CN100450492C - Composition and its use - Google Patents

Abstract

The present invention provides a kind of composition comprising aweto or its fermented culture 20-80 wt% and cryptoporu volvatus or its fermented culture 20-80 wt%. The composition can inhibit fibrosis of lung and fibrosis of cardiac and cerebral vascular tissue, and possesses tissue fibrosis inhibiting effect obviously higher than that of available medicine interferon gamma. The composition has synergistic tissue fibrosis inhibiting effects of both aweto and cryptoporu volvatus and has no any toxic side effect found.

Description

A kind of compositions and application thereof

(1) technical field

The present invention relates to a kind of compositions and the application in preparation prevention and treated tissue fibrosis medicine thereof.

(2) background technology

Tissue fibering (tissue fibrosis) is the common consequence of many different tissues damages, also is that the basic pathology of multiple serious threat human health disease changes.In fact tissue fibering involves nearly all organ of human body and system, is that numerous disease is disabled, lethal main cause.Data proves according to the relevent statistics, and the U.S. can belong to the tissue fibering disease because of among the lethal patient of various diseases near 45%.Really, tissue fibering plays an important role in the generation of each major organs disease of human body and evolution.For example the cardiovascular organization fibrosis causes in the cardiovascular organization reconstruct at hypertension and heart failure and has brought into play important function, also is atherosclerotic one of the main reasons; Interior external source is caused a disease and formerly can be caused hepatic fibrosis, as Type B and C type viral hepatitis, schistosomicide hepatopathy, chronic alcoholic liver disease and the non-alcoholic fatty liver disease hepatic fibrosis reason that become international; All that development takes place is closely related with tissue fibering for many acute and chronic kidney diseases, and particularly the renal fibrosis that causes of diabetic nephropathy and hypertension changes; The for example also Fibrotic in a organized way change of arthritis, whole body sclerosis and systemic lupus erythematosus (sle) of panimmunity and autoimmune disease.

Studies show that, agglutination after the tissue injury can be divided into tissue regeneration and two stages of tissue fibering, the former mainly comprises inflammatory reaction and normal structure agglutination that tissue injury causes, and the latter mainly is because the scarring process of the connective tissue hyperplasia that local various bioactie agents cause.These two stages are regulated by different molecular respectively, also are separable pathological processes.Whole body and local immune system have been brought into play pivotal role in the tissue fibering process causing, keep and suppress.Immune system can be divided into innate immune system and acquired immune system.Innate immune system is the first line of defence of human body to endogenous and exogenous cause a disease former generation immunne response and acute inflammatory reaction.Innate immune system mainly is made up of complement, polymorphonuclear cell (monokaryon-macrophage, mastocyte), natural killer cell (NK) and dendritic cell (DC) etc.Acquired immune system comprises humoral immune reaction and cell immune response mainly by leukocyte and cell mediated.Humoral immune reaction is excretory antibody-mediated by bone-marrow-derived lymphocyte, and cellular immunization is cytokine mediated by the T lymphocytic emiocytosis.The T cell comprises cytotoxic T (Tc), helps T (Th) and regulates T (Tr) cell.According to the difference of identification antigen presenting cell surface I type or II type MHC molecule conjugated antigen, the T cell can be divided into CD ₈ ⁺And CD ₄ ⁺Two kinds of hypotypes.According to producing different cytokines, CD ₄ ⁺The T cell can divide Th1, Th2 and natural killer T cell (NK-T) again.In this field, an important progress is, the several components of innate immune system impels particularly dendritic cell (DC) maturation of antigen presenting cell by different mechanism, has determined in the acquired immune system bone-marrow-derived lymphocyte to grow and the Th1 of T lymphocytic emiocytosis, the polarised direction of Th2 cytokine.Th1 of the acquired immune response or Th2 cytokine polarised direction are the key factors of tissue fibering and reconstructed tissue.For example, after the tissue injury, the important composition NK cell of innate immune system is activated.Activatory NK emiocytosis IFN-γ promotes antigen presenting cell DC to grow, and the acquired immune response is developed towards the Th1 direction.This Th1 polarised direction helps the reparation and the regeneration of tissue injury; Otherwise,, then promote the generation of tissue fibering and reconstructed tissue if the Th cell develops towards the Th2 direction.Really, clinical practice IFN-γ treatment lung fibroproliferative disease has obtained challenging PRELIMINARY RESULTS.Can expect that innate immune system and acquired immune system Study of Interaction will cause finding anti-tissue fibering new drug target, for the anti-fibrosis medicine of developing high-efficiency low-toxicity provides great opportunity.

