CN1454661A - Medicine for curing hepatitis B and preparing method thereof - Google Patents

Abstract

The invention is compound particles which is made up of glossy ganoderma, hedyotis diffusa, gardenia jasminoides, root of red rooted salvia, liquorice, Tuckahoe, the root of large-flowered skullcap, root of herbaceous peony, tulip according to a proportion. Through purifying, decocting with water, filtering, concentrating, drying, and the product are produced. It has functions of supplementing the vital energy and detoxicating, activating the spleen-energy and promoting vital energy circulation, dispersing blood stasis and alleviating pain. It applies to cure hepatitis B.

Description

A kind of medicine for the treatment of hepatitis B and preparation method thereof

Technical field

The present invention relates to a kind of is the medicine and preparation method thereof of raw material treatment hepatitis B with the Chinese herbal medicine.

Background technology

According to World Health Organization's statistics, the whole world has people more than 200,000,000 to be in chronic viral hepatitis B virus carrier state, and 70% is the Asia Chinese, and wherein about 25% finally will die from the sequela of hepatitis B, as liver cirrhosis, hepatocarcinoma etc.; China is high incidence of hepatitis, and according to the numeral that the professor Zhuan Hui of Beijing Medical University provides, China has 1.2 hundred million people to carry hepatitis B virus approximately at present; In the infectious disease of Chinese statutory report, the M ﹠ M of viral hepatitis all accounts for the first place, and the average year sickness rate is about one thousandth; According to investigations, existing about 1,200 ten thousand examples of chronic hepatitis patient of China, die from hepatopathy every year and be about 300,000 people, wherein half is a primary hepatoma, most and B-mode relevant with infection with hepatitis C virus, so the treatment to hepatitis B patient and the chronic carrier of hepatitis B virus has crucial meaning.

Summary of the invention

The purpose of this invention is to provide that a kind of therapeutic effect is good, side effect is low, the pure Chinese medicinal preparation of instant effect.

Another object of the present invention provides the preparation method of said preparation.

The present invention is under instruction of Chinese Medicine theory, accumulates the pathogenic characteristic of liver at the many stagnation of QI due to depression of the liver of chronic viral hepatitis, incoordination between the liver and stomach, stagnation of liver-QI with deficiency of the spleen, evil poison, on the folk remedy basis, adds to simplify in conjunction with the clinical experiences in more than 20 years and cuts out prescription and form.

Medicine of the present invention is made by following component: (consumption is a weight portion)

Ganoderma 308-420 Herba Hedyotidis Diffusae 392-840 Fructus Gardeniae 182-280

Radix Curcumae 182-336 Radix Paeoniae Alba 112-336 Poria 112-420

Radix Salviae Miltiorrhizae 84-420 Radix Scutellariae 84-280 Radix Glycyrrhizae 28-252

The optimum weight of medicine of the present invention (part) proportioning is:

Ganoderma 400 Herba Hedyotidis Diffusaes 400 Fructus Gardeniaes 200

Radix Curcumae 200 Radix Paeoniae Albas 166 Poria 166

Radix Salviae Miltiorrhizae 100 Radix Scutellariaes 100 Radix Glycyrrhizaes 50

Above-mentioned each component is made medicine production method of the present invention is:

1 gets Ganoderma, Herba Hedyotidis Diffusae, Radix Salviae Miltiorrhizae, be ground into coarse powder, add 70% alcohol reflux three times, the each extraction 2 hours adds the ethanol of 8 times of amounts for the first time, adds the ethanol of 6 times of amounts for the second time, for the third time, extracting solution filters, filtrate merges, and decompression recycling ethanol and to be concentrated into relative density be 1.02～1.05 clear paste is standby;

2 mix above-mentioned 1 gained medicinal residues with Radix Scutellariae, Fructus Gardeniae, the Radix Paeoniae Alba, Radix Curcumae, Radix Glycyrrhizae, Poria Six-element medical material, add for the first time 12 times of water gagings immersions 1.5 hours, decocted 2 hours, second and third time respectively adds 10 times of water gagings, decocts respectively 2 hours, decocting liquid merges, filter, be concentrated into the clear paste of relative density 1.05～1.10, add ethanol and make and contain the alcohol amount and reach 50%, stir evenly, leave standstill more than 24 hours, filter, filtrate recycling ethanol also is concentrated into the clear paste of relative density 1.02～1.05;

3 with above-mentioned 1,2 gained clear paste mix homogeneously, and spray drying is an amount of with sucrose and dextrin fine powder, mix homogeneously, and the alcohol granulation with 75%, drying, granulate promptly gets powder.

Wherein requiring inlet temperature during spray drying is 160 ℃～180 ℃, and spray tower temperature is 75～80 ℃, and leaving air temp is 70～75 ℃, and rotating speed is 2.5 ten thousand rev/mins; Be 5: 1 with the weight ratio of sucrose and dextrin.The present invention has following three aspect characteristics:

The one, can improve the chronic viral hepatitis B clinical symptoms in a short time, as through the medication in 1～February, patient's the pain over the hypochondriac region, the vexed abdominal distention in chamber, inappetence, fatigue and weakness, xerostomia symptom 85% patient such as feel sick disappears more.

The 2nd, former liver function deviant, normal through the many recoveries of medication in 2～March, especially ALT and AST recover obviously.

The 3rd, this side takes half a year above e antigen has 20% can cloudy change approximately.

Pharmacodynamics test

In order to further specify the mechanism of curing the disease of the present invention, we have carried out curing mainly relevant Pharmacodynamic test of active extract with pharmic function of the present invention, the influence of the chmice acute hepatic injury of comprise the vivo and vitro antivirus action, chemical factor being brought out, to CCl ₄Cause the influence of rat chronic hepatic injury and hepatic fibrosis, to effect of immunologic function etc., the result is as follows:

One, the pharmacodynamics test data and the documents and materials of anti-DHB

Be subjected to the reagent product: medicine of the present invention (lot number: 020406), with No. 2 capsulations.The medicine of the present invention of three kinds of dosage is adopted in test: 1.6g (crude drug in whole) kg ^-1D ^-1, 3.2g (crude drug in whole) kg ^-1D ^-1And 6.4g (crude drug in whole) kg ^-1D ^-1

The positive control medicine: lamivudine (lot number: B038238, the action company limited production of Glaxo Wellcome Britain) is purchased in heavily curing Annex II institute, and the crushing back is from the capsule of making the 5mg/ grain.Animal: 1 age in days duckling of the egg incubation that the Chongqing sheldrake of employing healthy adult produces, through abdominal cavity inoculation 0.1mlDHBV DNA positive-virus serum.After inoculating for 1 week, respectively external jugular vein blood drawing detects to filter out through dot blot hybridization with the DHBV dna probe of digoxigenin labeled and infects positive duck, raise to 3 all ages as laboratory animal.Route of administration: method and result irritate to be fed in the oral cavity: 1. method:

To infect 64 of positive ducks and be divided into 5 groups at random: (1) virus control group (12): use the starch capsule, the oral cavity was irritated and was fed 1 time every day, and dosage is 500mg/.(2) positive drug matched group (13): use the lamivudine capsule, the oral cavity was irritated and was fed 1 time every day, and dosage is 50mg.Kg ^-1.D ^-1(3) medicine small dose group of the present invention (13): the oral cavity was irritated and was fed 1 time every day, and dosage is 1.6g (crude drug in whole) .Kg ^-1.D ^-1(4) dosage group (13) in the medicine of the present invention: the oral cavity was irritated and was fed 1 time every day, and dosage is 3.2g (crude drug in whole) .Kg ^-1.D ^-1(5) the heavy dose of group of medicine of the present invention (13): the oral cavity was irritated and was fed 1 time every day, and dosage is 6.4g (crude drug in whole) .Kg ^-1.D ^-1

The experimental drug time is 28 days, and drug withdrawal was observed 7 days.2. experimental result:

(1) serum DHBV DNA titre changes situation before, during and after the medication:

Serum DHBV DNA titre does not have obvious reduction before and after virus control group (starch capsule) medication.The lamivudine group medication begins to have serum DHBV DNA titre and reduces (P＜0.05) after 7 days, DHBV DNA rise phenomenon is arranged after the drug withdrawal.The big or middle dosage group of medicine of the present invention reduces (P＜0.01 or P＜0.05) respectively at beginning to have serum DHBV DNA titre in 28 days, heavy dose organize in drug withdrawal after 7 days viral DNA rebound phenomenon appears.Medicine small dose group of the present invention during treating in the DHBV DNA titre of Sanguis Anas domestica in clear reduction trend is arranged, but not statistically significant relatively before and after the medication.See Table 1.

