CN101669938A - Nutrient composition for eliminating exercise-induced fatigue - Google Patents
Nutrient composition for eliminating exercise-induced fatigue Download PDFInfo
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- CN101669938A CN101669938A CN200810042767A CN200810042767A CN101669938A CN 101669938 A CN101669938 A CN 101669938A CN 200810042767 A CN200810042767 A CN 200810042767A CN 200810042767 A CN200810042767 A CN 200810042767A CN 101669938 A CN101669938 A CN 101669938A
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Abstract
The invention relates to a nutrient composition for eliminating exercise-induced fatigue, and discloses a mixture. The mixture comprises R-lipoic acid, acetylcarnitine, biotin, niacinamide, lactoflavin, pyridoxal, kreatine, coenzyme Q10, resveratrol and taurine. The mixture has a good effect on fatigue resistance, and has safe use, small toxic and side effects, stability and controllability.
Description
Technical field
The invention belongs to field of pharmacology, more specifically, the present invention relates to a kind of nutrient composition and the application in eliminating sports fatigue thereof.
Background technology
Along with the modern sports competition is more and more fierce, the athlete needs to bear bigger load in training, and the probability that sports fatigue occurs is also increasing.The motion of appropriateness and rational recovery means can promote the raising of athletes ' functions level, may cause the athlete overtired on the contrary, cause athlete's motor capacity and descend, and can cause the damage of organs such as body movement system, cardiovascular system, nervous system, respiratory system, hormonal system, tissue.Therefore, the mobility of solution over loading training is overtired is one of subject matter that is faced in the current competitive sport training.
Rationally, nourishing tonic for sport can be accelerated the physical efficiency recovery after training athlete, the match efficiently, thereby improve the efficient of training, effectively promote the raising of physical ability, function.Therefore, both at home and abroad the applied research of efficient nourishing tonic for sport is paid attention to gradually in recent years.But domestic its effect of employed nourishing tonic for sport is unsatisfactory at present.Trace it to its cause, the one, the research and development link weakness of domestic nourishing tonic for sport, and from the commercialization of external introduction, popular product can not be at the Chinese athlete; It two fails to carry out research and development of products targetedly from the overtired generation of mobility and the mechanism of reparation, to such an extent as to physiology of exercise, biochemical latest scientific research fail to be transformed into well the sport nutrition field.
Therefore, how to use the modern science and technology research means, the efficient targetedly sport nutrition product of development a new generation make the athlete to " higher, faster, stronger " horizontal impact, and are extremely urgent.
Summary of the invention
The object of the present invention is to provide a kind of antifatigue mixture, it contains R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine.
Another object of the present invention is to provide the compositions that contains described mixture.
In a first aspect of the present invention, a kind of purposes of mixture is provided, and described mixture contains: R-thioctic acid (LA), acetylcarnitine (ALCAR), biotin (Biotin), niacin amide (Nictinamide), riboflavin (VB2), 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. (VB6), creatine (Creatine), coenzyme Q10 (CoQ10), resveratrol (Resveratrol) and taurine (Taurine); Described mixture is used to prepare the antifatigue compositions.
In another preference, described mixture is gone up substantially by R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine and is formed.
In another preference, described mixture is made up of R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine.
In another preference, described compositions is used for:
Improve the mammal motor capacity;
Reduce mammalian blood serum blood urea nitrogen and creatinine content;
Reduce the apoptosis of peripheral blood lymphocyte;
Reduce the peripheral blood lymphocyte oxygen production;
Promote spleen lymphocyte proliferation;
Increase serum antioxidative activities and GST enzyme activity;
Increase the content of skeletal muscle muscle glycogen;
Increase mitochondria number in the skeletal muscle sarcostyle;
Increase the mitochondrion composite I, the proteic expression of II or III;
Promote the synthetic of mitochondrial DNA;
Raise the transcription factor PPARGC1A that the regulation and control mitochondrion generates, the expression of Nrf1 or Tfam; Or
Promote the mitochondrion of skeletal muscle to merge.
In another preference, described mixture contains:
R-thioctic acid 1-50 weight portion;
Acetylcarnitine 2-100 weight portion;
Biotin 0.002-0.1 weight portion;
Niacin amide 0.3-15 weight portion;
Riboflavin 0.12-6 weight portion;
2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. 0.12-6 weight portion;
Creatine 1-30 weight portion;
Coenzyme Q10 0.1-5 weight portion;
Resveratrol 0.1-5 weight portion;
Taurine 2-50 weight portion.
In another preference; in the described mixture, the part by weight between R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and the taurine is: (5-15): (15-25): (0.015-0.025): (2-4): (1-1.5): (1-1.5): (5-15): (0.8-1.2): (0.8-1.2): (15-25).
In a second aspect of the present invention, a kind of compositions is provided, described compositions is used for resisting fatigue, and described compositions contains: (a) R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine; (b) acceptable carrier on pharmacy or the bromatology.
In another preference, described compositions contains:
R-thioctic acid 1-50 weight portion;
Acetylcarnitine 2-100 weight portion;
Biotin 0.002-0.1 weight portion;
Niacin amide 0.3-15 weight portion;
Riboflavin 0.12-6 weight portion;
2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. 0.12-6 weight portion;
Creatine 1-30 weight portion;
Coenzyme Q10 0.1-5 weight portion;
Resveratrol 0.1-5 weight portion;
Taurine 2-50 weight portion;
Acceptable carrier 10-1000 weight portion on pharmacy or the bromatology.
In another preference, described compositions contains:
R-thioctic acid 1-25 weight portion;
Acetylcarnitine 2-50 weight portion;
Biotin 0.002-0.05 weight portion;
Niacin amide 0.3-7.5 weight portion;
Riboflavin 0.12-3 weight portion;
2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. 0.12-3 weight portion;
Creatine 1-15 weight portion;
Coenzyme Q10 0.1-2.5 weight portion;
Resveratrol 0.1-2.5 weight portion;
Taurine 2-25 weight portion.
Acceptable carrier 10-1000 weight portion on pharmacy or the bromatology.
In another preference, in the compositions, R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine sum account for the 5-100% of composition total weight; Preferable 10-60%.
In another preference; in the compositions, the part by weight between R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and the taurine is: (5-15): (15-25): (0.015-0.025): (2-4): (1-1.5): (1-1.5): (5-15): (0.8-1.2): (0.8-1.2): (15-25).
In a third aspect of the present invention; a kind of antifatigue method is provided, and described method comprises: the experimenter's effective dose that needs: R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Figure 1A. the measurement result of each rats in test groups body weight mass number.
Figure 1B. the rat motor treadmill runs Determination of distance; In the 5th week behind the treadmill exercise, write down the race distance (rice) that animal is finished every day; Wherein,
*Significant difference has been compared with quiet matched group in p＜0.05.
The measurement result of Fig. 1 C. serum glutamic pyruvic transminase and glutamic oxaloacetic transaminase, GOT.
Fig. 1 D. serum urea nitrogen, the measurement result of creatinine content; Wherein,
*P＜0.05,
*P＜0.01 significant difference of having compared with quiet matched group; #p＜0.05, ##p＜0.01 exhaust with power organizes the significant difference of having compared.
The measurement result of Fig. 2 A. treadmill exercise RBC number after 8 weeks.
