Food products rich in bioavailable plant-based iron for enhancing blood iron level

Abstract

Food product compositions are described herein. For example, this document provides food product compositions rich in bioavailable plant-based iron for enhancing blood iron levels.

Description

[0001] FOOD PRODUCTS RICH IN BIO A VAILABLE PLANT-BASED IRON FOR ENHANCING BLOOD IRON LEVEL

[0002] TECHNICAL FIELD

[0003] This document relates to the field of food product compositions rich in bioavailable plant-based iron. For example, this document relates to food product compositions useful for increasing human or animal blood iron levels, e.g., prevention and treatment of anemia in humans, especially iron deficiency anemia (IDA).

[0004] BACKGROUND

[0005] Globally, there is a vast ongoing burden of anemia. This is associated with increased mortality rate, poor pregnancy outcome, low quality of life of individuals and low work productivity causing huge economic losses. The most common type of anemia is iron-deficiency anemia (“IDA”), which is caused by lack of iron in the blood (e.g., low blood iron level). IDA is usually treated with orally administered artificial iron supplements. In general, relatively high doses and long duration of the treatment with oral iron supplements are needed to achieve a desired therapeutic effect. Also, oral administration of iron supplements is often accompanied by undesired side effects (including, for example, upset stomach, stomach-ache, constipation or diarrhea, nausea and vomiting). These side effects are particularly hard to bear for those at high risk of developing IDA, such as children, pregnant women and women in reproductive age. This greatly contributes to poor compliance to iron supplementation therapy and low success rate of IDA treatment.

[0006] SUMMARY

[0007] This document provides food product compositions (e.g., plant-based food product compositions). For example, this document relates to food product compositions useful for increasing human or animal blood iron levels, e.g., prevention and treatment of anemia in humans, especially IDA. The food product compositions provided herein can improve blood iron levels in human and animals. In general, the food product compositions provided herein include at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product; and may include one or more components selected from the group consisting of: less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; and less than about 50 mg of phytic acid per one serving or daily dose of product.

[0008] In general, the food product compositions described herein can improve the blood iron levels of a subject that was administered the composition. As provided herein, the food product compositions can include the following benefit. The combination of ingredients in the compositions, including high amounts of bioavailable iron (at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product) and low amounts of iron absorption inhibitors (e.g., less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; and less than about 50 mg of phytic acid per one serving or daily dose of product) offers an increase in blood iron level of a subject in need thereof.

[0009] In general, one aspect of this disclosure features a composition including at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product; and may include one or more components selected from the group consisting of: less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; and less than about 50 mg of phytic acid per one serving or daily dose of product.

[0010] Another aspect of this disclosure features a composition including white potato; honey; and one or more ingredients selected from the group consisting of: butternut squash, pineapple, cinnamon powder, melon, kiwi, and beetroot; wherein the food product composition comprises at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product.

[0011] Another aspect of this disclosure features a method of increasing blood iron level in a subject, the method including: administering an effective amount of the food product composition of this disclosure to a subject in need thereof. Another aspect of this disclosure features a method of treating IDA in a subject in need thereof, the method including: administering to a subject identified or diagnosed as having IDA an effective amount of the composition of this disclosure.

[0012] Another aspect of this disclosure features a composition for use in a method of treating IDA in a subject in need thereof, the method including: administering to a subject identified or diagnosed as having IDA an effective amount of the composition of the disclosure.

[0013] Unless otherwise defined, 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 invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0014] To facilitate understanding of the disclosure set forth herein, a number of additional terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Each of the patents, applications, published applications, and other publications that are mentioned throughout the specification are incorporated herein by reference in their entireties.

[0015] As used herein, “about” means ± 20% of the stated value, and includes, more specifically, values of ± 10%, ± 5%, ± 2% and ± 1% of the stated value.

[0016] The terms “treat,” “treating,” and “treatment,” in the context of treating a disease or disorder, are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof. As used herein, the term “substantially free of’ an ingredient(s) as provided throughout the disclosure is intended to mean that the composition contains less than about 0.01 wt% (percent by weight of the total weight of the composition), less than 0.001 wt%, less than 0.0001 wt%, or insignificant or negligible amounts of said ingredient(s) unless specifically indicated otherwise. In some embodiments, the composition (e.g., the food product composition) of the present disclosure is substantially free of one or more of phenolic acids, flavonoids, and phytic acid, meaning that the composition contains less than about 0.01 wt% of one or more of phenolic acids, flavonoids, and phytic acid. In some embodiments, the composition (e.g., the food product composition) of the present disclosure is substantially free of phenolic acids. In some embodiments, the composition (e.g., the food product composition) of the present disclosure is substantially free of flavonoids. In some embodiments, the composition (e.g., the food product composition) of the present disclosure is substantially free of phytic acids.

[0017] As used herein, the term “plant-based” refers to an ingredient(s) or composition(s) as provided throughout the disclosure is intended to mean that the ingredient(s) or composition(s) has been sourced or comes directly from a plant(s) and does not include synthetically manufactured chemicals or is substantially free of manufactured chemicals. For example, plant- based non- heme iron includes irons such as ubredoxins, ferredoxins, hemerythrin, aconitase, or the like, from a plant(s) (e.g., sourced from a plant(s) or is part of a plant(s)).

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG. 1 depicts photomicrographs of duodenal tissue of rats with iron deficiency anemia (IDA) after simultaneous treatment with food product compositions of the disclosure (ORG and RED) and iron supplementation (“Fe sup”) (H&E, 5 ). A - Fe sup; B - ORG + Fe sup; C - RED + Fe sup. FIG. 2 depicts photomicrographs of liver tissue of rats with IDA after simultaneous treatment with final food products (ORG and RED) and iron supplementation (H&E, 10x (A-C), 20 (D-F)). A, D - Fe sup; B, E - ORG + Fe sup; C, F - RED + Fe sup.

[0021] FIG. 3 depicts photomicrographs of spleen section of rats with IDA after simultaneous treatment with final food products (ORG and RED) and iron supplementation (H&E, 10x (A-C), 20x (D-F); Prussian blue, 40x (G-I)). A, D, G- Fe sup; B, E, H - ORG + Fe sup; C, F, I - RED + Fe sup.

[0022] FIG. 4 depicts photomicrographs of kidney section of rats with IDA after simultaneous treatment with final food products (ORG and RED) and iron supplementation. (H&E, 10x). A - Fe sup; B - ORG + Fe sup; C - RED + Fe sup.

[0023] FIG. 5 depicts photomicrographs of duodenal tissue of rats with IDA after treatment with final food product ORG alone and enriched with iron (H&E, 5x). A - Normal, B - Control, C - ORG, D - ORG + Fe enrich.

[0024] FIG. 6 depicts photomicrographs of liver tissue of rats from Normal group. A - Preserved structure of the lobule, devoid of inflammatory infiltrate both within the lobule and the portal space (H&E, 10x); B - hepatocyte structure exhibited maintained integrity (H&E, 20 x).

[0025] FIG. 7 depicts photomicrographs of liver tissue of rats from Control group. A - hepatocytes exhibited periportal damage (H&E, 10x), B - inflammatory infiltrate observed in the lobules (H&E, 20x), C - evident pyknosis of hepatocyte nuclei (H&E, 10x). D - inflammatory infiltrate present in portal spaces (H&E, 20x).

[0026] FIG. 8 depicts photomicrographs of liver tissue of rats from ORG group. A - the preserved structure of liver lobules without inflammatory infiltrate in both the lobule and portal space (H&E, 10x); B - mild perinuclear halo observed in hepatocytes within the periportal zone of the lobulus, without nuclear pyknosis (H&E, 20x).

[0027] FIG. 9 depicts photomicrographs of liver tissue of rats from ORG + Fe enriched group. A - preserved structure of liver lobules without any inflammatory infiltrate in both the lobule and portal space (H&E, 10x); B - individual hepatocytes exhibited a subtle perinuclear halo, and no nuclear pyknosis was evident (H&E, 20x). FIG. 10 depicts photomicrographs of spleen tissue of rats with IDA after treatment with final food product ORG alone and enriched with iron. A-D: Alterations of white and red pulp (H&E, 5x); E-H: e presence of megakaryocytes, serving as an indicator of extra medullary hematopoiesis (H&E, 20x); I-L: Visualization of iron depots within spleen cells detected as a blue-green hue in cytoplasm, Prussian blue staining (40x).

[0028] FIG. 11 depicts photomicrographs of kidney tissue of rats with IDA after treatment with final food product ORG alone and enriched with iron (H&E, 20x). A - Normal group displaying a preserved histological appearance of the kidney cortex; B - Control group exhibiting a reduced number of glomerular bodies, significantly smaller in size (circled), accompanied by marked peritubular infiltrate (asterisk) and vacuolization of the tubular epithelium (arrow); C - ORG group showcasing glomerular corpuscles slightly smaller in size (rounded) with no detectable Bowman's space, and an absence of peritubular infiltrate and vacuolization of the tubular epithelium; D - ORG + Fe enrich group depicting a histological appearance of the cortex without discernible differences when compared to the Normal group.

[0029] DETAILED DESCRIPTION

[0030] This document provides compositions (e.g., food product compositions) that include at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product; and may include one or more components selected from the group consisting of: less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; and less than about 50 mg of phytic acid per one serving or daily dose of product. For example, a food product composition provided herein can include about 1 mg to about 10 mg of plant-based non- heme iron per one serving or daily dose of product; less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; and less than about 50 mg of phytic acid per one serving or daily dose of product. A food product composition can be in the form of a liquid, solution, suspension, tablet, powder, cream, mist, atomized vapor, aerosol, soft gelatin capsule, hard gelatin capsule, a gel, a confectionary, a shake, a bar, and a supplemented food. For example, a food product composition can be formulated in a liquid form. In some embodiments, the food product composition can be formulated in solid or semi-solid form. In some embodiments, the food product composition can be formulated in the form of a juice, smoothie, porridge, muesli, bar, ice cream, jam, or soup.

[0031] As described herein, the food product compositions provided herein contains plant-based non-heme iron. A food product composition provided herein can contain a high amount of plant-based non-heme iron (e.g., 1 mg to about 10 mg per one serving or daily dose of product). For example, the food product composition comprises the plantbased non-heme iron in an amount in a range of about 1 mg to about 10 mg; about 2 mg to about 9 mg; about 3 mg to about 8 mg, about 2 mg to about 5 mg, about 3 mg to about 7.5 mg; about 1 mg to about 5 mg; about 1 mg to about 2.5 mg; per one serving or daily dose of product. In some embodiments, the food product composition comprises the plant-based non-heme iron in an amount in a range of about 1 mg to about 10 mg; about 1 mg to about 5 mg; about 1 mg to about 2.5 mg; per one serving or daily dose of product. In some cases, a food product composition provided herein can be formulated to include an amount of plant-based non-heme iron such that a daily dose of about 1 mg to about 10 mg (e.g., about 1 mg to about 5 mg or about 1 mg to about 2.5 mg) of plant-based non- heme iron can be conveniently administered.

