CN102327259A - Composite biological preparation with obvious chemical hepatic injury protecting function - Google Patents

Abstract

The invention relates to a composite biological preparation with an obvious chemical hepatic injury protecting function, which consists of the following raw materials in weight ratio: 15-18 parts of glycine, 12-15 parts of arginine, 15-18 parts of cysteine, 16-18 parts of glutamine, 7-10 parts of S-adenosylmethionine and 7-12 parts of alpha-lipoic acid. The composite biological preparation has favorable therapeutic action and prevention action to all hepatic injury caused by various reasons, and has the effective rate (effectual rate) of 98.6% and the cure rate of 76.8%.

Description

Composite biological agent with remarkable chemical liver injury defencive function

Technical field

The present invention relates to a kind of composite biological agent that chemical liver injury is had remarkable defencive function.

Background technology

China is hepatopathy big country occurred frequently, and annual medical expense of paying because of various hepatic disease is up to 50,000,000,000 yuans.The fatty liver that chemical liver injury and a variety of causes cause becomes China's common disease more and more occurred frequently, and epidemiological study shows that Chinese's pathogenesis of fatty liver rate is about 22～25%, its sickness rate Along with people's growth in the living standard and rising gradually.For example clinical epidemiology research shows: the pathogenesis of fatty liver rate that Shandong Province's population chemical liver injury and a variety of causes cause is about 33～35%; The pathogenesis of fatty liver rate that Beijing's population chemical liver injury and a variety of causes cause is about 25%; The pathogenesis of fatty liver rate that Shanghai City population chemical liver injury and a variety of causes cause is about 20%, and the pathogenesis of fatty liver rate that Shenzhen's population chemical liver injury and a variety of causes cause is about 17%.If the untimely treatment of hepatocellular degeneration moderate or severe fatty liver, the ratio that develops into hepatic fibrosis and liver cirrhosis in 10 years are up to 70%, the patient with liver cirrhosis of about 20%-30% develops into hepatocarcinoma patient the most at last.

Although utilizing the chemical synthetic drug protecting liver and detoxication is a potential approach, owing to must carry out metabolic conversion to this medicine, this will cause bigger load to the liver of this fragility in damaged condition.See from the clinical efficacy of the chemicals that adopted at present (like tiopronin etc.) and other health product (like Herba Silybi mariani etc.) and the effect of auxiliary protection chemical liver injury, generally all undesirable.From naturally occurring biochemical product, seek the liver protecting prevention ethanol or other chemical substance and poison, promote its metabolism, excretory novel endogenous biomacromolecule composite antioxidant to become prevention and treatment fatty liver and the syndromic optimum selection of metabolism disorder.International academic community is generally acknowledged; Stop or prophylactic activity oxygen-derived free radicals (Reactive Oxygen Species; ROS) and active nitrogen free radical (RNS) attack; Blocking-up nuclear factor NF-κ B (Nuclear transcipton factor; NF-κ B), the release of bringing out of cytokines such as tumor necrosis factor (Tumor necrosis factor-α, TNF-α), transferinggrowthingfactor (Transforming Growth Factor β, TGF-β) and interleukin is prevention and treatment fatty liver and the syndromic key of metabolism disorder; The metabolism of adjustment hepatocyte oxidoreduction, giving to imitate by force the antioxidant for clearing ROS and the toxic cell factor becomes treatment in future that international academic community generally acknowledges and corrects fatty liver and the syndromic main R&D direction of metabolism disorder that chemical liver injury and a variety of causes cause.

Up to the present; The hepatic injury that both at home and abroad different reasons is caused does not still have the good curing way; A series of result for retrieval shows, comprises that both at home and abroad the west main developed country U.S., Britain, Germany, Italy, France, Japan, Australia, Canada etc. still do not have the relevant efficient medicine of treating technology, the intellectual property of hepatic injury and passing through the FDA approval of registration.At present; The report that has some to use aminoacid auxiliary treatment hepatic injury or have other health-care effects both at home and abroad; The prescription that adopts one or more aminoacid to form mostly; As: the auspicious year board nutraceutical sheet of forming by lysine, tryptophan, methionine, arginine, leucine, isoleucine, aspartic acid etc.; Claim that mainly health care is to keep vigorous energy, improve sleep, improve immunity etc., not mentioned effective in cure to chemical liver injury, and also per 100 parts only contain aminoacid 43.6g part.Extra large Wang Jin cup sheet of selling on the market at present is a main component with oyster powder, vitamin C, L-cysteine, taurine, starch, sucrose etc. mainly; Functional component is coarse powder sugar and taurine; Wherein taurine is per hundred parts of only 0.846 part of content, and warp is market test in recent years, and certain sober-up function is arranged; This nutraceutical sheet mainly has certain auxiliary protection function to liver, but not obvious to the chemical liver injury therapeutic effect; Number of patent application is to disclose in 200610102355.7 the one Chinese patent application with any several seed amino acids in existing all 20 seed amino acids of nature to mix; Cooperating in all vitamins that nature exists arbitrarily again, several vitamins mixes, adds the medicine that several kinds of trace element are formula for treating fatty liver, alcoholic liver, blood fat reducing, transaminase lowering; It is characterized in that by weight proportion: aminoacid 30-60 part; Vitamin 10-30 part; Trace element 2-5 part is prescription; Find that through our experimental study for many years each seed amino acid cooperates the back that liver disease is not had actual effect with any several kinds, arbitrary proportion, more not reaching the obvious effective rate that this application declares is 83%.

The hepatic injury that both at home and abroad different reasons is caused does not still have the good curing way, and cure rate and effective percentage are lower.For example; In mid-June, 2006 at world's endocrine that Washington, DC is held and the academic conference latest report of diabetes; At present, in the U.S., 6 months effective percentage of fatty liver treatment is merely 43.6% (fatty liver is one of diabetes common complication); In current world's endocrine and the academic conference of diabetes, clearly classify as one of metabolism disturbance syndrome to fatty liver.Seeking prevention is one of hot research field of whole world Biomedical Science man focusing with treatment fatty liver and the syndromic active drug of metabolism disorder.

Summary of the invention

The object of the invention is primarily aimed at the shortcoming of prior art, provides a kind of through to the The Molecular Biology Mechanism and the potential approach research of molecular pathology, prevention and the therapeutical chemistry liver damage of fatty liver, fatty liver, the optimum formula of pharmacology, toxicology, molecular biology research experiment is carried out in active antioxidation active ingredient to multiple efficient natural.

Technical scheme of the present invention is: a kind of composite biological agent with remarkable chemical liver injury defencive function, form by the raw material of following part by weight:

Beneficial effect of the present invention is: the present invention has carried out pharmacology, toxicology, molecular biological research to the natural anti-oxidation effective ingredient; Several times screen, the combination of optimization and effective ingredient; Have the effects such as pathological process of extremely significant the liver protecting, antioxidation, adjusting metabolism, inflammation-inhibiting and reverse fatty liver, especially more remarkable to the alcoholic fatty liver therapeutic effect.The present invention stops active oxidation free radical (ROS) to be attacked through change and the hepatocellular metabolic pathway of multiple adjustment, multidigit point; Make oxidation resistance increase in the cell; Antioxidation GSH content in the obvious rising cell, inhibition liver spider cell, macrophage induce α-Zhong Liuhuaisiyinzi inflammatory factors such as (TNF-α) to discharge, inflammation-inhibiting reaction and apoptosis process; The hepatic injury that the inside and outside chemical toxicant of opposed body causes can significantly improve liver function.The present invention has extremely strong antioxidation and inflammation-inhibiting effect; S-adenosylmethionine and antioxidant cysteine induce TNF-α, interleukin I L-6, IL-10 that direct regulating action is arranged to lipopolysaccharide (LPS); Help lend some impetus to the drainage of nuisance (like chemical substances such as ethanol), stable and adjusting hepatocyte metabolism; Reduce alcohol damaged inductive liver transaminase (AST, ALT) and raise, reverse and repair liver structure and function.The present invention all has good curing and preventive effect to the hepatic injury that a variety of causes causes, and effective percentage (obvious effective rate) is 98.6%, and cure rate is 76.8%.

