Specific implementation mode
The main theory foundation for treating the illness caused by poultry mycoplasma has at 3 points:The first, clearing heat and detoxicating and promoting blood circulationization
The stasis of blood;The second, logical internal organs facilitaing lung, clearing liver-fire;Third, clear ripe removing toxic substances, the pure phlegm of facilitaing lung.Therefore, poultry mycoplasmosis needs to be treated
Have effects that clearing heat and detoxicating and heat-clearing and damp-drying drug Chinese medicine, such as the compatibilities coptis such as southern medicine Radix zanthoxyli, Pogostemon cablin, rhizoma et Radix Baphicacanthis Cusiae,
The Chinese medicines such as radix scutellariae, Cortex Phellodendri, Radix Glycyrrhizae, the root bark of shaggy-fruited dittany, corter pseudolaricis, folium artemisiae argyi.
The present invention isolated plurality of Chinese extract from above-mentioned Chinese medicine, and be serially diluted again by using 96 orifice plates two
Method obtains having the anti-active Chinese medical extract of poultry mycoplasma.China is the native place of Chinese herbal medicine, Chinese medicine resources of medicinal plant ten
It is point abundant, there is the Chinese medicine status that can not be ignored using Chinese herb prevention poultry mycoplasmosis, and from isolated in Chinese medicine
Medicament extract has the advantages that other antibiotic or chemicals are irreplaceable, such as with toxic side effect is small, is not likely to produce drug resistance
Property etc..
The Chinese medical extract contains Nitidine Chloride, ethoxychelerythrine, Chelerythrine, sanguinarine, Huang
Even any one or more in alkali, palmatine, epiberberine and enoxolone, the content range of each substance is:Chlorination two
Face needle alkali 1-512 μ g/mL, ethoxychelerythrine 1-512 μ g/mL, Chelerythrine 1-512 μ g/mL, sanguinarine 1-512 μ
G/mL, coptisine 1-512 μ g/mL, palmatine 1-512 μ g/mL, epiberberine 1-512 μ g/mL and enoxolone 1-512 μ g/
mL。
In above-mentioned Chinese medical extract, the enoxolone comes from Radix Glycyrrhizae, and the coptisine, palmatine and epiberberine come from
The coptis and Cortex Phellodendri, the Nitidine Chloride, ethoxychelerythrine, Chelerythrine and sanguinarine are all from Radix zanthoxyli.
The anti-active inhibition of poultry mycoplasma is different in vitro for the Chinese medical extract, wherein chlorination Radix zanthoxyli
These four Chinese medical extracts of alkali, ethoxychelerythrine, Chelerythrine and sanguinarine are to chicken virus mycoplasma, Mycoplasma bovis
And mycoplasma ovipneumoniae this 3 mycoplasma species all have stronger In-vitro Inhibitory Effect, coptisine, palmatine, epiberberine and
These four Chinese medical extracts of enoxolone only have stronger In-vitro Inhibitory Effect to chicken virus mycoplasma.
The systemic diseases medicines such as ox, sheep, the chicken caused by treatment poultry mycoplasma can be used alone in the Chinese medical extract
It, also can be according to the Chinese medical extract anti-poultry mycoplasma activity situation in vitro, by above-mentioned different Chinese medical extract phase in object
In the systemic diseases drugs such as ox, sheep, chicken of the mutual combination application caused by treatment poultry mycoplasma.Such as Nitidine Chloride with
The combination of ethoxychelerythrine, the combination of Nitidine Chloride and Chelerythrine, Nitidine Chloride and sanguinarine
Combination can be prevented or treated to poultry mycoplasmosis caused by chicken virus mycoplasma, Mycoplasma bovis and mycoplasma ovipneumoniae;
The combination of Nitidine Chloride and enoxolone can be prevented or controlled to chicken chronic respiratory disease caused by chicken virus mycoplasma
It treats.