Studies have shown that in a large number: the Mycophyta extract generally all has tangible immunoregulation effect; They or increase some immunoreation function or suppress some immunoreation function.For example, the Cordyceps fermentation culture medium both can enhance immunity reaction, also can suppress immunoreation and as the organ transplantation ancillary drug.According to inventor's previous research work, find that the Cordyceps fermentation culture medium is a Th1 immunoreation reinforce.On the other hand, the Cordyceps fermentation culture medium has been found and has suppressed hepatic fibrosis, the renal fibrosis that the various different causes of disease cause to some extent, but mechanism of action is still unclear.In addition, latent pore fungi fermentation culture medium also has obvious immunoregulation effect.Discover that further concealing the pore fungi fermentation culture medium obviously blocks Th2 immunoreation effect, have remarkable antiallergic, antasthmatic effect (irritated and asthma all is the disease that the Th2 immunoreation excessively causes).According to above-mentioned discovery and theory, on the animal model of several tissue fibering, launched research work.Through lot of experiments, find that Cordyceps fermentation culture medium and latent pore fungi fermentation culture medium can suppress the cardiovascular organization fibrosis that pulmonary fibrosis due to the bleomycin, hypertension cause very effectively.The effect of these two kinds of anti-tissue fibering in active ingredient of natural product combination back obviously exceeds anti-tissue fibering medicine--the interferon gamma of present extensive use, and does not find any toxic and side effects.PRELIMINARY RESULTS meets above-mentioned theory fully, and pointing out this Cordyceps fermentation culture medium and latent pore fungi fermentation culture medium compound recipe may be the active drug of the many tissue fibering of treatment, has huge DEVELOPMENT PROSPECT.

(3) summary of the invention

The present invention is for a kind of Cordyceps fermentation culture medium and the composite compositions of latent pore fungi fermentation culture medium are provided, and the application of this compositions in preparation prevention and treated tissue fibrosis medicine.

For reaching goal of the invention the technical solution used in the present invention be:

A kind of compositions, described composition quality is composed as follows: Cordyceps 20～80%, latent pore fungi 20～80%.

Further, described composition quality is composed as follows: Cordyceps fungus fermentation culture medium 20～80%, latent pore fungi fermentation culture medium 20～80%.

Described Cordyceps fungus fermentation culture medium is Hirsutella hepiali Chen et Shen Hiresutella hepialiChen et Shen sp.Nov. (1980) and different name of the same race--China pilose spore Hiresutellasinensis Lu, Guo, the fermentation culture medium of Yu et Zeng. (1989).Promptly latent pore fungi Crytoporus volvatus (Peck.) Hubbara. of described latent pore fungi fermentation culture medium comprises the latent pore fungi Crytoporus sinensis H.Wu ﹠amp of China; The M.Zang fermentation culture medium.

Described Cordyceps fungus fermentation culture medium is one of following or following both combination in any: (1) Cordyceps fungus fermentation mycelium; (2) cordyceps militeris fungus fermentation broth.

Described latent pore fungi fermentation culture medium is one of following or following both combination in any: (1) conceals the pore fungi fermentation mycelium; (2) latent pore fungi fermentation liquid.

Concrete, above-mentioned fermentation culture medium all can be made powder after extracting concentrate drying.Described composition quality is composed as follows: fermentation Cordyceps powder 20～80%, the latent hole of fermentation powder 20～80%.

Preferably, described composition quality is composed as follows: fermentation Cordyceps powder 50%, the latent pore fungi powder 50% of fermentation.

Latent pore fungi powder preparation step is as follows:

(1) actication of culture: with the cryptoporus volvatus kind of preserving under 4 ℃ of conditions, be transplanted in the following culture medium, under the conventional cultivation temperature of fungus, cultivated 3～10 days by the sterile working,

Nutrient media components and weight percentage are (%):

Glucose 0.5～2.0

Maltose 0.5～2

Peptone 0.1～0.2

Yeast powder 0.5～1

KH ₂PO ₄ 0.1～0.4

MgSO ₄·7H ₂O 0.01～0.5

Vitamin B ₁0.05～0.1

Agar 2.2～3.0

pH 4.5～5.0

All the other compositions are water

(2) shake-flask culture: adorn in above-mentioned activatory Cryptoporus volvatus (peck) Scnear mycelium moved in the 500mL conical flask of the following culture fluid of 150～200mL, frequency of oscillation be 100～200rpm, temperature be under 20～30 ℃ of conditions in shaking table shaken cultivation 6 days; As need add macrospecies amount can again mycelia liquid be moved in the 3000mL conical flask of the following culture fluid of dress 750mL, frequency of oscillation be 100～200rpm, temperature be under 20～30 ℃ of conditions in shaking table shaken cultivation 2～5 days.

Culture fluid component and quality (%) are:

Glucose 0.5～2.0

Maltose 0.5～2

Peptone 0.1～0.2

Yeast powder 0.5～1

KH ₂PO ₄ 0.1～0.4

MgSO ₄·7H ₂O 0.01～0.5

Vitamin B ₁0.05～0.1

pH 4.5～5.0

All the other compositions are water

(3) liquid fermentation and culture: packing in the fermentation tank of various capacity specifications is equal to or less than the following culture fluid of 70% tankage size, moving with pressure differential method and to connect the Cryptoporus volvatus (peck) Scnear mycelium liquid that is equivalent to tankage size 3～10% shake-flask culture, is that 1: 0.1～1.0 (v/v/ minutes), impeller mixing speed are that 100～200rpm, tank pressure are greater than 0.05kg/cm in ventilation ², 20～30 ℃ of condition bottom fermentations of temperature 1～10 day.

