CLIAMS:We claim:
1) An enrichment process for obtaining lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1, said process comprising steps of:
(e) identifying source of lutein and adsorbing it with a stationary phase;
(f) selecting appropriate column and mobile phase followed by packing the column with the stationary phase;
(g) loading the column, eluting, collecting and combining the fractions based on chromatographic monitoring; and
(h) concentrating, isolating and drying to remove residual solvent to obtain the mixture.
2) The process as claimed in claim 1, wherein said lutein source is marigold extract or regular lutein.
3) The process as claimed in claim 1, wherein said stationary phase is selected from a group comprising silica gel and alumina having a mesh profile 230/400.
4) The process as claimed in claim 1, wherein said mobile phase is a single solvent system, preferably chloroform.
5) The process as claimed in claim 1, wherein the mixture was dried under high vacuum at a temperature ranging from about 35oC to about 45oC, preferably at a temperature of about 40oC to remove chloroform as a residual solvent.
6) The process as claimed in claim 1, wherein said chromatographic monitoring is thin layer chromatography.
7) The process as claimed in claim 1, wherein fractions were combined based on thin layer chromatographic monitoring.
8) A lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as claimed in claim 1.
9) A formulation comprising lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as claimed in claim 1.
10) The formulation as claimed in claim 9, wherein said formulation is a pharmaceutical or herbal formulation.
,TagSPECI:FORM 2
The Patent Act 1970
(39 of 1970)
&
The Patents Rules, 2003
(See section 10 and rule 13)

Complete Specification

Title: An enrichment process for obtaining lutein and zeaxanthin mixture

Name of the Applicant:
Prakruti Products Pvt. Ltd., Sagar Complex, Ground Floor, Maruti Temple Road, Karwar – 581301, Karnataka, INDIA.
Nationality: Indian

The following specification particularly describes the nature of the invention and the manner in which it is to be performed.

TECHNICAL FIELD
The present disclosure relates to a chromatographic enrichment process for obtaining lutein and zeaxanthin mixture. In particular, the present disclosure relates to an enrichment process for obtaining lutein:zeaxanthin mixture at a ratio of about 10:2 and about 10:1 from a lutein source.
Background

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. As known to all, carotenoid is a kind of very important substance. Carotenoid is often composed through coupling of molecules of five carbon atoms, namely isoprene, and there are multiple unsaturated bonds in the molecular structure. The existence of these conjugated double bonds has endowed carotenoid with related color and some important physiological functions. While based on whether or not oxygen exists in the molecular structure, carotenoid can be further divided into two types, carotenes and xanthophyll, respectively, the former including .alpha.-, .beta.-, .gamma.-carotenes, and lycopene, etc, while the latter covers lutein, zeaxanthin, astaxanthin, and canthaxanthin, etc. These carotenoids are all biochemically and commercially quite important, and all have corresponding important physiological functions. The lutein and zeaxanthin of them have aroused increasing recognition from people in recent years, and new progresses have been made continually on research and product development related in this area.
The structural formulas of lutein and zeaxanthin are respectively as follows.

They are isomers and the only difference between the two structural formulas is the positions of double bonds on one (instead of two) end ring. The positions of double bonds on two end rings for the former are symmetrical, while those for the latter are asymmetrical. The entire linear chain part for each molecule of lutein and zeaxanthin is a conjugated structure, namely, provided with alternate double bonds and single bond. In the molecule of zeaxanthin, the conjugated structure extends to the first bond on the two end rings, while the conjugated degree for lutein is even lower, as the correct arrangement of complete conjugated structure has not yet been formed for double bonds on one of its end rings. This is the discrepancy in molecular structure that has caused certain difference in the functions of lutein and zeaxanthin.

As natural pigment matters lutein and zeaxanthin have a very wide distribution in natural world. They mainly exist in higher plants, alga, fishes, shells, and bacteria, and they always exist inside organisms in the form of esters. In these matters, marigold is a good source of lutein and zeaxanthin, and there is often about 2 g xanthophylls in 100 g marigold fresh flowers, mainly lutein wherein, accounting for more than 90%, while the rest are zeaxanthin and a few other carotenoids. The same as in marigold, among sources from other higher plants and alga, lutein has accounted a larger proportion relative to zeaxanthin, while the amount of zeaxanthin is larger than that of lutein in corn.
Regular Lutein contains the mixture of Lutein and zeaxanthin (91.85 % Lutein and 6.924 % zeaxanthin).

