CN101209257A - Medical use of baicalin and scutellarein derivatives - Google Patents
Medical use of baicalin and scutellarein derivatives Download PDFInfo
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Abstract
The invention discloses a baicalein derivative drug which can be used for the prevention and treatment of metabolic syndrome, obesity, type 2 diabetes and atherosclerotic cardiovascular disease. The baicalein derivative can increase the metabolic activity for the prevention and treatment of metabolic syndrome, obesity, type 2 diabetes and atherosclerotic cardiovascular disease by controlling and activating the activity of single adenylate activated protein kinase.
Description
Technical field
The present invention relates to the medical usage of baicalin and Scutellarein derivative, refer to more specifically that baicalin and such Scutellarein derivative are by regulation and control and activate the activity of simple gland thuja acid activated protein kinase in the tissue, increase metabolic activity and prevent and treat metabolism syndrome, obesity, type 2 diabetes mellitus and atherosclerotic cardiovascular and cerebrovascular disease.
Background technology
(1) metabolism syndrome is a complex set of metabolism disorder, comprises the copolymerization of risk factors such as obesity, hypertension, hyperglycemia, dyslipidemia, insulin resistant.Metabolism syndrome is the high risk factor of type 2 diabetes mellitus and cardiovascular disease, has become the problem that the world medicine field presses for solution at present.
(2) also do not have good treatment measure for fat and metabolism syndrome up to now, use antidiabetic drug usually, lipid lowerers and antihypertensive drug are treated metabolism syndrome, but these medicines lack effectiveness.At present, diabetes drug treatment is faced with great challenge, and the process of diabetes can not stop or reversing, and curative effect can not be lasting for a long time, and cardiovascular complication lacks effectively prevention and treatment measure.Estimate that global Remedies for diabetes was expected to be 20,000,000,000 dollars in 2006, China hypoglycemic medicine market is for being 10,000,000,000 yuan.At present, hypoglycemic medicament categories is more, except that insulin, and main having of using:
1, sulfonylureas: promote insulin secretion.Such medicine often has hypoglycemia and fat generation.
2, biguanides: increase the utilization of glucose, suppress the absorption of glucose, suppress the output of glycogen heteroplasia and glycogen.Phenformin (insoral): lactic acidosis; Metformin (metformin) energy blood fat reducing, hypoglycemia takes place few.
3, glucosidase inhibitor class medicine: suppress alpha-glucosidase, the absorption of slow down hydrolysis and glucogenic speed and delay glucose reduces post-prandial glycemia, also has certain effect for reducing blood fat.Commonly used have acarbose, a voglibose: gastrointestinal reaction.
4, non-sulfonylurea insulin secretion stimulators: promote the beta Cell of islet excreting insulin, do not bring out hypoglycemia.
5, euglycemic agent: PPAR α/γ dual agonists can improve type 2 diabetes mellitus patient's fat metabolic disturbance and strengthen insulin sensitivity, once is described as the new leap in the treating diabetes.But U.S. FDA was announced this class medicine before finishing rodent 2 term carcinogenic test in July, 2004, will not ratify any PPAR economic benefits and social benefits stimulant medicine and carry out human clinical research more than 6 months; Further, in JIUYUE, 2005, expert advisor's meeting of FDA is made assessment to PPAR α/γ dual agonists muraglitazar that U.S.'s Bristol Myers Squibb and Merck ﹠ Co., Inc. develop jointly, and requires enterprise to provide to have the relevant medicine long-term safety problem of cogency.
Medication of Diabetes Mellifus must lifelong medication.Security requirement to this class medicine is very high.The medicine that has gone on the market can't meet the requirement of long-term prescription.At new target spot, the antidiabetic drug of development of new, also blood fat reducing in blood sugar lowering reduces the medicine of diabetic vascular complications, remains the important directions of diabetes field drug research.
