CN101045048B

CN101045048B - Application of chondriosome nutrient composition

Info

Publication number: CN101045048B
Application number: CN2006101628884A
Authority: CN
Inventors: 刘健康; 沈伟利
Original assignee: Shanghai Institutes for Biological Sciences SIBS of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2006-03-27
Filing date: 2006-11-28
Publication date: 2011-05-18
Anticipated expiration: 2026-11-28
Also published as: CN101045048A

Abstract

本发明公开了一种混合物的用途，所述的混合物含有：(a)R-硫辛酸或其生理学可接受的盐；(b)一种或多种选自下组的线粒体营养素：乙酰肉碱或其生理学可接受的盐、维生素E或其生理学可接受的盐、生物素或其生理学可接受的盐、辅酶Q10或其生理学可接受的盐、或尼克酸或其生理学可接受的盐；所述的混合物可用于制备预防、改善或治疗糖尿病或胰岛素抵抗的药物或膳食添加剂，或用于制备预防、改善或治疗细胞线粒体代谢紊乱的药物或膳食添加剂。本发明还公开了一种用于预防、改善或治疗糖尿病或胰岛素抵抗的组合物，本发明的组合物能够显著预防、改善或治疗糖尿病或胰岛素抵抗，仅需很小的施用剂量即可获得显著的效果。The invention discloses a use of a mixture, the mixture contains: (a) R-lipoic acid or a physiologically acceptable salt thereof; (b) one or more mitochondrial nutrients selected from the following group: acetylcarnitine or a physiologically acceptable salt thereof, vitamin E or a physiologically acceptable salt thereof, biotin or a physiologically acceptable salt thereof, coenzyme Q10 or a physiologically acceptable salt thereof, or nicotinic acid or a physiologically acceptable salt thereof; The above mixture can be used for preparing medicine or dietary supplement for preventing, improving or treating diabetes or insulin resistance, or for preparing medicine or dietary supplement for preventing, improving or treating cell mitochondrial metabolic disorder. The invention also discloses a composition for preventing, improving or treating diabetes or insulin resistance. The composition of the invention can significantly prevent, improve or treat diabetes or insulin resistance, and only a small dosage can achieve significant Effect.

Description

The application of chondriosome nutrient composition

Technical field

The present invention relates to biotechnology and medical domain, more specifically, the present invention relates to the application in control insulin resistant and/or diabetes of chondriosome nutrient and compositions thereof.

Background technology

Diabetes (DM, Diabetes Mellitus) are now to one of disease of anthropogenic influence's maximum, mainly contain 1 type and 2 types, two major types.Type 2 diabetes mellitus is bigger to the mankind's harm, and its patient accounts for more than 90% of diabetics sum.Type 2 diabetes mellitus is usually expressed as hyperglycemia, causes the internal metabolism dysfunction, causes then to comprise that nervous system disease, nephropathy, retinopathy, hypertriglyceridemia, obesity and cardiovascular disease etc. are called as the complication of metabolic function syndrome.Because the glycosuria condition of disease causes multiple complications, it has become the fifth-largest fatal disease in the world at present.

Existing report, the mitochondrial function disorder is the major reason that causes insulin resistant and type 2 diabetes mellitus; And, mitochondrial metabolism, comprise sugar and fatty acid metabolism obviously impaired in insulin resistant and type 2 diabetes mellitus people (Lowell BB, Shulman GI. (2005) Mitochondrial dysfunction and type 2diabetes.Science.21 in muscle and the adipose cell; 307 (5708): 384-387).Have and much relate to the transcription factor that mitochondrion generates, comprise mitochondrion transcription factor A (mtTFA), mitochondrion transcription factor B1 (mtTFB1), B2, nuclear is breathed the factor (NRF1 and NRF2) receptor-α (GoffartS, Wiesner RJ. (2003) the Regulation and co-ordination of nuclear gene expression duringmitochondrial biogenesis.Exp Physiol.88 (1): 33-40) relevant with estrogen.Peroxide kinase transcription factor-γ activity factor-1 α (PGC-1 α) is the transcription factor with multiple regulatory function, be to regulate the key factor that mitochondrion generates, PGC-1 α stimulates the expression of NRF1 and mtTFA, thereby starts expression (the Wilson-Fritch L etc. (2003) of proteic karyogene of line of codes plastochondria and mitochondrial gene; Mitochondrial Biogenesis and Remodeling during Adipogenesis andin Response to the Insulin Sensitizer Rosiglitazone.Mol Cell Biol.23 (3): 1085-94).Effect between mitochondrial genome and the karyogene group depends on the regulation and control of NRF1 and mtTFA, and NRF1 regulates the expression of transcribing and raising mtTFA of the mitochondrial gene of nuclear coding, and nuclear mtTFA can raise transcribing of the interior mtTFA of mitochondrion.The thiazolidinediones medicine as can significantly raising the expression of PGC-1 α and the copy of mitochondrial DNA than lattice row ketone, promotes the oxidative phosphorylation of white adipose tissue and sensitivity (the Wilson-Fritch L etc. (2004) of increase insulin; Mitochondrial remodeling in adipose tissueassociated with obesity and treatment with rosiglitazone.J Clin Invest.114 (9): 1281-9).Therefore, the expression of promotion PGC-1 α, the mitochondrial generation of increase can improve the sensitivity of insulin and increase the utilization of glucose, and this is the effective measures of treatment and prevention insulin resistant and type 2 diabetes mellitus.

In addition, in type 2 diabetes mellitus, the mitochondrial deterioration that the mitochondrion oxidative stress can cause (Gerbitz, KD etc. (1996); Mitochondria and diabetes, Genetic, biochemical, and clinical implications of the cellularenergy circuit.Diabetes.45 (2): 113-126).Generation by the mitochondrion oxygen-derived free radicals that hyperlipemia caused can damage beta Cell of islet, because beta Cell of islet lacks the activity of antioxidase, easily produces oxidative damage.Therefore, prevent and treat the important measures that mitochondrial oxidative damage also is prevention and treatment type 2 diabetes mellitus.But although cause the beta Cell of islet function damage to receive much concern in recent years by the inductive oxidative stress of hyperlipemia at present, the damage that application effective anti-oxidants and nutrition are usually repaired due to the free fatty rarely has report.

R-thioctic acid (R-LA) is a kind of coenzyme that participates in the mitochondrion metabolic mechanism.Can be reduced to dihydrolipoic acid, be the coenzyme of lipoate acetyltransferase, it is easy to carry out redox reaction, so can protect the sulfydryl enzyme to avoid the murder by poisoning of heavy metal ion (Packer L etc. (1995); Alpha-Lipoic acid as a biological antioxidant.Free Radic BiolMed.19 (2): 227-50).Acetylcarnitine (ALC) is the acetyl derivative of L-carnitine, can transport long-chain fatty acid to the interior beta oxidation of mitochondrion, synthesizes ATP, and can remove the fatty acid of too much weak point-middle long-chain.Although R-LA is or/and ALC is proved can improve mitochondrial function in old and feeble and degenerative disease and delay mitochondrial decline.But people still understand very few for mitochondrial mechanism of action to R-LA, ALC and other nutrient, and also do not disclose R-LA, ALC and other nutrient so far for prevention and the effect that improves diabetes and insulin resistant aspect.

In addition, there are some researches show, take in vitamin E and can reduce the risk of suffering from type 2 diabetes mellitus that GK rat (type 2 diabetes mellitus model) is fed and contains 0,20 or during the food of 500mg/kg alpha-tocopherol, insulin secretion significantly increases, blood sugar level obviously descends.The raising of lipid and lipoperoxide level is the main hazard factor that causes the diabetics cardiovascular disease to take place in the blood, and diabetics oral vitamin E can obviously lower lipoperoxide and lipid level in the diabetics body.

In sum, correlational study only limits to the application of single nutrient mostly at present, do not see that for the complex treatment diabetes that these materials are formed report is arranged, and, single when using described nutrient in the prior art, application dosage is all at 200mg-2000mg/ days or more, the big and DeGrain of application dosage, therefore, press for the low and more obvious medicine that prevents and treat diabetes and insulin resistant of effect of exploitation application dosage at present.

Summary of the invention

The object of the present invention is to provide medicine or the dietary supplement of the mixture of chondriosome nutrient, or prepare prevention, improve or treat the medicine of cell mitochondrial metabolism disorder or the application in the dietary supplement at preparation prevention, improvement or treatment diabetes or insulin resistant.

In a first aspect of the present invention, a kind of purposes of mixture is provided, described mixture contains:

(a) R-thioctic acid or the acceptable salt of its physiology;

(b) one or more are selected from down the chondriosome nutrient of group: acetylcarnitine or the acceptable salt of its physiology, vitamin E or the acceptable salt of its physiology, biotin or the acceptable salt of its physiology, coenzyme Q10 or the acceptable salt of its physiology or nicotinic acid or the acceptable salt of its physiology;

Wherein, medicine or dietary supplement that described mixture is used to prepare prevention, improves or treat diabetes or insulin resistant, or be used to prepare the medicine or the dietary supplement of prevention, improvement or the metabolism disorder of treatment cell mitochondrial or be used to prepare medicine or the dietary supplement that strengthens oxidation resistance.

In another preference of the present invention, described component (b) is acetylcarnitine or the acceptable salt of its physiology.

In another preference of the present invention, described mixture contains:

(a) R-thioctic acid or the acceptable salt of its physiology;

(b) acetylcarnitine or the acceptable salt of its physiology, and one or more are selected from down the chondriosome nutrient of group: vitamin E or the acceptable salt of its physiology, biotin or the acceptable salt of its physiology, coenzyme Q10 or the acceptable salt of its physiology or nicotinic acid or the acceptable salt of its physiology.

In a second aspect of the present invention, a kind of compositions is provided, described compositions is used for prevention, improves or treatment diabetes or insulin resistant, and described compositions contains:

(a) R-thioctic acid or the acceptable salt of its physiology;

(b) one or more are selected from down the chondriosome nutrient of group: acetylcarnitine or the acceptable salt of its physiology, vitamin E or the acceptable salt of its physiology, biotin or the acceptable salt of its physiology, coenzyme Q10 or the acceptable salt of its physiology or nicotinic acid or the acceptable salt of its physiology.

In a preference of the present invention, described component (b) is acetylcarnitine or the acceptable salt of its physiology.

In another preference of the present invention, described compositions contains:

(a) R-thioctic acid or the acceptable salt of its physiology;

In another preference of the present invention, the content of component (a) R-thioctic acid or the acceptable salt of its physiology is the 10-200 weight portion;

And when one or more chondriosome nutrients of containing described in the component (b), described chondriosome nutrient content is: acetylcarnitine or the acceptable salt 10-200 of its physiology weight portion, vitamin E or the acceptable salt 20-200 of its physiology weight portion, biotin or the acceptable salt 0.2-2 of its physiology weight portion, coenzyme Q10 or its physiology acceptable salt 10-200 weight portion or nicotinic acid or the acceptable salt 5-300 of its physiology weight portion;

And described component sum accounts for the 10-100% of composition total weight.

(a) R-thioctic acid or the acceptable salt of its physiology: 10-200 weight portion; With

(b1) acetylcarnitine or the acceptable salt of its physiology: 10-200 weight portion;

And component (a)+(b1) sum accounts for the 25-100% of composition total weight.

In another preference of the present invention, also contain in the described compositions:

(c1) vitamin E or the acceptable salt of its physiology: 20-200 weight portion;

(d1) biotin or the acceptable salt of its physiology: 0.2-2 weight portion; With

(e1) coenzyme Q10 or the acceptable salt of its physiology: 10-200 weight portion.

In another preference, component (a) and weight ratio (b1) are 1: 10-10: 1; Preferred, component (a) and weight ratio (b1) are 1: 5-5: 1; Most preferred, component (a) and weight ratio (b1) they are 1: 3-3: 1, such as, component (a) and weight ratio (b1) they can be 1: 1.

In another preference, component (a)+(b1) sum accounts for the 40-99% of composition total weight; Preferred, component (a)+(b1) sum accounts for the 50-90% of composition total weight.

In another preference, contain in the described compositions:

(a) R-thioctic acid or the acceptable salt of its physiology: 10-200 weight portion;

(c1) vitamin E or the acceptable salt of its physiology: 20-200 weight portion;

In another preference, described compositions contains:

(a) R-thioctic acid or the acceptable salt of its physiology: 100 weight portions;

(b1) acetylcarnitine or the acceptable salt of its physiology: 100 weight portions.

