The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1 test material and method
1.1 trial drug
Chinese medicine Penthorum chinense 900g is purchased from Gulin Penthorum chinense cooperative society, and Radix Salviae Miltiorrhizae 900g, Herba Sedi 800g, Fructus Crataegi 800g, Rhizoma Polygoni Cuspidati 750g, Radix Curcumae 650g, Radix Glycyrrhizae 500g are purchased from Yaan Huimin hall pharmacy, and after being accredited as certified products medical material, 60 orders are pulverized respectively, pack standby.
Get above 7 tastes, wherein Penthorum chinense adds 6 times of amounts (ml/g, lower with) 65%(V/V) alcohol reflux 2 times, each 1.5 hours, merge extractive liquid,, filtered, and filtrate recycling ethanol, is concentrated into without ethanol taste standby; Radix Salviae Miltiorrhizae adds the 70%(V/V of 6 times of amounts) alcohol reflux 2 times, each 2 hours, filter, filtrate recycling ethanol, is concentrated into without ethanol taste; Fructus Crataegi, Rhizoma Polygoni Cuspidati add the 80%(V/V of 6 times of amounts) alcohol reflux 2 times, each 1.5 hours, filter, filtrate recycling ethanol, is concentrated into without ethanol taste; All the other Herba Sedis, Radix Curcumae, Radix Glycyrrhizae, add 6 times of water gagings decoctions 2 times with Penthorum chinense, Radix Salviae Miltiorrhizae, Fructus Crataegi, Rhizoma Polygoni Cuspidati medicinal residues after ethanol extraction, each 1.5 hours, collecting decoction, and add above-mentioned Penthorum chinense, Radix Salviae Miltiorrhizae, Fructus Crataegi, Rhizoma Polygoni Cuspidati extract, be diluted with water to about 5300mL, be equivalent to every mL medicinal liquid and contain crude drug 1g, stirring and dissolving, standing 12 hours, obtains (called after Plumbum preparatium liver protection decoction) for 2 times with 747 filter-cloth filtering.
1.2 experimental animal
The meat kind chicken of laying eggs of selecting doubtful chicken fatty liver hemorrhagic syndrome natural occurrence in Sichuan Agricultural University's poultry breeding field, is no less than 700.
1.3 equipment and instrument
Electronic balance; The freezing desk centrifuge of high speed; Cryogenic refrigerator; Optical microscope; Photographing unit; Baking oven.
1.4 test method
1.4.1 the diagnosis of case and inclusive criteria
According to chicken group's kind, feed nutrition, age of onset, chicken group situation and clinical symptoms, carry out comprehensive analysis and make tentative diagnosis.In conjunction with the eliminating of cuing open inspection pathological change, Serum Indexes detection, liver coefficient, liver fat rate, abdomen fat rate, laboratory diagnosis result and other case, be diagnosed as chicken fatty liver hemorrhagic syndrome again.In chicken group, fatty liver hemorrhagic syndrome sickness rate reaches 5%, judges that this chicken group obtained fatty liver hemorrhagic syndrome.
The ill chicken of clinical symptoms is too obese, and what have surpasses 25% of normal type, and egg production reduces, and laying rate declines, and conventionally by 75%-85%, is reduced to 35%-55%, at hypogastric region, can touch abundant fatty tissue.Chicken is sudden onset often, and sick chicken spirit is depressed, poor appetite, and wattle, cockscomb fade and even are pale, and happiness is sleeping, is reluctant to walk about, and abdominal part softness is sagging.Serious somnolence, paralysis, body temperature arrives 41.5-42.8 ℃, and then cockscomb, wattle and foot turn cold, can be dead within a few hours.
