CN1116891C - Hepatitis B treating medicine - Google Patents

Hepatitis B treating medicine Download PDF

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CN1116891C
CN1116891C CN01130956A CN01130956A CN1116891C CN 1116891 C CN1116891 C CN 1116891C CN 01130956 A CN01130956 A CN 01130956A CN 01130956 A CN01130956 A CN 01130956A CN 1116891 C CN1116891 C CN 1116891C
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medicine
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hepatitis
sage root
liver
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CN1336233A (en
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王旭中
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CHONGQING DUOPUTAI PHARMACEUTICAL Co Ltd
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Abstract

The present invention relates to a medicine for treating hepatitis B, which mainly comprises the following raw materials: ant, astragalus root, ginseng, wolfberry fruit, solomonseal rhizome, red sage root, white atractylodes rhizome, rehmannia root, red peony root, angelica, dandelion, giant knotweed rhizome, gentianae macrophyllae, atractylodes rhizome, polyporus, etc. Clinical tests prove that the curative rate of the medicine can reach 47.93% to 49.42%, and the total effective rate is from 86.98% to 88.80%.

Description

A kind of medicine for the treatment of hepatitis B and preparation method thereof
Invention field
The present invention relates to a kind of medicine for the treatment of hepatitis B, is the Chinese medicine preparation for the treatment of hepatitis B specifically.
Technical background
Hepatitis B is the high communicable disease of a kind of incidence of disease, and especially in China, hepatitis B patient and carrier are up to people more than 100,000,000. Chronic Hepatitis B is obstinate often, and part can develop into cirrhosis even liver cancer.
The open CN1194849A of Chinese patent discloses a kind of Chinese patent drug-medicinal powder for treatment of hepatitis B for the treatment of hepatitis B, mainly comprises the raw materials such as the fruit of Chinese magnoliavine, the red sage root, the radix paeoniae rubrathe, Radix Angelicae Sinensis, the Radix Astragali, leech, full worm, and the total effective rate of this medicine treatment acute and chronic hepatitis B is 84.64%.
The open CN1179323A of Chinese patent discloses a kind of Chinese medicine for the treatment of B-type hepatitis and preparation method thereof. This medicine component mainly comprises Radix Codonopsis, the Radix Astragali, the root of herbaceous peony, Danshen Tablets, globe amaranth, phoenix-tail fern etc., has clearing heat and detoxicating, soothing the liverly to be amusing, regulating qi-flowing for relieving pain, removing blood stasis for promoting tissue regeneration, removing body inner virus, to promote the effect such as liver cell regeneration.
Summary of the invention
The present invention is the improvement invention on same applicant's patent of invention ZL93 1 01758.0 bases. Therefore, the medicine that the purpose of this invention is to provide a kind of effective treatment hepatitis B, confirm through animal experiment, medicine of the present invention has significantly effects such as protecting the liver, promote liver regeneration, promotion liver BSP excretory function, enhancing immunologic function, can significantly suppress hepatitis B surface antibody and e antigen and significantly suppress duck hepatitis B virus infection.
Medicine of the present invention is made (by weight) by following raw material:
Ant 150-450 Radix Astragali 20-60 ginseng 10-30
Fruit of Chinese wolfberry 10-30 sealwort 10-30 red sage root 30-90
Bighead atractylodes rhizome 10-30 glutinous rehmannia 30-90 radix paeoniae rubrathe 10-30
Radix Angelicae Sinensis 10-30 dandelion 30-90 giant knotweed 15-45
Bark of ash 10-30 rhizoma atractylodis 10-30 umbellate pore furgus 10-30
Dried orange peel 10-30 hawthorn 10-30 Medicated Leaven 10-30
Fructus Hordei Germinatus 10-30 rascal 10-30
The preparation method of medicine of the present invention is: above-mentioned raw materials is ground into fine powder, sieves, mixing, per 1 kilogram of powder add refined honey 250-300 and restrain and an amount of water, make water-honeyed pill, and drying can obtain.
Perhaps, prepare in the following method medicine of the present invention: the ginseng pulverate is broken into fine powder, sieves; The red sage root merges extract with ethanol (preferred 90%) refluxing extraction secondary about the 2 hours first time about the 1.5 hours second time, filters, and filtrate recycling ethanol obtains red sage root extract; The bighead atractylodes rhizome, Radix Angelicae Sinensis, rhizoma atractylodis, dried orange peel, rascal extract volatile oil, and the aqueous solution after the distillation in addition device is collected; The red sage root dregs of a decoction behind 13 flavors such as the dregs of a decoction and all the other ants and the above-mentioned alcohol extract, boiling twice, 2 hours for the first time, 1.5 hours for the second time, filter, merging filtrate, filtrate are concentrated in right amount, are placed to room temperature, slowly stirring the lower ethanol that adds makes and contains alcohol and measure and reach about 60%, leave standstill, filter filtrate recycling ethanol; Merge this filtrate, it is the clear cream of 1.35-1.40 (50 ℃) that red sage root extract and the above-mentioned aqueous solution, reduced pressure concentration become relative density, adds above-mentioned ginseng fine powder mixing, granulation, drying are pulverized in low temperature drying, add above-mentioned volatile oil, mixing incapsulates and get final product.
The accompanying drawing summary
Fig. 1 is the duck serum DHBV-DNA dot hybridization autoradiograph of first experiment in the test three.
Fig. 2 is the duck serum DHBV-DNA dot hybridization autoradiograph of the second batch experiment in the test three.
Fig. 3 is the duck serum DHBV-DNA dot hybridization autoradiograph of the 3rd batch of experiment in the test three.
Preferred embodiment
Embodiment
Ant 300g Radix Astragali 40g ginseng 20g
Fruit of Chinese wolfberry 20g sealwort 20g red sage root 60g
Bighead atractylodes rhizome 20g glutinous rehmannia 60g radix paeoniae rubrathe 20g
Radix Angelicae Sinensis 20g dandelion 60g giant knotweed 30g
Bark of ash 20g rhizoma atractylodis 20g umbellate pore furgus 20g
Dried orange peel 20g hawthorn (Jiao) 20g Medicated Leaven (Jiao) 20g
Fructus Hordei Germinatus (Jiao) 20g rascal 20g
The ginseng pulverate is broken into fine powder, sieves; The red sage root extracts secondary with 90% alcohol reflux, and about 2 hours for the first time, about 1.5 hours for the second time, merge extract, filter, filtrate recycling ethanol obtains red sage root extract; The bighead atractylodes rhizome, Radix Angelicae Sinensis, rhizoma atractylodis, dried orange peel, rascal extract volatile oil, and the aqueous solution after the distillation in addition device is collected; The red sage root dregs of a decoction behind 13 flavors such as the dregs of a decoction and all the other ants and the above-mentioned alcohol extract, boiling twice, 2 hours for the first time, 1.5 hours for the second time, filter, merging filtrate, filtrate are concentrated in right amount, are placed to room temperature, slowly stirring the lower ethanol that adds makes and contains alcohol and measure and reach 60%, leave standstill, filter filtrate recycling ethanol; Merge this filtrate, red sage root extract and the above-mentioned aqueous solution, it is the clear cream of 1.35-1.40 (50 ℃) that reduced pressure concentration becomes relative density, adds above-mentioned ginseng fine powder mixing, low temperature drying, pulverize granulation, drying, add above-mentioned volatile oil, mixing incapsulates and get final product.
Certainly, medicine of the present invention can be made any formulation that is suitable for using clinically, such as pill, powder, oral liquid, condensed pill etc.Test oneThe pharmacodynamic study of medicine of the present invention
With CCl4Or immunological liver injury due to experimental acute and chronic liver injury and the BCG vaccine due to the D-galactosamine, gavage ALT, the AST vigor that this medicine can make unusual rising and descend, and alleviate fatty degeneration of liver, oedema, downright bad scope and degree; Can promote liver cell regeneration, reduce the hold-up of mouse BSP; To cell and humoral immunities such as macrophage phagocytic, T lymphocyte transformation, hemolytic antibody generations, humidification is also arranged simultaneously.
Test objective
The clinical testing of this medicine has certain curative effect to hepatitis B, now by animal experiment, observes the impact on hepatic injury pathological model and body's immunity.
Test material
1, animal: small white mouse has the Kunming kind, the NIH kind, and this institute breeds, BALB/C kind mouse and C57BL/6 kind mouse is available from Chinese medical courses in general institute institute of materia medica. Wistar kind big white mouse, this institute breeds.