The measurement result of back, Fig. 2 B. treadmill exercise 8 week back hemoglobin.
The measurement result of the red blood born of the same parents hematocrit in Fig. 2 C. treadmill exercise 8 week back.
Wherein,
*P＜0.01 significant difference of having compared with quiet matched group; ##p＜0.01 exhausts with power organizes the significant difference of having compared.
After 8 weeks of Fig. 3 A. treadmill exercise, the quantitative analysis of cell hypodiploid.Wherein,
*P＜0.05 significant difference of having compared with quiet matched group; #p＜0.05 exhausts with power organizes the significant difference of having compared.
After 8 weeks of Fig. 3 B. treadmill exercise, the mensuration of the mensuration of peripheral blood lymphocyte active oxygen and spleen lymphocyte propagation.The fluorescent quantitation of cell DCF dyeing back flow cytometry analysis.
Fig. 3 C. lymphocytic proliferation rate measurement result.Wherein,
*P＜0.05 significant difference of having compared with quiet matched group; #p＜0.05 exhausts with power organizes the significant difference of having compared.
Fig. 4. the measurement result of treadmill exercise peripheral blood oxidation resistance after 8 weeks.
A. MDA content;
B. Total antioxidant capacity;
The content of C.GSH;
D. serum GST enzyme activity;
Wherein,
*P＜0.05 significant difference of having compared with quiet matched group; #p＜0.05 exhausts with power organizes the significant difference of having compared.
Fig. 5. Musculoskeletal.
A. gastrocnemius and quadriceps femoris index;
B. the index of musculus soleus;
C. the activity of skeletal muscle creatine phosphokinase and lactic acid dehydrogenase;
D. skeletal muscle ATP and content of glycogen;
Wherein,
*P＜0.05,
*P＜0.01 significant difference of having compared with quiet matched group; #p＜0.05, ##p＜0.01 exhaust with power organizes the significant difference of having compared.
E. skeletal muscle transmission electron microscope picture (X1,0000);
F. the quantitative analysis of mitochondrion surface density;
Wherein, 0.05 significant difference of having compared with quiet matched group; #p＜0.05 exhausts with power organizes the significant difference of having compared;
G. the expression of western blot analysis mitochondrion complex proteins, #p＜0.05, ##p＜0.01 exhaust with power organizes the significant difference of having compared;
The content of H.PCR analytical line mitochondrial DNA;
I. the transcription factor Nrf1 of quantitative PCR analysis regulation and control mitochondrion generation and the expression of Tfam;
Wherein, p＜0.05, ##p＜0.01 exhaust with power and organize the significant difference of having compared.
J. the expression of epidemic disease western blot analysis skeletal muscle PPARGC1A and ERRa, #p＜0.05 exhaust with power organizes the significant difference of having compared;
K. throw Electronic Speculum and show that the skeletal muscle mitochondrion merges, and sees arrow indication (X2,0000 times);
L. the expression of western blot analysis skeletal muscle MFN1 and MFN2,
*Significant difference has been compared with the peace and quiet group in p＜0.05; #p＜0.05, ##p＜0.01 exhaust with power organizes the significant difference of having compared.
The specific embodiment
The inventor sums up and deep research through a large amount of groping; found a kind of mixture that has excellent effect for resisting fatigue first, the active component of described mixture is R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine.Mixture of the present invention can be coordinated body effectively, makes body reach optimum state, suppresses organism fatigue, and the big crowd of fatigable crowd or quantity of motion that is particularly suitable for uses.
As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by constituting ", " basically by constituting " and " by constituting "; " mainly by constituting ", " basically by constituting " and " by constituting " belong to the subordinate concept of " containing ", " having " or " comprising ".
As used herein, term " basically by constituting " refers in mixture or compositions, except containing neccessary composition or necessary component, also can contain a spot of and do not influence the submember and/or the impurity of effective ingredient.For example, can contain sweeting agent to improve taste, antioxidant in case oxidation, and other this areas additive commonly used.
As used herein, term " effective dose " is meant and can produces function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, the composition of term " pharmaceutically acceptable " or " acceptable on the bromatology " or " health care conduct and learning on acceptable " is applicable to people and/or animal and does not have excessive bad side reaction (as toxicity, stimulation and allergy), the material of rational benefit/risk ratio is promptly arranged.
As used herein, term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991) at Remington ' s Pharmaceutical Sciences.Acceptable carrier can contain liquid on combination of Chinese medicine is learned, as water, saline, glycerol and ethanol.In addition, also may there be complementary material in these carriers, as filler, disintegrating agent, lubricant, fluidizer, effervescent, wetting agent or emulsifying agent, correctives, pH buffer substance etc.
In mixture of the present invention or the compositions, each component also can be used with " the acceptable salt of physiology " or " acceptable acid of physiology or the deutero-salt of alkali " form.Described salt includes, but is not limited to: the salt that forms with following mineral acid: example hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid and the salt that forms with organic acid, organic acid then refers to acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid and maleic acid.Other salt comprise the salt that forms with alkali metal or alkaline-earth metal (as sodium, potassium, calcium or magnesium), with the form (when with this form administration, can change into active part in vivo) of " prodrug " of ester, carbamate or other routines.
As used herein, term " unit dosage form " is meant for taking convenience, becomes single to take required dosage form in mixture of the present invention or preparation of compositions, includes but not limited to various solid formulation (as tablet), liquid agent, capsule, slow releasing agent.
As used herein, " weight portion " or " parts by weight " is used interchangeably, described weight portion can be any one fixed with milligram, the gram number or the kilogram numerical table show weight (as 1mg, 1g, 2g, 5g or 1kg etc.).For example, a compositions that is made of 1 parts by weight of component a and 9 parts by weight of component b can be 1 gram component a+9 gram components b, also can be the compositions that 10 gram component a+90 gram components b etc. constitute.In described compositions, a certain percentages of ingredients content=(the parts by weight sum of the parts by weight/all components of this component) * 100%.Therefore, in the compositions that is made of 1 parts by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and components b is 90%.
Term " compositions of the present invention " includes but not limited to: pharmaceutical composition, food composition or Halth-care composition, as long as they contain R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine, or their physiology goes up acceptable salt; Or basically by R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine, or their physiology goes up acceptable salt composition.Also can comprise in the described compositions other pharmaceutically, acceptable carrier on the bromatology or on the health care conduct and learning; preferably; in compositions; described R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine or their physiology go up acceptable salt and account for 5～100% of composition total weight, preferably account for 10～60%.
As optimal way of the present invention, the consumption of each component that is used to prepare compositions of the present invention is as shown in table 1.