In another preference, described compositions contains:

(b1) acetylcarnitine or the acceptable salt of its physiology: 100 weight portions;

(c1) vitamin E or the acceptable salt of its physiology: 100 weight portions;

(d1) biotin or the acceptable salt of its physiology: 1 weight portion; With

(e1) coenzyme Q10 or the acceptable salt of its physiology: 100 weight portions.

In another preference, described compositions contains:

(c1) vitamin E or the acceptable salt of its physiology: 100 weight portions;

(d1) biotin or the acceptable salt of its physiology: 1 weight portion; With

(e1) nicotinic acid or the acceptable salt of its physiology: 100 weight portions.

In another preference, also contain in the described compositions:

(g) acceptable carrier or excipient pharmaceutically or on the bromatology: 50-1000 weight portion.

In another preference of the present invention, also contain in the described compositions: acceptable carrier or excipient pharmaceutically or on the bromatology.

In another preference of the present invention, the content of acceptable carrier or excipient is the 50-1000 weight portion on the pharmaceutically described or bromatology.

In another preference of the present invention, described compositions is a pharmaceutical composition; Or be food composition.

In another preference of the present invention, described compositions is a unit dosage form, and every dose contains 20-200mg component (a) and 20-200mg component (b); Preferred, every dose contains 30-150mg component (a) and 30-150mg component (b).

In another preference of the present invention, take the compositions 1-3 agent of described unit dosage form every day; Preferred, as to take described unit dosage form every day compositions 1-2 agent.

In another preference of the present invention, the dosage form of described compositions is selected from: granule, capsule, tablet, powder agent, oral liquid, suspension or Emulsion.

In another preference of the present invention, go to school acceptable carrier or excipient of pharmaceutically described or food is selected from: filler, disintegrating agent, lubricant, fluidizer, effervescent, correctives, clad material, meals goods or slow/controlled releasing agent.

In another preference of the present invention, described compositions and energy matter are used jointly, and described energy matter is a carbohydrate.

In another preference of the present invention, described compositions is a kind of dietary supplement.

In another preference of the present invention, described compositions is added in water, the non--orange juice beverage, in solid food, the dish-cooking.

In a third aspect of the present invention, a kind of purposes of mixture is provided, described mixture is by (i) R-thioctic acid or the acceptable salt of its physiology, (ii) acetylcarnitine or the acceptable salt of its physiology constitute, and component (i) and weight ratio (ii) are 1: 20～20: 1, the medicine that described mixture is used to prepare prevention, improves or treat the cell mitochondrial metabolism disorder; Or the medicine that is used to prepare prevention, improves or treat diabetes or insulin resistant; Or be used to prepare medicine or the dietary supplement that strengthens oxidation resistance.

In another preference of the present invention, the content of component (i) is the 10-200 weight portion, component content (ii) is the 10-200 weight portion, and described compositions contains the chondriosome nutrient that one or more are selected from down group: vitamin E or the acceptable salt of its physiology, biotin or the acceptable salt of its physiology, coenzyme Q10 or the acceptable salt of its physiology or nicotinic acid or the acceptable salt of its physiology.

In a fourth aspect of the present invention, the method for a kind of prevention, improvement or treatment diabetes or insulin resistant is provided, comprise that experimenter to needs applies effective dose:

(a) R-thioctic acid or the acceptable salt of its physiology;

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 has shown R-LA or/and ALC acts on adipose cell collecting cell after 24 hours, after using MitoTracker (100nM) probe and hatching 30 minutes, and the flow cytometry fluorescence intensity.Curve moves to right and represents the increase of mitochondria number.Wherein, a is the R-LA effect, and b is the ALC effect, and c is the R-LA+ALC combined effect.And i is R-LA or/and ALC0.1 μ M, and ii is R-LA or/and ALC 1 μ M, and iii is R-LA or/and ALC 10 μ M, and iiii is that R-LA is or/and ALC 100 μ M.Figure 1B has shown the R-LA of 3T3-L1 adipose cell and variable concentrations or/and ALC is hatched collecting cell after 24 hours, again after Mito Tracker (100nM) probe is hatched 30 minutes, and the flow cytometry fluorescence intensity; The result gets average ± standard deviation mapping of the result of 4 tests to represent with respect to the multiple of cellular control unit fluorescence intensity, ^*P＜0.05, ^*P＜0.01 compares with matched group that there were significant differences.

Fig. 2 A shown electronic microscope photos R-LA or/and ALC for the mitochondrial effect of adipose cell.Wherein, A is the mitochondrial form of adipose cell (amplification 10,000), and adipose cell broke up the 8th day, uses (ii) R-LA (10 μ M), (iii) ALC (10 μ M), and (iiii) R-LA (10 μ M)+ALC (10 μ M), matched group (i) does not add medicine.Fig. 2 B has shown mitochondrion sectional area and mitochondrion density, gets average ± standard deviation mapping of the result of 4 tests, ^*P＜0.05 compares with matched group that there were significant differences.

Fig. 3 has shown R-LA or/and ALC regulates and control mitochondrion transmission compound I, II, III albumen, the R-LA of adipose cell and variable concentrations is or/and the ALC effect after 24 hours, is used the expression of the methods analyst mitochondrion electron transport chain complex proteins of immune protein trace.Numerical value is represented with the multiple with respect to matched group, gets the average ± standard deviation of 3 tests and represents that β-actin is as confidential reference items. ^*P0.05, ^*P＜0.01 compares with matched group that there were significant differences.

Fig. 4 shown R-LA or/and ALC for the expression of PGC-1 α.The R-LA of adipose cell through using variable concentrations is or/and the ALC effect was compared with matched group after 24 hours, and numerical value is represented with the multiple with respect to matched group, gets the average ± standard deviation of 3 tests and represents that β-actin is as confidential reference items. ^*P＜0.05, ^*P＜0.01 compares with matched group that there were significant differences.

Fig. 5 has shown R-LA or/and ALC stimulates the expression of NRF1 and NRF2.Adipose cell uses the R-LA of variable concentrations or/and ALC handled 24 hours, extracts RNA.The expression of NRF1 and NRF2 mRNA in the RT-PCR analysis adipose cell.Numerical value is represented with the multiple with respect to matched group, gets the average ± standard deviation of 4 tests and represents that β-actin is as confidential reference items. ^*P＜0.05 expression is compared NRF1 with matched group, and there were significant differences.#p＜0.05 expression is compared NRF2 with matched group, and there were significant differences.

Fig. 6 has shown the protein-bonded activity of gel blocking electrophoretic analysis mitochondrion mtTFA.The adipose cell mitochondrion that the adipose cell mitochondrion B:ALC that the complex A:R-LA that the mtTFA probe of autoradiography 32P labelling and mitochondrion extracting protein binding form handles handles; Swimming lane 6 is shown as emulative reaction, uses 50 times of unlabelled probe sealing back retardance bands and disappears, and illustrates that this band is specificity retardance band.

Fig. 7 has shown that LA and ALC are to the excretory regulating action of MIN6 cell insulin.The MIN6 cell uses LA (10 μ M) or/and ALC (10 μ M) pretreatment 6 hours adds oleic acid (0.4mM) then and hatched altogether 72 hours.Insulin assay is that basal insulin (glucose 2.8mM) and glucose stimulate the insulin secretion of (glucose 30mM), insulin secretion is to represent with respect to the excretory multiple of the basal insulin of normal control group, the result represents the average (average ± standard deviation) of three independent trialss, and insulin secretion is with every milligram of protein quantification.* P＜0.05 expression oleic acid group compares with matched group that there were significant differences, #P＜0.05, and ##P＜0.01 expression adds LA, and the pharmaceutical intervention group of ALC compares with the oleic acid group that there were significant differences.

Fig. 8 has shown that the ATP that glucose stimulates generates.The MIN6 cell uses LA (10 μ M) or/and ALC (10 μ M) pretreatment 6 hours adds oleic acid (0.4mM) then and hatched altogether 72 hours.Cell was hatched in the buffer that contains glucose 2.8mM or 30mM 1 hour respectively.Cell lysis is used the content that ATP bioluminescence enzyme assay determination agent box is measured ATP then.Content is with the equal value representation (average ± standard deviation) of 5 tests.* P＜0.05 expression oleic acid group compares with matched group that there were significant differences, and the pharmaceutical intervention group that #P＜0.05 expression adds LA or ALC compares with the oleic acid group that there were significant differences

Fig. 9 has shown the expression of MIN6 cell UCP-2mRNA.Wherein, the A:MIN6 cell is used the oleic acid of variable concentrations and was handled 72 hours, extracts RNA, the expression of UCP-2mRNA in the quantitative PCR analysis MIN6 cell.Numerical value represents with the multiple with respect to matched group, gets the average ± standard deviation of 4 tests and represents, β-actin is as confidential reference items, and * P＜0.05 expression oleic acid group compares with matched group that there were significant differences.The B:MIN6 cell uses LA (10 μ M) or/and ALC (10 μ M) pretreatment 6 hours adds oleic acid (0.4mM) then and hatched altogether 72 hours.Use the expression of quantitative-pcr analysis UCP-2mRNA, numerical value is represented with the multiple with respect to matched group, getting the average ± standard deviation of 4 tests represents, β-actin is as confidential reference items, * P＜0.05 expression oleic acid group compares with matched group that there were significant differences, #P＜0.05 expression adds LA, and the pharmaceutical intervention group of ALC compares with the oleic acid group that there were significant differences.

Figure 10 has shown the proteic expression of immune protein engram analysis MIN6 cell UCP-2.Wherein, the A:MIN6 cell is used the oleic acid of variable concentrations and was handled 72 hours, extract total protein, use the proteic expression of immune protein engram analysis MIN6 cell UCP-2, numerical value is represented with the multiple with respect to matched group, get the average ± standard deviation of 3 tests and represent that β-actin is as confidential reference items, * P＜0.05 expression oleic acid group compares with matched group that there were significant differences.The B:MIN6 cell uses LA (10 μ M) or/and ALC (10 μ M) pretreatment 6 hours adds oleic acid (0.4mM) then and hatched altogether 72 hours.Use the proteic expression of immune protein engram analysis MIN6 cell UCP-2, numerical value is represented with the multiple with respect to matched group, getting the average ± standard deviation of 3 tests represents, β-actin is as confidential reference items, * P＜0.05 expression oleic acid group compares with matched group that there were significant differences, and the pharmaceutical intervention group that #P＜0.05 expression adds LA or ALC compares with the oleic acid group that there were significant differences.

Figure 11 has shown MIN6 cell mitochondrial transmembrane potential.Wherein, the A:MIN6 cell is used (0.4mM) oleic acid and is handled after 72 hours, and with JC-1 (10 μ g/ml), probe was hatched 30 minutes, uses DAPI transfect cell nuclear, and the Zeiss digital camera is taken the photograph sheet.The B:MIN6 cell is used the oleic acid of variable concentrations and is handled after 72 hours, with JC-1 (10 μ g/ml), probe was hatched 30 minutes, be resuspended in again in the KRBH buffer, fluorescence microplate reader is analyzed the ratio of (FL1/FL2), and numerical value is represented with the multiple with respect to matched group, gets the average ± standard deviation of 5 tests and represents, * P＜0.05, * * P＜0.01 expression oleic acid group compare with matched group that there were significant differences.The C:MIN6 cell is used the LA of variable concentrations, ALC pretreatment 6 hours, add then after oleic acid (0.4mM) hatches 72 hours altogether, with JC-1 (10 μ g/ml), probe was hatched 30 minutes, fluorescence microplate reader is analyzed the ratio of (FL1/FL2), numerical value is represented with the multiple with respect to matched group, getting the average ± standard deviation of 5 tests represents, * P＜0.05 expression oleic acid group compares with matched group that there were significant differences, #P＜0.05##P＜0.01 expression adds LA, and the pharmaceutical intervention group of ALC compares with the oleic acid group that there were significant differences.

Figure 12 has shown that transmission electron microscope takes the photograph sheet (amplification 10,000) and show that contrast (A) and oleic acid stimulate (B).Beta cell secretory granule and mitochondrion after oleic acid stimulates obviously reduce.The obvious swelling of mitochondrion lacks, and tangible pyknosis or chromosomal fragment do not appear in nucleus.

