CN103202897B - 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 treat and improve insulin resistant Herba Agrimoniae active component and application and its extracting method.
Background technology
Insulin resistant refers to that insulin promotes that the efficiency of glucose uptake and utilization reduces, and especially muscle, fatty tissue, to the Use barriers of glucose, to occur through type 2 diabetes mellitus, the overall process of development from start to finish to show as peripheral tissues.Particularly nineteen ninety-five Stern proposes 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 current clinical practice is in the drug main Studies of The Insulin Sensitizer Thiazolidinediones medicine (TZDs) (as pioglitazone and rosiglitazone) improving and treat insulin resistant, it is by activating PPAR γ, optionally promote transcribing of downstream gene further, can PECTORAL LIMB SKELETON be raised, promote its differentiation and at the more lipid of born of the same parents' inner accumulated.So this type of medicine often increases along with body weight, fat side effect, has also increased the weight of cardiovascular danger, and hypoglycemia, general edema, cardiac load, osteoporosis, cancer etc. are also obvious side effect simultaneously.Therefore, the PPAR activator of development of new then has important economy and social value, and middle medical instrument too many levels, Mutiple Targets and comparatively safe feature, find and develop the selectivity of PPAR γ or incomplete activator, while reduction insulin resistant, the side effect such as the risk of cardiovascular diseases caused because fat excessively accumulates can be reduced.The transcription factor of regulation and control PECTORAL LIMB SKELETON differentiation is mainly: PPAR γ, ADD1/SREBP-1 and C/EBP-α.PPAR γ can regulate and control C/EBP-α, lipid metabolism key enzyme, fatty secretory protein, the lipolytic activity factor expression thus participate in Adipocyte Differentiation process.In AP2 controllable cell, the transhipment of fatty acid and synthesis, be the crucial actuator of periphery insulin sensitivity and glycolipid metabolism.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 adipose cell, thus promotes the differentiation of adipose cell, but add the accumulation of born of the same parents' inner lipid simultaneously.Therefore AP2 overexpression, can cause the disorder that normal lipid metabolism balances.Research shows, uses the inhibitor of AP2 and the agonist of PPAR γ all can make 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 treatment and controls spitting of blood, spits blood, has blood in stool, bleeding not during menses, and impairment caused by overstrain takes off power, carbuncle, wound hemorrhage etc.Show the research of the chemical composition of Herba Agrimoniae at present, it contains polyphenol, flavone and flavone glycoside compounds, triterpenoid compound and agrimonolide etc.And not yet have Herba Agrimoniae active component to be used for the treatment of and improve the report of insulin resistant at present.
Summary of the invention
The present invention mainly for the intervention of Herba Agrimoniae active component to insulin resistant, find Herba Agrimoniae active component not only to the activation of PPAR γ and positive control medicine pioglitazone without significant difference, significantly can promote the differentiation of PECTORAL LIMB SKELETON; And activating the overexpression unlike positive control medicine pioglitazone sample induction AP2 while PPAR γ expresses, show that Herba Agrimoniae active component has the effect improving 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 comprises the compositions of flavonoid component or Herba Agrimoniae triterpene component or Herba Agrimoniae flavonoid component and Herba Agrimoniae triterpene component in Herba Agrimoniae.
Further, in the compositions of described flavonoid component and triterpene component, the mass ratio of flavonoid component and triterpene component is 4:11: 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 Herba Agrimoniae triterpene component total triterpene is not less than 40%;
Further, described Herba Agrimoniae is Rosaceae agrimony, comprises tooth grass, HUANGLONG tail, dredges the Radix Agrimoniae in hair Radix Agrimoniae, northeast Korea Radix Agrimoniae, the cognate dragon in northeast, the multiple tooth Radix Agrimoniae of Da Hinggan Mountains, Northeast China, little Hua Radix Agrimoniae, Flos Caryophylli Radix Agrimoniae, stipule Radix Agrimoniae and Europe.
