FIELD OF INVENTION The present invention relates to the process of isolation of carotenoids (xanthophyll) present in the marigold flower meal. The invention particularly involve isolation of pure crystals More, particulaty the content of lutein in the crystals is more than 80% and up to 98% while the remaining are Zeaxanthin and other related pigments. This invention further provides xanthophyll crystals of at least 80% by weight of xanthophylls and more particularly lutein content itself is more than 80%. The present invention also relates to the process of isolation of carotenoid crystals containing high percentage of Lutein and Zeaxanthin in a ratio of 10:1 to 4:1. The lutein prepared by the process of present invention is particularly useful as nutraceuticals application and other health benefits. BACKGROUND OF INVENTION Xanthophylls are a class of lipid soluble compound and generally insoluble in water. The xanthophylls belong to carotenoid family which are compounds having red, green, yellow and orange pigment and are widely found in the nature. The carotenoid is found in fruits, vegetables, egg yolk, poultry skin, fish like salmon, crustaceans. The subject of present invention marigold are rich in xanthophyll particularly lutein and Zeaxanthin. Carotenoid consists of two classes that are more polar xanthophyll and non-polar carotenoids like lycopene. The carotenoids contain conjugated double bonds which are responsible for the colour of these compounds. Their role in human health is also been investigated and they constitute important constituent of macular region of human eye. The marigold flower is rich source of xanthophylls basically in its ester form. The lutein is combined with fatty acid palmitic, stearic and oleic acid. It is estimated that marigold meal contain 1-5% xanthophylls and lutein ester constitute 90% of the total xanthophylls. From the xanthophylls present in marigold flower, the lutein is obtained by hydrolysis of lutein ester. 2
WO 2012098559A1: 2012: Uma Shankar Bharatia et al. :Single stage lutein ester extraction from tagets species, Describes the extraction of lutein from marigold meal by SCF-CO2 extraction at up to 625 bar to prepare 40-70% lutein ester and preparing various formulations thereof. US 8,425,948B2: 2004, Swaminathan et al. : Discloses a process of obtaining xanthophylls from marigold meal and hydrolysis of the same after enriching the oleoresin to lutein and Zeaxanthin. US 6.743953 (2004) S. Kumar: Discloses a process of extraction of xanthophyll from dried marigold flower using non-polar solvent hexane. Thereby, saponification of extracted oleoresin at 70 degree celsius and a time duration of 3 hours. The invention further states formation of degradative product if the reaction continued for longer duration. CN 2012CN-0424690: Discloses preparing lutein crystals from marigold ointment by catalytic saponification. A mixture of one or more of KOH, NaOH, sodium methoxide and sodium ethylate and alkaline ionic resins are used as saponification catalyst. US patent 5,382,714 (1995), Khachik: discloses saponified marigold oleoresin obtained from Kemin industries and prepared from extraction of dried marigold petals with n-hexane followed by saponification and purification. 2013-CN-0272825: Discloses method for preparing food grade lutein crystals from marigold extract with lower alcohol solvent by adding a solid alkali, diluting then cooling and crystallizing. The purity of crystals prepared by such a way is 80 to 90%. WO 2009019712 A1 (2009) Kumar T: The invention discloses preparation of novel xanthophyll compound containing trans isomeric forms of meso- Zeaxanthin, Zeaxanthin and lutein useful for nutrition and healthcare and process of its preparation. Phenyl carbinol used as an aromatic alcohol for the reaction. The process comprises heating the lutein containing material with aromatic alcohol and alkali hydride followed by purification of hydrolyzed reaction mass. US patent 20120107380 (2012) Joshua J.: Discloses method for producing xanthophyll enriched product from a xanthophyll ester source, comprising the steps of dissolving or suspending the xanthophyll ester source in isopropyl alcohol and water at a temperature of about 40-90 degree C to form a free flowing solution or mixture, hydrolyzing the dissolved xanthophyll ester source with an 3