Serum DHBV DNA titre change situation before and after table 1 medication (x ± S)

DHBV DNA (volume) group

7 days starch Capsules group 1523.33 ± 1571.15 ± 1617.52 ± 1536.734 ± 1542.45 ± 1494.04 of medication medication in 7 days medication in 14 days medication in 21 days drug withdrawal in 28 days before the medication ±

113.47 109.26 133.44 115.07 74.02 68.15 lamivudine group 1378.80 ± 1300.24 ± 1342.99 ± 1284.22 ± 1292.98 ± 1358.97 ±

130.03 119.71 ^*137.56 86.34 ^*60.81 ^*81.46 big dose 1468.75 ± 1438.70 ± 1435.60 ± 1421.10 ± 1351.81 ± 1378.63 of medicine of the present invention ±

Amount organizes 251.96 289.26 286.82 244.17 178.29 ^*196.82 agent 1467.72 ± 1491.76 ± 1558.29 ± 1437.37 ± 1334.07 ± 1324.98 in the medicine of the present invention ±

Amount organizes 202.50 305.83 245.73 323.28 164.29 ^*144.28 ^*Little dose 1529.57 ± 1547.78 ± 1479.37 ± 1466.81 ± 1510.87 ± 1472.45 of medicine of the present invention ±

Amount organizes 171.12 188.76 190.88 164.24 225.93 165.31

Annotate: go up table average and the standard deviation of respectively organizing DNA speckle volume value of classifying as, statistics adopts paired t-test (＂ ^*＂: P＜0.05; ＂ ^*＂: P＜0.01), is the comparison of DNA speckle value before each group different time DNA speckle value and the medication on the same group.(2) respectively organize serum DHBsAgO.D value change situation before, during and after the medication:

Serum DHBsAgO.D value does not have obviously change before and after each the dosage group medication of virus control group (starch capsule) and medicine of the present invention, the lamivudine group medication began to occur serum DHBsAgO.D value in 7 days and reduces (P＜0.01), the rise phenomenon is arranged to 7 days serum DHBsAgO.D of drug withdrawal value, but with medication before relatively still have and continue to reduce (P＜0.05).See Table 2.

Table 2 is respectively organized (preceding 7 days starch Capsules group 0.74 ± 0.39 0.65 ± 0.27 0.60 ± 0.34 0.56 ± 0.26 0.61 ± 0.30 0.63 ± 0.27 lamivudine group 0.64 ± 0.48 0.34 ± 0.25 of medication medication in 7 days medication in 14 days medication in 21 days drug withdrawal in 28 days of group medication of x ± S) of serum DHBsAgO.D value change situation before and after the medication ^*0.35 ± 0.26 ^*0.26 ± 0.17 ^*0.29 ± 0.25 ^*0.41 ± 0.27 ^*Dosage group 0.73 ± 0.53 0.71 ± 0.49 0.70 ± 0.50 0.64 ± 0.42 0.61 ± 0.49 0.58 ± 0.42 medicine small dose group 0.68 of the present invention ± 0.41 0.73 ± 0.48 0.68 ± 0.44 0.55 ± 0.35 0.56 ± 0.32 0.47 ± 0.34 in the heavy dose of group of medicine of the present invention 0.79 ± 0.59 0.88 ± 0.62 0.81 ± 0.61 0.77 ± 0.52 0.66 ± 0.47 0.68 ± 0.36 medicine of the present invention

Annotate: upward table institute classifies as and respectively organizes DHBsAgO.D value average and standard deviation, and statistics adopts paired t-test.(＂ ^*＂: P＜0.05, ＂ ^*": P＜0.01), be each group different time DHBsAgO.D value and the comparison of DHBsAgO.D value before the medication on the same group.

(3) liver HE dyeing pathological examination results:

Each group all has light degree hepatocyte cloudy swelling, has a liking for sour degeneration, and portion of tissue portal area, interlobular septum have gently～moderate inflammation cellular infiltration and hepatocyte spotty necrosis phenomenon, these pathological manifestations medication groups and matched group no significant difference.3. conclusion:

Study human hepatitis B pathogeny, virus replication and screen effective medicine as the hepatitis B animal model with the DHBV infected duck, generally acknowledged by Chinese scholars.1-3 age in days duckling infects DHBV and can keep long-term viremia and not have the tangible phenomenon of turning out cloudy naturally, therefore, we use 1 age in days duckling and set up the examination that the duck hepatitis-B animal model carries out medicine antiviral curative effect of the present invention through the method for abdominal cavity infection DHBV, and expectation filters out the medicine of anti-hepatitis B virus effect.And use and have obvious inhibiting nucleotide analog lamivudine to contrast as positive drug to hepatitis B virus.

This animal test results shows: in the medicine of the present invention, the DHBV DNA titre of heavy dose of group Sanguis Anas domestica in clear reduces has significantly or extremely remarkable meaning, medicine small dose group of the present invention during treating in the DHBV DNA titre of Sanguis Anas domestica in clear though reduction trend is arranged, but compare not statistically significant before and after the medication, illustrate that medicine of the present invention has certain dose-effect relationship to the inhibition effect of DHBV DNA and the drug dose of test and Selection.Medicine heavy dose of the present invention organize in drug withdrawal after 7 days viral DNA rebound phenomenon appears, illustrate that effect that medicine of the present invention suppresses virus is one to cross property, halfway.Except that lamivudine group, all the other are respectively organized, and DHBsAg does not all have tangible reduction in the serum, illustrate that medicine of the present invention is not obvious to DHB surface antigen inhibitory action, although lamivudine and the present invention have certain inhibitory action to DHB DNA, these inhibitory action still do not reach " the cloudy commentaries on classics " effect, so prolonging administration time, selecting suitable dosage is to be worth the further problem of discussion.

Because duck hepatitis-B animal model self, 1-3 age in days duckling immune system is grown imperfection, after infecting DHBV, simple viral carrier state only occurs, but the liver organization pathological changes is slight, the liver function Non Apparent Abnormality.Our experimental result finds that each dosage group hepatic tissue pathology of medicine of the present invention detects and the matched group no significant difference, though can not confirm this medicine the effect (this needs other animal checking) of improving liver function is arranged, can illustrate that 1 month medicine of the present invention of continuous use does not have the overt toxicity infringement to hepatic tissue.

Two, chemical factor is caused the protective effect of chmice acute hepatic injury

Purpose is observed and is subjected to the reagent thing to D-galactosamine (D-GlaN), thioacetamide (TAA) and carbon tetrachloride (CCl ₄) influence of induced mice acute liver damage.

Method D-GlaN, TAA and CCl ₄Cause the chmice acute hepatic injury Deng chemicals; with serum aspartate amino transferase (AST/GOT), alanine aminotransferase (ALT/GPT) regulating liver-QI histopathological examination etc. is index, observes to be subjected to reagent chemical factor to be caused the protective effect of chmice acute hepatic injury.

Medicine of the present invention as a result can reduce serum AST and ALT, alleviates the hepatic tissue pathology infringement, to D-GlaN, TAA and CCl ₄Has protective effect etc. the chmice acute hepatic injury due to the chemical factor.

(1), to the influence of the chmice acute hepatic injury due to the D-Gal

1, test objective: causing the chmice acute hepatic injury with D-GlaN, is index with serum AST, ALT regulating liver-QI histopathological examination, observes the influence of medicine of the present invention to acute liver damage due to the chemical factor.

2, test material

(1) experimental animal: healthy ICR kind is a mice, and is male, and body weight 20 ± 2 gram is provided by Xi'an Jiaotong University Medical College's Experimental Animal Center, the quality certification number: the moving word of doctor 08-004 number.Raising condition: ShanXi Chinese Medicine Academy pharmacology of Chinese materia medica laboratory (national traditional chinese medical science administration Chinese medicine scientific experiment chamber classification: secondary).