Description of drawings

Fig. 1 is the methionine metabolism approach

Fig. 2 is experiment one, SAMe, and the L-cysteine is to the contrast chart of the influence of inductive blood serum tumor transfer factor of LPS (TNF-α) and transaminase ALT

Fig. 3 is experiment two, SAMe, cysteine and the alpha-lipoic acid contrast chart to the influence of Mus RAW cell interleukin 6 (IL-6), interleukin-11 0 (IL-10)

The contrast chart of the influence that the RAW cell tumour necrosin (TNF-α) that Fig. 4 stimulates LPS for experiment three, SAMe and cysteine discharges

Fig. 5 be glutamine, arginine to the mice serum alanine aminotransferase (ALT, U/L) influence chart

Fig. 6 absorbs ripple at 532nm to measure the content chart of TBARS in hepatic tissue

Fig. 7 is experiment five, glycine is to transaminase in the inductive rat body of lipopolysaccharide (LPS) and tumor necrosis factor TNF-alpha generates and the contrast chart of the influence of release

Fig. 8 is the influence contrast chart of prescription of the present invention to mitochondrion GSH, TBARS, ALT

Fig. 9 is the influence contrast chart to rat blood serum ALT and TNF-alpha content

Figure 10 is that the present invention is to the metabolic chart that influences of Patients with Fatty Liver

Figure 11 is the influence chart of the present invention to the Patients with Fatty Liver liver function

Figure 12 is the influence chart of the present invention to renal function

Figure 13 is that the present invention is to the metabolic chart that influences of moderate fatty liver

Figure 14 is the influence chart of the present invention to moderate Patients with Fatty Liver liver function

Figure 15 is the influence chart of the present invention to severe Patients with Fatty Liver liver function

Figure 16 is the influence chart of the present invention to the special inflammatory factor of severe Patients with Fatty Liver

Figure 17 is the influence chart of the present invention to alcoholic fatty liver patients with hepatic function

Figure 18 is the influence chart of the present invention to non-alcoholic fatty liver disease patients with hepatic function

Figure 19 the present invention is to the dissimilar clinical comprehensive therapeutic effect charts of fatty liver in various degree that reach

The specific embodiment

Specific embodiment 1:

A kind of composite biological agent with remarkable chemical liver injury defencive function is made up of the raw material of following part by weight: 15 parts of glycine; 12 parts of arginine; 15 parts of cysteine; 16 parts of glutamine; 7 parts of S-adenosylmethionines; 7 parts of alpha-lipoic acids.

Specific embodiment 2:

A kind of composite biological agent with remarkable chemical liver injury defencive function is made up of the raw material of following part by weight: 18 parts of glycine; 15 parts of arginine; 18 parts of cysteine; 18 parts of glutamine; 10 parts of S-adenosylmethionines; 12 parts of alpha-lipoic acids.

Specific embodiment 2:

A kind of composite biological agent with remarkable chemical liver injury defencive function is made up of the raw material of following part by weight: 16 parts of glycine; 13 parts of arginine; 16 parts of cysteine; 17 parts of glutamine; 9 parts of S-adenosylmethionines; 10 parts of alpha-lipoic acids.

Glutathion (GSH) is a kind of tripeptides, attacks barrier as the antioxidation that body is main, is present in the cell with high concentration (m mol/L level).About 85%GSH is present in the Cytoplasm, and only 10～15%GSH is present in the oxidation place mitochondrion of cell.Because mitochondrion itself does not have the gamma-glutamic acid cysteine synthase, Intramitochondrial GSH active transport is taken from the GSH in the Cytoplasm.As everyone knows, mitochondrion is the place that produces reactive oxygen free radical (ROS), and oxidation is attacked responsive, so the barrier that the GSH topmost opposing ROS that is mitochondrion attacks.

It is one of key character of xenobiontics hepatic injury and alcoholic liver injury that the inducing of cytokine discharges.Clinical research is for many years found, TNF-α among steatosis hepatitis, liver cirrhosis and the alcoholic hepatitis patients serum, and interleukin 1 (IL-1), interleukin 6 (IL-6), interleukin 8 (IL-8) obviously raise.In a large amount of experimental liver injury models, find that also TNF-α, IL-1, IL-6, IL-8, TGF-β etc. bring into play central role in hepatocyte injury and downright bad process.Under normal circumstances, liver cell can be resisted the cytotoxicity of TNF-α.Reduce when being not enough to resist TNF-α yet antioxidant is a large amount of in TNF-α produces release or liver in a large number, hepatocyte is is promptly killed and wounded in a large number.TGF-β kills and wounds the hepatocyte effect simultaneously in performance, still can induce synthetic collagen in a large number, quickens liver fibrosis process.The a large amount of generations of TNF-α are one of key links of alcoholic hepatitis pathology incidence and development process with discharging.Because the ROS that the alcohol oxidation metabolism produces attacks and not only activates liver spider cell (Kupffer cell) release nuclear factor NF-κ B in a large number; Tumor necrosis factor TNF-alpha; And the oxidative metabolism of ethanol has seriously consumed antioxidant in the liver; Changed the normal redox equilibrium of hepatocyte, the liver that hepatocyte is caused TNF-α is poisoned responsive more.

The normal methionine of hepatocyte (Methionine) metabolic pathway is to keep the equilibrated important channel of antioxidant in the liver in the liver.Methionine is the medication of a kind of hepatic disease auxiliary treatment, also is one of a kind of essential amino acids of human body.The methionine metabolism approach particularly keeps in the human body antioxidation equilibrium process playing a significant role in normal human's physiological metabolism.Methionine is through methionine adenosyltransferase (Methionine Adenosyltransferase; MAT) catalysis; Can be metabolized to the important amino acid of multiple needed by human; Body weight for humans as the precursor synthesized human is essential is wanted physiologically substance, and like polyamines, choline, glutathion, cysteine etc., wherein glutathion, cysteine are most important antioxidant in human body and the liver cell.Another important function of methionine metabolism is for various important biochemical physiology methylation reactions in the body methyl (through methylferase) to be provided.The methionine metabolism approach is seen Fig. 1.

Under the fatty liver situation, methionine transferring enzyme (MAT) is active usually descends, and causes methionine to be piled up, and downstream metabolites significantly reduces simultaneously.

As can beappreciated from fig. 1, transform through methionine metabolism, human body can obtain many important physiological materials; Particularly glutathion (Glutathione, GSH), cysteine (Cysteine) etc.; In the normal oxidation reducing condition process that keeps cell, bringing into play epochmaking function, and S-adenosylmethionine (S-adenosylmethionine, SAMe); Be that the commentaries on classics reaction of Salmon-Saxl is necessary intermediate product; Through this approach, methionine just can be converted into cysteine, other key protein synthesis materials is provided and keeps in the cell and iuntercellular normal oxidation reducing condition.Behind the S-gland cysteine transmethylase, be metabolised to the S-adenosyl homocysteine (S-adenosylhomocysteine, SAH).(S-adenosylhomocysteine Hydrolase, SAHH) metabolism is homocysteine and adenosine to SAH by the S-SAHH.Homocysteine can be converted into cysteine on the one hand, and (Methionine Synthase MS) utilizes 5-methyl tetrahydrofolate (CH to the methionine synthetase that can be relied on by vitamin B12 again on the other hand ₃THF) synthetic again methionine.Then (Methylenetetrahydrofolate Reductase, MTHFR) reduction generates methyl tetrahydrofolate (CH to the tetrahydrofolic acid (THF) that produces again by the methyl tetrahydrofolate reductase again ₃THF); Homocysteine also can be by betanin (Betaine) homocysteine transferring enzyme (Bataine Homocysteine S-methyltransferase; BHMT) catalysis forms methionine, discharge simultaneously dimethylglycine (Dimethyl-glycine, DMG).

This shows that the methionine metabolism approach is a meticulous metabolic cycles approach in the body.In case a certain link of this approach is blocked, will produce the metabolism disorder of series, break original good redox state in the liver.

Under the normal condition, the adult can produce 6～8 gram SAMe every day, and big quantity research shows, has the methionine metabolism disturbance of circulation under most fatty liver states.For example, long-term drink ethanol can cause the active decline of methionine adenosyltransferase (MAT), cause methionine superfluous, and downstream metabolites significantly reduces, and makes body can't obtain to keep required aminoacid of normal function and antioxidant.Research confirms, human body lacks SAMe can cause that the TNF-alpha levels raises greatly in the blood, and can cause endotoxemia, and TNF-α can induce a series of release of cytokines again and activate inflammatory process.

1, S-adenosylmethionine, cysteine (Cysteine) antioxidation and liver injury protection

S-adenosylmethionine (SAMe) shortage is an extremely important link in fatty liver formation and the progression in the liver.As aforementioned methionine metabolism approach, SAMe is one of key link of metabolic pathway.Because SAMe is the synthetic precursor of important antioxidant in the bodies such as glutathion (GSH), cysteine.GSH is a kind of tripeptides, attacks barrier as the antioxidation that body is main, is present in the cell with high concentration (mmol/L level).About 85%GSH is present in the Cytoplasm, and only 10～15%GSH is present in the mitochondrion of the interior oxidation place of cell.Because mitochondrion itself does not have gamma glutamyl cysteine synthetase, Intramitochondrial GSH mainly takes from the GSH in the Cytoplasm through active transport.

As everyone knows, mitochondrion is the place that produces ROS, and oxidation is attacked responsive, so the barrier that the GSH topmost opposing ROS that is mitochondrion attacks.SAMe and cysteine are the synthetic precursors of GSH, replenish SAMe and cysteine can correct in the hepatocyte and in the mitochondrion because the GSH lowering of concentration, the attack damage that ROS causes.

It is one of key character of xenobiontics hepatic injury and alcoholic hepatic injury that the inducing of cytokine discharges.Clinical research is for many years found, TNF-α among steatosis hepatitis, liver cirrhosis and the alcoholic hepatitis patients serum, and interleukin 1 (IL-1), interleukin 6 (IL-6), plain 8 (IL-8) that are situated between certainly obviously raise.In a large amount of experimental liver injury models, find that also TNF-α, IL-1, IL-6, IL-8, TGF-β etc. bring into play central role in hepatocyte injury and downright bad process.