(3) simple gland thuja acid activated protein kinase (AMP-activated protein kinase, AMPK) and metabolism syndrome
People are also very limited to the understanding of metabolism syndrome at present.The cause of disease of metabolism syndrome and detectable component, at effective treatment measure of metabolism syndrome all components with and the influence of cardiovascular risk become the focus that the world medicine field is paid close attention to.Think that at present the factor that directly and indirectly causes metabolism syndrome and the factor that can be used for the treatment of comprise motion exercise, dietary habit, body weight, blood sugar level, triglyceride levels, cholesterol levels, insulin resistant, simple gland thuja acid activated protein kinase (malonyl coenzyme A concentration in the AMP-activated protein kinase, activity AMPK), adiponectin, leptin, estrogen level, gene mutation and body.But also do not have good medicine for metabolism syndrome and obesity up to now, clinical only is to use antidiabetic drug, and lipid lowerers and antihypertensive drug are treated, but these medicines lack effectiveness.Think that at present the effective and efficient manner of treatment metabolic syndrome and obesity still strengthens exercises, reduces body weight and diet control, and the common ground of all these measures is metabolism of acceleration energy, the storage of minimizing energy in cell.For reaching such purpose, need synthetic relevant approach of inhibition and material such as lipogenesis, glycogen to synthesize and glyconeogenesis, quicken approach relevant such as sugar consumption and oxidation, fat oxidation and mitochondrial biosynthesis with catabolism.And AMPK is exactly one of key factor of these a series of metabolic activities of regulation and control.
AMPK is the cellular energy sensor of high conservative, ubiquity in eukaryotic cell [FEBSLetters.546:113-120,2003].AMPK approximately finds with the cAMP deopendent protein kinase same time, but up to AMPK in recent years the important regulatory function ability of diabetes illustrated [Am.J.Physiol.277:E1-E10,1999; Curr Opin Cell Biol.17 (2): 167-73.2005.].AMPK is a class serine/threonine kinase, by a α of catalytic subunit and two heterotrimers [Biochem.J.346:659-669.2000] that on-catalytic subunit β, γ form.AMPK can be activated by the upstream tyrosine phosphorylation, also can regulate being activated simultaneously by allosteric.Under stressed conditions such as motion, energy expenditure, anoxia, ATP consumes increase, and AMP concentration raises, and makes AMP: the ATP ratio raises, and activates the AMPK kinase system.AMPK is in case activation can be raised the catabolism signal path that ATP generates, downward modulation consumes the anabolism path of ATP, participate in the regulation and control [Physiology.21 (1): 48-60.2006] of multiple metabolic activity, comprise and reduce the absorption of the generation of synthesizing, suppress hepatic glycogen of fat and cholesterol, the oxidation that increases fat and muscle, its effect and the effect very consistent [Biochem.Biophys.Res.Commun.273:1150-1155.2000] of moving and producing glucose.
1. to glycometabolic influence
In muscular tissue, AMPK can promote muscle contraction, accelerates glucose absorption, and induces the transposition of glucose transporter 4 (GLUT4) to cell membrane, increase glucose to intracellular transport, this process does not rely on activity [the J Appl Physiol.91 (3): 1073-83.2001 of insulin; ExpPhysiol.89 (5): 559-63.2004].When the glucose absorption increase, AMPK then activates hexokinase, promotes the glucose metabolism process, and it is synthetic to suppress glycogen simultaneously.Can regulate the transposition of GLUT4 by activating the NO-cGPT signal path at myocardium AMPK, increase the picked-up [Am J Physiol 287:E834-E841.2004] of myocardial cell glucose to cell membrane.In hepatocyte, the AMPK activation can suppress the activity of phosphoenolpy ruvate carboxy kinase (PEPCK) and glucose-6-phosphatase (G6Pase), and the two is the main rate-limiting enzyme of glyconeogenesis, thereby reduces the release of hepatic glycogen.Therefore, activate AMPK and can not rely on insulin and regulating and controlling blood sugar concentration [Diabetes.49 (6): 896-903.2000].
