CN103202897A - Agrimonia eupatoria active component for treating and improving insulin resistance, and application and extraction method thereof - Google Patents
Agrimonia eupatoria active component for treating and improving insulin resistance, and application and extraction method thereof Download PDFInfo
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Abstract
The invention discloses an Agrimonia eupatoria active component for treating and improving insulin resistance, and an application and an extraction method thereof. The Agrimonia eupatoria active component for treating and improving insulin resistance comprises an Agrimonia eupatoria flavone component and an Agrimonia eupatoria triterpene component, and the weight ratio of the Agrimonia eupatoria flavone component to the Agrimonia eupatoria triterpene component is 4:1-1:4. Results of the HLPC analysis of the Agrimonia eupatoria active component show that the Agrimonia eupatoria flavone component mainly contains vitexin, isovitexin, luteolin-7-O-glucoside, quercitrin and tiliroside; and the Agrimonia eupatoria triterpene component mainly contains ursolic acid, 4-hydroxyursolic acid, corosolic acid and oleanolic acid. Experiments about the intervention of the Agrimonia eupatoria active component to the insulin resistance show that the Agrimonia eupatoria active component has an almost same activation effect on PPARgamma with a positive contrast medicine pioglitazone, and can substantially promote the differentiation of preadipocytes; and the Agrimonia eupatoria active component does not have an AP2 overexpression inducing effect like the positive contrast medicine pioglitazone during the PPARgamma expression activation, so the Agrimonia eupatoria active component has the insulin resistance improvement and treatment effects, and can be processed to prepare corresponding formulations for preventing or treating the insulin resistance.
Description
Technical field
The present invention relates to a kind of active component of Herba Agrimoniae, particularly a kind of Herba Agrimoniae active component for the treatment of and improving insulin resistant and use with and extracting method.
Background technology
Insulin resistant refers to that insulin promotes the efficient of glucose uptake and utilization to reduce, show as peripheral tissues especially muscle, fatty tissue to the obstacle that utilizes of glucose, from start to finish through type 2 diabetes mellitus take place, the overall process of development.Particularly nineteen ninety-five Stern has proposed well-known " common soil " theory, enriched the intension of IR, namely obesity, hypertension, hyperlipemia, atherosclerosis, coronary atherosclerotic heart disease, type 2 diabetes mellitus, apoplexy etc. all have common pathologic basis---insulin resistant.If clinical practice at present is in the drug main euglycemic agent thiazolidinediones medicine (TZDs) (as pioglitazone and rosiglitazone) that improves and treat insulin resistant, it is by activating PPAR γ, further optionally promote transcribing of downstream gene, can raise preceding adipose cell, promote its differentiation to reach at the more lipid of born of the same parents' inner accumulated.Increase so this type of medicine often is accompanied by body weight, fat side effect has also increased the weight of cardiovascular danger, and hypoglycemia, general edema, cardiac load, osteoporosis, cancer etc. also are than significant side effects simultaneously.Therefore, the PPAR activator of development of new then has important economy and social value, and middle medical instrument too many levels, many target spots and comparatively safe characteristics, seek and develop selectivity or the incomplete activator of PPAR γ, can when reducing insulin resistant, reduce the side effect such as risk of cardiovascular diseases that cause because of the excessive accumulation of fat.The transcription factor of adipose cell differentiation is mainly before the regulation and control: PPAR γ, ADD1/SREBP-1 and C/EBP-α.Thereby PPAR γ can regulate and control the expression of C/EBP-α, lipid metabolism key enzyme, fatty secretory protein, fatty active factors participates in the adipose cell differentiation process.The transhipment of fatty acid and synthetic is the crucial actuator of periphery insulin sensitivity and glycolipid metabolism in the adjustable cell of AP2.PPAR γ is the important regulatory factor of AP2 gene expression, by regulating the PPAR response element (PPRE) in its gene promoter, increases the expression of AP2 in the adipose cell, thereby promotes the differentiation of adipose cell, but increased the accumulation of born of the same parents' inner lipids simultaneously.Therefore the AP2 overexpression can cause the disorder of normal lipid metabolism balance.Studies show that, use the inhibitor of AP2 and the agonist of PPAR γ all can make the periphery insulin sensitivity enhancing.
Herba Agrimoniae is the herb of Rosaceae herbaceos perennial Radix Agrimoniae (Agrimonia Pilosa Ledeb), and the traditional Chinese medical science is mainly used in controlling spitting of blood, spitting blood, have blood in stool, bleeding not during menses, and impairment caused by overstrain is taken off power, carbuncle, wound hemorrhage etc.The Chemical Constituents to Herba Agrimoniae shows that it contains polyphenol, flavone and flavone glycoside compounds, triterpenoid compound and agrimonolide etc. at present.And do not have the Herba Agrimoniae active component to be used for the treatment of and to improve the report of insulin resistant as yet at present.
Summary of the invention
The present invention is primarily aimed at the Herba Agrimoniae active component to the intervention of insulin resistant, and discovery Herba Agrimoniae active component does not only have significant difference to activation and the positive control medicine pioglitazone of PPAR γ, the differentiation of adipose cell before can significantly promoting; And the overexpression of when activating PPAR γ expression, inducing AP2 unlike positive control medicine pioglitazone sample, show that the Herba Agrimoniae active component has the effect that improves and treat insulin resistant.
