Background technology
Fatty liver is not a kind of independently disease, is the liver fat degeneration that is caused by the multiple disease and the cause of disease.The modal cause of disease has obesity, alcoholism, is nutritional disorder, diabetes, drug intoxication, gestation, heredity etc. secondly.The liver fat degeneration can influence liver function, and part patient also may further develop becomes hepatitis, hepatic fibrosis and liver cirrhosis.The sickness rate of China's fatty liver has the trend that increases gradually.Though the name of disease of the fat-free liver of Chinese medicine belongs to " disease is long-pending " " hypochondriac pain " categories such as " jaundice " mostly according to its clinical manifestation.The traditional Chinese medical science is thought, the fatty liver cause mostly is surfeit delicious food savoury, obesity, or insobriety, or experience the epidemic disease caused by damp-heat pathogen poison, or disorder of emotion, or weakness due to chronic disease and food stagnation, the stagnation of QI, pestilential pathogen etc. all can cause primary disease, its diseased region is liver, closely related with internal organs such as liver, gallbladder, spleen, stomach, kidneys, to its pathogenetic understanding, numerous scholars all think the liver failing to maintain the normal flow of QI, dysfunction of the spleen in transportation, retention of damp-heat in the interior, the turbid pent-up of expectorant, stagnation of blood stasis and finally form damp-phlegm stasis of blood resistance knot mutually, numbness resistance liver venation and form fatty liver.
Alcoholic liver is because the hepar damnification disease due to the long-term heavy drinking (being addicted to drink).Over past ten years, along with the raising of people's living standard and the expansion of social circle, the surge of the consumption figure of global wine.Simultaneously, the generation of alcoholic liver also significantly increases.Because the incidence rate that causes alcoholic liver of drinking also is obvious ascendant trend, become the latent type killer who can not be ignored in China.In American-European countries, alcoholic cirrhosis accounts for the 50-90% of whole liver cirrhosis.Long-term alcoholic also influences lipid metabolism, can cause fatty liver, and ethanol can make hepatocellular degeneration, necrosis, proliferation of fibrous tissue and cause liver cirrhosis.
The medicine of treatment fatty liver and alcoholic liver injury mostly is compound Chinese medicinal preparation at present, therefore, develop a clear and definite chemical component drugs thing that has with prevention and treatment alcoholic liver injury, fatty liver and hyperlipidemia safe and effective, that toxic and side effects is little, the patient is needed just.We belong to a kind of natural drug composition at the patent of invention, and chemical constituent is clear, and its purposes relates to the application to alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and blood fat reducing aspect.
Cortex Ilicis Rotundae (Ilicis routundae cortex) derives from the dry bark of holly plant Ilex rotunda Thunb. (Ilex rotunda Thunb.).Ilex rotunda Thunb. grows in deserted mountain sparse woods, hills or small stream limit, mainly is distributed in provinces and regions such as Guangdong, Guangxi, Fujian.Bitter in the mouth, cold in nature has effects such as heat-clearing and toxic substances removing, reducing swelling and alleviating pain, dampness removing, the expelling summer-heat that dispels the wind, and records in " south of the Five Ridges gather medicinal herbs record ", claim that " bitter in the mouth, heat clearing away poison " is China's southern area traditional herbal medicine (Huang Wenjian, the application among the people of Cortex Ilicis Rotundae, Jiangxi Chinese medicine, 1996,2:147).The Cortex Ilicis Rotundae bark contains flavonoid glycoside, phenols, tannin, triterpene glycosides.Be usually used in flu, tonsillitis clinically, pharyngolaryngitis, acute gastroenteritis, dysentery, osteodynia etc.Utilizing the medicine of Cortex Ilicis Rotundae medical material for raw material production at present, mainly all is crude extract, and preparation technology is mainly with water extract-alcohol precipitation, the extract drying, pulverizes the method for directly being used as medicine.Bibliographical information syringoside have to galactosamine cause hepatotoxic protective action (foreign medical science Chinese medicine fascicle 2000, the 22nd the volume, the 6th phase, 346-347); Find the patent (CN101342205) of Liu state firewood invention in the literature search, be to use the Cortex Ilicis Rotundae medical material, add ethanol extraction, filter, concentrate, after adding water and stirring evenly, the leaching precipitate, the dry extract that gets, containing Peltatin glucoside and rotundicacid total amount in the 1g extract dry product is 0.50~0.80g, wherein Peltatin glucoside and rotundicacid mass ratio are 12~1.5: 1, the two content only accounts for 50~80% of total extract in this extract, and wherein major ingredients is a Peltatin glucoside, and the effect of blood fat reducing is arranged, but do not contain syringoside in this extract, essential difference is arranged with our patent of invention.
