Encapsulated dihydrofolate formulation for dietary supplementation as effective nutraceutical supplement and method of preparation thereof

Abstract

Disclosed herein are stabilised dihydrofolate formulations intended for use as a dietary supplement. The formulations comprise active dihydrofolate / encapsulated active dihydrofolate, a stabilising agent to prevent degradation, and a carrier medium selected from powders, tablets, or capsules. The innovation provides an effective nutraceutical for oral administration, serving as a potent agent to enhance overall health and support specific bodily functions, especially in individuals with folate deficiency. The formulation remains substantially stable when stored in various temperature conditions (8-50 °C) for an extended period, and methods for preparing these formulations are illustrated.

Description

[0001] ENCAPSULATED DIHYDROFOLATE FORMULATION FOR DIETARY SUPPLEMENTATION AS EFFECTIVE NUTRACEUTICAL SUPPLEMENT AND METHOD OF PREPARATION THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the Dihydrofolate formulation for dietary supplementation as an effective nutraceutical supplement and method of preparation thereof. Particularly, the present invention discloses dihydrofolate formulation for supplementation, comprising dihydrofolate / encapsulated dihydrofolate as an active form with a stabilising agent to protect dihydrofolate from degradation and the carrier medium selected from the group consisting of powders, tablets, and capsules for delivery. More particularly the present invention provides a method of preparation of Dihydrofolate formulation and its utilization for enhancing nutritional and nutraceutical aspects for both humans and animals.

[0004] BACKGROUND OF THE INVENTION

[0005] Folate deficiency can arise from various factors, including inadequate dietary intake, increased demand (during pregnancy, nursing, or alcohol abuse), impaired absorption (celiac disease or sprue), concurrent use of antimetabolites (methotrexate or aminopterin, employed as competitive inhibitors of dihydrofolate reductase during chemotherapy, or sulfonamides, which act as antimetabolites of 4-aminobenzoic acid, thereby inhibiting folate biosynthesis), or genetic anomalies affecting one of the enzymes involved in folate metabolism, such as methylenetetrahydrofolate reductase.

[0006] In light of the aforementioned factors, it is recommended to increase folate intake through dietary means, achieved by fortifying food with folic acid or through oral folic acid supplementation. This recommendation aligns with guidelines set forth by regulatory bodies such as the U.S. Food and Drug Administration (FDA). Folic acid, in this case, is the oxidized form of folates in the form of monoglutamate (N-pteroyl-Lglutamic acid).

[0007] Synthetic folate, used for supplementation as powder, tablet and capsule, is underutilized in humans, as the enzyme dihydrofolate reductase in humans is less efficient (2% of that in comparison to rodents).

[0008] Unmetabolized synthetic folate is reported to be present in the serum of individuals in developed nations where mandatory folate fortification is adopted. Hence, folate fortification is shifting towards the utilization of biological folate derivatives such as 5- methyltetrahydrofolate (5-MTHF) and 5-formyltetrahydrofolate (5-FTHF).

[0009] Patent No.: US 6,921 ,754 B2 and WO 2009 / 103334 pertains to compositions containing folates encompassing dihydrofolate, and their uses, but lacks a stabilisation process.

[0010] Publication US 2015 / 0030676 A1 relates stabilised modified release folic acid derivative composition; detailed explanations are provided exclusively for methyltetrahydrofolate, leaving dihydrofolate and other folate derivatives.

[0011] Dihydrofolate preparations, as mentioned in the patents JP2004269416A, CN115177641A, CN115177644A, CN115177640A, CN115177732A, CN115702906A, and CN115177658A, were made for comparing the stability of dihydrofolate in these preparations with our preparation. No significant improvement in stability with respect to that of our control was observed for dihydrofolate in these preparations. Hence, the stability of dihydrofolate in these prior arts was also significantly lower in comparison to the stabilised forms of dihydrofolate in our preparations.

[0012] Folate derivatives such as dihydrofolate and tetrahydrofolate are comparatively less stable than other biological folate derivatives such as 5-MTHF and 5-FTHF that are used as folate supplements. Hence, dihydrofolate is not utilized for folate fortification purposes.

[0013] OBJECTIVES OF THE INVENTION

[0014] The main objective of the present invention is to provide a stabilised dihydrofolate formulation for dietary supplementation.

[0015] Another objective of the present invention is to provide a method of preparation for stabilised encapsulated dihydrofolate formulation that ensures an adequate supply of bioavailable folate in humans and animals.

[0016] Yet another objective of the present invention is to provide stabilised dihydrofolate formulation without encapsulation which ensures an adequate supply of bioavailable folate in humans and animals.

