CN101204501B - Application of anti-fatty liver Chinese traditional medicine and fistular onion stalk extractive on curing fatty liver - Google Patents
Application of anti-fatty liver Chinese traditional medicine and fistular onion stalk extractive on curing fatty liver Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine used for prevention and treatment of fatty liver as well as an application of fistular onion stalk extractive for prevention and treatment of fatty liver. The invention is application and medicine preparation of fistular onion stalk extractive used for prevention and treatment of fatty liver.The fistular onion stalk extractive is a mixture of the fat-soluble effective extractive and the neutral effective extractive of the fistular onion stalk. The traditional Chinese medicine used for prevention and treatment of fatty liver is the fistular onion stalk extractive.The invention is superior to Dongbao glucurolactone, a traditional Chinese medicine preparation on the market. The invention has the advantages of clear mechanism of action, controllable quality, low cost, little toxic and side effects and good compliance.The invention has better prevention and treatment of fatty liver.
Description
Technical field
The invention belongs to a kind of lipotropic Chinese medicine and application, be specifically related to a kind of Bulbus Allii Fistulosi extract control fatty liver Chinese medicine and Bulbus Allii Fistulosi extract in lipotropic application.
Background technology
(fatty liver disease FLD) is a kind of common chronic hepatopathy to fatty liver disease, and pathogenic process comprises the deposition of triglyceride in hepatocyte, hepatic necrosis, inflammatory reaction, and hepatic fibrosis, finally develops into liver cirrhosis and liver failure.Along with the change of people life style, the morbidity of fatty liver is ascendant trend year by year, and fatty liver has accounted for adult's 20%-30% in western countries' prevalence.Over nearly 20 years, the sickness rate of China and Asian-Pacific area primary disease also obviously increases, and is about 7%-40%.China's sickness rate accounts for 10% of population.Account for 50% of overweight people and diabetics, account for 57.5% of alcoholic.And closely half Patients with Fatty Liver can be developed to hepatic fibrosis, and about 15% is developed to liver cirrhosis, and 3% advances to liver failure.In recent years, its morbidity crowd has the trend of rejuvenation.Foreign literature report: have 2.6% normal type child and 52.8% Obese children to suffer from fatty liver, mostly be non-alcoholic fatty liver disease.
Because of its pathogenesis is failed to understand, the present clinical specific treatment medicine that still do not have, too high medical expense also makes many patients can not get treating timely and has delayed best treatment period simultaneously, if can find a kind of efficient, low toxicity, inexpensive medicine, will improve the Prevention and Management of Fatty Liver level, improve patient's quality of life.
The history in existing several thousand of Chinese medicine fatty liver relevant disease, accumulated abundant treatment experience, and the Chinese medicine emphasis is integrally-regulated, has the effect characteristics of multisystem, many target spots, become the focus of this area research, also demonstrate its lipotropic advantage.Present commercially available Chinese medicine preparation mostly is compound recipe, and quality controllability is relatively poor, has the high deficiency of cost equally.And the open Bulbus Allii Fistulosi extract of document is not arranged now as yet in lipotropic application.
Summary of the invention
The object of the present invention is to provide a kind of lipotropic Chinese medicine and Bulbus Allii Fistulosi extract in lipotropic application.
The technical scheme that realizes one of purpose of the present invention is: Bulbus Allii Fistulosi extract is in lipotropic application.
Described Bulbus Allii Fistulosi extract is the very light blue liposoluble effective extracts and the mixture of polarity effective extract.
Two the technical scheme that realizes the object of the invention is: lipotropic Chinese medicine, it is a Bulbus Allii Fistulosi extract.
Described Bulbus Allii Fistulosi extract is the very light blue liposoluble effective extracts and the mixture of polarity effective extract.
It is the drug oral preparation by the process for preparing medicine preparation.Described drug oral preparation is a capsule preparations, soft capsule preparation, oral liquid formulations, dropping pill formulation, tablet formulation.
Very light blue source is the fresh bulb of liliaceous plant shallot Allium fisturosum L.var.caespitosum Makio or chive Allium schoenoprasum.
This material is better than commercially available Chinese medicine preparation DONGBAO GANTAI, and mechanism of action is clear and definite, and is quality controllable, and cost is low, and toxic and side effects is little, and compliance is good.Fatty liver had the better prevention effect.
The specific embodiment
Here very light blue liposoluble effective extracts: it is after one or more the combination in exsiccant very light blue or dried Sucus Allii Fistulosi or the dry full Herba Alii fistulosi is pulverized, through CO
2The supercritical extraction separation and Extraction and material, described liposoluble extract is 0.8330～0.8350 20 ℃ of relative densities; Index of refraction is 1.3600～1.3700 at D20 ℃.
