CN107898779A

CN107898779A - Homo-orientin is being prepared and the purposes in insulin resistance relevant disease

Info

Publication number: CN107898779A
Application number: CN201711349429.1A
Authority: CN
Inventors: 王洪伦; 栾广祥; 马筱; 索有瑞
Original assignee: Northwest Institute of Plateau Biology of CAS
Current assignee: Northwest Institute of Plateau Biology of CAS
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2018-04-13
Also published as: AU2018101555A4

Abstract

The invention discloses a kind of homo-orientin to prepare and the purposes in insulin resistance relevant disease,The present invention extracts 7 kinds of compounds from faenum graecum,Activity research is carried out to these types of compound,Insulin resistance (IR) model is established using 3T3 L1 cells,In-depth study further is carried out to compound hypoglycemic activity from the aspect of mitochondrial dysfunction,Prevention and treatment mechanism of 7 kinds of compounds to diabetes is inquired into,The result shows that,Compound of the present invention can suppress the differentiation of 3T3 L1 cells,Suppress fat drips accumulation,Reduce TG contents,And the transcriptional level of Adipocyte Factor is reduced by associated signal paths,In addition,Level of the compound also by reducing ROS in cell,Recover 3T3 L1 cell mitochondrial membrane potentials,Promote the generation of ATP in cell,Prevent mitochondrial DNA damage,And promote the generation of mitochondria.

Description

Homo-orientin is being prepared and the purposes in insulin resistance relevant disease

Technical field

The present invention relates to the screening of reactive compound, and disease related to insulin resistance is being prepared more particularly to homo-orientin Purposes in disease.

Background technology

Fenugreek (Trigonella foenum-graecumL.) is pulse family, Trigonella, annual herb plant, again Name Trigonella foenum-graecum, vanilla etc., are mainly distributed on the countries such as China, India, African the north.Fenugreek is as a kind of integration of drinking and medicinal herbs Industrial crops, have long medicinal and edible history, on the books in many Chinese Ancient Books pharmacopeia.《Compendium of Materia Medica》Note Carry, fenugreek has the effect of notable for the disease such as treatment " disturbance of lower legs due to pathogenic cold and dampness ", " hernia ", " swelling of vulva pain ", " gas tap holder pain ". 《It is good to help book on Chinese herbal medicine》Record：Fenugreek " the dirty empty cold air of pivot；Monkshood, Sulfur control that kidney deficiency is cold, and abdomen side of body turgor greenish complexion is black；It must cherish Fragrant son, peach kernel, control bladder gas "；《Tibetan medicine will》Described in：" bitter, temperature；Control pulmonary abscess, and energy antidiarrheal ".Modern pharmacology is to Hu Lu Bar component and its chief active carried out relatively broad research, result of study finds that fenugreek contains a variety of active ingredients, Such as grease, combination of fenugreek gum, polysaccharide, saponin(e, flavones, terpene, trigonelline, cumarin, 4-hydroxyisoleucine.

Existing result of study shows that fenugreek has clear and definite hypoglycemic effect.(faenum graecum is to streptozocin by Zhou Jiuyao Induced diabetes rat model pharmacological research China traditional Chinese medicine academic periodical, 2007,25 (5):1005-1007.) etc. by rat Intraperitoneal injection 50mg/kg streptozocins (Streptozotocin, STZ) establish insulin-dependent diabetes mellitus (Insulin- Dependent diabetes mellitus, IDDM) animal model, gavage gives fenugreek decocting liquid respectively and gliclazide is molten Liquid is as treatment group and positive controls, successive administration 20 days；Measure rat fat, blood glucose value, hepatic tissue GSH, SOD in serum and NO contents.The result shows that fenugreek can substantially reduce IDDM rat blood sugars level, hence it is evident that HDL-C, TG, TC be reduced, in serum NO, SOD level are significantly raised, improve significantly to type II diabetes rat caused by STZ.(fenugreek is total by Sun Xiaofeng Saponin(e is to influence Xinjiang Medicine University journal of blood fat disorder rat model biochemical indicator, 2005,28 (2):101-103.) etc. Trigoneoside extract is fed by the rat got muddled to blood lipid metabolism, finds various dose trigoneoside extract Handle model group rats, compared with model group, its internal serum T G ﹑ low-density lipoproteins (LDL) ﹑ high-density lipoprotein (HDL) ﹑ Cholesterol (TC) metabolism content is in notable downward trend.Wei Youxia (influence s of the faenum graecum seed extract to diabetes rat Chinese patent drug, 2007,29 (3):450-451.) etc. also use SD rats high-calorie feed to be fed with for (8 weeks), with streptozocin (STZ) modeling, then using the water extract (main component is polysaccharide) and alcohol extract of fenugreek, (main component is unsaturated fat Acid) treated (6 weeks), detection blood glucose (GLU), T-CHOL (TC), triglycerides (TG) and high-density lipoprotein (HDL- C), each index significantly reduces compared with the control group for each treatment group, and HDL-C rises, water puies forward group and is better than alcohol extracting group, has the conspicuousness poor It is different, show that fenugreek Aqueous extracts are reducing rat blood sugar at the same time, moreover it is possible to play the role of adjusting blood lipid metabolism.Gupta(Effect of Trigonella foenum-graecum(fenugreek)seeds on glycaemic control and insulin resistance in type 2diabetes mellitus:a double blind placebo controlled study.Journal of the Association of Physicians of India,2001,49:1057-1061.) etc. It was found that the daily 1g fenugreeks water extract of type 2 diabetes patient is given, and after two months, compared with control group, fenugreek processing Group blood sugar level, serum TG and insulin concentration are decreased obviously, while patient's insulin sensitivity and high-density lipoprotein courage are consolidated Alcohol significantly raises.But result of study all carries out on the basis of fenugreek seed extract or extract part at present, monomer The effect of research report of compound be'ss still seldom, its is specific compound and its mechanism of action are also still not clear.

The content of the invention

The present invention provides following faenum graecum flavone compound to prepare prevention and/or treatment glucose -lipid metabolism disorder disease And the application in the medicine of association area, the compound be selected from orientin, homo-orientin, Vitexin, isovitexin wherein it More than one or both combination.

Structural formula of compound is as follows：

(I) --- homo-orientin, (II) --- Vitexin, (III) --- isovitexin, (IV) --- orientin.

Specifically, the present invention provides above-mentioned flavone compound to prepare the medicine of prevention and/or treatment insulin resistance Application in thing.

Further, the medicine is prevention and/or the medicine for treating glucose -lipid metabolism disorder relevant disease.

Further, the disease includes hyperlipidemia, cardiovascular disease, diabetes, hypertension or metabolic syndrome.

Further, the medicine is the medicine for suppressing Adipocyte Differentiation.

Further, the medicine is the medicine for suppressing PPAR γ, C/EBP α or/and FAS.

Further, the medicine is the medicine for improving mitochondrial function damage.

Further, the medicine is by regulating and controlling GAP-associated protein GAP table in insulin signaling pathway and/or AMPK signal paths Reach the purpose up to level.

