FIELD OF INVENTION
[001] The present disclosure in general relates to the field of nutritional formulations particularly compositions that enhance iron absorption. The disclosure also relates to methods of preparing such compositions.
BACKGROUND OF INVENTION
[002] Iron is an essential element required for various functions in the body. As an important component of haemoglobin, iron is required to supply oxygen to the tissues of the body, and thus it facilitates growth and developmental related activities of the body. Iron absorption is tightly regulated and is the only means by which the iron reserves of the body are replenished. Approximately 80% of total body iron is found in the red blood cells in haemoglobin, which accounts for approximately 3-4 gm of iron. Smaller reserves of iron are also stored in the liver, bone marrow, and spleen and some iron is also stored in myoglobin in muscle tissue.
[003] Dietary sources of iron include haem and non-haem sources. Haem sources primarily include meat, seafood and poultry, while non-haem sources include plant-derived sources including green leafy vegetables and legumes among others. The intake of iron is the only means of replenishing the iron stores in the body. [004] Iron is absorbed primarily in the gut: the duodenum and the upper jejunum. When inside the body, the bioavailability of different forms of iron differs within the body, the mechanisms of which are still not completely understood. Haem is absorbed most efficiently and is in general more robust to factors that affect the absorption of iron including pH. The soluble ferrous form of iron is also absorbed in the gut, but is affected by the gastric pH; a lower pH enhances the ferrous ion absorption while at physiological pH the ferrous ion is converted into the insoluble ferric form. Further, there are several inhibitors and competitors that hamper the absorption of iron in the body and many of these are found with the food sources of iron themselves, for

instance plant phyates and tannins act as chelators of iron and reduce the bioavailability of iron, even though green leafy vegetables may be recommended food sources of iron.
[005] Reduced bioavailability of iron is a major issue and often a cause of iron deficiency. The problem is greater for people with vegetarian diets who do not consume haem-based sources of iron. Iron supplements are often recommended to remedy this issue and the formulations often include ascorbic acid which is the most commonly used compound to enhance the absorption of iron. Besides ascorbic acid however, very few compounds are known to improve the dietary absorption of iron and therefore, mixed diets or mixed iron sources are the only other recommended means to improve the iron content in a diet.
[006] Thus, there remains a pressing need to identify other compounds that can increase the bioavailability of iron in the body and are suitable for all diets.
SUMMARY OF THE INVENTION
[007] In an aspect of the present disclosure there is provided a composition comprising (a) apigenin; (b) honey protein extract; and (c) at least one iron source; wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[008] In an aspect of the present disclosure there is provided, a method for preparation of a composition comprising: (i) apigenin; (ii) honey protein extract; and (iii) at least one iron source, said method comprising:
(a) obtaining apigenin;
(b) obtaining honey protein extract;
(c) obtaining at least one iron source; and
(d) contacting apigenin, honey protein extract, and at least one iron source to obtain a composition; wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.

[009] In an aspect of the present disclosure there is provided a method for preparation of a composition comprising: (i) apigenin; (ii) honey protein extract; (iii) at least one iron source and (iv) suitable carriers, diluents, and excipients, said method comprising:
(a) obtaining apigenin;
(b) obtaining honey protein extract;
(c) obtaining at least one iron source;
(d) obtaining suitable carriers, diluents, and excipients; and
(e) contacting apigenin, honey protein extract, at least one iron source, and suitable carriers, diluents, and excipients, to obtain a composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0010] 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 ACCOMPANYING DRAWINGS
[0011] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may be
better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0012] Figure 1 shows the cell toxicity of apigenin at different concentrations, in
accordance with an embodiment of the present disclosure.
[0013] Figure 2 shows the cell toxicity of honey protein extract at different
concentrations, in accordance with an embodiment of the present disclosure.