Described CO
2The pressure of supercritical extraction is 15～40Mpa, 20～60 ℃ of temperature.
Described CO
2Separating pressure behind the supercritical extraction is 4～10Mpa, 20～40 ℃ of temperature.
Described CO
2Separation behind the supercritical extraction comprises twice separation, and the pressure that separates I is 20～40 ℃ of 4～10Mpa temperature; The pressure that separates II is 20～40 ℃ of 4～10Mpa temperature.
Or utilize solvent method to extract: it is after one or more the combination in exsiccant very light blue or dried Sucus Allii Fistulosi or the dry full Herba Alii fistulosi is pulverized, adopt a kind of solvent cold soak or heat in ether, petroleum ether, chloroform, ethyl acetate, butanols and the ethanol to carry 1～24 hour, filtering extracting solution, extracting solution concentrated remove solvent, promptly get fat-soluble live part extract; The w/v of described raw material and solvent is: 1g: 6～15ml.
It is to adopt petroleum ether (30～60 ℃) to extract.
It is to adopt a kind of solvent in petroleum ether (60～90 ℃), chloroform, ethyl acetate, butanols and the ethanol to extract.
Described Semi-polarity class effective extract is: it is directly will be exsiccant very light blue or with exsiccant very light blue remainder after fat-soluble live part extraction, carry out reflux, extract, with ethanol, after the filtration, filtrate decompression is concentrated into the dried cream that relative density is 1.0～1.3g/ml, in dried cream, adds water, after the macroporous adsorptive resins absorption, can not know saponin component with alcohol flushing to inspection, reclaim solvent, drying obtains the polarity effective extract.Or directly with exsiccant very light blue or with exsiccant very light blue remainder after fat-soluble live part extracts, carry out reflux, extract, with ethanol, after the filtration, filtrate decompression is concentrated into the dried cream that relative density is 1.0～1.3/ml, in dried cream, add water, the full n-butyl alcohol that closes of water again, a kind of merceration that carries out in propanol or the ethyl acetate solution, seepage, a kind of processing the during heat is carried or extracted to water layer, inspection is known till the no obvious change color, tell extraction part be evaporated to again do after, dilute with water again, solvent is reclaimed in the macroporous resin column decolouring, drying obtains the polarity effective extract.
Two kinds of materials of said extracted are mixed fully, promptly all mix with a collection of very light blue two kinds of materials after liposoluble effective extracts and Semi-polarity class effective extract extracted twice.Or only adopt very light blue liposoluble effective extracts.
The said extracted thing is experimentized:
One, materials and methods
(1), experiment material
1, medicine and solution: lipophilic effective extract of Chinese green onion and the mixture of Semi-polarity class effective extract or very light blue liposoluble effective extracts; DONGBAO GANTAI PIAN (compound methionine choline sheet, lot number: 060106), use after being mixed with suspension with 0.5%CMC-Na; 10mgml-1 ethidium bromide: add 0.2g bromination second in the 20ml distilled water and form sediment, magnetic agitation a few hours, dissolve fully to guarantee it, then with lead foil parcel, room temperature preservation; 2.0% preparing gel (20ml): fine jade glycolipid 0.40g, 5 * TBE 2ml, distilled water 18ml, heating for dissolving, treat that temperature adds 2 μ l 10mgml-1 ethidium bromides when reducing to 60 ℃, add behind the mixing in the pre-prepd glue dish, placement after half an hour is extracted comb, and electrophoretic buffer is 5 * tbe buffer liquid; The electrophoresis sample-loading buffer: sucrose 25g, 1.2% bromjophenol blue 8.33ml, redistilled water 50ml, several of 1mol1-1NaOH are stored in-4 ℃; 5 * tbe buffer liquid: Tris alkali 10.8g, boric acid 5.5g, 0.5mol1-1EDTA 4ml adds water to 200ml, room temperature storage; Organize lysate: 50mmol1-1 Tris-Cl, 150mmol1-1NaCL, 10%Triton, X-100 or NP-40,0.02%Na3N faces with preceding adding 100 μ gml-1PMSF, and pH 8.0; 1 * sds gel electrophoresis buffer: Tris-base, 0.05 mol1-1, Glycerin 0.38mol1-1, SDS 0.1%; Transfering buffering liquid: glycine 14.4g, Tris-base 3g, 200ml methanol, adding distil water is to 1L; 10% polyacrylamide separation gel solution (ml): Acry: Bis (30: 0.8) 5.00,4 * TrisCl/SDS (PH8.8) 3.75, H2O6.25,10% Ammonium Persulfate 98.5 0.10, TEMED0.03; 4% polyacrylamide spacer gel solution (ml): Acry: Bis (30: 0.8) 1.30,4 * TrisCl/SDS (PH 6.8) 2.50, H2O6.10,10% Ammonium Persulfate 98.5 0.10, TEMED 0.02; Confining liquid: 1g closed protein dry powder is dissolved in 100ml 0.01mol.1-1PBST; Destaining solution: 30% methanol and the decolouring of 10% acetic acid mixed liquor; Dyeing liquor: methanol/acetum (45ml methanol, 45ml water and 10ml glacial acetic acid mixing), 0.25g Coomassie brilliant blue R250 is dissolved in the liquid; 0.01mol1-1PBST:NaCl9g, Na2HPO412H2O 7g, NaH2PO42H2O 0.5g, 0.5ml Tween-20, adding distil water is dissolved to 1000ml, pH 7.2-7.4.