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.
As a kind of carotenoid, lutein and zeaxanthin are initially and now still used as a pigment, such as used in the pigmention of egg yolk, the out skins of poultry (such as skin, leg, and beak) and hypodermal fat, muscle, as well as the outer skins of fishes and shells (skin, scale, and shell). Also it has recently begun to use lutein and zeaxanthin as a functional food colorant. However, as mentioned before, as there is one extra conjugated double bond in the molecular structure of zeaxanthin than that of lutein, zeaxanthin can show stronger golden yellow color and more lasting color than lutein, and on this account, zeaxanthin is preferred for use under many conditions.
Besides, lutein and zeaxanthin are the only two carotenoids existing in the retina of human body, and they are very important for curing and preventing age-related macular degeneration (ARMD) disease, able to effective in protecting the eyes from suffering ARMD and blindness there from. Since zeaxanthin is fully conjugated, it is able to certain degree to provide better protective function for trauma arising from resistance to luminous energy. In fact, some researches in the mid and late stages of the 80s have also proved that zeaxanthin exists mainly in the subzone at the positive macular center of human eyes, and the amount of zeaxanthin would gradually reduce deviating concentrically from the sunken part and approaching the outer macular circumference, while that of lutein will gradually increase alongside. In the macular periphery, lutein is the main yellow coloring matter.

As mentioned above, fruits and vegetables are the most important source of carotenoids in the human diet, and knowledge about this is important for preventive medicine. Several publications report the qualitative and quantitative content of carotenoids in different fruits and vegetables. However, many of the procedures used did not allow the isolation of lutein and zeaxanthin at a ratio as reported in the instant disclosure. In light of the above, it is very important to have lutein and zeaxanthin mixture in a ratio which is useful for various food and pharmaceutical purposes.

SUMMARY
Accordingly, the present invention provides an enrichment process for obtaining lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1, said process comprising steps of: identifying source of lutein and adsorbing it with a stationary phase; selecting appropriate column and mobile phase followed by packing the column with the stationary phase; loading the column, eluting, collecting and combining the fractions based on chromatographic monitoring; concentrating, isolating and drying to remove residual solvent to obtain the mixture; and is also provided for a lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as explained above; and is also provided for a formulation comprising lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as explained above.
DETAILED DESCRIPTION
Before explaining any one embodiment of the present disclosure by way of drawings, experimentation, results, and pertinent procedures, it is to be understood that the disclosure is not limited in its application to the details as explained in below embodiments set forth in the following description or illustrated in the drawings, experimentation and/or results. The disclosure is further capable of other embodiments which can be practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary--not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

The present disclosure is in relation to an enrichment process for obtaining lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1, said process comprising steps of:
(a) identifying source of lutein and adsorbing it with a stationary phase;
(b) selecting appropriate column and mobile phase followed by packing the column with the stationary phase;
(c) loading the column, eluting, collecting and combining the fractions based on chromatographic monitoring; and
(d) concentrating, isolating and drying to remove residual solvent to obtain the mixture.
In yet another embodiment of the present disclosure, said lutein source is marigold extract or regular lutein.
In still another embodiment of the present disclosure, said stationary phase is selected from a group comprising silica gel and alumina having a mesh profile 230/400.
In still another embodiment of the present disclosure, said mobile phase is a single solvent system, preferably chloroform.
In still another embodiment of the present disclosure, the mixture was dried under high vacuum at a temperature ranging from about 35oC to about 45oC, preferably at a temperature of about 40oC to remove chloroform as a residual solvent.
In still another embodiment of the present disclosure, said chromatographic monitoring is thin layer chromatography.
In still another embodiment of the present disclosure, fractions were combined based on thin layer chromatographic monitoring.
The present disclosure is in relation to a lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as detailed in the above embodiment.
The present disclosure is in relation to a formulation comprising lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as detailed in the above embodiments.
In yet another embodment, said formulation is a pharmaceutical or herbal formulation.
Additionally, the disclosure is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope of the present invention. On the contrary, it is to be clearly understood that various other embodiments, modifications, and equivalents thereof, after reading the description herein in conjunction with the drawings and appended claims, may suggest themselves to those skilled in the art without departing from the spirit and scope of the presently disclosed and claimed invention.

Example 1: Identifying source of lutein
The source of lutein and zeaxanthin are selected from wide variety of fruits and vegetables. It is herewith admitted by reference a journal publication entitled “Fruits and vegetables that are sources for lutein and zeaxanthin: the macular pigment in human eyes”, Br J Ophthalmol. 1998 Aug; 82(8): 907–910, which captures about various sources which are rich in lutein and zeaxanthin.