[0032] As described herein, the food product compositions provided herein contain or may not contain polyphenols. A food product composition provided herein can contain a low amount of polyphenols (e.g., less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product). For example, the food product composition comprises less than about 300 mg of polyphenols, less than about 150 mg of polyphenols, less than about 100 mg of polyphenols or substantially free of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product. In some embodiments, the food product composition comprises less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product or less than about 150 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product.

[0033] As described herein, the food product compositions provided herein contain or may not contain flavonoids. A food product composition provided herein can contain a low amount of flavonoids (e.g., less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product). For example, the food product composition comprises less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; less than 0.9 mg of flavonoids expressed as mg quercetin equivalents per one serving or daily dose of product; or substantially free of flavonoids expressed as mg quercetin equivalents per one serving or daily dose of product. In some embodiments, the food product composition is substantially free of flavonoids having a 3 -hydroxy group- and 4-carboxyl group; a 5- hydroxy group and 4-carboxyl group; or a 3’- and 4’ -hydroxyl groups. In some embodiments, the food product composition is substantially free of one or more flavonoids selected from the group consisting of: quercetin, genistein, and their glycosides, luteolin, apigenin, and epigallocatechin-3 -gallate.

[0034] As described herein, the food product compositions provided herein contain or may not contain phytic acid. A food product composition provided herein can contain a low amount of phytic acid (e.g., less than about 50 mg of phytic acid per one serving or daily dose of product). For example, the food product composition comprises less than about 50 mg of phytic acid per one serving or daily dose of product; less than about 25 mg of phytic acid per one serving or daily dose of product; or substantially free of phytic acid per one serving or daily dose of product.

[0035] In some embodiments, the food product composition is substantially free of phenolic acids having two hydroxyl groups in the ortho position of the phenolic acid or a hydroxyl and a carboxyl group in the ortho position of the phenolic acid.

[0036] As described herein, the food product compositions provided herein contain or may not contain ascorbic acid. In some embodiments, the food product compositions provided herein includes ascorbic acid. In some embodiments, a molar ratio of ascorbic acid to plant-based non- heme iron is in a range of about 2: 1 to about 4: 1, respectively.

[0037] Also provided herein are food product compositions including white potato; honey; and one or more ingredients selected from the group consisting of: butternut squash, pineapple, cinnamon powder, melon, kiwi, and beetroot; wherein the food product composition comprises about 1 mg to about 10 mg of plant-based non- heme iron per one serving or daily dose of product.

[0038] In some embodiments, the food product composition comprises white potato, butternut squash, pineapple, cinnamon powder, and honey. In some embodiments, about 60 g to about 150 g of white potato per 300 mL of food product composition; about 50 g to about 120 g of butternut squash per 300 mL of food product composition; about 40 mL to about 100 mL of pineapple juice per 300 mL of food product composition; about 0.5 g to about 2 g of cinnamon powder per 300 mL of food product composition; about 2.5 g to about 10 g of honey per 300 mL of food product composition; and about 50 mL to about 90 mL of water. In some embodiments, the food product composition comprises about 60 g of white potato per 300 mL of food product composition; about 100 g of butternut squash per 300 mL of food product composition; about 60 mL of pineapple juice per 300 mL of food product composition; about 1 g of cinnamon powder per 300 mL of food product composition; about 5 g of honey per 300 mL of food product composition; and about 70 mL of water.

[0039] In some embodiments, the food product composition comprises white potato, melon, kiwi, beetroot, and honey. In some embodiments, the food product composition comprises about 80 g to about 150 g of white potato per 300 mL of food product composition; about 80 mL to about 120 mL of melon juice per 300 mL of food product composition; about 50 mL to about 100 mL of kiwi juice per 300 mL of food product composition; about 2.5 mL to about 7.5 mL of beetroot juice per 300 mL of food product composition; and about 2.5 g to about 10 g of honey per 300 mL of food product composition. In some embodiments, the food product composition comprises about 100 g of white potato per 300 mL of food product composition; about 100 mL of melon juice per 300 mL of food product composition; about 70 mL of kiwi juice per 300 mL of food product composition; about 5 mL of beetroot juice per 300 mL of food product composition; and about 5 g of honey per 300 mL of food product composition.

[0040] In some embodiments, the white potato is a cubed or sliced white potato. In some embodiments, the white potato is a peeled white potato. In some embodiments, the white potato is a boiled white potato. In some embodiments, the white potato is a cubed or sliced, peeled, and boiled white potato.

[0041] In some embodiments, the butternut squash is a cubed or sliced butternut squash. In some embodiments, the butternut squash is a peeled butternut squash. In some embodiments, the butternut squash is a boiled butternut squash. In some embodiments, the butternut squash is a cubed or sliced, peeled, and boiled butternut squash.

[0042] In some embodiments, the pineapple juice is cold-pressed pineapple juice.

[0043] In some embodiments, the honey is acacia honey.

[0044] In some embodiments, the melon juice is cold-pressed melon juice.

[0045] In some embodiments, the kiwi juice is cold-pressed kiwi juice.

[0046] In some embodiments, the beetroot juice is cold-pressed beetroot juice.

[0047] In some embodiments, the food product composition includes an additional iron supplement. For example, the food product composition can include FeSCh and / or an iron equivalent (e.g., ferric sulfate, ferrous gluconate, ferric citrate, ferrous fumarate, iron sucrose, or the like). In some embodiments, the food product composition includes at least 2.72 mg of FeSC per one serving or daily dose of product. For example, the food product composition includes about 2.72 mg to about 240 mg of FeSCh per one serving or daily dose of food product composition. In some embodiments, the food product composition includes at least about 2.72 mg of FeSCh and / or an iron equivalent per one serving or daily dose of food product composition. In some embodiments, the food product composition includes about 2.72 mg to about 240 mg of FeSCh and / or an iron equivalent per one serving or daily dose of food product composition.

[0048] The food product compositions provided herein can be formulated for oral administration and can include one or more suitable excipients, flavorings, colorants, and / or other ingredients. When intended for oral use, for example, tablets, troches, lozenges, aqueous or oil suspensions, non-aqueous solutions, dispersible powders or granules (including micronized particles or nanoparticles), emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use can be prepared according to any appropriate method, including standard methods for the manufacture of pharmaceutical compositions. Such compositions can contain one or more agents such as, without limitation, sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. For oral administration, the compositions can be prepared with one or more excipients, such as binding agents, fillers, lubricants, disintegrants, and / or wetting agents. Liquid preparations for oral administration can take the form of, for example, smoothies, solutions, syrups, or suspension, or they can be presented as a dry product for reconstitution with saline or other suitable liquid vehicle before use. In some cases, liquid preparations can contain pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles, preservatives, buffer salts, flavoring agents, coloring agents, and sweetening agents as appropriate.

[0049] In some cases, the food product compositions provided herein can contain a pharmaceutically acceptable carrier for administration to a mammal. Suitable carriers include, without limitation, aqueous, or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include, without limitation, propylene glycol, polyethylene glycol, vegetable oils, and organic esters. Suitable aqueous carriers include, for example, water, alcohol, saline, and buffered solutions. Pharmaceutically acceptable carriers also can include physiologically acceptable aqueous vehicles (e.g., physiological saline) or other carriers appropriate for oral administration.

[0050] In some cases, the food product compositions provided herein can be in liquid form, by way of an example only, configured to have a unit dosage equal to the daily desired dosage for a particular mammal. For example, if a mammal desires 1 mg of a particular agent (e.g., plant-based non-heme iron), each liquid formulation can include about 1 mg in weight of that agent. As used herein, mammals generally refer to humans, but also can include domesticated mammals (e.g., dogs, cats, and livestock such as cows, horses, pigs, or sheep). The dosages of a particular food product compositions provided herein will depend on many factors, including the general health of a mammal. In some cases, a total daily dose (e.g., a full serving) may be prepared and administered in the form of one or more dosage units. For instance, in some cases, one liquid formulation (e.g., 300 m smoothie) can include a full serving (e.g., daily dosage) of the composition. In some cases, two liquid formulations in combination can include a full serving (e.g., daily dosage) of the composition. In some cases, three liquid formulations in combination can include a full serving (e.g., daily dosage) of the composition. Methods of Treatment

[0051] In some embodiments, methods of increasing blood iron levels in a subject in need thereof are provided, the methods comprising: administering an effective amount of the food product composition of the disclosure to a subject in need thereof are provided. In some embodiments, the blood iron levels of the subject increase by at least 10%, by at least 15%, by at least 20%, by at least 25% or more, compared to a control group that is not administered the food product composition. In some embodiments, the blood iron levels of the subject increase by at least 20%, compared to a control group that is not administered the food product composition. In some embodiments, the blood iron levels of the subject increase by about 10% to about 90%, about 15% to about 80%, about 20% to about 70%, about 10% to about 75%, or about 15% to about 50%, compared to a control group that is not administered the food product composition.

[0052] In some embodiments, methods of treating iron deficiency anemia in a subject in need thereof are provided, the methods comprising: administering to a subject identified or diagnosed as having iron deficiency anemia an effective amount of the food product composition of the disclosure are provided.

[0053] In some embodiments, at least one serving of the food product composition is administrated to the subject every day. In some embodiments, at least one serving of the food product composition is administrated to the subject more than once every day. In some embodiments, at least one serving of the food product composition is administrated to the subject every second day.

[0054] In some embodiments, the food product composition is administered to the subject on an empty stomach about an hour before a next meal. In some embodiments, the food product composition is administered to the subject every day for a period of at least two months. In some embodiments, the food product composition is administered to the subject every day for a period of at least two months.

[0055] In some embodiments, the food product composition is administered in order to prevent or reduce IDA, or symptoms of IDA during the periods when IDA is more likely to develop, e.g. during the reproductive period in women, pregnancy, periods of rapid growth in children, intensive sports activities, vegan diet, or the like. In some embodiments, when the food product composition is administered to prevent IDA, at least one serving of the composition, whether in its standalone form or enriched with iron, should be consumed every other day, on an empty stomach and an hour before next meal.

[0056] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0057] EXAMPLES

[0058] Example 1 - Formulation

[0059] The development of the nutritional formula was specifically guided by detailed chemical analysis of various edible plants, focusing on their content of iron and iron absorption inhibitors, particularly polyphenols and phytic acid. More specific, the development of the nutritional formula cantered on precise amounts of these compounds in the end product. The formulation was accomplished by elucidating the precise quantities of iron, specific polyphenols, and phytic acid that sustain adequate iron bioavailability.