The present invention also discloses the application that a kind of Herba Agrimoniae active component for the treatment of and improving insulin resistant is treated in preparation and improved in 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: comprise the following steps:
A. get Herba Agrimoniae herb at temperature is 105-115 DEG C, is dried to constant weight and is crushed to 35-45 order, then add 95% ethanol to filter after temperature is 85-95 DEG C of reflux, extract, 3 times, to extract 2 hours at every turn, filter, collect filtrate and filtering residue, the filtrate reduced in volume of collection is obtained extractum;
B. filter after the extractum in step a being refluxed 2 hours with the active carbon adding 10% extractum weight after 95% ethanol dispersing and dissolving and collect filtrate, with sample after collected filtrate is concentrated: the upper silicagel column of silica gel=1:30, use ethyl acetate successively: petroleum ether=1:1, ethyl acetate: petroleum ether=5:1 and neat ethyl acetate, each eluting 5 column volumes, collect to obtain eluent (I) respectively, eluent (II), eluent (III);
C. eluent (II) and eluent (III) being dissolved in volume ratio is chloroform: the mixed liquor of methanol=1:1, upper LH-20 gel column, detects and analyzes, obtain Herba Agrimoniae triterpene component and flavonoid component through thin layer chromatography;
D. by the filtering residue in step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the ethanol dispersing and dissolving of rear use 60%, PH is regulated to be 5.0, then the gelatin solution of 5.0% is slowly added to no longer producing precipitation, cross the ethanol adding 95% after filtering tannalbin again when concentration of alcohol is 85%, remove egg bletilla polysaccharide in excessive gelatin and extract, then filter and filtrate is concentrated, obtained extractum; This extractum is dissolved in the methanol of 80%, upper LH-20 gel column, detects through thin layer chromatography and analyze, obtain flavonoid component.
E. the flavonoid component in combining step c and steps d;
Further, get Herba Agrimoniae herb in step a and at temperature is 110 DEG C, is dried to constant weight and is crushed to 40 orders, then add 95% ethanol and filter after temperature is 90 DEG C of reflux, extract, 3 times;
Further, in step b, by the 95% ethanol dispersing and dissolving of the extractum in step a, filter after the active carbon then adding 10% extractum weight refluxes 2 hours and collect filtrate.
Beneficial effect of the present invention: treatment of the present invention and improve the Herba Agrimoniae active component of insulin resistant and application thereof and its extracting method, the active substance group of Herba Agrimoniae is flavonoid component and triterpene component.Analyzed by HPLC and learn: be main containing apigenin-8-C-glucoside, Saponaretin and luteolin-7-O-glucoside, Quercitroside, silver linden glycoside in the flavonoid component of Herba Agrimoniae; Main containing ursolic acid, 4-hydroxyl ursolic acid, Corosolic acid and oleanolic acid in Herba Agrimoniae triterpene component.For the intervention of Herba Agrimoniae active component to insulin resistant, find Herba Agrimoniae active component not only to the activation of PPAR γ and positive control medicine pioglitazone without significant difference, significantly can promote the differentiation of PECTORAL LIMB SKELETON; And activating the overexpression unlike positive control medicine pioglitazone sample induction AP2 while PPAR γ expresses, show that Herba Agrimoniae active component has the effect improving and treat insulin resistant, corresponding dosage form can be prepared into and carry out preventing or treating insulin resistant.
Accompanying drawing explanation
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-is undetermined;
Fig. 2 is the HPLC chromatogram of Herba Agrimoniae triterpene component (TC): No. 4 compounds: 4-hydroxyl ursolic acid, No. 6 compounds: maslinic acid, No. 7 compounds: section's sieve carboxylic acid, No. 9 compounds: oleanolic acid, No. 10 compounds: ursolic acid, compound 1,2,3,5,8 undetermined;
Fig. 3 is that Concentraton gradient Herba Agrimoniae flavonoid component (FC) processes 3T3-L1 PECTORAL LIMB SKELETON differentiation-inducing 7th day microscope figure (x200);
Fig. 4 Concentraton gradient Herba Agrimoniae triterpene component (TC) processes 3T3-L1 PECTORAL LIMB SKELETON differentiation-inducing 7th day microscope figure (x200);
Fig. 5 Concentraton gradient Herba Agrimoniae flavonoid component (FC) processes 3T3-L1 PECTORAL LIMB SKELETON differentiation-inducing 7th day oil red colored graph (x200);
Fig. 6 Concentraton gradient Herba Agrimoniae triterpene component (TC) processes 3T3-L1 PECTORAL LIMB SKELETON differentiation-inducing 7th day oil red colored graph (x200);
The different ratio component of Fig. 7 Herba Agrimoniae two active components and two components is on the impact of triacylglycerol accumulation in PECTORAL LIMB SKELETON atomization;
Fig. 8 Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) are to the regulation and control of the gene expression of PECTORAL LIMB SKELETON differentiation central transcription factor;
Fig. 9 Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) different ratio component are to the regulation and control of PECTORAL LIMB SKELETON differentiation central transcription factor gene expression;
The regulation and control that Figure 10 Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) are expressed PECTORAL LIMB SKELETON differentiation AP2;
The regulation and control that Figure 11 Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) different ratio component are expressed PECTORAL LIMB SKELETON AP2.
