CN101912456B - Novel application of rubus alceaefolius poir total alkaloid - Google Patents
Novel application of rubus alceaefolius poir total alkaloid Download PDFInfo
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Abstract
The invention provides application of rubus alceaefolius poir total alkaloid to preparing a medicament for treating non-alcoholic fatty liver and a pharmaceutical composition for treating the non-alcoholic fatty liver. The rubus alceaefolius poir total alkaloid is an effective component extracted from the root of a traditional Chinese medicine of rubus alceaefolius poir, has remarkable improving or reversing effect on the fatty degeneration of liver cells and the lipid accumulation index in a high-fat diet animal model, can reduce the contents of serum total cholesterol and the low-density lipoprotein cholesterol and improve the contents of high-density ipoprotein cholesterol. The invention shows that the rubus alceaefolius poir total alkaloid has better treatment function on the non-alcoholic fatty liver, in particular has more excellent pharmaceutical effect on the simple fatty liver and provides a new pharmaceutical selection for clinical application.
Description
Technical field
The present invention relates to the new purposes of Radix seu Folium Rubi alceaefolii total alkaloids, particularly, is the purposes in the medicine of preparation treatment non-alcoholic fatty liver disease, belongs to drug world.
Background technology
Crude Rubus (Rubus? Alceaefolius? Poir., RAP), as crude Rosaceae Rubus roots, leaves, also known as big-leaf snake bubble Jin, a large rag thorn tiger bubble, tiger palm Jin, September bubble etc., medicinal rich in resources, mainly distributed in China, Jiangsu, Jiangxi, Fujian, Taiwan, Hunan, Guangdong, Guangxi, Guizhou, Yunnan and other places, was born in 500 ~ 2000m above sea level on a sunny slope, valley weed or swamp thickets within and roadside rocks, its roots, leaves medicine, sweet and light and flat, with stasis, heat to stop bleeding and other effects, Indications of chronic hepatitis, dysentery, enteritis, mastitis, stomatitis, march hemoglobinuria, traumatic bleeding, hepatosplenomegaly, bruises, rheumatism and so on.
Mainly contain compositions such as triterpene, flavone, tannin, sterol, alkaloid, aminoacid in the bibliographical information Radix seu Folium Rubi alceaefolii.Chen Wang is etc., Radix seu Folium Rubi alceaefolii chemical constituent and will be from effect progress, " Asia-Pacific traditional medicine "; 2009 47 phases of volume, report separates the triterpenes components that obtains from the leaf of Radix seu Folium Rubi alceaefolii, be respectively 2 α, 3 beta-dihydroxies-Usu-12-alkene-28-acid, 2 α; 3 β, 19 α-trihydroxy-Usu-2-alkene-28-acid, 2 α, 3 β, 19 α; 23-tetrahydroxy-Usu-12-alkene-28-acid-28-O-β-D-glucoside, 2 α, 3 β, 19 α, 23; 24-penta hydroxy group-olive-12-alkene-18-acid-28-O-β-D-glucoside and 2 α, 3 β, 19 α-trihydroxy-Usu-12-alkene-23,28-diacid-28-O-β-D-glucoside; Isolating 2 flavones ingredients are respectively 3,5,7,3 from leaf
/, 4
/-pentahydroxyflavone-3-O-[(2-O-α-L-rhamanopyranosyl)]-(6-O-α-L-rhamanopyranosyl)-β-D-galactoside and hyperin.Isolating 3 tannin constituents are respectively sanguiin H-2ethyl ester, 1,2,3 from Radix seu Folium Rubi alceaefolii, 6-tetra-O-galloyl-β-D-glucopy-ranose, 1,2,3,4,6-penta-O-gal loyl-β-D-glucopy-ranose.To the report of Radix seu Folium Rubi alceaefolii active component, as: leaf luxuriant sesame, etc., Radix seu Folium Rubi alceaefolii is to the therapeutical effect research of experimental hepatic injury, and " Chinese medicine academic periodical " rolled up for 5 phases in 2005 23, with CCl
4Set up the rat acute liver injury model, investigate the therapeutical effect of Radix seu Folium Rubi alceaefolii decocting liquid to experimental hepatic injury, the result shows that Radix seu Folium Rubi alceaefolii decocting fluid power reduces CCl
4Unusually the MDA that increases unusually in the ALT that increases in the induced mice serum, AST and the hepatic tissue, the content of NO, while increased SOD content, and can improve the hepatic pathology damage.Huang Ling, etc., Radix seu Folium Rubi alceaefolii root extract to the influence of Rats with Acute Liver Injury serum NO level regulating liver-QI tissue SOD, MDA, " Chinese Chinese medicine science and technology ", 2008 15 1 phases of volume are through with CCl
