CN100341574C - Novel use of cell wall skeleton of red nocar-ray-fungus for treating liver diseases - Google Patents

Abstract

The present invention relates to the field of biologic medicines, more specifically an application of the cell wall skeleton of red nocardiosis actinomycete, namely a new application in the treatment of hepatopathy.

Description

Cell wall skeleton of red nocar-ray-fungus is used for the treatment of the new purposes of hepatic disease

Technical field

The present invention relates to biomedicine field, be specifically related to a kind of new medical use of cell wall skeleton of red nocar-ray-fungus, promptly treat the new purposes of hepatic disease.

Background technology

Red nocar-ray-fungus (Nocardia rubra) is from the isolating a kind of actinomycetes of soil, belongs to the antibacterial territory on the taxonomy, Firmicutes, Actinomycetes, Nocardia.

At present domestic existing researcher separates from this bacterium and prepares a kind of natural mix products---and red nocardiosis cell wall skeleton (Nocardia rubra cell wall skeleton, abbreviation N-CWS) also claims Lyopgized Nocardia rubra-cell Wall Skeleton.Main component is lipids (mycolic acids 17.1%), polysaccharide (arabinogalactan 46.3%) and mucopeptide (Zhang ZL such as (being mainly alanine, glutamic acid, meso diaminopimelic acid, glucamine and 3-O-.alpha.-carboxyethyl-D-glucosamine .), Lin S, Tang WL, et al.Studies on thephysico-chemical properties, composition and content determination of Nocardia rubra cell wallskeleton[J] .J China Antibiotic, 2002,27 (9): 532-534).The clinical treatment medicine that has been used for the treatment of the infection that external inflammation, gynaecopathia, herpesvirus cause clinically.At present, Chinese scholars is by tumor-bearing mice whole animal model, verified that N-CWS has remarkable prevention and suppresses tumor proliferative, and treat the effect of carcinous complication (cancerous hydrothorax, cancer ascites), comprise bladder cancer, breast carcinoma, pulmonary carcinoma, auxiliary treatment malignant melanoma, malignant lymphoma, late gastric cancer and esophageal carcinoma etc.In addition, the present report of not seeing relevant for other drug action of N-CWS.

In recent years, various chronic hepatitiss, viral hepatitis, alcoholic hepatitis and the fatty liver that causes because of obesity are obvious ascendant trend, hepatic disease has become one of major disease of serious harm human body health, but, do not see all kinds of Antihepatitis medicaments that the specially good effect low toxicity is arranged on the market as yet, find that more liver class treatment of diseases new drugs have become one of the emphasis in drug research field.

Summary of the invention

The invention discloses the new purposes of treatment of cell wall skeleton of red nocar-ray-fungus (hereinafter to be referred as N-CWS), be that it can be used for the treatment of hepatic disease, described hepatic disease comprises chronic hepatic injury, hepatic fibrosis, acute liver damage, chemical liver injury, hepatitis etc., also can be used for hepatoprotective.

N-CWS of the present invention can be prepared into it dosage form conventional on pharmaceutics when the treatment hepatic disease, carry out administration by the known administering mode of one of ordinary skill in the art, for example oral, rectum, Sublingual, pulmonary, transdermal, ion penetrate, vagina and intranasal administration.Preferred administering mode is oral.Dosage changes to some extent according to the situation of action time of dosage form and expectation and treatment target, the required amount of actual therapeutic can by the doctor according to practical situation (as, patient's the state of an illness, body weight etc.) and determine easily.For general adult, the amount of daily requirement administration 5-600mg.