2, reagent: total protein (TP), albumin (ALB), globulin (GLO) test kit; HDL-C (HDL-C) test kit, low-density lipoprotein cholesterol (LDL-C) test kit are equal; Glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) test kit.Superoxide dismutase (SOD), malonaldehyde (MDA), glutathione-S transferring enzyme (GSH-ST), nitric oxide (NO), nitricoxide synthase (NOS) detection kit; T-CHOL (TC), triglyceride (TG) test kit; ET-1 radioimmunoassay, RIA medicine box; DEPC, agarose, TRIZOL TagDNA polymerase is all purchased; M-MLV reverse transcriptase (MBI), PCRMarker); Paraformaldehyde; With preceding with formulated 4% solution of 0.1mol.L-1 phosphate buffer, PH7.4,4 ℃ of preservations; Gradient ethanol: 100%, 95%, 80%, 70%; Haematoxylin-Yihong solution; PBS:PH7.2-7.4, concentration 0.01mol.L-1; Reagent example hydrochloric acid, dimethylbenzene, dehydrated alcohol, sodium chloride etc. are all analyzed alcohol; ET-1 radioimmunoassay, RIA medicine box; The TNF-a:ELISA method; IL-6ELISA method test kit; The super quick test kit of SP; ICAM-1, NF-κ B, TGF-β antibody; Instant SABC immunohistochemical staining test kit; E.C. 3.4.21.64; DAB (3,3 '-diaminobenzidine); The TUNEL detection kit; Trizol total RNA extraction reagent, pyrocarbonic acid diethyl ester (DEPC), Oligo (dt) 18Primer, RNA enzyme inhibitor, TagDNA polymerase, agarose; PCRMarker; The M-MLV reverse transcriptase; Pvdf membrane; Trimethyl aminomethane (Tris) and tetramethylethylenediamine (TEMED), acrylamide (Acry), N, the two propylene phthalein amine (Bis) of N-methylene fork, glycine (Glycine) and sodium lauryl sulphate (SDS), Ammonium Persulfate 98.5 (AP), low-molecular-weight standard protein, the bright protein determination kit of coomassie and standard protein liquid, Western blot one are anti-and two resist.
3, animal: 60 of 150 ± 20g SPF level SD rats, male and female half and half;
4, instrument: full-automatic biochemical detector; Desk-top high temperature low speed centrifuge; FJ2003/50G type γ immunity enumerator; Full-automatic biological tissue dehydrator; Water-bath type biological tissue embedding machine; Histotome; Calorstat; BX-60 type Olympus optical microscope; Digital camera; Instrument Tu-1800s ultraviolet-visible spectrophotometer; Electronic Speculum; High speed centrifuge; Elx-800 type microplate reader; Constant Temp. Oven; The Tu-1800s ultraviolet-visible spectrophotometer; DFM-96 type multitube radioimmunity enumerator; Inverted biological microscope, vertical-type electrophoresis tank electrophresis apparatus and change film instrument, constant temperature shaking table, fully automatic digital imaging system and analytical system, PCR instrument (Hybaid thermal cycle, USA), 721 type spectrophotometer and refrigerated centrifuges;
(2) experimental technique
1, animal grouping: after normal diet is normally fed a week, be divided into 6 groups, 10 every group by the body weight stratified random.A group: blank group; B group: model control group; C group: extract low dose group of the present invention; D group: dosage group in the extract of the present invention; E group: extract high dose group of the present invention; F group: DONGBAO GANTAI PIAN group.
2, modeling method: want military reported method to duplicate rat fat liver model with reference to grandson: high lipid food (on the normal diet basis, adding 10% Adeps Sus domestica), drink 15% alcohol water, and weekly 40%CCL
4Salad oil solution, hind leg subcutaneous injection (initial dose 0.5mg/100g.BW is later on 0.3mg/100g.BW); The A group gives the standard particle forage feed, drinks light water;
3, medication: each group is organized and is given injection normal saline 8ml/kg, ig, qd with modeling while, A, B; C group, D group, E group are given Bulbus Allii Fistulosi extract, press 0.06g/kg, 0.12g/kg, 0.24g/kg respectively, ig, qd; The F group is given DONGBAO GANTAI suspension 0.7g/kgig, qd.
