TECHNICAL FIELD
[001] The subject matter described herein in general relates to the pharmaceutical and food supplement compositions, and in particular, relates to the field of immunomodulatory compositions.
BACKGROUND OF INVENTION
[002] Inflammation is a painful condition commonly associated with diseases such as rheumatoid arthritis. Such inflammation is typically tackled using non-steroidal anti-inflammatory drugs or NSAIDs. NSAIDs however, are known to have major side effects such as gastrointestinal ulcers and kidney complications. The usage of such drugs hence is to be avoided. US6410588 reveals the use of cannabinoids as anti-inflammatory agents. However, such molecules are difficult to isolate and also are known to have certain side-effects. Bio-active compounds or essential nutrients are a good alternative as they are known have high bio-availability with low toxicity.
[003] In light of various lifestyle related illness and diseases, daily intake of balanced nutrients (vitamins, minerals and amino acids etc.) are essential to all the age groups for both physical (Aline L. et.al, Journal de Pediatr 2008) and mental growth (Mrigen kr. Deka et.al, J. Family Med. Prim. Care. 2015). Some of the nutrients like vitamins and amino acids have non-traditional functions like anti-oxidative and anti-inflammatory potential (Maitreyi Raman et.al, Therap. Adv. Gastroenterol. 2011; Carvalho JTG et.al, PLoS one 2017). Furthermore, some of the studies illustrated that nutrients have good anti-inflammatory (Edwin Hoe et.al, Nutrients. 2016) as well as anti-oxidative potential (Joseph Lemire et.al FEMS microbiology letters, 2010). Hence, daily intake of these nutrients which has the additional potential can provide more health benefits along with their traditional benefits.
[004] EP3118215 reveals the use of anti-inflammatory peptides. However, the synthesis of such peptides is complicated and can lead to increased cost of production. Hence, there exists a need for a composition that is able to effectively

provide anti-inflammatory as well as anti-oxidant properties, while being beneficial to health as a supplement. SUMMARY OF THE INVENTION
[005] In an aspect of the present invention, there is provided a bio-active
composition comprising: a) cholecalciferol; and b) histidine, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800.
[006] In another aspect of the present invention, there is provided a process for
preparing the bio-active composition comprising: a) cholecalciferol; and b)
histidine, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200
to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b)
obtaining histidine; and c) contacting cholecalciferol and histidine to obtain the
bio-active composition.
[007] These and other features, aspects, and advantages of the present subject
matter will be better understood with reference to the following description and
appended claims. This summary is provided to introduce a selection of concepts in
a simplified form. This summary is not intended to identify key features or
essential features of the claimed subject matter, nor is it intended to be used to limit
the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE DRAWINGS
[008] The detailed description is described with reference to the accompanying
figures. The same numbers are used throughout the drawings to reference like
features and components.
[009] Figure 1 illustrates the cytotoxicity studies of active at different
concentrations, in accordance with an implementation of the present subject matter.
[0010] Figure 2 illustrates the evaluation of actives on anti-oxidative potential, in
accordance with an implementation of the present subject matter.
[0011] Figure 3 illustrates the effect of actives on lipopolysaccharide (LPS)
stimulated interleukin (IL-6) production in RAW264.7 cells, in accordance with an
implementation of the present subject matter.

[0012] Figure 4 illustrates the effect of actives on LPS stimulated TNF-a
production in RAW264.7 cells, in accordance with an implementation of the
present subject matter.
[0013] Figure 5 illustrates the evaluation of anti-oxidative potential by actives in
various ratios, in accordance with an implementation of the present subject matter.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Those skilled in the art will be aware that the present disclosure is subject to
variations and modifications other than those specifically described. It is to be
understood that the present disclosure includes all such variations and
modifications. The disclosure also includes all such steps, features, compositions,
and compounds referred to or indicated in this specification, individually or
collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0015] For convenience, before further description of the present disclosure,
certain terms employed in the specification, and examples are delineated here.
These definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the
meanings recognized and known to those of skill in the art, however, for
convenience and completeness, particular terms and their meanings are set forth
below.
[0016] The articles "a", "an" and "the" are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0017] The terms "comprise" and "comprising" are used in the inclusive, open
sense, meaning that additional elements may be included. It is not intended to be
construed as "consists of only".
[0018] Throughout this specification, unless the context requires otherwise the
word "comprise", and variations such as "comprises" and "comprising", will be
understood to imply the inclusion of a stated element or step or group of element or
steps but not the exclusion of any other element or step or group of element or
steps.

