DESC:Priority Claim:
[0001] This application claims priority from the provisional application numbered 202221071818 filed with Indian Patent Office, Chennai on 13th December 2022 and post-dated to 13th June 2023 entitled “A micelle formulation to enhance the bioavailability and efficacy of lutein”, the entirety of which is expressly incorporated herein by reference.
Preamble to the description
[0002] The following specification describes the invention and the manner in which it is to be performed:
Description of the invention
Technical field of the invention
[0003] The present invention relates to a formulation for enhancing the bioavailability, stability and the efficacy of lutein. More particularly, the invention relates to the formulation of lutein using micellization to complex lutein with other antioxidants to enhance the bioavailability, stability and efficacy of lutein. The invention also discloses a method of preparation of lutein micellar complex.
Background of the invention
[0004] Lutein is a major carotenoid commonly called as “eye vitamin” found in macula and retina of the human eye. Lutein acts as a light filter, protecting the eye tissues from sunlight damage and free radicals.
[0005] Humans are not capable of synthesizing carotenoids, and thus, their presence in human tissues is entirely of dietary origin. Lutein is protective against diseases such as age-related macular degeneration (ARMD). At present, data regarding bioavailability of lutein from various sources are insufficient.
[0006] The naturally available lutein exhibits low bioavailability and solubility due to hydrophobicity. Various formulations and strategies are employed to enhance the bioavailability of lutein. The use of drug delivery systems aids in enhancing the lipophilicity and bioavailability of lutein.
[0007] In order to overcome this, various technological strategies are reported including micronization, nano emulsions, solid dispersions and microencapsulation etc. Micronization is a process of reducing the average diameter of a solid material's particles. The conventional techniques for micronization focus on mechanical approaches such as milling and grinding. In contrast, the modern techniques make use of the properties of supercritical fluids and manipulate the principles of solubility.
[0008] The particle size is reduced to the micrometer, or in some cases nanometer, level using dynamic high-pressure homogenization. Micronization results in achieving the optimal particle size thus making the materials easier to handle and process, improve delivery of active ingredients and enhance various qualities or characteristics of raw materials.
[0009] Nano emulsions are nano-sized emulsions, which are manufactured for improving the delivery of active ingredients. These are the thermodynamically stable isotropic system in which two immiscible liquids are mixed to form a single phase by means of an emulsifying agent, i.e., surfactant and co-surfactant. Nano emulsions have small droplet size and are kinetically stable colloidal systems. They have enhanced functional properties in comparison to conventional emulsions. The composition and structure of the nano emulsions are controlled for the encapsulation and effective delivery of bioactive lipophilic compounds. Nano emulsions have potential application in the food industry for the delivery of nutraceuticals, coloring and flavoring agents, and antimicrobials. The nano emulsion formulations of active ingredients are used for developing biodegradable coating and packaging films to enhance the quality, functional properties, nutritional value, and shelf life of foods.
[0010] Nano emulsion is a simple and effective delivery system because of its safety, easy industrial adoption and widely accepted food and pharma applications. Thus, nano emulsion of bioactives such as lutein has received increasing attention as an effective approach to improving the bioavailability and bio efficacy.
[0011] Lutemax is a lutein extract standardized with lutein and zeaxanthin for eye health and vision performance, cognitive performance, mood support and sleep health. This is formulated using microencapsulation technology in a starch-based matrix for nutrient delivery system that improves product formulation and efficacy. The technology synergizes all benefits of Lutemax with flexibility, including improved stability, dispersibility and significantly improved bioavailability. Similarly, Floraglo is a lutein extract standardized with lutein and zeaxanthin for eye health and cognitive health prepared by employing the Actilease technology. This microencapsulation technology produces small particles of lutein within a readily dissolvable matrix that is capable of being compressed into product forms such as tablets without significant degradation. The formulation yields a highly bioavailable lutein due of the nature of the dissolvable matrix.
[0012] A micelle is an aggregate of surfactant amphipathic lipid molecules dispersed in a liquid, forming a colloidal suspension. It forms a fluid-like core formed by the nonpolar portion of the amphiphiles encased in a shell formed by the polar portion of the amphiphiles in contact with fluid water. Generally, micelles are biocompatible, enhances bioencapsulation efficacy, highly stable in vitro and in vivo, and can dissolve a broad variety of poorly soluble actives. Polymeric micelles, with stable, biocompatible and solubilizing properties, have drawn considerable attention for the oral administration of poorly water-soluble agents.
[0013] The Patent Application No. JP6563447B2 entitled “Carotenoid particles and uses thereof” discloses a pharmaceutical or nutritional composition suitable for oral administration comprising micelles, reverse micelles or a population of clusters thereof and excipients, in which each micelle, reverse micelle, or cluster thereof comprises the carotenoid compounds and cargo molecules. The carotenoids form an outer layer encapsulating an inner core comprising the cargo molecules and a pharmaceutical composition wherein the population does not contain whey protein or nutritional composition.