2, medicine: medicine of the present invention; Bifendate, Jinzhou City pharmaceutical factory, 930420 batches; YIGANNING CHONGJI is produced by Jiuzhitang Pharmaceutical Factory; Lot number 9504210; Levamisole hydrochloride, Yan'an Pharmaceutical Plant, Shanghai produces, lot number 930107; Tortoise order collection, the Shanxi pharmaceutical factory of traditional Chinese medicine, 9110060 batches; Freeze dried BCG vaccine, Ministry of Public Health institute of biological products, 3940308 batches.
One, hepatoprotective effect
1, to the effect of mouse carbon tetrachloride acute liver damage
50 of Kunming kind small white mouses, body weight 20 ± 2g, the male and female dual-purpose is divided into 5 groups at random. 1 group of normal control, 2 groups gavage physiological saline, 3,4 groups of aqueous suspensions that gavage respectively medicine 8g/kg of the present invention, 4g/kg, 5 groups of aqueous suspensions that gavage DDB 0.2kg/kg once a day, give 6 continuously. After the last administration 1 hour, rear 4 groups of lumbar injection 0.2%CCl respectively4Oil solution 10ml/kg. Fasting be can't help water after 20 hours, and each organizes the mouse orbit venous blood collection, measured ALT (ALT) by the reitman-frankel method of improvement, and won liver and do the Pathomorphology inspection.
As seen from Table 1, the ALT that each administration group all can make pathologic raise obviously reduces (P＜0.01), and microscopically sees that the lobuli hepatis structure is substantially clear, the rarely seen slight cloudy swelling of liver cell, and sinus hepaticus is slightly narrow, is dispersed in the spotty necrosis kitchen range, and CCl4Model group is the most of vacuolar degeneration of liver cell or extensive cloudy swelling then, and majority is dispersed in a sheet necrosis region, forms large sheet around indivedual blood vessels and infiltrates, and the portal area cell infiltration is obvious.
Table 1 couple mouse CCl4The effect of reducing enzyme levels of acute liver damage
Group Number of mice Dosage (g/kg) ALT(μ)
This medicine of this medicine of normal control model DDB  10  10  10  10  10  8  4  0.2 33.6±1.40*** 117.4±17.2 73.4±15.1*** 55.2±11.0*** 48.8±16.2***
Compare * * * P＜0.01 with model group
2, on the impact of mouse D-galactosamine hydrochloride (D-Gal) acute liver damage
60 of Kunming kind small white mouses, 18-22g, male and female have concurrently, be divided at random 6 groups, 1,2 group of physiological saline, 3,4,5 groups of aqueous suspensions that give respectively this medicine 8g/kg, 4g/kg, 2g/kg, 6 groups of aqueous suspensions that give DDB 0.2g/kg are gavage 1 time every day, totally 6 times. After the last gavage 1 hour, rear 5 groups of lumbar injection D-Gal 0.65g/kg physiological salt liquids. After 20 hours, the blood sampling of eye socket veniplex is got serum and is surveyed ALT and AST (L-aminobutanedioic acid aminotransferase). Cut open and get liver, fixing, send pathologic finding.
As seen from Table 2, the ALT of model group and AST, and Normal group is relatively, significantly raises, and illustrates that liver function is subject to grievous injury. Compare with model group, this medicine is high, middle dosage group can make ALT and AST obviously reduce (P＜0.01), and low dose group also significantly reduces the vigor of ALT.
Histopathology result shows that the pathological change of high, medium and low dosage medication group Mouse Liver is basically identical with Normal group, compares with the D-GAL model group, and hepatocellular degree of injury obviously alleviates.
The effect of reducing enzyme levels of table 2 pair mouse D-GAL acute liver damage
Group Number of mice Dosage (g/kg)  ALT(μ) AST(μ)
Normal control 10  17±10*** 46±16***
Model 10  107±41 138±10
This medicine 10  8  40±15*** 87±26***
This medicine 10  4  47±20*** 67±15***
This medicine 10  2  64±16*** 129±12*
DDB 10  0.2  40±16*** 90±20***
Compare * * * P＜0.01, P＞0.05 with model group
Table 3 is on the impact of mouse Galactosamine-induced Liver Damage pathology
Group The mouse number The microscopy finding
Normal control 10 Normal
Model
10 Necrosis region, and the zonal necrosis between visible adjacent central veins of hepatic lobules
This medicine high dose 10 Same Normal group has no obvious necrosis region
Dosage in this medicine 10 Same Normal group has no obvious necrosis region
This medicine low dosage 10 8 examples are normal, and 2 routine pathologies are like the simulation group
DDB
10 Same Normal group has no obvious necrosis region
3, on the impact of immunological liver injury
Get 50 of female BALB/C mices, body weight 22 ± 2g is divided into 5 groups at random. Except Normal group gives with the volume physiological saline, give every caudal vein injection freeze dried BCG vaccine 2.5mg, be dissolved as 0.2ml for all the other 4 groups. Gavage respectively simultaneously physiological saline or liquid, every day 1 time, totally 12 days. After the last administration, give every mouse intravenous injection bacterial endotoxin 5000Eu (0.2ml), the Normal group injecting normal saline. After 16 hours, the blood sampling of eye socket veniplex, separation of serum is surveyed ALT and AST. Cut open and get liver and fix, carry out pathologic finding. The result shows that the high low dosage of this medicine all can reduce ALT and the AST active (table 4) of unusual rising, and illustrating also has same protective effect to immunological liver injury.
Histopathology result shows that medicine high and low dose group of the present invention can reduce liver cell point, sheet necrosis, oedema, hyperplasia and inflammatory cell infiltration in various degree, and its effect is similar to the positive control drug DDB.
Table 4 is on the impact of immunological liver injury transaminase vigor
Group Dosage Number of mice  ALT(μ) AST(μ)
Normal control 10  31±14*** 86±21***
Model 10  60±17 137±19
This medicine 8 10  42±12** 105±10***
This medicine 4 10  44±13** 116±20**
DDB 0.2 10  30±14*** 86±15***
Compare * * P＜0.05, * * * P＜0.01 with model control group
Table 5 is on the impact of immunological liver injury lesion tissue
Group The mouse number The microscopy finding
Normal control 10 Tissue is normal, acellular oedema, necrosis
Model
10 10 examples all have necrosis; 6 routine sheets also have the bridge joint phenomenon; 3 routine focus necrosis have the chronic inflammation cellular infiltration, and take monocyte as main, 2 routine necrosis are lighter in addition, and the bridge joint phenomenon is not obvious, but visible sheet is downright bad, and with edema.
This medicine high dose 10 3 routine spotty necrosis, companion cell oedema 2 examples, wherein 1 example and the as seen little focal necrosis of minority, there is lighter chronic inflammation cellular infiltration this routine portal area, and 2 examples change not remarkable in addition; 1 routine edema.
This medicine low dosage 10 4 routine focal necrosis, 4 routine spotty necrosis.
DDB 10 The chronic inflammation cellular infiltration that 1 routine edema and portal area are slight, the visible minority special mess shape of 3 examples or focal necrosis, wherein 1 example has more living cell infiltration, wherein has 1 example that the bridge joint phenomenon is arranged.
4, on the impact of rat carbon tetrachloride chronic liver injury
49 of big white mouse, body weight 150-200g, 25 of ♂, 24 of ♀. Be divided at random 6 groups, i.e. the high, medium and low dosage of this medicine, positive control drug YIGANNING CHONGJI, physiological saline and Normal group. Every day, gastric infusion was 1 time, totally 8 weeks (56 days). Except Normal group, during all the other 5 groups of administrations every 3 days in every mouse nape hypodermic injection 40%CCl4Oil solution 3ml/kg, initial dose is 5ml/kg. After the last administration 24 hours, the blood sampling of eye socket veniplex, separation of serum is surveyed total protein, albumin content, and ALT, AST are active. Sacrificed by decapitation is got the hepatic tissue of every same leaf site of rat, and is fixing, send check pathological section.
The result shows, each dosage of this medicine can make the ALT vigor of unusual rising that obvious reduction is arranged, but the effect to the AST that raises only has high dose performance reduction to total protein and the albuminous trend that increase is arranged, but compares there was no significant difference (P＞0.05) with control group.
Table 6 is on the impact of chronic liver injury rat blood serum transaminase and protein
Group Dosage (g/kg) Number of rats  ALT(μ) AST(μ) Total protein (g/kg) Albumin (g/L)
Normal control  8  27±8*** 29±12*** 70.5±3.1*** 39.1±7.3*
Model  8  180±42 105±12 60.4±7.3 36.7±2.6
This medicine 8  9  92±46*** 82±24** 66.7±8.3* 39.2±2.4*
This medicine 4  8  106±42*** 186±24* 66.4±5.8* 36.4±2.6*
This medicine 2  8  121±21*** 104±2* 66.4±7.5* 37.2±2.1*
YIGANNING CHONGJI 17  8  105±13*** 89±7*** 66.3±11.2* 36.5±3.3*
Compare * * * P＜0.01, * * P＜0.05, * P＞0.05 with model group
Hepatic tissue section microscopy finding (table 7) compares with model group, the hepatocellular steatosis of administration group, and oedema and necrosis, obviously less and lighter.
