CN106265620A - The clever C of Aunar and the like application in the medicine of preparation preventing and treating metabolic disease - Google Patents

Abstract

The present invention relates to the clever C of Aunar and the like application in the medicine of preparation preventing and treating metabolic disease.Disclose the clever C of Aunar or its analog first, for the preventing and treating of metabolic disease, there is highly significant effect, thus provide to regulate CREB/CRTC2 and the SREBP multifunctional drug as target spot.

Description

The clever C of Aunar and the like application in the medicine of preparation preventing and treating metabolic disease

Technical field

The invention belongs to biomedicine field, more particularly it relates to the application that clever C of Aunar and the like is in the medicine of preparation preventing and treating metabolic disease.

Background technology

Liver processes (including absorbing blood glucose, glycogen conversion and glyconeogenesis and output) regulation and control and is in central status blood glucose.Hepatic gluconeogenic is hyperfunction is that type 2 diabetes mellitus glycogen exports the important pathology raised, and corrects hyperfunction glycogen and generates beneficially control onset diabetes process and secondary disease.So the inhibitor research that targeting glycogen produces is to controlling and treatment metabolic syndrome, including hyperglycemia, obesity and type 2 diabetes mellitus, significant.

The efficiency of hepatic gluconeogenic is largely dependent upon the transcriptional control to glyconeogenesis related gene.Time hungry, in blood, glucagon raises, glucagon receptor (Glucagon receptor, GCGR) it is activated, 3 are synthesized by the adenyl cyclase on G α s activated membrane, 5-ring AMP (cAMP), utilize cAMP-PKA signal path to activate CREB (the CRE response element-binding protein) transcriptional activators in hepatocyte and directly raise hepatic gluconeogenic pathway key enzyme: G-6-Pase (glucose-6-phosephatase, and the expression of PCK (PEPCK) G6Pase)；CREB is also by raising transcription factor PGC1 α (PPAR γ coactivator 1-α) and the transcribing of orphan nuclear receptor NR4A1 member, them are made to continue and indirectly raise the expression of glyconeogenesis indispensable gene, the body demand to blood glucose when meeting long-term hunger.Within 2003, it is found that a kind of new transcriptional co-factor can be combined with CREB and greatly strengthen CREB transcriptional activity, as first CREB co-activation factor being found, is named as TORC1 (Transducer of Regulated CREB).Because of similar with mammal rapamycin target spot mTORC (mammalian target of rapamycin) title, NCBI renames as CRTC1.

The CRTCs albumen of very high homology is all there is: include 3 members CRTC1, CRTC2 and CRTC3 from fruit bat, C. Elegans Automatic Screening to the mankind.They have similar protein molecular structure (as a example by CRTC2, Figure 11), all comprise N end CREB land (CBD, CREB binding domain), central regulator district (REG, regulatory domain), shear zone (SD, splicing domain) and C end transcriptional activity district TAD (transcription active domain).N end CBD forms coiled-coil structure and is combined with the ZIP district of CREB, is the feature structure of CRTCs family.Enter nuclear signal district NLS (nuclear leading signaling) district, go out nuclear signal district NES (nuclear export signal) and be positioned at central regulator district REG, the wherein nucleocytoplasmic shuttle process of the phosphorylation state regulation and control CRTC2 of 171 serines.C end TAD (transcription active domain) can be combined with TAF has transcriptional activity.3 kinds of hypotypes of CRTC have characteristic distribution, and CRTC1 mainly expresses in cerebral tissue, and CRTC2 expresses the highest in liver organization, and the high abundance expressive site of CRTC3 is lymphocyte and lung, and in the digestive system such as muscle, liver, pancreas, intestinal, expression is low.

In insulin-sensitive tissues, CREB/CRTCs path assume responsibility for various rolls in hormone and nutrition are to energy metabolism regulation process.The latest study proves that CRTC2 is the important transcriptional regulatory co-activation factor (Figure 12) in hepatic gluconeogenic approach.nullUnder the conditions of tranquillization，The S171 of CRTC2 is continued phosphorylation by SIK2 (Salk induced kinases 2)，Be combined with cytoplasmic protein 14-3-3 and be trapped in endochylema，When cAMP raises，The SIK2 phosphorylation abilities activated by PKA reduces，Cause CRTC2 phosphorylation level drastically to decline and depart from 14-3-3 subsequently entering nucleus and being combined with the CREB rested on CRE element，628 lysines of CRTC2 are by CBP/P300 acetylation simultaneously，CRTC2 recruits TAF4 again，Now the protein complexes with CBP/P300-CREB/CRTC2-ATF4 as main component effectively raises the target gene of CRE and transcribes，Transcribing of fast upregulation hepatic gluconeogenic related gene.Hungry or give to occur in hepatic parenchymal cells during glucagon above event.After feed, insulin makes SIK2 dephosphorylation by AKT, and the SIK2 that kinase activity strengthens makes CRTC2 phosphorylation again and 14-3-3 stable bond rest on outside core；On the other hand the CRTC2 in core is degraded by the ubiquitination mediated by COP1 immediately after SIRT1 deacetylation, thus suppresses rapidly transcribing of CREB downstream gene.Therefore, the modification level such as the phosphorylation of CRTC2, acetylation, ubiquitination plays a significant role in hepatic gluconeogenic regulates and controls.In vivo the expression reduction of CRTC2 has been compared clearly with liver product sugar minimizing relation.Multinomial independent achievement in research confirms cAMP-PKA-CREB/CRTC2 core status in hepatic gluconeogenic signal transduction, is a brand-new target spot of glycemic control.

Liver CRTC2 incorporates hormone, trophic factors (cAMP agonist, glucagon, insulin) and multiple control protein (CRY1, AMPK, SIK, SMEK1/2) and hepatic gluconeogenic is carried out transcriptional control.Research confirms that CRTC2 has also mediated anti-diabetic one line clinical medicine metformin and the blood sugar reducing function of thiazolidinediones (thiazolidinedione), and these medicines strengthen CRTC2 phosphorylation indirectly by the kinase activity of regulation AMPK, the suppression hyperfunction glycogen of type 2 diabetes mellitus people generates and finally improves hyperglycemia.Therefore the compound strengthening CRTC2 phosphorylation is likely to become new hepatic gluconeogenic inhibitor.

The glycogenetic specific inhibitor of targeting liver is an important directions of hypoglycemic medicine research and development.The target spot wherein belonging to hepatic gluconeogenic approach has glucagon receptor (Glucagon receptor, GCGR), fructose 1,6-diphosphatase (fructose-1,6-biphosphatase, and G-6-Pase (glucose-6-phosephatase, G6Pase) FBPase).(such as NNC92-1687, Bay-27-9955, and trisubstituted ureas) and the efficient monoclonal antibody of glucagon receptor have good hypoglycemic application prospect it has now been found that glucagon receptor micromolecular inhibitor.But these inhibitor are all the functions from suppression target spot starts with, and can't improve this kind of target spot expression and continue too high pathology.

Liver is again the vitals of lipid (fatty acid, cholesterol and phospholipid) metabolism simultaneously.Liver can decompose triglyceride to be become fatty acid and is secreted into blood circulation for the use of other tissue.Liver has also synthesized substantial amounts of lipoprotein simultaneously, to assist the secretion of fat.When excessive carbohydrate and protein occur, liver can be converted into fatty acid and triglyceride the carbohydrate of excess and protein, to proceed to store in fatty tissue.On the other hand, cholesterol synthesis, absorb and be converted into the metabolic process of cholic acid and also performed by liver.

Therefore, the hepatic gluconeogenic and the lipid synthesis that suppress overacfivity from expression source are the new approaches treating metabolic syndrome, and this area is necessary that the little molecule of screening regulation and control CREB/CRTC2 transcriptional control function is by novel Metabolism regulation medicine Development of Novel, machine-processed.

Summary of the invention

It is an object of the invention to provide the clever C of Aunar and the like application in the medicine of preparation preventing and treating metabolic disease.

In a first aspect of the present invention, it is provided that the clever C of Aunar or its analog or its pharmaceutically acceptable salt purposes in preparation prevents, alleviates or treat the medicine of metabolic disease.

In a preference, described metabolic disease includes: lipid metabolism disease, carbohydrate metabolism disease or metabolic syndrome.

In another preference, described lipid metabolism disease includes, but is not limited to: hyperlipidemia, fatty liver, obesity, atherosclerosis, coronary heart disease, hypertension, cerebral infarction, apoplexy, renal failure.

In another preference, described carbohydrate metabolism disease includes, but is not limited to: hyperglycemia, diabetes, obesity, Hyperinsulinism, atherosclerosis, coronary heart disease, hypertension, hyperthyroidism, diabetic ophthalmopathy, diabetic nephropathy.

In another preference, the clever C of described Aunar or its analog have such as following formula (I) structure:

Wherein, R1 is selected from H, isopentene group (prenyl)；

R2 is OH；

R3 is selected from H, isopentene group；

R4 is selected from H, CH₂Ph,Or CH₂CHPh。

In another preference, affiliated compound is selected from: the clever C of Aunar；

R1 be H, R2 be OH, R3 be H, R4 be the compound of H；

R1 be H, R2 be OH, R3 be isopentene group, R4 be the compound of H；

R1 be H, R2 be OH, R3 be H, R4 be CH₂The compound of Ph；Or

R1 be H, R2 be OH, R3 be that H, R4 areCompound.

