CN104523670B - A kind of method for examining protocatechuic acid soluble powder to be applied in terms of poultry diease treatment - Google Patents
A kind of method for examining protocatechuic acid soluble powder to be applied in terms of poultry diease treatment Download PDFInfo
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- CN104523670B CN104523670B CN201410811749.4A CN201410811749A CN104523670B CN 104523670 B CN104523670 B CN 104523670B CN 201410811749 A CN201410811749 A CN 201410811749A CN 104523670 B CN104523670 B CN 104523670B
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- protocatechuic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
Abstract
The present invention provides a kind of new method for examining protocatechuic acid to evaluate application in terms of poultry diease treatment, comprise the following steps：(1) dilution of protocatechuic acid soluble powder obtains at least two dilution factor protocatechuic acid soluble powder dilutions；(2) the protocatechuic acid soluble powder dilution obtains at least two mixtures with avian viruses immixture；(3) at least two mixture is put into 37 DEG C of 30 60min of effect, is gently shaken once every 5min；(4) at least two mixtures described in step (3) are inoculated in 9 10 age in days no-special pathogen SPF chicken embryos, each 6 10 pieces, 0.2ml/ pieces of dilution factor；(5) the SPF chicken embryos being inoculated with, which are placed in incubator, cultivates, daily to observe chicken embryo death situation, detects HA, makes detail statistics, operation is simple for this method, and cost is relatively low, saves the tedious work of artificial feeding test chicken.
Description
Technical field
The invention belongs to animal doctor's poultry diease therapy field, be related to a kind of inspection protocatechuic acid applied in terms of poultry diease treatment it is new
Method.
Background technology
Protocatechuic acid, alias 3,4-Dihydroxybenzoic acid, CAS registration numbers are [99-50-3], and molecular formula is (HO)
2C6H3COOH, molecular weight 154.12, character, to brown crystalline powder, change colour in atmosphere to be white.Density 1.54g/
cm3.About 200 DEG C of fusing point (decomposition).It is dissolved in 50 parts of water；It is dissolved in ethanol, ether.Protocatechuic acid is present in Lindsaeaceae plant crow
In the plant such as the leaf of fern, the leaf of holly plant Chinese ilex.With antibacterial action, during in vitro test to Pseudomonas aeruginosa, Escherichia coli,
Typhoid bacillus, shigella dysenteriae, Bacillus alcaligenes and hay bacillus and staphylococcus aureus have different degrees of bacteriostasis.Also
There are eliminating the phlegm, antiasthmatic effect.It is clinically used for treatment chronic bronchitis.In recent years, applying for protocatechuic acid is reported in the face research of people hospital
Road it is more, be CN102151256B in veterinary drug application aspect Authorization Notice No.) patent, its patent is by protocatechuic acid
In vivo studies describes application of the protocatechuic acid in prevention livestock and poultry virus infectious diseases.
H9N2 type bird flus, HoMee in 1966 are separated to first plant of H9N2 hypotype from the turkey for suffering from gentle respiratory diseases
Avian influenza virus, H9N2 subtype avian influenzas are worldwide rapid popular afterwards, and especially 1994~1999 years in world's model
Huge economic loss is caused in enclosing.The subtype avian influenza of autumn H9N2 in 1998 occurs in China, is swept across in short 2 months
Most of provinces.New research shows that the pathogenicity of H9N2 hypotypes part epidemic strain substantially makes a variation, dead especially to Broiler chicks
Rate can reach more than 30%；To the laying hen of egg-laying peak, its egg drop reduction amplitude is big, recovers difficult.
Bird flu H7N9 types are a kind of new bird flus, since 2 months 2013, Shanghai City, China, Anhui Province, Jiangsu Province
Unknown cause severe pneumonia case successively occurs with Zhejiang Province and other places, will on March 31st, 2013 after pathogen isolation is identified
It is made a definite diagnosis infects H7N9 subtype avian influenzas (Gao etc., 2013) for people.This is the disease for finding H7N9 subtype avian influenzas infection people first
Example.Current research the result shows that the virus be as entrained by the wild bird of East Asia Region with the ground such as Chinese Shanghai, Zhejiang, Jiangsu
Duck group and the genetic recombination of AIV entrained by chicken group form.
Newcastle disease (ND) is commonly called as checken pest, is a kind of high degree in contact, acute, strong of the chicken as caused by newcastle disease virus
Sexually transmitted disease.Septicemia is often presented to pass through, is mainly characterized by having difficulty in breathing, diarrhea, nerve disorder, mucous membrane and serous coat go out
Blood.The chicken at each breeder and various ages can infect, young chicken and middle chicken more easy infection, and the old chicken neurological susceptibility of more than 2 years reduces.
