CN102106465A - 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 ruminant viruses such as the anti-smelting ox of preparation, sheep, donkey, horse the present invention relates to Cai Yong perylene naphtoquinone compounds and adds the feed that is used to prepare control ruminant virus in the feed to.Belong to bioengineering and agricultural technology field.
Background technology
In recent years, along with Chinese economic development and growth in the living standard, milk industry and beef industry become the bright spot of animal husbandry structural adjustment.The sheep industry also is the pillar industry of China borderland and minority area economic development simultaneously.
But along with the continuous expansion of the scale of breed, also can bring large-scale epidemic disease outburst, cause tremendous loss to plant.Especially viral disease has the strong and high characteristics of the death rate of infectiousness.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), (Epuine morbillivirus, EMV) waiting all is pathogenies of the main diseases viral disease during ruminant is cultured for capripox virus (capripoxvirus), equine morbillivirus.
Patent CN200810166154.2, the method for employing vaccines such as CN03103300.8 and CN200680051113.8 is carried out the control of virus, however not only kind is many for animal virus, and variation often is hard to guard against rapidly.Amantadine, Rimantadine, Oseltamivir and zanamivir etc. are antiviral agents commonly used, but its curative effect of medication and imprecise, and have problems such as residual.
Patent CN200610072935.6 report hypericin is a kind of antiviral compound of wide spectrum, for viruses such as aftosas good killing effect is arranged.Hypericin is to extract a kind of phenanthro-quinones (Phenanthroperylenequinone) compound that obtains from the Chinese herbal medicine hypericum perforatum, and it has shown the good viral killing effect of wide spectrum.Yet the hypericin unstable chemcial property sees that light easily decomposes, and has quite a few hypericin to be used effectively in actual applications.
We Yan studies carefully Fa Xian perylene quinone (Perylenequinone) compounds and has the effect of broad-spectrum antiviral preferably.The perylene naphtoquinone compounds is compared with hypericin, has series of advantages such as the convenience of extraction, good stability, oral non-evident effect and metabolism are fast.Therefore the invention discloses Yi Zhong perylene naphtoquinone compounds is applied to the method for ruminant feed, thereby carries out the control of bird virus effectively.
Summary of the invention
The objective of the invention is the perylene naphtoquinone compounds is applied to be used in the feed prevention and treatment ruminant virus infections disease.
The correlated characteristic that the present invention relates to is as follows:
1,, perylene naphtoquinone compounds explanation
It is that three kinds of parent nucleus as follows are basic structure that Suo of the present invention is Zhied the perylene naphtoquinone compounds, and carries out the compound of base group modification with these three kinds of mother nucleus structures.Can be 2,3,6,7,11,12,17,18,26,28,29,30,32,36,37,45,46,53,55,56,58,62,63,71,72 positions are modified and are gone up various chemical groups, as CH
3, OCH
3, COCH
3, CH (OH) CH
3, CHO, OH, H etc. can also further form more complicated structure between these chemical groups.
Perylene naphtoquinone compounds parent nucleus
Special in hypocrellin (hypocrellin A Hypocrellin A, hypocrelline B Hypocrellin B), Elsinochrome element (the plain Elsinochrome A of Elsinochrome first, the plain Elsinochrome B of Elsinochrome second, the Elsinochrome third plain Elsinochrome C, the plain Elsinochrome D of Elsinochrome fourth), not come rhzomorph (Phleichrome), branch spore element (the plain Cladosporium A of branch spore first, the plain Cladosporium B of branch spore second, the branch spore third plain Cladosporium C, the plain Cladosporium D of branch spore fourth), 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 referring to 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: (1) is natural is longer than bamboo parasitic fungus (Shiraia bambusicola) fructification and the red bacterium of bamboo (Hypocrella bambusae) fructification on the bamboo; (2) adopt bamboo parasitic fungus to carry out liquid state fermentation according to the described method of patent CN200710132510.4 and CN200910182255.3 or solid state fermentation makes.
The tabasheer bacterial strain can be: Shiraia sp.SUPER-H168 preserving number is the Shiraia bambusicola bacterial strain that CCTCC NO:M 207104 or other can buy from culture presevation mechanism.
The plain available Elsinoe fawcettu ATCC 38162 of Elsinochrome or other can make by solid state fermentation or liquid state fermentation method from the Elsinoe fawcettu bacterial strain that culture presevation mechanism buys.
The plain available Cladosporium cucumerinum.ATCC 11279 of branch spore or other can make by solid state fermentation or liquid state fermentation method from the Cladosporium cucumerinum bacterial strain that culture presevation mechanism buys.
