NOVEL COMPLEXES COMPRISING COLLAGEN PEPTIDES AND CURCUMINOIDS AND COMPOSITIONS THEREOF

Abstract

Formulations comprising complexes of peptides with hydrophobic bioactive molecules are described herein. The invention more specifically relates to collagen peptide and curcuminoid complexes and compositions comprising the complexes. The compositions and formulations comprising the protein hydrolysate peptide-hydrophobic bioactive molecule complexes are water-soluble, stable at physiological and acidic pH, synergistically enhance the systemic bioavailability of the biomolecules and peptides, and are capable of delivering a high therapeutically effective amount of the bioactive molecules via oral route. The compositions and formulations comprising the collagen peptide-curcuminoid complexes provide significantly high levels of bioavailable curcuminoids that are water soluble and stable at physiological and acidic pH along with significant anti-inflammatory effects offered by collagen peptides. The invention further provides a process for preparing the peptide-bioactive molecule complexes, compositions and oral formulations comprising the collagen peptide-curcuminoid complex.

Claims

1. A water soluble pharmaceutical composition comprising: a) an effective amount of one or more hydrophobic bioactive molecules; b) water-soluble peptides derived from protein hydrolysates; and c) an emulsifying agent; wherein the water soluble protein hydrolysate peptides form an amorphous, nano-sized non-covalent complex with the hydrophobic bioactive molecules.

2. The pharmaceutical composition of claim 1, wherein the hydrophobic bioactive molecules are selected from the group consisting of curcuminoids, gingerol, flavonoids, stilbenes, carotenoids, saponins, terpenes, terpenoids, and chlorophyll.

3. The pharmaceutical composition of claim 1, wherein the hydrophobic bioactive molecules are curcuminoids.

4. The pharmaceutical composition of claim 1, wherein the hydrophobic bioactive molecules are present at a concentration in the range of 1 to 25% in the composition.

5. The pharmaceutical composition of claim 1, wherein the peptides are present at a concentration in the range of 10 to 90% in the composition.

6. The pharmaceutical composition of claim 1, wherein the protein hydrolysate peptides are 1000-5000 Da in size.

7. The pharmaceutical composition of claim 1, wherein the emulsifier is selected from the group consisting of lecithin, polysorbate, propylene glycol, sorbitol, glycerol, polyglycerol esters, Quillaja extract, and sugar esters.

8. The pharmaceutical composition of claim 1, wherein the protein hydrolysate peptides are collagen peptides.

9. The pharmaceutical composition of claim 1, wherein the curcuminoids are complexed and encapsulated in the collagen peptide matrix.

10. The pharmaceutical composition of claim 1, wherein the bioactive molecules comprising the composition exhibit enhanced solubility and stability as compared to unformulated bioactive molecules.

11. The pharmaceutical composition of claim 1, wherein the bioactive molecules comprising the composition exhibit increased bioavailability, increased absorption and longer half-life of the bioactive molecules after oral administration to a subject, as compared to unformulated bioactive molecules.

12. The pharmaceutical composition of claim 1, wherein the bioactive molecules comprising the composition exhibit increased cellular permeability, as compared to unformulated bioactive molecules.

13. The pharmaceutical composition of claim 1, wherein it exhibits enhanced anti-inflammatory as compared to unformulated bioactive molecules.

14. The pharmaceutical composition of claim 1, wherein it is orally administered either as capsules, tablets, softgels, beadlets, liquid solution, liquid suspension, liquid emulsion or as powder sachets for enhanced bioavailability of the bioactive molecules.

15. The pharmaceutical composition of claim 1, wherein it is administered orally at a dose of 100 to 500 mg/dose in the form of tablets, capsules or softgel.

16. The pharmaceutical composition of claim 1, wherein it is administered orally at a dose of 1000 mg to 5000 mg per sachet, once or twice a day, to deliver minimum 100 to 500 mg of bioactive molecules along with 1 to 10 g of peptides/dosage/day.

17. The pharmaceutical composition of claim 14, wherein the collagen bioactive molecule complex is amorphous.

18. A method of preparing water soluble pharmaceutical composition of claim 1, comprising the steps of: a. micronisation of curcuminoids followed by emulsification by homogenisation with an emulsifier; b. dissolving collagen peptides in water at a concentration range of 20 to 95%; c. complex formation of curcuminoids with the collagen peptide matrix by sonication with emulsified curcuminoids from step (a) with the dissolved collagen peptides from step (b) d. encapsulation of the complex from step (c) by making sub-micron sized micelle suspension by ultrasound mediated homogenisation; and e. drying the submicron sized micelles from step (d) by spray drying or freeze drying.

19. The method of claim 18, wherein the emulsifier is selected from the group consisting of lecithin, polysorbate, propylene glycol, sorbitol, glycerol, polyglycerol esters, Quillaja extract, and sugar esters.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 shows fluorescence analysis of the CCL complex. C95Curcuminoids with 95% purity isolated from turmeric rhizomes; CCLCurcuminoids-collagen complex.

[0043] FIG. 2 shows Ultraviolet scan data of the CCL complex. C95Curcuminoids with 95% purity isolated from turmeric rhizomes; CCLCurcuminoids-collagen complex; CP-collagen peptide.