[0019] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably. [0020] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, concentration ranges of about 120-130 nM should be interpreted to include not only the explicitly recited limits of about 120 nM to about 130 nM, but also to include sub-ranges, such as 120-125 nM, 125-130 nM, and so forth, as well as individual amounts, including fractional amounts, within the specified ranges, such as 122.2 nM, and 127.5 nM, for example.
[0021] The term "at least one" is used to mean one or more and thus includes individual components as well as mixtures/combinations.
[0022] The term 'essential fatty acid' refers to are fatty acids that humans and other animals must ingest because the body requires them for good health but cannot synthesize them. Only two fatty acids are known to be essential for humans: alpha-linolenic acid (an omega-3 fatty acid) and linoleic acid (an omega-6 fatty acid).
[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [0024] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

[0025] As mentioned previously, the existing food supplements suffer from drawbacks of toxicity and high cost. Therefore, there is a scope for exploration of synergistic bio-active compositions which can provide enhanced health benefits. Herbal or natural combinations could prove effective in providing such benefits, while being non-toxic. The present disclosure aims to solve the said problem by employing a combination consisting of cholecalciferol and histidine acid, which shows synergistic inhibition of reactive oxygen species (ROS) and inflammatory markers such as interleukin (IL-6) and tumor necrosis factor (TNF-a). [0026] In an embodiment of the present disclosure, there is provided a bio-active composition comprising: a) cholecalciferol; and b) histidine, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800. In another embodiment of the present disclosure, cholecalciferol to histidine weight ratio is in the range of 1:1500 to 1:3500. In yet another embodiment of the present disclosure, cholecalciferol to histidine weight ratio is in the range of 1:1800 to 1:3200. In yet another embodiment of the present disclosure, cholecalciferol to histidine weight ratio is in the range of 1:1900 to 1:2100.
[0027] In an embodiment of the present disclosure, there is provided a bio-active composition as described herein, wherein cholecalciferol to histidine weight ratio is 1:2000.
[0028] In an embodiment of the present disclosure, there is provided a bio-active composition comprising: a) cholecalciferol; and b) histidine, wherein cholecalciferol to histidine weight ratio is 1:2000.
[0029] In an embodiment of the present disclosure, there is provided a bio-active composition as described herein, wherein the composition further comprises at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof. In another embodiment of the present disclosure, the at least one additive is diluent. [0030] In an embodiment of the present disclosure, there is provided a bio-active composition comprising: a) cholecalciferol; b) histidine; and c) at least one additive selected from the group consisting of diluent, essential nutrient, preservative,

carrier, flavoring agent, colorant, and combinations thereof, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800.
[0031] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; and c) at least one additive
selected from the group consisting of diluent, essential nutrient, preservative,
carrier, flavoring agent, colorant, and combinations thereof, wherein
cholecalciferol to histidine weight ratio is 1:2000.
[0032] In an embodiment of the present disclosure, there is provided a bio-active
composition as described herein, wherein the diluent is selected from the group
consisting of dimethylsulfoxide, water, and combinations thereof; and the essential
nutrient is an essential fatty acid. In another embodiment of the present disclosure,
the diluent is dimethylsulfoxide. In yet another embodiment of the present
disclosure, the essential nutrient is linoleic acid. The linoleic acid may be
9(E),ll(E)-conjugated linoleic acid or 10(E), 12(Z)-conjugated linoleic acid.
Though some essential nutrients have been mentioned here, other known essential
nutrients may be included as part of the composition.
[0033] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; and c) diluent, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800.
[0034] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; c) diluent; and d) essential
nutrient, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200
to 1:3800.
[0035] In an embodiment of the present disclosure, there is provided a bio-active
composition as described herein, wherein the composition comprises
cholecalciferol having a concentration in a range of 120 - 130 nM. In another
embodiment of the present disclosure, the composition comprises cholecalciferol
having a concentration in a range of 122 - 128 nM.
[0036] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; and c) diluent, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800 and the