We prove experimentation, the pharmaceutical composition of forming by Peltatin glucoside (Pedunculoside) 30-70%, syringoside (Syringin) 6-25%, rotundicacid (rotundicacid) 1-5%, be from the naturally occurring Chinese crude drug Cortex Ilicis Rotundae of nature, to extract and get, do not pass through any chemical modification, effect with prevention and treatment alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidemia, the present invention other bibliographical information tools relatively have greatly improved and essential difference.
Before the present invention finishes, the pharmaceutical composition that does not also have bibliographical information to form by Peltatin glucoside, syringoside, rotundicacid, report with prevention and treatment alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidaemic conditions does not find that this compositions is used to prevent and treat the clinicing aspect application of alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidemia yet.The compositions of also not finding simultaneously from other natural Chinese medicines, to extract of forming by Peltatin glucoside, syringoside, rotundicacid.
Summary of the invention
Goal of the invention provides a kind of Cortex Ilicis Rotundae compositions, be used to prevent and treat the research and development of the medicine of diseases such as alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidemia, the present invention studies the effective ingredient of Chinese crude drug Cortex Ilicis Rotundae, seek new effective site, by further studies show that, the pharmaceutical composition of being made up of Peltatin glucoside, syringoside, rotundicacid has very strong pharmacological action to alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidemia etc.This natural pharmaceutical composition does not also find to have outstanding contribution in other medical materials.
Pharmaceutical composition of the present invention is to form by the Cortex Ilicis Rotundae medicinal material extract is refining, it is characterized in that, the Cortex Ilicis Rotundae pulverizing medicinal materials is become coarse powder, uses alcohol reflux, filters, and filtrate recycling ethanol filters, and gets the extract I; Filtrate is not waved to nothing alcohol flavor, and by the macroporous resin column of having handled well, first water is to colourless, and the reuse ethanol elution concentrates ethanol liquid, gets the extract II; Extract I and extract II are merged the dry Cortex Ilicis Rotundae pharmaceutical composition that gets.The Cortex Ilicis Rotundae compositions that the present invention extracts was with Cortex Ilicis Rotundae extract, the Cortex Ilicis Rotundae preparation of report had essential difference and significantly technological progress in the past.This natural pharmaceutical composition does not pass through any chemical modification simultaneously, has embodied the compound action of theory of Chinese medical science chemical constituent, has tangible novelty.
The invention has the beneficial effects as follows: filled up the effective site of preparation Cortex Ilicis Rotundae and comprise by saponins, the plain class of phenylpropyl alcohol, pentacyclic triterpene acids composition blank as compositions, simultaneously also expanded its range of application in field of medicaments, had very high using value the prevention and the treatment of alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidaemic conditions.Compositions is characterized in that, by weight: Peltatin glucoside 30-70%, syringoside 6-25%, rotundicacid 1-5%, this natural pharmaceutical formulation has clear and definite pharmacological action, in other Chinese crude drugs, also do not find to have very high value of exploiting and utilizing.
Another object of the present invention is that the pharmaceutical preparation of Cortex Ilicis Rotundae pharmaceutical composition is provided.Main drug form is tablet, capsule, pill, granule, suspensoid, drop pill, any in the injection.
