A COMPOSITION FOR TREATING ANEMIA

TECHNICAL FIELD

[01] The present disclosure relates to compositions used for substituting iron deficiency in living things, and more particularly to compositions that substitute iron deficiency in mammals through enhancing intestinal iron absorption.

BACKGROUND

[02] Several diseases and syndromes have been linked to deficiency in metals. Metals such as sodium, potassium, calcium, magnesium, iron and copper play a vital role in the living systems.

[03] Iron is a crucial mineral to several human body functions, such as DNA synthesis, respiration, and other metabolic functions. Iron deficiency may lead to anemia, a clinical condition where there is a significant decrease in the number of red blood cells and/or hemoglobin in the body.

[04] Iron deficiency is considered one of the most critical issues in medicine. Several causes have been identified for iron deficiency such as the lack of dietary iron or consumption of foods, or medications, that inhibit iron absorption including calcium, proton pump inhibitors and tannins. From another perspective, damaged intestine or duodenal surgery can reduce the surface area available for absorption.

[05] Usually, low amount of dietary iron is absorbed (from 5% to 35%) depending on many factors including the type of iron. However, best absorbed forms of iron come from animal products (heme iron). The duodenum is main absorption cite of iron through special cells called enterocytes that transport iron into the body. In order to be absorbed, dietary iron either can enter the blood a part of a protein such as heme protein or must be in its ferrous form (Fe ²⁺ ) in order to be transported across the enterocyte’s cell membrane via a divalent metal transporter type 1.

[06] One of the known iron absorption enhancers is ascorbic acid or vitamin C. It occurs naturally in vegetables and fruits, especially citrus. Ascorbic acid can also be synthesized for use in supplements. It is reported in the prior art that 20 mg of ascorbic acid is needed to enhance 3 mg of iron absorption. However, patients with kidney problems have to compromise their ascorbic acid intake. Additionally, iron is a toxic metal and its daily intake should be limited.

[07] Grewia tenax fruit juice or concentrate, on the other hand, has been traditionally used as a tonic drink and for its anti -anemic properties. However, the amount of iron present in this herb is low when measured. Grewia tenax dried fruit extract has been demonstrated in prior arts that it can enhance iron absorption. This might be attributed to the presence of ascorbic acid in this plant.

SUMMARY

[08] The present disclosure aims at providing a composition for enhancing intestinal iron absorption with minimized values for iron and acid intake.

[09] As an object of the present disclosure, there is provided a composition including a plant fruit from Malvaceae family, and an acid.

[010] In an aspect of the present disclosure, the plant may include Grewia tenax.

[011] In another aspect of the present disclosure, the plant fruit may include Grewia tenax dried fruit.

[012] In another aspect of the present disclosure, the plant fruit includes Grewia tenax hydrophilic and hydrophobic extracts.

[013] According to other aspects of the present disclosure, the composition may include a dose of 1-200 mg/kg Grewia tenax dried fruit.