(2) medicine and reagent

Be subjected to the reagent thing: fine drug powder of the present invention, 1g contain crude drug 8.33g, are provided lot number by Xi'an Green Valley Pharmaceutical Co., Ltd.: 020406.Be made into the drug suspension of the present invention of 45%, 22.5%, 11.25% (crude drug) with distilled water, shake up during administration.The oral 60g crude drug of per day for adults, promptly clinical human dosage are 0.86g/kg (body weight for humans is in 70kg).

Positive control drug: yiganning granules, produce by Guangxi Banyu Pharmaceutical Co., Ltd, authentication code: ZZ-5010-defends in osmanthus the accurate word (1990) of medicine No. 079023, product batch number 020120.Be made into the suspension of 37% (crude drug) with distilled water, this test mice dosage is 7.4g/kg.Bifendate drop pill, the 1.5mg/ ball, Dezhou, Shandong Province pharmaceutical factory produces, authentication code: the accurate word H37020113 of traditional Chinese medicines, product batch number: 020816.Be made into the suspension of 20mg/ml after grinding with distilled water, dosage is 200mg/kg.

Reagent: D-galactosamine (D-Glactosamine D-GlaN), Sigma produces, lot number: 83H0925.Facing the time spent is mixed with 10%D-DlaN solution with physiological saline solution.Lumbar injection 1000mg/kg body weight.D-GlaN is the agent interfering of hepatocyte phosphoric acid uridine, and the hepatic injury that causes is rather similar to human virus's hepatitis in biochemistry and Histological change ^[1,3]020031) and ALT test kit (lot number: 020301) produce AST test kit (lot number: by Beijing Zhongsheng Biological Engineering High Technology Company.

3. experimental technique and result

Get ICR kind male mice, be divided into 7 groups at random.The large, medium and small dosage group of medicine of the present invention is irritated stomach respectively and is given 45%, 22.5% and 11.25% drug suspension of the present invention, and dosage is followed successively by 9g crude drug/kg, 4.5g crude drug/kg and 2.25g crude drug/kg; The bifendate group is irritated stomach bifendate 200mg/kg; The YIGANNING group gives YIGANNING 7.4g/kg; Blank group and model group are irritated ordinary water.Administration every day 1 time, continuous 5 days, administration volume 0.2ml/10g.After the last administration 1 hour, the D-GlaN solution 0.1ml/10g body weight of lumbar injection 10%, posterior orbit was got fasting blood in 24 hours, separation of serum is measured serum AST (continuous detecting method) and ALT (continuous detecting method) with XD-811C type biochemistry analyzer (the Shanghai news reach Medical Instruments company and produce), gets leftlobe of liver 10% formalin solution simultaneously and fixes, the routine paraffin wax embedding, section, HE dyeing, microscopically is observed pathological change.The measurement data of test gained is through the analytical control of t value method, and ranked data carry out statistical analysis with the Ridit method, the significance of comparable group differences.

(1), to the influence of serum AST and ALT (table 2-1)

Table 2-1: to the influence of serum AST and ALT (X ± S)

Group dosage (g/kg) number of animals ALT (U/L) AST (U/L)

Normal control group-11 77.4 ± 18.1 ^*119.3 ± 15.6 ^*

Model group-10 132.2 ± 28.0 248.0 ± 88.0

Bifendate group 0.2 10 93.1 ± 31.1 ^*164.2 ± 44.1 ^*

YIGANNING group 7.4 9 118.2 ± 26.7 200.2 ± 59.0

Heavy dose of group 9 11 102.6 ± 20.5 ^*166.5 ± 62.7

Middle dosage group 4.5 11 120.8 ± 31.9 180.7 ± 61.0

Small dose group 2.25 10 138.3 ± 71.2 223.3 ± 126.3

Compare with model group: * P＜0.05 * * P＜0.01

As show shown in the 2-1, model group animal serum AST and ALT obviously raise, with the normal control group relatively, difference all has significance meaning (P＜0.01,＜0.01); The large, medium and small dosage of medicine of the present invention can reduce serum AST and ALT, and heavy dose of treated animal Serum ALT and model group compare, and difference all has significance meaning (P＜0.05); Positive control drug bifendate effect of reducing enzyme levels is (P＜0.01,＜0.05) significantly.

(2), the influence that liver histopathology is checked (table 2-2)

Table 2-2: to the influence of liver histopathology inspection

Lesion degree

Group

P

- + ++ +++

Normal group 8210＜0.05

Model group 0361

Bifendate group 2530＜0.05

YIGANNING group 1350

Heavy dose of group 4700＜0.05

Middle dosage group 2720＜0.05

Small dose group 1450 is annotated: degeneration classification standard-normal, no ballooning degeneration of liver cells+lobules of liver is less than 1/3 regional slight hepatic cell swelling, see amorphous cavity in the endochylema, the liver rope is clear substantially, sinus hepaticus is still as seen ++ between "+" and " +++" ++ and the extremely swelling of+hepatocyte, be the balloon sample and become, the disorder of liver rope, sinus hepaticus is unclear

4, brief summary

Cause the chmice acute hepatic injury with 10%D-GlaN with the 1000mg/kg lumbar injection, observe the protective effect of medicine of the present invention its damage.The result shows that medicine 9g/kg of the present invention can obviously reduce serum AST and ALT, alleviates hepatocellular degeneration, and the chmice acute hepatic injury due to the D-GlaN is had protective effect.

(2), thioacetamide is caused the influence of chmice acute hepatic injury

1, test objective: observe medicine of the present invention causes the chmice acute hepatic injury to thioacetamide TAA influence.

2, test material: experimental animal, be subjected to reagent thing and positive control drug with test 2-1.Thioacetamide (Thioacetamide TAA), analytical pure provides lot number by Shanghai chemical reagents corporation of Chinese Medicine group: 20011211.Be made into 2% solution with sterile distilled water, cause acute liver damage with the 50mg/kg lumbar injection.TAA can disturb PNA from examining cytoplasmic transport process, influences protein synthesis, and the infringement liver plasma membrane causes hepatic necrosis.Biochemical investigation reagent is with test 2-1.

3, test method and result: grouping and medication are with test 2-1.After the last administration 1 hour, except that the blank group, the TAA solution 0.025ml/10g of all the other each treated animal lumbar injections 2%, posterior orbit was got fasting blood in 16 hours, and separation of serum is measured serum AST and ALT, and liver carries out pathological examination.The measurement data of test gained is through the analytical control of t value method, and ranked data carry out statistical analysis with the Ridit method, the significance of comparable group differences.

(1), to the influence of serum AST and ALT (table 2-3)

Table 2-3: to the influence of serum AST and ALT (X ± S)

Group dosage (g/kg) number of animals ALT (U/L) AST (U/L)

Normal group-11 77.4 ± 18.1 ^*119.3 ± 15.6 ^*

Model group-15 148.8 ± 83.5 325.2 ± 118.1

Bifendate group 0.2 12 99.1 ± 27.0 ^*195.5 ± 81.8 ^*

YIGANNING group 7.4 17 114.6 ± 27.0 261.4 ± 84.1

Heavy dose of group 9 15 98.5 ± 20.6 ^*253.4 ± 94.1

Middle dosage group 4.5 15 105.0 ± 27.3 300.9 ± 71.2

Small dose group 2.25 15 122.3 ± 59.3 344.3 ± 62.9

Compare with model group: * P＜0.05 * * P＜0.01

The result shows that behind the injected in mice TAA, liver function injury is serious, and serum AST and ALT obviously raise, and compares with normal group, and difference has significance meaning (P＜0.01, P＜0.01); Heavy dose of treated animal Serum ALT and model group compare, and difference has significance meaning (P＜0.05); Bifendate can significantly reduce ALT (P＜0.05) and AST (P＜0.01).

