A stable composition of lutein and zeaxanthin and preparation method thereof

Abstract

Present invention discloses a stable composition of lutein and zeaxanthin and preparation method thereof. The disclosed composition comprises of free lutein, zeaxanthin as active pharmaceutical ingredients, carrier i.e., combination of olive oil and canola oil, marker compound i.e., oleocanthal derived from extra virgin olive oil and excipients preferably selected from tocopherol, thyme oil and ascorbyl palmitate. The resulting composition is highly stable and possesses a longer shelf life of about 6-12 months. It can be encapsulated preferably in soft-gel capsule form or in in the form of tablet or any other oral formulation including jelly, chewing gum, energy bar or patch form.

Description

[0001] A STABLE COMPOSITION OF LUTEIN AND ZEAXANTHIN AND PREPARATION

[0002] METHOD THEREOF

[0003] FIELD OF INVENTION

[0004] The present invention generally relates to stable lutein and zeaxanthin composition. More specifically it pertains to a stable composition of lutein and zeaxanthin (luteye) and preparation method thereof.

[0005] BACKGROUND OF THE INVENTION

[0006] Lutein and Zeaxanthin

[0007] Lutein and Zeaxanthin are powerful antioxidants belonging to Xanthophyll family of carotenoids pigments (yellow liposoluble pigments) produced by plants. These are polar and fat soluble carotenoids found in variety of green, yellow and orange fruits and vegetables including corn, nectarines, oranges, papaya, parsley, spinach, kale, marigold, egg yolk, maize, guava, collards, orange pepper, melons etc. Lutein generally coexists in nature with its stereoisomer zeaxanthin.

[0008] Chemically, lutein is an unsaturated polyene hydrocarbon having 40 carbon skeleton 8 isoprene residues forming long carbon chain with nine double bonds. Mainly in fruits and vegetables, lutein is found in trans isomer form and sum formula of lutein is C40H54O2. Zeaxanthin is sister isomer of lutein similar in structure with lutein but differing in location of double bond in cyclic ring with molecular formula C40H56O2. (https: / / www.ncbi.nlm.nih.gov / pmc / artides / PMC8874683 / )

[0009] Importance of Lutein and Zeaxanthin-

[0010] Lutein and Zeaxanthin are of great biological and pharmaceutical importance, as these are important in functioning of various organs of body including eyes, skin, heart, intestines etc. Lutein and zeaxanthin are only dietary carotenoids accumulating in retina of the eye, especially macula region that is why these pigments are also termed as macular pigments, and maintains eye vision. Further, various studies show the lutein and zeaxanthin possesses great antioxidant potential, thus plays an important role in management of disorders related to eyes such as age-related macular degeneration and also helps in cancer management and its action is associated with cognitive function in adults and is also essential for development of infant's brain. Role of Lutein and Zeaxanthin in Eye Vision-

[0011] Macula and eye lens are the two major tissues of eyes, playing vital role in mediating and managing vision. Macula is the central region of retina and a light sensitive tissue present at the back of the eye. Damage and degeneration of macula with ageing results in vision loss in adults and is termed as age related macular degeneration disorder. The macula region of retina is yellow in colour due to presence of macular pigments comprising 2 dietary xanthophylls lutein and zeaxanthin and another xanthophyll meso-zeaxanthin in axons of its photoreceptors. The effect of macular pigments on the retina is to protect it from oxidative processes when exposed to light.

[0012] Owning to antioxidant activity and capacity to absorb blue-light, lutein and zeaxanthin plays a crucial role in protecting outer retina rich in polysaturated fats from light induced free radicals, photo-bleaching of retinal pigments, age-linked macular degeneration, cataract and retinitis pigmentosa. Hence, adequate amount of these pigments is very essential for management of various eyes related disorders, thus lutein and zeaxanthin formulation are of great biological and pharmaceutical importance.

[0013] However, lutein and zeaxanthin suffers from the problem lower stability during storage and processing after extraction.

