CN102940650B - Propolis ethanol extract for dispeling the effects of alcohol, preparation method of propolis ethanol extract and application of propolis ethanol extract for producing enteric-coated tablets - Google Patents

Abstract

The invention discloses a propolis ethanol extract for dispelling the effects of alcohol, a preparation method of the propolis ethanol extract and application of the propolis ethanol extract for producing enteric-coated tablets and relates to the technical field of processes for extracting active substances from animals and plants, particularly propolis. The method comprises the following steps of: freezing, crushing and removing impurities of the propolis, soaking ethanol under normal-pressure normal-temperature and performing ultrasonic extraction, freezing and centrifuging to remove the solid impurities, and taking supernatant ethanol solution; and finally, separating the propolis ethanol extract with the molecular weight of 5,000D or below at the environment temperature of 1-5 DEG C, and performing distillation recovery on ethanol to obtain solid biological activity drying powder which has the molecular weight of less than 5,000D and the content of more than 90 percent which is extracted from the propolis through ethanol, and mixing the solid biological activity drying powder, the royal jelly freeze-dried powder, isomaltitol, magnesium stearate, enteric-coated powder and the like to prepare the enteric-coated tablets for dispelling the effects of alcohol. The enteric-coated tablets have the obvious effect of dispelling the effects of alcohol and have no side effect, and the method is low in cost, simple and feasible.

Description

For propolis extracted with alcohol, preparation method and the application at production enteric coatel tablets of relieving the effect of alcohol

Technical field

The present invention relates to from animals and plants and secretions thereof, particularly from propolis, extract the Technology field of active substance.

Background technology

China is the country of making wine in the world the earliest, and spirits culture is permeated in many Social Culture, becomes already the requisite part of daily life.Along with people's material, culture and growth in the living standard, the excessive consumption of alcohol social public health problem that become international, the incidence rate of the alcoholic hepatitis occurring therefrom also constantly increases, and has become the second largest hepatopathy after viral hepatitis in China; Due to reasons such as culture, historical or individual's hobbies, eliminate bad alcohol drinking patterns completely and still need and want the long-term endeavour of entire society.

Current various dinner party is more and more, because the sickness rate of the caused various hepatic disease of excessive drinking improves year by year, alcoholic fatty liver has become the major issue that affects modern's quality of life, and society is in the urgent need to the appearance of the natural disintoxicating product of novel cheapness.

Alcoholic strength heat and gas are strong, insobriety, and alcoholism is accumulated, and the many internal organs of liver taste are impaired and cause impairment of both QI and YIN.Chinese traditional treatment is worked as taking removing summer-heat alcoholism, removing heat-phlegm, supplementing QI and nourishing YIN as main; U.S. FDA has been ratified 3 medicines for alcoholism treatment, i.e. ester is adjoined in naltrexone, acamprosate and holder.In animal experiment and clinical research, find, to a certain degree improve crapulent effect as ubenimex, nimodipine, emetine etc. also have.

Propolis has very strong physiological regulatory action, propolis is the resinae material from different plants of being collected by Apis, just be used as Drug therapy by the mankind from the ancient times, it remains the most frequently used treatment article of Balkan Peninsula country now, is applied to the treatments such as wound, burn, sore throat, gastric ulcer.Over nearly 10 years, pharmacologically active and the chemical composition of researcher to propolis carried out comprehensive research, finds that propolis has pharmacologically active widely, as antibiotic property, antifungal, antiviral, antiinflammatory, protect the liver, antioxidation, anticancer etc.Propolis flavone is one of main effective ingredient contained in propolis, and its pharmacological action is very extensive, has the effects such as antiinflammatory, antiallergic, carcinogenesis, anti-hypertrophy, antiviral and anti-cell differentiation; Can alleviate reactive oxygen free radical to biomembranous decay and preservation SOD; Contained MDA in hepatic tissue be can reduce in a large number, and mouse liver SOD, CAT activity obviously improved; Prompting propolis flavone has stronger removing oxygen-derived free radicals, antioxidative effect.Mahran etc. study discovery, and propolis solution is dose dependent, and to protect hepatocellular damage, its mechanism may be that water extracts of propolis has repair to hepatocyte injury.Lin etc. study discovery, and the hepatic injury that propolis causes ethanol also has protective effect.Ethanol extracts of propolis can make serum transaminase and TG level significantly reduce, and hepatic steatosis degree obviously declines.In a word, that propolis has is antibacterial, antiviral, antiulcer, antiinflammatory, antitumor, blood pressure lowering, regulates blood-fat and blood sugar, enhancing immunity; protection cardiovascular, sedation anesthesia, the liver protecting; defying age, improves the effects such as memory function, and its preparation has been widely used in clinical.But propolis extracted with alcohol is applied to the research of relieving the effect of alcohol, remain at present blank, by this research in several years, we have made the molecular mechanisms of action that propolis relieves the effect of alcohol substantially clear, for researching and developing new solution drinks medicine based theoretical.

Current plant alcohol intoxication-alleviating preparation roughly comprises the ethanol absorption inhibitor taking Araliaceae as representative, the liver metabolism promoter taking Radix Puerariae, Flos puerariae lobatae as representative, and the latter is more paid attention to because it has hepatoprotective effect concurrently; In addition, Semen Hoveniae (Fructus Hoveniae) can, by activating the enzyme relevant to alcohol metabolism, accelerate the decomposition of body ethanol and acetaldehyde, also can play certain prevention and therapeutic effect to various chemical liver injury, is the good medicine of a class facilitating alcohol metabolism and protecting liver.

Alcoholic liver disease sickness rate is cumulative year after year trend, and the hepatic disease causing by drinking is day by day serious, therefore finds efficient, safe solution very urgent, generally believes by studying that at present relieving alcoholism and protecting the liver mainly can reach by following several approach:

1, reach relieving alcoholism and protecting the liver effect by raising ethanol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) activity

Focus mostly in improving the active aspect of ADH about the research of antialcoholic drugs at present, comprise the first pass metabolism (FPM) of stomach ADH and the oxidative metabolism of liver ADH.Radix Puerariae, Flos puerariae lobatae, Semen Hoveniae (Fructus Hoveniae) are the most representative solution drinks of Chinese medicine medicines, the effective ingredient of finding after deliberation their performance antialcoholism actions is Flavonoid substances, trifoliate orange yellow party (Semen Hoveniae (Fructus Hoveniae) and Radix Et Rhizoma Rhei) on the impact experiment of alcoholic liver injury in rats stomach ADHmRNA in, thereby trifoliate orange yellow party is proved and can improves ethanol and alleviate at the first pass metabolism of stomach the load that ethanol causes liver.Trifoliate orange yellow party and Qinggan Huoxue Recipe (Radix Bupleuri, Radix Scutellariae, Radix Salviae Miltiorrhizae, Carapax Trionycis, Radix Puerariae) all can improve the activity of hepatic tissue ADH and the expression of mRNA.In the situation of long-term heavy drinking, cause the active decline of ADH in liver, ethanol is mainly by CYP2E1 metabolism, but this approach can produce a large amount of acetaldehyde and active oxygen, and active oxygen makes redox state unbalance, produces response to oxidative stress and brings out liver organization damage.Therefore, for long-term alcoholic, improve its ADH activity and can slow down the oxidative stress being produced by CYP2E1 metabolism, also can play liver-protective effect.Trifoliate orange yellow party and Qinggan Huoxue Recipe as described above can play antialcoholism action, also can play liver protection effect simultaneously.In addition, acetaldehyde is one of principal element of alcoholic liver injury, and its metabolism is slow and toxicity is large.Research finds that acute alcoholism liver tissues of rats ADH, the ALDH specific activity model group after the intervention of Semen Hoveniae (Fructus Hoveniae) water extraction liquid all significantly increases (P<0.05), show that Semen Hoveniae (Fructus Hoveniae) water extraction liquid can effectively strengthen acute alcoholism liver tissues of rats ADH, ALDH activity, thereby the oxidation Decomposition metabolism of accelerating alcohol in liver, reduce the toxic action of its toxic metabolite (being mainly acetaldehyde) to liver, reach certain hepatoprotective effect.But, only improve ADH, ALDH activity can not slow down the ethanol toxic action of metabolism in vivo, also tackle the key enzyme that alcohol metabolism overall process is relevant, as CYP2E1, CAT carry out systematic study, ensure ethanol, the thorough metabolism of acetaldehyde and the balance of vivo oxidation reducing condition.

