CN102973609B - Propolis ethanol extract for treating alcoholic fatty liver, preparation method and applications in production of enteric-coated tablets - Google Patents
Propolis ethanol extract for treating alcoholic fatty liver, preparation method and applications in production of enteric-coated tablets Download PDFInfo
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- CN102973609B CN102973609B CN201210500236.2A CN201210500236A CN102973609B CN 102973609 B CN102973609 B CN 102973609B CN 201210500236 A CN201210500236 A CN 201210500236A CN 102973609 B CN102973609 B CN 102973609B
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Abstract
The invention discloses a propolis ethanol extract for treating alcoholic fatty liver, a preparation method and applications in the production of enteric-coated tablets, relating to the technical field of process for extracting effective matters from animals and plants especially propolis and using the royal jelly to prepare enteric-coated tablets. The preparation method comprises the following steps: freezing, crushing and removing impurities from the propolis, repeatedly soaking by three times with ethanol at normal pressure and normal temperature, then freezing, centrifuging and removing solid impurities to obtain ethanol supernatant solution, finally separating the propolis ethanol extract with molecular weight of 4000D below at the ambient temperature of 1-5 DEG C, distilling and recovering with ethanol to obtain dry powder, and mixing the propolis powder with royal jelly free-dried powder, iso-maltitol, magnesium stearate, coating powder and the like to obtain the propolis enteric-coated tablets for treating the alcoholic fatty liver. The propolis ethanol extract is obvious in effect for treating the alcoholic fatty liver, has not side effect, and is low in cost and simple and feasible.
Description
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
Through nearly 200,000,000 people of the WHO statistics long-term alcohol user of China, because the toxic action of ethanol causes liver injury, in alcoholic, approximately 2/3 can develop into alcoholic liver disease (ALD), it comprises alcoholic liver injury, fatty liver (AFL), hepatitis, hepatic fibrosis and liver cirrhosis, it is the progressive process of order, pathogenesis is more complicated, and Chinese medicine still can to AFL therapeutic effect.Alcoholic liver disease is one of common clinically hepatopathy, is mainly the hepatic injury due to the long-term heavy drinking of patient.According to abroad studies, alcoholic liver disease is the Etiological that causes liver cirrhosis, and almost 50% liver cirrhosis patient has the custom of drinking for a long time.
In recent years, along with China's living standards of the people improve, life style and dietary habit change, and alcoholic liver disease sickness rate obviously rises, and becomes the second largest hepatopathy that is only second to viral hepatitis, and gradually to womanlike, teenagerization development.Alcoholic liver disease has belonged to frequently-occurring disease and commonly encountered diseases clinically, so, the clinical research of alcoholic liver disease has been caused widely and paid close attention to.The age of sending out well of alcoholic liver disease is 3O-50 year, and its sickness rate increases year by year, and has rejuvenation and tendency faggoty.Find that by research the patients with alcoholic liver disease age is larger, history of drinking history is longer, and drinking amount is larger, and liver function injury is just heavier; Alcoholic liver disease is except outside the Pass having with duration of alcohol consumption and drinking amount, and also closely related with the quality of class that patient drinks and patient's alcohol drinking patterns, drinks quality is poorer, and its prevalence is higher; Drinking white spirit or drinking mixed multiple drinks on an empty stomach, prevalence also can obviously increase; Secondly, although male patient more than female patient, if men and women drinks the wine of same amount, women more easily suffers from alcoholic liver disease compared with male, and clinical symptoms is serious compared with male.Due to the impact being subject to from factors such as body quality, fat and gastric emptying times, women's alcoholic liver damage has high susceptibility.
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, society is in the urgent need to the appearance of the natural control new product of novel cheapness, and the bee product of pure natural has the effect of very strong control alcoholic fatty liver, promote the use of significant.
Excessive drinking can cause organa parenchymatosum's blood capillary to change and steatosis, and then develops into atrophy and the sclerosis of organ, can appear at first liver, lung, heart and brain; Obesity can make free fatty (FFAs) in body increase, and the fat toxicity that the latter causes except itself, oxidation, the inhibition glycogen that also can suppress glucose synthesize, affect islet beta cell function, accelerate that triglyceride is accumulated in hepatocyte etc.Ethanol and obesity can make outside above-mentioned all pathological changes increase the weight of, also can bring out fat hepatitis, and can promote active oxygen and 2-α-hydroxyethyl thiamine pyrophosphate-4-glycollic aldehyde base produce and increase, make alcoholic fatty liver (ASH) and non-alcoholic fatty liver disease (NASH) all be in high-caliber oxidative stress status, impel inflammation and fibrosis constantly to upgrade.Abnormality of Glycolipid Metabolism and excessive drinking can also show the synergism to insulin resistant (IR), IR is an important symbol of fat and Overweight people metabolism syndrome, also be the mark that FFAs assembles at liver simultaneously, FFAs in hepatocyte can be used as the carrier of non-TG metabolite, accelerates disorganization and inflammatory infiltration and fibrosis; Ethanol can mediate peripheral IR and stimulate liver fat acid metabolic, can also mediate oxidation of ethanol at ethanol dehydrogenase and become in acetaldehyde process, due to NAD
+transform and make NAD to NADH
+consume and increase, promote FFAs synthetic, suppress lipid and decompose, finally cause TG to accumulate in hepatocyte.In addition, cause obvious malnutrition different with weight loss from traditional alcohol user, carousing person may appetite increase, and more takes in the food such as fiber, high fat, the additional energy obtaining in ethanol can not be offset by reducing food intake dose, finally cause body weight to increase and obesity.