Culture fluid component and weight percentage (%) are:

Glucose 2.5～3.5

Maltose 0.5～2

Semen Maydis powder 0.3

Peptone 0.5

Yeast powder 0.5～1

KH ₂PO ₄ 0.1～0.4

MgSO ₄·7H ₂O 0.01～0.5

Vitamin B ₁0.05～0.1

pH 4.5～5.0

All the other compositions are water

(4) adopt the conventional filtration method to filter the cryptoporus volvatus fermented product in the above-mentioned fermentation tank and obtain mycelium and fermented liquid supernatant liquid.Mycelium or/and fermentation liquid is even can be as component of the present invention, gained mycelium and fermentation liquid can be separately separately or mixes or extract that making ferments conceals the pore fungi powder.The oven dry of cryptoporus volvatus filament filter cake is pulverized, and sieves, and obtains granule less than 60 purpose cryptoporus volvatus powders.Fermented supernatant fluid is concentrated into the thick paste shape, and add or do not add, above-mentioned gained powder, mixing, oven dry, pulverizing, or through extracting, spray promptly to get the latent pore fungi powder of described fermentation.

Fermentation Cordyceps powder preparation process is as follows:

China's Cordyceps asexual generation strain Hirsutella hepiali Chen et Shen liquid medium within top fermentation, obtain fermented supernatant fluid, mycelium and various metabolite wherein: fermentation process is aerobic culture in slant strains cultivation successively, shake-flask seed cultivation, one-level, secondary or three grades of seed tank culture, the liquid fermentation tank, and 10～18 ℃, pH6.0～7.5 were cultivated respectively 30～60,10,6,6,6,6～12 days.Fluid medium (percentage by weight) is: carbon source 0.5～5%, nitrogenous source 0.2～5%, trace element, vitamin a little, all the other are moisture, feed filtrated air in the sweat.Solid-liquid separation obtained mycelium and ferment filtrate after fermentation finished.Mycelium and ferment filtrate be even to can be used as compositions of the present invention, ferment filtrate can be concentrated also that the back be mixed with mycelium, homogenate, also can get described fermentation Cordyceps powder through extraction, spray drying.

Described Cordyceps and latent pore fungi compositions, Cordyceps fungus ferment and conceal pore fungi fermentation culture compositions and all can be applicable to preparation prevention and treated tissue fibrosis medicine.

Described prevention and treated tissue fibrosis medicine are used for the treatment of pulmonary fibrosis due to the bleomycin.

Described prevention and treated tissue fibrosis medicine are applied to treat the cardiovascular organization fibrosis that hypertension causes.

Described compositions effective dosage every day is: the animals administer amount is 0.1～15g/kg body weight, dosage 0.1～60g/kg body weight of conversion adult.Promptly for receptor, compositions effective dosage every day is: 0.1～60g/kg body weight.

The beneficial effect of compositions of the present invention and application thereof is mainly reflected in: (1) described compositions can effectively suppress the cardiovascular organization fibrosis that pulmonary fibrosis due to the bleomycin, hypertension cause; (2) effect of the anti-tissue fibering of compositions of the present invention obviously exceeds anti-tissue fibering medicine--the interferon gamma of present extensive use; (3) the Fibrotic inhibitory action of cardiovascular organization that when Cordyceps and latent pore fungi applied in any combination, pulmonary fibrosis due to the bleomycin and hypertension caused of compositions of the present invention, than using Cordyceps or latent pore fungi separately better effect is arranged, and do not find any toxic and side effects, have huge DEVELOPMENT PROSPECT.

(4) description of drawings

Fig. 1 is the effect (Masson ' s dyeing) of pulmonary fibrosis of bleomycin induced mice and various Drug therapys; A is normal group tissue pathological slice figure after 28 days, B is model group tissue pathological slice figure after 28 days, C is interferon group tissue pathological slice figure after 28 days, D is Cordyceps and latent pore fungi compositions tissue pathological slice figure after 28 days for the compositions group, E is that Dexamethasone group is treated two all one week of drug withdrawal back tissue pathological slice figure, and F is that Dexamethasone group is treated two all two week of drug withdrawal back tissue pathological slice figure;

Fig. 2 is the influences of various medicines to the Hypertensive Rats survival rate; Wherein CY is a compositions, and CHC is a Cordyceps fermented product, and YK is latent pore fungi fermented product, and DEX is a dexamethasone, and IFN is interferon (as follows).

Fig. 3 is that various medicines are to the Fibrotic influence of Hypertensive Rats cardiovascular organization; A: normal group cardiac muscular tissue (* 100); B: model group cardiac muscular tissue (* 100); C: positive controls cardiac muscular tissue (* 100); D: Cordyceps and latent pore fungi compositions (CY) treatment group cardiac muscular tissue (* 100); E: normal group thoracic aorta (* 400); F: model group thoracic aorta (* 400); G: positive controls thoracic aorta (* 400); H: Cordyceps and latent pore fungi compositions (CY) treatment group thoracic aorta (* 400).

Fig. 4 is various medicines to the regulating action of Hypertensive Rats body weight and cardiac index, left ventricle exponential sum index and spleen index.The A cardiac index; B left ventricle index; The C body weight change; The D index and spleen index.

Fig. 5 is the regulating actions of various medicines to the Hypertensive Rats haemodynamic function; *, compare p＜0.05 with the blank group; * compares p＜0.01 with the blank group; * * compares p＜0.001 with the blank group.

Fig. 6 is the regulating action (CY is the TGF-b1 mRNA expression of compositions, and GAPDH is the internal control index) to Hypertensive Rats cardiac muscle TGF-b1 mRNA expression.