Further, the medicine is by improving the phosphorylation level of AKT albumen in insulin signaling pathway, and/or, carry The phosphorylation level of AMPK albumen reaches the purpose in high AMPK signal paths.

In addition, present invention also offers application of the homo-orientin as AKT protein agonists or AMPK protein agonists.

In addition, should as PPAR γ antagonists or C/EBP alpha-2 antagonists present invention also offers homo-orientin, Vitexin With.

Homo-orientin, Vitexin can reduce the transcriptional level of Adipocyte Factor PPAR γ and C/EBP α, so as to fulfill to fat The histogenetic adjustment effect of fat.

In addition, present invention also offers above-mentioned flavone compound to prepare suppression Adipocyte Differentiation or/and repair line Application in the medicine of plastochondria damage.

It should be noted that term " activator " used refers to the egg that compound produces caused by its presence in the present invention The bioactivity of white matter is identical with bioactivity caused by the presence as the native ligand of protein.

Term " antagonist " used refers to that the biology for the protein that compound produces caused by its presence is living in the present invention Property intensity decreases.

The beneficial effects of the invention are as follows：The present invention extracts 7 kinds of compounds from faenum graecum, lives to these types of compound Journal of Sex Research, insulin resistance (IR) model is established using 3T3-L1 cells, further from the aspect of mitochondrial dysfunction to changing Compound hypoglycemic activity carries out in-depth study, has inquired into prevention and treatment mechanism of 7 kinds of compounds to diabetes, as a result table Bright, compound of the present invention can suppress the differentiation of 3T3-L1 cells, suppress fat drips accumulation, reduce TG contents, and pass through phase OFF signal path reduces the transcriptional level of Adipocyte Factor, in addition, the compound is also by reducing the level of ROS in cell, it is extensive Multiple 3T3-L1 cell mitochondrial membrane potentials, promote the generation of ATP in cell, prevent mitochondrial DNA damage, and promote mitochondria Occur.

Brief description of the drawings

Influence (x ± s, n=4) of Fig. 1 fenugreeks compound to 3T3-L1 PECTORAL LIMB SKELETON vigor；

The form (200 ×) of the 3T3-L1 cells of Fig. 2 induction different times, wherein, a figures are normal cell, and b figures are complete Normal cell after converging, c figures are the cell of induction the 4th day, and d figures are the cell of induction the 8th day；

The influence (200 ×) that Fig. 3 fenugreeks compound produces fat drips in 3T3-L1 PECTORAL LIMB SKELETON atomizations；

Fig. 4 fenugreeks compound to TG contents in 3T3-L1 PECTORAL LIMB SKELETON atomizations influence (^##P< 0.05vsCon；*P<0.05, * * P<0.01vs Mod)；

The influence that Fig. 5 fenugreeks compound expresses Adipocyte Factor in 3T3-L1 PECTORAL LIMB SKELETON atomizations；

Fig. 6 Dex handle intake (* * P of the different time 3T3-L1 cells to 2-NBDG<0.01)；

The phosphorylation level of AKT in Fig. 7 Dex processing different time 3T3-L1 cells；

Influence (x ± s, n=4, * P ＜ 0.05, * * the P ＜ that Fig. 8 fenugreeks compound absorbs 3T3-L1 cells 2-NBDG 0.01)；

The influence that Fig. 9 fenugreeks compound expresses p-AKT in 3T3-L1 cells；

The influence that Figure 10 fenugreeks compound expresses p-AMPK in 3T3-L1 cells；

Influence of Figure 11 fenugreeks compound to 3T3-L1 cell PPAR γ transcriptional levels；

Influence of Figure 12 fenugreeks compound to 3T3-L1 cell C/EBP alpha transcriptional levels；

Influence of Figure 13 fenugreeks compound to 3T3-L1 cell AP2 transcriptional levels；

Influence of Figure 14 fenugreeks compound to 3T3-L1 cell SREBP1c transcriptional levels；

Influence of Figure 15 fenugreeks compound to 3T3-L1 cell FAS transcriptional levels；

Influence of Figure 16 flow cytomery fenugreek compounds to 3T3-L1 cells ROS levels；

Figure 17 laser co-focusings detect influence of the fenugreek compound to 3T3-L1 cells ROS levels；

Figure 18 flow cytomery fenugreek compounds produce 3T3-L1 cell membrane potentials the influence of speed；

The influence that Figure 19 fenugreeks compound synthesizes 3T3-L1 cell ATPs；

Influence of Figure 20 fenugreeks compound to 3T3-L1 cell mitochondrial DNA damages；

Influence of Figure 21 fenugreeks compound to NRF1 transcriptional levels in 3T3-L1 cells；

Influence of Figure 22 fenugreeks compound to PGC-1 transcriptional levels in 3T3-L1 cells；

Influence of Figure 23 fenugreeks compound to Tfam transcriptional levels in 3T3-L1 cells.

It should be noted that Ori represents orientin, Iori represents homo-orientin, and Vit represents Vitexin, and Ivit represents different Vitexin, RHAc represent ponticin, and dRHAc represents deoxidation ponticin, and RHAg represents rhapontigenin, and respective packets represent Corresponding compound intervention group, Con groups represent normal group, and Mod groups represent model group, and Rosi (Rosiglitazone) groups represent positive right According to group, Insulin groups represent insulin incubation group, and vehicle groups represent insulin untreated fish group in normal group.

Embodiment

Orientin, homo-orientin, Vitexin and isovitexin are 4 kinds of flavone compounds, ponticin, deoxidation Rumex madaio Glycosides, rhapontigenin are 3 kinds of diphenylethylene compounds.

First, influence of the fenugreek compound to 3T3-L1 cell differentiations

(1) influence of the fenugreek compound to 3T3-L1 PECTORAL LIMB SKELETON vigor

Orientin, homo-orientin, Vitexin, isovitexin, ponticin, deoxidation ponticin, pellet are measured by mtt assay This influence of 7 kinds of faenum graecum compounds to the vigor of 3T3-L1 PECTORAL LIMB SKELETONs of leaf rheum emodin.3T3-L1 PECTORAL LIMB SKELETONs are connect Kind is to 96 well culture plates, per hole 1 × 10⁴A cell (150 μ L nutrient solutions), with the DMEM nutrient solution cultures containing 10% hyclone 12h converges completely to cell, changes the chemical combination for being respectively 0,0.1,1,10,50,100 μM with the concentration prepared with serum free medium When 150 μ L of thing processing cells 48 are small, each concentration sets 4 multiple holes.The end of 10 μ L Fresh is added after the completion of incubation per hole Concentration continues after cultivating 4h for 5mg/mL MTT solution, and the liquid absorbed in culture hole terminates reaction, and 100 μ L are added per hole DMSO, shake well 10min on plate shaker, makes crystallization fully dissolve, and measures absorbance in microplate reader immediately, detects ripple A length of 490nm.Cell survival rate is calculated as follows.