Ka Futading can make by solid state fermentation or liquid state fermentation method from the Cladosporium cladosporioides bacterial strain that culture presevation mechanism buys with Cladosporium cladosporioides CGMCC 34593 or other.
Cercosporin can make by solid state fermentation or liquid state fermentation method from the Cercospora kikuchiis bacterial strain that culture presevation mechanism buys with Cercospora kikuchii ATCC 42152 or other.
Not come rhzomorph can make by solid state fermentation or liquid state fermentation method from the Cladosporium phlei bacterial strain that culture presevation mechanism buys with Cladosporium phlei ATCC 36193 or other.
Scutiaquinone can adopt alcohol extract to obtain from the root to thorn Calamus plant Scutia myrtina.
More than the solid state fermentation conditions of all bacterial classifications all can be: employing grain is primary raw material, and it is crushed to the 50-80 order, adds 0.5%-2% glucose, 0.1%-0.5%KH in primary raw material in addition
2PO
4, 0.01%-0.08%MgSO
47H
2O.It is wet to add the water spice, and material-water ratio is controlled at 1: 1-1: 0.7,121 ℃ of sterilization 45-60min, the 5%-10% liquid seeds is inserted in the cooling back.Initial pH and sweat pH are nature, and fermentation temperature 24-32 ℃, sweat controlled 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.
More than the liquid state fermentation condition of all bacterial classifications all can be: 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 ℃ of sterilization 20min, bacterial classification is inserted in sterilization cooling back, and inoculum concentration is 5%-10%, and initial pH and sweat pH are nature, and fermentation temperature is 24-32 ℃, 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 soya-bean cake powder.
3. the adding method of perylene naphtoquinone compounds in feed
The dosage of pre-anti-virus is 1-10 μ mol perylene naphtoquinone compounds/kg body weight every day, the perylene naphtoquinone compounds is admixed in the feed fed, and adds all the time in the growth course of ruminant.
Therapeutic dose for the ruminant of infective virus is μ mol-100 μ mol perylene naphtoquinone compounds/kg body weight every days 10, and be 2 to 7 days the course of treatment.
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 the red mushroom entity of natural bamboo parasitic fungus or bamboo source De perylene naphtoquinone compounds: according to the body weight of ruminant with perylene naphtoquinone compounds demand, get the red mushroom entity of a certain amount of bamboo parasitic fungus or bamboo and be crushed to the 40-60 order, evenly sneak into then in the fine fodder of daily feed of ruminant.
(2) obtain De perylene naphtoquinone compounds for solid state fermentation: treat 50-60 ℃ of oven dry after the fermentation ends, be crushed to the 40-60 order then,, get a certain amount of crushed material and evenly sneak in the fine fodder of daily feed of ruminant with perylene naphtoquinone compounds demand according to the body weight of ruminant.
(3) obtain De perylene naphtoquinone compounds for liquid state fermentation: after treating fermentation ends, centrifugal removal liquid obtains solid contents such as mycelium, 50-60 ℃ of oven dry, be crushed to the 40-60 order then,, get a certain amount of crushed material and evenly sneak in the fine fodder of daily feed of ruminant with perylene naphtoquinone compounds demand according to the body weight of ruminant.
(4) for solid state fermentation or liquid state fermentation product: the ethanol that can in tunning, add 1 times of weight, cross the elimination solid content; crude extract is made in the extraction of perylene naphtoquinone compounds, 50-60 ℃ of oven dry, be crushed to the 40-60 order then,, get a certain amount of crushed material and evenly sneak in the fine fodder of daily feed of ruminant with perylene naphtoquinone compounds demand according to the body weight of ruminant.
(5) mainly be present in root 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, at first Scutiaquinone is extracted (ethanol: root=1: 0.5-1: 3 from root with ethanol, weight ratio), filter and remove rhizome, 50-60 ℃ of vacuum concentrates removes alcohol and oven dry, again it is crushed to the 40-60 order, measures according to demand in the fine fodder of the daily feed of evenly sneaking into ruminant then.
(6) Zhe Xie perylene naphtoquinone compounds can be monomer by the preparative chromatography purifying, sneak in the fine fodder of daily feed of ruminant.The condition of preparation purifying is as described in the document of before having delivered.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 De perylene naphtoquinone compounds can be pressed arbitrary proportion mixing use according to actual conditions.
