A method of isolating, purifying and recrystallizing substantially pure lutein, preferably from saponified marigold oleoresin in its pure free form, apart from chemical impurities and other carotenoids. lutein may be used as an analytical standard or in cancer prevention trials and as a safe and effective color additive for human food.
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5. A method of obtaining substantially pure, free form lutein crystals from saponified marigold extract, comprising the steps of:
(a) mixing the marigold extract with water/alcohol mixture remove soluble impurities;
(b) lowering the temperature of the mixture to a temperature to precipitate lutein crystals;
(c) washing the lutein crystals with water to remove water soluble impurities;
(d) washing the lutein crystals with an organic solvent mixture to remove organic impurities, and thereafter recrystallizing the lutein.
1. The carotenoid composition consisting essentially of substantially pure lutein crystals derived from plant extracts that contain lutein, said lutein crystals being of the formula:
##STR00003##
wherein the lutein is substantially free from other carotenoids and chemical impurities found in the natural form of lutein in the plant extract.
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3. The lutein carotenoid composition of
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Final purification of lutein is accomplished by recrystallization from a binary solvent system, preferably a 1:1 mixture of dichloromethane and hexane. This binary solvent system has never been used in the purification of lutein. The choice of dichloromethane is because of the excellent solubility of lutein in this solvent. Instead of dichloromethane other halogenated solvents such as chloroform or 1,2-dichloroethane can also be used; and instead of hexane one can also use other straight chain hydrocarbons such as pentane, heptane, and petroleum ether (b.p. 30°-60° C.). Therefore, partially purified lutein (70% pure) is dissolved in a minimum amount of dichloromethane preferably containing 1% triethylamine and hexane is added until the solution becomes cloudy. The 1% addition of triethylamine to dichloromethane neutralizes the trace amount of the acids that are normally present in halogenated solvents. These residual acids could result in conversion of lutein to another carotenoid known as 2′,3′-anhydrolutein. Thus, the 1% addition of triethylamine is preferred to prevent lutein from undergoing side reactions and to maximize the yield of pure lutein.
The above cloudy solution is kept within the range of from −20° C. to −10° C. to commence recrystallization. At this range of temperature, the recrystallization is most efficient and it is usually completed within 2 to 3 hours. Storage of the solution at 0° C. prolongs the recrystallization process which may take as long as 24 hours and the recovery of pure lutein is not optimum. The crystals of lutein are then filtered off and washed, preferably with cold (0° C.) hexane. The pure crystalline lutein is dried in vacuo, i.e., at 50° C. for three days.
The purity of the resulting lutein is usually greater than 90%, most often greater than 97% as determined by UV/visible spectrophotometry. Based on quantitative HPLC analysis, the purified lutein consists of 94.79% lutein, 3.03% of its geometrical isomers, and a total of 2.18% of 2′, 3′-anhydrolutein, zeaxanthin, α-cryptoxanthin, and β-cryptoxanthin. The presence of the low levels of these carotenoids is not of any concern since these carotenoids are of dietary origin and are found routinely at much higher concentrations relative to that of lutein in human serum or plasma. NMR analysis showed no residual solvents (i.e., dichloromethane, hexane, ethanol, triethylamine) or other non-carotenoid impurities. The lutein purified according to this method exists in substantially purer form in comparison with lutein found in the matrix of any naturally occurring plant. In comparison with multi-step chemical synthesis, the purification of lutein according to this method is much more economical. Furthermore, lutein isolated and purified from marigold flowers does not contain hazardous chemical impurities as a result of various organic reagents that are normally employed in the sequential synthesis of this compound.
The following examples are offered to illustrate but not limit the product and process of the present invention.
Saponified marigold oleoresin available commercially as “Kemin Yellow Oil” was obtained. It was previously processed in the following manner.