4, method of drawing material:
(1) rat fat hepatic tissue morphology: win liver and weigh.Calculate liver index (liver index=liver weight in wet base/body weight * 100%).After observing general appearance, get leftlobe of liver and make histopathological examination;
(2) blood biochemical detects: after the last administration, rat fasting 16h weighs earlier behind 8w, anaesthetize all animals with 3% pentobarbital sodium solution 40mg/kg, heart extracting blood is loaded in the calparine pipe, slowly put upside down mixing, after centrifugal blood plasma, get the biochemical indicator that supernatant is surveyed blood plasma after centrifugal;
(3) get liver organization and clean blood with 4 ℃ of pre-cold salines, weigh behind the filter paper suck dry moisture, every gram liver organization adds the 10ml normal saline, puts 10000r/min * 10s on the refiner * 3 time, prepares 10% homogenate; Get right lobe of liver 1g, make the liver tissue homogenate of 100g/L, get supernatant after centrifugal and survey biochemical indicator and protein expression in the hepatic tissue.
5, detect
(1), biochemical process detects: T-CHOL (TC): enzyme reagent method; Triglyceride (TG): enzyme reagent method; HDL-C (HDL-C): chemical modification enzyme process; Low-density lipoprotein cholesterol (LDL-C): chemical modification enzyme process; Glutamate pyruvate transaminase (ALT): ultraviolet-lactic dehydrogenase enzyme process; Glutamic oxaloacetic transaminase, GOT (AST): ultraviolet-malate dehydrogenase enzyme process; Total protein (TP): Coomassie brilliant blue method; Albumin (ALB): bromocresol green method.
(2) measured by radioimmunoassay blood plasma ET;
(3) adopt the quantitative assay of enzyme linked immunological absorption double antibody sandwich method to detect Plasma TNF-a, IL-6:
(4) the S-P immunohistochemical method is measured ICAM-1, NF-κ B, TGF-β;
(5) the TUNEL method is surveyed apoptosis;
(6) adopt RT-PCR to detect TNF-α, VEGF, the expression of MCP-1 mRNA;
(7) adopt Western blot to detect TGF-β, the expression of NF-KB;
6, statistical method: each is organized data and represents with x ± s, carries out One-way ANOVA with the SPSS11.0 software kit and analyzes (one factor analysis of variance);
Two, result:
(1), ordinary circumstance
1, normal observation: blank treated animal fur light is clean and tidy, and active flexibly, diet is normal, and body weight continues to increase, the no phenomena of mortality; All the other five groups of lethargy that all have in various degree, the movable minimizing, diet reduces, and fur is in disorder, performances such as weight loss.
2, liver cosmetic variation situation: the perusal of blank group liver is cerise, smooth surface.The blank model group naked eyes of fatty liver see that liver volume increases, and it is light yellow that color is, and matter is soft, and tangent plane has greasy shape, and immersing in the water has oil droplet.
3, basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group part liver are near normal, and the surface is aubergine, does not have greasy shape, and finger pressure is flexible, and the volume size is near the blank group.
(2) respectively organize the exponential comparison of rats'liver: blank model group rats'liver coefficient is significantly higher than blank group (P＜0.01), the model copy success; Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group liver coefficient significantly are lower than blank model group (P＜0.01), and this description of test extract formulation of the present invention has some improvement to the rat fat liver.
The results are shown in Table 1.
Table 1 is respectively organized the exponential comparison of rats'liver
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
(3) respectively organize rat liver histopathology morphological change
HE result: blank group lobules of liver structural integrity, hepatocyte does not see obvious degeneration; Blank model group liver leaflet structure disappears, and moderate becomes to moderate fat, and it is big that hepatocyte becomes, swelling, and size is uneven, and pathological changes is with the most obvious around the central vein; Basic, normal, high dosage group of medicine of the present invention and DONGBAO GANTAI PIAN group hepatic lesions have improvement in various degree, slightly become the master to severe fat, and the hepatocyte form is roughly normal, and size is consistent, and intrahepatic fat drips minimizing, and inflammatory cell infiltration alleviates.
Electronic Speculum result: blank group lobules of liver structural integrity, hepatocyte does not see obvious degeneration; Serious steatosis appears in blank model group, and fat drips not of uniform size, and the hepatic fibrosis phenomenon is arranged; Basic, normal, high dosage group of medicine of the present invention and DONGBAO GANTAI PIAN group fat drip alleviating in various degree, and the hepatic fibrosis phenomenon has the phenomenon of alleviating.