Detailed description of the invention
Embodiment one
A. get Herba Agrimoniae herb 1.0kg, at temperature is 110 DEG C, is dried to constant weight and is crushed to 40 orders, then add 95% ethanol and filter after temperature is 90 DEG C of reflux, extract, 3 times, each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate reduced in volume of collection is obtained extractum 140g;
B. filter after the extractum in step a being refluxed 2 hours with the active carbon adding 10% extractum weight after 95% ethanol dispersing and dissolving and collect filtrate, upper silicagel column (sample: silica gel=1:30) after collected filtrate is concentrated, use ethyl acetate successively: petroleum ether=1:1,5:1 and neat ethyl acetate, each eluting 5 column volumes, collect to obtain eluent (I) respectively, eluent (II), eluent (III);
C. eluent (II) and eluent (III) being dissolved in volume ratio is chloroform: the mixed liquor of methanol=1:1, upper LH-20 gel column, detects and analyzes, obtain Herba Agrimoniae triterpene component and flavonoid component through thin layer chromatography;
D. by the filtering residue in step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the ethanol dispersing and dissolving of rear use 60%, PH is regulated to be 5.0, then the gelatin solution of 5.0% is slowly added to no longer producing precipitation, cross the ethanol adding 95% after filtering tannalbin again when concentration of alcohol is 85%, remove egg bletilla polysaccharide in excessive gelatin and extract, then filter and filtrate is concentrated, obtained extractum; This extractum is dissolved in the methanol of 80%, upper LH-20 gel column, detects through thin layer chromatography and analyze, obtain flavonoid component.
E. the flavonoid component in combining step c and 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.
Be that 4:1 is conventional by obtained Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) with mass ratio to granulate, make capsule, tablet or granule etc.
Embodiment two
A. get Herba Agrimoniae herb 1.0kg, at temperature is 105 DEG C, is dried to constant weight and is crushed to 35 orders, then add 95% ethanol and filter after temperature is 85 DEG C of reflux, extract, 3 times, each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate reduced in volume of collection is obtained extractum;
B. filter after the extractum in step a being refluxed 2 hours with the active carbon adding 5% extractum weight after 95% ethanol dispersing and dissolving and collect filtrate, upper silicagel column (sample: silica gel=1:30) after collected filtrate is concentrated, use ethyl acetate successively: petroleum ether=1:1,5:1 and neat ethyl acetate, each eluting 5 column volumes, collect to obtain eluent (I) respectively, eluent (II), eluent (III);
C. eluent (II) and eluent (III) being dissolved in volume ratio is chloroform: the mixed liquor of methanol=1:1, upper LH-20 gel column, detects and analyzes, obtain Herba Agrimoniae triterpene component and flavonoid component through thin layer chromatography;
D. by the filtering residue in step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the ethanol dispersing and dissolving of rear use 60%, PH is regulated to be 5.0, then the gelatin solution of 5.0% is slowly added to no longer producing precipitation, cross the ethanol adding 95% after filtering tannalbin again when concentration of alcohol is 85%, remove egg bletilla polysaccharide in excessive gelatin and extract, then filter and filtrate is concentrated, obtained extractum; This extractum is dissolved in the methanol of 80%, upper LH-20 gel column, detects through thin layer chromatography and analyze, obtain flavonoid component.
E. the flavonoid component in combining step c and 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.
Be that 3:2 is conventional by obtained Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) with mass ratio to granulate, make capsule, tablet, granule etc.