Table 7 couple CCl4The impact of chronic liver injury rat tissue pathology
Group The mouse number The microscopy finding
Model
10 10 examples all have obvious steatosis, and 9 examples are edema obviously, and 2 examples are arranged with the collagenous fibres hyperplasia around the central vein, and 1 visible 2 the little focal necrosis of example and Fibrotic larger focal necrosis also has obvious collagenous fibres hyperplasia around the central vein in addition.
This medicine high dose 10 The visible steatosis of 6 examples, 5 routine liver fat become very light. 4 routine edemas, 2 routine spotty necrosis.
Dosage in this medicine 10 10 examples are visible hepatic cell fattydegeneration all, 6 routine edemas, 2 routine little focal necrosis.
This medicine low dosage 10 10 examples are visible hepatic cell fattydegeneration all, 3 routine little focal necrosis.
YIGANNING CHONGJI 10 3 examples are steatosis obviously, and 7 examples are lighter in addition; 7 routine edemas, 4 examples have little necrosis region.
Two, medicine of the present invention is on the impact of Mouse Liver power of regeneration
Kunming mouse, 18-22g, ♂ ♀ half and half is divided into 5 groups at random, i.e. control group, DDB (200mg/kg), this medicine high dose group (8g/kg), middle dosage group (4g/kg) and low dose group (2g/kg). Im cloxacillin sodium after the wrapping of experiment first day 1.5% calmine anesthesia (ip) excision left lobe of liver. Begin administration next day, administration is three altogether, and the last administration was weighed after 1 hour, puts to death, and claims liver heavy, and fixed preparation, and microscope inspection counting neonatal cell number calculates liver regeneration degree and liver coefficient. The results are shown in Table 8.
Figure C0113095600091
Liver weighs the ratio of 0.31 and former liver weight heavy for the excision liver after the heavy B regeneration of A excision liver
The impact that table 8 medicine of the present invention is heavy on the mouse Liver Regeneration (X ± SD)
Group Number of animals Dosage (g/kg) Regeneration degree (%) Liver index (%) The neonatal cell number
Control group 8  114.4±85.0 4.4±0.5  27.6±7.2
The DDB group 8  0.2  160.8±55.4* 4.7±0.4* 36.5±10.4**
This medicine high dose group 10  8  168.7±57.2* 4.6±0.3* 37.3±8.9**
Dosage in this medicine 10  4  162.5±79.7* 4.6±0.7* 34.5±11.9*
Group
This medicine low dose group 8  2  143.5±62.0* 4.4±0.5  33.6±8.6
Compare * P＞0.05, * * P＜0.05 with control group
By as seen from Table 8, medicine of the present invention has the trend that promotes liver regeneration, the liver regeneration degree is improved, but statistics is remarkable (P＞0.05) not, DDB is similar to its effect, DDB and medicine of the present invention all can make in the mouse liver leaflet neonatal cell count showed increased, and nuclear fission increases mutually, with control group significant (P＜0.05) is arranged relatively.
Three, on the impact of mouse liver BSP excretory function:
Kunming mouse, body weight 20 ± 1.2g is divided into 5 groups at random: medicine of the present invention high (8g/kg), in (4g/kg), low (2g/kg) three dosage groups, DDB group (200mg/kg) and blank group. Gastric infusion 3 days, once a day. After the last administration 1 hour, and tail iv BSP (sulfobromophthalein sodium) 100mg/kg (be made into 1% solution with physiological saline, 0.2ml/10g), behind the iv 15 minutes, the blood sampling of eye socket veniplex, centrifugal. Get 0.1ml serum, add 0.01mol/L HCl 5ml, survey absorption value (OD) in the 520nm place with UV-240 type spectrophotometer, add again one of 2mol/L NaOH, survey OD; The difference of twice OD is the storage allowance of BSP in blood, the results are shown in Table 9.
The result as seen, the high, medium and low dosage group of medicine of the present invention all has and promotes liver to drain, the equal contrast group of BSP retention is low.
Table 9 medicine of the present invention is drained the impact of BSP function on liver
Group Number of animals The residual absorbance of BSP The percentage that is equivalent to contrast
Control group 10  0.0318±0.0097 100％
This medicine high dose group 10  0.0206±0.0073*** 64.8％
Dosage group in this medicine 10  0.219±0.0054** 68.9％
This medicine low dose group 10  0.023±0.0084** 72.3％
DDB
10  0.0206±0.0056*** 64.8％
Compare * * P＜0.05, * * * P＜0.01 with control group
Four, on the impact of immunologic function
1, on the impact of mouse carbon particle clearance speed
Get 48 of Kunming mouses, 20 ± 2g divides 5 groups. Be this medicine high (8g/kg), in (4g/kg), low (2g/kg) three dosage groups, levamisol group and control group. Every day, gastric infusion was 1 time, totally 7 times. After the last administration 24 hours, tail vein injection india ink normal saline dilution liquid 10ml/kg. Rear 2 and 20 minutes of injection from eye socket veniplex blood sampling 20ul, adds among the 0.1% sodium carbonate liquor 2ml, shakes up. Measure trap in 680nm wavelength place with UV-240 type spectrophotometer; Index K and phagocytic index α are cleaned up in calculating. The result as seen, this medicine high dose can obviously improve the clearance rate of carbon granules foreign matter in the Mouse Blood, in, though low dosage increase that carbon is cleaned up index is not remarkable, but the phagocytic index of proofreading and correct by Unit Weight then is significantly improved (P＜0.05), illustrates that this medicine has the effect that strengthens the reticuloendothelial system phagocytic function. (table 10)
Table 10 is on the impact of mouse carbon particle clearance speed
Group Dosage (g/kg) The mouse number Clean up index K Phagocytic index α
Contrast  9  0.035±0.019  5.31±1.26
This medicine 8  9  0.060±0.017***  7.07±0.89***
This medicine 4  9  0.047±0.012*  6.48±0.78**
This medicine 2  10  0.040±0.007*  6.28±0.60**
Levamisol 0.01  10  0.064±0.022***  7.68±1.09***
Compare * * * P＜0.01, * * P＜0.05, * P＞0.05 with control group
2, on the impact of Phagocytosis By The Peritoneal Macrophages In Mice
100 of Kunming kind small white mouses, male, 22 ± 2g is divided into 10 groups at random. Every day, gastric infusion was 1 time, totally 10 days. The 6th～10 group in administration the 7th, 9 two days, respectively intraperitoneal injection of cyclophosphamide 70mg/kg. Each organizes mouse administration lumbar injection afternoon on the 10th 5% chicken red blood cell physiological saline 0.5ml, and the cervical vertebra dislocation method is put to death mouse after 16 hours, injects 2ml physiological saline in the abdominal cavity, gently rubs belly. The postabdomen center was cut off in 2 minutes, with the about 0.5ml of suction pipe sucking-off peritoneal fluid, dripped on slide, put 37 ℃ of incubators interior 30 minutes, used the not cell of paster of physiological saline flush away, airing. Fix with 1: 1 acetone methanol solution, the dyeing of Ji Mu Saruishi liquid, the chicken red blood cell sum of engulfing the macrophage number of chicken red blood cell in 200 macrophages of counting and being engulfed under the oily mirror is calculated phagocytic percentage and phagocytic index.
The results are shown in Table 11,12. Each dosage of this medicine all can obviously strengthen phagocytic rate and the phagocytic index of normal mouse macrophage, Immunodeficiency mouse for the endoxan inhibition, this medicine also can improve Peritoneal macrophage function and phagocytic index, only have low dosage and positive control drug tortoise to make the increase of set pair phagocytic index, statistics is without the difference of clear meaning.