[0044] FIG. 3 shows FTIR data for the CCL complex. C95Curcuminoids with 95% purity isolated from turmeric rhizomes; CCLCurcuminoids-collagen complex; CP-collagen peptide.

[0045] FIG. 4A shows .sup.1H NMR data of C95Curcuminoids with 95% purity isolated from turmeric rhizomes.

[0046] FIG. 4B shows .sup.1H NMR data of CP-collagen peptide.

[0047] FIG. 4C shows .sup.1H NMR data of CCLCurcuminoids-collagen complex.

[0048] FIG. 5A shows .sup.13C NMR data of C95Curcuminoids with 95% purity isolated from turmeric rhizomes.

[0049] FIG. 5B shows .sup.13C NMR data of CP-collagen peptide.

[0050] FIG. 5C shows .sup.13C NMR data of CCLCurcuminoids-collagen complex.

[0051] FIG. 6 shows DSC analysis of the CCL complex. C95Curcuminoids with 95% purity isolated from turmeric rhizomes; CCL-Curcuminoids-collagen complex; CP-collagen peptide.

[0052] FIG. 7 shows SEM analysis of the CCL complex. C95Curcuminoids with 95% purity isolated from turmeric rhizomes; CCL-Curcuminoids-collagen complex; CP-collagen peptide.

[0053] FIG. 8 shows particle size analysis of CCL solution (A), and particle size analysis of CCL solution filtered through a 420 nm filter (B).

[0054] FIG. 9 shows TEM data. CPCollagen peptide and CCLCurcuminoid-collagen complex.

[0055] FIG. 10 shows photographs of CCL and C95 water solutions. A. C95 in water, B. CCL in water and C. CCL in water after filtration through 200 nm filter.

[0056] FIG. 11 shows the plasma concentration of curcuminoids when supplemented with unformulated curcumin 95%, water dispersible physical mixture of curcumin and collagen peptides, and curcumin-collagen complex (CCL) in capsule and sachet format.

[0057] FIG. 12 shows cellular permeability of CCL in comparison with unformulated curcumin 95.

[0058] FIG. 13 shows cell proliferation inhibitory effect of CCL in SAS cell lines in comparison with unformulated curcumin 95.

[0059] FIG. 14 shows cytotoxicity of CCL investigated by flow cytometry in comparison with unformulated curcumin 95.

[0060] FIG. 15 shows anti-inflammatory effect of CCL in comparison with unformulated curcumin 95.

DETAILED DESCRIPTION

[0061] The invention is not limited to various embodiments given in this specification. The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention pertains. Certain terms used herein are described below, or elsewhere in the specification to provide additional guidance to a practitioner regarding description of the invention. In case of conflict, the present document, including definitions will control.

[0062] As used herein, the terms comprising, including, having, containing, involving and the like are to be understood to be open ended, i.e., to mean including but not limited to.

[0063] As used herein, the terms curcumin and curcuminoids are used interchangeably and indicate the biologic phenols obtained from the Curcuma plant. In the present application, curcuminoids refers to the standard curcumin with not less than 95% purity isolated from turmeric rhizomes by solvent extraction with a composition of curcumin 72 to 80% w/w, DMC (demethoxycurcumin) 12 to 15% w/w and BDMC (bisdemethoxycurcumin) 3 to 5% w/w.

[0064] As used herein, the term unformulated bioactive molecules refers to hydrophobic biomolecules not in complex with any carrier molecules or matrix. It refers to bare bioactive molecules, without any extraneously added carrier molecules.

[0065] The term unformulated curcuminoids refers to curcuminoids not in complex with any carrier or delivery molecules or matrix. It includes the curcuminoids that are isolated from the turmeric rhizome and not bound to any other extraneously added carrier molecules.

[0066] As used herein, the term effective concentration, effective amount means a concentration of the formulations described herein which can generate the desired effect in the subjects/patients, without leading to undesired levels of side effects. The effective concentrations of the formulations disclosed herein are given the various embodiments herein, as dosages of the formulation in different delivery formats.

[0067] As used herein the term C95 refers to Curcuminoids with 95% purity isolated from turmeric rhizomes; CP refers to collagen peptide, CCL refers to curcuminoids-collagen complex.

[0068] The current invention provides water-soluble formulations comprising peptides from protein hydrolysates, and hydrophobic bioactive molecules, and method of making this formulation.

[0069] One embodiment of the current invention is a water soluble pharmaceutical composition comprising: (a) an effective amount of one or more hydrophobic bioactive molecules; (b) water-soluble peptides derived from protein hydrolysates; and (c) an emulsifying agent, wherein the water soluble protein hydrolysate peptides form an amorphous and nano-sized non-covalent complex with the hydrophobic bioactive molecules and acts as a matrix for its effective encapsulation in solution.

[0070] In one embodiment, the hydrophobic bioactive molecules for the water soluble formulation described herein are selected from the group consisting of curcuminoids, flavonoids, stilbenes, carotenoids, saponins, terpenes, terpenoids, and chlorophyll. In one embodiment, the hydrophobic bioactive molecules are present at a concentration in the range of 1 to 25% in the formulation disclosed herein.

[0071] In one embodiment, the protein hydrolysate peptides are present at a concentration in the range of 10 to 90% in the composition. In another embodiment, such peptides have nutritional properties and are derived from food proteins.