2. to lipometabolic influence
The rising of free fatty acid levels causes liver and peripheral tissues's insulin resistant in the circulation.The oxidation of gathering the weakening glucose of free fatty induces glycogen to generate.Therefore, reducing free fatty may be useful to type 2 diabetes mellitus gathering of these tissues.Studies show that more and more AMPK regulates lipid metabolism by number of mechanisms.Activate AMPK and can make rate-limiting enzyme acetyl-CoA carboxylase (acetyl CoA carboxylase in the synthetic path of fatty acid, ACC) phosphorylation, thereby suppress the synthetic of fatty acid, and the concentration of malonyl coenzyme A (malonyl-CoA) in the reduction cell, malonyl coenzyme A is carnitine acyl transferase-1 (carnitine palmitoyltransferase 1, CPT-1) allosteric inbibitor, synthetic by ACC, CPT-1 regulates the speed that long-chain acyl coenzyme A (LCFA-CoA) enters the mitochondrion oxidation, therefore, suppress the ACC activity, reduce malonyl coenzyme A concentration and can promote fatty acid oxidation.The downstream target enzyme of AMPK also comprises hydroxyl first glutaryl CoA reductase (HMG-CoAreductase) and phosphoglyceride acyltransferase (Glycerol phosphate acyl transferase), the two is respectively cholesterol and the synthetic rate-limiting enzyme of triglyceride, but activate above-mentioned two rate-limiting enzymes of AMPK phosphorylation, thereby suppress synthetic [the J Biol Chem.277 (36): 32571-7.2002 of cholesterol and triglyceride; J Appl Physiol.92 (6): 2475-82.2002].After cholesterol regulation element conjugated protein (SREBP) is found to be the metabolic important nuclear factor of cell inner cholesterol [Cell.75 (1): 187-97.1993], carbohydrate response element binding protein (carbohydrate response element-binding protein is confirmed in up-to-date research, ChREBP) be the crucial nuclear factor of glucose induction lipogenesis enzyme gene expression, and do not rely on the activity [PNAS.98 (16): 9116-21.2001] of insulin.In liver organization, AMPK suppresses the gene expression [J BiolChem.277 (6): 3829-35.2002] of ACC, fatty acid synthetase and pyruvate kinase by phosphorylation carbohydrate response element binding protein.
Although exercise therapy is to cure metabolism syndrome and fat important measures for a long time always, clinical trial proves that also exercise can prevent and treat type 2 diabetes mellitus [Annu Rev Med49:235-261:235-261,1998; N Engl J Med 346:393-403,2002].Yet take exercise the molecular mechanism of diabetics generation positive role not fully aware of always.A large amount of in recent years animals and clinical diabetes people's research all shows, treadmill movement can activate AMPK in the muscle, participate in the adjusting of acute exercise to muscle metabolism, increase the skeletal muscle glucose absorption, remarkable blood sugar lowering, this kinases can also adapt to long-term active improvement of muscle metabolism [the J Biol Chem.277 (36): 32571-7.2002 that cause that takes exercise by the gene expression that influences multiple protein; Diabetes.50 (5): 921-7.2001; JPhysiol.564 (Pt 2): 563-73.2005; Diabetes.54 (4): 928-34.2005].
Pharmaceutical research shows that also strengthening the AMPK activity can become a New Policy for the treatment of type 2 diabetes mellitus.(5-aminoimidazole-4-carboxamideribotide is present unique non-specific agonist of AMPK AICAR) to 5-aminoimidazole-4-carbozamide nucleotide, is usually used in the research of vivo activation AMPK as the experimental tool medicine.To studies show that of obese diabetes Zucker Mus; take AICAR for a long time; can stop the generation of these Mus diabetes; improve the transhipment of glucose in the muscle; suppress the glycogen generation and increase lipolysis, significantly increase insulin sensitivity, the form and the function of protection islet cells; its curative effect consistent with the training group [J Appl Physiol 88:1072-1075,2000].Prior pharmacology evidence is from the study on mechanism result to present clinical widely used antidiabetic drug metformin (metformin).Metformin is the choice drug of treatment type 2 diabetes mellitus, especially to overweight and with patient's better efficacy of hyperinsulinemia.Although nearly 50 years of clinical use, the definite blood sugar lowering mechanism of biguanides is indeterminate always.Discovering in recent years, no matter at cellular level, still in whole animal and clinical patient, metformin can both strengthen the activity of AMPK, improve the opposing of liver to insulin, suppress glyconeogenesis, generate thereby suppress too much endogenous hepatic glucose, follow the glucose utilization rate to increase simultaneously, increase the transhipment and storage [the J Clin Invest.108:1167-1174.2001 of skeletal muscle glucose; Diabetes.51 (7): 2074-81.2002; Diabetes.53 (12): 3258-66.2004; J Biol Chem.279 (46): 47898-905.2004].AMPK is the target enzyme of the biguanides effect of present unique affirmation, but its biochemical mechanism that activates AMPK is still unclear.At present, academia to can improve insulin resistant, delay type 2 diabetes mellitus morbidity, the medicine that reduces cardiovascular danger has increasing interest.U.S.'s diabetes mellitus prevention project studies show that in 2005, adopted metformin can reduce the generation of metabolism syndrome the metabolism syndrome crowd, and prevention or delay onset diabetes [Ann Intern Med.142 (8): 611-9.2005].