[0014] The Patent Application No. US20190030048A1 entitled “Casein micelles for nanoencapsulation of hydrophobic compounds” discloses field of food technology and delivery of hydrophobic biologically active compounds, particularly nutrients, via food products and beverages. In particular, the invention provides isolated casein micelles useful for the encapsulation of hydrophobic nutrients, therapeutic and cosmetic compounds, compositions thereof and methods of preparing the micelles.
[0015] The Patent Application No. JP2012131768A entitled “Composition and application of carotenoid having improved absorption and bioavailability” discloses carotenoids or xanthophylls in a micro micellar form that act as a more effective and efficient antioxidant than the case of administering in a crystalline form. The carotenoids or xanthophylls in a micro micellar form are obtained as micro micelles after melting lutein diacetate or lutein dipropionate in their natural original lipid vegetable matrix, in the presence of lipids, phospholipids, fatty acids, emulsifiers and moisture without containing any of crystalline forms of carotenoids, and are ingested in a dosage between 2 to 50 mg, to provide an absorbed amount of the carotenoid solubilizate which is higher by at least 20% than that of crystalline carotenoids, and to provide a macular pigment deposit which exceeds by at least 10% of macular pigment optical density higher than the deposition obtained by ingesting crystalline lutein, thus prevent tissue degeneration, and UV light damage to skin and the retina.
[0016] The Patent Application No. CN114177162A entitled “Preparation method of pH-sensitive lutein-chitosan nano-micelle loaded with tripterine” discloses a preparation method of tripterine-loaded pH-sensitive lutein-chitosan nano-micelles, which can effectively solve the problems of low bioavailability of LU and Cel and reduction of toxicity of Cel to important organs such as heart, liver and nervous system, and adopts the technical scheme that the tripterine-loaded pH-sensitive lutein-chitosan nano-micelles can be used for preparing the tripterine-loaded pH-sensitive lutein-chitosan nano-micelles. The specific preparation method comprises steps of synthesizing a covalent coupling compound of the pH response auxiliary material and the xanthophyll, preparing a lutein-chitosan polymer which is sensitive to pH (Potential of Hydrogen), preparation of the pH-sensitive lutein-chitosan nano delivery system loaded with the tripterine, the preparation method is simple, the prepared pH-sensitive lutein-chitosan nano delivery system loaded with the tripterine can effectively respond to the acid environment of lysosome in renal tubular epithelial cells, the lutein and the tripterine are released, and the delivery effect is good. Kidney inflammation microenvironment can be rapidly improved, AKI can be improved, and the traditional Chinese medicine composition is an innovation of pharmaceutical preparations for treating AKI.
[0017] Hence, there is a need for a formulation of lutein in combination with antioxidants which overcomes the challenge of hydrophobicity to enhance the bioavailability thus executing the enhanced therapeutic activity of lutein.
Summary of the invention
[0018] The present invention overcomes the drawbacks of the existing system and the prior arts to provide a micellar composition of lutein for enhancing the bioavailability, stability and the efficacy of lutein.
[0019] The micellar composition of lutein comprises trans lutein at a concentration in a range between 10% to 90%, zeaxanthin at a concentration in a range between 10% to 90%, carnosic acid at a concentration in a range between 0.1% to 5%, tocopherol mixture at a concentration in a range between 1% to 10%, emulsifier at a concentration in a range between 0.1% to 5% and a food grade carrier at a concentration in a range between 10% to 90%, wherein the composition is prepared in a micelle form. The antioxidants present in the micellar composition increases the physicochemical properties, and lipophilicity of lutein, facilitating enhanced bioavailability, stability and efficacy.
[0020] The method of preparation of micellar composition of lutein comprises the steps of mixing trans lutein with antioxidant sources including zeaxanthin, forming a mixture and adding carnosic acid, tocopherol, along with an emulsifier. Further, the ingredients are mixed using a food grade carrier and alcohol and the mixture is subjected to homogenization at room temperature. The mixture is further dried at a temperature of 400C to 900C to obtain a lutein micellar complex. The micellar composition of lutein facilitates increased absorption of lutein and the antioxidants through the epithelial membrane. As the lutein is complexed with the antioxidants, the stability of these actives is enhanced so that the concentration of these actives is high in the blood.
[0021] The advantages of the present invention include enhanced physicochemical properties, and lipophilicity of lutein due to formation of micellar lutein composition in complex form or lutein micellar complex, further enhancing the bioavailability, stability and efficacy of lutein.
Brief description of the drawings
[0022] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0023] Figure 1 tabulates the composition according to an embodiment of the invention.