Table 1
| Component | Effective dose | Preferable amount |
| The R-thioctic acid | The 1-50 weight portion | The 1-25 weight portion |
| Acetylcarnitine | The 2-100 weight portion | The 2-50 weight portion |
| Biotin | 0.002-0.1 weight portion | 0.002-0.05 weight portion |
| Niacin amide | 0.3-15 weight portion | 0.3-7.5 weight portion |
| Riboflavin | 0.12-6 weight portion | 0.12-3 weight portion |
| 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. | 0.12-6 weight portion | 0.12-3 weight portion |
| Creatine | The 1-30 weight portion | The 1-15 weight portion |
| Coenzyme Q10 | 0.1-5 weight portion | 0.1-2.5 weight portion |
| Resveratrol | 0.1-5 weight portion | 0.1-2.5 weight portion |
| Taurine | The 2-50 weight portion | The 2-25 weight portion |
Some performances of each component and known main effect are as follows:
(A) R-thioctic acid: reported the infringement of thioctic acid energy the liver protecting and heart, the generation of the interior cancerous cell of inhibition body, and relaxed the caused allergy of body endogenous cause of ill inflammation, arthritis and asthma etc.The R-thioctic acid is a kind of coenzyme in the metabolic process, can treat alzheimer disease, diabetic neuropathy and other some diseases, and the R-thioctic acid also acts on lysosome as a kind of metal-chelator, suppress the generation of hydroxy radical and the accumulation of lipofuscin, keep lysosomal autophagy function, guarantee mitochondrial normal reparation, make cell avoid the self-dissolving death that lysosome membrane breaks and causes.
(B) acetylcarnitine: acetylcarnitine participates in mitochondrial fatty acid transhipment and beta oxidation, and mitochondrial membrane composition cardiolipin can also be provided.It can reduce the ratio of S-acetyl-coenzyme-A/coenzyme A, promotes the activity of pyruvic dehydrogenase, promotes the oxidation utilization of glucose.
(C) biotin: biotin is the coenzyme of plurality of enzymes in the human body, participates in the metabolism of intravital fatty acid and carbohydrate; Promote proteinic synthetic; Also participate in the metabolism of vitamin B12, folic acid, pantothenic acid; Promote urea synthesis and drainage.
(D) nicotinic acid or niacin amide: participate in the metabolism of carbohydrate; Participate in the metabolism of fat, the synthetic and decomposition of glycerol, oxidation of fatty acids and synthetic, the level of energy cholesterol reducing is a kind of endogenous antioxidant.
(E) riboflavin: the metabolism that riboflavin participates in carbohydrate, protein, nucleic acid and fat can improve human body to proteinic utilization rate, promotes growth promoter.Be the coenzyme F MN of oxphos enzyme complex I and II and the precursor of FAD.
(F) 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine.: as the coenzyme of many enzymes, known its except that the metabolism that participates in neurotransmitter, glycogen, sphingomyelin, haemachrome, steroid and nucleic acid, also participate in all amino acid metabolisms.
(G) creatine: known creatine has the supply cardiac muscle as exogenous phosphocreatine and the required energy of skeletal muscle cellular metabolism is organized effects such as microcirculation with promotion membrane stabilizing action and improvement.
(H) coenzyme Q10: coenzyme Q10 is repaired, increased the synthetic of hepatic glycogen to hepatocyte and strengthens liver all has certain effect to the detoxification ability of poisonous substance.
(I) resveratrol: known its can suppress hematoblastic cohesion, the expansion artery blood vessel, and microcirculation improvement is prevented and treated coronary heart disease, atherosclerosis, hyperlipidemia etc.
(J) taurine: taurine can play the maintenance myocardial function, improves effects such as liver function, blood pressure regulation and cholesterol reducing.
And the inventor thinks, each component may be passed through different approaches, collaborative effect, thereby can bring into play the antifatigue effect very effectively.
Dosage form for compositions of the present invention has no particular limits, and can be any dosage form that is applicable to that mammal is taken; Preferably, described dosage form can be selected from: capsule, granule, tablet, pill, oral liquid, soft capsule, suspension or Emulsion.From be easy to prepare, administration or the position of taking, preferred compositions is a solid-state composition, especially tablet, granule and solid are filled or the capsule of liquid filling.Oral administration is preferred.Preferably, compositions of the present invention can be made the dosage form of slow release.Preferably, compositions of the present invention can be made unit dosage form, is easy to carry and takes.
Needed various conventional carriers or adjuvant in the time of can adding the preparation different dosage form in the compositions of the present invention are as filler (as starch), correctives (as steviosin), antioxidant or coating material etc.Can adopt conventional method to be prepared into any dosage form commonly used, as tablet, powder, granule, capsule, pill.
The mixture or the compositions of the R-of containing thioctic acid of the present invention, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine can be directly used in alleviating physical fatigue, or can use jointly with other medicines or dietary supplement.
In an embodiment of the present invention, the inventor has proved mixture of the present invention or compositions alleviating physical fatigue effectively, particularly post exercise fatigue by sufficient animal experiment.A series of demonstration finds that mixture of the present invention or compositions can improve the mammal motor capacity; Reduce mammalian blood serum blood urea nitrogen and creatinine content; Reduce the apoptosis of peripheral blood lymphocyte; Reduce the peripheral blood lymphocyte oxygen production; Promote spleen lymphocyte proliferation; Increase serum antioxidative activities and GST enzyme activity; Increase the content of skeletal muscle muscle glycogen; Increase mitochondria number in the skeletal muscle sarcostyle; Increase the mitochondrion composite I, the proteic expression of II or III; Promote the synthetic of mitochondrial DNA; Raise the transcription factor PPARGC1A that the regulation and control mitochondrion generates, the expression of Nrf1 or Tfam; Or promote the mitochondrion of skeletal muscle to merge; Effective improvement of these aspects has produced good effect for resisting fatigue.
The consumption of mixture of the present invention or compositions can change with the pattern that gives, dosage form and experimenter's the tired order of severity.Yet, when compositions of the present invention every day gives with the dosage of about 0.01～0.75g/kg the weight of animals, can obtain gratifying effect usually, preferably give with the dosage that separates for 1～4 time every day, or with the slow release form administration.For most of large mammal, the accumulated dose of every day is about 0.05～50g, preferably is about 0.2～20g.This dosage of scalable is to provide optimum therapeuticing effect.For example, by the needs of treatment situation, but separately give the single dose of several times every day, or dosage is reduced pari passu.Certainly, concrete dosage also should be considered the factors such as health of method of application, subject, and these all are within the skill of this area.
Major advantage of the present invention is:
(1) finds first; the mixture or the compositions that contain R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine can be coordinated body very effectively; make body reach optimum state; suppress organism fatigue, the big crowd of fatigable crowd or quantity of motion that is particularly suitable for uses.
(2) contain pure natural components in mixture of the present invention or the compositions, safer, side effect is little.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise percentage ratio and umber calculate by weight.
I. material and method
A. material
Anti-PPARGC1A, MfN1, MfN2 and ERRa antibody purchase the Santa Cruz company in the U.S.; Anti-tubulin antibody and ATP enzyme testing cassete are purchased the Sigma company in the U.S.; TRIzol purchases the company in American I nvitrogen; Anti-ComplexI, II, the antibody of III is available from American I nvitrogen company; The reverse transcriptase test kit is purchased the Promega company in the U.S.; Thermal starting Taq enzyme is purchased the company in Japanese TaKaRa; D-loop, Nrf1, Tfam and 18S rRNA primer are synthetic by Shanghai Bo Ya company; Real-time quantitative PCR is measured test kit available from Japanese Japan mill biotech firm; BCA protein determination examination box and West Picochemiluminescent luminous substrate are from U.S. PIERCE company; Lymphocyte separation medium is purchased in last Haikang and is become biotech firm; The R-thioctic acid is given (or can available from the new high towering company limited in Shanghai) by German doctor K.Wessel, and acetylcarnitine is available from U.S. sigma company.Creatine, ubiquinone, riboflavin, niacin amide, taurine, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. and biotin are available from primary bio tech ltd difficult to understand in Shanghai; Resveratrol is available from times Xiang bio tech ltd in Shanghai.The muscle glycogen testing cassete, antioxidative activities testing cassete, malonaldehyde testing cassete, reductive glutathione testing cassete and glutathione S-transferase testing cassete are all available from build up bio-engineering research institute in Nanjing.
