CN102106465B - Application of perylenequinone compound in preparation of feed for preventing and controlling ruminant viruses - Google Patents
Application of perylenequinone compound in preparation of feed for preventing and controlling ruminant viruses Download PDFInfo
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Abstract
The invention discloses application of a perylenequinone compound in preparation of a feed for preventing and controlling ruminant viruses, and belongs to the technical field of bioengineering and agriculture. The perylenequinone compound is added into a ruminant feed to prepare the feed for preventing and controlling the ruminant viruses such as foot-and-mouth disease virus, cell pathogenetic bovine viral diarrhea virus-mucosal disease virus, bluetongue virus, capripoxvirus, epuine morbillivirus and the like. The perylenequinone compound comprises hypocrellin, elsinochrome, phleichrome, cladosporium, calphostin, cercosporin and the like; and the perylenequinone compound has a series of advantages of extracting convenience, good stability, no obvious side effect in oral administration, quick metabolism and the like. The dosage of the prepared feed for preventing the ruminant viruses is 1 to 10mumol perylenequinone compound per kg weight every day, the dosage of the prepared feed for treating the ruminant viruses is 10 to 100mumol perylenequinone compound per kg weight, the effect of preventing and treating the viruses is very remarkable, meanwhile, the perylenequinone compound can be obtained by modern biotechnology, and good economic and social benefits can be generated.
Description
Technical field
The application of perylene naphtoquinone compounds in the feed of the ruminant viruses such as preparation anti-smelting ox, sheep, donkey, horse, the present invention relates to Cai perylene naphtoquinone compounds and adds in feed for the preparation of the feed preventing and treating ruminant virus.Belong to bioengineering and agricultural technology field.
Background technology
In recent years, along with China's expanding economy and growth in the living standard, milk industry and beef industry become the bright spot of Structure Adjustment of Animal Husbandry.The pillar industry of Yang Yeyeshi China borderland and minority area economic development simultaneously.
But along with the continuous expansion of cultivation scale, large-scale epidemic disease also can be brought to break out, cause huge loss to plant.Especially viral disease has the advantages that infectiousness is strong and the death rate is high.As foot and mouth disease virus (Foot-and-mouth disease virus, FMDV), cytopathogenic effect type ox diarrhoea-bovine diarrhoea virus (Bovine viral diarrhoea virus, BVDV), blue tongue virus (Bluetongue virus, BTV), capripox virus (capripoxvirus), equine morbillivirus (Epuine morbillivirus, EMV) etc. are all pathogenies of the main diseases viral disease in ruminant cultivation.
Patent CN200810166154.2, CN03103300.8 and CN200680051113.8 etc. adopt the method for vaccine to carry out the control of virus, however animal virus not only kind is many, and variation is rapidly, is often hard to guard against.Amantadine, Rimantadine, Oseltamivir and zanamivir etc. are conventional antiviral agents, but its curative effect of medication imprecise, and there is the problems such as residual.
Patent CN200610072935.6 reports the antiviral compound that hypericin is a kind of wide spectrum, has good killing action for viruses such as aftosas.Hypericin is from Chinese herbal medicine hypericum perforatum, extract a kind of phenanthro-quinones (Phenanthroperylenequinone) compound obtained, and it shows the good viral kill effect of wide spectrum.But hypericin unstable chemcial property, see that light easily decomposes, have quite a few hypericin to be used effectively in actual applications.
We study the effect that Fa Xian perylene quinone (Perylenequinone) compounds has good broad-spectrum antiviral.Perylene naphtoquinone compounds, compared with hypericin, has and extracts the series of advantages such as convenience, good stability, oral non-evident effect and metabolism are fast.Therefore the invention discloses the method that Yi Zhong perylene naphtoquinone compounds is applied to ruminant feed, thus effectively carry out the control of avian viral.
Summary of the invention
The object of the invention is perylene naphtoquinone compounds to be applied in feed and be used for prevention and therapy ruminant virus infections disease.
The correlated characteristic that the present invention relates to is as follows:
1, perylene naphtoquinone compounds explanation
It is three kinds of parent nucleus as follows is basic structure that Suo of the present invention Zhis perylene naphtoquinone compounds, and carries out the compound of base group modification with these three kinds of mother nucleus structures.Can 2, upper various chemical group is modified in 3,6,7,11,12,17,18,26,28,29,30,32,36,37,45,46,53,55,56,58,62,63,71,72 positions, as CH
3, OCH
3, COCH
3, CH (OH) CH
3, CHO, OH, H etc., can also form more complicated structure between these chemical groups further.
Perylene naphtoquinone compounds parent nucleus
Special in hypocrellin (hypocrellin A Hypocrellin A, hypocrelline B Hypocrellin B), Elsinochrome element (Elsinochrome A Elsinochrome A, Elsinochrome B Elsinochrome B, Elsinochrome C Elsinochrome C, Elsinochrome D Elsinochrome D), Phleichrome (Phleichrome), branch spore element (Cladosporium A Cladosporium A, Cladosporium B Cladosporium B, Cladosporium C Cladosporium C, Cladosporium D Cladosporium D), Ka Futading (Calphostin A, Calphostin B, Calphostin C, Calphostin D), hypomycin A and hypomycin B, cercosporin (Cercosporin), Scutiaquinone A and Scutiaquinone B etc.Their chemical constitution is see following pertinent literature:
[1]Iida，T.，E.Kobayashi，M.Yoshida，and H.Sano(1989)Calphostins，novel and specific inhibitors of protein kinase C.II.Chemical structures.J.Antibiot.42：1475-1481.
