A bioactive peptide composition

Abstract

The present invention is related to a bioactive peptide composition More particularly, the present invention is particularly related to a bioactive peptide composition comprising Casein phosphopeptides, Isoleucyl-prolyl-proline and Valyl-prolyl-proline and one or more inactive excipients and process for preparing the same. The invention intended to enhance calcium absorption or improve calcium incorporation into bones.

Description

[0001] A BIOACTIVE PEPTIDE COMPOSITION

[0002] FIELD OF THE INVENTION

[0003] The present invention is related to a bioactive peptide composition. More particularly, the present invention is related to a bioactive peptide composition comprising Casein phosphopeptides, Lactotripeptides and one or more inactive excipients and process for preparing the same. The invention intended to synergistically enhance calcium absorption or improve calcium incorporation into bones.

[0004] BACKGROUND OF THE INVENTION

[0005] Bone is a strong tissue in the body made mostly of calcium. Calcium helps bones stay tough and keeps their shape. It's important to get enough calcium from your diet for bones to grow well and stay healthy, especially as you get older.

[0006] Calcium, a crucial mineral, is essential for numerous biological processes and plays a vital role. One of its main function is to help make bones strong. More than 99% of the calcium in body is found in bones, where it combines with phosphate to form a sturdy mineral. This gives its shape and toughness to skeleton, and also acts as a storage place for calcium that body can use for different functions.

[0007] Additionally, calcium is essential for the formation and upkeep of robust teeth and plays crucial roles in muscle function, nerve transmission, blood clotting, and numerous other essential bodily processes. The body necessitates significant amounts of calcium, with about 99% stored in bones and teeth, and the remaining 1% dedicated to critical physiological functions. Adequate calcium intake is crucial for proper muscle function, encompassing the essential processes of muscle contraction and relaxation.

[0008] Calcium plays a pivotal role in the body, being essential for muscle function, oocyte activation, maintaining bone and teeth strength, blood clotting, nerve signaling, regulating heartbeat, and managing cellular fluid balance. Its importance is heightened during periods of growth, pregnancy, and breastfeeding. Prolonged deficiency in calcium can lead to osteoporosis, a condition that intensifies the risk of bone fractures due to bone weakening over time.

[0009] Insufficient calcium levels can lead to various significant health concerns, including osteoporosis, fractures, dental issues, muscle cramps, tingling sensations, fatigue, heightened risk of heart disease, and alterations in mood. Several factors, such as low solubility, interactions with other nutrients, elevated parathyroid hormone levels, vitamin D deficiency, and aging, can impede calcium absorption. This inadequate absorption hampers the body's ability to maintain proper calcium levels in the bloodstream, underscoring the necessity for improved approaches to enhance calcium absorption.

[0010] To get enough calcium, it's best to eat foods that are rich in calcium. Different foods have different amounts of calcium, so it's important to choose ones that are high in calcium. Good sources of calcium include dairy products like milk and cheese, almonds, broccoli, kale, canned salmon (with bones), sardines, and soy products like tofu.

[0011] Not all ingested calcium is absorbed by the body. The absorption process occurs primarily in the small intestine, located immediately after the stomach in the digestive system. From there, calcium is transported via the bloodstream to bones and other tissues. Several factors influence how much calcium is absorbed like existing levels of calcium in the bloodstream, the form of calcium consumed, whether from dietary sources or supplements, and conditions within the small intestine, which affect absorption efficiency.

[0012] Excessive intake of calcium, coupled with poor absorption, can create a series of issues including deficiencies in vital minerals like zinc, copper, and magnesium. This interference in absorption can also trigger gastrointestinal discomfort such as constipation, gas, flatulence, and bloating. Several factors contribute to this poor absorption, such as low calcium solubility, interactions within the food matrix, elevated parathyroid levels, vitamin D deficiency, and age-related changes.

[0013] Calcium supplements are available in a variety of dosage forms, including chewable tablets, capsules, liquids, and powders. Individuals who have trouble swallowing tablets can use chewable or liquid calcium supplements. Health experts emphasize that nutrients in pill form do not undergo the same metabolic processes in the body as when obtained from natural food sources.

[0014] Simple calcium salts, such as phosphates and carboxylates, are only slightly soluble in water. In the stomach, calcium ions are released through acid dissolution. However, these calcium ions may precipitate as insoluble salts when the chime (partially digested food) moves from the stomach into the more alkaline environment of the intestine. The precipitation process, which results from supersaturated solutions formed during acid neutralization and rising pH, may reduce calcium bioavailability. The decreased bioavailability of calcium — and its associated health consequences — appears to be related to calcium precipitation at this stage of digestion, possibly due to the absence of inhibitors that prevent calcium salt precipitation.