[014] In other aspects of the present disclosure, the acid may include sulfurous acid, sulfuric acid, hyposulfurous acid, persulfuric acid, pyrosulfuric acid, disulfurous acid, dithionous acid, tetrathionic acid, thiosulfurous acid, hydrosulfuric acid, peroxydisulfuric acid, perchloric acid, hydrochloric acid, hypochlorous acid, chlorous acid, chloric acid, hyponitrous acid, nitrous acid, nitric acid, pernitric acid, carbonous acid, carbonic acid, hypocarbonous acid, percarbonic acid, oxalic acid, acetic acid, phosphoric acid, phosphorous acid, hypophosphous acid, perphosphoric acid, hypophosphoric acid, pyrophosphoric acid, hydrophosphoric acid, hydrobromic acid, bromous acid, bromic acid, hypobromous acid, hypoiodous acid, iodous acid, iodic acid, periodic acid, hydroiodic acid, fluorous acid, fluoric acid, hypofluorous acid, perfluoric acid, hydrofluoric acid, chromic acid, chromous acid, hypochromous acid, perchromic acid, hydroselenic acid, selenic acid, selenous acid, hydronitric acid, boric acid, molybdic acid, perxenic acid, silicofluoric acid, telluric acid, tellurous acid, tungstic acid, xenic acid, citric acid, formic acid, pyroantimonic acid, permanganic acid, manganic acid, antimonic acid, antimonous acid, silicic acid, titanic acid, arsenic acid, pertechnetic acid, hydroarsenic acid, dichromic acid, tetraboric acid, metastannic acid, hypooxalous acid, ferricyanic acid, cyanic acid, silicous acid, hydrocyanic acid, thiocyanic acid, uranic acid, diuranic acid, malonic acid, tartartic acid, glutamic acid, phthalic acid, azelaic acid, barbituric acid, benzilic acid, cinnamic acid, fumaric acid, glutaric acid, gluconic acid, hexanoic acid, lactic acid, malic acid, oleic acid, folic acid, propiolic acid, propionic acid, rosolic acid, stearic acid, tannic acid, trifluoroacetic acid, uric acid, ascorbic acid, gallic acid, acetylsalicylic acid, acetic acid, or a combination thereof.

[015] In other aspects of the present disclosure, the composition includes 0.1-40 mg/kg ascorbic acid.

[016] In yet other aspects, the composition further comprises an iron salt.

[017] According to other aspects of the present disclosure, the composition includes 0.1-5 mg/kg free iron.

[018] In accordance with other aspects of the present disclosure, the iron salt includes Iron (II) sulfate (FeSCri) also known as ferrous sulfate, or ferrous fumarate, ferrous ascorbate, ferrous aspartate, iron glycinate, ferrous gluconate, carbonyl iron or polysaccharide iron complex, or a combination thereof.

[019] In yet another aspect of the present disclosure, the disclosure relates to a composition according to the present disclosure for use as a medicament.

[020] In a further aspect of the present disclosure, the disclosure concerns the use of a composition according to the present disclosure for the manufacture of a medicament for the treatment of anemia.

[021] In yet other aspects, the disclosure concerns the use of a composition according to the present disclosure for the manufacture of a medicament for the treatment of Iron Deficiency Anemia.

[022] In one aspect, the use is for mammals. [023] In another aspect, the mammal is a human.

[024] In embodiments of the present disclosure, the said composition may be used as a nutraceutical, herbal, medicinal or a pharmaceutical product.

[025] In some aspects, the composition may be formulated with or as juice, energy drink, canned gaseous drink, flavored water.

[026] In other aspects, the composition may be formulated with fresh, cooked or unprocessed food, chocolates, sweets, jelly beans, gum, snacks, cereals, natural and processed potato or rice chips.

[027] In aspects of the present disclosure, the composition may include additional components such as, drugs, other plant extracts, vitamins, ionic minerals, and other substances purported to be of a health benefit.

[028] In yet another aspect, the disclosure relates to a pharmaceutical composition with natural composition for enhancing iron absorption in healthy and sick humans including geriatrics and pediatrics and in the case of pregnancy.

[029] In aspects of the present disclosure, the pharmaceutical composition includes the said composition and a pharmaceutically acceptable carrier and/or excipient.

[030] The pharmaceutical composition can be formulated in a delayed, sustained or targeted release formulations.

[031] In some aspects of the present disclosure, the pharmaceutical composition may be formulated in oral dosage.

[032] In some aspects, the oral dosage form may include liquid oral dosage form.

[033] The liquid oral dosage form may include emulsion, solution, suspension, syrup or elixir.

[034] In some aspects, the pharmaceutical composition may be formulated in effervescent preparations such as tablets, powders and sparkling drinks.

[035] In other aspects, the oral dosage form may include solid oral dosage form.