N-CWS of the present invention can be prepared by solid fermentation, the existing bibliographical information of concrete preparation method, and general method is as follows:

1, strain is preserved

Inclined-plane preservation method: preparation solid medium (0.5% Carnis Bovis seu Bubali cream, 1% peptone, 0.5% sodium chloride, 0.03%Na ₂HPO ₄.12H ₂O, 1% glycerol, 1.6% agar; With 10% sodium carbonate adjust pH to 7.2), bevel culture medium after 121 ℃ of sterilizations.Activate single bacterium colony with inoculating loop picking from flat board, the inclined-plane streak inoculation, behind 31 ℃ of cultivation 120h, 4 ℃ of preservations.

2, fermentation

Solid medium: Carnis Bovis seu Bubali cream 0.5%, peptone 1%, sodium chloride 0.5%, 0.03%Na ₂HPO ₄.12H ₂O, glycerol 1%, distilled water preparation; 10% sodium carbonate is regulated pH value to 7.5; After 121 ℃ of sterilizations, it is standby to pave plate.

Inoculation and fermentation: with the thalline in sterile distilled water or the fluid medium eluting slant strains preservation pipe, make certain density bacteria suspension after the piping and druming evenly, coat on the flat board of diameter 15cm, be inverted for 31 ℃ and cultivate 120h according to certain amount rubbing method.

Microorganism collection: the lawn that grows is scraped gently with the inoculation shovel, make bacteria suspension with the PBS buffer of certain volume then, 4 ℃ of centrifugal 5min of following 5000rpm, it is standby to weigh behind the collection thalline.

3, the preparation of N-CWS and analysis of physical and chemical property

The thalline that fermentation is obtained is resuspended in 10 times of volume PBS solution, batch process ultrasonication thalline under the condition of ice bath, and regulating frequency and power avoid taking place foam, and cracking is finished as early as possible.

Pyrolysis product is in 4 ℃, the centrifugal 15min of 10000g, get supernatant, an amount of DNase and RNase handle 1h with the nucleic acid in the digestion thalline, adding final concentration then is that 1%TritonX-100 handles 24h, and trypsin that reuse is an amount of and chain protease are handled 12h, handle 2h respectively with normal propyl alcohol, dehydrated alcohol/glacial acetic acid (1: 1) at last, promptly obtain the N-CWS of white behind the distilled water wash three times, weigh after 60 ℃ of oven dry.

Be configured to the finite concentration suspension with collecting the N-CWS that obtains, 0.07Mpa is sterilization 15min down, 4 ℃ of preservations.

Fermentation process (batch)	Seed OD 600	Inoculation volume (ml)	Fermentation volume (ml)	Wet bacterium heavy (g)	N-CWS measures (g)
						1 2 3	0.15 0.31 0.63	1 1 1	50 50 50	0.15 0.27 0.40	0.014 0.025 0.039

N-CWS does not have and smells tastelessly, and it is Powdered to be white in color, reagent such as water insoluble, ethanol, dimethyl sulfoxine, ether.

Description of drawings

Fig. 1 .N-CWS handles the dynamic change influence to ALT

Fig. 2 .N-CWS handles the dynamic change influence to AST

The pathology of Fig. 3 murine liver tissue change (HE dyeing, * 20), and A is the normal mouse hepatic tissue, and B is a 100mg/kg bifendate processed group, and C is a model group, and D, E, F are respectively 150,15 and 1.5mg/kg N-CWS processed group (n=10)

Fig. 4 .N-CWS is to the influence of the liver fiber rat blood serum AST of tetrachloro-methane induction

Fig. 5 .N-CWS is to the influence of the liver fiber rat blood serum ALT of tetrachloro-methane induction

The pathological change of Fig. 6 different disposal group liver tissues of rats (HE dyeing, * 400) A is a normal group, and B is a model group, and the positive matched group of C, D are the 40mg/kgN-CWS high dose group), E is a dosage group among the 8mg/kgN-CWS, F is the 1.6mg/kgN-CWS low dose group

For the ease of understanding, below will present invention is described by specific embodiment and accompanying drawing.It needs to be noted that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.