Under normal circumstances, liver cell can be resisted the cytotoxicity of TNF-α.Reduce when being not enough to resist TNF-α yet antioxidant is a large amount of in TNF-α produces release or liver in a large number, hepatocyte is is promptly killed and wounded in a large number.TGF-β kills and wounds the hepatocyte effect simultaneously in performance, still can induce synthetic collagen in a large number, quickens liver fibrosis process.The a large amount of generations of TNF-α are one of key links of alcoholic hepatitis pathology incidence and development process with discharging.Because the ROS that the alcohol oxidation metabolism produces attacks and not only activates Kupffer cell release TNF-α in a large number; And the oxidative metabolism of ethanol has seriously consumed antioxidant in the liver; Changed the normal redox equilibrium of hepatocyte, the liver that hepatocyte is caused TNF-α is poisoned responsive more.

Research shows, the hepatic injury that anti-TNF-Alpha antibodies therapy can stop ethanol and other chemical substances to cause effectively.Clinical research finds that anti-TNF-Alpha antibodies therapy can be improved arthritis, struvite disease of intestine etc. effectively and also confirm the effect of TNF-α in numerous disease in the side.Research shows that SAMe and cysteine all can be brought into play anti-hepatocyte injury effect through GSH in production of cytokines and release in the adjusting course of liver damage and the increase liver.Cysteine is to contain mercaptoamino acid, and it is the synthetic precursor of GSH on the one hand, and on the other hand, as antioxidant, it can directly remove interior free yl.

2, the influence of the antioxidation of SAMe and cysteine and the pair cell factor

The ROS oxidation is attacked can directly stimulate body to produce the secretion endotoxin; In the blood endotoxin raise [like MDA, lipopolysaccharide (LPS)] can induce and activate liver spider cell (Kupffer cell), make its secretion and discharge a large amount of α-Zhong Liuhuaisiyinzis (TNF-α) and interleukin IL-6, IL-8, IL-10 etc.TNF-α can directly cause hepatic cell fattydegeneration, inflammation and necrosis.TNF-α also can promote external all steatolysiss, makes free fatty get into liver through blood and accumulates.TNF-α also can reduce the removing of liver cell to low density lipoprotein, LDL.Therefore TNF-α plays an important role in the generation of fatty liver, liver cirrhosis, evolution.

Interleukin-11 0 (IL-10) is called as CSIF at first.It is inflammatory reaction and an immune hyperfunction reaction inhibitive factor important in a kind of body.Different cells comprise that mononuclear cell, macrophage, lymph B cell, T cell and natural killer cell etc. all can produce release IL-10; IL-10 has stronger anti-fibrosis effect, and IL-10 mainly transcribes and increases the collagen decomposition expression of enzymes through the inhibition glue protogene and brings into play its anti-fibrosis effect.Clinical research shows that patient with liver cirrhosis interleukin-11 0 (IL-10) growing amount and blood level all obviously reduce, and the inductive liver injury of IL-10 knock out mice Abensanil and ethanol is more responsive.Interleukin-6 is a kind of multifunctional cytokine, and its existing short inflammatory effect can be brought into play anti-inflammatory effect again, and up to the present, more evidence shows that IL-6 has anti-inflammatory and anti-apoptotic effect.The hepatic injury that interleukin-6 pretreatment rat can significantly protect ischemia-reperfusion to cause.Interleukin-6 also can be protected inner skin cell function and protection hepatocyte injury.Interleukin-6 is still closely related with the liver regeneration function in addition.The IL-6 knock out mice shows the liver regeneration dysfunction, so the IL-6 processing can be accomplished this liver regeneration obstacle of upset.

Thioctic acid (alpha lipoic acid) is a kind ofly can eliminate accelerated ageing and morbific free radical, belong to B family vitamin material; Thioctic acid is a kind of mitochondrial ferment that is present in, and thioctic acid gets into cell in vivo after intestinal absorption, has fat-soluble and water miscible characteristic concurrently; Therefore can go everywhere without any hindrance here at whole body; Arriving any one cell position, provide human body comprehensive usefulness, is unique fat-soluble and water miscible omnipotent biological anti-oxidant that has concurrently.The Parker doctor in California, USA university Berkeley branch school (Dr.Lester Packer) is top thioctic acid in the world and antioxidant authority.He finds that thioctic acid has only a specific task in vivo unlike other antioxidant, the identity of its tool " free body ", can be when the shortage of other antioxidant " in generation, beat " for it; That is to say that if lack vitamin e or C in the human body, thioctic acid will temporarily be taken over their work.And thioctic acid can also strengthen the effect of vitamin C and E greatly; What is; If Alpha-Lipoic Acid is processed compound recipe together with some other antioxidation composition; Possibly will improve the effect of these antioxidation compositions, the research report of minority show thioctic acid be used for prevention in addition treat some disease as: AIDS, diabetes and complication thereof, nervous system are degenerated and symptom such as hepatic insufficiency.But only exist on a small quantity in what Rhizoma Solani tuber osi, Herba Spinaciae and the meat by the what thioctic acid; Therefore come prevent disease if will replenish enough thioctic acid; The nutriment that had better select to have extracted on the market the dealer usually with thioctic acid and Semen Vitis viniferae, green tea, compositions such as vitamin C are processed compound product.Thioctic acid is a kind of antioxidant of super strong type; Be called as " universal antioxidant "; The free radical hunter is that body cell utilizes the required a kind of restricted essential nutrient substance of saccharide equal energy source material produce power especially, is widely used in multiple disease such as treatment and prevention of cardiac, diabetes etc.It is generally acknowledged its other antioxidant of preserving and regenerate, like vitamin C and E etc., and can balance blood sugar concentration.Effectively strengthen vivo immuning system, avoid destruction in free radical.It has therapeutic efficiency to various disease conditions: hepatopathy; Diabetes; The infull carrier of human acquired immunity; AIDS; The Corii Bovis seu Bubali moss; Eczema; Burn; Skin carcinoma; Multiple sclerosis; Parkinson's disease; The disease of department of neurology aspect; Rheumatism; Rheumatic arthritis; Lupus erythematosus; Scleroderma; The disease of autoimmune aspect; Cataract; Other ophthalmic diseases; Heart disease; Apoplexy; Arteriosclerosis; Be used for acute and chronic hepatitis; Hepatitis interstitialis chronica; Hepatic coma; Fatty liver diseases.

Be the regulating action of the research SAMe and the antioxidant cysteine pair cell factor, we have observed SAMe and cysteine to the influence of the inductive TNF-α of lipopolysaccharide (LPS) and to the influence of interleukin IL-6, IL-10.

Experiment one, SAMe, the L-cysteine is to the influence of inductive blood serum tumor transfer factor of LPS (TNF-α) and transaminase ALT

Normal male SD rat body weight 200-220g is divided into three groups, and 12 every group, normal control group, SAMe administration group, and cysteine administration group.Equal-volume normal saline of normal control group intramuscular injection every day, SAMe organizes SAMe of intramuscular injection every day (50mg/kg body weight), cysteine of cysteine group intramuscular injection every day, dosage is the 60mg/kg body weight.Successive administration 14 days, rat tail vein injection lipopolysaccharide (LPS) 2mg/kg body weight after 120 minutes, is got blood after 14 days, and separation of serum is measured TNF-alpha levels and alanine aminotransferase (ALT) level in the rat blood serum.The result is following.

^{※ ※}Compare with matched group, p＜0.001, ^{△ △}Compare p＜0.001 with matched group

Like Fig. 2, The above results shows that exogenous SAMe of giving and cysteine can significantly suppress the inductive TNF-α of LPS and produce and release.Results suggest SAMe and cysteine all have good protective action to inflammation and the cell injury that TNF-α causes.

Experiment two, SAMe, cysteine and alpha-lipoic acid are to the influence of Mus RAW cell interleukin 6 (IL-6), interleukin-11 0 (IL-10)

For observing SAMe and cysteine interleukin is produced and the influence that discharges; We have studied the influence to Mus mononuclear cell RAW264.7 cell strain IL-6 and IL-10 of SAMe and cysteine, and the RAW264.7 cell strain is widely used in studying cytokine and produces and discharge.

RAW cells cultivates with DMEM (containing 10% calf serum, 2mM glu famine, 5U/ml penicillin, 5 μ g/ml streptomycins), and RAW264.7 cell initial incubation density is 0.5 * 105 cell/ml, 37 ℃ of overnight incubation.After cultivating in 24 hours, add 0,0.1,0.5; 1.0mM SAMe (final concentration) or 0,10, the 50mM alpha-lipoic acid was cultivated 2 hours; In each culture dish, add 100ng/ml then, LPS (final concentration) cultivates after 16 hours the collecting cell culture fluid for 37 ℃; Removed cell and float in centrifugal 10 minutes for 3000rpm4 ℃, with IL-10, IL-6 concentration in IL-10, IL-6, the ELISA kit measurement culture fluid, IL-10, the sensitivity of IL-6 kit measurement are 4.0pg/ml.The result is following.