[2]Lousberg，R.J.，C.A.Salemink，U.Weiss，and Batterha.Tj(1969)Pigmentsof Elsinoe species.Part II.Structure of elsinochromes A，B，and C.Journal of theChemical Society C-Organic.1219-1227.
[3]Lousberg，R.J.，C.A.Salemink，and U.Weiss(1970)Pigments of Elsinoespecies.Part V.The structure of elsinochrome D.Journal of the Chemical SocietyC-Organic.2159-2162.
[4]Diwu，Z.J.，and J.W.Lown(1990)Hypocrellins and their use inphotosensitization.Photochem.Photobiol.52：609-616.
[5]Liu，W.Z.，Y.X.Shen，X.F.Liu，Y.T.Chen，and J.L.Xie(2001)A newperylenequinone from Hypomyces sp.Chinese Chemical Letters.12：431-432.
[6]Yoshihara，T.，T.Shimanuki，T.Araki，and S.Sakamura(1975)Phleichrome，a new phytotoxic compound produced by Cladosporium phlei.Agric.Biol.Chem.39：1683-1684.
[7]Ayers，S.，D.L.Zink，K.Mohn，J.S.Powell，C.M.Brown，T.Murphy，R.Brand，S.Pretorius，D.Stevenson，D.Thompson，and S.B.Singh(2007)Scutiaquinones A and B，perylenequinones from the roots of Scutia myrtina withanthelmintic activity.Journal of Natural Products.70：425-427.
[8]Lousberg，R.J.，U.Weiss，C.A.Salemink，A.Arnone，L.Merlini，andG.Nasini(1971)The structure of cercosporin，a naturally occurring quinone.Journalof the Chemical Society D-Chemical Communications.1463-&.
[9]Arnone，A.，G.Assante，V.Dimodugno，L.Merlini，and G.Nasini(1988)Perylenequinones from cucumber seedlings infected with Cladosporiumcucumerinum.Phytochemistry.27：1675-1678.
2, the source of perylene naphtoquinone compounds
Hypocrellin source has: bamboo parasitic fungus (Shiraia bambusicola) fructification that what (1) was natural be longer than on bamboo and the red bacterium of bamboo (Hypocrella bambusae) fructification; (2) method according to patent CN200710132510.4 and CN200910182255.3 adopts bamboo parasitic fungus to carry out liquid state fermentation or solid state fermentation obtains.
Tabasheer bacterial strain can be: Shiraia sp.SUPER-H168 preserving number is CCTCC NO:M 207104 or other Shiraia bambusicola bacterial strain that can buy from Spawn preservation organization.
The Elsinochrome available Elsinoe fawcettu ATCC 38162 of element or other Elsinoe fawcettu bacterial strain that can buy from Spawn preservation organization are obtained by solid state fermentation or liquid state fermentation method.
The branch spore available Cladosporium cucumerinum.ATCC 11279 of element or other Cladosporium cucumerinum bacterial strain that can buy from Spawn preservation organization are obtained by solid state fermentation or liquid state fermentation method.
The Cladosporium cladosporioides bacterial strain that Ka Futading can buy from Spawn preservation organization with Cladosporium cladosporioides CGMCC 34593 or other is obtained by solid state fermentation or liquid state fermentation method.
The Cercospora kikuchiis bacterial strain that cercosporin can buy from Spawn preservation organization with Cercospora kikuchii ATCC 42152 or other is obtained by solid state fermentation or liquid state fermentation method.
The Cladosporium phlei bacterial strain that Phleichrome can buy from Spawn preservation organization with Cladosporium phlei ATCC 36193 or other is obtained by solid state fermentation or liquid state fermentation method.
Scutiaquinone can adopt alcohol extract to obtain to the root of thorn Calamus plant Scutia myrtina.
The solid state fermentation conditions of all bacterial classifications all can be above: employing grain is primary raw material, and is crushed to 50-80 order, separately adds in 0.5%-2% glucose, the 0.1%-0.5%KH of primary raw material
2pO
4, 0.01%-0.08%MgSO
47H
2o.The spice that adds water wets, and material-water ratio controls 1: 1-1: 0.7,121 DEG C of sterilizing 45-60min, access 5%-10% liquid seeds after cooling.Initial pH and sweat pH is nature, fermentation temperature 24-32 DEG C, fermentation processes humidity 80%-90%, 5-15 days fermentation ends.Grain raw material can be: rice, millet, corn, wheat, buckwheat, Chinese sorghum, black rice, dregs of beans, soybean or red bean.