Embodiment three
A. get Herba Agrimoniae herb 1.0kg, at temperature is 115 DEG C, is dried to constant weight and is crushed to 45 orders, then add 95% ethanol and filter after temperature is 90 DEG C of reflux, extract, 3 times, each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate reduced in volume of collection is obtained extractum;
B. filter after the extractum in step a being refluxed 2 hours with the active carbon adding 15% extractum weight after 95% ethanol dispersing and dissolving and collect filtrate, upper silicagel column (sample: silica gel=1:30) after collected filtrate is concentrated, use ethyl acetate successively: petroleum ether=1:1,5:1 and neat ethyl acetate, each eluting 5 column volumes, collect to obtain eluent (I) respectively, eluent (II), eluent (III);
C. eluent (II) and eluent (III) being dissolved in volume ratio is chloroform: the mixed liquor of methanol=1:1, upper LH-20 gel column, detects and analyzes, obtain Herba Agrimoniae triterpene component and flavonoid component through thin layer chromatography;
D. by the filtering residue in step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the ethanol dispersing and dissolving of rear use 60%, PH is regulated to be 5.0, then the gelatin solution of 5.0% is slowly added to no longer producing precipitation, cross the ethanol adding 95% after filtering tannalbin again when concentration of alcohol is 85%, remove egg bletilla polysaccharide in excessive gelatin and extract, then filter and filtrate is concentrated, obtained extractum; This extractum is dissolved in the methanol of 80%, upper LH-20 gel column, detects through thin layer chromatography and analyze, obtain flavonoid component.
E. the flavonoid component in combining step c and 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.
Be that 2:3 is conventional by obtained Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) with mass ratio to granulate, make capsule or tablet etc.
Embodiment four
A. get Herba Agrimoniae herb 1.0kg, at temperature is 110 DEG C, is dried to constant weight and is crushed to 45 orders, then add 95% ethanol and filter after temperature is 85 DEG C of reflux, extract, 3 times, each extraction 2 hours, filter, collect filtrate and filtering residue, the filtrate reduced in volume of collection is obtained extractum;
B. filter after the extractum in step a being refluxed 2 hours with the active carbon adding 10% extractum weight after 95% ethanol dispersing and dissolving and collect filtrate, upper silicagel column (sample: silica gel=1:30) after collected filtrate is concentrated, use ethyl acetate successively: petroleum ether=1:1,5:1 and neat ethyl acetate, each eluting 5 column volumes, collect to obtain eluent (I) respectively, eluent (II), eluent (III);
C. eluent (II) and eluent (III) being dissolved in volume ratio is chloroform: the mixed liquor of methanol=1:1, upper LH-20 gel column, detects and analyzes, obtain Herba Agrimoniae triterpene component and flavonoid component through thin layer chromatography;
D. by the filtering residue in step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the ethanol dispersing and dissolving of rear use 60%, PH is regulated to be 5.0, then the gelatin solution of 5.0% is slowly added to no longer producing precipitation, cross the ethanol adding 95% after filtering tannalbin again when concentration of alcohol is 85%, remove egg bletilla polysaccharide in excessive gelatin and extract, then filter and filtrate is concentrated, obtained extractum; This extractum is dissolved in the methanol of 80%, upper LH-20 gel column, detects through thin layer chromatography and analyze, obtain flavonoid component.
E. the flavonoid component in combining step c and 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.
Be that 1:4 is conventional by obtained Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) with mass ratio to granulate, make capsule or agent etc.
Embodiment five
The HPLC substance characterization of Herba Agrimoniae flavonoid component (FC) and triterpene component (TC)
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 purchased from middle inspection institute
2. acetonitrile, methanol are import chromatographically pure
3. other reagent is domestic analytical pure
1.1.2 instrument
1. precision 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 Herba Agrimoniae flavonoid component (FC) 10.0mg, dissolve with methanol is dissolved in 10mL volumetric flask surely, to be measured.
