, ‘Marcel Dekker, Ine. 9-31 Shi, H., N. Noguchi, eta. (1999), “Formation of phospho- lipidnydroperoxidesandits inhibition by alpha-tceoplter {rat brain synaptosomes induced by peroxynitrite” Bio- ‘chem Biophys Res Commun. 257(3) 681-656. ‘Smith, S. (1998), “The molecular pharmacology of PPAR- ‘gamma’ Clinical Courier 16(48). 3-4, Sylvester, P.and A. Theriault (2003). “Role of Tocotrenolsin the Prevention of Cardiovascular Disease and Breast Can- cer” Current Topies in Nutraceutical Research (2): 121- 136. Sawergold B.,etal (2003), “Intracellular nonenzymatic gly cation of hemoglobin in human erythrocytes is eontallsd by enzymatic deglycation mechanisms” Diabetes '52(Supp. 1): A190 extendad abstract. 815P. 6s Tan, B. (1992) “Antitumor Eifets of Palm Carotenes and "Tocotrienols in HIRS/J Hairless Female Mice” Nutrition Research 12: 163-8173, 0 o US 8,586,109 B2 5 Theriault, A. et al (1999). “Tocotrienol: A Review of its ‘Therapeutic Potential” Clinical Biochemistry 32(uly 309.319, Tomeo,A., etal (1995). “Antioxidant Eects of Tocotrienols in Patients with Hyperlipidemia and Carotid Stenosis.” Lipids 30: 1179-1183, “Traber, M, etal (1997). “Diet derived and topically applied tocotrienols accumulate in skin and protect the tissue ‘against ulinviolet light-induced oxidative stress.” Asia Paeifie J. Clin. Nut. 6: 63-67, Traber, M., et al. (1998). “Peneinition and distribution of alpha-tocopherol, alpha- or gammas-tocorienols applied individoaly onto murine skin Lipids 33: 87-91 ‘Tsai A.J. Kelly. etal (1978). “Stay onthe Efect of Megs ‘Vitamin F Supplementation in Man.” Am. J. Clin. Nut. 31 831-837. Wallace, A. D. Chinn, etal, (2003), “Taking simvastatin in the moming compared with in the evening: randomised contol teal.” BMI 32707418): 788 Watkin, ., M. Geller, et al. (1999), “Hypecholesterolemic ‘and antioxidant efecto ree bran oil non-saponifiables in hhypercholesterolemie subjects” Environmental & Nutei= sional Iterations 3: 115-122. ‘Watkins, T, M Bierenbaum, etal. (1999). Tocotrienols: bio- Togical and ealth benefits: Antioxidant Stats, Diet, Nuei- > tion, und Health. A. M. Papas, CRC Press: 479-496, Weber, C, etal (1997), "Efficacy of Topieally Applied Toco- ‘pherosand Tocoirienols in Protection of Murine Skin fom (Oxidative Damage Induced by U'-Inadiation” Free Radi cal Biology and Medicine, 2: 761-769. Yap, S.. K. Yuen, et al.2001). "Pharmacokinetics and bio- availability ofalpha-,gamma-and delts-tocotrienolsunder sliferent food tats.” J. Pharm. Pharmacol. 53(1}: 67-71 Yoshida, etal. 2003). “Comparative Study on the Action ‘of Tocopherols and Tocotrienols as Antioxidant: Chemical "Chemistry and Physics of Lipids yons-Menchana, et al (1999), Apoptosis in Human Breast Cancer Cells by Tocopherols ‘and Tocotrieaols Nurtion and Cancer 39: 26.32, BACKGROUND OF THE INVENTION 1. Field ofthe Invention ‘The invention ison the ompenitionsand uses ofthe extract [350-450 Dalton molecular weight faction] srom the annatto ced and such extract that i annato oil or oleoresin eontain- ‘ng_on-saponifiables, especially non-saponifiable terpe- noid 2. Description ofthe Related Art Tocotriaols generally ae classified as farnesyated eho manols (FC) ane mixed terpenoids. Tocopherol and tocot- Fienol are believe to have benelical elects because ey aot fs antioxidants. Tocotrienols, in particular, have been doc- ‘mented to possess hypacholesterolemic effets a8 wel a8 a2 ability to reduce atherogenic apolipoprotein B and lipopro- tein plasma levels. Further, tocotrenols are believed 10 be usefl inthe treatment of cardiovascular disease and cancer (Theriault A, etal, 1999; Watkins, T. etal, 1999), Deltae ‘wcotrienol and gamma-tocotrinol, in paticula, have been ‘Mentifed as eflective suppressants of cholesterol activity (Qureshi ., tal, 1995), and in inducing apoptosis of breast ‘cancer calls (Yi, W. eta, 1999), "Tocols, whieh includes tocopherols and toeotienols, have several sources, including several vegetable ols, suc a rice bran, soynean, corn and palm. owever, each source of tocot- rienols and tocopherols generally contains more thana single 0 o 6 tocol homolog. For example, pal ol and rice bran ol gen- cnlly include both tocolrienols and tocopherols. Further, alpha-tocopherol has been reported to attenuate certain effects of tocotrionols, such as the cholesteral-suppressive activity of gamma-tocotrienol (Qureshi, A. ea, supra.) In addition, because oftheir structural similarity, tocotrienols ‘and tocopherols can be difficult to separate Tocotrienols (inching delt- and gamma-tocotrienols) and geranyl geraniols have been discovered inthe seeds of ‘Bisa orellan Linn, otherwise known as the achiote tec. lis ‘a member of the Bisaceae funily and is uative to wopical America. Is grown commercially inother pars ofthe word ‘generally within 20° ofthe equator or more preferably within 15° ofthe equator. The seeds of Bisa orellana are the source of @ reddish range colorant, known as annato, that contains bixin and ‘orellin, both of which are carotenoid pigments. The colorant js used commonly in foods, dyes and polishes. Typically, annato i extracted from dehusked seeds in an agueous eaus- tie solution, The coloraat is precipitated from the aqueous solution by addition of acid, and the precipitated colorant is removed by filtration. The oily phase is usually separated from the agucou solution, and discarded asa byproduct, ‘A “byproduct solution of Bisa orellana soe components” {sdefined herein as. solution derived fom Bisa orellana seed ‘components having a concentration of annatto colorant sig- nificantly reduced from that of Bixa orellana soeds them selves, Other common terms for byproduct solution used for commercial products include: oil-soluble annatto. color, Anata oil, annato oleoresia, or annatto extract, “Anno extracts contin predominantly tocotrenols, gera- ay geraniols, bixins