Background of invention:
Xanthophylls, the yellow natural pigments are one of the categories of carotenoids which occur widely in plant kingdom. These are the molecules responsible for yellow color of fruits, flowers and leaves. Xanthophylls are precious molecules, known for their various health benefits and have always been part of healthy diets. Our body can't prepare the xanthophylls for our needs and are dependent on our diets from plant sources. For this reason, there is always a demand of various xanthophylls as supplements in nutraceutical market.
Xanthophylls like Lutein and zeaxanthin in an appropriate ratio play a vital role for proper functioning of our eyes. They are absorbed from the food intake and accumulate in high concentration at eye retina and its role is to protect light sensitive part of retina from over exposure to sun rays. Lutein is widely used as supplement for age related macular degeneration (AMD), cataract and retinitis pigmentosa (Nutrients., 5: 1823-39, 2013). However, Lutein is also known for various other health benefits, like; anti oxidant (Trends Biotechnol., 21: 210-216, 2003), coronary heart disease and anti cancer (Food and Chemical Toxicology, 44: 1522-1529, 2006; Trends Food Sci. Technol., 8: 406-413, 1997; Science, 298:, 2149-2153, 2002; Exp. Biol.Med., 227: 920-923, 2003).
Marigold flower is the rich source of Lutein in form of their fatty acid esters. Along with Lutein esters, Zeaxanthin esters are also present in Marigold flower in the similar ratio as per the vital composition found in retina. Hence Marigold flowers turn out to be the best commercial source
to produce this combination for nutrational supplements.
»
Process of producing Lutein from Marigold flowers can be divided into three major steps. First step is the extraction of the plant material, to produce Lutein esters enriched oleoresins. Second step is saponification of oleoresin to give crude Lutein and finally purification and isolation of pure Lutein along with Zeaxanthin.
Marigold oleoresin contains about 20-30 % of Lutein and Zexanthin esters. Rest of the extracted material is composed of wax, gum, sterols, free fatty acids, anthocynincs and various other

xanthophylls. Out of them wax makes a big contribution. Just like Lutein and zeaxanthin esters, wax is also non polar in nature, which makes it difficult to remove selectively with simple methods. When oleoresin is saponified, Lutein esters present in.it are converted into product Lutein along with salts of fatty acids as byproduct. Here product Lutein still remains associated with significant amount of wax. Presence of wax not only decreases the potency of Lutein in finished product but also makes the Lutein sticky and waxy in nature.
There has been a lot of progresses word wide to produce Lutein from plant sources using different processes and made it available in market with a wide range of quality, specifications and formulations as dietary food supplements.
Patent No. 4048203 discusses the extraction of Lutein esters from plant material and its enrichment using alcohol at 75°C. This thermal exposure is sufficient to convert the active trans Lutein esters into cis geometrical isomers which is considered undesirable and less bio available for human nutritional supplement.
Patent 5382714, describes the process for purification of Lutein, with alcohol and water at 5° to -10°C followed with washing it with plenty of water to give crude Lutein and finally crystallizing it with halogenated solvent and hydrocarbon solvents. Need of lower temperature (5° to -10°C) in process adds the cost and use of plenty of washings with water ethanol and hexane is generating a lot of effluent. Additionally using Triethyl amine, halogen based solvent and hydrocarbon based solvents in final stage is always unwelcomed with respect to their toxicity and tight limits for the products for human consumption.
Patent US 5648564 discuss the process of saponification using propylene glycol at 65-80°C, preferably 70°C for 3 to 10 h, preferably 10 h and then isolating and washing the Lutein crystals with large volume of hot water of. temperature 60 to 80°C, preferably 70°C. This process exposes the Lutein at higher temperature for longer time, which promotes the conversion of Lutein into its undesired cis geometrical isomers. Additionally process needs a plenty of water for washing the Lutein crystals, which ultimately generates a huge effluent. Even though there is no test to guarantee that Lutein will be totally free of wax.
Patent US 6191293 provides the procedure to increase the concentration of trans xanthophylls by treating the xanthophylls esters with alcohols to remove the non-xanthophyll and cis