[0017] Yet another objective of the present invention is to provide stabilised dihydrofolate formulation for supplementation, comprising of dihydrofolate as an active form either in encapsulated form or with a stabilising agent to protect dihydrofolate from degradation and the carrier medium selected from the group consisting of powders, tablets, and capsules. Still another objective of the present invention is to replace synthetic folate with the stabilised dihydrofolate in the fortification program.

[0018] Still another objective of the present invention is to provide a method of utilization for enhancing nutritional and nutraceutical aspects for both humans and animals.

[0019] SUMMARY OF THE INVENTION

[0020] Accordingly the present invention provides a dihydrofolate formulation for dietary supplementation comprising a) dihydrofolate (0.005-32.25% w / w), b) stabilising agents selected from ascorbic acid (0.0006-5.88% w / w), sodium copper chlorophyllin (0.0104-66.78% w / w), c) an encapsulating agent polyethylene glycol 4000 (0.00-54.04% w / w), d) additives selected from dicalcium phosphate (0.00-99.98% w / w), microcrystalline cellulose (0.00-5% w / w), sodium starch glycolate (0.00-6% w / w), and magnesium stearate (0.00-1% w / w), wherein the said formulation is delivered through a carrier medium selected from the group consisting of powders, tablets, and capsules.

[0021] In an embodiment, the present invention provides a method of preparation of stabilised and encapsulated dihydrofolate formulation as claimed in claim 1, wherein the said method comprises steps, i. dissolving dihydrofolate (1-1.2 g / 10 mL) in an aqueous medium at a pH of 10; adding ascorbic acid (0.1-0.5% w / v) to the solution under subdued light and at a temperature of 8 °C and adding sodium copper chlorophyllin (2.00-2.5% w / v) into the solution under continuous stirring at 600 rpm to form a uniform dihydrofolate solution, ii. removing solvent from dihydrofolate solution obtained in step (i) by a rotary evaporator to obtain a dihydrofolate formulation powder, iii. mixing polyethylene glycol 4000 in acetone to obtain a PEG solution, iv. adding dihydrofolate solution obtained in step (i) to PEG solution of step (iii) dropwise while stirring with a homogenizer, followed by removing solvent from the mixture by a rotary evaporator to obtain a suspension, v. washing the suspension of step (iv) with water followed by water removal by centrifugation to obtain encapsulated suspension, vi. the encapsulated suspension of step (v) was freeze-dried to obtain the encapsulated dihydrofolate formulation powder, vii. mixing the dihydrofolate formulation powder (0.016-15.5% w / w) of step (ii) with dicalcium phosphate, microcrystalline cellulose (2-5% w / w), and sodium starch glycolate (4-6% w / w), followed by blending to achieve uniform mixing, followed by adding lubricant magnesium stearate (0.5-1% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight, viii. mixing encapsulated dihydrofolate formulation powder (0.025-20.5% w / w) of step (vi) with dicalcium phosphate, microcrystalline cellulose (2-5% w / w), and sodium starch glycolate (4-6% w / w), followed by blending to achieve uniform mixing, to obtain a mixture, followed by adding lubricant magnesium stearate (0.5-1% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight, ix. alternatively dihydrofolate formulation powder (0.016-15.5% w / w) of step (ii) and encapsulated dihydrofolate formulation powder (0.025-20.5% w / w) of step (vi) were mixed with dicalcium phosphate (79.5-99.98% w / w), and filled in gelatinous or non- gelatinous capsules of step (i) were filled with a capsule-filling machine to obtain capsules of 0.5-1 g weight.

[0022] In another embodiment, of present invention the Tablets obtained in step viii comprise active ingredient dihydrofolate in the range of 0.005-5% w / w and the tablets are configured with a diameter ranging from 0.2 to 1".

[0023] In yet another embodiment of present invention the capsules obtained in step ix comprise active ingredient dihydrofolate is in the range of 0.005-5% w / w and the capsule size ranges from 0.95-1.37 mL.

[0024] In still another embodiment of present invention the tablets have the hardness in Newton is in the range of 100 to 200 and the friability in the percentage is in the range of 0.5 to 0.8. In still another embodiment of present invention the said formulation is in stable form for consumption of for humans and animals and has an extended shelf life at temperatures in the range of 8 °C to 50 °C when stored in photoprotective amber bottles.