[0015] Hence, there is a need to provide an invention to improve the absorption of dietary calcium rather than high calcium intake as it is a better approach to achieving a normal blood calcium level.

[0016] Bioactive peptides (BPs) are chains of amino acids linked by peptide bonds, arising from the breakdown of proteins found in plants, animals, fungi, and microbes. These peptides are liberated through enzymatic processes or during food handling methods like fermentation, cooking, or ripening. Bioactive peptides generated from food proteins have great potential as functional foods and nutraceuticals. Bioactive peptides possess several significant functions, such as antioxidative, anti-inflammatory, anticancer, antimicrobial, immunomodulatory, and antihypertensive effects in the living body. Bioactive peptides derived from milk proteins are food components that, in addition to their nutritional value, retain many biological properties and have therapeutic effects in several health disorders, including cardiovascular disease.

[0017] However, none of the prior arts mentions the synergistic effect of peptides on absorption of calcium in to the body.

[0018] Therefore, the inventors of the present invention have formulated a bioactive peptide composition which will enhance calcium absorption or improve calcium incorporation into bones through synergistic effect.

[0019] After performing numerous research trials and experimentations, inventors of present invention arrived to a synergistic composition of bioactive peptides that is Casein phosphopeptides and Lactotripeptides to achieve the higher calcium absorption and serum calcium level.

[0020] The objectives of the present invention are as described herein.

[0021] OBJECTIVE OF THE INVENTION

[0022] The main objective of the invention is to provide bioactive peptide composition.

[0023] Other main objective of the invention is to provide bioactive peptide composition which enhance calcium absorption.

[0024] Other main objective of the invention is to provide synergistic bioactive peptide composition comprising Casein phosphopeptides and Lactotripeptides which enhance calcium absorption. Another objective of the invention is to provide bioactive peptide composition which improve calcium incorporation into bones.

[0025] Another objective of the invention is to provide a bioactive peptide composition which provides better patient compliance.

[0026] Yet another objective of the invention is to provide bioactive peptide composition which enhance calcium absorption with synergistic effect.

[0027] Another objective of the invention is to provide a bioactive peptide composition which is stable.

[0028] SUMMARY OF THE INVENTION

[0029] The main aspect of the present invention is to provide a bioactive peptide composition.

[0030] The other main aspect of the present invention is to provide a bioactive peptide composition comprising Casein phosphopeptides and Lactotripeptides.

[0031] Yet another aspect of the present invention is to provide a synergistic composition of bioactive peptides comprising therapeutically effective amount of Casein phosphopeptides and Lactotripeptides.

[0032] Another aspect of the invention is to provide a bioactive peptide composition and the process of preparation of the same.

[0033] The details of one or more aspects of the invention are outlined in the description below. Other features, objects, and advantages of the invention will be apparent from the description. BRIEF DESCRIPTION OF THE DRAWING

[0034] Fig.l: Represent the graph of Serum total calcium level

[0035] Fig.2: Represent the graph of Serum total calcium level AUC

[0036] Fig.3: Represent the graph of Serum PTH level

[0037] DESCRIPTION OF THE INVENTION

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0039] As used herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise.

[0040] The main embodiment of the present invention is to provide a bioactive peptide composition.

[0041] The term "therapeutically effective amount" as used herein means an amount or dose of the CPP and Lactotripeptides combination disclosed is sufficient to achieve the desired result which is increase in absorption of calcium in the body and reduce PTH (Parathyroid hormone) level.

[0042] An “effective amount of bioactive agent” is an amount sufficient to prevent, treat, reduce, and / or ameliorate the symptoms and / or underlying causes of cellular diseases.

[0043] As used herein the term “Lactotripeptides” means the combination of Isoleucyl- prolyl-proline (IPP) and Valyl -prolyl-proline (VPP) and in the whole specification where term Lactotripeptides is used needs to be interpreted as combination of IPP and VPP only.