The liquid state fermentation condition of all bacterial classifications all can be above: carbon source 5-50g/L, nitrogenous source 5-50g/L, potato 100-200g/L, diammonium hydrogen phosphate 1-2g/L, potassium dihydrogen phosphate 1-2g/L, magnesium sulfate 0.1-0.8g/L; 121 DEG C of sterilizing 20min, access bacterial classification after sterilizing cooling, inoculum concentration is 5%-10%, and initial pH and sweat pH is nature, and fermentation temperature is 24-32 DEG C, and fermentation period is 2-5 days; Carbon source can be glucose, fructose, sucrose or soluble starch.Nitrogenous source can be inorganic ammonium salt (as NH
4cl), inorganic nitrate is (as NaNO
3, NH
4nO
3), peptone, yeast extract, beef extract, urea, corn steep liquor or beancake powder.
3. the adding method of perylene naphtoquinone compounds in feed
The dosage of pre-anti-virus is 1-10 μm of ol perylene naphtoquinone compounds/kg body weight every day, is admixed in feed by perylene naphtoquinone compounds and feeds, add all the time in the growth course of ruminant.
Therapeutic dose for the ruminant infecting virus is 10 μm ol-100 μm ol perylene naphtoquinone compounds/kg body weight every day, and the course for the treatment of is 2 to 7 days.
Described ruminant virus is foot and mouth disease virus, cytopathogenic effect type ox diarrhoea-bovine diarrhoea virus, blue tongue virus, capripox virus or equine morbillivirus etc.
(1) for natural bamboo parasitic fungus or bamboo red mushroom entity source perylene naphtoquinone compounds: according to the body weight of ruminant with perylene naphtoquinone compounds demand, get a certain amount of bamboo parasitic fungus or the red mushroom entity of bamboo is crushed to 40-60 order, be then evenly mixed in the fine fodder of daily feed of ruminant.
(2) for the perylene naphtoquinone compounds that solid state fermentation must arrive: 50-60 DEG C of oven dry after fermentation ends, then 40-60 order is crushed to, according to the body weight of ruminant with perylene naphtoquinone compounds demand, get a certain amount of crushed material and be evenly mixed in the fine fodder of daily feed of ruminant.
(3) for the perylene naphtoquinone compounds that liquid state fermentation must be arrived: after fermentation ends, centrifugal segregation liquid obtains the solid contents such as mycelium, 50-60 DEG C of oven dry, then 40-60 order is crushed to, according to the body weight of ruminant with perylene naphtoquinone compounds demand, get a certain amount of crushed material and be evenly mixed in the fine fodder of daily feed of ruminant.
(4) for solid state fermentation or liquid state fermentation product: the ethanol that can add 1 times of weight in tunning, cross elimination solid content; crude extract is made in the extraction of perylene naphtoquinone compounds, 50-60 DEG C of oven dry, then 40-60 order is crushed to, according to the body weight of ruminant with perylene naphtoquinone compounds demand, get a certain amount of crushed material and be evenly mixed in the fine fodder of daily feed of ruminant.
(5) mainly root is present in for plant Scutia myrtina:Scutiaquinone, according to the body weight of ruminant with perylene naphtoquinone compounds demand, get a certain amount of Scutia myrtina root, first with ethanol, Scutiaquinone is extracted (ethanol: root=1: 0.5-1: 3 from root, weight ratio), filter and remove rhizome, 50-60 DEG C of Vacuum Concentration is removed alcohol and dries, be crushed to 40-60 order again, then measured according to demand in the fine fodder of the daily feed being evenly mixed into ruminant.
(6) Zhe Xie perylene naphtoquinone compounds can be monomer by preparative chromatography purifying, are mixed in the fine fodder of daily feed of ruminant.Prepare the condition of purifying as described in the document of previous publications.Xiao-Hui Lianga，Yu-JieCai，Xiang-Ru Liao，Kang Wu，Lei Wang，Da-Bing Zhang and Qiang Meng(2009).Isolation and identification of a new hypocrellin A-producing strain Shiraia spSUPER-H168.Microbiological Research 164(1)：9-17.
(6) separate sources perylene naphtoquinone compounds can be used in combination in any proportion according to actual conditions.
Beneficial effect of the present invention: the present invention relates to Cai perylene naphtoquinone compounds and add to for the preparation of the feed preventing and treating ruminant virus in ruminant feed, as foot and mouth disease virus, cytopathogenic effect type ox diarrhoea-bovine diarrhoea virus, blue tongue virus, capripox virus, equine morbillivirus etc.Perylene naphtoquinone compounds comprises: hypocrellin, Elsinochrome element, Phleichrome, branch spore element, Ka Futading, cercosporin etc., and perylene naphtoquinone compounds has the series of advantages such as extraction convenience, good stability, oral non-evident effect and metabolism are fast.Dosage in the feed of preparation prevention ruminant virus is 1-10 μm of ol perylene naphtoquinone compounds/kg body weight every day, dosage in the feed of preparation treatment ruminant virus is 10 μm ol-100 μm ol perylene naphtoquinone compounds/kg body weight, the effect highly significant Tong Shi perylene naphtoquinone compounds of control virus is produced by modern biotechnology and is obtained, and can produce good economic and social benefit.