and to lesser extent components of leoresinows materials, of which all these major and minor components (both saponiliables and non-saponifiables) are ‘unique to annato extracts, These extracts can be used 38 nutritional supplement, nutaceutical, functional food and beverage, animal ingredient and pharmaceutical, or as an admixture with other natural extracts. [380-450 Dalton ‘molecular weight froction} or nutrcats. ‘Vitamin E constitutes a clas of tocochromanols contain at east four tocopherol ada leas four tocotrienals. “Toca ‘means birth, “pheren” to bring forth, “rene” three double bonds, “ol” sleohol, and “chroman” the attached ring stc- ture, The chroman alcoho has consistently indicated hat all E vitamers are powerful membrane-sluble antioxidants (Serbinova, F., etal, 1991; Yoshida, ¥, etal, 2003). The “iene” refers wo the 3 doulble-boaded tail in a tootrienal whieh differentiates it froma tocopherols saturated tal, The “iene tail (also referred to as fares al) is about a third shorter than the saturate tail (also refered toa phy! tail. These vitamin E tocochromanols include the lesser abundant tocodienols (“diene with to double-bonded tail) and {ecomonoenols (‘monoene” with one double-bonded tail ‘The Grook alphabets “alpha”, “beta”, “gamma” and “lt refer tothe degree of methyl substitutions in the chroman structure (Table 1). TABLE 1 ea ei Toone stil a a at US 8,586,109 B2 7 Vitamin B, including tocopherols and tocorienols, are typically 390-430 Dalions in molecular weight or more broadly” 350-450 Daltons in molecular weight, which inchides tocopherols and teotrienols without any methylated troup in the lower range and tocopherols and toeoeienols ‘with fully methylated groups in the higher range. The reason ior the antioxidant scavenging efficiency of tocotrienol (13) is because ofits shorter larnesy tail The farnesylatod tl enabes thetocotienol to move with superior mobility across cell membranes, giving rise to greater efli- ‘ciency in free-radical scavenging activity (Serbinova, E. et al, 1991; Packer [etal 2001), The longer phytyltilof the tocopherol (T1), which anchors deeply’ito ipid membranes, renders tocopherol Test mobile and hereby making. it Tess ‘ellcien as a scavenger than T3. The faresol ails required to reduce cholesterol, Famesol down rogulates, a8 well 35, degrades HIMG CoA reductase, the enzyme that controls cholesterol biosynthesis, It is believed that the farmesyl tail of tocottienol works by this mechanism (Pearce, B. eta, 1992), a possibilty that does notexis with tocopherol. Fully occupied met ‘on the chromanol (eg. alpha isomer) prohibits any reaction fand unoccupied substitution on the ring (eg. delta isomer) makes available reactive nitrogen oxide trapping capability Giang, Q, et a, 2001). As shown in FIG. 2, when the carbon position 5 is unoccupied, TI or T3 becomes "C-5 unsubsti- tuted”. ‘Tocotrenol contains tive repeating isoprene units giving riseto the faresyl til. Gran geraniols (GG), both eis and trans isomers, contain four isoprene repeating units and sur- Prisingly the toeotrienolfarmesy! moiety s contained in the ‘GG wil, and therefore GG is believed to be an unique com= ponent in the annatto extract, among other valuable campo- nents In ft, the entire GG molecules contained inand used fr the biosynthesis ofa tocotrienol molecule where one of the four isoprene moieties s embedded insidethe 3 chroman ring (Plson, C., et a., 1995; Caboon, B. eal, 2003; Dor- ‘mann, P, 2003), See FIG, 2, Hence, both GG and tocorienol structures have a common moiety, farnesy! group. which is believed to modulate biological activities including some ‘overlopping setivities, Annatto carotenoids (with conjugated 8 ‘double bonds) of various chain lengths and existing as non- oxygenated carotenes, oxygenated xanthophyll, suchas free aleoiols, acids, aldehydes, Ketones, esterified or etherfied ‘with other annato extract components are inclusive of the said extracts, and ofthis invention, FIG. 1 shows the tocols compositions from different nati ral sources ate highly varied, which argues for the standaed- ination of tocols lo produce an address appropriate diseases and conditions, a concept that ‘surprisingly s aot been implemented, Annatte extract con- ‘ains tocols tht are consistent, typically 90% deltT3 and 10% gamma-T3, The rationale for the use of toeorienol con ‘aining annato see lipids is to increase the in vivo and ex vivo biological activites ofthese admixtures, and to increase the biological potency ofthese admixtures by decreasing the at of alpha-T1 consumed. Alpha-tocopherol has been shown to interfere with tocotrienols ability to sequester c lesterol biosynthesis (Qureshi, A. eta, 1996) and alpha-TI thas no effect on anticancer aetvity (Guthrie, Net a, 1997; ‘Yu, W, etal, 1999), Large doses ofalphs-T! has produced a marked hypertriglyceridemic effect in animals (Khor, H- and ‘Ng, 2000; Lehman, J, 1981) and in humans (Farell, P and J. Bies, 1978; Tsai, A. et, al, 1978). Consequenty, the increase of delt-T3 andor gammna-T3 presents superior bio- Jogial and antioxidant properties vissi-vis aphia-TI Table 2 shows a non-exhaustive sample af diverse heath ‘benefits and protection of the eight classically and individu- ally own vitamers. Aneedexists to develop arationale for ‘an “appropriate speciram”tocals prec that wold normal- je andor optimize biologie functions without the crossover mitigation of tocopherols, To date, only “fall spectrum ‘ocols (implied presence ofall eight tocols) are commercially available, espotsing to deliver the composite health benefits of the individualized effects of those found in Table 2. It ‘emins unsubstantiated that fll-specrum tocols wll deliver the complete elects ofthese individually identified proper- ties. Therefore, these full-spectrum tocol lack a compos ‘ona, technica andor