[0024] Figure 2 illustrates a method of preparation of composition of lutein in micelle form.
[0025] Figure 3 illustrates the graphical representation of the Differential Scanning Calorimetry (DSC) thermograms of free lutein and lutein micellar complex samples.
[0026] Figure 4 illustrates graphical representation of the thermogravimetric analysis (TGA) curves.
[0027] Figure 5A and Figure 5B illustrates the graphical representation of the Fourier Transform Infrared Spectroscopy (FTIR) spectra.
[0028] Figure 6 illustrates the images of free lutein and lutein micellar complex in Field Emission Scanning Electronic Microscopic (FE-SEM) analysis.
[0029] Figure 7 illustrates the high-resolution transmission electron microscopy (HRTEM) images of free lutein and lutein micellar complex.
[0030] Figure 8 illustrates the graphical representation of the Oxidation Test Reactor (OXITEST) studies.
[0031] Figure 9A illustrates the tabular representation of the cytotoxic properties of free lutein and lutein micellar complex against Caco-2 cell line.
[0032] Figure 9B illustrates the tabular representation of the invitro intestinal permeability data of free lutein and lutein micellar complex across Caco-2 cell monolayer.
[0033] Figure 10A illustrates the tabular representation of the assessment of antioxidant activity of test substances against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical.
[0034] Figure 10B illustrates the tabular representation of the antioxidant activity assessment of the test substances against 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical.
[0035] Figure 10C illustrates the tabular representation of the antioxidant activity assessment of the test substances against lipid peroxidation.
Detailed Description of the Invention
[0036] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0037] The term “Bioavailability” refers to the extent a substance or drug becomes completely available to its intended biological destination.
[0038] The term “Micelle” refers to aggregate of surfactant amphipathic lipid molecules dispersed in a liquid, forming a colloidal suspension.
[0039] The term “Hydrophobicity” refers to physical property of a molecule that is seemingly repelled from a mass of water.
[0040] The term “Lipophilicity” refers to the ability of a chemical compound to dissolve in fats, oils, lipids, and non-polar solvents such as hexane or toluene.
[0041] The present invention discloses a composition for enhancing the bioavailability, stability and the efficacy of lutein, wherein the micellar composition of lutein comprises antioxidants to increase the physicochemical properties, and lipophilicity, thus increasing the bioavailability, stability and efficacy of lutein.
[0042] Figure 1 tabulates the composition according to an embodiment of the invention. The composition comprises trans lutein at a concentration in a range between 10% to 90%, zeaxanthin at a concentration in a range between 10% to 90%, carnosic acid at a concentration in a range between 0.1% to 5%, tocopherol at a concentration in a range between 1% to 10%, emulsifier at a concentration in a range between 0.1% to 5% and a food grade carrier at a concentration in a range between 10% to 90%, wherein the composition is prepared in a micelle form.
[0043] The complexation of lutein with antioxidants forms a micelle and wherein the antioxidants are natural ingredients, wherein the micellar structure is same as the naturally existing micelles. The natural micelle structure overcomes the use of surfactants such as Tween 80 thus making the composition natural.
[0044] Figure 2 illustrates a method of preparation of composition of lutein in micelle form. The method (200) of preparation of micellar complex comprises the steps of adding trans lutein at a concentration in a range between 10% to 90% to zeaxanthin at a concentration in a range between 10% to 90% to form a mixture and further adding carnosic acid at a concentration in a range between 0.1% to 5%, emulsifier at a concentration in a range between 0.1% to 5%, tocopherol at a concentration in a range between 1% to 10%, in the step (201). Further, the ingredients are mixed using a food grade carrier and alcohol, in step (202). In the step (203), the mixture is subjected to homogenization at room temperature. The mixture is further dried at a temperature of 400C to 900C to obtain a micellar composition of lutein, as disclosed in the step (204).
[0045] According to the present invention, the composition exhibits increased absorption of lutein and antioxidants through an epithelial membrane. Further, as lutein is complexed with the antioxidants, the stability of these actives is also enhanced so that the concentration of these actives is high in the blood. The micellar complexation of lutein with antioxidants enhances the antioxidant efficacy of lutein in synergy with the antioxidants.
[0046] The properties of the lutein can be studied in detail using several examples. According to an embodiment of the invention, free lutein and lutein micellar complex were considered to determine their bioavailability, stability and efficacy.
Example 1: Characterization studies for free lutein and lutein micellar complex by Differential Scanning Calorimetry (DSC).