The preparation of nutrient composition
The inventor has also prepared compositions, each component of the following weight of precision weighing:
| |
|
Compositions 3 | |
Compositions 5 | |
| R-thioctic acid (g) | ??5 | ??10 | ??30 | ??25 | ??4 |
| Acetylcarnitine (g) | ??10 | ??15 | ??35 | ??50 | ??8 |
| Biotin (g) | ??0.01 | ??0.005 | ??0.08 | ??0.05 | ??0.01 |
| Niacin amide (g) | ??1.5 | ??3 | ??9 | ??7.5 | ??1.2 |
| Riboflavin (g) | ??0.6 | ??0.4 | ??5 | ??3 | ??0.4 |
| 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. (g) | ??0.6 | ??5 | ??1 | ??3 | ??0.5 |
| Creatine (g) | ??5 | ??8 | ??20 | ??15 | ??4 |
| Coenzyme Q10 (g) | ??0.5 | ??0.8 | ??3 | ??2.5 | ??0.8 |
| Resveratrol (g) | ??0.5 | ??4 | ??1 | ??2.5 | ??0.4 |
| Taurine (g) | ??10 | ??30 | ??6 | ??25 | ??6 |
The capsule preparation
To take by weighing component according to the prescription of last table pack thing 1, fully mix, place conventional capsule, fill in the capsule of routine every about 0.25g.
To take by weighing component according to the prescription of last table pack thing 4, fully mix, place conventional capsule, fill in the capsule of routine every about 0.25g.
The functional drink preparation
To take by weighing component according to the prescription of last table pack thing 2, and fully mix, with oligosaccharide 2000g, citric acid 2000g, Fructus Citri Limoniae essence 400g, sodium chloride 250g, potassium chloride 45g, sodium dihydrogen phosphate 45g, sodium bicarbonate 45g is assigned in the 200L water and makes beverage.
Feed formula
Normal feedstuff: provided by the Si Laike Experimental Animal Center, total amount of heat is 15.36KJ/g, wherein protein 21%, carbohydrate 55%, fat 6%.
Add nutrient categories: R-thioctic acid, acetylcarnitine, creatine, ubiquinone, resveratrol, riboflavin, niacin amide, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., biotin and taurine.
Adopt the content of the capsule of above-mentioned preparation to give animal, wherein compositions 1 is used to organize 1, and compositions 4 is used to organize 2.When giving, nutrient is made an addition in the animal normal feedstuff according to the dosage of setting, make the feedstuff that contains nutrient.Also nutrient can be mixed with solution, animal be carried out stomach raise.
Add medicine
The nutrient intervention group:
| Group 1 (low dosage) | Group 2 (high doses) | |
| R-thioctic acid (LA) | ??50mg/kg/d | ??250mg/kg/d |
| Acetylcarnitine (ALCAR) | ??100mg/kg/d | ??500mg/kg/d |
| Biotin (Biotin) | ??0.1mg/kg/d | ??0.5mg/kg/d |
| Niacin amide (Nictinamide) | ??15mg/kg/d | ??75mg/kg/d |
| Riboflavin (VB2) | ??6mg/kg/d | ??30mg/kg/d |
| 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. (VB6) | ??6mg/kg/d | ??30mg/kg/d |
| Creatine (Creatine) | ??50mg/kg/d | ??150mg/kg/d |
| Coenzyme Q10 (CoQ) | ??5mg/kg/d | ??25mg/kg/d |
| Resveratrol (Resveratrol) | ??5mg/kg/d | ??25mg/kg/d |
| Taurine (Taurine) | ??100mg/kg/d | ??250mg/kg/d |
The caffeine group:
| Caffeine | ??20mg/kg/d |
B. method
The experiment grouping
1) quiet group: normal control group SD rat;
2) training group: chronic power exhausts group (model control group);
3) training group: nutrient intervention group (low dose group);
4) training group: nutrient intervention group (high dose group);
5) training group: caffeine intervention group;
Wherein, the 1st treated animal is male 12, and the 2-5 group is initially male 20, after after the acclimatization training, screen 12 every group and carry out subsequent experimental.
Training Arrangement
The conventional raising do not carried out training in the quiet control rats cage.
Chronic power exhausts the group rat and trains on the electronic treadmill of DSPT-202 type.Motion mode with reference to Bedford (1979) according to increasing load motion model that rat body weight/the oxygen uptake regression equation is set up, undertaken by following program, comprise each 4 week of general training and the training of exhausting property of power, the animal treadmill gradient is 10 degree, trained Sunday no collection weekly 6 days.
The 1st week: finish 10 meters/minute every day, 10 minutes treadmill exercise;
The 2nd week: finish 10 meters/minute every day, after running in 10 minutes, accelerate to then 15 meters/minute 10 minutes;
The 3rd week: carry out 10 meters/minute, 15 meters/minute, 20 meters/minute each lasting treadmills of 10 minutes every day and run;
The 4th week: carry out 10 meters/minute, 15 meters/minute, 20 meters/minute, 25 meters/minute each 10 minutes persistent movements every day respectively.
The training of exhausting property of power is run from carrying out 4 all increasing load treadmills the 5th week, every day with 15 meters/minute, 20 meters/minute, 25 meters/minute each 10 minute campaigns after, accelerate to 30 meters/minute, 35 meters/minute each 20 minute campaigns, and constantly increase progressively running velocity, exhaust until rat power.
Power exhausts criterion: after button gave rat continuously and applies sound, light, mechanical stimulus, rat can not continue to run, and throws oneself on the ground to pant behind the following treadmill, does not temporarily have escape reaction.
Experiment is provided by Hangzhou Li Tai Science and Technology Ltd. with electric animal treadmill (PT98 type) 2 ones, and this treadmill can write down the rat training time automatically, the move distance and the speed of running.
Zoopery
(1) animal feeding: laboratory animal is provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center, SPF level, quality certification scxk (Shanghai) 2,003 0003.Rat is freely taken in drinking water, and the control circadian rhythm is 12h: 12h, and room temperature is 23 ± 1 ℃.Begin to give compositions of the present invention or contrast medicine after the ablactation of 3 weeks, begin treadmill exercise after about 4 weeks.
(2) administering mode: stomach is raised.
(3) processing time: in 12 weeks of administration time, administration number of times is every day 1 time.
(4) animal is handled: in the 5th week behind the treadmill exercise, write down the race distance (rice) that animal is finished every day, the body weight of all animals of participating in the experiment of weighing on every Mondays.5th, giving all animals dockings of participating in the experiment 7,9 weeks on every Mondays respectively gets blood and once measures content of hemoglobin (the about 0.2cm of each docking, and give the sterilization of broken ends of fractured bone iodine tincture and stop blooding).