2, reach relieving alcoholism and protecting the liver effect by slowing down oxidative stress effect

As mentioned above, ethanol 3 approach of metabolism in liver all can produce free radical and active oxygen, and initiated oxidation stress brings out liver organization and occurs to become the damage of principal character with fat.The factor relevant to oxidative stress has: malonaldehyde (MDA), superoxide dismutase (SOD), glutathion peroxidase (GSH-PX).The content of MDA embodies level of lipid, the strong and weak ability that embodies Scavenger of ROS of activity of SOD and GSH-PX, and they are the common counters in relieving alcoholism and protecting the liver research.In liver, exist the balance of antioxidant system and enzymatic oxidation system, after absorption ethanol, this balance will be broken, the activity decreased of SOD and GSH-PX.Therefore the activity of antioxidant system in protective, to remove interior free yl significant by the hepatic injury due to oxidative stress to control.At present relevant meals show the research of alcohol metabolism impact and anti-ALD with medicine: the antioxidant content in black tea can suppress the interior enzyme system of body that causes in alcohol metabolism process and the antioxidant activity reduction of non-enzyme system, contribute to body to maintain normal redox state, and research in this respect remain blank out for propolis.

3, by suppressing NF-kB activation and lower COX-2 expression to alleviate lipid peroxidation and inflammatory reaction

Current research shows, oxidative stress makes NF-κ B, COX-2 activation, NF-κ B be a kind of regulate and control a kind of transcription factor of inflammation and immunoreation, apoptosis and infection and stress, COX-2 is the inducible enzyme through stimulating rapid generation, is the key link of inflammatory reaction.COX-2 and synthetic product thereof can pass through to disturb the metabolism of fat, saccharide and protein, increase the weight of the release of oxidative stress and the promotion cytokine profiles of liver, thus the pathological changes such as the steatosis of participation liver, inflammation, fibrosis.This two factor is explored, become research alcohol metabolism and cause the new starting point of hepatic injury.Tea polyphenols, trifoliate orange yellow party all can, by suppressing the activation of NF-κ B in rat body and lowering the mechanism of action that COX-2 expresses, alleviate lipid peroxidation and inflammatory reaction, realize the target of protection hepatic tissue.

4, express by reducing level of endotoxin and CD-14

In blood plasma, level of endotoxin rising is also the key factor that causes ALD, but in modern relieving alcoholism and protecting the liver goods, how research affects the very few of level of endotoxin about goods, and bicyclol, as the novel Antihepatitis medicament of Chinese independent development, has been used for the treatment of slow virus hepatitis.Current research shows, bicyclol can reduce Endotoxin Levels and lower CD-14 and express, and has the effect of protection alcoholic liver injury, the formation of control ALD.Therefore, can express by reducing level of endotoxin and CD-14, realize the effect of the liver protecting tissue from suppressing Kupffer cell activation angle.

In sum, the pathogenesis of ALD is relevant with many factors, mainly comprises: the key enzyme in alcohol metabolism process, as ADH, ALDH, CYP2E1, CAT; Oxidative stress factor of influence, as MDA, SOD, GSH-PX; Signal conducts crucial nuclear factor, as NF-κ B, COX-2; Level of endotoxin and receptor CD-14 expression etc. thereof.

Therefore, also should launch around these key factors about the research of relieving alcoholism and protecting the liver mechanism of action.Simultaneously, China's hepatinica product that relieve the effect of alcohol, in health product, be main mainly with Chinese herbal medicine, about the exploitation of dietary ingredient little, therefore according to the theory of Chinese medicine " integration of edible and medicinal herbs ", the relieving alcoholism and protecting the liver composition that searches out high effect nontoxic evil from meals has innovative significance, also for nutritional intervention and the drug development of the collaborative anti-ALD of many target spots provide theoretical foundation, find the effectively damaging action of alleviation of alcohol to liver of propolis and study through us, successful relieves the effect of alcohol, its molecular mechanism adjusting stronger with it of relieving the effect of alcohol is multiple, and to have the gene of antialcoholism action relevant, be expected to be developed as a kind of health food that alleviates ethanol chemical liver injury.

Liver is the removing toxic substances organ that body weight for humans is wanted, and alcohol metabolism process is mainly carried out in liver.Take the photograph in a large number for a long time people's ethanol, can exceed liver normal physiological metabolic detoxification ability, cause the much important physiology of body, biochemistry and metabolism disorder, major injury hepatocyte, causes alcoholic liver disease of ZANG-organs (ALD).Alcoholic fatty liver (AFLD) is one of 3 kinds of histopathology forms of ALD, and pathogenesis is very complicated, and main manifestations is steatosis.Liver fat degeneration and fatty infiltration and disorders of lipid metabolism, minimizing and the mobilization of liver external fat etc. of the synthetic increase of the such as decline of fatty acid oxidation rate, triglyceride, fat output are closely related.Ethanol is particularly the following aspects to the damage main manifestations of liver to body: 1, enzymatic activity indicates the changing function in the metabolic processes of body basis: in hepatocyte, alcohol metabolism has 3 majors avenues of approach, and one of them is ethanol dehydrogenase (ADH) path of cytoplasm.In Ethanol Oxidation, in liver, oxygen consumption increases, in cytoplasm, be reduced with a large amount of nadide (NAD), form reducibility coenzyme I(NADH), cause NAD+/NADH ratio decline and unbalance, changed the redox state of cell, severe jamming the metabolism of cell, thereby tricarboxylic acid cycle is suppressed; In addition, because the mitochondria enzyme activities such as succinate dehydrogenase (SDH) weaken, oxidative phosphorylation ability is reduced, affect the breathing of cell, increase the weight of hepatocellular damage.Above-mentioned variation is to cause the topmost reason of AFLD.

Someone likens into two locusts on rope of hyperlipemia fatty liver and coronary heart disease because obesity, hyperlipemia, diabetes and excessive drinking be fatty liver commonly encountered diseases because of, and these factors are close with atherosclerosis and coronary heart disease too.There are obvious myocardial damage and hemodynamics disturbance, T cellular immune function decreased, liver biopsy studies show that, in these cases, 91.4% has hepatic cell fattydegeneration.Generally speaking, ethanol fatty liver belongs to reversibility disease, and early diagnosis treatment in time often can recover normal.Long-term edible high lipid food or heavy drinking, ethanol can directly be poisoned hepatocyte, affects its structure and function; Many people are exactly because insobriety is just suffered from alcoholic hepatitis, fatty liver causes liver damage.And hepatic injury can cause triglyceride (TG) to decline, triglyceride is the fat molecule that long-chain fatty acid and glycerol form.Be the lipid that people's in-vivo content is maximum, most tissues all can utilize triglyceride catabolite to supply with energy, is the main source of energy i (in vivo).Fatty liver can cause the significant quantities of fat of eating to absorb minimizing, and bile secretion deficiency, can not decompose the fat of eating into, increases the weight of the state of an illness thereby fat forms fat drop at liver.

Propolis is that Apis gathers the plumelet of glue source plant or the resinoid of callus secretion, and mixes a kind of colloid substance with stickiness that secretions, pollen and the Cera Flava etc. of self body of gland mix.Propolis is rich in various bioactivators, especially contains a large amount of Flavonoid substances, has multiple medicinal health care function, is described as " purple gold ".Propolis inevitably can be sneaked into the impurity such as wood flour, bee body, weeds, sandstone in recovery process, before for food, medicine etc., must be through purifying, otherwise can not take.