(5) specific embodiment

The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:

Embodiment 1: the latent pore fungi powder preparation of fermentation

Step is as follows:

(1) actication of culture: with the cryptoporus volvatus kind of preserving under 4 ℃ of conditions, be transplanted in the following culture medium, under the conventional cultivation temperature of fungus, cultivated 8 days by the sterile working,

Nutrient media components and weight percentage are (%):

Glucose 0.6

Maltose 1.5

Peptone 0.1

Yeast powder 0.8

KH ₂PO ₄ 0.1

MgSO ₄·7H ₂O 0.05

Vitamin B ₁0.07

Agar 2.7

pH 4.8

All the other compositions are water

(2) shake-flask culture: in above-mentioned activatory Cryptoporus volvatus (peck) Scnear mycelium moved in the 500mL conical flask of the following culture fluid of dress 200mL, frequency of oscillation be 200rpm, temperature be under 20～30 ℃ of conditions in shaking table shaken cultivation 6 days;

Culture fluid component and quality (%) are:

Glucose 0.7

Maltose 1.6

Peptone 0.2

Yeast powder 1

KH ₂PO ₄ 0.1

MgSO ₄·7H ₂O 0.05

Vitamin B ₁0.1

pH 5.0

All the other compositions are water

(3) liquid fermentation and culture: the following culture fluid of dress 14L in the 20L fermentation tank, moving with pressure differential method and to connect the Cryptoporus volvatus (peck) Scnear mycelium liquid that is equivalent to tankage size 3% shake-flask culture, is that 1: 0.1～1.0 (v/v/ minutes), impeller mixing speed are 200rpm, tank pressure greater than o.05kg/cm in ventilation ², 20～30 ℃ of condition bottom fermentations of temperature 10 days.

Culture fluid component and weight percentage (%) are:

Glucose 3.0

Maltose 1.8

Semen Maydis powder 0.3

Peptone 0.5

Yeast powder 1

KH ₂PO ₄ 0.1

MgSO ₄·7H ₂O 0.05

Vitamin B ₁0.05

pH 4.5

All the other compositions are water

(4) adopt the conventional filtration method to filter the cryptoporus volvatus fermented product in the above-mentioned fermentation tank and obtain mycelium and fermented liquid supernatant.Cryptoporus volvatus filament filter cake is dried under 80～90 ℃ of conditions, pulverizes, and sieves, and obtains granule less than 60 purpose cryptoporus volvatus powders; It is 1.4 grams per milliliters that 70～80 ℃ of rotary evaporations of fermented supernatant fluid are concentrated into density, and reheat is concentrated into the thick paste shape, adds above-mentioned gained mycopowder, stirs, and 60 mesh sieves are crossed in oven dry, pulverizing, promptly gets the latent pore fungi powder of described fermentation.

Embodiment 2: the preparation of fermentation Cordyceps powder

China's Cordyceps asexual generation strain Hirsutella hepiali Chen et Shen liquid medium within top fermentation, obtain fermented supernatant fluid, mycelium and various metabolite wherein: fermentation process is aerobic culture in slant strains cultivation successively, shake-flask seed cultivation, one-level, the cultivation of secondary seed jar, the liquid fermentation tank, and 10～18 ℃, pH6.0～7.5 were cultivated respectively 50,10,6,6,10 days.

Fluid medium (percentage by weight) is: dried silkworm chrysalis meal 2.0%, and peptone 1.5%, Semen Maydis powder 1.8%, sucrose 1.6%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, all the other are moisture, feed filtrated air in the sweat.

The fermentation back solid-liquid separation that finishes obtains mycelium and fermentation liquid, and fermentation liquid carries out the ferment filtrate that membrance separation obtains being rich in effective ingredient again, and ferment filtrate concentrates that the back is mixed with mycelium, homogenate, spray drying, promptly gets described fermentation Cordyceps powder.

Embodiment 3:

Prescription:

Embodiment 2 gained fermentation Cordyceps powder 5000g

The latent pore fungi powder 5000g of embodiment 1 gained fermentation;

Above-mentioned fermentation Cordyceps fungus powder and the latent pore fungi powder mix homogeneously of fermentation promptly get described compositions.

Embodiment 4:

Prescription:

Cordyceps 2000g

Latent pore fungi 2000g;

Method: Cordyceps is available from the original producton location, Qinghai, and latent pore fungi is picked up from the pine forest of Zhejiang Hangzhou area, is accredited as natural crude drugs through outward appearance, and pulverize separately mixes, and pulverizing must be crossed 80 order powder again, promptly gets described compositions.

Embodiment 5:

Prescription:

Press embodiment 2 method gained fermentation Cordyceps powder 8000g

Press the latent pore fungi powder 4000g of embodiment 1 method gained fermentation;

Above-mentioned fermentation Cordyceps fungus powder and the latent pore fungi powder mix homogeneously of fermentation promptly get described compositions.

Embodiment 6:

Prescription:

Press embodiment 2 method gained fermentation Cordyceps powder 2000g

Press the latent pore fungi powder 8000g of embodiment 1 method gained fermentation;

Above-mentioned fermentation Cordyceps powder is even with the latent pore fungi powder mixes of fermentation, promptly gets described compositions.