[0014] Lower stability of Lutein and zeaxanthin-

[0015] Both lutein and Zeaxanthin are not soluble in aqueous medium as these are fat soluble carotenoids and are quite susceptible to degradation. Lutein and Zeaxanthin polyene back chains are unstable and highly susceptible to thermal and oxidative degradation during processing and storage and the major factors affecting degradation of lutein and zeaxanthin during storage are temperature, oxygen, mechanical stress and light. Usually under high temperatures, lutein and zeaxanthin breakdowns into low molecular weight molecules. Lutein and Zeaxanthin contains long chains of conjugated carbon-carbon double bonds in trans form, which when exposed to heat undergoes conformational change to less stable cis isomers, hence these double bonds are quite susceptible to thermal and oxidative degradation, thus reduces the stability of lutein and zeaxanthin. Further, UV light induce breakage of carotenoids to low molecular weight compounds and oxidation via chemical or enzymatic reaction during drying process are majorly responsible for lutein and zeaxanthin degradation, in-stability and lower shelf life.

[0016] (https: / / www.ncbi.nlm.nih. ov / pmc / artides / PMC10258427 / ) (Indian patent application no. 202242056457) Need to enhance the stability-

[0017] As reported in certain literatures, lutein and zeaxanthin possesses potential anti-oxidant activity, due to which these are very essential and plays a crucial role in management of various disorders related to eyes and cancer etc. Further, Lutein and Zeaxanthin are the only dietary pigments that accumulates the retina and specifically macula and these pigments plays a very essential role in managing age related macular degeneration, thus prevent vision loss in elders. Lutein and zeaxanthin are not naturally produced within, however only incorporated via diet but intake of fruits and vegetables as normal diet is not sufficient to provide adequate amount of pigments lutein and zeaxanthin to the macular region of retina for management of such disorders, hence zeaxanthin and lutein are extracted from these sources and then utilized in form of capsules or eye drops for management of such disorders. Various researchers and scientists observed that these pigments are very susceptible to oxidative and thermal degradation during storage and processing of same, and due to this reason, this formulation has a very shorter shelf life and thus are not suitable to use for long time use. Hence, due to lesser stability and lower shelf life, it become very difficult to store and process this formulation for longer time durations. In view of this problem, there was an immense need to stabilize composition of zeaxanthin and lutein.

[0018] Existing commercial approaches to overcome the problem of poor stability of lutein / Zeaxanthin and their limitations-

[0019] Various researchers are continuously working in this field, in order to improve the stability, bioavailability as well as shelf life of the lutein and zeaxanthin. Some of the existing commercial approaches with their limitations are as below-

[0020] Stabilization of lutein by microencapsulation by spray drying using maltodextrin, Arabic gum and modified starch, however this method is quite complex, involves use of sophisticated spray drying technique and costly chemicals such as maltodextrin, modified starch etc, thus the same is not reliable.

[0021] Stabilization of lutein and zeaxanthin using soybean oil as carrier, however use of soybean oil is not that reliable, as same is insufficient to enhance the stability to a remarkable level, hence use of this technique is also restricted. Nano-emulsification and nano-encapsulation to stabilize lutein, a particle size of less than 1 pm help in lutein solubility, thus improving bioavailability and increasing stability. However, such techniques are cumbersome, expensive and time consuming, thus are not widely used for stabilization.

[0022] Problem solved by present invention-

[0023] Lutein and zeaxanthin, both suffer from a major problem i.e., instability and lower shelf life due to high susceptibility to thermal and oxidative degradation. To overcome this problem and to obtain highly stable lutein-zeaxanthin composition, use of various oils such as soybean oil, use of spray drying techniques, use of starch, antioxidants etc. are employed, but these techniques are not desirable as these are quite complex, cumbersome, expensive and do not suitably enhance the stability.

[0024] The above problems have been solved in very innovative manner in present invention.

[0025] The present invention discloses use of oleocanthal derived from extra virgin olive oil as a marker and stabilizing compound and combination of Olea europaea L extra virgin olive oil having approx. 67% of Oleic Acid and 10% Linoleic Acid and Brassica napus L. (canola / rapeseed) oil having 62% Oleic Acid and approx. 20% Linoleic Acid manufactured by BORGER, Spain as carrier / vehicle and stability enhancing agents, which synergistically enhances the stability, thus increases the shelf life of the lutein and zeaxanthin composition to desirable levels and overcome the problems associated with the use of existing techniques for enhancing the stability of the lutein and zeaxanthin.