The effective ingredient of the different gained of method for extracting propolis is not identical yet, and conventional extraction has at present: ethanol extraction method, water extraction method, super critical extraction, ultrasound wave assisted Extraction are followed the example of, enzyme extraction method, boron polyelectrolyte method, microwave-assisted extraction method.Find that by research Different Extraction Method, the different solvent that extracts can show different pharmacological effects, show to extract with pharmacological effect to there is obvious dependency.By comparing, SCF-CO 2 method, not only apparatus expensive, operating condition are difficult for grasping, and extract yield is low and bacteriostasis is low compared with alcohol steep method, but the method extract safety, noresidue, pollution-free, have certain DEVELOPMENT PROSPECT, main still for laboratory research at present.The organic solvent that organic solvent extraction is conventional has ethanol, ether, acetone, ethyl acetate, n-butyl alcohol etc., and wherein ethanol is the most conventional.Alcohol steep method equipment needed thereby is simple; reagent is easily buied; operate also very convenient; with ethanol as extract solvent; can be easy to make propolis dewaxing, and can protect some polyphenols, thereby obtain the refined propolis that is rich in polyphenols; therefore ethanol is the most frequently used extraction solvent, particularly 70% and 80% ethanol.Although sometimes also extract with 95% ethanol, compared with water-alcohol solution, water-alcohol solution extracts and can make Cera Flava dissociate out quickly, is also rich in a large amount of polyphenols in extracting solution simultaneously.Propolis is extracted with the ethanol of variable concentrations; and measure the absorption spectrum of Flavonoid substances in variable concentrations extracting solution; result is presented at 290nm place; in the propolis extract of 80% ethanol extraction, there is the highest absworption peak; this also just means and wherein contains maximum Flavonoid substances, is mainly the luxuriant and rich with fragrance alcohol of camphane, acacetin and isosakuranetin.But the maximum Flavonoid substances going out with 60% ethanol extraction is isosakuranetin, Quercetin and kaempferol, 70% ethanol can extract maximum pinocembrins and sakuranetin; Can extract some polyphenols soluble in water with pure water, methanol, normal hexane, chloroform are sometimes also used as extractant and use.The research of organic solvent lixiviate propolis total flavones is mainly processed to the aspect expansion such as ancillary method (as microwave treatment, ultrasonic Treatment) around its influence factor (as concentration of alcohol, extraction time, solid-liquid ratio, extraction temperature) and other both at home and abroad.Ultrasound wave assisted extraction can shorten extraction time greatly, has become in recent years the focus of propolis total flavones Study on extraction.Mostly studied around the aspect such as ultrasonic treatment time, ultrasonic power in the past, change about ultrasonic frequency the research report that extraction affects on propolis total flavones less, and research was not deep enough yet.

Lac regis apis is 5～15 age in days worker bee lingual glands and upper palatine gland secreted milky or faint yellow emulsion liquid, is whole period of development of queen bee nit and drone larva unique food in earlier stage, is similar to mammiferous milk, claims again Lac regis apis, royal jelly, Lac regis apis.Attention to Lac regis apis and attention, first be that the young honeybee of having found feed Mel can only grow up to worker bee, general 30～40 days of life-span, the longest 7～8 months, and the young honeybee of feed Lac regis apis can grow up to queen bee, life-span reaches 3～5 years, and these phenomenon prompting people utilize the food of Lac regis apis as life lengthening, but its nature and role mechanism is not understood.By 1888, just first carries out generality research to the component of Lac regis apis to Germanization scholar A Von Planta, so far people have started to inquire into the profoundness of Lac regis apis, but Lac regis apis is comprised to comprehensive research of biology, chemical pharmacology and clinical experiment, or nearest decades are inchoate, and people also do not study clear to some the micro-bioactive substances in Lac regis apis completely so far.At present, fixed functional factor totally 9 large classes: they are peptides and proteins classes; Functional sweetener; Functional grease; Free radical scavenger; Vitamin; Active trace element; Active polysaccharide; Flavone compound and micro-ecological factor are as lactobacillus etc.Find according to people's research, in Lac regis apis, contain these abundant functional factors, the mankind are had to extremely strong alimentary health-care function and medical function.

Modern study shows: Lac regis apis can alleviate hepatocellular damaging action in the ALD course of disease, liver organization after damage is had to the effect that promotes regeneration, its mechanism of action may be the generation by reducing or suppress COX-2, avoid excessive inflammatory reaction, reach and alleviate mitochondrion and lipid peroxidation injury, thus the level of reduction serum AST and ALT; Lac regis apis has reduced the expression of TNF-α in hepatocyte, and TNF-α is as inflammatory factor, has mediated hepatocellular inflammatory reaction, multiple pathology and the physiological activities such as immunoreation, hepatocellular apoptosis and amplification hepatic fibrosis.The reason that analysing royal jelly TNF-α reduces may be that Lac regis apis is natural oxygen free radical scavenger, can reduce the activation of the nuclear factor that ROS causes, thereby reduces the expression of the TNF-α gene that is subject to its regulation and control.Lac regis apis can press down the expression of COX-2 and TNF-α in the forming process of alcoholic liver disease, and alcoholic liver injury is had to certain protective effect.The Lac regis apis also hepatic fibrosis serological index to CC14 hepatic fibrosis rats and the equal tool of hepatic pathology morphology is significantly improved effect, but improve not obviously to forming liver tissue fibrosis after hepatic fibrosis, its possible mechanism is the Lac regis apis inflammation that can improve hepatic tissue, reduce the synthetic of ECM and suppress the secretion of TNF-α.And Lac regis apis and propolis have natural orthofunction and concordance, the two is combined with effect can be better.

Enteric coatel tablets, be a class under one's belt insoluble, in intestinal juice, soluble material is that main coating material carries out the tablet that coating is made.Tablet is enteric coated to be determined by pharmaceutical properties and medication object: 1. in order to prevent destruction to some drugs of acidity and enzyme or to prevent the intense stimulus of medicine to stomach; 2. for medicine is had an effect at enteral as anthelmintic, intestinal disinfectant etc., 3. wish some drugs in intestinal absorption or need to protect the long period with prolong drug effect at intestinal.Wrap after enteric coating, can keep under one's belt complete and in intestinal, discharge medicine.

People have also absorbed a large amount of food in heavy drinking, particularly high innage fat food makes people's stomach be high full abdomen state, the emptying general needs of its stomach are about 2 hours, if take the medicine relieving the effect of alcohol simultaneously, because dose differs too great disparity compared with stomach, be difficult to play a role and lost efficacy, if but adopt heavy dose of antialcoholic drugs, can be subject to the restriction of gastric capacity again and cannot implement, so the antialcoholic drugs using after meal must adopt the insoluble processing mode of stomach, Here it is, and why we will develop the main cause of enteric coatel tablets.

Summary of the invention

The present invention's the first object is to invent a kind of propolis extracted with alcohol that can efficiently fundamentally relieve the effect of alcohol.

The present invention is that the molecular weight that adopts ethanol to extract from propolis is less than the dry thing of biological activity that the solid content of 5000D is greater than 90% for the propolis extracted with alcohol that relieves the effect of alcohol.

Inventor is through evidence, and the bioactive substance that the molecular weight extracting from propolis is less than the dissolve with ethanol of 5000D has the effect of relieving the effect of alcohol preferably.

Second object of the present invention is the preparation method that proposes above-mentioned propolis extracted with alcohol.

The inventive method comprises the following steps:

1) propolis is placed under the ambient temperature conditions of-18 DEG C and pulverizes after freezing 20～40 hours;

2) edible ethanol that is 50%～90% by volumetric concentration under normal temperature and pressure mixes by the weight ratio of 1～10 ︰ 1 with the propolis after pulverizing, prior to low-frequency ultrasonic waves, process 10～40 minutes at 10～40 DEG C of temperature, be then airtight immersion 1～4 day under the condition of 10～40 DEG C in ambient temperature;

3) get the rear mixture of immersion and turn frozen centrifugation 10～15 minutes with 8000～16000, remove solid impurity, get the supernatant of dissolve with ethanol propolis;

4) by supernatant under 1～5 DEG C of ambient temperature conditions with 5000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 5000D;

5) propolis extracted with alcohol that above-mentioned molecular weight is less than to 5000D is placed in ethanol distillation recover, boils off edible ethanol, obtains the dry thing of biological activity that solid content that molecular weight is less than 5000D is greater than 90%.