A kind of Herba Agrimoniae active component for the treatment of and improving insulin resistant of the present invention, this active component comprise the compositions of flavonoid component in the Herba Agrimoniae or Herba Agrimoniae triterpene component or Herba Agrimoniae flavonoid component and Herba Agrimoniae triterpene component.
Further, the mass ratio of flavonoid component and triterpene component is 4:11 in the compositions of described flavonoid component and triterpene component: 4.
Further, the mass ratio of described Herba Agrimoniae flavonoid component and Herba Agrimoniae triterpene component is 4:1;
Further, the mass percentage content of described Herba Agrimoniae flavonoid component total flavones is not less than 30%, and the mass percentage content of the total triterpene of Herba Agrimoniae triterpene component is not less than 40%;
Further, described Herba Agrimoniae is the Rosaceae agrimony, comprises the Radix Agrimoniae in multiple tooth Radix Agrimoniae, Xiao Hua Radix Agrimoniae, Flos Caryophylli Radix Agrimoniae, stipule Radix Agrimoniae and the Europe in tooth grass, HUANGLONG tail, the cognate dragon of dredging a mao Radix Agrimoniae, northeast Korea Radix Agrimoniae, northeast, Daxing'an Mountainrange, northeast.
The present invention also discloses a kind of Herba Agrimoniae active component for the treatment of and improving insulin resistant in the preparation treatment and improves application in the insulin resistant medicine.
The present invention also discloses a kind of extracting method for the treatment of and improving the Herba Agrimoniae active component of insulin resistant, it is characterized in that: may further comprise the steps:
A. getting the Herba Agrimoniae herb is to be dried to constant weight under 105-115 ℃ and to be crushed to the 35-45 order in temperature, add 95% ethanol then and after temperature is 85-95 ℃ of reflux, extract, 3 times, filter, extracted 2 hours at every turn, filter, collect filtrate and filtering residue, the filtrate decompression of collecting is concentrated make extractum;
B. after refluxing 2 hours, the active carbon that the extractum among the step a is added 10% extractum weight after with 95% ethanol dispersing and dissolving filters and collects filtrate, collected filtrate is concentrated the back with the last silicagel column of sample: silica gel=1:30, use ethyl acetate: petroleum ether=1:1 successively, ethyl acetate: petroleum ether=5:1 and pure ethyl acetate eluting, 5 column volumes of each eluting, collect respectively eluent (I), eluent (II), eluent (III);
C. eluent (II) and eluent (III) are dissolved in the mixed liquor that volume ratio is chloroform: methanol=1:1, last LH-20 gel column detects analysis through thin layer chromatography, obtains Herba Agrimoniae triterpene component and flavonoid component;
D. with the filtering residue among the step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the back with 60% ethanol dispersing and dissolving, regulating PH is 5.0, slowly add 5.0% gelatin solution then till no longer produce precipitation, remove by filter that to add 95% ethanol to concentration of alcohol behind the tannalbin again be to remove excessive gelatin and the albumen in the extract and polysaccharide at 85% o'clock, filter then and filtrate is concentrated, make extractum; This extractum is dissolved in 80% methanol, and last LH-20 gel column detects analysis through thin layer chromatography, obtains flavonoid component.
E. the flavonoid component in combining step c and the steps d;
Further, getting the Herba Agrimoniae herb among the step a is to be dried to constant weight under 110 ℃ and to be crushed to 40 orders in temperature, adds 95% ethanol then and filters after temperature is 90 ℃ of reflux, extract, 3 times;
Further, among the step b, the extractum among the step a with 95% ethanol dispersing and dissolving, after refluxing 2 hours, the active carbon that adds 10% extractum weight is then filtered and collects filtrate.
Beneficial effect of the present invention: treatment of the present invention and improve the Herba Agrimoniae active component of insulin resistant and use with and extracting method, the active substance group of Herba Agrimoniae is flavonoid component and triterpene component.Learn by the HPLC analysis: mainly contain apigenin-8-C-glucoside, Saponaretin and luteolin-7-O-glucoside, Quercitroside, silver linden glycoside in the flavonoid component of Herba Agrimoniae; Mainly contain ursolic acid, 4-hydroxyl ursolic acid, Corosolic acid and oleanolic acid in the Herba Agrimoniae triterpene component.At the intervention of Herba Agrimoniae active component to insulin resistant, discovery Herba Agrimoniae active component does not only have significant difference to activation and the positive control medicine pioglitazone of PPAR γ, the differentiation of adipose cell before can significantly promoting; And the overexpression of when activating PPAR γ expression, inducing AP2 unlike positive control medicine pioglitazone sample, show that the Herba Agrimoniae active component has the effect that improves and treat insulin resistant, can be prepared into corresponding dosage form and prevent or treat insulin resistant.