The disease can occur throughout the year.In the chicken of nonimmune area or immunocompromised group, once there is the intrusion of anaphylactic type strain, can pass rapidly
Broadcast, in crushing prevalence, morbidity and mortality are up to more than 90%.
With raising of the China to the safe attention degree of biological food, in terms of livestock and poultry medication also can more specification, antibiosis
The situation of element abuse must be governed.Protocatechuic acid is as a kind of natural plant extracts, because it is with anti-bacteria and anti-virus work
With it can earn widespread respect in terms of livestock and poultry treatment use, especially to the bird flu of poultry pandemic, ewcastle disease etc.
Treatment in terms of application.
The content of the invention
The object of the present invention is to provide a kind of new method for examining protocatechuic acid to be applied in terms of poultry diease treatment, by external
Experiment, can Fast Evaluation protocatechuic acid to the killing effect of avian viruses, for carry out poultry diease treat in vivo studies provides theory according to
According to.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of new method for examining protocatechuic acid to be applied in terms of poultry diease treatment, comprises the following steps：
(1) by the protocatechuic acid soluble powder with the purified water containing antibiotic according to default at least two dilution ratio
Dilution, obtains the protocatechuic acid soluble powder dilution with least two dilution factors；
(2) the protocatechuic acid soluble powder dilution at least two dilution factors for obtaining step (1) respectively with avian viruses
Equal proportion mixes, and obtains at least two mixtures, while mixed with avian viruses equal proportion with the sterile saline containing antibiotic
As control；
(3) step (2) at least two mixture is put into 37 DEG C of effect 30-60min, one is gently shaken every 5min
It is secondary；
(4) 9-10 age in days no-special pathogens SPF will be inoculated in by least two mixture of step (3) processing
Chicken embryo, each 6-10 pieces, 0.2ml/ pieces of dilution factor；
(5) the SPF chicken embryos for the inoculation for obtaining step (4), which are placed in incubator, cultivates, daily to observe chicken embryo death situation,
Dead chicken embryo discards in 24h, and the chicken embryo of 24h-144h death is temporarily stored into 4 DEG C of preservations, and no matter chicken embryo is survival during more than 144h
Or it is dead all to take allantoic fluid, HA is detected, is counted.
Preferably, antibiotic described in step (1) is mycillin.
Preferably, the concentration described in step (1) in purified water is 1000IU/ml.
Preferably, default at least two dilution ratio described in step (1) is 1:100、1:1000、1:5000、1:
10000。
Preferably, avian viruses described in step (2) for newcastle disease virus (NDV), bird flu H9 subtype virus (H9N2) and
Any one in bird flu H7 subtype virus (H7N9) or any multiple combinations.
Preferably, each virus quantity is 200ELD50/0.1ml in step (2).
Preferably, mixture described in step (3) puts 37 DEG C of effect 45min.
Preferably, SPF ages in days described in step (4) are 10 ages in days.
Preferably, every kind of dilution factor is inoculated in 10 pieces of chicken embryos in step (4).
Preferably, observed once every 8h after inoculated into chick embryo 24h described in step (5).
The beneficial effects of the invention are as follows a kind of new method for examining protocatechuic acid to be applied in terms of poultry diease treatment is provided, pass through
In vitro test, can Fast Evaluation protocatechuic acid to the killing effect of avian viruses, treat in vivo studies to carry out poultry diease and reason be provided
By foundation.
Embodiment
The object of the present invention is to provide a kind of new method for examining protocatechuic acid to be applied in terms of poultry diease treatment, by external
Experiment, can Fast Evaluation protocatechuic acid to the killing effect of avian viruses, for carry out poultry diease treat in vivo studies provides theory according to
According to.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of new method for examining protocatechuic acid to be applied in terms of poultry diease treatment, comprises the following steps：
(1) by the protocatechuic acid soluble powder with the purified water containing antibiotic according to default at least two dilution ratio
Dilution, obtains the protocatechuic acid soluble powder dilution with least two dilution factors；
(2) the protocatechuic acid soluble powder dilution at least two dilution factors for obtaining step (1) respectively with avian viruses
Equal proportion mixes, and obtains at least two mixtures, while mixed with avian viruses equal proportion with the sterile saline containing antibiotic
As control；
(3) step (2) at least two mixture is put into 37 DEG C of effect 30-60min, one is gently shaken every 5min
It is secondary；
(4) 9-10 age in days no-special pathogens SPF will be inoculated in by least two mixture of step (3) processing
Chicken embryo, each 6-10 pieces, 0.2ml/ pieces of dilution factor；
(5) the SPF chicken embryos for the inoculation for obtaining step (4), which are placed in incubator, cultivates, daily to observe chicken embryo death situation,
Dead chicken embryo discards in 24h, and the chicken embryo of 24h-144h death is temporarily stored into 4 DEG C of preservations, and no matter chicken embryo is survival during more than 144h
Or it is dead all to take allantoic fluid, HA is detected, is counted.