At present research is found to affect propolis and is extracted active factor and mainly contain temperature, pH value, extraction time, solvent species, solvent strength, soak time, extraction time etc.The propolis that different extraction processes and solvent extract, its effective ingredient can be different, extracting method difference, the composition of extraction, content, solvent all may affect its function performance, cause kind of a species diversity, and then occur different pharmacological actions.

After the present invention is freezing by propolis, propolis is become fragile, while guaranteeing to pulverize propolis, accomplish that fineness is even, be convenient to the extraction of effective ingredient; Low-frequency ultrasonic waves before immersion is processed except realizing maximized separation, ethanol temperature can also keep extracting time is no more than 40 DEG C, guarantee that in alcoholic solution, extracting isolated material keeps active, and ensure that in propolis, effumability composition retains to greatest extent; The separation, dissolving and the maintenance activity that are beneficial to effective bioactive substance in propolis of the present invention's immersion; It is can obtain maximized dissolving in order to ensure ethanol soluble substance in propolis that suitable propolis and the ratio of ethanol are provided; After soaking, frozen centrifugation is that temperature while guaranteeing centrifugalize is lower, ensures that in propolis ethanol extract, Cera Flava is separated out better, makes the propolis that extracts purer, obtains abundant active substance while being beneficial to next step ultrafiltration; Get supernatant 5000D molecular sieve ultra-filtration and separation, obtaining molecular weight is the propolis ethanol soluble substance below 5000D, first find that through research the propolis ethanol soluble substance below molecular weight 5000D all has the effect of relieving the effect of alcohol, next guarantees that extracting solution is pure clear, transparent, make extract pure, controlling and extracting temperature is one of most critical measure of guaranteeing ethanol soluble substance purity under 1～5 DEG C of condition; Reclaim ethanol with ethanol distillation recover and obtain the dry thing that propolis solid content is greater than 90%, removing ethanol is the application for the ease of next step.

To sum up, the present invention adopts the ethanol extraction method of above improvement, and can extraction efficiency higher, speed be faster, particularly can significantly retain the effective composition that relieves the effect of alcohol, and shows the very strong effect of relieving the effect of alcohol.

The 3rd object of the present invention is the purposes that proposes the propolis extracted with alcohol for relieving the effect of alcohol.

One of purposes is: make for the propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and the coating powder that relieve the effect of alcohol the enteric coatel tablets that relieve the effect of alcohol for the preparation of propolis with described; Described propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and coating powder account for respectively 20～30%, 7～10%, 55～70%, 0.2～0.4%, 1～3% of enteric coatel tablets gross mass.

Through test, utilize propolis extracted with alcohol to merge the Lac regis apis lyophilized powder effect of relieving the effect of alcohol remarkable, and have no side effect, and expense is cheap, simple.Propolis is that Apis gathers the plumelet of glue source plant or the resinoid of callus secretion, and mixes a kind of colloid substance with stickiness that secretions, pollen and the Cera Flava etc. of self body of gland mix.Propolis is rich in various bioactivators, especially contains a large amount of Flavonoid substances, has multiple medicinal health care function, is described as " purple gold ".Lac regis apis is 5-1 5 age in days worker bee lingual glands and secreted milky or the faint yellow emulsion liquid of upper palatine gland, is whole period of development of queen bee nit and drone larva food in earlier stage, is similar to mammiferous milk, claims again Lac regis apis, royal jelly, Lac regis apis.Research is found, contains abundant functional factor in Lac regis apis, and the mankind are had to extremely strong alimentary health-care function and medical function.Propolis extracted with alcohol merges Lac regis apis can bring into play the pharmacological action of relieving the effect of alcohol on gene level, but to being that what material works current research seldom in propolis and Lac regis apis, the molecule mechanism that it is relieved the effect of alcohol imperfectly understands.

But the present inventor's research has solved propolis substantially merges the molecule mechanism that Lac regis apis relieves the effect of alcohol.The present invention utilizes the method for ethanol extraction from propolis, to isolate the composition that has therapeutic effect to relieving the effect of alcohol, and merges Lac regis apis and produces the enteric coatel tablets that relieve the effect of alcohol.

As the advantage of enteric coatel tablets be easy to use, absorb soon, directly absorb and enter liver and relieve the effect of alcohol effective, cheap; After drinking, gastric is full abdomen state, can have a negative impact to the effect of relieving the effect of alcohol, and can overcome well this unfavorable condition and be made into enteric coatel tablets, and the general solution wine product that enteric coatel tablets can be used as ordinary people uses, good and cheap.

Find in propolis extracted with alcohol, there are many bioactive substances under one's belt easily by stomach acids destroy through research, can protect better bioactive substance to exempt from destruction so make enteric coatel tablets, improve the result of use of product.

Brief description of the drawings

Fig. 1 is natural health group liver tissue slices figure.

Fig. 2 is natural health group renal tissue slice map.

Fig. 3 is excessive drinking model control group liver tissue slices figure.

Fig. 4 is excessive drinking model control group renal tissue slice map.

Fig. 5 is gavage enteric coatel tablets group of the present invention liver tissue slices figure.

Fig. 6 is gavage enteric coatel tablets renal tissue of the present invention slice map.

Detailed description of the invention

One, prepare the dry thing of biological activity that solid content that molecular weight is less than 5000D is greater than 90%:

1, propolis is placed under the ambient temperature conditions of-18 DEG C and pulverizes after freezing 20～40 hours.

2, the edible ethanol that is 50%～90% by volumetric concentration under normal temperature and pressure mixes by the weight ratio of 1～10 ︰ 1 with the propolis after pulverizing, prior to low-frequency ultrasonic waves, process 10～40 minutes at 10～40 DEG C of temperature, be then airtight immersion 1～4 day under the condition of 10～40 DEG C in ambient temperature.

3, get the rear mixture of immersion and turn frozen centrifugation 10～15 minutes with 8000～16000, remove solid impurity, get the supernatant of dissolve with ethanol propolis.

4, by supernatant under 1～5 DEG C of ambient temperature conditions with 5000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 5000D.

5, the propolis extracted with alcohol that above-mentioned molecular weight is less than to 5000D is placed in ethanol distillation recover, boils off edible ethanol, obtains the dry thing of biological activity that solid content that molecular weight is less than 5000D is greater than 90%.

Two, prepare the propolis enteric coatel tablets that relieve the effect of alcohol:

1, typical several quality proportioning table: (unit: kg)

?	Propolis powder	Lac regis apis lyophilized powder	Dextrinose	Magnesium stearate	Coating powder
						Proportioning 1	30	10	56.6	0.4	3
Proportioning 2	25	10	62.7	0.3	2
						Proportioning 3	20	10	67.7	0.3	2

2, granulating process: boiling pot is preheating to after 40～60 DEG C, first adds propolis extracted with alcohol dry powder, Lac regis apis lyophilized powder, hydroxyl isomaltulose in boiling pot, arranges according to following table parameter, opens peristaltic pump spray purified water and carries out one-step palletizing.Granulation finishes rear cold drying, after sieving, adds stearic acid, mix homogeneously.

Processing parameter:

Parameter	Inlet temperature	Leaving air temp	Air inducing frequency	Peristaltic pump	Atomizing pressure
						When whitewashing	60-80℃	40-60℃	40Hz	150rpm	0.2MPa
After whitewashing	70-80℃	50-60℃	35Hz	150rpm	0.2MPa

3, tablet forming technique: adopt ZP1100 tablet machine (29 punching) to carry out tabletting experiment, technological parameter: 30～40 revs/min of rotating speeds; Operating pressure 7～15kN; Sheet weighs 0.4～0.6g.

4, art for coating: coating powder adopts enteric coatings powder, and coating parameter is as following table.

Syrup concentration	50%
		Hot blast temperature	60-90℃
Drum rotation speed	5-8 rev/min
		Negative pressure	-0.5KPa
Tablet weightening finish	1-3%

Three, application:

(1) subjects:

The clean level of the Wistar male rat in 22 week age of gavage, body weight 344.43 ± 38.49.