Description of drawings
Fig. 1 is the HPLC chromatogram of Herba Agrimoniae flavonoid component (FC):
1, apigenin-8-C-glucoside 2, Saponaretin 3, hyperin 4, luteolin-7-O-glucoside 5, Quercitroside 8, Quercetin 9, silver linden glycoside 10, luteolin 11, rutin 12, apigenin 13, kaempferol 15, Taxifolin 16, catechin compounds 6,7,14-are undetermined;
Fig. 2 is the HPLC chromatogram of Herba Agrimoniae triterpene component (TC): No. 4 chemical compounds: 4-hydroxyl ursolic acid, No. 6 chemical compounds: maslinic acid, No. 7 chemical compounds: section's sieve carboxylic acid, No. 9 chemical compounds: oleanolic acid, No. 10 chemical compounds: ursolic acid, chemical compound 1,2,3,5,8 undetermined;
Fig. 3 handles the preceding adipose cell of 3T3-L1 for Concentraton gradient Herba Agrimoniae flavonoid component (FC) and induces the 7th day microscope figure (x200) of differentiation;
Adipose cell was induced the 7th day microscope figure (x200) of differentiation before Fig. 4 Concentraton gradient Herba Agrimoniae triterpene component (TC) was handled 3T3-L1;
Adipose cell was induced the 7th day oil red colored graph (x200) of differentiation before Fig. 5 Concentraton gradient Herba Agrimoniae flavonoid component (FC) was handled 3T3-L1;
Adipose cell was induced the 7th day oil red colored graph (x200) of differentiation before Fig. 6 Concentraton gradient Herba Agrimoniae triterpene component (TC) was handled 3T3-L1;
The different proportioning components of two active components of Fig. 7 Herba Agrimoniae and two components are to triacylglycerol effect of accumulation in the preceding adipose cell atomization;
Fig. 8 Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) are broken up the regulation and control of the gene expression of main transcription factor to preceding adipose cell;
The different proportioning components with Herba Agrimoniae triterpene component (TC) of Fig. 9 Herba Agrimoniae flavonoid component (FC) are broken up the regulation and control that main transcription factor gene is expressed to preceding adipose cell;
The regulation and control that Figure 10 Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) are expressed preceding adipose cell differentiation AP2;
The regulation and control that the different proportioning components with Herba Agrimoniae triterpene component (TC) of Figure 11 Herba Agrimoniae flavonoid component (FC) are expressed preceding adipose cell AP2.
The specific embodiment
Embodiment one
A. getting Herba Agrimoniae herb 1.0kg, is to be dried to constant weight under 110 ℃ and to be crushed to 40 orders in temperature, adds 95% ethanol then and filters after temperature is 90 ℃ of reflux, extract, 3 times, the each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate decompression of collecting is concentrated make extractum 140g;
B. after refluxing 2 hours, the active carbon that the extractum among the step a is added 10% extractum weight after with 95% ethanol dispersing and dissolving filters and collects filtrate, silicagel column on concentrating collected filtrate afterwards (sample: silica gel=1:30), use ethyl acetate: petroleum ether=1:1 successively, 5:1 and pure ethyl acetate eluting, 5 column volumes of each eluting, collect respectively eluent (I), eluent (II), eluent (III);
C. eluent (II) and eluent (III) are dissolved in the mixed liquor that volume ratio is chloroform: methanol=1:1, last LH-20 gel column detects analysis through thin layer chromatography, obtains Herba Agrimoniae triterpene component and flavonoid component;
D. with the filtering residue among the step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the back with 60% ethanol dispersing and dissolving, regulating PH is 5.0, slowly add 5.0% gelatin solution then till no longer produce precipitation, remove by filter that to add 95% ethanol to concentration of alcohol behind the tannalbin again be to remove excessive gelatin and the albumen in the extract and polysaccharide at 85% o'clock, filter then and filtrate is concentrated, make extractum; This extractum is dissolved in 80% methanol, and last LH-20 gel column detects analysis through thin layer chromatography, obtains flavonoid component.
E. the flavonoid component in combining step c and the steps d.
The general flavone content that spectrophotography records Herba Agrimoniae flavonoid component (FC) is 316.53 ± 6.37mg/g, and the total triterpene contents of Herba Agrimoniae triterpene component (TC) is 415.96 ± 5.15mg/g.
Being that 4:1 is conventional with the mass ratio with the Herba Agrimoniae flavonoid component (FC) that makes and Herba Agrimoniae triterpene component (TC) granulates, and makes capsule, tablet or granule etc.
Embodiment two
A. getting Herba Agrimoniae herb 1.0kg, is to be dried to constant weight under 105 ℃ and to be crushed to 35 orders in temperature, adds 95% ethanol then and filters after temperature is 85 ℃ of reflux, extract, 3 times, the each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate decompression of collecting is concentrated make extractum;
B. after refluxing 2 hours, the active carbon that the extractum among the step a is added 5% extractum weight after with 95% ethanol dispersing and dissolving filters and collects filtrate, silicagel column on concentrating collected filtrate afterwards (sample: silica gel=1:30), use ethyl acetate: petroleum ether=1:1 successively, 5:1 and pure ethyl acetate eluting, 5 column volumes of each eluting, collect respectively eluent (I), eluent (II), eluent (III);
C. eluent (II) and eluent (III) are dissolved in the mixed liquor that volume ratio is chloroform: methanol=1:1, last LH-20 gel column detects analysis through thin layer chromatography, obtains Herba Agrimoniae triterpene component and flavonoid component;
D. with the filtering residue among the step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the back with 60% ethanol dispersing and dissolving, regulating PH is 5.0, slowly add 5.0% gelatin solution then till no longer produce precipitation, remove by filter that to add 95% ethanol to concentration of alcohol behind the tannalbin again be to remove excessive gelatin and the albumen in the extract and polysaccharide at 85% o'clock, filter then and filtrate is concentrated, make extractum; This extractum is dissolved in 80% methanol, and last LH-20 gel column detects analysis through thin layer chromatography, obtains flavonoid component.