[0047] Lutein is a natural pigment and antioxidant found in various fruits and vegetables, that offers various health benefits, particularly in supporting eye health. Lutein is available in the form of a powder (herein called as free lutein and not in esterified form) or as part of micellar complex (herein called as lutein micellar complex). Free lutein and the lutein micellar complex were analysed to determine the physicochemical properties by Differential Scanning Calorimetry (DSC). Free lutein and lutein micellar complex (herein called samples without implying any limitations) were analysed using Differential Scanning Calorimetry (DSC) in order to identify their thermal behavior, wherein the samples were heated from room temperature to 500°C at a rate of 10°C/min under a controlled nitrogen atmosphere.
[0048] Figure 3 illustrates the graphical representation of the Differential Scanning Calorimetry (DSC) thermograms of free lutein and lutein micellar complex samples displayed an endothermic peak at around 160°C and 180°C, representing the melting point and phase transition. It was observed that free lutein and lutein micellar complex exhibited similar thermal behavior, wherein free lutein displayed the endothermic peaks at 172.31OC and 184.33OC.
Example 2: Characterization studies for free lutein and lutein micellar complex by Thermogravimetric Analysis (TGA).
[0049] Free lutein and the lutein micellar complex were analysed to determine the physicochemical properties using Thermogravimetric Analysis (TGA), wherein TGA was performed on the samples to observe the weight loss behavior due to thermal decomposition, where the samples were heated from room temperature to 600°C at a rate of 10°C/min under a controlled nitrogen atmosphere.
[0050] Figure 4 illustrates graphical representation of the thermogravimetric analysis (TGA) curves. With reference to Figure 4, the thermogravimetric analysis (TGA) curves revealed a gradual weight loss for both free lutein and lutein micellar complex samples, corresponding to the degradation of lutein. The free lutein exhibited a weight loss starting at approximately 250°C, while the lutein micellar complex exhibited a weight loss starting at around 280°C, suggesting that the lutein micellar complex may have a higher thermal stability than the free lutein.
Example 3: Characterization studies for free lutein and lutein micellar complex by Fourier Transform Infrared Spectroscopy (FTIR).
[0051] Free lutein and the lutein micellar complex were analysed by Fourier Transform Infrared Spectroscopy (FTIR) to investigate the functional groups present in the samples, where the samples were analyzed using a Fourier Transform Infrared Spectroscopy (FTIR) spectrometer in the range of 4000 to 400 cm-1.
[0052] Figure 5A and Figure 5B illustrates the graphical representation of the Fourier Transform Infrared Spectroscopy (FTIR) spectra. The Fourier Transform Infrared Spectroscopy (FTIR) analysis of free lutein and the lutein micellar complex indicated that they both display similar characteristic absorption peaks, indicating the presence of lutein in both forms. With reference to the graphical representation in Figure 5A disclosing the FTIR spectra of free lutein and Figure 5B disclosing the FTIR spectra of lutein micellar complex, wherein the major peaks were observed at around 3000-3600 cm-1 (O-H stretching), 1700-1750 cm-1 (C=O stretching), and 1500-1600 cm-1 (C=C stretching and aromatic ring vibrations) in lutein micellar complex with slight difference in free lutein structure. It was determined that 1724 cm-1 (C=O stretching) specific for carboxylic group may be attributed to tocopherol and carnosic acid that are absent in free lutein.
Example 4: Characterization studies for free lutein and lutein micellar complex by zeta potential studies.
[0053] Free lutein and the lutein micellar complex were analysed to determine the zeta potential using a zeta potential analyzer, where the samples were dispersed in an appropriate solvent (Ethanol: Water), and the electrophoretic mobility was measured to determine the zeta potential value. According to some embodiments of invention, the zeta potential measurements of free lutein and lutein micellar complex facilitated determination of the surface charge of the samples. The free lutein exhibited a negative zeta potential value of -25 mV, while the lutein micellar complex showed a slightly lower value of -30 mV, suggesting a slight increase in surface charge due to the complex formation.
[0054] The analysis of free lutein and the lutein micellar complex using DSC, TGA, FTIR, and zeta potential measurements provided insights into their physicochemical properties, and it was determined that free lutein and lutein micellar complex samples exhibited similar thermal behavior and characteristic FTIR peaks, confirming the presence of lutein. The TGA results indicated that the lutein micellar complex may have higher thermal stability compared to the free lutein. Furthermore, the zeta potential analysis suggested a slight increase in surface charge when lutein is part of a complex.
Example 5: Characterization studies for free lutein and lutein micellar complex by Field emission scanning electron microscopy (FE-SEM).
[0055] Free lutein and the lutein micellar complex were analysed using Field emission scanning electron microscopy (FE-SEM) to determine the microstructure image of the samples.