[0072] In one embodiment, the hydrophobic bioactive molecules are curcuminoids. In one embodiment, the protein hydrolysate peptides are 1000-5000 Da in size.

[0073] In one embodiment, the protein hydrolysate peptides are collagen peptides. In one embodiment, the curcuminoids are complexed with and encapsulated in the collagen peptide matrix, where peptides act as a protective coat of the water soluble bioactive micelle or liposomes or phytosomes formed between the emulsifiers and hydrophobic bioactive molecules.

[0074] In another embodiment, peptides provides a noodle like structure in which the micelles or liposomes or water miscible particles of the hydrophobic molecules are trapped and protected.

[0075] In one embodiment, it exhibits enhanced solubility and stability as compared to unformulated bioactive molecules. In another embodiment, the peptide network of the formulation swells in the gastrointestinal (GI) fluid and is acted upon by the GI enzymes to convert to smaller peptides and amino acids and the leached hydrophobic molecules get easily absorbed, thus enhancing bioavailability.

[0076] In one embodiment, the pharmaceutical composition disclosed herein exhibits increased bioavailability, increased absorption and longer half-life of the bioactive molecules after oral administration to a subject, as compared to unformulated bioactive molecules.

[0077] In one embodiment, the composition exhibits increased cellular permeability, as compared to unformulated bioactive molecules.

[0078] In one embodiment, the composition disclosed herein exhibits increased anti-inflammatory and antioxidant effects as compared to unformulated bioactive molecules.

[0079] In one embodiment, the composition disclosed herein is orally administered either as capsules, tablets, softgels, beadlets, liquid solution, liquid suspension, and liquid emulsion or as powder sachets (drinks) for enhanced bioavailability of the bioactive molecules.

[0080] In one embodiment, it is administered orally at a dose of 100 to 500 mg/dose in the form of tablets, capsules or softgel to deliver 25 to 100 mg of bioactive phytonutrient molecule in a bioavailable format.

[0081] In one embodiment, it is administered orally at a dose of 1000 mg to 5000 mg per sachet, once or twice a day, to deliver minimum 100 to 500 mg of bioactive molecules per day along with 1 to 10 g of peptides/dosage/day. In one embodiment, wherein the powder form of the formulation disclosed herein is amorphous.

[0082] In one embodiment, the method of making a water-soluble pharmaceutical composition comprising collagen peptides and curcumin, wherein the collagen peptides form a water soluble, stable, amorphous and nano-sized non-covalent complex with the curcuminoids, the method comprising the steps of: (a) micronisation of curcuminoids followed by emulsification by homogenisation with an emulsifier; (b) dissolving collagen peptides in water at a concentration range of 20 to 95%; (c) complex formation of curcuminoids with the collagen matrix by sonication with the emulsified curcuminoids from step (a) and the dissolved collagen peptides from step (b); (d) encapsulation of the complex from step (c) by making sub-micron sized micelle suspension by ultrasound mediated homogenisation; and (e) drying the submicron sized micelles from step (d) by spray drying or freeze drying.

[0083] In one embodiment, the emulsifier is selected from the group consisting of lecithin, polysorbate, propylene glycol, sorbitol, glycerol, polyglycerol esters, Quillaja extract, and sugar esters.

[0084] One embodiment of the pharmaceutical composition made by the method described above.

[0085] Collagen peptides support the integrity, elasticity, regeneration and strength of the connective tissues including skin, bones, cartilage, ligaments and tendons provide beneficial health effects to skin and bones, especially in improving joint mobility, ameliorating joint pain and the like associated with osteoarthritis.

[0086] In one embodiment, the present invention uses collagen hydrolysates or collagen peptides to prepare the complexes with hydrophobic bioactive molecules. In one embodiment, collagen peptides contains 19 amino acids. In one embodiment, the collagen hydrolysate or collagen peptides are prepared by enzymatic hydrolysis. In one embodiment, the collagen peptides used in the current invention are water soluble. Creating delivery forms with short peptides like collagen in solution is very difficult due to the fact that the short soluble peptides like collagen molecules does not have suitable conformation and 3dimensional structure with suitable hydrophobic pockets for engulfing hydrophobic molecules like curcuminoids.

[0087] In one embodiment, the short length water soluble collagen peptides used herein have molecular weight ranging from 2000 to 5000 Da. The water soluble peptides have a molecular weight ranging from 1000 to 20000 Da, more particularly 1000 to 5000 Da and more preferably 1000 to 3000 Da, obtained from the enzymatic hydrolysis of collagen, a high molecular weight animal protein of 300 kDa.

[0088] In one embodiment, the short collagen peptides act as carrier or vehicle for hydrophobic and poorly bioavailable small bioactive molecules like curcuminoids to convert them into water soluble, easily dispersible curcuminoids with enhanced bioavailability when delivered orally. These properties are achieved by a complex formulation where by curcuminoids are associated with collagen peptides in a non-covalent, complex formation.

[0089] In one embodiment, these short collagen peptides successfully form complexes without the formation of any chemical cross links.

[0090] In one embodiment, the complexes disclosed herein possess interaction of curcuminoids with collagen peptides and also create strong Van der Waals force of attraction such as hydrogen bonding between collagen peptide molecules and curcuminoids.