Discover that AMPK not only participates in the regulation and control of energy metabolism mechanism, also has important protective effect to cardiovascular system.Activating AMPK increases the myocardial cell glucose transport, and increases the phosphorylation of 6-phosphofructo-2-kinase, increases glycolysis, recovers the carbohydrate metabolism activity [AmJ Physiol.2006] of diabetic disease states heart.AMPK increases fatty acid oxidation by suppressing the activity of vascular endothelial cell ACC, thus the endothelial function imbalance [CircRes.88 (12): 1276-82.2001] that stops insulin resistant to cause.In addition, AMPK can promote NOS3 (eNOS) serl 177 site phosphorylations [FEBS Lett.443 (3): 285-9.1999], activate eNOS, it is synthetic to increase nitric oxide (NO), improve endothelial function, increase the picked-up [AmJ Physiol.287:E834-E841.2004] of cardiac muscle glucose.AMPK can also suppress the vascular smooth muscle hypertrophy [Circulation.110 (4): 444-51.2004] that angiotensin causes, hormone such as adiponectin and leptin also can activate AMPK[Nat Med.8 (11): 1288-95.2002], thereby AMPK also has great importance to preventing and treating atherosclerosis.
Summary of the invention
The object of the invention provides the medical usage to baicalin and Scutellarein derivative.
The present invention is by the biological study to baicalin and Scutellarein derivative, increase metabolic activity in cells by regulation and control and the activity that activates simple gland thuja acid activated protein kinase in the tissue and reach prevention and treatment disease thereby disclose them, can in the medicine of preparation prevention and treatment metabolism syndrome, obesity, type 2 diabetes mellitus and atherosclerotic cardiovascular and cerebrovascular disease, use.
What the present invention relates to is the Scutellarein derivative that following general structure is arranged:
(I) in the formula: R1, R2, R3 can be identical or different, and R1, R2, R3 represent hydrogen, alkyl, aryl, electron-donating group such as F or electron withdraw group such as OCH
3The cycloalkyl that replaces of substituted aryl, alkynyl, thiazolinyl, cycloalkyl, hetero atom, fatty acyl group, aromaticacyl radical ,-RNR``R` ,-RCOOR```; Wherein, R is an alkyl, R`, R`` can be identical or different, and R`, R`` and R``` represent hydrogen, alkyl, thiazolinyl, alkynyl, aryl.
Scutellarein derivative of the present invention adopts different route of synthesis to finish according to the replacement of diverse location, and synthetic method is open in Chinese patent application 200510112280.6 in detail.
According to an aspect of the present invention, it provides baicalin and the Scutellarein derivative HepG2 cell AMPK phosphorylation level (seeing Fig. 1, table 1) that all can raise.Wherein Scutellarein derivative refers to but is not limited to chemical compound in the table 2.
Table 1: the molecular structural formula of different Scutellarein derivatives, and compare their influences to p-AMPK protein expression in the HepG2 cell.
Table 2.
According to a further aspect of the invention, it provides in the insulin resistant HepG2 cell model that high sugar causes, the HepG2 cell AMPK phosphorylation level reduction that baicalin and Scutellarein derivative all can make high sugar cause is restored and improves (seeing Fig. 2, table 3).Wherein Scutellarein derivative refers to but does not limit HY101, HY66, HY30, HY118, HY04, HY40 (structure sees Table 2.).
Table 3: the molecular structural formula of different Scutellarein derivatives, and more different compound concentration is to the influence of p-AMPK protein expression in the sugared inductive insulin resistant HepG2 cell model of height.
According to a further aspect of the invention, it provides baicalin and Scutellarein derivative all can reduce the rising (see figure 3) of the HepG2 cell triglyceride that high sugar causes.Wherein Scutellarein derivative refers to but does not limit HY101, HY66, HY30, HY118, HY04, HY40 (structure sees Table 2.).
According to a further aspect of the invention, it provides at low dose of streptozotocin increases in the fat type 2 diabetes mellitus rat model of fat diet induced, baicalin and Scutellarein derivative all can reduce fasting glucose, serum cholesterol, triglyceride and low density lipoprotein, LDL, increase the ratio of high density lipoprotein/cholesterol, body weight is also decreased, and animal is not had overt toxicity, none death of animal (seeing Fig. 4-9).Wherein Scutellarein derivative refers to but does not limit HY66 (structure sees Table 2.).