Under chronic excessive drinking, diabetes and fat condition, the gut permeability that mucosa injury causes strengthens, make intestinal bacteria decompose the giant molecule material molecule as relevant in pathogen producing and enter enteral circulation, and enter people's liver by Portal system, activate hepatic stellate cell and express TLRs.After TLRs ligand stimulation, fibroblast is activated MAP signal pathway, activates NF-κ B, impels collagen muscle fiber archeocyte to secrete a large amount of proinflammatory cytokines and chemokines, makes blood vessel produce inflammatory reaction and fibrosis around.Especially tumor necrosis factor-alpha, interleukin-6, serum endotoxin/lipopolysaccharide and blood plasma collagen activation inhibitor-1 etc. can be used as the important biomolecule label that child NAFLD transforms to NASH.
Long-term excessive drinking, can make hepatocyte generation steatosis, necrosis and regeneration, causes alcoholic liver disease (ALD); On histopathology, main manifestations is three kinds of forms: alcoholic fatty liver, alcoholic hepatitis and alcoholic cirrhosis, these three kinds of forms can also can be mixed separately existence.Along with living condition's improvement, excessive drinking has the trend of increasing.Because China's hepatopathy is mainly caused by hepatitis virus, hepatitis virus carrier more, may cover and be actually the hepatopathy of ethanol as the cause of disease.Therefore, be correctly familiar with the hepatic injury that ethanol causes, diagnose in time and control has great importance.
According to the study, the cause of disease of alcoholic liver disease is the excessive absorption of ethanol, the ethanol of taking in mainly passes through simple diffusion absorption at duodenum and epimere ileum, stomach also can slowly absorb a small amount of ethanol, enter ethanol in blood circulation along with blood flow spreads rapidly, reach very soon balance at more organs of vascularity such as liver, lung and brains.Ethanol can not store, must be by metabolism, liver is the main organ of alcohol metabolism in body, wherein 90～95% carry out oxidative metabolism at liver by ethanol dehydrogenase (ADH) and microsome oxidation of ethanol enzyme system (MEOs), long-term heavy drinking, in increasing the weight of burden of liver, causes damage to liver to a great extent.Primary product after alcohol metabolism is acetaldehyde, also produces oxidative stress product simultaneously, and acetaldehyde is carbon dioxide and water by the final product of aldehyde dehydrogenase (ALDH) oxidative metabolism again subsequently.Alleviating alcohol addiction is that ALD treats one of crucial measure, and alleviating alcohol addiction can obviously improve ALD patient's life quality.After alleviating alcohol addiction, can make the abnormal liver function of ALD take a turn for the better rapidly, obviously improve survival rate; For slight ALD, after alleviating alcohol addiction, can not continue development; Liver cirrhosis fully forms, and has portal hypertension and esophageal varicosis person, and alleviating alcohol addiction is also difficult to reverse, but can improve active process, reduces the generation of complication.Patients with alcoholic liver disease merges heat/potein deficiency malnutrition conventionally, and malnutrition can aggravate alcoholic liver injury.Therefore, patients with alcoholic liver disease should be rich in the low fat soft diet of high-quality protein and vitamin(e) B group, high heat, and suitably supplementing if desired branched-chain amino acid is main compound amino acid supplement.Serious alcoholic liver disease should be carried out and lie up, and the stabilization sub stage of disease should be done muscular training, and as non-strenuous exercise, gymnastics, swimming, physical exercise simple in family is effective equally.
At present, about the definite mechanism of alcoholic fatty liver is not yet completely clear; It is generally acknowledged drinks for a long time can make acetaldehyde growing amount in body increase, and the balance of NADH/NAD changes and causes lipid metabolism and transhipment disorderly, and alcoholic fatty liver is because ethanol has weakened the metabolism of normal liver cell and the stability of liver plasma membrane.1. ethanol intermediate product acetaldehyde and acetic acid, can make the ratio of reducibility coenzyme I and codehydrogenase Ⅱ raise, thereby suppress mitochondrion tricarboxylic acid cycle, makes intrahepatic fat acid metabolic generation obstacle.2. ethanol has the effect that increases specifically choline requirement, and the deficiency of phospholipid has affected the synthetic of lipoprotein, thereby makes to transport generation obstacle.3. heavy drinking causes gastrointestinal dysfunction, affect the absorption of trace element, make vivo oxidation phosphorylation and fatty acid beta-oxidation impaired, blood free fatty acid increases, promote fatty liver to form, a large amount of ethanol stimulates adrenal gland and pituitary adrenal axis, increases fatty tissue resolution ratio, and triglyceride is synthesized to be increased and pile up.4. drink for a long time and can induce the activity of Cytochrome P450 in hepatomicrosome, its active increasing is more increased the weight of ethanol and the toxic action of metabolite to liver.The mechanism more complicated of the hepatic injury that alcoholism causes, except the direct toxic action of ethanol, in alcohol metabolism process, produces a large amount of oxygen-derived free radicals, causes hepatic lipid peroxidation, is a reason that forms hepar damnification.