Figure 13 has shown the mensuration of active oxygen.The A:MIN6 cell is used (0.4mM) oleic acid and is handled after 72 hours, hatches flow cytometry analysis 30 minutes with DCF-DA (10 μ M) probe. and curve moves to right and represents that oleic acid stimulates the back active oxygen to produce and increases a: negative control (not adding probe); B: normal control; C:0.2mM oleic acid; D:0.4mM oleic acid; E:0.8mM oleic acid.The B:MIN6 cell is used the oleic acid of variable concentrations and is handled after 72 hours, and flow cytometry analysis, numerical value are represented with the multiple with respect to matched group, gets the average ± standard deviation of 5 tests and represents, * P＜0.05, * * P＜0.01 expression oleic acid group and contrast

Group compares that there were significant differences.The C:MIN6 cell is used the LA of variable concentrations or/and ALC pretreatment 6 hours, add then after oleic acid (0.4mM) hatches 72 hours altogether, flow cytometry analysis, * P＜0.05 expression oleic acid group compares with matched group that there were significant differences, the pharmaceutical intervention group that #P＜0.05, ##P＜0.01 expression add LA or ALC compares with the oleic acid group that there were significant differences.

Figure 14 has shown application 2-D-[3H] method of glucose assessment nutrient complex is for the effect of the glucose transport of insulin stimulating.Nutrient complex (0.1-1000 μ M) respectively with adipose cell effect 0.5 hour, 6 hours, 12 hours, 24 hours and 48 hours, measure the glucose transport of insulin stimulating then.Numerical value to be representing (average ± standard deviation) with respect to the multiple of blank group, and * p＜0.05 shows that experimental group compares with the blank group that there were significant differences.

Figure 15 uses the determination of glucose oxidase fasting glucose after having shown that respectively organizing rat stands to try thing (chondriosome nutrient) or normal saline and handled for 8 weeks.Blood glucose value is represented with mean+SD.Compare * p＜0.05, * * P＜0.01 with the GK matched group.

After Figure 16 has shown that respectively organizing rat stands to try thing (chondriosome nutrient) or normal saline and handled for 8 weeks, the OGTT experiment.Fasting 16 hours before the experiment, 50% G/W 2g/kg irritates stomach, gets blood, the blood glucose determination of glucose oxidase from the posterior orbit venous plexus in 30,60,120 minutes after the kimonos sugar before clothes are sugared.Blood glucose value is represented with mean+SD.Compare * p＜0.05, * * P＜0.01 with the GK matched group.

Figure 17 has shown the measurement result of thymus index, and weight method is measured organ index, and the thymus index value is represented with average ± standard error.Compare * p＜0.05 with the GK matched group.

Figure 18 has shown spleen lymphocyte in-vitro multiplication ability, uses mtt assay and measures external lymphocytic multiplication capacity.Compare * p＜0.05 with the GK matched group.

Figure 19 has shown the mensuration of mda content in the blood plasma. the blood plasma lipide levels of peroxide is measured the content of TBARS in the blood plasma.Compare * p＜0.05 with the GK matched group.

Figure 20 has shown the mensuration of blood plasma Total antioxidant capacity.Compare * p＜0.05 with the GK matched group.

The specific embodiment

The inventor is through extensive and deep research, be surprised to find that, to 1. R-thioctic acid, and one or more chondriosome nutrients that 2. are selected from down group: acetylcarnitine, coenzyme Q10, biotin, vitamin E, or nicotinic acid use in conjunction, can significantly increase the mitochondrial generation of the type 2 diabetes mellitus adipose cell relevant with insulin resistant, and the mitochondrion oxidative damage of repairing pancreas β cell, thereby can be used for prevention and improve type 2 diabetes mellitus and insulin resistant, and the use in conjunction of above-mentioned chondriosome nutrient can reduce application dosage greatly than independent use.Thereby finished the present invention.

As used herein, term " contain " or " comprising " comprised " comprising ", " basically by ... constitute " and " by ... constitute ".

As used herein, term " basically by ... constitute " refer in compositions, except containing neccessary composition or necessary component, also can contain a spot of and 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, the composition of term " pharmaceutically acceptable " or " acceptable on the bromatology " 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 " 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, 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 the chondriosome nutrient composition of the present invention, each component also can be used with " the acceptable salt of physiology " or " acceptable acid of physiology or the deutero-salt of alkali " form, or uses with the form of " amide ".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 " compositions of the present invention " comprises pharmaceutical composition, food composition and/or dietary supplement, as long as they contain or are made of 1. R-thioctic acid+acetylcarnitine basically or contain or be made of in 2. R-thioctic acid+acetylcarnitine+coenzyme Q10, biotin, vitamin E or the nicotinic acid one or more basically.Usually, 1. the weight of one or more in R-thioctic acid+acetylcarnitine or 2.-thioctic acid+acetylcarnitine+coenzyme Q10, biotin, vitamin E or the nicotinic acid accounts for the 25-100% of composition total weight, preferably 40-99%, more preferably 50-90%.

As used herein, " ALC " and " ALCAR " is used interchangeably, and all is representative " acetylcarnitine ".

As used herein, term " unit dosage form " is meant for taking convenience, becomes single to take required dosage form preparation of compositions of the present invention, includes but not limited to various solid formulation (as tablet), liquid agent, capsule, slow releasing agent.Contain for prevention, treatment in the described unit dosage form or improve diabetes or the effective compositions of the present invention of insulin resistant.

As used herein, " chondriosome nutrient " is meant the nutrient that can the protective wire plastochondria avoid oxidative damage and can improve mitochondrial function, comprise those 1. the protective wire plastochondria exempt from oxidative stress, as antioxidant and metallo-chelate; 2. repair mitochondrial membrane; 3. functional reparation and prevent the mitochondrion oxidative damage is as the substrate or the coenzyme of cyclophorase; 4. induce the expression of 2 phase antioxidases.

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%.

Compositions of the present invention can be directly used in prevention, alleviate or treatment type 2 diabetes mellitus or insulin resistant, or can with other medicines or dietary supplement co-administered.

The compositions of chondriosome nutrient

In the present invention, the chondriosome nutrient composition that can be effective to prevent, improve and treat insulin resistant and type 2 diabetes mellitus comprises: R-thioctic acid+be selected from down one or more chondriosome nutrients of group: acetylcarnitine, coenzyme Q10, biotin, vitamin E or nicotinic acid.For the mammal that suffers from or easily suffer from insulin resistant and type 2 diabetes mellitus, use the compositions that above-mentioned two or more described chondriosome nutrient is prepared into, can obtain the effect of significantly promoting.Described compositions is a pharmaceutical composition; Or be food composition.

In a kind of preference of the present invention, adopt the form of one or more use in conjunction in R-thioctic acid+acetylcarnitine use in conjunction and R-thioctic acid+acetylcarnitine+coenzyme Q10, biotin, vitamin E or the nicotinic acid, improve greatly on effect than using a certain chondriosome nutrient separately, and on dosage, reduce greatly.In addition, should be understood that when adopting the combination of 1. R-thioctic acid+acetylcarnitine, with any in coenzyme Q10, biotin, vitamin E or the nicotinic acid or multiplely join 1., can reach more excellent effect.

In a kind of optimal way of the present invention, employing is selected from following component and prepares described compositions: the salt (niacin amide) of the salt of R-thioctic acid, acetylcarnitine (hydrochlorate), coenzyme Q10, biotin, vitamin E or nicotinic acid.

In a kind of optimal way of the present invention, the compositions that is used for preventing, improving or treat type 2 diabetes mellitus or insulin resistant of the present invention, contain:

And component (a)+(b1) sum accounts for the 25-100% of composition total weight; More particularly, component (a)+(b1) sum accounts for the 40-99% of composition total weight; Preferred, component (a)+(b1) sum accounts for the 50-90% of composition total weight.

In a kind of optimal way of the present invention, component (a) with (b1) exist in the proper ratio, be 1 (a): 10-10: 1 with (b1) weight ratio; Preferred, component (a) and weight ratio (b1) are 1: 5-5: 1; Most preferred, component (a) and weight ratio (b1) they are 1: 3-3: 1, such as, component (a) and weight ratio (b1) they can be 1: 1.

In a kind of optimal way of the present invention, also contain one or more that are selected from following component in the described compositions:

(c1) vitamin E or the acceptable salt of its physiology: 20-200 weight portion;

(d1) biotin or the acceptable salt of its physiology: 0.2-2 weight portion;

(e1) coenzyme Q10 or the acceptable salt of its physiology: 10-200 weight portion; Or

(f1) nicotinic acid or the acceptable salt of its physiology: 5-300 weight portion.

Also be, when containing component (a)+(b1) in the compositions, wherein can contain one or more of component (c1), component (d1), component (e1) or component (f1), such as, described compositions includes but not limited to: the combination of the compositions of compositions (a)+(b1)+(c1), (a)+(b1)+(d1), the compositions of (a)+(b1)+(d1), (a)+(b1)+(c1)+(d1), (a)+(b1)+(c1)+(d1)+(e1) or (a)+(b1)+(c1)+(d1)+(f1).

As a kind of particularly preferred compositions of the present invention, it is as follows that it contains component: (a) R-thioctic acid or the acceptable salt of its physiology: 10-200 weight portion; (b1) acetylcarnitine or the acceptable salt of its physiology: 10-200 weight portion; (c1) vitamin E or the acceptable salt of its physiology: 20-200 weight portion; (d1) biotin or the acceptable salt of its physiology: 0.2-2 weight portion; (e1) coenzyme Q10 or the acceptable salt of its physiology: 10-200 weight portion.

As a kind of particularly preferred compositions of the present invention, described compositions contains:

In another preference, described compositions contains:

(c1) vitamin E or the acceptable salt of its physiology: 100 weight portions;

(d1) biotin or the acceptable salt of its physiology: 1 weight portion; With

(c1) vitamin E or the acceptable salt of its physiology: 100 weight portions;

(d1) biotin or the acceptable salt of its physiology: 1 weight portion; With

As a kind of particularly preferred compositions of the present invention, also contain in the described compositions: acceptable carrier or excipient pharmaceutically or on the bromatology: 50-1000 weight portion.

The application dosage of chondriosome nutrient composition and dosage form

The present invention is with one or more chondriosome nutrients of R-thioctic acid+be selected from down group: acetylcarnitine, coenzyme Q10, biotin, vitamin E or nicotinic acid use in conjunction, more better than using single chondriosome nutrient effect separately, and the dosage that need use reduces greatly.

In a kind of optimal way of the present invention, when with chondriosome nutrient thioctic acid+acetylcarnitine use in conjunction during in human body, the application dosage of thioctic acid+acetylcarnitine compositions is 20-600mg/ days; Preferred, the application dosage of compositions is 30-400mg/ days; Most preferred, the application dosage of compositions is 50-200mg/ days.

In another kind of optimal way of the present invention, when with one or more use in conjunction in R-thioctic acid+acetylcarnitine+coenzyme Q10, biotin, vitamin E or the nicotinic acid during in human body, the application dosage of described compositions is 20-700mg/ days; Preferred, the application dosage of compositions is 30-500mg/ days; Most preferred, the application dosage of compositions is 50-200mg/ days.

When being used for pharmaceutical compositions or food composition, the effective dose of used chondriosome nutrient composition can change with the order of severity of pattern of using and disease to be treated.

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: granule, capsule, tablet, powder agent, oral liquid, suspension or Emulsion.

When taking described chondriosome nutrient, preferably take energy matter simultaneously, be a kind of carbohydrate such as described energy, thereby auxiliary energy is provided, promote the synthetic of mitochondrial ATP.

In another optimal way of the present invention, described compositions is added in water, non--beverage, solid food, the dish-cookings such as orange juice beverage, such as adding in Sucus Vitis viniferae or the Sucus Mali pumilae as a kind of dietary supplement.Preferably add in the meals that 50-150 caloric heat at least can be provided.

In another optimal way of the present invention, go to school acceptable carrier or excipient of described food is selected from: filler, disintegrating agent, lubricant, fluidizer, effervescent, correctives, clad material, meals goods or slow/controlled releasing agent.

In another optimal way of the present invention, described compositions is a unit dosage form, and every dose contains 20-200mg component (a) and 20-200m gram component (b); Preferred, every dose contains 30-150mg component (a) and 30-150m gram component (b).

When preparation of compositions is become unit dosage form, take the compositions 1-3 agent of described unit dosage form every day; Preferred, as to take described unit dosage form every day compositions 1-2 agent.