aqueous saponifying agent, cooling the saponified solution to about ambient temperature and allowing the solution to settle for at least 5 hours to 24 days. The crystals are purified by filteration and washing. The disadvantage of the process is degradation of xanthophyll due to long processing hours. US 7,812,198 (2010) Thomas: Discloses a process for manufacturing the composition containing xanthophylls by saponifying the marigold flower extract and isolating the xanthophyll in presence of quartenary organic base. US Patent7629007, 2009 Gustavo: Discloses a process for purification of free xanthophyll according to the disclosure xanthophyll containing plant derived oleoresin is saponified using alkali then the saponified oleoresin is washed with salt solution for several times before treating with oraganic solvent for further purification. Finally pure xanthophyll crystals are filtered and dried. US patent 7,622,599 (2009) Swaminathan: Discloses an isolation and purification method for carotenoids from marigold flower by hydrolyzing marigold oleoresin using alcoholic alkali. Followed by precipitation of carotenoid crystals with hydroalcoholic mixture, further crystals are washed to remove impurities. This patent uses numerous solvents stream that are costly on commercial scale to recover and separate US patent 7485738 (2009) Xinde Xu: The process discloses food grade Zeaxanthin through chemical isomerization from lutein by treating xanthophyll crystals with propylene glycol with organic alkali to carry out isomerization. Further, Zeaxanthin crystals are precipitated under deionized water and alcohol mixture to collect Zeaxanthin crystals. US patent 7,252,294 (2007) Hoffmann: Discloses isolation of lutein from alfalfa by saponifying green plant oleoresin to provide a saponified resin, which is treated with first volatile organic solvent to oily substance which is further treated with second volatile oraganic solvent to derive lutein crystals. The disadvantage of using multi volatile solvent makes process commercially non-viable. US patent 7,173,145 (2007) Khachik: Discloses process for extraction and purification of lutein, Zeaxanthin and other related carotenoids. This method employs Tetrahydro Furan and alcohol for 4
preferably ethanol for purification. The main disadvantage of this patent is use of two solvents particularly handling of tetrhydrofuran at commercial level. Indian patent 1379/CHE/2010: Discloses isolation and purification of carotenoid rich oleoresin derived from plants and micro-organisms without use of toxic chemicals and hazardous solvents. US patent 6,504,067 (2003) Olvera: Discloses cleaning the oleoresin with alkali and acid then the oleoresin is subjected to aqueous alkali saponification for 8hours in presence of an emulsifier at a temperature of 90 degree C The major disadvantage of the process is long saponification hours thus the prolonged exposure to heat might cause degradation of lutein crystals. US patent 6,380,442 (2002) Madhavi et al.: Discloses saponification process for carotenoids using isopropyl alcohol, water and alkali. US patent 6,262,284 (2001) Khachik: Describes a method which employs tetra hydro furan and alcoholic potassium or sodium hydroxide for isolating lutein and Zeaxanthin by simultaneous extraction and saponification at room temperature. US patent appl. 2010/0305366 (2010) Jaun Liu: Discloses lutein extraction process by first extracting lutein ester by oraganic solvent and ultrasound method then saponifying the lutein ester containing filtrate using alkali solution, Further, the alkali solution is washed and separated to collect crude crystals. US patent appl 2011/0065965 (2011) Mehta: Discloses a saponification process of marigold oleoresin using alkane hydrocarbon, alkanol solution and potassium hydroxide solution and further purification of the crystals. The major disadvantage of the process is its multi solvent uses for saponification and their commercial viability and recovery.
Saponification of carotenoid esters leads to decomposition at high temperature and high concentration of alkali. Lutein ester is collected from marigold flower. The saponification of purified lutein ester is studied at different parameters using UV-visible spectrophotometer, HPLC. Findings showed efficient saponification in 0.5 M KOH at 50 degree C for 30minutes.
