CN102526227A - Use of Rheum emodi extract for preparing drugs for preventing and treating fatty liver diseases - Google Patents

Abstract

The invention discloses the new use of Rheum emodi extract and in particular relates to the use of Rheum emodi extract for preparing drugs for preventing and treating fatty liver diseases. The study in an animal model of fatty liver disease proves that the Rheum emodi extract which is obtained by percolation-leaching Rheum emodi with ethanol and then eluting with ethanol and the compound 3,5,3',4'-tetrahydroxystilbene-4'-O-beta-D-glucopyranoside can significantly inhibit expression of CYP2E in liver cells and can inhibit lipogenesis in liver cells by down-regulating PPAR-gamma expression. Accordingly, the Rheum emodi extract has a significant protective effect on the experimental animals with fatty liver diseases due to different causes and can be applied to preparation of drugs for treating or preventing fatty liver diseases.

Description

The application of Radix Rhei emodi extract in preparation control fatty liver medicine

Technical field

The present invention relates to the application of Radix Rhei emodi extract, particularly relate to the application of Radix Rhei emodi extract in preparation control fatty liver medicine, belong to the therapeutic activity field of chemical compound or pharmaceutical preparation.

Background technology

Radix Rhei emodi (Rheum emodi Wall.) is root and the rhizome of Polygonaceae Rheum ripple leaf group plant, mainly is distributed in ground such as Tibet, Qinghai, Sichuan and Yunnan, all is wild.Radix Rhei emodi sour in the mouth, hardship, cold in nature, the Tibetan medicine claims bent prick (Quza), is traditional medical material of Tibetan medicine.Application number be 201110124226.9 Chinese invention patent applications that are called a kind of Radix Rhei emodi extract and preparation method thereof disclose a kind of Radix Rhei emodi through the ethanol percolation lixiviate again through the extract of ethanol elution.This extract contains the monomeric compound of structural formula suc as formula (I):

formula (I)

R wherein ₁Be Or H.Contain 30%～99% structural formula monomeric compound in this extract suc as formula (II):

Formula (II)

Structural formula can be through above-mentioned Radix Rhei emodi extract through further extraction, eluting prepare suc as formula the monomeric compound of (II); And through the NMR Analysis and Identification be 3,5,3 '; 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) (3; 5,3 ', 4 '-tetrahydroxystilbene 4 '-O-β-D-glucopyranoside).Based on the compare of analysis of existing chemical compound, tentatively confirm 3,5; 3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside has and Radix Polygoni Multiflori composition 2,3; 5; 4 '-tetrahydroxystilbene-2-O-β-similar biological activity of D-glucoside, comprise antitumor, cholesterol reducing, inhibition atherosclerosis, the liver protecting, vasodilator, control senile dementia, improving memory etc., can be applied to the exploitation of related drugs.

Reference material:

Tibet Golden Khada Medical Co., Ltd.. a kind of Radix Rhei emodi extract and preparation method thereof [P] .CN201110124226.9.

Lv Lishuan. the preparation of stilbene glucoside and antioxidation mechanism research [D] in the Radix Polygoni Multiflori. Wuxi: Southern Yangtze University, 2006.

Summary of the invention

The object of the present invention is to provide the Radix Rhei emodi extract to be used to prepare the application of prophylactic treatment fatty liver medicine, and the purposes of pharmaceutical composition in preparation treatment or prevention ischemic heart medicine that contains the Radix Rhei emodi extract.

The present invention also aims to provide 3; 5; 3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) is used to prepare the application of prophylactic treatment fatty liver medicine, and the purposes of pharmaceutical composition in preparation treatment or prevention ischemic heart medicine that contains PDG.

Fatty liver disease (Fatty liver disease; FLD) claim the intrahepatic fat degeneration again; Be the rational hepar damnification of a kind of clinical disease, mean that (being mainly triglyceride (Triglyceride, TG)) accumulates too much for liver fat due to a variety of causes; Surpass 5% of liver wet weights, the pathological state of liver metabolism dysequilibrium.FLD is the general name of a series of pathologic hepar damnifications, can show as simple fatty liver, fat hepatitis, and can develop into hepatic fibrosis and liver cirrhosis.Different according to pathogenic factor; Fatty liver can be divided into two types of alcoholic fatty liver and non-alcoholic fatty liver diseases, and the latter can be subdivided into multiclass such as obese fatty liver, nutritional disorder's property fatty liver, medicine property fatty liver, acute fatty liver of pregnancy, diabetic fatty liver again.Change according to liver histopathology, can fatty liver be divided into three periods: the I phase is the simple fatty liver without the liver tissues inflammatory reaction, and the II phase is with liver tissues inflammatory and Fibrotic fat hepatitis, and the III phase is a fatty cirrhosis.