xanthophylls esters. This process does not ensure for the removal of plant originated wax which makes a big contribution in the finished product if not removed selectively.
US Patent 6262284 describes the process using THF for extraction and saponification. Solvent THF has now been declared as use of solvent to be limited for its toxicity in goods for human consumption.
US patent 6380442 describes the saponification of Xanthophyl esters in Isopropannol using alkali in presence of water. After hydrolysis, product is precipitated with water. Precipitate is recovered by centrifugation and washed repeatedly with water. Here neither IPA nor Water can ensure for effective removal of wax or other wax like non polar impurities to provide Lutein with higher caretanoid contents.
Patent US 6504067 refers a process, which starts from refining of oleoresin by treating it first with diluted alkali and later with dilute acid to remove the un-desirable materials. After the saponification Patent describes the removal of remaining impurities using multiple washings of hexane in final stage to give the pure Lutein.
Processes reported in various patents are the result of successive work done worldwide. However there is still a good scope for further improving the process with respect to ease of production, yields, quality safety and manufacturing cost.
For the production of Lutein as supplement for human consumption, it is most desirable that product should be safe, with highest purity & potency, free of unwanted or inactive substances and low production cost. Additionally the finished product must be enough stable to be stored and used without any. degradation and use of preservatives. Lutein with the carotenoid skeleton is a sensitive molecule against thermal exposure, light exposure and contact of oxygen. Presence of unwanted substances may also affect the stability of Lutein.
Summary of invention:
Fatty acid esters of Lutein and Zeaxanthin are the constituents of interest present in Marigold oleoresin. Other than these desirable constituents, Marigold oleoresin is also composed with a bulk of undesirable polar and non polar constituents, which include free fatty acids, gums, sterols, other minor carotenoids or coloring constituents, lipids and a bulk of interfering waxy

mass. Presence of these impurities collectively makes the oleoresin a complex mixture and difficult for processing to isolate the free flowing solid product. Isolation of pure Xanthophylls out of this paste like mixture needs selective removal of impurities, without the loss of precious product.
It needed a systematic approach and comprehensive understanding of various components of oleoresin as well as saponified oleoresin. Individual components of oleoresin and saponified oleoresin both were physically separated into desirable and undesirable constituents by column chromatography.
This fractionation gave a preliminary idea about the contribution of major components present with the original material(s). Solubility studies of these individual components in different solvents helped to shortlist the best solvent for removal of undesirable constituents selectively.
Enriched product component(s) and wax so obtained were re-purified up to the level of analytical standard and used to quantify the desirable xanthophyll and undesirable wax in various stages of processing, using HPLC, GC and IR spectroscopy. Solubility studies and quantification of xanthophylls(s) and wax helped significantly in solvent selection and process optimization.
From all these studies, it was found that the presence of wax makes the entire mass greasy and difficult to handle. This not only creates emulsion like situations during processing but also chokes the filtration aids during operations.
Process suggested with this invention provides an effective method for the removal of this interfering waxy mass selectively, which makes it easy to isolate the free flowing and high purity Xanthophylls.
Present invention provides a simple, rugged manufacturing process to get high quality Lutein for human consumption. Efficient analytical methods have been deployed to monitor and quantify the presence or absence of wax substances and other impurities in ensuring their effective removal during the manufacturing stages.
One of the aspects of present invention is to provide an effective and easy method to remove the wax or other wax like non polar impurities using safe solvents. Invention is also capable for

effective removal of wax substances without the use of excessive volumes of solvents or hot water.
In addition to this, process of present invention is simple and doesn't face operational trouble in filtration. Present invention doesn't choke the filtration aids while removing the wax substances and one does not need specialized filtration equipments like hot water jacketed equipments or press filter to achieve the desired results. Additionally this process does not expose the product with high temperature for longer times, causing thermal degradation.
Present invention involves the effective removal of wax or wax like non polar substances by introducing the use of Ethereal solvent during or after the saponification as a single or co solvent. Use of Ethereal solvent selectively dissolves and removes the wax substances without the loss of yield, in terms of Xanthophyll(s). Merit of using Ethereal solvents over the hexane is its safer limits for human consumptions as stated in ICH guidelines. Ethyl ether and tert-Butylmethyl ether are categorized in Class 3 solvents in ICH guidelines (limit is up to 50 mg/day). Isopropyl ether is categorized in Table 4, for the solvents for which no adequate toxicological data was found, as stated in Q3C, ICH guidelines. However Hexane is categorized in class 2 solvents in Q3C, ICH guide lines (PDE: 2.9 mg/day). Additionally it has trouble free filtration without choking of filtration aids and need of comparatively low volumes and at safe temperature.
Detailed description of the invention:
Objective of this invention is to provide an improved process for producing high purity and high potency Xanthophyll(s), from crude or enriched oleoresin, originated from plant materials rich in xanthophylls ester(s). Considering the presence of a bulk of undesirable polar and non polar constituents with oleoresin, process of this invention is designed to remove the undesirable constituents in such a way to negate their interfering nature and provide high purity and high potency Xanthophylls, which is free from plant originate wax and other non polar and polar impurities.
Process of present invention is broadly composed of following operations.
1. Hydrolysis of crude or enriched oleoresin to convert the Xanthophyll esters into corresponding free Xanthophyll(s).