Purification of lutein fatty esters from marigold flower petals or from its oleoresin has been achieved by alkanol. The process is distinguished for the use of alkanol, P. Friederich, 1984. 5
During past decade, many researchers have quantified carotenoids bearing health supplementing properties. The studies have revealed that as many as 50 carotenoids may be available from diet and are metabolized by the human body. (Khachik et al, 1991, Pure appl chem., 63, 71-80)
Preparing lutein or lutein ester and used many complex processing methods involving used of hazardous aromatic hydrocarbon solvent, chlorinated solvents and other hazardous chemical substances treatment so make lutein either from marigold flower or other vegetable sources. However, the present investigation is taken as first ever art using aromatic alcohol and its sodium or potassium salt in presence of phase transfer catalyst and slight amount of water. The phase transfer catalyst are proven to enhance the reaction efficacy by multifold by bringing the two phases together. The process therefore is unique application of green technology providing high efficiency of conversion.
Other References are---
Chitta Ranjan Sarkar, B agmita Bhagawati, An efficient condition of Saponification of Lutein ester from marigold flower, Annals of Biological Research, 2012, 3 (3):1461-1466 F Khachik et al , “ separation, Identification and Quantification of carotenoids in fruits, Pure and Appl Chem (1991),63 (1) 71-80. F Khachik et al, Isolation structure elucidation and partial synthesis of lutein dehydration products, Journal of Chromatography (1995), 670, 219-233. F Khachik et al, Identification, quantification and relative concentration in Serum, Analytical Chem. (1997) 69,(10) 1873-1881. F. Khachik et al , Separation, Identification and Quantification of major carotenoids, J, of Agriculture and Food Chem (1989) 37(6) 1465-1473. F. Khachik et al. “Carotenoids content in thermally processed foods, Journal of Agriculture Food Chem (1995) 43 (3) 579-586. Nicole Harber, The impact of Lutein and Zeaxanthin on premature infant eye, Nutrition and Dietetics (2014), 6, 273-285. 6
Q. Aston Action, “Xanthophylls- Advances in Research and Applications (2013). Richard L. Robert “Lutein, Zeaxanthin and Skin health, American J. of Lifestyle Medicine , (2013), Feb 22 online. An Evidence bases systematic review of lutein by natural standard research collaboration, J. of Dietary Supplements (2015) 12 (4). Lutein and Zeaxanthin – Macular Degeneration Vitamins (2015)- Macular Degeneration-Vitamins.cm Christiana L. Sherry et al.” Lutein supplementation increase breast milk and plasma lutein concentration in lactating women, J Nutrn. (2014) 144 (8) 1256-1263. Cena et al., Lutein Concentration in Human milk during early Lactation and relationship with dietary Lutein uptake, (2009), Pub. Health Nutrn. (2009), 12, 1878-1884. -Achmad Subagio et al, Stability of Lutein and its Myristate Esters, Bioscience, Biotechnology and Biochemistry, (1999) 63 (10). -Elizabeth J. Johnson, Role of Lutein and Zeaxanthin in visual and cognitive functions, Nutritional Reviews, Oxford Journal (2014). -US 20140200374 A1, Phattanon Prasitchoke , Method of Extracting Lutein xanthophylls from natural material and highly purified lutein/xanthophylls (2014). -Jian Hao Lin, Lutein products from biomass, marigold flower verus Microalgae, Bioresource technol. (2014) online. -B. K. Tiwary, A study of optimization of marigold petal yield, pure lutein and formulations, J. of crop Science and Biotecnol. (2014) 17 (3) 175-181. -Research Delivers Insight into Global Lutein Industry, Report Stack, (2015) July 20 online. -Jochen U Ziegler , Lutein and Lutein ester in whole grain flour made from 75 genotypres of triticum, J. Agric. Food Chem. (2015) 103 (20) 5061-5071 7
-Wangian Wu, Lutein suppresses Inflammatory response through Nrf2 activation, Molecular Nutrition and Food research (2015) June 23, online -EP 2748144 A1, (2014), A method of extracting Lutein, Phattanon Prasitchoke -Namdeo R. Jadav, Herbal nanoparticle a patent review, Asian J, of Pharmaceutics (2014), 8 (1)58-69. SUMMARY OF THE INVENTION The present invention provides a process of isolating xanthophyll crystals from marigold flower extract. The Marigold flower is rich source of xanthophyll a carotenoid bearing yellow colour and finds its application nutraceuticals and dietary supplements particularly due its efficacy for vision improvement. The xanthophyll is present in its ester form in the extracted oleoresin. To make the extracted xanthophyll more bioavailable it needs to be saponified. The saponification which is an art involves preparation of carotenoid from its esters. Ester is hydrolyzed in alkaline condition releasing fatty acid and xanthophyll in its free form. The marigold meal oleoresin is prepared conventionally by solvent extraction with non-polar solvent or by green chemistry techniques of SCF-CO2. The present invention does not cover the extraction of oleoresin from the marigold flower petals. However, the process of invention could produce lutein from the lutein ester irrespective of whether it is processes by solvent or SCF-CO2.