[0025] ABBREVIATIONS USED:

[0026] AA: ascorbic acid

[0027] PEG: polyethylene glycol 4000

[0028] DHF: dihydrofolate

[0029] DHF-P: dihydrofolate powder mixture

[0030] DHF-E: dihydrofolate-encapsulated powder mixture

[0031] EE: encapsulation efficiency

[0032] SSG: sodium starch glycolate

[0033] MCC: microcrystalline cellulose

[0034] MS: magnesium stearate

[0035] ANOVA: Analysis of Variance

[0036] AOAC: Association of Official Analytical Chemists

[0037] ATCC: American Type Culture Collection

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows the optimization of the dihydrofolate encapsulation by response surface methodology

[0040] Figure 2 illustrates the storage stability of dihydrofolate

[0041] Figure 3 explains the optimization of the dihydrofolate tablet preparation by response surface methodology

[0042] Figure 4 illustrates the storage stability of dihydrofolate tablets

[0043] Figure 5 shows the storage stability of dihydrofolate capsules

[0044] DETAIL DESCRIPTION OF THE INVENTION

[0045] The invention aimed to provide stabilised dihydrofolate formulation as a dietary supplement. The stabilised form of dihydrofolate formulation for supplementation, comprising of dihydrofolate as an active form with a stabilising agent to protect dihydrofolate from degradation and the carrier medium selected from the group consisting of powders, tablets, and capsules. The present invention also aims to provide an effective nutraceutical for oral administration in the form of dihydrofolate formulation as an effective agent for improving overall health and supporting specific bodily functions, particularly for subjects with folate deficiency.

[0046] The present invention provides dihydrofolate formulations for dietary supplementation comprising a) dihydrofolate (0.005-32.25% w / w) (0.005 % w / w of dihydrofolate as reflected in final capsules / tablets 30-32.25% w / w of dihydrofolate as reflected in powder formulation) b) stabilising agents ascorbic acid (0.0006-5.88% w / w), sodium copper chlorophyllin

[0047] (0.0104-66.78% w / w), (0.0006 % w / w of ascorbic acid as reflected in final capsules / tablets 2.43-5.88% w / w of ascorbic acid as reflected in powder formulation) c) an encapsulating agent polyethylene glycol 4000 (0.00-54.04% w / w), d) additives dicalcium phosphate (0.00-99.98% w / w), microcrystalline cellulose (0.00- 5% w / w), sodium starch glycolate (0.00-6% w / w) and magnesium stearate (0.00-1 % w / w). wherein the said formulation is delivered through a carrier medium selected from the group consisting of powders, tablets, and capsules.

[0048] One of the stabilised dihydrofolate powder formulation for supplementation comprises dihydrofolate as an active form (30-32.25% w / w), ascorbic acid (3.22-4.24% w / w) and sodium copper chlorophyllin (63.51-66.78% w / w) as stabilising agents to protect dihydrofolate from degradation.

[0049] In another embodiment wherein the formulation is an encapsulated powder formulation consisting of dihydrofolate (20-24.39% w / w), ascorbic acid (2.43-5.88% w / w) and sodium copper chlorophyllin (23.53—48.78% w / w) wherein polyethylene glycol 4000 (20.95-54.04% w / w) as the encapsulation agent.

[0050] The carrier medium is a tablet consisting of stabilised dihydrofolate formulation (0.016- 15.50% w / w) dicalcium phosphate (77.50-88.484% w / w), microcrystalline cellulose (2-5% w / w), sodium starch glycolate (4-6% w / w) and magnesium stearate (0.5-1% w / w). The carrier medium is a tablet consisting of encapsulated dihydrofolate formulation (0.025- 20.5% w / w) dicalcium phosphate (67.50-88.475% w / w), microcrystalline cellulose (2-5% w / w), sodium starch glycolate (4-6% w / w) and magnesium stearate (0.5-1% w / w).

[0051] The carrier medium is a gelatinous or non-gelatinous capsule consisting of stabilised dihydrofolate formulation (0.016-15.50% w / w), and dicalcium phosphate (84.50-99.98% w / w).

[0052] The carrier medium is a gelatinous or non-gelatinous encapsulated capsule consisting of encapsulated dihydrofolate formulation (0.025-20.5% w / w) and dicalcium phosphate (79.5- 99.98% w / w).

[0053] The stabilised form of dihydrofolate can be used for direct consumption or as an additive to dietary components, an additive to processed foods, incorporated into health drinks, breakfast cereals and other edible substances and supplements. The dihydrofolate formulation mixture collectively contributes to an extended shelf life, ensuring the long-term integrity and efficacy of the supplemented dihydrofolate formulation.

[0054] The said dihydrofolate formulation is for preventing or treating a folate deficiency in a human or animal, which comprises administering an effective amount of the composition to said human or animal. The formulation is effective for infants, children, men and women and the said dihydrofolate formulation is a replacement of synthetic folic acid fortification for treating folate deficiency.

[0055] Encapsulation of dihydrofolate

[0056] A response surface methodology using a central composite design following the face- centred model was used to design the optimization experiments for dihydrofolate nanoencapsulation.