[0014] Figure 3 shows the cell toxicity of ferrous sulfate at different concentrations, in accordance with an embodiment of the present disclosure.
[0015] Figure 4 shows the effect of apigenin at different concentrations, in modulating the uptake of ferrous sulfate in cells, in accordance with an embodiment of the present disclosure.
[0016] Figure 5 shows the effect of honey protein extract at different concentrations, in modulating the uptake of ferrous sulfate in cells, in accordance with an embodiment of the present disclosure.
[0017] Figure 6 shows the effect of the combination of apigenin and honey protein extract at different concentrations, in modulating the uptake of ferrous sulfate in cells, in accordance with an embodiment of the present disclosure.
[0018] Figure 7 shows the effect of the combination of apigenin and honey protein extract at different concentrations, in modulating the uptake of ferrous gluconate in cells, in accordance with an embodiment of the present disclosure.
[0019] Figure 8 shows the effect of the combination of apigenin and honey protein extract at different concentrations, in modulating the uptake of ferric chloride in cells, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present disclosure relates to a composition to improve iron uptake. [0021] 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

[0022] 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.
[0023] 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.
[0024] 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”.
[0025] 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.
[0026] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
[0027] Apigenin, a naturally occurring plant flavone, abundantly present in common fruits and vegetables is recognized as a bioactive flavonoid shown to possess anti-inflammatory, antioxidant and anticancer properties (Shukla et al.; Pharm Res.; 27(6): 962–978; 2010).
[0028] 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.

[0029] Iron is an essential mineral in our diet and required throughout life. The dietary sources of iron are many, however, the absorption of iron is often limited by various factors, including the presence of inhibitors and chelators in food. The present disclosure overcomes this challenge by providing a unique composition of apigenin and honey protein extract which helps to significantly increase iron absorption by cells and can thus be used in formulations to address iron deficiencies.
[0030] 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.
[0031] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0032] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract comprising: (1) 50-60% major royal jelly protein 1; and (2) 30-40% major royal jelly protein 2; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0033] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract comprising: (1) 50-60% major royal jelly protein 1; and (2) 30-40% major royal jelly protein 2; and (c) at least one iron source selected from the group consisting of ferric pyrophosphate, ferric chloride, ferrous gluconate, ferrous sulfate, ferrous fumarate, anhydrous ferrous carbonate, carbonyl iron, and combinations thereof, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37.

[0034] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0035] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract comprising: (1) 50-60% major royal jelly protein 1; and (2) 30-40% major royal jelly protein 2; and (c) at least one iron source selected from the group consisting of ferric pyrophosphate, ferric chloride, ferrous gluconate, ferrous sulfate, ferrous fumarate, anhydrous ferrous carbonate, carbonyl iron, and combinations thereof, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0036] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0037] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract comprising: (1) 50-60% major royal jelly protein 1; and (2) 30-40% major royal jelly protein 2; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37
[0038] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin having a weight percentage in the range of 1% - 5%; (b) honey protein extract having a weight percentage in the range of 40%- 50%; and (c) iron source having a weight percentage in the range of 48%- 55%, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0039] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin in said composition is the range of 1 µg/ml – 2.5 µg/ml; (b)

honey protein extract having a concentration in the range of 28 µg/ml- 45 µg/ml; and (c) at least one iron source having a concentration in the range of 35 µg/ml – 45 µg/ml, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0040] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin in said composition is the range of 1.08 µg/ml – 2.16 µg/ml;
(b) honey protein extract having a concentration in the range of 30 µg/ml- 40 µg/ml;
and (c) at least one iron source having a concentration of 40 µg/ml, wherein apigenin
to honey protein extract to at least one iron source w/w ratio in said composition is in
the range of 1:13.8:18.5- 1:37:37.
[0041] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1% - 5%; (b) honey protein extract having a weight percentage in the range of 40%- 50%; and (c) at least one iron source having a weight percentage in the range of 48%- 55%, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0042] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin in said composition is the range of 1 µg/ml – 2.5 µg/ml; (b) honey protein extract having a concentration in the range of 28 µg/ml- 45 µg/ml; and
(c) at least one iron source having a concentration in the range of 35 µg/ml – 45
µg/ml, wherein apigenin to honey protein extract to at least one iron source w/w ratio
in said composition is 1:13.8:18.5.
[0043] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin in said composition is the range of 1.08 µg/ml – 2.16 µg/ml; (b) honey protein extract having a concentration in the range of 30 µg/ml- 40 µg/ml; and (c) at least one iron source having a concentration of 40 µg/ml, wherein apigenin