Pathological change is cutd open and is examined the chicken that dies of illness, and main manifestations is that intra abdominal fat deposits in a large number, and the periphery of particularly abdominal part, and muscular stomach and glandular stomach has the thick fat of one deck to exist.In addition, heart, mesentery etc. locate also to have the Fat Distribution of volume.The obvious enlargement of liver, is light brown to yellow, and matter is crisp frangible and have greasy feeling, and liver surface often has hemorrhage speckle, and has a large amount of blood clots around.Some liver disruption, have large go out clot or blood clot in liver surface and body cavity.Gallbladder enlargement or dwindle, cyst wall is because of lipidosis thickening.Spleen matter is soft, generally loses enlargement.Muscular stomach is due to the fat parcel being deposited, and its volume is only normal l/2-1/3, and muscular stomach content is few, and Endothelium Corneum Gigeriae Galli is thin and easily peel off the attenuation of flesh layer.Kidney is enlargement slightly, and matter is crisp.Some cases also has one piece of normal hard shelled egg forming in fallopian tube.
The Preliminary Determination of the detection of physiological and biochemical index Serum Indexes, liver coefficient, liver fat rate, abdomen fat rate is compared with normal kind chicken, and difference should be all remarkable.
Liver contact microscopy is got disease chicken liver contact microscopy, in visible hepatocyte, is full of fat vacuole, in endochylema, have a large amount of lipidosiss and differ in size hemorrhage; The visible local hepatic necrosis having, fat fills the air the whole lobules of liver that distributes, and makes lobules of liver lose normal structure, similar to general fatty tissue.
1.4.2 test grouping
To make a definite diagnosis and suffer from 672 of the syndromic laying hens of chicken fatty liver hemorrhagic, be divided at random 3 groups, every group of 4 repetitions, each repeats each 56.The 1st group is morbidity matched group, the 2nd group of positive medicine matched group, and choline chloride is pressed the dosed administration of 1g/kg feedstuff; The 3rd group is Plumbum preparatium liver protection decoction group, by the concentration of 0.3mL/L, concentrates drinking-water, every day 1 time; Two test group are first observed 1 week, then successive administration treatment all drug withdrawals after 21 days, and the whole test period is 4 weeks.
1.5 General observation
4 weeks experimental periods, observe chicken group situation, search for food, drink water.
1.6 detect index
4 weeks experimental periods, when first 1 week of on-test and off-test, respectively weigh 1 time (12h on an empty stomach), record the average weight of every group of chicken, the laying rate of every day, enter to incubate rate, mortality rate, cure rate after end, and carry out 1 Serum Indexes and detect, liver coefficient, liver fat rate, abdomen fat rate measured.
1.6.1 sample collecting
Each each repeat to randomly draw 3 chickens and carry out following processing: weigh every chicken body weight, wing venous blood collection (12h fasting before blood sampling), prepares serum.After blood sampling, cut open and kill, observe and record the naked eyes variation of respectively organizing chicken viscera tissue, the complete liver of cutting of cutting open the belly, takes liver weight; Peel off abdomen fat (stomach fat and muscular stomach be fat around), abdomen fat is weighed.
1.6.2 sample determination
Mensuration liver coefficient cuts open and kills after chicken, by the complete taking-up of liver, calculates liver coefficient.Liver coefficient=liver weight (g)/body weight (g) * l00.
The mensuration of Biochemical Indices In Serum with disposable syringe on an empty stomach wing venous blood collection 2~5ml in centrifuge tube, slant setting 30min at room temperature, the centrifugal 10min of 3500r/min, serum is placed on-20 ℃ of preservations.According to the explanation of diagnostic test box, measure the content of T-CHOL (TC), triglyceride (TG), glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) in serum.
The mensuration of liver fat rate
(1) liver of preserving under low temperature is thawed under room temperature.
(2) liver being put into the baking oven of 105 ℃ dries to constant weight; Fat-free filter paper is dried to constant weight in 105 ℃ of baking ovens simultaneously, with electronic balance, weigh up its weight (over dry filter paper weight).
(3) after sample is taken out from baking oven grinding, get 2g and wrap in filter paper, labelling is good, again puts into baking oven and dries to constant weight, and weighs up the accurate weight (sample Bao Chong before defat) of each filter paper packet on electronic balance.