ACC is a rate-limiting enzyme in the synthetic path of fatty acid, but activates AMPK phosphorylation ACC, thereby makes active reduction of ACC suppress the synthetic of fatty acid, and reduces the concentration of malonyl coenzyme A in the cell, promotes fatty acid oxidation.According to a further aspect of the invention, it provides the new pharmacological action of baicalin and Scutellarein derivative hypoglycemic, baicalin and Scutellarein derivative can significantly increase the expression of phosphorylation AMPK in fat type 2 diabetes mellitus rat liver and the skeletal muscle, also significantly increase simultaneously the expression (see Figure 10, table 4) of phosphorylation ACC in above-mentioned tissue.Wherein Scutellarein derivative refers to but does not limit HY66 (structure sees Table 2.).
Table 4: baicalin and Scutellarein derivative are to the influence of p-AMPK in STZ inducing obesity diabetes rat liver and the skeletal muscle and phosphorylation ACC (p-ACC) protein expression.
The present invention provides a new way for the treatment of metabolism syndrome, obesity, diabetes and cardio-cerebrovascular diseases, both by activating AMPK, increases the human body energy metabolism, promotes glucose absorption and fat oxidation, reduces insulin resistant.
Description of drawings
Fig. 1: the Scutellarein derivative of western blot determination different structure is to the influence of AMPK in the HepG2 cell and phosphorylation AMPK (p-AMPK) protein expression.Band (right certainly from a left side) is respectively cellular control unit (1 road), the cell of different compound treatment (2-8 road).
Fig. 2: the Scutellarein derivative of western blot determination different structure is to the influence of AMPK and p-AMPK protein expression in the sugared inductive insulin resistant HepG2 cell model of height.Band (right certainly from a left side) is respectively the control cells (1 road) that low concentration glucose is hatched; High sugared inductive insulin resistant cell (2 road); The cell (3-9 road) that high concentration glucose and different chemical compounds are hatched jointly.
Fig. 3: measure of the influence of the Scutellarein derivative of different structure to cell triglyceride (TG) rising in the sugared inductive insulin resistant cell model of height.Matched group: the HepG2 cell that low concentration glucose is hatched; High sugar group: high sugared inductive insulin resistant HepG2 cell model; Compound treatment group: the HepG2 cell that high concentration glucose and chemical compound are hatched jointly.#: compare p＜0.05 with matched group; *: compare p＜0.05 with high sugar group.
Fig. 4: Scutellarein derivative is to the influence of STZ inducing obesity blood glucose in diabetic rats.(metformin: 18mg/kg/day, n=4; Baicalin: 80mg/kg/day, n=7; HY66:20mg/kg/day, n=8).*: p＜0.05, compare with the STZ group.
Fig. 5: Scutellarein derivative is to the influence of STZ inducing obesity diabetes rat serum total cholesterol (TC).(metformin: 18mg/kg/day, n=4; Baicalin: 80mg/kg/day, n=7; HY66:20mg/kg/day, n=8).*: p＜0.05, compare with the STZ group.
Fig. 6: Scutellarein derivative is to the influence of the total triglyceride of STZ inducing obesity diabetes rat serum (TG).(metformin: 18mg/kg/day, n=4; Baicalin: 80mg/kg/day, n=7; HY66:20mg/kg/day, n=8).
Fig. 7: Scutellarein derivative is to the influence of STZ inducing obesity diabetes rat serum low-density LP (LDL-C).(metformin: 18mg/kg/day, n=4; Baicalin: 80mg/kg/day, n=7; HY66:20mg/kg/day, n=8).*: p＜0.05, compare with the STZ group.
Fig. 8: Scutellarein derivative is to the influence of STZ inducing obesity diabetes rat high density lipoprotein and ldl ratio value (HDL/TC).(metformin: 18mg/kg/day, n=4; Baicalin: 80mg/kg/day, n=7; HY66:20mg/kg/day, n=8).*: p＜0.05, compare with the STZ group.
Fig. 9: Scutellarein derivative is to the influence of STZ inducing obesity diabetes rat body weight.(metformin: 18mg/kg/day, n=4; Baicalin: 80mg/kg/day, n=7; HY66:20mg/kg/day, n=8).
Figure 10: total AMPK, p-AMPK albumen and the proteic expression of p-ACC in western blot determination STZ inducing obesity diabetes rat liver and the skeletal muscle.Band (right certainly from a left side) is respectively control rats (1 road), STZ inducing obesity diabetes rat (2,3 road), positive drug metformin treatment group (4,5 road), baicalin (6,7 road) and Scutellarein derivative HY66 treatment group (8 road).