Wherein, preferred Chinese medical extract is following five kinds combinations, is respectively:
Group unification:1 μ g/mL of Nitidine Chloride, 8 μ g/mL of ethyoxyl greater celandine;
Combination two:1 μ g/mL of Nitidine Chloride, 4 μ g/mL of Chelerythrine;
Combination three:2 μ g/mL of Nitidine Chloride, 2 μ g/mL of sanguinarine;
Combination four:1 μ g/mL of Nitidine Chloride, 1 μ g/mL of enoxolone;
Combination five:1 μ g/mL of Nitidine Chloride, 4 μ g/mL of coptisine.
The Chinese medical extract can be prepared into the preparation for treating or preventing poultry mycoplasmosis, and the preparation can be injection
Any one dosage form such as agent, spray, granule, oral solution, pill, powder.
The present invention also provides with the anti-active Chinese medicine composition of poultry mycoplasma, the Chinese medicine composition includes to match below
The bulk pharmaceutical chemicals of ratio:At least one and Baphicanthus cusia of 0-5g in Radix zanthoxyli 0-10g, coptis 0-10g, Cortex Phellodendri 0-10g and Radix Glycyrrhizae
At least one of root 0-30g, Pogostemon cablin 0-15g, radix scutellariae 0-10g, root bark of shaggy-fruited dittany 0-20g, corter pseudolaricis 0-5g, folium artemisiae argyi 0-5g.
Preferred Chinese medicine composition is following 7 kinds of compounds, is respectively:
Compound 1:Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g;
Compound 2:Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, Radix Glycyrrhizae 5g;
Compound 3:Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, coptis 10g, Cortex Phellodendri 10g;
Compound 4:Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, radix scutellariae 10g;
Compound 5:Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, root bark of shaggy-fruited dittany 20g;
Compound 6:Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, root bark of shaggy-fruited dittany 20g, coptis 10g, Cortex Phellodendri 10g;
Compound 7:Coptis 10g, radix scutellariae 10g, Cortex Phellodendri 10g, Radix Glycyrrhizae 5g;
Wherein it is more highly preferred to compound 3, compound 5 and compound 6.
It should be understood that the Chinese medicine composition can be prepared into the preparation for treating or preventing poultry mycoplasmosis, the system
Agent can be any one dosage form such as injection, spray, granule, oral solution, pill, powder.
Chinese medical extract provided by the invention is specifically described below by specific pharmacodynamic experiment.
1. experiment bacterial strain
Y-98 plants of PG45 plants of Mycoplasma bovis, Bc44T plants of chicken virus mycoplasma (MG) and mycoplasma ovipneumoniae are by Jilin University
Zoonosis research institute provides.
2. drug
Chinese medical extract Nitidine Chloride, ethoxy are extracted from Radix zanthoxyli, four taste Chinese medicine of the coptis, Cortex Phellodendri and Radix Glycyrrhizae
Base Chelerythrine, Chelerythrine, sanguinarine, coptisine, palmatine, epiberberine and enoxolone, purity reach
99.9%.Use DMSO (dimethyl sulfoxide, dimethyl sulfoxide (DMSO)) by traditional Chinese medicine extraction respectively above-mentioned 8 kinds of Chinese medical extracts
Object is made into the mother liquid medicine of final concentration of 40960 μ g/ml, dispenses 1.5mL sterile polypropylene centrifuge tubes, is sealed with sealed membrane, close
It is sealed at -60 DEG C or lower temperature.
3. culture medium
KM2The preparation of (Mycoplasma hyopneu-nK nlae Medium) basal medium:It is cultivated to the bases KM2
In base plus appropriate horse serum, 0.4% is phenol red and required things, adjustment pH value to 7.6,0.22 μm of filtering with microporous membrane such as penicillin remove
Bacterium is to get 4 DEG C save backup.
PPLO (pleuropneumonia-like organis, tissue cultures pollute l organisms) basal medium
Preparation:Into PPLO basal mediums plus appropriate cow's serum, 0.4% is phenol red and the required things such as penicillin, and adjustment pH value is extremely
7.8,0.22 μm of filtering with microporous membrane degermings are to get 4 DEG C save backup.