E. the flavonoid component in combining step c and the steps d.
The general flavone content that spectrophotography records Herba Agrimoniae flavonoid component (FC) is 316.53 ± 6.37mg/g, and the total triterpene contents of Herba Agrimoniae triterpene component (TC) is 415.96 ± 5.15mg/g.
Being that 3:2 is conventional with the mass ratio with the Herba Agrimoniae flavonoid component (FC) that makes and Herba Agrimoniae triterpene component (TC) granulates, and makes capsule, tablet, granule etc.
Embodiment three
A. getting Herba Agrimoniae herb 1.0kg, is to be dried to constant weight under 115 ℃ and to be crushed to 45 orders in temperature, adds 95% ethanol then and filters after temperature is 90 ℃ of reflux, extract, 3 times, the each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate decompression of collecting is concentrated make extractum;
B. after refluxing 2 hours, the active carbon that the extractum among the step a is added 15% extractum weight after with 95% ethanol dispersing and dissolving filters and collects filtrate, silicagel column on concentrating collected filtrate afterwards (sample: silica gel=1:30), use ethyl acetate: petroleum ether=1:1 successively, 5:1 and pure ethyl acetate eluting, 5 column volumes of each eluting, collect respectively eluent (I), eluent (II), eluent (III);
C. eluent (II) and eluent (III) are dissolved in the mixed liquor that volume ratio is chloroform: methanol=1:1, last LH-20 gel column detects analysis through thin layer chromatography, obtains Herba Agrimoniae triterpene component and flavonoid component;
D. with the filtering residue among the step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the back with 60% ethanol dispersing and dissolving, regulating PH is 5.0, slowly add 5.0% gelatin solution then till no longer produce precipitation, remove by filter that to add 95% ethanol to concentration of alcohol behind the tannalbin again be to remove excessive gelatin and the albumen in the extract and polysaccharide at 85% o'clock, filter then and filtrate is concentrated, make extractum; This extractum is dissolved in 80% methanol, and last LH-20 gel column detects analysis through thin layer chromatography, obtains flavonoid component.
E. the flavonoid component in combining step c and the steps d.
The general flavone content that spectrophotography records Herba Agrimoniae flavonoid component (FC) is 316.53 ± 6.37mg/g, and the total triterpene contents of Herba Agrimoniae triterpene component (TC) is 415.96 ± 5.15mg/g.
Being that 2:3 is conventional with the mass ratio with the Herba Agrimoniae flavonoid component (FC) that makes and Herba Agrimoniae triterpene component (TC) granulates, and makes capsule or tablet etc.
Embodiment four
A. getting Herba Agrimoniae herb 1.0kg, is to be dried to constant weight under 110 ℃ and to be crushed to 45 orders in temperature, adds 95% ethanol then and filters after temperature is 85 ℃ of reflux, extract, 3 times, the each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate decompression of collecting is concentrated make extractum;
B. after refluxing 2 hours, the active carbon that the extractum among the step a is added 10% extractum weight after with 95% ethanol dispersing and dissolving filters and collects filtrate, silicagel column on concentrating collected filtrate afterwards (sample: silica gel=1:30), use ethyl acetate: petroleum ether=1:1 successively, 5:1 and pure ethyl acetate eluting, 5 column volumes of each eluting, collect respectively eluent (I), eluent (II), eluent (III);
C. eluent (II) and eluent (III) are dissolved in the mixed liquor that volume ratio is chloroform: methanol=1:1, last LH-20 gel column detects analysis through thin layer chromatography, obtains Herba Agrimoniae triterpene component and flavonoid component;
D. with the filtering residue among the step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the back with 60% ethanol dispersing and dissolving, regulating PH is 5.0, slowly add 5.0% gelatin solution then till no longer produce precipitation, remove by filter that to add 95% ethanol to concentration of alcohol behind the tannalbin again be to remove excessive gelatin and the albumen in the extract and polysaccharide at 85% o'clock, filter then and filtrate is concentrated, make extractum; This extractum is dissolved in 80% methanol, and last LH-20 gel column detects analysis through thin layer chromatography, obtains flavonoid component.
E. the flavonoid component in combining step c and the steps d.
The general flavone content that spectrophotography records Herba Agrimoniae flavonoid component (FC) is 316.53 ± 6.37mg/g, and the total triterpene contents of Herba Agrimoniae triterpene component (TC) is 415.96 ± 5.15mg/g.
Being that 1:4 is conventional with the mass ratio with the Herba Agrimoniae flavonoid component (FC) that makes and Herba Agrimoniae triterpene component (TC) granulates, and makes capsule or agent etc.