[0060] Eight plant foods were identified as those with preferable chemical profile. The chosen plant-based foods were: white potato (boiled, WPB), beetroot (cold- pressed juice, BEE), kiwi (cold-pressed juice, KIW), pineapple (cold-pressed juice, PIN), butternut squash (boiled, BSB), melon (cold pressed juice, MEL), cinnamon powder (CIN) and acacia honey (HON). All chosen plant foods were purchased from different markets in Novi Sad, Serbia. The samples were collected randomly in sufficient quantities (at least 2 kg) to obtain representative samples. The samples were processed in the following ways: WPB and BSB were peeled and chopped in cubed slices, which were boiled in water for 30 min and then the water was removed. BEE, KIW, PIN and MEL were peeled and cold- pressed using an electronic juicer to obtain juices.

[0061] WPB, BSB, BEE, KIW, PIN, MEL, CIN and HON were analysed for their total phenols, flavonoids, phytic acid and iron contents and qualitative and quantitative polyphenol profiles by LC-MS / MS. Afterwards, two different final food products (smoothies) were made by mixing exact amounts of plant foods and homogenization was performed using electric blender. The first final food products, named orange smoothie (ORG), was made by mixing 60 g WPB, 100 g BSB, 60 mL PIN, 1 g CIN, 5 g HON and 70 mL of H2O, what was 300 mL in total and was marked as one serving.

[0062] The second final food product, named red smoothie (RED), was made by mixing 100 g WPB, 100 mL MEL, 70 mL KIW, 5 mL BEE and 5 g HON, what was 300 mL in total and was marked as one serving.

[0063] ORG and RED were made without adding artificial components. The smoothies had a unique sweet-sour taste, pleasant for consumption. ORG and RED were analysed for their total phenols, flavonoids, phytic acid and iron contents and qualitative and quantitative polyphenol profile by LC-MS / MS.

[0064] Chemical analysis of plant-based food stuffs and final food products

[0065] Phenolic profile determination

[0066] Phenolic profile of 8 plant-based food staffs and two final food products was determined by evaluation of total phenolic and flavonoid contents and HPLC-DAD- MS / MS analysis. Prior the analysis, all samples except CIN and HON were dehydrated in vacuo at 45 °C and the dry residue, or CIN and HON, were extracted with 70 % aqueous methanol, 15 min on ultrasonic bath followed by 1 hour on shaker (120 rpm / min), both at room temperature. Extracts of each sample were prepared in triplicate.

[0067] Total phenolic and flavonoid contents were determined according to spectrophotometric methods described previously (Lesjak et al., Juniperus sibirica Burgsdorf, as a Novel Source of Antioxidant and Anti-inflammatory Agents. Food Chemistry 2011, 124, 850-856).

[0068] Furthermore, filtered samples were used for qualitative and quantitative analysis of phenolic compounds using high-performance liquid chromatography (HPLC - Agilent Technologies 1200 Series) coupled with UV / VIS and tandem mass spectrometry with electrospray ionization (ESLQqQ-MS / MS - Agilent Technologies 6410A). Briefly, 5 pL of the sample were injected into the system, and compounds were separated on Zorbax Eclipse XDB-C18 (50 mm x 4.6 mm, 1.8 pm) rapid resolution column (Agilent Technologies) held at 50 °C. Mobile phase consisting of 0.05 % aqueous formic acid (A) and methanol (B) was delivered at flow rate of 0.5 mL / min in gradient mode (0 min 30 % B, 12 min 70 %, 18 min 100 %, 24 min 100 %, re-equilibration time 6 min). Eluted compounds were monitored at 340 nm in order to detect potential inhibitors of iron absorption, since flavonoids and most of the phenylpropanoids absorb light at that wavelength. For quantification of 44 selected phenolic compounds, data were acquired in dynamic MRM mode, using the optimised compound specific parameters (retention time, precursor ion, product ion, fragmentor voltage, collision voltage (Orcic et al., Quantitative determination of plant phenolics in Urtica dioica extracts by high- performance liquid chromatography coupled with tandem mass spectrometric detection. Food Chem. 2014 Jan 15;143:48-53). For all the compounds, peak areas were determined using Agilent MassHunter Workstation Software - Qualitative Analysis (ver. B.06.01). Calibration curves were plotted and samples’ concentrations calculated using the OriginLabs Origin Pro (ver. 8.0) software.

[0069] Total phytic acid content

[0070] Pythic acid content was measured using a commercial kit according to manufacturer’s instructions (Phytic Acid Assay Kit, Megazyme, Ireland).

[0071] Total iron content

[0072] Total iron content was examined by atomic absorption spectrophotometry (AAS). Before the analysis all samples, except CIN and HON, were dehydrated in vacuo at 45 °C and the dry residue, or CIN and HON (approx. 1 g), were digested with 10 mb ccHNOs and 2 mL CCH2O2, on a temperature of 180 °C, until the full mineralisation. After filtration and dilution with deionised H2O, samples were analysed on an atomic absorption spectrometer (Perkin Elmer, Atomic Absorption Spectrometer, AAnalyst 700") using the flame emission spectrometry in accordance with the standard method EPA 7000B. The practical limit of quantitation (PQL) for iron content in samples was 0.011 mg / L. Measurements were performed in triplicate.

[0073] Example 2 -In vivo Studies

[0074] After the nutritional formula was developed, two separate in vivo studies were carried out with food products based on the nutritional formula. In the first study, the aim was to estimate if the food product based on the nutritional formula affects iron absorption from artificial iron supplementation, in case it is taken simultaneously with supplementation. The aim was to conclude weather nutritional formula could be consumed together with oral iron supplements during IDA treatment in order to offset unpleasant side effects of iron supplementation, and at the same time not to decrease iron bioavailability and its efficacy. The aim of the second study was to estimate bioavailability of iron from the food product based on the nutritional formula when consumed alone. The aim was to conclude weather nutritional formula when consumed alone could be beneficial for prevention and treatment of mild IDA.

[0075] Both in vivo studies were performed on rats with IDA and bioavailability of iron was followed by measuring blood parameters and iron tissue levels, carrying out histology examination and estimation of gene expression of proteins of interest for iron homeostasis (duodenal cytochrome B (Dcytb), divalent metal transporter 1 (DMT1), hephaestin, ferroportin (FPN), ferritin light chain, hepcidin and glyceraldehyde 3- phosphate dehydrogenase (GAPDH)).

[0076] Experimental animals

[0077] All experimental procedures in animals were approved by the Ethics Committee of the University of Novi Sad, Republic of Serbia (approval No. EK: IV-E-2020-02) and were conducted in accordance with the European Commission legislation on the protection of animals used for scientific purposes Directive 2010 / 63 / EU and the Law on Animal Welfare (Official Gazette of Republic of Serbia No. 41 / 2009). In addition, during the entire research, the 3R principles were applied.

[0078] Male Wistar Rats (three weeks old) were supplied by the breeding centre from the Military Medical Academy, Belgrade, Republic of Serbia. The animals were kept in the vivarium at the Department of Pharmacology and Toxicology, Faculty of Medicine University of Novi Sad, Novi Sad, Republic of Serbia, and appropriate conditions were applied (temperature 25 °C, relative air humidity 30-50 %, daily lighting cycle, i.e. 12h of light / 12h of darkness). Animals were placed in collective cages (6 individuals per cage).

[0079] In vivo study on the effects of the final food products on absorption of iron from artificial oral iron supplementation when applied simultaneously

[0080] Study was divided in two stages. The aim of the first stage was to achieve IDA in all animals by dietary intervention. During the second stage animals were simultaneously orally treated with final food products ORG and RED and supplemented with iron. During the first stage, three weeks old rats were placed on a special diet in order to cause IDA in rats. Special diet was applied for 29 consecutive days following this regimen: from day 1 to day 18 (a total of 18 days), the animals were fed with a mixed diet composed of 50 : 50 = diet containing optimum iron content i.e. 270 mg Fe / kg (diet type Laboratory rats 20 %, manufacturer Veterinarski zavod Subotica, Stocna hrana doo, Subotica, Republic of Serbia) : diet with low iron content (5.9 mg Fe / kg) (type Iron deficient 5.9 mg / kg Fe, U8958P Version 0176, manufacturer SAFE, Augy, France); from day 19 to day 29 (a total of 11 days) the animals were fed only with rat diet with low iron content containing 5.9 mg Fe / kg. During this stage water and food were supplied ad libitum. On day 30 blood was taken from the tail vein and IDA was confirmed by hemeoglobin level (<13.7 g / dL). Additionally, analysis of blood samples was done as explained in text below.

[0081] Second stage of the experiment began right after IDA was confirmed on day 30. Animals were divided into three experimental groups and treated accordingly:

[0082] Fe sup (control group; 12 animals at the start, 10 animals at the end of experiment) was treated once a day orally by gastric tube with a 160 pL of mixture solution of FeSO4 and ascorbic acid (one dose contained 1.24 mg Fe and 6.576 mg ascorbic acid). Mixture solution was prepared as follows: 1.991 g of FeSCU x 7H2O and 2.055 g of ascorbic acid dissolved in 50 m of saline (0.9 % w / v of NaCl).

[0083] ORG + Fe sup (experimental group ORG + Fe sup; 12 animals at the start, 9 animals at the end of experiment) was treated twice a day orally by gastric tube with ORG supplemented with FeSO4 and ascorbic acid. The animals were treated in two doses, first one 1.3 mL and second one 1.26 mL (a total of 2.56 mL per day), with an interval of 3 hours between doses. FeSO4 x 7H2O and ascorbic acid were added to the ORG, so that the daily dose of 2.56 mL contained 1.24 mg of Fe and 6.576 mg of ascorbic acid.

[0084] RED + Fe sup (experimental group RED + Fe sup, 12 animals at the start, 7 animals at the end of experiment) was treated twice a day orally by gastric tube with RED supplemented with FeSO4 and ascorbic acid. The animals were treated in two doses, first one 1.3 mL and second one 1.26 mL (a total of 2.56 mL per day), with an interval of 3 hours between doses. FeSC x 7H2O and ascorbic acid were added to the RED, so that the daily dose of 2.56 mL contained 1.24 mg of Fe and 6.576 mg of ascorbic acid.

[0085] Described treatments in the second stage lasted 11 consecutive days. During the treatment, the animals were fed only with rat diet with low iron content containing 5.9 mg Fe / kg. Throughout the experiments, rats were afforded unrestricted access to water, while their dietary intake was meticulously regulated. Specifically, food provision was suspended on a nightly basis to facilitate gastric emptiness for the subsequent administration of two oral treatments in the morning. Following the treatment, food was reinstated to the rats, who underwent a total dietary restriction of approximately 15 hours. Subsequently, rats were granted unimpeded access to food until the subsequent evening period.

[0086] Sacrifice was performed on the 12thday of the second stage. Following administration of a terminal IP dose of pentobarbitone sodium (120 mg / kg body weight) a blood sample was removed via cardiac puncture and used for further analysis. Additionally, duodenum, liver, spleen and kidney were removed and rapidly frozen in liquid nitrogen before being stored at -80 °C, and subsequently used for determination of tissue non-heme iron content, gene expression levels and histology analysis.