[0057] Ascorbic acid (0.1-0.5% w / v), polyethylene glycol 4000 (PEG; 1-5% w / v) and sodium copper chlorophyllin (2-2.5% w / v) were used for the optimization reactions. All the experiments were conducted in a cold room set at 8 °C and under subdued light conditions. Dihydrofolate (1- 1.2 g) was solubilized in water (10 mL, pH 10), and ascorbic acid was added. To this, sodium copper chlorophyllin was added and continued stirring until mixed in the solution. A PEG solution was prepared by mixing it with acetone. The dihydrofolate solution was added dropwise into the PEG solution while stirring with a homogenizer. Later, a rotary evaporator was used to remove the solvent from the mixture. The resulting suspension was washed with water to remove the solvent, and excess water was removed by centrifugation. The optimization of the amount of the ingredients was determined by the RSM study. The encapsulated suspension was freeze-dried and used as the dihydrofolate supplement for the fortification, tablet, and capsule-making studies.

[0058] The stability of dihydrofolate in the final products was analysed by the microbiological assay using Lactobacillus rhamnosus (ATCC 7469) according to the AOAC method (AOAC Official Methods 944.12, 960.46). The assay was used as it is sensitive at picogram levels, and the degradation of dihydrofolate while sample preparation for other methods such as HPLC can be avoided.

[0059] The stability of dihydrofolate under various storage conditions involving temperature and time duration was also analysed by this method. The encapsulated dihydrofolate was dissolved in an acidic acetone solution (containing 0.1% HCI) to solubilize the PEG. The precipitated dihydrofolate was removed after centrifugation and used for quantification. A formulation of dihydrofolate containing only ascorbic acid and sodium copper chlorophyllin at the optimization values suggested was made and used for the storage stability studies as well. Dihydrofolate with 0.1% ascorbic acid was served as the control.

[0060] Dihydro folate formulation tablet making

[0061] To make the dihydrofolate tablets, the stabilised dihydrofolate powder formulation (0.016- 15.5% w / w) or the encapsulated dihydrofolate powder formulation (0.025-20.5% w / w) was mixed with dicalcium phosphate, microcrystalline cellulose (2-5% w / w), and sodium starch glycolate (4-6% w / w). After blending to achieve uniform mixing, the lubricant magnesium stearate (0.5-1% w / w) was added and mixed thoroughly. The final weight of the tablets was 0.5-1 g. The mixtures were loaded into a tablet-making machine, and the settings were optimized to obtain the desired tablet characteristics. The tablets were further analysed for their hardness and friability. RSM study was used to optimize the amount of the ingredients.

[0062] Tablets made with only dihydrofolate mixed with an appropriate quantity of dicalcium phosphate and 0.1% ascorbic acid were served as controls.

[0063] The stability of dihydrofolate during storage in the tablets was analysed by the microbiological assay using Lactobacillus rhamnosus (ATCC 7469) as the test organism according to the AOAC protocol. Tablets with the formulation of dihydrofolate, devoid of encapsulation but containing ascorbic acid, sodium copper chlorophyllin, dicalcium phosphate, microcrystalline cellulose, sodium starch glycolate and magnesium stearate at the optimization values suggested were made and used for the storage stability studies as well. Tablets with dihydrofolate, dicalcium phosphate, microcrystalline cellulose, sodium starch glycolate and magnesium stearate served as the control. Tablets were made with varying content of dihydrofolate (0.050-50 mg). Tablets containing 1 mg of dihydrofolate were used for the stability studies.

[0064] Dihydrofolate formulation capsule making

[0065] The dihydrofolate formulation powder (0.016-15.5% w / w) or the dihydrofolate-encapsulated formulation powder (0.025-20.5% w / w) were mixed with dibasic calcium phosphate. The mixture was blended thoroughly for uniform distribution of the particles. The capsules were filled with a capsule-filling machine. The final weight of the capsules was 0.5-1 g.

[0066] The stability of dihydrofolate during storage in the capsules was analysed by the microbiological assay using Lactobacillus rhamnosus (ATCC 7469) as the test organism according to the AOAC protocol. Capsules with the formulation of di hydrofol ate, devoid of encapsulation but containing ascorbic acid, sodium copper chlorophyllin, and dicalcium phosphate derived from the optimization study were made and used for the storage stability studies as well. Capsules with only dihydrofolate, 0.1% ascorbic acid and an appropriate quantity of dicalcium phosphate served as the control. Capsules were made with varying content of dihydrofolate (0.050-50 mg). Capsules containing 1 mg of dihydrofolate were used for the stability studies.

[0067] The definitions for the following terms will be used throughout the application:

[0068] The term “dihydrofolate” used herein is the natural form of vitamin folate, which is generally produced as an intermediary molecule in the folate metabolism pathway.

