Methods for improving physical performance and capsicum compositions used therein

Abstract

Methods of improving physical performance and endurance include administering a capsicum composition to a subject. More particularly the methods include administration of an effective amount of a capsicum composition including capsaicinoids alone and/or in combination with other nutrients for improvement in performance of physical activities. The methods described include administration of a capsicum composition to a subject undergoing physical activity to deliver daily dose of capsaicinoids in an effective amount. The methods include administering a capsicum composition to a subject in an effective amount to reduce blood lipids and oxidative stress. Methods for enhancing physical endurance and exhaustion time include administering an effective amount of a capsicum composition to a physically active subject, wherein body antioxidants are increased and muscle lactates are decreased, resulting in recovery from muscle fatigue arising due to such indoor and/or outdoor physical activities.

Claims

1. A method for improving physical performance, comprising: administering, to a subject undergoing physical activity, a capsicum composition comprising a combination of capsaicinoids in an effective amount, the combination of capsaicinoids consisting of capsaicin, dihydrocapsaicin, and nordihydrocapsaicin, wherein the effective amount is a daily dose of about 0.01 mg/kg body weight to 1 mg/kg body weight of the combination of capsaicinoids, wherein the physical activity is treadmill exercise and wherein said amount of the combination of capsaicinoids is effective in decreasing levels of muscle lactates and muscle malondialdehyde (MDA) and increasing levels of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the subject as compared to a subject that has not been administered with the capsicum composition.

2. The method as claimed in claim 1, wherein the subject is a human being.

3. The method as claimed in claim 1, wherein the capsicum composition contains the combination of capsaicinoids formulated with at least one pharmaceutically and/or nutraceutically acceptable excipient selected from the group consisting of a sugar, a surfactant, and a polymer.

4. The method as claimed in claim 1, wherein the amount of the combination of capsaicinoids is effective in increasing levels of IGF-1 and decreasing release of cortisol in the subject as compared to a subject that has not been administered with the capsicum composition.

5. The method as claimed in claim 1, wherein the amount of the combination of capsaicinoids is effective in increasing levels of pAMPK (phosphorylated AMP-activated protein) and Nrf2 (Nuclear factor [erythroid-derived 2]-like 2) in the subject as compared to a subject that has not been administered with the capsicum composition.

6. The method as claimed in claim 1, wherein the subject has a BMI ranging from 18.3 to 30.0 and wherein the effective amount is about 2 mg of the combination of capsaicinoids.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effect of capsicum composition on mitochondrial respiration.

(2) FIG. 2 shows the effect of capsicum composition on mitochondrial mass in cell-line study.

(3) FIG. 3 shows the effect of capsicum composition on IGF-1 in cell-line study.

(4) FIG. 4 shows the effect of capsicum composition on cortisol release as sports nutrition biomarker.

(5) FIG. 5 shows an evaluation of the effect of capsicum composition in protein levels in all 4 groups.

(6) FIG. 6 shows the effect of capsicum composition on relative gene expression.

DETAILED DESCRIPTION

(7) Methods and compositions herein are for improving exercise performance and endurance, such as by administering a capsicum composition in an effective amount to an exercising subject.

(8) It is to be appreciated that the term ‘capsicum composition’ can be interpreted to be within the scope of the compositions which are comprised of capsicum extract or the extract formulated in a solid dosage form using food grade or pharmaceutically acceptable excipients. The compositions are comprised of compounds such as capsaicinoids and may be further also comprised of non-capsaicinoids. Capsaicinoids may be selected from the group of, but not limited to, the compounds such as capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, dihydrohomocapsaicin, 6″,7″-dihydro-5′,5″′-dicapsaicin, 5,5′-dicapsaicin, and mixtures thereof. Non-capsaicinoid compounds may be selected from flavonoids and saponins, and mixtures thereof. Capsicum compositions herein include capsaicinoids and can be used in the form of an extract or formulated as an extended and sustained release stable, free flowing, solid composition suitable for formulating into tablets, capsules, blended powders, licaps, ointments, pastes, lotions, liniments, mouthwashes, gargles, consumable dry syrups, liquid syrups, health drinks, diet drinks, fruit juices, soft drinks and the like. A capsicum composition herein may be prepared by solvent extraction to get capsicum extract comprising of capsaicinoids. The capsicum extract may be administered or may be formulated in the form of a solid dosage form, convenient to administer to exercising subjects as per methods described herein. The formulation may be prepared as extended and sustained release beadlets or spray dried powder using the methods known to person skilled in the art.

(9) It is to be appreciated that the term ‘effective amount’ can be interpreted to be within the scope of amount of capsaicinoids comprised in capsicum composition, which is administered to exercising subjects, such that it is useful to bring out the desired effect and also avoids any adverse effect, over the time duration which is recommended for administration of the composition to the subjects. As per the methods described herein, the dose is delivered in the range of at or about 0.01 mg/kg body weight to at or about 200 mg/kg body weight of capsaicinoids, or at or about 10 mg/kg body weight/day to at or about 100 mg/kg body weight/day of capsaicinoids to a subject undergoing physical indoor and/or outdoor activities. The effective amount may vary depending upon the age and body weight of the subject or depending upon the type of activity the subject is undergoing such as sports. In such cases capsicum compositions may be administered with one or more other nutrients such as proteins, essential amino acids, or muscle stimulants, which may effectively lower the daily dose of the capsicum composition.

