Organic compounds -- part of the class 532-570 series – Organic compounds – Fatty compounds having an acid moiety which contains the...
Reexamination Certificate
2000-04-20
2001-07-17
Carr, Deborah D. (Department: 1621)
Organic compounds -- part of the class 532-570 series
Organic compounds
Fatty compounds having an acid moiety which contains the...
C554S013000, C554S019000, C554S020000, C568S830000
Reexamination Certificate
active
06262284
ABSTRACT:
1. FIELD OF THE INVENTION
A simultaneous process for extraction, isolation, and purification of lutein, zeaxanthin, and several rare carotenoids from Marigold flowers, Lycium Chinese Mill, and green plants.
2. BACKGROUND OF THE INVENTION
Carotenoids are amongst the most widespread of the naturally occurring groups of pigments and are found in all families of the plant and animal kingdoms. To date, as many as seven hundred carotenoids have been isolated from various sources and their chemical structures have been characterized. Numerous epidemiological studies in various populations have shown that consumption of substantial amounts of fruits and vegetables rich in carotenoids reduces the risk of acquiring several types of cancers. As a result, for nearly two decades, scientists have been focussing most of their attention on investigating the protective effect of beta-carotene in prevention of cancer, cardiovascular and eye diseases. This is despite the fact that beta-carotene is only one of the prominent carotenoids found in fruits and vegetables whose consumption has been associated with health benefits in humans. The reasons for such an intense focus can be attributed to the pro-vitamin A activity of beta-carotene and the lack of commercial availability of other prominent food carotenoids.
During the past decade, the author and coworkers have isolated, identified, and quantified carotenoids from fruits and vegetables commonly consumed in the U.S. These studies have revealed that as many as 40 to 50 carotenoids may be available from the diet and absorbed, metabolized, or utilized by the human body (Khachik et al. 1991, Pure Appl. Chem., 63: 71-80). However, among these, only 13 carotenoids and 12 of their stereoisomers are routinely found in human serum and milk (Khachik et al. 1997, Anal. Chem. 69:1873-1881). In addition, there are 8 carotenoid metabolites and one stereoisomer in human serum or plasma which result from a series of oxidation-reduction reactions of three dietary carotenoids, namely, lutein, zeaxanthin, and lycopene. These metabolites were first isolated and characterized by Khachik et al. (1992, Anal. Chem. 64: 2111-2122). In another study, the ingestion of purified supplements of dietary (3R,3′R,6′R)-lutein and (3R,3′R)-zeaxanthin was shown to not only result in an increase in the blood levels of these compounds in humans but also increased the concentration of their oxidative metabolites in plasma (Khachik et al. 1995, J. Cellular Biochem. 22:236-246). These findings, for the first time, provided preliminary evidence for the long standing hypothesis that carotenoids may function as antioxidants in disease prevention. In addition, these results also established the importance of non-vitamin A active dietary carotenoids, particularly, lutein, zeaxanthin, and lycopene.
In 1985 and 1993, Bone et al. (1985, Vision Res. 25: 1531-1535; 1993, Invest. Ophthalmol. Vis. Sci. 34: 2033-2040) elegantly demonstrated that the human macular pigment is a combination of lutein and zeaxanthin and speculated that these dietary carotenoids may play an important role in the prevention of an eye disease, namely, Age-Related Macular Degeneration (ARMD). This was later confirmed in a case-controlled epidemiological study in which the high consumption of fruits and vegetables, rich specifically in lutein and zeaxanthin, was correlated to a 43% lower risk of ARMD (Seddon et al. 1994, J. Am. Med. Assoc. 272: 1413-1420). More recently, in addition to lutein and zeaxanthin, the author and his co-workers reported on the isolation and identification of one major and several minor oxidation products of lutein and zeaxanthin in human and monkey retinas (Khachik et al. 1997, J. Invest. Ophthalmol. Vis. Sci. 38:1802-1811). The authors then proposed a metabolic pathways for these compounds which may play an important role in the prevention of ARMD. Therefore the commercial production of the purified forms of dietary carotenoids in general, particularly lutein and zeaxanthin, is of great importance. These carotenoids may be used, individually or in combination, as nutritional supplements and food colorants as well as in clinical trials where their potential health benefits in the prevention of ARMD and cancer can be investigated.
Although lutein and zeaxanthin may be obtained from certain fruits and vegetables, the isolation of lutein from extracts of marigold flowers and zeaxanthin from berries of
Lycium Chinese Mill
(LCM berries) proves to be most economical. In Marigold flowers lutein is the major carotenoid and is normally accompanied by about 3-6% zeaxanthin; in LCM berries zeaxanthin is the major carotenoid and is completely free from lutein. In both of these plants, lutein and zeaxanthin are esterified with fatty acids such as lauric, myristic, and palmitic acids. The purification of lutein fatty acid esters from marigold flowers was patented by Philip in 1977 (U.S. Pat. No. 4,048,203). However, dietary carotenol fatty acid esters, in general, have not been detected in human plasma or serum. Therefore, upon ingestion of purified lutein fatty acid esters by humans, these compounds partially undergo hydrolysis in the presence of pancreatic secretions in the small intestine to regenerate free lutein which is then absorbed [Khachik et al. Pure & Appl. Chem., 63(1): 71-80, 1991]. Since the most abundant dietary form of lutein is not its esterified form, a commercial process that could provide lutein free from fatty acids was needed.
A method for the purification of free lutein from extracts of marigolds was first reported in 1991 [Tyzkowski and Hamilton, Poultry Sci., 70(3): 651-654, 1991]. However because this method was extremely time-consuming, used harmful organic solvents, and produced poor yields, it could not be implemented commercially.
In view of the important biological activity of lutein and zeaxanthin, the author developed a process for isolation, purification, and recrystallization of lutein from saponified Marigold oleoresin which was patented in 1995 (Khachik, U.S. Pat. No. 5,382,714). The saponified Marigold oleoresin was obtained from Kemin Industries (Des Moines, Iowa) and is normally prepared from extraction of dried Marigold petals with n-hexane, followed by saponification and solvent evaporation. To date, this process is the only available method for isolation and purification of lutein (containing 3-6% zeaxanthin) from Marigolds in purities greater than 97%. Recently, another process for the isolation of lutein from a saponified Marigold oleoresin has been reported wherein lutien can be obtained in 70-85% purity (U.S. Pat. No. 5,648,564, 1997). This process employs propylene glycol (40.9% weight percent) and an aqueous alkali (18.2% weight percent) to saponify a hexane extract of dried Marigold petals (marigold oleoresin, 40.9% weight percent) containing lutein esters at 70° C. in 10 hours.
There are several major disadvantages with this process; these are discussed as follows. The Marigold oleoresin is prepared by extraction of dried marigold petals in boiling n-hexane for extended periods of time. Since lutein/zeaxanthin, and carotenoids in general, are sensitive to heat treatment, this procedure can result in degradation or isomerization of these compounds. Furthermore, according to the Food and Drug administration's (FDA) Federal Register Documents, Code of Federal Regulations & Food, Drug, and Cosmetic act, n-hexane is considered among the solvents whose levels in foods and pharmaceutical products should be limited. The classification of organic solvents by the FDA will be described later in this text.
In the next step of this process, the hydrolysis of lutein and zeaxanthin esters in the marigold oleoresin is conducted in an aqueous solution in the presence of alcohol and propylene glycol in which the fatty acid esters of lutein and zeaxanthin have very low solubility. As a result, this process requires high temperatures of up to 70° C. and 10 hours to complete the saponification. This can once again result in the degrada
Carr Deborah D.
Dykema Gossett PLLC
University of Maryland
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