Described chondriosome nutrient composition can give by approach such as oral.Solid-state carrier comprises: starch, lactose, dicalcium phosphate, microcrystalline Cellulose, sucrose and kaolin, and liquid carrier comprises: culture medium, Polyethylene Glycol, nonionic surfactant and edible oil (as Semen Maydis oil, Oleum Arachidis hypogaeae semen and Oleum sesami).Normally used adjuvant also can advantageously be comprised in pharmaceutical compositions, for example flavoring agent, pigment, antiseptic and antioxidant such as vitamin C, BHT and BHA.

From being easy to prepare the position with administration, preferred pharmaceutical composition is a solid-state composition, and especially tablet and solid are filled or the capsule of liquid filling.Oral administration is preferred.

Preferred dosage form is that those can guarantee that nutrient slowly and stably replenished to mitochondrion in 24 hours, and a kind of mode is to carry chondriosome nutrient composition with suitable carriers, makes chondriosome nutrient composition slowly discharge.Such as with excipient (as hydroxypropyl methylcellulose) as clad material, chondriosome nutrient is wrapped in wherein, form spherical particle (coating slow/controlled release preparation); Or chondriosome nutrient is written into (matrix type slow/controlled release preparation) in the matrix sustained release tablet (as hydroxyethyl-cellulose).

Should be understood that also can to contain other human body in the chondriosome nutrient composition of the present invention necessary or to human body beneficial's composition, such as containing a spot of vitamin C, nicotiamide, ubiquitin etc.In addition, according to doctor's guidance, chondriosome nutrient composition of the present invention also can be used jointly with other conventional diabetes or insulin resistant medicine.

The preparation method of chondriosome nutrient composition of the present invention decides according to the dosage form and the route of administration of required preparation, those skilled in the art adopt the conventional pharmaceutical composition or the preparation method of food composition can prepare compositions of the present invention after combination and proportioning with reference to chondriosome nutrient provided by the present invention.

The purposes of chondriosome nutrient mixtures

The inventor has confirmed medicine or the dietary supplement that chondriosome nutrient mixture of the present invention or compositions can be used for preparing prevention, improve or treat the cell mitochondrial metabolism disorder first; Perhaps, chondriosome nutrient mixture of the present invention or compositions can be used for preparing the medicine or the dietary supplement of prevention, improvement or treatment type 2 diabetes mellitus or insulin resistant; Perhaps, chondriosome nutrient mixture of the present invention or compositions can be used for preparing medicine or the dietary supplement that strengthens oxidation resistance.

In an embodiment of the present invention, use the cell model of type 2 diabetes mellitus, studied the mechanism of chondriosome nutrient improvement and treatment diabetes and insulin resistant.The inventor adopts cell (2 type) diabetes model from mitochondrion morphology, respiratory chain oxygen consumption activity, the mitochondrion key enzyme activity, and mitochondrion generates the variation of relevant transcription factor, investigate chondriosome nutrient for mitochondrion form in the cell, the regulation and control of number and function, for the effect of cell antioxidant system and adipose cell for the sensitivity of insulin and the variation of beta Cell of islet secretory function.

At first, the present invention confirms that R-thioctic acid and acetylcarnitine promote mitochondrial generation in the adipose cell.The inventor sets up the model of 3T3-L1 adipose cell, and two chondriosome nutrients of research alpha-lipoic acid and acetylcarnitine promote the single and cooperative effect that the adipose cell mitochondrion generates.Result of study show R-thioctic acid and acetylcarnitine associating low dose group (0.1 μ M-100 μ M) can the significant stimulation adipose cell in the increase of mitochondria number, and the effect of applied nutrition element is not remarkable separately.Use R-thioctic acid and acetylcarnitine simultaneously separately and fail to raise mitochondrion transfer chain composite I, the proteic expression of II; And associating dosage group (0.1 μ M), associating dosage group (1.0 μ M) can increase mitochondrion transfer chain composite I respectively, the proteic expression of II.The R-thioctic acid can raise the protein expression of mitochondrion transfer chain composite I II when 10,100 μ M concentration; Acetylcarnitine can increase the protein expression of composite I II when 1.0 μ M concentration; All can raise the proteic expression of composite I II and unite group (0.1-10 μ M).Further inquiring into its regulatory mechanism finds, R-thioctic acid (1-10 μ M), acetylcarnitine (0.1-10 μ M) raises the expression of transcription factor PGC-1 α respectively, R-thioctic acid and acetylcarnitine associating dosage group can promote the expression of PGC-1 α at (0.1-10 μ M), especially can significantly increase the expression of PGC-1 α in the concentration of 10 μ M, effect is better than the application of single dosage.Simultaneously find that also R-thioctic acid and acetylcarnitine can raise NRF1, the expression of NRF2 and mtTFA.

Secondly, the present invention confirms that R-thioctic acid and acetylcarnitine are repaired the mitochondrial injury that oleic acid is induced beta Cell of islet.The inventor carried out mitochondrion uncoupling protein 2 (UCP2) in the beta Cell of islet damage expression and with the research of insulin secretion relation, and thioctic acid, the single and cooperative effect of two chondriosome nutrients of acetylcarnitine are used in research.Adopt oleic acid to induce the external model of beta Cell of islet damage, observe and find that the oleic acid chronic stimulation has suppressed the insulin secretion that glucose stimulates, the result shows that the mitochondrion active oxygen produces obviously increase in the damage model, and the oxidative stress status that continues impels the mitochondrion transmembrane potential to descend; The expression that simultaneously can induce mitochondrial uncoupling protein-2 (UCP-2) causes oxidative phosphorylation decoupling zero connection in the mitochondrion, and Intramitochondrial ATP is synthetic to be reduced, and can think that in view of the above UCP-2 is the negativity regulatory factor of insulin secretion.And find that chondriosome nutrient R-thioctic acid and acetylcarnitine can remove the oxygen-derived free radicals that oleic acid causes and too much produce, repair mitochondrial membrane potential, on mRNA and protein level, suppress the rise of UCP-2, thereby ATP's is synthetic in the mitochondrion that the raising glucose stimulates, and increases the insulin secretion of glucose stimulation.And the inventor observes above-mentioned effect obviously is better than single nutrient at the action effect of R-thioctic acid (10 μ M) and acetylcarnitine (10 μ M) associating dosage group application.

And the present invention has verified further that also in chondriosome nutrient complex R-thioctic acid+acetylcarnitine+coenzyme Q10, biotin, vitamin E, the nicotinic acid one or more can promote the glucose transport of adipose cell insulin stimulating.Adipose cell becomes adipose cell before the external evoked 3T3-L1, observe variable concentrations chondriosome nutrient complex to adipose cell effect different time after, use 2-D-[ ³H] the method assessment nutrient complex of glucose is for the effect of the glucose transport of insulin stimulating.Nutrient complex (10-100 μ M) effect can significantly increase the glucose transport of insulin stimulating after half an hour; This effect is sustainable under 10 μ M concentration to reach 6 hours.Long-time effect (48 hours) shows under the 100 μ M concentration, can obviously increase the glucose transport of insulin stimulating.

In aforementioned cell model, the inventor adopts R-thioctic acid+acetylcarnitine of low dosage (as 0.1 μ M, 1 μ M, 10 μ M); Or in R-thioctic acid+acetylcarnitine+coenzyme Q10, biotin, vitamin E or the nicotinic acid one or more, can obtain significant effect.Therefore, when described chondriosome nutrient composition is used for mammal, only need uses and be lower than single chondriosome nutrient consumption 2-100 commonly used doubly (preferred 3-60 doubly; More preferably 5-30 is doubly) described chondriosome nutrient composition, can reach the good improvement and the effect of treatment diabetes.

Should understand, although in the specific embodiment, the inventor has enumerated the combining form of several chondriosome nutrients, but those skilled in the art also can derive thus: the combining form of other any chondriosome nutrient of the present invention also is to have outstanding effect equally.

Use the method for chondriosome nutrient composition prevention, improvement or treatment diabetes or insulin resistant

Chondriosome nutrient composition of the present invention can effectively prevent, improves or treat diabetes or insulin resistant, therefore, the present invention also provides the described compositions or the mixture that apply effective dose by the experimenter to needs to prevent, improve or treat the method for described disease.

When being used for prevention, improving or when treatment diabetes or insulin resistant, the effective dose of used chondriosome nutrient composition can change with the order of severity of pattern of using and disease to be treated.Concrete condition decides according to experimenter's individual instances, and this is in the scope that skilled practitioners or nutritionist can judge.

Major advantage of the present invention is: disclose 1. the R-thioctic acid first and 2. be selected from down one or more chondriosome nutrients of organizing: acetylcarnitine, coenzyme Q10, biotin, vitamin E or nicotinic acid use in conjunction, can prevent, improve and treat type 2 diabetes mellitus and insulin resistant.And the use in conjunction of above-mentioned chondriosome nutrient can reduce application dosage greatly than independent use, has produced outstanding effect.

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.

Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.

Material and universal method

1. material

Anti-PGC-1 α purchases the Santa Cruz company in the U.S.; Anti-β-actin purchases 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 U.S. invitrogen 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; NRF1, NRF2 and β-actin primer is synthetic by Shanghai Bo Ya company; ALC is available from Sigma company; R-LA is by K.Wessel, and doctor Variats gives (or also commercially available, as can available from the new high towering company limited in Shanghai); The reagent of remaining cell culture is all available from American I nvitrogen company.

The DMEM culture medium, hyclone, trypsin, JC-1 is available from American I nvitrogen company; The plain ELISA test kit of mouse islets is available from U.S. Linco company; Oleic acid and anti--β-actin are available from U.S. Sigma company; The UCP-2 polyclonal antibody of mouse-anti rabbit is available from U.S. Chemicon company; H2DCF-DA is available from U.S. Calbiochem company; The AMV reverse transcriptase is available from U.S. Promega company, and the PCR primer designs according to document, and is synthetic by company of Shanghai Bo Ya biotech company; The MIN6 cell is given in Kazutomo doctor Inoue of Kyoto Univ Japan (or can referring to U.S. Pat 5534404).

2. mitochondrion preparation

Cell is used LA or/and after the ALC processing, discard culture fluid, fully cleans the differential centrifugation separate mitochondria with PBS.Separating medium is a 0.21mol/L mannitol, 0.07mol/L sucrose, 1mmol/LEDTA, 5mmol/L Tris-HCl (pH7.4).All operations is all carrying out below 4 ℃.

3. the mensuration of mitochondrion volume

Use fluorescent probe Mito-Tracker Green FM to carry out mitochondrial dyeing specifically, measure mitochondrial volume.After cell is used the chondriosome nutrient effect, trypsinization, collecting cell, be resuspended in the KRB buffer, hatched 30 minutes in Mito-Tracker Green FM (100nM) again, centrifugal collecting cell is suspended from the KRB buffer, flow cytometry analysis fluorescence intensity (FACS Calibur Becton Dickinson).

4. statistical procedures

Two groups of data relatively adopt the t check, and multi-group data adopts ANOVA to take statistics and learns processing, and the result represents with mean+SD, with P＜0.05 expression significance of difference.

Embodiment 1 use in conjunction R-M and ALC promote the adipose cell mitochondria number to increase

1. adipose cell is cultivated and the adipose cell differentiation

Adipose cell before the 3T3-L1 (available from ATCC company, Cat.No.CL-173) (Wilson-Fritch L etc. (2003); Mitochondrial Biogenesis and Remodeling during Adipogenesis and in Response to the InsulinSensitizer Rosiglitazone.Mol Cell Biol.23 (3): 1085-94) with complete culture solution (DMEM+10%FBS+100u/ml penicillin and streptomycin), at 37 ℃ of incubator (5% carbon dioxide, 95% air) cultivates in, changed liquid 1 time in per 2 days.Treating that cell grows to merges back 2 days (the 0th day) fully, begins to induce differentiation, and concrete steps are: culture fluid is changed into the complete culture solution that contains 0.5mM IBMX, 0.25 μ M dexamethasone and 1 μ M insulin; After 48 hours, remove IBMX and dexamethasone, make and only contain 1 μ M insulin in the complete culture solution; Change liquid after 2 days, remove insulin, use the complete medium that does not contain any derivant; Changed liquid 1 time in per 2 days later on, be divided into sophisticated adipose cell until the 8th day 95% above cell.