(4) filter paper packet is put into exsiccator and be cooled to room temperature, with long forceps, filter paper packet is put into apparatus,Soxhlet's extracting barrel, inject the l.67 absolute ether doubly of primary rainbow pipette, sample bag is immersed in ether completely.Connect extractor each several part, connect condensed water current, in water bath with thermostatic control, carry out extracting, regulate water temperature between 70 ℃~80 ℃, make the ether of lower of condensation become beaded (120~150/min or reflux 7 times/more than h), extracting to extract ether in cylinder with filter paper drop inspection oil stains-less till (approximately needing 6~12h).
(5) after extracting, with long forceps, take out filter paper packet, in ventilation, make ether volatilization.Ether in extraction flask reclaims separately.After treating ether volatilization, filter paper packet is placed in to the dry 2h of l05 ℃ of baking oven, till putting into exsiccator and being cooled to constant weight, (sample Bao Chong after defat) finally weighs.
(6) calculate liver fat rate liver fat rate (%)=(sample Bao Chong after the front sample Bao Chong-defat of defat) (g)/(sample Bao Chong-over dry filter paper weight before defat) (g) * 100.
The mensuration of abdomen fat rate
The electronic balance that is placed with large plate is returned to zero; The chicken of slaughtering after weighing is cut open the chest from abdominal part, first peel off stomach fat, then muscular stomach fat is around peeled off out, be put in large plate; Weigh up and count record abdomen fat weight, calculate abdomen fat rate.Abdomen fat weight=stomach fat weight+muscular stomach fat weight around; Heavy (g) * 100 of heavy (the g)/live body of abdomen fat rate (%)=abdomen fat.
The statistical disposition of 1.7 test datas
Test data, all by statistics requirement, carries out statistical analysis with SPSS software, and all data represent with meansigma methods X ± standard deviation (SD), with variance analysis, carry out significance test of difference.
2 results and analysis
The selection of 2.1 cases
Be selected from Sichuan Agricultural University's poultry breeding field 350 age in days meat kind chickens, laying rate drops to current 43% by early stage 75%, and sick chicken spirit is depressed, poor appetite, and wattle, cockscomb fade and even are pale, and happiness is sleeping, is reluctant to walk about, and abdominal part softness is sagging.After dissecting, find that intra abdominal fat deposits in a large number, the periphery of particularly abdominal part, and muscular stomach and glandular stomach has the thick fat of one deck to exist.In addition, heart, mesentery etc. locate also to have the Fat Distribution of volume.The obvious enlargement of liver, is light brown to yellow, and matter is crisp frangible and have greasy feeling, and liver surface often has hemorrhage speckle, and has a large amount of blood clots around.Some liver disruption, have large go out clot or blood clot in liver surface and body cavity.Gallbladder enlargement or dwindle, cyst wall is because of lipidosis thickening.Spleen matter is soft, generally loses enlargement.Muscular stomach is due to the fat parcel being deposited, and its volume is only normal l/2~1/3, and muscular stomach content is few, and Endothelium Corneum Gigeriae Galli is thin and easily peel off the attenuation of flesh layer.Kidney is enlargement slightly, and matter is crisp.Serum Indexes detection, liver coefficient, liver fat rate, abdomen fat rate are compared with normal kind chicken, and difference should be all remarkable; Get disease chicken liver contact microscopy, in visible hepatocyte, be full of fat vacuole, in endochylema, have a large amount of lipidosiss.This chicken group of preliminary judgement suffers from fatty liver hemorrhagic syndrome, can be used as experimental animal.