From prevention effect, alleviating alcohol addiction is extremely important to the treatment prognosis of alcoholic liver disease, and alleviating alcohol addiction is treatment throughout one's life, and only this item can change hepatopathy process.Strengthen society and propagate and educate, change lifestyles, reduce strong ethanol consumption, early stage diagnosis and treatment alcoholic liver and improve alleviating alcohol addiction success rate etc., can gradually reduce the sickness rate of alcoholic liver disease.
Research shows that bicyclol has good function for protecting liver and reducing enzyme activity, obviously be better than polyene phosphatidylcholine group from resume speed and the degree of ALT, AST, ALP and GGT, its mechanism may be polyphenoils glutathion-s-transferring enzyme (GST) and reduced glutathion (GSH) in inductor, improve hepatocyte oxidation resistance, improve GSH level in liver cell mitochondria, bicyclol can also be protected hepatic mitochondria structure, resists all kinds of hepatic injury negative effects; Bicyclol is not inhibitor, but by removing oxygen-derived free radicals, protection liver plasma membrane, makes liver cell nuclear DNA avoid damage and reduce apoptotic generation and play hepatoprotective effect; Bicyclol can alleviate the hepatic injury that lipid peroxidation that ethanol causes causes, thereby improves inflammation necrosis, shows that bicyclol is a kind of better medicament selection for the treatment of alcoholic fatty liver.
Because fatty liver is not independent sexually transmitted disease (STD) kind, by causing the damage of liver function, cause the variation of blood parameters and enzyme value.In the test item of reaction hepatocyte injury, the most common with sero-enzyme value, as glutamate pyruvate transaminase (ALT), alkali phosphatase (ALP), lactic acid dehydrogenase (LDH) etc.In the time there is fat change in liver, because hepatocyte suffers damage, cytopathy, necrosis, the permeability of cell membrane fragmentation or cell membrane increases, and ALT contained in hepatocyte will be released in blood, and ALT activity in blood is increased.Because ALT is mainly present in hepatocyte, in the time that it obviously raises, normal prompting has hepatic injury; When fat change occurs liver, it is main raising mainly with ALT.Glutamate pyruvate transaminase (ALT) is the main project of diagnosis hepatocyte material injury, and its height often parallels with state of an illness weight.
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 extracting mode and pharmacological effect and have obvious dependency.By comparing, supercritical CO
2extraction, 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 or 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 ethanol of the most frequently used extraction solvent, particularly 70 % and 80%.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.We have found also to exist a class to have the material that relieves the effect of alcohol and prevent and treat alcohol fatty liver function in propolis through studying for many years, but adopt at present conventional propolis ethanol extraction method yield lower, thereby effect is not remarkable, or heavy dose of existing market propolis product on sale that uses just may achieve the goal, we propose this patent application for this reason, and the propolis dosage that the present invention uses is consistent with existing taking dose.
Lac regis apis is 5 to 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 3 0～4 0 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 years, 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, the large class of fixed functional factor approximately 9: 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.Past has worked out Lac regis apis and propolis has natural orthofunction and concordance, and 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 fundamentally prevent and treat efficiently alcoholic fatty liver.
The present invention is the dry thing of biological activity that the solid content that adopts the molecular weight that extracts from propolis of ethanol to be less than 4000D is greater than 90%.
Inventor is through evidence, and the bioactive substance that the molecular weight extracting from propolis is less than the dissolve with ethanol of 4000D has the effect of good control alcoholic fatty liver.
Another object of the present invention be propose above-mentioned for preventing and treating the preparation method of propolis extracted with alcohol of alcoholic fatty liver.
Comprise the following steps:
1) propolis is placed under the ambient temperature conditions of-18 ℃ 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 1～6 ︰ 1 with the propolis after pulverizing, airtight immersion 1～2 day under the temperature conditions of 10～40 ℃;
3) liquid after soaking is added to the propolis after pulverizing again, and repeat two to three times, obtain last soak, turn frozen centrifugation with 8000～12000 and remove solid impurity in 10～12 minutes, get the supernatant of dissolve with ethanol propolis;
4) by supernatant at room temperature with 4000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 4000D;
5) propolis extracted with alcohol that above-mentioned molecular weight is less than to 4000D 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 4000D 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; It is can obtain maximized dissolving in order to ensure effective ethanol soluble substance in propolis that propolis mixes with the proper ratio of ethanol; Be beneficial to separation, dissolving and the maintenance activity of effective bioactive substance in propolis by immersion, airtight is in order to prevent that in propolis, active component is lost activity by oxygen molecule oxidation in air, to prevent the loss of volatile ingredient in propolis; The technique of repeatedly soaking is to guarantee to extract in alcoholic solution the material of preventing and treating alcoholic fatty liver in isolated propolis to keep activity and content; Frozen centrifugation is that the temperature while guaranteeing centrifugalize is lower, guarantees 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; Find that through research the propolis ethanol soluble substance below molecular weight 4000D all has the effect of stronger control alcoholic fatty liver, by the molecular sieve of 4000D, guarantee 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; Reclaim ethanol by 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 improvement ethanol extraction method, and extraction efficiency is higher, and speed is faster, particularly can significantly improve in propolis and prevent and treat the effective composition of alcoholic fatty liver, demonstrates the effect of very strong control alcoholic fatty liver.
The 3rd object of the present invention is that proposition is for preventing and treating the propolis extracted with alcohol of alcoholic fatty liver in the application of production enteric coatel tablets.