(10) In an embodiment, a method for improving physical performance and endurance includes administering a capsicum composition in an effective daily dose including an amount of capsaicinoids, to a subject undergoing indoor or outdoor physical activities.

(11) It is also to be appreciated that the term ‘exercising subject or the subject undergoing physical activity’ as used herein can be interpreted to mean that it is the practical or experimental condition for evaluating effect of capsicum composition in terms of exercise performance and endurance, wherein the composition is administered to the subjects who are undergoing exercise at the time of evaluation. It is also the condition which is advised as per the methods described herein, to achieve the intended result from administration of capsicum compositions in effective dose. In an embodiment, subjects, who are administered the capsicum composition are also subjected to specific type and duration of exercise. The effect of capsicum composition and exercise in combination is evaluated according to the method described herein. The term can be also interpreted as the recommended condition for the subjects who are administered with capsicum composition, so that the desired effect is obtained. In an embodiment, the subjects who are doing physical indoor and/or outdoor activities such as sports, exercise, or strenuous other activities benefit more due to administration of capsicum compositions of the invention.

(12) Such benefits of capsicum composition administration are observed in exercising subjects as compared to those who are administered the composition but not doing any physical activity or exercise, and as compared to subjects who are exercising but not administered with capsicum composition.

(13) In an embodiment, the level of activity as shown for example in the in-vivo study, can include treadmill running for a specific time interval, e.g. about 15 to 20 minutes, can be called a minimum duration of exercise activity such as in an animal model. In an embodiment, walking for a human can include about 30 minutes, and can be considered as a minimum duration of exercise activity, and where such intensity can vary from person to person.

(14) The performance duration, endurance and stamina of such physically active subjects is enhanced because of administration of an effective amount of capsicum composition including capsaicinoids. However compositions described herein can be also administered to a subject at rest and can be asked to have their daily activities to get desired benefit or to increase stamina required for some physical strenuous activities. In its still wider meaning, the term ‘exercising subject’ also means that the subject is carrying out some type of physical indoor and/or indoor activity such as sports, exercise and other strenuous activities selected from the group of, but not limited to aerobic exercises, anaerobic exercises, fitness exercises, trekking, jogging, staircase climbing, jumping, walking and the like or the combinations thereof.

(15) Capsicum compositions as described herein may be administered alone or in combination with one or more other nutrients, health supplements, muscle enhancers, stimulants, essential amino acids and/or anabolic ingredients for improving the performance and endurance during indoor and/or outdoor activities such as for example exercise, sports or other activities.

(16) Capsicum compositions herein are obtained from natural resources by human intervention and are safe for administration.

(17) In an embodiment, a method for improving exercise performance and endurance includes administering an effective amount of capsaicinoids from a capsicum composition to an exercising subject. According to the method, when capsicum composition is administered to deliver an effective amount of capsaicinoids to a subject undergoing indoor or outdoor physical activity, such as for example exercise or sports, improvement in performance as well as endurance are observed. The method results in improvement in fatigue suppression during endurance exercise and post-exercise recovery from muscle fatigue and/or muscle soreness.

(18) In an embodiment, the composition as described herein may be comprised of capsicum extract alone or capsicum extract formulated as an extended and sustained release stable, free flowing, solid composition including, in some embodiments, capsaicinoids and/or derivatives, and employing at least one or more food grade excipients.

(19) By “derivatives” it is meant as compounds derived or obtained from parent compounds by replacement of one atom with another atom or group of atoms. For example, derivatives of capsaicinoids can be nor-capsaicinoids.

(20) In an embodiment, a capsicum composition includes a capsicum extract prepared for example from various varieties of paprika. In an embodiment, a capsicum composition includes chemical constituents including capsaicinoids and one or more other compounds such as non-capsaicinoids.

(21) In an embodiment, a capsicum extract is prepared from various varieties of paprika selected from the group of, but not limited to, varieties such as for example Teja and Namdhari. In an embodiment, the composition herein includes a capsicum extract obtained from varieties of Capsicum annum such as Teja and Namdhari. In an embodiment, an industrially viable process is provided for preparation of a capsicum composition by employing non-polar, semi-polar, polar solvents or combinations thereof in suitable ratios. In an embodiment, a capsicum composition is prepared using a combination of polar and non-polar solvents in a suitable ratio for an extraction cycle, followed by purification of the extract by using polar solvent(s) to obtain the extract. The composition is safe for human consumption. In an embodiment, the composition includes a biologically active chemical constituent including capsaicinoids and in some embodiments, further includes one or more other compounds such as non-capsaicinoids.