Nature control rats every day is by body weight 1% gavage pure water.

Model group rat every day is by body weight 1% gavage strong, colourless liquor distilled from sorghum simulation.

Gavage enteric coatel tablets experimental group rat every day is by 8 hours gavage enteric coatel tablets of the present invention after body weight 1% gavage strong, colourless liquor distilled from sorghum.

(2) using dosage of enteric coatel tablets:

50～150mg/ days, connects gavage 30～40 days.

(3) comparing result:

1, rats in test groups liver and the comparison of renal tissue section result:

(1) by nature matched group liver and renal tissue section, see Fig. 1,2.

From Fig. 1,2 visible male white rat liver tissue slices, hepatocyte clear in structure is polygon, and hepatic sinusoid is obvious, and hepatic cords marshalling does not have obvious fat vacuole in hepatocyte; Renal tissue section mesonephric glomerulus structure is clear, and glomerular capsule gap is obviously moderate, not obviously damage.

(2) by gavage Chinese liquor model group liver and renal tissue section, see Fig. 3,4.

From Fig. 3,4 visible hepatocyte obscure boundaries, become in circle and have cavity, hepatic sinusoid pressurized narrows, liver rope arrangement disorder; Glomerule structure is unclear, and damage is serious, glomerular capsule distortion.

(3) animal livers of gavage enteric coatel tablets of the present invention and renal tissue section, be shown in Fig. 5,6.

From Fig. 5,6 visible liver lobules of liver clear in structure, hepatic sinusoid is evenly distributed, and hepatocyte is polygon, marshalling; Renal glomerulus structure is obviously improved, and glomerular capsule gap is obvious, has the vestige of obvious glomerule reparation.

2, enteric coatel tablets intervene after to the comparative analysis of each rats in test groups Main Blood Biochemical Index:

Adopt anesthesia ventral aorta blood sampling, detect Main Blood Biochemical Index by Toshiba's fully automatic blood bio-chemical detector after getting serum.

Comparative analysis is as following table:

Group	Nature group	Model group	Enteric coatel tablets group
				ALT	73.00±16.67	91.25±29.33	46.60±7.09
ALP	102.50±11.90	146.75±21.70	78.00±22.93
				TG	1.25±0.18	1.84±0.37	1.19±0.20
BUN	5.89±0.65	4.78±0.71	5.25±0.65
				ALB	15.50±0.43	14.88±0.53	15.89±1.20

Through SPSS statistics, gavage enteric coatel tablets ALT and model group difference are extremely remarkable, with natural matched group significant difference; Transaminase extensively exists in histiocyte, and especially the strongest with activity in the tissue such as liver, cardiac muscle, brain, kidney, when normal, plasma content is very low, and ALT is mainly present in liver, and in the time of hepatic tissue disease damage, ALT is released in a large number blood and can causes that in blood, transaminase raises.This experiment gavage enteric coatel tablets group serum transaminase vigor is very low, illustrates that gavage enteric coatel tablets of the present invention are very strong to the repair of liver.

Healthy Human Serum ALP is mainly from liver and skeleton, and the ALP in liver is with the transhipment of material and drain relevant.And type 2 diabetes mellitus patient is because insulin secretion obstacle or insulin resistant cause, cause metabolism obstacles of blood glucose, also cause the obstacle of lipid metabolism and at intrahepatic deposition, even form fatty liver, and make liver damage cause the rising of ALP simultaneously.Through SPSS statistics, model group alkali phosphatase and other two experimental grouies difference are extremely remarkable; Enteric coatel tablets group alkali phosphatase and natural matched group significant difference, illustrate that enteric coatel tablets experimental group can improve hepatocyte injury, reduces alkaline phosphatase enzyme level, and the regulating and controlling effect effect of enteric coatel tablets is better than nature matched group.

Fat toxicity due to high triglyceride (HTG) has caused extensive concern in recent years, and it can cause and increase the weight of insulin resistant (IR), and the biological effect of insulin is reduced.Glycogen is synthetic by participating in for liver, glycogenolysis and glyconeogenesis maintain in glucostasis, play a part direct and important, particularly G-6-Pase plays a crucial role in glycogen generates, it is abnormal that hepatic insulin resistance also shows as lipid metabolism, increases and liver steatosis as triglyceride discharges.Through SPSS statistics, model group triglyceride and gavage enteric coatel tablets group difference are extremely remarkable, with natural matched group significant difference; From experiment situation, there is serum high triglyceride level in model group, and long-term excessive drinking meeting production hypertriglyceridemia is described, is one of paathogenic factor of bringing out type 2 diabetes mellitus; And enteric coatel tablets of the present invention have good regulating and controlling effect to the Triglyceride Metabolism bringing out due to excessive drinking, this regulating and controlling effect is also better than nature matched group.

Urea concentration is except being subject to Influence on kidney, is also subject to the impact of protein and organism metabolism state in diet, as bad in high protein diet, gastrointestinal function etc.Through SPSS statistics, model group blood urea nitrogen and natural matched group significant difference.Under this experiment condition, model group rat is due to excessive excessive drinking, drunk serious, causing feed intake to decline and protein metabolism disturbance, make blood urea nitrogen not rise counter falling, is minimum in all experimental grouies, and after enteric coatel tablets of the present invention are intervened, metabolism improving to hepatic protein is obvious, and urea nitrogen levels all gains momentum, and is to show that enteric coatel tablets have the function of very strong promotion protein metabolism.Through SPSS statistics, model group albumin level and enteric coatel tablets experimental group significant difference, also can find that from the variation of albumin level enteric coatel tablets have the ability of regulation and control to protein metabolism.

By the analysis to Main Blood Biochemical Index, we are not difficult to draw that such conclusion is after excessive drinking, and enteric coatel tablets not only can help body to relieve the effect of alcohol, and can recover rapidly the metabolism of body to three large materials, improve multinomial blood parameters, make organism metabolism functional recovery to normal condition.

3, gavage enteric coatel tablets of the present invention are expressed and are changed comparative analysis major gene in rats in test groups liver metabolism after intervening:

Study by biochip technology the important metabolic pathway of many impacts in the model group rat liver expressing gene of finding long-term excessive drinking (such as with the related gene that relieves the effect of alcohol, Genes Associated with Lipid Metabolism, hepar damnification is repaired gene) expression of key gene all shows the situation that is unfavorable for body homergy, and the variation of this gene expression has obtained good correction after the intervention of gavage enteric coatel tablets, from lower example table, we are not difficult to find that the ability that this being similar to " error correction " expressed is just embodying gavage enteric coatel tablets to the liver metabolism obstacle forming due to long-term excessive drinking, the reparation of tissue injury has very strong improvement effect, research by us finds that from gene level gavage enteric coatel tablets can relieve the effect of alcohol effectively, improve the metabolism of liver.

The impact that 3.1 gavage enteric coatel tablets are expressed key gene relevant to glycolipid metabolism in liver after intervening

Group	Ehhadh	Fasn	Scd	Scd1
					Model group/natural group	↓2.74	↑4.86	↑5.04	↑5.65
Enteric coatel tablets group/model group	↑2.77	↓2.24	↓14.73	↓26.25

Note: in table, ↓ 2.74 represent that Ehhadh genes model group in hepatocyte mRNA lowers 2.74 times with the expression ratio of natural matched group, ↑ 2.77 represent 2.77 times of the expression ratio rises of Ehhadh gene gavage enteric coatel tablets groups and model group.

