FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
1. Title of the invention: A COMPOSITION COMPRISING PTEROSTILBENE, AND
COENZYME Q10 AND USES THEREOF
2. Applicant(s)

NAME

NATIONALITY

ADDRESS

ITC LIMITED

Indian

ITC LIFE SCIENCE AND TECHNOLOGY CENTRE #3, 1st Main, Peenya Industrial Area, Phase 1, Bangalore 560 058, India

3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.

FIELD OF INVENTION
[0001] The present disclosure relates to a composition for enhanced levels of proteins involved in promoting metabolic health. In particular, the disclosure relates to a composition comprising pterostilbene, and coenzyme Q10 for enhancing levels of proteins involved in positive regulation of metabolic health.
BACKGROUND OF THE INVENTION
[0002] Coenzyme Q10, also known as ubiquinone, is a lipid-soluble benzoquinone having 10 isoprenyl units in the side chain. It is a key component of the mitochondrial respiratory chain for adenosine triphosphate (ATP) synthesis (Bor-Jen Lee et ah, The Scientific World Journal, 2012, 792756; Ernster et ah, Biochimica et Biophysica Acta., 1995, 1271(1), 195-204). Coenzyme Q10 is also known for its intracellular antioxidant property that protects the membrane phospholipids, mitochondrial membrane protein, and low-density lipoprotein-cholesterol (LDL-C) from damage due to respiratory chain impairment and free radical- induced oxidative damage (Orsucci et ah, Curr Med Chew., 2011, 18(26), 4053-4064; Bhagavan et ah, Free Radical Research, 2006, 40(5), 445-453). Mitochondrial respiratory chain impairment and increased production of reactive oxygen species have been implicated in neuromuscular, cardiovascular and other disorders. Scientific studies have proved the inverse relation between the plasma coenzyme Q10 concentration and risk of coronary artery diseases (CAD) (Alleva et ah, Proceedings of the National Academy of Sciences of The United States of America, 1995, 92(20), 9388-9391; Singh et ah, Nutrition Reviews, 2007, 65(6), 286-293; Littaru et ah, International Journal for Vitamin and Nutrition Research, 1972, 42(2), 291-305; Sarter, The Journal of cardiovascular nursing, 2002, 16(4), 9-20).
[0003] Pterostilbene is chemically related to resveratrol and it belongs to the group of phytoalexins, agents produced by plants to fight infections (Langcake et ah, Experientia, 1977, 33(2), 151-152). This compound is a double-methylated version of resveratrol exhibiting a higher bioavailability as it is more easily transported into the cell and more resistant to degradation and elimination. Animal studies showed anti-hypercholesterolemia and anti-hypertriglyceridemia properties, as well as the ability to fight off and reverse cognitive decline. It is believed that the compound also has anti-diabetic properties, but so far very little has been studied on this aspect. Pterostilbene has anti-inflammatory, antineoplastic, and antioxidant

actions via modulations of gene expression and enzyme activity (Kapetanovic et al, Cancer Chemotherapy and Pharmacology, 2010, 68(3), 593-601).
[0004] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bis-phosphoglycerate an important step in glycolysis (Saunders et al, J Neurochem., 1997, 69, 1820-1828; Hara et al, Biochim Biophys Acta, 2006, 1762, 502-509). Recent studies have shown that GAPDH is a protein with multiple cytoplasmic, membrane, and nuclear functions and is a major intracellular messenger mediating apoptosis of cells. Increased mitochondrial superoxide production inactivates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vascular endothelial cells, and inhibition of GAPDH is postulated to activate some of the key pathways that are associated with the development of diabetes complications, including increased formation of advanced glycation end products (AGEs) and activation of protein kinase C (PKC) and hexosamine pathway (Du et al, J Clin Invest, 2003, 112, 1049 -1057; Brownlee, Diabetes, 2005, 54, 1615-1625).
[0005] TNF receptor-associated protein 1 (TRAP 1) also known as HSP75, which belongs to Heat shock protein family and is known to reduce the risk of Cardiac hypertrophy, a major determinant of heart failure (studies on mice has showed that over expression of HSP75 prevented cardiac hypertrophy and fibrosis). Also known to prevent hypoxia induced damage to cardiomyocytes and acts as antagonist of ROS protecting cells from Granzyme M mediated apoptosis (Zhang et al., J Cell Biochem, 2011, Jul; 112(7), 1787-1794; Hua et al., J. Biol. Chem, 2007, 282, 20553-20560; Xiang etal, FEBSJournal, 2010, 277, 1929-1938). [0006] PCT/IB2012/052904 describes a process for production of a grape based, fermented alcoholic beverage with high resveratrol content.
[0007] PCT/EP2011/056645 describes nutritional compositions including branched chain fatty acids for wound healing.
SUMMARY OF INVENTION
[0008] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is