Embodiment 7:

Prescription:

Press embodiment 2 method gained fermentation Cordyceps powder 8000g

Press the latent pore fungi powder 2000g of embodiment 1 method gained fermentation;

Above-mentioned fermentation Cordyceps powder is even with the latent pore fungi powder mixes of fermentation, promptly gets described compositions.

Embodiment 8:

Prescription:

Press embodiment 2 method gained fermentation Cordyceps powder 4000g

Press the latent pore fungi powder 8000g of embodiment 1 method gained fermentation;

Above-mentioned fermentation Cordyceps powder is even with the latent pore fungi powder mixes of fermentation, promptly gets described compositions.

Embodiment 9: the mouse pulmonary fibrosis of prevention and reverse bleomycin induced

One. summary: the pulmonary fibrosis of bleomycin induced is the animal model of classics.Bleomycin (BLM) be from streptomyces one produce the one group of glycopeptide class that extracts in the antibiotic bacterial strain, be a kind of cancer chemotherapy preparation, be usually used in making the animal pulmonary fibrosis model.Think that at present BLM energy and ferrous ion form BLM Fe ²⁺Complex is at O ₂Exist down, this complex can provide electronics for oxygen molecule, forms midbody products such as peroxide and free hydroxyl group, makes the deoxyribose of DNA produce free radical.A large amount of radical pair lung tissues produces lipid peroxidation injury, promotes hypertrophy, propagation such as fibroblast, makes matter generation fibrosis between alveolar.

Main agents and medicine:

Interferon gamma (IFN γ): available from Shanghai clone Biology high technology Ltd;

Dexamethasone sodium phosphate injection: available from Tianjin gold credit aminoacid company limited, lot number 0407031;

The fermentation Cordyceps powder that Cordyceps fermented product: embodiment 2 makes;

The latent pore fungi powder of the fermentation that latent pore fungi fermented product: embodiment 1 makes;

Embodiment 3 resulting compositions;

Embodiment 4 resulting compositions.

Two. experimental technique: get male C57BL/6 (6～8 weeks of age week) mice, pentobarbital sodium (45mg/kg, i.p.) anesthesia, intratracheal injection bleomycin (3U/kg), cut skin of neck with the wound of trying one's best little, use behind the puncture trachea and inject about 50 μ l bleomycin in the microsyringe trachea, promptly upright and enatiozoa is so that make bleomycin enter the left and right sides lobe of the lung equably.

The normal saline of sham operated rats intratracheal injection equivalent; Establish 8 groups altogether, detailed protocol sees Table 1.Respectively by five time points: understood the pathological characteristic of pulmonary fibrosis due to the bleomycin in 3 days, 7 days, 14 days, 21 days, 28 days, observe anti-inflammatory drug dexamethasone, anti-fibrosis medicine interferon gamma and the present composition curative effect of medication to the different courses of disease of pulmonary fibrosis due to the bleomycin.

Table 1: the present composition is to the pharmacodynamic study of the different courses of disease of pulmonary fibrosis due to the bleomycin

Group	Dosage (continuous 14 days)	Solvent
			Sham operated rats (Con, n=33)	Equivalent solvent (p.o.)	H 2O
Model group (Mod, n=33)	Equivalent solvent (p.o.)	H 2O
			Dexamethasone group (DS, n=33)	3mg/kg((i.p.)	Normal saline
Interferon gamma group (IFN-γ n=33)	150,000 U/kg (i.p.)	Normal saline
			The Cordyceps fermented product group (CFC, n=33)	3g/kg (p.o.), every day is administration at twice	H 2O
Latent pore fungi fermented product group (CFS, n=33)	3g/kg (p.o.), every day is administration at twice	H 2O
			One group of compositions (CFX1, n=33)	Embodiment 3 resulting composition 3.0g/kg (p.o.)	H 2O
Two groups of compositionss (CFX2, n=33)	Embodiment 4 resulting composition 3.0g/kg (p.o.)	H 2O

Three. experimental result: represent with meansigma methods ± SD, user's difference analysis ANOVA, each group relatively selects the suitable method of inspection to carry out comparing in twos of many group sample averages in variant back, back.P as a result＜0.05 of statistical analysis thinks that there were significant differences, and p＜0.01 is thought significant differences.

(1) the pathological examination result of pulmonary fibrosis of bleomycin induced mice and Drug therapy effect

Trachea injection bleomycin can cause that tangible pulmonary fibrosis (sees Table 2, Fig. 1).In the observation period in 4 weeks, pulmonary fibrosis shows tangible phase characteristic: inflammatory reaction stage and fibrosis form and developmental stage.Pathological examination shows that the inflammatory reaction stage approximately continued for 2 weeks, and is the most obvious with the performance of first week: 3 days animal alveolar wall edema, broadening, thickening after the modeling, telangiectasis, macrophage increases in the hyperemia, alveolar space, and visible neutral grain and eosinophilic granulocyte are soaked into; After the modeling 7 days, the alveolar inflammation reached the peak, and visible a large amount of inflammatory cells ooze out in the alveolar space, and a pulmonary alveolar macrophage and a matter mononuclear cell number significantly increase; Individual animal generation kitchen range cell infiltration was still arranged after the modeling in 14 days.The beginning about about 7 days greatly of fibrosis phase: after the modeling 7 days, the animal alveolar septum thickened, and fibroblast and myofibroblast hypertrophy appear in interstitial lung and alveolar wall; After the modeling the 14th day, fibroblast showed increased in the alveolar septum, the substrate showed increased, alveolar structure destroys, withers; After the modeling 21 days, the alveolar septum fibrosis was more obvious, and the alveolar space stenosis is little, the large stretch of proliferation of fibrous tissue of lung tissue; 28 days pulmonary fibrosis degree further increase the weight of after the modeling, and a large amount of hypertrophy of fibroblast, fibrous tissue are bar rope sample and distribute, and alveolar structure further destruction even alveolar structure disappearance occur.