[0044] Lactotripeptides are naturally derived milk peptides and part of our daily diet. Lactotripeptides is a commonly used name for two naturally occurring milk peptides, isoleucyl-prolyl-proline (IPP) and Valyl -prolyl-proline (VPP). These Lactotripeptides are derived from casein, which is a milk protein found in many dairy products. While Lactotripeptides are present in normal dairy products, they are inactive in the original milk protein. They become active after being split off by enzymatic hydrolysis of the milk protein during digestion. In order to be useful as a functional ingredient, i.e. to be bioactive, it is necessary that Lactotripeptides reach their site of action intact. Lactotripeptides should (a) remain intact during gastrointestinal digestion and absorption, (b) be small enough to cross the intestinal barrier and (c), enter the circulation. Tripeptides containing the C-terminal prolineproline are generally resistant to degradation by digestive enzymes, thus remaining intact in the gastrointestinal tract. Since the Lactotripeptides are small peptides, they can be absorbed intact from the intestinal lumen into the blood circulation by paracellular and transcellular transport routes.

[0045] As per one embodiment, the concentration of IPP and VPP in the Lactotripeptides is in the range of 0.02 to 99.9%

[0046] As per one embodiment, the dosage range of Lactotripeptides is in the range from 1 mg to 600 mg, preferably 5 to 500 mg, preferably 5 to 400 mg, preferably 5 to 300 mg, preferably 5 to 200 mg, more preferably 10 to 100 mg, more preferably 10 to 50 mg, more preferably 20 to 45 mg.

[0047] Casein phosphopeptides (CPP) refer to milk peptides obtained from milk by means of enzymatic breakdown of milk protein. They reduce interactions between calcium and other minerals, prevent the formation of insoluble calcium salts, keep calcium in a soluble form, and enhance calcium absorption or improve calcium incorporation into bones.

[0048] As used herein Casein phosphopeptides (CPP) can also be termed as different names selected from but not limited to Casein derived peptides, Phosphorylated casein peptides, Casein derived phosphorylated peptides and Casein tryptic hydrolysate and all convey the same meaning with reference to present invention.

[0049] As per one embodiment, casein phosphopeptides (CPP), Isoleucyl-prolyl-proline (IPP) and Valyl-prolyl-proline (VPP) are obtained / isolated / derived from either milk or produced through fermentation or chemical process.

[0050] As per one embodiment, the ratio of CPP and Lactotripeptides plays a critical role to provide synergistic effect to enhance the calcium absorption.

[0051] As per one embodiment, the dosage range of Casein phosphopeptides is in the range from 10 mg to 500 mg, preferably 10 to 400 mg, preferably 20 to 300 mg, preferably 10 to 200 mg, preferably 10 to 150 mg, more preferably 20 to 150 mg, more preferably 40 to 150 mg, more preferably 50 to 120 mg.

[0052] As per one embodiment, the weight ratio of Lactotripeptides to CPP is in the range from 1:0.05 to 1:20, preferably 1: 0.5 to 1: 10, preferably 1: 0.5 to 1:8, preferably 1: 1 to 1:6, more preferably 1: 1.5 to 1:5, more preferably 1: 1.5 to 1:3.5.

[0053] As per one embodiment, the weight ratio of Lactotripeptides to CPP is in the range of 1: 1.5 to 1:3.5.

[0054] As per one embodiment the present composition is for administration through oral, nasal or topical, more preferably for oral administration. As per one embodiment, the composition of the present invention is selected from uncoated tablets, coated tablets, modified-release tablets, gastro-resistant tablets, orodispersible tablets, effervescent tablets, chewable tablets, soft capsules, hard capsules, modified-release capsules, gastro-resistant capsules; uncoated granules, effervescent granules, coated granules, gastro-resistant granules, modified- release granules, gummies, liquid filled gelcaps, powders, suspension solution and emulsion.

[0055] As per one embodiment, one or more pharmaceutically acceptable excipients are selected from but not limited to a diluent, a binder, a surfactant, a lubricant, a glidant or mixtures thereof.

[0056] As per one embodiment, diluent is selected from the group consisting of mannitol, lactose, microcrystalline cellulose, potassium sulfate, calcium phosphate, modified starch and mixtures thereof.

[0057] As per one embodiment, diluent can be used in the range from 1 to 90% w / w.

[0058] As per one embodiment, binder is selected from the group consisting of alginic acid, carbomer, sodium carboxymethylcellulose, dextrin, ethylcellulose, gelatin, glucose, guar gum, hydroxypropyl cellulose, hydroxypropyl methylcellulose, maltose, methylcellulose, microcrystalline cellulose, polyethylene oxide, starch, or povidone.

[0059] As per one embodiment, binder can be used in the range from 1 to 10% w / w.

[0060] As per one embodiment, lubricant is selected from the group consisting of calcium stearate, glyceryl behenate, magnesium stearate, mineral oil, polyethylene glycol, sodium stearyl fumarate, stearic acid, talc, sodium lauryl sulfate, and zinc stearate.