4. the preparation for the bacterium solution that works:
Freeze-drying standard mycoplasma strain is added in corresponding basal medium, PG45 plants of Mycoplasma bovis is added to PPLO culture mediums
In, Y-98 plants of Bc44T plants of chicken virus mycoplasma (MG) and mycoplasma ovipneumoniae are added separately in KM2 culture mediums, set 37 DEG C, 5%CO2
Cultivated 7 days in incubator, make its continuous passage, take third generation logarithmic growth bacterium solution, wherein PG45 plants of Mycoplasma bovis for 1 ×
108CCU/mL, Bc44T plants of chicken virus mycoplasma are 1 × 109CCU/mL, Y-98 plants of mycoplasma ovipneumoniae are 1 × 1010CCU/mL is used
Respective liquid culture medium is diluted to 1 × 104CCU/ml, as work bacterium solution.
5.MIC (Minimum inhibitory concentration, minimal inhibitory concentration) value measures:
Sterile 96 porocyte culture plates are taken, 195 μ L of culture medium are added in every the 1st hole of arranging, culture medium is added in the 2nd~12 hole
100μL;Then 5 μ L of mother liquid medicine are added to the 1st hole, be used in combination micro sample adding appliance since the 1st hole doubling dilution liquid to the 10th
Hole is sucked out 100 μ L in the 10th hole after mixing and discards;Then the 1st~11 hole is separately added into 100 μ L of work bacterium solution so that Suo Youkong
Total liquid volume be 200 μ L, the 11st hole be Positive control wells (i.e. growth control hole), the 12nd hole be negative control hole;Drug
Final concentration be followed successively by (unit from the 1st~10:μg/mL)512.00、256.00、128.00、64.00、32.00、16.00、
8.00,4.00,2.00,1.00, totally 10 dilution gradients, instill sterile liquid paraffin oil seal, cover plate lid, set constant temperature incubation
37 DEG C in case, 5%CO2Culture 3~4 days.
Judge result according to culture medium color change:Explanation has mycoplasma when culture medium becomes yellow or crocus from red
Growth.Culture is observed after 3~4 days as a result, (phenolic red indicator is by red i.e. in culture solution when color change occurs for Positive control wells
Become yellow or crocus) and when without muddiness, minimum drug concentration that color change no longer occurs in medicine feeding hole be it is minimum effectively
Inhibit mycoplasma growth concentration (MIC).Experiment is repeated 6 times, and takes the average value of 6 results.The external anti-fowl of 8 kinds of Chinese medical extracts
The MIC value of poultry mycoplasma specifically joins table one:
Table one
Currently, sentencing to minimal inhibitory concentration (MIC) of monomeric compound or antibiotics mycoplasma both at home and abroad
It is fixed, it is according to being judged according to the criterion of same experiments document report, i.e. when μ g/mL of MIC≤4 is more sensitive, and MIC is
It is medium sensitivity when 4~32 μ g/mL, is insensitive as the μ g/mL of MIC >=32.
As shown in Table 1:Above-mentioned 8 kinds of Chinese medical extracts all have the preferable external anti-active inhibition effect of poultry mycoplasma
Fruit, wherein enoxolone, this 4 Chinese medical extracts of coptisine, palmatine and epiberberine are only to chicken virus mycoplasma (MG) Bc44T
Strain has stronger In-vitro Inhibitory Effect;Nitidine Chloride, ethoxychelerythrine, Chelerythrine and sanguinarine are to this reality
The 3 mycoplasma species strains tested all have stronger In-vitro Inhibitory Effect.
The combination of Chinese medical extract provided by the invention is specifically described below by specific pharmacodynamic experiment.
Group unification:
It prepares and is combined by 1 part of Nitidine Chloride and 8 parts of Chinese medical extracts formed of ethyoxyl greater celandine, used DMSO
Be configured to final concentration be respectively 1 μ g/mL and 8 μ g/mL liquid it is spare.
Combination two:
It prepares and is combined by 1 part of Nitidine Chloride and 4 parts of Chinese medical extracts formed of Chelerythrine, it is matched with DMSO
Be made final concentration be respectively 1 μ g/mL and 4 μ g/mL liquid it is spare.
Combination three:
It prepares and is combined by 2 parts of Nitidine Chloride and 2 parts of Chinese medical extracts formed of sanguinarine, it is configured to DMSO
Final concentration is that the liquid of 2 μ g/mL is spare.