to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1% - 5%; (b) honey protein extract having a weight percentage in the range of 40%- 50%; and (c) at least one iron source having a weight percentage in the range of 48%- 55%, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0045] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin in said composition is the range of 1 µg/ml – 2.5 µg/ml; (b) honey protein extract having a concentration in the range of 28 µg/ml- 45 µg/ml; and (c) at least one iron source having a concentration in the range of 35 µg/ml – 45 µg/ml, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0046] In an embodiment of the present disclosure, there is provided a composition comprising (a) apigenin in said composition is the range of 1.08 µg/ml – 2.16 µg/ml; (b) honey protein extract having a concentration in the range of 30 µg/ml- 40 µg/ml; and (c) at least one iron source having a concentration of 40 µg/ml, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0047] In an embodiment of the present disclosure, there is provided a composition , comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein the iron source is selected from the group consisting of ferric pyrophosphate, ferrous gluconate, ferrous sulfate, ferrous fumarate, anhydrous ferrous carbonate, carbonyl iron, and combinations thereof, wherein apigenin to honey protein extract to

at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37.
[0048] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio is in the range of 1:13.8:18.5- 1:37:37, wherein said iron source is ferrous sulfate.
[0049] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio is in the range of 1:13.8:18.5- 1:37:37, wherein said iron source is ferric chloride.
[0050] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) at least one iron source, wherein apigenin to honey protein extract to at least one iron source w/w ratio is in the range of 1:13.8:18.5- 1:37:37, wherein said iron source is ferrous gluconate. [0051] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) ferrous sulfate, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0052] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) ferrous sulfate, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:13.8:18.5.
[0053] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) ferrous sulfate, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:37:37.
[0054] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; and (c) ferric chloride, wherein

apigenin to honey protein extract to ferric chloride w/w ratio in said composition is in
the range of 1:13.8:18.5- 1:37:37.
[0055] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) apigenin; (b) honey protein extract; and (c) ferric chloride, wherein
apigenin to honey protein extract to ferric chloride w/w ratio in said composition is
1:13.8:18.5.
[0056] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) apigenin; (b) honey protein extract; and (c) ferric chloride, wherein
apigenin to honey protein extract to ferric chloride w/w ratio in said composition is
1:37:37.
[0057] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) apigenin; (b) honey protein extract; and (c) ferrous gluconate, wherein
apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is
in the range of 1:13.8:18.5- 1:37:37.
[0058] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) apigenin; (b) honey protein extract; and (c) ferrous gluconate, wherein
apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is
1:13.8:18.5.
[0059] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) apigenin; (b) honey protein extract; and (c) ferrous gluconate, wherein
apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is
1:37:37.
[0060] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein said composition can be in the form of a liquid solution.
[0061] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein said composition can be in the form of a gel.
[0062] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein said composition can be in the form of a powder.

[0063] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be taken as a food supplement. [0064] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract, (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37.
[0065] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract comprising: (1) 50-60% major royal jelly protein 1; (b) 30-40% major royal jelly protein 2;(c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0066] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0067] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0068] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1 % - 5 %; (b) honey protein extract having a weight percentage in the range of 40 %- 50 %; (c) at least one iron source having a weight percentage in the range of 48 %- 55 %; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.

[0069] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1 µg/ml – 2.5 µg/ml; (b) honey protein extract having a weight percentage in the range of 28 µg/ml-45 µg/ml; (c) at least one iron source having a weight percentage in the range of 35 µg/ml – 45 µg/ml; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0070] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a concentration in the range of 1.08 µg/ml – 2.16 µg/ml; (b) honey protein extract having a concentration in the range of 30 µg/ml- 40 µg/ml; (c) at least one iron source having concentration in the range of 40 µg/ml; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37.
[0071] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1% - 5%; (b) honey protein extract having a weight percentage in the range of 40%- 50%; (c) at least one iron source having a weight percentage in the range of 48%- 55%; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0072] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1 µg/ml – 2.5 µg/ml; (b) honey protein extract having a weight percentage in the range of 28 µg/ml-45 µg/ml; (c) at least one iron source having a weight percentage in the range of 35 µg/ml – 45 µg/ml; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5.