[0056] Figure 6 illustrates the microstructure images of free lutein and lutein micellar complex in Field Emission Scanning Electronic Microscopic (FE-SEM) analysis. It was determined that when lutein forms a complex with other molecules, it can exhibit unique properties and structural changes. Figure 6 discloses the structure of free lutein and lutein micellar complex, wherein lutein particles appeared agglomerated when complexed with tocopherol and carnosic acid. The complexes are formed through the interactions such as hydrogen bonding, electrostatic attraction, or coordination compounds, wherein the change in characteristic in the surface features, such as the presence of additional structures or patterns, indicate the successful formation of the lutein micellar complex. With reference to Figure 6, the FESEM studies disclose that the microstructures of free lutein and the lutein micellar complex are different.
Example 6: Characterization studies for free lutein and lutein micellar complex by High-resolution transmission electron microscopy (HRTEM).
[0057] Free lutein and the lutein micellar complex were analysed by High-resolution transmission electron microscopy (HRTEM) to determine the crystallographic structure of the samples at an atomic scale.
[0058] Figure 7 illustrates the high-resolution transmission electron microscopy (HRTEM) images of free lutein and lutein micellar complex, wherein the high-resolution transmission electron microscopy (HRTEM) images indicated that the crystallographic structures of free lutein and lutein micellar complex are different.
Example 7: Characterization studies for free lutein and lutein micellar complex by Oxidation Test Reactor (OXITEST).
[0059] Free lutein and the lutein micellar complex were analysed by Oxidation Test Reactor (OXITEST) to monitor the oxygen uptake by reactive components in the samples and to determine their IP value.
[0060] Figure 8 illustrates the graphical representation of the Oxidation Test Reactor (OXITEST) studies. With reference to Figure 8, the Induction period (IP) determination was carried out, and the results are provided in the table.
[0061] The OXITEST studies indicated that the stability of the lutein micellar complex was improved compared to free lutein. The IP of lutein micellar complex was found to be 3 hours 10 minutes.
[0062] Further, according to the invention, the stability of free lutein and lutein micellar complex was assessed at -20OC for a period of 6 months, and the results indicated that free lutein and its micellar complex remained stable under these accelerated conditions, wherein lutein micellar complex was found to be more stable than regular lutein, and the results corroborate the OXITEST studies.
Example 9: Measurement of the permeability of lutein micellar complex in the Caco-2 cell line.
[0063] The permeation coefficient of lutein micellar complex molecules in the test substances are assessed and evaluated by Caco-2 permeability assay, wherein the test substances including free lutein and lutein micellar complex were evaluated for its in vitro permeation coefficient in Caco-2 cells. Initially, the test substances were tested for cytotoxicity and the non-toxic concentrations were selected for permeation studies on Caco-2 cells. The Caco-2 cells were seeded onto inserts (0.4µ) and were cultured for 25 days and the membrane integrity was examined and the test substances were loaded onto the receiver compartment at the pre-identified concentrations. After the specified time point, the apical and basolateral solutions were collected and were analyzed by HPLC.
[0064] For determining the cytotoxicity, 10 mg of test substances were weighed and separately dissolved in Dulbecco's Modified Eagle Medium (DMEM)-HG supplemented with 2% inactivated Fetal bovine serum (FBS), and the volume was made up with media to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Further, serial two-fold dilutions were prepared from the stock solution to perform cytotoxic studies and further efficacy studies. Further, the stock cells of the Caco-2 (Colon carcinoma) cell line were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ml), streptomycin (100 µg/ml) and amphotericin B (5 µg/ml) in a humidified atmosphere of 5% CO2 at 37°C until confluent. Additionally, the cells were dissociated with TPVG solution (0.2% trypsin, 0.02% Ethylenediaminetetraacetic acid (EDTA), 0.05% glucose in Phosphate buffer saline (PBS)). The stock cultures were grown in 25 cm2 culture flasks and all the experiments were carried out in multi-well plates, (for example: 96 well microtiter plates, 12 well plates).
[0065] The cytotoxicity studies were performed to assess the cell viability using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay in semi confluent monolayer cultures, wherein the drug solutions were added to cells and incubated at 37 °C in 5% CO2 atmosphere for 72 h. Upon incubation, the 100 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in DPBS was added to the drug solutions in the plurality of wells and further incubated for 3 h at 37 °C in 5% CO2 atmosphere. The mixture was further processed and the supernatant was separated, where 100 µl of Dimethyl sulfoxide (DMSO) was added to it to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 540 nm. The plates were protected from light throughout the process. The percentage growth inhibition was calculated using the standard formula and concentration of test substance, needed to inhibit the growth of the cell by 50% i.e., CTC50 values were generated from the dose-response curves.