Cell survival rate (%)=(medicine group OD/ blank group OD) × 100%

When cell survival rate is more than 80%, it is believed that medicine is small on cell viability influence, i.e. medicine is basic to culture cell Toxic action is not presented, and the standard of compound optimum concentration is chosen in this, as this research, and is the work of further experiment Concentration determines optimum range.Experimental result is as shown in Figure 1.

It will be seen from figure 1 that flavone compound does not have shadow substantially to 3T3-L1 PECTORAL LIMB SKELETON vigor within 100 μM Ring, cell viability is more than 90%；At 100 μM, cell viability is decreased obviously diphenylethylene compounds, especially deoxidation Ponticin, cell viability is down to less than 30%, but three kinds of compound on intracellular effect of vigor are smaller within 10 μM, exist More than 90%.Therefore, 10 μM of concentration for the treatment of as fenugreek compound of final choice study it.

(2) the induction differentiation of 3T3-L1 PECTORAL LIMB SKELETONs

Using " classical Cocktail Method " (Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers[J] .Journal of Natural Products,1998,61(1):71-76.) 3T3-Ll PECTORAL LIMB SKELETONs are induced, Induction the 8th day when more than 90% cell present adipocyte form, illustrate the differentiated mature fat cell of cell.3T3-L1 PECTORAL LIMB SKELETON is fibroblast, in typical fusiformis or sealene triangle, ability without secretion fat drips (a in Fig. 2 Figure), the growth retardation phase is in after cell converges completely, but after derivant induction differentiation, cell is become by fibroblast sample Circle, brightens, and being observed after the 4th day has scattered small fat drips (c schemes in Fig. 2), continues to cultivate, and by the 8th day, fat drips significantly increased, 95% cell differentiation is mature fat cell, and around around nucleus, it is thin for ripe fat in the structure of " finger ring " shape Born of the same parents' form (d schemes in Fig. 2), illustrates that 3T3-L1 PECTORAL LIMB SKELETONs induce differentiation into adipocyte completely.

(3) influence that faenum graecum compound accumulates 3T3-L1 cells fat drips

Set respectively in experiment normal group (Con groups), model group (Mod groups), positive controls (Rosi groups) and compound Treatment group, at the 0th day to the 4th day of induction, Con groups continued to cultivate in normal nutrient solution, and Mod groups are cultivated in inducing in liquid, And Rosi groups and compound treatment group are cultivated respectively in the induction liquid containing Rosi and 10 of 1 μM μM of fenugreek compound, Changing normal nutrient solution on the 4th day and continue to cultivate in induction, changes a nutrient solution in every 2 days.Until the 8th day of induction carries out oil Red O dyeing, micro- Microscopic observation, take pictures, and observes fat drips accumulation, and experimental result is as shown in Figure 3.

Fig. 3 shows, does not occur fat drips in Con groups, form and PECTORAL LIMB SKELETON it is substantially similar be shuttle-type or triangle, say The cell of bright Con groups is not broken up or differentiation degree is than relatively low.And there are substantial amounts of fat drips to assemble in model group in cytoplasm, The state of typical mature fat cell is presented in wherein more than 95% cell.Fat drips are more obvious in Rosi groups, or even compare mould It is more in type group, illustrate that Rosi can promote the accumulation of fat drips, accelerate cell differentiation as ripe adipocyte.At compound In reason group, formation of the homo-orientin to fat drips in flavone compound has obvious inhibitory action, can substantially reduce induction During fat drips accumulation, suppress the differentiation of cell, and the accumulation of orientin, Vitexin and isovitexin to fat drips is not obvious Inhibitory action；In diphenylethylene compounds, ponticin has stronger suppression 3T3-L1 PECTORAL LIMB SKELETON atomizations The effect of middle fat drips accumulation, can significantly inhibit the differentiation of cell, and the inhibitory action of deoxidation ponticin is weaker, in cell Also more fat drips produce, but rhapontigenin does not influence the differentiation of 3T3-L1 PECTORAL LIMB SKELETONs substantially, and its fat drips produces Quantity is substantially suitable with Mod groups.

(4) influence of the fenugreek compound to 3T3-L1 cell TG contents

Fat drips accumulate in fenugreek compound is studied to 3T3-L1 PECTORAL LIMB SKELETON atomizations using oil red O stain While influence, using TG assays kit (being purchased from the safe clinical reagent Co., Ltd of Beijing Northization) to TG in cell Content is qualitatively studied, and method to specifications surveys the TG contents in each processing cell pyrolysis liquid Fixed, experimental result is as shown in Figure 4.

Fig. 4 shows that the TG contents in Con groups are relatively low to only have 0.03mmol/gprot or so；And TG contents are shown in model group Work increases, and reaches 0.05mmol/gprot；TG contents reach 0.08mmol/gprot in Rosi treatment groups, illustrate that Rosi promotes The accumulation of TG in 3T3-L3 cell differentiation procedures；In drug-treated group, the shape of homo-orientin in flavone compound to fat drips Into there is obvious inhibitory action (P<0.05) accumulation of TG in Induction Process, can be substantially reduced, suppresses the differentiation of cell, and Polygonum Careless element, the accumulation of Vitexin and isovitexin to fat drips do not have obvious inhibitory action；In diphenylethylene compounds, Rumex madaio Glycosides has the function that TG is accumulated in stronger suppression 3T3-L1 PECTORAL LIMB SKELETON atomizations, can significantly inhibit point of cell Change, and the inhibitory action of deoxidation ponticin is weaker, also has more fat drips to produce in cell, but rhapontigenin is to 3T3- The differentiation of L1 PECTORAL LIMB SKELETONs has faint facilitation, this is also consistent into this with above-mentioned oil red O stain result.

(3) detection of 3T3-L1 cellular fats factor expression

During cell carries out induction differentiation, induction adds 10 μM of compound processing for the 0-4 days.Induced in cell Collect cell within the 8th day, extract plasmosin, protein content measured using BCA methods, SDS-PAGE electrophoresis is carried out after quantitative, grinds Faenum graecum compound is studied carefully to the expression of Adipocyte Factor PPAR γ, C/EBP α and FAS in 3T3-L1 PECTORAL LIMB SKELETON atomizations Influence, experimental result is as shown in Figure 5.

