Compositions for use in restoring muscle glycogen and/or muscle mass

Abstract

The present invention relates to compositions comprising a steviol glycoside for use in restoring muscle glycogen by increasing the rate of glycogen re-synthesis in muscles that are depleted in glycogen due to exhaustive exercise and/or for use in treatment of muscle mass by increasing the rate of protein synthesis in muscles that are depleted in protein mass.

Claims

1. A method for treatment of muscle glycogen depletion and/or loss of muscle mass in a human subject in need thereof, the method comprising the step of orally administering to the human subject a composition comprising an effective amount of a steviol glycoside and/or an aglycone thereof and a high glycemic carbohydrate, wherein said steviol glycoside and/or aglycone thereof is selected from the group consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, steviol, isosteviol, stevioside, steviolbioside, rubusoside, and mixtures thereof.

2. The method according to claim 1, wherein the steviol glycoside is stevioside.

3. The method according to claim 1, wherein the carbohydrate is maltodextrin.

4. The method according to claim 1, wherein the composition further comprises protein.

5. The method according to claim 4, wherein the protein is whey protein.

6. The method according to claim 1, wherein the composition further comprises electrolytes.

7. The method according to claim 1, wherein the composition is administered as a solid, frozen, semi-solid, or liquid composition.

8. The method according to claim 7, wherein the composition is a liquid composition selected from the group consisting of milk obtained from animals, milk products derived from soy, rice, coconut or other plant material, fermented milk products and drinking chocolates.

9. The method according to claim 7, wherein the composition is a liquid composition selected from the group consisting of sports drinks, beverages, refreshing beverages, carbonated water, flavoured water, carbonated flavoured water, drinks containing fruit or vegetable juice or nectar, vitamin enhanced sports drinks, high electrolyte sports drinks, highly caffeinated high energy drinks, coffee, decaffeinated coffee, tea, tea from fruit products, tea derived from herb products and decaffeinated tea.

10. The method according to claim 7, wherein the composition is a solid composition selected from the group consisting of chocolate and nutritional bars, drops, candies, cookies, cereals, snack bars and biscuits, or a frozen composition selected from the group consisting of frozen desserts, ice creams, ice sherbets and ice shavings, or a semi-solid composition selected from the group consisting of cream, jam and gels, yoghurt, pudding and jelly.

11. The method according to claim 1, wherein the composition is administered prior to, and/or during and/or following exercise.

12. The method according to claim 1, wherein the human subject is selected from the group consisting of endurance athletes, team sports athletes, athletes participating in weight class regulated sports, professional cyclists, professional football players, and ice hockey players.

13. The method according to claim 1, wherein the human subject is an elderly person.

14. The method according to claim 1, wherein the rate of glycogen re-synthesis is increased in muscles that are depleted in glycogen due to exhaustive exercise.

15. The method according to claim 14, wherein lost muscle mass is restored.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Glycogen concentration and glycogen synthesis rate for stevioside supplementation compared to placebo supplementation.

(2) FIG. 2. Plasma glucose response during the four hour recovery period, expressed as incremental area under the curve (iAUC). The figure illustrates the pattern in plasma glucose response for both of the interventionsplacebo and stevioside during the four hour recovery period of the trial.

(3) FIG. 3. Plasma insulin response during the four hour recovery period, expressed as incremental area under the curve (iAUC). Difference in plasma insulin concentration between stevioside and control at any times during the recovery period, expressed as incremental area under the curve (p=0.95).

(4) FIG. 4. Plasma glucagon response during the four hour recovery period, expressed as incremental area under the curve (iAUC).

DETAILED DESCRIPTION OF THE INVENTION

(5) The compositions of the present invention comprise a steviol glycoside. Preferably, the compositions of the present invention also comprise carbohydrate, protein and/or electrolytes. Additionally the compositions may also comprise further ingredients.

(6) The compositions may be formulated as solid, frozen, semi-solid or liquid compositions.

(7) Steviol Glycoside(s)

(8) The compositions of the present invention comprises a steviol glycoside and/or an aglycone thereof, which act as the active compound by increasing the rate of glycogen re-synthesis in muscles that are depleted in glycogen due to exhaustive exercise and/or by increasing the rate of protein synthesis in muscles that are depleted in protein muscle mass.

(9) Non-limiting examples of steviol glycosides and aglycones thereof that are suitable for use in the compositions of the present invention include rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, steviol, isosteviol, stevioside, steviolbioside, rubusoside, and combinations thereof. For example, steviol and isosteviol are aglycones of steviol glycoside.

(10) The general structure of steviol and its related glycosides are provided below. Glc, Xyl and Rha represent glucose, xylose and rhamnose sugar moieties, respectively.

(11) TABLE-US-00001 Compound name C.A.S. No. R1 R2 1 Steviol 471-80-7 H H 2 Steviolbioside 41093-60-1 H -Glc--Glc(2.fwdarw.1) 3 Stevioside 57817-89-7 -Glc -Glc--Glc(2.fwdarw.1) 4 Rebaudioside A 58543-16-1 -Glc 5 Rebaudioside B 58543-17-2 H 6 Rebaudioside C (dulcoside B) 63550-99-2 -Glc 7 Rebaudioside D 63279-13-0 -Glc-- Glc(2.fwdarw.1) 8 Rebaudioside E 63279-14-1 -Glc-- -Glc--Glc(2.fwdarw.1) Glc(2.fwdarw.1) 9 Rebaudioside F 438045-89-7 -Glc 10 Rubusoside 63849-39-4 -Glc -Glc 11 dulcoside A 64432-06-0 -Glc -Glc--Rha(2.fwdarw.1)

(12) The preferred steviol glycoside is stevioside.

(13) The steviol glycoside or its aglycone, such as steviol or isosteviol, is preferably present in the compositions in an amount of 50 mg to 1000 mg, such as for example 100 mg to 900 mg, such as for example 250 mg to 750 mg, such as for example 400 mg to 600 mg, preferably 500 mg.

(14) Preferably the steviol glycoside is stevioside and preferably the amount of stevioside in the composition lies in the range of 50 mg to 1000 mg, such as for example 100 mg to 900 mg, such as for example 250 mg to 750 mg, such as for example 400 mg to 600 mg, preferably 500 mg.

(15) In another preferred embodiment, the provided composition comprises an aglycone of a steviol glycoside, such as isosteviol and/or steviol, in the range of 50 mg to 1000 mg, such as for example 100 mg to 900 mg, such as for example 250 mg to 750 mg, such as for example 400 mg to 600 mg, preferably 500 mg.

(16) Carbohydrate

(17) The composition of the present invention must preferably comprise at least one carbohydrate in order to provide glucose to be stored in the glycogen-depleted muscles. The term carbohydrate as used herein refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups of the general formula (CH.sub.2O).sub.n, wherein n is 3-30, as well as oligomers and polymers. The carbohydrates of the present invention can in addition be substituted or deoxygenated at one or more positions.

(18) Non-limiting examples of carbohydrates include tagatose, trehalose, galactose, rhamnose, cyclodextrin (e.g. -cyclodextrin, -cyclodextrin and -cyclodextrin), maltodextrin (including resistant maltodextrins such as Fibersol), dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, leucrose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero-oligosaccharides, fucose, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars such as high fructose corn/starch syrup (e.g., HFCS55, HFCS42 or HFCS90), coupling sugars, soybean oligosaccharides or glycose syrup.