The pathogenesis of fatty liver is not clear and definite fully as yet so far; Present research think generate with abnormalities of sugar/lipid metabolism, reactive oxygen system (ROS) increase, liver lipid peroxidation increases, hepatic stellate cell activation and cytokine produce relevant unusually; And insulin resistant (insulin resistant, IR) and oxidative stress (oxidative stress) be considered to the key link of its morbidity.In recent years some scholar proposes to turn to oxidative stress and lipid peroxy " two-hit " hypothesis in axle center.Mainly be meant with the insulin resistant to be that the first strike of principal character is that hepatocyte fat occurs and becomes, hepatocyte increases the sensitivity of damage; Hit once more and the later oxidative stress and the enhancing of lipid peroxidation that is meant that a variety of causes causes that repeatedly hit, hepatocellular inflammation, necrosis and fibrosis change occur.Why fatty liver further develops, and main mechanism is the enhancing of oxidative stress and lipid peroxidation, and the factor that increases the weight of oxidative stress and lipid peroxidation is a lot.Some factors such as liver cell pigment p4502E1 (cytochrome p4502EI; CYP2E1) express increase, insulin resistant, ferrum, endotoxin and various cytokine cause lipid peroxidation and oxidative stress; Liver is carried out second strike; Promote lesion growth simultaneously, and the hepatocyte that causes fat to become is inflamed, necrosis and fibrosis.

The alleged fatty liver disease of the present invention comprises the fatty liver disease with liver fat accumulation, lobules of liver fatty pathological changes due to the multiple reason, and indication is by the high alcoholic fatty liver that causes of taking in of ethanol more targetedly.The present invention is the Radix Rhei emodi extract, particularly 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-drug effect principle that O-β-D-glucoside (PDG) is applied to prophylactic treatment fatty liver disease medication preparation mainly is two aspects:

(1) Radix Rhei emodi solution of extract and PDG aqueous solution can significantly suppress the expression of fatty liver disease hepatocyte CYP2E1, have the effect that prevents alcoholic fatty liver.

Ethanol (ethanol) metabolic process is in vivo mainly carried out in liver organ, and mainly through 3 metabolic pathways, one of them is the MEOS (MEOS) on the smooth endoplasmic reticulum.The main component of MEOS be liver cell pigment P4502E1 (cytochrome P4502E1, CYP2E1).CYP2E1 mainly shows both ways the damage of liver: on the one hand; In MEOS; Ethanol generates acetaldehyde and free radical by CYP2E1 catalysis, and free radical combines to cause that with CYP2E 1 on the cell membrane cell-mediated cell toxicant (ADCC) the generation liver toxicity that antibody relies on makes the hepatocyte injury damage.The principal character of this hepatocellular damage is to concentrate to be distributed in the centrilobular region of liver territory; On the other hand, why fatty liver further develops, and main mechanism is the enhancing of oxidative stress and lipid peroxidation, and the factor that increases the weight of oxidative stress and lipid peroxidation is a lot, and wherein CYP2E1 is one of principal element.CYP2E1 is abduction delivering during liver generation fatty pathological changes; Inductive CYP2E1 can increase the weight of and collaborative insulin resistant promotes the deposition of lipid in hepatocyte; Oxidative stress strengthens in the CYP2E1 metabolic process; Further increase abnormalities of sugar/lipid metabolism and increase the two-hit that fat becomes liver simultaneously, CYP2E 1 can also start and promote the hepatocyte fibrosis, so CYP2E1 has participated in the forming process of fatty liver disease.Research data shows that ethanol property or non-alcoholic FLD patient all exist the up-regulated expression of CYP 2E1 in the liver.

The animal experiment of PDG medication of the present invention shows; CYP2E1mRNA and albumen are in the expression enhancing in acinus 3 districts and to the disperse of 2 districts in the rat alcoholic fatty liver model hepatic tissue; Rising and the antioxidant SOD of simultaneous lipid peroxidation end-product MDA, GSH, the decline of VE content.Under PDG intervened, the water-soluble fluid power of PDG reduced the content of lipid in the fatty liver rat fat regulating liver-QI simultaneously, hindered the first effect that fatty liver is formed of hitting; Can significantly increase simultaneously polyphenoils SOD activity, GSH content and VE content; Inhibited oxidation metabolite MDA content; Significantly reduce the expression of CYP2E1, thereby can be from improving body antioxidative ability, increase the energy supply of liver and suppressing the progress of CYP2EI expression aspect blocking-up second strike to the hepatocyte fat lesion.

(2) the PDG aqueous solution has downward modulation PPAR γ expression, suppresses lipogenetic effect in the hepatocyte.