composition comprises cholecalciferol having a concentration in a range of 120 -
130 nM.
[0037] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; and b) histidine, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, and the
composition comprises cholecalciferol having a concentration in a range of 120 -
130 nM.
[0038] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; and b) histidine, wherein
cholecalciferol to histidine weight ratio is 1:2000, and the composition comprises
cholecalciferol having a concentration in a range of 120 - 130 nM.
[0039] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; and c) at least one additive
selected from the group consisting of diluent, essential nutrient, preservative,
carrier, flavoring agent, colorant, and combinations thereof, wherein
cholecalciferol to histidine weight ratio is 1:2000, and the composition comprises
cholecalciferol having a concentration in a range of 120 - 130 nM.
[0040] In an embodiment of the present disclosure, there is provided a bio-active
composition as described herein, wherein the composition comprises histidine
having a concentration in a range of 245 - 255 uM. In another embodiment of the
present disclosure, the composition comprises histidine having a concentration in a
range of 248- 252 uM.
[0041] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; and c) diluent, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, and the
composition comprises histidine having a concentration in a range of 245 - 255
uM.
[0042] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; and b) histidine, wherein
cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, and the

composition comprises histidine having a concentration in a range of 245 - 255
uM.
[0043] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; b) histidine; and c) diluent, wherein
cholecalciferol to histidine weight ratio is 1:2000, and the composition comprises
histidine having a concentration in a range of 245 - 255 uM.
[0044] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol; and b) histidine, wherein
cholecalciferol to histidine weight ratio is 1:2000, and the composition comprises
histidine having a concentration in a range of 245 - 255 uM.
[0045] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol having a concentration in a range of
120 - 130 nM; and b) histidine having a concentration in a range of 245 - 255 uM,
wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800.
[0046] In an embodiment of the present disclosure, there is provided a bio-active
composition comprising: a) cholecalciferol having a concentration of 125 nM; and
b) histidine having a concentration of 250 uM, wherein cholecalciferol to histidine
weight ratio is 12000.
[0047] In an embodiment of the present disclosure, there is provided a process for
preparing the bio-active composition comprising: a) cholecalciferol; and b)
histidine, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200
to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b)
obtaining histidine; and c) contacting cholecalciferol and histidine to obtain the
bio-active composition.
[0048] In an embodiment of the present disclosure, there is provided a process for
preparing the bio-active composition comprising: a) cholecalciferol; and b)
histidine, wherein cholecalciferol to histidine weight ratio is 1:2000, said process
comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; and c)
contacting cholecalciferol and histidine to obtain the bio-active composition.
[0049] In an embodiment of the present disclosure, there is provided a process for
preparing the bio-active composition comprising: a) cholecalciferol having a

concentration in a range of 120 - 130 nM; and b) histidine, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; and c) contacting cholecalciferol and histidine to obtain the bio-active composition. [0050] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol; and b) histidine having a concentration in a range of 245 - 255 uM, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; and c) contacting cholecalciferol and histidine to obtain the bio-active composition. [0051] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol having a concentration in a range of 120 - 130 nM; and b) histidine having a concentration in a range of 245 - 255 uM, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising the steps of: a) obtaining cholecalciferol; b) obtaining histidine; and c) contacting cholecalciferol and histidine to obtain the bio-active composition.
[0052] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol; b) histidine; and c) at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; c) obtaining at least one additive; and c) contacting cholecalciferol, histidine, and the at least one additive to obtain the bio-active composition. [0053] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol; b) histidine; and c) at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof, wherein cholecalciferol to histidine weight ratio is 1:2000, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; c) obtaining at least