Fig. 5 shows, in undifferentiated 3T3-L1 PECTORAL LIMB SKELETONs, PPAR γ, C/EBP α and FAS are all in the low of background Expression, and in the adipocyte of differentiation and maturation, its expression significantly raises.And 4 kinds of flavone compounds To reduce the expression of 3 kinds of Adipocyte Factors, but suppress horizontal different, wherein suppression effect of the orientin to FAS and PPAR γ Fruit is the most obvious, and expression of the homo-orientin to C/EBP α has inhibition strongly, while Vitexin is to PPAR γ Expression also there is significant impact.In diphenylethylene compounds, ponticin and rhapontigenin can reduce the table of FAS Up to level, and expression of the deoxidation ponticin on FSA does not influence；Ponticin and deoxidation ponticin can reduce PPAR The expression of γ, C/EBP α, and the effect of ponticin is more obvious.During 3T3-L1 PECTORAL LIMB SKELETONs break up, Adipocyte Factor plays important regulating and controlling effect, and as the differentiation of cell, the expression of Adipocyte Factor also occur to change accordingly. PPAR γ and C/EBP α are in very low expression in PECTORAL LIMB SKELETON, and are expressed in ripe adipocyte or tissue Level significantly increases, they play pass at the metabolism of formation and storage and glycolipid during Adipocyte Differentiation to lipid The effect of key.By adjusting the expression of PPAR γ and C/EBP α, the fatty atopen of atomization can be adjusted Expression change, such as AP2, SREBP1c, FAS etc., so as to adjust the differentiation of adipocyte.FAS is fatty acid synthase, directly negative The synthesis of aliphatic acid is blamed, the expression rise of FAS directly results in the accumulation of lipid.

2nd, the foundation of insulin resistance (IR) model

3T3-Ll PECTORAL LIMB SKELETONs are induced using " classical Cocktail Method " first, the fat for becoming maturation is thin Born of the same parents.Then IR models are established using the long-acting analog dexamethasone (Dex) of glucocorticoid, taken in experiment induce differentiation into it is ripe 3T3-L1 adipocytes, with 1 μM of Dex handle cell 48 it is small when, and blank group common DMEM in high glucose processing cell.Processing After the completion of detect 3T3-L1 cells under insulin stimulating to the intake of 2-NBDG and the phosphorylation level of AKT, and then evaluate Whether IR is successfully established.

Intake quantity measuring method of the cell to 2-NBDG：On the basis of the IR models of foundation, cell is divided into two groups, one group 15min is incubated with the sugar-free DMEM containing 100nM insulin, another group handles same time with sugar-free DMEM.Insulin is incubated After the completion of washed 2 times with DPBS, use instead the sugar-free DMEM containing 100 μM of 2-NBDG be incubated 1 it is small when, culture is discarded after the completion of incubation Liquid, DPBS are washed 2 times, and 500 μ L pancreatin, 37 DEG C of digestion 10min are added per hole, and (gently vibration is flat on testing stand at 5 minutes or so Plate), 3.5mL DPBS piping and druming is added per hole uniformly, in the round bottom centrifuge tube with liquid-transfering gun suction 5mL, 1000rpm centrifugations 10min, DPBS are washed 2 times.500 μ L DPBS resuspensions are eventually adding, streaming pipe, 488nm, 515~545nm wavelength are transferred to after crossing film Downflow system cell instrument detects.

The phosphorylation level detection method of AKT：On the basis of the IR models of foundation, the western when the 48th of processing the is small Blotting detects the phosphorylation level of AKT of the cell under insulin stimulating (100nM insulin is incubated 15min).

Experimental result is shown in Fig. 6 and Fig. 7, Dex processing 48 it is small when after, intake of the cell to 2-NBDG under insulin stimulating Ability is remarkably decreased, while the phosphorylation level of AKT also decreases.This explanation PI3K/AKT signal, which leads to, to be damaged so that Insulin is delivered to intracellular signal through signal transduction can not continue to reduce toward downstream transmission or transmission efficiency, so that cell The sensitiveness of insulin is reduced.Judge that IR models are successfully established accordingly.

3rd, improvement result of the faenum graecum compound to IR models

(1) detection of the 3T3-L1 cells to 2-NBDG intake abilities

The cell of differentiation and maturation is divided into blank control group, model group, positive controls and compound treatment group, except blank Group is with outside complete culture solution culture, remaining each group establishes IR models with 1 μM of Dex processing, and wherein positive controls are in Dex processing While add 1 μM of Rosi, and compound treatment group adds 10 μM of fenugreek compound while Dex is handled.Processing 48 it is small when after, be respectively divided into 2 groups inside blank control group and model group, one of which is handled with the insulin of 100nM 15min, and another group is used instead DMEM processing same times, positive controls and compound treatment group equally use the pancreas islet of 100nM Element processing 15min.After the completion of processing all experimental groups use instead the sugar-free DMEM containing 100 μM of 2-NBDG be incubated 1 it is small when, processing 488nm afterwards, 515~545nm wavelength downflow system cell instrument detect.

2-NBDG(2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) It is mainly used for detection of the cell to glucose uptake ability.After IR occurs for body or cell, since peripheral organization is to insulin Sensitiveness reduces, and the intake ability of adipose tissue and skeletal muscle to glucose declines, so as to cause the concentration of glucose in blood Rise.

This experiment establishes IR models using 3T3-L1 cells improves the action effect of IR, experiment to evaluate fenugreek compound After the completion of middle fenugreek compound processing cell, 3T3-L1 cells pair are detected using the glucalogue 2-NBDG of fluorescent marker Glucose uptake ability, experimental result are as shown in Figure 8.

Fig. 8 shows that intake ability of (Insulin groups) cell to 2-NBDG significantly increases after insulin stimulating, and IR groups Intake capacity variation of (Dex+Insulin groups) cell to 2-NBDG is little after middle insulin stimulating, poor compared with Insulin groups Heteropole significantly (P ＜ 0.01), illustrates the success of IR model foundations.And intake of the cell to 2-NBDG in Dex+Insulin+Rosi groups Ability is recovered, hence it is evident that higher than Dex+Insulin groups, and difference is extremely significantly (P ＜ 0.01), illustrates that Rosi can improve insulin thorn Swash lower intake ability of the 3T3-L1 adipocytes to 2-NBDG.In fenugreek compound treatment group, Polygonum orientale in flavone compound Plain (P ＜ 0.05) and homo-orientin (P ＜ 0.01) can promote under insulin stimulating 3T3-L1 adipocytes in various degree to 2- The intake of NBDG, and there is the significance of difference compared with Dex+Insulin groups.Ponticin also can in diphenylethylene compounds Intake of the 3T3-L1 adipocytes to 2-NBDG under insulin stimulating is enough enough improved, difference is extremely aobvious compared with Dex+Insulin groups Write.

In conclusion flavone compound and diphenylethylene compounds can improve insulin thorn in fenugreek compound Swash lower intake ability of the 3T3-L1 adipocytes to 2-NBDG, the wherein best results of compound homo-orientin and ponticin, Illustrate that homo-orientin and ponticin have improvement IR symptoms, treat the potentiality of diabetes.

(2) detection of 3T3-L1 cells AKT and AMPK phosphorylation

The present invention have studied expression of the fenugreek compound to AKT/pAKT and AMPK/pAMPK in 3T3-L1 cells Influence, experimental implementation finally detects related egg with the detection of ability " 2-NBDG absorb " described with western blotting White expression, experimental result is as shown in Figures 9 and 10.