In another preference, described medicine is additionally operable to:

Suppression hepatic gluconeogenic；

Reduce hepatic gluconeogenic key gene Pgc1 α, Pck1 (coding PEPCK), the level of G6pc；

Reduce hepatic glucose output；

Improve glucose tolerance；

Reduce body fat content；

Reduce serum triglycerides content；

Reduce total cholesterol level；

Reduce low-density lipoprotein cholesterol content；

Reduce HDL-C content；

Reduce liver blood fat output；

Suppression liver fat acid synthesis；

Suppression cholesterol biosynthesis；

The blood fat output of suppression liver and absorption；

Reduce SREBP and the expression of downstream target gene thereof；And/or

Improve insulin sensitivity.

In another aspect of this invention, a kind of method preparing medicine is provided, described medicine is for preventing, alleviate or treat lipid metabolism disease or the medicine of carbohydrate metabolism disease, and described method includes: clever for the Aunar of effective dose C or its analog or its pharmaceutically acceptable salt are mixed with pharmaceutically acceptable carrier.

In a preference, described metabolic disease includes: lipid metabolism disease, carbohydrate metabolism disease or metabolic syndrome.

In another preference, the clever C of described Aunar or its analog have such as following formula (I) structure:

Wherein, R1 is selected from H, isopentene group (prenyl)；

R2 is OH；

R3 is selected from H, isopentene group；

R4 is selected from H, CH₂Ph,Or CH₂CHPh。

The other side of the present invention, due to this disclosure, is apparent to those skilled in the art.

Accompanying drawing explanation

Fig. 1, Brazil's apiario silvestre improve glucose metabolism balance and the insulin sensitivity of diabetic mice.

A, oral Brazil apiario silvestre 1-2 days, the hunger 16 hours blood glucose level of db/db mice；

B, oral Brazil apiario silvestre are after 2 weeks, and the glucose tolerance (GTT) of db/db mice detects；

C, oral Brazil apiario silvestre are after 2 weeks, and the insulin sensitivity (ITT) of db/db mice detects；

D, oral Brazil apiario silvestre are after 2 weeks, and the acetone acid toleration (PTT) of DIO mice detects；

E, DIO mice takes Brazil's apiario silvestre after 3 weeks, the mRNA transcriptional level of QPCR detection hepatic gluconeogenic key gene.

F, DIO mice in vivo liver imaging analysis, DIO mice is after 13 weeks, tail vein injection adenovirus Ad-G6P-Luc, takes Brazil's apiario silvestre continuously 3 days, carry out living imaging detection after hungry 16 hours after 3 days；

In G, primary hepatocyte, reporter gene G6P-Luc transcriptional activity is analyzed, and liver primary cell glucagon (100nM) after propolis (0.2%) pretreatment 1 hour stimulates 6 hours；

In H, primary hepatocyte, reporter gene CRE-Luc transcriptional activity is analyzed, and liver primary cell glucagon (100nM) after propolis (0-0.2%) pretreatment 1 hour stimulates 6 hours；

The Brazilian apiario silvestre Ingredients Active of Fig. 2, CREB/CRTC2 two-hybrid system detection.

A, glucagon signal transduction pathway；

The phosphorylation of CREB and cAMP horizontal detection in B, primary hepatocyte, Artepillin C (10 μMs) pretreatment primary hepatocyte 1 hour, glucagon (100nM) stimulates 30 minutes, and in cell, cAMP content corrects with lysate protein content；

The foundation of C, CREB-CRTC2 Mammalian two-hybrid system, expression plasmid AD-hCRTC2-Ser171Ala, BD-hCREB, Gal4-Luc and RSV-Renilla corotation enters HEK293T Transient Expression；

D, the tracking activity of composition of Brazil's apiario silvestre, component Petr-ether, EtOAc, n-BuOH, H₂O；

E, the composition separation process of Brazil's apiario silvestre；

HPLC-UV chromatograph detection (254nm) of Artepillin C in F, Brazil's apiario silvestre；

G, CREB-CRTC2 two-hybrid system evaluates the biological activity of Brazil's apiario silvestre monomer, transfects 4 hours and adds test-compound (25 μMs) process 6 hours；

The molecular structure of H, Artepillin C and the analog in identical source.

The protein of Fig. 3, Artepillin C suppression CREB with CRTC2 be combined with each other.

A, GST-pull down analyzes the effect that Artepillin C suppression GST-CREB with FLAG-mCRTC2 be combined with each other, with the GST-CREB beads immunoprecipitation HEK293T cell pyrolysis liquid containing process LAN FLAG-mCRTC2, Artepillin C (10 μMs) pretreatment cell 1 hour also adds immunoprecipitation system overnight incubation altogether；

B, co-immunoprecipitation analyze the activity that Artepillin C suppresses endogenous CREB with FLAG-mCRTC2 to be combined with each other in liver primary cell, the liver primary cell of Artepillin C (10 μMs) pretreated expression HA-CRTC2 is after 1 hour, glucagon stimulates 30 minutes, is that an anti-co-immunoprecipitation system includes Artepillin C (10 μMs) overnight incubation altogether with anti-CREB；

The inhibitory activity that protein purification GST-CREB with HIS-CRTC2-Ser171Ala is be combined with each other by C, GST-pull down detection Artepillin C, GST-CREB beads immunoprecipitation protein purification HIS-CRTC2-Ser171Ala, co-immunoprecipitation system comprises Arepillin C (0,5,10,50 μMs)；

D, surface plasma resonance (SPR) detect little molecule Artepillin C and protein HIS-CREB directly in conjunction with, protein purification HIS-hCREB is fixed on NTA chip by HIS label, the Artepillin C (0-100 μM) of variable concentrations is contacted with each other with fixing HIS-CREB by flowing, composite molecular weight size variation on chip in detecting 300 seconds；

E, trace isothermal calorimetric titration (ITC) detect the combination heat release of little molecule Artepillin C and protein HIS-CREB, prokaryotic expression protein matter HIS-hCREB (100uM) of purification is added dropwise in the sample cell containing Artepillin C, measures the thermal discharge of system after every time dripping；

F, GST-CREB and N ' block the expression of body, including GST-CREB341 total length, GST-CREB Δ Q1 (102-341), GST-CREB Δ Q1 Δ KID (160-341), GST-CREB-bZIP (283-341), GST antibody test；

G, GST-pull down analyzes the calmodulin binding domain CaM of CREB Yu Artepillin C, GST beads fixes the GST-CREB containing different N ends and blocks body protein, pull-down protein purification HIS-CRTC2-Ser171Ala respectively, immunoprecipitation system includes Artepillin C (5 μMs), HIS antibody test pull-down eluate；

The dose curve that H, Artepillin C suppression CREB-CRTC2 be combined with each other, the IC50 of mammal CREB-CRTC2 two-hybrid system detection Artepillin C.

The hepatic gluconeogenic of glucagon induction in Fig. 4, Artepillin C suppression primary hepatocyte.

The impact that A, CHIP-QPCR detection Artepillin C CRTC2 and CREB on being induced by glucagon and CRE element is combined, the metainfective primary hepatocyte of adenovirus Ad-HA-CRTC2 was through Artepillin C (10 μMs) pretreatment 1 hour, and glucagon (100nM) carries out CHIP-QPCR detection after stimulating 30 minutes；

B, QPCR detection hepatic gluconeogenic key gene (G6P, Pck1, Pgc1 α) transcribe, and primary hepatocyte and Artepillin C (10 μMs) preculture are after 1 hour, and glucagon (100nM) stimulates 4 hours；

C, the glucose output detections of primary hepatocyte, primary hepatocyte and Artepillin C (10 μMs) preculture are after 1 hour, and glucagon (100nM) stimulates 4 hours, produce sugar reaction 8 hours；

D, MTT detect the Artepillin C impact on primary hepatocyte vigor, after the Artepillin C (0-100 μM) of primary hepatocyte and variable concentrations co-cultures 24 hours, and the vigor of MTT detection primary cell；

E, cAMP detect, and after the Artepillin C (10 μMs) of primary hepatocyte and variable concentrations co-cultures (0,0.5,1,2 hour), glucagon detects cAMP content after stimulating 30 minutes；

F, Western blot analysis Artepillin C is on CREB phosphorylation, the dephosphorylized impact of CRTC2 in primary hepatocyte, and Artepillin C (10 μMs) preculture is after 1 hour, and glucagon (100nM) stimulates 0.5 hour；

Primary hepatocyte (the CRTC2 of G, CRTC2 disappearance^-/-) glucose sugar output detections, Artepillin C (10 μMs) pretreatment CRTC2 disappearance primary hepatocyte (CRTC2^-/-) 1 hour, glucagon (100nM) stimulates 4 hours, produces sugar reaction 8 hours；

Liver primary cell (the CRTC2 of H, QPCR detection disappearance CRTC2^-/-The transcribing of hepatic gluconeogenic key gene in), the primary hepatocyte (CRTC2 of Artepillin C (10 μMs) pretreatment CRTC2 disappearance^-/-) 1 hour, glucagon (100nM) stimulates 4 hours.

Fig. 5, Artepillin C improves hyperlipidemia and fatty liver.

DIO mice (left, 0,10,20mg/kg) and db/db model mice (right, 20mg/kg) were through lumbar injection Artepillin C 5 weeks.

