CLIAMS:1. A food composition for serving as an exercise mimetic and reducing inflammation in mammalian cells, wherein the food composition comprises:
a) 0.2% to 2.0 % by weight of caffeic acid;
b) 8 % to 12 % by weight of carbohydrate; and
c) 12 % to 18 % by weight of protein
2. The food composition as claimed in claim 1, wherein the food composition comprises:
a) 0.2 % by weight of caffeic acid;
b) 10% by weight of carbohydrate; and
c) 16% by weight of protein.
3. The food composition as claimed in claim 1, wherein the composition further comprises vitamins , minerals, soluble fibres, sweeteners, flavouring agents and preservatives or combinations thereof.
4. The food composition as claimed in claim 3, wherein the vitamin is selected from a group comprising vitamin A, vitamin C, vitamin D, vitamin E, vitamin B complex and mixtures thereof.
5. The food composition as claimed in claim 3, wherein the mineral is selected from the group consisting of selenium, zinc, magnesium, iron, and calcium.
6. The food composition as claimed in claim 3, wherein the preservatives are selected from the group consisting of butylated hydroxyanisole, butylated hydroxytoluene , sulphur dioxide and sulphites, sorbic acid and its salts, benzoic acid and its salts.
7. The food composition as claimed in claim 1, wherein the composition is in the form of a solid.
8. The food composition as claimed in claim 1, wherein the composition is in the form of a liquid.
9. The food composition as claimed in claim 1, wherein the composition in the form of a bar, beverage or a confectionary.
10. The method for producing a food composition as claimed in any of the preceding claims.
,TagSPECI:As Attached

FIELD OF INVENTION
[0001] The present invention relates to food compositions comprising caffeic acid or analogs thereof having a dual role of serving as an exercise mimetic and reducing inflammation in body.
BACKGROUND OF THE INVENTION
[0002] The urban sedentary lifestyles have subjected people to increased risk of obesity, heart, and cardiovascular diseases among other health-related problems. In such a scenario, drugs that can replicate some fraction of the beneficial effects of exercise can serve as an attractive alternative. Exercise mimetic drugs or formulations are designed to chemically mimic and produce the benefits of exercise in humans without the need of any physical activity by the drug consumer. Exercise mimetics act by stimulating some of the molecular pathways that are activated by actual exercise.
[0003] Many proteins are known to modulate the benefits of physical exercise or activity. One of the major players is protein belonging to the sirtuins (silent information regulatory 2) family. The seven mammalian sirtuins, SIRT1 to SIRT7, have emerged as key metabolic sensors that directly link environmental signals to mammalian metabolic homeostasis and stress response.
[0004] SIRT1 is the best studied mammalian sirtuin and has been implicated in a variety of metabolic processes including hepatic lipid metabolism and gluconeogenesis, pancreatic insulin secretion, fat cell accumulation and maturation, central nutrient sensing, and circadian regulation of metabolism.
[0005] EP2548580 discloses a pharmaceutical composition containing an adiponectin receptor 1 agonist compound as an active ingredient for pseudo-exercise therapy capable of changing the physiological condition of a muscle to that after exercise without putting an exercise stress.
[0006] Although prior studies have identified a number of substances that can act as exercise mimetics, their development remains in the early stage and there exists a
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huge scope for exploring the plant derived compounds as the natural alternatives to chemical drugs.
SUMMARY OF THE INVENTION
[0007] This summary is not intended to identify essential features of the claimed subject matter nor is it intended for use in determining or limiting the scope of the claimed subject matter.
[0008] Accordingly it is an aspect of the present disclosure to provide a food composition for serving as an exercise mimetic and reducing inflammation in mammalian cells, wherein the food composition comprises 0.2% to 2.0 % by weight of caffeic acid; 8 % to 12 % by weight of carbohydrate complex; and 12 % to 18 % by weight of protein.
[0009] It is another aspect of the present invention to provide a method for producing the food composition according to the present disclosure.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[00010] The following drawings form a part of the present specification and are included to further illustrate various aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[00011] Figure 1 (a) shows the results of MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay for studying toxicity of caffeic acid on C2C12 lines. Also illustrated in figure 1 (b) is an image showing viable C2C12 cells in the presence of caffeic acid.
