CN102813688A - Anti-insulin resistance adenosine monophosphate-activated protein kinase agonist - Google Patents
Anti-insulin resistance adenosine monophosphate-activated protein kinase agonist Download PDFInfo
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- CN102813688A CN102813688A CN2012102871614A CN201210287161A CN102813688A CN 102813688 A CN102813688 A CN 102813688A CN 2012102871614 A CN2012102871614 A CN 2012102871614A CN 201210287161 A CN201210287161 A CN 201210287161A CN 102813688 A CN102813688 A CN 102813688A
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- aaloside
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- araliae chinensis
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Abstract
The present invention discloses an aralia chinensis saponin extract. According to the present invention, aralia chinensis root bark is subjected to reflux extraction with ethanol with a volume concentration percentage of 65-85%; the resulting extract liquid is filtered, and is subjected to merge concentration to obtain a material with a relative density of 1.01; the resulting material is extracted with water-saturated n-butyl alcohol; the resulting n-butyl alcohol layer is filtered, and is subjected to merge concentration to obtain an aralia chinensis saponin extract concrete with an aralia chinensis total saponin mass percentage of 60-80%; and the concrete is dried and crushed to obtain the aralia chinensis saponin extract, wherein the aralia chinensis saponin extract can be used for preparation of adenosine monophosphate-activated protein kinase (AMPK) agonists, the AMPK agonists can be used for a plurality of metabolic diseases such as diabetes, hypertension, dyslipidemia, obesity, and the like, and insulin resistance exists in the metabolic diseases. Compared with AMPK agonists in the prior art, the aralia chinensis saponin of the present invention has characteristics of strong AMPK agonism activity, less adverse reaction, low price, and easy availability.
Description
Technical field
The invention belongs to the biological medicine technology field, particularly single adenosine phosphate activated protein kinase agonist of one type of glucagon opposing.
Background technology
Insulin resistant is ubiquity in diabetic population, and through the overall process of diabetes incidence and development.The incidence rate of insulin resistant surpasses 80% among the type 2 diabetes mellitus patient, and the early prevention and treatment of insulin resistant type 2 diabetes mellitus Susceptible population is especially avoided ill key.In addition, insulin resistant also is present in the multiple metabolic diseases such as obesity, hypertension, dyslipidemia.At present, the medicine (euglycemic agent) that improves insulin resistant mainly comprises metformin and thiazolidinediones.Regrettably, metformin makes the decision of doctor's prescription become difficult further to the side effect of digestive system, blood system etc.And the side effect such as edema, weight increase and myocardial infarction that thiazolidinediones causes also strongly prompting need reappraise to the long-term safety of such medicine.Therefore, seek the novel targets of control insulin resistant and advanced subject and the hot fields that novel drugs is current research.
(adenosine monophosphate-activated protein kinase AMPK) is bringing into play very important effect as " energy sensor " to single adenosine phosphate activated protein kinase in glycolipid metabolism.Activation energy that there are some researches show AMPK in the animal model of insulin resistant is obviously improved insulin resistant.Epidemiological study shows only has 20% people to develop into type 2 diabetes mellitus among the overweight people; All the other people of 80%, (free fatty acid is FFA) to the stimulation of islets of langerhans because free fatty; Insulin secretion strengthens; Hyperinsulinemia occurs, but overcome insulin resistant, do not develop into type 2 diabetes mellitus.According to calendar year 2001 McGarry deciphering again to type 2 diabetes mellitus in ADA; The inventor infers this people of 80% except the influence of congenital gene, and higher activation degree or the stronger susceptibility of AMPK possibly be that they avoid ill major reason in the body.Though reduce the absorption of heat and increase the dystopy deposition that quantity of motion can reverse lipid, insulin resistant significantly improved, however few people can accept this therapeutic strategy for a long time.
Summary of the invention
One of the object of the invention is to provide Yi Zhong Aaloside extract; Prior theory, this invention Ti Gong Aaloside extract is as the purposes of glucagon opposing AMPK agonist.
Implementation procedure of the present invention is following:
Yi Zhong Aaloside extract is characterized in that obtaining through following method for preparing:
It is 65~85% alcohol reflux that the Cortex araliae chinensis root bark is used concentration of volume percent; After extracting liquid filtering, merging simmer down to relative density 1.01; The water saturated n-butanol extraction of reuse; N-butanol layer filtered, merges to concentrate De Cortex araliae chinensis total saponins quality percentage composition is 60-80% De Aaloside extract extractum, with extract dry, pulverize De Aaloside extract.