[0087] In vivo study on the effects of the final food products on iron absorption when applied alone

[0088] Study was divided in two stages. Aim of the first stage was to achieve IDA in three groups of animals (Control, ORG, ORG + Fe enrich). During the second stage, one group (Control) of animals was not treated and was used as a control, the second (ORG) was treated only with ORG, while the third group (ORG + Fe enrich) was treated with ORG enriched with iron. In parallel, during both stages of the study, separate group of animals (Normal) was fed with a diet containing optimum iron content (270 mg Fe / kg) and was not treated. This group was formed for comparison purposes.

[0089] During the first stage, three weeks old rats were placed on a special diet in order to cause IDA. Special diet was applied for 47 consecutive days following this regimen: from day 1 to day 7 (a total of 7 days), the animals were fed only with the diet containing optimum iron content (270 mg Fe / kg); from day 8 to day 31 (a total of 24 days), the animals were fed with a mixed diet composed of 50:50 = diet containing optimum iron content (270 mg Fe / kg) : diet with low iron content 5.9 mg Fe / k; from day 32 to day 47 (a total of 16 days) the animals were fed only with rat diet containing 5.9 mg Fe / kg. During this stage water and food were supplied ad libitum. On day 47 IDA blood was taken from the tail vein and IDA was confirmed by hemoglobin level (<13.7 g / dL). Additionally, analysis of blood samples was done as explained in text below.

[0090] The second stage of the experiment began right after IDA was confirmed in animals in groups Control, ORG and ORG + Fe enriched on day 47.

[0091] Animals were divided into four experimental groups and treated accordingly:

[0092] Control (control group, 11 animals at the start and at the end of experiment) had no treatment at all, but was fed only with diet with low iron content 5.9 mg Fe / kg.

[0093] Normal (Control group; 6 animals at the start and at the end of experiment) had no treatment at all and was fed with diet containing optimum iron content for feeding rats (270 mg Fe / kg).

[0094] ORG (experimental group ORG; 10 animals at the start, 9 animals at the end of experiment) was treated once a day orally by gastric tube with 1.2 mL of concentrated ORG enriched with ascorbic acid. Ascorbic acid was added to the ORG, so that the daily dose of 1.2 mL contained 0.58 mg of ascorbic acid. Compared to ORG in the previous experiment, this concentrated ORG had 24 % less water, so the dose of 1.2 mL in this experiment corresponds to the dose of 1.56 mL in the previous experiment. This reduction of water in ORG was introduced to avoid mass dying of rats due to the aspiration pneumonia as a result of aspiration of stomach contents.

[0095] ORG + Fe enrich (experimental group ORG + Fe enrich; 10 animals at the start and at the end of experiment) was treated once a day orally by gastric tube with 1.2 mL of concentrated ORG enriched with Fe and ascorbic acid. FeSO4 x 7H2O and ascorbic acid were added to the ORG, so that the daily dose of 1.2 mL contained 0.11 mg of Fe and 0.58 mg of ascorbic acid.

[0096] Described treatments in the second stage lasted 27 days. During treatments, the animals in groups Control, ORG and ORG + Fe enrich were fed only with diet with low iron content 5.9 mg Fe / kg. During this stage water and food were supplied ad libitum.

[0097] Sacrifice was performed the day after the end of the second stage. Following administration of a terminal IP dose of pentobarbitone sodium (120 mg / kg body weight), a blood sample was removed via cardiac puncture and used for further analysis. Additionally, duodenum, liver, spleen and kidney were removed and rapidly frozen in liquid nitrogen before being stored at -80 °C, and subsequently used for determination of tissue non-heme iron content, gene expression levels and histology analysis

[0098] Analysis of blood samples

[0099] Blood sampling was performed on two occasions during each study. First time blood sampling was performed from the tail vein after introduction of rats into IDA (before the second stage treatments). The second time blood sampling was performed during cardiac puncture, after the second stage treatments.

[0100] For biochemical analysis blood was collected by microtainers (from tail vein) or vacutainer (during cardiac puncture), both filled with separating gels and clot activator. Serum was separated from the clotted blood after centrifugation (15 min at 3000g, room temperature), 30 min after sampling. For hemeatological analyses blood was collected by EDTA microtainers (during cardiac puncture).

[0101] Serum iron content and unsaturated iron-binding capacity (UIBC) were determined on an automatic biochemical analyzer A25 (Biosystems, Spain).

[0102] Serum iron was determined by the spectrophotometric method. In brief Fe3+was liberated from transferrin by guanidine, followed by reduction of Fe3+to Fe2+by ascorbate. Reduced iron reacted with ferrozine to form a coloured complex whose concentration was determined spectrophotometrically.

[0103] UIBC was determined by adding a known amount of Fe2+to the samples, while the excess Fe2+reacted with ferrozine to form a coloured complex whose concentration was determined.

[0104] Total iron binding capacity (TIBC) was determined by summing values of serum iron and UIBC. The percentage of transferrin saturation (TS) with iron is calculated by dividing the serum iron concentration by the TIBC and multiplying by 100.

[0105] Hemeatological parameters (red blood cells (RBC), hemoglobin (Hgb), red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hematocrit (HCT), platelet count (PUT), white blood cell (WBC) and number / percentage of granulocytes, monocytes and lymphocytes) were determined on an automatic hematology analyser BC-2800 Vet (Mindray, China).

[0106] The concentrations of ferritin and transferrin were determined using commercial ELISA tests (Elabscience).

[0107] Histopathological analysis

[0108] The histological assessment was blind, performed by two researchers by light microscopy. The histological analysis was performed on a sample of liver, spleen, kidney and duodenal tissue sampled from each animal. Samples were fixed in Bouin’s solution for 24 hours. After that, samples were dehydrated in a graded series of isopropyl alcohol and embedded in paraffin blocks. For each sampled organ, 5 pm thick tissue sections were cut, using a rotation microtome (Sakura Finetek USA, Inc., Torrance, CA, USA). Sections were stained with routine hematoxylin and eosin (H&E) method, in addition to Prussian blue staining method applied to spleen tissue sections. All histological slides were analysed under a light microscope (Leica DM LB, Germany) and photographed using integrated camera (Leica DC 100).

[0109] Non-heme tissue iron

[0110] Quantitative of non-heme iron was carried out according to the method of Torrance and Bothwell (1980). Briefly, 20-50 mg of tissue sample (duodenum, liver, spleen and kidney) was dried at 55 °C for 72 h and subsequently weighed. Dried samples were digested in 1 mL acid mixture (30% (v / v) HC1 and 10 % (w / v) trichloroacetic acid) at 65 °C for 20 hours. 1 mL of chromogen reagent (0.1 % (w / v) bathophenanthrolinesulfonate; 1 % (v / v) thiogly colic acid in 50 % (w / v) sodium acetate solution) and samples were incubated at 37 °C for 10 min, after which the absorbance of samples and iron standards was measured at 535 nm. Results were calculated as pg iron / g of dry tissue weight (dw).

[0111] RNA extraction and RT-PCR

[0112] Gene expression of proteins of interest for iron homeostasis, such as Dcytb, DMT1, hephaestin, FPN, ferritin light chain, hepcidin and GAPDH, were measured according to procedure explained below.

[0113] Total RNA from tissues (duodenum, liver, spleen and kidney) was extracted with TRIzol® reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentration of extracted RNA was measured using a Qubit® 2 fluorometer and Qubit RNA BR Assay Kit according to manufacturer’s instructions (Thermo Fisher Scientific Inc).

[0114] Afterwards, total RNA (1 pg) was treated with recombinant deoxyribonuclease (DNase I, Thermo Fisher Scientific Inc) and reverse transcribed using the High capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions.

[0115] RT-PCR reactions were performed using a Mx3005P (Stratagene) and iTaq Universal SYBR Green Supermix kit (Bio Rad), according to the manufacturer’s instructions. Each reaction was performed in duplicate. Samples without cDNA were included as negative controls. The primer sequences used for each gene are given in Supplementary material Table SI. Quantitative measurements of iron transporter relative to GAPDH gene expression were derived using the ACt method.

[0116] Table SI.

[0117] Statistics All data are presented as mean ± standard error of the mean (SEM). Statistical significant difference between groups was determined using the Student’s two-tailed unpaired t test. Statistical significance was taken to be p < 0.05.

[0118] Chemical analysis of plant-based food staffs and final food products Phenolic profile of 8 plant-based food staffs and two final food products was determined by evaluation of total phenolic and flavonoid contents, LC-D D analysis at 340 nm, as well as LC-MS / MS analysis of 44 selected phenolic compounds. The results are presented in Tables 1 and 2. The analysis of total phenolics and flavonoid contents showed that all investigated samples are very poor in these compounds, with an exception of CIN, which contain them in considerable amounts (Table 2.). Furthermore, it can be seen that minimal number of small peaks are present in HPLC-DAD chromatograms of plant-based food stuffs, which is in accordance with the results of total flavonoid content assay. Only in CIN sample some higher peaks were found.

[0119] Table 1.

[0120]

[0121]

[0122]

[0123] ♦values are means ± SEM of three measurements.

[0124] **below instrument quantification limit (LoQ).

[0125] ***means within each row with different letters (a-b) differ significantly (p < 0.05).

[0126] Abbreviations: BEE: beetroot cold-pressed juice; BSB: butternut squash boiled; CIN: cinnamon powder; HON: acacia honey; KIW: kiwi cold-pressed juice; MEL: melon cold-

[0127] 5 pressed juice; ORG: final food product, named orange smoothie; PIN: pineapple cold-pressed juice; RED: final food product, named red smoothie; WPB: white potato boiled.

[0128] Analysed but not detected: amentoflavone, apigenin, baicalein, baicalin, daidzein, epigallocatechin gallate, genistein, isorhamnetin, kaempferol, luteolin, luteolin-7-O-glucoside, matairesinol, myricetin, quercetin, vitexin

[0129] Table 2.

[0130] 10 ’values are means ± SEM of three measurements.

[0131] "below detection limit (DL), which is 0.40 mg phytic acid / g.

[0132] ""means within each row with different letters (a-b) differ significantly (p < O.O5).

[0133] Abbreviations: BEE: beetroot cold-pressed juice; BSB: butternut squash boiled; CIN: cinnamon powder; HON: acacia honey; KIW: kiwi cold-pressed juice; MEL: melon cold- pressed juice; ORG: final food product, named orange smoothie; PIN: pineapple cold-pressed juice; RED: final food product, named red smoothie; WPB: white potato boiled.

[0134] LC-MS / MS analysis revealed that most of the tested individual polyphenols, especially those marked as potent iron chelators whose characteristics were described in above mentioned text (e.g. quercetin, myricetin, catechins, chi orogenic acid, etc.), were not detected in analysed samples or were detected in traces, such as in KIW (Table 1). The only exception was CIN sample. However, the content of CIN in the final product ORG is low (1 g CIN / 300 mL ORG), thus the concentration of polyphenols detected in ORG was also low. By LC-MS / MS analysis of the two final products, it was observed that the RED contains 1.7 times more polyphenols than the ORG, with the most significant disparity found in the content of phenolic acids, most of which do not inhibit iron absorption.