Research shows that AKT and AMPK signal paths play the effect of key in the pathogenic process of metabolic disease, especially During insulin Cascaded amplification regulates and controls glucose transport.The PI3K/ in the growth of cell, propagation and metabolic process Akt signal paths play an important role, it is adjusted be subject to a variety of factors, such as epidermal growth factor and insulin-like growth factor Deng positivity adjust and the important negative regulator of PTEN equimoleculars pair is to maintain the stable state of cell and life.PI3K/Akt signals lead to Road is the major downstream molecular pathway of insulin, when insulin exercises its metabolic function, it is considered that AKT2 is exercised Most important function, the phosphorylation level of AKT can dramatically increase after insulin stimulating, and body or cell enter IR states Afterwards, insulin insensitivity, AKT phosphorylation levels are reduced.Fig. 9 shows, AKT in cell after insulin stimulating in Insulin groups Phosphorylation level significantly raise, and in IR groups (Dex+Insulin groups) after insulin stimulating in cell AKT phosphorylation level Significantly reduced compared with insulin group, illustrate the success of IR model foundations, this is consistent with 2-NBDG intakes result of the test.And Dex+ The phosphorylation level of AKT is significantly raised compared with Dex+Insulin groups in Insulin+Rosi group cells, illustrates that Rosi can change Under kind insulin stimulating in 3T3-L1 adipocytes AKT phosphorylation level, and then improve IR symptoms, this also with document tune early period It is consistent to grind result.In fenugreek compound treatment group, in the homo-orientin and diphenylethylene compounds in flavone compound Ponticin can significantly improve the phosphorylation level of AKT in 3T3-L1 adipocytes under insulin stimulating.

Adenylate cyclase AMPK is a kind of important protein kinase, is sent out in terms of metabolism and energy balance is coordinated Wave important function.AMPK is increasing skeletal muscle to glucose uptake, enhancing insulin sensitivity, increase aliphatic acid oxygen after being activated Change and adjust genetic transcription etc. and play a significant role.Due to its adjust sugar and fatty acid metabolism in terms of effect, AMPK fat, insulin resistance and type II diabetes may provide new pharmacology target spot for treatment.Fenugreek compound pair The influence the result is shown in Figure 10 of AMPK phosphorylation levels in 3T3-L1 adipocytes, the results show that the Iroi in flavone compound AMPK can be activated with ponticin in diphenylethylene compounds, increases its phosphorylation level, so as to promote cell to grape Sugar intake, enhancing insulin sensitivity.

(3) detection of 3T3-L1 cells lipid metabolic gene transcriptional level

Experimental implementation is described with " 2-NBDG absorbs the detection of ability ", and cell, the extracting of Trizol methods are collected after the completion of processing Total serum IgE, then carries out reverse transcription synthesis cDNA, finally detects fenugreek compound to IR shapes using quantitative real-time PCR The change of 3T3-L1 cells lipid metabolism related gene transcriptional level under state.

During relative quantitative assay, the expression of target gene is using the method for comparing Ct values, with reference gene β-actin come school Positive variance, times quantity of change is 2^-△△Ct.Calculation formula is：Change multiple=2^-△△Ct, wherein, △ △ Ct=(Ct target bases Because of-Ct reference genes) compound treatment group-(Ct target gene-Ct reference genes) control.

Real-time fluorescence quantitative PCR actual conditions is as follows：

The gene order provided according to document, primer are synthesized by Shanghai bioengineering limited company, β-actin (GenBank sequence numbers:NC_000071.6, PPAR γ (GenBank sequence numbers:NM_001308354.1)、C/EBPα (GenBank sequence numbers:NM_001287523.1), aP2 (GenBank sequence numbers:M84651.1), SREBP 1c (GenBank sequences Row number:) and FAS (GenBank sequence numbers AB046200.1:NM_007988.3 primer sequence) is as shown in table 1.

1 real-time fluorescence quantitative PCR primer sequence of table

Pcr amplification reaction system is prepared, as shown in table 2.

2 real-time fluorescence quantitative PCR reaction system component of table

Above-mentioned reaction solution is put into fluorescence quantitative PCR instrument and is expanded, PCR reaction conditions are as shown in table 3.

3 real-time fluorescence quantitative PCR response procedures of table

Fluorescence signal is each gathered after circulation terminates, finally draws melting curve to determine that reaction product has primer free dimerization Body and non-specific amplification, when fluorescence signal intensity exceedes baseline, its domain period is recorded.In real time fluorescent quantitative In PCR, the initial concentration that Ct values are considered and are amplified gene is closely related.

Now research think adipose tissue be IR produce originate position, except can adjust body energy storage and nutrition put down Weighing apparatus is outer, or an active endocrine organ, can secrete cytokine profiles such as TNF-α, leptin, adiponectin and phylaxin Deng.Insulin resistance generation is often related to obesity, due to the accumulation of body fat, muscle and utilization of its hetero-organization to glucose Rate reduces, and causes insulin resistance and hyperinsulinemia.And the generation of adipose tissue is mainly close with the expression of Adipocyte Factor Correlation, this research pass through the technique study of real-time quantitative fluorescence PCR fenugreek compound to the 3T3-L1 cells under IR states The influence of the transcriptional level of Adipocyte Factor PPAR γ, C/EBP α, AP2, SREBP1c and FAS etc..

Figure 11 shows that the transcriptional level of PPAR γ significantly increases in IR groups (Dex treatment groups), illustrates thin in IR states The expression of PPAR γ rises in born of the same parents.The transcriptional level of PPAR γ is decreased obviously compared with Dex groups in Rosi groups, and fenugreek chemical combination In thing treatment group, ponticin in homo-orientin and Vitexin and diphenylethylene compounds in flavone compound and Deoxidation ponticin can reduce the transcriptional level of PPAR γ, and be respectively provided with the significance of difference compared with IR groups.

C/EBP α, that is, CCAAT/ enhancer binding protein α, in the stage great expression last eventually of Adipocyte Differentiation, with PPAR γ collective effects, open a series of expression of fat specific genes, so as to synthesize, absorb and store long chain fatty acids, and make Cell stops propagation, and complete differentiation state is presented.In order to study its expression in the adipocyte in IR states, this Invention have studied the change of its transcriptional level.The result is shown in Figure 12, Figure 12 is shown, compared with vehicle groups, (the Dex processing of IR groups Group) in the transcriptional level of C/EBP α significantly increase, and Rosi can strengthen the transcriptional level of C/EBP α, illustrate that Rosi can promote The metabolism of lipid, increases the synthesis of lipid in adipocyte.And in fenugreek compound treatment group, Iroi in flavone compound The expression of C/EBP α can be reduced with ponticin in Vitexin and diphenyl ethene compounds, and difference is extremely aobvious compared with IR groups Write.

AP2 is an important gene during lipid metaboli, and rise, therefore the present invention are expressed in mature fat cell It has studied the expression in its 3T3-L1 cell under IR states.Experimental result is as shown in figure 13, with vehicle group phases Than the transcriptional level of AP2 significantly increases in IR groups (Dex treatment groups), and Rosi can strengthen the transcriptional level of AP2, explanation Rosi can promote the metabolism of lipid, increase the synthesis of lipid in adipocyte, this is consistent with the above results.And fenugreek chemical combination In thing treatment group, Iroi and Vitexin in flavone compound can be reduced with the ponticin in diphenylethylene compounds The expression of AP2, and difference is extremely notable compared with IR groups.