[0026] Advantages offered by present invention i. Enhances the stability of lutein-zeaxanthin - The present invention uses combination of extra virgin olive oil and canola oil as carrier or vehicle in lutein and zeaxanthin composition, which synergistically enhance the stability of lutein and zeaxanthin in cost effective and simple manner. ii. Overcomes the problems of existing techniques - Present invention provides a cost effective and simple solution to stabilize lutein zeaxanthin composition using easily available olive oil and canola oil as carrier / vehicle and stability enhancers, hence overcomes the drawbacks of already existing techniques for lutein-zeaxanthin stabilization. iii. Use of oleocanthal as marker compound- The present invention involves the use of oleocanthal as a marker and stabilizing compound, which is a natural anti-inflammatory compound derived from extra virgin olive oil and is quite stable compound upon heating.

[0027] PRIOR ART

[0028] Indian Patent Application No. 202242056457 discloses "A stability enhanced composition of lutein and zeaxanthin comprising quercetagetin thereof"

[0029] The above cited patent discloses a composition of lutein and zeaxanthin comprising shelf life stability enhancing component i.e., quercetagetin extracted from defatted marigold flowers to enhance the shelf life as well as the stability of the lutein and zeaxanthin composition. The composition of the cited patent comprises of 60-80% lutein and zeaxanthin in combination, 20-40% diluents such as microcrystalline cellulose, maltodextrin, brown dextrin, starch etc, 0.5-1.0% combination of antioxidants consisting of Vitamin C, vitamin E, curcumin, green tea extract, rosemary extract, coffee bean extract, ascorbyl palmitate and 0.1-0.6% stability enhancing component i.e., quercetagetin. The disclosed composition has a shelf life of about 36 months at 25°C temperature and stability of about 12 months at higher temperatures i.e., about 40°C temperature.

[0030] The present invention discloses the use of carrier i.e., extra virgin olive oil and canola oil comprising moleculgr compound oleocgnthgl to enhgnce the stgbility of lutein gnd zeaxanthin, wheregs in gbove cited pgtent use of guercetggetin is disclosed, which is not anywhere mentioned in the present invention. Further, the use of diluents, antioxidants as disclosed in above cited patent is not disclosed in the present invention, making present invention different from the above cited invention. Oleocanthal / Virgin Olive Oil (VOO) has anti-inflammatory effects, therapeutic targets against many chronic inflammatory disease including neurodegenerative, and joint degenerative disease. Oleocanthal efficiency in decreasing markers of arthritis, disrupting processes vital to the formation of Alzheimer's and being a neuro-protective compound in vitro is confirmed. Furthermore oleocanthal reduces proliferation, migration.

[0031] Indian Patent Application no. 201921041676 discloses "Xanthophyll composition comprising lutein and zeaxanthin with enhanced bioavailability"

[0032] The above cited patent discloses a xanthophyll composition in the form of beadlets, powders, oils, granules, capsules etc. The disclosed composition comprises of 65-85% lutein, 10-30% zeaxanthin, 10-80% carrier / vehicle selected from medium chain triglyceride (MCT) oil, sunflower oil, coconut oil, corn oil, soybean oil, rapeseed oil and MCT oil or combinations thereof, 1-10% solubility enhancer selected from thyme oil, olive oil, linseed (flaxseed) oil or mixtures thereof, 1- 5% flavonoids (optional), 1-10% bioavailability enhancing agents selected from nigella (black cumin) oil, caraway oil, phosphatidyl choline or mixtures and 1-10% antioxidant agents selected from tocopherol and sodium ascorbate or mixtures there.

[0033] Present invention uses extra virgin olive oil having 67% of Oleic Acid (OA) & 10% Linoleic Acid. Whereas the above cited patent uses only regular olive oil and canola oil. OA has a non-unsaturated omega 9 fatty acids, and considered to be highly stable to oxidation and can enhance the activity of antioxidants and anti-polymerization agents. Evidences from epidemiological studies suggest that a higher proportion of Mono unsaturated Fatty Acids (MUFA) notably Oleic Acid in the diet is linked with a reduction in risk of Coronary Heart Diseases( CHD).