[0135] For the sake of comparison, broccoli, a well-known source of iron (9 pg Fe / g raw broccoli; https: / / fdc.nal.usda.gOv / fdc-app.html# / 7componentM089, November 2023), is also a rich source of many polyphenols which are known to chelate iron, such as quercetin and kaempferol (32 pg quercetin and 78 pg kaempferol per g of raw broccoli; USDA Database for the Flavonoid Content of Selected Foods. Release 3.2 (November 2015)), which when summed together present a 30 times greater quantity than that of flavonoids, for example, in KIW (3.8 pg flavonoids / mL KIW). Thus, broccoli and many other fruits and vegetables known to contain high amounts of iron are excluded from this study and nutritional formula design since their profile of iron absorption inhibitors is not favourable for efficient iron absorption.

[0136] Data on the total iron and phytic acid contents are provided in Table 2.

[0137] All analysed foodstuffs contained certain amount of iron, especially CIN and WPB. Final food products were prepared combining the chosen foodstuffs so that one human serving of final food product (e.g. 300 mL) contain at least 1 mg of iron, which was 1.5 and 1 mg / 300 mL for ORG and RED, respectively. Attained iron per serving of the nutritional formula was considered sufficient because recommended daily dose for the total iron intake is between 10-20 mg, from which only 10 % (up to 2 mg) is absorbed due to the presence of iron absorption inhibitors (Hurrell and Egli, (2010): Iron Bioavailability and Dietary Reference Values. Am J Clin Nutr. 91: 1461S-1467S). Also, reputable clinical trials regarding treatment of IDA tested iron-fortified food containing 0.63 mg Fe / 100 g of foodstuffs (Burgos et al., Total Iron Absorbed from Iron-Biofortified Potatoes Is Higher than that from Nonbiofortified Potatoes: A Randomized Trial Using Stable Iron Isotopes in Women from the Peruvian Highlands. J Nutr. 2023 Jun;

[0138] 153(6): 1710-1717), which is similar to iron quantity in ORG and RED.

[0139] All examined foodstuffs had low levels of phytic acid, with the exception of CIN and HON, which contained more than 2 mg of phytic acid per g. However, the final food products prepared by combining chosen foodstuffs contained less than 50 mg of phytic acid per serving, what is equivalent to 14 mg of phytic acid phosphorus per serving. Specifically, phytic acid content was 48 and 21 mg per serving (300 mL) for ORG and RED, respectively. Considering that only amounts of phytic acid exceeding 50 mg per serving have been associated with a reduction in iron absorption rates in vivo humans in previous human studies (Siegenberg et al., Ascorbic acid prevents the dose-dependent inhibitory effects of polyphenols and phytates on nonheme-iron absorption. Am J Clin Nutr. 1991 Feb;53(2):537-41), the concentrations of phytic acid in ORG and RED, should not inhibit iron absorption.

[0140] Furthermore, based on the existing literature it was not possible to predict the exact threshold above which polyphenols present in food will inhibit iron absorption. There is only a general consensus in scientific literature that high levels of polyphenols significantly reduce iron absorption.

[0141] Based on the evaluation of polyphenol profiles and phytic acid concentrations, it was determined that all chosen plant-based food stuffs, as well as two final food products (ORG and RED) had very low amount of polyphenols and adequate amounts of phytic acid. Consequently, ORG and RED would not inhibit iron absorption. This hypothesis was confirmed in the subsequent in vivo studies.

[0142] Effects of the final food products ORG and RED on absorption of iron from artificial oral iron supplementation when applied simultaneously in in vivo study

[0143] Rats with IDA were simultaneously orally treated with final food products ORG and RED supplemented with iron and the following parameters were measured: body weight, organ weight and selected blood parameters before and after the end of experiment (Table 3), histology analysis (FIG. 1-4), tissue iron levels (Table 4) and estimation of gene expression of proteins of interest for iron homeostasis (Table 5). The idea behind was to conclude weather final food products ORG and RED could be consumed together with oral iron supplements during IDA treatment in order to offset unwanted side effects of iron supplementation and at the same time not to decrease its efficacy.

[0144] Unfortunately, during the second stage of the study, 10 animals out of the initial 36 animals died, including two animals from Fe sup group, three animals from ORG +Fe sup group and five animals from RED + Fe sup group. The cause of death was assumed to be pneumonia as a result of aspiration of stomach contents after applied treatments. Namely, some animals vomited after treatment as they were unable to swallow the entire content, even though the volume of dose applied (first does 1.3 mL, second dose 1.26 mL (a total of 2.56 mL per day)) was according to good lab practices for animal research (Turner et al., Administration of substances to laboratory animals: routes of administration and factors to consider. J Am Assoc Lab Anim Sci. 2011 Sep; 50(5):600- 13). In order to avoid further loss of animals, study was stopped after 11 days of treatment, even though it was initially planned to last 14 days. However, the conducted study yielded valuable conclusions, contributing significantly to our understanding of the subject matter.

[0145] Specifically, obtained body and organ weights of all experimental groups were in accordance with reference values (Table 3). At the beginning of treatment severe IDA was confirmed in all animal groups by low Hgb, HCT, RBC, TS, serum iron and transferrin levels, as well as high RDW, UIBC and TIBC levels (Ning and Zeller, Management of iron deficiency. Hematology Am Soc Hematol Educ Program. 2019 Dec 6;2019(l):315-322; Camaschella. Iron-deficiency anemia. N Engl J Med. 2015 May 7; 372(19): 1832-43), as compared with reference values (Table 3). Interestingly, at the start of the treatment animals in all experimental groups showed increase in WBC counts, which is not typically caused by IDA. An increased WBC counts are more commonly associated with infections, inflammatory conditions, immune system responses, or certain disorders affecting the bone marrow. However, after 11 days of the treatment WBC counts in all experimental groups were significantly decreased and in Fe sup and ORG + Fe sup groups were in reference values, while for RED + Fe sup group was somewhat higher compared with reference values. Also, since serum ferritin is reliable blood IDA parameter only in the absence of inflammations / infections, serum ferritin was not considered as reliable for further discussion (Ning and Zeller, Management of iron deficiency. Hematology Am Soc Hematol Educ Program. 2019 Dec 6;2019(l):315-322; Camaschella. Iron-deficiency anemia. N Engl J Med. 2015 May 7;372(19): 1832-43).

[0146] Table3.

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] ’values are means ± SEM. Means within each row with different letters (a-c) differ significantly (p < O.O5).

[0155] "measurement was not performed due to low amount of serum sample.

[0156] Abbreviations: HCT: hemeatocrit, Hgb: hemeoglobin, MCH: mean corpuscular hemeoglobin, MCHC: mean corpuscular hemeoglobin concentration, MCV: mean corpuscular volume, PLT: platelet count, RBC: red blood cells, RDW: red cell distribution width, TIBC: total iron binding capacity, TS: transferrin saturation, UIBC: unsaturated iron-binding capacity, WBC: white blood cell.

[0157] After the treatment all animal groups showed improvement of main blood IDA parameters indicating that treatment was successful, independent of weather iron supplementation was applied alone or in combination with ORG and RED. Treatment was equally successful in Fe sup and ORG + Fe sup groups while it showed to some extent lower success in RED + Fe sup group. The highest increase was observed in TS and serum iron levels, 8.1 and 4.8 times higher compared to the start of the experiment, respectively. Also, Hgb, HCT and RBC levels increased between 1.2 to 1.5 times compared to the start of experiment, and was in most cases in reference range. However, even though RDW, UIBC and TIBC levels were increased at the end of experiment, their reduction trend was obvious when compared with the start of experiment. These parameters came to reference values if treatment lasted longer than 11 days. Also, after the treatment thrombocytosis occurred in all animal groups, which was not the case at the beginning of experiment. Nevertheless, it is established in the literature that IDA has been associated with the induction of reactive thrombocytosis (Kuku et al., (2009). Platelet counts in adults with iron deficiency anemia. Platelets, 20(6), 401-405) and this was confirmed in this study.

[0158] The evaluation of histological characteristics in the duodenum, liver, spleen, and kidney across all study groups revealed positive outcomes associated with the simultaneous administration of ORG and RED with iron supplementation. Specifically, the duodenal structure in all three groups exhibited well-preserved layered wall composition and the distinctive villous morphology of the mucosa. The epithelium of the intestinal villi was maintained, displaying characteristics typical of intestinal tissue. Variability in mucosal appearance was observed in the height of the villi, with slightly reduced villi height noted in the Fe sup and ORG + Fe sup groups (FIG. 1).

[0159] The hepatic tissue in Fe sup group had a typical lobular structure (FIG. 2A and 2D) with plates of hepatocytes radiating from the central vein to the periphery of the lobule. The hepatocytes cell borders were preserved, but cytoplasm exhibited signs of perinuclear halo and hydropic degeneration in 45.5 % of animals. Nuclei had regular shape and size. Within the portal spaces, scant connective tissue was present, containing blood vessels and bile ducts, along with some lymphocytes. Small clusters of lymphocytes were found in lobules, among hepatocytes.

[0160] The overall structure of the liver in ORG + Fe sup group maintained regular structure, with recognizable lobules featuring a characteristic shape (FIG. 2B and 2E). Hepatic plates radiated from the central veins, while sinusoids were discernible and unimpaired. Hepatocytes exhibited a perinuclear halo and signs of hydropic degeneration, indicating a damage of the cellular ultrastructure (FIG. 2E). In comparison to Fe sup group, hepatocytes displayed slightly more hepatocytes exhibiting perinuclear halo and hydropic degeneration, placed predominantly at the periportal area. Cell boundaries were clearly defined, while nuclei showed no indications of changes associated with cell death. In 37.5 % of animals, small clusters of lymphocytes were found among hepatocytes, and only in tissue of one animal (12.5 %) a small focus of necrosis was present. The portal spaces contained limited connective tissue housing elements of the portal triad.

[0161] The liver tissue of the animals in RED + Fe sup group preserved normal lobular arrangement (FIG. 2C). Hepatic plates radiated from central veins, containing hepatocytes which, in comparison to Fe sup and ORG + Fe sup groups, exhibited similar occurrence of perinuclear halo (71.43 % of animals) but the signs of hydropic degeneration was absent. Nuclear changes indicating cell death (karyopyknosis, karyorrhexis) were absent. Lymphocytic inflammatory infiltrate was rarely seen in the lobules (28 % of animals), and necrosis was absent. The portal spaces contained scarce connective tissue where elements of the portal triad were embedded.