Peroxisome proliferation-activated receptors γ (PPAR γ) is by the activated nuclear factor of part, belongs to the member of nuclear receptor superfamily.PPAR γ mainly is expressed in fatty tissue, as the main moderator that signal between adipose cell gene expression and insulin cell transmits, participates in regulating expression, the lipid of adipose cell specific gene and stores and metabolism, and is closely related with generation, the development of obesity.PPAR γ is topmost lipogenesis regulatory factor, the excretory gene expression signaling molecule of adipose cell such as adiponectin (Adiponectin) and phylaxin (Resistin) also by PPAR γ activate initiation.PPAR γ can be through the expression of other nuclear factors such as activated protein AP-1 and NF-κ B in the regulation and control liver; Inflammatory cytokine such as TNF-a; The generation of the generation regulating liver-QI extracellular matrix of IL-6 etc.; Influence liver tissues inflammatory, necrosis, apoptosis and Fibrotic generation and development, in the generation of FLD, development, play crucial effect, closely related with FLD pathogenetic " two-hit " theory.Normal liver cell has a certain amount of PPAR-γ to express, and when hepatic steatosis obviously reached fat hepatitis formation, the expression of PPAR-γ was stronger.PPAR γ plays an important role in the adipose cell differentiation at the fatty tissue high expressed, and the fatty acid that is transported to muscle and liver is reduced; Lipogenesis reduces; Thereby improve insulin resistant, suppress lipid metabolism, if overexpression then produces serious disorders of lipid metabolism.For example high fat diet causes the high expressed of PPAR-γ in the animal livers tissue, has promoted hepatocellular one-tenth fat property, thereby causes fatty liver.Animal PDG medication of the present invention test shows, intervenes down at PDG, and alcoholic fatty liver cell model triglyceride levels reduces, and PPAR γ expresses and obviously weakens in the hepatocyte, shows that PDG is through the interior lipogenesis of cell of having reduced PPAR γ expression inhibiting.

Based on the Radix Rhei emodi extract drug effect principle of PDG particularly, it is the pharmaceutical composition of active ingredient and be the pharmaceutical composition of active ingredient with PDG that the present invention provides with the Radix Rhei emodi extract.This two drug regimen is an active ingredient with Radix Rhei emodi extract and PDG respectively, adds pharmaceutically acceptable pharmaceutic adjuvant and also is prepared from according to the conventional method of this area.Prepared drug regimen can further prepare becomes pharmaceutical preparation.Pharmaceutical technology based on existing plant extract; Particularly existing pharmaceutical technology is to 2; 3; 5,4 '-application of tetrahydroxystilbene-2-O-β-D-glucoside, the pharmaceutical preparation of the present invention's preparation can be: the dosage form of oral administration such as tablet, capsule (comprising hard capsule, soft capsule, enteric coated capsule, microcapsule), powder, granule and syrup; The dosage form of non-oral administration such as injection, suppository, pill, gel and patch.In addition, can also oral fast release solid formulation (for example tablet, granule etc.) and the slow releasing preparation (tablet, granule, fine granular, pill, capsule, syrup, Emulsion, suspension, solution) that is used for oral or non-oral administration be used for the present invention.Above-mentioned preparation can be the coating or the form of coating not, depends on the needs.

The present invention proves that through experimental study Radix Rhei emodi extract and PDG all have significant protective effect to the laboratory animal fatty liver pathological changes due to the multiple reason, can be applied to treat or prevent the preparation of fatty liver pathological changes medicine.The crude drug Radix Rhei emodi is traditional Tibetan medicine's medical material, and is safe and reliable; PDG has medicinal safety through the same proof of animal toxicity test.

Description of drawings

Fig. 1-1, Fig. 1-2, Fig. 1-the 3rd, Test Example three probe medicines are looked for spectrum behavior figure.

Fig. 2-1, Fig. 2-2, Fig. 2-the 3rd, Test Example trisome giving drugs into nose-time curve chart.

The specific embodiment

Through the correlation test example content of the present invention is done below and further described.

Test Example one

This Test Example in order to explain 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) is through significantly suppressing the expression of alcoholic fatty liver liver cell pigment CYP2E1, plays the effect that prevents alcoholic fatty liver.

1, material and method

1.1 main agents and instrument:

3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG), from the Radix Rhei emodi medical material, extract purity 92.80% according to the open method of CN2011101722069.PDG adds normal saline and is configured to 20mgml ^-1Aqueous solution is hereinafter to be referred as the PDG aqueous solution; Exempt from anti-Mus CYP2E1 multi-resistance CYP2E1 plasmid (Chemicon company).Total length CYP 2 E1cDNA sequences (1.6kb) are assembled in the PGEM4 plasmid.Streptomycete avidin-peroxidase immunohistochemistry test kit (Foochow steps Newbiotics Inc); DIG-DNA labelling and detection kit (German Boebringer Mannheim company); Malonaldehyde (MDA), superoxide dismutase (SOD), glutathion (GSH), vitamin E (VE) test kit (biotech firm is built up in Nanjing).