[00012] Figure 2 shows the results obtained from western blot analysis of Sirtuin1 proteins in C2C12 cells treated with different concentrations of caffeic acid for different time periods.
[00013] Figure 3 shows the graphical representation of the effect of caffeic acid on LPS induced NFκB p65 ser536 phosphorylation in macrophages.
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[00014] Figure 4 shows the graphical representation of the effect of caffeic acid on the phagocytic activity in human monocytic U937 cells differentiated in presence of phorbol myristate acetate (PMA).
DETAILED DESCRIPTION OF THE INVENTION
[00015] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention 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 two or more of said steps or features.
Definitions
[00016] For convenience, before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.
[00017] 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.
[00018] 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.”
[00019] 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.
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[00020] The terms "including" and "including but not limited to" are used interchangeably. The term "including" is used to mean "including but not limited to.”
[00021] The term “caffeic acid” refers to phenolic phytochemical found in all plants which is chemically known as 3, 4 dihydroxycinnamic acid.
[00022] The term “inflammation” refers to a complex immune response of vascular tissues that is directed against harmful stimuli and foreign particles such as pathogens, damaged cells or irritants.
[00023] The term “exercise mimetic” refers to a drug or a formulation designed to chemically mimic and produce the benefits of exercise in humans without the need of any physical activity by the consumer of the drug.
[00024] The term “food” or “food product” refers to liquid, semi-solid and/or solid food products such as nutritional compositions suitable for human and/or animal consumption.
[00025] 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 invention, as described herein.
[00026] The present disclosure provides a food composition comprising caffeic acid or analogues thereof having a dual role of functioning as an exercise mimetic and reducing inflammation in body. Since, caffeic acid possesses properties like solubility and bioavailability; it can be incorporated in food products or beverages. Such a product can provide multiple health benefits including antiaging, skincare, memory enhancement, protection against chronic diseases besides functioning as an exercise mimetic.
[00027] It was surprisingly found in the present disclosure that caffeic acid, a naturally occurring organic compound has the ability to activate sirtuin expression in muscle cell line (C2C12 cells). Since the activation of sirtuins leads to enhanced
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metabolism, caffeic acid can function as an exercise mimetic. Caffeic acid can be formulated into an exercise mimetic composition for incorporation into various food products including beverages with minimized side effects.
[00028] The toxicity of caffeic acid on C2C12 mammalian cell line was studied by performing the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolim bromide) assay. Results of the toxicity studies as illustrated in Figure 1 indicate that concentrations of caffeic acid of up to 400µM did not affect cell viability, and thus were safe for human consumption.
[00029] The present disclosure describes a food composition comprising caffeic acid that serves as an exercise mimetic. The ability of the ingredient to function as an exercise mimetic was identified through demonstration of sirtuin expression in C2C12 muscle cell line.
[00030] Concentrations of 25µM and 50 µM of caffeic acid were chosen for studying the expression levels of sirtuins in C2C12 lines. Further, proteins were extracted from the cells and their concentrations were estimated using the Bradford assay. The proteins were separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis and the SIRT 1 protein was detected by following the Western Blot protocol.
[00031] Figure 2 illustrates increase in the level of sirtuins in cells treated with the selected concentrations of caffeic acid. Also shown in figure 2 are seven lanes that correspond to different samples. Lane 1 denotes the purified sirtuin proteins. Lanes 2 and 3 denote control samples incubated for 24 hrs and 48 hrs respectively. Lanes 4 and 5 denotes protein extracts of cells treated with 25 µM of caffeic acid for time periods of 24 hrs and 48 hrs respectively. Lanes 6 and 7 denotes protein extracts of cells treated with 50 µM of caffeic acid for time periods of 24 hrs and 48 hrs respectively.
[00032] The present disclosure also describes a food composition comprising caffeic acid that reduces inflammation in the mammalian cells. The anti-inflammatory potential of caffeic acid has been shown by performing Phospho NFκB p65 (Rel A) ser
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536 assay and phagocytosis assay. Figure 3 demonstrates that caffeic acid inhibits NFκB p65 ser 536 phosphorylation in macrophage cells challenged with lipopolysaccahrides and is thus effective in reducing inflammation.