[0066] According to some embodiments of the invention, Caco-2 permeability assay was performed wherein, for the intestinal permeability experiments, Caco-2 cells between passages 30 and 35 were seeded on PET membrane inserts (113.1 mm2, 0.4 µm pore size) at a density of 100,000 cells per insert and cultivated for 25 days, and the culture medium was replaced three times per week until the time of use. The permeability of the monolayers to Lucifer Yellow (LY) (100 µg/mL) was assessed using a microplate reader, as the permeability to the paracellular marker LY (Lucifer Yellow) is considered to be indicator for monolayer integrity.
[0067] The below equation was used to calculate the permeability coefficient (Papp).
………… Equation 1
where, dQ/dT is the rate of appearance of Lucifer yellow (LY) on the basolateral side (µg/sec); A= area of the semi-permeable membrane; and Cd= initial concentration of Lucifer yellow (LY) in the apical layer. The inserts with permeability coefficient (Papp) for Lucifer yellow (LY) less than 0.2x10-6 cm/sec were selected for the assay.
[0068] Further, Hank’s Balanced Salt Solution (HBSS) at pH 6.0 and pH 7.4 was used as transport buffer in the apical (AP) and basolateral (BL) compartments, respectively, in order to mimic in vivo conditions. Before the experiments, monolayers were washed twice with Hank’s Balanced Salt Solution (HBSS), pH 7.4, and incubated for 30 min at 37°C for membrane integrity measurements, followed by diluting the Dimethyl sulfoxide (DMSO) stock solutions of all test substances in Hank’s Balanced Salt Solution (HBSS) and added to the donor compartment, while fresh HBSS was added to the receiver compartment. Further, all experiments were performed in unidirectional manner as per the requirement, i.e., from AP to BL, for the period of 120 min, at 37°C in an orbital shaker (100 rpm). Upon incubation, 100µL aliquots were removed from the receiver compartment and replaced with an equal volume of fresh HBSS. At the end of the assessment, the samples were collected from donor compartments in order to measure the permeation coefficient of the marker molecules in Caco-2 cells. The permeability coefficient (Papp) is calculated by the following equation:
……….. Equation 2
where, ?Q/?T is the steady state flux (mols/sec); A is the surface area of the filter (mm2); and Co is the initial concentration of the donor compartment (mol/ml).
[0069] The cytotoxic properties of the free lutein and lutein micellar complex was evaluated against Caco-2 cell line, as disclosed in Figure 9A, and the invitro intestinal permeability data of the free lutein and lutein micellar complex across Caco-2 cell monolayer is disclosed in Figure 9B.
[0070] According to the invention, the free lutein and lutein micellar complex were initially examined for their in vitro cytotoxicity studies against Caco-2 cells by MTT assay by exposing the cells to different concentrations of the test substance; furthermore, the concentrations were selected for further permeability studies. It was determined that the transported free lutein and lutein micellar complex samples passed through the Caco-2 monolayer intact and non-metabolized, and was detected by HPLC in the basolateral or apical samples taken after the given time point exposure at the selected concentration of the test substances. The apparent permeability and %passage were calculated as a direct measure of concentration of the test substance and across the CaCo-2 monolayer. Further, the inserts with good membrane integrity were selected for the assay, where the integrity of the membrane was measured by LY dye.
[0071] In order to mimic the in vivo conditions, the pH of apical layer HBSS buffer was adjusted to pH 6.0 and the pH of the basolateral layer was adjusted to pH 7.4. The Dimethyl sulfoxide (DMSO) stock solutions of the compounds were dissolved in HBSS buffer at the pre-determined concentrations and were added to the apical layer, and were incubated for various time intervals. After 0 minute and 120 minutes, 100µl of each sample was collected from the apical layer and the basolateral layers and were substituted with equal volumes of HBSS, followed by analysis of the samples by HPLC. The permeability coefficient (Papp) of Free Lutein was found to be 3.91x10-6 and the permeability coefficient of lutein micellar complex was found to be 4.86x10-6, wherein among the free lutein and the lutein micellar complex samples, lutein micellar complex exhibited significantly higher permeability i.e. above 24% over lutein in Caco-2 cells.
Example 10: Evaluation of antioxidant activity of lutein micellar complex 50% and free lutein 70% by 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and Lipid peroxidation inhibitory assay.
[0072] The antioxidant activity of lutein micellar complex 50% and free lutein 70% was evaluated at concentrations 1000 to 62.5µg/ml against 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and Lipid peroxidation (LPO) inhibitory assays. In order to determine the antioxidant activity by DPPH assay, 21mg of test substance (lutein micellar complex, free lutein) was dissolved separately in 1ml of Dimethyl sulfoxide (DMSO) to obtain the concentration of 1000 µg/ml. Further, the stock solution was serially diluted to obtain 1000 - 62.5 µg/ml to get the lower concentrations. Further, the standard solution was prepared by dissolving 10mg of ascorbic acid in 1ml of Dimethyl sulfoxide (DMSO) to obtain concentration of 1000 µg/ml, and it was serially diluted to get lower concentrations of 62.5, 125, 250, 500 and 1000 µg/ml. Further, 2, 2-Diphenly 1-picryl hydroxyl solution (DPPH, 100 µM) was prepared by weighing 22 mg of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) accurately and dissolving it in 100 ml of methanol to get stock solution, wherein from the stock solution 18 ml was diluted to 100 ml using methanol to obtain 100µM 2, 2-diphenyl-1-picrylhydrazyl (DPPH) solution.