4Set up the rat acute liver injury model, investigate Radix seu Folium Rubi alceaefolii root total alkaloids, total polysaccharides, total flavones therapeutical effect acute liver damage.The result shows, Radix seu Folium Rubi alceaefolii root total alkaloids can obviously reduce MDA content (P＜0.01) in the NO that increases unusually in the rat blood serum and the hepatic tissue, improves SOD activities of liver, alleviates the rat hepatocytes degree of necrosis.Total polysaccharides, total flavones have certain influence to SOD, MDA in rat blood serum NO and the hepatic tissue, but do not have statistical significance.Hong Zhenfeng, etc., Radix seu Folium Rubi alceaefolii root extract to the influence of Rats with Acute Liver Injury TNF-α, IL-1 β, IL-6, " Chinese Chinese medicine academic periodical ", 2007 years 27 6 phases of volume are through with CCl
4Set up the rat acute liver injury model, investigate Radix seu Folium Rubi alceaefolii root total alkaloids, total polysaccharides, total flavones therapeutical effect acute liver damage; The result shows; Radix seu Folium Rubi alceaefolii root total polysaccharides can obviously reduce the content TNF-α, IL-1 β, the IL-6 (P＜0.01) that increase unusually in the rat blood serum and alleviate the rat hepatocytes degree of necrosis, and total alkaloids, total flavones have certain influence to rat blood serum TNF-α, IL-1 β, IL-6 content but do not have statistical significance.
The document of report all is the hepatic injury of Radix seu Folium Rubi alceaefolii root total alkaloids for treating at present; Acute liver damage is the lobules of liver structural deterioration that produced by the chemical substance acute injury; The general cloudy swelling of hepatocyte, appearance are that focal hepatocellular degeneration, necrosis and the cell infiltration at center is the inflammation performance of characteristic with the central vein.
The pathogenic factor of non-alcoholic fatty liver disease is under Long-term High-fat high-carbonhydrate diet condition, to cause excess fat acid in hepatocyte, to deposit, thereby causes hepatic cell fattydegeneration, promptly is defined as fatty liver when the liver inner lipid surpasses 5% of liver wet weights.Its pathological change is similar with alcoholic liver disease; But the patient does not have excessive history of drinking history, and paathogenic factor mainly is to lack moving life style, IR, metabolism syndrome and component thereof (obesity, hypertension, blood fat disorder and type 2 diabetes mellitus) by higher fatty acid high caloric diet structure, sit more; Its spectrum of disease comprise nonalcoholic fatty liver (nonalcoholic simple fatty liver, NAFL), non-alcoholic stellato-hepatitis (NASH) and relevant liver cirrhosis and hepatocarcinoma.The different diffusivity hepatic cell fattydegeneration of degree appears on the pathological section; Fat becomes extremely swelling of hepatocyte, and circular, volume obviously increases than normal group; Be full of the significant quantities of fat cavity in the endochylema; Cell boundary is unclear, the fatty change and ballooning degeneration, and all visible inflammatory cell infiltration or the point-like that is dispersed in or fusion are downright bad in all BIAO and BEN lobules of fatty liver rat.Wherein, Exist hepatic cell fattydegeneration and fat to store up in the lobules of liver of simple fatty liver; Account for the 1/3-1/2 of lobule during slight simple fatty liver, account for the 1/2-2/3 of lobule during moderate, account for during severe lobule 2/3 or more perhaps hepatocyte diffusivity steatosis be the fishnet shape; But there are not other obvious Histological changes, i.e. NIP, necrosis and fibrosis.
Therefore hepatic injury and non-alcoholic fatty liver disease have the difference of essence, and the medicine with treatment hepatic injury is not the effect that necessarily has the treatment non-alcoholic fatty liver disease.At present, there is not bibliographical information that Radix seu Folium Rubi alceaefolii total alkaloids is used to treat non-alcoholic fatty liver disease.