[036] The solid oral dosage form may include tablet, coated tablet, powder, powder for reconstitution, pellets, beads, mini-tablet, multilayer tablet, bilayered tablet, tablet-in-tablet, pill, micro-pellet, small tablet unit, multiple unit pellet system, disintegrating tablet, dispersible tablet, granules, microspheres, multiparticulates, capsule, sachet, or sprinkles.

[037] In yet other aspects, the pharmaceutical composition may be formulated for parenteral injection.

[038] In yet other aspects, the pharmaceutical composition may be formulated for for rectal administration.

[039] In other aspects, the disclosure provides a method of treating anemia, including administering a therapeutically effective amount of the pharmaceutical composition to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[040] The disclosure will now be described with reference to the accompanying drawings, which illustrate embodiments of the present disclosure, without limiting the scope thereof, and in which:

[041] FIG. 1 illustrates a column chart showing the effect of Grewia tenax powder on iron absorption inside everted gut sacs of adult male Sprague Dawley rats at different time intervals.

[042] FIG. 2 illustrates a column chart showing the effect Grewia tenax water and ethanol extracts on iron absorption inside everted gut sacs of adult male Sprague Dawley rats at different time intervals.

[043] FIG. 3 illustrates a column chart showing the effect of a composition for treating anemia configured in accordance with embodiments of the present disclosure and its components on iron absorption inside everted gut sacs of adult male Sprague Dawley rats at different time intervals.

[044] FIG. 4 illustrates a column chart showing the effect of a composition for treating anemia configured in accordance with embodiments of the present disclosure and its components on iron serum levels in adult male Sprague Dawley rats.

[045] FIG. 5 illustrates a column chart showing the effect of a composition for treating anemia configured in accordance with embodiments of the present disclosure and its components on serum hepcidin levels in adult male Sprague Dawley rats. [046] FIG. 6 illustrates a column chart showing the effect of a composition for treating anemia configured in accordance with embodiments of the present disclosure and its components on ferritin serum levels in adult male Sprague Dawley rats.

[047] FIG. 7A illustrates a High Performance Liquid Chromatogram of analyzed ascorbic acid.

[048] FIG. 7B illustrates a High Performance Liquid Chromatogram of analyzed Grewia tenax powder.

[049] FIG. 7C illustrates a High Performance Liquid Chromatogram of analyzed Grewia tenax water extract.

[050] FIG. 7D illustrates a High Performance Liquid Chromatogram of analyzed and Grewia tenax ethanol extract.

DETAILED DESCRIPTION

[051] Before the present disclosure is described in more detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[052] Throughout this specification and the claims which follow, unless the context requires otherwise, the words “comprise” and “include”, and variations such as “comprises”, “comprising”,“includes” and“including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integer or step.

[053] In one aspect of the present disclosure, there is provided composition including a plant fruit from Malvaceae family, and an acid.

[054] In embodiments of the present disclosure, the plant may include Grewia tenax. [055] In other embodiments of the present disclosure, the plant fruit may include Grewia tenax dried fruit.

[056] In yet other embodiments of the present disclosure, the plant fruit may include Grewia tenax hydrophilic and hydrophobic extracts.

[057] The composition may include a dose of 1-200 mg/kg Grewia tenax dried fruit.