1.2 the foundation and the medication of rat alcoholic fatty liver model

48 of cleaning level Kunming mouses, body weight (22 ± 2) g, male and female half and half (the Traditional Chinese Medicine Research Institute, Sichuan Province Experimental Animal Center provides) rat is divided into modeling group (24) and matched group (6) after normally feeding 1wk at random.Modeling method is: rat is arbitrarily drunk earlier 2% sucrose solution 3d, adds 5% ethanol (v/v) then, whenever increases by 5% until 15% at a distance from 4d, and then to increase by 5% weekly be 40% until final concentration.When rat is drunk 40% ethanol 16wk, randomly draw 2 and put to death the pathological changes situation of observing liver, after pathological examination confirms to have slight fat to become; Remaining 12 are divided into 6 of pathological model matched groups (I group) at random, and 6 of medical treatment groups (II group) are when continuing to drink 40% alcoholic solution; Every day, per 100 gram rat orals were irritated the PDG aqueous solution of clothes 1ml; Time is 4wk, and the III group gives the glucose solution of rat with heat for matched group.Three groups of all edible common foodstuffs (fat accounts for 5%) of rat, sacrificed by decapitation all animals behind the pharmaceutical intervention 4wk is got the liver living tissue specimen and is made LH biochemical measurement and pathological examination.

1.3 the biochemical measurement of the even oar of liver

Get right lobe of liver 1g, process 10% even oar under 4 ℃, measure MDA respectively, SOD, GSH, VE representes with the content of every gram liver weight in wet base tissue.Press the operation of test kit description.

1.4 pathological examination

Get leftlobe of liver, 10% formalin is fixed, conventional H E dyeing, the degree of observing fatty liver.CYP2E1, immunohistochemical staining adopt streptomycete avidin-peroxidase SABC method, the DAB colour developing; The working concentration of one anti-CYP2E1 multi-resistance 1: 400; Operating process is undertaken by the test kit description, and negative control is all established in each test, substitutes one with phosphate buffer and resists.

1.5 in situ hybridization

The section routine dewaxes to water, and E.C. 3.4.21.64 is handled, and under room temperature, carries out the back with 4% paraformaldehyde and fixes; Every drips (DIG labelling cDNA probe 0.5 μ g/ml, 50% first phthalein amine, Denhardit ' s liquid about hybridization solution 20 μ l; 5% dextran sulfate; The smart DNA of the frog of 200ng/L degeneration), and cover sizeable Parafilm film, hybridization is spent the night in 43 ℃ of wet boxes.Serial SSC washing in second day with the confining liquid sealing, places 1: 500 anti-digoxin monoclonal antibody with section, room temperature 6h, and the colour developing of NBT+BCIP lucifuge, TE liquid cessation reaction, the nuclear fast red is redyed the neutral gum mounting.Negative control is not for adding the hybridization solution of probe.

Statistical procedures: each is organized data and representes with

, carries out statistical disposition with SPSS12.0 software.Between each group with one factor analysis of variance (ANOVA), relatively checking in twos between each is organized with SNK-q.

2 results

2.1 the variation of MDA and SOD, GSH, VE content in the liver

I group is compared with the III group, and MDA content significantly raises, and SOD, GSH, VE content significantly descend; The content of II group MDA and SOD, GSH, VE returns to the level (table 1) of approximate III group again.

MDA and SOD, GSH, VE content

in three groups of rats'liver of table 1

^*The I group is compared with II, t=8.735～14.360, and P＜0.01, the I group is compared t=-9.127～15.320, P＜0.01 with the III group

2.2 the pathology of hepatic tissue change

(1) HE dyeing shows that the morphology of III treated animal hepatic tissue is acted normally under the light microscopic; The above hepatic cell fattydegeneration of moderate has all appearred in the I treated animal, and fat becomes attaches most importance to acinus III district, and a small amount of inflammatory cell infiltration is arranged, and visible spotty necrosis, and the hepatocyte marshalling of II group, form is organized near III.(2) the SP immunohistochemical staining shows under the light microscopic, and the hepatocyte CYP2E1 of III group expresses and is confined in the several hepatocyte of acinus 3 district central vein edge only a few; The hepatocyte CYP2E1 of I treated animal be expressed in that acinus 3 districts strengthen and to the expansion of 2 districts, its distribution that becomes with hepatocellular fat that distributes is consistent, and II group liver CYP2E1 expresses and organizes similarly with III, at the only a few fat drop the weak positive is arranged on every side.Negative control is not seen painted.