The specific embodiment

Embodiment 1

N-CWS is to CCl ₄Due to the protective effect of acute liver damage mice

1, drug dose:

The dosage of high dose group is: 150mg/kg, and compound concentration 7.5mg/ml irritates stomach 0.2ml/10g; Middle dosage group is 15mg/kg, and compound concentration 0.75mg/ml irritates stomach 0.2ml/10g; Low dose group is 1.5mg/kg, and compound concentration 0.075mg/ml irritates stomach 0.2ml/10g.

Experimental control:

The blank normal saline is by irritating the appetite clothes with administration group equal-volume.

Positive control drug is bifendate drop pill agent (a 1.5mg/ grain), and the Beijing XieHe medicine Factory produces.Facing the time spent, to add the dissolved in distilled water suspendible as required even, and 4 ℃ of preservations are standby.According to clinical consumption and animal tolerance capacity, the designing animal test dose: mice dosage 100mg/kg, compound concentration 5mg/ml irritates stomach 0.2ml/10g.

2, laboratory animal

Healthy Kunming mouse, complete male, body weight 20 ± 2g is available from animal reproduction field, Jiangning District Green Dragon mountain, Nanjing.

3, other experiment materials

Carbon tetrachloride, analytical pure, chemical reagent one factory in Nanjing produces.Formalin, analytical pure, chemical reagent one factory in Nanjing produces.The pure Oleum Arachidis hypogaeae semen of Shandong court cards, Shandong, Laiyang flower heavy fragrant peanut oil company limited.Sodium chloride injection, Pharmaceutical Zhejiang, Nanjing Tian Feng pharmaceutical factory produces.It is that Bioisystech Co., Ltd's product is built up in Nanjing that transaminase activity is measured test kit, and lot number is: 20040518.

4, experimental technique

4.1 N-CWS is to the protective effect of acute liver damage model mice due to the carbon tetrachloride

90 Kunming mouses are divided into six groups at random by body weight, every group 15, that is: normal control group (Control), model group (Model), positive drug control group (Bifendate, p.o 100mg/kg) and the high, medium and low dosage group of N-CWS (p.o, dosage are respectively 150,15 and 1.5mg/kg).The administration volume is 0.2ml/10g, and normal group and model group give the equal-volume normal saline respectively.

24h begins gastric infusion before the modeling, and respectively be administered once every day sooner or later, successive administration 4 days, and 1h after the 2d administration, except that the normal control group, all the other respectively organize the CCl with 0.2% ₄Peanut oil solution 0.2ml/10g lumbar injection is set up the chmice acute liver injury model.Mouse orbit is got blood behind the 48h, and the centrifugal 10min separation of serum of 3000rpm is measured serum transaminase with the improvement reitman-frankel method; The sacrificed by decapitation animal is got the liver lobus sinister with 10% formaldehyde fixed, and paraffin embedding is carried out pathological observation after the HE dyeing.

4.2 serum transaminase dynamic change experiment

Get 130 Kunming mouses and be divided into four groups at random by body weight, that is: the normal control group (Control, n=10), model group (Model, n=40), bifendate positive controls (Bifendate, p.o 100mg/kg, n=40) and the N-CWS high dose group (p.o 150mg/kg, n=40).The administration volume is 0.2ml/10g, and normal group and model group give the equal-volume normal saline.

Except that the normal control group, all the other respectively organize the CCl with 0.2% ₄Peanut oil solution 0.2ml/10g lumbar injection is set up the chmice acute liver injury model.1h begins gastric infusion after the modeling, is administered once every 12h, and 8h, 24h, 48h, 72h respectively get 10 mices from model group, positive controls, administration group after modeling respectively, and eye socket is got blood, and 3, the centrifugal 10min of 000rpm gets the determination of serum transaminase level.The normal group mice is only established a time point (0h) and as above operates.