Table 11 is on the impact of normal rat peritoneal macrophages phagocytic function
Group Dosage (g/kg) The mouse number Phagocytic rate (%) Phagocytic index
Contrast
 10  41.3±11.2 0.46±0.19
This medicine 8  10  69.1±10.1*** 0.66±0.21**
This medicine 4  10  64.2±9.9*** 0.76±0.11***
This medicine 2  10  55.7±11.6** 0.65±0.14**
Tortoise order collection 20  10  66.3±11.4*** 0.76±0.08***
Compare * * P＜0.05, * * * P＜0.01 with control group
Table 12 suppresses the impact of mouse phagocytic function on endoxan
Group Dosage (g/kg) The mouse number Phagocytic rate (%) Phagocytic index
Contrast
 10  34.4±6.6 0.42±0.17
This medicine 8  10  41.7±6.4** 0.63±0.17**
This medicine 4  10  42.7±5.8*** 0.59±0.18**
This medicine 2  10  42.4±4.6** 0.54±0.16*
Tortoise order collection 20  10  45.6±9.5*** 0.52±0.18*
Compare * P＞0.05, * * P＜0.05, * * * P＜0.01 with control group
3, on the impact of mouse delayed allergy (DTH)
Get 50 of NIH kind small white mouses, male and female half and half, 18～22g. Be divided at random the high, medium and low dosage of this medicine, 5 groups in levamisol and physiological saline etc. Every day, gastric infusion was 1 time, totally 8 times. Administration was given every caudal vein injection sensitization with 20% sheep red blood cell physiological saline suspension (SRBC) 0.2ml on 3rd, with 40% SRBC0.02ml, injected the left back sufficient sole of the foot of every mouse after the last administration; To inject the right back sufficient sole of the foot in contrast with volume physiological saline. After 24 hours, with the thickness of the sufficient sole of the foot about vernier caliper measurement, its difference large person of healing represents that the function of Cellular Immunity is stronger, as seen from Table 13, each dosage of this medicine all can significantly increase the thickness of the left back sufficient sole of the foot, and prompting has the effect of enhancing to the lymphocytic function of T.
Table 13 is on the impact of rat delayed allergy
Group Dosage The mouse number Left back sufficient sole of the foot thickness (mm)
Physiological saline  10  3.0±1.8
This medicine 8  10  6.9±2.0
This medicine 4  10  8.0±3.5***
This medicine 2  10  5.6±1.6***
Levamisol 0.01  10  6.1±1.8***
Compare * * * P＜0.01 with the salt solution group
4, on the impact of mouse spleen T lymphproliferation response
Get C57Plant 50 of mouse, 20 ± 2g, male and female half and half are divided into 5 groups at random. Gavage respectively liquid or physiological saline, every day 1 time, totally 7 days. Next day after the last administration, arteria carotis sacrificed by exsanguination mouse, the aseptic spleen of getting, preparation splenocyte suspension. NH with 0.155M4Cl liquid lysed erythrocyte, Hanks liquid are washed 2 times, use at last RPMI1640 complete nutrition liquid (containing 10% calf serum) resuspension, and adjusting cell concentration is 2.5 * 106/ ml expects the blue test of repelling through platform, and living cells is more than 95%.
The splenocyte suspension of getting this concentration adds in the 96 hole flat undersides, and every duplicate samples adds 6 holes, and every hole contains 5 * 105Individual cell. Add respectively the ConA in 4ug/ hole, and establish blank. Mixing is put 37 ℃, 5%CO2Cultivated 72 hours in the incubator. Stop cultivating front 6 hours, every hole adds respectively 1 μ Ci's3H-TdR, bull cell harvestor collecting cell are on glass fiber filter, and liquid scintillation counter is measured the cpm value.
The result shows: each dosage of this medicine all can promote the lymphocytic conversion of T that mitogen ConA stimulates, and improves its breeder reaction (table 14).
Table 14 is on the impact of mouse T lymphocyte breeder reaction
Group Dosage (g/kg) The mouse number   3H-TdR mixes cpm
Normal control  10  73099±18222
This medicine 8  10  1505414±42621***
This medicine 4  10  1496660±48421***
This medicine 2  10  128453±34980***
Levamisol 0.01  10  149316±27183***
Compare * * * P＜0.01 with control group
5, the impact that the mouse hemolytic antibody is generated
Get 50 of NIH kind small white mouses, male and female half and half are divided into 5 groups at random: this medicine high (8g/kg), in (4g/kg), low (2g/kg) dosage, levamisol (0.01g/kg) and physiological saline group. Every day, gastric infusion was 1 time, totally 7 days. After the 2nd administration, every mouse lumbar injection sheep red blood cell physiological saline suspension 0.2ml sensitization. After this administration 1 hour, the blood sampling of eye socket veniplex press literature method (Li Yikui, herbal pharmacology experimental methodology, 1991,159) and is measured the OD value, and calculating HD50 value (HC50). The result shows, the generation that this medicine is high, middle dosage all can obviously increase hemolytic antibody, and its effect is not as good as levamisol. (table 15)
The impact that table 15 generates the mouse hemolytic antibody
Group Dosage (g/kg) The mouse number  HC 50
Physiological saline  10  20.6±1.8
This medicine 8  10  26.1±2.7***
This medicine 4  10  24.6±2.4***
This medicine 2  10  21.8±1.2**
Levamisol 0.01  10  31.2±2.5***
Compare * * P＞0.05, * * * P＜0.01 with control groupTest two
Medicine of the present invention in 2215 cells to the inhibitory action of hepatitis b virus s antigen and e antigen
Materials and methods
One, medicine
Medicine of the present invention is brown ceramic powder, is made into 10mg/ml with nutrient solution during experiment, the aseptic filtration of 0.45um filter membrane.
Two, 2215 cells:
2215 clones of hepatitis type B virus (HBV) dna clone transfected with human HCC (Hep G2), U.S. Mount Sinai medical center makes up, the cultivation of going down to posterity voluntarily after the introduction.
Reagent, Eagles MEM dry powder, U.S. GIBCO company product; Hyclone, U.S. Hyclone Lab company product; G-418 (Geneticin), MEM preparation, U.S. Gibco company product; Glu, the import packing of Jing Ke chemical reagents corporation; HBsAg, HBeAg solid phase radioimmunoassay box is available from Beifang Inst. of Immune Reagents, Chinese Isotopes Co.'s product; Penicillin, streptomysin, North China Pharmaceutical Factory.
Experimental article and instrument
Blake bottle, Denmark Tunclon TM; Culture plate, 96 orifice plates, 24 orifice plates, U.S. Corning company product; The carbon dioxide incubator, U.S. Shel-lab product.
2215 cell culture fluids and reagent preparation
MEM nutrient solution 100ml: contain hyclone 10%, 3% glutamine 1%, G148 380/ml, each 100u/ml of penicillin and streptomycin adds 1ml, uses 5NaHCO3Transfer pH to 7.
Cell dissociation buffer: 0.25% pancreatin, with the preparation of Hanks liquid.
Four, experimental technique
(1) 2215 cell is cultivated
Add 0.25% pancreatin in the blake bottle that covers with 2215 cells, 37 ℃ digested 3 minutes, added nutrient solution piping and druming, went down to posterity at 1: 3, covered with in 10 days, and added the cell counting count board counting, be mixed with 100,000 every milliliter cell inoculated and cultured plates, the every hole 0.2ml of 96 orifice plates, the every hole 1ml of 24 orifice plates, 37 ℃ of 5%CO2Cultivated 24 hours, cell is tested after growing up to individual layer.
(2) medicine is to cell toxicity test
Medicine of the present invention is mixed with 10mg/ml solution with nutrient solution, is diluted to 0.156mg/ml with 2 times, adds 96 orifice plate Tissue Culture Plates, and every concentration 3 holes were changed same concentration liquid, and established without the drug cell control group in per 4 days. Because having pigment, medicine can not adopt decoration method, thus take the observation of cell pathology as index, 8 days microscopically observation of cell pathologies, destroying fully is 4,75% to be 3,50% to be 2,25% to be 1, anosisly becomes 0. Calculate every concentration liquid average cell lesion degree and suppress %. Press Reed Meuench method and calculate TC50, TC0.
Figure C0113095600141
A=log＜50% drug concentration B=log extension rate
(3) medicine is to HBeAg, HBsAg inhibition test
Every milliliter of 100,000 2215 cells are inoculated 24 porocyte culture plates, every hole 1ml, 37 ℃ of 5 %CO2Cultivated 24 hours, and added the following 2 times of dilution test liquids of non-toxic concn, 5 diluted concentrations are respectively 2.5mg/ml; 1.25mg/ml; 0.625mg/ml, 0.3125mg/ml; 0.156mg/ml, every concentration 3 holes, 37 ℃ of 5%CO2Cultivate, changed the cultivation of original content liquid in per 4 days, results nutrient solution in the time of the 8th day ,-20 ℃ of stored frozen, HBeAg and HBsAg are measured in three batches of experiments simultaneously. HBeAg, the HBsAg positive and negative control and cell contrast are established in experiment.
1, HBeAg, HbsAg measure: use Beifang Inst. of Immune Reagents, Chinese Isotopes Co.'s product, the solid phase radioimmunoassay box is measured, and method is seen specification, measures every hole cpm value with γ-calculating instrument.