Preferably, antibiotic described in step (1) is mycillin.
Preferably, the concentration described in step (1) in purified water is 1000IU/ml.
Preferably, default at least two dilution ratio described in step (1) is 1:100、1:1000、1:5000、1:
10000。
Preferably, avian viruses described in step (2) for newcastle disease virus (NDV), bird flu H9 subtype virus (H9N2) and
Any one in bird flu H7 subtype virus (H7N9) or any multiple combinations.
Preferably, each virus quantity is 200ELD50/0.1ml in step (2).
Preferably, mixture described in step (3) puts 37 DEG C of effect 45min.
Preferably, SPF ages in days described in step (4) are 10 ages in days.
Preferably, every kind of dilution factor is inoculated in 10 pieces of chicken embryos in step (4).
Preferably, observed once every 8h after inoculated into chick embryo 24h described in step (5).
In order to further illustrate the present invention, with reference to embodiments to a kind of inspection protocatechuic acid provided by the invention in fowl
The new method of application is described in detail in terms of sick treatment.
Below by way of specific example, to be described in detail to the present invention, but protection scope of the present invention is from reality
Apply the limitation of example.
The preparation of 1 test material
Protocatechuic acid soluble powder：Extracted and prepared by Shandong Sinder Technology Co., Ltd..
SPF chicken embryos：Purchased from Jinan Si Paifurui Qin Ye Science and Technology Ltd.s.
Purified water：It is prepared by Shandong Sinder Technology Co., Ltd. GMP workshops.
Strain：Bird flu H9N2, H7N9 and newcastle disease virus.
2 test methods
This experiment selection bird flu H9N2, H7N9 and newcastle disease virus are as virus is examined, and three kinds of viruses are under
Row operation carries out：
(1) mycillin is added into purified water, it is 1000IU/ml to make antibiotic concentration, and protocatechuic acid is used respectively
The purified water does 1:100、1:1000、1:5000、1:10000 dilutions.
(2) experiment is divided into 5 groups, and the 1st group is avian viruses and 1:100 times of diluted protocatechuic acid test groups, the 2nd, 3,4 components
Wei not avian viruses and 1:1000、1:5000、1:10000 times of diluted protocatechuic acid test groups, the 5th group to be viral with containing antibiotic
Purified water effect control group.Detailed packet situation is shown in Table 1.
(3) every kind of viral content is 200ELD50/0.1ml, and virus is with protocatechuic acid dilution group with 1:1 ratio is mixed
Close, after 37 DEG C act on 45min, be inoculated in 10 age in days SPF chicken embryos, every group 10 pieces, per embryo 0.2ml.
(4) observation each group chicken embryo death situation daily, dead in 24h to discard, dead chicken embryo is placed in 4 DEG C temporarily after 24h
Deposit, continuous observation to 144h, when 144h is not dead to take allantoic fluid to carry out HA detections at the same time with dead chicken embryo before, records every group of embryo
HA-HI test.
Table 1 tests packet situation
3 result of the tests
(1) it is inoculated with each test group result of H9N2 viruses
Table 2 is inoculated with H9N2 virus chicken embryo death situations
Table 3 is inoculated with H9N2 virus chicken embryo HA-HI tests
It is right by table 2 and table 3 as it can be seen that protocatechuic acid addition group has notable difference with being not added with control group chicken embryo death quantity
According to organizing dead 10 pieces, the addition group of protocatechuic acid has the trend of dead reduction as dosage increases chicken embryo, 1:100 and 1:1000
Survival rate 100% during dilution factor, 1:Survive 90% piece during 5000 dilution factor, but when dilution factor increment 1:When 10000, protective rate is bright
It is aobvious to decline；And dead chicken embryo has HA-HI test, dead chicken embryo is not without HA-HI test, illustrates protocatechuic acid in suitable concentration, 37 DEG C
H9N2 viruses can thoroughly be killed.
(2) it is inoculated with each test group of H7N9 viruses
Table 4 is inoculated with H7N9 virus chicken embryo death situations
Table 5 is inoculated with H7N9 virus chicken embryo HA-HI tests
It is right by table 4 and table 5 as it can be seen that protocatechuic acid addition group has notable difference with being not added with control group chicken embryo death quantity
According to organizing dead 10 pieces, the addition group of protocatechuic acid has the trend of dead reduction as dosage increases chicken embryo, 1:100 and 1:1000
Survival rate is 100%, 1 during dilution factor:5000 and 1:Protective rate is decreased obviously when 10000.And dead chicken embryo has HA-HI test, not
Dead chicken embryo illustrates in protocatechuic acid in suitable concentration, 37 DEG C can thoroughly kill H7N9 viruses without HA-HI test.