Combination four:
It prepares and is combined by 1 part of Nitidine Chloride and 1 part of Chinese medical extract formed of enoxolone, it is prepared with DMSO
Be at final concentration 1 μ g/mL liquid it is spare.
Combination five:
It prepares and is combined by 1 part of Nitidine Chloride and 4 parts of Chinese medical extracts formed of coptisine, it is configured to DMSO
Final concentration is respectively that the liquid of 1 μ g/mL and 4 μ g/mL is spare.
Liquid is made in above-mentioned five kinds combinations and carries out pharmacodynamic experiment, is existed using 96 orifice plate, two times of serial dilutions
The MIC value of the anti-poultry mycoplasma of external test, assay method are same as above.The MIC of the external anti-poultry mycoplasma of five groups of combinations
Value please specifically join table two.
Table two
It is learnt by table two:Combination, Nitidine Chloride and the greater celandine of Nitidine Chloride and ethoxychelerythrine
The combination of the combination of red alkali, Nitidine Chloride and sanguinarine all has stronger external suppression to 3 mycoplasma species strains of this experiment
It makes and uses;The combination of the combination of Nitidine Chloride and enoxolone, Nitidine Chloride and coptisine is only to chicken virus mycoplasma
(MG) there is stronger In-vitro Inhibitory Effect for Bc44T plants.
Chinese medicine composition provided by the invention is specifically described below by specific pharmacodynamic experiment.
Compound one:
It takes rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, 10 times of amount distilled water is added, impregnate 0.5h, refluxing extraction 3 times, each 1h,
Merge No. 3 extracting solutions, it is quantitative to filter, it is concentrated into 10mL (being equivalent to containing crude drug amount 2g per 1mL Aqueous extracts), finally uses 0.22
It is spare after μm filtering with microporous membrane.
Compound two:
It takes rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, Radix Glycyrrhizae 5g, 10 times of amount distilled water is added, impregnate 0.5h, refluxing extraction 3 times,
Each 1h merges No. 3 extracting solutions, quantitative to filter, and is concentrated into 10mL, (being equivalent to containing crude drug amount 2g per 1mL Aqueous extracts), finally
Using spare after 0.22 μm of filtering with microporous membrane.
Compound three:
Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, coptis 10g, Cortex Phellodendri 10g are taken, 10 times of amount distilled water are added, impregnates 0.5h, returns
Stream extraction 3 times, each 1h merges No. 3 extracting solutions, quantitative to filter, and is concentrated into 10mL and (is equivalent to containing crude drug per 1mL Aqueous extracts
Measure 2g), finally using spare after 0.22 μm of filtering with microporous membrane.
Compound four:
Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, radix scutellariae 10g are taken, 10 times of amount distilled water are added, impregnate 0.5h, refluxing extraction 3
Secondary, each 1h merges No. 3 extracting solutions, quantitative to filter, and is concentrated into 10mL (being equivalent to containing crude drug amount 2g per 1mL Aqueous extracts), most
Afterwards using spare after 0.22 μm of filtering with microporous membrane.
Compound five:
Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, root bark of shaggy-fruited dittany 20g are taken, 10 times of amount distilled water are added, impregnate 0.5h, refluxing extraction 3
Secondary, each 1h merges No. 3 extracting solutions, quantitative to filter, and is concentrated into 10mL (being equivalent to containing crude drug amount 2g per 1mL Aqueous extracts), most
Afterwards using spare after 0.22 μm of filtering with microporous membrane.
Compound six:
Rhizoma et Radix Baphicacanthis Cusiae 30g, Radix zanthoxyli 10g, root bark of shaggy-fruited dittany 20g, coptis 10g, Cortex Phellodendri 10g are taken, 10 times of amount distilled water, leaching is added
0.5h is steeped, refluxing extraction 3 times, each 1h merges No. 3 extracting solutions, quantitative to filter, and it is (i.e. suitable per 1mL Aqueous extracts to be concentrated into 10ml
In containing crude drug amount 2g), finally using spare after 0.22 μm of filtering with microporous membrane.