SREBP1c is the relevant gene of lipid metaboli in PPAR γ and C/EBP α metabolic pathways downstream during lipid metaboli, Express enhancing in ripe adipose tissue and cell, transcriptional level in its 3T3-L1 cell under IR states of this experimental study. Figure 14 shows that the transcriptional level of SREBP1c significantly raises in IR groups compared with normal group, and Rosi can strengthen it fatty thin Expression in born of the same parents.In fenugreek compound treatment group, homo-orientin and Vitexin and diphenylethylene in flavone compound Ponticin in compound can reduce the transcription of SREBP1c, and difference is extremely notable compared with IR groups.

FAS, that is, fatty acid synthase, is the important enzyme of synthetic fatty acid in adipocyte, is the downstream gene of SREBP1c, Expression raises in mature fat cell.Transcriptional level in its 3T3-L1 cell under IR states of this experimental study.Figure 15 show, the transcriptional level of FAS significantly raises in IR groups compared with normal group, and Rosi can strengthen it in adipocyte Expression.In fenugreek compound treatment group, in flavone compound in homo-orientin and Vitexin and styrene compound Ponticin can reduce the transcriptional level of FAS, and have the significance of difference compared with IR groups.

2-NBDG intakes result of the test shows that orientin, homo-orientin and ponticin can carry in fenugreek compound Hyperinsulinism stimulates lower intake ability of the 3T3-L1 cells to 2-NBDG, and homo-orientin and ponticin are optimal, with IR most phases It is extremely more notable than difference；And Western results of study are shown, homo-orientin and ponticin can be improved in insulin signaling pathway The phosphorylation level of AKT, while the phosphorylation level of AMPK can be improved, illustrate that homo-orientin and ponticin can improve IR diseases Shape, has the potentiality for the treatment of type II diabetes.Real-time fluorescence quantitative PCR research the result shows that, homo-orientin and ponticin energy The transcriptional level of enough lipid-metabolism correlation factors reduced at the same time in the adipocyte in IR states, this tests with glycometabolism and ties Fruit is consistent, and further demonstrates homo-orientin and ponticin adjusts glucose -lipid metabolism disorder, improve the potentiality of IR, for exploitation recklessly Lu Bayuan treatment type II diabetes medicines provide more favourable theories integration.

4th, influence of the fenugreek compound to 3T3-L1 mitochondrial functions

(1) detection of 3T3-L1 cells ROS levels

ROS in flow cytomery 2T3-L1 cells is horizontal：The 3T3-L1 cells of induced maturation in 6 orifice plates are taken, point For blank control group, model group, Rosi groups and compound treatment group, except blank group continues with addition to complete culture solution culture, remaining Each group with 1 μM of Dex handle cell 48 it is small when establish IR models, wherein Rosi groups add 1 μM of Rosi while Dex is handled, And compound treatment group adds 10 μM of fenugreek compound while Dex is handled.It is finally glimmering with FITC passage flow cytometer detections Luminous intensity.

ROS in laser co-focusing detection 3T3-L1 cells is horizontal：Experimental procedure is the same as " flow cytomery 2T3-L1 is thin ROS levels in the born of the same parents " step, PBS is washed 2 times after the completion of compound processing, and 200 μ L DCFH-DA are added per hole, and (DPBS matches somebody with somebody System, 10 μM) 37 DEG C of incubation 30min of fluorescence probe, PBS is washed 2 times after the completion of incubation, is seen in laser confocal microscope FITC passages Fluorescence intensity is examined, and is taken pictures.

ROS is the oxygen radical in organism, including oxygen and oxygen containing high reaction activity molecule (such as superoxides the moon from Son, tissue peroxide and free radical etc.), mitochondria is mainly derived from, is high during mitochondria is changed from state I II to state I V The respiratory chain of the environment of oxygen and high reduction-state makes a large amount of electronics leak out and reduce oxygen molecule and formed.Low-level ROS and Gao Shui Necessary to flat ATP is islet β cell insulin, on excessive ROS damages mitochondrial protein, mtDNA and mitochondrial membrane Lipid, so as to cause injury of mitochondria.But caused lasting, high yield ROS is generated on the contrary after mitochondrial function damage ATP generations can be reduced, damage β cells, suppress insulin releasing.DCFH-DA does not have fluorescence in itself, can pass freely through cell Film, into after intracellular, can generate DCFH by intracellular esterase hydrolyzed.And DCFH is unable to permeabilized cells film, so that visiting Pin is easy to be loaded onto intracellular.Intracellular ROS, which can aoxidize non-blooming DCFH generations, the DCF of fluorescence, detects DCF Fluorescence can in reacting cells active oxygen level.Fluorescence probe of this experiment using DCFH-DA as detection ROS, respectively The content of the active oxygen in 3T3-L1 cells is quantified and determined using two methods of flow cytometer and laser co-focusing Property analysis, streaming quantitative analysis results are as shown in figure 16, and the fluorescence intensity of Mod groups (IR groups) middle probe is (normal far above Con groups Group), and difference is extremely notable, illustrates the generation speed rise of ROS in IR cells.ROS produces speed and Mod in Rosi treatment groups Group is compared and significantly reduced, and difference is extremely notable, illustrates that Rosi can reduce the generation of ROS in cell.At fenugreek compound In reason group, the Rumex madaio in orientin, homo-orientin and isovitexin and diphenylethylene compounds in flavone compound Glycosides can significantly reduce the generation of ROS in cell, and have the significance of difference compared with Mod groups.

Laser co-focusing qualitative results are as shown in figure 17, and the fluorescence intensity of Mod group middle probes is far above Con groups, illustrates IR The generation speed rise of ROS in cell.The fluorescence intensity of Rosi treatment group middle probes significantly reduces compared with Mod groups, explanation Rosi can reduce the generation of ROS in cell.In compound treatment group, compound orientin, homo-orientin, isovitexin and soil Rheochrysin can reduce the fluorescence intensity of cell middle probe, and wherein compound isovitexin and ponticin effect is most obvious.

(2) detection of 3T3-L1 cell mitochondrials membrane potential

Same " ROS in flow cytomery 2T3-L1 cells the is horizontal " step of experimental procedure, 488nm, 529/ 590nm flow cytometer detections.