(22) The methods described herein include administering an effective amount of a capsicum composition in the form of a capsicum extract or in the form of the extract formulated using at least one pharmaceutically and/or nutraceutically acceptable excipient to obtain a stable dosage form, convenient for administration. The excipient may be selected from the group of, but not limited to a carrier, antioxidant, stabilizer, diluent, coating polymer, solvent alone, or a mixture thereof. Capsicum compositions as described herein are comprised of compounds, including capsaicinoids or capsaicinoids and/or non-capsaicinoids. The capsaicinoid compound(s) present in the capsicum composition may be selected from, but not limited to, the compounds such as capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, dihydrohomocapsaicin, 6″,7″-dihydro-5′,5″′-dicapsaicin, 5,5′-dicapsaicin, and mixtures thereof.

(23) In an embodiment, a capsicum composition as described herein is comprised of either capsicum extract alone containing pungent and oily capsaicinoids or the extract is formulated using at least one food grade excipient to entrap the extract at high shear pressure. Due to the high shear pressure extract containing capsaicinoids is entrapped in a carrier which delays or extends the release of the active ingredient thus reducing the pungency and irritation characteristic of the active ingredient, which would otherwise release in gastric region. The pungency can be further reduced or masked by coating the polymer entrapped substance with an excipient such as a polymer that can form an effective barrier between pungent active and the outside environment and thus further sustains the release of the active substance and makes the present formulation palatable and safe for human consumption.

(24) In an embodiment, an extended and sustained release capsicum composition herein includes a spheroidal nutrient core containing the active substance and at least one more excipient; and a protective polymeric enteric coat; wherein the coating facilitates gradual and uniform release of high dosage of the active substance to reduce irritation and minimize abdominal pain and gastric discomfort associated with its release.

(25) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is selected from a group consisting of capsicum extract; capsicum oleoresin; capsaicin crystals. It may be used either alone or is present in the range of at or about 0.1 to at or about 90% of the total weight of capsicum composition, formulated using at least one food grade excipient.

(26) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 0.1 to at or about 40% w/w of the capsicum composition.

(27) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 0.1 to at or about 20% w/w of the capsicum composition.

(28) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 0.1 to at or about 2% w/w of the capsicum composition.

(29) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 0.1 to at or about 1% w/w of the capsicum composition.

(30) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 1 to at or about 90% w/w of the capsicum composition.

(31) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 1 to at or about 40% w/w of the capsicum composition.

(32) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 1 to at or about 20% w/w of the capsicum composition.

(33) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 1 to at or about 2% w/w of the capsicum composition.

(34) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 2 to at or about 90% w/w of the capsicum composition.

(35) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 2 to at or about 40% w/w of the capsicum composition.

(36) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 2 to at or about 20% w/w of the capsicum composition.

(37) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 20 to at or about 90% w/w of the capsicum composition.

(38) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 20 to at or about 40% w/w of the capsicum composition.

(39) In an embodiment, the oily and pungent capsaicinoid used in the capsicum composition is present in the range of at or about 40 to at or about 90% w/w of the capsicum composition.

(40) In an embodiment, the excipient can be a carrier selected from microcrystalline cellulose, Avicel® PH 101, Avicel® PH 102, Avicel® PH 103, Avicel® PH 105, Avicel® PH 112, Avicel® PH 113, Avicel® PH300, Avicel® PH212, Avicel® PH 301, Avicel® PH 302, colloidal grades Carboxymethyl cellulose Sodium and other cellulose containing polymers and their derivatives or mixtures thereof.

(41) In an embodiment, the excipient is a sugar selected from the derivatives of sugar such as Mannitol, sucrose, xylitol, sorbitol, Maltitol, Lactitol, Isomalt or mixtures thereof.

(42) In an embodiment, a surfactant is selected from polysorbate, sodium lauryl sulfate, sorbitanmonooleate, and other surfactants of the same class or mixtures thereof. It is appreciated that in an embodiment, a surfactant is an acceptable excipient for the compositions herein.

(43) In an embodiment, a polymer is used for preventive coating and/or binders and may be are elected from Methyl Cellulose, Agar, Sodium Alginate, Hydroxy Propyl Methyl Cellulose, Hydroxy Propyl Cellulose, Microcrystalline Cellulose, Polyvinyl Pyrrolidone, Starch, Gum Arabic, Xanthan Gum, Polyethylene Glycols, preferably, Microcrystalline Cellulose, Hydroxy Propyl Cellulose, Methyl Cellulose, Hydroxy Propyl Methyl Cellulose, etc., more preferably, Hydroxy Propyl Methyl Cellulose, Methacrylates, Phthalate methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, polyvinyl acetate phthalate, Marcoat containing polymers and there derivatives or mixtures thereof.

(44) In an embodiment, the solvent employed may be selected from acetone, hexane, ethyl acetate, Isopropyl alcohol, ethanol, dichloromethane, methanol and the like or the combination thereof. In an embodiment, the solvent may be selected from acetone, ethanol, dichloromethane, Isopropyl alcohol. In an embodiment, the solvent may be selected from Dichloromethane and Isopropyl alcohol.

(45) In an embodiment, a free flowing, stable and solid capsicum composition as described herein is prepared by extrusion spheronization or by spray drying method.

(46) In an embodiment, the effect of capsicum composition is evaluated by administering an effective amount of capsaicinoids to an exercising subject, for improving exercise performance and endurance.