Detailed description of the invention
Embodiment 1: perylene naphtoquinone compounds antiviral study in vitro
(1) preparation of antiviral study in vitro cell
Grind study carefully the effect of perylene naphtoquinone compounds to foot and mouth disease virus (FMDV) with BHK-21 cell (young hamster kidney passage cell, Baby hamster kidney cells).Growth medium: by MEM (modified eaglemedium) culture medium buied, separately add NaHCO
32.0g/L, 2% hyclone, 1% penicillin, 1% streptomysin.By BHK-21 cell with after the trypsinization of 0.25%, piping and druming makes cell dispersal, is diluted to 1 × 10 with MEM growth medium
6cell/mL, adds in 96 porocyte culture plates, 37 DEG C, 5%CO
2cultivate in incubator, grow up to after cell monolayer until cell, carry out In vitro antibacterial test.
With Vero cell, (African green monkey kidney cell, Verda Reno) Yan Jiu perylene naphtoquinone compounds is to the effect of blue tongue rims (BTV).Growth medium: by the MEM culture medium buied, separately add 110mg/L Sodium Pyruvate, 300mg/L Glu, 200mg/L NaHCO
310% hyclone, 1% penicillin, 1% streptomysin.By Vero cell with after the trypsinization of 0.25%, piping and druming makes cell dispersal, is diluted to 1 × 10 with MEM growth medium
6cell/mL, adds in 96 porocyte culture plates, 37 DEG C, 5%CO
2cultivate in incubator, grow up to after cell monolayer until cell, carry out In vitro antibacterial test.
With BTC cell, (bovine testicle cell, Bovine testicular cells) Yan Jiu perylene naphtoquinone compounds is to the effect of cytopathogenic effect type ox diarrhoea-bovine diarrhoea virus (BVDV).Growth medium: DMEM (Dulbecco ' smodified eagle medium), containing 4500mg/L D-Glucose, 300mg/L Glu, 110mg/L Sodium Pyruvate, 5% hyclone, 1% penicillin, 1% streptomysin).By BTC cell with after the trypsinization of 0.25%, piping and druming makes cell dispersal, is diluted to 1 × 10 with DMEM growth medium
6cell/mL, adds in 96 porocyte culture plates, 37 DEG C, 5%CO
2cultivate in incubator, grow up to after cell monolayer until cell, carry out In vitro antibacterial test.
(2) the antiviral study in vitro mensuration of viral tissue culture infective dose (Tissue culture infectivedose, TCID50)
After cell grows up to individual layer, inoculation 10
-2-10
-1dilution virus liquid (0.1mL/ hole), each dilution factor inoculates 8 holes, in 5%CO
2in incubator 37 DEG C cultivate 120h, every day observation of cell pathology (CEP) situation, according to TCID50 (Reed, the L.J. of Reed-Muench formulae discovery virus; Muench, H. (1938) .A simplemethod of estimating fifty percent endpoints.The American Journal of Hygiene 27:493-497.).
(3) MTT decoration method calculates cell survival rate
Mtt assay (tetrazolium-based colorimetric assay) carries out cell dyeing (final concentration 0.5g/L) and detects cell survival and growth.The absorbance (OD) at 490nm place is measured, by following formulae discovery cell survival rate: cell survival rate (%)=[adding perylene quinone group OD/ normal cell group OD] × 100% with ELIASA.
(4) perylene naphtoquinone compounds are to the toxicity test of cell
With DMSO Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and Phleichrome be mixed with respectively 0.01,0.1,02,0.4mmol/mL, join in BHK-21, Vero and BTC cell grown up to respectively, and compare with BHK-21, Vero and BTC cell of not Han Jia perylene naphtoquinone compounds, each test 3 bottles, at 37 DEG C, 5%CO
2incubator cultivates 120 hours, and these are cultivated at darkroom (unglazed photograph) and carry out parallel laboratory test under having illumination condition, measures cell survival rate.
Experimental result find, the survival rate of all test cell all about 99.6%, therefore Xia above-mentioned Nong Du perylene naphtoquinone compounds to cell without obvious CDCC.
(5) perylene naphtoquinone compounds are to the direct deactivation of virus
Ce Dings perylene naphtoquinone compounds Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and Phleichrome to the direct deactivation of virus.In 100TCID50 virus liquid, Jia Ru perylene naphtoquinone compounds (is dissolved in DMSO [Dimethyl sulfoxide, dimethyl sulfoxide (DMSO)] in) Shi perylene naphtoquinone compounds in virus liquid concentration reach 0.05 respectively, 0.1,0.2,0.4mmol/L, at 37 DEG C, 5%CO
2after cultivating 1h in incubator, infect above-mentioned cultured corresponding cell.Observation of cell pathology situation under inverted microscope, when virus control group cytopathy reach 75%-100%, cell controls normal time, carry out interpretation of result by MTT decoration method.These experiments are respectively at darkroom (unglazed photograph) with carry out parallel laboratory test under having illumination condition, and light source used is the fluorescent lamp of wavelength at 470-590nm, and the light dosage that total irradiation enters is 9KJ (use photometric determination).In cell cultivation process, observation of cell pathology situation under inverted microscope, when virus control group cytopathy reach 75%-100% cell controls normal time, carry out interpretation of result by MTT decoration method.