[00033] Further, the capability of caffeic acid to activate inflammatory molecules
and reduce the pathogen load has been demonstrated using the specialized phagocytic
assay. Figure 4 illustrates the role of caffeic acid in enhancing phagocytosis in
differentiated human monocytic U937 cells.
[00034] In one embodiment, the present disclosure provides a food composition
for serving as an exercise mimetic and reducing inflammation in mammalian cells,
wherein the food composition comprises 0.2 to 2.0% by weight of caffeic acid; 8 to 12
% by weight of carbohydrate; and 12 to 18 % by weight of protein.
[00035] In one embodiment, the present disclosure provides a food composition
for serving as an exercise mimetic and reducing inflammation in mammalian cells,
wherein the food composition comprises 0.2% by weight of caffeic acid, 10% by weight
of carbohydrate and 16% by weight of protein.
[00036] In another embodiment, the food composition according to the present
disclosure comprises carbohydrates including but not limited to natural or chemically
modified starch, maltodextrin, glucose polymers, sucrose, fructose, lactose, corn syrup,
and mixtures thereof.
[00037] In another embodiment, the food composition according to the present
disclosure comprises proteins including but not limited to casein, whey protein, rice
protein, soy based protein, egg derived protein, and mixtures thereof.
[00038] In another embodiment, the food composition according to the present
disclosure further comprises vitamins, minerals, soluble fibres, sweeteners, flavouring
agents and preservatives or mixtures thereof.
[00039] In another embodiment, the food composition according to the present disclosure comprises vitamins including but not limited to vitamin A, vitamin C,
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vitamin D, vitamin E, vitamin B complex, vitamin K, niacin, pantothenic acid, biotin, vitamin c, salts and derivatives thereof, and mixtures thereof.
[00040] In another embodiment, the food composition according to the present disclosure comprises minerals including but not limited to calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, sodium, potassium, molybdenum, chloride, selenium, chromium, chloride, salts and derivatives thereof, and mixtures thereof.
[00041] In another embodiment, the food composition according to the present disclosure comprises preservatives including but not limited to butylated hydroxyanisole, butylated hydroxytoluene, sulphur dioxide and sulphites, sorbic acid and its salts, benzoic acid and salts of benzoic acid and mixtures thereof.
[00042] In still another embodiment, the food composition according to the present disclosure comprises flavouring agents including but not limited to vanilla, caramel, rum, coffee, chocolate, fruit flavour and mixtures thereof.
[00043] In another embodiment, the food composition according to the present disclosure is in the form of a solid.
[00044] In another embodiment, the food composition according to the present disclosure possesses a liquid.
[00045] In yet another embodiment, the food composition according to the present disclosure is in the form of a ready to eat storage-stable food bar.
[00046] In yet another embodiment, the food composition according to the present disclosure is in the form of a drink or a beverage.
[00047] In yet another embodiment, the food composition according to the present disclosure is in the form of a confectionary product.
[00048] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. As
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such, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiment contained therein.
EXAMPLES
[00049] 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 the 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.
Example 1
Toxicity studies on C2C12 cells (MTT Assay)
[00050] 3-(4, 5–Dimethylthiazol-2-yl)-2,5-dipheyl tetrazolium bromide (MTT) assay, a calorimetric assay that detects the formation of purple formazan in living cells was performed by following the assay protocol (Mosmann, Journal of Immunological Methods, 1983,65:55) .The C2C12 cells were cultured in Dulbeco’s minimum essential medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and streptomycin using T-25 cm2 cell culture flasks/6 and 96 well plates. The cells were treated with caffeic acid upon reaching a confluence of around 80%. The treated and control cells were allowed to grow in the incubator that was set optimally for mammalian cell culture. Concentrations of 25 μM, 50 μM, 100 μM, 200 μM, 400 μM and 800 μM were used for performing the MTT assay.
[00051] After the time periods of 24 and 48 hours of treatment with caffeic acid, the cells were treated with 2 mg/ml of MTT. The cells were then incubated at 37°C for 30 min. To dissolve the resultant formazan crystals, 500 μl of DMSO (Dimethyl
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Sulfoxide) was added. The absorbance was measured at 570 nm using ELISA reader (Thermo Fisher).
[00052] As illustrated in figure 1, results of the assay clearly indicated that cells were capable of active growth at concentrations of caffeic acid of up to 400µM.