Compare with 0mM SAMe matched group: ^{※ ※}P＜0.001, ^△P＜0.05, ^{△ △}P＜0.001

Compare with 0mM alpha-lipoic acid matched group: ^{※ ※}P＜0.001

Like Fig. 3, experimental result shows that SAMe can significantly promote the RAW cell to discharge interleukin-6 and IL-10 INTERLEUKIN-10 in 0.1mM concentration, when 1.0mM concentration, can stimulate the RAW cell to produce interleukin-6 and the IL-10 INTERLEUKIN-10 that discharges nearly two times of concentration.Yet alpha-lipoic acid discharges IL-10 INTERLEUKIN-10 to the RAW cell does not have obvious influence, and when high concentration (50mM), alpha-lipoic acid can obviously reduce the generation and the release of the interleukin-6 that LPS is stimulated.

The influence that the RAW cell tumour necrosin (TNF-α) that experiment three, SAMe and cysteine stimulate LPS discharges

The above results shows, SAMe and cysteine stimulate to LPS that to produce the influence that discharges interleukin different, and we further observe the influence of having compared the TNF-α release that SAMe and cysteine stimulate LPS.RAW cultivates with above-mentioned consistent.After 24 hours incubated overnight, add 0,0.1 respectively; 0.5,1.0mM SAMe or 0,10; The 50mM cysteine is cultivated after 2 hours in culture fluid, adds 100ng/mL (final concentration) LPS and cultivates 16 hours for 37 ℃; Collect culture fluid then, with the concentration of TNF-α EILSA kit measurement TNF-α in culture fluid.TNF-α test kit detection sensitivity is 13.5pg/mL, and result of the test is following.

Compare with 0mM SAMe matched group: ^※P＜0.05, ^{※ ※}P＜0.001

Compare with 0mM cysteine matched group: ^{※ ※}P＜0.001

Like Fig. 4, the result shows that SAMe and NAC all can significantly suppress LPS stimulates the TNF-α that causes to discharge.External, it is suitable with cysteine 10mM that 0.5mM SAMe suppresses the effect of TNF-α release.

Glutamine, arginic anti-oxidative damage effect and the compatibility synergism thereof of protecting the liver

Arginine is a kind of important semi-dispensable amino acid in the Human Physiology biochemical process; Arginine is brought into play the important physiological biochemical function in the histoorgan metabolic process; It is that protein synthesis and some have the synthetic precursor of critical function biomacromolecule, and these critical function biomacromolecules comprise: nitric oxide (NO), ornithine, polyamines, spermine, proline, glutamine, creatine, diethylarginine, carbamide etc.At growth and development stage, arginine be good growth promoter must ammonia need base acid.In the manhood, arginine is a kind of requisite modulability essential amino acids.Under some morbid state, for example wound, burn, resection of small intestine, pyemia, renal failure and tumor etc. can cause body to utilize arginine to increase greatly; When exceeding body self generation; Cause arginine to lack, when body nutrient Deficiency of Intake, will increase the weight of these diseases.Research shows that the picked-up of arginine external source increases can improve nitrogen balance, improves lymphocyte function, and can stimulate the transhipment of arginine in liver.In addition, take arginine and can improve cardiovascular, lung, immunity and digestive function, and early stage canceration is had the good preventing effect.Clinical research proves that arginine can effectively improve hypertension, hyperlipemia symptom, and can significantly reduce the diabetics level of lipid.

Recently research shows that arginine has obvious protection or improvement effect to non-ethanol fatty liver, ischemia-reperfusion acute liver damage and the circulation of fatty liver blood capillary.NO is well-known vessel dilator, and broad research confirms that at hepatic disease, particularly in fatty liver and the liver cirrhosis process, NO has effect of crucial importance to blood vessel kinetics and liver microcirculation in the adjusting liver.Oshita etc. study confirmation, and NO effect vessel dilator can significantly improve the inductive liver blood vessel of ethanol and shrink, and improves blood flow circulation in the liver.Discoveries such as Kakumitsu, giving the L-arginine can increase the portal vein blood flow of liver cirrhosis liver by selectivity, and obviously improves hemodynamics and liver microcirculation in the liver.

Bibliographical information is arranged recently; Rat feeding L-arginine (1.25g/kg body weight) all around; Can obviously alleviate nitrogen free radical; (NOS) damage, and can prevent or alleviate inductive renal calculus formation of ethylene glycol (EG) and nephrocyte damage, its main mechanism is for correcting the damage to the nephrocyte function of nitrogen free radical and chlorine radical (ROS) ^[82]The arginic above-mentioned effect of L-is had laid a good foundation for the exogenous L-arginine of clinical practice prevents and treat fatty liver and prevention renal calculus and cardiovascular disease.

Glutamine (GLN) is a non essential amino acid important in a kind of body, and glutamine changes in the amino metabolic process through KG and glutamic acid approach at aminoacid brings into play central role.Recently research shows that GLN has immunomodulating and cell regulating action.Research shows the synthetic precursor of glutamine effect glutathion and purine and the synthetic precursor of pyrimidine; Cellular function has many-sided regulating action; For example GLN can regulate arginine and NO metabolism; Can regulate cell volume and stimulating cytokine and form, regulate immune cell function, for example: stimulate interferon generate, suppress apoptosis, leukocyte increasing be situated between plainly generate, irritation cell RNA is synthetic etc.As the synthetic precursor of GSH, GLN is with cysteine, glycine, the synthetic GSH of N-acetylcystein, and glutamine and cysteine act in kidney, liver, the metabolism of small intestinal oxidoreduction and being even more important.

Research shows, all significantly reduces at body glutamine contents such as diabetes, uremia, liver cirrhosis, ulcerative colitis, chemotherapy patients, and non-insulin dependent patient free-radical generating obviously raises and increases the weight of the various complication of diabetics.Quiet notes GSH can significantly improve non-insulin-dependent diabetes mellitus patient's GSH: the GSSG ratio increases the Sugar intake utilization.Research confirms that already the reduction of GSH level has obviously weakened non-insulin-dependent diabetes mellitus patient's insulin sensitivity, has influenced the reaction of beta cell to blood glucose.In uremia's patient process, exogenous increase GSH can make the erythropoietin use amount reduce by 37%.In addition; GLN is the main metabolic fuel of small intestinal endotheliocyte; Can significantly protect the small intestinal ischemical reperfusion injury, external source increases GLN and can obviously promote the burn patient rehabilitation or reduce infection rate, and surgical operation and bone marrow is changeed plant postoperative recovery good facilitation is arranged.Discover that GLN all has the certain protection effect to the hepatic injury that the different chemical material causes, the main mechanism of its protective effect is antioxidation and increases GSH content.

Comprehensive Experiment four, glutamine and arginine and unite the coordinating protection effect of use to chemical liver injury

For inquiring into glutamine and the arginine protection therapeutical effect to fatty liver, we have studied glutamine and arginine and have united the protective effect of use to alcohol-induced hepatic damage.

Normal male mice body weight (18-22g); Be divided into 5 groups; 10 of every treated animals are divided into matched group, ethanol feed group, glutamine+ethanol feed group, arginine+ethanol feed group and glutamine+arginine+ethanol feed group, per 12 hours feed 6g/kg body weight of mice ethanol; Give altogether 4 times, the normal control group gave heat maltodextrin solutions such as equal-volume in per 12 hours.Glutamine+ethanol group is (6g/kg) lumbar injection 100mg/kg body weight glutamine before feed ethanol; Before arginine+ethanol group feed ethanol (6g/kg), lumbar injection 100mg/kg arginine; Glutamine+arginine+ethanol feed group, before the ethanol feed, lumbar injection 100mg/kg glutamine+100mg/kg body weight arginine.After ethanol and medicine give 6h the last time, get blood and separation of serum is subsequent use.Put to death mice, get the mouse liver homogenization rapidly, 4 ℃ of 600x g are centrifugal, pipette supernatant; Centrifugal 10 minutes of 4 ℃ of 7000x g, mitochondrion (deposition), the HMS buffer washed after 10 minutes; Carry out centrifugal (7000x g) again, abandon supernatant, the mitochondrion that suspends again is to be measured.

SAIT (ALT) is measured with diagnostic kit, and the result is following.

^{※ ※}Compare with matched group: p＜0.001, ^{△ △}Compare with matched group: p＜0.001

## and ethanol+L-glutamine group compares: p＜0.001, ++ compare with ethanol+arginine group: p＜0.001.

Glutamine, arginine are to mice serum alanine aminotransferase (ALT, influence U/L)

Like Fig. 5, the result shows that behind the ethanol feed, Serum ALT levels obviously raises.Give 100mg/kg glutamine and 100mg/kg arginine respectively and can significantly reduce the ALT rising that ethanol causes, glutamine can more obviously reduce the ALT rising that ethanol causes with the arginine Combined application than independent use.By above-mentioned (SAMe part) method; Measured the influence of glutamine, arginine to liver mitochondrion GSH content; And be index with thiobarbituric acid reaction thing (TBARS) content, observed the influence of the liver lipid peroxidation that glutamine and arginine cause ethanol.Mitochondrion GSH measures kit method with GSH and measures.TBARS presses literature method and absorbs the content of ripple mensuration TBARS in hepatic tissue at 532nm.The result is following.

^{※ ※}Compare with matched group: p＜0.001, ^△Compare with the ethanol group: p＜0.05, ^{△ △}P＜0.001,

⁺⁺Compare with ethanol+arginine group: p＜0.001, ^##Compare with ethanol+L-glutamine group: p＜0.001.