2. flow cytometry R-LA and ALC are to the influence of adipose cell mitochondria number

In order to determine that R-LA is or/and can ALC stimulate the mitochondrial hypertrophy of adipose cell, the inventor uses mitochondrion specificity fluorescent probe MitoTracker Green FM (available from Molecular Probes), this probe can accumulate in the mitochondrion, be not subjected to the interference of mitochondrial membrane potential, therefore the quantitative interior mitochondrion quantity of adipose cell, cell differentiation the 8th day, adipose cell and R-LA are or/and ALC effect 24 hours, R-LA or ALC can increase fluorescence intensity and present dose-dependence when 0.1-100 μ M, but there is not significant difference, yet, use in conjunction R-LA and ALC (0.1-100 μ M) can significantly increase fluorescence intensity, there were significant differences to use R-LA or ALC more separately, the results are shown in Figure 1A and Figure 1B.

Therefore, visible use in conjunction R-LA and ALC (0.1-100 μ M) can promote significantly that the adipose cell mitochondria number increases, and use in conjunction R-LA and ALC ratio independent application R-LA or ALC's is more effective.And very low coupling dosage (0.1 μ M R-LA+0.1 μ M ALC) can reach R-LA or the more effective effect of ALC than independent use higher dosage.

3. electron microscopic observation use in conjunction R-LA and ALC are to the influence of adipose cell mitochondrion form etc.

Adipose cell breaks up used R-LA (10 μ M) on the 8th day or/and ALC (10 μ M) effect after 24 hours, 2.5% glutaraldehyde spends the night fixing, and cell 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.Counting is also observed 10 adipose cells, quantitatively mitochondria number and mitochondrial sectional area.

The results are shown in Figure 2.Among Fig. 2 A, A is the mitochondrial form of adipose cell (amplification 10,000), and adipose cell broke up the 8th day, uses (ii) R-LA (10 μ M), (iii) ALC (10 μ M), and (iiii) R-LA (10 μ M)+ALC (10 μ M), matched group (i) does not add medicine.Fig. 2 A shows that R-LA (10 μ M) is or/and ALC (10 μ M) can change mitochondrial size and structure, in the cell of R-LA effect, mitochondrion is distributed in and presents fine and close and interconnective state in the whole cell, at the cell of ALC effect, mitochondrion demonstrates densification, but mitochondrion is shorter, fewer contacts, particularly contain abundant hypothallus, rare in normal cell, but comparatively general in the cell of R-LA or ALC processing.

Fig. 2 B has shown mitochondrion sectional area and mitochondrion density, gets average ± standard deviation mapping of the result of 4 tests, ^*P＜0.05 compares with matched group that there were significant differences, and visible use in conjunction R-LA and ALC can significantly increase mitochondrial sectional area and density to mitochondrion.

Simultaneously, inventor's analysis and observation 10 cells, the result shows in the cell of ALC effect, ALC does not increase mitochondrial area and number, R-LA has the tendency that increases mitochondria number and area, but does not have significant difference, and use in conjunction R-LA and ALC can significantly increase mitochondrial area; Increase average mitochondrial number (P＜0.05), increased by 187.8% ± 24 than matched group, P＜0.05.

R-LA and ALC can increase the expression of mitochondrion electron transport chain cpd in embodiment 2 adipose cells

In the present embodiment, adopt immune protein engram analysis R-LA or/and can ALC increase the composite I of mitochondrion electron transport chain, II, the protein expression of III.

Immune protein trace method is as follows: pair cell associating or use R-LA separately or/and ALC after 24 hours, discard culture fluid, fully clean with PBS, add cell pyrolysis liquid, scrape cell with the cell curet, hatched 1 on ice 30 minutes, 3000r/min4 ℃ centrifugal 10 minutes, collect supernatant and be the cell protein extracting solution.It is quantitative that application Bradford method is carried out total protein ,-80 ℃ of preservations.Albumen is transferred on the solid support nitrocellulose filter after using sds page (SDS-PAGE) electrophoretic separation, and sealing is 1 hour under the room temperature, add an anti-PGC-1 α (1: 1000), β-actin (1: 5000), complex I (1: 1000), complex II (1: 1000), complex III (1: 1000), 4 ℃ of overnight incubation are washed 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.Use NIH Image image analysis software the density of protein band is carried out semi-quantitative analysis.

The results are shown in Figure 3, immune protein engram analysis result shows, the R-LA of 10 μ M-100 μ M can increase the expression of composite I II, 1.0 μ M ALC can increase the expression of composite I II, use in conjunction R-LA and ALC (0.1+0.1 μ M) can significantly increase the expression (p＜0.05 is compared with matched group) of composite I, R-LA and ALC (1.0+1.0 μ M) can significantly increase the expression (p＜0.05 is compared with matched group) of composite I I, R-LA and ALC (0.1+0.1,1+1 is with 10+10 μ M) can significantly increase the expression (p＜0.05 is compared with matched group) of composite I II.

As seen the R-LA of low dosage and ALC can significantly increase the expression of composite I, II, III, so use in conjunction R-LA and ALC can reduce using dosage largely than independent use, and reach better effect.

Embodiment 3 R-LA and ALC are for the proteic expression of adipose cell PGC-1 α

PGC-1 α is the auxiliary enhancer that can promote mitochondrion generation and mitochondrion fatty acid oxidation, and adipose cell and R-LA are or/and the expression that the method (method is with described in the embodiment 2) of immune protein trace is observed PGC-1 α after 24 hours, is used in the ALC effect.

The results are shown in Figure 4, show that R-LA and ALC can raise PGC-1 α, from 1-100 μ M, ceiling effect occurs in 1.0+1.0 μ M, and R-LA can raise PGC-1 α from 1-100 μ M, and ceiling effect occurs in 10 μ M; ALC can raise PGC-1 α from 0.1-10uM; Use in conjunction R-LA and ALC raise PGC-1 α and demonstrate bell shaped curve, and ceiling effect occurs in 1.0+1.0 μ M; And 100+100 μ M does not demonstrate remarkable effect.

Therefore as seen, only need very the R-LA and the ALC use in conjunction of low dosage can bring into play significant effect, promote the proteic expression of PGC-1 α greatly, dosage is too high act on the contrary not obvious.

R-LA and ALC induce mitochondrion to generate expression of gene in embodiment 4 adipose cells

For the molecular mechanism of clear and definite R-LA or the generation of ALC promotion adipose cell mitochondrion, the inventor further investigates on transcriptional level and relates to the gene that mitochondrion generates.Use the method for RT-PCR and observe adipose cell NRF1 and the expression of NRF2 on transcriptional level.

Total RNA extracting and RT-PCR: press the test kit explanation,, measure purity and the content of RNA with the uv-spectrophotometric instrument with total RNA of TRIzol reagent extraction cell.According to the method design primer sequence of routine, primer is synthetic by match Parkson, Shanghai company, and concrete primer sequence is as follows:

The upstream sequence of NRF1 (GenBank No NM_010938):

5’-CGCAGCACCTTTGGAGAA-3’(SEQ?ID?NO：1)；

The downstream sequence of NRF1:

5’-CCCGACCTGTGGAATACTTG-3’(SEQ?ID?NO：2)；

The upstream sequence of NRF2 (GenBank No NM_010902):

5’-ATGGATTTGATTGACATCCTT-3；(SEQID?NO：3)；

The downstream sequence of NRF2:

5’-ATGTTTTTCTTTGTATCTGG-3’(SEQ?ID?NO：4)；

The upstream sequence of β-actin (GenBank NO NM_007):

5’-ACGGCCAAGTCATCACTATTG-3’(SEQ?ID?NO：5)；

The downstream sequence of β-actin:

5’-AGCCACCGATCCACACAGA-3’(SEQ?ID?NO：6)。

RT reacts the synthetic cDNA of the total RNA of 1 μ g.Cumulative volume 20 μ l, 42 ℃ of reaction 60min, 95 ℃ were reacted 5 minutes.Synthetic cDNA uses the DDR305A Ex Taq (Hot start version) of Takara company to carry out pcr amplification, pcr amplification reaction cumulative volume 50ul, 95 ℃ of 1min of degeneration; 58 ℃ of 1min anneal; Extend 72 ℃ of 1min; Carry out 30 circulations altogether, last circulation back extension 8min. gets amplification reaction solution 10ul and carries out 2% agarose gel electrophoresis, and Ultraviolet Detector is observed down and Taking Pictures recording.

The results are shown in Figure 5, use the method for above-mentioned RT-PCR and observe adipose cell NRF1 and the expression of NRF2 on transcriptional level, 1-100 μ M R-LA significant stimulation expression (Fig. 5 A of adipose cell NRF1 on transcriptional level, p＜0.01, compare with matched group that there were significant differences), 10-100 μ M R-LA significant stimulation the expression of adipose cell NRF2 mRNA (p＜0.05, compare with matched group there were significant differences).1-10 μ M ALC also can stimulate the expression of NRF1, but can not stimulate the expression (Fig. 5 B) of NRF2.

R-LA and ALC are for the regulation and control of transcription factor mtTFA in embodiment 5 adipose cells

The present invention adopts the electrophoretic method of gel blocking to measure R-LA and the ALC regulation and control for transcription factor mtTFA.

The gel blocking electrophoresis is according to (Kanazawa A, Nishio Y, Kashiwagi A, Inagaki H, Kikkawa R, HoriikeK (2002) .Reduced activity of mtTFA decreases the transcription in mitochondria isolated fromdiabetic rat heart.Am J Physiol Endocrinol Metab.282 (4): E778-85.) institute's reported method is carried out.Few nuclear probe 5 '-TTTCCTCCTAACTAAACCCTCTTTAC-3 ' (SEQ IDNO:7) of mtTFA (GenBank NO:AY769440), 5 '-GTAGGCAAGTAAAGAGGGTTTAGTTA-3 ' (SEQ ID NO:8).Radioactivity [α-32P] ATP (3,000Ci/mmol, Amersham Biosciences) labelling, in reaction system, hatch mitochondrial protein (100 μ g/ml polydI-dC altogether, 10mM Tris-HCl (pH7.5), 50mM NaCl, 0.5mM EDTA, 0.5mM dithiothreitol, 1mM MgCl2), by 4% non-denaturing polyacrylamide electrophoretic separation, behind the vacuum drying gel, use intensifying screen to spend the night with the X-ray film exposure at-70 ℃.

Transcription factor mtTFA can regulate and control the generation of the mitochondrial protein of nuclear gene encoding, and regulates and control transcribing and duplicating of mitochondrial DNA.The inventor has observed adipose cell and has used the expression that the EMSA method detects mtTFA through R-LA after 24 hours or/and ALC handles, 0.1-100 μ M R-LA significant stimulation the expression of adipose cell mtTFA; 0.1-100 μ M ALC significant stimulation the expression of adipose cell mtTFA; Be the specificity of conclusive evidence mtTFA, the inventor uses 50 times of unlabelled probe sealing back retardance bands and disappears, and illustrates that this band is special retardance band, sees Fig. 6.

The insulin secretion that embodiment 6 R-LA and ALC reverse the MIN6 cell glucose sugar due to the oleic acid to be stimulated descends

1.MIN6 the cultivation of cell and test grouping

The MIN6 cell is cultivated with high glucose medium, medium component is: Dulbecco ' s modified Eagle ' smedium (DMEM) contains 15% hyclone (FBS), 25mM glucose, 2mM glutamate, Glu, the penicillin of 100U/ml, the streptomycin of 100 μ g/ml.The MIN6 cell grows at 70% o'clock, carries out low sugar and spends the night, and the low sugar medium component of use is: DMEM contains the 11mM glucose, 1% bovine serum albumin (BSA), 2mM glutamate, Glu, the penicillin of 100U/ml, the streptomycin of 100 μ g/ml.After low sugar is spent the night, be grouped as follows:

1. normal control group: the low sugar serum-free medium was handled 72 hours;

2. normal dosing group: the low sugar serum-free medium contains the thioctic acid (0.1 μ M-1000 μ M) of variable concentrations and/or the acetylcarnitine (0.1 μ M-1000 μ M) of variable concentrations was handled 72 hours;

3. oleic acid group: the low sugar serum-free medium contains the oleic acid (0.2-0.8mM) of variable concentrations to be handled 72 hours;

4. oleic acid dosing group: contain acetylcarnitine (the 0.1 μ M-1000 μ M) pretreatment 6 hours of the thioctic acid (0.1 μ M-1000 μ M) of variable concentrations and/or variable concentrations earlier with the low sugar serum-free medium; Adding oleic acid (0.4mM) again handled 72 hours jointly.