[0061] As per one embodiment, lubricant can be used in the range from 0.5 to 5% w / w. As per one embodiment, glidant is selected from the group consisting of colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, and talc.

[0062] As per one embodiment, glidant can be used in the range from 0.5 to 5% w / w.

[0063] As per one embodiment, surfactant is selected from the group consisting of sodium lauryl sulfate, cetrimide, polysorbates such as polysorbate 80, poloxamers such as poloxamer 188 and poloxamer 407, sodium carboxymethylcelluloses, hydrogenated oils, polyoxyethylene glycols, polyoxypropylene glycols, polyoxyethylene sorbitan fatty acid esters, polyglycolized glycehdes available commercially such as GELUCIRE®40 / 14, GELUCIRE® 42 / 12, and GELUCIRE® 50 / 13, vitamin E TGPS, TWEEN® surfactants, SPAN® surfactants, and mixtures thereof.

[0064] As per one embodiment, glidant can be used in the range from 0.5 to 5% w / w.

[0065] As per one embodiment, the bioactive peptide composition comprising Casein phosphopeptides, Lactotripeptides is prepared as a composition without any inactive excipients and still achieve the desired therapeutic effect.

[0066] As per one more embodiment of the present invention, oral administration of an effective dose of the composition of the present invention improves calcium absorption by synergistic effect of bioactive peptide.

[0067] In another embodiment, the invention discloses bioactive peptide composition comprising synergistic combination of Lactotripeptides and CPP present in the ratio ranges from l:0.05to 1:20.

[0068] In another preferred embodiment, the invention discloses bioactive peptide composition comprising synergistic combination of Lactotripeptides and CPP present in the ratio ranges from l:0.05to 1: 10, preferably 1: 0.5 to 1:8, preferably 1: 1 to 1:6, more preferably 1: 1.5 to 1:5, more preferably 1: 1.5 to 1:3.5.

[0069] As per one more embodiment of the present invention, oral administration of an effective dose of the composition of the present invention increases serum calcium level by synergistic effect of bioactive peptides that is Casein phosphopeptides, Lactotripeptides.

[0070] As per one more embodiment of the present invention, oral administration of an effective dose of the composition of the present invention decreases serum parathyroid hormone (PTH) level by synergistic effect of bioactive peptide.

[0071] The following examples are given for the purpose of illustrating the present invention and shall not be construed as being limitations on the scope of the invention. It should be understood that the invention is not limited to the specific embodiments described herein. Various modifications, adaptations, and alternative implementations may be made without departing from the core concept or essential features of the invention. Accordingly, the present description is intended to be illustrative and not restrictive. The scope of the invention shall be defined by the claims that follow, and all equivalents and variations that fall within the meaning and range of the claims are intended to be included within that scope

[0072] EXAMPLES

[0073] EXAMPLE 1: BIOACTIVE PEPTIDE COMPOSITION

[0074] Composition A This composition refers the % w / w concentration of Casein phosphopeptides and Lactotripeptides which can be used for present invention.

[0075] Composition B This composition refers the dose range in mg of Casein phosphopeptides and Lactotripeptides which can be used for present invention.

[0076] EXAMPLE 2: BIOACTIVE PEPTIDE COMPOSITION

[0077] EXAMPLE 3: BIOACTIVE PEPTIDE TABLET COMPOSITION

[0078] All the ingredient mentioned above is used to prepare tablet with known techniques in the art. EXAMPLE 4 : BIOACTIVE PEPTIDE CAPSULE COMPOSITION

[0079] All the ingredient mentioned above is used to prepare capsule with known techniques in the art.

[0080] EXAMPLE 5: ANIMAL STUDY FOR BIOACTIVE PEPTIDE

[0081] COMPOSITION

[0082] Table: Animal study for bioactive peptide

[0083] All the groups of rats mentioned in the above table are standard group and treatment group (Tl, T2 and T3).

[0084] All the groups of rats mentioned in the above table are used for evaluation of serum total calcium levels, serum Parathyroid hormone (PTH) levels and serum total calcium area under curve parameter.

[0085] EXAMPLE 6: EFFECT OF BIOACTIVE PEPTIDE COMPOSITION ON SERUM TOTAL CALCIUM LEVEL

[0086] Serum total calcium levels were measured by Clinchem multi control level 1 Mindray using Calcium assay kit (of Shenzhen Mindray Bio-medical).

[0087] Table: Effect of bioactive peptide composition on serum total calcium level (mmol / 1)

[0088] The T3 group mentioned in the above table exhibits an incremental absorption of

[0089] 37.50% on serum total calcium levels which is higher than the other rest treatment groups.