[058] The acid may be selected from a group including sulfurous acid, sulfuric acid, hyposulfurous acid, persulfuric acid, pyrosulfuric acid, disulfurous acid, dithionous acid, tetrathionic acid, thiosulfurous acid, hydrosulfuric acid, peroxydisulfuric acid, perchloric acid, hydrochloric acid, hypochlorous acid, chlorous acid, chloric acid, hyponitrous acid, nitrous acid, nitric acid, pernitric acid, carbonous acid, carbonic acid, hypocarbonous acid, percarbonic acid, oxalic acid, acetic acid, phosphoric acid, phosphorous acid, hypophosphous acid, perphosphoric acid, hypophosphoric acid, pyrophosphoric acid, hydrophosphoric acid, hydrobromic acid, bromous acid, bromic acid, hypobromous acid, hypoiodous acid, iodous acid, iodic acid, periodic acid, hydroiodic acid, fluorous acid, fluoric acid, hypofluorous acid, perfluoric acid, hydrofluoric acid, chromic acid, chromous acid, hypochromous acid, perchromic acid, hydroselenic acid, selenic acid, selenous acid, hydronitric acid, boric acid, molybdic acid, perxenic acid, silicofluoric acid, telluric acid, tellurous acid, tungstic acid, xenic acid, citric acid, formic acid, pyroantimonic acid, permanganic acid, manganic acid, antimonic acid, antimonous acid, silicic acid, titanic acid, arsenic acid, pertechnetic acid, hydroarsenic acid, dichromic acid, tetraboric acid, metastannic acid, hypooxalous acid, ferricyanic acid, cyanic acid, silicous acid, hydrocyanic acid, thiocyanic acid, uranic acid, diuranic acid, malonic acid, tartartic acid, glutamic acid, phthalic acid, azelaic acid, barbituric acid, benzilic acid, cinnamic acid, fumaric acid, glutaric acid, gluconic acid, hexanoic acid, lactic acid, malic acid, oleic acid, folic acid, propiolic acid, propionic acid, rosolic acid, stearic acid, tannic acid, trifluoroacetic acid, uric acid, ascorbic acid, gallic acid, acetylsalicylic acid, acetic acid, or a combination thereof.

[059] In some embodiments, the composition may include 0.1-40 mg/kg ascorbic acid.

[060] In yet other embodiments, the composition may further include an iron salt

[061] In some embodiments, the composition may include 0.1-5 mg/kg free iron (i.e. iron without salt). [062] In other embodiments, the iron salt may include iron (II) sulfate (FeS0 ₄ ) also known as ferrous sulfate, or ferrous fumarate, ferrous ascorbate, ferrous aspartate, iron glycinate, ferrous gluconate, carbonyl iron or polysaccharide iron complex, or a combination thereof.

[063] In other embodiments, the composition may include additional components such as, drugs, other plant extracts, vitamins, ionic minerals, and other substances purported to be of a health benefit.

[064] The composition in embodiments of the present disclosure may be formulated with or as juice, energy drink, canned gaseous drink, flavored water.

[065] The composition in the present disclosure may also be formulated with fresh, cooked or unprocessed food, chocolates, sweets, jelly beans, gum, snacks, cereals, natural and processed potato or rice chips.

[066] The composition of the embodiments of the present disclosure may be used for the manufacture of a medicament for the treatment of anemia.

[067] The composition of the embodiments of the present disclosure may be used for the manufacture of a medicament for the treatment of Iron Deficiency Anemia.

[068] Embodiments of the present disclosure provides a pharmaceutical composition with natural composition for enhancing iron absorption in healthy and sick humans including geriatrics and pediatrics and in the case of pregnancy.

[069] In some embodiments, the pharmaceutical composition may include the composition described above and a pharmaceutically acceptable carrier and/or excipient.

[070] The term“pharmaceutical acceptable carrier”, as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil; cottonseed oil; safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgement of the formulator.

[071] The pharmaceutical composition of this disclosure can be formulated for oral administration in solid or liquid form, for parenteral injection or for rectal administration. The pharmaceutical composition can be administered to humans and other mammals orally, sublingually, rectally, pareneterally, intracisternally, intraurethrally, intraperitoneally, topically (as powder, ointment or drop), buccally or as an oral or nasal spray. The term "parenterally", as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, subcutaneous, intra-articular injection and infusion.

[072] Pharmaceutical compositions of this disclosure for parenteral injection comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[073] These compositions may also include adjuvants such as preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[074] In some cases, in order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[075] Suspensions, in addition to the active compounds, may contain suspending agents, as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and mixtures thereof.

[076] If desired, and for more effective distribution, the composition can be incorporated into slow-release or targeted-delivery systems such as polymer matrices, liposomes, and microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporation of sterilizing agents in the form of sterile solid compositions, which may be dissolved in sterile water or some other sterile injectable medium immediately before use.