one additive; and c) contacting cholecalciferol, histidine, and the at least one additive to obtain the bio-active composition.
[0054] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol having a concentration in a range of 120 - 130 nM; b) histidine; and c) at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; c) obtaining at least one additive; and c) contacting cholecalciferol, histidine, and the at least one additive to obtain the bio-active composition.
[0055] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol; b) histidine having a concentration in a range of 245 - 255 uM; and c) at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; c) obtaining at least one additive; and c) contacting cholecalciferol, histidine, and the at least one additive to obtain the bio-active composition.
[0056] In an embodiment of the present disclosure, there is provided a process for preparing the bio-active composition comprising: a) cholecalciferol having a concentration in a range of 120 - 130 nM; b) histidine having a concentration in a range of 245 - 255 uM; and c) at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800, said process comprising steps of: a) obtaining cholecalciferol; b) obtaining histidine; c) obtaining at least one additive; and c) contacting cholecalciferol, histidine, and the at least one additive to obtain the bio-active composition.

[0057] In an embodiment of the present disclosure, there is provided a bio-active composition as described herein, wherein the composition effectively inhibits the production of reactive oxygen species (ROS).
[0058] In an embodiment of the present disclosure, there is provided a bio-active composition comprising: a) cholecalciferol; b) histidine; and c) diluent, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800 and the composition effectively inhibits the production of reactive oxygen species (ROS). [0059] In an embodiment of the present disclosure, there is provided a bio-active composition as described herein, wherein the composition is an anti-inflammatory agent.
[0060] In an embodiment of the present disclosure, there is provided a bio-active composition comprising: a) cholecalciferol; b) histidine; and c) diluent, wherein the composition is an anti-inflammatory agent.
[0061] In an embodiment of the present disclosure, there is provided a bio-active composition as described herein, wherein the composition may be dispensed as part of commercial formulations such as health supplements, food supplements, topical formulations and health drinks. EXAMPLES
[0062] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply. [0063] As described above, there is a scope for exploration of active combinations which can provide health promoting benefits. The present disclosure reveals a

combination comprising a) cholecalciferol; and b) histidine, wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800. [0064] Source of Actives: cholecalciferol (Sigma), histidine (Sigma), conjugated linoleic acids- 9(E),ll(E)-conjugated linoleic acid (CLA-1) and 10(E), 12(Z> conjugated linoleic acid (CLA-2) (Cyaman). Example 1: Cytotoxicity studies
[0065] RAW 264.7 cells were seeded at 15000 cells/well density in 96-well plate and incubated at 37 °C, 5%C02. After 80% confluency, cells were treated with various bio-actives- cholecalciferol, histidine, 9(E),ll(E)-conjugated linoleic acid (CLA-1) and 10(E),12(Z)-conjugated linoleic acid (CLA-2) at different concentrations listed below for 24h. After incubation, cells were washed with calcium and magnesium free phosphate buffer solution pH 7. After washing, cell viability was analyzed by CCK-8 viability kit (cell counting kit- Dojindo). The list of actives tested for cytotoxicity studies and their concentrations are mentioned in Table 1 below. Table 1- List of different concentrations of actives used for cytotoxicity studies.
[0066] Safe dose of all actives was identified and the same doses were used in the identification of anti-oxidative and anti-inflammatory potential of actives (Figure 1). It was observed that histidine was non-toxic up to a concentration of 500 uM.