Table 2: the effect of pulmonary fibrosis of bleomycin induced mice and various Drug therapys

Group	7 days	14 days	21 days	28 days
					Normal group	-	-	-	2/7(+)
Model group	3/6(+)	4/6(+)	6/6(+)	2/7(++)，3/7(+++)
					The DS group	-	1/6(++)	3/6(+++)，1/6(+)	3/7(+)
The interferon group	2/6(+)	2/6(++)	1/6(+)	2/7(+)
					The CFC group	3/6(+)，1/6(++)	3/5(+)	4/6(+)	2/7(+)，1/7(++)
The CFS group	2/6(+)	3/6(+)	3/6(+)	1/9(+)，1/9(++)
					One group of CFX1 of compositions	1/6(+)，2/6(++)	2/6(+)	1/6(+)	2/9(++)
Two groups of CFX2 of compositions	2/6(+)，2/6(++)	3/6(+)	2/6(+)	2/9(++)

Annotate 1:Masson ' s dyeing standards of grading: (a) (b) mild fibrosis (+) of no fibrosis (-), the fibrosis area is less than 20%.(c) moderate fibrosis (++), the fibrosis area is 20% to 50%.(d) severe fibrosis (+++), the fibrosis area surpasses 50%

Annotate 2:CFC: Cordyceps fermented product, CFS: latent pore fungi fermented product, CFX1 (embodiment 3 resulting compositions): fermentation Cordyceps powder and latent 1: 1 the compositions of pore fungi powder of fermentation.CFX2 (embodiment 4 resulting compositions): Cordyceps fungus and 1: 1 compositions of latent pore fungi.

Annotate 3: shown in following examples table with annotate 1, annotate 2 identical.

Tissue pathological slice checks that display model treated animal alveolar structure disappears, and forms serious pulmonary fibrosis (seeing Figure 1B).Interferon gamma and compositions suppress the pulmonary fibrosis (seeing Fig. 1 C, Fig. 1 D) that bleomycin causes for one group significantly.Large stretch of proliferation of fibrous tissue (seeing Fig. 1 E) still appears in the animal two weeks back drug withdrawal of dexamethasone treatment after one week of drug withdrawal, but the lung tissue fibrosis alleviates (seeing Fig. 1 F) after two weeks of drug withdrawal.

Embodiment 10: suppress the inductive cardiovascular organization reconstruct of hypertension:

(1) main agents and medicine:

Interferon gamma (IFN γ): Shanghai clone Biology high technology Ltd

Dexamethasone sodium phosphate injection: Tianjin gold credit aminoacid company limited, lot number 0407031

Cordyceps fermented product: make Cordyceps fermented product, lot number CFC-16-040928 with embodiment 2 same procedure

Latent pore fungi fermented product: make latent pore fungi fermented product, lot number CFS-4-100L embodiment 3 resulting compositions with embodiment 1 same procedure.

(2) laboratory animal: the SD rat (male, 220～240g), available from Beijing Vital River Experimental Animals Technology Co., Ltd., animal quality certification SCXK (capital) 2002-0003.

(3) experimental technique:

1. animal grouping, modeling and administration situation:

Male SD rat (220～240g), lumbar injection pentobarbital sodium (50mg/kg) anesthetized rat [Kuwahara et al., 2002].Face upward and be positioned at the Mus plate and fix, cut off operation place fur, sterilization back median abdominal incision, cut skin and flesh layer, the about 1.5-2cm of otch behind the exposure internal organs, stretches into the abdominal cavity with finger, the abdominal cavity is aortal beats, to judge its position, then, push to right side such as the stomach of rat and mesentery with the antiseptic gauze that is soaked with normal saline and expose ventral aorta before kidney and the spinal column, it is long to separate ventral aorta 1cm above renal artery branch, be close to ventral aorta with No. 7 injection needles, parallel placement, and with the two ligation together, extract syringe needle then out, can the constriction ventral aorta.Postoperative is used penicillin 50,000 U/, one week an of lumbar injection every day, in order to avoid infect.Not ligation behind the sham operated rats separation ventral aorta.

Natural drug is to the therapeutical effect of the different courses of disease of caused by hypertension cardiovascular organization reconstruct, establish 8 groups (detailed protocol sees Table 3) altogether, 7 days, 14 days and 28 natural gift other places reason animal after the modeling, with immune change in the evolution that detects the reconstruct of caused by hypertension cardiovascular organization and the process thereof, detect dexamethasone, IFN-γ and Cordyceps fungus fermented product and the curative effect of medication of latent pore fungi fermented product to the different courses of disease of caused by hypertension cardiovascular organization reconstruct.