EXAMPLES

[077] The following examples illustrate the present disclosure without, however, limiting the same thereto.

Example 1:

Plant extraction

[078] Grewia tenax ripe fruits Ripe were washed in tap water, and then dried in in a heating oven. The dried fruit, without seeds, were milled in a blender, followed by sieving with 500pm sieves. Samples were taken from the dried fruit powder for quality control and microbial testing. Extraction solvent was added 0.5/L, 30% V/V Ethanol/Water, or distilled water, and left over night. The solvent was filtered through gauze, then froze dried by a freeze dryer. A carrier, Earosol in 20%, was added to the freeze dried powder for protection and ease of handling. Example 2:

In vivo testing

Animals

[079] Adult male Sprauge Dawley (SD) rats with an average weight of 240 ± 20g were used in the experiments. The rats were accommodated at the University of Petra’s Animal House (Amman, Jordan), under controlled temperature (22-24 °C), humidity (55-65%), and a 12-hour light/dark cycle. The rats were offered standard pellet diet (Jordan Food Co. Ltd., Amman, Jordan) and tap water ad libitum.

[080] All animal experiments were performed in compliance with FELASA guidelines (Federation of European Laboratory Animal Science Association).

Everted gut sacs

[081] The rats were fasted for 16 hours (ad libitum access to water) prior to the experiment, anesthetized with light ether, subjected to abdominal section, and exsanguinated by transection of the descending aorta. The whole small intestine was isolated and gently flushed with Krebs buffer. The entire small intestine was removed and everted over a silicone tube. The bottom portion was tied with thread, filled with Krebs buffer, and then segmented into 3cm long sacs. The filled sacs were then placed in lOOmL of Krebs buffer only (control group) or a solution of FeSCL (2 mg/mL) in Krebs buffer or/and solution of FeSCL (2 mg/mL) with Grewia tenax powder (20 mg/ml), solution of FeSCL (2 mg/mL) with ascorbic acid (20 mg/mL), solution of FeSCL (2 mg/mL) with Incubated at 37°C for 15, 30 or 45 minutes. After incubation, the solution on the serosal side (i.e. the outer part of the intestinal wall) of the segment was collected.

Experiment

[082] The animals were assigned into one of seven groups (n=7), all the animals were withdrawn from food and water access for 16 hours prior to the experiment. Group 1 served as control group and received only distilled water, Group 2-7, received FeSCL 20 mg/kg, Group 3 received Grewia tenax powder 20 mg/kg, Group 4 received ascorbic acid 20 mg/kg, Group 5 received Grewia tenax powder and ascorbic acid 20 mg/kg, Group 6 received Grewia tenax extract 20 mg/kg, Group 7 received Grewia tenax extract and ascorbic acid 20 mg/kg. [083] All animals were sacrificed half an hour after receiving their doses, and blood samples were collected from the heart. The samples from each animal were divided into three tubes, one heparinized tube (Becton Dickinson) for CBC analysis, and the other two centrifuged at 6000rpm for 10 minutes. Serum from one tube was analyzed for serum iron. Serum from the second tube was transferred to a -80 _° C freezer and eventually analyzed for ferritin and hepcidin serum levels.

Iron determination

[084] Iron concentration inside the sacs and in serum was measured using commercial Iron- Ferrozine kits.

[085] The absorbance was measured at 546 nm wavelength using a commercial chemistry analyzer.

Ferritin and Hepcidin serum levels analysis

[086] Serum ferritin and hepcidin levels were measured using enzyme-linked immune sorbent assay (ELISA) kits.

CBC Analysis

[087] Whole blood collected from rats in heparinized tube were analyzed using a commercial hematology analyzer.