2, the influence that liver histopathology is checked

Table 2-4: to the influence of liver histopathology inspection

Lesion degree

Group

P

- + ++ +++

Normal group 10 100＜0.05

Model group 0663

Bifendate group 2550＜0.05

YIGANNING group 2861＜0.05

Heavy dose of group 6720＜0.05

Middle dosage group 2661

Small dose group 0672 is annotated: degeneration classification standard-normal, no ballooning degeneration of liver cells+lobules of liver is less than 1/3 regional slight hepatic cell swelling, see amorphous cavity in the endochylema, the liver rope is clear substantially, sinus hepaticus is still as seen ++ between "+" and " +++" ++ and the extremely swelling of+hepatocyte, be the balloon sample and become, the disorder of liver rope, sinus hepaticus is unclear

4, brief summary

Cause the chmice acute liver injury model with the TAA50mg/kg lumbar injection, observe the protective effect of medicine of the present invention damage.As a result, heavy dose of obviously reduction Serum ALT (P＜0.05), and obviously alleviate liver cell lesions (P＜0.05) such as cloudy swelling, necrosis.Prompting, medicine of the present invention has protective effect to the chmice acute hepatic injury due to the TAA.

(3), carbon tetrachloride is caused the influence of chmice acute hepatic injury

1, test objective: observe medicine of the present invention causes the chmice acute hepatic injury to carbon tetrachloride influence.

2, test material: experimental animal, be subjected to reagent thing and positive control drug with test 2-1.Carbon tetrachloride (CCl ₄), analytical pure, chemical experimental factory, Laiyang produces (GB688-92), lot number: 20011030.Face with preceding 0.1% the oil solution of being made into olive oil.Biochemical reagents are with test 2-1.

3, test method and result: grouping and administration are with test 2-1.After the last administration 1 hour, except that the blank group, all the other each treated animal lumbar injection 0.1%CCl ₄Olive oil solution 10ml/kg.Fasting 18 hours, eye socket is got blood, and separation of serum is measured serum AST and ALT, and liver carries out pathological examination.The measurement data of test gained is through the analytical control of t value method, and ranked data carry out statistical analysis with the Ridit method, the significance of comparable group differences.

(1), to the influence of serum AST and ALT (table 2-5)

Table 2-5: to the influence of serum AST and ALT (X ± S)

Group dosage (g/kg) number of animals ALT (U/L) AST (U/L)

Normal group-17 77.7 ± 17.2 ^*120.3 ± 18.5 ^*

Model group-17 125.4 ± 33.4 213.2 ± 50.7

Bifendate group 0.2 16 81.5 ± 15.7 ^*139.7 ± 38.6 ^*

YIGANNING group 7.4 15 96.1 ± 25.6 ^*181.4 ± 67.1

Heavy dose of group 9 17 89.1 ± 25.2 ^*168.4 ± 51.5 ^*

Middle dosage group 4.5 16 97.5 ± 26.7 ^*173.1 ± 71.3

Small dose group 2.25 11 113.2 ± 27.0 179.0 ± 78.6

Compare with model group: * P＜0.05 * * P＜0.01

The result shows that model group serum AST and ALT obviously increase, with the normal control group relatively, difference all has significance (P＜0.01,＜0.01); Each dosage group of medicine of the present invention can reduce serum AST and ALT to some extent, and heavy dose of group and model group comparing difference all have significance meaning (P＜0.01,＜0.05).Middle dosage group Serum ALT and model group comparing difference all have significance meaning (P＜0.05).

(2), the influence that liver histopathology is checked (table 2-6)

Table 2-6: to the influence of liver histopathology inspection

Lesion degree

Group

p

- + ++ +++

Normal group 16 100＜0.05

Model group 1376

Bifendate group 4642＜0.05

YIGANNING group 2751＜0.05

Heavy dose of group 4 10 30＜0.05

Middle dosage group 2482

Small dose group 1442 is annotated: ballooning degeneration classification standard-normal, no ballooning degeneration of liver cells+lobules of liver is less than 1/3 regional slight hepatic cell swelling, see amorphous cavity in the endochylema, the liver rope is clear substantially, sinus hepaticus is still as seen ++ between "+" and " +++" ++ and the extremely swelling of+hepatocyte, be the balloon sample and become, the disorder of liver rope, sinus hepaticus is unclear

The normal group animal livers organizes finding with test 2-1; A large amount of hepatocyte cavity changes and atrophy around the lobules of liver portal area of model group liver and/or the central vein, necrosis and with inflammatory cell infiltration, with normal control group comparing difference remarkable (P＜0.05), the subregion cell is the loose and water sample change of endochylema in various degree, the hepatocyte arrangement disorder, but sinus hepaticus is still as seen; The big or middle dosage group of medicine of the present invention cell is slightly swollen long, and water sample change, necrosis and inflammatory cell infiltration alleviate, and heavy dose of group has significance meaning (P＜0.05) with the model group comparing difference.

4, brief summary

The heavy dose of group of medicine of the present invention can reduce CCl ₄Cause chmice acute liver injury model serum AST and ALT, with the model group comparing difference significance meaning (P＜0.01,＜0.05) is arranged all, it is slightly swollen long to alleviate cell, water sample change, necrosis and inflammatory cell infiltration, and heavy dose of group has significance meaning (P＜0.05) with the model group comparing difference.Prompting, medicine of the present invention is to CCl ₄Cause the chmice acute hepatic injury protective effect is arranged.

(4), conclusion

Medicine 9g/kg of the present invention, 4.5g/kg can resist D-GlaN, TAA and CCl to some extent ₄Deng due to the chmice acute hepatic injury, reduce serum AST and ALT, alleviate the pathologic lesion of hepatic tissue, the chmice acute hepatic injury due to the chemical factor is had protective effect.

Three, to CCl ₄Cause the influence of rat chronic hepatic injury and hepatic fibrosis

Purpose is observed medicine of the present invention to CCl ₄Cause the influence of rat chronic hepatic injury and hepatic fibrosis, inquire into the pharmacology of Chinese materia medica basis of its treatment chronic hepatitis.

Continuous 24 the CCl of method to rat skin lower injection 25% ₄Oil solution, induce rats'liver damage and hepatic fibrosis, irritate stomach medicine of the present invention simultaneously, with serum aspartate amino transferase (AST/GPT), alanine aminotransferase (ALT/GOT), total protein (TP), albumin (Alb), laminin (LN), hyaluronic acid (HA), III procollagen type albumen (PC-3) and liver organ coefficient and of science inspection of hepatopathy is index, observes being subjected to the reagent thing to CCl ₄Cause the influence of rat chronic hepatic injury and hepatic fibrosis.

Medicine of the present invention as a result can reduce serum AST, ALT; Reduce serum LN, HA and PC-3, obviously alleviate various pathological changes and hepatic fibroplasias such as swelling of liver cell in the lobules of liver, loose, the fatty change of endochylema, the change of balloon sample.Prompting, medicine of the present invention is to CCl ₄Inductive rat chronic hepatic injury and hepatic fibrosis have protective effect.

(1), test objective

Use CCl ₄Injection causes rat chronic hepatic injury and hepatic fibrosis repeatedly, irritate stomach medicine of the present invention simultaneously, with serum biochemistry and liver organization pathological examination is index, observes the influence of medicine of the present invention to this model, inquires into the pharmacology of Chinese materia medica basis that it treats acute and chronic hepatitis.

(2), test material

(1) experimental animal: 120 of SD kind rats, male, body weight 150g ± 10g is provided by Xi'an Jiaotong University Medical College's Experimental Animal Center, the quality certification number: the moving word of doctor 08-005 number.Raising condition: ShanXi Chinese Medicine Academy pharmacology of Chinese materia medica laboratory (national traditional chinese medical science administration Chinese medicine scientific experiment chamber classification: secondary).

(2) medicine and reagent

Be subjected to the reagent thing: fine drug powder of the present invention, 1g contain crude drug 8.33g, are provided lot number by Xi'an Green Valley Pharmaceutical Co., Ltd.: 020316.Be made into 64%, 32%, 16% drug suspension of the present invention with distilled water, shake up during administration.The oral 60g crude drug of per day for adults, promptly clinical human dosage are 0.86g/kg (body weight is in 70kg).

Positive control drug: yiganning granules, produce by Guangxi Banyu Pharmaceutical Co., Ltd, authentication code: ZZ-5010-defends in osmanthus the accurate word (1990) of medicine No. 079023, product batch number 020120.This test rat dosage is 5.3g crude drug/kg (a human dosage 5 times), is made into the suspension of 53g crude drug/100ml with distilled water.