scientifie basis or rationale, Curent, fo present ar teaches compositions and methods of use in ‘humans, nor teaching so effieaciously and by simply adding satura tools extracts in appropriate combinations, TABLE 2 Tl er ant eff of nil ope ant ssl Gannett Denstsaper ih anes non Roney coton snl igo Vin sein ViaminE cins inode sens High evel of tla mate loa of cate. "hats acm, op eng atin seat ‘Aine nsay antwhes gem, Bath dei andar ae xii foc ystems eed cope reson ot), buns ce anda Rewer anoxia cope phe TL {aniN insane biog mens) Fit alienate ebexe, Tari we oc abbas neunsoue el sling ghana i ie, pala anes. Nat nits Highsbntace in anetn, Tc oe ane compost song tines peter thin pi Dla ep nee Saag nine US 8,586, 9 Is penerally desirable to target diseases with specifi isomers of tocols. For example, to lower lipids itis desirable to have the highest levels of delta-T3 and gamma-T3 and lowest levels of tocopherols, especially alpha-T! (Qureshi A. et al, 1996). Such compositional specificity which is. 5 exited to lover cholesterol is presently tnatainable. Table 3 presenis the natural compositional abundance typically ound in plant sourees. The natural abundance of palm and rice sources favors relatively large amounts ofalpha-T and amnia-T3, as well as, large amounts of tocopherols. C ‘quently, the disclosed use of palm and rice TRFS to lower Tipid has limited uty. Tis is because these TRF are high Jn alpha-T3, low or absence in delta-T3 and high in toco- phervls (30-50%), especially alpha-T1. Such compositional ‘ariabilty in TRF fctions have boca responsible fr several ‘equivocal clinical study outcomes (Mustad,V:, et al, 2002; Mensink, Rot a. 1999), Soy nd’ com ils eonlain exclusively tocopherols although they tend to be highest in the CS unsubstituted tocopherols (70968 delia-T1 and gamma-Tl) (oe, Shep- pard, A. eta, 1993) Such high levels of C5 unsubstituted ‘dlta-TI and pamma-T! from soy and com (Table 3) have ‘unique admixture application, which unexpectedly ave not been implemented TABLES, Socceofmteal Ain Canna Det Alpha Gama Det Seam ot “ino mu Nb ND as Rice ban ol MI oe ot 4S. 13 Rector “7 4027 Sta 2» bet ey ty a Comal ND__ND_ND_ si Ay ‘The effectiveness of cholesterol reduction is due to the ‘iamesyted tail of tootrienols where the isomeric potency of tive materials orchestrated by inflammatory stimuli and pre- vent the tethering of circulating monocytes an leukocytes ‘onto endothelial cells. The break of this restraint onto the rulating cells by the C5 unsubstituted tals is one ential Jntervention in proteting the integrity ofthe vasculature, and therefore athersclerosis TDL is well known for is role in eiculating cholesterol back tothe liver: Moreover, the HDL. particles (“good cho- Jester”) have antiinflammatory and anti-trombatie prop- ‘erties and suppress surface bioactive materials, as well a, markedly inhibit oxidized LDL formation and NFKB aetiva- tion (Robbesyn, B, et al, 2003; Jonkins, A. and T. Lyons, 2000), Lipidemia and Diabetic Dyslipidemia Tocotrienols have been ised for treatment of Fipidemia and lactic dyslipidemia, through tocotrcnol. inhibition of hepatic cholesterol biosyathesis, spevfically via the inibi- tionof HMGR, the rate limiting step in cholesterol synthesis. However, diabetes represents a plethora of pathological ‘evens besides cholesterol dysfunetion where 13 has not been represented to work, especially the CS unsubstituted tocols Sterol regulatory clement binding protein-1 (SREBP-1) is @ teanscription factor that responds to nutritional status and regulates metabolic gene expression in various organs, Including liver, adipose and muscle. It has been shown that insulin and glucose induced de novo fatty acid synthesis leading o arapidinerease in ipogenie xin skeletal musele. ‘Such lipid accumulation is associated with muse IR in obe- sity and T2DM, and is stimulated mediated via the SREBP-1 ‘expression (Guille-Deniau, [, eal, 2003). As discussed ‘earlier, TR. is tightly associated with increased lipids (MeLaughlin, Tet al, 2003) and increased insulin or HI (Defronzo, R, 1998) Diabetes Diabetes may be considered a hypercoagulable state. betic platelets ure hypersensitive 10 platelet ageregating gens, andthe vasoconstrictor TXB is a poweril platelet ‘aggregator. Excess TXBS release in diabetics has been as50- sé with CVD in these patients. These matters have been well documented and is herein referenced ia is entirety (Col- ‘well, J, 1997 & 2004), Aspicin specifically blocks the ‘omei-6 arachidonic aeid-derived thromboxane TXBS s thesis, which dramatically reduces platelet aggregation, and has been used as. primary and secondary strategy to prevent cardiovascular events in patients Aspirin’s major risks are gastric mucosal injury, G:l, hemorchoge and hemorrhagic stroke (Colwell, J, 1997 & 2004). It is known that gamma- TS, delia-T3 and gamma-T! inhibit platelet aggregation, ‘TXB4 and PGE2 (Qureshi, tal, 2002; 1999) and that delt-T3 preferentially absorbs 0 o 2 {ng human platelets (Hayes, Ket al, 1993), Gamnma-T3 and szamma-T' both metabolize in mammalian issues to gamuma- carboxyetbyl hydroxy chromans (7-CEHC), essentially the chromato] ring without the faruesy] and phyty wl. It has ‘een shown that theirpareat moieties as well as the y-CEHC setaboite inhibit PGE2 and COX? (iang. Qt al, 2001) ‘whieh farther supports that C5 unsubstituted tocols play & roe in inhibition of vasoconsrction, coapulation/toting, and chemotaxis, Therefore, C$ unsubstituted tocols should help to reverse the hypereoagulable sate of diabetes in safe ‘manner without side effects Diabetes isa disease of frank hyperglycemia and the con- two of sugar is always a standing goal. Is now recognized that glycation of lips and proteins contributes t diabetic macrovascular and microvascular diseases. For example, y= coxidized LDLs ineteased binding to extrocelilar