Mitochondrial function is mainly reflected in the ability for maintaining oxidative phosphorylation, and this ability can use mitochondrial inner membrane film Potential is evaluated.Because interior negative outer positive membrane potential had not only ensured energy of position of the electronics in electron transport chain, but also ensured Hydrogen ion enters the energy of position of mitochondria interior room, so as to provide necessary proton stream for ATP synthesis.Therefore, mitochondrial membrane potential The height for being is one of important indicator for evaluating mitochondrial function.JC-1 is a kind of reason for being widely used in detection mitochondrial membrane potential Think fluorescence probe, the change of mitochondrial membrane potential can be easily detected by the transformation of fluorescence color.In mitochondria When film potential is higher, JC-1 is gathered in the matrix of mitochondria, forms polymer (J-aggregates), can be produced red glimmering Light；When mitochondrial membrane potential is relatively low, JC-1 cannot be gathered in the matrix of mitochondria, and JC-1 is monomer (monomer) at this time, Green fluorescence can be produced.Fluorescence probe of this experiment using JC-1 as detection mitochondrial membrane potential, using flow cytometer pair The content of active oxygen in 3T3-L1 cells is analyzed, and experimental result is as shown in figure 18, the ratio of red/green in Mod groups Value is far above Con groups, illustrates the reduction of IR cells mitochondrial potential change.Red/green ratio and Mod group phases in Rosi treatment groups Than significantly reducing, illustrate Rosi can raise IR caused by 3T3-L1 cell mitochondrials membrane potential reduce.The processing of fenugreek compound In group, the ponticin in orientin, homo-orientin, isovitexin and diphenylethylene compounds in flavone compound It can recover 3T3-L1 cell mitochondrial membrane potentials to a certain degree with rhapontigenin, wherein compound isovitexin effect is most bright It is aobvious.

(3) detection of 3T3-L1 cellular ATP contents

Experimental procedure is with " ROS in flow cytomery 2T3-L1 cells the is horizontal " step, using enhanced ATP Detection kit (the green skies bio tech ltd in Shanghai) carries out ATP content detections, and ATP is the main energy of living organism Material, produces inner membrance proton gradient, proton promotes atp synthase to synthesize ATP when flowing back by mitochondrial inner membrane oxidative phosphorylation.ATP Generation ability be mitochondrial function a leading indicator, if mitochondrial inner membrane damage produces proton leak or oxidative phosphorylation Impaired, the synthesis of ATP can be damaged.This experiment utilizes ATP assay kit measurements fenugreek compound to 3T3-L1 The influence that ATP is produced in cell, experimental result are as shown in figure 19.Figure 19 shows that the content of ATP is significantly low in cell in Mod groups In Con groups, and difference is extremely notable, illustrates that IR can cause the synthesis of ATP in 3T3-L1 adipocytes to decline.Compared with Mod Cellular ATP content increase in Rosi treatment groups, and significant difference.And in fenugreek fenugreek compound treatment group, flavonoids Homo-orientin and Vitexin in compound can increase containing for ATP in cell with the ponticin in diphenylethylene compounds Amount, has the significance of difference compared with Mod groups.

(4) detection of 3T3-L1 cell mitochondrials DNA damage

Experimental procedure extracts cytogene with " ROS in flow cytomery 2T3-L1 cells the is horizontal " step Group DNA, the integrality of agarose electrophoresis identification of dna, the complete glue of electrophoresis is put into ultraviolet imagery system and is observed, if band Clearly and without conditions of streaking, show that DNA integralities are preferable.

It is as follows using the damage of Long PCR detection 3T3-L1 cell mitochondrials DNA, specific method：

A. gene order (the mitochondria full-length genome GenBank provided according to document:J01420.1), primer is given birth to by Shanghai Thing Engineering stock Co., Ltd synthesizes, and the primer sequence of mitochondria related gene is as shown in table 4：

4 mitochondria Long PCR primer sequences of table

B. pcr amplification reaction system is prepared, as shown in table 5：

Table 5PCR reaction system components

C. above-mentioned reaction solution is put in PCR instrument and is expanded, PCR reaction conditions are as shown in table 6：

6 long segment real-time fluorescence quantitative PCR response procedures of table

7 short-movie section real-time fluorescence quantitative PCR response procedures of table

DNA electrophoresis is carried out after the completion of d.PCR reactions immediately, long segment is left with 1% Ago-Gel 120V electrophoresis 30min The right side, short-movie breaks treats that purpose band is completely separable with 0.5% Ago-Gel 120V electrophoresis 40-60min or so, immediately after in Observed, taken pictures in gel imaging system.

The long segment target gene and the ratio of short-movie section target gene that the degree of mitochondrial DNA damage is gone out using PCR amplification Value represents.Wherein DNA damage degree is bigger in long segment, and PCR amplification will be obstructed, and amplification is slower.Therefore DNA damage degree Difference, the amount that same time amplifies the target gene come is also just different, and short-movie section is since gene order is shorter, DNA damage PCR amplification is influenced smaller.Therefore the ratio of long segment and short-movie section can be used for representing the degree of mitochondrial DNA damage.

Genome contained by mitochondrial DNA is all, its sequential coding 2 related with the function of mitochondrial electron transport chain RRNA, 22 tRNA and 13 polypeptide for participating in mitochondrial inner membrane respiratory enzyme composition.Lost when active oxygen is formed with the balance decomposed Timing, body are in oxidative stress status.No matter the increase of active oxygen is endogenous or exogenous, mitochondria is responsible for The oxidative damage of DNA.The structure and functional characteristics and place physiological location of mitochondrial DNA itself determine that it is more easy to than core DNA Attacked in be subject to oxidative damage, and the Mitochondrial DNA Mutation or its gene outcome that oxidative damage is led to lose meeting further Promote the release of active oxygen.This experiment uses the technique study of longPCR fenugreek compound to 3T3-L1 cell center line grains The influence of body DNA damage, the experimental result of mitochondrial DNA damage are as shown in figure 20.

Figure 20 shows that the target gene that long segment amplifies in mitochondria Mod groups is reduced compared with Con groups, illustrates IR moulds Type Mitochondria DNA is damaged, and Rosi group Mitochondria DNA damage degree substantially mitigates.Fenugreek compound treatment group In, the ponticin group Mitochondria DNA damage in the homo-orientin group and diphenylethylene compounds in flavone compound It is suppressed, illustrates that homo-orientin and ponticin have certain protective effect to mitochondrial DNA.

(5) detection of 3T3-L1 cell mitochondrials producer transcriptional level

The culture of cell is identical with the horizontal test experiences of ROS with chemical combination substance treating method, is collected after the completion of compound processing Cell, Trizol method extracted total RNAs, are then carried out reverse transcription synthesis cDNA, are finally detected using quantitative real-time PCR The change of 3T3-L1 cell Mitochondria related gene transcriptional levels, real-time fluorescence quantitative PCR specific method are as follows：

A. the gene order provided according to document, primer are synthesized by Shanghai bioengineering limited company, β-actin (GenBank accession number:NC_000071.6), DRP1 (GenBank accession number:AB079133.1), (GenBank is logged in mfn1 Number:NM_024200.4), mfn2 (GenBank accession number:AY123975.1), NRF1 (GenBank accession number: AF098077.1), PGC-1 (GenBank accession number:) and TFam (GenBank accession number AF049330.1:AH003701.2) Primer sequence is as shown in table 8：

8 real-time fluorescence quantitative PCR primer sequence of table

B. pcr amplification reaction system is prepared, as shown in table 9：

9 real-time fluorescence quantitative PCR reaction system component of table

C. above-mentioned reaction solution is put into fluorescence quantitative PCR instrument and is expanded, PCR reaction conditions are as shown in table 10：

10 real-time fluorescence quantitative PCR response procedures of table

The expression of target gene corrects difference, times of change using the method for comparing Ct values with reference gene β-actin Quantity is 2^-△△Ct.Calculation formula is：Change multiple=2^-△△Ct, wherein, △ △ Ct=(Ct target gene-Ct reference genes) medicine Thing treatment group-(Ct target gene-Ct reference genes) control.