[0034] Indian Patent Application No. 201841039166 discloses "Novel dosage form of lutein-zeaxanthin complex for treatment of age-related macular degeneration"

[0035] The above cited patent discloses a sachet, film coated tablet and gummy dosage of lutein-zeaxanthin complex for management of age related macular degeneration (AMD). The said composition comprises of binders selected from microcrystalline cellulose gelatin, sugars, polyethylene glycol, waxes, natural and synthetic gums, polyvinylpyrrolidone, cellulosic polymers etc, diluents selected from anhydrous lactose, lactose monohydrate and dibasic calcium phosphate, lubricants selected from magnesium stearate, calcium stearate, zinc stearate, stearic acid, talc, polymers etc, disintegrant selected from starch, clay, cellulose, aligns, gums, cross-linked polymers etc, flavors selected from aromatics, natural oils or artificial essences such as cinnamon oil, Wintergreen oil, peppermint oil, clove oil, bay oil, anise oil, thyme oil, eucalyptus oil, vanilla essence etc. The composition further comprises of natural or artificial colors and glidants selected from magnesium trisilicate, cellulose, starch, talc, tribasic calcium phosphate etc.

[0036] The above cited patent does not discloses the utilization of combination of olive oil and canola oil for stabilization of lutein and zeaxanthin composition.

[0037] Patent Application No. EP2433640A1 discloses "Composition comprising SOD, lutein and zeaxanthin"

[0038] The above cited patent discloses a composition comprising 100-500 mg enzyme i.e., sodium dismutase in association with 0.5 to 25 mg lutein and 0.1 to 5 mg zeaxanthin. The disclosed composition is suitable for oral administration and is suitable for treating , preventing and stabilizing disorders of eye associated to oxidative stress such as Age related macular degeneration (AMD).

[0039] The above cited patent does not discloses the utilization of combination of olive oil and canola oil for stabilization of lutein and zeaxanthin composition.

[0040] Patent Application No. EP1471898B1 discloses "Lutein / zeaxanthin for glare protection"

[0041] The above cited patent discloses a soft gelatin capsule for reducing glare and promoting recovery from glare, thus helps in improvement of visual performance, particularly of visual performance in the darkness. The said soft gel comprises of about lOmg lutein, 50mg lecithin, lOmg zeaxanthin and 200mg soybean oil. It further also comprises of other excipients such as Vitamin E, Vitamin C, zinc, 0-carotene etc. The same can also be available in other forms conventional oral administration form such as effervescent tablets, hard shell capsules, liquid formulation etc.

[0042] The above cited patent discloses use of soybean oil as carrier, in which, the lutein and zeaxanthin is unstable, whereas, in present invention soybean oil is replaced with combination of olive oil and canola oil, enhancing the stability of lutein zeaxanthin composition.

[0043] Journals and Publications

[0044] Zohreh Sattar, Milad Iranshahy, Mehrdad Iranshahi; Lutein is Four Times More Stable in Sunflower Oil than Olive Oil at 40 °C; European Journal of Lipid Science and Technology; 28 November 2018.

[0045] The above cited article investigates the degradation of lutein in sunflower oil and olive oil at temperature (40 °C) for 3 months. Samples of pigment are incubated in oils for 3 months and amount of lutein is monitored by spectrophotometric analysis. The / r values of lutein degradation in sunflower oil and olive oil are estimated to be 44 x lQ-4and 186 x 1Q-4, respectively. In the presence of vitamin E (15 mg mb1), the / r values are decreased and reach 24 x 10“4and 72 x 10“4in sunflower and olive oils, respectively. These results suggest that lutein is approximately four times more stable in sunflower oil than olive oil.

[0046] From the above cited article, it is evident that olive oil has role in stabilization of lutein, but combination of olive oil and canola oil to enhance stabilization of lutein-zeaxanthin composition as disclosed in present invention is not disclosed in the above cited article. Javaria Zafar, Amna Aqeel et.al; Biochemical and Immunological implications of Lutein and Zeaxanthin; International Journal of Molecular sciences; October 2021

[0047] The above cited article discloses various matrices for stabilization of lutein and zeaxanthin such as use of fish oil, sea buckthorn oil and water emulsion based matric, formation of nanoparticles, microcapsules, glycyrrhizic acid, arabinogalactan etc. Further, stability of lutein in olive oil is also mentioned and it is mentioned that in olive oil, lutein is more stable compared to 0-carotene and 0-cryptoxanthin in virgin olive oil.