The body weight of A, Artepillin C suppression diabetic mice (left side, DIO, the right side, db/db) increases；

B, Artepillin C reduces the in vivo fat content of diabetic mice (left side, DIO, the right side, db/db)；

C, Artepillin C reduces diabetic mice (left side, DIO, the right side, db/db) serum levels of triglyceride (TG) content；

D, Artepillin C reduces the serum cholesterol content of diabetic mice (left side, DIO, the right side, db/db), including T-CHOL (TC), low density cholesterol (LDLC) and HDL-C (HDLC)；

E, Artepillin C reduces triglyceride (TG) content of diabetes model (DIO) mouse liver；

F, DIO mouse liver, adipose tissue sections analysis.

Fig. 6, Artepillin C reduces lipid by the expression of suppression SREBPs and synthesizes.

DIO mouse peritoneal injection Artepillin C (0,10,20mg/kg) is after 5 weeks, and the mouse liver tissue freely taken food is for detecting the expression of Srebps；

Srebp-1a, 1c and the mRNA level in-site detection of 2 in A, DIO mouse liver

SREBP1 in B, DIO mouse liver, the protein level detection of 2；

In C, DIO mouse liver, the target gene of SREBPs and the mRNA level in-site of lipid metabolism related gene detect；

The mRNA level in-site detection of related gene in D, DIO mice white adipose tissue (WAT)；

The nuclear receptor related gene transcriptional level detection of the liver organization of E, DIO mice；

The expression of LXR α and SREBP1 in F, primary hepatocyte, after primary hepatocyte and Artepillin C (10 μMs) co-culture 1 hour, insulin (100nM) stimulates (0,3,18 hour).

Fig. 7, Artepillin C improves glucose metabolism balance and the insulin sensitivity of diabetic mice.

A, living imaging analyze liver G6P-luc reporter gene expression in DIO mice, DIO mice infects adenovirus Ad-G6p-luc through tail vein injection, lumbar injection Artepillin C (20mg/kg) for three days on end after 3 days, carries out living imaging analysis after hungry 16 hours；

B, DIO mouse peritoneal injection Artepillin C (20mg/kg) is after 5 weeks, and in QPCR detection DIO mouse liver tissue, hepatic gluconeogenic key gene transcribes；

C, DIO mouse peritoneal injection Artepillin C (20mg/kg) is after 5 weeks, and Western blot analyzes glyconeogenesis key protein matter PEPCK (being shown as PCK1 in figure) and the protein expression level of PGC1 α in DIO mouse liver tissue；

D, wild type normal mouse blood sugar level detect, and wild type normal mouse lumbar injection Artepillin C (20mg/kg) is after 3 days, glucagon (100 μ g/kg) induce 4 hours after blood sugar level；

E, DIO mice hungry blood glucose monitor, high fat induced diabetes Mus (DIO) lumbar injection Artepillin C (20mg/kg) is after 1 week, and within 16 hours, hungry blood sugar level detects；

F, db/db mice hungry blood glucose monitor, db/db mouse peritoneal injection Artepillin C (20mg/kg) is after 1 week, and within 16 hours, hungry blood sugar level detects；

G, db/db mouse peritoneal injection Artepillin C (20mg/kg) is after 5 weeks, and serum insulin levels detects；

After H, db/db mice (5 weeks) and DIO mice (4 weeks) lumbar injection Artepillin C (20mg/kg), glutamic oxaloacetic transaminase, GOT (Aspartate transaminase in serum, and glutamate pyruvate transaminase (Alanine transaminase, ALT) horizontal detection AST).

Fig. 8, Artepillin C improves glucose metabolism balance and the insulin sensitivity of diabetic mice.

DIO mice (left) and within db/db mice (right) lumbar injection Artepillin C (20mg/kg) 1-5 week, glucose tolerance (GTT, A), acetone acid toleration (PTT, B) and insulin sensitivity (ITT, C) detection.

Fig. 9, Artepillin C improves the molecular mechanism of overall insulin sensitivity.

The inhibitory activity evaluation of Figure 10, APC analog.

The activity rating that A, APC analog suppression CREB/CRTC2 be combined with each other；

B, APC analog IC50 tests.

Figure 11, transcribe the protein structure of co-activation factor CRTC2.

The hepatic gluconeogenic molecular mechanism of Figure 12, CREB/CRTC2 mediation.

Detailed description of the invention

The present inventor's process extensively in-depth study, the announcement clever C of Aunar (Artepillin C, APC) first or its analog have highly significant effect for the preventing and treating of metabolic disease.The invention provides to regulate CREB/CRTC2 and the SREBP multifunctional drug as target spot.

Compound

The invention provides the clever C of Aunar (Artepillin C, APC) or its analog, it has such as the chemical constitution of following formula (I):

The compound of structure shown in structure formula (I) forms a kind of applicable compound steric configuration, the compound with this structure can be with CREB/CRTC2 approach as target spot, interacted by the related locus with CREB, thus regulate and control a series of mechanism of action in downstream.Namely: the clever C of described Aunar or its analog are the inhibitor of a kind of CREB/CRTC2 approach.

Most preferably, described compound is the clever C of Aunar, and its structural formula is as follows:

Present invention additionally comprises the pharmaceutically acceptable salt of above-claimed cpd, hydrate or precursor, as long as they also have prevention, alleviate or treat the effect of metabolic disease.Described " pharmaceutically acceptable salt " refers to that a compounds reacts, with mineral acid, Organic Acid and Base metal or alkaline-earth metal etc., the salt generated.These salt include, but is not limited to: the salt that (1) is formed with following mineral acid: example hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid；(2) salt formed with following organic acid, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid or arginine.Other salt includes the salt formed with alkali metal or alkaline-earth metal (such as sodium, potassium, calcium or magnesium), with the form of " prodrug " of ester, carbamate, or other routine.

Described " precursor of compound " refers to after taking by suitable method, the precursor of this compound carries out metabolism or chemical reaction in patient body and is transformed into a kind of compound with structure shown in structure formula (I), or the salt that formed of a compound of structure shown in structure formula (I) or solution.

Present invention additionally comprises the isomer of above-claimed cpd, racemic modification, as long as they also have the effect of preventing and treating metabolic disease.Term used herein " isomer " including: geometric isomer, enantiomer, diastereomer (such as cis-trans-isomer, conformer).Compound has one or more asymmetric center.So, these compounds can exist as racemic mixture, single enantiomer, single diastereomer, non-enantiomer mixture, cis or trans isomer.

Those skilled in the art it should be understood that, after knowing the structure of the compounds of this invention, can be by multiple method well known in the art, the known raw material of utilization, obtain the compound of the present invention, such as chemosynthesis or from biological (such as animal or plant) method of extraction, these methods are included in the present invention.

Synthesis chemical improvement, protection functional group's methodology (protect or deprotect) are helpful to synthesis application compound; and the technology being well known in; such as R.Larock; Comprehensive Organic Transformations, VCH Publishers (1989)；T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3^rdEd., John Wiley and Sons (1999)；L.Fieser and M.Fieser, Fieser and Fieser ' s Reagents for Organic Synthesis, John Wiley and Sons (1994)；And L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) has disclosure.

Some have the compound of structure shown in formula (I) also can in biological (such as animal or plant) extracting and developing and purification.

Purposes

The inventors discovered that, the clever C of Aunar by with the N end of CREB directly in conjunction with, suppress be combineding with each other of CRTC2 with CREB after core, reduce the expression of the downstream gene driven by CREB/CRTC2, main component PGC1 α including hepatic gluconeogenic, and important rate-limiting enzyme G6PC and PEPCK (PCK1), the glucose output of suppression hepatic gluconeogenic and liver, reduce hungry blood glucose.On the other hand, APC is by suppressing the expression of nuclear receptor LXR α, work in coordination with and transcribe the co-activation repressed state of factor PGC1 α, reduce the expression of the SREBPs driven by LXRE, the downstream target gene of suppression SREBPs, reduce the cholesterol regulated and controled by SREBPs and the synthesis of fatty acid, absorption, fat content in final reduction blood.

New discovery based on the present inventor, the invention provides compound or the purposes of its isomer, racemic modification, pharmaceutically acceptable salt, hydrate or precursor with structure shown in formula (I), for preparing the medicine (or compositions) of preventing and treating metabolic disease.

As the optimal way of the present invention, described metabolic disease includes: described metabolic disease includes: lipid metabolism disease or carbohydrate metabolism disease.Described lipid metabolism disease includes: described lipid metabolism disease includes: hyperlipidemia, fatty liver, obesity, atherosclerosis, coronary heart disease, hypertension, cerebral infarction, apoplexy, renal failure etc..Described carbohydrate metabolism disease includes: hyperglycemia, diabetes, obesity, Hyperinsulinism, atherosclerosis, coronary heart disease, hypertension, hyperthyroidism, diabetic ophthalmopathy etc..

In a word, the compound of the present invention passes through brand-new mechanism regulating lipid metabolism and carbohydrate metabolism, proves through animal experiment, lipid metabolism disease or carbohydrate metabolism disease are had significant curative effect.

Compositions

As used herein, term " compositions of the present invention " is typically pharmaceutical composition, and it contains the clever C of Aunar or its analog or their isomer, racemic modification, pharmaceutically acceptable salt, hydrate or the precursor active component as preventing and treating metabolic disease；And pharmaceutically acceptable carrier or excipient.

In the present invention, term " contains " and represents that various composition can be applied in mixture or the compositions of the present invention together.Therefore, term " mainly by ... composition " and " consist of " are included in during term " contains ".

In the present invention, " pharmaceutically acceptable " composition applies to people and/or animal and i.e. has the material of rational benefit/risk ratio without excessive bad side reaction (such as toxicity, stimulation and allergy).