Compound seven:
Coptis 10g, radix scutellariae 10g, Cortex Phellodendri 10g, Radix Glycyrrhizae 5g are taken, 10 times of amount distilled water are added, impregnate 0.5h, refluxing extraction 3
Secondary, each 1h merges No. 3 extracting solutions, quantitative to filter, and is concentrated into 10mL (being equivalent to containing crude drug amount 2g per 1mL Aqueous extracts), most
Afterwards using spare after 0.22 μm of filtering with microporous membrane.
Liquid is made in above-mentioned seven kinds of compounds and carries out pharmacodynamic experiment, is existed using 96 orifice plate, two times of serial dilutions
The MIC value of the anti-poultry mycoplasma of external test, assay method are same as above.The MIC value of the external anti-poultry mycoplasma of 7 kinds of compounds
It please specifically join table three:
Table three
Herein it should be noted that MIC 1 is MIC value when culture medium first time there is color change, i.e. starting is minimum
Mlc, for the Mlc of the growth and breeding of apparent inhibition poultry mycoplasma;MIC2 is after judging result for the first time after 5
Its MIC value determined, i.e., final minimal inhibitory concentration, for the Mlc of the persistently growth of effective inhibition poultry mycoplasma.
This is because the mechanism of action that Chinese medicine composition is cured the disease is to adjust organism balance using the Various Complex active ingredient of Chinese medicine, improve
Immunity of organisms, rather than all the components directly act on pathogen and achieve the purpose that cure disease, utilize trace liquid diluting
Method do Chinese medicine composition extracorporeal bacteria inhibitor test be only capable of indicating in surveyed Chinese medicine whether active ingredient containing certain direct antibacterials or
The size of number and its antibacterial activity containing the antimicrobial component, therefore the MIC value of Chinese medicine composition is often than Chinese medical extract
Big more of MIC value, it usually needs judge the fungistatic effect of Chinese medicine by judging result twice.
Currently, both at home and abroad to the minimal inhibitory concentration (MIC) of Chinese medicine composition mycoplasma still without no unified standard, therefore root
Criterion according to same experiments document report is more sensitive, MIC when i.e. MIC≤7.81mg crude drugs/mL according to being judged
For 7.81~250mg crude drugs/mL when be medium sensitivity, as MIC > 250mg crude drugs/mL to be insensitive.
As shown in Table 2,1~7 body of compound all has the good external anti-active effect of livestock and poultry mycoplasma, wherein with compound
3, compound 5 and 6 effect of compound are preferable.
The present invention on the basis of theory of traditional Chinese medical science, provide with the good anti-active Chinese medical extract of poultry mycoplasma and
Chinese medicine composition.The active Chinese medical extract of anti-poultry mycoplasma provided by the invention and Chinese medicine composition have following excellent
Point:
1. the active Chinese medical extract of inhibition poultry mycoplasma provided by the invention is to extract to be made from Chinese medicine, with
Chinese medicinal compound all has that the traditional Chinese medicine medicinal herbs resource is extensive, toxic side effect is small, safe to use, and is not likely to produce drug resistance, and poultry
The advantages of medicament residue being not likely to produce in meat, egg.These advantages are all that antibiotic etc is incomparable.
2. Chinese medical extract provided by the invention and Chinese medicinal compound have effects that clearing heat and detoxicating, heat-clearing and damp-drying drug, to poultry
Mycoplasma activity has apparent inhibition, has good antipyretic, analgesia, anti-inflammatory, cough-relieving, resolving sputum and antiasthmatic effect,
Respiratory symptom caused by poultry mycoplasma can be effectively relieved, it is especially former by chicken virus mycoplasma, Mycoplasma bovis and sheep pneumonia branch
Poultry mycoplasmosis caused by one or more mycoplasmas in body.
3. above-mentioned Chinese medical extract and Chinese medicine composition can prepared by chicken virus mycoplasma, Mycoplasma bovis and sheep pneumonia
Application in poultry mycoplasmal diseases drug caused by mycoplasma etc. has good in the treatment and prevention of poultry mycoplasmosis
Development prospect.
Those skilled in the art can also do other forms and details in the claims in the present invention scope of disclosure and spirit
On various modifications, addition and replacement.Certainly, the changes such as these spirit is done according to the present invention various modifications, addition and replacements
Change, all should include within scope of the present invention.