Beneficial effect of the present invention: the present invention relates to Cai Yong perylene naphtoquinone compounds and add in the ruminant feed preparation to and be used to prevent and treat the feed of ruminant virus, as foot and mouth disease virus, cytopathogenic effect type ox diarrhoea-bovine diarrhoea virus, blue tongue virus, capripox virus, equine morbillivirus etc.The perylene naphtoquinone compounds comprises: hypocrellin, Elsinochrome element, not come rhzomorph, branch spore element, Ka Futading, cercosporin etc., the perylene naphtoquinone compounds has series of advantages such as the convenience of extraction, good stability, oral non-evident effect and metabolism are fast.Dosage in the feed of preparation prevention ruminant virus is 1-10 μ mol perylene naphtoquinone compounds/kg body weight every day, dosage in the feed of preparation treatment ruminant virus is 10 μ mol-100 μ mol perylene naphtoquinone compounds/kg body weight, the effect highly significant Tong Shi perylene naphtoquinone compounds of control virus can obtain by modern biotechnology production, can produce good economic and social benefit.
The specific embodiment
The experiment of embodiment 1: perylene naphtoquinone compounds extracorporeal antivirus effect
(1) the extracorporeal antivirus effect experiment preparation of cell
(young hamster kidney passage cell, Baby hamster kidney cells) grinds and studies carefully the effect of perylene naphtoquinone compounds to foot and mouth disease virus (FMDV) with the BHK-21 cell.Growth medium: with MEM (modified eaglemedium) culture medium of buying, other adds NaHCO
32.0g/L, 2% hyclone, 1% penicillin, 1% streptomysin.With the BHK-21 cell with 0.25% trypsinization after, piping and druming disperses cell, is diluted to 1 * 10 with the MEM growth medium
6Cell/mL adds in the 96 porocyte culture plates, and 37 ℃, 5%CO
2Cultivate in the incubator, treat that cell grows up to cell monolayer after, carry out extracorporeal antivirus effect test.
(African green monkey kidney cell, Verda Reno) Yan Jiu perylene naphtoquinone compounds is to the effect of blue tongue rims (BTV) with the Vero cell.Growth medium: with the MEM culture medium of buying, other adds the 110mg/L Sodium Pyruvate, 300mg/L L-glutamine, 200mg/L NaHCO
310% hyclone, 1% penicillin, 1% streptomysin.With the Vero cell with 0.25% trypsinization after, piping and druming disperses cell, is diluted to 1 * 10 with the MEM growth medium
6Cell/mL adds in the 96 porocyte culture plates, and 37 ℃, 5%CO
2Cultivate in the incubator, treat that cell grows up to cell monolayer after, carry out extracorporeal antivirus effect test.
(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) with the BTC cell.Growth medium: DMEM (Dulbecco ' smodified eagle medium) contains 4500mg/L D-glucose, 300mg/L L-glutamine, 110mg/L Sodium Pyruvate, 5% hyclone, 1% penicillin, 1% streptomysin).With the BTC cell with 0.25% trypsinization after, piping and druming disperses cell, is diluted to 1 * 10 with the DMEM growth medium
6Cell/mL adds in the 96 porocyte culture plates, and 37 ℃, 5%CO
2Cultivate in the incubator, treat that cell grows up to cell monolayer after, carry out extracorporeal antivirus effect test.
(2) extracorporeal antivirus effect is tested with viral tissue culture infective dose (Tissue culture infectivedose, mensuration TCID50)
After cell grows up to individual layer, inoculation 10
-2-10
-1Dilution viral liquid (0.1mL/ hole), each dilution factor is inoculated 8 holes, in 5%CO
2Cultivate 120h for 37 ℃ in the incubator, observation of cell pathology every day (CEP) situation is calculated viral TCID50 (Reed, L.J. according to the Reed-Muench formula; Muench, H. (1938) .A simplemethod of estimating fifty percent endpoints.The American Journal of Hygiene 27:493-497.).
(3) the MTT decoration method is calculated cell survival rate
Mtt assay (tetramethyl azo azoles salt colorimetric method) carries out cell dyeing (final concentration 0.5g/L) and detects cell survival and growth.Absorbance (OD) with ELIASA mensuration 490nm place calculates cell survival rate by following formula: cell survival rate (%)=[adding perylene quinone group OD/ normal cell group OD] * 100%.
(the toxicity test of 4) perylene naphtoquinone compounds pair cells
With DMSO with Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and Phleichrome be mixed with 0.01,0.1,02 respectively, 0.4mmol/mL, join respectively among BHK-21, the Vero and BTC cell that grows up to, and so that BHK-21, Vero and the BTC cell of Han Jia perylene naphtoquinone compounds do not compare, 3 bottles of each tests, at 37 ℃, 5%CO
2Incubator was cultivated 120 hours, and these are cultivated in the darkroom (unglazed photograph) and have and carry out parallel laboratory test under the illumination condition, the mensuration cell survival rate.