3.1.1 Ehhadh: difunctional peroxidase (peroxisomalbifunctionalenzyme), enoyl-CoA hydratase, peroxisome, participates in lipid metabolism, fatty acid metabolism.Peroxisome is unique place that saturated/undersaturated very-long-chain fatty acid carries out beta-oxidation, D-3 hydroxyl acyl CoA dehydratase/D-3 hydroxyl acyl CoA dehydrogenase is that D-bifunctional protein (D-BP) is a kind of enzyme of the participation peroxisome fatty acid beta-oxidation of reported first, in the beta-oxidation of polyunsaturated fatty acid, 2-methyl branch fatty acid and D-isomer hydroxy fatty acid, plays vital effect.Under diabetic disease states the rising of blood plasma very-long-chain fatty acid and levels of polyunsaturated fatty acids whether with the beta-oxidation of peroxisome fatty acid, particularly the change of D-BP activity is relevant.Eukaryotic mitochondrion and peroxisome all carry out the beta-oxidation of fatty acid, carbon chain lengths is less than the mainly beta-oxidation energy supply in mitochondrion of fatty acid of 20 carbon, mainly in peroxisome, be oxidized and carbon chain lengths is greater than saturated/unsaturated very-long-chain fatty acid, dimethyl branched chain fatty acid and the bile acid biosynthesis metabolic intermediate of 2O carbon, then the Short-Chain Fatty Acids of generation proceeds to mitochondrion exhaustive oxidation.Second step and the three-step reaction of peroxisome fatty acid beta-oxidation approach, it is alkene acyl CoA generates 3-ketoacyl CoA reaction through hydration, dehydrogenation, by a kind of bifunctional protein catalysis with alkene acyl CoA hydrase/3-hydroxyl acyl CoA dehydrogenase activity, two kinds of bifunctional proteins are confirmed to exist in peroxisome: L-bifunctional protein (L-BP) and D-bifunctional protein.The trans alkene acyl of L-BP catalysis CoA is converted into L-type 3-hydroxyl acyl CoA, and then dehydrogenation is 3-ketoacyl CoA; And the intermediate product that the trans alkene acyl of D-BP catalysis CoA hydration generates is D type 3-hydroxyl acyl CoA, and taking it as its dehydrogenation of substrate catalysis.Substratspezifitaet research shows, L-BP mainly works in the oxidation of satisfied fatty acid, non-branched chain fatty acid; And D-BP is not only and is uniquely oxidized relevant enzyme with the alkene acyl CoA of polyunsaturated fatty acid, 2-methyl branch fatty acid, and D-BP defect patient's research is also found, D-BP participates in utmost point long-chain straight chain fatty acid, for example Major Enzymes of lignoceric acid beta-oxidation.In liver, the content of D-BP and L-BP is almost equal, in every gram of protein, approximately containing 2.5mg, illustrates that two kinds of bifunctional proteins play a part in the beta-oxidation of hepatocellular peroxisome fatty acid of equal importance.In higher organism body, there are two kinds of multifunctional enzymes to there is L-3-hydroxyl acyl CoA dehydrogenase activity, be respectively the L-BP being present in peroxisome, and in mitochondrion, there are three functional proteins of enoyl-CoA hydratase/L-3-hydroxyl acyl CoA dehydrogenase/3-ketoacyl-CoA thiolase activity.Known, when diabetes, the fatty acid beta-oxidation activity of mitochondrion and peroxisome increases simultaneously, research is found, the beta-oxidation of diabetes rat liver peroxisome fatty acid is active also to raise with liver L-3-hydroxyl acyl CoA dehydrogenase activity simultaneously, and when diabetes are described, the beta-oxidation of mitochondrion and peroxisome linear saturated fatty acids, L-isomer hydroxy fatty acid strengthens; But, the protein content and the enzymatic activity that have research to observe D-BP significantly reduce, thereby the beta-oxidation of polyunsaturated fatty acid, branched chain fatty acid and D-isomer long-chain hydroxy fatty acid probably weakens, cause the intermediate product D-isomer of unsaturated very-long-chain fatty acid, the accumulation of branched chain fatty acid.This discovery conforms to the phenomenon that arachidonic level obviously raises with the patients with NIDDM blood plasma very-long-chain fatty acid of observing, and prompting D-BP activity decreased may be that while causing diabetes, blood plasma fatty acid is composed one of abnormal reason.

The model group of indulging in excessive drinking under this experiment condition and natural matched group are than 2.74 times of the expression of lowering difunctional peroxidase gene, illustrate model group rat indulge in excessive drinking for a long time can by lower this gene, fatty acid beta-oxidation ability in peroxisome and mitochondrion is weakened, promoting liver fat to become, is one of the main molecules mechanism of bringing out liver fat change of indulging in excessive drinking for a long time.

Under this experiment condition, gavage enteric coatel tablets group of the present invention has raised 2.77 times of difunctional peroxidase genes with model group ratio, promote the fatty acid beta-oxidation in liver organization peroxisome and mitochondrion, having positive effect to preventing and treating the fatty liver causing of indulging in excessive drinking, is that one of main molecules mechanism of liver fat change is brought out in the long-term excessive drinking of gavage enteric coatel tablets control.

3.1.2 Fasn: fatty acid synthetase, participates in fatty acid biological synthetic.In mammal, fatty acid synthetase plays vital effect in fatty acid is synthetic, be responsible for all catalytic steps of a kind of long-chain palmitic acid of S-acetyl-coenzyme-A and malonyl coenzyme A de novo synthesis (cetylate), the expression of gene directly affects fatty acid synthetase number and has great importance to controlling body fat deposition.

This experiment condition drag group leader's phase indulge in excessive drinking make in hepatocyte fatty acid synthetase gene with natural matched group than 4.86 times of up-regulateds, illustrate: it is the arch-criminal who causes hepatocyte fat excess deposition that model group can make synthetic the increasing of fatty acid synthetase, is to form one of fatty liver molecule mechanism.

Under this experiment condition, gavage enteric coatel tablets group of the present invention and model group are lowered 2.24 times of the gene expressions of fatty acid synthetase, illustrate: enteric coatel tablets group can be by reducing the synthetic of fatty acid synthetase, reduce hepatic tissue fatty acid synthetic, become significant to alleviating liver fat.

3.1.3 Scd: sterin coenzyme A desaturase, participates in fatty acid biological synthetic.Stearyl-coenzyme A desaturase (SCD) can be introduced in fatty acid carbon chain cis-9 position two keys, is catalysis butterfat cis-9, trans-11CLA and trans-7, the key enzyme that cis-9 CLA is synthetic.

3.1.4 Scd1: sterin coenzyme A desaturase 1, participates in fatty acid biological synthetic.Stearyl-coenzyme A desaturase 1(Scd-1) be the biosynthetic rate-limiting enzyme of monounsaturated fatty acid, in fatty acid metabolism, play center adjustment effect, be also one of genes of interest of leptin (Leptin) effect.Leptin and diabetes, obesity, fatty liver close relation are one of hot fields of metabolic disease in recent years.Type 2 diabetes mellitus patient, usually with insulin resistant, hyperleptinaemia, there is fatty liver in approximately 50% type 2 diabetes mellitus patient simultaneously.Scd-1st, the key enzyme that catalysis satisfied fatty acid changes to monounsaturated fatty acid, with the generation of a series of metabolism syndrome such as fat, fatty hepatic lesions and insulin resistant, develop closely related, thereby expression, the regulation and control of studying Scd-1 may, for the monitoring of the diagnosis of clinical disorders of lipid metabolism relevant disease and therapeutic effect provides a very important lab index, have broad application prospects in clinical medicine.Because Scd-1 is mainly present in endoplasmic reticulum, at present the detection of Scd-1 in body is mainly adopted and measures blood fat index of unsaturation (unsaturated fatty acid/satisfied fatty acid ratio) or by molecular biology method, Scd-1mRNA expression in cell detected.In sum, Scd-1 has been play a part important in energy metabolism.By the research to Scd-1 and energy metabolism relation, can clearerly understand the mechanism of Scd-1 in energy metabolism, thereby be clinical targeting adjusting energy metabolism and avoid fat toxicity, a series of metabolic syndromes such as prevention or treatment are fat, fatty hepatic lesions and diabetes provide basic.

The model group of indulging in excessive drinking under this experiment condition raises 5.04 times of sterin coenzyme A desaturase (Scd) gene expressions with natural matched group ratio, sterin coenzyme A desaturase 1(SCD-1) raise 5.65 times, illustrate: two gene up-regulated is one of main molecule mechanism causing alcohol fatty change, lipotropic is the pathological index of most critical in the process of relieving the effect of alcohol, processing mode effectiveness is just whether can control fatty liver, model group has raised two gene, and there is serious fatty liver in model group liver section display model group rat, even reach the state of hepatic fibrosis.