Described Cortex Ilicis Rotundae preparation of pharmaceutical compositions method of the present invention is as follows:
The Cortex Ilicis Rotundae pulverizing medicinal materials is become coarse powder, doubly measures alcohol reflux 1-3 time of the 50-95% of medical material weight with 5-10, each 1-3 hour, filter, filtrate recycling ethanol filters, the extract I; Filtrate is not waved to nothing alcohol flavor, and by the macroporous resin column of having handled well, first water is to colourless, and reuse 30-80% ethanol elution concentrates ethanol liquid, gets the extract II; Extract I and extract II are merged the dry Cortex Ilicis Rotundae pharmaceutical composition that gets.
Extracting method of the present invention has with short production cycle, workable, does not use inflammable and explosive chemical reagent, safety, and cost is low, can satisfy big need of industrial production.In view of the present invention has clear and definite pharmacologically active, the Cortex Ilicis Rotundae compositions of technology gained of the present invention can prepare in industrialization, has broad application prospects.
Peltatin glucoside, syringoside, rotundicacid high-efficient liquid phase chromatogram process measuring method:
With the octadecylsilane chemically bonded silica is filler; With acetonitrile-0.05% phosphate aqueous solution is mobile phase; Concrete ratio sees the following form, and the detection wavelength is 210nm.Number of theoretical plate calculates by syringoside and is not less than 3000.
The preparation precision of reference substance solution takes by weighing syringoside, Peltatin glucoside, rotundicacid reference substance, puts in the volumetric flask, makes every ml with dissolve with methanol and contains syringoside 0.05mg, Peltatin glucoside 0.15mg, rotundicacid 0.008mg mixed solution.
Compositions 20mg is got in the preparation of need testing solution, and accurate the title decides, and puts in the 50ml measuring bottle, adds methanol 40ml, and ultrasonic 30min is placed to room temperature, and methanol constant volume shakes up to scale, as need testing solution.
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of assay method inject chromatograph of liquid, measure, promptly.
The effect that Cortex Ilicis Rotundae pharmaceutical composition (syringoside accounts for 21.8%, Peltatin glucoside accounts for 57.6%, rotundicacid account for 3.6%) has prevention and treatment alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidemia, these pharmacological actions are confirmed by following pharmacodynamics test example.
1, the Cortex Ilicis Rotundae compositions is to the protective effect of alcoholic liver injury
50 of mices are divided into 5 groups at random, i.e. basic, normal, high three dosage groups, ethanol model group and blank group of Cortex Ilicis Rotundae compositions.The Cortex Ilicis Rotundae composition component is not pressed Cortex Ilicis Rotundae compositions 50,100 and 200mg/kg filling stomach mice, and 1 time/d, be total to 14d, ethanol model group and blank group matched group are irritated stomach equal-volume distilled water respectively, 1 time/d, are total to 14d.Fasting is 12 hours after the last administration, except that the blank group is still irritated stomach equivalent distilled water, other 4 treated animals (basic, normal, high three dosage groups of Cortex Ilicis Rotundae compositions, the ethanol model group) irritates stomach 50% ethanol 12mL/kg respectively, vena ophthalmica is got blood behind the 6h, measure alanine aminotransferase (ALT) and aspartate amino transferase (AST) content in the serum, getting mouse liver weighs, measure liver device index, get lobus dexter and measure (SOD) of superoxide dismutase, glutathione peroxidase (GSH-PX) activity, malonaldehyde (MDA) content.
Table 1 Cortex Ilicis Rotundae compositions is to the influence of alcohol damaged mice serum ALT, AST and liver coefficient
Compare with the blank group:
*P<0.05,
*P<0.01,
* *P<0.001; The ethanol group is △ p<0.05 relatively, △ △ p<0.01.
As known from Table 1, irritate stomach ethanol the Mouse Liver index is significantly increased, irritate stomach Cortex Ilicis Rotundae compositions alcohol damaged Mouse Liver index is significantly reduced.Irritate stomach ethanol mice serum ALT, AST are significantly increased, irritate stomach Cortex Ilicis Rotundae compositions alcohol damaged mice serum ALT, AST are significantly reduced.
Table 2 Cortex Ilicis Rotundae compositions is to the influence of alcohol damaged mouse liver MDA, GSH-PX, SOD
Compare with the blank group:
*P<0.05,
*P<0.01,
* *P<0.001; The ethanol group is △ p<0.05 relatively, △ △ p<0.01.