This present invention employs the marigold extract extracted by SCF-CO2 as well as solvent extraction and further its saponification procedure for marigold flower lutein and its isomer Zeaxanthin. The marigold extract prepared by Green SCF-CO2 extraction and or by extraction with hydrocarbon solvent is taken with 1 to 5 volume of phenyl ethyl alcohol( PEA), preferably 1 to 3 volume of PEA and more Preferably with 1-2 volume of PEA. After addition of phenyl ethyl alcohol to the lutein ester water in a quantity 40-50% of marigold oleoresin is added and stirred for half an hour. To this mixture potassium hydroxide 100-200% of the lutein content in the oleoresin is added. The heating of the reaction mass started and temperature raised to 70-80 degree celsius for a duration of 2-6 hours more preferably 1.7 to 3.0 hours. The pH of reaction mixture for conducting the saponification is maintained above 10, more preferably more than 12 for 1.7 to 3.0 hour. Further the phase transfer catalyst maintains a bridge between the organic and aqueous phase and catalyze 8
the reaction to forward direction. This process not only leads to saponification but also maintain a ratio of Lutein and its isomer Zeaxanthin.
In the next step, the mixture is filtered off, the solvents (PEA) is evaporated/distilled. The residue is washed with water or ethanolic water to remove the soap from it. The washing is continued till the pH is achieved neutral (pH=7.0. The washing step removes the salt of fatty acid esters, the base (potassium or sodium hydroxide) present and used in saponification reaction and the water soluble plant components. The crystals after this washing step are 60-70% lutein and Zeaxanthin and contain minor quantities of other carotenoids. For further improving the crystals quality washing of the crystals with ethanolic water is taken up. The crystals are finally dried under vacuum at low temperature to protect the crystal quality.
The process described above is a convenient and commercially viable method for saponification of lutein ester extracted from marigold flower where the lutein is present in its esterified form with fatty acids. The production of carotenoids according to this process can be conducted under controlled and mild conditions at room temperature or below 80°C to control the degradation of heatlabile compounds. The only organic solvent used is PEA which, can be easily removed by washing with water by distillation.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for isolation of carotenoid crystals from the saponified marigold extract. The marigold meal extract is prepared by supercritical fluid extraction or by solvent extraction and is not a part of present invention. The process of invention comprises saponifying the marigold meal extract a temperature ranging from 50-90degree Celsius more preferably 60-80 degree Celsius for duration of 1.7 to 5 hours. The alkali used for saponification is in the range of 1-3 part of the marigold meal extract. The alkali is added more preferably in a quantity 200-300% of the active lutein present in the oleoresin determined spectrophotometrically. The alkali used is potassium hydroxide, sodium hydroxide, ammonium hydroxide and calcium hydroxide, more preferably potassium hydroxide in aqueous solution. The alkali is dissolved in similar quantity of water (1:1 alkali: water) and added to the marigold meal extract. The alcohol used comprises benzyl alcohol, phenyl ethyl alcohol, diethylene glycol and Polyethylene glycol. 9
Preferably, phenyl ethyl alcohol which has limited water miscibility is used for the saponification for solubilizing meal extract. Further for carrying out the saponification phase transfer catalyst most preferably tetra butyl ammonium is used.