Summary of the invention
The new purposes that the purpose of this invention is to provide Radix seu Folium Rubi alceaefolii total alkaloids, i.e. purposes in the medicine of preparation treatment non-alcoholic fatty liver disease.Another object of the present invention has provided a kind of pharmaceutical composition of treating non-alcoholic fatty liver disease.
The invention provides the purposes of Radix seu Folium Rubi alceaefolii total alkaloids in the medicine of preparation treatment non-alcoholic fatty liver disease.
Further preferably, described medicine is the medicine of treatment simple fatty liver.
Wherein, contain total alkaloids in berberine hydrochloride in the described Radix seu Folium Rubi alceaefolii total alkaloids, greater than 95%w/w.
Wherein, described Radix seu Folium Rubi alceaefolii total alkaloids derives from rosaceous plant Radix seu Folium Rubi alceaefolii Rubusalceaefolius Poir. root extract.
Wherein, described extract is the 70-95% ethanol extraction.
Wherein, described extract is 85% ethanol extraction.
The present invention also provides a kind of pharmaceutical composition of treating non-alcoholic fatty liver disease, and it is that Radix seu Folium Rubi alceaefolii total alkaloids by effective dose is an active component, adds that acceptable accessories or complementary composition are prepared into preparation pharmaceutically commonly used.
Wherein, described preparation is an oral formulations.
Radix seu Folium Rubi alceaefolii total alkaloids of the present invention is the effective ingredient that extracts from Chinese crude drug Radix seu Folium Rubi alceaefolii root; Hepatic cell fattydegeneration in the high fat diet animal model and the gathering of liver lactone class are improved significantly or reversing effect; And can reduce serum total cholesterol and low-density lipoprotein cholesterol content; The high density lipoprotein increasing cholesterol level; Show that Radix seu Folium Rubi alceaefolii total alkaloids has therapeutical effect preferably to non-alcoholic fatty liver disease, especially preferable to the drug effect of simple fatty liver, provide a kind of new medication to select for clinical.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Below, foregoing of the present invention is remake further detailed description through the specific embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1: normal group HE dyeing hepatic tissue (lobus sinister) pathological section light microscopic view, amplification be 40 *.
Fig. 2-3: fatty liver model group HE dyeing hepatic tissue (lobus sinister) pathological section light microscopic view, amplification be followed successively by 40 *, 100 *.
Fig. 4-5:PPC matched group HE dyeing hepatic tissue (lobus sinister) pathological section light microscopic view, amplification be followed successively by 40 *, 100 *.
Fig. 6-7:TARAP low dose group HE dyeing hepatic tissue (lobus sinister) pathological section light microscopic view, amplification be followed successively by 40 *, 100 *.
Dose groups HE dyeing hepatic tissue (lobus sinister) pathological section light microscopic view among Fig. 8-9:TARAP, amplification be followed successively by 40 *, 100 *.
Figure 10-11:TARAP high dose group HE dyeing hepatic tissue (lobus sinister) pathological section light microscopic view, amplification be followed successively by 40 *, 100 *.
Figure 12-17: each organizes the freezing hepatic tissue of fat stains (lobus sinister) pathological section, amplification 40 * and, be followed successively by dose groups, TARAP high dose group among normal group, model group, PPC matched group, TARAP low dosage, the TARAP.
The specific embodiment
The preparation of embodiment 1 Radix seu Folium Rubi alceaefolii total alkaloids of the present invention
Take by weighing the 200g Radix seu Folium Rubi alceaefolii and place the 2000ml round-bottomed flask, add 1200ml85% alcoholic solution (solid-to-liquid ratio 1: 6) and place the electronic thermostatic electric jacket to reflux each 30 minutes 3 times for 80 ℃; Merge extractive liquid, reclaims ethanol, gets concentrated solution; Drying promptly gets Radix seu Folium Rubi alceaefolii total alkaloids.
The quality standard of Radix seu Folium Rubi alceaefolii total alkaloids adopts acid-dye colorimetry for being contrast with the berberine hydrochloride, under the 415nm wavelength, measures, and contains total alkaloids in the Radix seu Folium Rubi alceaefolii total alkaloids in berberine hydrochloride, greater than 95%w/w.
Except adopting the said method extraction, also can adopt the method for present bibliographical information to extract, as: Hong Zhenfeng, etc., Radix seu Folium Rubi alceaefolii root total alkaloid contents is measured, " Colleges Of Traditional Chinese Medicine Of Fujian's journal ", 2008 18 5 phases of volume.