Example 3:

High Performance Liquid Chromatography (HPLC) analysis of Grewia tenax extract

[088] An instrument equipped with an L-7100 pump, an L-7400 UV diode array detector, an L-7200 Lachronm auto-sampler and a D-7000 interface was used for the analysis. The liquid chromatogram was equipped with 245 nm detector and 250x4.6 mm, C18, 5pm analytical column. The mobile phase was an adjusted purified water to pH 2.2 using phosphoric acid. The flow rate was about 0.7ml/min.

[089] The standard was prepared by adding 50 mg ascorbic acid standard in 50 ml diluent, then dilute 1ml to 200ml using the same diluent. The diluent was prepared by adding 1.76 ml of phosphoric acid (85.0%) and 50 mg Sodium Metabisulfite to 500 ml purified water. [090] Samples were prepared by weighing lg of Grewia tenax powder, Grewia tenax ethanol extract or Grewia tenax water extract. Then dissolved in 50ml diluent, sonicated for 15 minutes, and centrifuged for 10 minutes at 3000rpm. Standard and samples were injected separately and in equal volumes (20m1).

Example 4:

Effect of Grewia tenax powder, and Grewia tenax water and ethanol extracts on iron absorption in vitro

[091] In this example, reference is being made to FIGS. 1 - 3.

[092] Data in FIGS. 1- 3 is presented as mean ± standard error of the mean and assessed by using one-way Analysis of Variance (ANOVA) followed by a Tukey’s test (95% confidence).

[093] Everted Rat Intestinal Sac (ERIS) in vitro method was used to investigate the effect of Grewia tenax powder on iron absorption and transfer and compare absorbed iron concentration versus time. As depicted in FIG. 1, a solution of Grewia tenax powder (20mg/ml) and FeSCE (2mg/ml) have significantly enhanced iron absorption inside the everted gut sacs after 30 minutes of incubation time, compared to FeSCE (2mg/ml) alone.

[094] As illustrated in FIG. 2, a solution of Grewia tenax water or ethanol extracts (20mg/ml) and FeSCE (2mg/ml) have significantly enhanced Iron absorption inside the everted gut sacs in all incubation time intervals, when compared to FeSCE (2mg/ml) alone.

[095] As illustrated in FIG. 3, a solution of Grewia tenax powder and/or ascorbic acid (20mg/ml) and FeSCE (2mg/ml) have significantly enhanced iron absorption inside the everted gut sacs in all incubation time intervals, when compared to FeSCE (2mg/ml) alone.

Example 5:

Effect of Grewia tenax powder and Grewia tenax water and ethanol extracts on iron absorption in vivo

[096] In this example, reference is being made to FIG. 4, wherein the data in such a figure is presented as mean ± standard error of the mean and assessed by using one-way Analysis of Variance (ANOVA) followed by a Tukey’s test (95% confidence). [097] To confirm the in vitro results, an in vivo model was designed by giving iron orally with Grewia tenax powder and/or ascorbic acid. As depicted in FIG. 4, Grewia tenax powder significantly increased iron serum levels after the administration of Grewia tenax , ascorbic acid and their combination while Grewia tenax ethanol extract did not show a similar effect.

Example 6:

Effect of Grewia tenax powder on hepcidin and ferritin serum levels

[098] Reference in this example is made to FIGS. 5 and 6, in which the data is presented as mean ± standard error of the mean and assessed by using one-way Analysis of Variance (ANOVA) followed by a Tukey’s test (95% confidence).

[099] To determine Grewia tenax powder effect on biological parameters that regulate blood iron concentration, serum hepcidin and ferritin levels were tested.

[0100] As depicted in FIG. 5, Grewia tenax powder, ascorbic acid and their combination significantly increased the expression of hepcidin.

[0101] As shown in FIG. 6, Grewia tenax powder, ascorbic acid and their combination significantly increased serum ferritin levels.

Example 7:

Effect of Grewia tenax powder on hematological parameters

[0102] Whole blood screening was performed to determine Grewia tenax powder effect on hematological parameters in rats. The hematological profile is illustrated in Table (1).