In the present invention, " pharmaceutically acceptable carrier " is acceptable solvent, suspending agent or the excipient pharmaceutically or on food for clever for the Aunar of present invention C or its analog or their isomer, racemic modification, pharmaceutically acceptable salt, hydrate or precursor send to animal or people.Carrier can be liquid or solid.The pharmaceutically acceptable carrier being applicable to the present invention includes (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.

The method that present invention also offers the compositions of preparation preventing and treating metabolic disease, including using the compositions that clever for the Aunar of effective dose C or its analog or their isomer, racemic modification, pharmaceutically acceptable salt, hydrate or precursor can mix the acquisition present invention with pharmaceutically acceptable carrier, active component part by weight in the composition can be such as 0.0001-50wt%；Can be preferably 0.001-20wt%.

The dosage form of pharmaceutical composition of the present invention can be diversified, as long as the dosage form that active component can be made effectively to arrive mammal affected part is all possible.In terms of position that is easily prepared and that be administered, preferred pharmaceutical composition is a kind of oral or the preparation of injection.Such as it is selected from: granule, tablet, capsule, solution or suspension, powder agent.The compositions of the present invention can add various conventional carrier required when preparing different dosage form or adjuvant, such as filler, correctives, antioxidant, spice, pigment, lubricant, fluidizer, wetting agent, emulsifying agent, pH buffer substance etc..These additives are all known to those skilled in the art.

Present invention also offers a kind of method preventing and treating metabolic disease, including step: to the clever C of Aunar of subject effective amounts needed or its analog or their isomer, racemic modification, pharmaceutically acceptable salt, hydrate or precursor.The dosage of active component is therapeutically effective amount.When outer used time, the safe and effective amount of compound of the present invention normally about 0.1ng-100mg/kg body weight；The most about 1ng-10mg/kg body weight.Certainly, concrete dosage is it is also contemplated that the factor such as route of administration, medication person's health status, within the scope of these are all skilled practitioners technical ability.

Additionally, compound of the present invention also can be used together with other active component or therapeutic agent (such as other blood lipid-lowering medicine, anticholesteremic agent, diabetes medicament etc.).

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally writes according to normal condition such as J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.

I, material and method

1, Detection of Brazilian Propolis compound separates

500mL Brazil apiario silvestre 60% ethanol just extracting solution concentrating under reduced pressure is obtained extractum, after the suspendible that adds water, successively with petroleum ether, ethyl acetate and n-butanol extraction 3 times, obtains each position extractum.Ethyl acetate layer, through MCI column chromatography, successively with methanol-water difference gradient elution, obtains 40%, 60%, 80% and 100% 4 segmentation.Ethyl acetate-40% position (3.2g) is separated with anti-phase ODS post by normal phase column chromatography；Second-60% (21.8g) separates through separation means such as normal phase column repeatedly, ODS post (methanol-water solution), Sephadex LH-20 gel columns (methanol-water solution eluting)；Ethyl acetate-80% position (37.2g) separates through methods such as normal phase column, recrystallization, ODS post (methanol-water solution), Sephadex LH-20 gel columns (methanol-water solution eluting)；Normal phase column chromatography and recrystallization are passed through in ethyl acetate-100% position (29.6g)；Water layer is separated by ODS post.Obtain the monomeric compound of Brazil's apiario silvestre.

2, mammal double cross screening

Build CREB-CRTC2 Mammalian two-hybrid system, including people source CREB Yu BD (Gal4-Binding domain), the fusion expression vector of people source CRTC2 Yu AD (Gal4-Active domain), the double reporter gene expression carrier of zygotic induction Gal4-Luciferase and composition RSV-Renilla, thus set up a complete CREB/CRTC2 Mammalian two-hybrid system.

In Mammalian two-hybrid system fused protein expression vector with plasmid 804 and 811 (available from Tongji University) as skeleton.Cloning site and primer such as table 1.Double cross reporter gene GAL4-Luciferase is purchased from (Promega), builds such as table 1 with reference to reporter gene RSV-Renilla, i.e. RSV promoter sequence replaces the CMV promoter sequence of CMV-Renilla (NEB).

Fusion protein HIS-CRTC2-S 171A expression vector skeleton is pET28C (purchased from Addgene), CREB total length with GST label and the prokaryotic expression carrier of N end truncate fused protein are with pGEX5X-1 (GE) as skeleton, and concrete cloning site and pcr amplification primer thing are shown in Table 1.

Table 1, the structure of expression vector

The growth of HEK293T cell causes to be inoculated into overnight incubation on 24 well culture plates during exponential phase, liposome (Lipo 2000, Invitrogen) mediate mammalian double cross pUC pUC transfection, testing compound and solvent control (1%DMSO) is added after 6h, each compound sets 3 repetitions, examining report gene expression amount after cultivating 18 hours, evaluates the expression of Gal4-luciferase after correcting, and utilizes suppression screening model to be analyzed test-compound.

3, liver primary cell double reporter gene detection

It is that transgenic medium proceeds to the Ad-RSV-Renilla reporter gene of inductivity Ad-CRE-Luciferase and constructive expression to the primary hepatocyte of short term culture by high-purity adenovirus, build double reporter gene detecting system, with glucagon as agonist, with test-compound as inhibitor, evaluate the compound inhibitory activity to CRE-luciferase reporter gene activity.

The recombinant adenovirus of induced expression Ad-CRE-Luciferase is available from the Dr.Marc R.Montminy of U.S. Salk Institute.Express the construction method of the recombinant adenoviral vector of the Ad-RSV-Renilla reporter gene of constructive expression: promoter CMV of CMV-Renilla expression vector is replaced as promoter RSV by clone, pass through sub-clone, fragment RSV-Renilla is made to be cloned into adenovirus shuttle vector pshutter, positive pshutter-RSV-Renilla and plasmid Ad-easy recombinates, it is thus achieved that Ad-easy-RSV-Renilla plasmid.Positive recombiant plasmid Ad-easy-RSV-Renilla becomes linearisation Ad-easy-RSV-Renilla through restriction endonuclease pmeI enzyme action, then continue at adenovirus packaging cell system MGH293 (available from the Dr.Marc R.Montminy of U.S. Salk Institute), after 7-14 days, occur that plaque shows that adenovirus is packed successfully.Adenopathy poisons expand and cesium chloride is interior for Mice Body after purification and cell experiment.

4, Mouse Liver primary cell separates and cultivates

Fix after about 25g wild type C57 mouse anesthesia, trans-portal vein-postcava circulation carries out perfusion in situ, blood during perfusate removes liver before HBSS, collagenase (Sigma) digestion in site extracellular collagen protein is to separate cell, after 100 mesh cells are sieved through filter fragment of tissue, cleaning liver primary cell 2 times by serum-free M199 culture medium, the liver primitive cell culture after process is in the M199 culture medium containing serum.Front hunger 16 hours in serum-free M199 culture medium of experiment.

5, cell viability detection MTT

Primary hepatocyte is inoculated in 96 porocyte plates (3000 cells/well), hunger 16 hours in serum-free M199, add the test-compound of variable concentrations, cultivate 1-2 days, after going culture medium, add 20ul MTT reagent (5 μ g/ml, PH 7.4) with co-culture of cells 5 hours, fully dissolve MTT, the light absorption value of detection OD490 after adding 150 μ l DMSO, evaluate the test-compound impact on primary hepatocyte vigor.

6, immunoblotting Western Blot

Cell or cryopreserved tissue after process crack in RIPA buffer (25mM Tris-HCl, 150mM NaCl, 1%NP-40,1% NaTDC (sodium deoxycholate), 0.1%SDS, pH 7.6), go to take supernatant after fully centrifugal after cracking, sample total protein concentration is measured by BCA method after appropriateness dilution, the sample taking equivalent total protein adds after 5 × SDS in 95 DEG C of degeneration 5 minutes, separate different protein with certain density SDS-PAGE glue, forward pvdf membrane the most in the electric field to.Utilize the phosphorylation of target protein in antibody test gross protein or the change of expression.

7, co-immunoprecipitation

Cell or cryopreserved tissue after process crack in IP buffer, remove to take supernatant, measure sample total protein concentration by BCA method after appropriateness dilution after being fully centrifuged after cracking, and the lysate taking 10% is Input group.Take the lysate containing 1 μ g gross protein, add Protein A/G beads (Millipore) and identify that the one of target protein is hatched after resisting at 4 DEG C.Clean the beads after precipitation, add 3 × SDS buffer at 95 DEG C, heat the protein complex be combineding with each other with antibody mediated immunity albumen under eluting for 6 minutes.Western blot analyzes the change of target protein in immunoprecipitated samples.FLAG-mCRTC2 plasmid is available from the Dr.Marc R.Montminy of U.S. Salk Institute.

8, immunostaining

Liver primary cell is inoculated on the sterile cover slips in 24 orifice plates cultivation, after corresponding process, fixes with paraformaldehyde, and PBS washes away paraformaldehyde, closes and makes cell membrane penetration (PBS, 0.3%TritonX-100,5%BSA).Hatch one anti-(PBS, 0.3%TritonX-100,5%BSA) to clean afterwards, hatch the anti-Alex of fluorescence two (Invitrogen) and clean afterwards, mounting after DAPI dye core and cleaning.The change of object observing protein under fluorescence microscope.