[0048] Above cited publication discloses the stabilization of lutein in virgin olive oil, however, presence of canola oil not disclosed anywhere.

[0049] Marfa Victoria Alvarez-Henao, Nataly Saavedra, Sonia Medina, Claudio Jimenez Cartagena, Luz Maria Alzate, Julian Londono-Londono; Microencapsulation of lutein by spray-drying: Characterization and stability analyses to promote its use as a functional ingredient; Food Chemistry Volume 256, 1 August 2018, Pages 181-187

[0050] In above cited article, the stability of lutein is enhanced and improved using maltodextrin, arabic gum and a modified starch, to obtain micro-particles using spray-drying.

[0051] Above cited publication discloses the stabilization of lutein using maltodextrin, starch and Arabic gum, however, use of olive oil in combination of canola oil is nowhere disclosed.

[0052] Commercial Products

[0053] ZEALUTEIN® the Stabilized Lutein Composition; Sabinsa Corporation- Feb to March, 2022

[0054] The Sabinsa Corporation discloses lutein and zeaxanthin composition comprising mixture of natural xanthophyll carotenoid pigments and bioperinea. The disclosed composition comprises of 5% lutein, 1% zeaxanthin, 1% capsanthin, 1-2% natural xanthophyll blend and about 1-2% bioperine comprising 95% piperine and the same is used as a bioavailability enhancing compound.

[0055] The above article does not discloses the utilization of combination of olive oil and canola oil for stabilization of lutein and zeaxanthin composition.

[0056] OBJECT OF THE PRESENT INVENTION The main object of the present invention is to disclose a stable composition of lutein and zeaxanthin and preparation method thereof.

[0057] Another object of the present invention is to disclose a lutein and zeaxanthin composition comprising combination of olive oil and canola oil for enhancing the stability of the same.

[0058] SUMMARY OF THE INVENTION

[0059] Lutein and zeaxanthin, both suffer from a major problem i.e., instability and lower shelf life due to high susceptibility to thermal and oxidative degradation. To overcome this problem and to obtain highly stable lutein-zeaxanthin composition, use of various oils such as soybean oil, use of spray drying techniques, use of starch, antioxidants etc are employed, but these techniques are not desirable as these are quite complex, cumbersome, expensive and does not suitably enhance the stability.

[0060] The above problems have been solved in very innovative manner in present invention. The present invention discloses use of oleocanthal derived from extra virgin olive oil as a marker and stabilizing compound and combination of Olea europaea L extra virgin (olive) oil having approx. 67% of Oleic Acid and 10% Linoleic Acid and Brassica napus L. (canola / rapeseed) oil having 62% Oleic Acid and approx. 20% Linoleic Acid manufactured by BORGER, Spain as carrier / vehicle and stability enhancing agents, which synergistically enhances the stability, thus increases the shelf life of the lutein and zeaxanthin composition to desirable levels and overcomes the problems associated with the use of existing techniques for enhancing the stability of the lutein and zeaxanthin.

[0061] Present invention discloses a stable composition of lutein and zeaxanthin and preparation method thereof. The disclosed composition is formulated comprising of 20% free lutein, 4% zeaxanthin as active pharmaceutical ingredients, 67% carrier i.e., combination of olive oil and canola oil, 0.1-0.15% marker ingredient i.e., oleocanthal and 4-5% excipients selected from tocopherol, thyme oil and ascorbyl palmitate. The resulting formulation is highly stable and possesses a longer shelf life.

