FORM 2
THE PATENT ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003
COMPLETE SPECIFICA TION
(See section 10 and rule 13)
1. Title of the Invention
A NUTRACEUTICAL FORMULATION AND A PROCESS FOR PRODUCTION THEREOF
2. Applicant(s)
Name Nationality Address
TATA CHEMICALS INDIA BOMBAY HOUSE, 24 HOMI MODY
LIMITED STREET, MUMBAI - 400001
3. Preamble to the description
COMPLETE SPECIFICA TION
The following specification particularly describes the invention and the manner in which it is to
be performed.

The present disclosure relates to a nutraceutical formulation of molecular complex of Curcumin and Epigallocatechin-3-gallate and process for production thereof. In particular the invention relates to a nutraceutical formulation of molecular complex of Curcumin and Epigallocatechin-3-gallate having improved curcumin water solubility and bioavailability and process for production thereof.
BACKGROUND
Curcumin is the major yellow pigment of turmeric, a commonly used spice, derived from the rhizome of the herb Curcuma Longa. Curcumin has been widely used as to promote health and treat and/or prevent disease and/or illnesses. The herb belongs to ginger family and turmeric widely used and cultivated in South Asia, particularly in Indian subcontinent. Turmeric is widely known for its medicinal activity for inflammation, skin wounds, pain relief etc. Curcumin is the active ingredient of the turmeric. Curcumin is not soluble in water and hence bioavailability of this compound is very poor.
Epigallocatechin-3-gallate (EGCG), the main and most important polyphenol in green tea (Camellia sinensis), has shown numerous health promoting effects. Epigallocatechin gallate belongs to the family of catechins. It contains 3 phenol rings and has very strong antixoidant properties.
SUMMARY
A nutraceutical formulation of molecular complex of Curcumin and Epigallocatechin-3-gallate is disclosed. A nutraceutical formulation comprises a molecular complex of Curcumin and

Epigallocatechin-3-gallate. The molecular complex further comprises the improved water solubility and improved bioavailability.
A process for preparing a molecular complex of Curcumin and Epigallocatechin-3-gallate is disclosed. The process comprises mixing curcumin and Epigallocatechin-3-gallate in any molar ratios between 1:48 to 48: 1, followed by grinding to form a dry mixture; adding a solvent to the dry mixture followed by grinding to obtain a wet mixture; and air drying the wet mixture to obtain the molecular complex.
BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
Figure 1 illustrates a comparison powder X-ray diffraction (PXRD) profile of various samples of molecular complex of Curcumin and Epigallocatechin-3-gallate having different molar ratios of Curcumin and Epigallocatechin-3-gallate.
Figure 2 illustrates the comparison Differential Scanning Calorimetry (DSC) profile of various samples of molecular complex of Curcumin and Epigallocatechin-3-gallate having different molar ratios of Curcumin and Epigallocatechin-3-gallate.
Figure 3 illustrates the comparison infrared (IR) absorption profile of various samples of molecular complex of Curcumin and Epigallocatechin-3-gallate having different molar ratios of Curcumin and Epigallocatechin-3-gallate.
Figure 4 illustrates the standard known concentration curve of Curcumin solubility using Ultra Violet-visible spectroscopy
Figure 5 illustrates the standard known concentration curve of Curcumin solubility using HPLC instrument

DETAILED DESCRIPTION
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the disclosed process, and such further applications of the principles of the invention therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to "one embodiment" "an embodiment" or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase "in one embodiment", "in an embodiment" and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
Definitions:
Molecular complex: herein refers to a substance in a solid form or solid formulations, which comprises at least two pure substances which interact with each other through hydrogen bonding or any other non-covalent interactions to form molecular complex including co-crystals, solvates, hydrates or eutectic combinations or solid solutions, in which at least one of the pure substance is present in the solid form.