[0069] The compound discussed in this embodiment can be created from synthetic folate and can be made using methods familiar to those with expertise in the field, as outlined in the current information provided and the methods described in international publications.

[0070] Formulations described herein are stabilised nutritional composition for supplementation that includes the active ingredient as dihydrofolate, ascorbic acid and sodium copper chlorophyllin as the stabilising agent, a carrier medium to protect dihydrofolate from degradation and a carrier medium selected from the group consisting of powders, tablets, and capsules.

[0071] In the stabilised dihydrofolate formulation, the carrier medium can include encapsulated powder, tablet, capsule, encapsulated tablet and encapsulated capsule. Nutraceutical agent, the active molecule / compound / drug / nutraceutical / medication disclosed by reference herein is a chemical substance that is used to diagnose, cure, treat, or prevent a disease or medical condition.

[0072] In a preferred embodiment, the nutraceutical agent dihydrofolate can be administered in multiple doses daily.

[0073] The total molar amount of the dihydrofolate present in the composition can be between 5% and 200% of a human daily requirement for folate per a customarily consumed quantity of the composition.

[0074] The preferred doses of dihydrofolate described range from 50 to 1000 pg with optimal doses being between 300 and 500 pg.

[0075] In embodiments disclosed herein, solid dosage forms, comprising tablets, capsules, powders, and granules, may be equipped with coatings, including enteric coatings and other coatings known in the pharmaceutical formulating art. These solid dosage forms may be formulated to release the active ingredient(s) selectively in a particular part of the intestinal tract, potentially in a delayed manner. Compositions suitable for embedding, such as polymeric substances and waxes, can be utilized.

[0076] Additionally, the solid dosage form may undergo coating processes to mask or enhance taste, improve appearance, or modify the release rate.

[0077] Tablets may comprise a quantity varying from 0.3 to 2g, with a preferred range of 0.4 to 1g and a more preferred range of 0.5 to 1g. The tablets are configured with a diameter ranging from 0.2 to 1", with a preference for 0.8 to 1" and a more specific preference for 0.4 to 0.6".

[0078] Capsules may comprise a quantity varying from 0.3 to 2g, with a preferred range of 0.4 to 1 g and a more preferred range of 0.5 to 0.8 g. The tablets are configured with a diameter ranging from 0.2 to 1", with a preference for 0.8 to 1" and a more specific preference for 0.4 to 0.6".

[0079] The number of tablets or capsules per serving to deliver sufficient quantity of dihydrofolate is .

[0080] EXAMPLES

[0081] The following examples are given by way of illustration and, therefore, should not be construed to limit the scope of the invention. Example 1

[0082] A method of preparation of stabilised and encapsulated dihydrofolate formulation wherein the said method comprises step (i) dissolving dihydrofolate ( 1g / 10 mL) in an aqueous medium at a pH of 10; adding ascorbic acid (0.1% w / v) to the solution under subdued light and at a temperature of 8 °C and adding sodium copper chlorophyllin (2.00% w / v) into the solution under continuous stirring at 600 rpm to form a uniform dihydrofolate solution. Followed by step (ii) removing solvent from dihydrofolate solution obtained above by a rotary evaporator to obtain a dihydrofolate formulation powder. Step (iii) PEG solution is obtained by mixing polyethylene glycol 4000 in acetone. Followed by step (iv) adding dihydrofolate solution obtained to PEG solution dropwise while stirring with a homogenizer, and removing solvent from the mixture by a rotary evaporator to obtain a suspension and washing the suspension of step (iv) with water and water removal by centrifugation to obtain encapsulated suspension. The encapsulated suspension was freeze-dried to obtain the encapsulated dihydrofolate formulation powder.

[0083] Example 2

[0084] The dihydrofolate formulation powder was made into capsuled by mixing the dihydrofolate powder (0.016 % w / w) with dicalcium phosphate, microcrystalline cellulose (2% w / w), and sodium starch glycolate (4% w / w), followed by blending to achieve uniform mixing, followed by adding lubricant magnesium stearate (0.5% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight,

[0085] The encapsulated dihydrofolate formulation powder (0.025% w / w) was mixed with dicalcium phosphate, microcrystalline cellulose (2% w / w), and sodium starch glycolate (4% w / w), followed by blending to achieve uniform mixing, to obtain a mixture, followed by adding lubricant magnesium stearate (0.5% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight, alternatively dihydrofolate formulation powder (0.016% w / w) and encapsulated dihydrofolate formulation powder (0.025% w / w) were mixed with dicalcium phosphate (79.5% w / w), and filled in gelatinous or non-gelatinous capsules of step (i) were filled with a capsule-filling machine to obtain capsules of 0.5-1 g weight.