(4), respectively organize rat plasma TC, TG, HDL-C, the comparison of LDL-C: blank model group rat plasma TC, TG are significantly higher than blank group (P＜0.01), and blank model group TG value surpasses the twice of blank group.Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat plasma TC, TG significantly is lower than blank model group (P＜0.05); Though the LDL value is no difference of science of statistics between the group respectively, the trend of reduction is arranged.This shows that pharmaceutical preparation of the present invention has good effect for reducing fat, acts on suitable with DONGBAO GANTAI PIAN.The results are shown in Table 2:
Table 2 is respectively organized rat plasma TC, TG, and HDL-C, the comparison of LDL-C (mmol/L) (x ± s)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
(5), respectively organize the comparison of rat plasma ALT, AST: blank model group rat liver organizes ALT (GPT) value to compare with the blank group, and significant difference (P＜0.05) is arranged; Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN class value are compared with blank model group, and no difference of science of statistics shows that extract formulation of the present invention is to the liver avirulence.The results are shown in Table 3:
Table 3 is respectively organized the comparison of rat plasma ALT, AST, (x ± s)
Annotate: compare with the blank group,
*P＜0.05,
*P＜0.01; Compare #P＜0.05, ##P＜0.01 with blank model group
(6), respectively organize the comparison of rat plasma TP, ALB, GLO, A/G: blank model group rat liver tissue T P, ALB, GLO, the A/G value is compared with the blank group, there is not significant difference, compare with blank model group, extract formulation high dose group of the present invention has the effect (P＜0.05) of raising albumin, infers that it has immunoregulation effect.The results are shown in Table 4:
Table 4 is respectively organized the comparison (x ± s) of rat plasma TP, ALB, GLO, A/G
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
(7), respectively organize rat plasma SOD, MDA, the comparison of GST: blank model group rat liver tissue SOD, the GST value significantly is lower than the blank group, and the MDA value is significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat liver tissue SOD, the GST value often is significantly higher than blank model group, the MDA value significantly is lower than blank model group (P＜0.05), show that extract formulation of the present invention has the ability of enhancing body removing free radical significantly, can significantly reduce the snperoxiaized degree of body inner lipid, detoxification ability that can enhancing body.The results are shown in Table 5:
Table 5 is respectively organized rat plasma SOD, MDA, the comparison of GST (x ± s)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
(8), respectively organize rat liver and organize NO, the comparison of NOS: blank model group rat liver organizes the NO value to be significantly higher than the blank group, and total NOS value is significantly higher than blank group (P＜0.01).Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group significantly reduce NO content and NOS activity, show that extract formulation of the present invention has the minimizing fatty liver to form the effect of middle active nitrogen damage.The results are shown in Table 6:
Table 6 is respectively organized rat liver and is organized NO, and the comparison of NOS (x ± s)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
(9), respectively organize blood plasma ET-1, TNF-α, IL-6 level relatively: blank model group rat plasma ET, TNF-α, IL-6 value are significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat plasma value significantly are lower than blank model group (P＜0.05).Show that extract formulation of the present invention can alleviate the damaging action that fatty liver forms middle ET, TNF-α, IL-6.The results are shown in Table 7
Respectively organize relatively (x ± s) of blood plasma ET-1, TNF-α, IL-6 level after table 7 treatment
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
(10), respectively organizing ICAM-1, NF-κ B, TGF-β expresses: the model control group rat liver organizes endochylema pale brown color to occur in each group, and the brown yellow granule that basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN organize endochylema to occur has alleviating in various degree.Three index IOD values more than the imagepro-plus5.0 statistical analysis, model control group rat liver organize highly significant to be higher than blank group (P＜0.01); Basic, normal, high dosage group of extract formulation of the present invention and DONGBAO GANTAI PIAN group IOD value significantly are lower than model control group value (P＜0.05), show that extract formulation of the present invention can alleviate the damaging action of cytokine.The results are shown in Table 8:
Table 8 is respectively organized ICAM-1, NF-κ B, TGF-β and is expressed (x ± s)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with model control group
(11), the respectively apoptotic influence of forming a team: the model control group rat liver organizes karyon pale brown color to occur in each group, and the brown yellow granule that basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN organize karyon to occur has alleviating in various degree.Through the above IOD value of imagepro-plus5.0 statistical analysis, the model control group rat liver organizes highly significant to be higher than blank group (P＜0.01); Basic, normal, high dosage group of extract formulation of the present invention and DONGBAO GANTAI PIAN group IOD value significantly are lower than model control group value (P＜0.05), show that preparation of the present invention can have anti-effect of apoptosis.The results are shown in Table 9:
Table 9 is respectively organized relatively (x ± s) of apoptosis
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare # P＜0.05, ##P＜0.01 with model control group