Example 2
Extraction of proteins from C2C12 cells
[00053] The C2C12 cells were prepared for protein extraction by removing the DMEM culture medium and transferring the cells to ice. The cells were then washed twice with ice cold 350 mM sucrose solution. Thereafter, the cells were scraped in 4 ml of 350 mM sucrose solution using a sterile cell scraper. The cells were pelleted by centrifugation at 4,000 rpm for 5 min. The resultant pellet was used for protein extraction.
[00054] The extraction was carried out by dissolving the cell pellet in 200 μl of RIPA buffer (1X RIPA Buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate). The mixture was then sonicated (at the rate of 3-4 bursts of 30 seconds each). The resultant cell lysate was then centrifuged at 10,000 rpm for 10 minutes. The supernatant was used for carrying out the proceeding experiments.
Example 3
Estimation of protein concentration and SDS-PAGE
[00055] The concentration of protein in the supernatant obtained by following the steps as explained in example 2 was estimated using the Bradford assay. Bradford assay was carried out using Cat.# B6916; Sigma Bradford reagent by following a 96 well plate assay protocol as recommended in the kit. After determination of the protein concentration, SDS-PAGE of the extracted proteins was carried out. An 8% polyacrylamide gel was prepared for separating the proteins and 30 μg of protein was
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loaded per well. The composition of the stacking and resolving gel is provided in Table 1 and Table 2.
Table 1: Composition of 8% resolving gel

Components Volume
30% Acryl amide solution 1.3ml
1.5M Tris-HCl pH 8.8 1.3ml
10% APS 50µl
10% SDS 50µl
TEMED 3µl
Milli-Q Water 2.3ml
Total 5.0 ml
Table 2: Composition of 5% stacking gel

Components Volume
30% Acryl amide solution 500µl
1.0M Tris-HCl pH 6.8 380µl
10% APS 30µl
10% SDS 30µl
TEMED 3µl
Milli-Q Water 2.1ml
Total 3.04ml
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Example 4
Western Blot of Sirt1 proteins
[00056] The proteins after being separated on polyacrylamide gels were transferred to nitrocellulose membranes. The nitrocellulose membrane, gel and filter papers were stacked and then blotted using a semi-dry blotting machine (TE 77 PWR) at constant voltage of 100 mA for 80 min. The membrane was blocked with 5% skim milk in tris buffered saline (pH 7.5) containing 0.05% Tween 20 (TBST) for 2 h. The membrane was then washed 3 times for 5 min with tris-buffered saline and Tween 20 (TBST).
[00057] The membrane was then probed with primary antibody rabbit polyclonal anti Sirt1. The antibody was mixed in 0.5% skim milk with a dilution of 1:1000 for 2 hours at 4°C. Following incubation with primary antibody, the blot was washed with TBST three times for 5 minutes each. The blot was then probed with secondary antibody goat anti rabbit IgG for 1 hour. The secondary antibody was also prepared in 0.5% skim milk in a dilution of 1:1000. After 1 hour of probing with secondary antibody, the blot was developed using the commercially available Western Lightening Plus-ECL kit. The components of the Lightening Plus-ECL kit namely enhanced luminol reagent plus and the oxidizing reagent plus were mixed in equal proportion (1:1) and probed on the membrane, covering it completely and kept in dark for 5 minutes. After 5 minutes, the membrane was tilted to run-off the mixture and the membrane were visualized in the Chemidoc XRS (BioRad) gel documentation system (molecular weight of Sirt1: 97 kiloDalton).
[00058] An increase in the level of sirtuins in cells treated with caffeic acid was observed as is illustrated in Figure 2.
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Example 5
Performance of Phospho-NFκB p65 (RelA) ser 536 assay:
[00059] NFκB p65 ser 536 phosphorylation was stimulated with LPS (1 μg/ml). This treatment increases the inflammatory conditions within 90 min. Detection was done using sandwich ELISA. The media was removed from the cells and rinsed with ice cold 1x PBS. Cells were lysed with 0.5 ml ice cold 1x cell lysis buffer and incubated on ice for 5 minutes. The scraped cells were transferred to a tube and lysed. The lysate was then spun at 14,000 rpm at 4°C and transferred to a new tube. Cell lysates when needed were diluted with sample diluents. The lysates were then transferred to 96 well plates and incubated overnight at 37°C. Next, washing of the wells was done with 1 x wash buffer. To each well, 100 μl of primary antibody was added and the plate was incubated at 37°C for 1 hour. The wells were then washed again and 100 µl of HRP-Linked secondary antibody was added to each well. The plate was then incubated for 30 minutes at 37°C. The wash step was repeated and 100 µl of TMB (3, 3, 5, 5’-tetramethylbenzidine) buffer was added. Finally the stop solution was added and absorbance read at 450 nm.