(19) The preferred carbohydrate is maltodextrin. Maltodextrin is an oligosaccharide that is used as a food additive. Maltodextrin consists of D-glucose units connected in chains of variable length. The glucose units are primarily linked with (1.fwdarw.4) glycosidic bonds. Maltodextrin is typically composed of a mixture of chains that vary from three to seventeen glucose units long. It is produced from starch, such as barley, wheat, and potato, by partial hydrolysis and is usually found as a white hygroscopic spray-dried powder. Maltodextrin is easily digestible, being absorbed as rapidly as glucose, and might be either moderately sweet or almost flavorless. It is commonly used for the production of sodas and candy. It can also be found as an ingredient in a variety of other processed foods.

(20) The carbohydrate is preferably present in the compositions in an amount of 20 to 150 g, such as for example 50 to 100 g. When the carbohydrate is maltodextrin, it is preferably present in the compositions in an amount of 20 to 150 g, such as for example 50 to 100 g.

(21) Polyol additives may also be included in the compositions of the present invention as sweet taste improving additives. Polyols are also known as sugar alcohols. The term polyol and sugar alcohols as used herein refers to the hydrogenated form of carbohydrate.

(22) Non-limiting examples of polyol additives include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol (glycerine), threitol, galactitol, palatinose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup and reduced glucose syrup.

(23) The composition of the present invention may also comprise a sweet taste improving sugar acid additive. Non-limiting examples of such sugar acid additives include aldonic, uronic, aldaric, alginic, gluconic, glucuronic, glucaric, galactaric, galacturonic or salts thereof (e.g. sodium, potassium, calcium, magnesium salt or other physiologically acceptable salts) and combinations thereof.

(24) Protein

(25) The compositions of the present invention also preferably comprise a protein source. The term protein source as used herein refers to amino acids, peptides, natural protein sources and combinations thereof.

(26) The protein source may also be in the form of amino acids. By the term amino acid as used herein refers to organic compounds comprising both NH.sub.2 and COOH groups. Non-limiting examples of amino acids include aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, phenylalanine, valine, tyrosine, tryptophane, leucine, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, creatine, glucuronolactone, inositol, aminobutyric acid (alpha-, beta-, and gamma-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine and their salt forms such as sodium or potassium salts or acid salts. The amino acids may be in their D- or L-configuration. Additional the amino acids may be in their -, -, -, - and -isomers if appropriate. The amino acids may be natural or synthetic. The amino acids may also be modified, wherein at least one atom has been added, removed or substituted, e.g. N-alkyl amino acid, N-acyl amino acid or N-methyl amino acid. Non-limiting examples of modified amino acids include trimethyl glycine, N-methyl-glycine and N-methyl-alanine.

(27) The protein source may be in the form of peptides, such as dipeptides, tripeptides, tetrapeptides, pentapeptides etc, such as for example glutathione and L-alanyl-L-glutamine. An important peptide source include spray-dried combination of casein hydrolysate and malic acid, known as PeptoPro (DSM), which is a protein hydrolysates derived from casein protein fraction of cow's milk.

(28) The protein source may also be in the form of vegetable proteins. The vegetable proteins may be present either in their native state or as hydrolysates. Non-limiting examples of vegetable proteins include soya protein, soya protein isolate, soy protein concentrate, pea protein, rice protein, soy flour, wheat protein, whey protein, corn protein, nut protein or a combination comprising at least one of the foregoing proteins.

(29) In a preferred embodiment the protein source is whey protein.

(30) Whey protein is the collection of globular proteins isolated from whey, a by-product of cheese manufactured from cow's milk. The protein in cow's milk is 20% whey protein and 80% casein protein, whereas the protein in human milk is 60% whey and 40% casein. Whey protein is typically a mixture of beta-lactoglobulin (65%), alpha-lactalbumin (25%), and serum albumin (8%), which are soluble in their native forms, independent of pH. The protein fraction in whey (approximately 10% of the total dry solids within whey) comprises four major protein fractions and six minor protein fractions. The major protein fractions in whey are beta-lactoglobulin, alpha-lactalbumin, bovin serum albumin and immunoglobulins. Whey protein typically comes in three major forms: concentrate (WPC), isolate (WPI), and hydrolysate (WPH). Concentrates have typically a low (but still significant) level of fat and cholesterol but, in general, higher levels of bioactive compounds, and carbohydrates in the form of lactose. Isolates are processed to remove the fat, and lactose, but are usually lower in bioactivated compounds as well. Like whey protein concentrates, whey protein isolates are mild to slightly milky in taste. Hydrolysates are whey proteins that are predigested and partially hydrolyzed for the purpose of easier metabolizing, but their cost is generally higher. Highly-hydrolysed whey may be less allergenic than other forms of whey. The isolate (WPI) is suitable used in compositions prepared for human beings who need to restore their muscle glycogen that are depleted in glycogen due to exhaustive exercise. Hydrolysate (WPH) is suitable used in composition prepared for human beings, who need to restore muscle mass by increasing the rate of protein synthesis.

(31) Other non-limiting examples of proteins sources that can be included in the compositions of the present invention include bovine serum albumin, egg albumin, yeast concentrate or a combination comprising at least one of the foregoing proteins.

(32) The protein source is generally present in the composition in an amount of about 15 g to 150 g.

(33) The compositions of the present invention may also comprise sweet taste improving nucleotide additives. Non-limiting examples of such nucleotide additives include inosine monophosphate (IMP), guanosine monophosphate (GMP), adenosine monophosphate (AMP), cytosine monophosphate (CMP), uracil monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil diphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil triphosphate and their alkali or alkaline earth metal salts and combinations thereof.

(34) Electrolytes

(35) The compositions of the present invention also preferably comprise electrolytes. Sodium, potassium, magnesium, calcium and chloride are some of the more important electrolytes/minerals that are involved in filling body fluid compartments. It is further believed that electrolytes and minerals play an important role in rehydration by possibly affecting fluid replacement and fluid retention. In response to fluid loss during dehydration, water is distributed between fluid compartments so that both the extracellular and intracellular compartments share the water deficit.

(36) Non-limiting examples of sodium compounds include sodium chloride, sodium acetate, acidic sodium citrate, acidic sodium phosphate, sodium bicarbonate, sodium bromide, sodium citrate, sodium lactate, sodium phosphate, sodium pyruvate, anhydrous sodium sulphate, sodium sulphate, sodium tartrate, sodium benzoate and sodium selenite.

(37) Non-limiting examples of potassium compounds include potassium chloride, potassium acetate, potassium bicarbonate, potassium bromide, potassium citrate, potassium-D-gluconate, potassium monophosphate, potassium diphosphate, potassium tartrate, potassium sorbate and potassium iodide. Potassium monophosphate is the preferred potassium compound.

(38) Non-limiting examples of magnesium compounds include magnesium acetate, magnesium chloride, magnesium diphosphate, magnesium triphosphate, magnesium oxide, magnesium sulphate, magnesium carbonate, magnesium aspartate and magnesium silicate. Magnesium oxide is the preferred magnesium compound.

(39) Non-limiting examples of chloride compounds include sodium chloride, potassium chloride, magnesium chloride and mixtures thereof. Sodium chloride preferred.

(40) Calcium may also be present in the composition. Non-limiting examples of calcium compounds include calcium lactate, calcium carbonate, calcium chloride, calcium phosphate salts, calcium citrate. Calcium lactate is the preferred calcium compound.