(47) In an embodiment, a capsicum composition herein may be administered to a subject in the form of an extract or as a solid oral dosage formulation, so as to provide daily dose of at least 0.01 mg/kg body weight to at or about 200 mg/kg body weight of capsaicinoids, or at a daily dose of at or about 10 mg/kg body weight to at or about 100 mg/kg body weight of capsaicinoids.

(48) In an embodiment, a method includes evaluating the effect of capsicum composition through an in-vitro cell-line study to understand the effect on sports nutrition biomarkers, mitochondrial oxygen consumption, mitochondrial mass, IGF (Insulin like growth factor) and cortisol release. In an embodiment, a capsicum extract may be used in such in-vitro study and the effects monitored.

(49) Mitochondria represent the principal energy source in cells, converting nutrients to energy via cellular respiration. The function and content of mitochondria increase with physical training and decrease with physical inactivity. An alteration in the rate of oxygen consumption can serve as a useful indicator of mitochondrial dysfunction. By measuring oxygen consumption, a direct and specific assessment of the functioning of the electron transport chain (the key element of oxidative phosphorylation and cellular metabolism) may be obtained. Mitochondrial abundance (mass) can also be used as an indicator of mitochondrial biogenesis.

(50) As a direct result of aerobic exercise, elevated oxygen consumption contributes to an increase in the circulation of glucocorticoids, such as cortisol. During exercise training, cortisol causes rapid mobilization of fat, protein, and carbohydrates, providing the body with resources to manage an imbalance in homeostasis. However, cortisol is catabolic, decreasing muscle growth hormones, such as insulin-like growth factor (IGF)-1. Conversely, is anabolic, promoting protein synthesis and is thus considered as important biomarker for fitness and exercise training.

(51) In an embodiment, methods described herein including evaluating the effect of a capsicum composition through an in-vivo study in animal models and human volunteers through plasma, muscle protein analysis and checking effect on physical performance, endurance, exhaustion time and actual run time. In an embodiment, a capsicum composition in the form of a capsicum extract or beadlets may be administered in such study.

(52) In an embodiment, compositions as described herein are studied in experimental animal models to investigate the effect of administration on changes in serum parameters, antioxidant status of muscles in rats after exhaustive exercise. The effects can also be investigated on exercise performance, exercise time of exhaustion and changes in cardio-metabolic health markers such as HO-1 and silent mating type information regulation 2 homolog) 1 (SIRT1) pathways in the skeletal muscles of exercise-trained and sedentary rats.

(53) The methods as described herein promote exercise performance and endurance through effective utilization of lipids such as cholesterol and triglycerides. The method as described herein also increases phosphorylated AMP activated protein kinase (pAMPK) and Nuclear erythroid derived factor-like 2 (Nrf2), thus reducing inflammatory process and improving exercise performance. The method also reduces lactate amount in muscles, enhances running performance, increases heme-oxygenase (HO-1) and decreases interleukin (IL-10) and thus enhances exhaustion time as well as running performance in exercising subject.

(54) Exercise results in increased oxidative stress and capsaicinoids from capsicum compositions help to reduce oxidative stress and enhance antioxidant and anti-inflammatory effects. Activation of AMPK promotes glucose uptake, fatty acid oxidation, mitochondrial biogenesis, and insulin sensitivity, AMPK is activated by low energy status (increased AMP/ADP: ATP) such as during exercise, and regulates metabolic process and energy homeostasis by switching off ATP consuming pathways (fatty acid and cholesterol synthesis) and switching on ATP generating processes (glucose uptake and fatty acid oxidation).

(55) In an embodiment, methods described herein lower cholesterol and triglyceride levels and exhibit increased antioxidant capacity. In an embodiment, a method for enhancing exercise endurance and exhaustion time includes administering an effective amount of a capsicum composition to an exercising subject, which enhances an antioxidant amount in the subject's body and decreases lactates, thus resulting in recovery from muscle fatigue during exercise and/or post exercise.

(56) In an embodiment, a method described herein includes administering an effective amount of capsaicinoids of a capsicum composition to a subject who is undergoing physical activity, wherein it also lowers oxidative stress. Capsicum compositions as described herein are stable and safe for oral administration and are prepared using conventional equipment.

(57) The Examples below are provided to illustrate examples of the compositions and methods described herein.

EXAMPLES

Example 1: In-Vitro Cell Line Study for Effect of Capsicum Compositions on Sports Nutrition Biomarkers

(58) Preparation of C2C12 Cell Line

(59) C2C12 cells (ATCC® CRL-1772) were seeded in 24- or 96-well culture plates as undifferentiated myoblasts and grown to 100% confluency in ATCC-formulated Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Upon reaching confluency, cells were induced to differentiate from myoblasts into multinucleated, fused myotubes, which exhibit similar characteristics to mature muscle cells.

(60) Cell culture conditions were maintained at 37° C. in a humidified atmosphere of 5% CO.sub.2/95% air. After 5 days of differentiation, cells were ready for incubation with capsicum composition as test inputs and without the composition as controls.