matrix, have procosgulant effects extravasate into glomeruli retinae and atheroma. Also glycoxiized albumin aeresto the aor tie wall, According to one research study, there was an approximately 6cfold accumulation of plyeoxidized N-(car boxymethyl lysine (CML) in the hearts of diabetic patients sseomparedto normal subjects (Schallwij,C. etal, 2003). Other advanced glycation end-products (AGE) including Amadori modified proteins, all of which are sugar mediated oxidation to proteins are’ herein referenced by way of examples (Jaleel,., & al, 2003; Arai, Yet a, 2003; Sverwold, B, ei al, 2008), The measurement of plyeated hemoglobin (FIbATe) inthe blood isa standard marker to measure the history of sugar damage to tissues, Tocotrienols, specially ganuma-U3 inhibit protein oxidation (Kamat. al, 1997). Further, tocotrienols effectively: prevented an increase in AGE in normal rats, and decreased blood glucose and HDALe in diabetic rats (Nazaimoon, W. and B, Khalid, 2002), Peroxisomal Proliferator Activated Receptor Peroxisomal profifertor activated receptors (PPAR) are ‘members ofthe nuclear receptor transcription factors. The metabolic consequences of PPARY sctnation have been ‘mostly researched on adipose tstue where it is largely expressed (Smith, S, 1998: Kraegen, E1998) The met bolic effects of thiglidinediones (TZD) are: a) reduce hyperalycemis and hyperinsulinemia, b) lower FFA and TG Jevels,c) enhance IS and lower I states, and d use insulin to lower elucose. T2D are known PPARY agonists or activators ‘Many of PPAR activator functions are similar to PPAR& activator funetions. PPAR« has been actively researched on liver tissue, especially with regards 10 lipid se (eg. uptake and beta-oxidation). Even though the seton sits of PPAR (predominant in adipose) and PPAR (mainly in liver) are dhfferent thee activations have many overlapping clinical ‘outcomes. Typically [2D and ibratsaflet the activation of PARy and PPAR respectively. Tocotienls inthis inven- tion behave primarily ke a TZD (and secondarily like @ fihrate)asT3 metabolic effeetsmatch those fou listed above {or TZD. Surprisingly, the ehromano! ring structure found in "TS fs the same moiety found in troglitazone, a TZD. Put together, C5 unsubstituted T3 atvate or agonize the nclear transcription factor PPAR (7, cor mixed) and thereby cary fot the metabolic effets similar to those of TZDs and fibrates, in many common tissue sites (adipose, skeletal ruscle, and kidney, macrophage, VSMC, endothelial cell an ferent sites for PPAR (ear, gu) and PPAR (iver). ‘These various PPAR expressions share more common sites tan difereat ones. Mixed PPAR activation, besides PPAR and PPARar also inludes PPARA whose expression is ubigy ‘tous inal Gisves US 8,586,109 B2 13 Nervous Syste ‘Reversing damage tothe neurons and brain, whether acute ‘or chronic is an important heath sue. Potential neuropotent hrients ave to adress the issue of the blood bran barrier (BBB), over which the nutrients must cross over to enter the min. All tocotrienols enter the bin in general, and they protect glutamate-indveed neurotoxicity (Sen, C, et al 2000). As well, these CS unsubstituted tocols, both tocol” rienols (Meiniyre, Bet, 2000) and tocopherols (Lit, M, ‘etal, 2002) have particular bioavailability ito cellular tis sues, Bran cells are typically rich in PUFAs, especially the ‘omegi-3 DHA and FPA, and hence they are very susceptible 'o oxidation In sties with brain mitochondrial organelles, tocotrienols and TRF effectively prevented oxidative dam- ages to both lipids, as well as, proteins. Studies of brain mitochondria and rat microsomes indicate gamma-T3 i the most effective in oxidative protection followed by alpha-T3 and delta-T3 (Kamat, J. and T: Devasagayam, 1995; Kamat J etal, 1997). The gamma-TI is mostly Toeated in the biomembrunes of brain homogenates, and it markedly inhib- it lipid peroxidation inthe brain (Shi, H., ct al. 1999) In an extreme form of neurodegenerative genetic disease {amilial dysantonomis (FD), the development and survival of neurons (eg. sensory. sympathetic, parasympathetic) are seriously impaired (Mezey, E., eal, 2003). Delta-T3 increases the IKBKAP pene transcription 35-fold, and all ‘wcotrienols increase the IKAP transcripts and proteins (dela-T3 and gamma-T3 producing more than beto-T3 and alpha-T3) as muchas fold (Anderson, S.eta.,2003).None ‘of the tocopherols have any effec. "Toootrcnols have heen shown to reverse nerve damageand repair, genetic disposition, acute brain damage, glumate induced damage, chronie nervebrain damage, Alzheimer’s, Parkinson's, and Hatiagtons, Statin Drugs Statin drugs are known to derease isoprenoid pool (IP) products, including intenmediate and distal metabolites of ‘CoQIO, dolichol, heme, protein synthesis, and cholesterol (Goldstein, J. Land M.S. Brown, 1990) Topical Applications ‘The skin is an unique site for tocotrienals foe both topical and dietary tocotienol application (Traber, M. etl, 1998: Ikeda, S.. et al, 2008). Tocotrienols protect UV-induced ‘eqythean and also prevent the loss of skin vitamin F (Weber, C, etal, 1997; Traber, M, et al, 1997), Immune System Dietary tocotrenols given to immunodeficient mice pro- Jonged their survival, presumably via an immune system boost (Tan, B., 1992): Dietary tocotrenols increased imm- noglobuti (Ig, IgG and IgM) in ra spleen and MLN Iym- phocytes where the extent was generally more marked in the 1 group than the alpha-T1 group (Gu, J.,etal., 1997; Kaku, S., etal, 1999), Composiionaly, C-S unsubstituted toeot Fienols, composed of delta-T and gamma-T3, accounted for 7% of the toools used in these cited studies. Bone Mineralization Is known tht tocotrenols prevent the loss of bone min- ‘eral density, and improve the bone ealeium content of grow= jing aisle and Female, however, alpha-tl supplementtir ‘does not improve