(12), MCP-1, VEGF, TNF-α gel electrophoresis: the model group rat liver organizes MCP-1, TNF-α, VEGF value to be significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat liver organize MCP-1, TNF-α, VEGF value significantly to be lower than model group, illustrate that extract formulation of the present invention can suppress the expression of the mRNA of MCP-1, TNF-α, VEGF.The results are shown in Table 10
Table 10 is respectively organized the expression (x ± s) of MCP-1, VEGF, TNF-α
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
The Western of NF-KB, TGF-β in (13), the hepatic tissue: the Western of NF-KB, TGF-β in the hepatic tissue; The model group rat liver organizes NF-κ B, TGF-β value to be significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat liver organize NF-κ B, TGF-β value significantly to be lower than model group, illustrate that extract formulation of the present invention can suppress the protein expression of NF-κ B, TGF-β.The results are shown in Table 11:
Table 11 is respectively organized NF-κ B, TGF-β protein expression (x ± s)
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group; Compare #P＜0.05, ##P＜0.01 with blank model group
Three conclusions
One) extract formulation of the present invention can be protected the fatty liver liver tissues of rats, and fatty liver is had certain therapeutical effect:
This experiment empty model group rats'liver coefficient is significantly higher than blank group (P＜0.01), the model copy success; Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group liver coefficient significantly are lower than blank model group (P＜0.01), illustrate that extract formulation of the present invention has some improvement to the rat fat liver.HE result shows blank group lobules of liver structural integrity, and hepatocyte does not see obvious degeneration; Blank model group liver leaflet structure disappears, and moderate becomes to moderate fat, and it is big that hepatocyte becomes, swelling, and size is uneven, and pathological changes is with the most obvious around the central vein; Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group hepatic lesions have improvement in various degree, slightly become the master to severe fat, and the hepatocyte form is roughly normal, and size is consistent, and intrahepatic fat drips minimizing, and inflammatory cell infiltration alleviates.Electronic Speculum result shows blank group lobules of liver structural integrity, and hepatocyte does not see obvious degeneration; Serious steatosis appears in blank model group, and fat drips not of uniform size, and the hepatic fibrosis phenomenon is arranged; Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group fat drip alleviating in various degree, and the hepatic fibrosis phenomenon has the phenomenon of alleviating.
Low albumen high lipid food has been adopted in this research, and assistant is with ethanol, and subcutaneous injection of carbon tetrachloride is made rat fat liver animal model.Inflammatory cell infiltration in this model hepatic tissue, pathological changes such as the serious degeneration of fat have all reached the main observation index of fatty liver, model ideal.No matter from the liver index, HE and Electronic Speculum result show that extract formulation of the present invention all has some improvement to the experimental rat fatty liver.
Two), extract formulation of the present invention is influential to the fatty liver lipid metabolism in rats
This experiment empty model group rat plasma TC, TG is significantly higher than blank group (P＜0.01), and the blank model group TG value twice that is the blank group.Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat plasma TC, TG significantly is lower than blank model group (P＜0.05); Though the LDL value is no difference of science of statistics between the group respectively, the trend of reduction is arranged.This shows that extract formulation of the present invention has good effect for reducing fat, acts on suitable with DONGBAO GANTAI PIAN; This experiment empty model group rat liver organizes ALT (GPT) value to compare with the blank group, and significant difference (P＜0.05) is arranged; Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN class value are compared with blank model group, no difference of science of statistics, show that extract formulation of the present invention is to the liver avirulence, in addition, extract formulation of the present invention also has the effect of slight raising albumin, infers that the Herba Alii fistulosi preparation has immunoregulation effect.
The pathological examination of this experiment is a core index, and it reflects the pathological changes situation of liver objectively, alleviates the steatosis of liver.HE and Electronic Speculum result show that extract formulation of the present invention can obviously improve the hepatic pathology situation of fatty liver rat, alleviate the steatosis of liver, and this experimental result provides the pharmacology foundation for its clinical practice.
Three), extract formulation of the present invention has anti-oxidative damage, the effect of protection hepatic tissue
This experiment empty model group rat liver tissue SOD, the GST value significantly is lower than the blank group, and the MDA value is significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat liver tissue SOD, the GST value often is significantly higher than blank model group, the MDA value significantly is lower than blank model group (P＜0.05), show that extract formulation of the present invention has the ability of enhancing body removing free radical significantly, can significantly reduce the snperoxiaized degree of body inner lipid, detoxification ability that can enhancing body.Blank model group rat liver organizes the NO value to be significantly higher than the blank group, and total NOS value is significantly higher than blank group (P＜0.01).Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group significantly reduce NO content and NOS activity, show that extract formulation of the present invention has the minimizing fatty liver to form the effect of middle active nitrogen damage.