Under this experiment condition, gavage enteric coatel tablets group is lowered 14.73 times of sterin coenzyme A desaturase (Scd) gene expressions with model group ratio, lower sterin coenzyme A desaturase 1(Scd-1) 26.25 times of gene expressions, illustrate: the fatty liver tool that enteric coatel tablets group forms the bad habit of excessive drinking is significantly improved effect, and consistent with the result of liver section, this change is one of main molecule mechanism of enteric coatel tablets control fatty liver of the present invention.

After 3.2 gavage enteric coatel tablets in liver to hepatic injury, repair the impact that relevant key gene is expressed

Group

Igfbp6

Il7r

Irs2

Socs2

Srd5a1

Tgfb2

Relieve the effect of alcohol model group/natural group

↓3.16

↓3.21

↓21.42

↓33.58

↑4.0

↑3.63

Enteric coatel tablets group/wine model group

↑4.46

↑4.12

↑3.92

↑4.18

↓4.25

↓2.48

Note: in table, ↓ 3.16 represent that Igfbp6 genes model group in hepatocyte mRNA lowers 3.16 times with the expression ratio of natural matched group, ↑ 4.46 represent 4.46 times of the expression ratio rises of Igfbp6 gene gavage enteric coatel tablets groups and model group.

3.2.1 Igfbp6: IGFBP (insulin-like growth factor binding protein) 6, regulating cell growth, signal transduction, negative regulation cell proliferation.Rise is conducive to Igf1 and plays a role, and control fatty liver and hepatic fibrosis are had to fine effect.

Study and confirm, IGFs is relevant with the formation development of hepatic fibrosis, liver cirrhosis.Having isolated at present two kinds of IGFs is IGF-l and IGF-2.They act on target organ with autocrine, paracrine and endocrine mode.It is the complex of 150kb or 50kb that nearly all endocrine IGF-l or IGF-2 all form relative molecular mass with insulin-like growth factor binding protein (IGFBPs) in body fluid.The single chain polypeptide that IGF-l is made up of 70 amino acid residues, mainly synthetic at liver, be the extensive cell mitogen existing of body and the promoter of differentiation and maturation, affect the adjusting of growth, differentiation and the metabolism of many organ inner cells.The above-mentioned functions of IGF-l is by IGF-l receptor (IGF-lR) mediation, and the effectiveness that IGF-l is combined with IGF-lR is subject to the adjusting of IGFBPs.Liu Lixins etc. studies show that, express obviously and strengthen, the formation of prompting IGF and receptor participation hepatic fibrosis thereof in the HSC-T6 that IGFBP (insulin-like growth factor binding protein) 6 (IGFBP6) activates in IGF-β l induction.Because IGFBPs and IGFs have high-affinity and can regulate the biological activity of IGFs, these protein-bonded inductions produce and also can affect developing of hepatic fibrosis.But separately there are some researches show, IGF-1 has the effect of anti-hepatic fibrosis in body, can suppress the activation of HSC, its may with improve superoxide dismutase and catalatic activity, improve the ability that hepatocyte is removed free radical, the stimulating factor of minimizing HS activation etc. are relevant.

This experiment condition drag group has been lowered 3.16 times of IGFBP (insulin-like growth factor binding protein) 6 genes with natural matched group than model group rat, illustrate: model group is by lowering the expression of IGFBP (insulin-like growth factor binding protein) 6 gene, cell cultured supernatant transforms to fibrosis direction, has promoted hepatocyte fat to become and fibrosis.

Under this experiment condition, enteric coatel tablets group raises 4.46 times of IGFBP (insulin-like growth factor binding protein) 6 genes with model group ratio, illustrate: enteric coatel tablets can be by the expression of regulation and control IGFBP (insulin-like growth factor binding protein) 6 gene after intervening, slow down or prevent and treat the Fibrotic process of hepatocyte, control alcoholic fatty liver is had to positive effect.

3.2.2 Il7r: interleukin-17, receptor, participates in regulating DNA restructuring, immunne response, cell surface receptor signal connects conduction, antimicrobial humoral response.

This experiment condition drag group has been lowered 3.21 times of interleukin-17 genes with natural matched group than model group, illustrate: the long-term excessive drinking of model group rat causes hepatocyte immunne response ability to decline, the expression of lowering interleukin-17 gene is exactly to mean to increase the weight of hepatocellular inflammatory reaction, promotes hepatocyte fat to become and Fibrotic process.

Under this experiment condition, enteric coatel tablets group raises 4.12 times of interleukin-17 genes with model group ratio, illustrate: after enteric coatel tablets are intervened, strengthen hepatocellular immunne response ability by the expression of raising interleukin-17 gene, alleviate inflammation reaction, have positive effect to preventing and treating the fatty liver and the fibrosis that cause due to long-term excessive drinking.

3.2.3 Irs2: IRS 2, Irs2 expresses at liver and pancreatic beta cell, participation glucose metabolism, signal transduction, positive regulating cell propagation, Irs2 protein expression is lowered and is made the active attenuating of PI3-K, can increase the weight of liver insulin resistant.Use gene Knockout confirmed that the defect of Irs2 gene can cause the discoveries such as insulin resistant, Kubota, knock out after Irs2, liver shows obvious insulin resistant, and to the insulin sensitivity of skeletal muscle without effect.

This experiment condition drag group has been lowered 21.42 times of IRS 2 genes with natural matched group than model group, illustrate: model group rat has been lowered 21.42 times because excessive drinking causes IRS 2 genes, must make IRS synthesize is greatly affected, obviously can have influence on the sensitivity of hepatocyte to insulin response, impel model group to produce serious insulin resistant, bring out a series of metabolism syndromes, the final fatty liver that forms is that one of main molecules mechanism of type-II diabetes and fatty liver is brought out in long-term excessive drinking.

Under this experiment condition with enteric coatel tablets intervene after with model group ratio, 3.92 times of the expression of Irs2 gene are raised, illustrate: gavage enteric coatel tablets of the present invention can improve the sensitivity of hepatocyte to insulin response by the expression of raising Irs2 gene, and enteric coatel tablets of the present invention can play certain alleviation Insulin Resistance.

3.2.4 Socs2: Suppressor of Cytokine Signaling is named according to 4O amino acid whose Socs frame by discoveries such as Starr R for 2,1997 years.Found that at present this class negative regulator molecule has 6 kinds, the negative feedback that most members participate in JAK/STAT path regulates.There are 8 members in this family containing SH2 domain, i.e. CIS and Socs-1～7, this structure directly with phosphotyrosine interaction, be that inhibiting key plays in Socs family.Extensively, to lipidosis, skeletal development, central nervous system's effect, immunne response, cancer occurs all to play an important role in Socs 2 effects.Socs 2 can regulate the hormone secretion such as insulin, prolactin antagonist in addition, and the cytokine signaling paths such as GH/1GF, insulin, interleukin are risen and promoted or inhibitory action.Cytokine profiles is by lipometabolic balance in complicated signal network regulating cell and body, and most research at present concentrates growth hormone (GH) on lipometabolic impact, and Socs 2 is crucial negative feedback inhibition factors of growth hormone signal path.

This experiment condition drag group and natural matched group ratio, the down-regulated expression of Socs2 gene 33.58 times, illustrate: model group excessive drinking for a long time can cause metabolism " brake " regulator control system in hepatocyte seriously malfunctioning, will inevitably exert an influence to normal lipid metabolism and insulin action in hepatocyte, form dysbolismus, finally impelling hepatocyte fat to become, is that one of main molecules mechanism of fatty liver is brought out in long-term excessive drinking.

After intervening with enteric coatel tablets of the present invention under this experiment condition with intervene before model group ratio, the up-regulated of Socs2 gene 4.18 times, illustrate: enteric coatel tablets of the present invention can be by improving the expression of Socs2 gene, repair in the hepatocyte causing due to excessive drinking " brake " regulator control system malfunctioning, significant to improving hepatocyte lipid metabolism and insulin action.