[0162] In the histological analysis of the spleen of Fe sup group, both red and white pulp were distinguishable (FIG. 3A). The white pulp was preserved in 50 % of animals, while in others it was significantly reduced to a narrow band of lymphoid tissue around the centrofollicular artery (FIG. 3A and 3D). The surrounding mantle zone was also notably reduced and in cases absent. Opposite to white, the red pulp was more extensive and voluminous. Red pulp contained megakaryocytes, indicating the extramedullary hematopoiesis (FIG. 3D). In 16.7 % of animals megakaryocytes were abundant, while in 83.3 % they were detectable, but rare. The Prussian blue method (FIG. 3G), revealed iron deposits evident as greenish-bluish cytoplasmic staining in a limited number of cells (7 cells per 10 high power fields (HPF)). This limited number of iron- containing cells, suggested iron depletion in the spleen of this group. Spleen of ORG + Fe sup group of animals (FIG. 3B) had regular appearance in 75 % of animals, and only a quarter of cases had reduced, impaired appearance of white pulp. The red pulp was notably pronounced and voluminous. In 75 % of cases megakaryocytes were numerous, indicative of the intense extramedullary hematopoiesis (FIG. 3E). Utilizing the Prussian blue staining method, an increased number of cells with greenish-bluish dotted staining (11 / 10 HPF) was observed compared to Fe sup group, suggesting increase in iron deposition due to the applied treatment (FIG. 3H). FIG. 3C exhibited the histological appearance of spleen of RED + Fe sup group. The white pulp was reduced in only 29 % of animals, less than in Fe sup and ORG + Fe sup groups. The red pulp was moderately voluminous, with frequent megakaryocytes in 86 % of animals, which is much more compared to other groups (FIG. 3F). Application of the Prussian blue staining method (FIG. 31), an increase in iron-containing cells (16 / 10 HPF) was evident, and this increase was more pronounced compared to Fe sup and ORG + Fe sup groups. It can be inferred that the applied treatment led to an increase in the iron depot.

[0163] In Fe sup group samples of kidney tissue, both the cortex and medulla were identifiable, with the most of tissue damage was detected in cortex. Renal corpuscles are not reduced in number, but 25 % of them (semi quantitative assessment) had impaired structure (reduced size, narrow or absent Bowmans space) (FIG. 4A). The tubular epithelium did not exhibit vacuolization in the tubular epithelial cells. In the kidney tissues of ORG + Fe sup group, the cortex and medulla were clearly distinguishable, with the most notable changes occurring in the kidney cortex. The number of renal corpuscles in the cortex does not appear to be reduced. In contrast to Fe sup group, a slightly smaller subset of renal corpuscles displayed a reduced size and narrow or absent Bowmans space. There was no observable vacuolization in the tubular epithelial cells (FIG. 4B). Tissue samples of the kidney obtained from RED + Fe sup group exhibited the least tissue impairment. Cortex and medulla were clearly demarcated, with preserved number of renal corpuscles. Only about 10 % showed impairment in structure, while tubular structure and epithelium was intact (FIG. 4C).

[0164] Iron levels in liver, spleen and duodenum was shown in Table 4. Liver and spleen iron pools were found to be the highest in ORG + Fe sup group, what was not in accordance with histological observations, while levels of duodenal iron was highest and same in groups were ORG and RED were administrated simultaneously with iron supplementation.

[0165] Table 4.

[0166] ‘values are means ± SEM.

[0167] "means within each column with different letters (a-b), differ significantly (p < 0.05).

[0168] Gene expression of proteins of interest for iron homeostasis in liver, spleen, duodenum and kidney was shown in (Table 5). It is evident that gene expression was somewhat consistent in all study groups, except for expression of mRNA for DMT1 in duodenum and hepcidin in liver. Namely, in groups ORG + Fe sup and RED + Fe sup expression of DMT1 mRNA was significantly increased, 2.2 and 6.6 times, respectively, when compared with Fe sup group. This occurrence was probably due to IDA in rats (Recalcati et al., (2010): Iron Regulatory Proteins: From Molecular Mechanisms to Drug Development. Antioxidants & Redox Signaling. 13: 1593-1616), which was most pronounce in RED + Fe sup group. Hepcidin mRNA in liver was greatly increased in ORG + Fe sup compared with all other groups from this study (10.9 times when compared with Fe sup, and 14.7 times when compared with RED + Fe sup). This occurrence could be explained by high iron in liver of animals from ORG + Fe sup group, since high iron could be a signal for iron overload and call for hepcidin expression, primarily by liver. Also hepcidin production is induced during the inflammation process, which was confirmed at the start of the study in all animals groups, but this increase was absent in other groups herein (Viatte and Vaulont, (2009): Hepcidin, the iron watcher. Biochimie. 91: 1223-1228).

[0169] Table 5. ‘values are means ± SEM. Means within each row with different letters (a-c), differ significantly (p < 0.05).

[0170] Abbreviation: Dcytb: duodenal cytochrome B; DMT1: divalent metal transporter 1; FPN: ferroportin; GAPDH: glyceraldehyde 3-phosphate dehydrogenase

[0171] Amount of iron applied daily in all animal groups within supplementation in this study was 1.24 mg of Fe, from FeSCh, which is equivalent to daily human dose of 60 mg of Fe from FeSC , which is usual dose for IDA treatment in humans (Reagan-Shaw et al., Dose translation from animal to human studies revisited. FASEB J. 2008 Mar;22(3):659- 61).

[0172] Amount of ascorbic acid applied daily in all animal groups in this study was 6.576 mg, which is equivalent to daily human dose of 305 mg of ascorbic acid. This aligns with the recommended 2: 1 to 4: 1 molar ratio concerning ascorbic acid to iron, as advised for the treatment of IDA in humans. (Lynch et al., Biomarkers of Nutrition for Development (BOND)-Iron Review. J Nutr. 2018 Jun l;148(suppl_l): 1001S-1067S). Furthermore, amounts of ORG and RED applied daily in all animal groups was 2.56 mL, which is equivalent to the daily human dose of 120 mL of ORG and RED. This is 2.5 times less than recommended volume for one serving (300 mL).

[0173] Within this study, it was shown that during oral treatments of rats having IDA with iron supplementation, equivalent to human recommended dose, simultaneously with innovative plant- based food products ORG and RED, in a daily dose which was 2.5 times lower than recommended volume for one serving, during 11 consecutive days, led to the same improvement in IDA blood parameters as when oral iron supplementation was applied alone. The dose of iron consumed by oral supplementation was 60 times higher than iron amount in ORG and RED. Therefore, the ORG and RED in combination with oral iron supplementation increased iron absorption and blood IDA parameters compared to when iron supplementation was applied alone. However, this study showed that the final food products ORG and RED do not lead to a reduction of positive effect of iron supplementation and thus could be safe for consumption together with iron supplementation in order to reduce unwanted side-effects and contribute to compliance to iron supplementation therapy.

[0174] Effects of the final food product ORG on iron absorption when applied alone in in vivo study

[0175] Since the final food product ORG expressed better effect in treatment of rats with IDA, in the above described study, than product RED, ORG was chosen to be further investigated. It was given alone to rats with IDA (ORG group), and the results were compared with control group having no treatment and consuming the diet with low level of iron (Control), group consuming diet with optimum level of iron (Normal) and with a group receiving ORG enriched with iron (ORG + Fe enrich).

[0176] The results are presented as follows: body weight, organ weight and selected blood parameters before and after the end of experiment (Table 6), histology analysis (FIG. 5-11), tissue iron levels (Table 7) and estimation of gene expression of proteins of interest for iron homeostasis (Table 8). The idea behind was to conclude weather the final food product ORG alone or enriched with iron could enhance blood iron levels and improve other IDA parameters. Table 6.

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] ’values are means ± SEM. Means within each row with different letters (a-d) differ significantly (p < O.O5).

[0184] Abbreviations: HCT: hemeatocrit, Hgb: hemeoglobin, MCH: mean corpuscular hemeoglobin, MCHC: mean corpuscular hemeoglobin concentration, MCV: mean corpuscular volume, PLT: platelet count, RBC: red blood cells, RDW: red cell distribution width, TIBC: total iron binding capacity, TS: transferrin saturation, UIBC: unsaturated iron-binding capacity, WBC: white blood cell

[0185] 5 Table 7.

[0186]

[0187] ’values are means ± SEM.

[0188] "means within each column with different letters (a-d), differ significantly (p < O.O5).

[0189] Table 8.

[0190] ’values are means ± SEM. Means within each row with different letters (a-c), differ significantly (p < 0.05).

[0191] Abbreviation: Dcytb: duodenal cytochrome B; DMT1: divalent metal transporter 1; FPN: ferroportin; GAPDH: glyceraldehyde 3-phosphate dehydrogenase

[0192] During the second stage of the study 1 animal, from ORG group, out of the initial 37, died. The aetiology of mortality was not readily discernible.

[0193] Specifically, body and organ weights of all experimental groups were in accordance with reference values, but group which was consuming diet with optimum level of Fe (Normal group) had significantly highest body weight compared to other animal groups (Table 6).

[0194] At the beginning of the treatment, moderate IDA was confirmed in treated animal groups fed by low iron diet (Control, ORG, ORG + Fe enrich) by low Hgb, HCT, RBC, TS and serum iron, as well as high RDW, UIBC and TIBC levels (Ning and Zeller, Management of iron deficiency. Hematology Am Soc Hematol Educ Program. 2019 Dec 6;2019(1): 315-322), as compared with reference values (Table 6). IDA did not occur in animals from Normal group.

[0195] Initially, at the start of the treatment, animals across all experimental groups (Control, Normal, ORG, ORG + Fe enrich) exhibited rise in WBC counts, with a notable increase observed in monocytes and granulocytes. Such an elevation, typically uncharacteristic for IDA, is more commonly associated with conditions such as infections, inflammatory responses, immune system activation, or certain pathologies affecting the bone marrow. However, following 28 days of the administered treatments, WBC counts in all experimental animals displayed a significant reduction, reverting to reference values. Despite the marked decrease in WBC counts, this phenomenon was not subjected to further analysis. Additionally, it was noteworthy that serum ferritin, a reliable parameter for assessing IDA, was considered reliable only in the absence of concurrent inflammations or infections. Consequently, serum ferritin was deemed unreliable for further deliberation in the present context (Ning and Zeller, Management of iron deficiency. Hematology Am Soc Hematol Educ Program. 2019 Dec 6;2019(1 ):315- 322).

[0196] After the treatment, animal groups ORG and ORG + Fe enrich showed significant increase in IDA blood parameters, comparing with Control group, indicating that treatment was successful, independent of weather ORG was applied alone or enriched with iron. However, trend of ID A parameters levels was decreasing from start to the end of the treatment in each group (Control, ORG and ORG + Fe enrich). The treatment lasted 28 days, and during that period animals were fed with food with greatly reduced amount of iron. Amount of iron in applied food was 45 times lower than in regular food which was applied in Normal group. Thus ORG alone or enriched with iron could not compensate such a low amount of dietary iron. Nevertheless, ORG alone or enriched with iron was successful in neutralising negative effect of low intake of dietary iron and managed to increase IDA blood parameters compared with Control.