Whereas, cholecalciferol, CLA-1 and CLA-2 were found to be non-toxic up to concentrations of 125 nM, 3.125 uM and 3.125 uM, respectively. Example 2: Effect of active combinations on LPS mediated pro-inflammation markers (IL-6 and TNF-a)
[0067] After incubation with actives, supernatants were analyzed for the LPS stimulated secreted cytokine production- IL-6 by ELISA from R&D systems (Duo set ELIS catalog no: IL-6 DY406) and TNF- a by ELISA from R&D systems (Duo set ELIS catalog no: TNF-a DY410).
[0068] Preparation of assay plates for IL-6: Capture antibody of IL-6, 0.2 ug/well were used for the coating of 96-well microplate and incubated overnight at room temperature. After incubation, the samples were aspirated and the capture antibody was washed with wash buffer containing 0.05% tween 20 in PBS (pH-7.2). The washing was repeated four times. Then the plates were blocked by treating plates with 300ul of reagent diluent containing 1% BSA in PBS (pH-7.2) for lhr at room temperature. After incubation, the reagent diluent was aspirated and washed with wash buffer (x4 times). These plates were then used for estimation of IL-6.
[0069] Preparation of IL-6 standards: Recombinant IL-6 were reconstituted with distilled water and allowed to stand for 15min and final concentration was made up to 120ng/ml. Recombinant IL-6 (120ng/ml) was diluted with reagent diluent to prepare the gradient of IL-6 concentrations (0, 9.375, 18.75, 37.5, 75, 150, 300 and 600 pg/ml).
[0070] Assay procedure for estimation of IL-6 production in supernatants: Standard solution (as described above) and supernatants of different active compositions were added in aliquots of 100 ul to the 96 well containing capture antibody IL-6 and incubated at room temperature for 2hrs. After incubation, the solution was aspirated and plate was washed with wash buffer (x4 times). Then the detection antibody was added (5ng/well) to the 96-well plate and incubated at room temperature for 2hrs. After incubation, the solution was aspirated and plate was washed with wash buffer (x4 times). lOOul of 1:200 diluted streptavidin-HRP was added to the 96-well plate and incubated at room temperature for 20 min (in dark).

The washing step was repeated and lOOul of substrate solution containing hydrogen peroxide and tetramethylbenzidine in 1:1 ratio was added to each well and incubated for 20min (in dark). After incubation, 50ul of 2N sulfuric acid was added to stop the reaction. Then the calibration standards and samples were measured at 450nm. A calibration curve was recorded with the OD values against the respective cytokines IL-6 concentration. Cytokine production in supernatants were calculated using standard curve.
[0071] A number of bio-active compositions were attempted involving cholecalciferol, histidine, CLA-1 and CLA-2, alone and in combination. Among the all actives and their combinations, the composition comprising cholecalciferol (125nm) and histidine (250uM) showed synergistic inhibition of LPS induced IL-6 (-69%) as compared to other combinations and individual actives (Figure 3). [0072] Preparation of assay plates for TNF-a: Capture antibody of TNF-a, 0.2ug/well were used for the coating of 96-well microplate and incubated overnight at room temperature. After incubation, the capture antibody was aspirated and washed with wash buffer containing 0.05% tween 20 in PBS (pH-7.2) (x4 times). The plate was blocked by treating the plate with 300ul of reagent diluent containing 1% BSA in PBS (pH-7.2) for lhr at room temperature. After incubation, the reagent diluent was aspirated and washed with wash buffer for 4 times. The plates were then used for estimation of TNF- a.
[0073] Preparation of TNF-a standards: Recombinant TNF-a were reconstituted with distilled water and allowed to stand for 15min and final concentration was made up to 120ng/ml. Recombinant TNF-a (120ng/ml) was diluted with reagent diluent to prepare a gradient of TNF-a concentrations (0, 9.375, 18.75, 37.5, 75, 150, 300 and 600 pg/ml).
[0074] Assay procedure for estimation of TNF-a production in supernatants: A procedure similar to the one followed for IL-6 estimation was done using the standards mentioned above. A number of bio-active compositions were attempted involving cholecalciferol, histidine, CLA-1 and CLA-2, alone and in combination. Among the all actives and their combinations, the composition comprising cholecalciferol (125nm) and histidine (250uM) showed synergistic inhibition of