2.3 in situ hybridization detects

The I group is the fine and closely woven granule of hyacinthine in the endochylema in the positive signal of mRNA level hybridization, is dispersivity in acinus 3 districts and distributes, and is consistent with the liver cell fatty degeneration distribution, and the II group is similar with the III group, does not see the expression of CYP2E1mRNA positive signal.Negative control is not seen painted.

This animal experiment result of study shows; The hepatic tissue steatosis of PDG aqueous solution intervention recovers normal basically; Immunohistochemistry and in situ hybridization proof PDG can significantly suppress the expression of fatty liver liver cell pigment CYP2E1, and MDA and SOD, GSH, VE content return near normal in the liver simultaneously.

Test Example two

This Test Example explanation 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG) downward modulation PPAR γ expression, lipotropic drug effect.

1 material and method

1.1 material

3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (the PDG aqueous solution is with Test Example one); Hyclone (Hangzhou Sijiqing Biological Engineering Material Co., Ltd.); L02 people's normal liver cell (Medical University Of Chongqing's hepatopathy institute provides); RPMI-1640 culture medium (U.S. GBICO company); PPAR γ goat-anti people polyclonal antibody, Santa Cruz company), SP test kit (Beijing Zhong Shan biotech company).

1.2 method

1.2.1 cell culture and experiment are divided into groups with the RP-MI-1640 culture fluid cultivating people's normal liver cell strain L02 that contains 10% hyclone.The sublethal dose concentration ethanol that filters out with mtt assay acts on the L02 cell, and 6d goes down to posterity in the cycle, is passaged to for the 30th generation to be labeled as Ld30.PDG is made into final concentration is respectively 0.568,1.136, the working solution of 2.273,4.545,9.090 μ g/ml adds 96 well culture plates that ethnic group is implanted with the Ld30 cell successively, and mtt assay filters out PDG the best use of concentration.Experimental group: 1. the PDG processed group adds the PDG of the best use of concentration in the Ld30 cell culture fluid, and every 3d gives 1 medicine, and 6d goes down to posterity 1 time, observes 30d; 2. break away from liquor-saturated group not administration of second and ethanol, only cultivate the Ld30 cell with the RPMI-1640 culture fluid that contains 10% hyclone, 6d goes down to posterity 1 time, observes 30d.

1.2.2 it is that the exponential phase cell 20ml of 5 * 104/ml is inoculated in the culture bottle that the transmission electron microscope observing cell ultrastructure is got concentration; Peptic cell behind the cultivation 72hr, the centrifugal 5min of 1500r/min abandons supernatant; 4 ℃ of cold glutaraldehyde solution fixed cell agglomerate 2hr, the BIAO and BEN Electronic Speculum detects.

1.2.3 full automatic biochemical apparatus detects 106 of the interior horizontal counting cells of TG of cell; The centrifugal 5min of 1500r/min, cold PBS wash 5min * 3 time, need 200 μ l lysate (Urea8mol/L with reference to 2 * 105 cells of cracking fully; CHAPS 40g/L; Tris-HCl 40mmol/L, sodium azide 0.02%, pH 7.4) this standard cell lysis.4 ℃ of thermal agitations, cracking 30min, low-temperature centrifugation 30min collects supernatant, censorship.

1.2.4 immunocytochemical method is observed PPAR γ and is expressed that to get concentration be that the exponential phase cell inoculation of 5 * 104/ml is in 24 orifice plates that are placed with coverslip; After cultivating 72hr; Take out coverslip PBS liquid flushing 5min * 3 time, 95% ethanol is 45min fixedly, presses SP test kit description and operates.Adopt Morphology photo chromic microimage analytical system that expression of results is analyzed, 5 visuals field are selected in the following 100 times of amplifications of light microscopic at random, detect positive staining average optical density value (MOD), positive expression index (=MOD * positive cell percentage * 100).

1.2.5 statistical analysis is handled with SPSS 10.0 for Windows statistical softwares.Detecting data representes with mean ± standard deviation (

); Measurement data adopts the t check; P＞0.05 expression there was no significant difference; P＜0.05 expression has significant difference, and P＜0.01 expression has utmost point significant difference.

2 results

2.1 the PDG of variable concentrations acts on the MTT result behind the Ld30 cell 72h

The PDG of different effects concentration is different to Ld30 cell growth effect.The PDG of each working concentration all has cell growth inhibiting effect to a certain degree; Respectively organize the size back of suppression ratio and find,, suppress to take the lead in showing as increase trend with the increase of PDG activity; When activity is 2.273 μ g/ml, descend to some extent, and then increase with activity.Therefore, choose the activity (table 1) of 2.273 μ g/ml as PDG in this experiment.

The MTT result of the PDG effect ld30 cell 72hR of table 1 variable concentrations

Annotate:

^*The best use of concentration.