(1) it is inoculated with each test group of ND viruses
Table 6 is inoculated with ND virus chicken embryo death situations
Table 7 is inoculated with ND virus chicken embryo HA-HI tests
By table 6 and table 7 as it can be seen that protocatechuic acid addition group is 1:100 survival rates are 90%, 1:1000 and following dilution factor
When, survival rate is not higher than 30%, illustrates protocatechuic acid in higher concentrations, and 37 DEG C, with that can kill part ND viruses, are killed
Effect of going out is obvious not as bird flu H9N2 and H7N9.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the principle of the present invention, some improvement and change can also be made, such modifications and variations also should
It is considered as protection scope of the present invention.
Claims (6)
- A kind of 1. method for examining protocatechuic acid to be applied in terms of poultry diease treatment, it is characterised in that comprise the following steps：(1) it is the protocatechuic acid soluble powder is dilute according to default at least two dilution ratio with the purified water containing antibiotic Release, obtain the protocatechuic acid soluble powder dilution with least two dilution factors, the antibiotic is mycillin, described pure The concentration for changing mycillin in water is 1000IU/ml；(2) the protocatechuic acid soluble powder dilution at least two dilution factors for obtaining step (1) respectively with avian viruses grade ratio Example mixing, obtains at least two mixtures, at the same mixed with avian viruses equal proportion by the use of the sterile saline containing antibiotic as Control, each virus quantity are 200ELD50/0.1ml, and the avian viruses is newcastle disease virus (NDV), bird flu H9 subtype virus (H9N2) any one and in bird flu H7 subtype virus (H7N9) or any multiple combinations；(3) step (2) at least two mixture is put into 37 DEG C of effect 30-60min, is gently shaken once every 5min；(4) 9-10 age in days no-special pathogen SPF chickens will be inoculated in by least two mixture of step (3) processing Embryo, each 6-10 pieces, 0.2ml/ pieces of dilution factor；(5) the SPF chicken embryos for the inoculation for obtaining step (4), which are placed in incubator, cultivates, daily to observe chicken embryo death situation, 24h Interior dead chicken embryo discards, and the chicken embryo of 24h-144h death is temporarily stored into 4 DEG C of preservations, during more than 144h no matter chicken embryo be survival or It is dead all to take allantoic fluid, HA is detected, is counted.
- 2. the method for inspection according to claim 1, it is characterised in that default at least two dilution described in step (1) Ratio is 1：100、1：1000、1：5000、1：10000.
- 3. the method for inspection according to claim 1, it is characterised in that mixture described in step (3) puts 37 DEG C of effects 45min。
- 4. the method for inspection according to claim 1, it is characterised in that SPF ages in days described in step (4) are 10 ages in days.
- 5. the method for inspection according to claim 1, it is characterised in that every kind of dilution factor is inoculated in 10 pieces of chickens in step (4) Embryo.
- 6. the method for inspection according to claim 1, it is characterised in that every 8h after inoculated into chick embryo 24h described in step (5) Observation is once.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792739A (en) * | 2010-03-26 | 2010-08-04 | 广西大学 | Optimum condition for proliferating Guangxi epidemically representative strains of infectious bursal disease virus (IBDV) in chicken embryo |
| CN102151256A (en) * | 2011-02-23 | 2011-08-17 | 中国农业大学 | Application of protocatechuic acid in preparation of drugs for preventing and controlling livestock and poultry virus infectious diseases |
| CN102391366A (en) * | 2011-11-08 | 2012-03-28 | 哈药集团生物疫苗有限公司 | Preparation method of chicken infectious bronchitis virus HA antigen |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792739A (en) * | 2010-03-26 | 2010-08-04 | 广西大学 | Optimum condition for proliferating Guangxi epidemically representative strains of infectious bursal disease virus (IBDV) in chicken embryo |
| CN102151256A (en) * | 2011-02-23 | 2011-08-17 | 中国农业大学 | Application of protocatechuic acid in preparation of drugs for preventing and controlling livestock and poultry virus infectious diseases |
| CN102391366A (en) * | 2011-11-08 | 2012-03-28 | 哈药集团生物疫苗有限公司 | Preparation method of chicken infectious bronchitis virus HA antigen |
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Denomination of invention: A method for testing the application of protocatechuic acid soluble powder in the treatment of poultry diseases Effective date of registration: 20211222 Granted publication date: 20180427 Pledgee: Shandong Zhucheng rural commercial bank Limited by Share Ltd. Pledgor: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Registration number: Y2021980015708 |
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Denomination of invention: A method for testing the application of Protocatechuic acid soluble powder in the treatment of poultry diseases Effective date of registration: 20230608 Granted publication date: 20180427 Pledgee: Shandong Zhucheng rural commercial bank Limited by Share Ltd. Pledgor: SHANDONG SINDER TECHNOLOGY Co.,Ltd. Registration number: Y2023980043376 |
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