The preparation of embodiment 2 medicinal tablets of the present invention
Get the Radix seu Folium Rubi alceaefolii total alkaloids of embodiment 1 preparation, add tablet and use adjuvant, granulation, tabletting promptly get tablet.
Below prove beneficial effect of the present invention through pharmacodynamics test.
Experimental example 1 medicine Radix seu Folium Rubi alceaefolii total alkaloids treatment non-alcoholic fatty liver disease of the present invention experiment
1) animal grouping, modeling and administration
At random 60 SD rats are divided into 6 groups; Every group 10; Every group of equal male and female half and half; Promptly being divided into normal group, fatty liver model group, Essentiale N/Essentiale Forte N is polyene phosphatidylcholine (polyene phosphatidylcholine, PPC) 3 treatment groups of the high, medium and low dosage of matched group and Radix seu Folium Rubi alceaefolii total alkaloids (TARAP).2 rats are incorporated model group in addition, use as sampling check modeling effect.
Except that normal group, all the other are respectively organized rat and all replace normal feedstuff to feed for 12 weeks with high lipid food.High lipid food proportioning: normal feedstuff 87.3%, Adeps Sus domestica 10%, cholesterol 2%, Fel Sus domestica salt 0.7%.In getting each 1 of female, the great and mighty or powerful Mus of model group at random in the 10th week of modeling, observe the modeling effect.
The 10th week beginning administration of modeling success back.When continuing modeling (preventing animal spontaneous recovery); Normal group, fatty liver model group are given 0.9% sodium chloride injection; The PPC matched group is with administration 76mg/kgd, and the high, medium and low administration of alkaloid is respectively: 2.6g/kgd, 1.3g/kgd and 0.65g/kgd (by the preparation of embodiment 1 method).All irritate the administration of stomach mode with solvent, dosage is 2ml/250g.Below all in 9-11 point execution in the morning, every day 1 time, continuous 14 days.
2) observation index and test item
The hepatic tissue HE check pathological section that dyes
(1) cuts 1cm * 1cm * 1cm piece of tissue in leftlobe of liver, preserved in 10% formalin fixing 24 hours.
(2) take out hepatic tissue blocking, carry out gradient alcohol dehydration, transparent, waxdip and FFPE.
(3) the tissue wax disk(-sc) is with cycle type microtome transverse section serial section, and thickness is 4 μ m, drags for sheet, baking box oven dry.
(4) tissue slice is with 100% xylene dewaxing 10min * 2 time, and dehydrated alcohol, 95%, 80% gradient ethanol soak 5min * 1 time successively.
(5) tissue slice is washed through 1% haematoxylin immersion 15min, water logging respectively successively and is returned blue 15min, 5% hydrochloric acid color separation 5sec and 1% Yihong dyeing 30sec, oven dry after the water flushing, resin mounting.
(6) classification of fatty liver pathology is built high pathological diagnosis standard with reference to model.Under the light microscopic, with "-", "+", " ++ " and " +++" four grades, with corresponding " normally ", " slightly fatty liver ", " moderate fatty liver " and " severe fatty liver " clinically respectively as the fatty liver degeneration.Normal or the accidental fat vacuole of hepatocyte structure is "-" level; 1/3 hepatic cell fattydegeneration in the lobules of liver involves lobules of liver quantity＜50% and is "+" level; 2/3 hepatic cell fattydegeneration in the lobules of liver involves lobules of liver quantity＜50%, or 1/3 hepatic cell fattydegeneration in the lobules of liver, involves lobules of liver quantity＞50% and is " ++ " level; In the lobules of liver＞and 2/3 hepatic cell fattydegeneration, involve lobules of liver quantity＞50% and be " +++" level.
Fat stains of hepatic tissue frozen section and image quantitative analysis
(1) hepatic tissue cuts the about 1cm * 1cm of size * 1cm,, is stored in-20 ℃ of refrigerators with 10% formaldehyde fixed to make frozen section.
(2)-20 ℃ descending frozen section, slice thickness is 8 μ m, to speckle with the slide bonding die of collodion.
(3) 70% ethanol is put in section and washed 1min, a Sudan IV 1～2min that dyes is dripped in the back, and reuse 70% ethanol is washed slightly, and flowing water is washed again, and redye with haematoxylin the back, and flowing water washes to returning indigo plant, the neutral glycerine mounting.