Testing index:
After nutrient composition is intervened,
1. power is exhausted the mensuration of motion rat matter energy metabolism and metabolic capacity index;
2. power is exhausted the index determining of motion rat oxygen transport system;
3. power is exhausted the index determining of motion rat body immune system;
4. power is exhausted the index determining of motion rat body anti-oxidative damage ability;
5. power is exhausted every index determining of motion rat skeletal muscle system.
At treadmill exercise after 8 weeks, it is 10% chloral hydrate-normal saline anesthesia that rat power exhausts immediate postexercise lumbar injection mass fraction, takes the about 8ml of blood under the paracentesis pericardii direct-view, after a part leaves standstill and waits to coagulate, with the centrifugal 10min separation of serum of 3000r/min, it is standby to put 4 ℃ of refrigerators; Other gets 4ml blood with lymphocyte separation medium isolated lymphocytes from fresh blood, after the PBS washing, with the RPMI1640 culture fluid cell is transferred to 1.5 * 10
6Individual/ml.
The detection of malonaldehyde
The blood plasma lipide levels of peroxide is measured the content of TBARS in the blood plasma.Get the blood plasma 80 μ l of fresh separated, operation is measured the light absorption value at A532nm place in strict accordance with test kit description (building up biotech firm available from Nanjing).
The detection of blood plasma Total antioxidant capacity
The power of the oxidation resistance of body defense system and the degree of disease exist close ties, measuring principle is that body has many antioxidant, can make Fe3+ be reduced to Fe2+, the latter can form firm complex with luxuriant and rich with fragrance quinoline class material, can measure its oxidation resistance by colorimetric.Get the blood plasma 40 μ l of fresh separated, operation is measured the light absorption value at A520nm place in strict accordance with test kit (building up biotech firm available from Nanjing) description.
Blood plasma reduced glutathion Determination on content
Get the blood plasma 40u l of fresh separated, carry out the GSH assay according to the test kit description, the principle of this test kit is that dithio dinitrobenzoic acid and sulfhydryl compound reaction produce a kind of yellow compound, thereby carries out colorimetric determination.
The active detection of blood plasma glutathione S-transferase (GST)
Assay method is the blood plasma 40 μ l that get fresh separated, and at 1mM GSH, 1mM CDNB in the reaction system among the 3mg/mlBSA, measures the light absorption value at A340nm place, measures the GST enzymatic activity.
The test of periphery lymphocyte active oxygen and apoptosis
Isolating lymphocyte with contain 10 μ M DCF dyestuffs and hatched 30 minutes, discard the culture medium that contains dyestuff, give a baby a bath on the third day after its birth time with cold PBS, censorship flow cytometer (excitation wavelength 488nm, emission wavelength 530nm) in back reads the level that fluorescent value reflects intracellular reactive oxygen.
Isolating lymphocyte adds 70% ethanol of ice pre-cooling to be fixed, and 4 ℃, 1-2 hour, the centrifugal fixative that discards, resuspended 5 minutes of 3ml PBS, 400 purpose screen filtrations 1 time, centrifugal 5 minutes of 500-1000r/min discards PBS.With the dyeing of 1ml PI dye liquor, 4 ℃ of lucifuge 30min.Flow cytometer detection laser optical wavelength is 488nm, and the emission optical wavelength is greater than 630nm, and on the rectangular histogram of PI fluorescence, apoptotic cell a hypodiploid peak occurred at G1/G0 before the phase.
ConA is to the influence of rat spleen lymphocyte proliferation
Aseptic taking-up thymus is made the lymphocyte suspension behind the rat anesthesia, and this suspension is made into 2 * 10 with RPMI 1640 complete culture solutions
6The lymphocyte suspension of/mL adds 12 multiple holes (100 μ l/ hole), and wherein every hole, 8 holes contains the RPMI-1640 culture fluid 100 μ l of Con A (final concentration 20 μ g/ml), mixing rearmounted 37 ℃, 5%CO
2After cultivating 48h in the incubator, every hole adds 5mg/mL MTT liquid 40 μ L, continues to cultivate 4h, adds the every hole 100 μ l of 10%SDS liquid (0.01mol/L HCl preparation), surveys 570nm light absorption value, the every hole of reference wavelength 630nm. A value=A570nm-A630nm after spending the night; Cell proliferation multiple=(measuring hole A value-control wells A value)/control wells A value.
The preparation of skeletal muscle mitochondrion
Rat last immediate postexercise sacrificed by decapitation, rapid complete excision bilateral quadriceps femoris, 4 ℃ of pre-cold salines are cleaned blood, filter paper blots the muscular tissue surface moisture, after claiming wet quality, take by weighing the 1g specimen, eye scissors shreds, and presses W: V=I: 9 add the homogenate medium, after the homogenate, on the low-temperature and high-speed centrifuge, make differential centrifugation and separate endochylema and mitochondrion, more than operate under 0～4 ℃ and carry out, get rapidly about each 1g of quadriceps femoris red muscle, make 10% tissue homogenate, with the centrifugal 5min of 3000r/min, getting supernatant, to put 4 ℃ of refrigerators standby, to measure muscle glycogen.The mitochondrion preparation method adopts Nanjing to build up the method that bio-engineering research is recommended.
The skeletal muscle body mass index is measured
Get the right side gastrocnemius, quadriceps femoris or the musculus soleus weighing quality that wets is calculated itself and the ratio of body constitution amount respectively.Muscle quality is calculated as follows with the ratio of body constitution amount:
Muscle quality (mg)/body constitution amount (g) * 100%.
ATP Determination on content in the skeletal muscle endochylema
The mensuration of ATP is used the assay determination kit measurement of ATP luciferase in the endochylema.
The 50mg skeletal muscle tissue adds 0.42mol/l perchloric acid 600 μ l, homogenate, the vortex mixer mixing, 3000rpm/ divided low temperature (4 ℃) centrifugal 5 minutes, got supernatant 200 μ l and added 0.1mol/l KOH, and transferring pH is 6.24, the vortex mixer mixing, centrifugal 10 minutes of low temperature (4 ℃), the microplate reader reading numerical values is used in the reactant liquor that contains luciferase (10mg/ml) effect of getting supernatant 100 μ l and 100 μ l.
Skeletal muscle muscle glycogen Determination on content
The hind leg quadriceps femoris blots with filter paper after the rinsing of ice normal saline, accurately takes by weighing weight, and is according to the test kit description operation, using sulfated-anthrone colorimetric method for determining muscle glycogen content.
Electron microscopic observation
The observation of skeletal muscle mitochondria ultrastructure: test every group of each treated animal and randomly draw 6, getting the part musculus soleus prepares by transmission electron microscopy, 2.5% glutaraldehyde spends the night fixing, and is fixing behind the 10g/L osmic acid, acetone dewater, soak into, resin embedding, ultrathin section, uranium-lead are redyed, transmission electron microscope observation down.(10000 times) take the photograph 20 of sheets to containing mitochondrial skeletal muscle under the Electronic Speculum, according to the morphology metering method mitochondrion volume and area in the skeletal muscle endochylema being carried out relative measurement, is reference frame slotted line plastochondria surface density (Sv: unit volume contains the surface area that certain branch that coordinates has in the space) with the endochylema.
The mitochondrial DNA Determination on content
Total DNA uses the DNA extraction test kit to extract (the excellent brilliant bio-engineering corporation in Shanghai).Real-time quantitative PCR uses Master Premix (Japanese Japan mill biotech firm).