Table (1): Hematological profile of rats’ blood.

Wherein

WBC is white blood cell count;

RBC is red blood cell count;

Hb is hemoglobin;

PCV is packed cell volume;

MCV is corpuscular volume; MCH is corpuscular hemoglobin;

MCHC is corpuscular hemoglobin concentration; and

Pit is platelet count.

Example 8:

Identification of ascorbic acid levels in Grewia tenax powder and extracts

[0103] In this example, reference is being made to FIGS. 7A-7D.

[0104] In order to determine if the effect of Grewia tenax powder and extracts on iron absorption is due to an ascorbic acid content, an identification of ascorbic acid content in Grewia tenax powder and extracts by High Performance Liquid Chromatography Ultra Violet (HPLC-UV).

[0105] The amount of ascorbic acid present in Grewia tenax powder, Grewia tenax water extract and Grewia tenax ethanol extract were calculated based on their retention time in HPLC analysis.

[0106] FIG. 7A shows the ascorbic acid standard chromatogram.

[0107] As depicted in FIG. 7B, lOOg of Grewia tenax powder was equivalent to 0.71 mg of ascorbic acid, while lOOg of Grewia tenax water extract and Grewia tenax ethanol extract were equivalent to 1.3mg and 1.4mg of ascorbic acid, respectively, as shown in FIGS. 7C and 7D.

Example 9:

Preparation of a pharmaceutical composition with tablet preparation formula

Table (2): Tablet Preparation Formula

[0108] The active ingredients were weighed and transferred to a suitable-sized polyethylene bag and mixed for 15 minutes. Calculated amount of Mycrocrystalline Celluose and Cross Caramellose were accurately weighed and added to the active ingredients and mixed for another 10 minutes. Later, Aerosil was added to the mixture and mixed for another 10 minutes. Finally, Magnesium Stearate was added to the mixture and mixed for another 5 minutes. The prepared powder was then compressed into tablets.

Example 10:

Preparation of a pharmaceutical composition with tablet preparation formula

Table (3): Tablet Preparation Formula

[0109] The active ingredients were weighed and transferred to a suitable-sized polyethylene bag and mixed for 15 minutes. Calculated amount of Mycrocrystalline Celluose and Cross Caramellose were accurately weighed and added to the active ingredients and mixed for another 10 minutes. Later, Aerosil was added to the mixture and mixed for another 10 minutes. Finally, Magnesium Stearate was added to the mixture and mixed for another 5 minutes. The prepared powder was then compressed into tablets.

Example 11:

Preparation of a pharmaceutical composition with orally disintegrating tablet preparation formula

Table (4): Disintegrating Tablet Preparation Formula

[0110] Accurately weighed amounts of the Grewia tenax powder and Ascorbic acid were dry mixed with Sucrose and Mannitol in a granulator bowl. Ethanol was added to the powder mixer and mixed for 120 seconds. The mixture was then transferred to a Fluid Bed Dryer and granules were prepared and passed through a 2mm sieve. After checking the weight of the produced granules, Aerosil and Magnesium Stearate were mixed with the granules. Finally, the granules were compressed into tablets. Example 12:

Preparation of a pharmaceutical composition with orally disintegrating tablet preparation formula

Table (5): Disintegrating Tablet Preparation Formula

[0111] Accurately weighed amounts of the Grewia tenax powder, Iron (II) sulfate, and Ascorbic acid were dry mixed with Sucrose and Mannitol in a granulator bowl. Ethanol was added to the powder mixer and mixed for 120 seconds. The mixture was then transferred to a Fluid Bed Dryer and granules were prepared and passed through a 2mm sieve. After checking the weight of the produced granules, Aerosil and Magnesium Stearate were mixed with the granules. Finally, the granules were compressed into tablets.

[0112] While embodiments of the present disclosure have been described in detail and with reference to specific embodiments thereof, it will apparent to one skilled in the art that various additions, omissions, and modifications can be made without departing from the spirit and scope thereof.