Reagent: carbon tetrachloride (CCI ₄), analytical pure, chemical experimental factory, Laiyang produces (GB688-92), lot number: 20011030, face with preceding 25% the oil solution that is diluted to olive oil.020031), ALT test kit (lot number: 010071), total protein test kit (product batch number: 020301) and albumin reagent box (product batch number: 020302), produce AST test kit (lot number: by Beijing Zhongsheng Biological Engineering High Technology Company.Hyaluronic acid (HA) radioimmunoassay kit, Henan Province's Jiaozuo City liberation immune diagnostic reagent institute is produced lot number: 02022; Laminin (LN) radioimmunoassay kit, Henan Province's Jiaozuo City liberation immunologic diagnosis institute is produced lot number: 020225; III procollagen type (PC-3) radioimmunoassay kit (counterbalanced procedure), lot number: 020201, provide by Shanghai INM biotechnology center.

(3), test method

Get SD kind rat, be divided into 6 groups immediately.Except that the normal control group, all the other are respectively organized according to literature method, subcutaneous injection 25%CCl ₄Olive oil solution 0.2ml/100g, 2 times weekly, continuous 12 weeks, totally 24 times.At injection CCl ₄The time, the large, medium and small dosage group of medicine of the present invention is irritated the drug suspension of the present invention that stomach gives 64%, 32%, 16% (crude drug) respectively, and dosage is followed successively by 6.4g crude drug/kg, 3.2g crude drug/kg and 1.6g crude drug/kg; The YIGANNING group gives YIGANNING 5.3g/kg; Model group gavages ordinary water.Each organizes administration every day 1 time, continuous 12 weeks, administration volume 1ml/100g.After the last administration 24 hours, use the pentobarbital sodium anesthetized animal, anatomic observation liver outward appearance and other visible pathological changes, through abdominal aortic blood, separation of serum carries out blood biochemical and learns inspection.It is fixed to win the accurate title of liver, gets leftlobe of liver and carries out pathological examination.

(4), detect index and method

1, blood biochemical is learned and is checked: measure serum AST (continuous detecting method), ALT (continuous detecting method), total protein (TP, biuret method) and albumin (Alb, bromocresol green method) with XD-811C type biochemistry analyzer.

2, serum laminin, hyaluronic acid, III procollagen type measured by radioimmunoassay.

3, hepatic pathology: liver is weighed, and calculates liver coefficient (g/100g body weight), and get in the leftlobe of liver 1/3 and use 10% formalin fixed, the routine paraffin wax embedding, section, HE dyes, the pathological change of observation by light microscope hepatic tissue.

(5), statistical method

The measurement data of test gained is through the analytical control of t value method, and ranked data carry out statistical analysis with the Ridit method, the significance of comparable group differences.

(6) result

1, general situation: the model group animal is than the obvious flavescence of blank group fur, fluffy and disorderly, lethargy, and body weight gain is slow, and the general situation of other each treated animal has in various degree improvement than model group, but does not see significant difference.Test each treated animal stochastic sampling of later stage cuts open inspection and shows that Hepar Mus has enlargement in various degree after the modeling, and medicine of the present invention is to CCl ₄Cause the rat chronic hepatic injury certain curative effect is arranged.

2, to the influence of serum AST, ALT, Alb and TP (table 3-1)

Table 3-1: to the influence of serum AST, ALT, Alb and TP

Group number of animals ALT (U/L) AST (U/L) TP (g/L) Alb (g/L)

Normal control group 14 49.6 ± 7.5 ^*148.9 ± 28.8 ^*62.5 ± 6.5 31.6 ± 5.0

Model group 20 180.7 ± 108.5 291.2 ± 109.7 66.3 ± 6.5 30.6 ± 8.5

YIGANNING group 15 52.7 ± 9.4 ^*149.5 ± 27.6 ^*71.9 ± 7.7 ^*31.8 ± 10.3

Small dose group 17 50.9 ± 17.0 ^*149.6 ± 33.1 ^*66.8 ± 5.5 30.3 ± 9.2

Middle dosage group 20 53.2 ± 10.4 ^*146.1 ± 19.7 ^*66.8 ± 9.1 29.8 ± 12.6

Heavy dose of group 20 52.9 ± 8.9 ^*156.4 ± 26.5 ^*73.3 ± 9.4 ^*27.5 ± 10.8

Annotate: compare * P＜0.05 * * P＜0.01 with model group

It is as shown in the table, and model group animal serum AST and ALT obviously raise, and with the normal group comparing difference significance meaning (P＜0.01) arranged all, and CCl is described ₄Cause the success of rat chronic liver damage animal model; Medicine 6.4g crude drug/kg of the present invention, 3.2g crude drug/kg and 1.6g crude drug/kg irritates stomach can obviously reduce animal serum AST and ALT, with the model group comparing difference significance meaning (P＜0.01) is arranged all, the yiganning granules agent also has significant effect of reducing enzyme levels (P＜0.01).

Model group total serum protein and albumin and normal control group be no significant difference (P＞0.05) relatively, illustrates not cause hypoproteinemia as yet; The heavy dose of group of medicine of the present invention compares with model group, and total serum protein is higher than model group, and difference has significance meaning (P＜0.05).

3, to the influence of serum LN, HA and PC-3 (table 3-2)

Blood-serum P C-3, LN, HA level and the hepatic fibrosis order of severity are high-positive correlation, are cirrhotic early diagnosis of hepatic fibrosis and curative effect of medication, the significant index of post-evaluation more.The result shows that HA and PC-3 obviously raise in the model group animal serum, and relatively there were significant differences (P＜0.01,＜0.01) with normal group; Medicine 3.2g crude drug/kg of the present invention can obviously reduce LN, HA and PC-3, with the model group comparing difference significance meaning (P＜0.05,＜0.01,＜0.01) is arranged all; Medicine 6.4g crude drug/kg of the present invention can obviously reduce LN (P＜0.05).Prompting injects CCl for rat continuously ₄Can cause hepatic fibrosis, medicine of the present invention can suppress liver fibrosis process.

Table 3-2: to the influence of serum LN, HA and PC-3 (X ± S)

Animal

Group LN (ng/ml) HA (ng/ml) PC-3 (ug/L)

Number

Normal control

Organize 14 84.72 ± 10.16 61.92 ± 42.80 ^*35.92 ± 9.44 ^*

Model group 20 86.33 ± 7.78 173.38 ± 132.18 49.98 ± 4.86

YIGANNING group 15 81.35 ± 9.69 78.94 ± 35.28 ^*44.59 ± 5.74 ^*

Small dose group 17 80.58 ± 10.52 108.87 ± 36.55 47.41 ± 5.33

Middle dosage group 20 81.10 ± 7.45 ^*84.88 ± 35.76 ^*44.56 ± 5.98 ^*

Heavy dose of group 20 79.32 ± 5.58 ^*121.40 ± 51.76 47.41 ± 3.27

Annotate: compare * P＜0.05 * * P＜0.01 with model group

5, liver organization pathological examination results

(1) perusal: do not see ascites at each treated animal intraperitoneal when cuing open inspection.The normal rat liver surface is smooth, and color is dark red and matter is soft; The model group animal livers is thick, and is rough, and tuberosity is in various degree arranged, and color is reddish brown, edge seal circle, and quality is hard, and the liver enlargement is obvious.Each dosage group liver color of medicine of the present invention, edge, quality have improvement in various degree.Each treated animal liver coefficient (g/100g body weight) the results are shown in Table 3-3.

Table 3-3: to the influence of liver coefficient (g/100g body weight)

Group number of animals dosage (g/kg) liver coefficient (g/100g body weight)

Normal control group 14-2.73 ± 0.30 ^*

Model group 20-4.08 ± 0.43

YIGANNING group 15 5.3 2.94 ± 0.31 ^*

Small dose group 17 1.6 3.34 ± 0.36 ^*

Middle dosage group 20 3.2 3.08 ± 0.18 ^*

Heavy dose of group 20 6.4 3.18 ± 0.23 ^*

Annotate: compare * * P＜0.01 with model group

The result shows that model group animal livers coefficient (g/100g body weight) is significantly higher than normal control group (P＜0.01), and CCl is described ₄When causing the rat chronic hepatic injury, liver enlargement phenomenon is arranged.Medicine 6.4g crude drug/kg of the present invention, 3.2g crude drug/kg and 1.6g crude drug/kg all can obviously reduce the animal livers coefficient, with the model group comparing difference all have the significance meaning (P＜0.01,＜0.01,＜0.01).