Like Fig. 6, the result shows, behind the ethanol feed; The GSH level obviously reduces in the mouse liver; Liver inner lipid peroxidating product improves greatly, gives glutamine and the arginine GSH content in the hepatic mitochondria that all can partly raise respectively, reduces liver inner lipid peroxidating product growing amount.Glutamine and arginine are united use almost can make the interior GSH of hepatic mitochondria be raised to normal range, and has reduced the liver inner lipid peroxide content that alcohol oxidation produces than single more significantly with glutamine or arginine.

3, protection of glycine and liver oxidative damage and treatment

Glycine is a kind of human body non essential amino acid, participates in the biochemical adjusting of the multiple important physical of human body.For example glycine involved in sugar metabolism, protein synthesis, purine are synthetic, bile acid association reaction etc.Glycine can be stablized macrophage and stellate cells (Kupffer cell) and suppress TNF-α release through suppressing flow of calcium ions.Glycine can prevent and alleviate law during ischemia damage, alcoholic liver injury, fatty liver, chemicals liver damage well.In addition, have report to point out, glycine can prevent hepatocarcinoma to take place and some melanoma is had the good curing effect.Other there are some researches show that glycine can be through anti-histanoxia, and prevention is exhausted ATP and protect renal proximal tubules and hepatocyte effectively that glycine can also be through promoting that microcirculation alleviate the liver reperfusion injury in the liver.

The main pathological characters of fatty liver is hepatic cell fattydegeneration and necrosis; Wherein liver inner stellate cell (Kupffer cell) produces the cytokine that discharges, and for example tumor necrosis factor (TNF-α) plays a major role, vivo oxidation reducing condition disequilibrium; When producing excessive free radicals; (comprising ROS, NOS etc.), level of endotoxin in the blood (for example LPS) obviously raises, and activates Kupffer cell release TNF-α in the liver.Recently research shows; Glycine protection hepatocyte, the mechanism that stops the Kupffer cell to discharge TNF-α are that glycine can activate the chloride channel (Glycine-gated Chloride Channel) of glycine mediation in the open Kupffer cell membrane; Increase stream (raising 2～3 times) in the chloride ion; Cell membrane polarity is raise, stablized the Kupffer cell membrane, the Ca++ ion channel can't be opened; Thereby blocked the synthetic and secretion (endotoxin activates flow of calcium ions release TNF-α mainly through stimulating the Kupffer cell membrane) of TNF-α that flow of calcium ions causes.

Experiment five, glycine generate and the influence that discharges transaminase in the inductive rat body of lipopolysaccharide (LPS) and tumor necrosis factor TNF-alpha

Be the influence that secretion discharges to TNF-α of research glycine, we have observed glycine TNF-α in the inductive rat body of lipopolysaccharide (LPS) have been generated and the influence that discharges.

The normal male rat, body weight 200～220g establishes 3 groups, and 10 of every treated animals are divided into normal control group, 60mg/kg body weight glycine group, 120mg/kg body weight glycine group.Normal rats lumbar injection every day equal-volume normal saline 1 time; The different metering of glycine rat lumbar injection every day glycine 1 time, successive administration 14 days, rat tail vein injection lipopolysaccharide (LPS) 2mg/kg body weight after 14 days; After 120 minutes; Get blood, separation of serum is measured alanine aminotransferase (ALT) and TNF-alpha content in the rat serum.The result is following.

^{※ ※}Compare with matched group: p＜0.001, ^##Compare with 60mg/kg glycine group: p＜0.001

Like Fig. 7, The above results shows, generates and release but glycine highly significant ground reduces the inductive TNF-α of LPS.It is more obvious that 120mg/kg glycine administration group TNF-α discharges inhibitory action.Glycine also raises to the inductive intensive transaminase of LPS has stronger protective effect.

4, the establishment of efficient natural antioxidation synthetic prescription and to the joint synergy of the protection of hepatic injury treatment

According to above lot of experiments result and the progress that obtains at this area research in the world, we established through repeatedly optimize the compatibility experiment screening, comprehensive, multipath protects the liver the antioxidation prescription.This prescription is according to the molecular pathology principle of chemical liver injury and fatty liver incidence and development, or multiple blocking oxide attacks or multiple active antioxidation biology precursor is provided, or promotes the rapid drainage of nuisance.We with animal experimental observation combination and cooperation protective effect and the mechanism thereof of antioxidation synthetic prescription to hepatic injury.

Comprehensive Experiment six, efficient natural antioxidation synthetic prescription are treated joint synergy and mechanism research to the protection of hepatic injury

Normal male mice body weight 18～22g is divided into 3 groups, and 12 of every animals are divided into normal control group, ethanol feed group, synthetic prescription group+ethanol feed group.Ethanol group mice feed every day ethanol 1 time; The 6g/kg body weight; Synthetic prescription group+ethanol feed group; At feed ethanol preceding 1 hour, feed 0.15mL synthetic prescription (glycine 60mg/kg, L-arginine 50mg/kg, glutamine 60mg/kg, L-cysteine 70mg/kg, S-adenosylmethionine 35mg/kg, alpha-lipoic acid 35mg/kg).Feed ethanol 6g/kg then, normal group gives equal-volume (0.15mL) normal saline, gives ethanol or 2 weeks of medicine+ethanol continuously.Get blood, separation of serum is subsequent use.Method for preparing mitochondrion and liver tissue homogenate measure GSH content and liver tissue homogenate's MDA (TBARS) in the mitochondrion.The result is following:

^{※ ※}Compare with the normal control group: p＜0.001, ^{△ △}Compare with the ethanol group: p＜0.001, ^△P＜0.05

Like Fig. 8, The above results shows that synthetic prescription has the anti-oxidation protection liver effect of highly significant.The ethanol feed is after 2 weeks; Mouse Liver mitochondrion GSH content extremely reduces; MDA growing amount (TBARS) significantly raises (10 times), and ALT also significantly raises, and ethanol+2 week of synthetic prescription group back mitochondrion GSH and normal control group content are similar; The TBARS growing amount is a little more than normal control, and ALT also only slightly raises.

We have also further observed synthetic prescription TNF-α in the inductive rat body of lipopolysaccharide (LPS) have been generated and the influence that discharges.Normal male SD rat, body weight 200～220g is divided into 2 groups; Matched group and synthetic prescription group; 12 of every treated animals, control rats lumbar injection every day equal-volume normal saline 1 time (0.2mL), the synthetic prescription group is injected the 0.2mL prescription drug every day; Prescription element dosage identical with above-mentioned experiment (cysteine, L-arginine, glutamine, glycine, S-adenosylmethionine, alpha-lipoic acid), successive administration is 14 days altogether.Rat tail vein injection lipopolysaccharide (LPS) 2mg/kg body weight was got blood after 120 minutes after 14 days, and separation of serum is measured rat blood serum ALT and TNF-alpha content and changed.The result is following:

^{※ ※}Compare with matched group: P＜0.001

Like Fig. 9, synthetic prescription has extremely significantly inhibitory action to the inductive rat TNF-of LPS α, the LPS control rats ALT that can significantly raise, and synthetic prescription group ALT only slightly raises.Results suggest: synthetic prescription has extremely strong combination and cooperation protective effect to the hepatic injury of endotaxin induction.

The result of study of the The Molecular Biology Mechanism of above-mentioned prevention and treatment to chemical liver injury and fatty liver and to multiple efficient natural active antioxidation active ingredient carry out pharmacology, toxicology, molecular biology experiment result of study and show; Stoping or prophylactic activity oxygen-derived free radicals (ROS) is attacked of our research design from multiple hepatocyte metabolic pathway, multidigit point; Blocking-up α-Zhong Liuhuaisiyinzi (TNF-α) brings out and discharges; What prevention chemical liver poisoming liver-protective natural anti-oxidation polymolecular synthetic prescription had a highly significant improves the hepatocyte oxidation resistance; Can stabilizing cell membrane, significantly suppress or blocking-up α-Zhong Liuhuaisiyinzi (TNF-α) discharges; The hepatic injury that the inside and outside chemical toxicant of opposed body causes, synthetic prescription has extremely strong combination and cooperation protective effect to hepatic injury.

Clinical efficacy of the present invention and security ststem are observed and the result of study data

(A) diagnostic criteria of fatty liver:

1, ultrasonic Diagnosis of Fatty Liver standard: the high echo of (1) near field, hepatic region diffusivity point-like.Echo intensity is higher than spleen and kidney, and minority shows as the high echo of kitchen range property; (2) far field echo attenuation, luminous point is sparse; (3) the liver flexible innerduct structure shows unclear; (4) the slight or moderate swelling of liver, the margo anterior hepatis rust.Only possesses (1) item person as doubtful diagnosis; Possessing (1) adds all the other 1 above persons and can be diagnosed as fatty liver.

2, fatty liver deciding degree standard: (Fan Jiangao, Ceng Minde, the chief editor, fatty liver, publishing house of Shanghai Medical Univ, 2000,115-188)

(1) slight: luminous point is fine and closely woven, and near field echo strengthens, and far field echo is slightly decayed, blood vessel, and blood vessel structure is clear.

(2) moderate: luminous point is fine and closely woven, and front court echo strengthens, and far field echo is obviously decayed, and blood vessel structure is unclear.

(3) severe: luminous point is fine and closely woven, and front court echo significantly strengthens, and far field echo is significantly decayed, and blood vessel structure can not be recognized.