(41) Electrolytes are generally present in the composition in an amount of 1 to 300 mmol/l

(42) Other Ingredients

(43) The compositions of the present invention may further comprise other ingredients such as food-grade organic acids, food-grade inorganic acids, bitter compound additives, flavour additives, taste improving polymer additives, emulsifier additives, thickening additives, preservatives, comprise vitamins or vitamin precursors, minerals, micronutrients, phytochemicals, stimulants, cognitive enhancing additives and relaxants.

(44) The compositions of the present invention may also comprise food-grade organic acids. Non-limiting examples of suitable food-grade organic acids for use in the compositions include acetic acid, adipic acid, alginic acid, ascorbic acid, benzoic acid, bile acids, butyric acid, caffeic acid, chlorogenic acid, citric acid, erythorbic acid, formic acid, fruitaric acid (a blend of malic, fumaric and tartaric acids), fumaric acid, glyconic acid, glucoheptonic acid, hydroxycitric acid, lactic acid, maleic acid, malic acid, phosphoric acid, polyglutamic acid, oxalic acid, salicylic acid, succinic acid, tannic acid, tartaric acid and combinations thereof. The food-grade acids can be added as acidulant to control the pH of the composition and also to provide preservative properties or to stabilise the composition. Compositions of the present invention preferably have a pH of from about 2.5 to about 6.5, preferably 2.5 to 4.5, more preferably 3 to 4. In addition citric acid and the like add tartness to the beverage.

(45) The compositions of the present invention may also comprise food-grade inorganic acids. Non-limiting examples of suitable food-grade inorganic acids for use in the compositions include phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulphuric acid, carbonic acid, sodium dihydrogen phosphate and combinations thereof.

(46) The compositions of the present invention may also comprise bitter compound additives. Non-limiting examples of suitable bitter compound additive include caffeine, quinine, urea, orange oil, naringin, quassia, salts thereof and any combinations thereof.

(47) The compositions of the present invention may also comprise flavour additives. The use of flavour additives is to provide an enhanced aesthetic quality to the nutritional composition, which will increase the user's appeal in using the product. The flavour additives may be water soluble natural or artificial additives. Non-limiting examples of suitable flavour additives include almond, apple, banana, cherry, chocolate, cinnamon, citrus, coconut, cola, cranberry, ginger, grape, honeydew, honey, kiwi, lemon, lime, mango, menthol, orange, peach, peppermint, pineapple, raspberry, tangerine, vanilla, viridiflorol, watermelon, wild cherry and equivalents and combinations thereof.

(48) The compositions of the present invention may also comprise taste improving polymer additives. Non-limiting examples of such taste improving polymer additives include chitosan, pectin, pectic acid, pectinic acid, polyuronic acid, polygalacturonic acid, starch, food hydrocolloid or crude extracts thereof (e.g., gum acacia Senegal, gum acacia seyal, carrageenan), poly-L-lysine, poly-L-ornithine, polyarginine, polypropylene glycol, polyethylene glycol, poly(ethylene glycol methyl ether), polyaspartic acid, polyglutamic acid, poly ethyleneimine, alginic acid, sodium alginate, propylene glycol alginate, sodium hexametaphosphate and its salts or other cationic and anionic polymers.

(49) The semi-solid and liquid compositions of the present invention may also comprise emulsifier additives in order to prevent separation of the composition components by keeping the ingredients dispersed. Emulsifier additives include molecules which have both a hydrophilic part and a hydrophobic part. Emulsifier additives operate at the interface between hydrophilic and hydrophobic materials of the semi-solid or liquid composition to prevent separation of the components of the composition. Non-limiting examples of suitable emulsifier additives for use in the compositions include lecithin (e.g. soy lecithin); mono and di-glycerides of long chain fatty acids, specifically saturated fatty acids, and more specifically, stearic and palmitic acid mono- and diglycerides; mono- and di-glycerides of acetic acid, citric acid, tartaric acid or lactic acid, egg yolks; polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80), propylene glycol esters (e.g. propylene glycol monostearate); propylene glycol esters of fatty acids, sorbitan esters (e.g. sorbitan monostearates, sorbitan tristearates, sorbitan monolaurate, sorbitan monooleate), Acacia (gum Arabic), sucrose monoesters; polyglycerol esters; polyethoxylated glycerols; and combinations thereof. Compositions of the present invention may also comprise omega-3 and/or omega-6 fatty acids.

(50) The compositions of the present invention may also comprise thickening additives. Thickening additives which can impart added mouth-feel to the composition include natural and synthetic gums for example locust bean gum, guar gum, gellan gum, xanthan gum, gum ghatti, modified gum ghatti, tragacanth gum, carrageenan and the like; natural and modified starches, for example pregelatinized starch (corn, wheat, tapioca), pregelatinized high amylose-content starch, pregelatinized hydrolysed starches (maltodextrins, corn syrup solids), chemically modified starches such as pregelatinized substituted starches (e.g. octenyl succinate), and the like; cellulose derivatives for example carboxymethylcellulose, sodium carboxymethylcellulose and the like, polydextrose; whey or whey protein concentrate; pectin; gelatin and a combination thereof.

(51) The compositions of the present invention may also comprise preservatives. Such preservatives can be added to the composition to provide freshness and to prevent the unwanted growth of bacteria, moulds, fungi or yeast. The addition of preservatives, including antioxidants, may also be used to maintain the composition's colour, flavour or texture. Any suitable preservatives for use in food and beverage products can be incorporated into the compositions. Examples include benzoic acid alkali metal salts (e.g. sodium benzoate), sorbic acid alkali metal salts (e.g. potassium sorbate), ascorbic acid (Vitamin C), citric acid, calcium propionate, sodium erythorbate, sodium nitrite, calcium sorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), tocopherols (Vitamin E), di-alpha-tocopheryl phosphate, tocotrienols, alpha lipoic acid, dihydrolipoic acid, straight chain polyphosphates and combinations thereof. The compositions may also comprise natural preservatives, which include rosemary extracts comprising carnosic, rosemarinic and ursolic acid. Preservatives also include well-known antioxidants such as for example polyphenols (preferably cocoa), xanthophylls, beta cryptoxanthin, lycopene lutein, zeaxanthin, astaxanthin, beta-carotene, carotenes, mixed carotenoids, resveratrol, flavonoids and combinations thereof.

(52) The compositions of the present invention may also comprise vitamins or vitamin precursors. Non-limiting suitable examples of such vitamin or vitamin precursors include ascorbic acid (Vitamin C), beta carotene, niacin (Vitamin B3), riboflavin (Vitamin B2), thiamine (Vitamin B1), niacinamide, folate or folic acid, alpha tocopherols or esters thereof, Vitamin D, retinyl actate, retinylpalmitate, pyridoxine (Vitamin B6), folic acid (Vitamin B9), cyanocobalimin (Vitamin B12), pantothenic acid, biotin and combinations thereof. Some of the vitamins are fat soluble such as vitamin A, vitamin D, vitamin E and vitamin K, whereas other of the vitamins are water soluble, such as vitamin C (ascorbic acid), the B vitamins (thiamine or B1, riboflavin or B2, niacin or B3, pyridoxine or B6, folic acid or B9, cyanocobalamin or B1, pantothenic acid, biotin.

(53) The compositions of the present invention may also comprise minerals. Non-limiting suitable examples of minerals include iron, zinc, chromium, calcium, copper and magnesium.