2. accurately take Herba Agrimoniae triterpene component (FC) 10.0mg, dissolve with methanol is dissolved in 10mL volumetric flask surely, 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 substance characterization of Herba Agrimoniae flavonoid component
Chromatographic column: Welch Materials Ultimate XB-C184.6x250mm, 5 μm; Mobile phase: mobile phase A is 0.1% phosphate aqueous solution, Mobile phase B: acetonitrile; Determined wavelength: 350nm, column temperature: 35 DEG C, flow velocity: 1.0mL/min, sample size: 20 μ L
Condition of gradient elution is as table 1:
The HPLC eluent gradient table of table 1 Herba Agrimoniae flavonoid component (FC)
1.2.2 the HPLC substance characterization of Herba Agrimoniae triterpene component
Chromatographic column: Welch Materials Ultimate XB-C184.6x250mm, 5 μm
Mobile phase: mobile phase A is 0.1% phosphate aqueous solution, Mobile phase B is acetonitrile: methanol=2:1
Determined wavelength: 210nm, column temperature: 30 DEG C, flow velocity: 1.0mL/min, sample size: 20 μ L
Condition of gradient elution is as table 2:
The HPLC eluent gradient table of table 2 Herba Agrimoniae triterpene component (TC)
1.3 results and analysis
The HPLC chromatogram of Herba Agrimoniae flavonoid component (FC) is shown in Fig. 1.By reference substance additive process determination Herba Agrimoniae flavonoid component (FC) mainly containing 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 shown in Fig. 2, by reference substance additive process determination Herba Agrimoniae TC mainly containing ursolic acid, 4-hydroxyl ursolic acid, section's sieve carboxylic acid, maslinic acid and oleanolic acid.
Embodiment six
In Herba Agrimoniae, active component flavonoid component (FC) and triterpene component (TC) and the two different ratio are to the facilitation of Adipocyte Differentiation
1, experiment material and method
1.1 reagent and instrument
1.1.1 reagent D MEM high glucose medium, hyclone, calf serum, dual 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, purchased from sigma company; Tissue Culture Plate (6 holes, 12 holes, 96 holes), Hyclone company
1.1.2 instrument precision electronic balance FA2004 type, Shanghai Precision Scientific Apparatus Co., Ltd; CO2 cell culture incubator, THERMO company of the U.S.; Microplate reader Model680, Bio-Rad, USA; Quantitative real time PCR Instrument CYMMZ-EPXFY-052, Bio-Rad, USA; Eppendorf refrigerated centrifuger-5417R, gene company limited; RNA/DNA ultraviolet spectrophotometer, beckman company of the U.S.; Just putting microscope CYMML-EPTXCJ-004, OLYMPUS
1.2,3T3-L1 PECTORAL LIMB SKELETON induction and drug treating
Experimental group is Herba Agrimoniae sample variable concentrations process (each sample arranges 1,5,25 and 125 μ g/mL, tetra-gradients), and positive controls is pioglitazone (10 μMs), and blank group is blank solvent.To the cell of 90% be fused to, be inoculated in 6 orifice plates or 12 orifice plates, and cultivate with the DMEM high glucose medium containing 10% dual anti-calf serum, every other day change a subculture, cultivate 2 days, treat the fusion of cell 100%; The first phase is differentiation-inducing: change MDI induced liquid, adds sample and the positive control solution of the Herba Agrimoniae of variable concentrations, cultivates 2 days; The second phase is differentiation-inducing: abandon culture fluid, changes insulin-induced liquid, adds sample and the positive control solution of the Herba Agrimoniae of variable concentrations, continues cultivation 2 days; The third phase is differentiation-inducing: abandon culture fluid, and the high sugar of DMEM changed containing 10% hyclone is cultivated, and adds sample and the positive control solution of the Herba Agrimoniae of variable concentrations simultaneously, continues to cultivate 2-4 days, wait 3T3-L1 PECTORAL LIMB SKELETON differentiation and maturation, observation of cell form.
1.3, Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) are on the impact of cell differentiation form
To break up the 3T3-L1 PECTORAL LIMB SKELETON of the 7th day in 6 orifice plates, careful absorption supernatant culture medium, then discards.Get 1mLPBS, inject along culture plate hole wall, jiggle standing 5min with hands, then draw PBS, wash 3 times by PBS solution; After cleaning up culture medium and medicine, formaldehyde fixative (the every hole 1mL of 24 orifice plate) the fixed cell 30-60min with 10%; Cell is dried 30min after cleaning by PBS; Add the every hole 0.5mL of the oil red O working solution (oil red storage liquid: distilled water=3:2) configured, dye under room temperature 40-60min(lucifuge, sealing) oil red O dye liquor, remove unnecessary dyestuff for 2 times with 60% isopropyl alcohol wash cell and wash 3 times with tri-distilled water again.With glycerol or glycerin gelatine mounting; Observed result under inverted microscope, takes pictures.