[0086] Example 3 A method of preparation of stabilised and encapsulated dihydrofolate formulation wherein the said method comprises step (i) dissolving dihydrofolate ( 1.2 g / 10 mL) in an aqueous medium at a pH of 10; adding ascorbic acid (0.5% w / v) to the solution under subdued light and at a temperature of 8 °C and adding sodium copper chlorophyllin (2.5% w / v) into the solution under continuous stirring at 600 rpm to form a uniform dihydrofolate solution. Followed by step (ii) removing solvent from dihydrofolate solution obtained above by a rotary evaporator to obtain a dihydrofolate formulation powder. Step (iii) PEG solution is obtained by mixing polyethylene glycol 4000 in acetone. Followed by step (iv) adding dihydrofolate solution obtained to PEG solution dropwise while stirring with a homogenizer, and removing solvent from the mixture by a rotary evaporator to obtain a suspension and washing the suspension of step (iv) with water and water removal by centrifugation to obtain encapsulated suspension. The encapsulated suspension was freeze-dried to obtain the encapsulated dihydrofolate formulation powder.

[0087] Example 4

[0088] The dihydrofolate formulation powder was made into capsuled by mixing the dihydrofolate powder (15.5 % w / w) with dicalcium phosphate, microcrystalline cellulose (5 % w / w), and sodium starch glycolate (6% w / w), followed by blending to achieve uniform mixing, followed by adding lubricant magnesium stearate (1 % w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight,

[0089] The encapsulated dihydrofolate formulation powder (20.5% w / w) was mixed with dicalcium phosphate, microcrystalline cellulose (5% w / w), and sodium starch glycolate (4% w / w), followed by blending to achieve uniform mixing, to obtain a mixture, followed by adding lubricant magnesium stearate (1% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight, alternatively dihydrofolate formulation powder (0.15.5% w / w) and encapsulated dihydrofolate formulation powder (20.5% w / w) were mixed with dicalcium phosphate (99.98% w / w), and filled in gelatinous or non-gelatinous capsules of step (i) were filled with a capsule-filling machine to obtain capsules of 0.5-1 g weight.

[0090] Example 5.

[0091] Encapsulation of dihydrofolate optimization using response surface methodology. Varying concentrations of polyethylene glycol 4000 (PEG, 1-5%) and ascorbic acid (AA, 0.1-0.5%) served as the factors. The encapsulation efficiency (EE) and dihydrofolate stability after encapsulation were analysed as responses.

[0092] Table 1 shows the central composite experimental design for the encapsulation of the dihydrofolate

[0093] Example 6

[0094] The dihydrofolate (DHF), along with sodium copper chlorophyll and ascorbic acid was encapsulated in polyethylene glycol 4000 (PEG). The percentage encapsulation efficiency and percentage stability of dihydrofolate in the final product were estimated. Table 2 shows the analysis of variance (ANOVA) for the encapsulation of di hydrofol ate.

[0095] The factors are polyethylene glycol 4000 (PEG) and ascorbic acid (AA).aThe correlation coefficient (r2) of the Encapsulation efficiency (EE) is 97.91bThe correlation coefficient (r2) of the dihydrofolate stability (DHF) is 91 .73

[0096] Example 7

[0097] The surface plots illustrate the effect of factors AA and PEG on the responses EE and dihydrofolate stability.

[0098] Figure 2 shows the optimization of the dihydrofolate encapsulation by response surface methodology. A dihydrofolate mixture containing sodium copper chlorophyllin was mixed with ascorbic acid (AA, 0.1-0.5%) and encapsulated in polyethylene glycol 4000 (PEG, 1- 5%). Encapsulation efficiency (EE) and dihydrofolate retention of the final products were analysed. A. Surface plot showing the effect of AA and PEG on encapsulation efficiency. B. Surface plot showing the effect of AA and PEG on dihydrofolate retention. C. The optimization plot shows a value of 5% PEG and 0.10% AA as the optimum values for dihydrofolate encapsulation.

[0099] Example 8

[0100] The optimization plot has shown a value of 5% PEG and 0.1% AA for obtaining the maximum desired values for EE and dihydrofolate stability.

[0101] Figure 3 shows the effect of storage on the stability of dihydrofolate. Dihydrofolate with 0.1% ascorbic acid (DHF), and dihydrofolate mixture with ascorbic acid and sodium copper chlorophyllin (DHF-P) and dihydrofolate mixture with ascorbic acid and sodium copper chlorophyllin encapsulated in polyethylene glycol 4000 (DHF-E) were stored for up to three months in various temperature conditions (-20, 8, 23 and 50 °C) in amber bottles. The content of dihydrofolate was estimated at one-month intervals. The percentage stability was calculated. The values represent mean ± 3 with S.D. The significance was analysed by oneway ANOVA followed by Tukey’s post hoc test. * represent a significant difference between DHF and DHF-P or DHF-E.#represents a significant difference between DHF-P and DHF-E. * and * P < 0.05, ** and " P < 0.01 , *** and “ P < 0.001 , **** and ™ p < 0.0001.