[0090] EXAMPLE 7: EFFECT OF BIOACTIVE PEPTIDE COMPOSITION ON SERUM PARATHYROID HORMONE (PTH) LEVELS.

[0091] Serum PTH levels were measured by Multimode Reader (Biotek Instruments), Cytation 5 model Serial no. 1909161E using respective ELISA kit (of Krishgen Biosystems) leaflet under biosafety cabinet.

[0092] Table: Effect of bioactive peptide composition on serum parathyroid levels (pg / ml)

[0093] The T3 group mentioned in the above table exhibits an incremental improvement by 38.76% on Serum Parathyroid hormone (PTH) levels which is higher than the other rest treatment groups.

[0094] EXAMPLE 8: EFFECT OF BIOACTIVE PEPTIDE COMPOSITION ON SERUM TOTAL CALCIUM AREA UNDER CURVE (AUC)

[0095] Serum total calcium Area under curve (AUC) was calculated through graphPad prism.

[0096] Table: Effect of bioactive peptide composition on serum total calcium AUC (mmol / 1)

[0097] The T3 group mentioned in the above table exhibits an incremental absorption of 32.76% on serum total calcium Area under curve (AUC) which is higher than the other rest treatment groups.

[0098] The present invention is related to a bioactive peptide composition comprising Casein phosphopeptides, Lactotripeptides and one or more inactive excipients which is effective in providing the incremental absorption of calcium.

Claims

CLAIMSWe Claim;1. A bioactive peptide composition comprising Casein phosphopeptides, Lactotripeptides, wherein the ratio of Lactotripeptides to CPP is 1:0.05 to 1:20.

2. The bioactive peptide composition as claimed in claim 1, wherein Lactotripeptides is the mixture of Isoleucyl-prolyl-proline (IPP) and Valyl- prolyl-proline (VPP).

3. The bioactive peptide composition as claimed in claim 1, wherein the weight ratio of Lactotripeptides to CPP is in the range from 1:0.05 to 1:20, preferably 1: 0.5 to 1: 10, preferably 1: 0.5 to 1:8, preferably 1: 1 to 1:6, more preferably 1: 1.5 to 1:5, more preferably 1: 1.5 to 1:3.5.

4. The bioactive peptide composition as claimed in claim 1, wherein the dosage range of Casein phosphopeptides is in the range from 10 mg to 500 mg, preferably 10 to 400 mg, preferably 20 to 300 mg, preferably 10 to 200 mg, preferably 10 to 150 mg, more preferably 20 to 150 mg, more preferably 40 to 150 mg, more preferably 50 to 120 mg.

5. The bioactive peptide composition as claimed in claim 1, wherein the dosage range of Lactotripeptides is in the range from 1 mg to 600 mg, preferably 5 to 500 mg, preferably 5 to 400 mg, preferably 5 to 300 mg, preferably 5 to 200 mg, more preferably 10 to 100 mg, more preferably 10 to 50 mg, more preferably 20 to 45 mg.

6. The bioactive peptide composition as claimed in claim 1, wherein the composition is for oral administration.

7. The bioactive peptide composition as claimed in claim 1, wherein the composition is selected from uncoated tablets, coated tablets, modified-releasetablets, gastro-resistant tablets, orodispersible tablets, effervescent tablets, chewable tablets, soft capsules, hard capsules, modified-release capsules, gastro-resistant capsules; uncoated granules, effervescent granules, coated granules, gastro-resistant granules, modified-release granules, gummies, liquid filled gelcaps, powders, suspension solution and emulsion.

8. The bioactive peptide composition as claimed in claim 1, wherein the composition is in the form of immediate release composition.

9. The bioactive peptide composition as claimed in claim 1, wherein the composition may further comprise one or more inactive excipients.

10. The bioactive peptide composition as claimed in claim 9, wherein the inactive excipients are selected from a diluent, a binder, a surfactant, a lubricant, a glidant or mixtures thereof.

11. The bioactive peptide composition as claimed in claim 10, wherein the amount of diluent ranges from 1-90%; the amount of binder ranges from 1-10%; the amount of surfactant ranges from 0.5-5%; the amount of lubricant ranges from 0.5-5%; the amount of glidant ranges from 0.5-5%.

12. The bioactive peptide composition as claimed in claim 1, wherein the administration of effective amount of the composition increases the serum calcium levels.

13. The bioactive peptide composition as claimed in claim 1, wherein the administration of effective amount of the composition decreases the serum PTH level.

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