Table 3

Group

Dosage

Solvent

Sham operated rats (Con, n=30)	Sham
			Model group (Mod, n=36)	AC
IFN-γ organizes (n=36)	150,000 U/kg/day (i.p.)	NS
			Cordyceps fermented product CFC organizes (n=36)	3g/kg/day(p.o.)	H 2O
Latent pore fungi fermented product YK group (n=36)	3g/kg/day(p.o.)	H 2O
			Embodiment 3 compositionss (n=36)	3g/kg/day(p.o.)	H 2O
Dex organizes (n=36)	1mg/kg/day(i.p.)	NS

7d after administration, 14d, 28d handle animal respectively.Before handling animal, fasting overnight, the pneumoretroperitoneum of weighing injection pentobarbital sodium (50mg/kg) anesthetized rat is measured the total arterial blood extracting of hemodynamic index collare.Take out heart and kidney respectively, the back of weighing is divided into 4 parts to left ventricle and renal tissue, and is a with fixing in 4% paraformaldehyde solution, does the analysis of pathology tissue slice; Two parts place liquid nitrogen rapidly, are used for protein expression and detect; Get an amount of left and right other portion respectively and place RNAwait to preserve liquid rapidly, detect the expression of mRNA in the tissue.Take out thymus and spleen respectively, place RNAwait to preserve liquid after weighing rapidly, to detect mRNA expressions such as Th1/Th2 relevant cell factor and TGF-β 1.

The immunohistochemistry interpretation of result:

Utilize immunohistochemical method, carry out the scarlet or Masson dyeing of day wolf respectively, to observe I, the distribution of III Collagen Type VI and the influence of drug treating; The infiltration of inflammatory cell is observed in HE dyeing; Detect fibroblast proliferation, monocyte infiltration and IFN-γ, IL-13 and TGF-β 1 respectively at the partial production of cardiac muscular tissue, nephridial tissue and vascular tissue with the antibody of anti-Brdu, ED-1, IFN-γ, IL-13 and TGF-β 1.

Statistical method:

Experimental result is represented with meansigma methods (Mean) ± SD, user's difference analysis ANOVA, and each group relatively selects the suitable method of inspection to carry out comparing in twos of many group sample averages in variant back, back.P as a result＜0.05 of statistical analysis thinks variant, and p＜0.01 is thought significant difference.

(4) experimental result:

1. to the influence of Hypertensive Rats survival rate: various medicines are seen Fig. 2 to the protective effect of Hypertensive Rats existence;

2. to the Fibrotic regulating action of Hypertensive Rats cardiovascular organization: see Fig. 3; The ligation ventral aorta causes rat hypertension, puts to death animal after 14 days, gets its heart and thoracic aorta, through 10% formaldehyde fixed, and paraffin embedding, HE dyeing, the change of its fibroplasia is analyzed in microphotograph.A: normal group cardiac muscular tissue (* 100); B: model group cardiac muscular tissue (* 100), to compare with normal group, its fibroplasia extremely significantly increases (p＜0.001); C: positive controls cardiac muscular tissue (* 100), to compare with model group, its fibroplasia obviously alleviates (p＜0.01); D:CY treatment group cardiac muscular tissue (* 100) compares with model group, and its fibroplasia obviously alleviates (p＜0.01); E: normal group thoracic aorta (* 400); F: model group thoracic aorta (* 400), to compare with normal group, the degree of its vascular hypertrophy extremely significantly increases (p＜0.001); G: positive controls thoracic aorta (* 400), to compare with model group, the degree of its vascular hypertrophy obviously alleviates (p＜0.01); H: combination treatment group thoracic aorta (* 400), to compare with model group, the degree of its vascular hypertrophy obviously alleviates (p＜0.05).

3. to the regulating action of Hypertensive Rats body weight and cardiac index, left ventricle exponential sum index and spleen index: see Fig. 4; The A cardiac index; B left ventricle index; The C body weight change; The D index and spleen index.

4. to the regulating action of Hypertensive Rats haemodynamic function: medicine to the ventral aorta ligation after the influence of different time (7 days, 14 days and 28 days) narcotism rat aorta systolic pressure see Fig. 5; *, compare p＜0.05 with the blank group; * compares p＜0.01 with the blank group; * * compares p＜0.001 with the blank group.

5. to the regulating action of Hypertensive Rats cardiac muscle TGF-β 1 mRNA expression: 7 days Hypertensive Rats cardiac muscle TGF-β 1 mRNA changes of expression level are seen Fig. 6 after the administration.

The hemodynamics testing result shows, promptly apparently higher than matched group (p＜0.05), the blood pressure of model group continued raise (p＜0.001), tend towards stability in the time of 28 days (p＜0.01) to the pressure value of model group in the time of 14 days back 7 days of experiment.Positive controls and combination treatment group are not all found remarkable hypotensive activity (p＞0.05) (Fig. 4).Cardiac index is meant the weight (g) of every 100g rat body weight cardiac, and the left ventricle index is meant the left ventricular mass (g) and the ratio of weight (g) whole-heartedly, and both directly reflect heart, especially the plump degree of left ventricle.IFN-γ group and treatment group all can reduce because cardiac index (Fig. 2 A) and the exponential increase of left ventricle (Fig. 2 B) that hypertension causes.Proliferation of fibrous tissue is to increase with fibrocyte between cardiac muscle between cardiac muscle, the kitchen range proliferation of fibrous tissue or around the myocardium small artery proliferation of fibrous tissue and big lamellar proliferation of fibrous tissue be main.The result shows, modeling the 7th day, and positive controls and combination treatment group cardiac muscle fiber hamartoplasia are lighter, compare with the model model group and do not see notable difference.The 14th day and 28 days, positive controls and combination treatment group cardiac muscle fiber hamartoplasia obviously alleviated (p＜0.01) (Fig. 3 A～D).The thoracic aorta proliferation of fibrous tissue mainly shows film in the arterial wall, and collagen fiber and smooth muscle cell proliferation between middle film elastic fibers make that the crack broadens between the elastic fibers, and film thickens in the arterial wall.Made film the 14th day, positive controls and the proliferation of fibrous tissue of combination treatment group thoracic aorta obviously alleviate, and compare difference highly significant (p＜0.01) or significant difference (p＜0.05) respectively with model group; Made film the 28th day, positive controls and combination treatment group have also shown similar therapeutic effect (Fig. 3 G～H).