[0073] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1 µg/ml – 2.5 µg/ml; (b) honey protein extract having a weight percentage in the range of 28 µg/ml-45 µg/ml; (c) at least one iron source having a weight percentage in the range of 35 µg/ml – 45 µg/ml; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:37:37.
[0074] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin having a weight percentage in the range of 1 % - 5 %; (b) honey protein extract having a weight percentage in the range of 40 %- 50 %; (c) at least one iron source having a weight percentage in the range of 48 %- 55 %; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0075] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37, wherein said iron source is selected from the group consisting of ferric pyrophosphate, ferrous gluconate, ferrous sulfate, ferrous fumarate, anhydrous ferrous carbonate, carbonyl iron, and combinations thereof.
[0076] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37, wherein the iron source is ferrous sulfate.
[0077] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at

least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37, wherein the iron source is ferric chloride.
[0078] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) at least one iron source; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37, wherein the iron source is ferrous gluconate.
[0079] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferrous sulfate; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37. [0080] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferrous sulfate; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:13.8:18.5.
[0081] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferrous sulfate; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:37:37.
[0082] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferric chloride; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37. [0083] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferric chloride; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is 1:13.8:18.5.

[0084] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferric chloride; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is 1:37:37.
[0085] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferrous gluconate; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37. [0086] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferrous gluconate; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is 1:13.8:18.5.
[0087] In an embodiment of the present disclosure, there is provided a composition comprising: (a) apigenin; (b) honey protein extract; (c) ferrous gluconate; and (d) carriers, diluents, and excipients, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is 1:37:37.
[0088] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0089] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract, wherein honey protein extract comprises: (1) 50-60% major royal jelly protein 1; and (2) 30-40% major royal jelly protein 2; (c) obtaining at least

one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5-1:37:37.
[0090] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[0091] In an embodiment of the present disclosure, provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0092] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin having a weight percentage in the range of 1% - 5% in said composition; (b) obtaining honey protein extract having a weight percentage in the range of 40%- 50% in said composition; and (c) obtaining at least one iron source having a weight percentage in the range of 48%-55% in said composition, and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein

extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0093] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin in said composition is the range of 1 µg/ml – 2.5 µg/ml in said composition; (b) obtaining honey protein extract having a concentration in the range of 28 µg/ml- 45 µg/ml in said composition; and (c) obtaining at least one iron source having a concentration in the range of 35 µg/ml – 45 µg/ml in said composition, and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0094] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin in said composition is the range of 1.08 µg/ml – 2.16 µg/ml in said composition; (b) obtaining honey protein extract having a concentration in the range of 30 µg/ml- 40 µg/ml in said composition; and (c) obtaining at least one iron source having a concentration of 40 µg/ml in said composition, and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[0095] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin having a weight percentage in the range of 1% - 5% in said composition; (b) obtaining honey protein extract having a weight percentage in the range of 40%- 50% in said composition; and

(c) obtaining at least one iron source having a weight percentage in the range of 48%-55% in said composition, and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5. [0096] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin in said composition is the range of 1 µg/ml – 2.5 µg/ml in said composition; (b) obtaining honey protein extract having a concentration in the range of 28 µg/ml- 45 µg/ml in said composition; and (c) obtaining at least one iron source having a concentration in the range of 35 µg/ml – 45 µg/ml in said composition, and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5.
[0097] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin in said composition is the range of 1 µg/ml – 2.5 µg/ml in said composition; (b) obtaining honey protein extract having a concentration in the range of 28 µg/ml- 45 µg/ml in said composition; and (c) obtaining at least one iron source having a concentration in the range of 35 µg/ml – 45 µg/ml in said composition, and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:37:37.
[0098] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin having a weight percentage in the range of 1% - 5%; (b) obtaining honey protein extract having a

weight percentage in the range of 40%- 50%; and (c) obtaining at least one iron source having a weight percentage in the range of 48%- 55%, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[0099] In an embodiment of the present disclosure, there is provided a method of preparing a composition, comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein the w/w ratio of apigenin: honey protein extract: at least one iron source in the range of 1:13.8:18.5- 1:37:37, wherein said iron source is selected from the group consisting of ferric pyrophosphate, ferrous gluconate, ferrous sulfate, ferrous fumarate, anhydrous ferrous carbonate, carbonyl iron, and combinations thereof.
[00100] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein the w/w ratio of apigenin: honey protein extract: at least one iron source in the range of 1:13.8:18.5- 1:37:37, wherein said iron source is ferrous sulfate.
[00101] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein the w/w ratio of apigenin: honey protein extract: at least one