2.2 cell ultrastructure under the transmission electron microscope

The Ld30 cell surface microvilli obviously reduces, nucleus major malformotion, endochylema cohesion; The homogeneity fat that number differs, differs in size drips to fill the air and is dispersed in distribution; Observe the PDG group: cell surface has microvillus, and still visible fat drips in the endochylema, but quantity reduces relatively.

2.3 TG level in the cell

Compare with the Ld30 group, breaking away from ethanol group TG level has reduction, but difference not statistically significant (P＞0.05); And the minimizing of TG amount has utmost point significant difference (P＜0.01) (table 2) in the PDG group cell.

TG level in table 2 cell

^*The Ld30 group compares P＜0.01 with the PDG processed group; ^◆The Ld30 group compares P＞0.05 with disengaging ethanol group ^▲The Ld30 group compares P＜0.01 with the L02 group

^*The best use of concentration.

2.4 immunocytochemistry is observed PPAR γ and is expressed

The power that PPAR γ expresses in the experiment is judged according to measured MOD value of Morphology photo chromic microimage analytical system and positive expression index.The result shows: compare with the L02 group, Ld30 group PPAR γ expresses obviously and strengthens; Compare with Ld30, PDG group PPAR γ expresses and obviously weakens (table 3).

Table 3PPAR γ expresses index

^{* ★}Than Ld30 group, P＜0.01

Above-mentioned experiment selected ursodeoxycholic acid acts on external alcoholic fatty liver cell model, observes 30d.Observe expiration and detect TG level in the cell.The result shows that TG obviously reduces in the PDG processed group cell, explain PDG can ameliorate body the steatosis of alcoholic fatty liver cell model outward; Result of the test shows that simultaneously breaking away from the interior TG level of ethanol group cell also decreases, and conforms to the clinical treatment measure of eliminating the cause of disease.Test shows that further L02 compares with people's normal liver cell, and PPAR γ expresses significantly and strengthens in the external evoked alcoholic fatty liver cell model.Intervene through PDG, then can reduce PPAR γ and express, suppress lipogenesis in the cell, the effect that the outer inductive alcoholic fatty liver cellular fat of ameliorate body becomes.

Test Example three

This Test Example can significantly suppress the expression of alcoholic fatty liver liver cell pigment CYP2E1 in order to explanation Radix Rhei emodi extract.

1 materials and methods

1.1 trial drug Radix Rhei emodi extract specifically is a Radix Rhei emodi through the ethanol percolation lixiviate again through the extract of ethanol elution, from the Radix Rhei emodi medical material, extracts acquisition according to the said method of CN2011101722069; These article contain 30%3 approximately; 5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (PDG); Be chocolate brown powder, be mixed with 40mgml with normal saline during experiment ^-1Aqueous solution is through using behind the 0.22 μ m aperture filter membrane EK, to call the Radix Rhei emodi solution of extract in the following text.

1.2 reagent probe medicine: caffeine (coffine), dapsone (dapsone), chlorzoxazone (chlorzox azone) (sigma-Acdrich company); Interior mark: phenazone (antipyrine, Tianjin recovery fine chemistry industry institute); Acetonitrile, methanol are chromatographically pure (Tianjin section close europeanized reagent development centre), and other reagent are analytical pure; Experimental water is a redistilled water.

1.3 animal is with Test Example one.

1.4 instrument high performance liquid chromatograph: Agilent 1100, diode array UV-detector DAD; Agilent chromatograph chem workstation (U.S. Agilent Technologies); TGL-16G High speed refrigerated centrifuge (Anting Scientific Instrument Factory, Shanghai); The quick vortex mixer of XK96-A (Jiangyan City, Jiangsu Xin Kang instrument plant).

1.5 method

1.5.1 chromatographic condition chromatographic column: EclipesXDB-C18 post (46mm * 250mm, 5 μ m); Mobile phase: acetonitrile-water (25: 75), flow velocity 1.0mlmin ^-1Detect wavelength: 280nm; Column temperature: room temperature; Sample size: 20 μ l; Interior mark: phenazone (20mgl ^-1).

1.5.2 being formulated as concentration, the Radix Rhei emodi solution of extract is equivalent to crude drug in whole 0.31gml ^-116 of rats are divided into administration group and blank control group at random.The administration group is irritated stomach early morning every day on an empty stomach and is given the Radix Rhei emodi solution of extract and irritate gastric juice once, dosage 1.1ml100g ^-1, the blank group gives the normal saline of equal volume, induces 10 days.