In another embodiment of the present invention the ratio of marigold meal oleoresin to phenyl ethyl alcohol used is in a ratio of 1:5 and more preferably 1:3 w/v and more preferably 1:2. The use of equi- proportion of alcohol conversion of lutein ester to lutein took place but the purification was problematic.
Therefore minimum 1.75 volume of phenyl ethyl alcohol was used. The conversion of lutein ester to lutein conducted with calcium hydroxide and ammonium hydroxide resulted in purification complexities. With calcium hydroxide a thick mass formed which was difficult to filter thereby further reaction were studied with potassium hydroxide and sodium hydroxide only
Use of benzyl alcohol resulted in 34-41% conversion of lutein ester to lutein while polyethylene glycol 200 and 600 gave better results.ie up to 42-48% conversion. The Saponification with Benzyl alcohol and polyethylene glycol were carried out to study the efficacy for saponification. However, detailed experiments were conducted with phenyl ethyl alcohol. The results of benzyl alcohol and polyethylene glycols are only indicative.
As presently practiced, the conventional method of preparing marigold crystals containing lutein and Zeaxanthin , such as blending crystals after purifying lutein crystals and Zeaxanthin crystals individually result in changing and different ratio of the integral part of marigold flower i.e. lutein and Zeaxanthin. Preparation of high purity Zeaxanthin crystals result in poor recovery and thereby making the process commercially non-viable. To overcome this situation there is an urgent need to process marigold meal for preparation of carotenoid crystals which contain both lutein and Zeaxanthin. The ratio of both carotenoid s varies from a range 5:1 to 10:1 as achieved in the present invention.
In the present process of invention for preparing carotenoid crystals involve, process for isolation of carotenoid crystals having carotenoid crystals Lutein and Zeaxanthin in a weight ratio of 9:1 to 10:1. The process for preparing above quality crystals consist of— 10
Contacting the marigold meal oleoresin extracted by SCF-CO2 and or by solvent extraction with aromatic alcohol or polyethylene glycol, diethylene glycol in a ratio of 1:5 in presence of aqueous solution of alkali. The alkali is used in 2-3 part of the marigold meal oleoresin and saponified at a temperature ranging fro 50-80 degree celsius. The saponification reaction is carried out for 2-5 hours more preferably for 4 to 5 hours at temperature below 80 degree Celsius. Once ensuring the conversion of lutein ester to lutein by HPLC the reaction mixture is filtered. Alternatively the aqueous layer is separated and reaction mass filtered to remove the impurities in the filtrate.
In yet another embodiment b the alkali used for present invention consists of alkali metals and alkaline earth metals particulaty hydroxide of sodium, potasium and calcium. For washing the filtered crystals water in a ratio of 1:1 to 1:4, more preferably 1:2 is used.
The present invention provides and effective process for saponification of marigold oleoresin extracted by SCF-CO2 and or by solvent extraction. The carotenoid crystals produced are rich in Lutein and Zeaxanthin predominantly rich in lutein.
In yet another embodiment of the present invention relates to the process for isolation of carotenoid crystals having lutein and Zeaxanthin in a weight ration of 5:1 to a ratio of 10:1, where the above carotenoid crystals comprises of about 75% to 85% of trans lutein and 5-10% of trans Zeaxanthin and not more than 5.0% of other carotenoids when analyzed by HPLC.
In yet another embodiment applying 100-150% alkali for saponification resulted in partial conversion of lutein ester to lutein indicating that for removal of fatty acids more quantity of bases is absolutely necessary and further experiments were conducted with higher concentration of alkali used for conversion.
Attempt to carry out the conversion of lutein ester to lutein by using potassium hydroxide 200-300% of the active lutein present and excluding phase transfer catalyst resulted in 4-5% lesser recovery of active as compared when phase transfer catalyst is used. Further, the reaction without phase transfer catalyst took more time for conversion of lutein ester to lutein. For, example this took more than 4-5 hours for reaction conversion. 11
In yet another embodiment of present invention deal with the process of isolation of carotenoid crystals having lutein and zeaxanthin in a ratio of 9:1 to 10:1 where the carotenoid crystals comprises of 80-90% of trans lutein, 5-18% of trans zeaxanthin and 2-5% of other related carotenoids.