Nutraceuticals also known as phytochemicals, are natural, bioactive chemical compounds that provide numerous physiological benefits, including, inter alia, disease/illness prevention/treatment and health promotion. Nutraceuticals are used to achieve both long-term and short-term health objectives. Bioavailability
The U.S. Food and Drug Administration (FDA) define bioavailability as "the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of drug action". . Because in practice it is rare that active ingredient concentrations can be determined at the site of action.
The therapeutic effectiveness of an active ingredient depends upon the ability of the dosage form to deliver the active ingredient to its site of action at a rate and amount sufficient to elicit the desired pharmacological response. This attribute of the dosage form is referred to as physiologic availability, biologic availability or simply bioavailability.
For most active ingredients, the pharmacologic response can be related directly to the plasma levels. Thus the term bioavailability is defined as the rate and extent (amount) of absorption of unchanged active ingredient from its dosage form. It can also be defined as the rate and the extent to which the ingredients or active moiety is absorbed from the product and becomes available at the site of action. Blood level studies are the most common type of human or animal bioavailability studies, and are based on the assumption that there is a direct relationship between the concentration of active ingredient in blood or plasma and the concentration of active ingredient at the site of action. By monitoring the concentration in the blood, it is thus possible to obtain an indirect measure of active ingredient response. Following the administration of a single dose of an active ingredient, blood samples are drawn at specific time intervals and analyzed for

active ingredient content. A profile is constructed showing the concentration of active ingredient in blood at the specific times the samples were taken. The key parameters to note are:
1. AUC, The area under the plasma concentration-time curve., The AUC is proportional to the total amount of active ingredient reaching the systemic circulation, and thus characterizes the extent of absorption.
2. Cmax, The maximum active ingredient concentration. The maximum concentration of active ingredient in the plasma is a function of both the rate and extent of absorption. Cmax will increase with an increase in the dose, as well as with an increase in the absorption rate.
3. Tmax, The time at which the Cmax occurs. The Tmax reflects the rate of active ingredient absorption, and decreases as the absorption rate increases.
Bioavailability (the rate and extent of active ingredient absorption) is generally assessed by the determination of these three parameters. Since the AUC is representative of, and proportional to, the total amount of active ingredient absorbed into the circulation, it is used to quantitate the extent of active ingredient absorption. Thus, the faster the absorption of an active ingredient the higher the maximum concentration will be and the less time it will take to reach the maximum concentration.
Improved solubility: as employed herein refer to a form of the curcumin that has increased solubility or dissolution rate relative to the least soluble form of the curcumin known.

The disclosure relates to nutraceutical formulation of molecular complex of Curcumin and Epigallocatechin-3-gallate. In accordance with an aspect of the invention the nutraceutical formulation is the molecular complex of Curcumin and Epigallocatechin-3-gallate having improved curcumin water solubility and a process for production thereof. Chemically, curcumin is a bis-R,-unsaturated-diketone (commonly called diferuloylmethane), which exhibits keto-enol tautomerism having a predominant keto form in acidic and neutral solutions and stable enol form in alkaline medium. Commercial curcumin contains approximately 77% diferuloylmethane, 17% demethoxycurcumin, and 6% bisdemethoxycurcumin.
Curcumin refers to all polymorphs, solvates, and hydrates of the all curcuminoids, having the formula I, II and III, alone or in combination in any ratio:


Epigallocatechin-3-gallate refers to all polymorphs, solvates, and hydrates of the substance having the formula (IV):

In accordance with an aspect, the green tea extract may be used in place of Epigallocatechin-3-gallate in making the molecular complex of the proposed invention.
In accordance with an aspect, the nutraceutical formulation comprising curcumin and Epigallocatechin-3-gallatein of the proposed invention is having improved curcumin solubility in water.
In accordance with an aspect, the stoichiometric ratio of Curcumin and Epigallocatechin-3-gallatein the molecular complex is any molar ratios between 1:48 to 48: 1, preferably 1:4 to 4:1.
In accordance with an aspect, the curcumin water solubility in Curcumin and Epigallocatechin-3-gallatein molecular complex is at least 1 microgram per milliliter (|ag/ml) of

water. In accordance with an aspect the water solubility was checked in substantially pure water. The maximum water solubility of the curcumin has been reported 11 nano gram per milliliter by Ucisik M H et al {Journal of Nanobiotechnology 2013, 11:37).
In accordance with an aspect, molecular complex of curcumin and Epigallocatechin-3-gallate in of the proposed invention is having differential scanning calorimetry melting peak in the range of 162 °C to 166 °C. Differential scanning calorimetry analysis determines the temperature and heat flow associated with material transitions as a function of time and temperature. The change in the differential scanning calorimetry melting peak determines the formation of molecular complex of the proposed invention.
In accordance with an aspect, the nutraceutical formulation comprising curcumin and Epigallocatechin-3-gallatein of the proposed invention is having improved bioavailability. In accordance with an aspect of the invention the molecular complex of curcumin and Epigallocatechin-3-gallatein has 991% of bioavailability compared to curcumin as 100% bioavailability.
In accordance with an aspect, the invention provides a pharmaceutical, foodstuff, nutritional supplement, and nutritional composition comprising molecular complex of Curcumin and Epigallocatechin-3-gallate as described herewith.
The process comprises of preparing a mixture of Curcumin and Epigallocatechin-3-gallate in different molar stoichiometric ratios, grinding the mixture, heating the ground mixture, cooling the heated mixture and grinding it to obtain molecular complex of Curcumin and Epigallocatechin-3-gallate.
The grinding may be carried out in any suitable apparatus for grinding solids. Such apparatus includes but is not limited to mortar mills, vibrator mills or ball mills.