[0102] Example 9

[0103] The response surface methodology used for the optimization of the tablet-making showed that the factors, different concentrations of microcrystalline cellulose (MCC), sodium starch glycolate (SSG) and magnesium stearate (MS) affect the responses, hardness and friability.

[0104] Table 3 shows the central composite experimental design for the dihydrofolate tablet optimization Example 10

[0105] The dihydrofolate tablets were made with dicalcium phosphate as the filler along with different concentrations of microcrystalline cellulose (MCC), sodium starch glycolate (SSG) and magnesium stearate (MS). The hardness in Newton and the friability in the percentage of the tablets are shown.

[0106] Table 4 shows the analysis of variance (ANOVA) of the responses, hardness and friability as a function of the factors, microcrystalline cellulose (MCC), sodium starch glycolate (SSG) and magnesium stearate (MS).

[0107] The factors are microcrystalline cellulose (MCC), sodium starch glycolate (SSG) and magnesium stearate (MS). aThe correlation coefficient (r2) for the Hardness model is 99.47 bThe correlation coefficient (r2) for the Friability model is 90.68 Example 11

[0108] The surface plots show the influence of the factors on friability.

[0109] Figure 4 shows the optimization of the dihydrofolate tablet preparation by response surface methodology. Dihydrofolate with ascorbic acid and sodium copper chlorophyllin encapsulated in polyethylene glycol 4000 mixed with dicalcium phosphate was used for making tablets with microcrystalline cellulose (MCC, 5-7%), sodium starch glycolate (SSG, 4-6%) and magnesium stearate (MS, 0.5-1%). A. Surface plot showing the effect of MCC and SSG on friability. B. Surface plot showing the effect of MS and SSG on friability. C. Surface plot showing the effect of MS and MCC on friability. D. Surface plot showing the effect of MCC and SSG on hardness. E. Surface plot showing the effect of MCC and MS on hardness. F. Surface plot showing the effect of SSG and MS on hardness. G. The optimization plot shows the values of 7% for MCC, 4% for SSG and 0.5% for MS for the preparation of dihydrofolate tablets.

[0110] Example 120

[0111] Storage stability studies of dihydrofolate tablet and capsule.

[0112] Figure 5 shows the effect of storage on the stability of dihydrofolate tablets. Tablets were made with dihydrofolate mixed with ascorbic acid, dicalcium phosphate, microcrystalline cellulose, sodium starch glycolate and magnesium stearate (DHF), dihydrofolate mixed with ascorbic acid, sodium copper chlorophyllin, dicalcium phosphate, microcrystalline cellulose, sodium starch glycolate and magnesium stearate (DHF-P) and dihydrofolate mixture with ascorbic acid and sodium copper chlorophyllin encapsulated in polyethylene glycol 4000, dicalcium phosphate, microcrystalline cellulose, sodium starch glycolate and magnesium stearate (DHF-E) were stored for up to three months in various temperature conditions (-20, 8, 23 and 50 °C) in amber bottles. The content of dihydrofolate was estimated at one-month intervals. The percentage stability was calculated. The values represent mean ± 3 with S.D. The significance was analysed by one-way ANOVA followed by Tukey’s post hoc test. * represent a significant difference between DHF and DHF-P or DHF-E.#represents a significant difference between DHF-P and DHF-E. * and * P < 0.05, ** and * P < 0.01 , *** and “ P < 0.001, **** and ™ p < 0.0001.

[0113] Figure 6 shows the effect of storage on the stability of dihydrofolate capsules. Capsules were filled with dihydrofolate mixed with ascorbic acid and dicalcium phosphate (DHF), dihydrofolate mixed with ascorbic acid, sodium copper chlorophyllin and dicalcium phosphate (DHF-P) and dihydrofolate mixture with ascorbic acid and sodium copper chlorophyllin encapsulated in polyethylene glycol 4000 and dicalcium phosphate (DHF-E) were stored for up to three months in various temperature conditions (-20, 8, 23 and 50 °C) in amber bottles. The content of dihydrofolate was estimated at one-month intervals. The percentage stability was calculated. The values represent mean ± 3 with S.D. The significance was analysed by one-way ANOVA followed by Tukey’s post hoc test. * represent a significant difference between DHF and DHF-P or DHF-E.#represents a significant difference between DHF-P and DHF-E. * and * P < 0.05, ** and * P < 0.01 , *** and “ P < 0.001, 0.0001.