the mineralization of bone i female rats (lina-Nirwana S., etal, 2000, Norazlina, M., etal, 2002; Norazlina, M. eta, 2007). Hypertension ‘Gamma-T3 has been shown to prevent the development of ‘increased blood pressure in spontaneous hypertensive rat (SHR) and thatthe lowest dose of 1S mpg Teed (approxi mately translating to 75 mg T3dlay for humans) was best i 0 o 14 preventing hypertension (Newaz, M. and N. Nawal, 1999). Gamm-TS is also shown to be a vodium excreting agent, as ‘well a, increasing endothelial nitric oxide synthase (NOS) activity to teat essential hypertension (Igareshi, O, et a. 2003; Newaz, Metal, 2003). The water-soluble metaboli 2-CEHC (.e., ehromanol ring without the famesol til; see FIG.2)isthe same metabolite for gamma-T3 and for gamma TL. Sucha metabolite has also been identified for alpha @-CEHC. Accordingly, the metabolite for delis-T3 und del This CHC. Cholesters Biosynthesis ‘Some 80% of cholesterol in the human body is endog cenously produced in the liver, and the remaining 20% fro dietary sources. Physiological studies show that cholestes biosynthesis is nocturnal when dietary intake is at its Io ‘When statin is taken in the evening versus morning, lipids drop about 10% more (Willae, A. etal, 2008). Tocotienols taken with food more than double their bsorption and their ‘maximum concentrations peak 46 hours alter the supple- smeatation (Yap, S.,et al, 2001; Farus, Seta, 2003). Deni Annattoextract—A source of material known aka hyped: vet solution of Bika orellana seed components, which is jobiained as an vily oleoresinous material after the bulk of annato color, is Largely removed from ether the aqueous extract oF solvent extract of annatto seeds, Further, this byproduct contains a tocotrenol component and a geranyl geraniol component and ean be used as a source Tor the recovery of a tocotrienol component and a geranyl geraniol component "Annatto Extract Oil—The oily product from the annatto seed extoet containing the tocorienols and tocopherols, col Ieetively tocochromanols (Vitamin E), which are inthe 350- 4450 Dalton MW fraction of the extract. Vegetable cooking oils, commonly knownas trilyeerides, which havea rangeot ‘molecular weights of 500-1000 Daltons, and are essentially {ce or free tocotrienols and tocopheros (typically 0.05% ‘Vitamin E) are not included within this defition, ‘Appropriate Spectrum of Tacals-Mixtures of annatto ‘ocotrenols with other plantextactstoachieve efficacy ofthe newly constitted tocols composition. Annaito tocotienols satisfy this definition by having the highest amount of CS ‘unsubstituted tocorienols aad the lowest amount of toeo- pherls, especially alpha-T1 ‘Chemotactic Bioactive Materials Biochemical. mol- ecules involved in any oxidativesinfammatory process that leads to loss of arterial vasculature ‘Com Oil Thecily product from the com exteact contain- ing the tocotrienols and tocopherols, collectively tocochro- ‘manols (Vitamin E), which are in the 350-450 Dalton MW Iraction of the exact, Vegetable cooking oils, commonly known as triglycerides, which have a range of molecular ‘Weights of 500-1000 Dalton, and are essentially ree or free ‘of tocotrenols and tocopherols (¢ypically “0.1% Vitamin F) fare not included within this definition Cottonseed Oil—The ily product from the cottonseed extract containing the focotriens and tovopherols, collee- tively ocochromanols (Vitamin E), which rein the 350-450 Dalton MW fraction ofthe extract, Vegetable cooking oils, commonly known as triglycerides, which have a range of ‘molecular weights of 500-1000 Daltons, and ate essentially {ee or fee of tootrienols and tocopherols (¢pically 0.1% Vitamin F) are not incloded within this definition, (Cranberry Seed Oil-—The ily prot from the cranberry seed extoet containing the tecatienols and tocopherols, col US 8,586,109 B2 ), which aren the 350- 4450 Dalton MW ffaction ofthe extract. Vegetable cooking oils, commonly knownas tiglycerides, which havea range of molecular weights of 500-1000 Daltons, andl are essentially fee or fee of tocotrienols and tocopherols (typically <0.1% Vitamin E) are not included within this definition. "Essentially Free of Bixins-—A composition that contains Jess than 0.1% by weight of bins. Litehi Seed Oil—The oily produet from the hitehi seed ‘exact containing the tocotrienols and tocopherols, cole tively oeoehomanols (Vitamin E), which are in the 350-450 Dalton MW fraction ofthe exirci. Veyelable cooking ols ‘commonly known as triglycerides, whieh have a range of ‘molecular weights of 500-1000 Daltons, and are essentially fee r fee oftocotrienols and tocopherols (typically <0.1% Vitamin F) are not included within this definition, MW (Molecular Weight) Fraction—Refers to the part (Graction) of a substance (ie. natural extract that has chemi cals of that molecular weight. A standard analytical tool in Biochemistry or Chemistry isthe separaion of a substance into its various individual chemicals by their molecular Weight. Typical methods include column chromatography, HPLC, and SDS-PAGE. Fach ofthese analytical tools will separate a complex substance (Le, natural extract) So the individual chemicals will ravel at diferent rates though the mediuns (silica or coated silica, sepharose beads or poly- mmerizedl gos). In the case of column chromatography oF HPLC, th cari solution is collected (ees tubes) as it ‘comes off the column into “fractions”. There isa detector on theend ofthe column which devets the presence of materi “The detector charts the “peaks” and the corresponding frac tion that contains this material, The moleculie weights of these peaks can be calculated sing standards with known molecular weights (eg, Keyhole Limpet Femoeyanin)orthe pure compounds with previously identified MW "Natural Exteact -Nonsyathetie (natal. substance tat js derived from mineral, plant, or