[0091] In one embodiment, the strong interaction between emulsified curcuminoids and short collagen peptides results in a protective coating of the curcuminoids micelles or liposomes or phytosomes with collagen peptides to attain better stability in vivo. The interaction also results in an increase in the water solubility of hydrophobic curcuminoids, increased bioavailability of curcuminoids, and hence improved therapeutic efficacy.

[0092] In one embodiment, the present invention of collagen peptide-curcumin complex claim superiority a mere physical mixture or water dispersible physical mixture formulated by mixing the collagen peptides and curcuminoids, in terms of its solubility, stability, bioavailability and bio-efficacy. The physical mixture results in a water-insoluble, indispersible (not-dispersible) mixture of curcumin that settles to the bottom within few seconds thereby making its consumption difficult. Such a mixture will have low bioavailability without any synergistic effect, and will lack in sensory appeal necessary for an edible product.

[0093] In one embodiment, compositions and formulations comprising the unique collagen peptide-curcuminoid complexes disclosed herein provide significantly high levels of bioavailable curcuminoids that is water soluble and stable at physiological and acidic pH along with significant anti-inflammatory effects offered by collagen peptides.

[0094] In one embodiment, the collagen peptide-curcuminoid complex is in a water soluble, stable, solid dosage form having a concentration of 100 to 250 mg curcuminoids in every 3 g of collagen peptides, preferably in 2 g collagen peptides, or more preferably in 1 g collagen peptides suitable for oral consumption which renders collagen peptides and curcuminoids bioavailable for enhanced efficacy on skin, bone, joint and gastrointestinal health.

[0095] In one embodiment, compositions and formulations comprising the collagen peptide-curcuminoid complexes can be conveniently made to deliver effective dose of curcuminoids orally. In one embodiment, the collagen peptide-curcuminoid complex in the composition disclosed herein leads to increase in the water solubility of curcuminoids and their in vivo stability.

[0096] In one embodiment, the collagen-curcumin compositions and formulations exhibit better absorption and bio-availability when consumed orally.

[0097] In one embodiment, the collagen peptide-curcuminoid complex disclosed herein can be formulated for oral consumption in solid and liquid dose forms such as pills, capsules, tablets, soft-gels, emulsions, solutions, syrups and the like for delivering effective dose of collagen peptides and curcuminoids orally.

[0098] In an embodiment, the collagen peptide-curcuminoid complex disclosed herein can be formulated into food and drink edible compositions like powder mixes, granules, ready-to-drink liquids, beverage powders, drink and dairy mixes, confectionaries, and dietary supplements to name a few, for delivering effective dose of collagen and curcuminoids orally.

[0099] In an embodiment, powder formulations of curcuminoid-collagen peptide complexes suitable for supplementation at 1 to 10 g/serving of collagen peptides and 250 to 500 mg curcuminoids/serving is achieved as single ready to drink water soluble powder. The formulation not only provides bioavailable forms of bioactive molecules like curcuminoids, but also provides therapeutically significant dosage of peptides like collagen peptides together for synergic effect.

[0100] In another embodiment, the oral formulation of curcuminoid collagen peptide complexes suitable for supplementation as a sachet/to consume along with water, milk or yogurt, soups, ice creams, etc.

[0101] Although the present invention has been illustrated and described herein with reference to the embodiments and examples disclosed herein, it will be readily apparent to those skilled in the art that other embodiments and examples may perform similar functions and/or achieve like results. All such equivalent embodiments and examples are within the spirit and scope of the present invention, are contemplated thereby, and are intended to be covered by claims put forth in the application.

EXAMPLES

[0102] The examples below illustrate the present invention and are non-limiting. The below exemplify representative embodiments of the invention, where all parts, proportions and percentages are by weight unless otherwise indicated. All fine chemicals, reagents, solvents and the like were commercially procured.

[0103] Collagen peptides from bovine, chicken and fish were purchased from Nitta Gelatin India Limited, Kochi, Kerala State, India. Phospholipids were purchased from Lipoid AG, Steinhausen, Switzerland. Curcumin 95% was taken from the quality assurance laboratory of Akay Flavours and Aromatics Pvt Ltd., Kochi, Kerala, India.

Example 1: Preparation of Curcumin-Collagen Complex: Preparation of Curcuminoid Suspension

[0104] About 9.2 g of commercially procured curcumin with not less than 90% purity was mixed with 4.5 g of MCT oil and with 4.5 g of glycerine and heated for 30 min to get a uniform mass. 9 g of lecithin having a phosphatidyl choline content of 20 to 90%, most preferably 25 to 45%, was then dissolved in 50 mL of ethanol/water mixture by homogenization or sonication. The water dispersible curcumin mass was then added slowly to ethanol/water by sonication and further suspended in 200 mL water with high power ultrasound (1000 KW) as pulses of 1 to 3 seconds or homogenization. Ethanol was then evaporated under vacuum to obtain a water suspension of curcuminoids having an approximate particle size not more than 2 m.

[0105] Preparation of Collagen Peptide-Curcuminoid Complex:

[0106] 70 g of commercially obtained collagen peptides was separately dissolved in 400 mL water by homogenization. The solution was slowly mixed with the curcuminoids suspension (275 mL) by high shear mixing, optionally heating. The solution was homogenized and then spray dried or freeze dried to obtain free-flowing, water-soluble, stable, collagen peptide-curcuminoids complex having total curcuminoids content of 10.2%.