LPS induced TNF-a (-62%) as compared to other combinations and individual actives (Figure 4).
Example 3: Evaluation of actives on anti-oxidative potential [0075] RAW 264.7 cells were seeded at 15000 cells/well density in 96-well plate and incubated at 37 °C, 5% CO2. After 80% confluency, cells were treated with different actives and their combinations within their safe dose (as established from Example 1) for 24hr. After incubation, the cells are washed with IX PBS with no Ca and Mg or with HBSS without Ca and Mg, and then incubated with 25 uM DCFH-DA (2',7'- dichlorofluorescin diacetate) in serum free DMEM for 30min. Post incubation, DFFIDA is removed and the cells are washed with PBS/HBSS and incubated in PBS with lOOuM H2O2 for 30min and the fluorescence is measured at 480/530nm. The fluorescence intensity (arbitrary unit) was calculated by the formula mentioned below
[(Ft30-s - Ft30-c)/Ft30-c x 100)] Ft30-s = fluorescence at time 30 (min) after the addition of hydrogen peroxide of the treated well with actives; Ft30-c= fluorescence of cells added after the addition of hydrogen peroxide without treatment.
[0076] Among the all active and their combinations, histidine and cholecalciferol combination showed synergistic inhibition of reactive oxygen species (ROS) production (-94%) as compared to other individual active and combinations (Figure 2). Also, various ratios of cholecalciferol and histidine were tested for establishing the extent of synergism. As can be observed from Figure 5, the synergy was observed for the weight ratio of cholecalciferol to histidine- 1:2000 (histidine- 250 uM + cholecalciferol- 125 nM). The ratios of 1:1000 and 1:4000 were found to be non-synergistic over the individual components. Hence, it is clear that the synergistic weight ratio range of cholecalciferol to histidine- 1:1200 to 1:3800 was obtained by due experimentation and was not obvious. Advantages of the present disclosure:
[0077] The present disclosure reveals a bio-active composition comprising a) cholecalciferol; and b) histidine. The present combination showed synergistic anti-

oxidative and anti-inflammatory potential. Furthermore, composition can be easily adapted to obtain useful commercial formulations such as health supplements.

I/We Claim:
1. A bio-active composition comprising:
a) cholecalciferol; and
b) histidine,
wherein cholecalciferol to histidine weight ratio is in the range of 1:1200 to 1:3800.
2. The bio-active composition as claimed in claim 1, wherein cholecalciferol to histidine weight ratio is 1:2000.
3. The bio-active composition as claimed in claim 1, wherein the composition further comprises at least one additive selected from the group consisting of diluent, essential nutrient, preservative, carrier, flavoring agent, colorant, and combinations thereof.
4. The bio-active composition as claimed in claim 3, wherein the diluent is selected from the group consisting of dimethylsulfoxide, water, and combinations thereof; and the essential nutrient is an essential fatty acid.
5. The bio-active composition as claimed in claim 3, wherein the composition comprises cholecalciferol having a concentration in a range of 120 - 130 nM.
6. The bio-active composition as claimed in any one of the claims 3-5, wherein the composition comprises histidine having a concentration in a range of 245- 255 uM.
7. A process for preparing the bio-active composition as claimed in claim 1, said process comprising the steps of: a) obtaining cholecalciferol; b) obtaining histidine; and c) contacting cholecalciferol and histidine to obtain the bio-active composition.
8. A process for preparing the bio-active composition as claimed in claim 3, said process comprising the steps of: a) obtaining cholecalciferol; b) obtaining histidine; c) obtaining at least one additive; and d) contacting cholecalciferol, histidine, and the at least one additive to obtain the bio-active composition.

9. The bio-active composition as claimed in any one of the claims 1-6, wherein the composition effectively inhibits the production of reactive oxygen species (ROS).
10. The bio-active composition as claimed in any one of the claims 1-6, wherein the composition is an anti-inflammatory agent.