Primer sequence:
Mitochondrion D-loop:
Upstream sequence: 5 AATCTACCATCCTCCGTG-3 (SEQ ID NO:1),
Downstream sequence: 5-GACTAATGATTCTTCACCGT-3 (SEQ ID NO:2);
18S?rRNA
Upstream sequence: 5-CATTCGAACGTCTGCCCTATC-3 (SEQ ID NO:3),
Downstream sequence: 5-CCTGCTGCCTTCCTTGGA-3 (SEQ ID NO:4).
The PCR condition: 95 ℃, 10min, carrying out 40 circulations then is 95 ℃, 1min, 55 ℃, 1min, last 72 ℃, 1min.The application standard curve carries out relative quantification respectively, compares with the ratio of mitochondrion D-loop/18SrRNA.
Total RNA extracting and qRT-PCR
Press the test kit explanation, extract the total RNA that organizes, measure purity and the content of RNA with the uv-spectrophotometric instrument with TRIzol reagent.The design reference document of primer sequence, primer is synthetic by match Parkson, Shanghai company, and primer sequence is:
The Nrf1 upstream sequence: 5 '-GCAAACGCAAACACAGGCC-3 ' (SEQ ID NO:5),
Nrf1 downstream sequence: 5-CTGCATCTCCCTGAGAAGC-3 (SEQ ID NO:6);
The Tfam upstream sequence: 5 '-AAAAAGGAAAGCTATGAC-3 ' (SEQ ID NO:7),
The Tfam downstream sequence: 5 '-AGCACCATATTTTCGTTG-3 ' (SEQ ID NO:8);
18S rRNA upstream sequence: 5-CATTCGAACGTCTGCCCTATC-3 (SEQ ID NO:9),
18S rRNA downstream sequence: 5-CCTGCTGCCTTCCTTGGA-3 (SEQ ID NO:10);
The RT reaction: the total RNA of 1 μ g synthesizes cDNA.Cumulative volume 20 μ l, 42 ℃ of reaction 60min, 95 ℃ of reaction 5min.Synthetic cDNA, use the DDR305A Ex Taq (Hot startversion) of Takara company to carry out pcr amplification, PCR condition: 95 ℃, 10min carries out 95 ℃ of 40 circulations, 1min, 55 ℃, 1min then; Last 72 ℃, 1min.
Data are expressed as: 2
-Δ Ct, Δ Ct=target gene Ct-18S Ct.
The immune protein trace
Behind the tissue homogenate, hatched on ice 30 minutes, 1, centrifugal 10 minutes of 4 ℃ of 3000r/min collect supernatant and are the histone extracting solution.It is quantitative that application BCA method is carried out total protein ,-80 ℃ of preservations.After albumen is used sds page (SDS-PAGE) electrophoretic separation, be transferred on the solid support nitrocellulose filter, sealing is 1 hour under the room temperature, add an anti-PPARGC1A (1: 1000), tubulin (1: 5000), complexI (1: 1000), complex II (1: 1000), complex III (1: 1000), Mfn1 (1: 1000), Mfn2 (1: 1000) and ERRa (1: 1000), 4 ℃ of overnight incubation, wash film, add two anti-(antibody of horseradish peroxidase-labeled), incubated at room 1 hour, ECL is luminous, the light reaching the film of X line is used NIH Image image analysis software the density of protein band is carried out semi-quantitative analysis.
Statistical analysis
All numerical value represent with mean ± standard error, carry out t check or variance analysis (ANOVA), and when P＜0.05, thinking has significant difference between processed group and the matched group, and p＜0.01 is considered to have utmost point significant difference.
II. embodiment
The observation of embodiment 1. ordinary circumstances
Each treated animal is carried out the observation of ordinary circumstance, finds the peace and quiet of quiet matched group expression, the time and vivaciously active, fur is bright and clean neat, eyes have god; Chronic power exhausts the group rat and generally has a weary expression, and afraid of one's shadow, fur comes off, and the bodily form is thin and weak, and eyes are intensely dark.
Each organizes the subtrahend situation: the death that meets accident when the training of two rats is respectively arranged of each training group, during the 8th weekend, each group respectively has 10 to be used for experiment and to draw materials.
Body weight
In the 8th week behind treadmill exercise, each group rat is weighed.Found that quiet group body weight gain is fast than each exercise group, presents positivity and increases.Power exhausts the exercise rats body weight and significantly is lower than quiet group (P＜0.5), and nutrient intervention group (comprising high dose group, low dose group) rat body weight exhausts group with chronic power and compares all increases to some extent of body weight, but difference is all not remarkable, sees Figure 1A; And caffeine group and chronic power exhaust group and compare zero difference.
Move distance
The 5th week is measured the race distance that animal is finished every day behind the treadmill exercise.Figure 1B result shows: nutrient intervention group low dose group obviously increases (p＜0.05) than the move distance that power exhausts exercise group, and the caffeine group exhausts group with power and compares there was no significant difference.Show and give the motor capacity that nutrient of the present invention can significantly improve rat.
Serum glutamic pyruvic transminase and glutamic oxaloacetic transaminase, GOT
Serum glutamic pyruvic transminase and glutamic oxaloacetic transaminase, GOT also increase with the increase of sports load, simultaneously as the index of evaluating tissue injury.
At treadmill exercise after 8 weeks, animal serum glutamate pyruvate transaminase and glutamic oxaloacetic transaminase, GOT measurement result such as Fig. 1 C.The result shows: power exhausts the group serum glutamic pyruvic transminase to be compared with quiet matched group with glutamic oxaloacetic transaminase, GOT, presents the trend of rising, but there was no significant difference; The caffeine group exhausts exercise group with power to be compared, and serum glutamic pyruvic transminase and glutamic oxaloacetic transaminase, GOT also are downward trend, exhausts group with power and has compared significant difference (p＜0.05); The glutamate pyruvate transaminase content of nutrient intervention group (high dose group, low dose group) also reduces, and compares zero difference but glutamic oxaloacetic transaminase, GOT exhausts group with power.
Serum urea nitrogen, creatinine content
Body blood urea nitrogen content increases with the increase of sports load; Body is poor more to load performance, and the serum urea nitrogen increase is obvious more.
At treadmill exercise after 8 weeks, the measurement result of animal serum blood urea nitrogen such as Fig. 1 D.The result shows: power exhausts the group serum urea nitrogen, and creatinine content obviously increases (p＜0.01) with quiet matched group; Nutrient intervention group (high dose group, low dose group) exhausts exercise group with power to be compared, and serum urea nitrogen obviously reduces (p＜0.01); The urea nitrogen content of caffeine group also presents downward trend, compares no difference of science of statistics but exhaust group with power.The creatinine content that nutrient is intervened low dose group also significantly reduces (exhaust group with power and compare p＜0.01); The creatinine content of high dose group and caffeine group also presents downward trend, exhausts group with power and compares no difference of science of statistics.
This result shows, can play a good protection the anti-fatigue ability of enhancing body after nutrient gives to body.