The cell survival rate of FMDV, BTV and BVDV virus control group of Bu Jia perylene naphtoquinone compounds is respectively 8.4%, 8.0 and 8.1%.The cell survival rate of Jia perylene naphtoquinone compounds is as shown in table 1.
Table 1: cell survival rate (%)
(6) perylene naphtoquinone compounds are to the blocking effect of Virus entry cell
Ce Dings perylene naphtoquinone compounds Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and Phleichrome to the blocking effect of Virus entry cell.Get the culture plate growing up to cell monolayer, outwell nutrient solution ， Jiang perylene naphtoquinone compounds with 0.05,0.1,0.2,0.4mmol/L concentration (being dissolved in DMSO), be added to respectively on Tissue Culture Plate, 0.1mL/ hole, each trial degree repeats 3 holes.37 DEG C of effect 4h, discard liquid, every hole adds 100TCID50 virus liquid 0.1mL, separately establishes cell controls and virus control.5%CO
2, 120h cultivated by incubator 37 DEG C.These experiments are respectively at darkroom (unglazed photograph) with carry out parallel laboratory test under having illumination condition, and light source used is the fluorescent lamp of wavelength at 470-590nm, and the light dosage that total irradiation enters is 9KJ (use photometric determination).In cell cultivation process, by observation of cell pathology situation under inverted microscope, when virus control group cytopathy reach 75%-100%, cell controls normal time, carry out interpretation of result by MTT decoration method, and calculate cell and deposit rate.
The cell survival rate of FMDV, BTV and BVDV virus control group of Bu Jia perylene naphtoquinone compounds is respectively 9.3%, 9.0 and 9.5%.The cell survival rate of Jia perylene naphtoquinone compounds is as shown in table 2.
Table 2: cell survival rate (%)
(7) perylene naphtoquinone compounds are to the inhibitory action of virus multiplication
Ce Dings perylene naphtoquinone compounds Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and Phleichrome to the inhibitory action of virus multiplication.Get the culture plate growing up to cell monolayer, outwell nutrient solution, the virus liquid of inoculation 100TCID50,100 μ L/ holes, put 37 DEG C, 5%CO
24h is adsorbed in incubator.Discard virus liquid, add respectively variable concentrations be 0.05,0.1,0.2,0.4mmol/L perylene naphtoquinone compounds (being dissolved in DMSO), 0.1mL/ hole, each concentration repeats 3 holes, establishes normal cell controls and virus control simultaneously.5%CO
2incubator 37 DEG C cultivation.These experiments are respectively at darkroom (unglazed photograph) with carry out parallel laboratory test under having illumination condition, and light source used is the fluorescent lamp of wavelength at 470-590nm, and the dosage that total irradiation enters is 9KJ (use photometric determination).In cell cultivation process, observation of cell pathology situation under inverted microscope, when virus control group cytopathy reach 75%-100% cell controls normal time, carry out interpretation of result by MTT decoration method.
The cell survival rate of FMDV, BTV and BVDV virus control group of Bu Jia perylene naphtoquinone compounds is about 8.4%.The cell survival rate of Jia perylene naphtoquinone compounds is as shown in table 3.
Table 3: cell survival rate (%)
(8) conclusion
Can know that ， perylene naphtoquinone compounds all has killing action for test by ruminant virus by above experiment, be the antiviral compound of wide spectrum.Wherein the effect of Calphostin C, Hypocrellin A, Hypocrellin B and Elsinochrome A is best, and Qi Ta perylene naphtoquinone compounds is slightly weak.In addition under illumination condition, antiviral effect is also better than unglazed condition, this mainly You Yu perylene naphtoquinone compounds can produce the products such as superoxide ion under light illumination, its photosensitized oxidation product can strengthen anti-virus ability.
Embodiment 2: the anti-sheep FMDV that solid state fermentation hypocrellin makes an addition in feed tests
In experiment, the basal diet formula of sheep is:
Concentrated feed is filled a prescription: corn 46%, wheat bran 20%, cotton dregs or the dish dregs of rice 30%, stone flour 1%, calcium monohydrogen phosphate 1%, salt 1%, premix 1% (can be every kg feed and provides vitamin A 1200IU, vitamin D 32500IU, vitamin E 30IU, vitamin K 3mg, riboflavin 4mg, nicotinic acid 40mg, pantothenic acid 15mg, Choline Chloride 400mg.Folic acid 700g, thiamine VB 1.5mg, Cobastab 3mg, biotin 100g, Zn 80mg, Mn 20mg, Fe 83mg, Cu 25mg), particle diameter 1-2 millimeter.In the daily ration of final sheep: grass hay accounts for 45%, silage corn 23%, fine fodder 32%.