[0197] The ORG group exhibited the highest increase in TS and serum iron levels, with increments of 1.3 times (29 %) and 1.24 times (25 %), respectively, compared to the Control group. Additionally, the ORG + Fe enrich group showed even greater increases, with TS and serum iron levels rising by 2.52 times (252 %) and 2.64 times (264 %), respectively, compared to the Control group. Moreover, Hgb and HCT levels experienced a rise of 7.5 % and 12 %, respectively, in the ORG group, while the ORG + Fe enrich group demonstrated more substantial increases of 30 % and 35 %, respectively, compared to the Control group.

[0198] Moreover, the efficacy of treatment was notably higher in the ORG + Fe enrich group compared to the ORG group, as anticipated due to the additional iron administered in the former. The daily iron dose in the ORG + Fe enrich group amounted to 0.11 mg of Fe from the FeSO4 source, equivalent to a daily human dose of 0.29 mg of Fe from the FeSO4 source. This dosage is 200 times lower than the recommended dose for the treatment of IDA in humans (Reagan-Shaw et al., Dose translation from animal to human studies revisited. FASEB J. 2008 Mar;22(3):659-61). Nevertheless, it was proven to be sufficient for the effective treatment of IDA. This finding serves as additional confirmation that the ORG does not impact the bioavailability of iron when consumed simultaneously with iron supplementation.

[0199] The assessment of the histological features of the duodenum, liver, spleen and kidney of all groups in this study showed beneficial effects of ORG. Namely, in the duodenum, all groups maintained the layered wall composition, with comparable mucous membrane thickness except for the Control group. The liver morphology varied, with the Control group exhibiting signs of damage, while the ORG and ORG + Fe enrich groups displayed reduced damage, indicating the efficacy of the treatment with nutritional formula. In the spleen, the ORG and ORG + Fe enrich groups demonstrated improved morphology compared to the Control group, again suggesting a positive effect of the treatment. The kidney examination revealed that the treatment mitigated the adverse effects of IDA on kidney morphology in the ORG and ORG + Fe enrich groups, as observed in the cortex and medulla. In further text examination of histological samples was explained in details.

[0200] Namely, in the structural examination of the duodenum (FIG. 5), the layered structure of the wall remained intact in all experimental groups. The thickness of the mucosa membrane, in ORG and ORG Fe +enrich groups was comparable to that of the Normal group, as opposed to Control group where it was thinner. In all groups mucosa exhibited preserved villous structure. The intestinal villi, lined by intact intestinal epithelium, exhibited proper morphology, alignment and height. Intestinal glands (crypts of Liberkin) were present in adequate numbers and exhibited a regular arrangement, while the lamina propria contained a typical mononuclear inflammatory infiltrate.

[0201] The hepatic tissue in Normal group exhibited a typical structure, characterized by lobules with distinctive hexagonal shapes (FIG. 6). One cell thick plates of hepatocytes radiate from the central vein to the periphery of the lobule, as do sinusoids. The hepatocytes displayed well-defined cell borders, homogeneous and abundant cytoplasm, and one to two nuclei, each featuring prominent nucleoli. Within the portal spaces, there is scant connective tissue containing blood vessels and bile ducts along with rare lymphocytes.

[0202] The overall architecture of the liver in Control group of anaemic rats was maintained, with recognizable lobules featuring a characteristic shape (FIG. 7). Hepatic plates radiated from the central veins, centrally positioned within each lobule, served as radial points from which hepatocytes extend. Separated by sinusoids which were well visible, although discernible, displaying a slightly narrower lumen. Hepatocytes exhibited a pronounced perinuclear halo and signs of hydropic degeneration, indicating a damage of the cellular ultrastructure (FIG. 7A). In some areas of the lobule, cell boundaries were indistinct, and individual nuclei displayed signs of karyopyknosis (FIG. 7C), suggesting a process of cell death. Focal regions within the lobules revealed clusters of inflammatory cells, including lymphocytes and neutrophilic granulocytes (FIG. 7B). In the portal spaces at the lobule corners, there was sparse to moderately abundant connective tissue containing elements of the portal triad (interlobular artery, interlobular veins and interlobular bile ducts). A moderately abundant mononuclear inflammatory infiltrate, composed of lymphocytes and granulocytes, was evident in the connective tissue of the portal spaces (FIG. 7D). Notably, a significant number of portal space blood vessels, as well as certain sinusoids within the lobules, appeared dilated and filled with blood from the liver tissue.

[0203] The overall structure of the liver in ORG group remained intact, with normal structure (FIG. 8). In comparison to Control group, hepatocytes displayed a notably reduced occurrence of perinuclear halo and hydropic degeneration, primarily evident at the lobule's periphery (periportal), while mild hepatocyte edema was observed in the remainder of the lobule. Cell boundaries were clearly defined, and nuclei exhibited no signs of karyopyknosis or karyorrhexis, indicating the absence of processes associated with cell death. Rare blood vessels within the portal spaces appeared dilated and filled with blood. The portal spaces contained limited connective tissue housing elements of the portal triad, and a mononuclear inflammatory infiltrate was within physiological limits.

[0204] The liver tissue of the animals in ORG + Fe enrich group remained intact, with normal structure (FIG. 9). In comparison to Control and ORG groups, hepatocytes exhibited a significantly reduced occurrence of perinuclear halo, while hydropic degeneration was absent, along with the absence of signs indicating cell death (karyopyknosis, karyorrhexis). The liver structure closely resembled that of the Normal group, with no dilation observed in the portal space vessels. The portal spaces contained scarce connective tissue housing elements of the portal triad and a mononuclear inflammatory infiltrate within physiological limits. It can be deduced that the administered treatment has effectively mitigated liver damage caused by IDA, as evidenced by the close resemblance of the liver tissue to that of the Normal group.

[0205] In the histological image of the spleen of Normal group, both red and white pulp were distinguishable (FIG. 10). The white pulp was prominently developed around the centrofollicular arteries, featuring a broad mantle zone (FIG. 10A). The red pulp was moderately expressed, and upon H&E staining, no evidence of extramedullary hematopoiesis was observed, with the absence of megakaryocytes (FIG. 10E). Utilizing the Prussian blue staining method (FIG. 101), sporadic individual cells are identified, characterized by intensely blue-stained cytoplasm indicating of substantial iron depots. In the histological image of the spleen from Control group, both red and white pulp were identifiable. In comparison to the Normal group, the white pulp was diminished to a narrow band of lymphoid tissue around the centrofollicular artery (FIG. 10B). The surrounding mantle zone was also notably reduced. Conversely, the red pulp was accentuated and voluminous. FIG. 10F revealed the presence of numerous megakaryocytes in the red pulp, indicative of the extramedullary hematopoiesis. With the application of the Prussian blue method (FIG. 10 J), iron deposits were evident as greenish-bluish cytoplasmic staining in a limited number of cells (2 / 10 HPF). This limited number of iron- containing cells, suggested pronounced iron depletion in the spleen of this group. In the spleen of ORG group animals (FIG. 10C) the white pulp appeared reduced compared to Normal group, however, when compared to Control group, the white pulp and mantle zone were more clearly defined and more abundant. The red pulp was notably pronounced and voluminous, featuring numerous megakaryocytes indicative of the extramedullary hematopoiesis (FIG. 10G). Utilizing the Prussian blue staining method, an increased number of cells with greenish-bluish dotted staining (7 / 10 HPF) was observed compared to Control group, suggesting a rise in the iron pools due to the applied treatment (FIG. 10K). Similarly, in FIG. 10D, the histological image reveals recognizable red and white pulp of ORG + Fe enrich group. The white pulp exhibited no discernible differences compared to the Normal group. When compared to Control group, both the white pulp and mantle zone were more distinctly defined and abundant. The red pulp was moderately expressed, with noticeable signs of extramedullary hematopoiesis (FIG. 10H). Employing the Prussian blue staining method (FIG. 10L), an increase in iron-containing cells (20 / 10 HPF) was evident, and this increase was more pronounced compared to Control group as well as ORG group. It can be inferred that the applied treatment led to an increase in the iron depot.

[0206] In the kidney tissue of Normal group, both the cortex and medulla can be distinguished. Within the cortex, an appropriate number and regular histological appearance of renal corpuscles was observed. Notably, there was an absence of vacuolization in the tubular epithelial cells (FIG. 11 A). In Control group both the cortex and medulla of kidney were identifiable. However, in this instance (FIG. 11B), a significant reduction in the number of renal corpuscles within the cortex was evident when compared to the Normal group. Additionally, a substantial proportion of the remaining corpuscles displayed reduced size, lacking a discernible Bowman's space along with bleeding and sclerosis in the glomerular tuft. Concurrently, the nephron tubules exhibited vacuolization in the tubular epithelial cells. In the kidney tissues of ORG and ORG + Fe enrich groups, the cortex and medulla also exhibited distinguishable regions, with the most notable changes occurring in the kidney cortex. The number of renal corpuscles in the cortex, assessed using a semiquantitative method, does not differ significantly when compared to the Normal group. In contrast to Control group, a smaller subset of renal corpuscles within the cortex displayed a reduced dimensions and morphological aberrations, along with evidence of hemeorrhages in the glomerular tuft. Importantly, there was no observable vacuolization in the tubular epithelial cells (FIG. 11C and 11D). This suggested that the adverse impact of IDA on kidney morphology was mitigated by the administered treatment with ORG nutritional formula alone and enriched with iron.

[0207] Table 7 displayed the iron levels in the liver, spleen, and duodenum. The Normal animal group exhibited the highest iron levels in all examined tissues, followed by tissues from the ORG + Fe enrich group. Iron levels in tissues from the ORG and Control groups were comparable but remained significantly lower than those in the ORG + Fe enrich group. Iron levels determined in spleen tissue were in accordance with histological observations herein.

[0208] This study investigated the gene expression of proteins associated with iron homeostasis in the liver, spleen, duodenum and kidney, as outlined in Table 8. The gene expression patterns were relatively consistent across the Control, ORG and ORG + Fe enrich study groups. In contrast, the Normal group exhibited significant reductions in the expression of most examined mRNAs, except for hepcidin, which showed a marked increase compared to the other groups.

[0209] Specifically, in the Control, ORG, and ORG + Fe enrich groups, the expression of DMT1 and Dcytb mRNA in the duodenum was significantly elevated, surpassing 100 and 60 times, respectively, compared to the Normal group. Additionally, FPN mRNA levels in the duodenum, liver and spleen were significantly higher in the Control, ORG, and ORG + Fe enrich groups, exceeding 5.4, 3, and 2.1 times, respectively, in comparison to the Normal group. These observations are likely attributed to the presence of IDA in rats, a condition that was more pronounced in these groups (Zoller et al., (2003): Duodenal cytochrome b and hephaestin expression in patients with iron deficiency and hemochromatosis. Gastroenterology. 125: 746-754). In the liver, hepcidin mRNA exhibited a substantial increase in the Normal group, approximately 10 000 times higher than all other groups in this study. However, this should be interpreted as a significant decrease in the Control, ORG, and ORG + Fe enrich groups, considering that hepcidin levels are considered regular in the Normal group. This phenomenon aligns with the occurrence of IDA in these three groups, wherein during IDA, hypoxia and an increased demand for erythropoiesis lead to a reduction in hepcidin expression to facilitate efficient iron mobilization. Increase in hepcidin mRNA expression was observed in the Normal group in kidney and spleen tissues, although the difference was less pronounced (Viatte and Vaulont, (2009): Hepcidin, the iron watcher. Biochimie. 91: 1223-1228).