Make the propolis enteric coatel tablets for the preparation of control alcoholic fatty liver with described for preventing and treating propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and the coating powder of alcoholic fatty liver; Described propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and coating powder account for respectively 20～30%, 10～20%, 50～68%, 0.2～0.6%, 1～3% of enteric coatel tablets gross mass.
Through test, utilize propolis extracted with alcohol to merge Lac regis apis control alcoholic fatty liver effect 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 control alcoholic fatty liver on gene level, but to being that what material works research at present seldom in propolis and Lac regis apis, the molecule mechanism of its control alcoholic fatty liver is imperfectly understood.
But the present invention has solved the molecule mechanism of propolis merging Lac regis apis control alcoholic fatty liver substantially, for this reason, files an application patent of the present invention.The present invention utilizes the method for ethanol extraction from propolis, to isolate the composition that control alcoholic fatty liver is had to therapeutic effect, merges Lac regis apis and produces the enteric coatel tablets of preventing and treating alcoholic fatty liver.
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 enteric coatel tablets can be used as control alcoholic fatty liver product and use, good and cheap.
Find in Lac regis apis and 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.
Accompanying drawing explanation
Fig. 1 is natural health group liver tissue slices figure.
Fig. 2 is natural health group renal tissue slice map.
Fig. 3 is alcoholic fatty liver model group liver tissue slices figure.
Fig. 4 is alcoholic fatty liver model 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 group of the present invention renal tissue slice map.
The specific embodiment
One, preparation control alcoholic fatty liver ethanol extraction dry powder:
1, propolis is placed under the ambient temperature conditions of-18 ℃ 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 1～6 ︰ 1 with the propolis after pulverizing, airtight immersion 1～2 day under the temperature conditions of 10～40 ℃.
3, again continue to soak propolis after new pulverizing with the soak after soaking for the first time, after waiting and soaking and finish, again soak propolis after new pulverizing.
4, get soak after repeating to soak for the third time and turn frozen centrifugation with 8000～12000 and remove solid impurity in 10～12 minutes, get the supernatant of dissolve with ethanol propolis.
5, by supernatant at room temperature with 4000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 4000D.
6, the propolis extracted with alcohol that above-mentioned molecular weight is less than to 4000D 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 4000D is greater than 90%.
Two, prepare the enteric coatel tablets of Prevention And Cure Using Propolis alcoholic fatty liver:
1, typical several quality proportioning table: (unit: kg)
| ? | Propolis powder | Lac regis apis lyophilized powder | Dextrinose | Magnesium stearate | Coating powder |
| Proportioning 1 | 30 | 15 | 51.5 | 0.5 | 3 |
| Proportioning 2 | 25 | 10 | 62.6 | 0.4 | 2 |
| Proportioning 3 | 20 | 10 | 66.6 | 0.4 | 3 |
2, granulating process: boiling pot is preheating to after 40～60 ℃, 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, sieves, and adds magnesium stearate mix homogeneously.Technological 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.
Model group rat every day is by body weight 0.5% gavage strong, colourless liquor distilled from sorghum and body weight 0.5% gavage Adeps Sus domestica simulation.
Gavage enteric coatel tablets experimental group: rat adopts the mode of gavage propolis Lac regis apis enteric coatel tablets on an empty stomach to test by body weight 0.5% gavage strong, colourless liquor distilled from sorghum and body weight 0.5% gavage Adeps Sus domestica every day after 8 hours.
(2) the gavage using dosage of enteric coatel tablets:
50～100mg/ days, connects and takes 30～60 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.
Be polygon from Fig. 1,2 visible male white rat liver hepatocyte clear in structure, hepatic sinusoid is obvious, and hepatic cords marshalling does not have obvious fat vacuole in hepatocyte; Kidney group section mesonephric glomerulus structure is clear, and glomerular capsule gap is obvious, moderate, not obviously damage.
(2) by alcoholic fatty liver 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, and liver rope arrangement disorder has certain Fibrotic sign; Glomerule structure is unclear, and damage is serious, glomerular capsule distortion.
(3) gavage enteric coatel tablets liver 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, propolis Lac regis apis enteric coatel tablets intervene after to the comparative analysis of rats in test groups Main Blood Biochemical Index:
Adopt the blood sampling of anesthetized rat ventral aorta, 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 | Gavage enteric coatel tablets group |
| ALT | 73.0±16.67 | 81.8±14.81 | 55.5±28.69 |
| ALP | 102.50±11.90 | 179.40±32.39 | 103.25±24.72 |
| GRE | 55.78±2.97 | 48.02±4.44 | 50.73±3.53 |
| CHO | 1.23±0.08 | 1.57±0.18 | 1.03±0.07 |
| TG | 1.25±0.18 | 1.46±0.31 | 1.11±0.21 |
Through SPSS statistics, model group ALT and gavage enteric coatel tablets difference are extremely remarkable; 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 enteric coatel tablets are very strong to the repair effect 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 due to insulin secretion obstacle or insulin resistant, 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 serum levels of ALP level simultaneously.Through SPSS statistics, model group alkali phosphatase and other two experimental grouies difference are extremely remarkable; Illustrate that enteric coatel tablets of the present invention can improve hepatocyte injury, reduce alkaline phosphatase enzyme level, rising and the hepatocellular damage of alkali phosphatase (ALP) are closely related, illustrate that this experimental model group excessive drinking high fat diet are very serious to hepatocyte injury, and after gavage enteric coatel tablets, can improve the untoward reaction that long-term excessive drinking high fat diet cause.