animal matter and does not undergo a synthetic process as defined in section 6502(21) of 7 USC. 680221) (the Act) For the purposes ofthis part rnonsynthetie i used asa synonym for natural as the term is used in the Act. (21) Syneti: The term “synthetic” means substance thats formlated or manufactured by chemical process or by a process that chemically changes a substance ‘exrcied [fom naturally occurring plant animal, or mineral sources, excep that sue tem shall not apply wo substances ‘created by naturally occurring biological processes] (Oat Bran Oil—The oily product from the oat bran extract ‘containing the tocotrienols and tocopherols, collectively tocochromanols (Vitamin F), which are in the 350-450 Dal- ton MW fraction ofthe extract. Vegetable cooking oils, com monly kaownas triglycerides, which havea range of molecu Jar weights of 500-1000 Daltons, and are essentially foe oF {reeof tocotrenolsand tocopherols (typically 0.1% Vitamin ) are nat included within this definition Olive Oil—The oily product from the olive extract contain- ing the tocotrenols an tocopherols, collectively tocochro- ‘manols (Vitamin F), which are i the 380-480 Dalton MW faction of the extract. Vegetable cooking oils, commonly known as tglyeerides, which have a range of molecul ‘weights of 500-1000 Daltons, and are essentially free or free ‘of tcotrienols and tocopherols (typically <0.1% Vitamin E) fare not included within this definition. Palm Oil—Theoily product from the palm extract eontain- ing the tocotrienols and tocopherols, collectively tocochro- ‘manols (Vitamin E), which are i the 380-480 Dalton MW faction of the extract. Vegetable cooking vils, commonly known as tiglyeerides, which have a range of molecule 0 o 16 ‘weights of 500-1000 Dalons, and are essentially free or free of oootrienols and tocopherols (ypically 0.19% Vitamin E) ‘are not included within this definition Purification of Annatto Extract—A process to obtain iso- mers of tocolienol and geranylgeraniol where the ratios are different from found in nature. Purified Annatio Geranylgeraniol Composition—A come positon purified from an snpatto extract that contains one or ‘more of the isomers of geranylgeraniol in a ratio that is ilerent From the natral ratio foued in an aato extract. ule Annatto Tacotrienel Composition —A compos ‘ion purified from an annatto extract that contains one ormore ofthe isomers of tocotrienol in arti that is different fom the ‘tural ratio Found in an anaatto extra. ‘Rice Fin Oil-—The oly pretoc fromthe rice bran extract containing the tocotrienols and tocopherols, collectively ‘ocochromanols (Vitamin E), which are in the 350-450 Dal- ton MW fiaetion ofthe extract, Vegetable cocking oils, com- vonly known as triglycerides, which have arange of molec Jar weights of 500-1000 Daltons, and are essentially fee or {ree of tocotrienolsand tocopherols (typically <0.1% Vitamin are not included within this definition. Soy Oil—The oily product from the soybean extract con- taining the tocotrienols and tocopherols, collectively toeo- chromanols (Vitamin F), which are inthe 350-480 Dalton (MW fraction of the extract. Vegetable cooking oils, com- ‘monly known as triglycerides, which have a range of moleen- Jar weights of 500-1000 Daltons, and are essentially foe oF {ree of tocotrienolsand tocopherols typically 0.1% Vitamin are not included within this definition. Sunflower Seed Oil—The oily product from the sunflower soed extract containing the tecorienols and tocopherols, col- Ieetively tocochromanols (Vitamin), which ar in the 350- 4450 Dalton MW fraction ofthe extract. Vegetable cooking oils, commonly knowitas triglycerides, which have a range ot ‘molecular weights of 500-1000 Daltons, and are essentially {tee of fee oftocotrienols and tocopherols (¢ypically <0.1% ‘Vitamin ) ae not included within this definition, ‘Toools A general temn for toeotsienols, tocopherol, ‘ocochromanols, vitamin Fs, mixed tocopherols and toeo sienols, TRFs including any additionally separated fraction- ated forms, admixtures of anato tocotrienols and other plant-derived TREs, appropriate spectrum tocol, admintare ‘of annatotocotrienols with other tcolsin onder to standard- ‘ne the amount and type of tocotrienols and/or tocopherols and the amount or ratio of alpha-tocopherol or other toca pherols present in the admixture “Tocopherol -Achnomanol with any degree of substitution ‘with saturated phyty] tal Substitution in the chromanol is taken to mean any adduet of the alcobo! and/or the ring moiety "Tocopheral-Free—A preparation of tactrienols and the ‘ocotrenols are predominantly deka-T3 andor gamma-T3 where the tocopherols are to 8 composition where the dela-to-gamma ratio of toc0 Fienolsisbetween I:510$:1, Ina more preferred embodiment the invention is drawn toa composition where the delta-to- amma rato oftcotrienols is 1:1 none embodiment the invention is drawn toacomposition ‘of tocotrienols comprising a mixture of an annatto extract [350-480 Dalton molecular weight fraction) and a 350-480 Dalton moleculae weight fraction of natural extract where the mixture has standardized low levels of tocopherols. In & preferred embodiment the invention is drawn toa composi- tion where the standardized level af tocopherols is =50% In ‘2 more preferred embodiment the invention is drawn to 2 ‘composition where the standardized level of tocopherols is 20%, In a more prefered embodiment the invention is ‘drawn to composition where the standardized level of toco- pherolsis «10%, Ina most prefered embodiment the inven- fiom sdrasen to composition where the standardized level of tocopherols is 1% In another embodiment the invention is ‘dria 10 a composition of tocotrienols where the natural ‘extract [350-450 Dalton molecular weight fraction} is, scloted from the group consisting of oils from rice bran, palm, eranbery see and