Embodiment five
The HPLC material of Herba Agrimoniae flavonoid component (FC) and triterpene component (TC) characterizes
1. experiment material method
1.1 reagent and instrument
1.1.1 reagent
1. reference substance: Quercitroside, ursolic acid, kaempferol, silver linden glycoside, Quercetin, luteolin, apigenin, luteoloside, Du chaste tree glycosides, different Du chaste tree glycosides, Taxifolin, rutin, section's sieve carboxylic acid, oleanolic acid, 4-hydroxyl ursolic acid and maslinic acid are available from middle inspection institute
2. acetonitrile, methanol are the import chromatographically pure
3. other reagent is homemade analytical pure
1.1.2 instrument
1. precise electronic balance FA2004 type, Shanghai Precision Scientific Apparatus Co., Ltd
2. high performance liquid chromatograph Agilent1200, Anjelen Sci. ﹠ Tech. Inc
1.1.3 the preparation of sample and reference substance solution
1. accurately take by weighing Herba Agrimoniae flavonoid component (FC) 10.0mg, dissolve with methanol is dissolved in the 10mL volumetric flask surely, and is to be measured.
2. accurately take by weighing Herba Agrimoniae triterpene component (FC) 10.0mg, dissolve with methanol is dissolved in the 10mL volumetric flask surely, and is to be measured.
3. Quercitroside, ursolic acid, kaempferol, silver linden glycoside, Quercetin, luteolin, apigenin, luteoloside, Du chaste tree glycosides, different Du chaste tree glycosides, Taxifolin, rutin, section's sieve carboxylic acid, oleanolic acid, 4-hydroxyl ursolic acid and each personal dissolve with methanol of maslinic acid reference substance are mixed with the solution for later use of 0.5mg/mL.
1.2 method
1.2.1 the HPLC material of Herba Agrimoniae flavonoid component characterizes
Chromatographic column: Welch Materials Ultimate XB-C184.6x250mm, 5 μ m; Mobile phase: mobile phase A is 0.1% phosphate aqueous solution, Mobile phase B: acetonitrile; Detect wavelength: 350nm, column temperature: 35 ℃, flow velocity: 1.0mL/min, sample size: 20 μ L
Condition of gradient elution such as table 1:
The HPLC eluent gradient table of table 1 Herba Agrimoniae flavonoid component (FC)
1.2.2 the HPLC material of Herba Agrimoniae triterpene component characterizes
Chromatographic column: Welch Materials Ultimate XB-C184.6x250mm, 5 μ m
Mobile phase: mobile phase A is 0.1% phosphate aqueous solution, and Mobile phase B is acetonitrile: methanol=2:1
Detect wavelength: 210nm, column temperature: 30 ℃, flow velocity: 1.0mL/min, sample size: 20 μ L
Condition of gradient elution such as table 2:
The HPLC eluent gradient table of table 2 Herba Agrimoniae triterpene component (TC)
1.3 result and analysis
The HPLC chromatogram of Herba Agrimoniae flavonoid component (FC) is seen Fig. 1.Determine that by the reference substance additive process Herba Agrimoniae flavonoid component (FC) mainly contains apigenin-8-C-glucoside, Saponaretin, hyperin, Quercitroside, Quercetin, silver linden glycoside, luteolin, rutin, apigenin, kaempferol, Taxifolin, catechin and luteolin-7-O-glucoside.
The HPLC chromatogram of Herba Agrimoniae triterpene component (TC) is seen Fig. 2, determines that by the reference substance additive process Herba Agrimoniae TC mainly contains ursolic acid, 4-hydroxyl ursolic acid, section's sieve carboxylic acid, maslinic acid and oleanolic acid.
Embodiment six
Active component flavonoid component (FC) and triterpene component (TC) and the two different proportioning are to the facilitation of adipose cell differentiation in the Herba Agrimoniae
1, experiment material and method
1.1 reagent and instrument
1.1.1 reagent D MEM high glucose medium, hyclone, calf serum, two anti-, pancreatin, Hyclone company; Oil red O, Sigma company; Dexamethasone, IBMX, insulin, sigma company; Trizol, invitrogen company; CDNA transcript reagent box, Takara company; DEPC removes enzyme water, green skies company; Bio-Rad SYBgreen, Bio-Rad quantitative fluorescent PCR pipe/pipe lid, Bio Rad Laboratories; MTT is available from sigma company; Tissue Culture Plate (6 holes, 12 holes, 96 holes), Hyclone company
1.1.2 instrument precise electronic balance FA2004 type, Shanghai Precision Scientific Apparatus Co., Ltd; The CO2 cell culture incubator, U.S. THERMO company; Microplate reader Model680, Bio-Rad, USA; Quantitative real time PCR Instrument CYMMZ-EPXFY-052, Bio-Rad, USA; Eppendorf refrigerated centrifuger-5417R, the gene company limited; The RNA/DNA ultraviolet spectrophotometer, U.S. beckman company; Just putting microscope CYMML-EPTXCJ-004, OLYMPUS
1.2, adipose cell induction and drug treating before the 3T3-L1
Experimental group is that Herba Agrimoniae sample variable concentrations is handled (each sample arranges four gradients of 1,5,25 and 125 μ g/mL), and positive controls is pioglitazone (10 μ M), and the blank group is blank solvent.With the cell that is fused to 90%, be inoculated in 6 orifice plates or 12 orifice plates, cultivate with the DMEM high glucose medium that contains two 10% calf serums that resist, every other day change a subculture, cultivated 2 days, treat the fusion of cell 100%; The first phase is induced differentiation: change the MDI induced liquid, add sample and the positive control solution of the Herba Agrimoniae of variable concentrations, cultivated 2 days; The second phase is induced differentiation: abandon culture fluid, change insulin-induced liquid, add sample and the positive control solution of the Herba Agrimoniae of variable concentrations, continue to cultivate 2 days; The third phase is induced differentiation: abandon culture fluid, change the DMEM height sugar that contains 10% hyclone and cultivate, add sample and the positive control solution of the Herba Agrimoniae of variable concentrations simultaneously, continue to cultivate 2-4 days, wait the preceding adipose cell differentiation and maturation of 3T3-L1, the observation of cell form.