[0062] BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 - HPLC analysis of Lutein extract after IM

[0064] Figure 1(a) - HPLC analysis of Blank sample

[0065] Figure 1(b) - HPLC analysis of Standard Aztec Marigold Zeaxanthin sample (data 002. led) Figure l(c)-HPLC analysis of Standard Aztec Marigold Zeaxanthin sample (data 003. led)

[0066] Figure 1(d) - HPLC analysis of Standard Aztec Marigold Zeaxanthin sample (data 004. led)

[0067] Figure 1(e) - HPLC analysis of marigold extract lutein sample i.e., CR / LUT / 2023 / AUG / 003 IM 25 / 60 sample.

[0068] Figure 2 - HPLC analysis of Marigold extract lutein after 2M i.e., CR / LUT / 2023 / AUG / 003 2M 25 / 60 sample.

[0069] Figure 3 - HPLC analysis of lutein extract after 3M

[0070] Figure 3(a) - HPLC analysis of Blank sample

[0071] Figure 3(b) - HPLC analysis of marigold extract lutein- Standard R1

[0072] Figure 3(c) -HPLC analysis of Marigold extract lutein sample i.e., CR / LUT / 2023 / AUG / 003 3M 25 / 60% sample.

[0073] Figure 4 - HPLC analysis of lutein extract after 6M

[0074] Figure 4(a) - HPLC analysis of Blank sample

[0075] Figure 4(b) - HPLC analysis of Marigold extract lutein- Standard-Rl

[0076] Figure 4(c) - HPLC analysis of Marigold extract lutein Standard-R2

[0077] Figure 4(d) - HPLC analysis of Marigold extract lutein Standard-R3

[0078] Figure 4(e) - HPLC analysis of Marigold extract (lutein) CR / LUT / 2023 / AUG / 003 6M 25 / 60

[0079] Figure 5- HPLC analysis of lutein extract after 9M

[0080] Figure 5(a) - HPLC analysis of Blank sample

[0081] Figure 5(b) - HPLC analysis of Marigold extract lutein- Standard-Rl

[0082] Figure 5(c) - HPLC analysis of Marigold extract lutein Standard-R2

[0083] Figure 5(d) - HPLC analysis of Marigold extract lutein Standard-R3

[0084] Figure 5(e) - HPLC analysis of Marigold extract (lutein) CR / LUT / 2024 / AUG / 003

[0085] Figure 6- HPLC analysis of lutein extract after 9M

[0086] Figure 6(a) - HPLC analysis of Blank sample Figure 6(b) - HPLC analysis of Marigold extract lutein- Standard-Rl

[0087] Figure 6(c) - HPLC analysis of Marigold extract lutein Standard-R2

[0088] Figure 6(d) - HPLC analysis of Marigold extract lutein Standard-R3

[0089] Figure 6(e) - HPLC analysis of Marigold extract (lutein) CR / LUT / 2023 / AUG / 003 12M 25 / 60

[0090] DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention discloses a stable composition of lutein and zeaxanthin and method for preparation thereof. The present invention discloses use of combination of Olea europaea L. extra virgin (olive) oil and Brassica napus L (canola / rapeseed) oil as carrier / vehicle and stability enhancing agents, which synergistically enhances the stability, thus increases the shelf life of the lutein and zeaxanthin composition to desirable levels. The disclosed composition is easy to formulate and cost effective and overcomes the drawbacks associated with existing techniques available to enhance the stability of lutein and zeaxanthin.

[0092] The present invention discloses use of combination of Olea europaea L. extra virgin (olive) oil having approx. 67% of Oleic Acid and 10% Linoleic Acid and Brassica napus L. (canola / rapeseed) oil having 62% Oleic Acid and approx. 20% Linoleic Acid sourced from BORGER, Spain as carrier / vehicle and stability enhancing agents, which synergistically enhances the stability, thus increases the shelf life of the lutein and zeaxanthin composition to desirable levels and overcomes the problems associated with the use of existing techniques for enhancing the stability of the lutein and zeaxanthin

[0093] Composition of present invention

[0094] In present invention, lutein and zeaxanthin can be formulated in any suitab e form preferably in the form of a soft gel capsule and the same comprises of free lutein and zeaxanthin as active pharmaceutical ingredients (APIs), combination of canola oil and olive oil as carrier / vehicle and stability enhancing agent, oleocanthal as marker ingredient and excipients.