2.2 overview
Before test, respectively organizing main manifestations is that appetite declines, astasia, and happiness is sleeping, and enlarged abdomen softness is sagging, and severe patient is drowsiness, paralysis, one-sided sleeping ground, body temperature rises to 42 ℃; Minority case cockscomb, wattle are pale, and accidental cockscomb increases.During off-test, matched group still keeps weak state, without taking a turn for the better; Choline group and Plumbum preparatium liver protection decoction group medication after 10 days mortality rate reduce gradually, cockscomb starts to redden, during off-test, chicken group substantially recovers normal drinking-water and searches for food, laying rate and enter the rate of incubating and have certain rising.
2.3 fertility performance situations
As can be seen from Table 1, before test, respectively organize indices without significant difference (P > 0.05).Within 3 weeks, while finishing, as seen from Table 2, matched group increases by 13.88%, 13.25% difference extremely significantly (P < 0.01) than choline group, Plumbum preparatium liver protection decoction group body weight respectively.Laying rate comparative control group difference choline group and Plumbum preparatium liver protection decoction group reduce by 46.70%, 67.11% difference extremely significantly (P < 0.01); Choline group reduces by 13.91% significant difference (P < 0.05) than Plumbum preparatium liver protection decoction group.Enter to incubate rate comparison, matched group difference choline group and Plumbum preparatium liver protection decoction group reduce by 32.84%, 38.92% difference extremely significantly (P < 0.01).Mortality rate comparison, matched group difference choline group and Plumbum preparatium liver protection decoction group increase by 48.99%, 68.59% difference extremely significantly (P < 0.01); Choline group increases by 38.42% difference extremely significantly (P < 0.01) than Plumbum preparatium liver protection decoction group.Cure rate comparison, matched group difference choline group and Plumbum preparatium liver protection decoction group reduce by 79.64%, 98.05% difference extremely significantly (P < 0.01).After medication, choline group and Plumbum preparatium liver protection decoction group chicken group recover gradually, it is normal that body weight is tending towards, laying rate, enter to incubate rate and rise, mortality rate reduces, and the higher state of an illness of cure rate is effectively controlled, and matched group production achievement continues to decline, showing as body weight increases, and laying rate, enters to incubate rate and declines, and mortality rate rises.
First 1 week interior chicken public sentiment condition of table 1 administration
Chicken public sentiment condition in 3 weeks after table 2 administration
With the different capitalizations of column data, represent difference extremely significantly (p < 0.01), different lowercase alphabets show significant difference (p < 0.05), same letter or without letter representation without significant difference (P > 0.05).
2.4 liver index variation
Cut open and kill after chicken, by the complete taking-up of liver, calculate liver coefficient.Liver coefficient=liver weight (g)/body weight (g) * l00.Result is as follows:
Table 3 liver index variation situation n=12
From upper table, find out, before test, each organizes difference not remarkable (p > 0.05).When administration finishes, Plumbum preparatium liver protection decoction group and choline group all with matched group significant difference (p < 0.05), reduce respectively 12.65%, 13.71%.After use medicine is described, can reduce liver coefficient, eliminate the swelling of liver.
The mensuration of 2.5 Biochemical Indices In Serums
With disposable syringe on an empty stomach wing venous blood collection 2~5mL in centrifuge tube, slant setting 30min at room temperature, the centrifugal 10min of 3500r/min, serum is placed on-20 ℃ of preservations.With automatic blood analyzer, measure the content of T-CHOL (TC), triglyceride (TG), glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) in serum.As seen from Table 4, during on-test, each organizes difference not remarkable (P > 0.05).After administration 3 weeks time, Plumbum preparatium liver protection decoction group and matched group glutamate pyruvate transaminase difference difference is (P < 0.05) extremely significantly, reduce by 25.00%, choline group and matched group glutamate pyruvate transaminase difference is (P < 0.01) extremely significantly, reduces by 29.17%; Glutamic oxaloacetic transaminase, GOT relatively Plumbum preparatium liver protection decoction group, choline group respectively with matched group difference extremely significantly (P < 0.01), reduce by 21.26%, 23.21%; T-CHOL is respectively organized difference not remarkable (P > 0.05), and medicine group is lower than matched group; Triglyceride Plumbum preparatium liver protection decoction group, choline group respectively with matched group difference extremely significantly (P < 0.01), reduce by 48.35%, 57.46%.