1.4, oil red staining observes the deposition degree of triglyceride
To plant 24 orifice plates having cell, carry out inducing culture, oil red dyeing is carried out in taking-up in the 7th day; Remove excess dyestuff 2 times with 60% isopropyl alcohol wash cell, wash 3 times with tri-distilled water, add 100% isopropyl alcohol 1mL, rifle head is blown and beaten repeatedly, and then vibrate 20min on shaker; By microplate reader, measure light absorption value at 520nm place.
2, result
2.1, Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) are on the impact of cell differentiation form
As Fig. 3 and Fig. 4, not differentiation-inducing 3T3-L1 PECTORAL LIMB SKELETON is the state of long shuttle shape, after cell induction breaks up 7 days, can find out that differentiation and maturation adipose cell is for circular, there is obvious lipid accumulation simultaneously in born of the same parents, and in born of the same parents, have significantly " finger ring fat drips " enrichment; As can be seen from the form of cell relatively, Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) have the effect significantly promoting cell differentiation, and promote the differentiation of PECTORAL LIMB SKELETON in the mode of dose dependent; And compare from the size of fat drop, comparatively Herba Agrimoniae triterpene component (TC) under Herba Agrimoniae flavonoid component (FC) process and positive control little.
2.2, oil red staining observes the deposition degree of triglyceride
Can be learnt by oil red colored graph 5 and 6: along with the differentiation of 3T3-L1 PECTORAL LIMB SKELETON, the triacylglycerol content in born of the same parents builds up, and the size and number that fat drips organizes process with blank obvious difference; Herba Agrimoniae flavonoid component (FC) and Herba Agrimoniae triterpene component (TC) all present the generation promoting triacylglycerol in the mode of dose dependent.As Fig. 7, wherein, * for have significant difference (p<0.5) compared with blank, and * * for have pole significant difference (p<0.1) compared with blank; A is triacylglycerol content effect after the drug treating of the variable concentrations of flavonoid component, B is the triacylglycerol content effect after the drug treating of the variable concentrations gradient of triterpene component, C figure is the effect of component when 50 μ g/mL to triacylglycerol content of the different ratio of flavonoid component and triterpene component: I is FC:TC=1:4, II is FC:TC=1:1, III is FC:TC=4:1.A shows the effect that Herba Agrimoniae flavonoid component (FC) tool urgees triacylglycerol content, and 5-25 μ g/mL concentration range, with blank tool significant difference, when reaching 125 μ g/mL, has pole significant difference, but not as positive control; B shows the effect that Herba Agrimoniae triterpene component (TC) tool promotes triacylglycerol accumulation, during 5 μ g/mL, has significant difference compared with blank, when reaching 25-125 μ g/mL, and tool pole significant difference, but all not as the effect of positive control; C shows, has remarkable facilitation as FC:TC=4:1 compared with blank, and when FC:TC=1:4 tool pole significant difference, TC has the effect of stronger promotion triacylglycerol deposition than FC.
3, conclusion
1. classical to 3T3-L1 PECTORAL LIMB SKELETON cocktail revulsion is carried out differentiation-inducing, observe flavonoid component (FC) and triterpene component (TC) two active component synchronous drug treating cellular morphology of differentiation-inducing 7th day of Herba Agrimoniae, cell can be observed and become circular by long shuttle shape, can observe in born of the same parents significantly " finger ring fat drips ", illustrate that Herba Agrimoniae active component FC and TC has the effect obviously promoting cell differentiation.2. by oil red colored graph result and the display of triacylglycerol assay, in the mode of concentration dependent, FC and TC all promotes that the triacylglycerol content in adipose cell increases; After the drug treating of the compounding ingredients of three different ratio simultaneously, data analysis is learnt, the facilitation that TC is stronger to triacylglycerol than FC.The differentiation of above description of test Herba Agrimoniae active component FC and TC to PECTORAL LIMB SKELETON has facilitation.