Upon receipt the flowers were tested for herbicides and pesticides in order to ensure that they meet qualifications for food ingredients. The completed oleoresin extract (200 g) was then subjected to saponification with aqueous potassium hydroxide. This was accomplished through continuous mixing under heat (65°14 70° C.) of food grade aqueous potassium hydroxide (45%) and the oleoresin until greater than 98% of lutein was free from fatty acid esters. The saponification was normally completed within 35 minutes. The product was then homogenized with a mixture of distilled water (700 mL)/ethanol (300 mL, food grade):2.3/1 at room temperature for 30 minutes. The mixture was filtered off and the filtrate was discarded. The retained orange precipitate of lutein was washed with distilled water until the filtrate was almost colorless and the pH was neutral. The precipitate was then washed sequentially with cold (0° C.-5° C.) ethanol (200 mL) and hexane (200 mL), respectively. The resulting lutein obtained as orange crystals from three to be 70% pure by spectrophotometric analysis.
Final purification was accomplished by recrystallization from a 1:1 mixture of dichloromethane and hexane by dissolving the 70% pure crystals in about 550 ml of dichloromethane containing 1% of triethylamine. The hexane was added until the solution became cloudy. The cloudy solution was kept at −20° C. to −10° C. to commence recrystallization. This was completed within about 3 hours resulting in orange crystals of lutein. The crystals were then filtered off and washed with cold (0° C.) hexane (200 ml) and dried in vacuo at 50° C. for 3 days. The purity of lutein in this instance was greater than 97%.
Saponified marigold oleoresin (200 g) was homogenized with a mixture of distilled water/ethanol (food grade) at various ratios at 0° C.-10° C. for 30 minutes. The mixture was filtered off and the filtrate was discarded. The retained orange precipitate of lutein was washed with distilled water until the filtrate was almost colorless and the pH was neutral. The precipitate was then washed sequentially with cold (0° C.-5° C.) ethanol (200 mL) and hexane (200 mL), respectively. The yield of lutein obtained as orange crystals from three experiments employing different ratios of water/ethanol are shown below. The lutein obtained in all three experiments was shown to be about 70% pure by spectrophotometric analysis.
Weight (g) of Crude Lutein
Isolated from 200g Saponi-
Ratio of Water/Ethanol
fied Marigold Oleoresin
2.3/1
28
3.0/1
22
1.0/2
15
This example demonstrates the highest yield of lutein is obtained with 2.3/1 ratio of water/ethanol. The final purification step by recrystallization was accomplished as in Example 1, to give lutein in excess of 97% purity.
The purity and the constituents of the lutein isolated from “Kemin Yellow Oil” have been determined by UV/visible spectrophotometry as well as high performance liquid chromatography-photodiode array detection/mass spectrometry (HPLC/MS). In addition proton nuclear magnetic resonance (NMR) spectrum of the purified lutein has been obtained in order to determine the presence of trace amounts of residual solvents and other impurities.
UV/Visible Spectrophotometric Analysis: The purity of lutein is greater than 97% as determined from the absorption spectrum of this compound in ethanol, which exhibits maxima at 422, 446, and 476 nm and extinction coefficient of 1% E=2550 in ethanol at 446 nm.
HPLC/MS Analysis: The purified lutein has been examined by HPLC equipped with a photodiode array detector on both a C18-reversed phase column and a silica-based nitrile bonded column. The HPLC system was interfaced with a Hewlett-Packard particle beam mass spectrometer. The analysis of the purified lutein with HPLC/MS system in addition to HPLC peak purity determination at various wavelengths also provided further evidence for purity of this compound from the total ion chromatogram obtained by mass spectrometry analysis.
The HPLC profile of the purified lutein on a C18-reversed phase column was observed. 1). As revealed in chromatograms, the purified lutein also contains four other carotenoids as minor impurities. In the order of chromatographic elution on a C18-reversed phase column, the carotenoids in the purified and recrystallized lutein are: lutein+zeaxanthin (coeluting HPLC peaks, 99.31%), 2′, 3′-anhydrolutein (0.23%), α-cryptoxanthin (0.34%), and β-cryptoxanthin (0.10%). Since lutein and its geometrical isomers as well as zeaxanthin (present as a minor component) are not separated on the C18-reversed phase column, a nitrile bonded column was employed. The HPLC profile of the purified lutein on the nitrile bonded column revealed that under these chromatographic conditions 2′, 3′-anhydrolutein, α-cryptoxanthin, and β-cryptoxanthin appear as one HPLC peak, while lutein and zeaxanthin and several of their geometrical isomers are well separated. Based on Quantitative HPLC analysis of purified lutein on C18-reversed phase and nitrile bonded columns, the chemical composition of this compound isolated and purified from extracts of marigold flowers are shown in Table I.