In accordance with an embodiment the ground mixture is heated at 60 to 80°C using rotatory evaporator but not limited to temperature control water bath with or without vacuum. The heating may be carried out under an inert atmosphere.
Any known method may be used for preparing molecular complex of Curcumin and Epigallocatechin-3-gallate. By way of a specific example the process comprises of admixing Curcumin and Epigallocatechin-3-gallate in a 1:1 molar stoichiometric ratio to form a dry mixture, grinding dry mixture for a predetermined period of time, adding few drops of solvent to make it wet and grinding the wet mixture for a predetermined period of time under inert condition, heating this ground mixture at around 75 °C temperature water bath for a period 10 to 15 minutes under inert condition and cooling the heated ground mixture to room temperature to obtain the molecular complex of Curcumin and Epigallocatechin-3-gallate.
The wet grinding and heating may be carried out under nitrogen atmosphere.
The grinding of the dry mixture may be carried out for 1 to 20 minutes. The grinding of the wet mixture may be carried out for 1-20 minutes.
The grinding may be carried out in any suitable apparatus for grinding solids. Such apparatus includes but is not limited to mortar mills, vibrator mills or ball mills.
In accordance with an embodiment the solvent is any suitable solvent including but not limited to water, acetonitrile, ethanol, methanol, ethyl acetate, acetone or their mixture. The amount of solvent added is in a range of 0.1 ml to 5 ml per 1 gram of combined weight of Curcumin and Epigallocatechin-3-gallate.
In accordance with an embodiment the nutraceutical formulation comprising the molecular complex of Curcumin and Epigallocatechin-3-gallate of the present disclosure as an active ingredient may be provided as a pharmaceutical composition for preventing or treating

inflammation, skin wounds, pain relief, indigestion or other common aliment. The composition of the present disclosure may comprise molecular complex of Curcumin and Epigallocatechin-3-gallate of the present disclosure in an amount of 0.001-99.999% (w/w) as well as an excipient as balance.
In accordance with an embodiment the nutraceutical formulation comprising the molecular complex of Curcumin and Epigallocatechin-3-gallate of the present disclosure may be formulated into powder, granule, tablet, pill, sugar-coated tablet, capsule, liquid, gel, syrup, suspension, wafer, or the like together with an adequate excipient for oral administration according to a method known in the art.
In accordance with an embodiment the nutraceutical formulation comprising the molecular complex of Curcumin and Epigallocatechin-3-gallate of the present disclosure is a food additive.
EXAMPLES
Example 1: Molecular Complex preparation:
Curcumin (Sigma-Aldrich®) and Epigallocatechin-3-gallate (Teavigo®) were weighed in 1:1, 1:2, 3:1 and 4:1 molar ratios as separate samples for different experiments and ground using mortar and pestle for 5 minutes to make a homogenous mixture followed making paste under inert condition (Nitrogen atmosphere) with few drops of water added to it. Heating the mixture around 70 °C temperature in a control water bath for 5-10 min under vacuum in a rotary evaporator. The resulted material was cooled to room temperature to obtain the molecular complex.
Example 2: Powder X-ray Diffraction

Technical Details:
Powder X-ray Diffraction (PXRD) profiles were obtained using PANalytical "X"pertPRO diffractometer. PXRD profile data presented for the region where significant peaks were observed. Analysis:
Molecular complexes of Curcumin and Epigallocatechin-3-gallate gave different PXRD profile than pure Curcumin and Epigallocatechin-3-gallate. Figure 1 illustrates a comparative PXRD profile of samples of molecular complexes of Curcumin and Epigallocatechin-3-gallate.
Example 3: Infrared Spectroscopy Technical Details:
Fourier transformed infrared spectra (FT-IR) were collected on a Bruker Vertex 70 model. Analysis:
FT-IR spectra of all the molecular complexes obtained from examples 1 were compared with individual compounds and it was found that there are significant changes in IR spectral band of functional group regions to confirm the formation of novel molecular complexes. Figure 3 exhibits the major IR peaks of the sample of molecular complexes of curcumin and Epigallocatechin-3-gallate.
Example 4: Differential Scanning Calorimetry Technical Details:

Differential Scanning Calorimetry (DSC) thermograms of all the samples including samples from Example 1 were recorded on a Mettler DSC1 instrument. Analysis:
Molecular complexes of Curcumin and Epigallocatechin-3-gallate gave completely different DSC profile. Figure 2 illustrates the comparative DSC profile of samples of molecular complexes of Curcumin and Epigallocatechin-3-gallate.
Example 5: Solubility study of molecular complex:
Water solubility of curcumin in molecular complex of curcumin and Epigallocatechin-3-gallate was checked in water. 150 mg of Molecular complexes of Curcumin and Epigallocatechin-3-gallate of different ratios were weighed and transferred in different flasks containing 50 mL water. Flasks were kept on shaker at 100 round per minute (RPM) of speed. 2 ml aliquot from each sample (of different ratios) was withdrawn separately from the flasks at the time interval of 24 hours followed by the aliquots were filtered through 0.45 \x PTFE filter. Filtered samples were analyzed using Carry5000 UV/Vis instruments at 420 nm for the determination of Curcumin concentration, which provide solubility of Curcumin formulations. Water solubility of Curcumin in molecular complexes of curcumin and Epigallocatechin-3-gallate was determined from the known concentration standard curve of Curcumin in methanol illustrated with Figure 4. The curcumin solubility of different samples in water has been enumerated in table 1.
Table 1. Table 1: UV Absorbance of pure Curcumin in known concentration

Concentration (u.g/ml)
Absorbance
1 0.119
2 0.253
3 0.419
4 0.572
5 0.732
Table 2: Water Solubility study of molecular complex

Sr. NO. Molecular Complex
(Curcumin and
Epigallocatechin-3-gallate
Ratio) Curcumin water Solubility (24 hours)
Hg/ml
1 1:4 1.656
2 1:2 1.636
3 1:1 2.273
4 2:1 1.753
5 4:1 1.971
Example 6: Solubility study of molecular complex in water ethanol mixture:

Solubility of curcumin in molecular complex of curcumin and Epigallocatechin-3-gallate was checked in water containing 5% Ethanol. 150 mg of Molecular complexes of Curcumin and Epigallocatechin-3-gallate was weighed and transferred in a flask containing 50 mL of 5% ethanol water solution. Flask was kept on shaker at 100 rotation per minute (RPM) speed. 2 ml aliquots for different experiments were withdrawn from the flask at the time intervals of 2 hours, 24 hours and 48 hours followed by the aliquots were filtered through 0.45 u PTFE filter. Filtered sample was analyzed using Themo scientific, Ultimate 3000Ultra high performance liquid chromatography (UHPLC) for the determination of Curcumin concentration, which provide solubility of Curcumin formulations. Solubility of molecular complexes Curcumin-EGCG was compared with the solubility of standard Curcumin using standard curve prepared from the known concentration shown in table 3 and Figure 5. The table 4 illustrates the solubility of different ratios of Curcumin and Epigallocatechin-3-gallate molecular complexes.
Table 3. Area Under the Curve (AUC) in HPLC profile of pure Curcumin in known concentration

Curcumin Concentration, (ng) Area Under the Curve (AUC)
0.8 0.06
1.6 0.10
2.4 0.14
3.2 0.17

Table 4: Solubility study of molecular complex in water ethanol mixture

Sr. No. Sample Solubility (u.g/ml)
1 Curcumin 0.25
2 Molecular complex of Curcumin and EGCG (1:1 ratio) 6.42
Example 7: Bioavailability study of molecular complex in Rat Model:
Bioavailability (pharmacokinetics) of curcumin test was conducted as per OECD guidelines. Pure curcumin as control sample and Molecular complexes of Curcumin and Epigallocatechin-3-gallate as exemplary product of the proposed invention was taken as test sample for the bioavailability study was conducted by orally feeding to male Sprague Dawley rats. Rats were taken in two groups the first group was for control group and the second group was for test sample group. In each group three male Sprague Dawley rats were taken for study. For control group dose of 300 mg/kg of Curcumin was administrated to each Rat and similarly an equivalent dose of 300 mg/kg of Curcumin was administrated to each Rat in test sample group. Blood samples were collected at pre dose and at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h post-dose from each animal within ± 2 min of schedule time. At each time point, approximately 0.25 mL of blood was withdrawn and transferred to a labeled microfuge tube containing 200 mM K2EDTA solution (20 uL per mL of blood). Following sampling, equal volume of heparinized saline was replaced into the catheter. Blood was processed to collect plasma and stored below - 60 °C until their bioanalysis. Each plasma sample was analyzed for curcumin using a fit-for purpose liquid chromatographic tandem mass spectrometric detection (LC-MS/MS) method with a lower limit