1.5.3 the processing of blood serum sample is induced back fasting in evening on the 10th with mensuration Radix Rhei emodi solution of extract; The 11st day morning under etherization; Do the intubate operation in rats with left femoral artery place, treat to give after animal revives Cocktail probe medicine caffeine, dapsone, chlorzoxazone.Caffeine, dapsone, chlorzoxazone respectively by 10,10,20mgkg ^-1Dosage add a small amount of Tween 80 and grind and add normal saline again and process the emulsion drug administration by injection, and after administration 0.17,0.5,0.83; 1.17,1.5,2.0,2.5; 3.0,5.0,8.0,12.0hr gets the about 0.25ml of blood immediately with the centrifugal 5min of 8000rpm by the intubate place; Accurately take out 100 μ l serum, add phenazone working solution 20 μ l mixings, add 500 μ l chloroforms again, suspendible concussion 2min; With the centrifugal 8min of 6000rpm, get and add acetonitrile-water (1: 1) solution 100 μ l dissolving after organic facies chloroform 400 μ l volatilize, get 20 μ l sample introductions.

1.5.4 pharmacokinetic parameter calculates and the analytical method pharmacokinetics calculates the completion of employing DAS 2.0 softwares, selects for use non-compartment analysis method-statistical moment to carry out match.

2 results

2.1 A, B, C are respectively the serum of blank serum, adding probe medicine and the serum behind the interior injection of the body probe medicine is analyzed gained under above-mentioned chromatographic condition liquid chromatogram among the chromatographic behavior figure.Among Fig. 1-1, Fig. 1-2, Fig. 1-3, A---blank serum; B---add the serum of probe medicine; Serum in C---the body behind the injection probe medicine; 1---caffeine; 2---phenazone (interior mark); 3---dapsone; 4---chlorzoxazone.Separated well with interior target by the visible caffeine of this figure, dapsone, 3 kinds of probe medicines of chlorzoxazone, interference is not seen in blank serum experiment yet.

Add 3 kinds of probe medicines 2.2 standard curve is got blank serum 100 μ l, make that the concentration of caffeine is respectively 0.5,1.0,2.0,4.0,8.0,12.0 in the serum, 16.0mg μ l ^-1, the concentration of dapsone is respectively 0.25,0.5,1.0,2.0,4.0,8.0,12.5mg μ l ^-1, the concentration of chlorzoxazone is respectively 0.5,1.0,2.0,4.0,8.0,16.0,25.0mg μ l ^-1, mark liquid 20 μ l press the blood serum sample disposal methods in adding, with the ratio S of probe medicine and interior target peak area concentration C (the mg μ l to the probe medicine ^-1) do linear recurrence, get regression equation, caffeine: S=0.4079C+0.068, r=0.9999; Dapsone: S=1.4066C-0.2481, r=0.9998; Chlorzoxazone: S=0.3069C-0.0039, r=0.9971.Minimum quantitative limit is respectively: 0.5,0.25, and 0.5mg μ l ^-1

2.3 precision is prepared basic, normal, high three kinds of concentration: caffeine (1.0,4.0,16.0) mg μ l ^-1, dapsone (0.5,2.0,12.5) mg μ l ^-1, chlorzoxazone (1.0,4.0,25.0) mg μ l ^-1Each 5 parts of serum analog samples, according to the blood serum sample disposal methods, calculate withinday precision; METHOD FOR CONTINUOUS DETERMINATION 5 days is calculated day to day precision, three kinds of probe medicines in a few days, the RSD value of day to day precision is all less than 5.60%.

2.4 the response rate is got basic, normal, high three kinds of concentration: caffeine (1.0,4.0,16.0) mg μ l ^-1, dapsone (0.5,2.0,12.5) mg μ l ^-1, chlorzoxazone (1.0,4.0,25.0) mg μ l ^-1The serum analog sample each 5 parts, record average recovery, be respectively: caffeine (103.70 ± 2.95; 101.33 ± 1.22; 99.91 ± 1.14) %, dapsone (107.39 ± 4.42,96.26 ± 4.78,100.45 ± 1.48) %, chlorzoxazone (105.72 ± 3.25; 95.69 ± 3.08,102.20 ± 1.85) %.

2.53 behind the Wistar male rat injection Cocktail probe medicine of the drug-time curve matched group of kind probe medicine, PDG group; Each time point blood sampling; By 2.1 chromatographic condition determination and analysis; Blood drug level ± the time graph of 3 kinds of probe medicines is like Fig. 2-1, Fig. 2-2, Fig. 2-3, A among the figure---caffeine; B---dapsone; C---chlorzoxazone.

Adopt the DAS2.0 program 2.63 plant the pharmacokinetic parameters of probe medicine, different time blood drug level data are carried out match to the time, adopt the statistical moment method to calculate pharmacokinetic parameters, see table 1.Caffeine, dapsone and chlorzoxazone be respectively by liver drug enzyme CYP1A2, CYP3A4, CYP2E1 metabolism, and as probe medicine, the pharmacokinetic parameters of these three kinds of medicines changes especially the reacting condition of half-life and the influence of PDG to the rats'liver drug metabolizing enzyme.Can find out under the influence of Radix Rhei emodi solution of extract, to have only chlorzoxazone half-life and matched group from last table, equal not statistically significants of half-life of other each groups than significant prolongation.