itch see, ‘In atother embodiment the invention is drawa to acompo- sition comprising an annatto extract [350-450 Dalton ‘molecular weight fraction] containing tocopherol, where the tocopherol isalpha-TI. Ina preferred embodiment the inven- tion drawn toa composition comprising an annato extract [350-450 Dalion molecular weight fraction} containing 10c0- pherol, where the amount of the alpha-T! is =80% of the total tocopherols. Ina more preferred embodiment the invention is ‘dna o a composition comprising an annatto extract [350- 450 Dalton molecular weight fraction] containing toco- pherol, where the amount ofthe alphs-TI i 225% of the total tocopherols. Ina more preferred embodiment the invention is ‘drain to & Composition comprising an annatto extract [350- 4450 Dalton molecular weight fraction) containing toco- pherol, where the amount ofthe alpha-T! is 510% ofthe total tocopherols. Ina most preferred embodiment the invention is ‘drain {o a Composition comprising an annatto extract [350- 4450 Dalton molecular weight fraction) containing toco- pherol, where the amount of the alpha-T1 is 21% ofthe total tocopherols Tone embodiment the invention isdrawntoacomposition ‘comprising @ mixture of annatto extract (350-480 Dalton ‘molecular weight fraction} and a 380-480 Dalton molecular ‘weight fraction of # natural extract that is an appropriate spectrum. Ina prefered embodiment the invention is drawn 0 o 18 toa composition where 250% ofthe tocotienols are dlta-T3 ‘and gamma-T3, In a more preferred embodiment the inven- tion s draintoacomposition where 250% ofthe tocotrienols aredelts-T3. Ina most preferred embodiment the invention is {drawn toa composition where iis tocophero- fee, nome embodiment the invention isdeawn toa composition ‘whore the CS unsubstituted tocotrenols are 260%, and toe0- pherosare 15%. Ina preferred embodiment the invention is drawn t0 a composition where the CS unsubstituted tocot- ienols are 270% C5 unsubstituted tocotienols and <10% tocopherols, Ina more preferred one embodiment the inven- tion is drawn to a composition where the C5 unsubstituted tocotrienols are 280% CS unsubstituted tocotrienols and =5% tocopherols, nome embodiment the invention isdrawa toa composition comprising annatto extract [380-480 Dalton molecular ‘weight fetion), where blood evel o triglyceride decreases Ina prefered embodiment the invention is drawn to @com- position comprising anatto extract [350-450 Dalton molecw- Jar weight action}, where the decrease inthe blood level of the triglyceride has an effect selected rom the group consi ing of reversal of insulin resistance, metabolic syndrome, prodiabetes, diabetes und diabetes-related cardiovascular di Tn one embodiment the invention is dawn to a method to reverse insulin resistance, comprising aiministering annatto exttat [350-450 Dalton moteeulae weight tration} contain- ‘ng tocotrienols and potentiating insulin. In 2 more preferred embodiment the invention i drawn to method lowering the Fisk ofa disease selected fom the group consisting of CVD, ‘T2DM, hypertension, PCOS and ft liver disease none embodiment he invention isdrawa toa composition comprising annatto extract [350-480 Dalton molecular ‘weight fraction] where the tocotrienols lower CRP. In a pre- {ered embodiment the invention is drawn to composition comprising annatto extract [380-480 Dalton molecular ‘weight fraction] where the tocorienols lower CRP and pro- ‘eet against inflammation. Ia a more prfeered embodiment the invention is drawa t a composition comprising anna «extract [350-450 Dalton molecular weight fraction] where the tocotrenols elevate HDL. In # more preferred embodiment the invention is dren to a composition comprising annatto extract [350-450 Dalton molecular weight faction} where the tocotrienols lower cholesterol and decrease cardiovascular risk index. Ina more prefered embodiment the invention is {dawn toa composition comprising annatto extract [350-450 Dalton molecular weight fraction) where the tocotienols lower cholesterol and metabolic risk inde. Tnone embodiment the invention isdeawn toa composition comprising annatto extract [350-480 Dalton molecular ‘weight fraction) where the composition is tocopherol-free ‘with 298% tocotrienols, and tocatrienols ae predominantly delta and gamma 13. Ina more preferred embodiment the invention isdrawn toa composition where the composition is tocopherol-free with 298% tocotrienols and woeotienols are predominantly delta-T3, Tnoae embodiment the inventions deawn toa composition comprising. annatto extract [380-480 Dalton molecular ‘weight fraction] where the composition produces beneficial effets listed in Tables 2 & 4. In a preferred embodiment the invention is drawn to @ composition comprising. annatto «extract [380-480 Dalton molecular weight fraction} where CS ‘unsubstituted tocols produce the beneficial effets in Tables 2&4, US 8,586,109 B2 19 TARLI 4 xiion “Arata Roe Intanmation,asssihypereasion Aaa aloe ‘Coleen skin damage, ‘sto sleoe Iyperensioncstopaons ‘ast sh al ‘Anat and Rise Dslpsemia bypespyeeienis Atte lve Bll ‘atte soe ‘stan Soy Siyalone Intestin eel an ‘isto slooe ‘hemor biactvematent, Ata and Soy inoeconpiston,vaculhreless— Sayalone, Taman dyeknctin Resiace aman le ‘atte an Soy Bainter nage and Sonat ‘Ato an Soy Clycondtion tbAtSAE nate {nT dines persion Ueremion imei sss es none embodiment the invention isdnwwa toacomposition ‘comprising. annatto extract [350-450 Dalton mole ‘weight faction} where the tocopherol-fee anna tocot riegols, dlta-T3 and gamma-T3, lower lipids. Ina prefered ‘embodiment the invention is deswn to a composition com: prising annatto extract [350-450 Dalton molecular weight fraction} where the tocopherol-free annatte tocotrienols, dlia-T3 and gamaa-T3, lower lipids, particularly wiglyeer- ‘des. In a more preferred embodiment the invention