The result shows from table 2, and ethanol can cause that mouse liver MDA content significantly raises, causes GSH-PX enzyme, the active significantly reduction of SOD, and the Cortex Ilicis Rotundae compositions can significantly reduce MDA content, and GSH-PX enzyme, SOD activity can significantly raise.
2, Cortex Ilicis Rotundae compositions fatty liver that hyperlipidemia and hyperlipidemia are caused improves function
Get 60 of male rats, be divided into 6 groups at random, except that the blank group, all the other are respectively organized and give high lipid food every day: 2% cholesterol, 10% Adeps Sus domestica, 0.5% sodium cholate, 0.2% propylthiouracil, 87% normal feedstuff.Except that the blank group was freely drunk water, the hyperlipidemia model group gave the equivalent normal saline, and all the other are respectively organized per os and irritate stomach, every day 1 time, and according to table 1 grouping, administration, in 10 weeks of successive administration, fasting 12h can't help water after the last medication, the sacrificed by decapitation all animals.Broken end is got blood, surveys T-CHOL (TC), triglyceride (TG) in the blood, high density lipoprotein (HDL-C), low-density protein (LDL-C), and measurement result sees Table 3; Get right lobe of liver 1g, make 10% homogenate under 4 ℃, 3000rpm 10min gets supernatant, measures the content of TC and TG respectively, and measurement result sees Table 4; Get fresh liver tissue and make 1% liver homogenate with normal saline, press the test kit operating instruction and measure SOD, GSH, VitE, adopt the thiobarbituricacid method to measure the content of hepatic tissue MDA, measurement result sees Table 5.
Table 3 Cortex Ilicis Rotundae compositions is to the result that influences of hyperlipidemia rats blood lipids index
Compare with model control group,
*P<0.05;
*P<0.01; Compare △ P<0.05 with the blank group; △ △ P<0.01;
The result shows, model control group serum TC, TG, LDL-C value obviously raise, HDL-C descends, can obviously reduce serum TC, TG content with model control group comparison Cortex Ilicis Rotundae compositions, significantly reduce serum low-density LP cholesterol (LDL-C) content, the content of serum high-density LP cholesterol (HDL-C) that significantly raise.
Table 4 Cortex Ilicis Rotundae compositions is to the influence of TC, TG in the rats'liver
Compare with the blank group:
*P<0.05,
*P<0.01; Model group is △ p<0.05 relatively, △ △ p<0.01.
As known from Table 4, model control group is compared with the blank group, and TC, TG content significantly raise.Compare with model control group, the high, medium and low dosage group of Cortex Ilicis Rotundae compositions TC content all has significance to reduce, and Cortex Ilicis Rotundae combination object height, middle dosage group TG content also have significance to reduce.
Table 5 Cortex Ilicis Rotundae compositions is to the influence of liver tissues of rats SOD, MDA, GSH, VitE content
Compare with the blank group:
*P<0.05,
*P<0.01,
* *P<0.001; Model group is △ p<0.05 relatively, △ △ p<0.01, △ △ △ p<0.001.
As known from Table 5, model control group and blank group compare, the decline of the active significance of SOD, and MDA content obviously increases.Each group of medication is compared with model control group, and the SOD activity obviously increases, and MDA content obviously reduces, and returns to the level of approximate normal control group substantially.Model group and normal control group comparison GSH and VitE content obviously reduce, and Cortex Ilicis Rotundae medication group can obviously improve the content of GSH and VitE.