[0073] The process involved in the antioxidant assay includes a multi-well plate, for example: 96 well microtiter plate, where 0.01ml of different concentrations of test substance (lutein micellar complex, free lutein) and standard was added separately in the test and test blank wells. Additionally, instead of test substance 0.01ml of Dimethyl sulfoxide (DMSO) was taken for control and control blank, and 0.2 ml of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) was added to the test and control, whereas to the test blank and control blank, 0.2ml of methanol was added in place of 2, 2-diphenyl-1-picrylhydrazyl (DPPH). The same process was repeated for standard by replacing test substance with standard. The microtiter plate was incubated at 37°C for 30 minutes, and the absorbance was measured at 490 nm using microplate reader.
[0074] Further, the antioxidant activity of lutein micellar complex 50% and free lutein 70% by 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) Radical Assay was performed, wherein the standard test substance at 1000µg/ml concentration was prepared by dissolving 13.6 mg of test sample in 2ml of Methanol and further serially diluting the solution from 1000 to 62.5 µg/ml to obtain lower concentrations. Additionally, ABTS solution was prepared by dissolving 5.48mg of 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in 5 ml of distilled water to achieve 2mM concentration and to this solution 17mM of 0.03ml potassium persulphate was added. The reaction mixture was incubated at room temperature overnight in dark environment.
[0075] Further, the antioxidant activity of lutein micellar complex 50% and free lutein 70% was determined using ABTS radical assay, by taking 0.2 ml of different concentration of test substance and standard separately in Eppendorf tube, to which 1.0 ml of Phosphate buffer saline (PBS) and 0.16 ml of 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) solution were added to reach the final volume of 1.36 ml. For control, 0.2ml of methanol was added in the place of test substance and 0.16ml of distilled water was taken for test blank and control blank. All the tubes were incubated for 20 minutes, and upon incubation, 0.1ml of reaction mixture was pipetted out to microtiter plate and absorbance was measured in ELISA reader at 734nm and values were recorded. Further, the same procedure was repeated for standard by replacing test sample with standard.
[0076] Further, the inhibitory activity of lutein micellar complex 50% and free lutein 70% was determined by lipid peroxidation (LPO) assay, where the test substance was prepared by dissolving 4.5mg of test substance in 0.4ml of Dimethyl sulfoxide (DMSO) to obtain the concentration of 1000µg/ml, and the stock solution was further serially diluted to obtain 1000 - 62.5µg/ml to get the lower concentration. The standard solution was prepared by dissolving 4.5mg of Butylated Hydroxy Anisole (BHA) in 0.4ml of Dimethyl sulfoxide (DMSO) to obtain solutions of 1000µg/ml, and further serially diluting it to get lower concentrations containing 62.5, 125, 250, 500 and 1000µg/ml.
[0077] Further, egg lecithin was prepared by boiling an egg and separating the egg yolk, followed by washing the egg yolk with acetone until the yellow color is removed. The creamy white powder obtained after wash is egg lecithin. 75mg of egg lecithin was added in 25ml of Phosphate buffer saline (PBS) followed by sonication to achieve complete dissolution to get egg lecithin solution with 3mg/ml concentration. Further, 200mM ascorbic acid was prepared by dissolving 35.23 mg of ascorbic acid in 1ml of distilled water. Furthermore, 200mM ferric chloride was prepared by dissolving 32.44 mg of ferric chloride in 1ml of distilled water. Other solutions including 0.375% thiobarbituric acid (TBA) was prepared by dissolving 187 mg of thiobarbituric acid (TBA) in 50 ml distilled water and 15% Trichloroacetic acid (TCA) was prepared by dissolving 7.5 g of Trichloroacetic acid (TCA) in 50ml of distilled water. Further, 0.25N Hydrochloric acid (HCl) solution was prepared by dissolving 1.3ml Hydrochloric acid (HCl) in 50ml mixture of thiobarbituric acid (TBA) and trichloroacetic acid (TCA).