Shang Shu Aaloside extract can be used for preparing single adenosine phosphate activated protein kinase agonist, comprises being used for the multiple metabolic disease that insulin resistant exists, as: diabetes, hypertension, dyslipidemia, obesity etc.
Shang Shu Aaloside extract is the root bark extract of Tai Bai Cortex araliae chinensis, Liao Dong Cortex araliae chinensis or Aralia decaisneana Hance, is preferably Tai Bai Radix araliae chinensis (Cortex Araliae Chinensis) peel extract.
The present invention uses with the form of conventional pharmaceutical formulation.Described conventional pharmaceutical formulation contain as active component in preparation with pharmaceutically acceptable carrier, this pharmaceutical formulation can be solid form such as tablet, capsule, powder, pill, granule etc.; Also can be liquid form such as injection, Emulsion, suspensoid, microsphere, liposome, nanoparticle etc.
Can contain auxiliary substance, stabilizing agent, wetting agent and other additive commonly used in the above-mentioned preparation, like lactose, citric acid, tartaric acid, magnesium stearate, Gypsum Fibrosum powder, sucrose, corn starch, Pulvis Talci, gelatin, agar, pectin, Oleum Arachidis hypogaeae semen, olive oil, cocoa butter, ethylene glycol, ascorbic acid, mannitol etc.
Above-mentioned preparation can be according to the conventional preparation technology's preparation of various preparations.
Compare with AMPK agonist in the past, the Aaloside of Ben Faming has stronger AMPK agonist activity, and untoward reaction is few, and is cheap, obtains easily.
Description of drawings
Figure 1 Wei Aaloside is to C2C12 myocyte AMPK and target protein acetyl-CoA carboxylase (acetyl-CoA carboxylase, effect ACC).Data all are expressed as mean ± SD (n=3), * *
P<0.01vs. 0min group; ATA: Aaloside.
Figure 2 is the effect of Aaloside to C2C12 myocyte's glucose uptake and fatty acid oxidation, can declare disconnected Aaloside and whether normal myocyte brought into play the effect that increases glucose uptake and promotion fatty acid oxidation through activating AMPK.Left side figure is the glucose uptake amount, and right figure is that (carnitine palmityl transferase-I, CPT-I) activity is if the active increase of CPT-I can promote fatty acid oxidation to Carnitine palmitoyltransferase-I.Data all are expressed as mean ± SD (n=3), *
P<0.05vs. Veh group; Veh: solvent, ATA: Aaloside.
Figure 3 Wei Aaloside are to the effect of the C2C12 myocyte AMPK and the target protein ACC thereof of insulin resistant.Data all are expressed as mean ± SD (n=3), * *
P<0.01vs. 0min group; ATA: Aaloside.
Figure 4 Wei Aaloside is to the C2C12 myocyte's glucose uptake of insulin resistant and the effect of fatty acid oxidation, and whether Ke Pan Duan Aaloside is brought into play the insulin resistant myocyte and increased glucose uptake and the effect that promotes fatty acid oxidation through activating AMPK.Left side figure is a glucose uptake, and right figure is that CPT-I is active, if the active increase of CPT-I can promote fatty acid oxidation.Data all are expressed as mean ± SD (n=3), *
P<0.05vs. Veh group (left figure), *
P<0.05vs. IR group (right figure); Veh: solvent, ATA: Aaloside, Ins: insulin, CON: normal control, IR: insulin resistant.
Figure 5 Wei Aaloside are to the effect of 3T3-L1 adipose cell AMPK.Data all are expressed as mean ± SD (n=5), * *
P<0.01vs. IR group; ATA: Aaloside, AICAR:5-amino-4-Methanamide imidazole nucleus thuja acid (AMPK specific agonist), CON: normal control, IR: insulin resistant.