2, effect of drugs calculates: calculate cell contrast and every concentration c pm average and standard deviation, P/N value and inhibition percentage (%), medium effective concentration (IC50) and therapeutic index (TI). (3) calculate medicine and suppress antigen medium effective concentration (IC50)
A=log＜50% drug concentration B=log extension rate
(4) medicine of the present invention to the therapeutic index (TI) of HBsAg and HBeAg, calculates cytotoxicity index cytopathy (TIc) by it in 2215 cell culture incubators.
Figure C0113095600154
(5) calculate the difference of cpm between each diluted concentration HBsAg, HBeAg and control group with the t method of inspection.
The result
One, the cytotoxicity of medicine of the present invention in 2215 cells are cultivated
Be to observe medicine of the present invention to the toxicity of people's liver cancer 2215 cells of hepatitis B virogene transfection, behind inoculation 2215 cells 24 hours, add 2 times of dilute liquid medicines. Experiment is from 10mg/ml, and 7 dilution factors are respectively 10; 5; 2.5; 1.25; 0.625; 0.3125; 0.156mg/ml every concentration 3 holes. Change liquid in 4 days one time, kept 8 days, use the microscope observing cell pathology, by formula calculate the poisonous concentration of half (TC50) and maximum non-toxic concn (TC0), see Table 16. Microscope inspection CPE as a result, three batches of experiments of TC50 are respectively 5mg/ml, 5mg/ml, 5.57mg/ml, and average median toxic concentration is 5.19 ± 0.33mg/ml; Non-toxic TC0 is 2.5mg/ml.
Two, the effect in 2215 cells are cultivated, HBeAg and HBsAg expressed of invention medicine
Medicine 2.5mg/ml of the present invention; 1.25mg/ml; 0.625mg/ml; 0.3125mg/ml and 0.156mg/ml adds 2215 cells and cultivates, the 8th day to HBeAg and HBsAg effect see Table 17, table 18, table 19.
1, to the inhibiting rate of HBeAg: three batches of each the concentration groups of experiment medicine of the present invention are cultivated the 8th day supernatant to 2215 cells the average inhibiting rate (table 19) of HBeAg is respectively: 2.5mg/ml suppresses 62.5 ± 3.2%, 1.25mg/ml suppresses 49.3 ± 3.6%, 0.625mg/ml suppresses 37.8 ± 7.5%, 0.3125mg/ml suppresses 26 ± 4.4%, 0.156mg/ml suppresses 18.5 ± 3.8%. The HBeAg P/N value 2.5mg/ml of three batches of experiments is 4.97 ± 0.06, relatively suppresses 8.4 ± 1.3 with control group; 1.25mg/ml be 6.73 ± 0.21, relatively suppress 6.6 ± 1.14 with control group; 0.625mg/ml be 8.23 ± 0.29, relatively suppress 5.1 ± 1.48 with control group; 0.3125mg/ml be 9.83 ± 0.5 with control group relatively suppress 3.5 ± 0.87; 0.156mg/ml be 10.87 ± 0.68, relatively suppress 2.47 ± 0.71 with control group. Three crowdes of experiment IC50 respectively are (table 19): 0.87mg/ml; 0.86mg/ml; 1.14mg/ml. Average 0.96 ± 0.16mg/ml.
2, to the inhibiting rate of HBsAg: each concentration group of three batches of experiments of medicine of the present invention to the average inhibiting rate (table 18) of HBsAg be respectively that kind of 2.5mg/ml suppresses 28.3 ± 2.96%, 1.25mg/ml suppresses 28.03 ± 4.1%, 0.625mg/ml suppresses 12.4 ± 10.6%, 0.3125mg/ml suppresses 10.6 ± 5.0%, 0.156mg/ml suppresses 6.2 ± 4.8%. HBsAg P/N value 2.5mg/ml is 2.78 ± 0.18, relatively suppresses 1.07 ± 0.12 with control group; 1.25mg/ml be 2.77 ± 0.11, relatively suppress 1.08 ± 0.18 with control group; 0.625mg/ml be 3.33 ± 0.32 with control group relatively suppress 0.52 ± 0.39; 0.3125mg/ml be 3.43 ± 0.15, relatively suppress 0.42 ± 0.19 with control group; 0.156mg/ml be 3.6 ± 0.1, relatively suppress 0.25 ± 0.19 with control group. Three crowdes of experiment IC50 respectively are 1.9; 2.21 and＞2.5mg/ml. On average＞2.21 ± 0.3mg/ml.
3, therapeutic index [TI]: to the on schedule cytopathy calculating of therapeutic index [TI] of HBeAg, three batches of experiment IC50 values are respectively (1) medicine of the present invention: 5mg/ml, 5mg/ml, 5.57mg/ml, average out to 5.1 ± 0.33mg/ml in 2215 cells are cultivated. IC50 is respectively: 0.87; 0.86; 1.14mg/ml. Average 0.96 ± 0.16. TI respectively is: 5.97,6.03,4.55. Average 5.52 ± 0.84.
(2) medicine of the present invention in 2215 cells are cultivated to three crowdes of the inhibition % of HBsAg test 2.5mg/ml respectively be 28%, 31.4% and 25.5%, 1.25mg/ml respectively be 31.7%; 28.8%
Be respectively with 23.6%, IC50: 1.9; 2.21 and＞2.5mg/ml. Therapeutic index (TI) respectively is: 2.37,2.35 and 2.08. On average＜2.4 ± 0.33.
Table 16, the toxicity of medicine of the present invention in 2215 cells are cultivated
The experiment batch Experimental technique Different pharmaceutical concentration mg/ml/ cytopathy TC50 (ug/ml)  TC0 (ug/ml)
  10  5  2.5  1.25  0.625  0.313  0.516   0
 I  CPE   4  2  0  0  0  0   0   0  5  2.5
  4  2  0  0  0  0   0   0
  4  2  0  0  0  0   0   0
Destroy %   100  50  0  0  0  0   0   0
 II  CPE   4  2  0  0  0  0   0   0  5  2.5
  4  2  0  0  0  0   0   0
  4  2  0  0  0  0   0   0
Destroy %   100  50  0  0  0  0   0   0
 III  CPE   4  2  0  0  0  0   0   0  5  2.5
  4  2  0  0  0  0   0   0
  4  2  0  0  0  0   0   0
Destroy %   83.   3  50  0  0  0  0   0   0
Three batches average  5.19±  0.33  2.5
Table 17 medicine of the present invention is the 8th day inhibitory action to HBeAg (three batches of experiments) in 2215 cells
The experiment batch Drug concentration (mg/ml)   cpm(X±SD)               HBeAg   ED50   mg/ml   TI
Suppress %   P/N P/N suppresses
  I   2.5   2772±57.2***   64.1   5.0   9   0.87   5.97
  1.25   3796.7±133.0**   50.4   6.9   7.1
  0.625   4320.7±653.2*   43.4   7.9   6.1
  0.3125   5361.3±134*   29.5   9.9   4.1
  0.156   6166±372.4   18.8   11.4   2.6
The cell contrast   7570±881.5   14.0
  II   2.5   2766±23.1***   64.6   5.0   9.2   0.86   6.03
  1.25   3708±197.4**   52.2   6.8   7.4
  0.625   4588.7±535.9*   40.6   8.4   5.8
  0.3125   5592.7±887.9   27.3   10.3   3.9
  0.156   5990±978.9   22.1   11.1   3.1
The cell contrast   766.27±1585.1   14.2
  III   2.5   2684±72.6***   58.8   4.9   6.9   1.14   4.55
  1.25   3536±270***   45.3   6.5   5.3
  0.625   4551.3±464.1**   29.3   8.4   3.4
  0.3125   5065.3±471.1*   21.1   9.3   2.5
  0.156   5479.3±307*   14.6   10.1   1.7
The cell contrast   6398.7±272.6   11.8
Three batches average   0.96±   0.16   5.55±   0.84
Positive control   53365±   1776
Negative control   618.7±   100.3
Blank   84.3
Annotate: experimental group and control group be * * * P＜0.001 relatively, * * P＜0.01, * P＜0.05
Table 18 medicine of the present invention is the 8th day inhibitory action to HBsAg (three batches of experiments) in 2215 cells
The experiment batch Drug concentration (mg/ml)   cpm(X±SD)                HBeAg   ED50   mg/ml   TI
Suppress %   P/N P/N suppresses
  I     2.5   2462±96.6***   28   2.95     1   1.9   2.73
    1.25   2342±97***   31.7   2.71     1.25
    0.63   2699.3±113.2**   *   20.8   3.1     0.86
    0.31   2926±360.8   14   3.4     0.56
    0.16   3002±27.1   11.7   3.5     0.46
The cell contrast   7570±881.5   3.96
  II     2.5   2263.3±136.1**   31.4   2.6     1.2   2.21   2.35
    1.25   2346.7±106.8**   28.8   2.7     1.1
    0.63   2760±124.9*   15.8   3.2     0.6
    0.31   3111.3±96.5   4.8   3.6     0.2
    0.16   3161.3±119.6   3.2   3.7     0.1
The cell contrast   3262.7±283.7   3.8
  III     2.5   2418±123.2***   25.5   2.8     1   ＞2.5   ＜2.08
    1.25   2477.3±152.3**   *   23.6   2.9     0.9
    0.63   3199.3±219.2**   0.5   3.7     0.1
    0.31   2811.3±184.9   12.9   3.3     0.5
    0.16   3103.3±111.6   3.6   3.6     0.2