[0095] Example 1: Composition in the form of soft-gel capsule consists of the ingredients are disclosed in table 1 below:

[0096] Table 1- Composition of soft-gel capsule

[0097] Apart from composition formulated in soft gel capsule form, the composition can be formulated in any other suitable form such as in the form of liquid formulation or capsule or tablet or any other oral formulation including jelly, chewing gum, energy bar etc. It can also be formulated as patch formulation.

[0098] Specific use of olive oil and canola oil-

[0099] Olea europaea L (olive) oil- Olive oil is oil extracted by pressing whole olive Olea europaea L. fruits. Olive oil serves as an excellent source of various bioactive phytochemical compounds. Olive oil possesses wide antioxidant properties due to presence of various antioxidants including phenol alcohols and acids, secoiridoids, lignans and flavones etc. Oleocanthal is a type of natural phenolic compound majorly present in virgin olive oil (VOO), which is a potent and natural non-steroidal and anti-inflammatory and anti-oxidant agent. Oleocanthal / Virgin Olive Oil (VOO) has anti-inflammatory effects, therapeutic targets against many chronic inflammatory disease including neurodegenerative, and joint degenerative disease. Oleocanthal efficiency in decreasing markers of arthritis, disrupting processes vital to the formation of Alzheimer's and being a neuro-protective compound in vitro is confirmed. Furthermore oleocanthal reduces proliferation, migration. This molecular compound is particularly responsible for stabilization of carotenoids lutein and zeaxanthin owning to their antioxidants properties.

[0100] Brassica napus L (canola / rapeseed) oil- Canola oil also known as rapeseed oil is extracted from the seeds of Brassica napus L (Canola / rapeseed) plant. Canola oil is also a rich source of various phytochemicals including antioxidant properties, anti-inflammatory properties, anti-ageing properties etc. Further, canola oil is rich source of oleic acid (mono-saturated fats) and antioxidants, allowing canola oil to act as stability enhancing agent. https: / / openagriculturejournal.com / VOLUME / 10 / PAGE / 69 / FULLTEXT / #:~:text=Due%20to%20high%20oleic %20ocid,control%2C%20blood%20pressure%20ond%20concer.)

[0101] Both canola oil and olive oil synergistically enhances the stability of the lutein and zeaxanthin composition owning to presence of antioxidants and other molecular compounds such Oleocanthal, oleic acid etc.

[0102] Combination of olive oil and canola oil (100%) stock solution is prepared by mixing 15% olive oil to 85% canola oil and further from stock solution, 67% solution of olive oil and canola oil is added to active pharmaceutical ingredients i.e., lutein and zeaxanthin.

[0103] Oleocanthal is a natural anti-inflammatory compound derived from secoiridoids found in extra-virgin olive oil. It is phenylethanoid and natural phenolic compound and in EVOO, it is present in low i.e., 0.2mg / kg to high i.e., 498mg / kg concentrations and oleocanthal is quite stable upon heating. (https: / / www.internationaloliveoil.org / oleocanthal-a-magic-phenolic-compound / )

[0104] Stability study results

[0105] Stability study results are summarized in table 2 below:

[0106] Table 2: Stability of lutein and zeaxanthin for a period of 6 months

[0107] The composition is kept at temperature of 25°C±2°C and at relative humidity of 60% ±5%.

[0108] From table 2, it was observed that the color and texture of the composition remain constant for about 6 months and further, the content of both lutein and zeaxanthin also remains similar, which indicates that the disclosed composition is stable for about 6 months. Further testing of stability is under progress.

[0109] HPLC ANALYSIS FOR STABILITY STUDIES

[0110] HPLC analysis for stability of blank samples, standard lutein extracts and lutein and zeaxanthin extract extracted in present invention after 1 month, 2 months, 3 months, 6 months, 9 months and 12 months are provides in figures 1 to 6. The analysis of retention time (RT) for same is provided in table 3 and 4 below:

[0111] Table 3- Retention time (RT) for standard lutein extract and lutein extracted in present invention.