3, non-alcoholic fatty liver disease diagnostic criteria

Non-alcoholic fatty liver disease is a kind of there not to be excessive history of drinking history; The hepatic parenchymal cells steatosis is stored up the clinical pathology syndrome for characteristic with fat. and disease differs with the progress performance of the course of disease; Comprise that simple fatty liver, fat hepatitis are (except that above-mentioned simple fatty liver Radiologic imaging; Liver parenchyma density and signal change can occur, spleen thickens or enlargement, thickening of capsule wall of gallbladder or the change of gallbladder form etc.), fatty liver fiber and liver cirrhosis.

All possess following 1-5 item and the 6th or the 7th each person can be diagnosed as non-alcoholic fatty liver disease.

1. easy trouble factor is arranged, like obesity, type 2 diabetes mellitus, hyperlipemia and women etc.;

2. do not have history of drinking history or drink amount to the ethanol amount weekly less than 40 the gram;

3. viral hepatitis, drug induced hepatitis, Wilson disease, total parenteral nutrition and autoimmune hepatitis except;

4. outside the protopathy clinical manifestation, symptoms such as weak, pain in the hepatic region can appear, and can be with hepatosplenomegaly;

5. clear transaminase can raise, and is main with ALT, can increase with γ-GT, ferritin and uric acid etc.;

6. the ultrasonic diagnosis foundation is arranged.

The simple fatty liver diagnostic criteria: (1) liver function test is normal basically; (2) ultrasonics performances meets light, moderate, severe fatty liver.

The non-alcoholic fatty liver disease hepatitis diagnostic criteria: (1) Serum ALT with (or) γ-GT is higher than 1.5 times of the normal value upper limit, the persistent period is greater than 4 weeks (2) ultrasonics diagnosis basis.

4, alcoholic fatty liver diagnostic criteria

The alcoholic fatty liver diagnostic criteria: surpass male's ethanol intake regular every day 40g, women's ethanol intake surpasses 20-30g regular every day.Perhaps once property or repeatedly super amount history of drinking history (can directly cause acute alcoholism and grievous injury liver, human body vitals such as kidney) in the recent period.Symptoms such as weak, pain in the hepatic region can appear, and can be with hepatosplenomegaly; Usually follow serum transaminase to raise, and be main, can increase with γ-GT, ferritin and uric acid etc. with AST, ALT; The imaging diagnosis foundation is arranged.

(B) materials and methods:

1, object of study and grouping

This project from Qingdao City endocrine diabetes hospital be in hospital, outpatient service prescription on individual diagnosis patient and the Physical Examination crowd; Select through colored multispectral Diagnosis of Fatty patient 862 examples (male 558 examples of reining in; Woman's 304 examples) 52 ± 19 years old mean age (40-71 year) is divided into 2 groups with reference to " Chinese Medical Association's hepatology branch alcoholic fatty liver and non-alcoholic fatty liver disease diagnostic criteria " with 862 routine Patients with Fatty Liver: (1) alcoholic fatty liver group (290 example), (2) non-alcoholic fatty liver disease group (572 example).

2, observation item

All fatty liver experimenters (treatment group and matched group) all are responsible for investigating through the medical speciality technical staff of special training, inquiry and record, age, sex, occupation, drink, diabetes, hypertension and history of medications etc.All accomplish that following physical examinations and laboratory and colour are multispectral reins in inspection.

(1) clinical examination

With desk-top scale measure the examinee height (centimetre, cm), on an empty stomach body weight (kilogram, kg), the examinee should take off one's shoes, raise one's hat, unlined garment during measurement, unlined trousers is measured.With tape measure waistline and hip circumference (centimetre, cm).The measurement of waistline directly is as the criterion with intermarginal Xiao Zhou on costal margin and the crista iliaca, and before the measurement of hip circumference through pubic symphysis, both sides through greater trochanter of femur, after the most outstanding position of buttocks is a standard, calculate Body Mass Index (BMI)=body weight (Kg)/height (m) ², waist-to-hipratio (WH)=waistline (cm)/hip circumference (cm).The measuring method of blood pressure (BP) adopts national hypertension sampling survey unified standard, uses hydrargyrum to lean on the formula sphygomanometer without exception.The examinee need sit quietly before measurement and have a rest 5 minutes, gets seat then, measures the right upper extremity blood pressure.Systolic pressure (SBP) is as the criterion to hear the 1st phase hear sounds, and diastolic pressure (DBP) is as the criterion with the 5th phase disappearance sound.2 minutes at interval, its meansigma methods was got in continuous measurement twice.Weighing machine, sphygomanometer are all through overcorrect before measuring.More than operation is special messenger's special plane and carries out.

(2) lab testing

The fasting all overnight of full-fledged research object is 12 hours before the treatment; Draw blood inferior everyday early morning on an empty stomach; Adopt the full-automatic biochemical instrument, survey fasting glucose (FBG), cholesterol (TC), triglyceride (TG), HDL-C (HDL-C), low-density lipoprotein cholesterol (LDL-C); Gamma glutamyl transpeptidase (GTT), glutamate pyruvate transaminase (AST), glutamic oxaloacetic transaminase, GOT (ALT), blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA) etc.Adopt U.S. Johnson & Johnson full-automatic dry chemistry appearance to measure tumor necrosis factor (TNF-α), C-reactive protein (CRP).

(3) liver color Doppler inspection

Liver color ultra use U.S. PHILIPS M2540A type CDFI appearance, frequency 10MHz, fixedly special messenger's operation.Experimenter's fasting overnight 12 hours, early morning on the same day, the clinostatism of making even, transverse scan combines with vertical disconnected scanning, the clear liver structure that shows.

All object of study all began to take prescription of the present invention (use relevant pharmaceutic adjuvant to process tablet with above-mentioned efficient synthetic prescription, tablet technology details in the lower part), 0.6g/ sheet, 4/inferior, 3 times/day, continuous use 8-10 week the same day.The back 4-5 and 8-10 week check These parameters and lab testing respectively that takes medicine, observe and the record patient to drug reaction etc.

3, therapeutic evaluation standard

Observation index and therapeutic evaluation standard: extract venous blood on an empty stomach before and after the treatment respectively; Measure fasting glucose (FBG), cholesterol (TC), triglyceride (TG), HDL-C (HDL-C), low-density lipoprotein cholesterol (LDL-C); Gamma glutamyl transpeptidase (GTT), glutamate pyruvate transaminase (AST), glutamic oxaloacetic transaminase, GOT (ALT); Blood urea nitrogen (BUN), creatinine (Cr), uric acid (UA), tumor necrosis factor (TNF-α), C-reactive protein (CRP) are measured blood pressure, the inspection of row liver color Doppler; Undertaken by same position doctor before and after the treatment, the paired observation ultrasonography of liver changes.

Cure: symptom, sign disappear basically, and it is normal that liver function each item index is recovered, and ultra liver ultrasonogram recovers normal.

Produce effects: symptom, sign are obviously improved; Liver function recovery is normal or descend more than 50%; Ultrasonic liver image pattern obviously improves (1 grade descends), and blood fat, uric acid (UA), tumor necrosis factor each item indexs such as (TNF-α) have at least more than 1 to be recovered normal or descend more than 50%

Effectively: healing and produce effects person all are judged to be effectively;

Invalid: symptom, sign do not have obvious improvement, and liver function, blood fat each item index descend below 20%, and B ultrasonic liver ultrasonogram does not have significant change.

(3) interpretation of result and statistical procedures

Statistical analysis adopts the spss11.5 software kit to carry out, and group difference adopts the t check, and a plurality of sample averages are relatively used variance analysis; In addition; By fatty liver light and heavy degree sorting technique the fat hepatopath is divided into: slight fatty liver group (220 example), moderate fatty liver group (386 example), severe fatty liver group (256 example); So that further analyze the typical Pathophysiology characteristic of summing up all kinds of fatty livers, the clinical effectiveness of different reason fatty livers of the clear extract for treating of assessment U.S.-China and different light and heavy degree fatty livers.

(4) result

We have observed 862 examples and have suffered from the Patients with Fatty Liver that different reasons cause; Use of the influence of efficient natural antioxidation polymolecular compound prescription, so that relatively efficient natural antioxidation polymolecular compound prescription U.S.-China is clear plain to dissimilar, the clinical efficacy of fatty liver in various degree to clinical indices such as fatty liver, blood glucose, blood fat, T-CHOL, uric acid, liver function, renal functioies.

1, use the present invention to treat front and back each item index relatively

The clinical physical data influence of Patients with Fatty Liver (table 1A)

Project	Before the treatment	After the treatment	The P value
				Example number (man/woman)	862(558/304)	862(558/304)	?
Age	52.10±12.19	52.10±12.19	?
				Height (m)	1.68±0.87	1.68±0.87	?
Body weight (Kg)	78.38±14.09	78.05±13.28	0.56
				Body Mass Index (BMI)	27.75±3.51	27.65±3.27	0.87
Waistline (cm)	94.86±7.73	94.51±6.88	0.70
				Hip circumference (cm)	101.28±5.57	100.95±5.38	0.94
Waist/buttocks	0.94±0.06	0.94±0.05	0.825
				Systolic pressure (mmHg)	130.31±15.56	129.60±14.86	0.410
Diastolic pressure (mmHg)	83.73±7.60	83.15±7.42	0.447
				Body fat content (%)	34.52±3.83	32.45±3.47	0.56

After the treatment, body weight, BMI, waistline, hip circumference descend before the treatment to some extent, but significant difference is not remarkable, and body fat content obviously descends.