(2) pathological examination result: see Table 3-4 (to the influence of liver degeneration) and table 3-5 (to the outgrowth influence of hepatic fibrosis).

Table 3-4: to the influence of liver degeneration

Animal pathological changes degeneration classification

Group

P

Number-±+++ +++

Normal control group 14 12 1100＜0.05

Model group 20 0124 13

YIGANNING group 15 03831＜0.05

Heavy dose of group 17 13562＜0.05

Middle dosage group 20 14564＜0.05

Small dose group 20 02684＜0.05 is annotated: degenerative disease variation grade standard-hepatocyte and lobules of liver structure are normal, no pathological changes ± accidental individual cells degeneration pathological changes+droplet fat is preapred for an unfavorable turn of events and the hepatocyte cloudy swelling accounts for below 1/3 of lobules of liver ++ and big droplet fat attenuates, and born of the same parents are assorted to deposit, simultaneously drip the fat born of the same parents that attenuate greatly and spread all over lobules of liver, destroy the lobules of liver normal configuration fully with the loose 1/3～2/3+++ that accounts for lobules of liver of hepatocyte bag slurry

Shown in the table 3-4, almost the different cloudy swelling of degree appears to model group in all animals liver, endochylema is loose and the balloon sample becomes (comprising that fat becomes).Each dosage group pathological changes of medicine of the present invention obviously alleviates, and with the model group comparing difference significance meaning (all P＜0.05) is arranged all.

Table 3-5 is to the outgrowth influence of hepatic fibrosis

The fibroplasia classification

The group number of animals

P

0 I II III IV V

Normal control group 14 14 00000＜0.01

Model group 20 10 53110

YIGANNING group 15 923100

Heavy dose of group 17 13 31000＜0.05

Middle dosage group 20 11 42300

Small dose group 20 10 33310 grade scales: 0 grade of hepatocyte is normal, fibroplasia I level collagen fiber slightly stretch out the extension of II level collagen fiber obviously from the portal area or around the central vein, but not holding lobules of liver III level collagen fiber extends obviously, interconnection, hold lobules of liver but not too complete IV level collagen fiber extend obviously, interconnection, form pseudolobuli, based on big pseudolobuli.V level lobules of liver structure is destroyed fully, forms the pseudolobuli that varies in size, and collagen fiber are thick.

Table 3-5 shows that the model group animal livers has fibroplasia in various degree, with normal group animal comparing difference significance meaning (P＜0.01) is arranged, but fibroplasia formation rate and degree is abundant not enough.Heavy dose of treated animal hepatic fibrosis hypertrophy of medicine of the present invention and model group compare, and difference has significance meaning (P＜0.05).

(7), conclusion

Use CCl ₄Injection causes rat chronic hepatic injury and hepatic fibrosis repeatedly, irritates stomach medicine of the present invention simultaneously, is index with serum biochemistry and liver organization pathological examination, observes the influence of medicine of the present invention to this model.The result represents that medicine 6.4g crude drug/kg of the present invention, 3.2g crude drug/kg and 1.6g crude drug/kg can obviously reduce animal serum AST and ALT, with the model group comparing difference significance meaning (P＜0.01) is arranged all; Medicine 3.2g crude drug/kg of the present invention can obviously reduce LN, HA and PC-3, with the model group comparing difference significance meaning (P＜0.05,＜0.01,＜0.01) is arranged all; Medicine 6.4g crude drug/kg of the present invention can obviously reduce LN (P＜0.05).Pathological research shows that medicine of the present invention can obviously reduce the liver coefficient of animal chronic hepatic injury, alleviates inflammatory disorderses such as cloudy swelling in the lobules of liver, endochylema are loose, balloonization, alleviates hepatic fibroplasia.Medicine of the present invention is to CCl ₄Inductive rat chronic hepatic injury and hepatic fibrosis have protective effect.

Four, to effect of immunologic function

(1), to the influence of mice carbon clearance

Purpose adopts the carbon clearance method, observes the influence of medicine of the present invention to mice reticuloendothelial system phagocytic function.

Method is divided into 7 groups with animal, gastric infusion 7 days, and tail vein injection 10% india ink, the injection back was got blood in 2,10 minutes respectively, measured optical density OD value in wavelength 600nm place.

Medicine of the present invention as a result (9g/kg) is accelerated mice carbon clearance speed, and prompting this product can strengthen the phagocytic function of reticuloendothelial system.

1, test objective

Observe the influence of medicine of the present invention with the carbon clearance method to mice reticuloendothelial system phagocytic function.

2, test material

(1) experimental animal: healthy ICR kind is a mice, each property of male and female, and body weight 20 ± 2 gram is provided by Xi'an Jiaotong University Medical College's Experimental Animal Center, the quality certification number: the moving word of doctor 08-004 number.Raising condition: ShanXi Chinese Medicine Academy pharmacology of Chinese materia medica laboratory (national traditional chinese medical science administration Chinese medicine scientific experiment chamber classification: secondary).

(2) medicine and reagent

Be subjected to the reagent thing: fine drug powder of the present invention, 1g contain crude drug 8.33g, are provided lot number by Xi'an Green Valley Pharmaceutical Co., Ltd.: 020406.Be made into the drug suspension of the present invention of 11.25%, 22.5%, 45% (crude drug) with distilled water, shake up during administration.

Positive control drug: yiganning granules, produce by Guangxi Banyu Pharmaceutical Co., Ltd, authentication code: ZZ-5010-defends in osmanthus the accurate word (1990) of medicine No. 079023, product batch number 020120.This test mice dosage is 7.4g/kg, is made into the suspension of 37% (crude drug) with distilled water.Levamisole hydrochloride tablets, the 25mg/ sheet, Nanjing second pharmaceutical factory produces, authentication code: No. the 254011st, the accurate word (1998) of Su Wei medicine, product batch number: 20020401, be made into suspension with distilled water, mouse stomach dosage is 30mg/kg.

3. experimental technique and result

Get ICR kind male mice, be divided into 6 groups at random.The large, medium and small dosage group of medicine of the present invention is irritated stomach respectively and is given 45%, 22.5% and 11.25% drug suspension of the present invention, and dosage is followed successively by 9g crude drug/kg, 4.5g crude drug/kg and 2.25g crude drug/kg; Levamisole is irritated stomach levamisole 200mg/kg; The YIGANNING group gives YIGANNING 7.4g/kg; The blank group is irritated ordinary water.Administration every day 1 time, continuous 10 days, administration volume 0.2ml/10g.After the last administration 2 hours, give mouse tail vein injection 10% india ink 0.2ml/ only.Blood 20ul was got from the eyeball rear vein beard respectively in 2 and 10 minutes in the injection back, join lysed erythrocyte in the test tube that the 4ml distilled water is housed, measure optical density OD value in wavelength 600nm place with 721 type spectrophotometers then, be calculated as follows the speed K of carbon clearance in the interior serum of unit interval.The results are shown in Table 4-1. $K=\frac{l_{0}g{\mathrm{OD}}_2-l_{0}g{\mathrm{OD}}_{10}}{t_{10}-t_2}$ OD ₂And OD ₁₀Be respectively the OD value of 2 and 10 minutes institute's blood samplings behind the injection india ink, t ₂And t ₁₀For getting the blood time.The result is through the t check diversity between each group and blank group relatively.

Table 4-1: to the influence of mice carbon clearance (X ± S)

Group number of animals dosage (g/kg) carbon clearance index (K)

Blank group 11-0.00829 ± 0.00695

Levamisole group 10 0.2 0.01853 ± 0.00548 ^*

YIGANNING group 11 7.4 0.1032 ± 0.00550

Small dose group 13 2.25 0.00888 ± 0.00446

Middle dosage group 12 4.5 0.00953 ± 0.00585

Heavy dose of group 13 9 0.01396 ± 0.00565 ^*

Annotate: compare * P＜0.05 * * P＜0.01 with model group

The result shows, levamisole group carbon clearance index significantly increases (P＜0.01) than the blank group, (the 9g crude drug/kg) the carbon clearance index significantly increases (P＜0.05) than the blank group to the heavy dose of group of medicine of the present invention, though other two dosage group carbon clearance speed are fast than the blank group, difference does not have significance meaning (P＞0.05).Point out medicine of the present invention that mice reticuloendothelial system phagocytic function is had potentiation.