Adopt solid state fermentation to produce hypocrellin, corn is primary raw material, separately adds with 2% glucose, the 0.2%KH of corn weighing scale
2pO
4, 0.04%MgSO
47H
2o.The spice that adds water wets, and material-water ratio controls 1: 0.7,121 DEG C of sterilizing 45-60min, connects 10% liquid bamboo parasitic fungus Shiraia sp.SUPER-H168 seed after cooling.Initial pH and sweat pH is nature, fermentation temperature 30 DEG C, fermentation processes humidity 90%, end in 10 days of fermenting.Dry for 60 DEG C and remove moisture content, measure wherein hypocrellin A and account for 8.4%, hypocrelline B accounts for 1.3%.It accounts for 0.4% at Ta perylene naphtoquinone compounds total amount.
The weaned lamb that average weight is 10 kilograms 40, close according to body weight, male and female half and half, random packet principle is divided into 4 groups, often organizes 10 repetitions, each repetition 1 bellwether.Feed mode is free choice feeding and drinking-water.Experiment is by being divided into four groups as follows:
Test group 1: blank group, whole experimentation is only fed basal diet.
Test group 2: experiment is approximately hay and the silage corn of 300g fine fodder and corresponding proportion by the one day appetite of sheep.In its 300g fine fodder, be evenly mixed into the hypocrellin solid state fermentation thing 54.1mg of drying and pulverizing according to its body weight, make the daily intake of sheep be about 1 μm of ol/kg like this.Later day by day according to appetite and the body weight increase solid state fermentation thing mixed volume of sheep.Whole experimentation all eats the feed that with the addition of hypocrellin solid state fermentation thing.
Test group 3: experiment is approximately hay and the silage corn of 300g fine fodder and corresponding proportion by the one day appetite of sheep.In its 300g fine fodder, be evenly mixed into the hypocrellin solid state fermentation thing 270.3mg of drying and pulverizing according to its body weight, make the daily intake of sheep be about 5 μm of ol/kg like this.Later day by day according to appetite and the body weight increase solid state fermentation thing mixed volume of sheep.Whole experimentation all eats the feed that with the addition of hypocrellin solid state fermentation thing.
Test group 4: experiment is approximately hay and the silage corn of 300g fine fodder and corresponding proportion by the one day appetite of sheep.In its 300g fine fodder, be evenly mixed into the hypocrellin solid state fermentation thing 540mg of drying and pulverizing according to its body weight, make the daily intake of sheep be about 10 μm of ol/kg like this.Later day by day according to appetite and the body weight increase solid state fermentation thing mixed volume of sheep.Whole experimentation all eats the feed that with the addition of hypocrellin solid state fermentation thing.
The all sheep of test group 2,3,4 eat the feed that with the addition of containing hypocrellin after 10 days continuously, with 1 × 10 of 1mL
6tCID50/mL FMDV virus-culturing fluid carries out injection to all sheep of all groups and attacks poison, then measures every bellwether Temperature changing every day, observes every bellwether clinical symptoms, and record death toll, observes 21 days.Observed result is as follows:
Test group 1: all sheep are attacked poison and to occur between mucous membrane of mouth, gums, lip, tongue and toe after 2 days etc. bubble or rotten to the corn symptom occur, and serious symptom gradually, all sheep body temperature is elevated to about 40 DEG C.Had 8 sheep death to 14 days, other 2 symptoms alleviate gradually and recover normal.
Test group 2:2 bellwether body temperature after attacking malicious latter 2nd day starts to be elevated to 40 DEG C, and continue 5 days, and occur bubble symptom occurs between oral cavity and toe, after 5 days, symptom recovers normal gradually.Other sheep is observed still to be good for for 21 days and lives.
Test group 3: all sheep are observed all to be good for for 21 days and live.
Test group 4: all sheep are observed all to be good for for 21 days and live.
Experiment finds out that hypocrellin serves extraordinary protective effect for sheep thus.
Embodiment 3: liquid state fermentation hypocrellin makes an addition to the anti-ox BVDV Viral experiment in feed
Experiment basis daily ration: corn 48%, wheat bran 14%, cottonseed cake 35%, stone flour 1.0%, bone meal 0.5%, salt 1.0%, sodium acid carbonate 0.5%.
Adopt liquid fermentation method to produce hypocrellin, often liter of culture medium consists of: glucose 20g, peptone 50g, potato 200g, (NH
4)
2hPO
42g, KH
2pO
42g, MgSO
40.5g/L, 121 DEG C of sterilizing 20min, connect 10% liquid bamboo parasitic fungus seed after cooling.Initial pH and sweat pH is nature, fermentation temperature 30 DEG C, and fermentation 72h terminates.Centrifugal segregation zymotic fluid obtains mycelium, 60 DEG C of oven dry, and measure wherein hypocrellin A and account for 8.1%, hypocrelline B accounts for 1.3%, and it accounts for 0.2% at Ta perylene naphtoquinone compounds total amount.
The calf 40 that average weight 100kg wean body condition is good, close according to body weight, male and female half and half, random packet principle is divided into 4 groups, often organizes 10 repetitions, each repetition 1 calf.Feed mode is free choice feeding and drinking-water.Experiment is by being divided into four groups as follows:
Test group 1: blank group, whole experimentation is only fed basal diet.