[0210] The daily administration of ORG alone or enriched with iron (ORG and ORG + Fe enrich groups) in this in vivo study amounted to 1.2 mL of concentrated ORG. This dose is equivalent to 1.56 mL of the non-concentrated, standard ORG serving, applied in previous experiment described herein. Translated to a human dose, this dose corresponds to a daily human dose of 41 mL of ORG, which is 7.3 times less than the quantity found in a single serving (300 mL).

[0211] Regarding iron supplementation, the nutritional formula ORG + Fe enrich provided an additional daily iron intake of 0.11 mg of Fe from FeSO4. Translated to a human dose, this is equivalent to a daily dose of 2.72 mg of Fe from FeSO4, which is 22 times less than the established dose for the treatment of IDA in humans, set at 60 mg of Fe from FeSO4 (Reagan-Shaw et al., Dose translation from animal to human studies revisited. FASEB J. 2008 Mar;22(3):659-61). Thus, ORG + Fe enrich could be considered as iron fortified drink (Angeles-Agdeppa et al., Fortified juice drink improved iron and zinc status of schoolchildren. Asia Pac J Clin Nutr. 2011 ;20(4):535-43).

[0212] The quantity of ascorbic acid administered in this study was 0.58 mg, corresponding to a daily human dose of 15 mg of ascorbic acid. This aligns with the recommended 2: 1 to 4: 1 molar ratio concerning ascorbic acid to iron, as advised for the treatment of IDA in humans. (Lynch et al., Biomarkers of Nutrition for Development (BOND)-Iron Review. J Nutr. 2018 Jun l;148(suppl_l): 1001S-1067S).

[0213] In this study, it was demonstrated that the oral administration of the innovative final plant-based food product ORG, both in its standalone form or enriched with iron, over a period of 28 consecutive days, yielded significant improvements in blood parameters associated with IDA in rats. Notably, the daily dosage administered to the rats was 7.3 times lower than the equivalent one serving for humans recommended herein. It was anticipated that extended duration of the treatment would result in further enhancement in blood parameters. Consequently, this study highlights that the innovative plant-based food product contains highly bioavailable iron and have the substantial potential as a viable alternative for the enhancement of blood iron level and improvement of biochemical parameters associated with IDA. Thus, the innovative plant-based food product could be useful in treatment of mild IDA in humans or for protective measures during periods when the likelihood of IDA occurrence is heightened, such as during reproductive phases in women, pregnancy and growth periods in children.

[0214] Conclusion

[0215] The examination of foodstuffs and final food products made from them (ORG and RED), revealed a favourable polyphenol profile, sufficient iron content, and low phytic acid levels, aligning with the initial study objectives. Consequently, ORG and RED were further investigated in in vivo studies as promising candidates for addressing IDA. The initial in vivo study suggested that the food products ORG and RED can be safely administered concurrently with artificial oral iron supplementation, without interfering with iron bioavailability, which could mitigate adverse side effects of supplementation without compromising its efficacy. In a subsequent investigation, the oral administration of the innovative plant-based food product ORG, whether in its standalone form or enriched with iron, demonstrated a significant improvement in blood parameters associated with IDA. These findings support the notion that this plant-based nutritional product holds considerable potential as a viable alternative for treating mild IDA in humans or as a preventive measure during periods susceptible to IDA, such as reproductive phases in women, pregnancy, and growth periods in children. This study affirmed the possibility of developing a nutritional formula that serves as a versatile foundation for creating various final food products, including juices, bars, smoothies, ice creams, soups, etc., for the prevention and treatment of IDA.

[0216] OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A food product composition, comprising: at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product; and one or more components selected from the group consisting of: less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; and less than about 50 mg of phytic acid per one serving or daily dose of product.

2. The food product composition of claim 1, wherein the food product composition comprises the plant-based non-heme iron in an amount in a range of: about 1 mg to about 10 mg; about 1 mg to about 5 mg; about 1 mg to about 2.5 mg; per one serving or daily dose of product.

3. The food product composition of claim 1 or 2, wherein the food product composition comprises: less than about 300 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product; or less than about 150 mg of polyphenols, expressed as mg gallic acid equivalents per one serving or daily dose of product.

4. The food product composition of any one of claims 1-3, wherein the food product composition comprises: less than about 1.8 mg of flavonoids, expressed as mg quercetin equivalents per one serving or daily dose of product; or less than 0.9 mg of flavonoids expressed as mg quercetin equivalents per one serving or daily dose of product.

5. The food product composition of any one of claims 1-4, wherein the food product composition is substantially free of flavonoids having a 3-hydroxy group- and 4-carboxyl group; a 5-hydroxy group and 4-carboxyl group; or a 3’ - and 4’ -hydroxyl groups.

6. The food product composition of any one of claims 1-5, wherein the food product composition is substantially free of one or more flavonoids selected from the group consisting of: quercetin, genistein, and their glycosides, luteolin, apigenin, and epigallocatechin-3 -gallate.

7. The food product composition of any one of claims 1-6, wherein the food product composition is substantially free of phenolic acids having two hydroxyl groups in the ortho position of the phenolic acid or a hydroxyl and a carboxyl group in the ortho position of the phenolic acid.

8. The food product composition of any one of claims 1-7, wherein the food product composition comprises: less than about 50 mg of phytic acid per one serving or daily dose of product; or less than about 25 mg of phytic acid per one serving or daily dose of product.

9. The food product composition of any one of claims 1-8, wherein the food product composition is substantially free of one or more of phenolic acids, flavonoids, and phytic acid.

10. The food product composition of any one of claims 1 -9, further comprising ascorbic acid.

11. The food product composition of claim 10, wherein a molar ratio of ascorbic acid to plant-based non- heme iron is in a range of about 2: 1 to about 4: 1, respectively.

12. A food product composition, comprising: white potato;honey; and one or more ingredients selected from the group consisting of: butternut squash, pineapple, cinnamon powder, melon, kiwi, and beetroot; wherein the food product composition comprises at least about 1 mg of plant-based non-heme iron per one serving or daily dose of product.

13. The food product composition of claim 12, wherein the food product composition comprises white potato, butternut squash, pineapple, cinnamon powder, and honey.

14. The food product composition of claim 13 comprising: about 60 g to about 150 g of white potato per 300 mL of food product composition; about 50 g to about 120 g of butternut squash per 300 mL of food product composition; about 40 mL to about 100 mL of pineapple juice per 300 mL of food product composition; about 0.5 g to about 2 g of cinnamon powder per 300 mL of food product composition; about 2.5 g to about 10 g of honey per 300 mL of food product composition; and about 50 mL to about 90 mL of water.

15. The food product composition of claim 14 comprising: about 60 g of white potato per 300 mL of food product composition; about 100 g of butternut squash per 300 mL of food product composition; about 60 mL of pineapple juice per 300 mL of food product composition; about 1 g of cinnamon powder per 300 mL of food product composition; about 5 g of honey per 300 mL of food product composition; and about 70 mL of water.

16. The food product composition of claim 12, wherein the food product composition comprises white potato, melon, kiwi, beetroot, and honey.

17. The food product composition of claim 16 comprising: about 80 g to about 150 g of white potato per 300 mL of food product composition; about 80 mL to about 120 mL of melon juice per 300 mL of food product composition; about 50 mL to about 100 mL of kiwi juice per 300 mL of food product composition; about 2.5 mLto about 7.5 mL of beetroot juice per 300 mL of food product composition; and about 2.5 g to about 10 g of honey per 300 mL of food product composition.

18. The food product composition of claim 17 comprising: about 100 g of white potato per 300 mL of food product composition; about 100 mL of melon juice per 300 mL of food product composition; about 70 mL of kiwi juice per 300 mL of food product composition; about 5 mL of beetroot juice per 300 mL of food product composition; and about 5 g of honey per 300 mL of food product composition.

19. The food product composition of any one of claims 12-18, wherein the white potato is a cubed or sliced, peeled, and boiled white potato.

20. The food product composition of any one of claims 12-16 and 19, wherein the butternut squash is a cubed or sliced, peeled, and boiled butternut squash.

21. The food product composition of any one of claims 12-16 and 19, wherein the pineapple juice is cold-pressed pineapple juice.

22. The food product composition of any one of claims 12-21, wherein the honey is acacia honey.

23. The food product composition of any one of claims 12-14, 17-19, or 22, wherein the melon juice is cold-pressed melon juice.

24. The food product composition of any one of claims 12-14, 17- 19, or 22-23 , wherein the kiwi juice is cold-pressed kiwi juice.

25. The food product composition of any one of claims 12-14, 17-19, or 21 -23 , wherein the beetroot juice is cold-pressed beetroot juice.

26. The food product composition of any one of claims 1-25 further comprising about 2.72 mg to about 240 mg of FeSCh and / or an iron equivalent per one serving or daily dose of food product composition.

27. The food product composition of any one of claims 1 -26, wherein the food product composition is in liquid form.

28. The food product composition of any one of claims 1 -26, wherein the food product composition is in solid or semi-solid form.

29. The food product composition of any one of claims 1 -26, wherein the food product composition is in the form of a juice, smoothie, porridge, muesli, bar, ice cream, jam, or soup.

30. The food product composition of any one of claims 1-29 for use to improve blood iron levels in the treatment of iron deficiency anemia.

31. A method of increasing blood iron levels in a subject, the method comprising: administering an effective amount of the food product composition of any one of claims 1-30 to a subject in need thereof.

32. The method of claim 31 , wherein the blood iron levels of the subject increase by at least 20% compared to a control group that is not administered the food product composition.

33. A method of treating iron deficiency anemia in a subj ect in need thereof, the method comprising: administering to a subject identified or diagnosed as having iron deficiency anemia an effective amount of the food product composition of any one of claims 1-30.

34. The method of any one of claims 31-33, wherein at least one serving of the food product composition is administrated to the subject every day or every second day.

35. The method of any one of claims 31-34, wherein the food product composition is administered to the subject on an empty stomach about an hour before a next meal.

36. A food product composition of any one of claims 1-30 for use in a method of treating iron deficiency anemia in a subject in need thereof, the method comprising: administering to a subject identified or diagnosed as having iron deficiency anemia an effective amount of the food product composition.

37. The food product composition for use of claim 36, wherein at least one serving of the food product composition is administrated to the subject every day or every second day.

38. The food product composition for use of claim 36 or 37, wherein the food product composition is administered to the subject on an empty stomach about an hour before a next meal.

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