not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
[0009] An aspect of the present disclosure relates to a composition comprising coenzyme Q10, and pterostilbene, wherein said composition enhances levels of proteins involved in promoting metabolic health.
[00010] Another aspect of the present disclosure relates to a method of enhancing levels of proteins involved in promoting metabolic health, said method comprising (a) obtaining a composition comprising pterostilbene, and coenzyme Q10, and (b) contacting said composition from (a) with a host, wherein w/w ratio of coenzyme Q10 to pterostilbene in said composition is in the range of approximately 5:1-2:1.
[00011] Yet another aspect of the present disclosure relates to a formulation for oral ingestion, said formulation comprising a composition comprising coenzyme Q10, and pterostilbene, wherein said composition enhances levels of proteins involved in promoting metabolic health. [00012] An aspect of the present disclosure relates to a method of enhancing levels of proteins involved in promoting metabolic health, said method comprising (a) obtaining a formulation comprising a composition, said composition comprising i) pterostilbene, and ii) coenzyme Q10, and (b) contacting said composition from (a) with a host, wherein w/w ratio of coenzyme Q10 to pterostilbene in said formulation is in the range of approximately 5:1-2:1.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[00013] The following drawings form part of the present specification and are included to
further illustrate aspects of the present disclosure. The disclosure may be better understood by
reference to the drawings in combination with the detailed description of the specific
embodiments presented herein.
[00014] Figure 1 depicts the quantitative fold change in levels of GAPDH upon treatment of
host cells with composition comprising pterostilbene, and coenzyme Q10, in accordance with an
embodiment of the present disclosure.
[00015] Figure 2 depicts the quantitative fold change in levels of HSP75 upon treatment of host
cells with composition comprising pterostilbene, and coenzyme Q10, in accordance with an
embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[00016] Those skilled in the art will be aware that the present disclosure is subject to variations
and modifications other than those specifically described. It is to be understood that the present
disclosure includes all such variations and modifications. The disclosure also includes all such
steps, features, compositions and compounds referred to or indicated in this specification,
individually or collectively, and any and all combinations of any or more of such steps or
features.
Definitions
[00017] For convenience, before further description of the present disclosure, certain terms
employed in the specification, and examples are collected here. These definitions should be read
in the light of the remainder of the disclosure and understood as by a person of skill in the art.
The terms used herein have the meanings recognized and known to those of skill in the art,
however, for convenience and completeness, particular terms and their meanings are set forth
below.
[00018] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at
least one) of the grammatical object of the article.
[00019] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning
that additional elements may be included. It is not intended to be construed as "consists of only".
[00020] Throughout this specification, unless the context requires otherwise the word
"comprise", and variations such as "comprises" and "comprising", will be understood to imply
the inclusion of a stated element or step or group of element or steps but not the exclusion of any
other element or step or group of element or steps.
[00021] The term "including" is used to mean "including but not limited to". "Including" and
"including but not limited to" are used interchangeably.
[00022] 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 this disclosure
belongs. Although any methods and materials similar or equivalent to those described herein can
be used in the practice or testing of the disclosure, the preferred methods, and materials are now
described. All publications mentioned herein are incorporated herein by reference.
[00023] A composition comprising "synergistic activity" or a "synergistic composition" is a
combination of compounds which exhibits increased biological or functional activity as a non-