Figure 6 Wei Aaloside are to the influence of insulin resistance rat food intake dose and body weight thereof, and whether Ke Pan Duan Aaloside the side effect of influential rats eating amount.Left side figure is the body weight increment of insulin resistance rat, and right figure is the food intake dose of insulin resistance rat.Insulin resistance rat is irritated stomach and is given normal saline Huo Aaloside (high dose: 80mg/kg, middle dosage: 160mg/kg, high dose: 320mg/kg) three weeks.Veh: solvent, ATA: Aaloside, CH: standard feed, HF: high lipid food, data all are expressed as mean ± SD (n=8), *
P<0.05; *
P<0.01vs.HF+Veh group.
Figure 7 Wei Aaloside are to the influence of insulin resistance rat serum triglycerides and FFA, and Ke Pan Duan Aaloside is to the therapeutical effect of insulin resistance rat.Left side figure is the serum triglyceride level of insulin resistance rat, and right figure is the FFA level of insulin resistance rat.Insulin resistance rat is irritated stomach and is given normal saline Huo Aaloside (high dose: 80mg/kg, middle dosage: 160mg/kg, high dose: 320mg/kg) three weeks.Veh: solvent, ATA: Aaloside, CH: standard feed, HF: high lipid food, data all are expressed as mean ± SD (n=8), *
P<0.05; *
P<0.01vs.HF+Veh group.
Figure 8 Wei Aaloside are to the influence of insulin resistance rat insulin sensitivity, and Ke Pan Duan Aaloside is to the therapeutical effect of insulin resistance rat.Insulin resistance rat is irritated stomach and is given normal saline Huo Aaloside (high dose: 80mg/kg, middle dosage: 160mg/kg, high dose: 320mg/kg) three weeks.Veh: solvent, ATA: Aaloside, CH: standard feed, HF: high lipid food, data all are expressed as mean ± SD (n=8), *
P<0.05; *
P<0.01vs.HF+Veh group.
The specific embodiment
The exsiccant Tai Bai of 100g Radix araliae chinensis (Cortex Araliae Chinensis) PIFEN is broken and cross 80 mesh sieves, obtain Tai Bai Cortex araliae chinensis medical material fine powder, reuse weight 6 times of amounts, mass percent concentrations are that 70% ethanol extracts backflow 3 times, each 1 h down at 80 ℃; After extracting liquid filtering, merging and simmer down to relative density 1.01; The water saturated n-butyl alcohol of reuse extracts 3 times with the volume ratio of 1:1; With the quality percentage composition of n-butanol layer filtration, merging and concentrated De Cortex araliae chinensis total saponins is the Tai Bai Cortex araliae chinensis extract extractum of 60-80%; Extractum is put drying in 50 ℃ of vacuum desiccators, pulverize De Aaloside dry extract, be used for following experimentation.
Embodiment 1: Aaloside is to the effect of C2C12 myocyte AMPK and target protein ACC thereof
Accomplish the C2C12 myocyte of differentiation, be divided into 5 groups at random: (1) matched group (being the 0min group), C2C12 myocyte; (2) 15min group, C2C12 myocyte gives 1 μ g/ml De Aaloside 15min and collects; (3) 30min group, C2C12 myocyte gives 1 μ g/ml De Aaloside 30min and collects; (4) 60min group, C2C12 myocyte gives 1 μ g/ml De Aaloside 60min and collects; (5) 120min group, C2C12 myocyte gives 1 μ g/ml De Aaloside 120min and collects.
Cell cleans the back with the PBS (pH 7.4) of pre-cooling to 0 ℃ and moves into the lysate of pre-cooling [ TrisHCl 20, and NaCl 50, and NaF 50, Na
4P
2O
750, Sucrose 250, Na
3VO
42, DTT 1 (mmol/l) and 1% compound albumen enzyme inhibitor ] in.BSA protein quantification test kit carries out protein quantification.The capable SDS-PGEA electrophoresis of sample, with half-dried electric commentaries on classics method with albumen transfer to PVDF membrane (PVDF) go up (Bio-Rad, USA).5% defatted milk powder room temperature sealing 1h.Pvdf membrane and one anti-is hatched together, and 4 ℃ are spent the night; With the rinsing of PBST buffer, film is hatched 1h (two anti-be horseradish peroxidase target goat anti-rabbit antibodies) for anti-37 ℃ with coupling two, flush away all unconjugated two anti-after, use the ECL-plus immunity detection reagent to carry out chemiluminescence detection.One anti-anti-AMPK, the anti-phospho-AMPK-Thr of being respectively
172(Cell Signaling Technology, Beverly, MA) specific antibody of anti-ACC and anti-phospho-ACC-Ser79 (Upstate Biotech).After experimental result Biao Ming Aaloside was handled 15min, (see figure 1) was obviously increased in the expression of the AMPK of phosphorylation and ACC among the C2C12 myocyte, and Ti Shi Aaloside is the AMPK agonist to the activation of Skeletal Muscle Cell AMPK and the inhibitory action , Aaloside of ACC.