2. Removal of wax and other non-polar impurities, by using Ethereal solvent(s) during hydrolysis, after hydrolysis or in final purification as a single solvent or as a co-solvent.
3. Final purification xanthophylls in Alcoholic solvent(s), water or their combination, yielding free flowing xanthophylls with high purity and assay.
Here, use of Ethereal solvent for the removal of wax and other non-polar impurities in step-2, can optionally be performed individually or can be clubbed either with step-1, i.e. hydrolysis or with step-3, i.e. final purification.
In one of the conditions of this invention, oleoresin is hydrolyzed using a suitable alkali metal hydroxide in a suitable alcoholic solvent under hot condition. Later, reaction mass is cooled and filtered, yielding crude Lutein. Crude Lutein so obtained is then leached with Ethereal solvent to remove the wax and other non polar impurities. Wax free Lutein so obtained can finally be purified in suitable alcoholic solvent, in water or in their combination, yielding free flowing xanthophylls with high purity and assay.
In one of the other conditions of this invention, oleoresin is hydrolyzed using a suitable alkali metal hydroxide in a suitable alcoholic solvent in combination with Ethereal solvent under hot condition. Later, reaction mass is cooled and filtered, yielding wax free xanthophylls, which is finally purified in a suitable alcoholic solvent, in water or in their combination, yielding free flowing xanthophylls with high purity and assay.
In one of the other conditions of this invention, oleoresin is hydrolyzed using a suitable alkali metal hydroxide in a suitable alcoholic solvent under hot condition. After completion of saponification, reaction mass is diluted with Ethereal solvent and homogenized at hot conditions. Later, reaction mass is cooled and filtered, yielding wax free xanthophylls, which is finally purified in a suitable alcoholic solvent, in water or in their combination, yielding free flowing xanthophylls with high purity and assay.
In one of the other conditions of this invention, oleoresin is hydrolyzed using a suitable alkali metal hydroxide in a suitable alcoholic solvent under hot condition. Later, reaction mass is cooled and filtered, yielding crude xanthophylls. Crude product so obtained is then purified with alcoholic solvent in combination with Ethereal solvent, water in combination with Ethereal

solvent or aqueous alcoholic solvent in combination with Ethereal solvent, yielding free flowing xanthophylls with high purity and assay.
In step -1, hydrolysis can be performed either using crude oleoresin or enriched one. Enrichment of oleoresin can be achieved by leaching it with alcoholic solvent in presence or absence of water, at hot condition. Later, suspension mass is cooled and filtered, yielding enriched oleoresin.
Hydrolysis is carried out using alkali metal hydroxide. Choice of metals hydroxide is among lithium hydroxide, sodium hydroxide or potassium hydroxide, most preferably potassium hydroxide. Hydrolysis is carried out in alcoholic solvent. Most preferable alcoholic solvent used in hydrolysis is among methanol, ethanol or isopropanol.
Step-2 involves the removal of wax from product using Ethereal solvent(s). Choice of Ethereal solvents used in Step-2 may be diethyl ether, diisopropyl ether, methyl-tert-butyl ether.
Step-3 involves purification of xanthophyll in Alcoholic solvent in presence or absence of water. Preferable alcoholic solvents used in this step are among ethanol, methanol or isopropanol.
Step-3 involves the final crystallization of Lutein, which involves purification of wax free Lutein in Alcohol, water or their mixture. Choice of alcohol is ethanol, methanol or isopropanol, most preferably ethanol.
Following are the examples, provided to describe the invention, which are not intended to limit the scope of invention
Exam pie-1
Solution of Potassium hydroxide (175 gm) dissolved in Methanol (700 mL) was charged into the suspension of marigold oleoresin (700 gm) with total xanthophylls content 35.5 % (by UV) in Ethanol (700 mL). Heated the stirred reaction mass at 60-65°C for 30-60 min and monitored the progress of reaction by TLC. Diluted the stirred reaction mass with Ethanol (700 mL) at 60-65°C. Cooled the reaction mass to 25-30°C and stirred at this temperature for 60 min. Filtered the reaction mass and washed with Ethanol (700 mL). Drying of wet product under vacuum at