[0107] Alternatively emulsifiers including lecithin, polysorbate, propylene glycol, sorbitol, glycerol, and polyglycerol esters, Quillaja extract, or sugar esters, can be used with and without lecithin to make collagen peptide-curcuminoids complexes.

[0108] Fatty oils including but not limited to sunflower oil, coconut oil, polyunsaturated fatty oils, olive oil or fish oil may be used instead of MCT oil.

[0109] The composition of ethanol/water can vary from 70:30 v/v to 95:5 v/v.

[0110] Temperature during formulation can vary from 50 to 80 C., depending on the stability of the bioactive substance.

[0111] Peptides can be derived from any food-grade proteins having water solubility and molecular weight ranging from 1000 to 10000, preferably 1000 to 5000, more preferably 1000 to 3000 Da.

[0112] The amount of curcumin can vary from 1 to 25% to produce curcumin-collagen complex with varying levels of curcuminoids content preferably less than 20%.

Example 2: Preparation of Curcumin-Fava Peptide Complex

[0113] Curcumin-fava peptide complex can be used by using the similar process disclosed in Example-1. The fava protein hydrolysate (i.e. fava peptide with molecular weight of around 3000 Da) and polysorbate 3 g was used instead of Glycerol.

Example 3: Preparation of Collagen Peptide-Gingerol Complex

[0114] Gingerol is the bioactive molecule from Ginger and is water insoluble.

[0115] Supercritical ginger extract containing 40% of gingerols was used for the formulation. It is obtained as an oil soluble paste form (Oleoresin). No oils were used in this formulation as specified in example 1. Briefly, about 18 g of oleoresin was mixed with and 18 g of lecithin with heating in ethanol/water medium. The ethanol was then evaporated and emulsified in water containing 3% Quillaja extract. The water-soluble form is then mixed with the collagen peptide with molecular weight 5000 and again sonicated. The uniform solution is then kept for 2 h. The supernatant solution is separated and freeze dried to fine water-soluble powder. Gingerol content was 6.3%.

Example 4: Characterisation of Collagen Peptide-Curcuminoids Complex

[0116] Spectroscopic techniques such as Fluorescence, Ultraviolet, Infrared, and NMR were used to confirm the non-covalent complex formation of curcuminoids with peptides. The shifts in the characteristic peaks due to hydrogen bonding between the peptide bonds, amino acid side chains and curcuminoids were confirmed. Proton (.sup.1H) and .sup.13C NMR studies show the inclusion of curcuminoids in the collagen network. Powder X-ray diffraction studies and differential scanning calorimetric confirms the amorphous nature of the complex whilst electron microscopic studies provide information on particle size, surface morphology and molecular arrangements in the complex.

[0117] Solid-state Fourier-transformed infrared spectra (FTIR) was recorded on Shimadzu spectrophotometer 8700 using potassium bromide pellets, prepared by compressing the powder at 20 psi for 10 min on a KBr press (Shimadzu Analytical Pvt. Ltd., Mumbai, India). The spectra were scanned over the wave number range of 3600-400 cm.sup.1. Thermogram was recorded using a differential scanning calorimeter (DSC) (Mettler-Toledo India Pvt. Ltd., Mumbai, India), by heating the samples (3-5 mg) in the aluminum crimp pan at a rate of 10 C./min from 30 to 300 C. under nitrogen atmosphere. Powder X-ray diffraction studies (PXRD) were performed on a Bruker D8 Advance instrument: target Cu, k1.54 A, filter Ni, voltage 40 kV, time constant 5 min/s; scanning rate 1 C./min (Bruker AXS GmbH, Karlsruhe, Germany). Scanning electron microscopic analysis (SEM) was done on SEM Jeol 6390 LA equipment (JEOL Ltd., Tokyo, Japan).

[0118] The enhanced bioavailability of curcuminoids presented in the form of collagen peptide-curcuminoid complexes was confirmed by measuring the plasma concentrations over 24 h time period as compared to unformulated curcuminoids with 95% purity (C95).

Example 4A: Fluorescence Analysis

[0119] The fluorescence analysis (FIG. 1) shows that, upon complexation CCL has an increased intensity of absorption. Curcumin exhibited an emission spectra max at 580 nm which on complex formation with collagen, decreased to 505 nm, indicating the inclusion of curcuminoids in a hydrophobic environment with hydrophobic interactions. Hence the blue shift in the complex indicates higher energy requirement for the excitation of molecule which further supports a stronger interaction (complexation) between the curcuminoids and peptides.

Example 4B: Ultraviolet/Visible Spectroscopy Analysis

[0120] The Ultraviolet/Visible spectroscopy (FIG. 2) of C95, CP and CCL indicated an interaction between the curcumin moiety and the collagen in CCL. The absorption maxima of curcumin has shifted from 420 nm to 408 nm, a blue shift. The absorption peak of curcumin at 420 nm has also been altered to a hill like plot spread out from 190 to 500 nm in CCL. This is due to the influence of the peptide which along with the curcumin exhibits uniform UV absorption, a multipoint attraction where a wide range of chromospheres are available.