(54) The compositions of the present invention may also comprise micronutrients. Non-limiting examples of such micronutrients include L-carnitine, choline, coenzyme Q10, alpha-lipoic acid, omega-3-fatty acids (preferably long chain polyunsaturated fatty acids), pepsin, phytase, trypsin, lipases, proteases, lactotripeptide, Isoleucine-Proline-Proline (IPP), cellulases and combinations thereof.

(55) The compositions of the present invention may also comprise phytochemicals (phytonutrients). Phytochemicals are plant derived compounds which may provide a beneficial effect on the health or well-being of the consumer. Phytochemicals include plant derived antioxidants, phenolic compounds including monophenols and polyphenols and the like. Non-limiting examples of such phytochemicals include lutein, lycopene, carotene, anthocyanin, capsaicinoids, flavonoids hydroxycinnamic acids, isoflavonols, isothiocyanates, monoterpenes, chalcones, coumestans, dihydroflavonols, flavonoids, flavanols, quercetin, flavanones, flavones, flavan-3-ols (catechins, epicatochin, epigallocatechin, epigallocatechingallate and the like), flavonals (anthocyanins, cyanidine and the like); phenolic acids, phytosterols, saponins, terpenes (carotenoids) and combinations thereof.

(56) The compositions may further include one or more stimulants in order to reduce physical and mental impairment of the human being during and following exercise. Non-limiting examples of suitable stimulants include taurine, caffeine and green tea and combinations thereof.

(57) The compositions of the present invention may also comprise cognitive enhancing additives. Non-limiting examples of such cognitive enhancing additives include green tea extracts, L-theanine, phosphatidyl serine, acetyl carnitine, CDP-choline and combinations thereof.

(58) The compositions of the present invention may also comprise relaxants such as melatonin.

(59) Formulation of the Compositions

(60) The compositions of the present invention may be formulated as solid, frozen, semi-solid and liquid compositions.

(61) The composition of the present invention must comprise at least one steviol glycoside and preferably at least one carbohydrate in order to provide glucose to be stored in the glycogen-depleted muscles.

(62) In alternative embodiments of the present invention the composition does not comprise a carbohydrate. In such cases it is necessary to administer the composition comprising steviol glycoside together with another composition comprising carbohydrate. Such embodiments where the steviol glycoside composition and the carbohydrate composition is stored and sold separately the human being to be treated is free to combine the compositions so that an optimal combination of steviol glycoside and carbohydrate is obtained. Hence, different amount and mixtures of steviol glycosides may be combined with different amounts, types and mixtures of carbohydrates.

(63) Solid compositions include, but are not limited to, chocolate and nutritional bars, drops, candies, cookies, cereals, snack bars and biscuits. Solid compositions also include tablets, sachets, capsules, powders and concentrates to be reconstituted before use by addition of water or an appropriate liquid.

(64) Frozen compositions include, but are not limited to, frozen desserts, ice creams, ice sherbets and ice shavings.

(65) Semi-solid compositions include, but are not limited to, cream, jam and gels, yoghurt, pudding and jelly.

(66) Liquid compositions include ready-to drink compositions and concentrates to be reconstituted before use by addition of water or an appropriate liquid. Suitable examples of liquid compositions include, but are not limited to, sports drinks, beverages, refreshing beverages, carbonated water, flavoured water, carbonated flavoured water, drinks containing juice (juice derived from any fruit or any combination of fruits, juice derived from ant vegetable or any combination of vegetables) or nectar, vitamin enhanced sports drinks, high electrolyte sport drinks highly caffeinated high energy drinks, coffee, decaffeinated coffee, tea, tea from fruit products, tea derived from herb products decaffeinated tea, milk obtained from animals, milk products derived from soy, rice, coconut or other plant material, fermented milk products and drinking chocolates.

(67) Human Beings to be Treated

(68) In some aspects, human beings to be treated by the compositions according to the present invention include any human being in need of restoring their muscle glycogen content. The depletion of muscle glycogen may be caused by physical activity of the human being. By the term physical activity as used herein is meant vigorous exercise and in particular physical exercise for a period that results in exhaustion. Suitable forms of exercise include running, football, rugby, cycling, jogging, biathlons, triathlons, marathons, tennis, basketball, squash, housework, dancing and the like. Preferably the duration of the exercise is at least 20 minutes, more preferably 30 minutes or more.

(69) In some embodiments the human beings are athletes, such as endurance and team sports athletes as well as athletes participating in weight class regulated sports, such as for example professional cyclists and professional football player and ice hockey players. In particular during competitions that continue for more than one day, such as for example Tour de France and the World Championship in Football or Ice Hockey, where the players are competing every day or almost every day, it is important that the restoration of muscle glycogen is optimized and that the rate of re-synthesise of muscle glycogen is proceedings as fast as possible.

(70) In some aspects, human beings to be treated also include elderly people, whose muscles are depleted in protein muscle mass due to lack of exercise. It is known that aging is associated with a loss of muscle mass, at a rate of 1% per year, after the age of 50. This loss in muscle mass often results in a loss of independence in elderly, together with an increased risk of falling and premature death. Also elderly people who in a period has been ill in bed or has been bedridden because of surgery may take advantage of oral intake of the compositions of the present invention during their period of rehabilitation, because their depleted muscle mass may be restored during a shorter period of time and thereby improving the elderly's mobility, activity and well-being.

(71) In some embodiments the group of human beings to be treated includes all healthy and non-healthy human beings. In some embodiments, the group of human beings to be treated does not include subjects suffering of diabetes type 2. In some embodiments, the group of human beings to be treated does not include subjects suffering of type 2 diabetes or metabolic syndrome. In other embodiments the group of human beings to be treated does not include subjects suffering of insulin resistance.

(72) In some embodiments, the group of human beings to be treated includes subjects in need of treatment of muscle glycogen depletion due to exhaustive exercise. Examples of such human beings include athletes, such as endurance and team sports athletes as well as athletes participating in weight class regulated sports. Other examples include professional cyclists and professional football player and ice hockey players.

(73) In some embodiments, the group of human beings to be treated includes subjects in need of treatment of loss of muscle mass. Examples of such human beings include elderly people. Other examples include elderly people, who have been bedbound for a period due to illness or surgery. Other examples include elderly people, who have not been physical active for a period. In yet other embodiments, the group of human beings to be treated includes subjects of all ages during their period of rehabilitation. In other embodiments the group to be treated includes elderly people during their period of rehabilitation.

(74) Administration Regime

(75) The composition of the present invention may be taken prior to and/or during and/or after an exercise.

(76) The daily dosage of steviol glycoside, or its aglycone, such as steviol or isosteviol, lies in the range of 50 to 2000 mg, such as for example 500 to 1500 mg, such as for example 900 to 1100 mg, such as for example 1000 mg.

(77) The daily dosage of carbohydrate, such as maltodextrin, lies in the range of 5 to 500 g/day, such as for example 10 to 250 g/day, such as for example 20 to 150 g/day.

(78) The daily dosage of protein, such as whey protein, lies in the range of 5 to 500 g/day, such as for example 10 to 250 g/day, such as for example 15 to 150 g/day.

EXAMPLES

(79) The examples herein will serve to test if stevioside possess a beneficial effect with regard to the rate of resynthesis of glycogen, and demonstrate a positive effect of stevioside on the rate of resynthesis of glycogen.