3, Cortex Ilicis Rotundae compositions fatty liver that ethanol is caused improves function
120 of test rats are divided into the normal control group at random; The fatty liver model group; Positive control drug DONGBAO GANTAI 600mg/kg dosage group: the Cortex Ilicis Rotundae compositions is established 100mg/kg, 50mg/kg and three dosage groups of 25mg/kg.Totally 6 groups, 20 every group.Except that the normal control group, all the other are respectively organized rat 8:30 in morning every day filling stomach and give (56% ethanol+0.5mL fish oil) 1 time, and ethanol dosage begins to be 7g/kg, increases progressively 0.2g/kg subsequently weekly, continuous 4 weeks.Rats in normal control group is irritated stomach and is given isocaloric 56% glucose+0.5mL fish oil.Be subjected to the reagent thing to be mixed with suspension with distilled water, 1:00 filling in afternoon every day stomach gives 1 time, and normal control group and model control group rat oral gavage give the distilled water of equivalent, irritate the long-pending 10ml/kg/d of being of body of stomach.The rat drinking-water of freely ingesting is weighed in weekly 1 time in the process of the test, to adjust dosage.The dead animal in end around administration the.Collection of specimens and observation index, result of the test is added and subtracted standard deviation with mean
Expression, t check between statistical procedures method employing group.Experimental result sees Table 6 and table 7.
Table 6 Cortex Ilicis Rotundae compositions is to alcoholic fatty liver rat blood serum ALT, AST and the active influence of LDH
Compare with the normal control group:
*P<0.05,
*P<0.01,
* *P<0.001; Model group is △ p<0.05 relatively, △ △ p<0.01, △ △ △ p<0.001.
Cortex Ilicis Rotundae combination object height and middle administration treated animal Serum ALT and AST activity very significantly are lower than model group; this result of the test shows; test sample Cortex Ilicis Rotundae compositions can obviously reduce the unusual ALT that raises, the activity of AST in the alcoholic fatty liver rat blood serum, illustrates that the Cortex Ilicis Rotundae compositions has good protective action to hepatocyte.Cortex Ilicis Rotundae combination object height and middle administration group have tangible reduction effect to the unusual rising of alcoholic fatty liver animal serum LDH level, and this result of the test shows, the Cortex Ilicis Rotundae compositions has good protective effect to fatty liver due to the ethanol.
Table 7 is rescued the heart and is answered the influence of compositions to alcoholic fatty liver Serum TC, TG and MDA content and hepatic tissue TG content
Compare with the normal control group:
*P<0.05,
*P<0.01,
* *P<0.001; Model group is △ p<0.05 relatively, △ △ p<0.01.
After giving three different dosing dosage of Cortex Ilicis Rotundae compositions 100mg/kg, 50mg/kg and 25mg/kg respectively and carrying out Drug therapy, wherein, MDA content is starkly lower than model group (for P<0.01) in the Cortex Ilicis Rotundae compositions 100mg/kg administration treated animal serum.Show that the Cortex Ilicis Rotundae compositions can reduce fatty liver animal pattern Content of MDA, hepatocellular lipid peroxidation when suppressing fatty liver, thereby the effect that performance protection hepatocyte avoids damaging.Cortex Ilicis Rotundae combination object height and in two administration treated animal serum TC, TG content and hepatic tissue TG content significantly be lower than model control group (being respectively P<0.01 or P<0.05).Show that the Cortex Ilicis Rotundae compositions can reduce the rising of fatty liver rat fat content and liver lipid content due to the ethanol, has the effect that liver fat is piled up that suppresses.
From above pharmacodynamic experiment as can be known, the Cortex Ilicis Rotundae compositions has the effect of prevention and treatment alcoholic liver injury, alcoholic fatty liver, non-alcoholic fatty liver disease and hyperlipidemia, and takes for a long time and be free from side effects.
The present invention executes example by following experiment to be achieved (confirmation), but does not mean that this present invention just is limited to this, and specific implementation method is as follows.
Example 1
2kg is ground into coarse powder with the Cortex Ilicis Rotundae medical material, with 70% alcohol reflux of 8 times of amount medical material weight 3 times, and each 2 hours, filter, merging filtrate reclaims ethanol, to 2500ml, filters, and gets filter cake and gets the extract I; Filtrate is not waved to there not being pure flavor, by the D101 macroporous resin column of having handled well, washes with water earlier to colourless, and reuse 50% ethanol elution 4BV collects ethanol elution, reclaims ethanol, concentrates, and gets the extract II; Extract I and extract II are merged, and drying gets Cortex Ilicis Rotundae pharmaceutical composition 158.6g.Utilize high effective liquid chromatography for measuring, calculate, syringoside accounts for 23.8%, Peltatin glucoside accounts for 59.1%, rotundicacid accounts for 2.9%.