1.3, Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) be to the influence of cell differentiation form
With adipose cell before the 7th day the 3T3-L1 of differentiation in 6 orifice plates, carefully draw the supernatant culture medium, discard then.Get 1mLPBS, inject along the culture plate hole wall, leave standstill 5min with the rolling of have gentle hands jog, draw PBS then, wash 3 times with PBS solution; Clean up after culture medium and the medicine formaldehyde fixed liquid with 10% (the every hole 1mL of 24 orifice plates) fixed cell 30-60min; PBS dries 30min with cell after cleaning; Add the oil red O working solution configure (oil red storage liquid: the every hole 0.5mL of distilled water=3:2), dyeing 40-60min(lucifuge under the room temperature, sealing) oil red O dye liquor, wash cell with 60% isopropyl alcohol and remove unnecessary dyestuff for 2 times and wash 3 times with tri-distilled water again.With glycerol or glycerin gelatine mounting; Observed result under the inverted microscope is taken pictures.
1.4, the oil red staining deposition degree of observing triglyceride
Kind there are 24 orifice plates of cell, carry out inducing culture, took out in the 7th day and to carry out oil red dyeing; Wash cell 2 times with 60% isopropyl alcohol and remove excess dyestuff, with tri-distilled water washing 3 times, add 100% isopropyl alcohol 1mL, the rifle head is blown and beaten repeatedly, then at the shaker 20min that vibrates; Use microplate reader, measure light absorption value at the 520nm place.
2, result
2.1, Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) be to the influence of cell differentiation form
As Fig. 3 and Fig. 4, do not induce the preceding adipose cell of 3T3-L1 of differentiation to be the state of long shuttle shape, after cell induction broke up 7 days, the differentiation and maturation adipose cell was circular as can be seen, tangible lipid accumulation is arranged simultaneously in born of the same parents, and significantly " finger ring fat drips " enrichment is arranged in born of the same parents; More as can be seen, Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) have the effect of tangible promotion cell differentiation from the form of cell, and the differentiation of adipose cell before promoting in the mode of dose dependent; And come comparison from the size of fat drop, little than Herba Agrimoniae triterpene component (TC) and positive control under Herba Agrimoniae flavonoid component (FC) is handled.
2.2, the oil red staining deposition degree of observing triglyceride
Can learn by oil red colored graph 5 and 6: along with the differentiation of adipose cell before the 3T3-L1, the triacylglycerol content in the born of the same parents builds up, and the size that fat drips and quantity and blank group are handled evident difference; Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) all present the generation that promotes triacylglycerol in the mode of dose dependent.As Fig. 7, wherein, * has significant difference (p＜0.5) for comparing with blank, and * * has utmost point significant difference (p＜0.1) for comparing with blank; A is triacylglycerol content effect after the drug treating of variable concentrations of flavonoid component, B is the triacylglycerol content effect after the drug treating of variable concentrations gradient of triterpene component, C figure is the effect to triacylglycerol content when 50 μ g/mL of the component of flavonoid component and the different proportionings of triterpene component: I is FC:TC=1:4, II is FC:TC=1:1, and III is FC:TC=4:1.A shows the effect of the short triacylglycerol content of Herba Agrimoniae flavonoid component (FC) tool, and 5-25 μ g/mL concentration range with blank tool significant difference, when reaching 125 μ g/mL, has utmost point significant difference, but not as positive control; B shows that Herba Agrimoniae triterpene component (TC) tool promotes the effect of triacylglycerol accumulation, during 5 μ g/mL, compares with blank and to have significant difference, and when reaching 25-125 μ g/mL, tool utmost point significant difference, but all not as the effect of positive control; C shows, compares with blank to have remarkable facilitation when FC:TC=4:1, and when FC:TC=1:4 tool utmost point significant difference, TC has the effect of stronger promotion triacylglycerol deposition than FC.
3, conclusion
1. adipose cell before the 3T3-L1 is induced differentiation with classical cocktail revulsion, observe flavonoid component (FC) and two synchronous drug treating of active component of triterpene component (TC) of Herba Agrimoniae and induce the 7th day cellular morphology of differentiation, can observe cell and become circle by long shuttle shape, in born of the same parents, can observe significantly " finger ring fat drips ", illustrate that Herba Agrimoniae active component FC and TC have the effect of obvious promotion cell differentiation.2. show by oil red colored graph result and triacylglycerol assay that FC and TC all promote the triacylglycerol content in the adipose cell to increase in the mode of concentration dependent; Data analysis learns that TC is than the stronger facilitation of the triacylglycerol of FC after the drug treating of the compounding ingredients of the two three different proportioning simultaneously.Above description of test Herba Agrimoniae active component FC and TC have facilitation to the differentiation of preceding adipose cell.