of quantification of 0.5 ng/mL for curcumin. The pharmacokinetic parameters for free curcumin were calculated using the non-compartmental analysis tool of the validated Phoenix WinNonlin software (Version 6.3). Mean pharmacokinetic parameters of Curcumin-EGCG formulation and standard Curcumin given in below Table 2: Table 2: Mean pharmacokinetic parameters of Curcumin

Treatment/ Group Sample Tmax (h) Cmax (ng/mL) AUClast (ng.h/mL) Relative bioavailability b
Control group Curcumin 6.0 (4.0 - 6.0) 9.09
± 1.88 35.8
± 5.61 NA
Test sample group Curcumin-EGCG 24.0 (6.0 - 24.0) 36.1
± 38.5 355
±
331 991
In the above table a Tmax has been reported as median (min-max), AUClast and nominal doses were used for bioavailability (F) calculation with respect to control group. NA: not applicable

INDUSTRIAL APPLICABILITY
The molecular complex of Curcumin and Epigallocatechin-3-gallate has synergistic effect. It provides better Curcumin solubility in water and better EGCG stability. A soluble and stable variant of Curcumin and EGCG would allow enhancement of bioavailability. The enhanced bioavailability leads to better absorption in body for better bio-efficacy of the active ingredient. It is used as nutraceutical.

We Claim:
1. A nutraceutical formulation comprising a molecular complex of Curcumin and Epigallocatechin-3-gallate having a water solubility at least 1 fig/ml; and improved bioavailability.
2. The nutraceutical formulation as claimed in claim 1, wherein Curcumin and Epigallocatechin-3-gallate in the molecular complex are in a stoichiometric ratio in a range of 1: 48 to 48 : 1.
3. The nutraceutical formulation as claimed in claim 2, wherein Curcumin and Epigallocatechin-3-gallate in the molecular complex are in a stoichiometric ratio in a range of 1: 4 to 4 : 1.
4. The nutraceutical formulation as claimed in claim 1, wherein differential scanning calorimetry melting peak of molecular complex of Curcumin and Epigallocatechin-3-gallate are in the range of 162 °C to 166 °C.
5. A process for preparing a molecular complex of Curcumin and Epigallocatechin-3-gallate, the process comprising:
mixing Curcumin and Epigallocatechin-3-gallate in a stoichiometric ratio in a range
of 1: 48 to 48 : 1, followed by grinding to obtain a dry mixture;
adding a solvent to the dry mixture followed by grinding to obtain a wet mixture; and

air drying the wet mixture to obtain the molecular complex having a improved curcumin water solubility and bioavailability.
6. The process as claimed in claim 5, wherein the process further comprises heating the wet mixture at about 70°C prior to air drying.
7. The process as claimed in claim 6, wherein the wet mixture is heated under a nitrogen atmosphere.
8. The process as claimed in claim 5, wherein the solvent is added to the dry mixture in a range of 0.5 ml to 10 ml with respect to each gram of combined weight of Curcumin and Epigallocatechin-3-gallate.
9. The process as claimed in claim 5, wherein the solvent is selected from water, acetonitrile, ethanol, methanol, ethyl acetate, acetone and mixtures thereof.
10. The process as claimed in claim 5, wherein the solvent is added to the dry mixture in a range of 0.1ml to 5ml per 1 gram of combined weight of Curcumin and Epigallocatechin-3-gallate.
11. The process as claimed in claim 5, wherein the molecular complex of Curcumin and Epigallocatechin-3-gallate having curcumin water solubility at least 1 (ig/ml.

12. The process as claimed in claim 5, wherein the differential scanning calorimetry melting peak of molecular complex of Curcumin and Epigallocatechin-3-gallate are in the range of 162 °C to 166 °C.