Embodiment 3. oxygen transport system---erythrocyte, the mensuration of hemoglobin and hematocrit
Treadmill exercise is after 8 weeks, animal erythrocyte, and the hemoglobin and hematocrit measurement result is seen Fig. 2 A-C.The result shows: power exhausts the group RBC number, and the quieter matched group of the content of hemoglobin obviously increases (p＜0.01), and packed cell volume also is significantly higher than quiet matched group (p＜0.01); The nutrient intervention group exhausts exercise group with caffeine group and power and compares erythrocyte, the content and the packed cell volume of hemoglobin obviously reduces (p＜0.01 respectively), show that power exhausts the group possibility owing to anoxia, the erythrocyte compensatory increases, and the hemoglobin compensatory raises, though can improve erythrocytic oxygen carrying capacity to a certain extent, improve histanoxia, but because the increase of packed cell volume, blood viscosity increases, and then influence the ability of red blood cell deformation, increase the weight of histanoxia.
The test of embodiment 4. immune systems
But the motion enhancing human body immunity power of appropriateness, excessive or vigorous exercise then can reduce immunity of organisms.In the present embodiment, with the periodically running of extra long distance repeatedly of endurance events in the rat treadmill exercise simulation track and field sports, cause rat power to exhaust motion model, from the generation of lymphocyte activity oxygen, Lymphocyte Apoptosis and proliferation test are estimated the motion rat body's immunity of the adjusting nutrient intervention exhausts to(for) power.
The analysis in peripheral blood lymphocyte cycle
The test of periphery lymphocyte active oxygen and apoptosis shows that power exhausts the apoptosis rate and the apparent in view increase of quiet matched group (p＜0.05) of group peripheral blood lymphocyte, and the nutrient intervention group exhausts exercise group with power and compares apoptosis rate decline (p＜0.05); Compare the apoptosis rate there was no significant difference and the caffeine group exhausts exercise group with power, see Fig. 3 A.
The mensuration of peripheral blood lymphocyte active oxygen
The mensuration of peripheral blood lymphocyte active oxygen is shown in Fig. 3 B.The result shows: power exhausts group peripheral blood lymphocyte oxygen production and the apparent in view increase of quiet matched group (p＜0.05), and the nutrient intervention group exhausts exercise group with power and compares active oxygen generation decline (p＜0.05); And exhausting exercise group with power, the caffeine group compares there was no significant difference.
The mensuration of spleen lymphocyte proliferation experiment
The measurement result of spleen lymphocyte proliferation experiment is shown in Fig. 3 C.The result shows: power exhausts group spleen lymphocyte proliferation and the apparent in view reduction of quiet matched group (p＜0.05), and nutrient intervention group and power exhaust exercise group and compares spleen lymphocyte proliferation and obviously increase (p＜0.05); And exhausting exercise group with power, the caffeine group compares there was no significant difference.
The above results shows that the nutrient intervention group can reduce the peripheral blood lymphocyte oxygen production, reduces lymphocytic apoptosis, promotes the propagation of spleen lymphocyte, and giving of demonstration nutrient can enhancing body's immunological function.
Free radical and sports fatigue have confidential relation, are the major reasons that causes sports fatigue.For a long time, people are always the oxidation resistance of generation that suppresses free radical and raising body, and the effective means of basic metabolic balance as delay fatigue that affranchise.For this reason, the inventor is from the peripheral blood MDA content, the serum antioxidative activities, and reductive glutathione, the evaluation of measuring nutrient of GST enzyme activity is intervened the effect for the rat antioxidant ability of organism.
The serum mda content
Malonaldehyde is the product of reactive oxygen free radical to polyunsaturated fatty acid oxidation on the biological cell membrane, malonaldehyde generates the space structure that increases the plurality of enzymes of inlaying on the biological cell membrane that disturbs double-deck phospholipid to arrange, and then crosslinked, polymerization formation lipofuscin takes place in the lipid, the protein that cause lipid bilayer, thereby produce damage of cell membrane popularity and pathological changes, influence the normal function of cell membrane, represent the quantity of free-radical generating usually with malonaldehyde.
Serum mda content measurement result is shown in Fig. 4 A.The result shows: power exhausts the trend that group serum mda content and quiet matched group relatively have rising, and nutrient intervention group and caffeine group and power exhaust exercise group and compares the serum mda content and have a declining tendency.
The serum antioxidative activities, reductive glutathione, the mensuration of GST enzyme activity
Serum antioxidative activities measurement result such as Fig. 4 B; Reductive glutathione assay result such as Fig. 4 C; GST enzyme activity determination result is shown in Fig. 4 D; Power exhausts group and compares obvious reduction (p＜0.05) with quiet matched group; The nutrient intervention group exhausts with power that exercise group is compared the serum antioxidative activities and the GST enzyme activity significantly increases (p＜0.05), and reductive glutathione has the trend of increase.The caffeine group exhausts group with power and compares no change.
Above result shows that the nutrient intervention group can be removed free radical effectively, stops lipid peroxidation, improves antioxidant ability of organism, and the protection body cell is avoided motional injury.
The influence of 6. pairs of Musculoskeletals of embodiment
The generation of skeletal muscle movement fatigue, development are all directly related with the variation of the structure of Skeletal Muscle Cell, function.These variations mainly comprise: the exhaustion of energy substance in the Skeletal Muscle Cell, and the inhibition of Skeletal Muscle Cell self-energy metabolic enzyme activity, the change of skeletal muscle fiber structure, the Skeletal Muscle Cell ultrastructure changes, mitochondria dysfunction etc.
The skeletal muscle body mass index
Power exhausts the one-sided gastrocnemius index of group rat skeletal muscle, and quadriceps femoris index (Fig. 5 A) and musculus soleus index (Fig. 5 B) are compared with the peace and quiet group and all presented downward trend; Nutrient intervention group, caffeine group and power exhaust group every index of comparing and all rise, and especially the quadriceps femoris index rises and demonstrates significant difference (p＜0.05).
Serum lactate dehydrogenase (SLD) vigor and creatine kinase vigor
Along with the increase of sports load, behind the rat motor damage of skeletal muscle also serious more, therefore more muscle marker enzyme can leak in the blood, and then causes the further increase of the corresponding enzyme activity level of blood.In addition, the sero-enzyme activity also can reflect the damage and the reparation situation of tissue.CPK and LDH are the important metabolic enzymes of reflection muscle physiologically active.
Blood plasma enzyme activity determination result is shown in Fig. 5 C.The result shows: the vigor that power exhausts group serum creatine kinase and lactic acid dehydrogenase is significantly higher than quiet matched group, the nutrient intervention group, caffeine group and power exhaust group to be compared the activity of two enzymes of serum and all descends, and the vigor of caffeine group creatine kinase descends significant difference (p＜0.01); And the nutrient intervention group, the vigor of caffeine group lactic acid dehydrogenase descends all significant difference (p＜0.01).
Skeletal muscle inose unit and ATP Determination on content
During sports fatigue, the concentration of Ca raises, and mitochondrial membrane potential reduces, and the synthetic level of ATP descends, and the result of Fig. 5 D shows: power exhausts the content of group flesh skeletal muscle ATP and compares reduction with quiet matched group, replenishes the content that the low dosage nutrient can increase ATP.
The accumulation of the generation of sports fatigue and energy expenditure, metabolite is relevant with the factors such as change of interior environment.Muscle glycogen is an important energy supply material in the motion, and its reserves size and tired delaying have direct relation.The result of Fig. 5 D shows: power exhausts group muscle glycogen content and compares no change with quiet matched group, and the plain content (p＜0.01) that can significantly increase muscle glycogen supplements the nutrients.