Embodiment seven
The regulating and controlling effect that in Herba Agrimoniae, active component is expressed Adipocyte Differentiation and Genes Associated with Lipid Metabolism
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, the extraction of the mRNA of 3T3-L1 PECTORAL LIMB SKELETON, RT-PCR and real-time PCR
1. get the cell ice bath of differentiation-inducing 7th day, PBS washes 2 times.Add 0.9mL Trizol lysate.Each hole lysate is collected in 1.5mL EP pipe respectively.In each EP pipe, all add 0.2mL chloroform, vibration 15S, treats as milky white shape, leaves standstill 3min.4 DEG C of 12000g leave heart 15min.Draw supernatant, add 0.5mL isopropyl alcohol, mixing, leave standstill 10min.4 DEG C of 12000g leave heart 10min.Abandon supernatant, add the ethanol 1mL of 75%, 4 DEG C of 7500g leave heart 5min.Drying at room temperature, adds 25 μ LDEPC water dissolution precipitations.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 RNA extracting solution, add DEPC water and make cumulative volume be 20 μ L.Carry out reverse transcription, cDNA is in-20 DEG C of preservations.Get the cDNA solution that 2 μ L dilute 10 times, mix with 8 μ L and transcribe liquid (upstream and downstream primer 1.4 μ L/ hole, 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, the extraction of the mRNA of 3T3-L1 PECTORAL LIMB SKELETON, RT-PCR and real-time PCR
2.1.1, central transcription factor C/EBP-α, the PPAR γ of the differentiation of PECTORAL LIMB SKELETON and the expression regulation of ADD1/SREBP-1
As Fig. 8, wherein, * (p<0.05) * * (p<0.01): refer to comparing of FC experimental group and its blank group; #(p<0.05) ##(p<0.01): refer to comparing of TC experimental group and its blank group; & & (p<0.01): refer to that FC compares with its positive controls; $ $ (p<0.01): refer to comparing of TC and its positive controls.The expression of gene and the concentration of FC and TC are 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 pole significant difference, when TC is 125 μ g/mL, expresses and have significant difference; When FC and TC is 125 μ g/mL, it is expressed without significant difference; During 125 μ g/mL, the expression of the C/EBP-α mRNA of FC and TC is compared with blank group remarkable increase, and has compared with positive control and reduce extremely significantly; During 125 μ g/mL, the mrna expression amount of SREBP-1 is maximum, and having significance compared with blank group increases, and without remarkable statistical discrepancy compared with the expression of positive control.As Fig. 9, * (p<0.05) * * (p<0.01): refer to that the drug treating group of the component of the different ratio of FC and TC and the C/EBP-α mrna expression of positive controls compare with blank group; & (p<0.05): refer to the drug treating group of the component of the different ratio of FC and TC and positive control SREBP-1mRNA express and compare with blank group; $ (p<0.05) $ $ (p<0.01): refer to that the drug treating group of the component of the different ratio of FC and TC and the PPAR γ mrna expression of positive controls compare with blank group.C/EBP-α mRNA is when FC and TC proportioning is 1:150 μ g/mL, and tool increases significantly compared with blank group, and under proportioning 1:4 and 4:1, does not have significant difference; For SREBP-1, three kinds of proportionings, when 50 μ g/mL, more all do not have remarkable significant difference with blank group; Known to the expression analysis of PPAR γ, as FC:TC4:150 μ g/mL, have compared with blank group and increase the most significantly.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 comparing of FC experimental group and its blank group; #(p<0.05) ##(p<0.01): refer to comparing of TC experimental group and its blank group; & & (p<0.01): refer to that FC compares with positive controls; $ $ (p<0.01): refer to comparing of TC and positive controls; The expression of AP2 presents metering dependency increases relation.During TC25 μ g/mL, expression and the blank group tool of AP2mRNA significantly strengthen, during 125 μ g/mL, FC and TC expresses and compares tool significance strengthen (p<0.05) with equal blank group, but all shows and be reduced to positive controls (p<0.01).As Figure 11, wherein, & (p<0.05) & & (p<0.01): refer to that the AP2mRNA of the drug treating group of the component of the different ratio of FC and TC expresses and compare with blank group; When FC:TC is 1:4, the expression ratio blank group of AP2mRNA has significance to increase (p<0.05), and another 2 kinds of proportionings express no difference of science of statistics to it.