The specific embodiment
Pharmacology's test:
One. experiment in vitro
1. material and reagent
The human hepatoma HepG2 cell is available from Shanghai Inst. of Life Science, CAS cell resource center.MEM culture medium dry powder, 0.25% trypsin-EDTA, hyclone, 100 * Sodium Pyruvate, 100 * mycillin are available from Invitrogen company.The protein quantification test kit is available from Bio-Rad company.Anti-AMPK α 2, phosphorylation AMPK α (p-AMPK α), ACC and phosphorylation ACC (p-ACC) antibody are available from Cell signaling company.β-actin one is anti-available from Lab vision company.Horseradish peroxidase-labeled two is anti-available from Jackson company.The protease inhibitor complex is available from Calbiochem company.Other reagent all are analytical pure.
2. cell culture
Human liver cancer cell HepG2 in the MEM culture medium that contains 10% hyclone, 100U/ml penicillin, 100g/ml streptomycin and normal concentration glucose (5.5mM D-glucose), in 37 ℃, 5%CO
2Cultivate in the incubator, cell went down to posterity with 0.25% trypsinization in per 3 days.Experiment is grown in the 100mm culture dish with cell, is used for experiment when cell reaches 80% density.
3. high sugared inductive insulin resistant HepG2 cell model
Pressing document [J Biol Chem.279 (46): 47898-905,2004] carries out.The HepG2 cell was cultivated 24 hours in not containing the MEM culture medium that hyclone, concentration of glucose be respectively 5.5mM and 30mM, and the cell that high concentration glucose is hatched reduces insulin response, and cytolipin raises.
4. compound treatment
4.1.HepG2 cell is that the MEM culture medium culturing of 5.5mM spends the night at no hyclone, concentration of glucose, the Scutellarein derivative (10 μ M) of baicalin (10 μ M) and different structure continued cultured cell 4 hours, collecting cell.
4.2.HepG2 cell is to cultivate collecting cell 24 hours with the Scutellarein derivative (10,100 μ M) of baicalin (10,100 μ M) and different structure in the MEM culture medium of 30mM at no hyclone, concentration of glucose.
5. western blot determination baicalin and Scutellarein derivative are in the influence of cellular level to AMPK and p-AMPK expression
Cold PBS washing is 3 times behind the collecting cell, adds 60 μ l cell pyrolysis liquid (20mM Tris, pH7.5; 150mM NaCl; 1mM EDTA; 1mM EGTA; 1% Triton X-100; 2.5mMsodium pyrophosphate; 1mM β-Glycerolphosphate; 1mM Na3VO3; 1 μ g/mlLeupeptin; 1mM PMSF) cell lysis, and carry out ultrasonic, centrifugal 15 minutes of 4 ℃ of 4000g, with Bio-Rad protein quantification kit measurement protein concentration [Anal Biochem.72:248-254,1976], bovine serum albumin is standard substance.Get the capable 10%SDS-PAGE electrophoresis of 60-80 μ g albumen, electrotransport is in nitrocellulose membrane, and anti-AMPK α 2 and p-AMPK α one anti-hatching are carried out enzyme chromogenic reaction and graphical analysis (Bio-Rad image analysis software) (seeing Fig. 1-2, table 1, table 3).
6. triglyceride determination in the cell
Cell triglyceride determination method is oxidation enzyme process [Clin Chem.28 (10): 2077-80,1982], and its content is expressed as μ g lipid/mg albumen.The HepG2 cell is to cultivate 24 hours with the Scutellarein derivative (10 μ M) of baicalin (10 μ M) and different structure in the MEM culture medium of 30mM at no hyclone, concentration of glucose, and cell pyrolysis liquid prepares as stated above and measures protein concentration.Cell triglyceride Shanghai famous classic bio-engineering corporation kit measurement (see figure 3).
Two. animal experiment
With low dose of streptozotocin (Streptozotocin, STZ) increase the fat type 2 diabetes mellitus rat model (hereinafter to be referred as the STZ model) of fat diet induced, utilized this Research of Animal Model for Study baicalin and Scutellarein derivative HY66 is to the ratio of blood glucose, triglyceride, cholesterol, low density lipoprotein, LDL, high density lipoprotein/cholesterol and the influence of body weight.
1. material and reagent
Cleaning level SD rat, male, body weight 250-300g is available from Chinese Academy of Sciences's animal center; Streptozotocin (STZ) is available from Sigma company (98%HPLC is pure); Metformin is from medicine-feeding group; Blood glucose, triglyceride, cholesterol, low density lipoprotein, LDL, high density lipoprotein test kit are available from Shanghai famous classic bio-engineering corporation.