iron source in the range of 1:13.8:18.5- 1:37:37, wherein said iron source to obtain said composition, wherein the iron source is ferric chloride.
[00102] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; and (d) contacting apigenin, honey protein extract and at least one iron source to obtain said composition, wherein the w/w ratio of apigenin: honey protein extract: at least one iron source in the range of 1:13.8:18.5- 1:37:37, wherein said iron source is ferrous gluconate.
[00103] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferrous sulfate, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous sulfate; and (d) contacting apigenin, honey protein extract and ferrous sulfate to obtain said composition, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00104] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferrous sulfate, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous sulfate; and (d) contacting apigenin, honey protein extract and ferrous sulfate to obtain said composition, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:13.8:18.5. [00105] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferrous sulfate, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous sulfate; and (d) contacting apigenin, honey

protein extract and ferrous sulfate to obtain said composition, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:37:37. [00106] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferric chloride, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferric chloride; and (d) contacting apigenin, honey protein extract and ferric chloride to obtain said composition, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00107] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferric chloride, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferric chloride; and (d) contacting apigenin, honey protein extract and ferric chloride to obtain said composition, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is 1:13.8:18.5. [00108] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferric chloride, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferric chloride; and (d) contacting apigenin, honey protein extract and ferric chloride to obtain said composition, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is 1:37:37. [00109] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c ferrous gluconate, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous gluconate; and (d) contacting apigenin, honey protein extract and ferrous gluconate to obtain said composition, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.

[00110] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferrous gluconate, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous gluconate; and (d) contacting apigenin, honey protein extract and ferrous gluconate to obtain said composition, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is 1:13.8:18.5.
[00111] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) ferrous gluconate, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous gluconate; and (d) contacting apigenin, honey protein extract and ferrous gluconate to obtain said composition, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is 1:37:37.
[00112] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00113] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract comprising: (1) 50-60% major royal jelly protein 1; and (2) 30-40% major royal jelly protein 2; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at

least one iron source, and carriers, diluents, and excipients to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00114] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5.
[00115] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37.
[00116] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, comprising: (a) obtaining apigenin having a weight percentage in the range of 1% - 5% in said composition; (b) obtaining honey protein extract having a weight percentage in the range of 40%- 50% in said composition; (c) obtaining at least one iron source having a weight percentage in the range of 48%-55% in said composition; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers,

diluents, and excipients, to obtain said composition wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00117] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin ; (b) honey protein extract, and (c) at least one iron source, comprising: (a) obtaining apigenin having a concentration in said composition in the range of 1 µg/ml – 2.5 µg/ml; (b) obtaining honey protein extract having a concentration in said composition in the range of 28 µg/ml- 45 µg/ml; (c) obtaining at least one iron source having a concentration in said composition in the range of 35 µg/ml – 45 µg/ml; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00118] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin ; (b) honey protein extract, and (c) at least one iron source, comprising: (a) obtaining apigenin having a concentration in said composition in the range of 1.08 µg/ml – 2.16 µg/ml; (b) obtaining honey protein extract having a concentration in said composition in the range of 30 µg/ml-40 µg/ml; (c) obtaining at least one iron source having a concentration in said composition of 40 µg/ml; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00119] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin ; (b) honey protein extract, and (c) at least one iron source, comprising: (a) obtaining apigenin having a concentration in

said composition in the range of 1 µg/ml – 2.5 µg/ml; (b) obtaining honey protein extract having a concentration in said composition in the range of 28 µg/ml- 45 µg/ml; (c) obtaining at least one iron source having a concentration in said composition in the range of 35 µg/ml – 45 µg/ml; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:13.8:18.5.
[00120] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin ; (b) honey protein extract, and (c) at least one iron source, comprising: (a) obtaining apigenin having a concentration in said composition in the range of 1 µg/ml – 2.5 µg/ml; (b) obtaining honey protein extract having a concentration in said composition in the range of 28 µg/ml- 45 µg/ml; (c) obtaining at least one iron source having a concentration in said composition in the range of 35 µg/ml – 45 µg/ml; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is in the range of 1:37:37.
[00121] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, said method comprising: (a) obtaining apigenin having a weight percentage in the range of 1% - 5% in said composition; (b) obtaining honey protein extract having a weight percentage in the range of 40%- 50% in said composition; (c) obtaining at least one iron source having a weight percentage in the range of 48%-55% in said composition; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers,

diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:13.8:18.5. [00122] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, and (c) at least one iron source, comprising: (a) obtaining apigenin having a weight percentage in the range of 1% - 5% in said composition; (b) obtaining honey protein extract having a weight percentage in the range of 40%- 50% in said composition; (c) obtaining at least one iron source having a weight percentage in the range of 48%-55% in said composition; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract at least one iron source, and carriers, diluents, and excipients, to obtain said composition wherein apigenin to honey protein extract to at least one iron source w/w ratio in said composition is 1:37:37. [00123] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) at least one iron source; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition, wherein w/w ratio of apigenin: honey protein extract: at least one iron source in said composition is in the range of 1:13.8:18.5- 1:37:37, wherein the at least one iron source is selected from the group consisting of ferric pyrophosphate, ferrous gluconate, ferrous sulfate, ferrous fumarate, anhydrous ferrous carbonate, carbonyl iron, and combinations thereof. [00124] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) at least one iron source; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e)

contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition, wherein w/w ratio of apigenin: honey protein extract: at least one iron source in said composition is in the range of 1:13.8:18.5- 1:37:37, wherein the at least one iron source is ferrous sulfate. [00125] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) at least one iron source; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition, wherein w/w ratio of apigenin: honey protein extract: at least one iron source in said composition is in the range of 1:13.8:18.5- 1:37:37, wherein the iron source is ferric chloride.
[00126] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) at least one iron source; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining at least one iron source; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, at least one iron source, and carriers, diluents, and excipients, to obtain said composition, wherein w/w ratio of apigenin: honey protein extract: at least one iron source in said composition is in the range of 1:13.8:18.5- 1:37:37, wherein the iron source is ferrous gluconate.
[00127] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferrous sulfate; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous sulfate; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferrous sulfate and carriers, diluents, and excipients, to obtain said

composition, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00128] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferrous sulfate; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous sulfate; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferrous sulfate, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:13.8:18.5.
[00129] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferrous sulfate; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous sulfate; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferrous sulfate and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferrous sulfate w/w ratio in said composition is 1:37:37.
[00130] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferric chloride; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferric chloride; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferric chloride and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37.
[00131] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferric

chloride; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferric chloride; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferric chloride, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is 1:13.8:18.5.
[00132] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferric chloride; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferric chloride; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferric chloride, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferric chloride w/w ratio in said composition is 1:37:37.
[00133] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) at ferrous gluconate; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous gluconate; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferrous gluconate, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is in the range of 1:13.8:18.5- 1:37:37. [00134] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferrous gluconate; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous gluconate; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferrous gluconate, and carriers, diluents, and excipients, to