Example 1

(80) Effect of Stevioside Intake During Physical Activity on Time of Exhaustion

(81) The aim of this experiment is to investigate if intake of stevioside in addition to a carbohydrate-containing composition during exhaustive bicycling work will prolong the period of time before exhaustion as compared with intake of the carbohydrate-containing composition alone.

(82) Test Subjects

(83) 15-20 healthy and well trained male subjects will be selected for the study. The subjects will be completing the test procedure twice, on two separate days at least 7 days apart in a crossover design, where interventioni.e. the additional intake of stevioside together with the carbohydrate-containing composition during the bicycling workis blinded and randomised for both the test subjects and the scientific staff.

(84) Inclusion Criteria:

(85) Well trained, healthy exhaustive trained males, age 18 to 40, having a maximal oxygen uptake (VO2-max) of at least 55 ml O.sub.2 per kg body weight per minute.

(86) Exclusion Criteria:

(87) Persons suffering of a metabolic disease, which is related to the carbohydrate metabolism, such as diabetes type 1 or 2, insulin resistance and the like, cannot be included in the test. Also persons, who are prescribed a medical drug or diet supplement that could affect the carbohydrate metabolism, will be excluded from the study. Finally persons that are not capable of completing the fasting period or the test protocol will be excluded from the study.

(88) Before Testing

(89) Before testing the VO2-max and the Watt-max will be determined for each test person. The test will be performed on a bicycle ergometer as a step test where the workload will be increased with 20 watt every 60 seconds, while the test person will be exercising continuous bicycling work. The test will be discontinued when the test subject experiences exhaustion. VO2 will be measured every 15 seconds. A discharge of intake of oxygen despite an increasing working load and a respiratory coefficient (RER)1.15 will be taken as the criterion for obtainment of maximum VO2.

(90) The test will be performed at least 72 hours before the first duration-before-exhaustion-test (DBET).

(91) Experimental Design:

(92) The test persons will be completing the test twice on two separate days at least 7 days apart in a crossover design, where the interventioni.e. the additional intake of stevioside together with the carbohydrate-containing composition during the bicycling workis blinded and randomised for both the test subjects and the scientific staff.

(93) The test will start by a starving period of 12 hours. Thereafter the test will commence by a 5 minutes warming-up period corresponding to 50% watt-max workload and then the test person will pedal for 120 minutes at 75% Watt-max. This will be followed by 10 minutes of rest and then work to exhaustion will be performed at 90% watt max. Exhaustion will be defined as the point in time where the test person will no longer be capable of maintaining a pedal frequency of 80 revolutions per minute (RPM) at the workload of 90% watt max.

(94) Intervention

(95) During the bicycling exercise the test persons will be given 3.5 ml per kg body weight of a solution comprising 6% carbohydrate (maltodextrin) per 15 minutes. Additionally, the test persons will be given either 500 mg stevioside or 500 mg corn flower at the beginning of the bicycling exercise.

(96) Blood Samples

(97) Blood samples will be collected 0, 30, 60, 90, 120, 130 minutes from start of the bicycling exercise and at the time of exhaustion. After collection the samples will be centrifuged and the serum will be stored at 20 C. Parameters that will be evaluated in the blood samples will be content of glucose, insulin, glycogen, kreatine kinase, lactate dehydrogenase and myoglobin.

Example 2

(98) Effect of Stevioside Intake on Muscle Glycogen Re-Synthesis During Physical Activity

(99) The aim of this experiment is to investigate if intake of stevioside in addition to a carbohydrate-containing composition after work related depletion of muscle glycogen will increase the rate of glycogen re-synthesis as compared with intake of the carbohydrate-containing composition alone.

(100) Test Subjects

(101) 15 healthy and well trained male subjects will be selected for the study. The subjects will be completing the test procedure twice, on two separate days at least 7 days apart in a crossover design, where interventioni.e. addition of stevioside to the restitution mealis blinded and randomised for both the test subjects and the scientific staff.

(102) Inclusion Criteria:

(103) Well trained, healthy exhaustive trained males, age 18 to 40.

(104) Exclusion Criteria:

(105) Persons suffering of a metabolic disease, which is related to the carbohydrate metabolism, such as diabetes type 1 or 2, insulin resistance and the like, cannot be included in the test. Also persons, who are prescribed a medical drug or diet supplement that could affect the carbohydrate metabolism, will be excluded from the study. Finally persons that are not capable of completing the fasting period or the test design will be excluded from the study.

(106) Before Testing

(107) Before testing the VO2-max and maximal pulse will be determined for each test subject. The results of this test will be used for determination of the working load for each test subject during the test.

(108) The test will be performed for at least 72 hours before the bicycling test is commenced.

(109) Experimental Design:

(110) The bicycling test is designed as a working session having the purpose of completely depleting the muscle glycogen storage of the working muscles. The depletion of muscle glycogen will be further facilitated by a twelve hours fasting period prior to commencement of the bicycling test.

(111) The test persons will be completing the test twice on two separate days at least 7 days apart in a crossover design, where the interventioni.e. the additional intake of stevioside together with the carbohydrate-containing composition during the bicycling workis blinded and randomised for both the test subjects and the scientific staff.

(112) During the working session the test subjects will be exercising for 2 hours on a bicycle ergometer at 65-75% of VO2-max. Afterwards these two hours of bicycling exercise, the persons will be performing a number of one minute intervals at maximum workload. Every interval will be followed by a one minute break, where the subjects pedal at low intensity of own choice. This interval exercise will be continued until the test subject's plasma glucose is below 3.89 mmol/l. This value is selected to ensure that the glycogen storage in the liver is depleted to identical extent in each test.

(113) Immediately after the working session the test subject will be given a carbohydrate-containing composition that comprises 2 g carbohydrate per kg of body weight. Also 2 hours after completion of the working session the test persons will be given a carbohydrate-containing composition that comprises 2 g carbohydrate per kg of body weight. These compositions will be added either 500 mg stevioside or placebo (starch corn).

(114) Tissue and Blood Samples

(115) Before the working session will be commenced blood samples will be collected together with muscle biopsies in order to determine the content of plasma glucose, plasma insulin, and muscle glycogen before commencement of the bicycling workload. Further blood samples will be collected at 0, 30, 60, 90, 120, 150, 180, 210 and 240 minutes after completion of the working session, and further muscle biopsies will be collected at 0, 120 and 240 minutes after completion of the working session.

(116) After collection the blood samples will be centrifuged and the serum will be stored at 20 C. Parameters that will be evaluated in the blood samples will be content of glucose, insulin, glycogen, kreatine kinase, lactate dehydrogenase and myoglobin.

(117) After collection of the muscle biopsies the samples will be stored under liquid nitrogen. The parameter evaluated in the muscles biopsies will be glycogen.

Example 3

(118) Effect of Stevioside Intake on Increase of the Muscle Mass

(119) The aim of this experiment is to investigate if intake of stevioside in addition to a protein-containing composition will have a positive effect by increasing the muscle mass.

(120) Test Subjects

(121) Elderly human being of an age ranging from 65 to 95, both males and females, will be selected for the study.

(122) Inclusion Criteria:

(123) Elderly and healthy people, age 65 to 95. Also test subjects suffering of hypertension, hyperlipidaemia or type 2 diabetes may be included in the test.