2.STZ inducing obesity diabetes rat
The SD rat (male, 250-300g) week is observed in precuring, animal feeding environment SPF level system, and 22-24 ℃ of room temperature control, the ad lib normal diet is freely intake.Begin to feed high lipid food (feed ingredient: moisture 5.91%, crude protein 18.57%, crude fat 16.88%, crude fibre 3.95%, ashes 6.00%, calcium 1.37%, phosphorus 0.83%) second week.After high fat fed for 6 weeks, water 12h was can't help in fasting, and tail vein injection STZ 45mg/kg surveyed fasting glucose after 3 days, and blood glucose value is fat type 2 diabetes mellitus model greater than 6.7mmol/L.Animal is divided into STZ model group, positive drug metformin group, baicalin and Scutellarein derivative HY66 treatment group at random.Every morning begins administration after the modeling success, and medicine is lumbar injection.Metformin is dissolved in normal saline, dosage is 18mg/kg/ days, and baicalin and Scutellarein derivative HY66 are dissolved in the 0.5%CMC-normal saline, and dosage was respectively 80mg/kg/ days and 20mg/kg/ days, the solvent 0.5%CMC-normal saline of STZ model group lumbar injection equal volume, administration is totally 6 weeks.Eye socket venous plexus blood sampling before the modeling, after the modeling, before the administration, in 1 week after the administration, in 3 weeks, each monitoring of 5 weeks once.Animal fasting, drug withdrawal before each blood sampling, can't help more than the water 12h.Collect serum ,-80 ℃ of preservations.After experiment finished, the animal fasting be can't help more than the water 12h, 12% chloral hydrate 360mg/kg intraperitoneal injection of anesthesia, and the heart blood sampling is also got liver rapidly, skeletal muscle places liquid nitrogen, ℃ preservation then-80.
3. the blood plasma biochemical indicator detects
Triglyceride, cholesterol and glucose assays method are oxidation enzyme process [Clin Chem.28 (10): 2077-80,1982; Clin Chem.20 (4): 470-5,1974; J Clin Pathol.22 (2): 158-61,1969]; Low density lipoprotein, LDL and high density lipoprotein adopt enzyme process and phosphotungstic acid magnesium precipitate to measure [Chinese journal of medical examination .14 (2): 66-70,1991; Circulation.55 (5): 767-72,1977].Above-mentioned biochemical indicator all detects (seeing Fig. 4-8) with Shanghai famous classic bio-engineering corporation test kit.
4. AMPK albumen, p-AMPK and p-ACC horizontal analysis in the tissue
With the variation of AMPK, p-AMPK and p-ACC protein level in the liver of western blot determination STZ rat model and the muscular tissue, and relatively baicalin and Scutellarein derivative HY66 to its influence.Liver or muscle sample are put on ice and are thawed, and tissue and homogenate added by 1: 10, and 70 rev/mins of homogenate 5min of Syrup-homogenizing instrument put and leave standstill 1h on ice, 3000g, and 4 ℃ of centrifugal 15min get supernatant, and protein concentration is (Bio-Rad Protein Assay) quantitatively.Get the capable 10%SDS-PAGE electrophoresis of 60-80 μ g protein sample, electrotransport is in nitrocellulose membrane, and anti-AMPK α 2, p-AMPK α and p-ACC one anti-hatching are carried out enzyme chromogenic reaction and graphical analysis (Bio-Rad image analysis software) (seeing Figure 10, table 4).
Claims (5)
1. the baicalin that a class formation formula is following, Scutellarein derivative (I)
(I) in the formula: R1, R2, R3 can be identical or different;
R1, R2, R3 represent cycloalkyl that hydrogen, alkyl, aryl, substituted aryl, alkynyl, thiazolinyl, cycloalkyl, hetero atom replaces, fatty acyl group, aromaticacyl radical ,-RNR``R` ,-RCOOR```;
Wherein, R is an alkyl, R`, R`` can be identical or different, and R`, R`` and R``` represent hydrogen, alkyl, thiazolinyl, alkynyl, aryl;
Medical usage, in preparation blood sugar lowering, blood fat reducing and regulate in the medicine of metabolism syndrome and use.
2. the medical usage of baicalin according to claim 1, Scutellarein derivative is characterized in that, regulates in the medicine that metabolism syndrome comprises obesity, type 2 diabetes mellitus and vascular complication and atherosclerotic cardiovascular and cerebrovascular disease in preparation and uses.