Example 4C: Fourier Transform Infrared Spectroscopy (FTIR)

[0121] Since, the complexation reaction was performed in water at almost neutral pH, the curcumin exists in an equilibrium between the keto and enol forms. In the keto form, the oxygen atom attached to the carbonyl group has a higher electronegativity due to the adjacent unsaturation. This electronegativity of carbonyl oxygen attracts the lone pairs of the amide bond in the peptide chains, leading to intermolecular hydrogen bonding. This is evident from the shift of the 1647 cm.sup.1 of the amide bond to 1631 cm.sup.1 in CCL. The small peaks due to the stretching vibrations of enol-carbonyl groups at 1625 cm.sup.1 and 1599 cm.sup.1 were widened and observed as a shoulder of 1631 cm.sup.1 in CCL (FIG. 3). The peaks at 1426 cm.sup.1 in curcumin due to its CC vibration is shifted to 1405 cm.sup.1 in CCL, indicating the presence of keto form and its hydrogen bonding. The absorption peak at 3510 cm.sup.1, which is the typical peak of the OH group of the phenolic structure and the absorption peak at 3410 cm.sup.1 which is the typical peak of the normal amine group has fused together and got shifted to 3443 cm.sup.1 indicating an interaction between groups. Thus, the two OH groups in the aromatic rings and the OH and keto form of the keto-enolic form in curcumin has interactions with peptide bonds and terminal groups of peptides. Yet another possible reason for the shifts may be the hydrophobic interactions due to the inclusion of the curcuminoids in the hydrophobic pockets formed by the entanglement of random coiled peptides to form noodle-like structure for CCL.

Example 4D: NMR Studies

[0122] NMR studies revealed the extent of complexation in CCL. The .sup.1H NMR data (FIG. 4A, B, C) of curcumin indicated the presence of a high shielded hydrogen atom (hydrogen of the enol form) with chemical shift at 16.053 ppm. Due to low shielding effect, the OCH.sub.3 group in the aromatic ring of curcumin exhibited sextet at 3.949 ppm. Upon compexation, the higher shielding effect of the enol form was lost. Similarly, the sextet at 3.949 ppm got shifted towards 3.629 ppm which indicated a further lowering of the shielding effect. This is due to the association of the peptide fractions at the OH group adjacent to it. The hydrogen splitting raised from 6 to 7.55. 3.629 ppm which indicated a further lowering of the shielding effect. This is due to the association of the peptide fractions at the OH group adjacent to it. The hydrogen splitting raised from 6 to 7.55.

[0123] As per the .sup.13C NMR data (FIG. 5A, B, C), the high shielded carbon of the keto-enolic form in curcumin was observed at 184.521 ppm. Upon CCL formation, the chemical shift lowered to 172.366 ppm. Similarly, the carbon atom of the OCH.sub.3 group, which was observed at 57.222 ppm in curcumin got further lowered to 46.085 ppm which indicated lowering of shielding effect. This too indicates the non-covalent force of attraction that may have formed in between the aromatic OH group and the terminal NH.sub.2 of the peptide.

Example 4E: Differential Scanning Calorimeter (DSC) Analysis

[0124] DSC analysis (FIG. 6) analysis exhibited a sharp endothermic shift at 175.74 C. for curcumin due to its melting. The collagen peptide exhibited a slight endothermic shift at 74.60 C. and 194.23 C. Upon complexation, the CCL exhibited a shift at 71.77 C., 179.49 C. and 196.33 C., indicating an adduct/bonding between the two entities. However, no sharp shift was observed, due to the amorphous nature of CCL.

Example 4F. Scanning Electron Microscopy (SEM)

[0125] The scanning electron microscopy analysis of the powder samples (SEM) (FIG. 7) indicated the change in surface nature of the curcuminoids and collagen peptides. The curcuminoids were crystalline as evident from the needle shapes. Collagen peptide on the other hand is amorphous and forms spherical forms. The CCL complex is also spherical with smooth surface indicating the encapsulation of crystalline curcuminoids into the collagen network and its change into amorphous form.

Example 5: Particle Size and Transmission Electron Microscopy (TEM) Analysis of CCL in Water

Example 5A: Particle Size Analysis

[0126] Particle size/zeta potential of CCL in solution was analyzed by a dynamic light scattering Mastersize 3000 equipment of Melvern Pananalytical, USA. About 100 mg of curcuminoid-collagen complex was suspended in 10 mL distilled water for 1 h and shaken in a rotatory shaker. The supernatant solution was subjected to particle size analysis, which indicated two fractions having a mean particle size of less than 200 nm (150 to 200 nm) and relatively bigger particles of around 700 nm (600 to 700 nm). The solution was also filtered through a 420 nm Nylon filter and again subjected to particle size analysis. The particles were of around 176 nm (FIG. 8A, 8B).

Example 5B: TEM Studies

[0127] The morphology of particles in solution was visualized under transmission electron microscopy (JEM-2100, JEOL, Tokyo, Japan). Aqueous solution of curcuminoid-collagen complex was placed (20 L) on a copper grid and allowed to dry at room temperature. After staining with 2% phosphotungstic acid, the thin film formed was subjected for TEM observation. TEM indicated the agglomeration of curcumin with collagen peptides, with strong association of both to observe as a single molecular solution (FIG. 9).