is drawa toa composition comprising annatto extract [380-480 Dalton ‘molecular weight fraction} where the tocopherol-freeannatto tocotrienols, delta-T3 and gamma-T’, lower lipids, pariew- Jaely teiglyeerides and do not eause a drop in CoQUO level. La a more preferred embodiment the invention is drawn to 2 ‘composition comprising annatto extract [380-450 Dalton ‘molecular weight fraction] where the tocopherol-freeannatto ‘ocotrienols, deta-T3 and gamma-T3, lower lipids, particu- Jaely trglyeerdes, and eause an increase in CoQHO level. Ina ‘more prefered embodiment, the invention i drawn to a.com position comprising anatto extract [350-450 Dalton molecu lar eight friction] where the tocopherol-free sna toca rionols, deia-I3 and gamma-T3, lower lipids, particularly triglycerides, and cause an increase in CoQ1O level up 10 20%, Ina more preferred embodiment the invention isdrawn twa composition comprising anatto extract [380-480 Dalton ‘molecular weight fraction} where the oeophero-frseannatto ‘ocotrienols, deta-T3 and gamma-T3, lower lipids, particu- larly triglycerides, andl causean increase in CoQIO level more than 20%, none embodiment the invention is dwn toacomposition ‘comprising annatio extract [350-450 Dalton molecular ‘weight faction) where the tocopherol-free annatto tocot Fienols, deia-T3 and gamma-T3, lower lipids, particularly triglycerides, in normal weight, overweight and obese sub- Jeet. In a preferred embodiment the invention is drawn to & ‘composition comprising annatto extract (380-450 Dalton ‘molecular weight fraction] where the tocopherol-freeannatto 0 o 20 ‘ocotrenols, delta-T3 and gamma-T3, ower lipids, particw- larly triglycerides, in animals of both sexes. na more pre- ‘ered embodiment the invention is drawa toa composition comprising annatto extract [380-480 Dalton molecular ‘weight fraction] where the tocopherol-ffee annato toeot- senols, delta-T3 and gamma-I3, lower lipid, particularly teglyeerides, in humans of both sexes Ta one emboxliment the invention is dnwwa to a method ‘comprising administering annatto extract [350-450 Dalton ‘molecular weight lraction| where the dose of tocopherol-free ‘annato toeotrienos, dlia-T3 and gammy-T3, is given in a range fom 10t0 1000 mg per day. Ina preferred embodiment ‘the invention is caw to a method eompeising administering ‘annato extroct [350-480 Dalton molecular weight fraction] where the dose of tocopherol-free annattotocotrienos, dear Sand gamma-T3, i given in a range from 20 0 500 mg per day. Ina more prefered embodiment the invention isarasento ‘method comprising administering anatto extract [350-450 Dalton molecular weight fraction] where the dose of toe0- pherl-irve annatto tocotrenols, delia-T3 and gamnms-T3, is {given ina range from 50 to 150 mg per day Tnone embodiment he invention isdrawa toa composition comprising annatto extract [350-480 Dalton molecular ‘eight fraction] where the annato C5 unsubstituted toeot- rienols potentiate insulin to promote insulin sensitivity and reverse insulin resistance. In a preferred embodiment the invention is drawn 10 a composition comprising annatto extract [350-450 Dalton molecular weight fraction] where the fannatio CS unsubstituted tocotrienals potentiate insalin to romote insulin sensitivity and reverse insulin resistance with Supplementation of varying duration, In a more preferred embodiment the invention is drawn to a composition com- prising annatta extract [360-450 Dalton molecular weight {action} where the annatto CS unsubstituted tocotienols potentiate insulin to promote insulin sensitivity and reverse insulin resistance with supplementation in normal weight, ‘overcigh, and obese subjces. Ina more profered embod ‘meat the inveation is drawn to a compestion comprising annato extract [350-480 Dalton molecular weight fraction) ‘where the annatto C5 unsubstituted tocotrenols potentiate insulin to promote insulin sensitivity and reverse insulin resistance With supplemeatation in animals of both sexes. In ‘4 more preferred embodiment the invention is drawn to composition comprising annatto extract [350-450 Dalton ‘molecular Weight lraction] where the ocopherol-free anna ‘ocotrienols, deta-T3 and gamma-T3, lower lipids, pariew- Jarl triglycerides, in humans of both Sexes Tone embodiment he invention dean toa composition comprising annatto extract [380-480 Dalton molecular ‘weight fraction] where the annatotocotrenols reverse inst- linresistanceand potentiating insulin. Ina preferred embodi- ‘meat the invention is drawn to a compestion comprising ‘annato exioet [350-450 Dalton molecular weight fraction) ‘where the snnato tosotrienls reverse insulin resistance and potentiating insulin, and lower the risk of a disease selected trom the group consisting of CVD, T2DM, hypertension, PCOS anil Taly liver disease, In a more preerred embodi- ‘meat the invention is drawn to a compesition comprising anato extract [350-450 Dalton molecular weight fraction) ‘where the annato toeotrienols reverse insulin resistance and potentiating insulin, and reluce conditions i prediabetic and ‘iabetes patients Tone embodiment the invention isdeawn toa composition comprising annatto extract [350-480 Dalton molecular ‘eight fraction] where C5 unsubstituted tocols inhibit sur- fice chemotactic bioactive materials (CIM), In a preferred ‘embodiment the invention is drawn to a composition com- US 8,586,109 B2 2 prising annatto extract [350-450 Dalton molecular weight Fraction} where annatto CS unsubstituted TS inhibit surface chemotactic bioactive materials, Ina more preferred embod rent the invention is dria to a composition comprising annatto extract [350-450 Dalton molecular weight fraction] ‘where annato CS unsubstituted T3 inhibit surface chematac= tie bioactive materials and prevent the tether or adhesion of

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