4, brief summary

Medicine of the present invention can promote mice carbon clearance speed, and mice reticuloendothelial system phagocytic function is had potentiation.

(2), Turnover of Mouse Peritoneal Macrophages is engulfed the influence of sheep red blood cell

Purpose is observed medicine of the present invention Turnover of Mouse Peritoneal Macrophages is engulfed the sheep red blood cell effect, and detection of drugs is to the influence of body non-specific immunity.

Method is given mice perfusion 10 days continuously, lumbar injection 5% sheep red blood cell (SRBC) 0.2ml/10g, after 10 hours, put to death animal, the flushing peritoneal fluid, smear, oily mirror are engulfed the macrophage number of sheep red blood cell and the sheep red blood cell sum of being engulfed in 200 macrophage numbers of meter down, calculate phagocytic index and phagocytic percentage.

(9g crude drug/kg) Turnover of Mouse Peritoneal Macrophages is engulfed the sheep red blood cell function has facilitation to medicine of the present invention as a result.

1, test objective: observe medicine of the present invention is engulfed the sheep red blood cell function to Turnover of Mouse Peritoneal Macrophages influence.

2, test material: experimental animal, be subjected to reagent thing, yiganning granules and levamisole with the test 4-1.

3, experimental technique and result

Get ICR kind male mice, be divided into 6 groups at random.The large, medium and small dosage group of medicine of the present invention is irritated stomach respectively and is given 45%, 22.5% and 11.25% drug suspension of the present invention, and dosage is followed successively by 9g crude drug/kg, 4.5g crude drug/kg and 2.25g crude drug/kg; The levamisole group is irritated stomach levamisole 30mg/kg; The YIGANNING group gives YIGANNING 7.4g/kg; The blank group is irritated ordinary water.Administration every day 1 time, continuous 10 days, administration volume 0.2ml/10g.After the last administration 1 hour, every Mus lumbar injection 5% sheep red blood cell (SRBC) 0.2ml/10g took off cervical vertebra and puts to death after 10 hours, and it is fixing to face upward the position, and the sterilization abdominal part is cut off skin, injects normal saline or A Shi liquid 2～2.5ml through the abdominal cavity.Rotated 1 minute, and extracted abdominal cavity washing liquid 1ml, drip and be applied on the clean slide, every 0.2ml placed 37C incubator incubation 30 minutes, took out slide, dropped into rinsing in the normal saline, to remove the not cell of paster.Dry, fix 5 minutes with acetone-methanol (1: 1), dyeed 3～5 minutes with 4% Ji Mu Sa-Rui Teshi liquid, the distilled water rinsing is dried.Under oily mirror, engulf the macrophage number of sheep red blood cell and the sheep red blood cell sum of being engulfed in 200 macrophage numbers of meter, be calculated as follows phagocytic index and phagocytic percentage.

Table 4-2: to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function (X ± S)

Group number of animals phagocytic percentage (%) phagocytic index

Blank group 12 29.5 ± 10.4 0.433 ± 0.168

Levamisole group 13 39.4 ± 8.6 ^*0.687 ± 0.141 ^*

YIGANNING group 10 47.5 ± 6.2 ^*1.015 ± 0.175 ^*

Small dose group 15 36.4 ± 12.4 0.648 ± 0.220 ^*

Middle dosage group 11 30.7 ± 10.7 0.534 ± 0.221

Heavy dose of group 10 40.9 ± 12.9 ^*0.689 ± 0.283 ^*

Compare with the blank group: * P＜0.05 * * P＜0.01

The result shows that (9g crude drug/kg) can improve phagocytic percentage (%) and the phagocytic index of Turnover of Mouse Peritoneal Macrophages being engulfed sheep red blood cell compares with the blank group medicine of the present invention, and difference has significance meaning (P＜0.05).Medicine of the present invention (2.25g crude drug/kg) can improve phagocytic index.Prompting, medicine of the present invention can strengthen the function that Turnover of Mouse Peritoneal Macrophages is engulfed sheep red blood cell.

(3), to 2, the 4-dinitrofluorobenzene causes the influence of mice delayed allergy (DTH)

Purpose observe medicine of the present invention to 2,4-dinitrofluorobenzene (DNFB) causes the influence of mice delayed allergy.

Each treated animal of method is total to administration the 9th day, administration the 3rd day, be applied in 1 sensitization of abdominal part depilation place with 1%DNFB acetone soln 50ul, sensitization the 5th day, smear DNFB20ul attacks in the auris dextra two sides, after 24 hours, the cervical vertebra dislocation is put to death, and cuts left and right sides auricular concha, sweep away diameter 8mm auricle with card punch, weigh, calculating left and right sides auricle weight difference is the swelling degree, compares.

Big or middle two the dosage groups of medicine of the present invention as a result all obviously reduce by the swollen degree of the caused ear of delayed allergy (P＜0.01,＜0.05).

1, test objective: with 2, the 4-dinitrofluorobenzene is that antigen is given mouse sensitization, observe medicine of the present invention and attack the influence of the delayed allergy (DTH) that causes to accepting this antigen once more.

2, test material

(1) laboratory animal: with experiment 4-1.

(2) medicine and dosage: with experiment 4-1.

3, test method: animal is divided into attack group, sensitization attack group, levamisole group, YIGANNING group and the large, medium and small dosage group of medicine of the present invention at random.Each group is by dosage and the method gastric infusion of experiment 4-1, every day 1 time, continuous 8 days.Administration the 3rd day, except that the attack group, all the other each groups are applied in 1 sensitization of abdominal part depilation place with 1%DNFB acetone soln 50ul.Sensitization the 5th day smears 1%DNFB acetone soln 20ul for whole mouse right ears two sides, attacks.After 24 hours, mice is put to death in cervical vertebra dislocation, cuts left and right sides auricular concha, sweeps away the auricle of diameter 8mm with card punch, weighs.The difference of left and right sides auricle weight is the swelling degree, in order to the degree of reflection delayed allergy (DTH).The result adopts the T check to compare the significance of each administration group and sensitization attack group group difference.The results are shown in Table 4-3.

It is as shown in the table, and the swelling degree of sensitization attack group auricle significantly improves (P＜0.01) than the attack group, illustrates 2,4-dinitrofluorobenzene (DNFB) causes the success of mice delayed allergy model.Medicine of the present invention is little, in, heavy dose of group all can significantly suppress the Mus ear swelling that DNFB causes the mice delayed allergy (P＜0.01,＜0.01,＜0.01).

Table 4-3: to 2,4-dinitrofluorobenzene (DNFB) causes the influence (X ± S) of mouse DTH

Group dosage (g/kg) number of animals swelling degree (mg)

Attack group-11 0.63 ± 0.64 ^*

Sensitization attack group-10 2.25 ± 0.61

YIGANNING group 5.3 13 0.60 ± 0.39 ^*

Small dose group 2.25 12 0.61 ± 0.70 ^*

Middle dosage group 4.5 12 0.44 ± 0.29 ^*

Heavy dose of group 9 11 0.34 ± 0.34 ^*

Annotate: compare * P＜0.05 * * P＜0.01 with sensitization attack group

4, brief summary

Medicine of the present invention has inhibitory action to the mice delayed allergy that DNFB causes.

Toxicological study 1, studies on acute toxicity:

According to the requirement of " Chinese medicine (new drug) research guide ", observe the acute toxicity of medicine mouse stomach of the present invention, measure LD50 or maximum dosage-feeding.Method is in trial test, and (during g crude drug/100ml), carry out maximum dosage-feeding then and measure by 5 none death of animal give medicine Cmax 562.8% of the present invention with the 0.4ml/10g single for mice.During test, get 20 of ICR mices, male and female half and half are irritated stomach and are given 562.8% drug suspension of the present invention, every 4 hours once, continuous 2 times, observe a week after the administration.Do not see in observation period as a result that animal has acute toxic reaction.The maximum dosage-feeding of this product mouse stomach is 450g crude drug/kg, and this dosage is 523.3 times of clinical people's consumption, and (people is in 70kg, and the dosage of every day is 0.86g crude drug/kg).2, long term toxicity research:

According to the requirement of " Chinese medicine (new drug) research guide ", observe 6 months toxic reactions of rat oral gavage medicine of the present invention to body.