Embodiment 2: Aaloside are to the effect of C2C12 myocyte's glucose uptake and fatty acid oxidation
Give C2C12 myocyte 1ug/ml Aaloside and handle 15min, utilize the glucose 2-deoxy-[1-of labelled with radioisotope
3H]-glucose detects the picked-up of achievement cell to glucose, and utilize the rate of release of CoA-SH to estimate the activity of CPT-1.Jie fruit Xian Shi Aaloside significantly increases the C2C12 sarcoplast to the picked-up (seeing Fig. 2 left side) of glucose and the activity (seeing that Fig. 2 is right) of CPT-1.
Embodiment 3: Aaloside are to the effect of the C2C12 myocyte AMPK and the target protein ACC thereof of insulin resistant
Set up the C2C12 myocyte of insulin resistant, be divided into 5 groups at random: (1) matched group (being the 0min group), the C2C12 myocyte of insulin resistant; (2) 15min group, the C2C12 myocyte of insulin resistant gives 1 μ g/ml De Aaloside 15min and collects; (3) 30min group, the C2C12 myocyte of insulin resistant gives 1 μ g/ml De Aaloside 30min and collects; (4) 60min group, the C2C12 myocyte of insulin resistant gives 1 μ g/ml De Aaloside 60min and collects; (5) 120min group, the C2C12 myocyte of insulin resistant gives 1 μ g/ml De Aaloside 120min and collects.
The detection method of AMPK and ACC is with embodiment 1; It is the AMPK agonist to the activation of the Skeletal Muscle Cell AMPK of insulin resistant and the inhibitory action , Aaloside of ACC that experimental result Biao Ming Aaloside makes the expression of AMPK and the ACC of phosphorylation among the C2C12 myocyte of insulin resistant obviously increase (see figure 3) Ti Shi Aaloside.
Embodiment 4: Aaloside are to the effect of C2C12 myocyte's glucose uptake and fatty acid oxidation
The C2C12 myocyte 1ug/ml Aaloside that gives insulin resistant is handled 15min, utilizes the glucose 2-deoxy-[1-of labelled with radioisotope
3H]-glucose detects the picked-up of achievement cell to glucose, and utilize the rate of release of CoA-SH to estimate the activity of CPT-1.Jie fruit Xian Shi Aaloside significantly increases the C2C12 sarcoplast of insulin resistant to the picked-up (seeing Fig. 4 left side) of glucose and the activity (seeing that Fig. 4 is right) of CPT-1.
Embodiment 5: Aaloside are to the activation of 3T3-L1 adipose cell AMPK
Set up the 3T3-L1 adipose cell model of insulin resistant, be divided into 4 groups at random: (1) normal control group, the 3T3-L1 adipose cell of normal cultured; (2) insulin resistant model matched group, the 3T3-L1 adipose cell of insulin resistant; (3) Aaloside groups, the 3T3-L1 adipose cell of insulin resistant give 10 μ g/ml De Aaloside; (4) AICAR group, add in the 3T3-L1 adipose cell of insulin resistant AICAR (the AMPK specific agonist, 0.5mmol/l).The detection method of AMPK and ACC can significantly strengthen 3T3-L1 adipose cell AMPK phosphorylation (see figure 5) with embodiment 1 experimental result Biao Ming Aaloside; Prompting AMPK signal path possibly participated in Aaloside, and Aaloside is the AMPK agonist to the regulating action , of adipose cell glycolipid metabolism Er.