Add up through SPSS, model group creatinine (GRE) is extremely remarkable with natural matched group difference, illustrate under this experiment condition that excessive drinking high fat diet meeting decline the utilization of body exogenous protein, and gavage enteric coatel tablets of the present invention can improve the utilization of body exogenous protein, creatinine level rises to some extent.
Type 2 diabetes mellitus is the class disease take insulin resistant and islet beta cell function obstacle as principal character, and wherein the loss of beta Cell of islet and dysfunction play a decisive role in the generation of disease and development.Research in recent years shows, cholesterol metabolism and islet beta cell function are closely related.Under normal circumstances, beta Cell of islet can be controlled by the series of receptors of its surface expression and transport molecule absorption and the outflow of cell inner cholesterol, makes the content of cell inner cholesterol maintain a dynamic equilibrium.After the metabolism of beta Cell of islet inner cholesterol gets muddled, can affect by number of ways the carbohydrate metabolism of β cell, finally induce β apoptosis.Therefore, cholesterol metabolism disorder may be a new mechanism that causes type 2 diabetes mellitus patient β cell dysfunction.Through SPSS statistics, model group cholesterol and natural matched group and gavage enteric coatel tablets group difference are extremely remarkable, natural matched group CHO and gavage enteric coatel tablets group significant difference; Illustrate aspect regulation and control cholesterol metabolism, gavage enteric coatel tablets of the present invention can significantly be lowered the cholesterol levels causing due to excessive drinking and raise, its regulating effect is even better than nature matched group, takes enteric coatel tablets of the present invention significant to the generation that prevents to bring out type 2 diabetes mellitus due to long-term excessive drinking.
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 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 take enteric coatel tablets of the present invention to thering is good regulating and controlling effect due to excessive drinking the Triglyceride Metabolism of Induced by High Fat Diet.
Above result shows: enteric coatel tablets of the present invention become and have sizable therapeutical effect the rat liver fat that also high fat diet causes due to excessive drinking, from blood parameters and liver tissue slices, the improvement effect of enteric coatel tablets of the present invention is very clear and definite, and the renal tissue of damage is also had to repair.
3, enteric coatel tablets of the present invention are expressed and are changed comparative analysis major gene in alcoholic fatty liver 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 high fat diet (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 gavage enteric coatel tablets intervention of the present invention, 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 of the present invention to the liver metabolism obstacle because excessive drinking high fat diet form for a long time, the reparation of liver organization damage has very strong improvement effect, research by us finds that from gene level gavage enteric coatel tablets of the present invention can relieve the effect of alcohol effectively, improve the metabolism of liver, the generation of control alcoholic fatty liver.
The impact of after 3.1 gavage enteric coatel tablets, key gene relevant to lipid metabolism in alcoholic fatty liver being expressed
| Group | Fasn | LpL | Scd | Scd1 |
| Model group/natural group | ↑4.17 | ↓3.12 | ↑6.76 | ↑6.47 |
| Enteric coatel tablets group/model group | ↓2.33 | ↑2.19 | ↓13.28 | ↓12.86 |
Note: in table, ↑ 4.17 represent that Fasn genes model group in hepatocyte mRNA raises 4.17 times with the expression ratio of natural matched group, ↓ 2.33 represent 2.33 times of the expression ratio downwards of Fasn gene gavage enteric coatel tablets groups and model group.
3.1.1 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 and high fat diet make in hepatocyte fatty acid synthetase gene with natural matched group than 4.17 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, causes fatty liver generation primary factor.
Under this experiment condition, gavage enteric coatel tablets group and model group are lowered 2.33 times of the gene expressions of fatty acid synthetase, illustrates: enteric coatel tablets group can, by reducing the synthetic of fatty acid synthetase, reduce hepatic tissue fatty acid synthetic, significant to alleviating liver fat change.
3.1.2 LpL: lipoprotein lipase, participate in fatty acid metabolism, be one of lipometabolic key enzyme.LpL is synthetic at the rough endoplasmic reticulum of parenchyma, and new synthetic LpL non-activity, after glycosylation, just changes into active LpL.The heparin sulfate glycoprotein that is present in cell membrane outer surface makes enzyme keep a kind of unvital enrichment stage, after being stimulated by heparin, in blood plasma, obtain the LpL of activation, be distributed in containing in the lipoprotein of triacylglycerol, it is mainly the triacylglycerol that decomposes Chylomicron and very low density lipoprotein (VLDL), and combination and being attached in these remnant lipoproteins, may absorb as liver the signal of these granules.LpL acts on lipoprotein, mainly decomposes the triacylglycerol in lipoprotein, is the rate-limiting enzyme of removing triacylglycerol in blood plasma, and impels lipoprotein to shift cholesterol, phospholipid and apolipoprotein.LpL also has the Chylomicron of increasing and is attached to the ability on LP receptor, promotes Chylomicron picked-up.
This experiment condition drag group, illustrates than lowering 3.12 times of lipoprotein lipase gene expressions with natural matched group: model group can make lipoprotein lipase content decline, and causes triacylglycerol to decompose and reduces, and acceleration model group rat liver fat becomes.