3 kinds of probe medicine pharmacokinetic parameters in table 1 rat blood serum ( n=8)

Compare with matched group ^*P＜0.05.

Above-mentioned animal experiment adopts Cocktail probe medicine method to estimate the influence of Radix Rhei emodi extract to CYP450.The probe medicine comprises caffeine, dapsone, chlorzoxazone, detects 3 kinds of main metabolic enzyme CYP1A2, the CYP3A4 of CYP450 enzyme system, the activity of CYP2E1 respectively.Table 1 result shows that the metabolism of chlorzoxazone in the Radix Rhei emodi solution of extract group slows down t _1/2Prolong, the metabolism of caffeine, dapsone and matched group change not obvious.This shows that the Radix Rhei emodi extract is inhibited to CYP2E1, and is not obvious to CYP1A2, CYP3A4 effect; Therefore when the Radix Rhei emodi extract when passing through the drug combination of CYP2E1 enzymes metabolism, need be careful the variation of blood drug level closely, to avoid contingent drug interaction.

Test Example four

3,5,3 ', 4 '-tetrahydroxystilbene-4 '-test of the toxicity of compound of O-β-D-glucoside.

1, experiment material

Laboratory animal: the ICR mice, male, body weight 18～22g provides the (quality certification number: 1037764) by Sichuan University's West China Experimental Animal Center.

Experimental compound: 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside (from from the Radix Rhei emodi medical material, extracting acquisition, content 92.80%) according to embodiment two methods.

2, test method and result

The compounds of this invention is carried out acute toxicity test as test specimen to mouse gavaging.The result shows: the maximum tolerated dose of mouse gavaging The compounds of this invention is 20gkg ^-124hr ^-1, be about 2,3,5,4 '-(the 60kg body weight becomes human oral consumption per day 1.2gkg for the taking dose standard of tetrahydroxystilbene-2-O-β-D-glucoside medicine ^-124hr ^-1) 1000 times; The LD50 of the disposable filling clothes of mice is 15.2g/kg, and 95% confidence limit is 22.60～34.23g/kg.

Long term toxicity test shows, rat filling every day clothes The compounds of this invention 0.4,0.8,1.6gkg ^-1(be equivalent to 2; 3; 5; 4 '-20,40,80 times of tetrahydroxystilbene-2-O-β-D-glucoside clinical drug amount), 24 week back hematologys, blood biochemical are learned relatively all no significant differences of index, organ coefficient and matched group, and pathological examination results does not also see that the pathologic that health food causes changes.Stop using the invention chemical compound after 2 weeks, and each item index of each administration group and blank group compare, and all no significant difference points out this chemical compound not have obvious retardance toxicity.Explain that it also is safe that The compounds of this invention is taken for a long time.

Claims (8)

1. the application of Radix Rhei emodi extract in preparation control fatty liver disease disease medicament, said Radix Rhei emodi extract contains the monomeric compound of structural formula suc as formula (I):

formula (I)

R wherein ₁Be Or H; Said R ₁Be

Monomeric compound be 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside, its structural formula is suc as formula (II):

formula (II).

2. contain the application of pharmaceutical composition in preparation control fatty liver medicine of Radix Rhei emodi extract, said Radix Rhei emodi extract contains the monomeric compound of structural formula suc as formula (I):

formula (I)

R wherein ₁Be

Or H; Said R ₁Be

Monomeric compound be 3,5,3 ', 4 '-tetrahydroxystilbene-4 '-O-β-D-glucoside, its structural formula is suc as formula (II):

formula (II).

3. application according to claim 2 is characterized in that: said pharmaceutical composition contains Radix Rhei emodi extract and pharmaceutically acceptable pharmaceutic adjuvant.

4. according to claim 1,2,3 arbitrary described application, it is characterized in that: said medicine is to suppress liver cell pigment P4502E1 active medicine.

5. according to claim 1,2,3 arbitrary described application, it is characterized in that: said medicine is to suppress PPAR γ expression medicine.

6. according to claim 1,2,3 arbitrary described application, it is characterized in that: said medicine is an anti-oxidation medicine.

7. according to claim 1,2,3 arbitrary described application, it is characterized in that: said fatty liver disease is the alcohol fatty hepatopathy.

8. application according to claim 7 is characterized in that, said medicine is to suppress liver cell pigment P4502E1 active medicine, or suppresses PPAR γ expression medicine, or anti-oxidation medicine.

Images