obtain said composition, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is 1:13.8:18.5.
[00135] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apigenin; (b) honey protein extract, (c) ferrous gluconate; and (d) carriers, diluents, and excipients, said method comprising: (a) obtaining apigenin; (b) obtaining honey protein extract; (c) obtaining ferrous gluconate; (d) obtaining carriers, diluents, and excipients; and (e) contacting apigenin, honey protein extract, ferrous gluconate, and carriers, diluents, and excipients, to obtain said composition, wherein apigenin to honey protein extract to ferrous gluconate w/w ratio in said composition is 1:37:37.
[00136] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[00137] 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 vary.
Example 1
Material and Methods
[00138] Materials: Caco-2, human epithelial colorectal adenocarcinoma cells (ATCC HTB-37) were obtained from American Type Culture Collection Rockville, MD, USA (ATCC). Cells were cultured in complete Dulbecco's Modified Eagle's medium (DMEM) (Sigma Aldrich) with 10% Fetal Bovine Serum (FBS) (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO2. Apigenin (A3145), ferrous sulphate (414913) were purchased from Sigma-Aldrich. Honey was purchased from a local vendor.
[00139] Honey protein extraction: 100 gm of the honey sample was subjected to protein precipitation. Four volumes of ice cold acetone was added to 100 gm of honey and was incubated at -20°C overnight. The next day the contents were centrifuged at 10, 000 RPM and the precipitate obtained was washed in ice cold acetone and centrifuged once again to get a clear precipitate. The precipitate so obtained was then air dried and dissolved in 1 ml of deionized distilled water and stored at -80°C. [00140] Protein estimation: The honey protein extracted was subjected to protein estimation using Pierce BCA protein estimation kit form Thermo fisher (Catalogue number-23225). Protein estimation was performed as described in the product manual using micro plate method. The proteins identified in the honey sample were: Major Royal Jelly Protein1 (MRJP1) (280kDa) at approximately 50-55%, MRJP2 (72kDa) at approximately 30-33% and MRJP3,4,5 & others at approximately 12-20% (Tamura, S. et al.; Food Chemistry 114; 1491-1497; 2009).
[00141] Differentiation of Caco2 cells: Caco-2 cells were seeded at a density of 5 × 105 cells per well. The cells were grown in DMEM medium supplemented with 1% non-essential amino acids, 1% of penicillin-streptomycin and 10% FBS. The medium

was changed every alternate day. Cellular uptake studies were carried out with cell monolayers that were 21 days old.
[00142] Cytotoxicity assay: Caco2 cells were treated with various concentrations of apigenin, honey protein and ferrous sulphate. Cytotoxicity was determined by reduction of Thiazolyl Blue Tetrazolium Blue (MTT, Sigma) to formazan. Cells were seeded in 48 wells and differentiated. They were then treated with various concentrations of apigenin, honey protein and ferrous sulphate and incubated for 24 hours. After 24 hours, the cells were analyzed for cytotoxicity by MTT assay. MTT (2 mg/ml in PBS, 100 μl) was added to each well. Cells were incubated at 37˚C for 30 min and DMSO (100 μl) was added to dissolve the formazan crystals. The absorbance was measured at 560 nm using an ELISA reader (Thermo Fisher, Germany). [00143] Cellular uptake of ferrous sulphate in Caco-2 cells: Caco-2 cells were seeded at a density of 5 × 105 cells per well. The cells were grown in DMEM medium supplemented with 1% non-essential amino acids, 1% of penicillin-streptomycin and 10% FBS. The medium was changed every alternate day. Cellular uptake studies were carried out with cell monolayers that were 21 days old. The cells were treated in triplicate with apigenin, honey protein and ferrous sulphate alone and in combinations for 16 hours. After the treatment, the cells were washed with 0.9% saline and uptake of ferrous sulphate was estimated using Prussian blue spectrophotometric method. To quantify cellular iron uptake, cell layers were dissolved at room temperature for 1 hour in 125 μL of 6 N HCl, then 125 μL of a 5% solution of K4[Fe(CN)6] • 3H2O dissolved in water was added and the suspension was incubated for 10 minutes. The absorbance was read at 450 nm in a ELISA plate reader (Thermo Fisher, Germany). A standard curve of ferrous sulphate in 6 N HCl treated under the same conditions was used to quantify the amount of iron uptake by Caco2 cells.

Results of cytotoxicity assays of apigenin, honey protein extract and ferrous
sulfate.
[00144] The cell toxicity of apigenin and honey protein was tested individually to determine the concentrations that would be used for further experiments. The three compounds were added to the cells at different concentrations and tested according to the protocol described above.
[00145] Apigenin was found to be toxic after 20µM (5.4 µg/ml) concentration as at higher concentrations the viability of the cells dropped significantly (Figure 1). Honey protein showed the least toxicity at 50 µg/ml. At higher concentrations, the viability of the cells decreased gradually (Figure 2). Finally, similar tests for ferrous sulfate indicated that concentrations higher than 40 µg/ml were cytotoxic (Figure 3). [00146] Thus, based on these results, 5.4 µg/ml of apigenin, 50 µg/ml honey protein and 40 µg/ml of ferrous sulfate were chosen as the concentrations for further experiments.
Example 3
A combination of apigenin and honey protein enhances the bioavailability iron. [00147] To determine whether iron absorption could be increased by either apigenin or honey protein, the compounds were tested individually using ferrous sulfate as the source of iron.
[00148] Apigenin at concentrations varying from 1.35 µg/ml to 8.10 µg/ml were tested in combination with 40 µg/ml ferrous sulfate (FeSO4) (Figure 4). In isolation, ferrous sulfate at 40 µg/ml did not show any absorption by CaCo2 cells. Surprisingly, it was found that when apigenin was added iron was taken up by the cells, with an 8% increase at the lowest concentration of 1.35 µg/ml. The absorption of ferrous sulfate increased proportionally with an increase in apigenin concentration, until a concentration of apigenin at 5.4 µg/ml (20 µM apigenin). At concentrations higher than 5.4 µg/ml apigenin, the absorption of iron dropped and plateaued at around 20%.