Example 6: Water Solubility and Stability of Collagen Peptide-Curcuminoid Complex

[0128] About 20 mg of curcumin 95% which contains 19 mg of pure curcumin by HPLC was weighed into a 100 mL standard flask and marked as C95. 100 mg of CCL which contains 19 mg of pure curcumin by HPLC was weighed into another 100 mL standard flask and marked as CCL. Both were made up to the volume using HPLC grade water and were sonicated in a bath sonicator for 5 min. It was centrifuged and the supernatant was subjected to HPLC analysis to measure the dissolved curcumin content. It is observed that curcuminoid content in C95 had 14 ng/mL and that in CCL was 2040 g/mL, indicating enhancement in solubility of curcuminoids (FIG. 10).

Example 7: Storage Stability of Collagen Peptide-Curcuminoid Complex

[0129] Storage stability studies were carried out as per the guidelines of International Conference on Harmonization (ICH) of technical requirements for registration of pharmaceuticals for human use (ICH, 2003). Briefly, the sample packets (10 g) of curcuminoid-collagen complex was sealed in a double layered polyethylene bags and were incubated at 402 C. and 705% relative humidity for a period of 6 months in a stability chamber (Remi, Mumbai, India). The samples were withdrawn at 0, 1, 2, 3, and 6 months and subjected to analysis for various physical and chemical parameters. Curcuminoids content, moisture content and microbial parameters comprising total plate count, yeast and mold, Escherichia coli, Salmonella and coliforms were measured, results are shown in Table 1.

TABLE-US-00001 TABLE 1 Parameters Specification 0 month 3 month 6 month Appearance Free flowing Complies Complies Complies powder Identification HPLC Complies Complies Complies Colour Yellowish Complies Complies Complies orange Curcumin >20% 20.68% 20.35% 20.19% content Density 0.4-0.8 g/mL 0.42 g/mL 0.42 g/mL 0.42 g/mL Microbiology US-FDA (BAM) Total plate <3000 cfu/g 100 cfu/g 110 cfu/g 100 cfu/g count Yeast & Mold <100 cfu/g <10 cfu/g <10 cfu/g <10 cfu/g Coliforms <3 MPN/g <3 MPN/g <3 MPN/g <3 MPN/g Escherichia Absent/g Absent/g Absent/g Absent/g coli Salmonella Absent/25 g Absent/25 g Absent/25 g Absent/25 g

Example 8: Bioavailability Studies

[0130] The enhanced bioavailability of curcuminoids presented in the form of collagen peptide-curcuminoid complexes is confirmed by measuring the plasma concentration of curcuminoids over 12 h time period as compared to curcuminoids 95%. Human studies were conducted in accordance with the clinical research guidelines Healthy adult human volunteers (5 males and 3 females; aged between 24 and 46 years), who were not under any medication or dietary supplements were selected for the study. Subjects are assigned with a three digit unique randomization code. Ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometer (UPLC-ESI-MS/MS) (6460 Mass spectrometer, Agilent India Pvt Ltd, Bangalore, India) was employed for the analysis of curcuminoids in plasma or serum or whole blood collected after various post-administration time intervals (0, 1, 3, 5, 8 and 12 h).

[0131] Extraction efficiency of curcuminoids from plasma was confirmed by spiking 10 g/mL of standard solution in 1 mL of blank plasma followed by analysis. Retention time was confirmed by 10 repeated analyses at 20 g/mL level on the same column under identical conditions. Measurement of the contents in plasma was validated by spiking standard the actives in plasma at 10 and 20 g/mL concentrations. Extraction efficiency from plasma was 94% with a linear response of R.sup.2 value of 0.998. The identity was established by analytical standards and by multiple reaction monitoring (MRM) of their MS/MS spectra in mass spectrometry.

[0132] In a typical protocol followed in the present study, each volunteer was first given one capsule of 500 mg (500 mg1) of unformulated curcuminoids 95% or Curcuminoid-collagen complex (500 mg of Curcuminoid-collagen complex containing 20% curcuminoids as capsules); blood samples were withdrawn; plasma was separated and frozen at 20 C. till analysis. After 1 week, the subjects were cross-covered and provided with either one capsule of either 500 mg of Curcuminoid-collagen complex containing 20% curcuminoids or unformulated curcuminoid 95%. The protocol of collection of blood and plasma was repeated exactly the same as above. Plasma concentration verses time plot was constructed for the detailed analysis of pharmacokinetics of curcuminoids and their respective formulations to deduce bioavailability or relative absorption.

[0133] The same procedure was also repeated with the supplementation of 3 g of collagen-curcuminoids complex containing 8.5% of curcuminoids as a ready-to-drink sachet. Here the powder complex was dissolved in 200 mL of drinking water allowed to drink in such a way that one sachet provides 250 mg of curcuminoids in a soluble form. The blood and plasma samples at various post-administration time points was collected and subjects to LC-MS/MS analysis as before.