To the metabolic influence of Patients with Fatty Liver (table 1B)

Project	Before the treatment	After the treatment	The P value
				FBG(mmol/L)	6.35±2.02	6.24±1.71	0.583
TC(mmol/L)	5.77±1.41	5.36±1.54	0.066
				TG(mmol/L)	2.62±2.27	1.37±0.61	0.000*
LDL(mmol/L)	3.45±1.63	3.51±1.48	0.38
				HDL(mmol/L)	1.13±0.48	1.22±0.46	0.365

Influence (table 1C) to the Patients with Fatty Liver liver function

Project	Before the treatment	After the treatment	The P value
				GGT(U/L)	54.66±21.40	34.40±9.81	0.000*
ALT(U/L)	54.53±24.05	33.19±10.33	0.000*
				AST(U/L)	51.08±27.72	26.15±9.32	0.000*

[0154]?

Influence (table 1D) to renal function

Project	Before the treatment	After the treatment	The P value
				BUN(mmol/L)	5.19±1.51	5.17±1.25	0.620
Cr(umol/L)	74.80±13.88	75.45±17.10	0.352
				UA(umol/L)	364.2±86.18	358.1±62.62	0.368

Influence (table 1E) to the special inflammatory cytokine of Patients with Fatty Liver

Project	Before the treatment	After the treatment	The P value
				TNF(pg/ml)	17.82±6.84	9.58±3.17	0.000*
CRP(mg/L)	3.12±0.91	2.42±0.68	0.000*

Above-mentioned statistical result shows that the treatment group is compared before and after using the present invention, and body fat content, TG, GGT, ALT, AST all obviously descend, and tumor necrosis factor, CRP level reduce significantly, and remarkable significant difference is arranged before and after the treatment.

2, the present invention is to the influence and the efficacy analysis of Patients with Fatty Liver in various degree

According to the ultrasonic fatty liver deciding degree of coloured silk standard, this organizes slight Patients with Fatty Liver 220 examples (man/woman 131/89); Moderate 386 examples (man/woman 221/165); Severe 256 (man/woman 206/50).Statistical analysis compound prescription of the present invention is to the influence and the efficacy analysis of fatty liver in various degree.

(1) to the influence of slight fatty liver

The variation of physical data (table 2A) before and after the slight fatty liver treatment

Project

Before the treatment

After the treatment

The P value

[0166]

Example number (man/woman)	220 (131/89)	220 (131/89)
				Age	51.90 ± 12.83	51.90 ± 12.83
Height (m)	1.65 ± 0.10	1.65 ± 0.10
				Body weight (Kg)	73.01 ± 13.22	73.06 ± 12.78	0.834
Body Mass Index (BMI)	26.65 ± 3.58	26.67 ± 3.44	0.747
				Waistline (cm)	89.90 ± 7.83	90.19 ± 7.03	0.432
Hip circumference (cm)	100.50 ± 4.66	100.30 ± 4.52	0.522
				Waist-to-hipratio	0.89 ± 0.06	0.90 ± 0.05	0.138
Systolic pressure (mmHg)	129.75 ± 11.75	130.00 ± 14.33	0.895
				Diastolic pressure (mmHg)	83.00 ± 7.33	82.00 ± 8.34	0.560
Body fat content (%)	34.80 ± 4.51	34.11 ± 3.46	0.67

To the metabolic influence of slight Patients with Fatty Liver (table 2B)

Project	Before the treatment	After the treatment	The P value
				FBG(mmol/L)	5.99±1.56	6.16±1.18	0.543
TC(mmol/L)	6.30±1.52	5.95±1.62	0.362
				TG(mmol/L)	2.20±1.36	1.52±0.74	0.023
LDL(mmol/L)	4.17±1.52	4.03±1.52	0.710
				HDL(mmol/L)	1.13±0.45	1.23±0.49	0.226

Variation (table 2C) to slight Patients with Fatty Liver treatment back liver function

Project	Before the treatment	After the treatment	The P value
				GGT(U/L)	41.25±12.37	36.95±11.17	0.026
ALT(U/L)	33.30±11.59	30.65±7.45	0.206
				AST(U/L)	36.75±12.11	29.45±8.03	0.001

Renal function before and after the slight Patients with Fatty Liver treatment is changed (table 2D)

Project	Before the treatment	After the treatment	The P value
				BUN(mmol/L)	5.35±1.91	5.31±1.60	0.753
Cr(umol/L)	73.22±13.96	83.03±24.40	0.155
				UA(umol/L)	366.12±78.93	359.49±55.04	0.641

Influence (table 2E) to the slight Patients with Fatty Liver special cells factor

Project	Before the treatment	After the treatment	The P value
				TNF(pg/ml)	13.89±5.86	8.85±3.30	0.000

Above-mentioned statistical result shows that slight fatty liver group is used before and after the present invention, and TG, GGT, AST, body fat content all are clearly better or reduce, and the tumor necrosis factor level obviously reduces, and remarkable significant difference is relatively arranged before and after the treatment.

(2) the present invention is to the influence of moderate fatty liver

Influence (table 3A) to the general clinical data of moderate fatty liver

Project	Before the treatment	After the treatment	The P value
				Example number (man/woman)	386(221/165)	386(221/165)	?
Age	52.10±14.24	52.10±14.24	?
				Height (m)	1.69±0.08	1.69±0.08	?
Body weight (Kg)	79.85±13.83	79.57±13.18	0.279
				Body Mass Index (BMI)	27.92±3.88	27.80±3.64	0.314
Waistline (cm)	96.81±5.78	96.63±5.35	0.485
				Hip circumference (cm)	101.22±5.90	101.07±5.65	0.351
Waist-to-hipratio	0.96±0.04	0.96±0.04	0.884
				Systolic pressure (mmHg)	131.10±16.77	130.00±14.92	0.480
Diastolic pressure (mmHg)	83.52±7.37	84.52±7.05	0.401
				Body fat content (%)	34.29±3.52	32.76±3.65	0.067

To the metabolic influence of moderate fatty liver (table 3B)

Project	Before the treatment	After the treatment	The P value
				FBG(mmol/L)	6.77±2.22	6.14±1.44	0.066
TC(mmol/L)	5.46±1.00	5.04±1.34	0.325
				TG(mmol/L)	3.03±3.44	1.27±0.59	0.028
LDL(mmol/L)	2.89±1.82	3.14±1.34	0.639
				HDL(mmol/L)	1.19±0.50	1.32±0.47	0.124

Influence (table 3C) to moderate Patients with Fatty Liver liver function

Project	Before the treatment	After the treatment	The P value
				GGT(U/L)	52.86±19.86	33.62±8.47	0.000*
ALT(U/L)	55.59±14.36	33.95±12.30	0.000*
				AST(U/L)	48.05±17.80	24.86±11.06	0.000*

[0184]?

Influence (table 3D) to moderate fatty liver renal function

Project	Before the treatment	After the treatment	The P value
				BUN(mmol/L)	4.90±1.13	5.07±1.06	0.374
Cr(umol/L)	77.65±13.49	73.97±10.30	0.166
				UA(umol/L)	351.50±91.98	358.38±65.71	0.674

Influence (table 3E) to the special inflammatory cytokine of moderate fatty liver

Project	Before the treatment	After the treatment	The P value
				TNF(pg/ml)	18.03±5.95	9.80±3.44	0.000*

Above-mentioned statistical result shows; Moderate fatty liver group is used before and after the present invention, and body fat content, TG, GGT, ALT, AST all are clearly better or reduce, and the tumor necrosis factor level obviously reduces; Nearly 1 times of reduction amplitude relatively has remarkable significant difference before and after the treatment.

(3) to the influence of severe Patients with Fatty Liver

Influence (table 4A) to the general clinical data of severe Patients with Fatty Liver

Project	Before the treatment	After the treatment	The P value
				Example number (man/woman)	256(206/50)	256(206/50)	?
Age	52.29±9.70	52.29±9.70	?
				Height (m)	1.69±0.09	1.69±0.09	?
Body weight (Kg)	82.03±14.25	81.29±13.07	0.62
				Body Mass Index (BMI)	28.64±2.89	28.41±2.55	0.79
Waistline (cm)	97.64±7.35	96.49±6.42	0.2
				Hip circumference (cm)	102.07±6.16	101.47±6.01	0.196
Waist-to-hipratio	0.96±0.06	0.95±0.05	0.326
				Systolic pressure (mmHg)	130.05±18.03	128.81±15.96	0.242
Diastolic pressure (mmHg)	84.62±8.34	82.80±6.99	0.102
				Body fat content (%)	34.40±3.60	33.50±3.34	0.12

To the metabolic influence of severe Patients with Fatty Liver (table 4B)

Project	Before the treatment	After the treatment	The P value
				FBG(mmol/L)	6.29±2.23	6.42±2.32	0.752

[0195]?