Under this experiment condition, gavage enteric coatel tablets group, with model group than 2.19 times of up-regulateds, illustrates: enteric coatel tablets group can make the synthetic increase of lipoprotein lipase in experimental group rat hepatocytes, is conducive to hepatocyte and reduces fat, and alleviates liver fat and becomes.
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.
Under this experiment condition, the model group of long-term excessive drinking high fat diet raises 6.76 times of sterin coenzyme A desaturase (Scd) gene expressions with natural matched group ratio, sterin coenzyme A desaturase 1(SCD-1) raise 6.47 times, illustrate: two gene up-regulated is one of main molecule mechanism causing alcohol fatty change, what prevent and treat alcoholic fatty liver 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 of the present invention is lowered 13.28 times of sterin coenzyme A desaturase (Scd) gene expressions with model group ratio, lower sterin coenzyme A desaturase 1(Scd-1) 12.86 times of gene expressions, illustrate: the alcoholic fatty liver tool that enteric coatel tablets group forms the bad habit of long-term excessive drinking high fat diet 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 alcoholic fatty liver of the present invention.
The impact of after 3.2 gavage enteric coatel tablets, key gene relevant to hepatic injury, reparation and metabolism in liver being expressed
| Group | Cd2 | Il7r | Pla2g2a | Tnfrsf10b |
| Model group/natural group | ↑29.6 | ↓2.76 | ↓2.47 | ↑4.14 |
| Enteric coatel tablets group/model group | ↓32.89 | ↑2.36 | ↑3.91 | ↓3.94 |
Note: in upper table, ↑ 29.6 represent that Cd2 genes model group in hepatocyte mRNA raises 29.6 times with the expression ratio of natural matched group, ↓ 32.89 represent 32.89 times of the expression ratio downwards of Cd2 gene gavage enteric coatel tablets groups and model group.
3.2.1 Cd2:Cd2 molecule, receptor, protein binding, participates in Lipid Rafts polar, guiding apoptosis, cell surface receptor connects signal transduction, cell attachment, natural killer cell activates, and is is just regulating and controlling dendritic cell-stimulating, t cell activation, modulating T cell differentiation.Lipid Rafts refers to that membrane lipid bilayer contains the microcell of special lipid and protein, and diameter is about 50～350nm, and microcell caves in and can form vesicle (caveola or alveole), and Lipid Rafts does not exist only on cytoplasma membrane, and also has on Golgi membrane.Different Lipid Rafts have specific protein separately, and contained lipid is also incomplete same, and has different functions.Lipid Rafts is made up of sphingomyelins, ganglioside and cholesterol, because sphingomyelins contains chain saturated fatty acids, interact into a kind of lipid phase in order with cholesterol, (Tm) is higher for its phase transition temperature, the mobility of adipose membrane is reduced and stability increase, Lipid Rafts contains more unsaturated fatty acid in Mo district around, and Tm temperature is lower, and phase-splitting has just appearred in the Lipid Rafts district of film and non-Lipid Rafts district like this.The function of dendritic cell in human body is just equivalent to a precaution device, and as the hardcore in human immunocyte's system, they find external invader (antigen) from virus, antibacterial or other organs, and information then gives a warning.Their shake antigen, as a kind of signal, makes T cell can enter timely and disperse invader.
This experiment condition drag group raises 29.6 times of Cd2 genes with natural matched group than model group, illustrate: model group, by raising Cd2 gene, activates dendritic cell, impels immunocyte excessive activation, cause excessive immunoreation, bring out hepatocyte inflammation and fibrosis.
Under this experiment condition, enteric coatel tablets group, with model group than lowering 32.89 times of Cd2 genes, illustrates: enteric coatel tablets can, by lowering Cd2 gene expression, be alleviated immunocyte excessive activation after intervening, and prevent because hepatocyte inflammation and fibrosis are brought out in excessive immunoreation.
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 2.76 times of interleukin-17 genes with natural matched group than model group, illustrate: model group rat is because the long-term also high fat diet meeting of indulging in excessive drinking causes hepatocyte immunne response level to rise, by lowering the expression of interleukin-17 gene, increase the weight of hepatocellular inflammatory reaction, promote hepatocyte fat to become and Fibrotic process.
Under this experiment condition, enteric coatel tablets group raises 2.36 times of interleukin-17 genes with model group ratio, illustrate: after enteric coatel tablets of the present invention are intervened, by raising the expression of interleukin-17 gene, anti-hepatocellular immunne response, alleviate inflammation reaction, control alcoholic fatty liver and fibrosis are had to positive effect.
3.2.3 Pla2g2a: phospholipase A2, base 2A, calcium relies on, and participates in fat catabolism.
This experiment condition drag group and natural matched group ratio, the down-regulated expression of Pla2g2a gene 2.47 times, illustrate: the long-term excessive drinking of model group high fat diet, by lowering the expression of Pla2g2a gene, reduce the catabolism to fat in hepatocyte, the process of weight fat liver.
Under this experiment condition with experimental group rat after enteric coatel tablets intervention of the present invention and intervene before alcoholic fatty liver model group ratio, the up-regulated of Pla2g2a gene 3.91 times, illustrate: enteric coatel tablets of the present invention are by having raised the expression of this gene, promote the catabolism of fat in hepatocyte, favourable to control alcoholic fatty liver.