(4) the quantitative test sample book of form is selected 5 examples for every group at random from 60 routine BIAO and BEN, and optional 2 kiernan's space visuals field are clapped 5 under mirror, does not repeat, and every group of photo has the lobules of liver visual field 5 * 5 * 2=50.Every is all amplified 40 times.
(5) with the positive area of the Motic Med.6.0 of Mike Audi computer software measurement, gray scale, AO and integral optical density.
(6) adopt SPSS16.0 to carry out the statistical computation of data.
The mensuration of serum TG, TC, HDL-C and LDL-C
After the rat anesthesia success, advance abdomen through median incision of lower abdomen, the about 6ml of whole blood is got in the ventral aorta blood sampling, behind the centrifugal 10min of low speed 1500rpm, gets supernatant in-20 ℃ of preservations, measures serum TG, TC, HDL-C and LDL-C respectively.Adopt SPSS 16.0 to carry out the statistical computation of data.
3) result
HE dyeing hepatic tissue (lobus sinister) pathological section result
The analysis of table 1 pathological section relatively
Image quantitative analysis table 2 result as a result shows; In positive area, gray scale, AO and 4 indexs of integral optical density; The TARAP high dose group is compared model group and all is significantly improved; The improvement level all is better than the PPC matched group, and along with the rising of TARAP dosage, scope and degree that hepatic tissue cell is got involved all make moderate progress.Each is organized the freezing hepatic tissue of fat stains (lobus sinister) pathological section light microscopic image and sees Figure 12-17.
The positive area of table 2, gray scale, AO, integral optical density fat stains image quantitative analysis between each group compares
* for relatively to have significant difference (P＜0.05) with model group
Serum TG, TC, HDL-C and LDL-C measure as a result that table 3, table 4 result show that PPC can significantly improve fatty liver rat serum HDL-C concentration (P＜0.05), but TC, TG and LDL-C influenced not statistically significant (P＞0.05).Compare with each dose groups of TARAP, though the significant difference on the not statistically significant, PPC is better than low dose group to the approaching middle and high dose groups level of the rising effect of Serum HDL-C level; The effect of falling serum LDL-C then is inferior to the middle and high dose groups of TARAP.
Table 3 serum TC, TG analyze relatively
Table 4 Serum HDL-C, LDL-C analyze relatively
* for relatively to have significant difference (P＜0.05) with model group
Above-mentioned experimental result shows; Radix seu Folium Rubi alceaefolii total alkaloids of the present invention is the effective ingredient that extracts from Chinese crude drug Radix seu Folium Rubi alceaefolii root; Hepatic cell fattydegeneration in the high fat diet animal model and the gathering of liver lactone class are improved significantly or reversing effect; And can reduce serum total cholesterol and low-density lipoprotein cholesterol content; The high density lipoprotein increasing cholesterol level shows that Radix seu Folium Rubi alceaefolii total alkaloids has therapeutical effect preferably to non-alcoholic fatty liver disease, and is especially preferable to the drug effect of simple fatty liver.
Claims (2)
1. the purposes of Radix seu Folium Rubi alceaefolii total alkaloids in the medicine of preparation treatment non-alcoholic fatty liver disease; Wherein, described medicine is the medicine of treatment simple fatty liver.
2. purposes according to claim 1 is characterized in that: contain total alkaloids in berberine hydrochloride in the described Radix seu Folium Rubi alceaefolii total alkaloids, greater than 95%w/w.
3. purposes according to claim 1 and 2 is characterized in that: described Radix seu Folium Rubi alceaefolii total alkaloids derives from the rosaceous plant Radix seu Folium Rubi alceaefolii
Rubus alceaefolius Poir. the root extract.
4, purposes according to claim 3 is characterized in that: described extract is the 70-95% ethanol extraction.
5, purposes according to claim 4 is characterized in that: described extract is 85% ethanol extraction.
6, purposes according to claim 1 is characterized in that: said medicine is that the Radix seu Folium Rubi alceaefolii total alkaloids by effective dose is an active component, adds that acceptable accessories or complementary composition are prepared into preparation pharmaceutically commonly used.
7, purposes according to claim 6 is characterized in that: described preparation is an oral formulations.
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| 陈文列等.粗叶悬钩子总生物碱抑制肝细胞凋亡的超微结构与TUNEL染色观察.《中国中药杂志》.2010,第35卷(第8期),1060-1063. * |
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