The ultrastructure of skeletal muscle
The ultrastructure of electron microscopic observation skeletal muscle, the result is shown in Fig. 5 E.The result shows: after rat power exhausted motion, musculus soleus z line was arranged unusual, and mitochondria number reduces, and behind the extra-nutrition element, saw that the mitochondria number in the sarcostyle increases, and exhausted group z line than power and arranged unusual the improvement.Through image analysis, calculate the mitochondrion surface density, show that power exhausts the quiet group of group mitochondrion surface density and reduces, and the nutrient intervention group, the mitochondrion surface density has significance to increase (p＜0.05), sees Fig. 5 F.
The mensuration of skeletal muscle tissue mitochondrion index
Mitochondrial biogenous adaptive change can strengthen the oxidability and the fatigue resistance of muscle in the Skeletal Muscle Cell, relate to the transcription factor that mitochondrion generates, comprise mitochondrion transcription factor A (Tfam), nuclear is breathed the factor (Nrf and the Nrf2) receptor-α (ERR α) relevant with estrogen.PPARGC1A is the transcription factor with multiple regulatory function, is to regulate the key factor that mitochondrion generates, and PPARGC1A stimulates the expression of Nrf1 and Tfam, thereby starts the expression of proteic karyogene of line of codes plastochondria and mitochondrial gene.Effect between mitochondrial genome and the karyogene group depends on the regulation and control of Nrf1 and Tfam, Nrf1 regulates the expression of transcribing and raising Tfam of the mitochondrial gene of nuclear coding, nuclear Tfam can raise transcribing of the interior Tfam of mitochondrion, promotes mitochondrial DNA and proteic synthetic.
The result of Fig. 5 G-J shows that the nutrient intervention group can significantly increase the mitochondrion composite I, and the proteic expression of II and III promotes the synthetic of mitochondrial DNA, has raised simultaneously and has participated in the transcription factor PPARGC1A that the regulation and control mitochondrion generates, the expression of Nrf1 and Tfam.Show that the nutrient group can promote the mitochondrion of skeletal muscle to generate, and improves mitochondrial function.
The skeletal muscle mitochondrion merges the mensuration of index
Mitochondrion merges for cell function perfect and has crucial meaning, and mitochondrion merges can promote mitochondrial cooperation.Mitochondrion connects into network and helps energy transmission therein; Help information communication, comprise that transmembrane potential is transmitted fast and the exchange of mitochondrion content.Fusion can also make between the mitochondrion by the sufficient DNA complementation of mitochondrial genome exchange carrying out.Mfn1/2 plays a significant role in the mitochondrion fusion process.
Fig. 5 K observes mitochondrial adventitia and inner membrance is local closely arranged side by side at some, and inner membrance and adventitia are close together, and other local inner membrance then inwardly is folded to form " ridge ".The expression of MFN1 and MFN2 can be significantly raised in the nutrient intervention, shows that the nutrient intervention group can promote the mitochondrion of skeletal muscle to merge, and improves mitochondrial function, shown in Fig. 5 L.
To sum up; the compositions that contains R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine of inventor's exploitation has excellent resisting fatigue; improve the mitochondrion effect; and its resisting fatigue effect of empirical tests is significantly higher than single with material or their combinations such as taurine, acetylcarnitines, also is higher than existing other antifatigue preparation.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Institute of Sports Science Research; Shanghai Inst. of Life Science, CAS
＜120〉nutrient composition of elimination sports fatigue
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Claims (10)
1. the purposes of a mixture, described mixture contains:
R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine;
Described mixture is used to prepare the antifatigue compositions.
2. purposes as claimed in claim 1 is characterized in that, described compositions is used for:
Improve the mammal motor capacity;
Reduce mammalian blood serum blood urea nitrogen and creatinine content;
Reduce the apoptosis of peripheral blood lymphocyte;
Reduce the peripheral blood lymphocyte oxygen production;
Promote spleen lymphocyte proliferation;
Increase serum antioxidative activities and GST enzyme activity;
Increase the content of skeletal muscle muscle glycogen;
Increase mitochondria number in the skeletal muscle sarcostyle;
Increase the mitochondrion composite I, the proteic expression of II or III;
Promote the synthetic of mitochondrial DNA;
Raise the transcription factor PPARGC1A that the regulation and control mitochondrion generates, the expression of Nrfl or Tfam; Or
Promote the mitochondrion of skeletal muscle to merge.
3. purposes as claimed in claim 1 is characterized in that, described mixture contains:
R-thioctic acid 1-50 weight portion;
Acetylcarnitine 2-100 weight portion;
Biotin 0.002-0.1 weight portion;
Niacin amide 0.3-15 weight portion;
Riboflavin 0.12-6 weight portion;
2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. 0.12-6 weight portion;
Creatine 1-30 weight portion;
Coenzyme Q10 0.1-5 weight portion;
Resveratrol 0.1-5 weight portion;
Taurine 2-50 weight portion.
4. purposes as claimed in claim 1; it is characterized in that the part by weight between R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and the taurine is: (5-15): (15-25): (0.015-0.025): (2-4): (1-1.5): (1-1.5): (5-15): (0.8-1.2): (0.8-1.2): (15-25).
5. a compositions is characterized in that, described compositions is used for resisting fatigue, and described compositions contains:
(a) R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine; With
(b) acceptable carrier on pharmacy or the bromatology.
6. compositions as claimed in claim 5 is characterized in that, described compositions contains:
R-thioctic acid 1-50 weight portion;
Acetylcarnitine 2-100 weight portion;
Biotin 0.002-0.1 weight portion;
Niacin amide 0.3-15 weight portion;
Riboflavin 0.12-6 weight portion;
2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. 0.12-6 weight portion;
Creatine 1-30 weight portion;
Coenzyme Q10 0.1-5 weight portion;
Resveratrol 0.1-5 weight portion;
Taurine 2-50 weight portion;
Acceptable carrier 10-1000 weight portion on pharmacy or the bromatology.
7. compositions as claimed in claim 6 is characterized in that, described compositions contains:
R-thioctic acid 1-25 weight portion;
Acetylcarnitine 2-50 weight portion;
Biotin 0.002-0.05 weight portion;
Niacin amide 0.3-7.5 weight portion;
Riboflavin 0.12-3 weight portion;
2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine. 0.12-3 weight portion;
Creatine 1-15 weight portion;
Coenzyme Q10 0.1-2.5 weight portion;
Resveratrol 0.1-2.5 weight portion;
Taurine 2-25 weight portion.
Acceptable carrier 10-1000 weight portion on pharmacy or the bromatology.
8. as the arbitrary described compositions of claim 5-7, it is characterized in that R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine sum account for the 5-100% of composition total weight.
9. compositions as claimed in claim 5; it is characterized in that the part by weight between R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and the taurine is: (5-15): (15-25): (0.015-0.025): (2-4): (1-1.5): (1-1.5): (5-15): (0.8-1.2): (0.8-1.2): (15-25).
10. an antifatigue method is characterized in that, the experimenter's effective dose that needs: R-thioctic acid, acetylcarnitine, biotin, niacin amide, riboflavin, 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine., creatine, coenzyme Q10, resveratrol and taurine.
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