3.2.4 Tnfrsf10b: Tumor Necrosis Factor Receptors surpasses family, member 10b, Guang winter enzyme activity factor, TRAIL combination, participates in Guang winter enzyme and activates, and signal transduction activates NF-κ B guiding kinases, and regulating cell apoptosis, is is just regulating and controlling NF-κ B cascade.
This experiment condition drag group and natural matched group ratio, the up-regulated of Tnfrsf10b gene 4.14 times, illustrate: model group activates NF-κ B cascade by raising the expression of Tnfrsf10b gene, promotes hepatocyte lipid peroxidation and inflammatory reaction, increases the weight of liver fat and becomes and fibrosis.
Under this experiment condition with experimental group after enteric coatel tablets intervention and intervene before alcoholic fatty liver model group ratio; the down-regulated expression of Tnfrsf10b gene 3.94 times; illustrate: enteric coatel tablets of the present invention can be by lowering Tnfrsf10b gene expression; suppress the activation of NF-κ B in rat hepatocytes; alleviate lipid peroxidation and inflammatory reaction, realize the target of protection hepatic tissue.
Be not difficult to analyze from above expression conditions, alcoholic fatty liver model group rat, due to long-term excessive drinking high fat diet, can be increased the weight of hepatocyte injury, promotes hepatocyte inflammatory reaction, the damage to liver and repair extremely unfavorable; And contrary situation appears in the expression of above these genes after gavage enteric coatel tablets of the present invention, to the reparation of liver organization, control fatty liver and hepatic fibrosis have positive effect.
The impact that 3.3 enteric coatel tablets are expressed key gene relevant to controlling normal activity in liver after intervening
| Group | Adh4 | Hspb1 | Igfbp2 | Igfbp6 | Srd5a1 |
| Model group/natural group | ↓2.05 | ↓2.65 | ↓6.24 | ↓3.97 | ↑2.6 |
| Enteric coatel tablets group/model group | ↑2.18 | ↑3.45 | ↑3.89 | ↑3.15 | ↓2.81 |
Note: in upper table, ↓ 2.05 represent that Adh4 genes model group in hepatocyte mRNA lowers 2.05 times with the expression ratio of natural matched group, ↑ 2.18 represent 2.18 times of the expression ratio rises of Adh4 gene gavage enteric coatel tablets groups and model group.
3.3.1 Adh4: alcoholdehydrogenase 4, participate in alcohol metabolism and aldehyde metabolism, oxidation of ethanol is decomposed.It just works in the time of ethanol high concentration, different from other Adh in immunology, and has different substrate specificities.Adh4 also participates in the metabolism of retinol, has the physiological action of obvious degradation epinephrine and norepinephrine.
This experiment condition drag group and natural matched group ratio, alcoholdehydrogenase 4(Adh4) gene down-regulated expression 2.05 times, illustrate: the model group of long-term excessive drinking high fat diet has been lowered alcoholdehydrogenase 4 genes, the expression that reduces this enzyme just means that model group downward is by the approach of ethanol dehydrogenase system decomposition ethanol, make liver by ethanol dehydrogenase system relieve the effect of alcohol function decline, be the one of the main reasons that causes rat hepatocytes alcohol metabolism obstacle.
Under this experiment condition experimental group with enteric coatel tablets intervene after with alcoholic fatty liver model group ratio, alcoholdehydrogenase 4(Adh4) up-regulated 2.18 times, illustrate: enteric coatel tablets of the present invention can pass through to raise alcoholdehydrogenase 4(Adh4) gene expression, recover hepatocyte by the function of relieving the effect of alcohol of alcohol dehydrogenase enzyme system, reaching and improve the object of relieving the effect of alcohol, is that enteric coatel tablets of the present invention have one of the effect of relieving the effect of alcohol reason.
3.3.2 Hspb1: much research shows, no matter body in stress state or normal physiological state, all can participate in complicated and important biological process.As sHSP and other albumen interact, stop unnecessary interaction between albumen, maintain various cell proteins stable; Carry out the maintenance of film, and relevant with the mobility of film fat; There is driving functions in core, or Molecular regulator process, or protected protein; Stabilized cell skeleton, participates in regulating growth and the differentiation of cell, suppresses the apoptosis of cell; Also there is the functions such as immunology; All play a significant role maintaining in the physiological status of body and the pathophysiological process of some disease.
This experiment condition drag group and natural matched group ratio, the down-regulated expression of Hspb1 gene 2.65 times, illustrate: model group, by lowering the expression of Hspb1 gene, declines to damaging the hepatocellular ability of maintenance, is unfavorable for hepatocellular reparation.
Under this experiment condition with experimental group after enteric coatel tablets intervention and intervene before alcoholic fatty liver model group ratio, 3.45 times of the up-regulateds of Hspb1 gene, illustrate: after enteric coatel tablets are intervened, improve the hepatocellular ability of maintenance by the expression of raising Hspb1 gene, promote the hepatocellular reparation of damage.
3.3.3 Igfbp2: IGFBP2, participates in regulating cell growth.Rise is conducive to Igf1 and plays a role, and enhances metabolism, and is conducive to glycolipid metabolism, favourable to prevention fatty liver.
3.3.4 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, IGFBP2,6(IGFBP2,6) in the HSC-T6 of IGF-β l induction activation, to express obviously and strengthen, prompting IGF and receptor thereof participate in the formation of hepatic fibrosis.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 6.24 times of IGFBP2 genes, 3.97 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 IGFBP2,6 digenic expression, cell cultured supernatant transforms to fibrosis direction, has promoted hepatocyte fat to become and fibrosis.