(61) The effect of Capsicum composition (also abbreviated as Cap or capsimax in graphs and figures) was assessed using the following assays:

(62) Cell Viability Measurement:

(63) In order to determine appropriate dosages of capsaicinoids from capsicum composition for endurance/stamina related assays, differentiated C2C12 myotubes were treated with a range of capsaicinoids concentrations. Cell viability was assessed by MTT (3-(4, 5-desethyithiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) assay, a common measurement of the in vitro cytotoxicity of capsicum composition (called as test inputs in this study). Conversion of MTT reagent (yellow colour) to formazan (purple colour) by living cells provides an indication of mitochondrial activity, which is directly related to cell viability. C2C12 cells were seeded in 96-well culture plates at a density of 2×10.sup.4 cells/mL and induced to differentiate. After a 24-hour pre-treatment with a range of select test input concentrations, used medium was removed, replaced with MTT labeling reagent (5 mg/mL in phosphate buffered saline), and incubated for 4 hours. The purple coloured formazan crystals formed in the intact cells were then dissolved overnight with MTT solubilisation solution (10% SDS in 0.01 M HCl). After solubilisation of the formazan crystals, absorbance was measured at 570 nm with a microplate reader. Data obtained from this cytotoxicity testing allowed for dose range optimization of capsicum composition for further testing.

(64) A. Effect of Capsicum Composition on Mitochondrial Oxygen Consumption

(65) Extracellular oxygen consumption in differentiated C2C12 cells was measured by assessing phosphorescence of a porphyrin-based, water soluble, oxygen sensitive probe (MitoXpress®-Xtra-HS, Luxcel Biosciences). Probe fluorescence is quenched by molecular oxygen (O.sub.2), resulting in lower probe signal. As cellular respiration reduces the concentration of O.sub.2, probe signal increases. The rate of this increase is related to the rate of cellular oxygen consumption. C2C12 cells were seeded in 96-well culture plates at a density of 2×10.sup.4 cells/mL, induced to differentiate, and incubated with MitoXpress probe (1 μM), in the presence or absence of select test inputs. High sensitivity mineral oil was added (100 μL/well) to increase assay sensitivity by minimizing interference from ambient O.sub.2. Probe fluorescence was measured (excitation 380 nm, emission 645 nm) using a fluorescence plate reader.

(66) B. Effect of Capsicum Composition on Mitochondrial Mass

(67) The effect of treatment with capsicum compositions on mitochondrial mass was assessed by measuring changes in fluorescent intensity in differentiated C2C12 cells. The nonyl acridine orange (NAO) probe binds to cardiolipin in mitochondria, regardless of their energetic state, providing a measure of mitochondrial mass and an indication of mitochondrial biogenesis. C2C12 cells were seeded in 96-well culture plates at a density of 2×10.sup.4 cells/mL, induced to differentiate, and pre-treated with capsicum composition or used as controls. Following treatment, media was replaced with NAO probe (100 ng/mL) and incubated for 30 mins at 37° C. in a humidified atmosphere of 5% CO.sub.2/95% air. Probe fluorescence was measured (excitation 380 nm, emission 645 nm) using a fluorescence plate reader. Fluorescent intensity relative to untreated control was then calculated. To standardize probe fluorescence to protein content of the cells, total protein content (in μg) was assessed by bicinchoninic acid (BCA) using bovine serum albumin as standard.

(68) C. Effect of Capsicum Composition on Insulin-Like Growth Factor-1 (IGF-1)

(69) To evaluate the effect of capsicum composition treatment on IGF-1 concentrations in differentiated C2C12 cells, the mouse IGF-1 enzyme-linked immunosorbent assay (ELISA) kit (Sigma) was utilized. The kit provides a quantitative measurement of mouse IGF-1 in cell culture supernatants by employing an antibody specific coated 96-well plate. Standards and test samples were added to the coated plate and any IGF-1 present in the sample gets bound to the immobilized antibody. After washing away any unbound antibody, HRP-conjugated streptavidin was added to the wells. The wells were washed again, followed by addition of a colorimetric reagent. Colour was developed in proportion to the amount of bound IGF-1. The colour intensity was read at wavelength 450 with a microplate reader. Blank-corrected unknown sample protein concentrations were then extrapolated from a known standard curve.

(70) D. Effect of Capsicum Composition on Cortisol Release in H295R Cell Line

(71) H295R cells (ATCC® CRL-2128) were seeded in 24-well culture plates and grown to confluence in ATCC-formulated Dulbecco's Modified Eagle's Medium (DMEM). Cell culture conditions were maintained at 37° C. in a humidified atmosphere of 5% CO.sub.2/95% air. Cells were incubated in the presence or absence of a capsicum composition and controls.

(72) To evaluate the effect of capsicum composition on cortisol concentrations in H295R cells, the DetectX® (Arbor Assays) immunoassay was utilized. A cortisol standard was provided to generate a standard curve for the assay and all samples were read off the standard curve. Standards or samples (capsicum composition) were pipetted into a clear microtiter plate coated with an antibody. A cortisol-peroxidase conjugate was added to the standards and samples in the wells. The binding reaction was initiated by the addition of a monoclonal antibody to cortisol in each well. After one hour incubation, the plate was washed and substrate was added. The substrate was reacted with the bound cortisol-peroxidase conjugate. After a short incubation, the reaction was stopped and the intensity of the generated color was detected in a microtiter plate reader capable of measuring 450 nm wavelength. The concentration of cortisol in the samples was then calculated by extrapolating from the standard curve.