Table 4 administration is biochemical indicator situation in the time of first 1 week
Biochemical indicator situation 3 weeks time after table 5 administration
The mensuration of 2.6 liver fat rates
Adopt method noted earlier to record liver fat rate, as seen from Table 6, liver fat rate 1 week each group after medication does not have significant difference (P > 0.05), but all lower than matched group.In the time of 2 weeks, Plumbum preparatium liver protection decoction group and choline group all with matched group difference difference extremely significantly (P < 0.01), reduce respectively 14.82%, 23.04%.In the time of 2 weeks, Plumbum preparatium liver protection decoction group and choline group all with matched group difference significant difference (P < 0.05), reduce respectively 27.66%, 23.17%.
Table 6 liver fat rate measurement result
The mensuration of 2.7 abdomen fat rates
From following table 7, find out, before test, it is not remarkable that each organizes difference.When administration finishes, Plumbum preparatium liver protection decoction group and matched group difference is (P < 0.01) extremely significantly, reduces by 23.99%, and choline group and matched group difference significant difference (P < 0.05) reduce by 18.87%.
Table 7 abdomen fat rate measurement result
3 discuss
3.1 overview
After using choline and Plumbum preparatium liver protection decoction, chicken group's outward appearance be improved significantly, searching for food drinking-water is replied, successful.
3.2 fertility performance situations
Matched group fertility performance continuous decrease, and the body weight of medicine group is alleviated, laying rate, enters to incubate rate and rises gradually, and wherein Plumbum preparatium liver protection decoction group effect is better than matched group and choline group.
3.3 liver coefficients
After medication, liver coefficient decreases, and medicine group is obvious, with improve liver circulation, certain relation diminishes inflammation.Prescription Chinese medicine can improve microcirculation disturbance, but also can improve because of the dysbolismus due to cell hypoxic-ischemic, can the metabolite Accelerating Removal in liver is external, improve hepatocellular fibrosis simultaneously, diminish inflammation, this is also the reason that affects fertility performance, liver index variation.
3.4 biochemical indicator
T-CHOL (TC), triglyceride (TG), glutamate pyruvate transaminase (ALT), glutamic oxaloacetic transaminase, GOT (AST) in the index serum of reflection liver health, be improved significantly.Hepatocyte be protected and be repaired to Chinese drug-treated group can, maintains hepatocellular normal function, and the permeability of liver plasma membrane is reduced, and in cell, transaminase discharges reduction.Li Kunwu etc. (2003) reported ' ' JD-tri-levies liver Thailand " the chicken FLS being caused by high lipid food is had to remarkable therapeutical effect; can significantly reduce TG in hepatic fat content and serum, TC resultant; alleviate hepatocyte edema, downright bad lesion degree, reduce serum GPT, GOT active.
3.5 liver fat rates and abdomen fat rate
Use after medicine, the blood circulation of liver part improves, and nutrition supply is obviously supplied with, and hepatocyte metabolism is restored and strengthens, and can promote the outward transport of liver fat, accelerates the beta oxidation in hepatocyte, so liver fat rate is reduced; Abdomen fat rate is also obviously improved simultaneously, Plumbum preparatium liver protection decoction successful.