Example 2: Preparing for column chromatography to obtain the mixture
Loading of the column using proper stationary phase (silica gel or alumina) is a vital aspect. Proper stationary phase is the key factor for the ideal separation of the two molecules, lutein and zeaxanthin, and the enrichment of respective molecule. Column was selected, the length of the column is 20 times more than actual diameter of the column and the diameter should be proportionally match with loaded material weight like 1gm of the regular Lutein was loaded in 2cm diameter and 40cm length. Loading was done with aluminum oxide / silica gel which have a mesh profile 230/400 and the quantity of the stationary phase was 20 times more than the weight of the loaded material and for binding 3 times more than the loaded material. Adsorption of the regular Lutein with a stationary phase was done using stationary phase, which is further mixed and loaded. The choice of the mobile phase was critical for the good separation of Lutein and zeaxanthin. Both the molecules are having same polarity and slight separation was observed on the double development of the Thin Layer Chromatography (TLC) system. The choice of the solvent was more challenging because the separation from mixture of solvent was not recommendable due to reusable factor was not possible in this case. Single solvent was the best answer for the development of this product because of the feasibility of easy handling and recovery of the same. Each fraction was collected and monitored by thin layer chromatography using silica gel plate. Based on the TLC pattern the fractions were combined, concentrated and isolated. This step is followed by removal of residual solvent under vacuum oven. Dried zeaxanthin rich material contains the residual solvent as chloroform, which can be removed by drying the product under high vacuum at 40oC.
Example 3: Process for lutein and zeaxanthin mixture at a ratio of 10:2 and 10:1
The source of lutein and zeaxanthin are selected from wide variety of fruits and vegetables. It is herewith admitted by reference a journal publication entitled “Fruits and vegetables that are sources for lutein and zeaxanthin: the macular pigment in human eyes”, Br J Ophthalmol. 1998 Aug; 82(8): 907–910, which captures about various sources which are rich in lutein and zeaxanthin. Loading of the column using proper stationary phase (silica gel or alumina) is a vital aspect. Proper stationary phase is the key factor for the ideal separation of the two molecules, lutein and zeaxanthin, and the enrichment of respective molecule. Column was selected, the length of the column is 20 times more than actual diameter of the column and the diameter should be proportionally match with loaded material weight like 1gm of the regular Lutein was loaded in 2cm diameter and 40cm length. Loading was done with aluminum oxide / silica gel which have a mesh profile 230/400 and the quantity of the stationary phase was 20 times more than the weight of the loaded material and for binding 3 times more than the loaded material. Adsorption of the regular Lutein with a stationary phase was done using stationary phase, which is further mixed and loaded. The choice of the mobile phase was critical for the good separation of Lutein and zeaxanthin. Both the molecules are having same polarity and slight separation was observed on the double development of the Thin Layer Chromatography (TLC) system. The choice of the solvent was more challenging because the separation from mixture of solvent was not recommendable due to reusable factor was not possible in this case. Single solvent was the best answer for the development of this product because of the feasibility of easy handling and recovery of the same. Each fraction was collected and monitored by thin layer chromatography using silica gel plate. Based on the TLC pattern the fractions were combined, concentrated and isolated. This step is followed by removal of residual solvent under vacuum oven. Dried zeaxanthin rich material contains the residual solvent as chloroform, which can be removed by drying the product under high vacuum at 40oC.
Example 3a: 100g of the regular lutein was binded with 300g of silica gel. Silica gel having mesh profile of 230/400 was used to pack the column using chloroform as a mobile phase. The binded/ adsorbed regular lutein was loaded followed by running the column with chloroform. Carefully collecting the elute of up to 1ltr by parallel monitoring with Thin Layer chromatography. The pure elute was collected and concentrated. The isolated solid was vacuum dried to get 38g of Lutein rich material. The subsequent two fractions were collected, concentrated and processed to obtain a zeaxanthin and lutein as 1:10 and 2:10. But analytical data proves that the formation of large quantity of ipoxide due to the instability in silica column.
Example 3b: 100g of the regular lutein was binded with 300g of alumina. Alumina was then packed in the column with chloroform as a mobile phase. The binded/ adsorbed regular lutein was loaded followed by running the column with chloroform. Carefully collecting the elute of up to 1ltr by parallel monitoring with thin Layer chromatography. The pure elute was collected and concentrated. The isolated solid was vacuum dried to get 37g of Lutein rich material. The subsequent two fractions were collected, concentrated and processed, which gave a zeaxanthin and lutein as 1:10 and 2:10.
In conclusion, the yield with lutein rich material was 37gm. Lutein: Zeaxanthin-10:1 material was 25g and Lutein: Zeaxanthin-10:2 material was 20g.