(73) Results:

(74) A. Evaluation of Mitochondrial Respiration (Oxygen Consumption) as Sports Nutrition Biomarker

(75) TABLE-US-00001 TABLE 1 Effect of capsicum composition on mitochondrial respiration Mitochondrial respiration Concentration Fluorescent Intensity Relative to Product (ug/mL) n = 1 n = 2 n = 3 AVG SD SEM control control N/A 28.8 30 29.9 29.6 0.6 0.4 99.9 Capsicum 10 38.5 38.5 38.5 38.5 0 0.0 130.2 composition

(76) Capsicum compositions increased mitochondrial oxygen consumption (respiration) significantly as compared to control. The results of Tables 1 to 5 are of an in-vitro cell line study, where the composition is in the form of a capsicum extract not formulated using an excipient.

(77) FIG. 1 shows the effect of capsicum composition on mitochondrial respiration.

(78) TABLE-US-00002 TABLE 2 Effect of Capsicum composition on mitochondrial mass in C2C12 cells Mitochondrial Mass Concentration Fluorescent Intensity Relative to Product (ug/mL) n = 1 n = 2 n = 3 AVG SD SEM control control N/A 13673.0 13705.3 13769.3 13175.9 49.0 28.3 100 Capsicum 10 16271.7 16468.0 19181.7 17307.1 1626.4 939.0 126.2 composition

(79) Table 2 indicates that capsicum compositions enhanced mitochondrial mass in cell-line study as compared to control cells.

(80) FIG. 2 shows the effects of capsicum composition on mitochondrial mass in cell-line study.

(81) TABLE-US-00003 TABLE 3 Effect of Capsicum composition on IGF-1 in cell-line study n = 1 IGF-1 Concentration Concentration Relative to Product (ug/mL) (pg/ml) n = 2 n = 3 AVG SD SEM control control N/A 36.3 34.7 31.9 34.3 2.22 1.28 100 Capsicum 10 42.5 43.2 36.2 40.6 3.82 2.20 118.5 composition

(82) Table 3 indicates that IGF-1 concentrations increased significantly when the cells were treated with capsicum compositions.

(83) FIG. 3 shows the effect of capsicum composition on IGF-1 in cell-line study.

(84) TABLE-US-00004 TABLE 4 Effect of capsicum composition on cortisol release in cell-line study n = 1 Cortisol Concentration Concentration Relative to Product (ug/mL) (pg/ml) n = 2 n = 3 AVG SD SEM control control N/A 868.0 693.3 1200.9 920.7 257.9 148.9 100.0 cAMP 1 mM 1446.1 1042.2 2183.3 1557.2 578.6 334.1 169.1 (negative control) Capsicum 10 1073.7 827.3 1659.8 1186.9 427.7 246.9 76.2 composition

(85) Capsicum compositions reduced cortisol release relative to control as indicated by cell-line study and thus enhance sport performance.

(86) FIG. 4 shows the effect of capsicum composition on cortisol release as sports nutrition biomarker.

(87) TABLE-US-00005 TABLE 5 Effect of capsicum composition on sports nutrition biomarkers in cell-line study Mitochondrial Mitochondrial Product Mass Respiration IGF-1 Cortisol Control 100 100.1 100 100 cAMP(control) 169.1 Capsicum 126.2 130.2 118.5 76.2 composition

(88) Improvements in endurance and stamina are related to both the content and function of the mitochondrion. Mitochondrial mass can be used as an indicator of mitochondrial biogenesis. Similarly, oxygen consumption rate, a direct measurement of the functioning of the electron transport chain, is indicative of cellular metabolism. The cell-line study indicates that capsicum composition enhanced mitochondrial mass as well as mitochondrial oxygen consumption (respiration) significantly as compared to control.

(89) Mitochondrial efficiency enhancement also results in increased glucocorticoids such as cortisol which is catabolic and can lead to decreases in protein synthesis and muscle growth hormones, such as IGF-1. In the sports nutrition market, controlling the deleterious catabolic effects of cortisol and enhancing the levels and activity of muscle building proteins such as IGF-1 is desired effect. Capsicum compositions showed good effect in all 4 assays related to endurance/stamina and thus represents a potentially beneficial natural supplement for this segment of the sports nutrition market.

Example 2: In-Vivo Study for Evaluation of Capsicum Composition

(90) Animals and Exercise Protocol:

(91) 8-10 male Wistar rats per treatment arm (age: 8 week, weight: 180±20 g) were housed in a controlled environment and were provided with rat chow and water ad libitum. All experiments were conducted under the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the Veterinary Control Institute. Following a 7-day acclimatization period, rats of both the control and exercise groups were sub-divided into groups by matched body weight.