[0114] ADVANTAGES OF INVENTION

[0115] 1. The invention provides stabilised dihydrofolate formulation for supplementation in the form of powders, tablets, and capsules.

[0116] 2. The stabilised form of dihydrofolate can be used for direct consumption or as an additive to dietary components, as an additive to processed foods, incorporated into health drinks and breakfast cereals, and as an additive to other edible substances and supplements.

[0117] 3. The dihydrofolate formulation mixture collectively contributes to an extended shelf life, ensuring the long-term integrity and efficacy of the supplemented dihydrofolate formulation.

[0118] 4. The said dihydrofolate formulation is for preventing or treating a folate deficiency in a human or animal, which comprises administering to said human or animal an effective amount of the composition.

[0119] 5. The formulation is effective for infants, children, men and women, and the said dihydrofolate formulation is a replacement of synthetic folic acid fortification for treating folate deficiency.

Claims

We claim,1. A dihydrofolate formulation for dietary supplementation comprising a) dihydrofolate (0.005-32.25% w / w), b) stabilising agents selected from ascorbic acid (0.0006-5.88% w / w), sodium copper chlorophyllin (0.0104-66.78% w / w), c) an encapsulating agent polyethylene glycol 4000 (0.00-54.04% w / w), d) additives selected from dicalcium phosphate (0.00-99.98% w / w), microcrystalline cellulose (0.00-5% w / w), sodium starch glycolate (0.00-6% w / w), and magnesium stearate (0.00-1% w / w), wherein the said formulation is delivered through a carrier medium selected from the group consisting of powders, tablets, and capsules.

2. A method of preparation of stabilised and encapsulated dihydrofolate formulation as claimed in claim 1, wherein the said method comprises steps, i. dissolving dihydrofolate (1-1.2 g / 10 mL) in an aqueous medium at a pH of 10; adding ascorbic acid (0.1-0.5% w / v) to the solution under subdued light and at a temperature of 8 °C and adding sodium copper chlorophyllin (2.00-2.5% w / v) into the solution under continuous stirring at 600 rpm to form a uniform dihydrofolate solution, ii. removing solvent from dihydrofolate solution obtained in step (i) by a rotary evaporator to obtain a dihydrofolate formulation powder, iii. mixing polyethylene glycol 4000 in acetone to obtain a PEG solution, iv. adding dihydrofolate solution obtained in step (i) to PEG solution of step (iii) dropwise while stirring with a homogenizer, followed by removing solvent from the mixture by a rotary evaporator to obtain a suspension, v. washing the suspension of step (iv) with water followed by water removal by centrifugation to obtain encapsulated suspension, vi. the encapsulated suspension of step (v) was freeze-dried to obtain the encapsulated dihydrofolate formulation powder,vii. mixing the dihydrofolate formulation powder (0.016-15.5% w / w) of step (ii) with dicalcium phosphate, microcrystalline cellulose (2-5% w / w), and sodium starch glycolate (4-6% w / w), followed by blending to achieve uniform mixing, followed by adding lubricant magnesium stearate (0.5-1% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight, viii. mixing encapsulated dihydrofolate formulation powder (0.025-20.5% w / w) of step (vi) with dicalcium phosphate, microcrystalline cellulose (2-5% w / w), and sodium starch glycolate (4-6% w / w), followed by blending to achieve uniform mixing, to obtain a mixture, followed by adding lubricant magnesium stearate (0.5-1% w / w) and loading in tablet making machines to obtain tablets of 0.5-1 g weight, ix. alternatively dihydrofolate formulation powder (0.016-15.5% w / w) of step (ii) and encapsulated dihydrofolate formulation powder (0.025-20.5% w / w) of step (vi) were mixed with dicalcium phosphate (79.5-99.98% w / w), and filled in gelatinous or non- gelatinous capsules of step (i) were filled with a capsule-filling machine to obtain capsules of 0.5-1 g weight.

3. The method of preparation of dihydrofolate formulation as claimed in claim 2, wherein the Tablets obtained in step viii comprise active ingredient, dihydrofolate in the range of 0.005-5% w / w and the tablets are configured with a diameter ranging from 0.2 to 1".

4. The method of preparation of dihydrofolate formulation as claimed in claim 2, wherein the capsules obtained in step ix comprise active ingredient dihydrofolate in the range of 0.005-5% w / w and the capsule size ranges from 0.95-1.37 mL.

5. The dihydrofolate formulation as claimed in claim 1, wherein tablets have the hardness in Newton is in the range of 100 to 200 and the friability in the percentage is in the range of 0.5 to 0.8.

6. The dihydrofolate formulation as claimed in claim 1 , wherein the said formulation is in stable form for consumption of for humans and animals and has an extended shelf life at temperatures in the range of 8 °C to 50 °C when stored in photoprotective amber bottles.

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