Embodiment seven
Active component is to the regulating and controlling effect of adipose cell differentiation and Genes Associated with Lipid Metabolism expression in the Herba Agrimoniae
1, experiment material and method
1.1, reagent and instrument
1.1.1 reagent is with embodiment one
1.1.2 instrument is with embodiment one
1.2, extraction, RT-PCR and the real-time PCR of the mRNA of adipose cell before the 3T3-L1
1. get and induce the 7th day cell ice bath of differentiation, PBS washes 2 times.Add 0.9mL Trizol lysate.Each hole lysate is collected into respectively in the 1.5mL EP pipe.All add the 0.2mL chloroform in each EP pipe, vibration 15S treats to leave standstill 3min into milky white shape.4 ℃ of 12000g leave heart 15min.Draw supernatant, add the 0.5mL isopropyl alcohol, mixing leaves standstill 10min.4 ℃ of 12000g leave heart 10min.Abandon supernatant, add 75% ethanol 1mL, 4 ℃ of 7500g leave heart 5min.Drying at room temperature adds 25 μ LDEPC water dissolutioies precipitation.Ultraviolet spectrometry detectable concentration and purity RNA concentration determination.Get 7 μ L mixing transcript reagents, represent RNA concentration by V=1/C(C) add the RNA extracting solution, it is 20 μ L that adding DEPC water makes cumulative volume.Carry out reverse transcription, cDNA is in-20 ℃ of preservations.Get the cDNA solution of 10 times of 2 μ L dilutions, mix with 8 μ L and transcribe liquid (upstream and downstream primer 1.4 μ L/ holes, SYB green μ L/ hole, DEPC water μ L/ hole) mixing.Undertaken by table 4 reaction condition
Table 3 primer sequence
The Protocol of table 4 quantitative fluorescent PCR
2, result
2.1, extraction, RT-PCR and the real-time PCR of the mRNA of adipose cell before the 3T3-L1
2.1.1, main transcription factor C/EBP-α, the PPAR γ of the differentiation of preceding adipose cell and the expression regulation of ADD1/SREBP-1
As Fig. 8, wherein, * (p＜0.05) * * (p＜0.01): refer to the comparison of FC experimental group and its blank group; #(p＜0.05) ##(p＜0.01): refer to the comparison of TC experimental group and its blank group; ﹠amp; ﹠amp; (p＜0.01): refer to that FC and its positive controls are relatively; $$(p＜0.01): refer to the comparison of TC and its positive controls.The concentration of expression of gene and FC and TC is the increase of dose dependent.When FC is 25 μ g/mL and 125 μ g/mL, the expression of PPAR γ respectively with matched group significant difference and utmost point significant difference, when TC was 125 μ g/mL, expressing had significant difference; When FC and TC were 125 μ g/mL, it expressed no significant difference; During 125 μ g/mL, the expression of the C/EBP-α mRNA of FC and TC relatively has remarkable increase with blank group, and has compared reduction extremely significantly with positive control; During 125 μ g/mL, the mRNA expression of SREBP-1 is maximum, compares with the blank group to have the significance increase, and compares no remarkable statistical discrepancy with the expression of positive control.As Fig. 9, * (p＜0.05) * * (p＜0.01): the C/EBP-α mRNA that refers to the drug treating group of component of different proportionings of FC and TC and positive controls express with the blank group relatively; ﹠amp; (p＜0.05): refer to the drug treating group of component of different proportionings of FC and TC and positive control SREBP-1mRNA express with the blank group relatively; $(p＜0.05) $$(p＜0.01): the PPAR γ mRNA that refers to the drug treating group of component of different proportionings of FC and TC and positive controls express with the blank group relatively.C/EBP-α mRNA is when FC and TC proportioning are 1:150 μ g/mL, and comparing tool with the blank group increases significantly, and under proportioning 1:4 and the 4:1, does not have significant difference; For SREBP-1, three kinds of proportionings more all do not have remarkable significant difference with the blank group when 50 μ g/mL; To the expression analysis of PPAR γ as can be known, when FC:TC4:150 μ g/mL, compared the most significant increase with the blank group.2.1.2, the expression regulation of Genes Associated with Lipid Metabolism AP2
As Figure 10, wherein, * (p＜0.05) * * (p＜0.01): refer to the comparison of FC experimental group and its blank group; #(p＜0.05) ##(p＜0.01): refer to the comparison of TC experimental group and its blank group; ﹠amp; ﹠amp; (p＜0.01): refer to that FC and positive controls are relatively; $$(p＜0.01): refer to the comparison of TC and positive controls; The expression of AP2 presents the metering dependency increases relation.During TC25 μ g/mL, the expression of AP2mRNA and blank group tool significantly strengthen, and during 125 μ g/mL, FC and TC express and all relatively tool significance enhancings (p＜0.05) of blank group, are reduced to positive controls (p＜0.01) but all show.As Figure 11, ， ﹠amp wherein; (p＜0.05) ﹠amp; ﹠amp; (p＜0.01): the AP2mRNA of drug treating group of component that refers to the different proportionings of FC and TC express with the blank group relatively; When FC:TC was 1:4, the expression ratio blank group of AP2mRNA had significance to increase (p＜0.05), and 2 kinds of proportionings are expressed no difference of science of statistics to it in addition.