5, result

5.1 N-CWS is to CCl ₄Cause the influence of acute liver damage mice serum transaminase activity

N-CWS can obviously suppress CCl ₄The unusual rising of inductive mice serum transaminase activity, and present tangible dose-effect relationship (seeing Table 1).Compare with the positive drug bifendate, the N-CWS of 15mg/kg is suitable to the drug effect of the inhibitory action of transaminase activity and 100mg/kg, but dosage only is the former 15%.

Table 1.N-CWS is to CCl ₄The influence of inductive acute liver damage mice serum transaminase activity

Group	Dosage (mg/k g)	N	ALT(A 505)	ALT(IU/L)	AST(A 505)	AST(IU/L)
							Normal group model group bifendate N-CWS	/ / 100 150 15 1.5	15 15 15 15 15 15	0.153±0.008 0.491±0.055 0.300±0.060 0.186±0.046 0.295±0.061 0.483±0.038	31.33±4.02 280.02±26.25 △△ 110.30±24.12 ** 52.51±15.24 ** 114.88±44.40 ** 273.53±33.14	0.091±0.015 0.233±0.027 0.160±0.032 0.077±0.013 0.155±0.030 0.192±0.027	30.51±7.01 80.34±17.96 △△ 45.22±10.12 ** 25.47±12.29 ** 42.02±14.09 ** 60.9±15.18 *

^*P＜0.05， ^**P＜0.01 compared with Model group， ^△△P＜0.01 compared with Normal group.

In view of by CCl ₄Inductive serum aminotransferase activity was recovered in the later stage, observed the dynamic change of mice serum aminotransferase activity in the different time (seeing Fig. 1 and Fig. 2) after the modeling, the result shows: model group mice serum transaminase activity recovers normal in 72h, the N-CWS of 150mg/kg and positive drug matched group are close to the trend that influences of serum aminotransferase activity, they all can reduce, and (0～72h) transaminase's activity, the former effect is particularly remarkable in the whole process.

5.2 the influence that N-CWS changes the mouse liver histology

Observed the pathological change (Fig. 3) of liver organization by paraffin section, the result shows: compare the obvious swelling of hepatocyte around the central vein of model group Mouse Liver lobule, endochylema puffing, the visible a large amount of neutrophil infiltration in portal area with the normal control group; The sinus hepaticus dilatation and congestion, the visible hepatocyte spotty necrosis of some cases, hepatic cell fattydegeneration.And after the N-CWS of 150mg/kg and the 15mg/kg processing, the lobules of liver structure is clear, though hepatocyte has Mild edema, does not have downright bad phenomenon; Rarely seen a small amount of neutrophil infiltration in the portal area of liver, lesion degree are light far beyond model group, and can stop because CCl ₄The swelling of liver cell that causes, the endochylema puffing, steatosis, finally to the pathological process of spotty necrosis, the liver protecting organizational structure is impaired; The result shows that simultaneously after the N-CWS of 150mg/kg handled, mitosis took place hepatocyte, and hepatocyte differentiation and propagation are on the point of numerous, and liver regeneration is vigorous.

Embodiment 2

N-CWS is to the protective effect of the rat liver fibrosis of tetrachloro-methane induction

1, is subjected to the reagent thing

Drug dose:

High dose group is: 40mg/kg, compound concentration 2mg/ml, p.o 0.2ml/10g; Middle dosage group is 8mg/kg, compound concentration 0.4mg/ml, p.o 0.2ml/10g; Low dose group is 1.6mg/kg, compound concentration 0.08mg/ml, p.o 0.2ml/10g.

Experiment contrast

The blank normal saline is by irritating the appetite clothes with administration group equal-volume.

The positive control Malotilate is Shanxi Yabao Pharmaceutical Co., Ltd.'s product.Facing the time spent, to add the dissolved in distilled water suspendible as required even, and 4 ℃ of preservations are standby.

2, laboratory animal

The SD rat, complete male, body weight 150-180g is available from animal reproduction field, Jiangning District Green Dragon mountain, Nanjing.