The cell contrast   3215.3±52.6   3.8
Three batches average   ＞2.21±     0.30   ＞2.39   ±0.33
Positive control     23531±57     1
Negative control     981.3±47     .7
Blank     84.3
Table 19 medicine of the present invention in 2215 cells are cultivated to the inhibition analysis of HBeAg and HBsAg
Viral antigen Medicine mg/ml Toxicity CPE Antigen suppresses % P/N suppresses
 1  2  3  X±SD  1  2  3  X±SD
 HbeAg  2.5  0  64.1  64.6  58.8  62.5±3.2  9  9.2  6.9  8.4±1.3
 1.25  0  50.4  52.2  45.3  49.3±3.6  7.1  7.4  6.5  6.6±1.1
 0.625  0  43.4  40.6  29.3  37.8±7.5  6.1  5.8  3.4  5.1±4.8
 0.3125  0  29.5  27.3  21.1  26±4.4  4.1  3.9  2.5  9.8±0.5
 0.156  0  18.8  22.1  14.6  18.5±3.8  2.6  3.1  1.7  2.5±0.7
 HBsAg  2.5  0  28  31.4  25.5  28.3±3.0  1  1.2  1  1.1±0.1
 1.25  0  31.7  28.8  23.6  28.0±4.1  1.3  1.1  0.9  1.8±0.2
 0.625  0  20.8  15.8  0.5  12.4±10.6  0.9  0.6  0.1  0.5±0.4
 0.3125  0  14  4.8  12.9  10.6±5.0  0.6  0.2  0.5  0.4±0.2
 0.156  0  11.7  3.2  3.6  6.2±4.8  0.5  0.1  0.2  0.5±0.2
Conclusion
One, medicine of the present invention toxicity that 2215 cells are cultivated: medicine of the present invention adds 2215 cells to be cultivated 8 days, and take cytopathy as index, three batches of empirical averages: half toxic concentration (TC50) is 5.19 ± 0.33mg/ml. Maximum non-toxic concn (TC0) is 2.5mg/ml.
Two, the medicine of the present invention inhibitory action of in 2215 cells are cultivated HBeAg and HBsAg being secreted: medicine of the present invention was cultivated 8 days in 2215 cells, three batches of empirical averages: maximum non-toxic concn 2.5mg/ml is 62.5 ± 3.2, IC to the inhibiting rate of HBeAg50Be 0.96 ± 0.16mg/ml. Therapeutic index 5.52 ± 0.84. Inhibitory action to HBsAg: maximum non-toxic concn 2.5mg/ml inhibiting rate is that 28.3 ± 2.96%, IC is 2.21 ± 0.3mg/ml, and therapeutic index is＜2.39 ± 0.33.
Three, at present the clinical arabinofuranosyl adenine monophsphate (Ara-AMP) that is used for the treatment of hepatitis type B virus, acyclovir (ACV), phosphorus formic acid (PFA) though etc. in 2215 cells, can suppress HBV-DNA, but the HBeAg expression inhibiting is lower than 50%, can not calculates IC50. This experiment positive control drug.
Test three medicines of the present invention result for the treatment of to duck hepatitis B virus infection in the duck body
Be hepatitis virus resisting effect in the checking medicine body of the present invention, this experiment adopts the oral medicine of the present invention of duck hepatitis B virus infection duckling to treat, observe it to the impact of duck serum DHB dna level, and with acyclovir (ACV) relatively, the toxicity of having observed simultaneously the oral medicine of the present invention of duck.
Materials and methods
(1) medicine 1, medicine of the present invention
2, positive drug: acyclovir (ACV), available from benefit drugmaker of Wuhan section.
(2) DHB DHB DNA (DHBV-DNA) strong positive serum picks up from the Shanghai sheldrake ,-70 ℃ of preservations.
(3) animal:
1 age in days Beijing duck is available from Institute of Medical Plants duckery of China Concord Medical Science University of the Chinese Academy of Medical Sciences.
(4) reagent:
α-P-dCTP is available from the auspicious biotechnology of Beijing good fortune engineering company. The nick translation box is available from Promega Co.; Sephadex G-50, Ficoll PVP is available from Sweden Pharmacia company; SDS West Germany Merck company product; Milt DNA, bovine serum albumin(BSA) are biophysics institute of Chinese Academy of Sciences product; Nitrocellulose filter 0.45um Amersham company product.
(5) experimental technique:
1, duck hepatitis B virus infection:
1 age in days Beijing duck is through the positive duck serum of leg shin intravenous injection Shandong sheldrake DHBV-DNA, and every 0.2ml will get blood in infection the day after tomorrow, separation of serum, and-70 ℃ of preservations are to be checked.
2, drug therapy test:
DHBV infect duckling after 7 days random packet carry out. The drug therapy test, every group of 5-7 only do not wait; Every batch of administration group experiment minute 3 dosage groups, the 1st crowd minute 2,4 and 8g/kg organize 10 days oral 1 day 2 times. 2nd, 3 crowdes of experiments minutes 2.5,5,10g/kg organize 10 days oral 1 day 2 times. If virus control group (DHBV) is with the physiologic saline for substitute medicine. Positive drug acyclovir (ACV), oral administration 100mg/kg, 1 day 2 times, 10 days; The 7th day is (T0) before the medication after infection, medication the 5th day (T5), and after medication the 10th day (T10) and the drug withdrawal the 3rd day (P3), from duck shin venous blood sampling, separation of serum ,-70 ℃ of preservations are to be checked.
3, detection method:
Get above-mentioned duck serum to be checked, every batch with the time point film, measure DHBV-DNA level in the duck serum dynamically. Press nick translation kit specification method, with P mark DHBV-DNA probe, and make duck serum dot hybridization, autoradiograph diaphragm spot, measure OD value (optical filter is 490nm) at enzyme mark detector, calculate serum DHBV-DNA density, as sample DHBV-DNA level value, the results are shown in accompanying drawing 1,2 and 3 with hybridization spot OD value.
(6) drug effect is calculated:
1, calculates the mean value (X ± SD) and with different time (T5 after every group of duck medication of every group of duck different time serum DNA OD value, T10) (T0) OD value comparison before the 3rd day (P3) serum DHBV-DNA level and the administration on the same group and after the drug withdrawal, adopt paired t-test, calculate t1, P1 value. Take statistics to learn and process, analyze the conspicuousness of difference, judge that medicine is to the inhibition of virus infections.
2, the inhibition % of different time (T5, T10) and front the 3rd day (P3) serum DHBV-DNA of drug withdrawal after every group of duck medication of calculating, and mapping respectively organizes the dynamic of duck serum HBV-DNA inhibiting rate.
Figure C0113095600221
3, with drug treatment group different time DHBV-DNA inhibiting rate relatively, adopt in groups t check, take statistics to learn and process, calculate t2, P2 value, analyze the conspicuousness of difference, judge drug effect.
The result
After (one) 1 age in days Beijing duck infects DHBV, DHBV-DNA total positives behind the dynamic DHBV-DNA infected duck of the serum DHBV-DNA oral normal saline. The 7th day serum DHBV-DNA total positives after 18 ducklings of virus control group infect, 3 batches of experimental infection injection DHBs, it is on the rise to infect the 7th, 12,17 and 20 day the 1st, 3 batches of serum DHBV-DNA level, the 2nd batch has downward trend, but there was no significant difference is natural fluctuation.
(2) positive drug acyclovir (ACV) is on the impact of DHBV infected duck serum DHBV-DNA
The 3rd crowd of experiment DHBV infected duck oral positive drug acyclovir 100mg/kg 1 day 2 times, 10 days, the results are shown in Table 1, table 2. 6 ducks, administration the 5th day (T5) and the 10th day (T10) is compared with (T0) before the administration, and the pairing statistics obviously descends, and difference has very significant (P＜0.01), remarkable inhibition DHBV effect has been described, positive contrast. Suppress % after the drug withdrawal and reduce, rebound phenomenon is arranged, though still have certain inhibition, not statistically significant.