Example 2
2kg is ground into coarse powder with the Cortex Ilicis Rotundae medical material, with 50% alcohol reflux of 6 times of amount medical material weight 3 times, and each 3 hours, filter, merging filtrate reclaims ethanol, to 2500ml, filters, and gets filter cake and gets the extract I; Filtrate is not waved to there not being pure flavor, by the D101 macroporous resin column of having handled well, washes with water earlier to colourless, and reuse 60% ethanol elution 4BV collects ethanol elution, reclaims ethanol, concentrates, and gets the extract II; Extract I and extract II are merged, and drying gets Cortex Ilicis Rotundae pharmaceutical composition 168.4g.Utilize high effective liquid chromatography for measuring, calculate, syringoside accounts for 19.7%, Peltatin glucoside accounts for 55.3%, rotundicacid accounts for 2.2%.
Example 3
2kg is ground into coarse powder with the Cortex Ilicis Rotundae medical material, with 80% alcohol reflux of 8 times of amount medical material weight 2 times, and each 3 hours, filter, merging filtrate reclaims ethanol, to 2500ml, filters, and gets filter cake and gets the extract I; Filtrate is not waved to there not being pure flavor, by the AB-8 macroporous resin column of having handled well, washes with water earlier to colourless, and reuse 40% ethanol elution 4BV collects ethanol elution, reclaims ethanol, concentrates, and gets the extract II; Extract I and extract II are merged, and drying gets Cortex Ilicis Rotundae pharmaceutical composition 148.6g.Utilize high effective liquid chromatography for measuring, calculate, syringoside accounts for 23.4%, Peltatin glucoside accounts for 60.1%, rotundicacid accounts for 3.4%.
Example 4
2kg is ground into coarse powder with the Cortex Ilicis Rotundae medical material, with 50% alcohol reflux of 8 times of amount medical material weight 3 times, and each 2 hours, filter, merging filtrate reclaims ethanol, to 2500ml, filters, and gets filter cake and gets the extract I; Filtrate is not waved to there not being pure flavor, by the AB-8 macroporous resin column of having handled well, washes with water earlier to colourless, and reuse 35% ethanol elution 4BV collects ethanol elution, reclaims ethanol, concentrates, and gets the extract II; Extract I and extract II are merged, and drying gets Cortex Ilicis Rotundae pharmaceutical composition 141.6g.Utilize high effective liquid chromatography for measuring, calculate, syringoside accounts for 23.9%, Peltatin glucoside accounts for 62.1%, rotundicacid accounts for 3.1%.
Example 5, (capsule)
Cortex Ilicis Rotundae pharmaceutical composition 100g, medical starch is an amount of, and mix homogeneously is granulated, drying, granulate, the capsule of packing into No. 1 is made 1000, promptly.Each 1, every day 2 times.
Embodiment 6, (tablet)
Cortex Ilicis Rotundae pharmaceutical composition 100g, medical starch is an amount of, and dextrin is an amount of, and mix homogeneously is granulated, drying, granulate, drying, the adding lubricant is an amount of, and tabletting is made 1000, promptly.Each 1, every day 2 times.
Embodiment 7, (drop pill)
Take by weighing the 15g Macrogol 4000, the 15g polyethylene glycol 6000 melts in water-bath, Cortex Ilicis Rotundae pharmaceutical composition 10g, stir, in the impouring insulating tube, regulate thermostat, make medicinal liquid under 80-90 ℃, splash in the liquid paraffin that cooled off, after dripping off, to blot paraffin oil on the pill impouring filter paper, make 1000, promptly.Each 10, every day 2 times.
Embodiment 8, (granule)
Cortex Ilicis Rotundae pharmaceutical composition 100g, dextrin 400g, lactose 500g, mix homogeneously is used an amount of alcohol granulation, and drying, granulate, packing are made 1000 bags, promptly.Each 1 bag, every day 2 times.