This experiment shows: extract formulation of the present invention significantly increases SOD and GST activity, reduces the NOS activity, reduces MDA and NO content, can reduce fatty liver effectively and form middle active oxygen and active nitrogen damage, and its effect is suitable with DONGBAO GANTAI PIAN.
Four), extract formulation of the present invention is influential to rat fat hepatitis inflammation factor ET-1, TNF-α, IL-6:
Originally studies show that: blank model group rat plasma ET value is significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat plasma value significantly are lower than blank model group (P＜0.05).Show that extract formulation of the present invention can alleviate the damaging action that fatty liver forms middle ET.Blank model group rat plasma TNF-α, IL-6 value are significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat plasma value significantly are lower than blank model group (P＜0.05).Show that extract formulation of the present invention can alleviate the damaging action that fatty liver forms middle TNF-α, IL-6, its effect is suitable with DONGBAO GANTAI PIAN.
Five), extract formulation of the present invention is by regulating the expression control rat fat liver of ICAM-1, NF-κ B, TGF-β
Through imagepro-plus 5.0 statistical analysis ICAM-1, NF-κ B, TGF-β and four index IOD of apoptosis figure value, blank model group rat liver organizes highly significant to be higher than blank group (P＜0.01); Basic, normal, high dosage group of extract formulation of the present invention and DONGBAO GANTAI PIAN group IOD value significantly are lower than blank model class value (P＜0.05), illustrate that extract formulation of the present invention can alleviate the damaging action of cytokine.The model group rat liver organizes MCP-1, TNF-α, VEGF value to be significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat liver organize MCP-1, TNF-α, VEGF value significantly to be lower than model group, illustrate that extract formulation of the present invention can suppress the expression of the mRNA of MCP-1, TNF-α, VEGF.The model group rat liver organizes NF-κ B, TGF-β value to be significantly higher than blank group (P＜0.01); Basic, normal, high dosage group of extract of the present invention and DONGBAO GANTAI PIAN group rat liver organize NF-κ B, TGF-β value significantly to be lower than model group, illustrate that extract formulation of the present invention can suppress the protein expression of NF-κ B, TGF-β.
Originally studies show that: extract formulation of the present invention can suppress the activation of NF-KB, reduces the expression of ICAM-1, MCP-1, TNF-α, VEGF, TGF-β etc., and its effect is suitable with DONGBAO GANTAI PIAN, thereby plays a significant role in the control fatty liver.
Six), extract formulation of the present invention can reduce rat fat hepatocellular apoptosis protection hepatocyte
This experiment finds that the positive hepatocyte of TUNEL method is more rare in the normal group hepatic tissue, and blank model group rat is along with the increasing the weight of of fatty liver degree and hepatocyte inflammation, degree of necrosis, and its mechanism may cause hepatocellular apoptosis and downright bad generation for approach such as oxidative stress and lipid peroxidation injury and Kupffer cell activation
[5], the generation development of prompting apoptosis and fatty liver is closely related, and hepatocellular apoptosis may be high fat diet, ethanol, the early stage pathological change of rat hepatocytes damage of tetrachloro-methane induction fatty liver and inflammation, necrosis.
Through imagepro-plus 5.0 statistical analysis apoptosis values, the model control group rat liver organizes highly significant to be higher than blank group (P＜0.01); Basic, normal, high dosage group of extract formulation of the present invention and DONGBAO GANTAI PIAN group IOD value significantly are lower than model control group value (P＜0.01), show that extract formulation of the present invention can have anti-apoptotic effect, wherein the effect of high dosage effect is better, and extract formulation effect fundamental sum DONGBAO GANTAI PIAN of the present invention is suitable.
Claims (4)
1. the application of Bulbus Allii Fistulosi extract in the lipotropic medicine of preparation, it is characterized in that: Bulbus Allii Fistulosi extract is the very light blue liposoluble effective extracts and the mixture of Semi-polarity class effective extract; Described very light blue liposoluble effective extracts is after one or more the combination in exsiccant very light blue or dried Sucus Allii Fistulosi or the dry full Herba Alii fistulosi is pulverized, through CO
2The supercritical extraction separation and Extraction and material, or utilize solvent method to extract: it is after one or more the combination in exsiccant very light blue or dried Sucus Allii Fistulosi or the dry full Herba Alii fistulosi is pulverized, adopt a kind of solvent cold soak or heat in ether, petroleum ether, chloroform, ethyl acetate, butanols and the ethanol to carry 1～24 hour, filtering extracting solution, extracting solution concentrated remove solvent, promptly get fat-soluble live part extract and promptly obtain very light blue liposoluble effective extracts; It is directly will be exsiccant very light blue or with exsiccant very light blue remainder after fat-soluble live part extraction for described Semi-polarity class effective extract, carry out reflux, extract, with ethanol, after the filtration, filtrate decompression is concentrated into the dried cream that relative density is 1.0～1.3g/ml, in dried cream, adds water, after the macroporous adsorptive resins absorption, can not know saponin component with alcohol flushing to inspection, reclaim solvent, drying obtains the polarity effective extract and promptly obtains Semi-polarity class effective extract; Or directly with exsiccant very light blue or with exsiccant very light blue remainder after fat-soluble live part extracts, carry out reflux, extract, with ethanol, after the filtration, filtrate decompression is concentrated into the dried cream that relative density is 1.0～1.3/ml, in dried cream, add water, the full n-butyl alcohol that closes of water again, a kind of merceration that carries out in propanol or the ethyl acetate solution, seepage, a kind of processing the during heat is carried or extracted to water layer, inspection is known till the no obvious change color, tell extraction part be evaporated to again do after, dilute with water again, solvent is reclaimed in the macroporous resin column decolouring, drying obtains the polarity effective extract and promptly obtains Semi-polarity class effective extract.