(3) medicine of the present invention in DHBV infected duck body on the impact of duck serum DHBV-DNA
Three batches of experimental results are seen Fig. 1,2,3, table 1,2 and hybridization photo 1,2,3, the result shows:
1, three dosage groups are selected in first experiment, are respectively 2,4 and 8g/kg group, before administration after (T0) and the administration after the 5th day (T5), the 10th day (T10) and the drug withdrawal 3 days (P3), get duck blood, separation of serum detects DHBV-DNA OD value, makes self paired comparisons. 8g/kg group duck serum DHBV-DNA OD value after administration, with comparison before the administration, obvious inhibitory action is arranged, statistics shows significant (P＜0.05), and 4 and the inhibition % of 8g/kg group 3 days duck serum DHBV-DNA after drug withdrawal, with relatively (T0) relatively before the administration, form contrast with virus control, obvious inhibitory action is arranged, and statistics shows significantly or very significant meaning (P＜0.01), has no toxic reaction. The 2g/kg group makes group analysis with infecting control group inhibition %, without significant difference.
2,3 dosage groups of second batch experiment are respectively 2.5,5 and the 10g/kg group, the result shows: 2.5 and 5g/kg group in the 10th day duck serum DHBV-DNA of administration OD value, compare with (T0) before the administration, obvious inhibitory action is arranged, statistics shows significant (P＜0.05). 10g/kg group 3 days duck serum DHBV-DNA after administration the 10th day and drug withdrawal suppress % and the contrast of virus control composition group, and very significantly inhibitory action is arranged, and the statistics demonstration is very significant (P＜0.01).
3, the 3rd batch of repeated experiments, result show 2.5, the 5g/kg group relatively has remarkable inhibition (P＜0.05) before the 5th day horizontal OD value of duck serum DHBV-DNA of administration and administration. 5, the inhibition % of the 5th day duck serum DHBV-DNA and the contrast of virus control composition group after the administration of 10g/kg group has significantly or highly significant suppresses. Acyclovir 100mg/kg 1 day 2 times 10 days, had highly significant to suppress in the 5th day, 10 days after administration. Knock-on is arranged, with many experiments was consistent in the past after the drug withdrawal.
Table 20 medicine of the present invention in the duck body to the inhibitory action of DHBV-DNA OD value
Experiment refers to inferior Group The duck number The OD490 value
  T0   T5   T10   P3
  I Virus control   5   0.25±0.05   0.22±0.02   0.26±0.06   0.30±0.03
Medicine of the present invention
  2.0g/kg   5   0.27±0.03   0.25±0.04   0.19±0.06   0.26±0.07
  4.0g/kg   5   0.25±0.06   0.33±0.04   0.24±0.09   0.19±0.04
  8.0g/kg   6   0.38±0.06   0.30±0.11**   0.31±0.09*   0.13±0.06**
  II Virus control   6   0.63±0.06   0.57±0.11   0.53±0.00   0.46±0.10**
Medicine of the present invention
  2.5g/kg   6   0.67±0.14   0.49±0.18**   0.41±0.17**   0.41±0.12**
  5.0g/kg   6   0.76±0.19   0.65±0.36   0.68±0.18   0.53±0.25**
  10g/kg   5   1.03±0.15   0.93±0.23   0.21±0.12**   0.40±0.13**
  III Virus control   7   0.28±0.07   0.22±0.02   0.26±0.06   0.30±0.03
Medicine of the present invention
  2.0g/kg   6   0.31±0.06   0.25±0.04   0.19±0.06   0.26±0.07
  4.0g/kg   6   0.31±0.09   0.33±0.04   0.24±0.09   0.19±0.04
  8.0g/kg   5   0.45±0.09   0.30±0.11**   0.31±0.09*   0.13±0.06**
  ACV0.1g/kg   6   0.45±0.17   0.08±0.06**   0.15±0.07*   0.28±0.17
(T0) OD value comparison (paired t-test) * P＜0.05 before different time after the administration (T5, T10, P3) OD value and the administration on the same group, * * P＜0.01.
Table 21 invention medication therapy groups and virus control group in the duck body to the comparison of DHBV-DNA inhibiting rate
The experiment batch Group The duck number Inhibiting rate (%)
  T5   T10   P3
  I Virus control   5   11.75   -4.95   -24.26
The invention medicine
  2.0g/kg   5   6.05   28.58   0.61
  4.0g/kg   5   -42.21*   -5.95   22.59*
  8.0g/kg   6   23.88   18.80   63.89***
  II Virus control   6   9.37   14.78   26.55
The invention medicine
  2.5g/kg   6   28.20   40.02*   39.43
  5.0g/kg   6   19.00   8.99*   32.74
  10g/kg   6   8.98   80.99**   60.58**
  III Virus control   7   -0.27   -1.53   -25.62
The invention medicine
  2.5g/kg   6   -113.69**   -133.08*   0.03
  5.0g/kg   6   -35.76**   11.99   -5.88
  10g/kg   5   49.99*   24.88   40.72
  ACV0.1g/kg   6   79.52**   61.58**   22.68
Drug treatment group different time DHBV-DNA inhibiting rate compares respectively (in groups t check), * P＜0.05, * * P＜0.01 with virus control group same time DHBV-DNA inhibiting rate.Clinical testing one
Divide treatment group and control group, treatment group medicine of the present invention, control group hepatitis B nourishing yin and activating blood electuary. Follow up a case by regular visits to comprehensive therapeutic effect after a year: the treatment group cure rate is 49.42%, and efficient is 39.38%, and total effective rate is 88.80%, obviously is better than control group (P＜0.05), sees Table 22:
Table 22 is followed up a case by regular visits to 1 year efficacy analysis
Group n The basic % that cures Effective % Invalid % Total effective rate
Treatment group 259  128(49.42) 102(39.38) 29(11.20) 230(88.80)
Control group 81  18(22.22) 21(25.90) 42(51.80) 39(48.12)
The tcm syndrome curative effect: total treatment group dry is bitter, weak, abdominal distension, hypochondriac pain, the improvement of the purple symptom such as dark of poor, soreness and weakness of waist and knees, complexion received, and its curative effect is better than control group (P＜0.05), sees Table 23:
Table 23 treatment group and control group traditional Chinese medical science cardinal symptom integral contrast
Project n Treatment group Control group
Before the treatment After the treatment Before the treatment After the treatment
Dry is bitter 232  1.62±±0.61  0.59±0.35  1.62±0.56  0.90±0.72
Abdominal distension 228  1.81±0.56  0.56±0.42  1.82±0.45  1.12±0.69
Weak 217  1.72±0.45  0.77±0.51  1.59±0.43  0.98±0.58
Soreness and weakness of waist and knees 188  1.62±0.54  1.02±0.43  1.57±0.61  1.22±0.67
It is poor to receive 240  1.86±0.43  0.61±0.61  1.59±0.41  0.96±0.53
Hypochondriac pain 245  1.83±0.62  0.76±0.58  1.69±0.65  1.11±0.49
Complexion is purple dark 220  1.34±0.41  1.15±0.62  1.41±0.42  1.24±0.46
Self comparing difference remarkable (P＜0.05) before and after Hepatic function improvement: ALT, AST, SB treatment group and the treatment of control group. Liver function normalization rate treatment group is better than control group, has significant difference (P＜0.05), sees Table 24:
Table 24 liver function again normal time, normalization rate compares
Project The average multiple normal time (day) Normalization rate % Time limit (day)
The S treatment group   39.2±10.9   75.4   90-15
The B control group   49.2±15.6   54.4   90-15
  ALT Treatment group   36.5±0.92   84.8   90-15
Control group   42.6±11.2   60.7   90-15
  AST Treatment group   41.8±9.5   83.2   90-15
Control group   55.1±12.5   57.8   90-15
HBV-M:HBsAg negative conversion rate treatment group is 35.9% (after taking 2 courses for the treatment of), and the HBeAg negative conversion rate is that 58.7%, HBV-DNA negative conversion rate is 57.1%, with control group significant difference (P＜0.05) is arranged more all, sees Table 25.