9、GST-pull down

The structure (table 1) of HIS tag fusion people source CRTC2-Ser171Ala recombinant protein prokaryotic expression carrier: people source CRTC2-Ser171Ala expressed sequence clones prokaryotic expression carrier pET28C by EcoRI/HindIII, order-checking identifies that CRTC2-Ser171Ala Yu HIS merges and expressed intact.The structure of the recombiant protein prokaryotic expression carrier of GST tag fusion CREB such as table 1.People source CREB (hCREB) is cloned into prokaryotic expression carrier pGEX5X1 by EcoRI/NotI, and order-checking is identified and merged and expressed intact with label G ST.

Prokaryotic expression carrier proceeds in E.coli, at 16 DEG C IPTG abduction delivering overnight after, collect crack after thalline thalline, ultrasonic, centrifugal after take supernatant, with GST beads or HIS beads purification tag protein.

The lysate supernatant of the recombiant protein that GST merges CREB adds GST beads incubated at room 30 minutes, cleans the non-specific binding albumen removed on beads, be prepared as GST-CREB beads.

HIS tag fusion people source CRTC2-Ser171Ala recombinant protein (HIS-CRTC2-Ser171Ala) is induced to express in antibacterial, remove imidazoles with desalination after HIS beads purification HIS-CRTC2-Ser171Ala protein, it is thus achieved that protein purification HIS-CRTC2-Ser171Ala for pull down.

Pull down system at 500 μ l includes the HIS-CRTC2-Ser171Ala protein purification of 800ng, 40 μ l GST-CREB beads and test-compounds, hatches in 4 DEG C.Clean the beads after combining, remove non-specific binding albumen, add the protein example that 3XSDS buffer 95 DEG C processes 6 minutes and obtains after combining.Western blot analyzes the change of protein in elution samples.

10, cAMP detection

With the primary hepatocyte of short term culture as experiment material, with test-compound pretreatment 1 hour, 100nM glucagon removes supernatant after stimulating 30 minutes subsequently, cell lysis after PBS washing, according to the explanation operation of cAMP ELISA (R&D) test kit, evaluate the impact of the cAMP synthesis that glucagon is induced by test-compound.

11, hepatic glucose output

With M199 as culture medium, short term culture primary hepatocyte overnight starvation, add test-compound preculture 1 hour, after 100nM glucagon stimulates 4 hours, change sugar-free, without Sodium Pyruvate, without phenol red medium (Sigma) and add Sodium Pyruvate and sodium lactate is hatched 8 hours, collect culture medium and be also centrifuged, supernatant is according to test kit (GO kit, Sigma) measure the glucose content in culture medium, evaluate test-compound and primary hepatocyte is produced the impact of sugar ability.

12, gene transcription level detection

Cell after cultivation or frozen Tissue Lysis in Trizol, isopropanol precipitating method extracted total RNA, 80% ethanol (DEPC) washing, be dissolved in EDPC water after drying.With the total serum IgE of 0.5-1 μ g for masterplate reverse transcription synthesis cDNA (TAKARA).With cDNA as masterplate, utilize QPCR method amplification target gene (IBA7900), analyze each target gene mRNA copy number by detection Ct value and change.

13, surface ion resonance (SPR)

Surface plasma resonance (SPR, Surface plasmon resonance) the biosensory analysis technology little molecule of detection be combined with each other with protein.People source recombinant protein HIS-CREB is by the Ni ion specific bond captured with NTA chip, thus be fixed on NTA chip, the little molecule of variable concentrations flows through the protein of chip surface by flowing with certain speed, as mobile phase molecule be combined with each other with fixing protein, the molecular weight of chip surface binding molecule changes, cause chip that the refractive index (RU) of incident illumination is changed accordingly, the sensing figure of the refractive index (RU) of chip under instrument (Biacore 100, GE) detection different condition in real time.Setting capture Ni during detection but the passage of conjugated protein is not for reference to passage, under the same terms, the RU value on experiment channel is initial data after reducing reference passage RU value.The RU value that mobile phase molecule concentration responds with it is by single location pattern matching binding curve, to obtain kinetic parameter when molecule is be combined with each other by this.

The acquisition of HIS tag fusion people source CREB recombinant protein: people source CREB encoding gene is cloned into prokaryotic expression carrier pET28C by homologous recombination means, order-checking identifies that CREB Yu HIS merges and expressed intact.HIS-CREB proceeds to, in escherichia coli statement bacterial strain BL21, after abduction delivering, collect thalline, ultrasonic, separating and cracking liquid supernatant, with HIS beads affinity purification HIS-CREB, HIS-CREB under high eluting salt removes imidazoles through dialysis, for the affine experiment of little molecule Yu protein.

14, isothermal titration calorimetric detection (ITC)

Isothermal titration Calorimetric Techniques (ITC, isothermal titration calorimetry, GE) by be combineding with each other and the thermal change (heat release or heat absorption) that produces between monitoring composition, with measure between little molecule and protein directly in conjunction with.It is dissolved in PBS (8.0 after the protein HIS-CREB desalination and concentration of purification, NaCL500mM), instill in little molecular dilution liquid to be measured, detection (Micro Cal iTC200, GE) thermal discharge of system after titration adds every time, obtain the thermal change of per injection, map with injecting molecule mol ratio with thermal change peak integral area.Isothermal line is combined with single location pattern matching, it is thus achieved that binding constant, stoichiometry, entropy and enthalpy parameter by ORIGIN7 software.

15, mouse glucose tolerance (GTT), acetone acid toleration (PTT) and insulin sensitivity (ITT) detection

Experiment mice is after treatment, overnight starvation before GTT experiment, every mice according to body weight lumbar injection sterile dextrose (2g/kg), the most after injection 0,15, tail venous blood sampling when 30,60,120 minutes, blood sugar concentration is tested by blood glucose meter (Roche), with the time as transverse axis, blood sugar concentration is vertical coordinate, does the blood sugar concentration change curve of different disposal group mice.PTT experiment injected material is Sodium Pyruvate (1.5g/kg).Hungry 3 hours of mice before ITT experiment, injected material is insulin (1.5U/kg).

16, reporter gene living imaging in Mice Body

nullExperiment mice is after treatment，Tail vein injection is with the adenovirus of reporter gene，Including induced expression Ad-G6P-Luciferase reporter gene and the reporter gene of composition Ad-RSV-β-gal，Living imaging experiment is carried out after 3 days，Mice overnight starvation before experiment，Subcutaneous injection luciferase substrate Luciferin (Biosynth AG) after isoflurane (Abbott) anesthetized mice，By activity imager (IVIS lumina after 15 minutes，Xenogen) signal of mouse liver luciferase bioluminescence is gathered，Analyze the luciferase signal after β-gal value correction，Evaluate the test-compound impact in vivo on the G6P-Luciferase relative activity that hungry glucagon is induced.

17, adenovirus construction and purification

Adenovirus Ad-HA-mCRTC2, Ad-G6p-luc, Ad-CRE-Luc.Ad-β-Gal；It is all obtained from the Dr.Marc R.Montminy. of U.S. Salk Institute

Express the construction method of the recombinant adenoviral vector of the Ad-RSV-Renilla reporter gene of constructive expression: promoter CMV of CMV-Renilla expression vector is replaced as promoter RSV by clone, pass through sub-clone, fragment RSV-Renilla is made to be cloned into adenovirus shuttle vector pshutter, positive pshutter-RSV-Renilla and plasmid Ad-easy recombinates, it is thus achieved that Ad-easy-RSV-Renilla plasmid.Positive recombiant plasmid Ad-easy-RSV-Renilla becomes linearisation Ad-easy-RSV-Renilla through restriction endonuclease pmeI enzyme action, then continues at adenovirus packaging cell system MGH293, occurs that plaque shows that adenovirus is packed successfully after 7-14 days.Adenopathy poisons expand and cesium chloride is interior for Mice Body after purification and cell experiment.

After adenovirus amplifies is cultivated, collecting culture medium, centrifugal collecting cell multigelation 3 times, precipitate virus granule under conditions of saturated ammonium sulfate, then by the resuspended virus of PBS.Through twice purified virus in CsCl density gradient, dialysed overnight in PBS-glycerol (10%) buffer subsequently, remove the inorganic salt of virus, be stored in-80 DEG C.

18. tissue slices

It is fixing that mouse experiment takes out tissue after terminating, and cuts into slices after paraffin embedding, and HE dyes observation of cell form.Olympus camera is taken pictures.

19, chromatin immune co-precipitation (CHIP)

Primary hepatocyte scrapes cell after formaldehyde crosslinking processes in PBS, collects nucleus, ultrasound wave 50% output pulses 1 second, 2 seconds, gap, shear treatment 3 minutes in cracking buffer, obtains supernatant for subsequent experimental.Chromosome solution after shearing hatches 2H with IgG (Santa Cruz), CREB (Cell Signal) antibody and CRTC2 (Calbiochem) antibody 4 DEG C respectively, is subsequently added Protein A beads (Milliopre) in 4 DEG C of overnight incubation.The chromatin of immunoprecipitation Chelex 100 (Biored) purification reclaims, and quantitative by QPCR (ABI7900) method.Antibody CREB and CRTC2 immunoprecipitation-QPCR signal are corrected with corresponding IgG signal for internal standard, and PCR primer used is listed in table 2.

Table 2, CHIP-QPCR analysis primer

20, triglyceride (TG), the detection of cholesterol (Cholesterol)

Eye socket blood is centrifugal (3000rpm after EDTA anticoagulant processes, 15 minutes) supernatant that obtains afterwards is for detecting serum total triglyceride (TG, triglyceride), T-CHOL (TC, total cholesterol), low density cholesterol (LDLC, LDL cholesterol) and HDL-C (HDLC, HDL cholesterol) content, according to test kit (northization is safe) description operate.In liver organization, triglyceride, the extracted rear content of cholesterol illustrate operation, fatty consistency sample protein matter concentration correction in its tissue sample according to test kit (Puli comes).