Experimental result finds that all about 99.6%, therefore perylene naphtoquinone compounds pair cell does not have obvious CDCC to the survival rate of all test cell Xia above-mentioned Nong Du.
(5) perylene naphtoquinone compounds are to the direct deactivation of virus
Ce is Dinged perylene naphtoquinone compounds Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and the Phleichrome direct deactivation to virus.Jia Ru perylene naphtoquinone compounds in the 100TCID50 virus liquid (be dissolved in DMSO[Dimethyl sulfoxide, dimethyl sulfoxide (DMSO)] in) ， Shi perylene naphtoquinone compounds in viral liquid concentration reach 0.05,0.1,0.2 respectively, 0.4mmol/L, at 37 ℃, 5%CO
2After cultivating 1h in the incubator, infect above-mentioned cultured corresponding cell.Observation of cell pathology situation under the inverted microscope when virus control group cytopathy reaches 75%-100%, cell contrasts just often, is carried out interpretation of result with the MTT decoration method.These experiments are in the darkroom (unglazed photograph) and have and carry out parallel laboratory test under the illumination condition respectively, and used light source is the fluorescent lamp of wavelength at 470-590nm, and the light dosage that total irradiation enters is 9KJ (using photometric determination).In the cell cultivation process, observation of cell pathology situation under the inverted microscope when virus control group cytopathy reaches the contrast of 75%-100% cell just often, is carried out interpretation of result with the MTT decoration method.
FMDV, the BTV of Bu Jia perylene naphtoquinone compounds and the cell survival rate of BVDV virus control group are 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 invaded the blocking effect of cell to virus
Ce is Dinged perylene naphtoquinone compounds Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and Phleichrome and virus is invaded the blocking effect of cell.Get the culture plate that grows up to cell monolayer, outwell nutrient solution ， Jiang the perylene naphtoquinone compounds with 0.05,0.1,0.2,0.4mmol/L concentration (being dissolved among the DMSO), be added to respectively on the Tissue Culture Plate, the 0.1mL/ hole, each trial degree repeats 3 holes.37 ℃ of effect 4h discard liquid, and every hole adds 100TCID50 virus liquid 0.1mL, and other establishes cell contrast and virus control.5%CO
2, incubator is cultivated 120h for 37 ℃.These experiments are in the darkroom (unglazed photograph) and have and carry out parallel laboratory test under the illumination condition respectively, and used light source is the fluorescent lamp of wavelength at 470-590nm, and the light dosage that total irradiation enters is 9KJ (using photometric determination).In the cell cultivation process,,, carry out interpretation of result, and calculate cell and deposit rate with the MTT decoration method when virus control group cytopathy reaches 75%-100%, cell contrasts just often with observation of cell pathology situation under the inverted microscope.
FMDV, the BTV of Bu Jia perylene naphtoquinone compounds and the cell survival rate of BVDV virus control group are 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 is Dinged perylene naphtoquinone compounds Calphostin C, Hypocrellin A, Hypocrellin B, Cladosporium A, Elsinochrome A, Scutiaquinone A, Cercosporin and the Phleichrome inhibitory action to virus multiplication.Get the culture plate that grows up to cell monolayer, outwell nutrient solution, the viral liquid of inoculation 100TCID50,100 μ L/ holes are put 37 ℃, 5%CO
2Adsorb 4h in the incubator.Discard viral liquid, add variable concentrations respectively and be 0.05,0.1,0.2,0.4mmol/L De perylene naphtoquinone compounds (being dissolved among the DMSO), the 0.1mL/ hole, each concentration repeats 3 holes, establishes normal cell contrast and virus control simultaneously.5%CO
237 ℃ of cultivations of incubator.These experiments are in the darkroom (unglazed photograph) and have and carry out parallel laboratory test under the illumination condition respectively, and used light source is the fluorescent lamp of wavelength at 470-590nm, and the dosage that total irradiation enters is 9KJ (using photometric determination).In the cell cultivation process, observation of cell pathology situation under the inverted microscope when virus control group cytopathy reaches the contrast of 75%-100% cell just often, is carried out interpretation of result with the MTT decoration method.
FMDV, the BTV of Bu Jia perylene naphtoquinone compounds and the cell survival rate of BVDV virus control group are 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 by above experiment ， perylene naphtoquinone compounds all has killing action for test with ruminant virus, be the antiviral compound of wide spectrum.Wherein the effect of Calphostin C, Hypocrellin A, Hypocrellin B and Elsinochrome A is best, Qi Ta perylene naphtoquinone compounds slightly a little less than.Under illumination condition, antiviral effect also is better than unglazed condition in addition, this mainly Shi You Yu perylene naphtoquinone compounds under illumination, can produce products such as superoxide ion, its photosensitized oxidation product can strengthen anti-virus ability.