linear multiple of the biological or functional activity of the individual compounds. In other words, the combined biological or functional activity of two or more compounds being tested is significantly greater than the expected result based on independent effects of the compounds when tested separately. Synergy may be apparent only at some ranges or concentrations. [00024] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
[0001] In an embodiment of the present disclosure, there is provided a composition comprising coenzyme Q10, and pterostilbene, wherein said composition enhances levels of proteins involved in promoting metabolic health.
[0002] In an embodiment of the present disclosure, the protein involved in promoting metabolic health is HSP75.
[0003] In an embodiment of the present disclosure, the protein involved in promoting metabolic health is GAPDH.
[0004] In an embodiment of the present disclosure, the w/w ratio of coenzyme Q10 to pterostilbene is in the range of approximately 5:1-2:1.
[0005] In a preferred embodiment of the present disclosure, the w/w ratio of coenzyme Q10 to pterostilbene is approximately 3.4:1.
[0006] In an embodiment of the present disclosure, the molar ratio of coenzyme Q10 to pterostilbene is in the range of approximately 0.25:1-1.5:1.
[0007] In a preferred embodiment of the present disclosure, the molar ratio of coenzyme Q10 to pterostilbene is 1:1.
[0008] In an embodiment of the present disclosure, there is provided a method of enhancing levels of proteins involved in promoting metabolic health, said method comprising (a) obtaining a composition comprising coenzyme Q10, and pterostilbene as described herein, and (b) contacting said composition from (a) with a host.
[0009] In an embodiment of the present disclosure, the host is a mammal.
[00010] In an embodiment of the present disclosure, the weight percentage of pterostilbene in a composition as described herein is in the range of 1.6]ig/ml-4.8]ig/ml of said composition.

[00011] In a preferred embodiment of the present disclosure, the weight percentage of
pterostilbene in a composition as described herein is 3.2]ig/ml of said composition.
[00012] In an embodiment of the present disclosure, the weight percentage of coenzyme Q10 in
a composition as described herein is in the range of 5.4|ig/ml-16.2|ig/ml of said composition.
[00013] In a preferred embodiment of the present disclosure, the weight percentage of
coenzyme Q10 in a composition as described herein is 10.79]ig/ml of said composition.
[00014] In an embodiment of the present disclosure, there is provided a composition as
described herein, further comprising suitable carriers, diluents, and excipients for oral ingestion.
[00015] In an embodiment of the present disclosure, coenzyme Q10 at a concentration of
12.5]iM enhances protein levels of GAPDH by approximately 1.4 fold.
[00016] In an embodiment of the present disclosure, pterostilbene at a concentration of 12.5]iM
enhances protein levels of GAPDH by approximately 1.75 fold.
[00017] In an embodiment of the present disclosure, coenzyme Q10 at a concentration of
10.79]ig/ml enhances protein levels of GAPDH by approximately 1.4 fold.
[00018] In an embodiment of the present disclosure, pterostilbene at a concentration of
3.2]ig/ml enhances protein levels of GAPDH by approximately 1.75 fold.
[00019] In an embodiment of the present disclosure, coenzyme Q10 at a concentration of
12.5]iM enhances protein levels of HSP75 by approximately 0.75 fold.
[00020] In an embodiment of the present disclosure, pterostilbene at a concentration of 12.5]iM
enhances protein levels of HSP75 by approximately 1.6 fold.
[00021] In an embodiment of the present disclosure, coenzyme Q10 at a concentration of
10.79]ig/ml enhances protein levels of HSP75 by approximately 0.75 fold.
[00022] In an embodiment of the present disclosure, pterostilbene at a concentration of
3.2]ig/ml enhances protein levels of HSP75 by approximately 1.6 fold.
[00023] In an embodiment of the present disclosure, there is provided a composition comprising
coenzyme Q10,and pterostilbene that synergistically enhances protein levels of GAPDH.
[00024] In an embodiment of the present disclosure, there is provided a composition comprising
coenzyme Q10,and pterostilbene that synergistically enhances protein levels of HSP75.
[00025] In a preferred embodiment of the present disclosure, there is provided a composition
comprising coenzyme Q10, and pterostilbene, wherein the w/w ratio of coenzyme Q10 to