21, biochemical index

Insulin (Millipore) in mice serum, alanine aminotransferase (ALT, China of section), the assay of arginine transaminase (AST, China of section) operate according to test kit.

22, live body fat content detection

NMR instrument (The Minispec, Bruker) is used to measure mice in vivo fat content, muscle and fatty ratio (Lean/Fat) under conditions of clear-headed, nothing invade.

II, experimental result

Embodiment 1, Brazil's apiario silvestre suppression diabetic mice hepatic gluconeogenic

In order to study the blood sugar lowering mechanism of propolis, in db/db diabetic mice, first have detected the hypoglycemic activity of Brazil's apiario silvestre.Result shows that the short-term oral Brazil apiario silvestre (250mg/kg) of 1-2 days substantially reduces the fasting glucose (Fig. 1 .A) of diabetic mice.Db/db diabetic mice takes Brazil's apiario silvestre after 2 weeks, and the sugared toleration (Fig. 1 .B) of db/db diabetic mice and insulin sensitivity (Fig. 1 .C) obtain significance raising.

DIO (high fat inducing obesity model, diet induced obesity, DIO) mice takes Brazil's apiario silvestre after 2 weeks, carries out acetone acid toleration detection.Result shows, Brazil's apiario silvestre substantially inhibits the hepatic gluconeogenic ability (Fig. 1 .D) of high fat induced diabetes mice (DIO).

DIO mice takes Brazil's apiario silvestre and finds after 3 weeks, the transcriptional level (Fig. 1 .E) of hepatic gluconeogenic rate-limiting enzyme G6pc and Pck1 during propolis significantly suppress DIO mouse liver on transcriptional level.

DIO mice takes Brazil's apiario silvestre after 3 days, and mice in vivo liver image checking further demonstrates that, oral Brazil apiario silvestre significantly reduces the liver G6p-LUC reporter gene activity (Fig. 1 .F) of hungry induction.G6p-LUC (Fig. 1 .G) and the biological activity (Fig. 1 .H) of CRE-LUC reporter gene transcription that propolis glucagon suppression induces again is detected in primary hepatocyte.

These results suggest that, Brazil's apiario silvestre can improve diabetic mice overall glucose metabolism and insulin sensitivity, and this activity has close ties with the biological activity of the hepatic gluconeogenic of propolis glucagon suppression induction.

The Brazilian apiario silvestre Ingredients Active of embodiment 2, CREB/CRTC2 two-hybrid system detection.

Glucagon induction hepatic gluconeogenic depends on GCGR-cAMP-PKA-CREB/CRTC2-CRE signal path (Fig. 2 .A).

Owing to Brazil's apiario silvestre stimulates the phosphorylation of cAMP and CREB produced do not make significant difference (Fig. 2 .B) to glucagon, illustrate that Brazil's apiario silvestre is likely to be at the end of pancreas hyperglycemia signal path to the inhibitory action target spot of hepatic gluconeogenic.CRTC2 is activated after dephosphorylation by entering be combineding with each other of core and CREB by glucagon, can effectively promote the transcribing of related gene of hepatic gluconeogenic, construct CREB:CRTC2-Ser171Ala Mammalian two-hybrid system (Fig. 2 .C) (native system utilizes mutant CRTC2-Ser171Ala to simulate by dephosphorylation, the CRTC2 state of activation) based on this principle, utilize the impact that CREB-CRTC2 is combined by this system detection propolis.

The detection display of double cross reporter gene, propolis (0.2% (V/V)) inhibits the transcriptional activity (Fig. 2 .C) of two-hybrid system reporter gene very significantly.This result shows, Detection of Brazilian Propolis by suppressing be combineding with each other of CREB with CRTC2, and then can suppress hepatic gluconeogenic on transcriptional level.In order to find effective blood-sugar decreasing active from Brazil's apiario silvestre, inventor has carried out tracking activity to the separated component of propolis.

First, utilize opposed polarity solvent that propolis aqueous suspension is carried out liquid-liquid extraction, obtain petroleum ether (Petroleum ether), ethyl acetate (EtOAc), n-butyl alcohol (n-BuOH) and four parts of water layer (Water), and the activity of each position suppression G6p-Luc reporter gene is evaluated.Result shows, the activity of ethyl acetate layer is preferably (Fig. 2 .D).Owing to ethyl acetate composition of layer is the abundantest, the present inventor carries out further segmentation by MCI post to ethyl acetate layer, and gradient elution obtains 4 positions, and wherein the eluting position activity of 60% and 80% methanol-water is preferably.Emphatically the two part is finely divided, finally obtains 18 compounds from 60% and 80% position isolation identification.The most also from ethyl acetate layer 40% and 100% layer of isolated P1, P9 and P8 (Fig. 2 .E).21 compounds separated have 8 cinnamic acids, 2 Caffeic acids, 1 benzofurans, 6 flavonols, 2 flavanones and 2 flavanone alcohol compounds (Fig. 2 .E).Wherein compounds derived from phenyl acrylic acid P3 (the clever C of Aunar, Artepillin C) is one of high-load composition in Detection of Brazilian Propolis.

In order to identify the propolis monomer of the hepatic gluconeogenic activity with suppression CREB/CRTC2 mediation, by external CREB-CRTC2 Mammalian two-hybrid system, the Brazilian apiario silvestre monomeric compound after separating is carried out activity rating.Primary dcreening operation experimental selection single concentration of 25 μMs.Primary dcreening operation result shows, 0.2% propolis concentrated solution can significantly inhibit the combination of CRTC2:CREB；Compared with solvent control group (1%DMSO), 8 compounds (P1, P2, P3, P5, P6, P11, P16 and P18) demonstrate stronger inhibitory activity；Wherein the activity of P3 is the strongest, compound P1, and the activity of P2, P5, P6, P11 and P16 is compared the most weak, and compound P4, P7, P10, P12, P14, P15, P17 and P19 under 25 μMs of concentration almost without inhibitory action (Fig. 2 .G).P1, P2, P3, P5 have similar structure (Fig. 2 .H) with P6, are all phenolic acids derivants.By CREB-CRTC2 two-hybrid system, Brazil's apiario silvestre Object Evaluation result is shown, the activated monomer of propolis is concentrated mainly on the methanol-eluted fractions position of ethyl acetate layer, and P3 (Artepillin C) is to have the active bee glue monomeric compound that suppression CREB/CRTC2 be combined with each other.

The protein interaction of embodiment 3, Artepillin C suppression CREB Yu CRTC2

In order to study the molecular mechanism that Artepillin C suppression CREB-CRTC2 be combined with each other further, the present inventor utilizes the inhibitory activity of the method detection Artepillin C of immunoprecipitation.In the HEK293T cell pyrolysis liquid of process LAN FLAG-mCRTC2, Artepillin C significantly reduces be combined with each other (Fig. 3 .A) of GST-hCREB beads Yu FLAG-mCRTC2.Due to hepatocyte energy metabolism exogenous drugs and the function of metabolic intermediate, the most whether checking Artepillin C can keep inhibitory activity in hepatocyte.With primary hepatocyte as experiment material, co-immunoprecipitation detection CREB with CRTC2 be combined with each other, and utilizes this system to detect Artepillin C inhibitory activity in hepatocyte.Result shows that Artepillin C (10 μMs) significantly suppress the combination of CREB Yu CRTC2, and the Artepillin C again added during immunoprecipitation has higher inhibitory activity (Fig. 3 .B).This explanation Artepillin C in hepatocyte also has the biological activity that suppression CREB-CRTC2 protein be combined with each other.

It is directly to act on or indirect action to study the inhibitory activity of Artepillin C further, utilizes protein purification to carry out ion vitro immunization precipitation analysis.First recombinant protein GST-hCREB and HIS-hCRTC2-Ser171Ala is obtained at expression in escherichia coli purification.The state that the point mutation Ser171Ala that HIS-hCRTC2-Ser171Ala carries simulates after CRTC2 is activated by glucagon dephosphorylation and then is trapped in nucleus is therefore higher with the affinity of CREB.Utilizing these 2 kinds of protein purifications to carry out GST-Pull down analysis, result display Artepillin C inhibits be combineding with each other and having dosage effect (Fig. 3 .C) of CREB (being shown as GST-CREB in figure) and HIS-hCRTC2-Ser171Ala significantly.This result shows that Artepillin C can directly suppress be combineding with each other of CREB with CRTC2, without other auxiliary element.

In order to determine the direct target spot of Artepillin C inhibitory activity, need further detection Artepillin C and action protein matter CREB, the CRTC2 whether can be directly in conjunction with.The present inventor utilizes surface plasma resonance (SPR, Surface plasmon resonance) biosensory analysis technology directly to detect be combineding with each other of little molecule Artepillin C (APC) and protein C ERB.Purification HIS-CREB protein is fixed on NTA sensing chip (GE) by N end HIS label, carries Artepillin C mutually with flowing and flows through HIS-CREB protein surface.SPR analyzes (Biacore, GE) result shows that the RU value (sensing figure response value) of censorchip surface when flowing through of Artepillin C increases, and the concentration sensing figure response value (RU) and mobile phase A rtepillin C is proportionate (Fig. 3 .D).SPR result display Artepillin C can with CREB directly in conjunction with and there is dosage effect, this combination is likely to the molecular basis of Artepillin C inhibitory activity.