Still another embodiment of present invention related to the process of preparation of carotenoid crystals having lutein and zeaxanthin in a ratio of 92:5% where the carotenoid crystals are formulated in the form of beadlets, powder, granules and oil. And water dispersible powder and suspension. Still another embodiement of present invention leads to the process of isolation of carotenoid crystals having lutein and zeaxanthin in a weight ratio of 10:1where the carotenoids lutein is present 55-95% w/w of the carotenoid crystals.
Although the present invention has been detailed with number of possible embodiment still more embodiment could be possible. Most importantly the current invention emphasizes the use of green solvents and conducting the reaction within 1.75 hours to 3 hours while or patented process until now envisages a reaction time of 4-5hours, preferably 4 hours. The reaction is carried out by using alkali with minimum water and phase transfer catalyst which enable the reaction completion in shortest time. The current invention prohibits the use of aromatic hydrocarbons which are not safe. The use of primary extract prepared by SCF-CO2 or solvent used in the current process provides high recoveries due to reaction completion in short time thereby leading to minimum product degradation. The lutein crystals prepared in such a way which are, highly pure are used for preparing different variants form by diluting with diluents or adding excipients to get 5-40% lutein granular powder, 5-20 % beadlets and 10-20 and 40% oleoresin. Further the product prepared by present invention could be source material for preparing water dispersible formulation as required. The processes totaly avoid the use of chlorinated solvents and other hazardous solvents and therefore find application for different nutraceutical applications having better stability and bioavailability.
Since among the three solvents used for conversion of lutein ester to lutein only Phenyl ethyl alcohol gave more than 90% conversion therefore all examples are present with this solvent. However, conversion with all three solvents used is presented in Fig 1: The Lutein Content in the final crystals prepared from three solvent of present invention is shown in Fig 2: As the quality 12
of crystals was not very good the further work was conducted with initial saponification with phenyl ethyl alcohol and further purification of crystals with ethanol: water. The other two solvents were studied and preliminary results are present in Fig 1 and Fig 2: The lutein crystals from phenyl ethyl alcohol contain upto 98% lutein However in the Fig 2 optimum lutein content 90% is presented as obtained in maximum number of experiments.
The present invention now will deal with example below.
EXAMPLES
Example 1:
1.000 Kgs Lutein oleoresin solvent extract 20-25% and SCF-CO2 extract 40-70% lutein ester is taken in a 5.0 Liter three neck flask. 200 Kgs Phenyl ethyl alcohol is added along with 20% water that of phenyl ethyl alcohol. The oraganic and aqueous phase are mixed under agitation. Inorganic alkali i.e. Potassium hydroxide is added 750g and mixed thoroughly. Phase transfer catalyst is added at a concentration of 0.1-0.15% of lutein ester used in the reaction. The mass is heated at a temperature of 70-75 degree Celsius, preferably 70 degree celsius for 3 hours. The reaction progress is observed by HPLC monitoring.
The reaction mass is filtered while hot thereby removing the mother liquor containing oil and other minor carotenoids and enriching lutein. The crystals are washed with acidic aqueous ethanolic mass to remove the impurities and base from the crystals. The process yield 130-150g crystals having lutein content of 55-65%. Re-crystallization of the crystals with acidic aqueous ethanol containing 0.5-1% hydrochloric acid yield pure crystals having lutein content above 80.0%.
Example 2:
1.000 Kgs Lutein oleoresin solvent extract 20-25% lutein ester is taken in a 5.0 Lutein three neck flask. 4.000 Kg Polyethylene glycol is added along with 40% water to the quantity of polyethylene glycol. The reaction mass is stirred and under agitation Inorganic alkali i.e. Potassium hydroxide 600-800g and mixed thoroughly. Phase transfer catalyst is added at a concentration of 0.15-0.2% of lutein ester used in the reaction. The mass is heated at a temperature of 70-75 degree Celsius,
13
preferably 75 degree Celsius for 1.5-3 hours, preferably 3 hours. The reaction progress is observed by HPLC monitoring. After ensuring the 48% conversion of lutein ester to lutein heat is closed. A trial to enhance the conversion does not result even on increasing the temperature.