Embodiment 2: the solid state fermentation hypocrellin makes an addition to the anti-sheep FMDV experiment in the feed
The basal diet prescription of sheep is in the experiment:
Concentrated feed prescription: corn 46%, wheat bran 20%, the 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 vitamin A 1200IU is provided, 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 a primary raw material, adds 2% glucose, 0.2%KH in corn weight in addition
2PO
4, 0.04%MgSO
47H
2O.It is wet to add the water spice, and material-water ratio was controlled at 1: 0.7, and 121 ℃ of sterilization 45-60min connect 10% liquid bamboo parasitic fungus Shiraia sp.SUPER-H168 seed after the cooling.Initial pH and sweat pH are nature, 30 ℃ of fermentation temperatures, sweat controlled humidity 90%, the end in 10 days of fermenting.Moisture content are removed in 60 ℃ of oven dry, measure wherein that hypocrellin A accounts for 8.4%, and hypocrelline B accounts for 1.3%.It accounts for 0.4% at Ta perylene naphtoquinone compounds total amount.
40 of the wean lambs that average weight is 10 kilograms, the principle of, male and female half and half close according to body weight, random packet is divided into 4 groups, every group of 10 repetitions, each repeats 1 bellwether.The mode of feeding is free choice feeding and drinking-water.Experiment is divided into four groups by following:
Test group 1: blank group, the whole experiment basal diet of only feeding.
Test group 2: hay and silage corn that experiment approximately is 300g fine fodder and corresponding proportion with the one day appetite of sheep.In its 300g fine fodder, evenly sneak into the hypocrellin solid state fermentation thing 54.1mg that oven dry is pulverized according to its body weight, make intake every day of sheep be about 1 μ mol/kg like this.Later on increase solid state fermentation thing mixed volume according to appetite and the body weight of sheep day by day.Whole experiment is all eaten the feed that has added hypocrellin solid state fermentation thing.
Test group 3: hay and silage corn that experiment approximately is 300g fine fodder and corresponding proportion with the one day appetite of sheep.In its 300g fine fodder, evenly sneak into the hypocrellin solid state fermentation thing 270.3mg that oven dry is pulverized according to its body weight, make intake every day of sheep be about 5 μ mol/kg like this.Later on increase solid state fermentation thing mixed volume according to appetite and the body weight of sheep day by day.Whole experiment is all eaten the feed that has added hypocrellin solid state fermentation thing.
Test group 4: hay and silage corn that experiment approximately is 300g fine fodder and corresponding proportion with the one day appetite of sheep.In its 300g fine fodder, evenly sneak into the hypocrellin solid state fermentation thing 540mg that oven dry is pulverized according to its body weight, make intake every day of sheep be about 10 μ mol/kg like this.Later on increase solid state fermentation thing mixed volume according to appetite and the body weight of sheep day by day.Whole experiment is all eaten the feed that has added hypocrellin solid state fermentation thing.
Test group 2,3,4 all sheep eat has continuously added the feed after 10 days that contains hypocrellin, with 1 * 10 of 1mL
6TCID50/mL FMDV virus-culturing fluid is injected all sheep of all groups and is attacked poison, measures every bellwether body temperature then every day and changes, and observes every bellwether clinical symptoms, and the record death toll was observed 21 days.Observed result is as follows:
Test group 1: all sheep are attacked poison and occur after 2 days between mucous membrane of mouth, gums, lip, tongue and toe etc. bubble or rotten to the corn symptom take place, and serious symptom gradually, and all sheep body temperature are elevated to about 40 ℃.Had 8 sheep death to 14 days, other 2 symptoms alleviate gradually and recover normal.
Test group 2:2 bellwether in attack poison back after the 2nd day body temperature begin to be elevated to 40 ℃, continue 5 days, and occur the bubble symptom takes place between oral cavity and toe, symptom recovers normal gradually after 5 days.Other sheep is observed 21 days still strong living.
Test group 3: all sheep are observed 21 days all strong living.
Test group 4: all sheep are observed 21 days all strong living.
Experiment finds out that hypocrellin has played extraordinary protective effect for sheep thus.
Embodiment 3: the liquid state fermentation hypocrellin makes an addition to the anti-ox BVDV virus experiment in the 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, every liter of culture medium consists of: glucose 20g, peptone 50g, potato 200g, (NH
4)
2HPO
42g, KH
2PO
42g, MgSO
40.5g/L 121 ℃ of sterilization 20min connect 10% liquid bamboo parasitic fungus seed after the cooling.Initial pH and sweat pH are nature, 30 ℃ of fermentation temperatures, and fermentation 72h finishes.Centrifugal removal zymotic fluid obtains mycelium, and 60 ℃ of oven dry measure wherein that hypocrellin A accounts for 8.1%, and hypocrelline B accounts for 1.3%, and it accounts for 0.2% at Ta perylene naphtoquinone compounds total amount.