Under this experiment condition, enteric coatel tablets group raises 3.89 times of IGFBP2 genes, 3.15 times of IGFBP (insulin-like growth factor binding protein) 6 genes with model group ratio, illustrate: enteric coatel tablets can be by the above digenic expression of regulation and control after intervening, slow down or prevent and treat the Fibrotic process of hepatocyte, control alcoholic fatty liver is had to positive effect.
3.3.5 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 2.6 times, illustrate: model group causes serum cortisol clearance rate to increase by improving Srd5a1 gene expression enhancing Srd5a1 activity, makes model group occur MS, the final fat that forms.
Under this experiment condition with enteric coatel tablets intervene after with intervene before alcoholic fatty liver model group ratio, the down-regulated expression of Srd5a1 gene 2.81 times, illustrate: enteric coatel tablets of the present invention can be by lowering this gene expression, improve due to the excessive drinking metabolism syndrome that also high fat diet brings, this has explained the reason why enteric coatel tablets of the present invention can prevent and treat metabolism syndrome from another point of view.
In a word, show by above test: product of the present invention can effectively be prevented and treated alcoholic fatty liver, the liver fat that control excessive drinking high fat diet cause becomes.
Claims (3)
1. for preventing and treating the propolis extracted with alcohol of alcoholic fatty liver, for the molecular weight that adopts ethanol to extract from propolis is less than the dry thing of biological activity that the solid content of 4000D is greater than 90%; The extraction step of the dry thing of described biological activity is:
1) propolis is placed under the ambient temperature conditions of-18 ℃ 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 1～6 ︰ 1 with the propolis after pulverizing, airtight immersion 1～2 day under the temperature conditions of 10～40 ℃;
3) liquid after soaking is added to the propolis after pulverizing again, and repeat two to three times, obtain last soak, turn frozen centrifugation with 8000～12000 and remove solid impurity in 10～12 minutes, get the supernatant of dissolve with ethanol propolis;
4) by supernatant at room temperature with 4000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 4000D;
5) propolis extracted with alcohol that above-mentioned molecular weight is less than to 4000D 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 4000D is greater than 90%.
2. as claimed in claim 1 for preventing and treating the preparation method of propolis extracted with alcohol for alcoholic fatty liver, it is characterized in that comprising the following steps:
1) propolis is placed under the ambient temperature conditions of-18 ℃ 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 1～6 ︰ 1 with the propolis after pulverizing, airtight immersion 1～2 day under the temperature conditions of 10～40 ℃;
3) liquid after soaking is added to the propolis after pulverizing again, and repeat two to three times, obtain last soak, turn frozen centrifugation with 8000～12000 and remove solid impurity in 10～12 minutes, get the supernatant of dissolve with ethanol propolis;
4) by supernatant at room temperature with 4000D molecular sieve ultra-filtration and separation, obtain the propolis extracted with alcohol that molecular weight is less than 4000D;
5) propolis extracted with alcohol that above-mentioned molecular weight is less than to 4000D 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 4000D is greater than 90%.
3. as claimed in claim 1 for preventing and treating the propolis extracted with alcohol of alcoholic fatty liver in an application for production enteric coatel tablets, it is characterized in that making the propolis enteric coatel tablets for the preparation of control alcoholic fatty liver with described for preventing and treating propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and the coating powder of alcoholic fatty liver; Described propolis extracted with alcohol, Lac regis apis lyophilized powder, hydroxyl isomaltulose, magnesium stearate and coating powder account for respectively 20～30%, 10～20%, 50～68%, 0.2～0.6%, 1～3% of enteric coatel tablets gross mass.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360897A (en) * | 2000-12-28 | 2002-07-31 | 上海大承生物制品有限公司 | Bee glue purifying technological process |
| CN1683011A (en) * | 2005-03-04 | 2005-10-19 | 辽宁天成生物制药研究所有限公司 | Human vaccine containing bee glue adjuvent and its preparing method |
| CN102224892A (en) * | 2011-05-09 | 2011-10-26 | 大连海尔思科技有限公司 | Propolis extraction process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1360897A (en) * | 2000-12-28 | 2002-07-31 | 上海大承生物制品有限公司 | Bee glue purifying technological process |
| CN1683011A (en) * | 2005-03-04 | 2005-10-19 | 辽宁天成生物制药研究所有限公司 | Human vaccine containing bee glue adjuvent and its preparing method |
| CN102224892A (en) * | 2011-05-09 | 2011-10-26 | 大连海尔思科技有限公司 | Propolis extraction process |
Non-Patent Citations (3)
| Title |
|---|
| The Hepatoprotective and Therapeutic Effects of Proplis Ethanol Extract on Chronic Alcohol-induced liver Injuries;Song-Chow Lin, et al.;《American Journal of Chinese Medicine》;19971231;第25卷(第3-4期);325-332 * |
| 慢性酒精性肝损伤大鼠COX-2、TNF-a的表达及蜂王浆的干预作用;徐小冬等;《健康研究》;20110228;第31卷(第1期);第4-8页 * |
| 蜂胶萃取方法对蜂胶有效成分影响的研究;杨明等;<<中国蜂业>>;20060131;第57卷(第1期);6-8 * |
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| CN102973609A (en) | 2013-03-20 |
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