Statistical analysis is carried out using statistics software SPSS16.0, the comparison of mean is using independent sample t inspection between two groups Test, the comparison of multigroup mean uses one-way analysis of variance (one-way ANOVA), and Exemplary statistical data is with average value ± mark It is accurate poorRepresent,^*Represent P<0.05 explanation has statistical significance,^**Represent P<0.01, which illustrates that its difference has, extremely significantly unites Meter learns meaning.

Mitochondria biology occurs have weight in terms of mitochondrial function and number is maintained to meet eukaryotic physiological requirements Act on.Under morbid state, if after injury of mitochondria, enhancing, which occurs, for mitochondria biology helps to maintain mitochondrial function and thin Born of the same parents' energy state, and then promote histoorgan reparation；Conversely, if impaired and mitochondria dysfunction occurs for mitochondria biology at the same time Occur, then mitochondrial function can be made further to deteriorate, aggravate cell, function of organization's damage.Determined in this research using real-time fluorescence Amount PCR have studied fenugreek compound and generation related gene NRF1, PGC-1 and Tfam of 3T3-L1 cell Mitochondrias transcribed Horizontal influence, experimental result are as shown in figure 21：

Figure 21 shows that the transcriptional level of NRF1 reduces in Mod groups, and otherness is notable compared with Con groups, illustrates that IR causes NRF1 expressions reduce in 3T3-L1 cells.In Rosi treatment groups, NRF1 transcriptional levels significantly increase, and compared with Mod Difference is extremely notable, illustrate Rosi can improve IR NRF1 transcriptional levels extremely reduction.It is yellow in fenugreek compound treatment group Homo-orientin, Vitexin and isovitexin in ketone compounds can make with the ponticin in diphenylethylene compounds NRF1 transcriptional levels significantly increase, wherein homo-orientin group and isovitexin the group significant difference compared with Mod, Vitexin group and soil Rheochrysin group difference compared with Mod is extremely notable.

Figure 22 shows that the transcriptional level of PGC-1 reduces in Mod groups, and otherness is extremely notable compared with Con groups, illustrates IR shapes The 3T3-L1 cell Mitochondria producer PGC-1 expressions of state reduce, and mitochondria is likely to occur obstacle.At Rosi In reason group, PGC-1 transcriptional levels significantly increase, and difference is extremely notable compared with Mod, illustrate Rosi can improve IR extremely The reduction of mitochondria producer PGC-1 transcriptional levels.In fenugreek compound treatment group, flavone compound and talan Class compound influences all unobvious, wherein orientin, homo-orientin and ponticins to PGC-1 transcriptional levels slightly to raise PGC-1 transcriptional levels, but do not have the significance of difference compared with Mod.

Figure 23 shows that the transcriptional level of Tfam reduces in Mod groups compared with Con groups, has pole significant difference, illustrates IR The 3T3-L1 cell Mitochondria producer Tfam expressions of state reduce, and mitochondria is likely to occur obstacle.Rosi In treatment group, Tfam transcriptional levels significantly increase, and difference is extremely notable compared with Mod, illustrate that Rosi can improve IR institutes extremely Mitochondria producer Tfam transcriptional levels reduction.In fenugreek compound treatment group, the Polygonum orientale in flavone compound Element can lower its transcriptional level, and ponticin can raise in homo-orientin and isovitexin and diphenyl ethene compounds Its transcriptional level, and all there is difference pole conspicuousness compared with Mod groups.

Mitochondrial function damages and insulin resistance has close relationship, and more and more evidences show that IR is at present Induce one of principal element of insulin resistance.Reactive oxygen species produce number, the height of mitochondrial membrane potential, ATP Generation, generation of mitochondrial DNA damage situation, mitochondria etc. can influence the function of mitochondria, one index of any of which The function for the mitochondria that goes wrong can be affected.Above indices are studied respectively in experiment.Mitochondria work( Energy result of study shows that orientin, homo-orientin, isovitexin and ponticin can be reduced in cell in fenugreek compound The generation of ROS, this may be related with position with the number of the phenolic hydroxyl group in fenugreek compound structure；At the same time orientin, Homo-orientin, isovitexin and ponticin can also recover 3T3-L1 cell mitochondrial membrane potentials to a certain degree, increase in cell The generation of ATP, enough protects mitochondrial DNA to be damaged, and can promote mitochondria to a certain extent.Illustrate fenugreek chemical combination Thing can improve the function damage of 3T3-L1 cells Mitochondria caused by IR to a certain extent, and by alleviating mitochondria work( Cell metabolism state can be improved when and can promote the mitochondria to occur, and then improve the effect of glycolipid metabolism.

The foregoing is merely the embodiment of the present invention, is not intended to limit the scope of the invention, every to utilize this hair The equivalent structure or equivalent flow shift that bright description is made, is directly or indirectly used in other relevant technology necks Domain, is included within the scope of the present invention.

Sequence table

<110>Northwest Plateau-organisms Research Inst. of Chinese Academy of Sciences

<120>Homo-orientin is being prepared and the purposes in insulin resistance relevant disease

<160> 0

<170> SIPOSequenceListing 1.0

Claims

1. application of the homo-orientin in the medicine for preparing prevention and/or treatment insulin resistance.

2. application according to claim 1, it is characterised in that the medicine is prevention and/or treatment glucose -lipid metabolism disorder Relevant disease medicine.

3. application according to claim 1 or 2, it is characterised in that the disease include hyperlipidemia, cardiovascular disease, Diabetes, hypertension or metabolic syndrome.

4. application according to claim 1 or 2, it is characterised in that the medicine is the medicine for suppressing Adipocyte Differentiation.

5. the application according to claim 1 or 4, it is characterised in that the medicine be suppress PPAR γ, C/EBP α or/and The medicine of FAS.

6. application according to claim 1, it is characterised in that the medicine is the medicine for improving mitochondrial function damage.

7. application according to claim 1 or 2, it is characterised in that the medicine is by regulating and controlling insulin signaling pathway And/or in AMPK signal paths correlative protein expression level medicine.

8. application according to claim 7, it is characterised in that the medicine is to improve AKT eggs in insulin signaling pathway The medicine of white phosphorylation level, and/or, the medicine of AMPK protein phosphorylations level in raising AMPK signal paths.

9. application of the homo-orientin as PPAR γ antagonists, C/EBP alpha-2 antagonists, AKT activators or AMPK activators.

10. homo-orientin is preparing the application in suppressing Adipocyte Differentiation or/and repairing the medicine of injury of mitochondria.