40 of the good calves of average weight 100kg wean body condition, the principle of, male and female half and half close according to body weight, random packet is divided into 4 groups, every group of 10 repetitions, each repeats 1 calf.The mode of feeding is free choice feeding and drinking-water.Experiment is divided into four groups by following:
Test group 1: blank group, the whole experiment basal diet of only feeding.
Test group 2: experiment approximately is 3kg with Niu Yitian appetite, evenly sneaks into the oven dry liquid state fermentation thing 568.8mg that oven dry is pulverized according to its body weight in its 3kg basal diet, makes intake every day of ox be about 1 μ mol/kg like this.Later on increase liquid state fermentation thing mixed volume according to appetite and the body weight of ox day by day.Whole experiment is all eaten the feed that has added hypocrellin liquid state fermentation thing.
Test group 3: experiment approximately is 3kg with Niu Yitian appetite, evenly sneaks into the oven dry liquid state fermentation thing 2.8g that oven dry is pulverized according to its body weight in its 3kg basal diet, makes intake every day of ox be about 5 μ mol/kg like this.Later on increase liquid state fermentation thing mixed volume according to appetite and the body weight of ox day by day.Whole experiment is all eaten the feed that has added hypocrellin liquid state fermentation thing.
Test group 4: experiment approximately is 3kg with Niu Yitian appetite, evenly sneaks into the oven dry liquid state fermentation thing 5.7g that oven dry is pulverized according to its body weight in its 3kg basal diet, makes intake every day of ox be about 10 μ mol/kg like this.Later on increase liquid state fermentation thing mixed volume according to appetite and the body weight of ox day by day.Whole experiment is all eaten the feed that has added hypocrellin liquid state fermentation thing.
Test group 2,3,4 all Niu Lianxu eat the feed after 7 days that has added hypocrellin, with 1 * 10 of 1mL
6TCID50/mL BVDV virus-culturing fluid is attacked poison to all Niu Jinhang injections of all groups, measures every ox body temperature then every day and changes, and observes every ox clinical symptoms, observes 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.Blood and mucus occur in the 6th day subsequently ight soil, maxilla has shallow blue or green ulceration.Similar symptom is arranged in the observation period all the time.
Test group 2: all Niu Tiwen and clinical symptoms have no significant change, and observe 14 days all strong living.
Test group 3: all Niu Tiwen and clinical symptoms have no significant change, and observe 14 days all strong living.
Test group 4: all Niu Tiwen and clinical symptoms have no significant change, and observe 14 days all strong living.
Experiment finds out that hypocrellin has played extraordinary protective effect for ox thus.
Embodiment 4: liquid state fermentation Ka Futading makes an addition to the anti-sheep BTV virus experiment in the feed
The experiment basis daily ration is identical with embodiment 2.
Adopt liquid fermentation method to produce Ka Futading, every 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 ℃ of sterilization 20min connect 10% liquid Cladosporium cladosporioides CGMCC, 34593 bacterial classification after the cooling.Initial pH and sweat pH are nature, 28 ℃ of fermentation temperatures, and fermentation 96h finishes.Centrifugal removal zymotic fluid obtains mycelium, 60 ℃ of oven dry, and wherein Calphostin A accounts for 0.029%, and Calphostin B accounts for 0.025%, and Calphostin C accounts for 0.85%, and Calphostin D accounts for 0.02%, and Calphostin I accounts for 0.01%.
40 of the wean lambs that average weight is 10 kilograms, the principle of, male and female half and half close according to body weight, random packet is divided into 4 groups, every group of 10 repetitions, each repeats 1 bellwether.The mode of feeding is free choice feeding and drinking-water.Experiment is divided into four groups by following:
Test group 1: blank group, the whole experiment basal diet of only feeding.
Test group 2: hay and silage corn that experiment approximately is 300g fine fodder and corresponding proportion with the one day appetite of sheep.In its 300g fine fodder, evenly sneak into the Ka Futading liquid state fermentation thing 0.8g that oven dry is pulverized according to its body weight, make intake every day of sheep be about 1 μ mol/kg like this.Later on increase liquid state fermentation thing mixed volume according to appetite and the body weight of sheep day by day.Whole experiment is all eaten the feed that has added Ka Futading liquid state fermentation thing.