3, other experiment materials

Carbon tetrachloride, analytical pure, chemical reagent one factory in Nanjing produces.Formalin, analytical pure, chemical reagent one factory in Nanjing produces.The pure Oleum Arachidis hypogaeae semen of Shandong court cards, Shandong, Laiyang flower heavy fragrant peanut oil company limited.Sodium chloride injection, Pharmaceutical Zhejiang, Nanjing Tian Feng pharmaceutical factory produces.Transaminase activity, liver hydroxyproline, serum A/G are that Bioisystech Co., Ltd's product is built up in Nanjing than detection kit, and lot number is: 20050318.

4. experimental technique

120 SD rats are divided into six groups at random by body weight, every group 20, that is: normal control group (Control), model group (Model), positive controls (Bifendate, p.o 50mg/kg) and the high, medium and low dosage group of N-CWS (p.o, dosage are respectively 40,8 and 1.6mg/kg).

On every Mondays, four mornings, all the other each groups were with 20%CCl except that the normal control group ₄Peanut oil solution 2.5ml/kg lumbar injection is set up the rat liver fibrosis model; On every Mondays to gastric infusion in afternoon Saturday, administration volume 2.5ml/kg, be administered once every day, and normal group and model group give the equal-volume normal saline, and testing total cycle was 10 weeks.

The rat eye socket is got blood behind the last administration 12h, and serum adopts the improved method reitman-frankel method to measure ALT and AST activity.Sacrificed by decapitation animal, liver lobus sinister are with 10% formaldehyde fixed, and paraffin embedding is carried out pathology after the HE dyeing and detected; Middle period quick-freezing in liquid nitrogen is placed on-80 ℃ of preservations, carries out the liver hydroxyproline, the detection of serum albumin and globulin ratio related biochemical indicator such as (A/G).

5, result

5.1 serum aminotransferase activity changes

Utilize CCl ₄Inductive hepatic fibrosis injured animal model has been estimated the drug action of N-CWS to anti-hepatic fibrosis, and the result shows: the N-CWS of high, medium and low various dose (40mg/kg, 8mg/kg and 1.6mg/kg) all shows remarkable reduction CCl ₄Due to the activity that raises of serum transaminase ALT and AST morbid state; and present tangible dose-effect relationship (Fig. 4 and Fig. 5); wherein the N-CWS of 1.6mg/kg dosage group is suitable to the effect of the drug effect of liver injury protection effect and positive drug Malotilate, but dosage only is the latter's 3.2%.

5.2 the pathological change of hepatic tissue

Liver tissues of rats pathological change situation to the different disposal group is analyzed (Fig. 6), compare with the normal control group, significantly steatosis takes place in the model group rat hepatocytes, cellular swelling is serious, the visible significantly cell infiltration in portal area, the fibrous connective tissue moderate generates, and hepatic fibrosis forms.And after the N-CWS of the 40mg/kg processing, the lobules of liver structure is clear; Rarely seen a small amount of neutrophil infiltration in the portal area of liver only has the visible swelling of liver cell of a routine specimen, steatosis, and lesion degree is light far beyond model group.After the N-CWS of 8mg/kg and 1.6mg/kg handled, inflammatory cell infiltration and hepatic cell fattydegeneration were all more serious in the rat liver, but did not see significantly fibrous connective tissue hypertrophy, and hepatic fibrosis does not form.

5.3 the ratio of albumin and globulin in the serum

Utilize CCl ₄Inductive hepatic fibrosis injured animal model is estimated the drug action of N-CWS to anti-hepatic fibrosis, and the result shows: each dosage group N-CWS all can significantly reverse CCl ₄Cause the reduction of serum A/G ratio, and drug effect presents significantly dose-effect relationship (table 2).