(124) Exclusion Criteria:

(125) Elderly people suffering of a disease relating to low cognitive function, orthopaedic surgical disease and pharmacological treatment where exogenous testosterone or any other active substance known to affect muscle mass is administered. Moreover, the test subjects must not suffer of any musculoskeletal disorder and the like.

(126) Test Design:

(127) The test will be performed during 12 weeks, where the test subjects will subjected to exercise 3 days a week, where each exercise is separated by at least one day. The test will be performed in groups of 20-30 subjects. During the first week the test subjects will be taught how to perform the exercises correctly on the machines at low weight, where the test subject. Thereafter the weight is increased to such an extent that the test person is able to repeat exercise 6-8 times. In the following weeks the weight is increased (approximately 5-10% per week) so that the number of repetitions are kept at 6-8. The test subjects will be subjected to ten different exercises, all of which will be performed in machines with weights for strengthening the muscles.

(128) The test subjects will be given a liquid composition immediately after completion of the exercises. Two different liquid compositions will be tested. One liquid composition of approximate 250 ml will comprise whey protein, maltodextrin and steviol glycosides (500 mg) and the other liquid composition of approximate 250 ml will comprise whey protein and maltodextrin together with a sweetener. The test will be blinded and randomised.

(129) Body Composition:

(130) The body composition of each test person will be determined by use of dual energy x-ray absorption (DXA, Hologic QDR-2000 plus, Hologic Inc., Waltham, Mass., USA) and MR scanning of the muscles in the legs and arms.

(131) Muscle Strength:

(132) The muscle strength and knee extensor muscle strength will be evaluated. Quadriceps strength will be tested using an isokinetic dynamometer (kin-Com 500H Chattanooga).

(133) Biochemical Analysis:

(134) The test persons will be instructed to avoid exhausting exercise and intake of alcohol the day before collection of fasting blood samples. The blood samples will be centrifuged and stored at 80 C. until the samples are analyzed. Parameters that will be evaluated in the blood samples will be content of glucose, insulin, triglycerides, total cholesterol, high-density lipoprotein, insulin growth factor I (IGF-1).

Example 4

(135) Effects of Stevioside on Glycogen Restitution after Long-Term Exercise

(136) In this example, the effect is examined of a 500 mg steviol glycosides supplementation together with post-exercise oral carbohydrate (1.5 g/kg/h), versus an isocaloric carbohydrate supplementation on muscle glycogen resynthesis, following glycogen depletive exercise. Fifteen trained male cyclists performed two cycling sessions of 120 min at 75-85% Vo2-max, with the post exercise supplementation of stevioside organized in a double blinded crossover study design. Over the course of a 4 h of recovery period, muscle biopsies were obtained from the vastus lateralis immediately and 240 minutes after exercise, to measure post-recovery glycogen concentration and rate of post-exercise glycogen synthesis. In order to measure plasma glucose, insulin and glucagon, blood samples were drawn before and immediately after exercise, and at every half hour during the four hour recovery period. Results A significant increase in total glycogen concentration was seen following the 4 hour of recovery in both trials (p<0.005). Glycogen resynthesis following 4 h of recovery showed a clear tendency in the rate of glycogen repletion (27.962.60 mmol/kg/h and 37.843.72 mmol/kg/h for placebo and stevioside supplementation p<0.002). Plasma glucose and glucagon levels also show a reduction when stevioside was supplied. Conclusion: The addition of stevioside to oral CHO feedings increase significantly post-exercise muscle glycogen resynthesis or rate of glycogen repletion, p<0.022.

INTRODUCTION

(137) Based on the above information the aim of the present study was to investigate the hypothesis that the acute supplementation of the steviol glucosidestevioside, to post exercise carbohydrate feeding, will further increase the glycogen resynthesis rate compared to intake of carbohydrate alone.

(138) Methods

(139) Subjects:

(140) In this example, nine healthy and well trained male participants was included, age 253 years, weighing 77.57.7 kg, were recruited for the study through advertising on the webpages of local cycling, triathlon and mountain bike clubs in the area of Aarhus, Denmark. Comprehensive verbal and written explanation, of the aim and content of the study as well as the potential risks and discomforts associated with participating in the study were given, before all subjects gave their written informed consent to be enrolled in the study. The experimental protocol was approved by The Central Denmark Region Committees on Health Research Ethics.

(141) The following criteria were set for inclusion in the study: Subjects should be male, between 18 and 40 years of age, be accustomed to cycle training at low and high intensity workloads, have a VO2-max of 50 ml.Math.kg-1.Math.min-1 or higher at the initiation of the study (measured by an initial incremental VO2-max test on an ergometer bike). Furthermore the subjects should be free of any metabolic diseases relating to carbohydrate metabolism, and not be taking medication and/or food supplements in any form that could affect carbohydrate metabolism. The subjects' performance characteristics are presented in table 1a. Female subjects were not included in the study, as we wanted to rule out any gender dependent physiological differences that could bias the results of the study. All of the nine subjects completed the entire protocol, and all data from all subjects have been included in the analysis. Subject's performance characteristics are presented in table 1.

(142) TABLE-US-00002 TABLE 1 Anthropometric and performance characteristic of study participants n = 9 All of the nine subjects completed the entire protocol. Anthropometric characteristics Mean SD Age [years] 25 3 Weight [kg] 77.5 7.7 VO.sub.2-max [ml/min.sup.1] 4765 331 VO.sub.2-max [ml .Math. kg.sup.1 .Math. min.sup.1] 62 7 Watt-max 379 24
Experimental Design

(143) In the present study, a double blinded crossover protocol was used to examine the effect of post exercise carbohydrate ingestion supplemented with or without stevioside supplementation on muscle glycogen resynthesis. The double blinded crossover protocol was chosen to ensure a satisfactory statistical power with the relative few subjects that were included in this study. After completing a preliminary anthropometric assessment incremental and VO2-max test, each subject completed two randomized experimental trials; separate by at least seven days.

(144) Preliminary Testing

(145) An incremental exercise test (20 W.Math.min-1) was performed on a computer-controlled electromagnetically-braked cycle ergometer (Excalibur Sport, Lode, Groningen, NL) to voluntary exhaustion. Prior to conducting the incremental test procedure the subjects performed a thorough warm-up. The warm-up consisted of a 10 minutes bike ride on the ergometer bike used for the VO2-max test starting with a fixed workload of 100 Watt. Immediately after completion of the warm-up the subject starts the incremental VO2-max test, with an initial work load of 100 watt where after the work load are increased by 20 watts a minute until voluntary exhaustion, at which point the test was terminated. VO2-max and watt max was determined as the highest 15 seconds average during the test. Strong verbal encouragement was given throughout the test Throughout the incremental test inspired and expired volumes (bi-directional turbine, Jaeger TripleV, Hoechberg, Germany) and gas concentrations (chemical fuel cell (O2) and infrared (CO2) analyzers; Jaeger Oxycon Pro, Hoechberg, Germany) were sampled at 50 Hz, with the time-aligned volume and gas concentration signals allowing online calculation of breath-by-breath pulmonary gas exchange and ventilatory variables (e.g. O2 uptake (VO2), CO2 output (VCO2) and ventilation (VE)). Prior to each test the gas analyzers were calibrated with one precision-analyzed gas mixture and room air to span the concentration range observed during exercise, with the turbine volume sensor calibrated using a 3-liter syringe (Hans Rudolph, Kansas City, Mo.).