25-30°C, afforded the crude Lutein product (568 gm) with 13.98 % of total xanthophylls content (byUV).
ExampIe-2
Suspended the crude saponified xanthophyll (568 gm) with 13.98 % of total xanthophylls content (by UV) in Di-isopropyl ether (2840 mL) and stirred at 45-50°C for 30-60 min. Gradually cooled , the suspension to 25-30°C and stirred at this temperature for 30-60 min Filtered the suspension, washed with Di-isopropyl ether (568 mL) and dried the mass under vacuum at 25-30°C. Wax free product (481.6 gm) with 29.5 % of total xanthophylls content (by UV), so obtained was suspended in mixture of Ethanol (1926mL) and water (1926 mL) and stirred at 50-55°C for 30 min. Cooled the suspension to 40-45°C, filtered the product under vacuum at 40-45°C and washed with hot water (2x963 mL) at 75°C. Wet product was dried under vacuum at 45-50°C affording the Lutein (54.0 gm), with 92.06 % of total xanthophylls content by UV with 91.67 % of Lutein and 6.75 % of Zeaxanthin by HPLC.
Example-3
Solution of Potassium hydroxide (5 gm) dissolved in Methanol (20 mL) was charged into the suspension of marigold oleoresin (20 gm) with total xanthophylls content 35.5 % (by UV) in Methanol (20 mL). Heated the stirred reaction mass at 60-65°C for 60 min and monitored the progress of reaction by TLC. Diluted the stirred reaction mass with Disopropyl ether (40 mL) and water (40 mL) and continued stirring at 60-65°C for 15 min. Cooled the reaction mass to 40-45°C and stirred at this temperature for 60 min. Filtered the reaction mass and washed with water (3x20 mL). Drying of wet product under vacuum at 25-30°C, afforded the Lutein product (2.0 gm) with 89.83 % of total xanthophylls content (by UV) with 90.64 % Lutein and 6.72% Zeaxanthin.
Example-4
Solution of Potassium hydroxide (5 gm) dissolved in Methanol (20 mL) was charged into the suspension of marigold oleoresin (20 gm) with total xanthophylls content 35.5 % (by UV) in Methanol (20 mL). Charged Diisopropyl ether (40 mL) and stirred the reaction mass at 60-65°C for 30-60 min. Monitored the progress of reaction by TLC. Cooled the reaction mass to 40-45°C

and stirred at this temperature for 60 min. Filtered the reaction mass and washed with water (3x20 mL). Drying of wet product under vacuum at 25-30°C, afforded the Lutein product (1.7 gm) with 91.13 % of total xanthophylls content (by UV) with 90.75 % Lutein and 6.91% Zeaxanthin.

We Claim:
1. We claim for the process for preparation of free flowing, high potency Xanthophyll(s) from
oleoresins, obtained from xanthophylls(s) rich plant sources by introducing the use of
Ethereal solvent in process for effective and selective removal of wax and other non polar
impurities followed by use of alcoholic solvent, water or their combination for the removal of
polar impurities, which comprises following steps.
a. Saponification of oleoresin having Xanthophyll ester(s) in suitable alcoholic solvent
using a suitable alkali metal hydroxide at practical feasible temperature required for
smooth conversion.
b. Cooling the reaction mass to room temperature and filtering to yield crude saponified
xanthophyll(s).
c. Treating the crude saponified xanthophyll(s) with ethereal solvent during or after
saponification as single solvent or as co-solvent to remove the wax and other non polar
impurities.
d. Treating the product with xanthophyll(s), using alcoholic solvent, water or their
combination to remove the polar impurities affording free flowing, high potency
Xanthophyll(s)
2. Process as described in claim* 1, wherein oleoresin used may be crude extract or enriched extract, originated from plant materials rich in xanthophylls ester(s) with commercial worth.
3. Process as described in claim-1, wherein choice of alkali metal hydroxides is among Lithium hydroxide, Sodium hydroxide or Potassium hydroxide.
4. Process as described in claim-1, wherein choice of alcoholic solvent for saponification is among methanol, ethanol, isopropanol, n-propanol, n-butanol, tert-butanol, isobutanol or sec-butanol.
5. Process as described in claim-1, wherein choice of Ethereal solvents is among di alkyl ethers, preferably with the number of carbon atoms ranging from CI to C4, for example diethyl ether, di-n-propyl ether, diisopropyl ether, n-butylmethyl, tert-butylmethyl ether etc, most preferably diisopropyl ether.
6. Process as described in claim-1, wherein Ethereal solvent can be used with the reaction mass of saponification, as co-solvent with alcoholic solvent, either from the beginning of saponification or after completion of saponification.

7. Process as described in claim-1, wherein Ethereal solvent can also be used alone to treat the crude saponified xanthophyll(s) isolated after saponification.
8. Process as described in claim-1, wherein Ethereal solvent can also be used in final purification as co solvent with alcoholic solvent, water or their combination.
9. Process as described in claim-2, wherein oleoresin used is marigold oleoresin.