Table 2 N-CWS is to the influence of A/G ratio in the inductive hepatic fibrosis rats serum of CCl4

Group	Dosage (mg/kg)	Number of animals	A/G(％)
				Normal group model group Malotilate	/ / 50	15 12 12	16.26±3.25 14.56±4.33 ** 15.89±1.92

N-CWS

40 8 1.6

14 14 12

15.66±8.04 * 15.28±2.48 * 14.60±2.21 *

^*P＜0.05 compared with Model group， ^**P＜0.05 compared with Normal group.

5.4 the variation of hydroxyproline content in the liver organization (HYP)

Utilize CCl ₄Inductive hepatic fibrosis injured animal model has been estimated the drug action of N-CWS to anti-hepatic fibrosis, and the result shows: the N-CWS of 40mg/kg, 8mg/kg and 1.6mg/kg dosage group can significantly reverse CCl ₄Increasing of the liver hydroxyproline content that causes, the drug effect of three dosage group N-CWS present significantly dose-effect relationship (table 3).

Table 3 N-CWS is to the influence of hydroxyproline content in the inductive hepatic fibrosis rats hepatic tissue of CCl4

Group	Dosage (mg/kg)	Number of animals	Hyp(ug/mg tissue weight)
				Normal group model group Malotilate N-CWS	/ / 50 40 8 1.6	15 12 12 14 14 12	3.36±0.62 4.72±1.11 ** 3.46±1.57 4.22±1.08 * 4.27±0.47 4.57±1.51

^*P＜0.05 compared with Model group， ^**P＜0.05 compared with Normal group.

6, discuss

Serum transaminase (ALT and AST) is considered to reflect the sensitive indicator of hepatic injury, and the evaluation index that is used as liver function is widely used in the clinical diagnosis process of hepatopathy.ALT is positioned at the hepatocyte endochylema, and AST then mainly is distributed in the liver cell mitochondria, and small part is positioned at endochylema.Liver is impaired when light, permeability of cell membrane increases, and ALT and AST discharge into blood in the endochylema, and Intramitochondrial AST does not then discharge, when hepatocyte impaired when serious AST just discharge into blood, so the level of serum transaminase can reflect the extent of damage of liver plasma membrane to a certain extent.

Hydroxyproline is the basic substance of one of the main component of synthetic cell epimatrix (ECM) collagen protein, thereby the content of hydroxyproline can directly reflect the degree of hepatic fibrosis in the liver.N-CWS can significantly reduce CCl ₄The increasing unusually of hydroxyproline in the liver due to inducing illustrates that N-CWS can be to anti-hepatic fibrosis, and this result is also consistent with the histopathology result.

Mainly synthetic and secretion of albumin by hepatocyte, can keep colloidal osmotic pressure, and can produce the change of extensive reversible configuration in conjunction with multiple materials such as cation, aniones, so can carry many nutrient substance, as fatty acid, hormone, trace metal ion, enzyme, vitamin and medicine etc.In addition, albumin is transported to metabolism organ and detoxifcation organ after can also being combined into complex with sl. sol. metabolite and noxious substance, excretes then; Globulin is the immune protein that produces in the immune response process.The ratio of albumin and globulin amount is an important prognostic indicator of chronic hepatic injury treatment.And N-CWS can recover CCl to a certain extent ₄The reduction of inductive A/G ratio and reach and be tending towards normal level shows that N-CWS is better to the prognosis effect for the treatment of liver fibrosis.

In sum, N-CWS can suppress the activity and the inflammatory reaction of serum transaminase, and promotes hepatocellular division and proliferation, and chemical liver injury and chronic hepatic injury are had the good protection effect.Simultaneously, N-CWS can significantly reduce accumulating of hydroxyprolin levels and ECM in the liver, have significant effect of anti hepatic fibrosis, and the prognosis effect is better.

Claims (1)

1, cell wall skeleton of red nocar-ray-fungus is used to prepare the purposes of treatment hepatic fibrosis or hepatitis disease medicine.

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