Test group 3: hay and silage corn that experiment approximately is 300g fine fodder and corresponding proportion with the one day appetite of sheep.In its 300g fine fodder, evenly sneak into the Ka Futading liquid state fermentation thing 4.0g that oven dry is pulverized according to its body weight, make intake every day of sheep be about 5 μ mol/kg like this.Later on increase liquid state fermentation thing mixed volume according to appetite and the body weight of sheep day by day.Whole experiment is all eaten the feed that has added Ka Futading liquid state fermentation thing.
Test group 4: hay and silage corn that experiment approximately is 300g fine fodder and corresponding proportion with the one day appetite of sheep.In its 300g fine fodder, evenly sneak into the Ka Futading liquid state fermentation thing 8.0g that oven dry is pulverized according to its body weight, make intake every day of sheep be about 10 μ mol/kg like this.Later on increase liquid state fermentation thing mixed volume according to appetite and the body weight of sheep day by day.Whole experiment is all eaten the feed that has added Ka Futading liquid state fermentation thing.
Test group 2,3,4 all sheep eat has continuously added the feed after 7 days that contains Ka Futading, with 1 * 10 of 1mL
6TCID50/mL BTV virus-culturing fluid is injected all sheep of all groups and is attacked poison, measures every bellwether body temperature then every day and changes, and observes every bellwether clinical symptoms, and the record death toll was observed 21 days.Observed result is as follows:
Test group 1: attack poison after 3 days all sheep body temperature raise and reach 40～42 ℃, down in spirits, hydrostomia, mucous membrane of mouth and tongue hyperemia, tongue cyanosis.Dead 2 sheep in the time of 7 days, the 8th day dead 2 sheep.Other sheep is death, has 2 in recovery in the 10th day normally, and other has 3 in recovery in the 12nd day normally, and 1 in recovery in the 14th day normally.
Test group 2: all sheep are observed 21 days all strong living.
Test group 3: all sheep are observed 21 days all strong living.
Test group 4: all sheep are observed 21 days all strong living.
Experiment sees that card release Fu Tading has played extraordinary protective effect for sheep thus.
Embodiment 5: the plain smelting of the Elsinochrome of liquid state fermentation is treated ox FMDV and is infected.
40 of the good calves of average weight 100kg wean body condition, the principle of, male and female half and half close according to body weight, random packet is divided into 4 groups, every group of 10 repetitions, each repeats 1 calf.The mode of feeding is free choice feeding and drinking-water.
Adopt liquid fermentation method to produce the Elsinochrome element, every 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 ℃ of sterilization 20min connect 5% liquid Elsinoe fawcettii ATCC, 38162 bacterial classification after the cooling.Initial pH and sweat pH are nature, 24 ℃ of fermentation temperatures, and fermentation 96h finishes.Centrifugal removal zymotic fluid obtains mycelium, and 60 ℃ of oven dry measure wherein that Elsinochrome A accounts for 0.012%, and Elsinochrome B accounts for 0.021%, and Elsinochrome C accounts for 0.034%, and Elsinochrome D accounts for 0.05%.With with the alcohol of weight such as mycelia, the extraction mycelium, alcohol are removed in 60 ℃ of oven dry then, obtain the plain crude extract of Elsinochrome, wherein the plain total content of Elsinochrome is 11.7%, and the plain ratio of various Elsinochromes does not become, and is used for the antiviral therapy of calf with this Elsinochrome element that obtains.
With 1 * 10 of 1mL
6TCID50/ml FMDV virus-culturing fluid is attacked poison to all little beef injections of all groups.Concentrate full mass-sending sick in 3 days, test body temperature reaches 41 ℃, broad bean takes place to the big bubble of walnut on inner aspect of lip, gums, lingual surface and the cheek bifurcation mucous membrane of ox.After the plain and a small amount of basal diet of Elsinochrome mixed, the feed of feeding interpolation Elsinochrome element at first when calf is hungry then gavaged as if no feed ability.
Test group 1: blank group, the whole experiment basal diet of only feeding.
Test group 2: dried alcoholic extract 4.7g is evenly mixed disposable feeding with the 50g basal diet.Make intake every day of calf be about 10 μ mol/kg like this.Eat the feed 3 days that contains the Elsinochrome element continuously.
Test group 3: dried alcoholic extract 23.3g is evenly mixed disposable feeding with the 50g basal diet.Make intake every day of calf be about 50 μ mol/kg like this.Eat the feed 3 days that contains the Elsinochrome element continuously.
Test group 4: dried alcoholic extract 46.6g is evenly mixed with the 50g basal diet, and disposable feeding makes intake every day of calf be about 100 μ mol/kg like this.Eat continuously and contain the plain feed of Elsinochrome 3 days.