2. lipotropic Chinese medicine, it is the very light blue liposoluble effective extracts and the mixture of Semi-polarity class effective extract; Described very light blue liposoluble effective extracts is after one or more the combination in exsiccant very light blue or dried Sucus Allii Fistulosi or the dry full Herba Alii fistulosi is pulverized, through CO
2The supercritical extraction separation and Extraction and material, or utilize solvent method to extract: it is after one or more the combination in exsiccant very light blue or dried Sucus Allii Fistulosi or the dry full Herba Alii fistulosi is pulverized, adopt a kind of solvent cold soak or heat in ether, petroleum ether, chloroform, ethyl acetate, butanols and the ethanol to carry 1～24 hour, filtering extracting solution, extracting solution concentrated remove solvent, promptly get fat-soluble live part extract and promptly obtain very light blue liposoluble effective extracts; It is directly will be exsiccant very light blue or with exsiccant very light blue remainder after fat-soluble live part extraction for described Semi-polarity class effective extract, carry out reflux, extract, with ethanol, after the filtration, filtrate decompression is concentrated into the dried cream that relative density is 1.0～1.3g/ml, in dried cream, adds water, after the macroporous adsorptive resins absorption, can not know saponin component with alcohol flushing to inspection, reclaim solvent, drying obtains the polarity effective extract and promptly obtains Semi-polarity class effective extract; Or directly with exsiccant very light blue or with exsiccant very light blue remainder after fat-soluble live part extracts, carry out reflux, extract, with ethanol, after the filtration, filtrate decompression is concentrated into the dried cream that relative density is 1.0～1.3/ml, in dried cream, add water, the full n-butyl alcohol that closes of water again, a kind of merceration that carries out in propanol or the ethyl acetate solution, seepage, a kind of processing the during heat is carried or extracted to water layer, inspection is known till the no obvious change color, tell extraction part be evaporated to again do after, dilute with water again, solvent is reclaimed in the macroporous resin column decolouring, drying obtains the polarity effective extract and promptly obtains Semi-polarity class effective extract.
3. as lipotropic Chinese medicine as described in the claim 2, it is characterized in that it is a drug oral preparation by the process for preparing medicine preparation.
4. as lipotropic Chinese medicine as described in the claim 3, it is characterized in that described drug oral preparation is a capsule preparations, soft capsule preparation, oral liquid formulations, a kind of in dropping pill formulation or the tablet formulation.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1058529A (en) * | 1991-08-28 | 1992-02-12 | 上海中华日用化工实验所 | Scar-removing beauty cream and method for making thereof |
| CN1907420A (en) * | 2005-08-01 | 2007-02-07 | 武汉市中西医结合医院 | Medicine for treating myocardial ischemia disease and its preparing process |
| CN1965982A (en) * | 2005-11-29 | 2007-05-23 | 孙连芳 | Chinese medicine for lowering blood fat and protecting liver and method for preparing same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058529A (en) * | 1991-08-28 | 1992-02-12 | 上海中华日用化工实验所 | Scar-removing beauty cream and method for making thereof |
| CN1907420A (en) * | 2005-08-01 | 2007-02-07 | 武汉市中西医结合医院 | Medicine for treating myocardial ischemia disease and its preparing process |
| CN1965982A (en) * | 2005-11-29 | 2007-05-23 | 孙连芳 | Chinese medicine for lowering blood fat and protecting liver and method for preparing same |
Non-Patent Citations (4)
| Title |
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| 脂肪肝病人饮食原则.长寿 4.2005,(4),37. |
| 脂肪肝病人饮食原则.长寿 4.2005,(4),37. * |
| 谭江.脂肪肝患者不宜吃什么?.中华养生保健 9.2004,(9),27. |
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