2.MIN6 the insulin secretion of cell is measured

The MIN6 cell is removed culture medium, with KRB buffer (118mM NaCl, 4.7mM KCl, 2.5mM CaCl after different pharmaceutical is handled ₂, 1.2mM KH ₂PO ₄, 1.2mM MgSO ₄, 24mM NaHCO ₃) wash twice, again with KRB buffer incubation 30 minutes, add glucose, be respectively 2.8mM or 30mM to the glucose final concentration, to measure the insulin secretion that basal insulin secretion and glucose stimulate, collect supernatant after 1 hour, use the plain ELISA test kit of mouse islets and carry out quantitatively.

3.R-LA and the influence of ALC insulin secretion that MIN6 cell glucose sugar due to the oleic acid is stimulated

The MIN6 cell is after 0.4mM oleic acid is handled 72 hours, and basal insulin secretion (stimulation of 2.8mM glucose) does not have very big change.Under the 30mM glucose stimulated, normal insulin secretion had increased about 4 times than the basal insulin secretion, and the insulin secretion that the sugar of the MIN6 grape cell after the oleic acid effect stimulates obviously descends, and has only increased by 2 times of left and right sides (see figure 7)s than the basal insulin secretion.After using R-thioctic acid (10 μ M) or acetylcarnitine (10 μ M) pretreatment, can improve the decline (p＜0.05 is compared with the oleic acid group) of the GSIS that causes by oleic acid.Use in conjunction R-thioctic acid can significantly improve the insulin secretion (p＜0.01 is compared with the oleic acid group) that glucose stimulates with acetylcarnitine (10 μ M).

Therefore as seen, the insulin secretion (GSIS) of the MIN6 cell glucose sugar stimulation due to R-thioctic acid and the acetylcarnitine reverse oleic acid descends.

The ATP that embodiment 7 R-thioctic acid and acetylcarnitine improve MIN6 grape cell sugar to stimulate generates

1. the ATP of glucose stimulation generates

The mensuration of ATP is used assay determination kit measurement (the Patane G etc. of ATP luciferase in the endochylema; Diabetes.; 51 (9): 2749-56,2002).Cell grows in 96 orifice plates, after said medicine is handled, supernatant is abandoned in suction, the buffer balance of application sugar-free 2 hours, again respectively with contain glucose (2.8mM or 30mM) and hatched 1 hour, cell lysis, the microplate reader reading numerical values is used in the reactant liquor that contains luciferase (10mg/ml) effect of getting 80 μ l cracking supernatant and 100 μ l.

The results are shown in Figure 8, hatched 1 hour in glucose (2.8mM) level of arm's length basis, the oleic acid processed group does not change the content of ATP in the endochylema, and the content of ATP does not change yet in using R-thioctic acid and acetylcarnitine pretreated group endochylema.Normal MIN6 cell is after hatching 1 hour under high sugar (30mM) condition, the content that can significantly improve ATP in the endochylema is (by 2.30 ± 0.57 to 4.10 ± 0.43nmol/mg albumen, but the content (by 2.27 ± 0.34 to 2.53 ± 0.52nmol/mg albumen) that high sugar does not stimulate ATP in the oleic acid group endochylema P＜0.01).After using R-thioctic acid (10 μ M) or acetylcarnitine (10 μ M) pretreatment, can significantly improve the content of oleic acid group ATP, use in conjunction R-thioctic acid is compared effect slightly by force with the effect of acetylcarnitine (10 μ M) with independent application.

Embodiment 8 R-LA and ALC are for the influence of MIN6 cell UCP-2

1. extraction and the quantitative PCR of total RNA

Press the test kit explanation,, measure purity and the content of RNA with the uv-spectrophotometric instrument with total RNA of TRIzol reagent extraction cell.The design reference document of primer sequence, primer is synthetic by match Parkson, Shanghai company.UCP-2 (GenBank NO:NM_011671) primer upstream sequence 5 '-GTC GGA GAT ACC AGA GCA CT-3 ' (SEQ ID NO:9); UCP-2 downstream sequence 5 '-GTG ACC TGC GCT GTG GTA CT-3 ' (SEQ ID NO:10); β-actin (GenBank NONM_007) downstream sequence 5 '-ACG GCC AAG TCA TCA CTA TTG-3 ' (SEQ ID NO:11); And β-actin primer upstream sequence 5 '-AGC CAC CGA TCC ACA CAG A-3 ' (SEQ ID NO:12).The synthetic cDNA of the total RNA of RT reaction 1ug.Cumulative volume 20ul, 42 ℃ of reaction 60min, 95 ℃ of reaction 5min.Synthetic cDNA uses the DDR305A Ex Taq (Hot start version) of Takara company to carry out pcr amplification (Bio-rad iCycler quantitative PCR instrument).The PCR product detects quantitatively by SYBR Green I dye fluorescence.

2. the analysis of immune protein trace

After the effect of various processing factor, discard culture fluid, fully clean, add cell pyrolysis liquid, scrape cell, hatched on ice 30 minutes with the cell curet with PBS, 1, centrifugal 10 minutes of 4 ℃ of 3000r/min, the collection supernatant is the cell protein extracting solution.It is quantitative that application Bradford method is carried out total protein ,-80 ℃ of preservations.After albumen is used 12%SDS polyacrylamide gel (SDS-PAGE) electrophoretic separation, be transferred on the solid support nitrocellulose filter, sealing is 1 hour under the room temperature, adds one anti-(UCP-2 polyclonal antibody 1: 1000, β-actin monoclonal antibody 1: 5000), 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.Use NIH Image image analysis software the density of protein band is carried out semi-quantitative analysis.

3.R-LA and ALC is for the influence of MIN6 cell UCP-2

The MIN6 cell is through using the expression that stimulates UCP-2 mRNA after oleic acid (0.2-0.8mM) is handled 72 hours.0.4mM can produce ceiling effect after OA stimulates (312% ± 5%, p＜0.05 oleic acid group is compared with the normal control group) taking place, sees Fig. 9 A.Reach 117% ± 11% (p＜0.05 is compared with the oleic acid group) through using the expression that significantly to reduce the UCP-2mRNA of oleic acid stimulation after chondriosome nutrient (R-LA 10 μ M) is handled, see Fig. 9 B.The expression that can significantly reduce the UCP-2mRNA of oleic acid stimulation after ALC 10 μ M handle reaches 77.5% ± 4% (p＜0.05 is compared with the oleic acid group).Yet use in conjunction R-thioctic acid and acetylcarnitine (10 μ M) group does not change the expression of UCP-2mRNA, sees Fig. 9 B.

Observe the change of UCP2 on protein level synchronously, use the method for immune protein trace, observing oleic acid can increase the proteic expression of UCP-2, and the oleic acid of 0.4mM can produce maximum effect, sees Figure 10 A.Can reduce the proteic expression of UCP-2 after using R-LA and ALC 10,100 μ M pretreatment respectively, and present dosage effect, the use in conjunction group has also significantly suppressed the proteic expression of UCP-2, sees Figure 10 B.And use in conjunction is bigger than using R-LA or ALC 10 inhibition degree separately.

Therefore as seen, single R-LA or ALC regulate and control the expression of UCP-2mRNA on transcriptional level, and use in conjunction R-LA and ALC regulate and control the proteic expression of UCP-2 on translation skill.

Embodiment 9 R-LA and ALC can repair mitochondrial membrane potential

1. mitochondrial membrane potential analysis

JC-1 is the specific probe of reflection mitochondrial membrane potential, and the ratio of red fluorescence and green fluorescence can be used as the index of mitochondrial membrane potential.The MIN6 cell is cultivated after 72 hours in 24 orifice plates, supernatant discarded, PBS washes twice, harvesting is in the 1.5ml EP pipe of cleaning, add the JC-1 dyestuff to final concentration 10 μ g/mL, after lucifuge is hatched 15 minutes, add centrifugal the washing twice of equivalent PBS 500g, supernatant discarded is analyzed with flow cytometer with the resuspended back of 0.5ml PBS.

2.R-LA and ALC is to the influence of mitochondrial membrane potential

The MIN6 cell is used the JC-1 probe in detecting and is descended to the mitochondrion transmembrane potential through oleic chronic stimulation in 72 hours, sees Figure 11 A, and 0.2mM has suppressed the mitochondrion transmembrane potential in various degree to the oleic acid of 0.8mM, sees Figure 11 B.

By Figure 11 B as can be known, the MIN6 cell makes the ratio decline of JC-1 reach 40% (P＜0.05 oleic acid group is compared with the normal control group) after the oleic acid of 0.4mM is handled 72 hours.And the protective effect of R-LA and ALC is seen Figure 11 C, uses R-LA and ALC (1uM-100 μ M) pretreated group and can repair mitochondrial transmembrane potential, and associating dosage group also demonstrates protective effect, and especially the 100 μ M effect of uniting group more is better than single medication.

As seen, use in conjunction R-LA and ALC can repair mitochondrial membrane potential well, and use in conjunction embodies better mitochondrial membrane potential repairing performance than using separately.

Embodiment 10 R-LA or ALC can reverse the mitochondrion volume that causes because of the oleic acid processing and reduce

1. transmission electron microscope observing

The MIN6 cell is after different pharmaceutical is handled, 2.5% glutaraldehyde spends the night fixing, cell is fixing behind the 10g/L osmic acid, acetone dewater, soak into, resin embedding, ultrathin section, uranium-lead are redyed, transmission electron microscope observation (Hayakawa T down, et al 1998). the secretory granule of pancreatic beta cell performance characteristic, counting is also observed 5 MIN6 cells, quantitatively mitochondria number and mitochondrial sectional area.

2.R-LA or ALC handles the unusual influence of mitochondrion that causes for oleic acid

The MIN6 cell after oleic acid is handled 72 hours, the obvious swelling of mitochondrion, high and steep disappearance, lysosome enlarges and occurs the mitochondrion of medicated cap shape or cyclic fragmentation, sees Figure 12 A and 12B.

Quantitative analysis shows that the normal matched group of oleic acid group cell mitochondrial sectional area reduces obviously that (normal control group beta cell sectional area is 2.4mm ², and the oleic acid stimulating group is 1.7mm ²P＜0.05), cell size and number do not have change between two groups; The average mitochondria number of normal control group beta cell is 10.9, and the oleic acid stimulating group is 9.7; The average endochylema area of normal control group beta cell is 22.4mm ², and the oleic acid stimulating group is 23.5mm ²Therefore, normal control group beta cell average line plastochondria volume is big by 29.2% than oleic acid stimulating group average line plastochondria volume.Using R-LA or ALC pretreated group can impel average line plastochondria volume to increase 15% or 17% than the oleic acid stimulating group.

Therefore as seen, the MIN6 cell being carried out R-LA and ALC pretreatment can reverse and handle the reduction of mitochondrion number of volumes and the volume that cause because of oleic acid and reduce.

Embodiment 11 R-LA and ALC can remove the oxygen-derived free radicals that oleic acid causes and too much produce

1. the mensuration of active oxygen

Use molecular probe 2 ', 7 '-dichlorofluorescein diacetate (H ₂DCF-DA) labelling adventitia fibroblast, H ₂The free permeate through cell membranes of DCFDA energy, resolved into dihydro-dichlorofluorescein by esterase in endochylema, the latter is converted into fluorescigenic 2 ', 7 '-dichlorofluorescein (DCF) by hydrogen peroxide, by the fluorescence intensity of cells were tested by flow cytometry fluorescence-causing substance, carry out relative quantitative assay.The MIN6 cell is after different disposal, again with H ₂After DCFDA (10 μ M) was hatched 30 minutes, trypsinization after the collection, was counted 10,000 living cells, and flow cytometer detects and analyze (FACS Calibur Becton Dic kinson).

2.R-LA and ALC the too much influence of oxygen-derived free radicals that oleic acid is caused

The MIN6 cell is after the oleic acid of variable concentrations is handled 72 hours, and DCF-DA detection of active oxygen generates increase and presents dose-dependence, sees Figure 13 A.1.0-1000 the active oxygen that the R-thioctic acid of μ M suppresses to be stimulated by oleic acid produces; The acetylcarnitine of 1000 μ M also can suppress active oxygen and produce; Use in conjunction R-LA and ALC can suppress oxygen production, effect and the independent applications similar of R-LA, and during 1000 μ M couplings, effect is remarkable, sees Figure 13 C, and use in conjunction is better than independent effect.Therefore as seen, R-LA and ALC can significantly remove the oxygen-derived free radicals that oleic acid causes and too much produce.