TC(mmol/L)	5.56±1.56	5.11±1.55	0.223
				TG(mmol/L)	2.61±1.35	1.32±0.47	0.000
LDL(mmol/L)	3.31±1.32	3.39±1.51	0.842
				HDL(mmol/L)	1.07±0.49	1.12±0.41	0.594

Influence (table 4C) to severe Patients with Fatty Liver liver function

Project	Before the treatment	After the treatment	The P value
				GGT(U/L)	69.24±21.24	32.76±9.67	0.000*
ALT(U/L)	73.71±24.33	34.86±10.57	0.000*
				AST(U/L)	67.76±37.14	24.29±8.08	0.000*

Influence (table 4D) to the severe fatty liver renal function

Project	Before the treatment	After the treatment	The P value
				BUN(mmol/L)	5.33±1.44	5.15±1.10	0.390
Cr(umol/L)	73.46±14.41	69.70±11.19	0.267
				UA(umol/L)	375.1±89.30	356.7±69.00	0.134

Influence (table 4E) to the special inflammatory factor of severe Patients with Fatty Liver

Project	Before the treatment	After the treatment	The P value
				TNF(pg/ml)	21.35±6.79	10.08±2.76	0.000*
CRP(mg/L)	7.12±1.91	3.42±0.68	0.000*

Above-mentioned statistical result shows that the severe fatty liver group is used before and after the present invention, and waistline, body fat content, TG, GGT, ALT, AST all obviously improve or reduce, and the tumor necrosis factor level obviously reduces, and remarkable significant difference is relatively arranged before and after the treatment.

3, the present invention is to the influence and the efficacy analysis of alcoholic fatty liver

Influence (table 5A) to the general clinical data of alcohol fatty hepatopath

Project

Before the treatment

After the treatment

The P value

[0208]

Example number (man/woman)	290 (251/39)	290 (251/39)
				Age	50.34 ± 9.04	50.34 ± 9.04
Height (m)	1.71 ± 0.67	1.71 ± 0.67
				Body weight (Kg)	81.84 ± 12.87	81.33 ± 11.97	0.68
Body Mass Index (BMI)	27.71 ± 3.18	27.55 ± 2.94	0.98
				Waistline (cm)	96.28 ± 7.69	95.92 ± 6.60	0.283
Hip circumference (cm)	100.788 ± 5.08	100.70 ± 4.89	0.498
				Waist-to-hipratio	0.96 ± 0.06	0.95 ± 0.05	0.375
Systolic pressure (mmHg)	127.52 ± 13.22	126.55 ± 12.89	0.527
				Diastolic pressure (mmHg)	84.48 ± 8.06	83.45 ± 8.67	0.424
Body fat content (%)	35.80 ± 3.35	33.53 ± 2.92	0.062

To the metabolic influence of alcohol fatty hepatopath (table 5B)

Project	Before the treatment	After the treatment	The P value
				FBG(mmol/L)	6.36±2.08	6.53±1.85	0.606
TC(mmol/L)	5.48±1.45	4.97±1.53	0.095
				TG(mmol/L)	2.49±1.54	1.67±0.69	0.001
LDL(mmol/L)	3.28±1.53	3.12±1.43	0.648
				HDL(mmol/L)	1.07±0.42	1.17±0.48	0.224

Influence (table 5C) to alcoholic fatty liver patients with hepatic function

Project	Before the treatment	After the treatment	The P value
				GGT(U/L)	60.59±24.57	35.38±8.29	0.000
ALT(U/L)	59.24±27.44	34.31±11.70	0.000
				AST(U/L)	61.48±31.40	27.62±8.02	0.000

Influence (table 5D) to alcohol fatty hepatopath renal function

Project	Before the treatment	After the treatment	The P value
				BUN(mmol/L)	5.25±1.33	5.05±0.98	0.160
Cr(umol/L)	74.68±12.30	76.20±20.97	0.753
				UA(umol/L)	397.98±82.46	378.64±58.37	0.141

Influence (table 5E) to the special inflammatory factor of alcohol fatty hepatopath

Project	Before the treatment	After the treatment	The P value
				TNF(pg/ml)	20.44±5.43	10.02±3.15	0.000

Above-mentioned statistical result shows that the alcoholic fatty liver group is used before and after the present invention, and body fat content, TG, GGT, ALT, AST all are clearly better or significantly reduce, and the tumor necrosis factor level obviously reduces, and remarkable significant difference is relatively arranged before and after the treatment.

4, the present invention is to the influence and the efficacy analysis of non-alcoholic fatty liver disease

Influence (table 6A) to the general clinical data of non-alcoholic fatty liver disease patient

Project	Before the treatment	After the treatment	The P value
				Example number (man/woman)	572(268/304)	572(268/304)	?
Age	53.64±14.37	53.64±14.37	?
				Height (m)	1.64±0.09	1.64±0.09	?
Body weight (Kg)	75.34±14.58	75.17±13.88	0.431
				Body Mass Index (BMI)	27.79±3.83	27.73±3.58	0.470
Waistline (cm)	93.62±7.67	94.26±6.98	0.130
				Hip circumference (cm)	101.71±6.01	101.18±5.84	0.125
Waist-to-hipratio	0.92±0.06	0.92±0.05	0.739
				Systolic pressure (mmHg)	132.76±17.18	132.27±16.11	0.607
Diastolic pressure (mmHg)	83.06±7.24	83.88±6.25	0.841
				Body fat content (%)	33.39±3.92	32.50±3.68	0.162

To the metabolic influence of non-alcoholic fatty liver disease patient (table 6B)

Project	Before the treatment	After the treatment	The P value
				FBG(mmol/L)	6.35±2.00	5.99±1.55	0.142
TC(mmol/L)	6.01±1.35	5.70±1.48	0.334
				TG(mmol/L)	2.73±2.78	1.28±0.52	0.004*
LDL(mmol/L)	3.59±1.73	3.85±1.47	0.495
				HDL(mmol/L)	1.17±0.52	1.26±0.44	0.154

Influence (table 6C) to non-alcoholic fatty liver disease patients with hepatic function

Project	Before the treatment	After the treatment	The P value
				GGT(U/L)	49.45±16.89	33.54±11.04	0.000*
ALT(U/L)	50.38±20.15	32.21±9.03	0.000*
				AST(U/L)	41.94±20.44	24.85±10.28	0.000*

[0226]?

Influence (table 6D) to non-alcoholic fatty liver disease patient renal function

Project	Before the treatment	After the treatment	The P value
				BUN(mmol/L)	5.14±1.67	5.28±1.46	0.310
Cr(umol/L)	74.91±15.33	74.78±13.09	0.962
				UA(umol/L)	334.5±79.17	340.1±61.48	0.582

Non-alcoholic fatty liver disease is used the present invention and is treated front and back specific factor relatively (table 6E)

Project	Before the treatment	After the treatment	The P value
				TNF(pg/ml)	15.52±7.19	9.20±3.19	0.000*

Use before and after the present invention, average body fat content, TC, TG, GGT, ALT, AST, TNF all have significant difference.Average body fat content descends 1.07%, TG decline 1.25mmol/L, GGT decline 20.26mmol/L, ALT decline 21.33mmol/L, AST decline 24.94mmol/l, comparing difference highly significant before and after the TNF-α decline 8.24pg/mL treatment.

5, the present invention treats fatty liver clinical efficacy overall merit

1, treatment group is used before and after the present invention, and body fat content, T-CHOL TC, triglyceride TG, gamma glutamyl transpeptidase (GTT), glutamate pyruvate transaminase (AST), glutamic oxaloacetic transaminase, GOT (ALT), TNF all have significant difference.Wherein body fat content descends 1.07%, TG decline 1.25mmol/L, GGT decline 20.26mmol/L, ALT decline 21.33mmol/L, AST decline 24.94mmol/l, TNF-α decline 8.24pg/mL.

2, with the treatment group according to fatty liver light in severe classification back row statistical analysis, the result shows that each item index was all obviously improved or reduced significantly before and after slight fatty liver group was used the present invention, TG, GGT, AST, TNF difference have statistical significance.Moderate fatty liver group is with before and after the present invention, and body fat content, TG, GGT, ALT, AST, TNF obviously improve or reduce, and remarkable significant difference is relatively arranged before and after the treatment.The severe fatty liver group is with before and after the present invention, and waistline, body fat content, TG, GGT, ALT, AST, TNF have remarkable significant difference.Wherein light, in, severe fatty liver patient TNF-α drop-out value is respectively 11.27pg/mL, 8.23pg/mL, 5.04pg/mL.

3, no matter carry out statistical analysis after the treatment group is divided into groups according to alcoholic fatty liver and non-alcoholic fatty liver disease, be ethanol property and non-alcoholic fatty liver disease, and before and after the treatment, body fat content, TG, GGT, ALT, AST, TNF difference have statistical significance.Alcoholic fatty liver group each item index all descends significantly than the non-alcoholic fatty liver disease group.Wherein, non-alcoholic fatty liver disease group TNF-α decline 6.32pg/mL, alcoholic fatty liver group decline 10.42pg/ml.

The present invention to clinical comprehensive therapeutic effect dissimilar and fatty liver in various degree with estimate as follows:

Claims (1)

1. composite biological agent with remarkable chemical liver injury defencive function, it is characterized in that: the raw material by following part by weight is formed:

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