[0134] Pharmacokinetic parameters like the maximum curcumin concentration in the blood (C.sub.max), the time taken to reach the maximum concentration (T.sub.max) and concentration of curcumin in blood after 12 hr (C.sup.12.sub.max) were analysed. It can be seen that the curcuminoids in the new formulation absorbs almost 53 times for in Sachet form and 36 times in capsule form more than the normal curcumin, on an average. Moreover, T.sub.max for curcumin-collagen complex was 4.2 hr for Sachet and 7 hr for in capsule administration as compared to the normal curcumin.

[0135] FIG. 11 shows the plasma concentration verses time graph for curcuminoids when supplemented with curcumin 95%, water dispersible physical mixture of curcumin and collagen peptides, and curcumin-collagen complex in capsule and sachet format. The table 2 provides the pharmacokinetic parameters indicating the higher absorption, bioavailability and longer duration of existence in the blood when curcumin-collagen was consumed as compared to the curcumin alone or its physical mixture with collagen peptides.

TABLE-US-00002 TABLE 2 Cmax Tmax T Sample Dosage (ng/mL) (hr) (hr) AUC Folds CCL Capsule 500 mg 74.08 5 7 348.4 36.7899 CCL Sachet 3000 mg 118.84 3 4.2 506.8 53.5164 WD Physical 500 mg 10.25 0.5 0.9 16.69 1.76241 mix* Standard 500 mg 4.35 0.5 0.7 9.47 1 Curcumin *WD Physical mixwater dispersible physical mixture of curcumin and collagen peptides

Example 9: Cell Permeability of CCL

[0136] Cell permeability using Inverted Fluorescence Microscope. Fluorescent microscopy was employed to examine the cellular uptake and distribution changes of LFCSNs into HepG2 cells. The HepG2 cells approximately (110.sup.5) were cultured in a six well plate in a medium made up of EMEM and treated with of LFCSNs for 24 and 48 h with technical triplicates, untreated control and treated cells were observed under inverted microscope. For each experiment, nuclei from 10 random fields of well were examined at 200 magnification (FIG. 12). From the result (FIG. 12), cellular uptake of CCL is clear as compared to C95.

Example 10: Cell Toxicity of CCL

Example 10A: MTT Assay for Cell Proliferation

[0137] The MTT assay is used to measure cellular metabolic activity as an indicator of cell viability, proliferation and cytotoxicity. This colorimetric assay is based on the reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT) to purple formazan crystals by metabolically active cells. The proliferation study was conducted in SAS cell line. FIG. 13 shows the relative cellular toxicity of CCL in comparison with C95 in SAS cell lines.

Example 10B: Cytotoxicity Assay Using PI FACS

[0138] Flow cytometry provides a rapid and reliable method to quantify viable cells in a cell suspension. Live cells have intact membranes that exclude a variety of dyes that easily penetrate the damaged, permeable membranes of non-viable cells. Propidium iodide (PI) is a membrane impermeant dye that is generally excluded from viable cells. It binds to double stranded DNA by intercalating between base pairs. PI is excited at 488 nm and, with a relatively large Stokes shift, emits at a maximum wavelength of 617 nm. In this study 25 ug/mL concentration of C95 in DMSO and CCL in water were used. FIG. 14 shows the flow cytometery data for CCL vs C95.

Example 11: Anti-Inflammatory Effect of CCL

[0139] Carrageenan-Induced Mouse Paw Edema Model: Male Swiss albino mice were divided into four groups. First group was kept as vehicle control; second group treated with unformulated curcumin at a dose of 50 mg/kg b.Math.wt; third group was treated with CCL at a dose of 50 mg/kg b.Math.wt. Fifth group was treated with standard drug diclofenac at a dose of 25 mg/kg b.Math.wt. The carrageenan-induced mouse hind paw edema test was employed using the method described anteriorly with some modifications. Briefly, after administration for 60 min, each animal except the intact control was injected with 25 L of 1% freshly prepared carrageenan suspension at the plantar side of right hind paw. The paw edema values were evaluated before as the basal volume (tC.sub.0) and 1, 2, 3, 4, 5, or 6 h as the pathological volume (tC.sub.n) after carrageenan injection by the MK101CMP paleothermometer (Muromachi Kikai Co., Ltd., Japan). The percentage degree of swelling and inhibition of paw edema were calculated using the following formulas (FIG. 15):

[00001]$\text{\%inhibition}.Math.=\frac{\left({\mathrm{tC}}_n-{\mathrm{tC}}_0\right)-\left({\mathrm{tT}}_n-{\mathrm{Tt}}_0\right)1.Math.0.Math.0}{\left({\mathrm{tC}}_n-{\mathrm{tC}}_0\right)}$

Where, tCn=paw thickness at particular time point of control animal;
tC.sub.0=paw thick ness of control animals before induction;
tT.sub.n=paw thickness at particular time point of treated animal; and
Tt.sub.0=paw thickness of treated animals before induction

[0140] It is observed that CCL has offered higher inhibition of paw edema compared to unformulated curcumin and collagen peptide (FIG. 15).

Example 12

[0141] Delivery forms comprising collagen peptide-curcuminoid complex compositions comprising the collagen peptide-curcuminoid complexes formulated as capsules, tablets, soft-gels, powders, ready-to-consume mixes, granules, liquids for consumption as such, in aqueous beverages, incorporated in dairy products, candies like gummies, milk powder, protein shakes etc., demonstrated the stability and increased bioavailability of various dosage forms at 100 to 500 mg/serving.