TABLE I
Carotenoid Composition of Lutein Isolated and
Purified from Extracts of Marigold Flowers (Tagetes
erecta, variety orangeade)
Carotenoids
Composition (%)
all-E Lutein
94.79
9Z-Lutein
0.14
9′Z-Lutein
0.15
13 + 13′Z-Lutein
0.29
Poly Z-Lutein
2.45
Total of Lutein + Z-Isomers
97.82
2′,3′-Anhydrolutein
0.23
All-E-Zeaxanthin
1.51
α-Cryptoxanthin
0.34
β-Cryptoxanthin
0.10
Total of Other Carotenoids
2.18
From these data it appears that the purified lutein from marigold flowers consists of 94.79% of all E-lutein, 3.03% of its geometrical isomers (Z-luteins), and a total of 2.18% of 2′, 3′-anhydrolutein, zeaxanthin, α-Cryptoxanthin, and β-Cryptoxanthin. The presence of the low levels of these carotenoids in the purified lutein product should not be of any concern since these carotenoids are of dietary origin and they are found routinely at much higher concentrations relative to that of lutein in human serum/plasma. Individual carotenoids separated by the two HPLC columns described above have also been characterized from comparison of their absorption and mass spectra determined by a photodiode array detector interfaced into a mass spectrometer with those of standards characterized previously.
NMR Analysis: The proton NMR spectrum of the purified lutein was in agreement with the spectrum of this compound reported previously and no residual solvents (i.e., dichloromethane, hexane, methanol, triethylamine) or other non-carotenoid impurities (plant sterols and fatty acids) could be detected in this sample.
No substantially pure form of lutein derived from plant extract having these levels of purity has heretofore been available.
Carotenoids are considered among the fat soluble nutrients and are usually associated with the lipoprotein fractions of human blood. Therefore, the absorption and bioavailability of carotenoids is significantly increased if these compounds are orally ingested with a small amount of an oil or foods that contain certain amounts of lipids. In a human study that was conducted the following preparation of lutein resulted in excellent absorption of this compound by the subjects as determined from the analysis of their plasma carotenoid profile. In the following, the procedure employed for the preparation of a small batch (100 dose, each 10 mg) of purified lutein is described.
Purified lutein from marigold flowers extract (1 g) was added to absolute alcohol (75 mL). To this solution α-tocopherol (50 mg) and food grade polysorbate 80 (an emulsifier, 4 g) was added and the mixture was sonicated for 10 minutes. The addition of α-tocopherol (0.01% by weight) was to stabilize lutein and prevent this compound from possible oxidation during long term storage. The above suspension was then mixed with 350 g of light and mild olive oil (saturated fat/polyunsaturated fat=2/1) and the mixture was sonicated for 5 minutes. This resulted in a suspension of lutein in olive oil which was stored under nitrogen in a refrigerator. 7 mL aliquots of this suspension was shown by spectrophotometric analysis to contain 10 mg of lutein. At various intervals, the stability and the purity of lutein suspension in the olive oil was determined by spectrophotometric and HPLC analysis. The stability studies revealed that lutein prepared and stored under the conditions described above is stable for up to one month. In one study each 7 mL aliquot of the olive oil (containing 10 mg lutein) was spread on a bagel prior to ingestion. Alternatively, this small volume of oil can be readily taken with other foods. Although this preparation of lutein dose is appropriate for conducting human studies with a limited number of subjects, other preparations of this compound into tablets or capsules can be accomplished. The formulation of β-carotene into tablets and capsules which has been developed by Hoffmann-La Roche Inc. (Nutley, N.J.), may also be found suitable for lutein with some minor modifications.
From the above examples it can be seen that applicant has prepared the first ever substantially pure lutein as derived initially from plant extracts. The product is substantially free of impurities, substantially free of other carotenoids, and contains only very low levels of certain carotenoids of dietary origin. The presence of these carotenoids are of no concern since they are found routinely at much higher concentrations in human serum/plasma relative to that of lutein.
It can therefore be seen that the invention accomplishes at least all of its stated objectives.
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