Embodiment 12 chondriosome nutrient compositions promote the glucose transport of adipose cell insulin stimulating

In the present embodiment, with the R-thioctic acid, acetylcarnitine, coenzyme Q10, biotin and vitamin E are made compositions, observe the glucose transport that chondriosome nutrient composition promotes the adipose cell insulin stimulating.Described compositions is divided into into some groups, sees Table 1.

Table 1

?	0.1 μ M group	1.0 μ M group	10 μ M group	100 μ M group	1000 μ M group
						The R-thioctic acid	0.1μM?	1.0μM?	10μM?	100μM?	1000μM?
Acetylcarnitine	0.1μM?	1.0μM?	10μM?	100μM?	1000μM?
						Coenzyme Q10	0.1μM?	1.0μM?	10μM?	100μM?	1000μM?
Biotin	0.1μM?	1.0μM?	10μM?	100μM?	1000μM?
						Vitamin E	0.1μM?	1.0μM?	10μM?	100μM?	1000μM?

Adipose cell becomes adipose cell before the external evoked 3T3-L1, observe variable concentrations chondriosome nutrient complex to adipose cell effect different time after, use 2-D-[ ³H] the method assessment nutrient complex of glucose is for the effect of the glucose transport of insulin stimulating.

The results are shown in Figure 14, nutrient complex (10-100 μ M) effect can significantly increase the glucose transport of insulin stimulating after half an hour; This effect is sustainable under 10 μ M concentration to reach 6 hours.Long-time effect (48 hours) shows under the 100 μ M concentration, can obviously increase the glucose transport of insulin stimulating.

The prescription of embodiment 13 chondriosome nutrient compositions

The inventor has also prepared other multiple chondriosome nutrient composition, and it is as shown in table 2 to fill a prescription.

Table 2 (unit: weight portion)

?

The R-thioctic acid

Acetylcarnitine

Coenzyme Q10

Biotin

Vitamin E

Nicotinic acid

Prescription

1

100?

0?

Prescription 2

60?

1.0?

0?

Prescription 3

40?

0.5?

40?

0?

Prescription 4

30?

0.5?

30?

20?

Adopt the chondriosome nutrient composition of prescription 1, prescription 2, prescription 3, prescription 4, the inventor has carried out foregoing glucose transport test, ATP generates test, and measured the variation that they produce mitochondrion quantity, volume etc., the compositions of finding above-mentioned prescription can produce cooperative effect, particularly fashionable in low dose group, effect significantly is better than the application of single nutrient.

In addition, above-mentioned each prescription can be used separately, perhaps with an amount of pharmaceutically or on the food acceptable carrier mix, be prepared into medicine or dietary supplement.

Embodiment 14 chondriosome nutrient compositions are for the effect of type ii diabetes GK Mus

GK Mus (Shanghai Slac Experimental Animal Co., Ltd. provides) is a spontaneous type ii diabetes model, and its feature has: the insulin secretion that glucose stimulates is impaired, and the β cell number reduces, and glycogen generates too much, muscle and fatty tissue moderate insulin resistant.

Experimental technique: select 48 of male 4 all GK rats, be divided into model control group, pioglitazone group, chondriosome nutrient compound recipe low dose group and chondriosome nutrient compound recipe high dose group at random, every group each 12 by blood sugar level; The Wistar rat that other establishes coupling in age in week is the normal control group.Each treated animal is irritated corresponding thing or the normal saline of being tried of stomach respectively every day, simultaneously the feed normal diet.Concrete grouping situation is as follows:

1) normal control group Wistar Mus (male 12): irritate the stomach normal saline

2) model control group GK Mus (male 12): irritate the stomach normal saline

3) low dosage nutrient group (male 12):

R-thioctic acid 50mg/kg/d

Acetylcarnitine 100mg/kg/d

Biotin 0.1mg/kg/d

Niacin amide 15mg/kg/d

4) high dose nutrient group (male 12):

R-thioctic acid 500mg/kg/d

Acetylcarnitine 1000mg/kg/d

Biotin 1mg/kg/d

Niacin amide 150mg/kg/d

5) pioglitazone group (male 12): 10mg/kg/d

Each is organized rat and freely takes in drinking water, and the control circadian rhythm is 12h: 12h, and room temperature is 18～25 ℃, simultaneously the feed normal diet.Feedstuff is provided by the Si Laike Experimental Animal Center, total amount of heat is 15.36KJ/g, and wherein protein 21%, carbohydrate 55%, fat 6% reach micro-vitamin (A, D, B1, B2, B12, K3, E, (biotin additive capacity 0.01mg/kg in the feedstuff, the feedstuff food-intake of rat every day is 10-20g to biotin.The amount that from feedstuff, obtains biotin actual every day be about the combination of nutrient low dosage 1/100)).

Detect in the experimentation and respectively organize glucose tolerance experiment (OGTT), thymus index, ConA malonaldehyde (MDA) and blood plasma Total antioxidant capacity in lymphopoietic influence of mouse thymus and the blood plasma.

OGTT experiment: fasting 16h before the test, 50% G/W 2g/kg irritates stomach, before clothes sugar after the kimonos sugar 30,60,120min gets blood from the posterior orbit venous plexus, the blood glucose determination of glucose oxidase of routine.

Thymus index is measured: weight method is measured organ index.Organ index=(organ weights/body weight) * 1000.

The influence that ConA breeds mice spleen/thymic lymphocytes: pentobarbital sodium anesthetized rat, aseptic taking-up thymus, make the lymphocyte suspension, this suspension is made into the lymphocyte suspension of 2 * 106/mL with RPMI 1640 complete culture solutions, add 12 multiple holes (100ul/ hole), wherein every hole, 8 holes contains the RPMI-1640 culture fluid 100ul of Con A (final concentration 20ug/ml), rearmounted 37 ℃ of mixing, after cultivating 48h in the 5%CO2 incubator, every hole adds 5mg/mL MTT liquid 40 μ L, continue to cultivate 4h, add the every hole 100 μ L of 10%SDS liquid (0.01mol/L HCl preparation), survey 570nm light absorption value, the every hole of reference wavelength 630nm. A value=A570nm-A630nm after spending the night; Cell increment multiple=(measuring hole A value-control wells A value)/control wells A value.

The detection of malonaldehyde (MDA): the blood plasma lipide levels of peroxide is measured the content of TBARS in the blood plasma.Get the blood plasma 80ul 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, and measuring principle is that body has many antioxidant, can make Fe ³⁺Be reduced to Fe ²⁺, 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 40ul 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.

The result

(1) blood glucose and OGTT before the pharmaceutical intervention: get blood from the posterior orbit venous plexus, the blood glucose determination of glucose oxidase.The random blood sugar of GK rat is significantly higher than normal control group Wistar rat (p＜0.01) before the pharmaceutical intervention, OGTT clothes sugar back 2h＞11.1mmol/L, and according to international rat diabetes diagnostic criteria, decision model is set up successfully.

(2) blood glucose and OGTT after the pharmaceutical intervention: each is organized after the rat pharmaceutical intervention every month and detects fasting glucose and carbohydrate tolerance experiment, result such as Figure 15 and Figure 16.The result shows: the GK model group is compared with the normal control group, fasting glucose is significantly higher than normal control group wistar rat (p＜0.05), the GK rat is after the mitochondrion nutrient handled for 8 weeks, low dosage nutrient group fasting glucose is compared obvious reduction (p＜0.01) with the GK model group, the carbohydrate tolerance of diabetic model group rat is badly damaged, compared significant difference (p＜0.01) with the wistar matched group, after chondriosome nutrient handled for 8 weeks, low dosage nutrient group, high dose nutrient group is compared with the GK matched group, behind the

glucose load

30,60, blood sugar level significantly reduced (P＜0.01=in 90,120 minutes.

(3) rat chest gland lymphocyte function after the pharmaceutical intervention: each detects the thymic lymphocytes function after organizing the rat pharmaceutical intervention.The results are shown in Figure 17 and Figure 18, show: the GK model group significantly reduces (p＜0.05 than the Detection of thymocyte proliferation in vitro ability and the thymus index of Wistar matched group, p＜0.01), the peripheral T lymphocyte subgroup distributes unusual simultaneously, the CD4/CD8 ratio increases, and has compared significant difference (p＜0.01) with the Wistar matched group.Low dose group has significantly increased Detection of thymocyte proliferation in vitro ability (comparing p＜0.01 with the GK group), and low dose group also significantly increases (comparing p＜0.05 with the GK group) with pioglitazone group thymus index, and high dose group does not demonstrate effect; Pioglitazone group and high dose group do not have influence for the Detection of thymocyte proliferation in vitro ability.Above-mentioned data show that low dose group closes the cellular immunization situation that can improve diabetes rat.

(4) rat blood oxidation resistance after the pharmaceutical intervention: each organizes the oxidation resistance that detects blood plasma after the rat pharmaceutical intervention.The results are shown in Figure 19 and Figure 20, show: lipid peroxidation product (malonaldehyde) showed increased (p＜0.05) that produces in the blood plasma of GK model group than the Witar matched group, Total antioxidant capacity significantly reduces (p＜0.01).After the pharmaceutical intervention, low dose group is compared with the GK model group, and the generation of lipid peroxidation product obviously reduces (p＜0.05) in the blood plasma, and Total antioxidant capacity increases (p＜0.05); The oxidation resistance of pioglitazone group also significantly improves, but the oxidation resistance of high dose group blood plasma does not have remarkable improvement.Show that low dose group can strengthen intravital oxidation resistance.

Discuss

Although it is the main paathogenic factor of insulin resistant and type 2 diabetes mellitus that the disorder of research report mitochondrial function is arranged, yet further about mitochondrial function is prevented and treated insulin resistant and the type 2 diabetes mellitus generation does not see that but report is arranged by regulating, for clear and definite mitochondrion is created on effect in type 2 diabetes mellitus and the insulin resistant, seek the generation that effective medicine comes prevent diabetes, the inventor adopts and is widely used in the adipose cell differentiation and observes the cell model 3T3-L1 cell strain of insulin effect, has furtherd investigate the use in conjunction of R-thioctic acid and acetylcarnitine, and R-thioctic acid, acetylcarnitine and other coenzyme Q10, biotin, the effect of nutrient use in conjunction such as vitamin E in 3T3-L1 adipose cell mitochondrion generates.

It is relevant with the morbidity of old and feeble and a lot of diseases that mitochondrion generates the reduction that reduces with mitochondria enzyme activity, such as diabetes and complication (Chabi B thereof, Adhihetty PJ, Ljubicic V, Hood DA 2005.How is mitochondrial biogenesisaffected in mitochondrial disease? Med Sci Sports Exerc.37 (12): 2102-10).Mitochondrion generates and transcribing and duplicating of mitochondrial genome followed in the activation of oxidative phosphorylation, is the regulation and control person of cell differentiation.The molecular mechanism of mitochondrial generation is not still set forth clearly in the fatty tissue, has research report PGC-1 α to participate in the mitochondrion generation of regulation and control fat and muscular tissue.PGC-1 α plays a significant role in oxidative phosphorylation metabolism and mitochondrion generation, and can on transcriptional level, regulate and control mitochondrion anti-oxidative defense system, the downward modulation of PGC-1 α can reduce expression (Valle I etc., the 2005.PGC-1alpha regulates the mitochondrial antioxidant defense system in vascular endothelial cells.Cardiovasc Res.66 (3): 562-73) that mitochondrion " is separated toxalbumin ".PGC-1 α is considered to induce the key of mitochondrion generation, and it can start the expression of the transcription factor that promotes that mitochondrion generates.PGC-1 α can stimulate the gene expression of NRF1 and NRF2, particularly PGC-1 α can be combined in the promoter site of mtTFA with NRF1, mtTFA can the direct regulation and control mitochondrial DNA transcribing and duplicating (Choi YS etc. (2004), Regulation of mitochondrial transcription factor A expression by high glucose.Ann NY Acad Sci.1011:69-77).PGC-1 α can regulate and control the transportation of fatty acid and the beta oxidation of fatty acid, and (Patti ME etc. (2003), Coordinated reduction of genes of oxidative metabolism in humans with insulin resistanceand diabetes:Potential role ofPGC1 and NRF1.Proc Natl Acad Sci U S is (14) A.100: 8466-71).In muscular tissue, the activation of persistent endurance training and beta-adrenaline can stimulate the expression of PGC-1 α, increases the oxidation of mitochondrial generation and lipid.