Embodiment 6: Aaloside are to the pharmacodynamic study of insulin resistance rat
With the adult Spragure-Dawley rat of healthy male, be divided into 6 groups at random: (1) normal control group, irritate stomach by the dosage of 2ml/kg and give normal saline, once a day, continuous 3 weeks; (2) insulin resistant model matched group, the modeling success was irritated stomach by the dosage of 2ml/kg in back second day and is given normal saline, once a day, continuous 3 weeks; (3) Di Ji Liang Aaloside group, the back dosage of pressing 80mg/kg in second day of modeling success is irritated stomach Gei Yu Aaloside, once a day, continuous 3 weeks; (4) Ji Liang Aaloside group in, the back dosage of pressing 160mg/kg in second day of modeling success is irritated stomach Gei Yu Aaloside, once a day, continuous 3 weeks; (5) Gao Ji Liang Aaloside group, the back dosage of pressing 320mg/kg in second day of modeling success is irritated stomach Gei Yu Aaloside, once a day, continuous 3 weeks.The variation of monitoring body weight and food ration.Monitoring blood glucose, serum insulin calculate insulin sensitivity index.The variation of triglyceride and FFA in the detection serum.Experimental result Biao Ming Aaloside significantly reduces the body weight increment (see figure 6) of insulin resistance rat on the basis that does not influence food ration; Reduce insulin resistance rat serum triglycerides and the horizontal (see figure 7) of FFA, and then improve the insulin resistant (see figure 8).
The result of embodiment 1-6 shows that the: Aaloside can be through activating the effect of AMPK performance treatment insulin resistant; AMPK agonist for the glucagon opposing; Comprise being used for the multiple metabolic disease that insulin resistant exists, as: diabetes, hypertension, dyslipidemia, obesity etc.Aaloside passes through to activate the AMPK signal path and increases glucose uptake, suppresses triglyceride simultaneously and synthesizes, promotes fatty acid oxidation, and then improve insulin resistant, has the excellent development prospect as the AMPK agonist of treating insulin resistant.
Claims (9)
1. Yi Zhong Aaloside extract is characterized in that obtaining through following method for preparing:
It is 65~85% alcohol reflux that the Cortex araliae chinensis root bark is used concentration of volume percent; After extracting liquid filtering, merging simmer down to relative density 1.01; The water saturated n-butanol extraction of reuse; N-butanol layer filtered, merges to concentrate De Cortex araliae chinensis total saponins quality percentage composition is 60-80% De Aaloside extract extractum, with extract dry, pulverize De Aaloside extract.
2. state the Aaloside extract according to claim 1, it is characterized in that: institute's Cortex araliae chinensis root bark of stating is Cortex araliae chinensis, distant eastern Cortex araliae chinensis or Aralia decaisneana Hance root bark too in vain.
3. state the Aaloside extract according to claim 2, it is characterized in that: institute's Cortex araliae chinensis root bark of stating is Cortex araliae chinensis root bark too in vain.
4. the application of one of any Suo Shu Aaloside extract of claim 1 to 3 in the single adenosine phosphate activated protein kinase agonist of preparation.
5. the application of one of any Suo Shu Aaloside extract of claim 1 to 3 in preparation insulin resistant medicine.
6. the application of one of any Suo Shu Aaloside extract of claim 1 to 3 in preparation treatment diabetes medicament.
7. the application of one of any Suo Shu Aaloside extract of claim 1 to 3 in preparation treatment hypertension drug.
8. the application of one of any Suo Shu Aaloside extract of claim 1 to 3 in preparation treatment dyslipidemia medicine.
9. the application of one of any Suo Shu Aaloside extract of claim 1 to 3 in preparation treatment antiobesity agents.
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RU2591081C1 (en) * | 2015-05-19 | 2016-07-10 | Государственное бюджетное образовательное учреждение высшего профессионального образования "Самарский государственный медицинский университет" Министерства здравоохранения Российской Федерации | Method for producing total saponins from manchurian aralia roots |
CN105859675A (en) * | 2016-05-18 | 2016-08-17 | 江西海富生物工程有限公司 | Preparation method of high-purity anthocyanins by extracting from roselle |
CN115806483A (en) * | 2021-09-13 | 2023-03-17 | 深圳大学 | Phenolic heteroterpene compound (+) -Dayaolingzhiol M and application thereof in pharmacy and food |
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CN105859675A (en) * | 2016-05-18 | 2016-08-17 | 江西海富生物工程有限公司 | Preparation method of high-purity anthocyanins by extracting from roselle |
CN115806483A (en) * | 2021-09-13 | 2023-03-17 | 深圳大学 | Phenolic heteroterpene compound (+) -Dayaolingzhiol M and application thereof in pharmacy and food |
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