Test group 2: experiment Niu Yitian appetite is approximately 3kg, is evenly mixed into the oven dry liquid state fermentation thing 568.8mg of drying and pulverizing, makes the daily intake of ox be about 1 μm of ol/kg like this in its 3kg basal diet according to its body weight.Later day by day according to appetite and the body weight increase liquid state fermentation thing mixed volume of ox.Whole experimentation all eats the feed that with the addition of hypocrellin liquid state fermentation thing.
Test group 3: experiment Niu Yitian appetite is approximately 3kg, is evenly mixed into the oven dry liquid state fermentation thing 2.8g of drying and pulverizing, makes the daily intake of ox be about 5 μm of ol/kg like this in its 3kg basal diet according to its body weight.Later day by day according to appetite and the body weight increase liquid state fermentation thing mixed volume of ox.Whole experimentation all eats the feed that with the addition of hypocrellin liquid state fermentation thing.
Test group 4: experiment Niu Yitian appetite is approximately 3kg, is evenly mixed into the oven dry liquid state fermentation thing 5.7g of drying and pulverizing, makes the daily intake of ox be about 10 μm of ol/kg like this in its 3kg basal diet according to its body weight.Later day by day according to appetite and the body weight increase liquid state fermentation thing mixed volume of ox.Whole experimentation all eats the feed that with the addition of hypocrellin liquid state fermentation thing.
The all oxen of test group 2,3,4 eat the feed after 7 days that with the addition of hypocrellin continuously, with 1 × 10 of 1mL
6tCID50/mL BVDV virus-culturing fluid carries out injection to all oxen of all groups and attacks poison, then measures every ox Temperature changing every day, observes every ox clinical symptoms, observe 14 days.Observed result is as follows:
Test group 1: all oxen body temperature after 3 days slightly raises, the thinning deliquescing of ight soil.Occur blood and mucus in the 6th day subsequently ight soil, maxilla has shallow blue or green ulceration.All the time similar symptom is had in observation period.
Test group 2: all ox body temperature and clinical symptoms have no significant change, observes all to be good for for 14 days and lives.
Test group 3: all ox body temperature and clinical symptoms have no significant change, observes all to be good for for 14 days and lives.
Test group 4: all ox body temperature and clinical symptoms have no significant change, observes all to be good for for 14 days and lives.
Experiment finds out that hypocrellin serves extraordinary protective effect for ox thus.
Embodiment 4: liquid state fermentation Ka Futading makes an addition to the anti-sheep BTV Viral experiment in feed
Experiment basis daily ration is identical with embodiment 2.
Adopt liquid fermentation method to produce Ka Futading, often liter of culture medium consists of: glucose 5g, yeast extract 5g, potato 100g, (NH
4)
2hPO
41g, KH
2pO
41g, MgSO
40.1g/L, 121 DEG C of sterilizing 20min, connect 10% liquid Cladosporium cladosporioides CGMCC 34593 bacterial classification after cooling.Initial pH and sweat pH is nature, fermentation temperature 28 DEG C, and fermentation 96h terminates.Centrifugal segregation zymotic fluid obtains mycelium, 60 DEG C of oven dry, and wherein Calphostin A accounts for 0.029%, Calphostin B and accounts for 0.025%, Calphostin C and account for 0.85%, Calphostin D and account for 0.02%, Calphostin I and account for 0.01%.
The weaned lamb that average weight is 10 kilograms 40, close according to body weight, male and female half and half, random packet principle is divided into 4 groups, often organizes 10 repetitions, each repetition 1 bellwether.Feed mode is free choice feeding and drinking-water.Experiment is by being divided into four groups as follows:
Test group 1: blank group, whole experimentation is only fed basal diet.
Test group 2: experiment is approximately hay and the silage corn of 300g fine fodder and corresponding proportion by the one day appetite of sheep.In its 300g fine fodder, be evenly mixed into the Ka Futading liquid state fermentation thing 0.8g of drying and pulverizing according to its body weight, make the daily intake of sheep be about 1 μm of ol/kg like this.Later day by day according to appetite and the body weight increase liquid state fermentation thing mixed volume of sheep.Whole experimentation all eats the feed that with the addition of Ka Futading liquid state fermentation thing.
Test group 3: experiment is approximately hay and the silage corn of 300g fine fodder and corresponding proportion by the one day appetite of sheep.In its 300g fine fodder, be evenly mixed into the Ka Futading liquid state fermentation thing 4.0g of drying and pulverizing according to its body weight, make the daily intake of sheep be about 5 μm of ol/kg like this.Later day by day according to appetite and the body weight increase liquid state fermentation thing mixed volume of sheep.Whole experimentation all eats the feed that with the addition of Ka Futading liquid state fermentation thing.
Test group 4: experiment is approximately hay and the silage corn of 300g fine fodder and corresponding proportion by the one day appetite of sheep.In its 300g fine fodder, be evenly mixed into the Ka Futading liquid state fermentation thing 8.0g of drying and pulverizing according to its body weight, make the daily intake of sheep be about 10 μm of ol/kg like this.Later day by day according to appetite and the body weight increase liquid state fermentation thing mixed volume of sheep.Whole experimentation all eats the feed that with the addition of Ka Futading liquid state fermentation thing.