Embodiment 10: Cordyceps and latent pore fungi compositions (CFX) are to the best proportioning test of pulmonary fibrosis therapeutical effect due to the bleomycin

In the best proportioning test of compositions, establish 10 groups altogether, group and dosage regimen see Table 3.Experimental technique is with embodiment 8.(ratio of Cordyceps fermented product and latent pore fungi fermented product was respectively 1: 4 to observe the Cordyceps fermented product proportioning different with latent pore fungi fermented product, 1: 2,1: 1,2: 1,4: 1), dexamethasone acute inflammation stage administration (DS1), Dexamethasone group long term administration (DS2), interferon long term administration (IFN-γ) be to the effect of pulmonary fibrosis due to the bleomycin.

Table 4: compositions is to the best proportioning test of pulmonary fibrosis therapeutical effect due to the bleomycin

Annotate: the example in the table: CF4 represents that Cordyceps fermented product and latent pore fungi fermented product weight are 4: 1

CF2 represents that Cordyceps fermented product and latent pore fungi fermented product weight are 2: 1

CFX represents that Cordyceps fermented product and latent pore fungi fermented product weight are 1: 1

CF1 represents that Cordyceps fermented product and latent pore fungi fermented product weight are 1: 2

CF0 represents that Cordyceps fermented product and latent pore fungi fermented product weight are 1: 4.

Table 5: the effect of pulmonary fibrosis of bleomycin induced mice and various Drug therapys

Group	Group	The fibrosis classification
			Sham operated rats	Matched group	1/8(+)
Model group	Model group					3/10(+)，5/10(+++)
			Dexamethasone life-time service group	DS28			1/9(+)，1/9(++)，1/9(+++)
Dexamethasone short-term use group	DS3					6/9(+)，1/9(++)
			CFC∶CFS＝4∶1	CFX4	2/11(+)，3/11(++)，1/11(+++)
CFC∶CFS＝2∶1	CFX2	1/11(+)，1/11(++)，2/11(+++)
			CFC∶CFS＝1∶1	CFX	3/11(+)
CFC∶CFS＝1∶4	CFX0	2/11(+)，1/11(++)，2/11(+++)
			CFC∶CFS ＝1∶2	CFX1	4/9(+)，2/9(++)

Annotate: 1. the CFC in the table is a Cordyceps fermented product, the promptly latent pore fungi fermented product of CFs; 2. fibrosis and mortality rate results suggest: CFX organize, and the percentage by weight of promptly ferment Cordyceps fungus powder and the latent pore fungi powder of fermentation is 1: 1, is a reasonable compatibility group of curative effect.

Claims (9)

1. compositions that is used for the treatment of tissue fibering is characterized in that described composition quality is composed as follows:

Cordyceps 20～80%

Latent pore fungi 20～80%.

2. compositions that is used for the treatment of tissue fibering is characterized in that described pharmaceutical composition quality is composed as follows:

Cordyceps fungus fermentation culture medium dry weight 20～80%

Latent pore fungi fermentation culture medium dry weight 20～80%.

3. compositions as claimed in claim 2 is characterized in that described Cordyceps fungus fermentation culture medium dry weight is one of following or the combination in any of following both dry weights: (1) Cordyceps fungus fermentation mycelium; (2) cordyceps militeris fungus fermentation broth.

4. compositions as claimed in claim 2, it is characterized in that described latent pore fungi fermentation culture medium dry weight is one of following or the combination in any of following both dry weights: (1) conceals the pore fungi fermentation mycelium; (2) latent pore fungi fermentation liquid.

5. as the described compositions of one of claim 2～4, it is characterized in that described fermentation culture medium makes powder.

6. compositions as claimed in claim 5 is characterized in that described pharmaceutical composition quality is composed as follows:

Fermentation Cordyceps powder 50%

The latent pore fungi powder 50% of fermentation.

7. the application of compositions as claimed in claim 1 or 2 in preparation prevention and treated tissue fibrosis medicine.

8. the application of compositions as claimed in claim 7 in preparation prevention and treated tissue fibrosis medicine is characterized in that described prevention and treated tissue fibrosis medicine are applied to treat pulmonary fibrosis due to the bleomycin.

9. the application of compositions as claimed in claim 7 in preparation prevention and treated tissue fibrosis medicine is characterized in that described prevention and treated tissue fibrosis medicine are applied to treat the cardiovascular organization fibrosis that hypertension causes.

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