[00060] As illustrated in figure 3, the results clearly demonstrate that caffeic acid can reduce inflammation in cells challenged with lipopolysaccharides by inhibiting NFκB p65 ser536 phosphorylation.
Example 6
Performance of phagocytosis assay
Cell culture
[00061] U937 human monocyte cells (1 – 5 x 105cells/mL) were cultured in RPMI 1640 medium (containing 10% heat-inactivated fetal calf serum, 1% of penicillin/streptomycin) in a 96-well plate and incubated overnight. Macrophage
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differentiation was induced by the addition of phorbol-12-myristate-13-acetate (PMA) (10-20 ng/ml) to the culture medium.
Performance of the assay
[00062] The phagocytosis assay was carried out using the components of Cytoselect 96 well phagocytosis assay kit (Cat. # CBA-224; Cell Biolabs Inc.)
[00063] After 4 days of treatment with phorbol-12-myristate-13-acetate, the adherent, differentiating macrophages were treated with caffeic acid (25µM) and cytochalasin D inhibitor (1µM). Further, 10μL of zymosan suspension was added to each well and incubated for 2 hours. The culture medium was removed and 200 μL of cold, serum-free medium was added to each well. 100 μL of fixation solution as provided in the kit, was then added to each well and incubated for 5 minutes at room temperature. The fixation solution was then removed and washed twice with 1X PBS. 100μL of prediluted 1X blocking reagent as provided in the kit was added to each well and the plate was incubated for 60 minutes at room temperature on an orbital shaker. The blocking was performed quickly and plate washed three times with 1X PBS. 100μL of prediluted 1X permeabilization solution as provided in the kit, was added to each well which was then incubated for 5 minutes at room temperature. The cells were washed once with 1X PBS. 100 μL of prediluted 1X detection reagent was added to each well and incubated for 60 minutes at room temperature on an orbital shaker. The detection reagent as provided in the kit was removed and washed three times with 1X PBS. 50μL of detection buffer as provided in the kit was added to each well and the plate was incubated for 10 minutes at room temperature on an orbital shaker. The readings were taken at 405 nm. The results of the assay clearly demonstrate that caffeic acid increases the phagocytic activity of the differentiated human monocytic U937 cells, as illustrated in figure 4.
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Example 7
Components in the food composition
[00064] In view of the exercise-mimetic and anti-inflammatory properties shown by caffeic acid as described in previous examples, it was combined with other nutritional products to prepare a food composition. In one embodiment, the food composition according to the present disclosure has a composition as provided in Table 3.
Table 3: Major components of the food composition

S.No. Component Weight (g)/45 grams Percentage Composition
1. Total Carbohydrates 3.6 – 5.4 8.0% – 12.0%
2. Total Fat 1.0 – 2.0 2.2% – 4.4%
3. Protein 5.4 – 8.1 12.0 %– 18.0 %
4. Caffeic acid 0.09 – 0.9 0.2% – 2.0%
5. Plant based extracts including
Resveratrol, Pterostilbene,
Quercetin, Guarana 0.225 – 0.9 0.5% – 2.0%
6. Vitamins 0.0115 – 0.023 10.0% – 20.0%
7. Minerals 2.7 – 4.05 6.0 %– 9.0%
8. Dietary fibers 3.6 – 5.4 8.0 %– 12.0 %
9. Water/Flavouring agents/ Preservatives including butylated hydroxyanisole , butylated hydroxytoluene , sulphur dioxide and sulphites, sorbic acid and its salts, benzoic acid and its salts 28.373 – 18.227 53.1% – 20.6%
TOTAL 45 grams 100%
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[00065] It is provided that in another embodiment of the food composition according to the present disclosure, the combination of products as described above can be varied to suit the health and body weight of the consumer.
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