This might have occurred as apigenin concentrations above 20 µM were found to be cytotoxic (Figure 1). Thus, this data reveals the novel finding that apigenin within the range of (1.35 µg/ml- 5.4 µg/ml) enhances the bioavailability of the iron source ferrous sulfate.
[00149] In a similar manner, honey protein extract was tested in combination with ferrous sulfate to determine whether it too could improve the uptake of iron. Concentrations ranging from 5 µg/ml- 100 µg/ml of honey protein extract were tested with 40 µg/ml ferrous sulfate (Figure 5). As seen previously, ferrous sulfate could not be absorbed by the cells in isolation and showed 0% uptake. However, surprisingly, it was found that iron uptake improved considerably in combination with the honey protein extract and increased proportionally with an increase in the concentration of the honey protein extract. The lowest concentration of the protein extract increased the uptake of iron by 12.3%. The highest percentage uptake was obtained using 50 µg/ml honey protein extract after which the uptake percentage declined. Thus, it was found that honey protein extract could also increase iron uptake.
[00150] As both apigenin and honey protein extract could improve the uptake of iron by cells, the compounds were then tried in combination to determine the combined effect of both. The combination of both apigenin and honey protein extract was supplied to cells at different concentrations in addition to 40 µg/ml of iron, which was kept constant throughout the concentration range tested. As seen in Figure 6, the trend shows that the combination of apigenin and honey protein extract is able to promote iron uptake, however, a few of the combinations did not show any significant increase as compared to the actives in isolation. Surprisingly, synergy was observed at the specific combination of 1.08 µg/ml apigenin and 40 µg/ml, which represents a 1:37 w/w ratio of apigenin: honey protein extract, and 2.16 µg/ml apigenin and 30 µg/ml honey protein extract, which represents a 1:13.8 ratio of apigenin: honey protein. At a ratio of 1:37 apigenin: honey protein extract, the concentration is increased to 62.1% and at a 1:13.8 ratio of apigenin: honey protein extract, the iron uptake is increased to

50.6% (Figure 6). The increase observed at both these ratios is more than what is expected by the mere addition of apigenin and honey protein; for instance, the addition of the two constituents at a 1:37 ratio of apigenin and honey protein extract gives an expected percentage uptake at approximately 40.95%, however, the observed iron uptake is 62.1% which represents a 51.6% increase from the expected value. [00151] The effect of the combination of apigenin and honey protein extract in increasing the bioavailability of iron is not limited to iron sulfate, as seen in Figures 7 and 8, the bioavailability of iron in the form of ferrous gluconate (Figure 7) and ferric chloride (Figure 8) are both substantially increased.
[00152] Surprisingly, the same w/w ratios of 1:13.8 and 1:37 apigenin to honey protein demonstrate synergism in increasing the uptake of ferrous gluconate and ferric chloride. For instance, at 65.9%, the 1:37 ratio of apigenin: honey protein shows an approximately 36.7% increase in ferrous gluconate uptake from the values that would be expected from an admixture of the two constituents at the same concentrations. [00153] Overall, the present disclosure provides a composition comprising apigenin, honey protein extract and an iron source, which at particular concentrations and w/w ratios exhibits a surprising and unexpected synergistic effect in improving the bioavailability of iron in cells. It is to be appreciated that the observed synergism in particular instances in not a result of mere aggregation of the individual properties of apigenin, honey protein extract and the iron source as apigenin or honey protein extract alone at any concentration, or in combination at various other w/w ratios and concentrations do not show any significant enhancement of increasing iron uptake.