(92) Animals were randomly divided into the following groups:

(93) TABLE-US-00006 Group I - Control (No exercise or capsicum composition) Group II-Control + capsicum composition(100 mg/kg body weight/day capsaicinoids) Group III - only Exercise(No capsicum composition) Group IV: Exercise + capsicum composition 100 mg/kg body weight/day capsaicinoids)

(94) Similar to Example 1, the capsicum composition in this experiment is administered to animals in the form of the extract alone, so the dose is 100 mg/kg body weight/day of capsicum extract composition containing capsaicinoids.

(95) Capsicum compositions were administered to group II and IV animals daily as an oral supplement for 8 weeks to deliver specific dose of capsaicinoids.

(96) The exercise protocols were performed on a motor-driven rodent treadmill equipped with an electric shock grid on the rear barrier to provide exercise motivation to the animals. All exercise tests were performed during the same time period of the day to minimize diurnal effects. The animals in the chronic exercise groups were habituated by treadmill exercise over a 5-d period such as: 1st day 10 m/min, 10 min; 2nd day 20 m/min; 10 min, 3rd day 25 m/min, 10 min; 4th day 25 m/min, 20 min, and 5th day 25 m/min, 30 min. Thereafter, the animals were exercised at 25 m/min, 45 min/d, 5 d per week for 8 weeks (Liu et al. 2000.) To minimise diurnal effects, all animals were exercised at the same time.

(97) Sample Collection

(98) The rats were killed 24 h after the last exercise in the chronic exercise group by cardiac puncture. To minimize diurnal effects, all animals were killed at the same hours. Plasma samples were stored at −80° C. until the time of analysis. Muscle samples (100 mg) were collected and frozen at −80° C. for further analyses.

(99) Laboratory Analyses

(100) Plasma was used for the determination of glucose, lipid profile, cortisol, serotonin, testosterone, creatine kinase activity (CK), aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH) activity, urea, creatinine) with an automatic analyser (Olympus). The serum and muscle malondialdehyde (MDA) levels were measured by HPLC (Shimadzu). The total superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were measured using a commercially available assay kit (Cayman Chemical, Ann Arbor, Mich., USA) according to the manufacturer's instructions. Inflammatory cytokines (TNFα, IL6 and IL10), C-reactive protein (CRP) were analyzed by ELISA. All proteins (AMP-activated protein kinase, SIRT1, mitochondrial transcription factor A, NF-κB, I-κB, Nrf2, HO-1) for pathways were analyzed by Western blot methods in muscle samples.

(101) Histological Analysis

(102) Samples of skeletal muscle (vastus lateralis) were collected from each rat in each experimental condition and fixed with a solution of 2% glutaraldehyde in phosphate buffer at 4° C. for 2 h. Samples were then washed several times in phosphate buffer and post fixed with 1% OsO4 (Osmium tetroxide) in phosphate buffer for 1 h at room temperature. After being washed in phosphate buffer, samples were dehydrated in a graded series of ethanol and embedded in Epon 812 resin (Fluka, Sigma-Aldrich). From each sample, sections of 500 nm were obtained with ultra microtome and subsequently stained with a solution of 1% toluidine blue buffered with borate. They were finally observed under light microscopy, and images were recorded by software.

(103) Statistical Analyses

(104) Data are given as mean±SEM. Sample size were calculated based on a power of 85% and a p value of 0.05. Given that assumption, a sample size of seven per treatment was calculated. Data analysis was done between control vs exercise vs control+capsicum composition vs exercise+effect of capsicum composition. The data were analyzed using the procedure of SAS (SAS Institute: SAS User's Guide: Statistics). The treatments were compared between control vs exercise vs control+capsicum vs exercise+product capsicum using ANOVA and student's unpaired t test; P<0.05 was considered statistically significant.

(105) Results:

(106) TABLE-US-00007 TABLE 6 Effect of capsicum composition on distance run avearage per day and run to exhaustion ↑ Distance run average per day and ↑ run to exhaustion with CAPs supplementation Groups Exercise + Item Control CAPs Exercise CAPs SEM --P-- Distance run average per — — 1037.14 1099.86 9.88 0.004 day, m Run to exhaustion, min 77.31.sup.c 80.49.sup.c 169.34.sup.b 201.11.sup.a 3.28 0.0001 Data are means the standard error of the mean (SEM). Different superscripts (a-d) Indicate group mean differences (p < 0.05).

(107) Capsicum compositions when administered to exercising subjects increased distance run average per day and run to exhaustion as compared to the subjects not administered with capsicum composition. Table 6 shows the effect of capsicum composition on exercise duration and time to exhaustion.

(108) TABLE-US-00008 TABLE 7 Effect of capsicum composition on lipid profile in exercising subjects Groups Exercise + Item Control CAPs Exercise CAPs SEM --P-- Glucose. mg/dL 101.57 99.14 91.43 93.86 5.14 0.512 T-C. mg/dL 75.14.sup.a 70.57.sup.b 74.00.sup.a 47.86.sup.c 0.70 0.0001 TG. mg/dL 103.71.sup.a 100.14.sup.ab 84.29.sup.b 60.57.sup.c 3.62 0.0001 T-C: Total Cholesterol; TG: Triglycerides Data are means the standard error of the mean (SEM). Different superscripts (a-d) indicate group mean differences (p < 0.05).