3. the medical usage of baicalin according to claim 2, Scutellarein derivative, it is characterized in that, specifically refer to but be not limited to obesity, abdominal obesity, hypertension, hyperinsulinemia, hyperglycemia, type 2 diabetes mellitus, dyslipidemia comprise in the medicine of hypercholesterolemia, hypertriglyceridemia, low hdl mass formed by blood stasis and insulin resistance disease and using at the preparation metabolism syndrome; Described cardiovascular and cerebrovascular disease is meant soldier, cerebrovascular dementia disease in the myocardial infarction that comprises the atherosclerosis relevant with dyslipidemia, macroangiopathy and cause, myocardial ischemia, the brain; Described diabetic vascular complications is meant the diabetic nephropathy that microangiopathies caused, retina injury and the diabetes B lower limb pathological changes that comprises hyperglycemia and diabetes and cause.
4. the medical usage of baicalin according to claim 2, Scutellarein derivative, it is characterized in that, in preparation treatment atherosclerotic cardiovascular disease comprises the medicine of soldier in the myocardial infarction that caused by atherosclerosis and macroangiopathy, myocardial ischemia, the brain or cerebrovascular dementia, use.
5. the medical usage of baicalin according to claim 2, Scutellarein derivative is characterized in that, uses in the medicine of preparation prevention, treatment hyperglycemia, diabetic nephropathy, retina injury and diabetes B lower limb pathological changes.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2013542229A (en) * | 2010-11-12 | 2013-11-21 | 天津医科大学 | Flavonoid derivatives, production methods and uses as pharmaceuticals |
| CN104840962A (en) * | 2015-02-13 | 2015-08-19 | 大连理工大学 | Pharmaceutical composition for improving complications of high-fat and high-sugar diet and application thereof |
| CN110144005A (en) * | 2019-05-29 | 2019-08-20 | 兰州大学 | Two oxidative phosphorylations access key protein relevant to baicalein anticancer activity |
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| WO2021107730A1 (en) * | 2019-11-29 | 2021-06-03 | 기초과학연구원 | Synthetic-baicalein-derivative as chemotherapeutic agent |
| CN113181169A (en) * | 2021-06-23 | 2021-07-30 | 上海市中医医院 | Application of baicalein in preparing medicine for treating hypothalamic obesity |
| CN116162077A (en) * | 2023-02-28 | 2023-05-26 | 苏州普瑞森生物科技有限公司 | Baicalein derivative and preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2421887A1 (en) * | 2002-10-22 | 2004-04-22 | Jenken Biosciences, Inc. | Baicalein and baicalein analogs and uses thereof |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013542229A (en) * | 2010-11-12 | 2013-11-21 | 天津医科大学 | Flavonoid derivatives, production methods and uses as pharmaceuticals |
| CN104840962A (en) * | 2015-02-13 | 2015-08-19 | 大连理工大学 | Pharmaceutical composition for improving complications of high-fat and high-sugar diet and application thereof |
| CN104840962B (en) * | 2015-02-13 | 2017-12-05 | 大连理工大学 | A kind of pharmaceutical composition for improving high fat high-carbonhydrate diet complication and its application |
| CN110538187A (en) * | 2018-05-28 | 2019-12-06 | 北京大学 | CPT1 activator |
| CN110144005A (en) * | 2019-05-29 | 2019-08-20 | 兰州大学 | Two oxidative phosphorylations access key protein relevant to baicalein anticancer activity |
| CN110144005B (en) * | 2019-05-29 | 2021-07-16 | 兰州大学 | Two oxidative phosphorylation pathway key proteins related to baicalein anticancer activity |
| WO2021107730A1 (en) * | 2019-11-29 | 2021-06-03 | 기초과학연구원 | Synthetic-baicalein-derivative as chemotherapeutic agent |
| CN113181169A (en) * | 2021-06-23 | 2021-07-30 | 上海市中医医院 | Application of baicalein in preparing medicine for treating hypothalamic obesity |
| CN116162077A (en) * | 2023-02-28 | 2023-05-26 | 苏州普瑞森生物科技有限公司 | Baicalein derivative and preparation method and application thereof |
| CN116162077B (en) * | 2023-02-28 | 2024-02-02 | 苏州普瑞森生物科技有限公司 | Baicalein derivative and preparation method and application thereof |
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| CN101209257B (en) | 2011-01-19 |
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