The processing of reaction mass is carried in a similar way as given under example 1 to yield 60-80g crystals having UV assay 50-65%. Further recrystallization with 80% ethanol containing 20% water and 0.5% acid yield crystals having more than 72.0% lutein content once measured Spectrophotometrically.
Example 3:
1.000 Kgs Lutein oleoresin solvent extract 20-25% and SCF-CO2 extract 40-70% lutein ester is taken in a 5.0 Liter three neck flask. 2.00 Kg Phenyl ethyl alcohol is added along with 50% water that of phenyl ethyl alcohol. The oraganic and aqueous phase are mixed under agitation. Inorganic alkali i.e. Potassium hydroxide is added 700-750 g and mixed thoroughly. Phase transfer catalyst is added at a concentration of 0.15-0.2% of lutein ester used in the reaction. The mass is heated at a temperature of 70-75 degree Celsius, preferably 70 degree Celsius for 1.5-2 hours, preferably 2 hours. The reaction progress is observed by HPLC monitoring. After ensuring the 90% conversion of lutein ester to lutein heat is closed.
The processing of reaction mass is carried in a similar way as given under example 1 to yield 100-120 crystals having UV assay 80-85%. Further recrystallization with 80% ethanol containing 20% water and 0.5% acid yield crystals having more than 88.0% lutein content.
Example 4:
4.000 Kg Lutein oleoresin solvent extract 20-25% and SCF-CO2 extract 40-70% lutein ester is taken in agitated SS. reactor of 50 L volume. To this 7.50 Kgs phenyl ethyl alcohol is used for solubilizing the oleoresin. To this mass 40% water (1.6 L) was added and agitated under heat. Caustic potash 2.5-3.000 kg is added along with 8gram of phase transfer catalyst preferably tetra butyl ammonium bromide. The reaction mixture is put under heating at a temperature of 70-80 14
degree Celsius. The reaction progress is monitored by HPLC analysis of the reaction mass frequently. The reaction is terminated once the reaction completion is ensured by HPLC.
The reaction mass is filtered under hot condition to separate the mother liquor from the crystals. The crystals are freed of soap and all basic mass on washing with copious amount of water. The material is dried under vacuum for 6-10 hours and quality of crystal is checked again by HPLC. From 4.000 Kg luetin ester (25%) 270-300g crystals with 80-90% lutein content are obtained. The recovery of active crystals ranges from 52.0-60.0%
Example 5:
The Lutein prepared by above process (Assay from 80-98%) is taken in hot water/steam jacketed reactor and diluted with diluents to make luetin oil. The diluent like vegetable oils, Medium chain triglyceride, Glycerol Mono-oleoate individually or a mixture thereof are added for dilution of lutein to make lutein oil 5-50% Flow able liquid. For stability rosemary extract, natural tocopherol (mixture of Alpha, Beta, delta and Gamma tocopherrol) is added at a concentration of 0.2 to 2.0% on the weight of lutein crystals used for storage stability of the diluted lutein oil. For preparing 20% Lutein oil, the crystals and diluents are added in a proprtion of 1:3.5.