[0078] According to an embodiment of the invention, the inhibitory activity of lutein micellar complex 50% and free lutein 70% was determined by taking 0.1ml of different concentrations of free lutein 70% and standard as test and test blank samples. In the control and control blank sample, 0.1ml of Dimethyl sulfoxide (DMSO) was taken in place of test sample. Further, to the test and control sample, 1ml of egg lecithin was added whereas to the test blank and control blank samples 1ml of Phosphate buffer saline (PBS) was added. Additionally, to the test and control sample, 0.01ml of Ferric Chloride (FeCl3) and ascorbic acid was added, and to the test blank and control blank 0.01ml of distilled water was added. All the tubes were incubated at 370C for 1hr, and upon incubation, 2ml of 0.25N Hydrochloric acid (HCl) was added to all the tubes and incubated in boiling water bath (900C) for 15 minutes. The tubes were then allowed to cool and then subjected to centrifugation at 5000 RPM for 5 minutes. The resultant supernatant liquid was collected and 0.1ml of reaction mixture was pipetted out to microtiter plate, and the absorbance was measured at 490 nm using microtiter reader. The same procedure was repeated for standard by replacing test sample with standard.
[0079] The results of assessment of antioxidant activity of test substances against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical, is shown in the table in Figure 10A. The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay revealed that the IC50 value of lutein micellar complex 50% and free lutein 70% was found to be 143.02 ±1.59 and 427.44±29.52 µg/ml, respectively. IC50 value of ascorbic acid was found to be 80.85±6.01µg/ml. Among the two substances, free lutein 70% exhibited better antioxidant activity against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical.
[0080] The results of the antioxidant activity assessment of the test substances against 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical is shown in the table in Figure 10B. The ABTS assay revealed that lutein micellar complex 50% and free lutein 70% exhibited antioxidant activity with IC50 values 93.75±2.08 and 119.99±1.68µg/ml, respectively, whereas the standard ascorbic acid showed activity with an IC50 value 64.97 ± 0.06 µg/ml. Among the test substances, lutein micellar complex 50% exhibited better inhibition against ABTS with lower IC50 value.
[0081] The antioxidant activity assessment of the test substances against Lipid peroxidation is disclosed in the table, in Figure 10C. The lipid peroxidation inhibitory assay revealed that lutein micellar complex 50% and free lutein 70% exhibited antioxidant activity with IC50 values of 139.58±4.30 and 235.42±15.73µg/ml, respectively, whereas the standard butylated hydroxyanisole (BHA) exhibited antioxidant activity with IC50 value 117.09 ± 1.18µg/ml. Among the test substances, lutein micellar complex 50% exhibited better inhibition against LPO with lower IC50 value. According to the invention, the antioxidant activity of lutein micellar complex 50% was determined to be high against 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2-axino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and Lipid peroxidation (LPO) inhibitory assays.

,CLAIMS:We Claim:
1. A composition of micelle complexation of lutein, the composition comprising:
a. trans lutein at a concentration at a concentration range of 10% to 90% w/w;
b. at least one antioxidant source; and
c. an emulsifier at a concentration range of 0.1% to 5%, with a food grade carrier at a concentration range of 10% to 90%;
wherein, the antioxidants, in combination with trans lutein in the composition forms a lutein micellar complex exhibiting structural changes.
2. The composition as claimed in claim 1, wherein the antioxidant source comprises zeaxanthin at a concentration range of 10% to 90%, carnosic acid at a concentration range of 0.1% to 5%, and tocopherol at a concentration range of 1% to 10%.

3. The composition as claimed in claim 1, wherein the free lutein displays agglomeration when it interacts with tocopherol and carnosic acid, whereby the lutein micellar complex is derived through interactions including hydrogen bonding, electrostatic attraction, and/or coordination compounds.

4. The composition as claimed in claim 1, wherein the antioxidants present in the lutein micellar complex increases the physicochemical properties, and lipophilicity, further increasing the bioavailability, stability and efficacy of lutein.

5. The composition as claimed in claim 1, wherein the lutein micellar complex exhibits high thermal stability than free lutein based on the thermogravimetric analysis (TGA).

6. The process of preparation of lutein micellar complex, the method comprising the steps of:
a. combining trans lutein and zeaxanthin with antioxidants carnosic acid and tocopherol, along with an emulsifier;
b. adding a food grade carrier and alcohol to the mixture obtained in Step a;
c. subjecting the obtained mixture to homogenization at room temperature;
d. drying the homogenized mixture at a temperature of 400C to 900C to obtain the lutein micellar complex.

7. The process as claimed in claim 6, wherein the micellar composition of lutein exhibits increased absorption of lutein and antioxidants through the epithelial membrane.

8. The process as claimed in claim 6, wherein the micellar composition of lutein exhibits higher permeability over free lutein by over 24%.

9. The process as claimed in claim 6, wherein the lutein micellar complex with atleast one antioxidant enhances the antioxidant efficacy of lutein in synergy with the antioxidants.