The all sheep of test group 2,3,4 eat the feed that with the addition of containing Ka Futading after 7 days continuously, with 1 × 10 of 1mL
6tCID50/mL BTV virus-culturing fluid carries out injection to all sheep of all groups and attacks poison, then measures every bellwether Temperature changing every day, observes every bellwether clinical symptoms, and record death toll, observes 21 days.Observed result is as follows:
Test group 1: after attacking malicious 3 days, all sheep body temperature raises and reaches 40 ~ 42 DEG C, down in spirits, salivation, mucous membrane of mouth and tongue hyperemia, tongue cyanosis.Dead 2 sheep when 7 days, the 8th day dead 2 sheep.Other sheep is not dead, has 2 to recover normal in the 10th day, separately has 3 to recover normal in the 12nd day, and 1 was recovered normal in the 14th day.
Test group 2: all sheep are observed all to be good for for 21 days and live.
Test group 3: all sheep are observed all to be good for for 21 days and live.
Test group 4: all sheep are observed all to be good for for 21 days and live.
Experiment sees that card release Fu Tading serves extraordinary protective effect for sheep thus.
Embodiment 5: the Elsinochrome element smelting of liquid state fermentation is treated ox FMDV and infected.
The calf 40 that average weight 100kg wean body condition is good, close according to body weight, male and female half and half, random packet principle is divided into 4 groups, often organizes 10 repetitions, each repetition 1 calf.Feed mode is free choice feeding and drinking-water.
Adopt liquid fermentation method to produce Elsinochrome element, often liter of culture medium consists of: glucose 25g, beef extract 25g, potato 100g, (NH
4)
2hPO
41.5g, KH
2pO
41.5g, MgSO
40.8g/L, 121 DEG C of sterilizing 20min, connect 5% liquid Elsinoe fawcettii ATCC 38162 bacterial classification after cooling.Initial pH and sweat pH is nature, fermentation temperature 24 DEG C, and fermentation 96h terminates.Centrifugal segregation zymotic fluid obtains mycelium, 60 DEG C of oven dry, measures wherein Elsinochrome A and accounts for 0.012%, Elsinochrome B and account for 0.021%, Elsinochrome C and account for 0.034%, Elsinochrome D and account for 0.05%.With the alcohol with the weight such as mycelia, extraction mycelium, then dries and removes alcohol for 60 DEG C, obtain Elsinochrome element crude extract, wherein Elsinochrome element total content is 11.7%, and various Elsinochrome element ratio does not become, and the Elsinochrome element obtained with this is for the antiviral therapy of calf.
With 1 × 10 of 1mL
6tCID50/ml FMDV virus-culturing fluid attacks poison to all little beef injection of all groups.Concentrate in 3 days and entirely mass-send disease, test body temperature reaches 41 DEG C, the inner aspect of lip of ox, gums, lingual surface and cheek bifurcation mucous membrane occurs broad bean to the large bubble of walnut.After being mixed with a small amount of basal diet by Elsinochrome element, when calf is hungry, first feeding adds the feed of Elsinochrome element, if without feed ability, gavage.
Test group 1: blank group, whole experimentation is only fed basal diet.
Test group 2: by dried alcoholic extract 4.7g and 50g basal diet Homogeneous phase mixing, disposablely feeds.The daily intake of calf is made to be about 10 μm of ol/kg like this.Eat feed containing Elsinochrome element continuously 3 days.
Test group 3: by dried alcoholic extract 23.3g and 50g basal diet Homogeneous phase mixing, disposablely feeds.The daily intake of calf is made to be about 50 μm of ol/kg like this.Eat feed containing Elsinochrome element continuously 3 days.
Test group 4: by dried alcoholic extract 46.6g and 50g basal diet Homogeneous phase mixing, disposablely feeds, and makes the daily intake of calf be about 100 μm of ol/kg like this.Eat continuously containing Elsinochrome element feed 3 days.
Observe every calf clinical symptoms, observe 21 days.Observed result is as follows:
Test group 1: after attacking poison, has 3 calf self-healings for about 8 days, within about 10 days, has 4 calf death, and about 14 have 3 calf self-healings.
Test group 2: after starting treatment, about 3 days all calf transference cures, rehabilitation in 5 days.
Test group 3: after starting treatment, about 3 days all calf transference cures, rehabilitation in 5 days.
Test group 4: after starting treatment, have calf transference cure year in about 3 days, rehabilitation in 5 days.
Experiment finds out that Elsinochrome element has extraordinary therapeutic action for little cattle infected virus thus.
Above-described embodiment 1-5 is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and implement according to this; can not limit the scope of the invention with this; all equivalence changes done according to flesh and blood of the present invention become or modify, and all should be encompassed within protection scope of the present invention.
Claims (1)
1. the application of perylene naphtoquinone compounds in the feed of preparation control ruminant virus, is characterized in that Suo Shu perylene naphtoquinone compounds is selected: hypocrellin A Hypocrellin A or hypocrelline B HypocrellinB; The virus of described control is foot and mouth disease virus, cytopathogenic effect type ox diarrhoea-bovine diarrhoea virus, blue tongue virus.
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