Increasing mitochondrial generation by the path of regulating and control PGC-1 α is considered to prevention and improves insulin resistant, diabetes and fat key (McCarty MF. (2005), Up-regulation of PPARgamma coactivator-1 alpha as a strategyfor preventing and reversing insulin resistance and obesity.Med Hypotheses.64 (2): 399-407).The existing tested research of several drugs, such as metformin and 5-aminoimidazole-4-carboxamideribonucleoside, the ratio lattice row ketone in the plug oxazolidinedione class medicine, beta-2 adrenoceptor agonist CL-316,243, and estrogen-related receptor-a.

The additive of food may be that prevention and a kind of of treatment diabetes replenish, and particularly is directed to the complication of preventing and treating diabetes.Below be at test or confirmed clinically effective mineral, vitamin and coenzyme: vanadium, chromium, magnesium, zinc, selenium, copper, vit E, vitC, coenzyme Q10, niacin amide, riboflavin, flavonoid.The inventor thinks that R-LA and ALC are important chondriosome nutrients.Find in the clinical trial that in recent years R-LA and ALC can prevent and treat diabetes and complication thereof.Two kinds of medicines can improve the sensitivity of insulin, and recent research also shows can improve the concurrent cardiovascular symptom of metabolism syndrome.Yet, promote mitochondrion to generate for R-LA and ALC and do not see that report is arranged, particularly for both in the synergitic research aspect the mitochondrion generation.

The white adipose tissue is important endocrine organ, participates in the metabolism of regulation and control whole machine body and the sensitivity of insulin.It may be the key of regulation and control organism metabolism and insulin sensitivity that mitochondrion generates.The inventor discovers, adipose cell or/and ALC can significantly increase mitochondrial volume after handling 24 hours, increases the composite I of mitochondrion electron transport chain, II through using R-LA, the proteic expression of III, these variations are accompanied by the expression of the transcription factor that promotes that mitochondrion generates.Comprise PGC-1 α, mtTFA, NRF1 and NRF2.

Use in conjunction R-LA and ALC 0.1 and the 1uM low dosage demonstrate significant cooperative effect, almost be to use R-LA or ALC 10 times separately, R-LA and ALC can raise the expression of mtTFA, the part of transcribing of mtTFA is subjected to the adjusting of NRF1 and NRF2, and NRF1 and the NRF2 rise on mRNA is consistent with it.

Mitochondrial generation and reinventing can increase oxidation of fatty acids and improve oxygen consumption in the white adipose tissue, and the effect of R-LA and ALC can be contemplated as the energy metabolism of direct or indirect change body and the sensitivity of insulin.The inventor's result of study display application R-LA and ALC act on the sensitivity that the 3T3-L1 adipose cell not only can increase insulin, increase mitochondrial density and change mitochondrial structure, the cooperative effect of R-LA and ALC has shown the effect that short mitochondrion generates, but how interactional mechanism still needs further discussion between the two.

The inventor observes R-LA or/and ALC can increase PGC-1 α, the expression of NRF1 and NRF2, and according to document and experiment, prompting R-LA and ALC impel the mitochondrial generation of fatty tissue by raising the expression of PGC-1 α; Also be that R-LA promotes the synthetic of mitochondrial generation and mitochondrial protein or/and ALC by activating PGC-1 α, works in coordination with then.R-LA can increase the ability of white adipose tissue oxidizing phosphorylation or/and ALC increases expression and the proteic expression of mitochondrion transfer chain of PGC-1 α.It should be noted that the ability that increases the mitochondrion fatty acid oxidation parallels with the synthetic of fatty tissue lipid.

On the other hand, the insulin secretion that free fatty can suppress the beta Cell of islet glucose and stimulate descends, and follows the increase of mitochondria activity oxygen, the decline of transmembrane potential, and the ATP that UCP-2 proteic upward mediation reduction glucose stimulates is synthetic.These results show that the mitochondrial function disorder that oleic acid causes is the main cause of the beta Cell of islet dysfunction of type 2 diabetes mellitus, use chondriosome nutrient R-LA and the preventative intervention of ALC and can repair the beta Cell of islet function.The inventor's research reported first R-LA and ALC for the protective effect of the beta Cell of islet of type 2 diabetes mellitus, point out the above-mentioned chondriosome nutrient of daily interpolation can effectively prevent and delay the beta Cell of islet dysfunction of type 2 diabetes mellitus.

But the electron leak of for a long time excessive free fatty heavy weight line plastochondria electron transport chain makes the mitochondrial membrane hyperpolarization produce active oxygen, and a large amount of oxygen-derived free radicals can damage beta Cell of islet and may cause the death of cell.Hyperglycemia, hyperlipidemia and the fat insulin secretion that can cause the glucose stimulation descend, the beta Cell of islet damage, this is the principal character of type 2 diabetes mellitus.The inventor's result of study shows that the rise of active oxygen and UCP-2 occupies important effect in the inductive β cell injury of free fatty.This conclusion is being confirmed but also using in the protective effect of chondriosome nutrient R-LA and ALC and also be confirmed in the disorder of the mitochondrial function that high free fatty acid caused on not only.

The inventor studies confirm that, can suppress MIN6 cell secretion of insulin for a long time under the condition of hyperglycemia, hyperlipidemia.Use the insulin secretion that R-LA and ALC intervention treatment can improvement oleic acid suppresses.The ATP of beta Cell of islet 98% derives from mitochondrial supply, and mitochondrial ATP is the insulin secretion of glucose stimulation and the regulation and control person of mitochondrion oxidative phosphorylation.Directly measure intracellular ATP content and confirm that the oleic acid long-time stimulus can cause synthetic decline of ATP that glucose stimulates and uses R-LA and ALC can improve the synthetic of ATP, the result shows that the disorder of mitochondrial function can suppress secretion of insulin.

Fat toxicity has increased beta Cell of islet mitochondrion oxidative phosphorylation decoupling zero connection, and uncoupling protein (UCP) is positioned at mitochondrial inner membrance, participates in the synthetic of the interior ATP of regulating cell.Make proton when UCP-2 is activated outside mitochondrial matrix is leaked to mitochondrial inner membrane, and then make carbohydrate metabolism and the synthetic uncoupling of ATP.Oxidative stress can impel UCP-2 to express and increase, and is the adaptability mechanism that beta Cell of islet is resisted oxidative damage.Yet on the other hand, for beta Cell of islet, the rise of UCP-2 makes the decoupling zero of mitochondrion oxidative phosphorylation join, and the ATP that glucose stimulates is synthetic to descend.And the insulin secretion that making beta Cell of islet high expressed UCP-2 can reduce glucose stimulates descends; In the mice of UCP-2 gene knockout, insulin secretion can increase, and can repair decline (the Joseph JW etc. (2002) of the insulin secretion of glucose stimulation; Uncoupling Protein 2 KnockoutMice Have Enhanced Insulin Secretory Capacity After a High-Fat Diet.Diabetes 51 (11): 3211-9).Therefore, UCP-2 is the negativity regulatory factor of insulin secretion.

The inventor's experiment is found, oleic long-time stimulus can raise the expression of UCP-2 on mRNA and protein level, UCP-2 makes the oxidative phosphorylation decoupling zero join, the ATP that the UCP-2 that raises has suppressed the glucose stimulation synthesizes, thereby suppressed the insulin secretion that glucose stimulates, the intervention of R-LA and ALC is handled, and can increase the synthetic of ATP, repairs the insulin secretion that glucose stimulates.The intervention of R-LA or ALC is handled, can effectively suppress the rise of UCP-2 on mRNA and protein level that oleic acid stimulates, what is interesting is that use in conjunction R-LA and ALC can not suppress the expression of UCP-2 on the mRNA level, but effectively suppressed its expression on protein level, the single nutrient of indication wire plastochondria is regulated and control on transcriptional level, and the use in conjunction nutrient is regulated and control on translation skill.

Oxidative stress is relevant with the high free fatty acid level.Especially active oxygen is the main cause of bringing out type 2 diabetes mellitus beta Cell of islet function damage.The mitochondrion electron transport chain is the main position that mammal produces oxygen-derived free radicals, simultaneously also be reduce oxidative damage and remove oxygen-derived free radicals main target spot (Poitout, Vincent DVM. (2002) Lipid partitioning in thepancreatic[beta] cell:physiologic and pathophysiologic implications.Current Opinion inEndocrinology ﹠amp; Diabetes.9 (2): 152-159).Previous test card open-wire line plastochondria is the main position that the MIN6 cell produces active oxygen; the inventor detects MIN6 cell mitochondrial oxygen production and mainly depends on oleic long-time stimulus; the pretreatment of also observing simultaneously R-LA and ALC can reduce the inductive oxygen production of oleic acid, thereby the MIN6 cell is had protective effect.The oleic acid chronic stimulation can significantly increase oxygen production, and this is the main cause of bringing out mitochondrial function disorder (ATP is synthetic to descend, and transmembrane potential descends, and structure of mitochondria changes, and is accompanied by the decline of insulin secretion).

Chondriosome nutrient R-LA and ALC repair oleic acid inhibition MIN6 cell insulin secretion and mainly are to remove the generation of mitochondrion oxygen-derived free radicals and impel the synthetic of energy.R-LA is used for the treatment of the complication of diabetes, and the neuropathy that is mainly used in diabetes is by improving the transhipment and the metabolism of pancreas and peripheral tissues's glucose.Yet; use in conjunction R-LA and ALC treatment diabetes do not see that report is arranged; the inventor studies show that ALC be similar to R-LA can effectively protect MIN6 cell oleic acid inductive mitochondrial oxidative damage and suppress the insulin secretion that glucose stimulates, the The combined effect is better.

To sum up, the insulin secretion that free fatty can suppress the beta Cell of islet glucose and stimulate descends, and follows the increase of mitochondria activity oxygen, the decline of transmembrane potential, and the ATP that UCP-2 proteic upward mediation reduction glucose stimulates is synthetic.These results show that the mitochondrial function disorder that oleic acid causes is the main cause of the beta Cell of islet dysfunction of type 2 diabetes mellitus, use chondriosome nutrient R-LA and the preventative intervention of ALC and can repair the beta Cell of islet function.The inventor's research reported first R-LA and ALC for the protective effect of the beta Cell of islet of type 2 diabetes mellitus, point out the above-mentioned chondriosome nutrient of daily interpolation can effectively prevent and delay the beta Cell of islet dysfunction of type 2 diabetes mellitus.

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 Inst. of Life Science, CAS

＜120〉application of chondriosome nutrient composition in control insulin resistant and diabetes

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Claims

1. the purposes of a kind of mixture in the preparation prevention, improvement or the medicine of type 2 diabetes or dietary supplement, it is characterized in that, described mixture contains:

10-200 parts by weight of R-lipoic acid or its physiologically acceptable salt, 10-200 parts by weight of acetylcarnitine or its physiologically acceptable salt, 0.2-2 parts by weight of biotin or its physiologically acceptable salt and nicotinic acid 5-300 parts by weight of its physiologically acceptable salt; the sum of the components accounts for 10-100% of the total weight of the mixture.

2. purposes as claimed in claim 1, is characterized in that, described mixture also contains: vitamin E or its physiologically acceptable salt 20-200 weight part and coenzyme Q10 or its physiologically acceptable salt 10-200 weight share.

3. A composition, characterized in that, the composition is used to prevent, improve or treat type 2 diabetes, and the composition contains:

10-200 parts by weight of R-lipoic acid or its physiologically acceptable salt, 10-200 parts by weight of acetylcarnitine or its physiologically acceptable salt, 0.2-2 parts by weight of biotin or its physiologically acceptable salt and nicotinic acid 5-300 parts by weight of its physiologically acceptable salt; the sum of the components accounts for 10-100% of the total weight of the composition.

4. composition as claimed in claim 3, is characterized in that, also contains vitamin E or its physiologically acceptable salt 20-200 weight part and coenzyme Q10 or its physiologically acceptable salt 10- 200 parts by weight.

5. The composition according to claim 3, further comprising: a pharmaceutically or food acceptable carrier or excipient.

6. A mixture is used in the preparation of medicines or dietary additives for preventing, improving or treating type 2 diabetes, characterized in that, the mixture consists of 10-200 parts by weight of R-lipoic acid or its physiologically acceptable salt, 10 - 200 parts by weight of acetylcarnitine or its physiologically acceptable salt, 0.2-2 parts by weight of biotin or its physiologically acceptable salt, and 5-300 parts by weight of nicotinic acid or its physiologically acceptable salt.