Observe every calf clinical symptoms, observed 21 days.Observed result is as follows:
Test group 1: after attacking poison, 3 calf self-healings were arranged about 8 days, 4 calf death were arranged about 10 days, about 14 have 3 calf self-healings.
Test group 2: behind the begin treatment, all calf transference cures about 3 days, rehabilitation in 5 days.
Test group 3: behind the begin treatment, all calf transference cures about 3 days, rehabilitation in 5 days.
Test group 4: behind the begin treatment, there are calf transference cure, rehabilitation in 5 days in year about 3 days.
Experiment finds out that the Elsinochrome element has extraordinary therapeutic action for the calf infective virus thus.
The foregoing description 1-5 only is explanation technical conceive of the present invention and characteristics; its purpose is to allow the personage who is familiar with this technology can understand content of the present invention and enforcement according to this; can not limit protection scope of the present invention with this; all equivalences that flesh and blood is done according to the present invention change and become or modification, all should be encompassed within protection scope of the present invention.
Claims (5)
1. the application of perylene naphtoquinone compounds in the feed of preparation control ruminant virus.
2. according to the application of claim 1 Suo Shu De perylene naphtoquinone compounds in the feed of preparation control ruminant virus, it is characterized in that Tian Jia perylene naphtoquinone compounds is used to prepare the feed that prevents ruminant virus in ruminant feed, addition is a 1-10 μ mol perylene naphtoquinone compounds/kg body weight every day, adds all the time in the feed of using in growth period.
3. according to the application of claim 1 Suo Shu De perylene naphtoquinone compounds in the feed of preparation control ruminant virus, it is characterized in that Tian Jia perylene naphtoquinone compounds is used to prepare the feed of preventing and treating ruminant virus in feed, addition is a 10-100 μ mol perylene naphtoquinone compounds/kg body weight every day, and the time that the feed of this control ruminant virus is used the ruminant of infective virus is 2-7 days.
4. according to the application of claim 1 Suo Shu De perylene naphtoquinone compounds in the feed of preparation control ruminant virus, it is characterized in that 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.
5. according to the application of claim 1 Suo Shu De perylene naphtoquinone compounds in the feed of preparation control ruminant virus, it is characterized in that Suo Shu De perylene naphtoquinone compounds comprises: hypocrellin: hypocrellin A Hypocrellin A, hypocrelline B Hypocrellin B; Elsinochrome element: the plain Elsinochrome A of Elsinochrome first, the plain Elsinochrome B of Elsinochrome second, the Elsinochrome third plain Elsinochrome C, the plain Elsinochrome D of Elsinochrome fourth; Not come rhzomorph Phleichrome; Branch spore element: the plain Cladosporium A of branch spore first, the plain Cladosporium B of branch spore second, the branch spore third plain Cladosporium C, the plain Cladosporium D of branch spore fourth; Ka Futading: Calphostin A, Calphostin B, Calphostin C, Calphostin D; Cercosporin Cercosporin, Scutiaquinone A, Scutiaquinone B, hypomycin A, hypomycin B.
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| CN102302089A (en) * | 2011-07-18 | 2012-01-04 | 江南大学 | Application of perylenequinone compound as feed coloring agent |
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|---|---|---|---|---|
| US20040029975A1 (en) * | 2001-10-24 | 2004-02-12 | Trevor Castor | Inactivation of viral infections agents by chemiluminescence activated light-sensitive compounds |
| WO2009146859A2 (en) * | 2008-06-02 | 2009-12-10 | Universiteit Gent | Methods and compositions in the treatment of coronaviruses |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040029975A1 (en) * | 2001-10-24 | 2004-02-12 | Trevor Castor | Inactivation of viral infections agents by chemiluminescence activated light-sensitive compounds |
| WO2009146859A2 (en) * | 2008-06-02 | 2009-12-10 | Universiteit Gent | Methods and compositions in the treatment of coronaviruses |
| EP2285975A2 (en) * | 2008-06-02 | 2011-02-23 | Universiteit Gent | Methods and compositions in the treatment of coronaviruses |
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| Title |
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| 《Photochemistry and Photobiology》 19971231 J. B. Hudson et.al Antiviral Activities of Photoactive Perylenequinones 352-354 1-5 第65卷, 第2期 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102302089A (en) * | 2011-07-18 | 2012-01-04 | 江南大学 | Application of perylenequinone compound as feed coloring agent |
| CN102302089B (en) * | 2011-07-18 | 2013-03-13 | 江南大学 | Application of perylenequinone compound as feed coloring agent |
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