pterostilbene is approximately 3.4:1, and wherein said composition synergistically enhances
protein levels of GAPDH.
[00026] In a preferred embodiment of the present disclosure, there is provided a composition
comprising coenzyme Q10, and pterostilbene, wherein the w/w ratio of coenzyme Q10 to
pterostilbene is approximately 3.4:1, and wherein said composition synergistically enhances
protein levels of HSP75.
[00027] In a preferred embodiment of the present disclosure, there is provided a composition
comprising coenzyme Q10, and pterostilbene, wherein the molar ratio of coenzyme Q10 to
pterstilbene is 1:1, and wherein the molar concentration of coenzyme Q10, and pterostilbene is
12.5]iM, wherein said composition synergistically enhances levels of GAPDH.
[00028] In a preferred embodiment of the present disclosure, there is provided a composition
comprising coenzyme Q10, and pterostilbene, wherein the molar ratio of coenzyme Q10 to
pterstilbene is 1:1, and wherein the molar concentration of coenzyme Q10, and pterostilbene is
12.5]iM, wherein said composition synergistically enhances levels of HSP75.
[00029] In a preferred embodiment of the present disclosure, there is provided a composition
comprising coenzyme Q10, and pterostilbene, wherein the concentration of coenzyme Q10 is
10.79]ig/ml, and the concentration of pterstilbene is 3.2]ig/ml, wherein said composition
synergistically enhances levels of GAPDH.
[00030] In a preferred embodiment of the present disclosure, there is provided a composition
comprising coenzyme Q10, and pterostilbene, wherein the concentration of coenzyme Q10 is
10.79]ig/ml, and the concentration of pterostilbene is 3.2]ig/ml, wherein said composition
synergistically enhances levels of HSP75.
[00031] In an embodiment of the present disclosure, there is provided a formulation for oral
ingestion, said formulation comprising a composition comprising pterostilbene, and coenzyme
Q10, wherein said composition enhances levels of proteins involved in promoting metabolic
health.
[00032] In an embodiment of the present disclosure, there is provided a formulation as
described herein, said formulation further comprising of suitable carriers, diluents, and
excipients.
[00033] In an embodiment of the present disclosure, there is provided a method of enhancing
levels of proteins involved in promoting metabolic health, said method comprising (a) obtaining

a formulation comprising a composition, said composition comprising i) pterostilbene, and ii) coenzyme Q10, and (b) contacting said composition from (a) with a host, wherein w/w ratio of coenzyme Q10 to pterostilbene in said formulation is in the range of approximately 5:1-2:1. [00034] In a preferred embodiment of the present disclosure, there is provided a method of enhancing levels of proteins involved in promoting metabolic health, said method comprising (a) obtaining a formulation comprising a composition, said composition comprising i) pterostilbene, and ii) coenzyme Q10, and (b) contacting said composition from (a) with a host, wherein w/w ratio of coenzyme Q10 to pterostilbene in said formulation is in the range of approximately 3.4:1.
EXAMPLES
[00035] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. Example 1
Materials and Methods
[00025] Cell Culture: HepG2 cells (ATCC Catalog number HB-8065) grown in DMEM media (Gibco, Life Technologies Catalog number 12800-017) supplemented with 10% FBS (Gibco, Life Technologies Catalog number 10100-147) were seeded in T 25 flasks and the treatment with test ingredients was given overnight and incubated at 37°C with 5% CO2. A final concentration of 25uM of individual compounds [coenzyme Q10 (CoQIO and pterostilbene] and combination of both (12.5uM of CoQIO + 12. 5uM of pterostilbene) were added to the complete medium and further incubated for 24-hours. The cells were then washed with IX PBS and trypsinised and collected in 1.5ml centrifuge tubes for further protein extraction.

[00026] Protein extraction and SDS page gel analysis: The cells were lysed and total protein was extracted using Mike's special lysis buffer (25mM Tris pH 7.4, 150mM NaCl, 1% Triton X100, ImM EDTA pH 8, ImM DTT, ImM PMSF) supplemented with Roche protease cocktail inhibitor complex (Catalog number 04693159001). The treated cells were lysed by adding 250 jil of above buffer followed by sonication for three cycles of pulsing for 1 minute with an interval of five minutes each. Cell debris was removed by centrifuging at 14000g for 10 minutes and the supernatant was collected for proteomic study.
[00027] The protein content in the samples was determined using Pierce BCA protein estimation kit. Equal amounts of protein were loaded on to the SDS-PAGE gel and proteins were electrophoretically separated using 90V current. The gel was then stained with Gel code stain from Sigma to visualize protein bands.
[00028] Protein analysis using LTQ ORbitrap Velos: Protein analysis using LTQ ORbitrap Velos: The gel was then destained with distilled water and protein bands in the gel were cut into small pieces for protein in-gel digestion and extraction. The proteins in the gel were digested using MS grade trypsin (Promega Catalog number VS280) and extracted. The peptides were then subjected to LTQ orbitrap analysis using Ion trap method and the collected signals were analyzed using proteome discover software to get the protein ID's using Bioinformatics tools. Quantitative approach of analysis was done and the results are shown for our protein of interest in the results section. The proteomics workflow is summarized below in Scheme 1. [00029] Table 1