3.2.5 Srd5a1: steroid 5 alpha-reductases, α peptide 1, distribution endoplasmic reticulum, microsome, 3-C-5 α steroid 4 dehydrogenases, participate in cell signalling, Sex determination, cell differentiation.Can be converted into another kind of androgen-dihydrotestosterone by catalysis testosterone, therefore play a significant role in property differentiation and androgen physiology, this enzyme has 1,2 two isomers of Srd5a.Mainly be expressed in skin and fatty tissue and liver, can catalysis the overwhelming majority the 4th, the reduction reaction of unsaturation steroid on 5 carbon atoms, makes activated glucocorticoid (GC) be converted into the metabolite of non-activity.Research find, glucocorticoid effect not only with blood circulation in GC Horizontal correlation, also have the GC concentration of physiologically active closely related with tissue local.The Srd5a1 activity strengthening in liver causes serum cortisol (COR) clearance rate to increase, and then compensatory hypothalamo-pituitary-adrenal axis (HPA) increased activity, COR secretes increase, further cause abdominal obesity and MS(MS to refer to a series of metabolism disorders and the gathering of cardiovascular and cerebrovascular vessel risk factor on same individuality, be the syndrome of the multiple Developmental and Metabolic Disorder clinical signs such as obesity, hypertension, dyslipidemia and impaired glucose tolerance).

This experiment condition drag group and natural matched group ratio, the up-regulated of Srd5a1 gene 4.0 times, illustrate: model group causes serum cortisol clearance rate to increase by improving Srd5a1 gene expression enhancing Srd5a1 activity, makes model group occur MS, finally makes hepatocyte that fat occurs and becomes.

Under this experiment condition with enteric coatel tablets intervene after with intervene before model group ratio, the down-regulated expression of Srd5a1 gene 4.25 times, illustrate: enteric coatel tablets of the present invention can be by lowering this gene expression, improve the metabolism syndrome bringing due to excessive drinking, this has explained the reason why enteric coatel tablets of the present invention can prevent and treat metabolism syndrome from another point of view, is also that enteric coatel tablets control excessive drinking causes one of molecule mechanism of fatty liver.

3.2.6 Tgfb2: transforming grouth factor beta 2, participate in cell death, just regulating and controlling process, regulating cell propagation, regulation and control immunne response, dopamine biosynthesis.Studies confirm that in cytokine, to only have Tgf-β energy HSC stimulated rubber polymer fibril, other cytokines are a proliferation of HSC stimulated.In process of hepatic fibrosis, KC produces a large amount of Tgf-β by the mode of autocrine or paracrine, and it not only can promote that HSC is converted into fibroblast, promotes the activation of HSC, and can strengthen the synthetic of ECM, suppresses its degraded.Tgf-β can increase the expression of the upper pdgf receptor of HSC simultaneously, promotes HSC autocrine Tgf-β, PDGF, obviously suppresses the apoptosis of activation HSC.PDGF and Tgf-β have formed the Autocrine of activation HSC, are the important mechanisms of the sustained activation of HSC.

This experiment condition drag group and natural matched group ratio, the up-regulated of Tgfb2 gene 3.63 times, illustrate: long-term excessive drinking causes hepatocyte injury, raise the expression of Tgfb2 gene, HSC stimulated rubber polymer fibril, promotes the formation of hepatic fibrosis, consistent with section result.

Under this experiment condition with experimental group after enteric coatel tablets intervention and intervene before model group ratio, the down-regulated expression of Tgfb2 gene 2.48 times, illustrate: enteric coatel tablets of the present invention can be prevented and treated by lowering this gene expression the process of hepatic fibrosis, great to safeguarding hepatocyte homergy Functional Significance.

By the mutation analysis in model group gene expression, be not difficult to find that the consequence of these variations causes lipogenesis to rise exactly, fatty acid oxidation ability declines, and glyconeogenesis weakens, and produces and is similar to insulin and leptin resistance effect, and liver occurs that serious fat becomes; And the change of the gene expression causing due to long-term excessive drinking after gavage enteric coatel tablets of the present invention is adjusted, liver organization function is restored, fatty liver and hepatic fibrosis are effectively controlled, can either bring into play the effect of relieving the effect of alcohol so take enteric coatel tablets of the present invention, can prevent and treat well fatty liver again, be worthy to be popularized.

In a word, show by above test: product of the present invention can effectively relieve the effect of alcohol, the liver fat that control excessive drinking causes becomes, and the good effect of enteric coatel tablets.The research aspect that China is used propolis and Lac regis apis to relieve the effect of alcohol is at present at the early-stage, by experiment, finding that propolis merges Lac regis apis enteric coatel tablets to relieving the effect of alcohol and prevent and treat the lipotropism that excessive drinking causes effective, is the antialcoholic drugs proposition new approaches of exploitation propolis and Lac regis apis dosage form.Propolis and Lac regis apis are China's Traditional health care products, can improve hepatocyte activity, improve multiple blood parameters, and prevention fatty liver is significant to the liver health care of maintenance and raising Chinese.Propolis and Lac regis apis product can be used as the first-selected health product that relieve the effect of alcohol, promotion and application in addition.

Claims (3)

1. the propolis extracted with alcohol for relieving the effect of alcohol, the dry thing of biological activity that is greater than 90% for the solid content that adopts the molecular weight that extracts of ethanol to be less than 5000D from propolis; The preparation method of the dry thing of described biological activity comprises the following steps:

1) propolis is placed under the ambient temperature conditions of-18 DEG C and pulverizes after freezing 20～40 hours;

2) edible ethanol that is 50%～90% by volumetric concentration under normal temperature and pressure mixes by the weight ratio of 1～10 ︰ 1 with the propolis after pulverizing, prior to low-frequency ultrasonic waves, process 10～40 minutes at 10～40 DEG C of temperature, be then airtight immersion 1～4 day under the condition of 10～40 DEG C in ambient temperature;

3) get the rear mixture of immersion and turn frozen centrifugation 10～15 minutes with 8000～16000, remove solid impurity, get the supernatant of dissolve with ethanol propolis;

4) by supernatant under 1～5 DEG C of ambient temperature conditions with 5000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 5000D;

5) propolis extracted with alcohol that above-mentioned molecular weight is less than to 5000D is placed in ethanol distillation recover, boils off edible ethanol, obtains the dry thing of biological activity that solid content that molecular weight is less than 5000D is greater than 90%.

2. as claimed in claim 1 for a preparation method for the propolis extracted with alcohol that relieves the effect of alcohol, it is characterized in that comprising the following steps:

1) propolis is placed under the ambient temperature conditions of-18 DEG C and pulverizes after freezing 20～40 hours;

2) edible ethanol that is 50%～90% by volumetric concentration under normal temperature and pressure mixes by the weight ratio of 1～10 ︰ 1 with the propolis after pulverizing, prior to low-frequency ultrasonic waves, process 10～40 minutes at 10～40 DEG C of temperature, be then airtight immersion 1～4 day under the condition of 10～40 DEG C in ambient temperature;

3) get the rear mixture of immersion and turn frozen centrifugation 10～15 minutes with 8000～16000, remove solid impurity, get the supernatant of dissolve with ethanol propolis;

4) by supernatant under 1～5 DEG C of ambient temperature conditions with 5000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 5000D;

5) propolis extracted with alcohol that above-mentioned molecular weight is less than to 5000D is placed in ethanol distillation recover, boils off edible ethanol, obtains the dry thing of biological activity that solid content that molecular weight is less than 5000D is greater than 90%.

As claimed in claim 1 for the propolis extracted with alcohol that relieves the effect of alcohol in an application of producing enteric coatel tablets, it is characterized in that making for the propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and the coating powder that relieve the effect of alcohol the enteric coatel tablets that relieve the effect of alcohol for the preparation of propolis with described; Described propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and coating powder account for respectively 20～30%, 7～10%, 55～70%, 0.2～0.4%, 1～3% of enteric coatel tablets gross mass, and each constituent content percent sum is 100%.

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