(146) This test used for determination of maximal oxygen uptake (VO2-max), from the average VO2 for an integral number of breaths over the final 15 s of the incremental phase. The preliminary test protocol was carried out at least 72 hours before initiation of the experimental protocol, and served to 1) ensure that the subjects adhered to the inclusion criteria, 2) determine individual values for VO2-max, watt-max and maximal heart rate which were used to calculate the work load to be used during the experimental protocol. All preliminary testing was carried out by Sren Lavrsen

(147) Experimental and Supplementation Protocol

(148) After the preliminary testing, each subject underwent two experimental trials, with the experimental supplementation randomized using a randomizing software (Research randomizer) and blinded to both subject and researchers by supplementing the stevioside concealed in capsules. The capsules were color coded according to their content, codes of the supplementation were not revealed to the researchers or laboratory personnel until all data analysis was completed. The two experimental trials were separated by at least seven days. The subjects were instructed to refrain from any intense exercise 24 hour before both the preliminary test and the two experimental trials. The subjects were also instructed to follow their normal diet during the last 48 hours leading up to both the preliminary test as well as the 12 hour fast preceding the experimental trials.

(149) As illustrated the table below, the subjects arrived fasting at 8:00 o'clock a.m. in the lab and completed a glycogen depletion ride consisting of 120 minutes cycling at 75%-80% of their maximal heart rate reserve, followed by a series of five 30 seconds sprint interval at an all-out intensity interspaced with a rest period of 60 seconds. Workload and target heart rate zone was calculated for the subjects, based on the results presented in table 1, performance characteristics of the subjects. The depletion ride was done on a mechanically breaked SRM ergometer bike (Schoberer Rad MestechnikSRMGmbH Jlich, Germany). The subjects were allowed only water during the depletion ride.

(150) Illustration of the Experimental Protocol

(151) TABLE-US-00003 12 hour fast Depletion Time [minutes] protocol 0 30 60 90 120 150 180 210 240 Cabohydrate supplementation Muscle biopsy Plasma glucose Plasma insulin Plasma glucagon

(152) Before the subjects started the depletion ride, blood was drawn from a medial antecubital vein to establish baseline values for plasma glucose, plasma insulin and plasma glucagon concentrations. After completion of the depletion ride and 240 minutes later, a muscle biopsy was obtained from the vastus lateralis muscle by the use of a percutaneous needle biopsy technique, using the protocol described by Bergstrm (Bergstrm 1962). The biopsy sampling was performed by a trained physician with extensive experience in sterile technique. An area of anterolateral side of one thigh was disinfected with chlorhexidine, and local anesthetized with lidocaine (10 mg/ml) in the skin, subcutaneous tissue and muscle fascia. After the area was covered with a sterile hole-piece, a small incision in the skin and fascia of the leg was made with a scalpel and a tissue sample was taken with a Bergstrm biopsy needle with the aid of suction. The biopsy procedure was managed by Mikkel Overgaard, and assisted by Sren Lavrsen. Approx. 100 mg of muscle tissue was taken from the vastus lateralis muscle. The muscle tissue samples were weighted and immediately placed in liquid nitrogen, and then transferred to a freezer (80 C.) for storage until analysis for determination of glycogen concentration would be made. At the same time a polyethylene catheter was placed subcutaneous into a medial antecubital vein from which blood samples were collected. 10 ml of blood were drawn for the analysis of plasma glucose and insulin, and 7.5 ml of blood was drawn for the analysis of plasma glucagon. After the first sample at 0 minutes, blood was drawn at 30, 60, 90, 120, 150, 180, 210 and 240 minutes after the subject has finished the glycogen depletion protocol. Blood samples were centrifuged before 2 ml of the plasma was pipetted into standard analyzing tubes and stored at 80 C. for later analysis.

(153) Immediately after the muscle biopsy procedure and initial blood sampling, the subjects ingested either 500 mg of steviol glucosides, or placebo (500 mg of corn starch) Supplementation, together with 1.5 gr/body weight of liquid maltodextrine solution (Pure Power, Harre Denmark). The sequence of stevioside and placebo supplementation was randomized in a balanced order. At 60, 120 and 180 minutes after termination of the depletion ride, the subjects received another carbohydrate rich feeding (1.5 gr/body weight/hour of Carbohydrates/kg body weight/hour), in accordance with the guidelines recommended by (Ivy et al. 1988).

(154) Supplementation:

(155) Carbohydrate Source

(156) The carbohydrates were derived from a mixture of liquid maltodextrines (PurePower, Harre, Denmark) and a standardized meal of solid high glycemic foods (90 gr of white bread and 15 gr sugar sweetened fruit spread). The carbohydrate drink was prepared in accordance with the manufactures guidelines in order to ensure an identical fluid volume at both trials, as change in volume could alter gastrointestinal absorption rate. Supplying part of the carbohydrates as solid food was chosen as we found through a pilot study, that supplying the subjects with only liquid carbohydrates, after 12 hours of fasting and completion of the depletion protocol, could cause gastrointestinal distress, nausea and vomiting in the subject, which could interfere with the results of the study, through other means than the processes which we wanted to examine.

(157) Stevioside

(158) For this study steviol glycosides containing: 91% of stevioside, 4% rebaudioside A and 5% of other steviol glucosides (99.8% pure from Steviafarm Industrial S/A, Maringa, Parana, Brazil) was supplemented to the subjects, together with liquid maltodextrine immediately after the first muscle biopsy. As steviol glycosides used in this study has a taste 300-350 times sweeter than sucrose, it was chosen to supplement it concealed in capsules, in order to blind the interventions for both researchers and study subjects.

(159) Muscle Tissue Preparation, Glycogen Analysis and Calculations

(160) The Glycogen content was be determined by an acid-hydrolysis method as described by Passonneau and Lowry (Passonneau & Lowry 1972. As illustrated below, glucose, the hydrolysis product of glycogen, is converted into glucose-6-phosphate (G-6-P) by hexokinase in the presence of ATP. In the presence of nicotinamide adenine dinucleotide (NAD), G-6-P is oxidized by the enzyme glucose-6-phosphate dehydrogenase (G-6-PD) to 6-phosphogluconate and reduced nicotinamide adenine dinucleotide (NADH). The increase in NADH concentration is directly proportional to the glucose concentration and can be measured spectrophotometrically at 340 nm using a Beckmann spectrophotometer (at 340 nm) to express the glycogen content of the analyzed muscle sample.

(161) ##STR00007##
Preparatory Procedure:

(162) In preparation for glycogen analysis, a portion of each muscle sample was freeze-dried in a desiccator for 48 hours. Once removed from the desiccator non-muscle elements such as blood and connective tissue was removed. Muscle material was weighed and placed in heat resistant tubes and covered with 0.5 ml 1M HCL. Tubes containing the freeze-dried muscle sample and HCL were then heated in a water bath for 150 minutes at 100 C. to hydrolyze the glycogen into glucose unites. Thereafter, the samples were cooled in an ice bath for 20 minutes, before they were whirl-mixed and then centrifuged at 3500 g for 10 minutes at 4 C. Hereafter, 20 l of the supernatant was transferred to the analysis cuvettes by use of an air displacement pipette. 500 l of a reagent was then added to all cuvettes.

(163) Analysis

(164) The initial light absorbency rate of the solution (A1) was recorded using a Beckmann spectrophotometer at 340 nm (a H2O filled cuvette is used as reference). Hereafter 5 l of diluted Hexokinase was the added to the solution and the cuvettes. After 60 minutes the light absorbency rate of the solution is the recorded again (A2) using the spectrophotometer at 340 nm. Sren Lavrsen assisted with in analysis procedure.