Table 25 is on the impact of HBV-M negative conversion rate
Group n  HBsAg％  HBeAg％ Anti--HBc  HBV-DNA
Treatment group 295  93(259)(35.9)  134(228)(58.7)  78/238(32.9)  113/199(57.1)
Control group 81  4/31(4.9)  14/72(19.4)  5/72(6.9)  12/73(16.4)
The result shows: medicine of the present invention is higher than 81.3% of CAH to the curative effect 92.2% of chronic persistant hepatitis, sees Table 26:
Table 26 chronic persistent hepatitis, chronic active hepatitis efficacy analysis
Somatotype n The basic % that cures Effective % Invalid % Total effective rate %
Chronic persistent hepatitis 179  97(54.2) 68(38.0) 14(7.8) 165(92.2)
Chronic active hepatitis 80  31(38.80) 34(42.5) 15(18.7) 65(81.3)
The above results shows, drug therapy chronic viral hepatitis type B of the present invention is suppressing virus replication, improving the aspects such as cardinal symptom, liver function normalization rate index and significantly be better than control group.Clinical testing two
Clinical data
1, the choice criteria of research object
The Western medicine diagnose standard of chronic active, chronic persistent hepatitis B (hereinafter to be referred as " chronic active hepatitis ", " chronic persistent hepatitis ") is by " new Chinese medicine guideline of clinical investigations (1993 the 1st volume) pertinent regulations are formulated; The differential diagnosis in tcm standard is: spiritlessness and weakness, hypochondriac pain, low-heat, indigestion and loss of appetite, dry, the soreness of waist, dizziness, tinnitus, order are colored, complexion dimly stagnates, tongue is yellow or stripping, dimly red tongue or ecchymosis is arranged, and small and stringy pulse or thin string belong to deficiency of both qi and yin, wet stasis blocking network card.
2, physical data
Be in hospital and clinic case totally 400 examples by above Standard Selection. Treatment group 300 examples, inpatient 258 examples wherein, out-patient's 42 examples, the male sex's 242 examples, women's 58 examples, 38.6 years old mean age, average course of disease 2.8 years, chronic active hepatitis 197 examples, chronic persistent hepatitis 103 examples; Control group 100 examples, inpatient 81 examples wherein, out-patient's 19 examples, the male sex's 84 examples, women's 16 examples, 38.5 years old mean age, average course of disease 2.9 years, chronic active hepatitis 75 examples, chronic persistent hepatitis 25 examples; Two groups of above projects relatively show the no significant difference statistical significance through the Chi-square Test result.
Research method
1, group technology
Adopt Casio (fx-3600p) calculator random packet, be divided into treatment group, control group; Adopt double-blind study at 1: 1 in the check experiment, adopt single blind method in 2: 1 check experiments.
2, observe medication
Medicine adopts double-dummy technique to process, and makes its placebo, observes medicine, the contrast medicine is in full accord in appearance. Observing medicine is medicine of the present invention, and the contrast medicine is selected the hepatitis B electuary (defending the accurate word Z-60 of medicine number) that nourishes blood.
3, administrated method and consumption
Treatment group gives medicine of the present invention, and is oral, and three times on the 1st, each 12g, 3 months was 1 course for the treatment of; And simulate the placebo that contrasts medicine, and oral, three times on the 1st, each 12g. The hepatitis B that the gives control group electuary that nourishes blood, oral, three times on the 1st, each 10g, 3 months was 1 course for the treatment of; And observe the placebo of medicine, and oral, three times on the 1st, each 12g.
4, observation item
(1) symptom and sign: the method that adopts scoring.
(2) general health check-up project and routine test inspection item.
(3) serum SB, ALT, AST, total protein, A/G, serum HbsAg, anti--HBc, HbeAg, HBV-DNA etc.
5, criterion of therapeutical effect
According to " new Chinese medicine guideline of clinical investigations (1993 the 1st volume) related content regulation is formulated.
Result and discussion
Result of study shows that comprehensive therapeutic effect: the basic cure rate for the treatment of group is 47.93%, and is efficient 39.0%, and total effective rate is 86.9%, obviously is better than control group (P＜0.05); Main tcm syndrome curative effect: treatment group dry hardship, abdominal distension, weak, hypochondriac pain, the improvement poor, dizzy, the tinnitus symptom of receiving are better than control group (P＜0.05), and soreness and weakness of waist and knees, the purple dark improvement of complexion and control group compare significantly statistical significance of nothing; Between the group of difference remarkable statistical significance (P＜0.05) is arranged relatively before and after the treatment group of Hepatic function improvement ALT, AST, SB and the treatment of control group; Liver function normalization rate treatment group ALT normalization rate is 85.6%, the AST normalization rate is that 82.1%, SB normalization rate is 73.5%, is better than control group and significant difference (P＜0.05) is arranged; HBV-M: treatment group HbeAg negative conversion rate is that 44.9%, HBV-DNA negative conversion rate is 44.8%, with control group significant difference (P＜0.05) is arranged more all. Medicine of the present invention is higher than CAH (80.6%) to the curative effect (90.6%) of chronic persistant hepatitis, and significant differences (P＜0.01) is arranged.
Result of study shows, medicine of the present invention to improve cardinal symptom, liver function again normal, suppress the aspect such as Viral replicative marker and all be better than control group. In the observation process, do not find chronic toxic and side effect.

Claims (4)

1, a kind of medicine for the treatment of hepatitis B is characterized in that it is made by following raw material by weight:
Ant 150-450 Radix Astragali 20-60 ginseng 10-30
Fruit of Chinese wolfberry 10-30 sealwort 10-30 red sage root 30-90
Bighead atractylodes rhizome 10-30 glutinous rehmannia 30-90 radix paeoniae rubrathe 10-30
Radix Angelicae Sinensis 10-30 dandelion 30-90 giant knotweed 15-45
Bark of ash 10-30 rhizoma atractylodis 10-30 umbellate pore furgus 10-30
Dried orange peel 10-30 hawthorn 10-30 Medicated Leaven 10-30
Fructus Hordei Germinatus 10-30 rascal 10-30.
2, according to claim 1 medicine, wherein the weight proportion of each raw material is:
Ant 300 Radixs Astragali 40 ginsengs 20
The fruit of Chinese wolfberry 20 sealworts 20 reds sage root 60
The bighead atractylodes rhizome 20 glutinous rehmannia 60 radix paeoniae rubrathe 20
Radix Angelicae Sinensis 20 dandelions 60 giant knotweeds 30
Bark of ash 20 rhizoma atractylodis 20 umbellate pore furgus 20
Dried orange peel 20 hawthorn 20 Medicated Leavens 20
Fructus Hordei Germinatus 20 rascal 20
3, the method for the medicine of preparation claim 1 comprises described raw meal is broken into fine powder, sieves, and mixing, per 1 kilogram of powder add refined honey 250-300 gram and water, make water-honeyed pill, and is dry again.
4, the method for the medicine of preparation claim 1 comprises: the ginseng pulverate is broken into fine powder, sieves; The red sage root extracts secondary with alcohol reflux, merges extract, filters, and filtrate recycling ethanol obtains red sage root extract; The bighead atractylodes rhizome, Radix Angelicae Sinensis, rhizoma atractylodis, dried orange peel, rascal extract volatile oil, and the aqueous solution after the distillation in addition device is collected; The red sage root dregs of a decoction behind the dregs of a decoction and all the other ants etc. 13 flavor and the above-mentioned alcohol extract, boiling twice filters, merging filtrate, filtrate is concentrated, is placed to room temperature, slowly stirs the lower ethanol that adds and makes and contain the alcohol amount and reach 60%, leaves standstill filtration, filtrate recycling ethanol; Merge this filtrate, it is the clear cream of 1.35-1.40 that red sage root extract and the above-mentioned aqueous solution, reduced pressure concentration become relative density, adds above-mentioned ginseng fine powder mixing, and low temperature drying is pulverized, granulation, and drying adds above-mentioned volatile oil, and mixing incapsulates and get final product.
CN01130956A 2001-08-28 2001-08-28 Hepatitis B treating medicine Expired - Lifetime CN1116891C (en)

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Publication number Priority date Publication date Assignee Title
CN100387271C (en) * 2004-07-26 2008-05-14 高广法 Medicine for treating hepatitis B
CN100356958C (en) * 2006-07-06 2007-12-26 张砚 Synergistic medicinal composition for treating hepatitis
CN100353981C (en) * 2006-07-06 2007-12-12 汪甬伟 Medicine for auxiliary treating hepatitis
CN103977245A (en) * 2014-04-18 2014-08-13 北京化工大学 Drug for treating hepatitis B and preparation method thereof
CN104958660A (en) * 2015-07-01 2015-10-07 陈维玉 Traditional Chinese medicine for treating liver Qi stagnation type chronic hepatitis B and preparation method for traditional Chinese medicine
CN107050352A (en) * 2017-03-08 2017-08-18 重庆多普泰制药股份有限公司 A kind of Chinese medicine composition for treating chronic hepatitis B

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