3, conclusion
1. two of Herba Agrimoniae FC and TC active components obviously promote PPAR γ, C/EBP-α, SREBP-1 to activate expression at the finite concentration tool; But the downstream of FC and TC controlling gene AP2 has certain facilitation is not strong with respect to the positive controls significance.2. the FC of Herba Agrimoniae and TC nature proportioning (4:1) has good advantage in the effect proportioning.Found through experiments, Herba Agrimoniae active component flavone and triterpene part have comparatively significantly effect in improvement and opposing insulin resistant.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not breaking away from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (9)
1. Herba Agrimoniae active component for the treatment of and improving insulin resistant is characterized in that: this active component comprises the compositions of flavonoid component in the Herba Agrimoniae or Herba Agrimoniae triterpene component or Herba Agrimoniae flavonoid component and Herba Agrimoniae triterpene component.
2. treatment according to claim 1 and improve the Herba Agrimoniae active component of insulin resistant is characterized in that:
The mass ratio of flavonoid component and triterpene component is 4:1-1:4 in the compositions of described flavonoid component and triterpene component.
3. treatment according to claim 2 and improve the Herba Agrimoniae active component of insulin resistant, it is characterized in that: the mass ratio of described Herba Agrimoniae flavonoid component and Herba Agrimoniae triterpene component is 4:1.
4. treatment according to claim 3 and improve the Herba Agrimoniae active component of insulin resistant, it is characterized in that: the mass percentage content of described Herba Agrimoniae flavonoid component total flavones is not less than 30%, and the mass percentage content of the total triterpene of Herba Agrimoniae triterpene component is not less than 40%.
5. according to the described treatment of the arbitrary claim of claim 1-4 with improve the Herba Agrimoniae active component of insulin resistant, it is characterized in that: described Herba Agrimoniae is the Rosaceae agrimony, comprises the Radix Agrimoniae in multiple tooth Radix Agrimoniae, Xiao Hua Radix Agrimoniae, Flos Caryophylli Radix Agrimoniae, stipule Radix Agrimoniae and the Europe in tooth grass, HUANGLONG tail, the cognate dragon of dredging a mao Radix Agrimoniae, northeast Korea Radix Agrimoniae, northeast, Daxing'an Mountainrange, northeast.
6. the described treatment of claim 1 and the Herba Agrimoniae active component of improving insulin resistant are in the preparation treatment with improve application in the insulin resistant medicine.
7. the described treatment of claim 1 and improve the extracting method of the Herba Agrimoniae active component of insulin resistant is characterized in that: may further comprise the steps:
A. getting the Herba Agrimoniae herb is to be dried to constant weight under 105-115 ℃ and to be crushed to the 35-45 order in temperature, add 95% ethanol then and after temperature is 85-95 ℃ of reflux, extract, 3 times, filter, extracted 2 hours at every turn, filter, collect filtrate and filtering residue, the filtrate decompression of collecting is concentrated make extractum;
B. after refluxing 2 hours, the active carbon that the extractum among the step a is added 10% extractum weight after with 95% ethanol dispersing and dissolving filters and collects filtrate, collected filtrate is concentrated the back with the last silicagel column of sample: silica gel=1:30, use ethyl acetate: petroleum ether=1:1 successively, ethyl acetate: petroleum ether=5:1 and pure ethyl acetate eluting, 5 column volumes of each eluting, collect respectively eluent (I), eluent (II), eluent (III).
C. eluent (II) and eluent (III) are dissolved in the mixed liquor that volume ratio is chloroform: methanol=1:1, last LH-20 gel column detects analysis through thin layer chromatography, obtains Herba Agrimoniae triterpene component and flavonoid component;
D. with the filtering residue among the step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the back with 60% ethanol dispersing and dissolving, regulating PH is 5.0, slowly add 5.0% gelatin solution then till no longer produce precipitation, remove by filter that to add 95% ethanol to concentration of alcohol behind the tannalbin again be to remove excessive gelatin and the albumen in the extract and polysaccharide at 85% o'clock, filter then and filtrate is concentrated, make extractum; This extractum is dissolved in 80% methanol, and last LH-20 gel column detects analysis through thin layer chromatography, obtains flavonoid component.
E. the flavonoid component in combining step c and the steps d.
8. treatment according to claim 7 and improve the extracting method of the Herba Agrimoniae active component of insulin resistant, it is characterized in that: getting the Herba Agrimoniae herb among the step a is to be dried to constant weight under 110 ℃ and to be crushed to 40 orders in temperature, adds 95% ethanol then and filters after temperature is 90 ℃ of reflux, extract, 3 times.
9. treatment according to claim 8 and improve the extracting method of the Herba Agrimoniae active component of insulin resistant, it is characterized in that: among the step b, extractum among the step a with 95% ethanol dispersing and dissolving, after refluxing 2 hours, the active carbon that adds 10% extractum weight is then filtered and collects filtrate.
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