(165) Calculations

(166) The light absorbency rates, recorded by use of spectrophotometer and weight of the muscle sample were used to calculate the glycogen concentration using following equation (all calculation was made by Sren Lavrsen):

(167) $c=\frac{VF}{edv}A=\frac{\mathrm{mmol}}{\mathrm{kg}}\mathrm{muscle}$$c=\frac{0.5250.51000}{6.310.020\mathrm{muscle}\mathrm{weight}}A=\frac{\mathrm{mmol}}{\mathrm{kg}}\mathrm{muscle}$$c=A\frac{2083}{\mathrm{muscle}\mathrm{weight}}=\frac{\mathrm{mmol}}{\mathrm{Kg}}\mathrm{muscle}$ Where: A=A.sub.1-A.sub.2 V=total volume[ml] V=sample volume[ml] D=light distance [cm] e=extinction coefficient for NADH at 3.40 nm=6.3 [1 mmol.sup.cm.sup. 1] F=dilution factor (0.500 ml 1M HCL/2 mg muscle tissue) Reagent:

(168) TABLE-US-00004 1. Tris-buffer 1M 15.0 ml 2. Distilled H.sub.2O 67.5 ml 3. ATP 100 mM 450 l 4. MgCl.sub.2 900 l 5. NAD 100 mM 90 l 6. G-6-PD 60 l Enzyme:

(169) TABLE-US-00005 1. Hexokinase 30 l 2. Distilled H.sub.2O 900 l
Plasma Analysis

(170) Blood samples were collected for the measurements for measurement of the plasma glucose, insulin and glucagon values before initiation of the glycogen depletion protocol and at 0, 30, 60, 90, 120, 150, 180, 210 and 240 minutes after the subject has finished the glycogen depletion protocol.

(171) 10 ml of blood were drawn for the analysis of plasma glucose and insulin, and 7.5 ml of blood was drawn for the analysis of plasma glucagon. All blood was drawn from the antecubital vein. For glucose and insulin the drawn blood was collected in lilthium heparinized tubes (Venosafeplasma VF-109SHL, Terumo Europe N.V., Leuven, Belgium). For glucagon the drawn blood was collected in EDTA treated tubes (Venosafe). With all samples the tubes were turned over 10 times, to avoid coagulation, before they were centrifuged at 1500 G for 10 minutes at 5 C. using a Sigma 3-18 centrifuge (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). 2 ml of the plasma was pipetted into standard analyzing tubes and stored at 80 C. for later analysis. After all testing sessions were finished; all blood samples were packed, according to regulations, in an icebox at 20 C., and transported to the Diabetes laboratory 4, at Aarhus University Hospital, Tage-Hansens Gade (Aarhus, Denmark) for analysis of plasma glucose, insulin and glucagon concentration.

(172) Insulin Assay

(173) Insulin was analyzed by radioimmunoassay using guinea pig anti-porcine insulin antibody (Novo Nordisk, Bagsvrd, Denmark) and mono-125I-(Tyr A14)-labeled human insulin (Novo Nordisk) as tracer and rat insulin as standard (Novo Nordisk). Bound and free radioactivity was separated by ethanol. The inter- and antra-assay variation coefficients were both less than 5%. Stevioside did not interfere with insulin assay at the studied concentrations.

(174) Glucagon Assay

(175) Glucagon was analyzed by radioimmunoassay kit (Millipore Research, Park Drive, St Charles, Mo. USA) according to manufacturer's instructions. The glucagon antibody is specific for pancreatic glucagon and has no cross-reaction with other islet polypeptides. The limit of sensitivity for glucagon assay is 20 pg/ml.

(176) Statistical Analysis

(177) Glucose, insulin and muscle glycogen were analyzed using 2-tailed dependent T-test. The difference in the overall rate of muscle glycogen resynthesis was analyzed with a 2-tailed dependent T-test. Statistical significance was established using an alpha level of p<0.05. Data were analyzed using Grafpad, Prism4 for Windows. All data are presented as meansSE.

(178) Results

(179) Power Output and Heart Rate Response

(180) There were no differences in average relative power output between the two protocols: 69%1% for the placebo protocol and 72%2% for the stevioside protocol. These workloads caused an average heart rate response of 73%2% in the placebo protocol and 74%2% of hr-max in the stevioside protocol. There were no significant differences between the placebo and stevioside protocol.

(181) Muscle Glycogen Concentration and Glycogen Resynthesizes Rate

(182) A clear difference was observed in muscle glycogen concentration among the two intervention groups at 240 minutes after initiation of the recovery protocol. 240 minutes after completion of the depletion protocol the muscle glycogen concentration for the placebo intervention reached 246.420 mmol/kg/dw and 281.529 mmol/kg/dw for the stevioside intervention, cf. FIG. 1 top panel.

(183) Difference in glycogen resynthesis rate during the recovery period, measured as mmol/kg/h, was interestingly significantly higher for the stevioside supplementation compared to placebo supplementation, FIG. 1 bottom panel (p<0.022). For the placebo the glycogen synthesis rate was 27.962.6 mmol/kg/h and for the stevioside supplementation the rate was 37.843.72 mmol/kg/h. The glycogen resynthesis rate was increased 35%, which is very unique (see FIG. 1).

(184) Plasma Glucose (Nine Participates, not all Samples Analyzed)

(185) Plasma glucose response during the four hour recovery period, expressed as incremental area under the curve (iAUC), tend to be affected by stevioside ingestion, as the glucose level was lower for the stevioside group. Due to the low power (not all participants was included in this study), the difference did not show statistical significance. It is expected that the difference will be significant when all participants are included. FIG. 2 illustrates the pattern in plasma glucose response for both of the interventionsplacebo and stevioside during the four hour recovery period of the trial.

(186) Plasma Insulin (Nine Participates, not all Samples Analyzed)

(187) There was no significant difference in plasma insulin concentration between stevioside and control at any times during the recovery period, expressed as incremental area under the curve (p=0.95). Mean area under the curve was 5579012660 ng/ml*4 hours and 5672011950 ng/ml*4 hours for placebo and stevioside respectively. As shown in FIG. 3; 30 minutes after ingesting the first carbohydrate supplement, the plasma insulin concentration increased significantly, reaching the highest concentration at 60 minutes after the first CHO supplementation. Another increase in insulin plasma concentration follows the third CHO feeding at 120 minutes with the peak concentration reached at 150 minutes for the placebo protocol and at 180 for the stevioside protocol respectively. From here the insulin plasma concentration decline steadily for both the placebo and the stevioside supplementation protocol.

(188) Plasma Glucagon (Nine Participates, not all Samples Analyzed)

(189) The mean plasma glucagon concentration expressed as area under the curve was clearly reduced in the stevia group; cf. FIG. 4 left panel. Not all participants was included in this example, which reduce the statistical power, however, it is expected that the difference will be significant when all participants are included.

DISCUSSION

(190) The primary finding of the present example is that supplementation of stevioside, in addition to carbohydrate, after exhaustive glycogen depleting exercise, increase muscle glycogen resynthesis significantly (p<0.022) over a four hour recovery period (all fifteen participants). In addition, stevioside supplementation over this period appears to alter blood levels of glucose and glucagon but not insulin levels.