In order to obtain the kinetic parameter that Artepillin C with CREB be combined with each other, utilize thermal change after the combination of microtitration Calorimetric Techniques (Isothermal titration calorimetry, ITC) detection Artepillin C Yu CREB further.ITC result shows when HIS-CREB instills Artepillin C solution, a large amount of heat that combines is had to discharge, the combination release heat detected fits to thermomechanical curve, the equilibrium dissociation constant K that curvilinear regression prompting Artepillin C with CREB is combined with the relativeness of titrimetric substance molar concentration according to unijunction conjunction location pattern_DIt is about 2-5 μM (Fig. 3 .E).ITC detects that the combination release heat of Artepillin C Yu CREB further determined that little molecule APC can directly be combined with CREB, and to point out CREB be the protein target of Artepillin C.

In order to determine the calmodulin binding domain CaM of CREB Yu Artepillin C, utilizing immunoprecipitation means to carry out Deletional analysis, the present inventor detects the Artepillin C impact on different CREB truncates with total length CRTC2 protein binding capacity respectively.People source CREB total length (Genbank ID:M34356；Unigene ID:Hs.516646；Uniprot ID:P16220) and truncate include: CREB Δ Q1 (the 102-341 amino acids of total length)；CREB Δ Q1 Δ KID (the 160-341 amino acids of total length)；CREB-bZIP (the 283-341 amino acids of total length), expresses (see Fig. 3 .F) with GST tag fusion respectively, and fixes with GST beads.

Result shows, after the Q1 region of CREB lacks, Artepillin C then can not suppress be combined with each other (Fig. 3 .G) of CREB Yu CRTC2.CREB afunction result of the test shows that Artepillin C may be combined by the Q1 region with CREB, thus suppresses the interaction of CREB-CRTC2.In Mammalian two-hybrid system, Artepillin C suppression CREB-CRTC2 be combined with each other 50 3nhibitory dose (IC50) be about 2 μMs, show that the Artepillin C of micromolar levels can play biological activity (Fig. 3 .H) in living cells.

In sum, Artepillin C is combined by the Q1 region with CREB, and suppression CREB with CRTC2 be combined with each other, and the inhibitory activity of Artepillin C is according to there being dosage effect, and can play inhibitory activity in hepatocyte.

The hepatic gluconeogenic of glucagon induction in embodiment 4, Artepillin C suppression primary hepatocyte

Based on Artepillin C, there is the biological activity that suppression CREB-CRTC2 be combined with each other, have detected the Artepillin C effect at liver primary cell further.

Use the activity that CHIP-QPCR detection Artepillin C suppression CRTC2 with CRE is combined.Result shows, by infecting adenovirus Ad-HA-CRTC2, in the liver primary cell of process LAN HA-CRTC2, the CRTC2 that Artepillin C inhibits glucagon to induce raises to CRE element, but the impact that CREB combines CRE is not notable (Fig. 4 .A).This result shows, Artepillin C can suppress CRTC2 to be attached on CRE element, and this result and Artepillin C can suppress the immunoprecipitation of CREB Yu CRTC2 and the effect of CREB/CRTC2 double cross reporter gene to be identical.

On transcriptional level, Artepillin C significantly reduces the mRNA level in-site (Fig. 4 .B) of hepatic gluconeogenic key gene G6pc, Pck1 and Pgc1a that glucagon is induced.

In cell integral level, the Artepillin C process of 1-5 μM significantly suppress glucose output (Fig. 4 .C) that glucagon stimulates, and this shows that Artepillin C can significantly inhibit the glucose output of liver primary cell.

Detected by MTT, the Artepillin C of up to 100 μMs there is no obvious toxicity (Fig. 4 .D) to the vigor of liver primary cell, illustrating that Artepillin C is little to the damage of liver primary cell, Artepillin C suppression liver primary cell glucose inputs not Artepillin C reduction cell viability to be caused.

The detection Artepillin C impact on glucagon signal path further, result display Artepillin C does not affect glucagon in liver primary cell stimulates the cAMP level (Fig. 4 .E) produced, and the phosphorylation of CREB and the dephosphorylation of CRTC2 are not had significant impact (Fig. 4 .F) yet.Illustrate that the signal path upstream of hepatic gluconeogenic transcriptional control is had no significant effect by Artepillin C.

At the end of glucagon signal path, CRTC2, as transcribing the co-activation factor, can remarkably promote the transcriptional efficiency of CRE target gene, and therefore the present inventor utilizes deficiency model to detect whether Artepillin C inhibitory activity depends on the function of CRTC2 further.Result shows at CRTC2^-/-In primary hepatocyte, the inhibitory activity that hepatic glucose is exported by Artepillin C disappears (Fig. 4 .G).Now, Artepillin C can not glucagon suppression induction hepatic gluconeogenic key gene Pgc1 α, Pck1 transcribe (Fig. 4 .H).This explanation Artepillin C depends on the existence of CRTC2 to the inhibitory activity of hepatic gluconeogenic.

Result above shows, Artepillin C is by the glucagon suppression hepatic gluconeogenic function that mediated by CREB/CRTC2 of induction thus the glucose reducing liver primary cell exports.

Embodiment 5, Artepillin C improve hyperlipidemia and fatty liver

In order to detect the Artepillin C effect in vivo to metabolism, the present inventor with high-fat adiposity mice (DIO) and db/db mice as experimental subject, through lumbar injection (DIO:0,10,20mg/kg；Db/db:20mg/kg) it is administered.

Result shows, Artepillin C can significantly inhibit the body weight of DIO and db/db model mice and increase (Fig. 5 .A, left DIO, right db/db), and in DIO mice, Artepillin C minimizing DIO body weight growth has dosage effect (Fig. 5 .A).Artepillin C also reduces the fat content (Fig. 5 .B, left DIO, right db/db) in Mice Body simultaneously, alleviates obesity.Corresponding, Artepillin C reduces two kinds of model mice triglyceride in serum (TG) (Fig. 5 .C, left DIO, right db/db), reduce serum cholesterol and (include T-CHOL TC, low density cholesterol LDLC and high density HDLC) content (the left DIO of Fig. 5 .D, right db/db), also reduce the content (Fig. 5 .E) of triglyceride (TG) in DIO mouse liver tissue simultaneously.

Tissue slice result display Artepillin C makes the cell of liver organization and fatty tissue diminish, and in cell, fat drips and reduces (Fig. 5 .F), shows that Artepillin C alleviates the fatty liver symptom of diabetic mice.

Artepillin C not only reduced lipids contents but also reduced liver organization in fat content, this shows that Artepillin C reduces the effect of blood lipid level and may be mostly derived from and reduce liver blood fat output, and therefore the blood fat of liver is synthesized, exports and absorb and have inhibitory action by prompting Artepillin C.

Embodiment 6, Artepillin C are synthesized by the expression inhibiting lipid reducing SREBPs

In view of Artepillin C can significantly improve hyperlipidemia and fatty liver symptom, and the transcription factor SREBPs transcription regulatory factor that to be lipid metabolism important, therefore the present inventor have detected Artepillin C further to SREBP1,2 impacts expressed.The present inventor with high-fat adiposity mice (DIO) and db/db mice as experimental subject, through lumbar injection (DIO:0,10,20mg/kg；Db/db:20mg/kg) it is administered intervention.

Result shows, Artepillin C significantly reduces the mRNA level in-site (Fig. 6 .A) of Srebp1a, 1c and Srebp2 in liver, reduces liver SREBP1, the protein content (Fig. 6 .B) of 2 simultaneously.QPCR testing result shows, Scap gene expression in Artepillin C mono-aspect suppression liver, on the other hand promote that Insig1's and Insig2b transcribes (Fig. 6 .C), this phenomenon shows that Artepillin C may be by increasing INSIG protein expression, strengthen the stability of INSIG-SREBPs complex, suppression SREBPs precursor protein matter moves to Golgi body from endoplasmic reticulum, thus the protease suppressing SREBPs to carry out on Golgi body cuts through journey, and then reduce the protein content of ripe SREBPs.Artepillin C is by reducing the crucial rate-limiting enzyme Hmgcl in cholesterol biosynthesis approach simultaneously, Hmgcs expression, reduce low-density protein cholesterol receptors (Ldlr) to transcribe, the expression (Fig. 6 .C) of suppression Ldlr receptor degradation protein Pcsk9, the synthesis of suppression hepatic cholesterol and absorption, reduce synthesis and the content of cholesterol in liver；In terms of fatty acid metabolism, Artepillin C is by suppression fatty acid synthesis key enzyme Fasn, Acc transcribes reduction fatty acid synthesis, secreted by the reduction fatty acid of transcribing of suppression ApoE, by over oxidation and the synthesis (Fig. 6 .C) of long-chain fatty acid of suppression Acox1 and Acsl1 expression inhibiting fatty acid.In white adipose tissue, Artepillin C significantly suppress the transcriptional level of lipid regulative transcription factor Srebp1c and Srebp2, inhibiting its target gene, Fans and Acc transcribes simultaneously, reduces synthesis and the content (Fig. 6 .D) of triglyceride of fat acid in liver organization.Meanwhile, Artepillin C significantly suppress multiple lipid metabolism genes such as LPL-1 in white adipose, SCD-1, ApoB, Res (Resistin), it is suppressed that fatty acid and the secretion of Adipocyte Factor,

Owing to Artepillin C can suppress transcribing of SREBPs and downstream target gene very significantly, and PPAR γ, PPAR α and LXR α is the important positive regulative transcription factor of SREBPs, and therefore the present inventor detects the expression of these nuclear receptor genes further.Result shows, Artepillin C can significantly inhibit transcribing of nuclear receptor Lxra in liver, but transcribing of PPAR γ, PPAR α is had no significant effect (Fig. 6 .E).In Artepillin C the most also suppression white adipose tissue, Lxra's transcribes (Fig. 6 .D).