[0112] From table 3 above, it is concluded that the Retention time (RT) for lutein standard sample after 1 month is 22.451, after 3 months is 24.831, after 6 months is 18.063, after 9 months is 17.439 and after 12 months the RT is 23.973, whereas the retention time (RT) for extracted lutein samples after 1 months is 22.415, after 2 months is 20.417, 3months is 25.381, after 6 months is 18.061, after 9 months is 17.457 and after 12 months, the RT is 23.925. This indicates that the RT values and standard values of RTs are almost identical, suggesting stability of lutein upto 12 months. The retention times of the extracted sample closely align with the standard retention times at most time points, indicating that lutein remains relatively stable over a 12- month period, with only minor variations that do not suggest significant degradation.

[0113] Table 4- Retention time (RT) for standard zeaxanthin extract and zeaxanthin extracted in present invention.

[0114] The Retention time (RT) for zeaxanthin standard sample after 1 month is 25.665, after 3 months is 28.236, after 6 months is 20.178, after 9 months is 19.507 and after 12 months the RT is 27.487, whereas the retention time (RT) for extracted zeaxanthin samples after 1 months is 25.603, after 2 months is 23.045, after 3months is 28.846, after 6 months is 20.175, after 9 months is 19.523 and after 12 months, the RT is 27.436. Overall, the retention times of the extracted zeaxanthin sample are very close to the standard RTs across most time points, suggesting that zeaxanthin remains stable over the 12-month period.

[0115] The preliminary studies indicate that the present composition exhibits increased bioavailability as compared to earlier existing compositions and further studies in this regard are in progress.

[0116] Present invention offers a significant advantage as the composition possesses higher stability as well as shelf life and the same is cost effective and easy to formulate. The same is technological advanced as well as economical over the existing methods / techniques available to enhance the stability of lutein and zeaxanthin. Moreover, the product is highly stable, hence is of great economical and pharmaceutical importance.

[0117] The stable composition of lutein and zeaxanthin as disclosed in present invention is novel and the same is evident from non-disclosure in the prior art.

[0118] The inventive step by way of technological advancement lies in utilization of combination of olive oil and canola / rapeseed oil as carrier / vehicle and marker compound oleocanthal as stability enhancing agents, which stabilizes the lutein and zeaxanthin effectively in superior manner as compared to other existing techniques available for stabilization. Further, the disclosed technique has significant economic advantages being simple, cost-effective and less time consuming alternative of protecting lutein and zeaxanthin from thermal, light and oxidative degradation. Industrial application is duly clear as stable lutein and zeaxanthin composition has crucial role in reduction of age related macular degeneration, hence the same is of great pharmaceutical importance.

Claims

I CLAIM:

1. A stable composition of Lutein and Zeaxanthin wherein the same consists of free lutein, Zeaxanthin, combination of olive oil and canola / rapeseed oil as carrier / stability enhancing agent, oleocanthal derived from in the extra virgin olive oil as marker compound and excipients / stability enhancing agents.

2. The stable composition of Lutein and Zeaxanthin as claimed in claim 1 wherein the same consists of: free lutein : 20-23%Zeaxanthin : 4-4.5% combination of olive oil and canola / rapeseed oil : 65-70% oleocanthal derived from in the extra virgin olive oil : 0.1-0.15% tocopherol, thyme oil and ascorbyl palmitate : 4-5%3. The method of preparation of composition as claimed in claim 1 wherein the same consists of following steps: preparation of 100% stock solution of olive and canola oil by mixing 15% olive oil in 85% canola oil; mixing 20-23% lutein with 4-4.5% Zeaxanthin; adding 67% of mixture of olive oil and canola oil to mixture of lutein and zeaxanthin; adding 0.1-0.15% oleocanthal as marker compound; adding excipients and stabilizing agents for formulating the composition in suitable form.

4. The stable composition of Lutein and Zeaxanthin as claimed in claim 1 wherein the stability of said formulation is 12 months at 25oC±2oC temperature and 60% ±5% relative humidity.

5. The stable composition of Lutein and Zeaxanthin as claimed in claim 1 wherein the same is encapsulated in soft gel capsule.

6. The stable composition of Lutein and Zeaxanthin as claimed in claim 1 wherein the same is in the form of tablet or any other oral formulation including jelly, chewing gum, and energy bar.

7. The stable composition of Lutein and Zeaxanthin as claimed in claim 1 wherein the same is in the patch form.

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