Example 2
Results of proteomics workflow
[00030] The combinational studies using coenzyme Q10 and pterostilbene on HepG2 cells was
performed using two-dimension LC MS/MS approach. A total of 6475 [Control-1835, CoQIO
treated-1402, Pterostilbene treaed-1570 and combination (COQ10+Ptrostilbene)-1668] proteins
were identified across all four samples tested. Out of the total proteins identified, 467 proteins
were common across all of the samples. GAPDH and TRAP1/HSP75, which are known to play
key role in maintaining metabolic health, showed upregulation of expression in comparison with
others (control and individual treatments).

[00031] Table 2 depicts the quantitative fold change in GAPDH protein levels under various experimental conditions. Figure 1 depicts the graphical representation of GAPDH protein level changes under various experimental conditions. As seen in Figure 1, both coenzyme Q10 and pterostilbene can both individually enhance expression of GAPDH protein. However, surprisingly, a combination of coenzyme Q10, and pterostilbene synergistically enhances the levels of GAPDH. The combination enhances the levels of GAPDH by approximately 4.4 fold compared to the additive effect of coenzyme Q10, and pterostilbene alone (additive effect is approximately 3.1 fold increase).
[00033] Table 3 depicts the quantitative fold change in HSP75 protein level under various experimental conditions. Figure 2 depicts the graphical representation of HSP75 protein level changes under various experimental conditions. As seen in Figure 2, pterostilbene can enhance expression of HSP75. However, surprisingly, a combination of coenzyme Q10, and pterostilbene synergistically enhances the levels of HSP75. The combination enhances the levels of HSP75 by approximately 4 fold compared to the additive effect of coenzyme Q10, and pterostilbene alone (additive effect is approximately 2.3 fold increase).
[00035] Overall, the data suggest that a combination of pterostilbene and coenzyme Q10 at specific ratios as described within the specification, surprisingly exhibits a synergistic effect in

enhancing the levels of proteins involved in promoting metabolic health, specifically GAPDH, andHSP75.

I/We claim:
1. A composition comprising
a. coenzyme Q10; and
b. pterostilebene,
wherein said composition enhances levels of proteins involved in promoting metabolic health.
2. The composition as claimed in claim 1, wherein the w/w ratio of coenzyme Q10 to pterostilbene is in the range of approximately 5:1-2:1.
3. The composition as claimed in claim 1, wherein the molar ratio of coenzyme Q10 to pterostilbene is in the range of approximately 0.25:1-1.5:1.
4. The composition as claimed in claim 1, wherein the protein is selected from the group consisting of GAPDH, and HSP75.
5. A formulation for oral ingestion, said formulation comprising a composition as claimed in claim 1.
6. The formulation as claimed in claim 5, further comprising of suitable carriers, diluents, and excipients.
7. The formulation as claimed in claim 5 or 6, wherein w/w ratio of coenzyme Q10 to pterostilbene is in the range of approximately 5:1-2:1.
8. A method of enhancing levels of proteins involved in promoting metabolic health, said method comprising:
a. obtaining a composition as claimed in claim 1; and
b. contacting said composition from (a) with a host,
wherein w/w ratio of coenzyme Q10 to pterostilbene in said composition is in the range of approximately 5:1-2:1.
9. A method of enhancing levels of proteins involved in promoting metabolic health, said
method comprising:

a. obtaining a formulation as claimed in claim 5; and
b. contacting said formulation from (a) with a host,
wherein w/w ratio of coenzyme Q10 to pterostilbene in said formulation is in the range of approximately 5:1-2:1.

The present disclosure relates to a composition comprising pterostilbene and coenzyme Q10 for enhanced expression levels of proteins involved in promoting metabolic health. The disclosure also provides methods and formulations for promoting metabolic health by enhancing the levels of proteins involved in the same.