Example 4: HPLC determination of lutein zeaxanthin mixture
Lutein and zeaxanthin mixtures are quantified as per USP monograph-Dietary supplements.
Zeaxanthin and other related compounds— [NOTE—Use low-actinic glassware.]
Solvent, Mobile phase, Standard solution, Test solution, and Chromatographic system— Proceed as directed under Content of lutein.
Procedure— Inject a volume (about 10 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak responses. The peak area of zeaxanthin is not more than 9.0% of the total detected area of peaks in the chromatogram of the Test solution. Calculate the percentage of zeaxanthin in the portion of Lutein taken by the formula:
T(ri / rs)
in which T is the content, in percentage, of total carotenoids as determined in the test for Content of total carotenoids; ri is the individual peak response of zeaxanthin; and rs is the sum of the responses of all the peaks. Calculate the percentage of other related compounds in the portion of Lutein taken by the formula:
100(ri / rs)
in which ri is the individual peak response of any other peak in the chromatogram (excluding zeaxanthin and lutein); and rs is the sum of the responses of all the peaks: not more than 1.0% of any other single related compound is found.
Content of lutein—
Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).
Mobile phase— Prepare a filtered and degassed mixture of hexane and ethyl acetate (75:25). Standard solution— Dissolve a suitable quantity of USP Lutein RS in Mobile phase to obtain a solution containing about 150 µg per mL.
Test solution— Transfer about 1 mL of Test stock solution from the test for Content of total carotenoids, and evaporate under a stream of nitrogen to dryness. Add 1 mL of Mobile phase, and sonicate to dissolve.
Chromatographic system: The liquid chromatograph is equipped with a 446-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L3. The flow rate is about 1.5 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed forProcedure: the relative retention times are about 1.05 for zeaxanthin and 1.0 for lutein; the resolution, R, between lutein and zeaxanthin is not less than 1.0; the tailing factor is not more than 2; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure— Inject a volume (about 10 µL) of the Test solution into the chromatograph, record the chromatogram, and measure the peak area responses. The peak area of lutein is not less than 85.0% of the total detected area of peaks in the chromatogram. Calculate the percentage of Lutein taken by the formula:
T(ri / rs)
in which T is the content, in percentage, of total carotenoids as determined in the test for Content of total carotenoids; ri is the individual peak response of lutein in the Test solution; and rs is the sum of the responses of all the peaks.
Content of total carotenoids— [NOTE—Use low-actinic glassware.]
Solvent: a mixture of hexanes, acetone, toluene, and dehydrated alcohol (10:7:7:6).
Test stock solution— Transfer about 30 mg of Lutein to a 100-mL volumetric flask, and dissolve in and dilute with Solvent to volume.
Test solution— Quantitatively dilute the Test stock solution (1 in 100) with dehydrated alcohol to obtain a solution having a final concentration of about 3.0 µg per mL.
Procedure— Determine the absorbance of the Test solution at the wavelength of maximum absorbance at about 446 nm, with a suitable spectrophotometer, using dehydrated alcohol as a blank. Calculate the percentage of total carotenoids as lutein (C40H56O2) by the formula:
10,000A / 2550W
in which A is the absorbance of the Test solution; W is the weight, in g, of Lutein taken to prepare the Test stock solution; and 2550 is the absorptivity of the lutein in alcohol.

Fig: 1 HPLC chromatogram of Lutein and Zeaxanthin

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

We claim:
1) An enrichment process for obtaining lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1, said process comprising steps of:
(e) identifying source of lutein and adsorbing it with a stationary phase;
(f) selecting appropriate column and mobile phase followed by packing the column with the stationary phase;
(g) loading the column, eluting, collecting and combining the fractions based on chromatographic monitoring; and
(h) concentrating, isolating and drying to remove residual solvent to obtain the mixture.
2) The process as claimed in claim 1, wherein said lutein source is marigold extract or regular lutein.
3) The process as claimed in claim 1, wherein said stationary phase is selected from a group comprising silica gel and alumina having a mesh profile 230/400.
4) The process as claimed in claim 1, wherein said mobile phase is a single solvent system, preferably chloroform.
5) The process as claimed in claim 1, wherein the mixture was dried under high vacuum at a temperature ranging from about 35oC to about 45oC, preferably at a temperature of about 40oC to remove chloroform as a residual solvent.
6) The process as claimed in claim 1, wherein said chromatographic monitoring is thin layer chromatography.
7) The process as claimed in claim 1, wherein fractions were combined based on thin layer chromatographic monitoring.
8) A lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as claimed in claim 1.
9) A formulation comprising lutein and zeaxanthin mixture at a ratio of about 10:2 and about 10:1 obtained using the process as claimed in claim 1.
10) The formulation as claimed in claim 9, wherein said formulation is a pharmaceutical or herbal formulation.

ABSTRACT

Title: An enrichment process for obtaining lutein and zeaxanthin mixture
The present disclosure provides a chromatographic process to obtain lutein and zeaxanthin mixture at a ratio of about 10:2 and 10:1. This mixture has got wide applications in both food and pharmaceutical sectors.