Example 6:
The lutein of present invention (assay 80-98%) are used in a suitable mixing pan/Rota cone blender having arrangement for heating and cooling . For stability rosemary extract, Vitamin C, Vitamin C-palmitate, natural tocopherol either individually or a mixture thereof at a concentration of 0.5-1.0%, 0.5-1.0% and 0.5-20% respectively are used as stabilizers. Food grade excipients like, nusilin, starch ester, microcrystalline cellulose, Hydroxy propyl methyl cellulose, magnesium oxide and carbonate, tribasic calcium phosphate, magnesium stearate and mixture thereof are added and blended by though mixing. The Mixing is done at 50-60 degree celsius until perfect granular powder is prepared. In such a way powder from a concentration 5-40% could be prepared by adjusting the proportion of excipients. For example for preparing 20% concentration of lutein, the crystals and excipient are added in a proprtion of 1:3.5. 15
Example 7:
Lutein crystals (80-98%) are taken in a granulator. Excipients like starch ester, micro crystalline cellulose, Hydroxy propyl methyl cellulose, Silicon dioxide, natural tocopherol powder , tri basic calcium phophate, Vitamin C, are added in a proportion 0.5-10%; 0.5-10% and 0.5 to 2.0% respectively used for stabilization. For granulation stir the blended mass in a ribbon blender for 1-2 hours and add water purified by reverse osmosis (quantity sufficient) till the mass become damp. Pass the damp mass through extruder fitted with 1-2 mm mesh to form the filaments or needles of the blended mass. These filaments/needles are further put for spherodization to get 1 or 2 mm beadlets as required. Excipient ratio selected for the lutein crystal of 85% to get 20% lutein beadlets is 3 .5:1 while for 5% beadlets excipients to lutein ration is 14:1.
Example 8:
Water dispersible powder is prepared by the luetin crystals prepared in present invention in a stainless Steel blender. Adding emulsifier, gum acacia, polysorbate along with the stabilizers and mixing the same .Diluents/excipients like water soluble starch ester, hydroxy propyl methyl cellulose are added. The mixture is blended to distribute the mass by homogenization.
ADVANTAGES OF INVENTION:
i) An ecofriendly process using only minimum quantity of aromatic alcohol for the conversion of lutein ester to lutein while all other processes use 4-5 volume of solvent.
ii) The reaction is carried out in short time 1.75 to 3.00 hours which prevent degradation of the product leading to higher recovery.
iii) The reaction is carried out at mild temperature thereby resulting in better recovery of the lutein.
iv) Phase transfer catalysts assist the reaction for better recovery and faster reaction.
v) The quality of the crystals produced is highly pure having lutein content more than 90% is produced by the process. 16
Fig 1: % Conversion In the Reaction from Lutein Ester with Three Solvents
Fig 2: Lutein Content in Final crystals Prepared from Lutein Ester
0
10
20
30
40
50
60
70
80
90
Benzyl Alcohol
Polyethylene Glycol
Phenyl Ethyl Alcohol
1.75 Hrs
2.500 Hrs
3.00 Hrs
0
10
20
30
40
50
60
70
80
90
Benzyl alcohol
Polyethylene Glycol
Phenyl Wethyl Alcohol
% Lutein Content 17
Table 1: DATA OF LUTEIN ESTER CONVERSION TO LUTEIN
Quantity of Lutein ester
Assay,%
(Lutein)
Active, g
Quantity of Lutein
Content,%
Recovery,%
500
12.5
62.5
55
81.0
71.00
1000
11.6
116
142
57.0
69.80
1000
12.5
125
110
84.0
73.90
4000
11.0
440
530
52.0
62.60
4000
13.0
520.
600
64.0
73.80
Table 2: QUALITY OF THE CRYSTALS OBTAINED AFTER PURIFICATION/ RECRYSTALLIZATION
Quantity of crystals, g
Assay,%
Active, g
Quantity of Lutein after recrystallization, g
Content,%
Recovery,%
Overall Recovery,%
55
81.0
39.10
32
95.0
78.00
55.30
142
57.0
80.94
94
80.0
92.90
64.80
110
84.0
92.40
95
88.0
90.40
66.80
530
52.0
275.6
278
84.0
84.70
53.00
600
64.0
384.0
380
92.0
91.00
67.10
18

We Claim, 1. A method of saponification of marigold extract prepared by supercritical fluid extraction and /or with solvent hexane/ethanol extraction in Higher aromatic alcohol, the process comprising: i) Adding 1-5 volume aromatic alcohol to the marigold meal extract, more preferably 1-3 volume aromatic alcohol.