3.6 about medicament composing prescription
In side, Penthorum chinense is monarch drug, Ming Dynasty < < Herbal for Relief of Famines > > begins to be loaded in, warm in nature, sweet in the mouth, nontoxic, tool heat clearing away, diuresis elimination of urine, detoxify, invigorate blood circulation, suppressing the hyperactive liver, spleen invigorating, the effects such as jaundice of dispelling, be Miao ethnic group's treatment hepatopathy among the people through proved recipe, be called " Radix Rhodiolae ", cure mainly jaundice, edema, amenorrhea, metrorrhagia, leukorrhagia, traumatic injury, and various hepatitis, cholecystitis, fatty liver etc.Modern study shows can spleen invigorating soothing liver-QI, protects the liver and treats liver, and dredging collateral is invigorated blood circulation, and reduces transaminase, for fatty liver, and alcoholic liver, medicine liver, hepatic injury, hepatic fibrosis, liver cirrhosis and metabolic gout etc. has prevention and repairs blocking effect.Radix Salviae Miltiorrhizae is ministerial drug, can invigorate blood circulation and adjust liver, stasis-dispelling and pain-killing, removing heat from blood eliminating carbuncle, the relieving restlessness that clears away heart-fire, nourishing blood to tranquillize the mind, hepatocellular degeneration when modern study proof can suppress and alleviate acute and chronic liver injury, necrosis and inflammatory reaction, accelerating fibers is organized heavily and is absorbed, and has anti-hepatic fibrosis, improves the effect that liver blood circulates, prevents liver cirrhosis.Herba Sedi is adjuvant drug, is extremely wide conventional medicinal herbs of folklore simply, and all herbal medicine has the effect of heat-clearing and toxic substances removing, swelling diuretic, evacuation of pus granulation promoting etc.; Sarmentosin has the effect of obvious reduction serum glutamic pyruvic transminase, and effect rapidly and lastingly, is also the active drug for the treatment of hepatitis.Fructus Crataegi is adjuvant drug, can appetite-stimulating indigestion-relieving, change stagnant removing food stagnancy, promoting blood circulation to remove blood stasis, the circulation of qi promoting of reducing phlegm; For carnivorous stagnant long-pending, lump in the abdomen, abdominal distention feeling of fullness, stasis blocking stomachache, phlegm retention, have loose bowels, discharging fresh blood stool etc.; Studies confirm that Fructus Crataegi can significantly reduce serum cholesterol and triglyceride, effectively prevents and treats atherosclerosis; Fructus Crataegi can also be by strengthening myocardial contraction, increasing cardiac output.Rhizoma Polygoni Cuspidati is adjuvant drug, has the hemostasis of Huoxue San " expectorant, the removing toxic substances of dispeling the wind, anti-inflammatory analgetic, remuval of damp and hot jaundice, controls chronic tracheitis, antibacterial, antiviral, protects the liver, reduces the effects such as blood fat, blood glucose.Radix Curcumae is for making medicine, can promoting QI circulation for relieving depression, and removing heat from blood removing blood stasis with potent drugs, soothing the liver strongly fragrant all medicine people Liver Channel that draws.Radix Glycyrrhizae invigorating the spleen and replenishing QI, heat-clearing and toxic substances removing, expelling phlegm for arresting cough, relieving spasm to stop pain, coordinating the actions of various ingredients in a prescription, modern pharmacology proof have the effect of adrenocortical hormone sample, arrhythmia have protect the liver, the effect such as analgesia, antipyretic, sedation, blood fat reducing, cholesterol reducing, resisting pathogenic microbes.All medicines share the more aobvious magical effect, are total to long memorial liver spleen dampness removing, repercussive intestinal stasis relieving, the merit of disappear fat liver heat removing, heat-clearing and toxic substances removing.
4 conclusions
Known by this test, the fatty liver that Plumbum preparatium liver protection decoction occurs meat kind chicken has good therapeutical effect, under doses, can obviously play and improve fertility performance, reduces mortality rate, improves cure rate; The liver protecting; reduce liver coefficient; reduce glutamate pyruvate transaminase, glutamic oxaloacetic transaminase, GOT, cholesterol, triglyceride; can also reduce the effect of liver fat rate, abdomen fat rate; Plumbum preparatium liver protection decoction has had the soothing liver and strengthening spleen of moving back dampness removing by reasonable formula; repercussive intestinal stasis relieving, the merit of disappear fat liver heat removing, heat-clearing and toxic substances removing, can be for the treatment of clinical meat kind chicken fatty liver.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.