Method is divided four groups at random with 120 of SD kind rats, 30 every group.Heavy dose of group is irritated stomach medicine 51.6g of the present invention crude drug/kg (be equivalent to human dosage 60 times), in the dosage group irritate stomach medicine 25.8g of the present invention crude drug/kg (be equivalent to human dosage 30 times), small dose group gives medicine 12.9g crude drug/kg of the present invention (be equivalent to human dosage 15 times), and matched group gives the isometric(al) ordinary water.The administration cycle is 6 months, observes general situation every day, weighs weekly 1 time, and food ration and amount of drinking water are measured 5 days weekly.After the administration 3 months, get 10 animals for every group and carry out electrocardiogram, hematology, blood biochemical and pathological examination.All the other each treated animals continue administration, behind the administration end cycle, check These parameters by preceding method, and the residue animal stops administration, observes for 2 weeks, carry out electrocardiogram, hematology, blood biochemical and pathological examination again.

The result respectively organizes rat during administration and in convalescent period, and any and medicine all not occurring phenomenons such as causal toxic reaction and death; The overview of each administration treated animal, electrocardiogram, hematology, blood biochemical and pathological examination and matched group relatively, difference there are no significant meaning.

Conclusion: 51.6g crude drug/kg, 25.8g crude drug/kg and 12.9g crude drug/kg continuous irrigation stomach did not have long term toxicity to SD kind rat in 6 months and reacts, the body weight of animal, the water yield of ingesting, hematology, blood biochemical and electrocardiogram etc. there is not obviously influence, to main organs or the no tangible pathologic lesions of tissue such as liver, kidney, bone marrow, gastrointestinals.

In sum, main pharmacodynamics studies show that medicine of the present invention has anti-DHB effect; Can resist D-GlaN, TAA and CCl to some extent ₄Deng due to the chmice acute hepatic injury, reduce serum AST and ALT, alleviate the pathologic lesion of hepatic tissue; Chmice acute hepatic injury due to the chemical factor is had protective effect, can alleviate by various pathological changes in the inductive rat liver of CCI4, and can suppress hepatic fibrosis and hepatitis interstitialis chronica formation; The mice delayed allergy that DNFB is caused has inhibitory action.

Toxicological study shows: the maximum dosage-feeding of this product mouse stomach is 450g crude drug/kg, and this dosage is 523.3 times of clinical people's consumption, and (people is in 70kg, and the dosage of every day is 0.86g crude drug/kg).SD kind rat not being had long term toxicity in 6 months with this product 51.6g crude drug/kg, 25.8g crude drug/kg and 12.9g crude drug/kg continuous irrigation stomach reacts, the body weight of animal, the water yield of ingesting, hematology, blood biochemical and electrocardiogram etc. there is not obviously influence, to main organs or the no tangible pathologic lesions of tissue such as liver, kidney, bone marrow, gastrointestinals.

Description of drawings Fig. 1 is a preparation method process chart of the present invention

It is as follows that specific embodiment takes by weighing raw material Ganoderma 400g Herba Hedyotidis Diffusae 400g Fructus Gardeniae 200g Radix Curcumae 200g Radix Paeoniae Alba 166g Poria 166g Radix Salviae Miltiorrhizae 100g Radix Scutellariae 100g Radix Glycyrrhizae 50g production method by following proportioning;

Get Ganoderma, Herba Hedyotidis Diffusae, Radix Salviae Miltiorrhizae, be ground into coarse powder, add 70% alcohol reflux three times, the each extraction 2 hours adds the ethanol of 8 times of amounts for the first time, adds the ethanol of 6 times of amounts for the second time, for the third time, extracting solution filters, filtrate merges, and decompression recycling ethanol also is concentrated into the clear paste that relative density is 1.02～1.05 (60 ℃ of surveys), and is standby; Above-mentioned gained medicinal residues are mixed with Radix Scutellariae, Fructus Gardeniae, the Radix Paeoniae Alba, Radix Curcumae, Radix Glycyrrhizae, Poria Six-element medical material, add for the first time 12 times of water gagings immersions 1.5 hours, decocted 2 hours, second and third time respectively adds 10 times of water gagings, decocted respectively 2 hours, decocting liquid merges, filter, be concentrated into the clear paste of relative density 1.05～1.10 (60 ℃ of surveys), adding ethanol makes and contains the alcohol amount and reach 50%, stir evenly, leave standstill more than 24 hours, filter, filtrate recycling ethanol also is concentrated into the clear paste of relative density 1.02～1.05 (60 ℃ of surveys); With above-mentioned gained clear paste mix homogeneously, spray drying adds sugarcane 200 g and dextrin 40g fine powder to total amount 1000g, mix homogeneously, and the alcohol granulation with 75%, drying, granulate, promptly.

Inlet temperature is 168 ℃ during spray drying, and spray tower temperature is 78 ℃, and leaving air temp is 72 ℃, and rotating speed is 2.5 ten thousand rev/mins.

Claims (7)

1 one kinds of pharmaceutical compositions for the treatment of hepatitis B is characterized in that it is the medicament of being made by following materials of weight proportions:

Ganoderma 308-420 Herba Hedyotidis Diffusae 392-840 Fructus Gardeniae 182-280

Radix Curcumae 182-336 Radix Paeoniae Alba 112-336 Poria 112-420

Radix Salviae Miltiorrhizae 84-420 Radix Scutellariae 84-280 Radix Glycyrrhizae 28-252

2 pharmaceutical compositions according to claim 1, wherein the weight proportion of each raw material is:

Ganoderma 400 Herba Hedyotidis Diffusaes 400 Fructus Gardeniaes 200

Radix Curcumae 200 Radix Paeoniae Albas 166 Poria 166

Radix Salviae Miltiorrhizae 100 Radix Scutellariaes 100 Radix Glycyrrhizaes 50

3 pharmaceutical compositions according to claim 1 and 2 is characterized in that described medicament is a said dosage form on any pharmaceutics.

4 pharmaceutical compositions according to claim 3 is characterized in that described medicament is a powder.

The described preparation of drug combination method of 5 claim 1 is characterized in that

(1) gets Ganoderma, Herba Hedyotidis Diffusae, Radix Salviae Miltiorrhizae, be ground into coarse powder, add 70% alcohol reflux three times, the each extraction 2 hours adds the ethanol of 8 times of amounts for the first time, adds the ethanol of 6 times of amounts for the second time, for the third time, extracting solution filters, filtrate merges, and decompression recycling ethanol and to be concentrated into relative density be 1.02～1.05 clear paste is standby;

(2) above-mentioned (1) gained medicinal residues are mixed with Radix Scutellariae, Fructus Gardeniae, the Radix Paeoniae Alba, Radix Curcumae, Radix Glycyrrhizae, Poria Six-element medical material, add for the first time 12 times of water gagings immersions 1.5 hours, decocted 2 hours, second and third time respectively adds 10 times of water gagings, decocts respectively 2 hours, decocting liquid merges, filter, be concentrated into the clear paste of relative density 1.05～1.10, add ethanol and make and contain the alcohol amount and reach 50%, stir evenly, leave standstill more than 24 hours, filter, filtrate recycling ethanol also is concentrated into the clear paste of relative density 1.02～1.05;

(3) with above-mentioned (1), (2) gained clear paste mix homogeneously, spray drying, an amount of with sucrose and dextrin fine powder, mix homogeneously, the alcohol granulation with 75%, drying, granulate promptly gets powder.

6 according to the described preparation of drug combination method of claim 5, and requiring inlet temperature when it is characterized in that spray drying is 160 ℃～180 ℃, and spray tower temperature is 75～80 ℃, and leaving air temp is 70～75 ℃, and rotating speed is 2.5 ten thousand rev/mins.

7 according to the described preparation of drug combination method of claim 6, it is characterized in that be 5: 1 with the weight ratio of sucrose and dextrin.