In primary hepatocyte, the inventors discovered that, Artepillin C significantly reduces the protein level of nuclear receptor LXR α, and significantly suppress by the protein level (Fig. 6 .F) of insulin-induced SREBP1, and this result matches with QPCR result.

Result above shows, Artepillin C is transcribed by suppression nuclear receptor LXR α's, thus suppression is by the Srebps genetic transcription of LXRE element drives, and then reduce SREBPs and the expression of the most multiple target genes, it is suppressed that the cholesterol of SREBPs regulation and control, fatty acid synthesis and secretion.

Embodiment 7, Artepillin C improve the glucose metabolism in diabetic rat model body

Phenomenon based on Artepillin C suppression primary hepatocyte hepatic gluconeogenic, the present inventor the most in vivo detects the function of reducing blood sugar bioactivity of Artepillin C.

DIO mouse peritoneal injection Artepillin C (20mg/kg) is after 3 days, and DIO mouse liver living imaging analysis display Artepillin C inhibits G6P-LUC reporter gene transcription activity (Fig. 7 .A) of hungry induction.The mRNA level in-site (Fig. 7 .B) of hepatic gluconeogenic key gene Pgc1 α, Pck1 and G6pc during Artepillin C significantly reduces DIO liver organization simultaneously.In DIO mice live donor liver tissue, the protein level of hepatic gluconeogenic rate-limiting enzyme PEPCK and transcription factor PGC1 α is significantly inhibited (Fig. 7 .C) by Artepillin C.

Inventor detects the Artepillin C effect to mice overall glucose metabolism further.First, with healthy adult Mus as object, after lumbar injection Artepillin C (20mg/kg) 3 times, the blood sugar level that pancreas hyperglycemia stimulates raises and is substantially suppressed (Fig. 7 .D) by Artepillin C.Detect the Artepillin C impact on glucose metabolism under the conditions of insulin resistant the most further.Result shows, the Artepillin C being administered a week significantly suppress DIO mice (Fig. 7 .E) and the hungry blood sugar level of db/db mice (Fig. 7 .F).After being administered 5 weeks, the serum insulin levels of db/db mice is had no significant effect (Fig. 7 .G) by Artepillin C.Owing to Artepillin C does not affect glutamic oxaloacetic transaminase, GOT (Aspartate transaminase in DIO mice serum, and glutamate pyruvate transaminase (Alanine transaminase AST), ALT) level (Fig. 7 .H), illustrate the dosage of Artepillin C (20mg/kg) to liver without overt toxicity, also illustrate that the Artepillin C of this dose is not from hepar damnification to the inhibitory activity of hepatic gluconeogenic simultaneously.

Experiment made on the living result explanation Artepillin C can significantly improve the hyperglycemia of diabetic mice.

Embodiment 8, Artepillin C improve the raising insulin sensitivity of diabetic rat model

Owing to insulin resistant is not only pathogenesis and the diagnosis basis of type 2 diabetes mellitus, also it is the main common bond of multiple metabolic disease, so insulin resistant is the common pathophysiological basis of obesity, hyperglycemia, hyperlipidemia and metabolic syndrome.In view of Artepillin C is significantly improved effect to body weight, fat content, hyperlipidemia and the hyperglycemia of DIO and db/db diabetic mice, inventor detects the Artepillin impact on diabetic mice insulin sensitivity further.With DIO mice and db/db mice as experimental subject, lumbar injection (20mg/kg) carries out tolerance test after being administered 5 weeks.Result shows, Artepillin C mono-aspect significantly improves glucose tolerance (GTT) (Fig. 8 .A, a left side, the DIO of diabetic mice；The right side, db/db), on the other hand enhance acetone acid toleration, significantly inhibit ability (PTT) (Fig. 8 .B, a left side, DIO that diabetic mice conversion acetone acid is glucose；The right side, db/db).In integral level, Artepillin C significantly enhances insulin sensitivity (ITT) (Fig. 8 .C, a left side, the DIO of diabetic mice；The right side, db/db), improve the diabetic mice opposing to insulin.

In sum, Artepillin C significantly improves the glycolipid metabolism balance of diabetic mice in integral level, significantly improves the insulin sensitivity of diabetic mice.

Embodiment 9, Artepillin C improve the molecular mechanism of insulin sensitivity

The present inventor test result indicate that, micromolecular compound Artepillin C (APC) by with the N end of CREB directly in conjunction with, suppress be combineding with each other of CRTC2 with CREB after core, reduce the expression of the downstream gene driven by CREB/CRTC2, main component PGC1 α including hepatic gluconeogenic, and important hepatic gluconeogenic rate-limiting enzyme G6PC and PEPCK, the glucose output of suppression hepatic gluconeogenic and liver, reduce hungry blood glucose.On the other hand, APC is by suppressing the expression of nuclear receptor LXR α, work in coordination with and transcribe the co-activation repressed state of factor PGC1 α, eventually reduce the expression of the SREBPs driven by LXRE, the downstream target gene of suppression SREBPs, reduce the cholesterol regulated and controled by SREBPs and the synthesis of fatty acid, absorption, fat content (Fig. 9) in final reduction blood.

Owing to APC significantly improves hyperglycemia and the hyperlipidemia of diabetic mice in live body, decreasing fat, therefore APC have adjusted glycolipid metabolism stable state on the whole, alleviates the insulin resistant of body, enhances the insulin sensitivity of entirety.

Embodiment 10, the structure optimization of Artepillin C

In order to transform further and optimize the micromolecular compound Artepillin C regulating and controlling effect to target protein CREB/CRTC2, multiple analogs of Artepillin C are tested and evaluate by the present inventor.

It was found that compound 63#, 65# also have the activity that suppression CREB/CRTC2 be combined with each other, such as Figure 10.Wherein 65# inhibitory activity is relatively strong, is tested by double cross, and the IC50 of 65# compound is about 15.9uM.

Compound 65# structural formula is as follows:

The all documents mentioned in the present invention are incorporated as reference the most in this application, are individually recited as with reference to like that just as each document.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.

Claims (10)

1. the clever C of Aunar or its analog or its pharmaceutically acceptable salt prevent in preparation, alleviate or control Treat the purposes in the medicine of metabolic disease.

2. purposes as claimed in claim 1, it is characterised in that described metabolic disease includes: fat Metabolic disease, carbohydrate metabolism disease or metabolic syndrome.

3. purposes as claimed in claim 2, it is characterised in that described lipid metabolism disease includes: high Blood fat disease, fatty liver, obesity, atherosclerosis, coronary heart disease, hypertension, cerebral infarction, apoplexy, Renal failure.

4. purposes as claimed in claim 2, it is characterised in that described carbohydrate metabolism disease includes: high Blood glucose disease, diabetes, obesity, Hyperinsulinism, atherosclerosis, coronary heart disease, hypertension, Hyperthyroidism, diabetic ophthalmopathy, diabetic nephropathy.

5. purposes as claimed in claim 1, it is characterised in that the clever C of described Aunar or its be similar to Thing has such as following formula (I) structure:

Wherein, R1 is selected from H, isopentene group (prenyl)；

R2 is OH；

R3 is selected from H, isopentene group；

R4 is selected from H, CH₂Ph,Or CH₂CHPh。

6. purposes as claimed in claim 5, it is characterised in that affiliated compound is selected from:

The clever C of Aunar；

R1 be H, R2 be OH, R3 be H, R4 be the compound of H；

R1 be H, R2 be OH, R3 be isopentene group, R4 be the compound of H；

R1 be H, R2 be OH, R3 be H, R4 be CH₂The compound of Ph；Or

R1 be H, R2 be OH, R3 be that H, R4 areCompound.

7. purposes as claimed in claim 1, it is characterised in that described medicine is additionally operable to:

Suppression hepatic gluconeogenic；

Reduce hepatic gluconeogenic key gene Pgc1 α, the level of Pck1, G6pc；

Reduce hepatic glucose output；

Improve glucose tolerance；

Reduce body fat content；

Reduce serum triglycerides content；

Reduce total cholesterol level；

Reduce low-density lipoprotein cholesterol content；

Reduce HDL-C content；

Reduce liver blood fat output；

Suppression liver fat acid synthesis；

Suppression cholesterol biosynthesis；

The blood fat output of suppression liver and absorption；

Reduce SREBP and the expression of downstream target gene thereof；And/or

Improve insulin sensitivity.

8. the method preparing medicine, described medicine is used for preventing, alleviate or treat lipid metabolism disease Or the medicine of carbohydrate metabolism disease, it is characterised in that described method includes: by clever for the Aunar of effective dose C Or its analog or its pharmaceutically acceptable salt mix with pharmaceutically acceptable carrier.

9. method as claimed in claim 8, it is characterised in that described metabolic disease includes: fat Metabolic disease, carbohydrate metabolism disease or metabolic syndrome.

10. method as claimed in claim 8, it is characterised in that the clever C of described Aunar or its class Have such as following formula (I) structure like thing:

Wherein, R1 is selected from H, isopentene group (prenyl)；

R2 is OH；

R3 is selected from H, isopentene group；

R4 is selected from H, CH₂Ph,Or CH₂CHPh。