3, conclusion
1. at finite concentration tool, two active components of Herba Agrimoniae FC and TC obviously promote that PPAR γ, C/EBP-α, SREBP-1 activate expression; It is still it is not strong relative to positive controls significance that FC and TC has certain facilitation to Downstream regulatory Gene A P2.2. FC and TC nature proportioning (4:1) of Herba Agrimoniae has good advantage in effect proportioning.Found through experiments, Herba Agrimoniae active component flavone and triterpene part have and comparatively significantly act in improvement and opposing insulin resistant.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (7)
1. treat and improve the Herba Agrimoniae active component of insulin resistant for one kind, it is characterized in that: this active component is the compositions of Herba Agrimoniae flavonoid component or Herba Agrimoniae triterpene component or Herba Agrimoniae flavonoid component and Herba Agrimoniae triterpene component, main containing apigenin-8-C-glucoside, Saponaretin, luteolin-7-O-glucoside, Quercitroside and silver linden glycoside in the flavonoid component of Herba Agrimoniae; Main containing ursolic acid, 4-hydroxyl ursolic acid, Corosolic acid and oleanolic acid in Herba Agrimoniae triterpene component, this Herba Agrimoniae active component is extracted by the extracting method comprised the following steps:
A. get Herba Agrimoniae herb at temperature is 105-115 DEG C, is dried to constant weight and is crushed to 35-45 order, then add 95% ethanol to filter after temperature is 85-95 DEG C of reflux, extract, 3 times, to extract 2 hours at every turn, filter, collect filtrate and filtering residue, the filtrate reduced in volume of collection is obtained extractum;
B. filter after the extractum in step a being refluxed 2 hours with the active carbon adding 10% extractum weight after 95% ethanol dispersing and dissolving and collect filtrate, with sample after collected filtrate is concentrated: the upper silicagel column of silica gel=1:30, use ethyl acetate successively: petroleum ether=1:1, ethyl acetate: petroleum ether=5:1 and neat ethyl acetate, each eluting 5 column volumes, collect to obtain eluent (I) respectively, eluent (II), eluent (III);
C. eluent (II) and eluent (III) being dissolved in volume ratio is chloroform: the mixed liquor of methanol=1:1, upper LH-20 gel column, detects and analyzes, obtain Herba Agrimoniae triterpene component and flavonoid component through thin layer chromatography;
D. by the filtering residue in step a with 50% alcohol reflux 3 times, each 1.5h, extracting solution concentrates the ethanol dispersing and dissolving of rear use 60%, pH is regulated to be 5.0, then the gelatin solution of 5.0% is slowly added to no longer producing precipitation, cross the ethanol adding 95% after filtering tannalbin again when concentration of alcohol is 85%, remove egg bletilla polysaccharide in excessive gelatin and extract, then filter and filtrate is concentrated, obtained extractum; This extractum is dissolved in the methanol of 80%, upper LH-20 gel column, detects through thin layer chromatography and analyze, obtain flavonoid component;
E. the flavonoid component in combining step c and steps d.
2. treatment according to claim 1 and improve the Herba Agrimoniae active component of insulin resistant, is characterized in that:
In the compositions of described flavonoid component and triterpene component, the mass ratio of flavonoid component and triterpene component is 4:1-1:4.
3. treatment according to claim 2 and improve the Herba Agrimoniae active component of insulin resistant, 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%, the mass percentage content of Herba Agrimoniae triterpene component total triterpene is not less than 40%.
5. the treatment according to the arbitrary claim of claim 1-4 and improve the Herba Agrimoniae active component of insulin resistant, is characterized in that: described Herba Agrimoniae is Rosaceae agrimony.
6. treatment according to claim 1 and improve the Herba Agrimoniae active component of insulin resistant, it is characterized in that: get Herba Agrimoniae herb in step a and at temperature is 110 DEG C, is dried to constant weight and is crushed to 40 orders, then add 95% ethanol and filter after temperature is 90 DEG C of reflux, extract, 3 times.
7. a treatment according to claim 1 and the Herba Agrimoniae active component of improving insulin resistant are in preparation treatment and the application that improves in insulin resistant medicine.
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| 仙鹤草对糖尿病大鼠血糖胰岛素水平的影响;周晓蓉等;《职业与健康》;20120331;第28卷(第6期);688-689,692 * |
| 仙鹤草降糖活性部位筛选;陈优生等;《安徽医药》;20100731;第14卷(第7期);765-766 * |
| 陈优生等.仙鹤草降糖活性成分研究(Ⅱ).《中药材》.2010,第33卷(第5期), * |
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