(109) Capsicum compositions decreased total cholesterol and triglycerides, when administered to exercising subjects.

(110) TABLE-US-00009 TABLE 8 Effect of capsicum composition on lactates and muscle antioxidant activity Groups Exercise + Item Control CAPs Exercise CAPs SEM --P-- Lactate mg/dL 9.66.sup.a 8.77.sup.b 7.47.sup.c 5.43.sup.d 0.17 0.0001 Muscle MDA (nmol/mg protein) 78.84.sup.a 60.50.sup.b 74.16.sup.a 44.79.sup.c 2.07 0.0001 Muscle SOD (U/mg protein) 0.22.sup.d 0.43.sup.b 0.34.sup.c 0.56.sup.a 0.01 0.0001 Muscle GPx (U/mg protein) 143.29.sup.c 172.57.sup.b 155.71.sup.bc 203.86.sup.a 5.13 0.0001 MDA: Malondialdehyde; SOD: superoxide dismutase; GPx: Glutathione peroxidase. Data are means the standard error of the mean (SEM). Different superscripts (a-d) indicate group mean differences (p < 0.05).

(111) When Capsicum compositions were administered to exercising subjects, this resulted in decreased muscle lactate and muscle malonaldehyde, which indicates decreased oxidative stress. On the other hand SOD and GPx values increased in exercising subjects with capsicum compositions (Group IV), which suggested increased muscle antioxidant activity.

(112) FIG. 5 shows an evaluation of the effect of capsicum composition in protein levels in all 4 groups.

(113) It was observed that oxidative stress marker such as NFkBand Interleukin-10 (IL-10) was decreased in group II and IV.

(114) Protein levels of phosphorylated AMP-activated protein kinase (pAMPK), Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), and heme oxygenase-1 (1101) were increased in Group IV. There were no significant differences in any of the end points in Group I and II.

(115) The results suggested that dietary capsaicinoids enhance running performance and decreased oxidative stress. These results suggest that administration of capsicum compositions to the exercising subjects may enhance the effect of exercise by effective utilization of lipids by increasing pAMPK and Nrf2. Group IV significantly increased running performance and exhaustion time. In Group IV, significant decrease in triglycerides and cholesterol was observed compared with other treatments. A significant decrease in lactate, muscle oxidative stress and increase in muscle antioxidant activity were observed in Group IV. No significant changes in liver and kidney functions were observed in any of the treated groups.

Example 3: Human Umbilical Endothelial Cells Line Model to Study Nitric Oxide Effects by Capsicum Composition

(116) Human Umbilical Vein Endothelial Cells (HUVEC) were purchased from Clonetics (Cambrex, Inc., Walkersville, Md., USA) as a frozen stock (passage 3), defrosted and cultured in endothelial basal medium 2 growth media plus bullet kit (Cambrex, Inc.) containing 2% fetal calf serum, antibiotics and growth factor supplements at 37 C and 5% CO2. The cells were passaged three times to provide a pool of cells to be used for experiments. Experiments were performed in six-well plates (Corning, Inc., Corning, N.Y., USA) when the cells were 80% confluent, at which stage the endothelial basal medium 2 growth media (2% fetal calf serum, with growth factors) was replaced with endothelial basal medium 2 control media (0.8% fetal calf serum, no growth factors) and incubated for 24 h (37C, 5% CO2). Treatments were prepared in endothelial basal medium 2 containing 0.8% fetal calf serum and antibiotics but no other supplements. Capsicum composition was dissolved in water at three concentrations, as shown. Eighteen hours after treatment RNA was extracted. Real-time PCR was performed on selected list of genes using standard protocols.

(117) TABLE-US-00010 Name CONC Untreated (DMSO only) Control 0 Capsimax beadlets 6.25 Capsimax beadlets 12.5 Capsimax beadlets 25 Rosiglitazone 10

(118) FIG. 6 shows the effect of capsicum composition on relative gene expression.

(119) It was observed that capsicum compositions upregulated eNOSat higher concentration and down regulated ET1 in human umbilical endothelial cells. This indicates effect of exercise on decreasing reactive oxygen species, thus reducing oxidative stress in the system, as beneficial effect of administration of capsicum compositions in exercising subjects.

Example 4: Evaluation of Capsicum Composition as Sports Nutrition in Human Volunteers

(120) Capsicum formulation was administered to 152 normal to overweight females and males having BMI ranging from 18.3 to 30.0. Daily dosage of 2 mg capsaicinoids from 2% capsimax beadlets was administered for 7 days. Heart rate, general health status, and life style factors including exercise intensity were recorded at baseline and after 7 days. Effect of capsicum composition was evaluated and recorded in terms of duration of exercise and time to exhaustion at the end of 7 days.

(121) At the end of 7 days it was observed that duration of exercise and time to exhaustion was significantly increased in individuals administered with capsicum composition. Subjects work out intensity also increased after capsicum supplementation.