KR20160118506A

KR20160118506A - Pharmaceutical Composition Comprising an Extract of Celosia for Preventing and Treating Fish Disease Caused by Virues

Info

Publication number: KR20160118506A
Application number: KR1020150046721A
Authority: KR
Inventors: 문치웅; 최태진
Original assignee: 구을리주식회사; 문치웅
Priority date: 2015-04-02
Filing date: 2015-04-02
Publication date: 2016-10-12

Abstract

The present invention relates to a pharmaceutical composition for preventing fish virus, containing a cockscomb extract. According to the present invention, the pharmaceutical composition is produced by the following steps: acquiring the cockscomb extract by carrying out distillation under reduced pressure after hot-water extracting stems and roots of cockscomb; and adding a daikon extract as an excipient. By injecting 2.5-10 g per one fish, it is possible to produce a pharmaceutical composition for preventing and treating fish virus infection, containing the cockscomb extract for preventing and treating fish viral hemorrhagic septicemia.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing and treating fish virus infection,

The invention is cockscomb (Celosia it relates to a pharmaceutical composition for preventing and treating fish virus comprising a cockscomb extract, more particularly and distilled under reduced pressure, to hot water extraction in the stems and roots of cristata), radish (Raphanus The present invention relates to a pharmaceutical composition for preventing and treating viral infection in fishes, which comprises an extract of Bombyx mori, which is prepared by using a root extract of Bombyx mori L. sativus as an excipient.

Recently, in the aquaculture industry, large-scale deaths occur frequently due to viral diseases, causing many economic losses, but due to the failure to find appropriate countermeasures for prevention and treatment, there has been repeated damage every year. There is a trend.

Particularly, the flounder, which occupies a large proportion of the seafood cultivated in Korea, is produced by the seaweed meat belonging to the flounder of the flounder, and more than 40 thousand tons are produced annually only in the aquaculture. Of the 29,297 tons of flounder produced in 351 onshore farms in Jeju Island in 2013, it is estimated that 6928 tons, or 30.1%, of the flounder produced due to various diseases, amounted to 52.9 billion won.

Although the cause of our disease was predominantly bacterial diseases such as Edwards and Streptococci until the mid-2000s, viruses and parasites were spreading rapidly in the late 2000s. Recently, viral diseases such as viral hemorrhagic septicemia (VHS) and sciticacid disease (70%) are estimated to be caused by bacterial diseases such as Edward and Streptococcus.

In particular, the disease occurring in the flounder was not caused by viral hemorrhagic septicemia until 2006, but after that, the number of flounder killed due to viral hemorrhagic septicemia is increasing, and the number of flounder in the Jeju Special Self- According to a laboratory report, the overall rate of virus detection for viral hemorrhagic sepsis increased to 32% in 2007 and to 62% in 2008.

Viral haemorrhagic septicaemia (VHS) was first discovered in a rainbow trout farm in Denmark in 1949 and was named Egtved disease, which is the name of a village with a disease site. In 1962, Hemorrhagic sepsis.

Symptoms of VHS are usually of three types: acute, chronic, and nervous. Rapid progression of symptoms and rapid mortality rate is very high. Infected sickles darken body color, protruding eyes, gills, eyeballs, pectoral fin and bleeding shows. The chronic type is seen in the early stages of the epidemic, the progress of the symptoms is delayed and the mortality rate is low. The body of the infected body is blackened, anemic is remarkable, the gill is almost discolored, and the protrusion of the eyeball is prominent and individuals with pluralities are present, but the hemorrhage of the body is not as remarkable as the rapid molding.

The nervous type appears at the end of the fashion, but the body does not exercise the body, or it tilts to one side to swim and rush. These strange behaviors and the abdominal wall seem to be shrunken, but not so much with healthy individuals. These three types are not strictly distinguishable, but some of them are intermediate, and the three types are also mixed in the same disease group.

In the flounder, when the VHS is present, color darkening, abdominal distension, and hernia are the most common, and when the anatomy is observed, fading of the fingertips is observed, and clear ascites is found in the abdomen. In contrast to other viral diseases, It is invisible and has a very good food intake. In addition, there is no specific symptom at the early stage or latency period, or there is no other symptom other than color darkening. However, once VHS occurs in fish farms, an average of 35 ~ 90% of the fish are killed.

The causes of VHS are factors such as fish species, size of fish body, nutrition status, aquaculture density, water temperature and water quality, but water temperature is the most important environmental factor. It can occur regardless of the season, but it usually occurs in spring where the water temperature usually rises or water temperature fluctuates. In flounder, it occurs mainly at 9 ~ 13 ℃, and the mortality rate is high and the rate of progression is high at around 12 ℃. However, the mortality rate drops sharply even when the temperature is 15 ℃ and the rise of water temperature at 18 ℃ is not observed . In other words, in Korea, the symptoms of infectious diseases appear from late November to April - May in the aquaculture method.

VHS was known to be a virus predominantly affecting rainbow trout or some freshwater fish species in Europe until the early 1980s, but was also found to be found in marine fishes when found in coyotes and king salmon migrating to the western Pacific coast of the United States in 1988 . Currently, about 50 kinds of marine fish and freshwater fish, including flounder, cod, sardine, pollack and rainbow trout, have been infected with VHS and the causative virus has been isolated.

VHS (VHSV) is a single-stranded RNA virus with an outer membrane of Rhabdoviridae and Novirhabdovirus. It is a virus with a size of 180 ~ 240 ㎚.

Diagnostic methods include cell culture methods, immunochemical methods, and PCR detection methods using molecular biology. The susceptible fish cell lines are known to be BF-2, RTG-2, EPC, FHM and CHSE-214, but the cell line that is most sensitive to seawater-derived VHSV is known as EPC.

The primary method of preventing the occurrence of such VHS is mainly by blocking the infection route. A method of disinfecting the breeding water with ultraviolet rays or a disinfectant such as chlorine, hypochlorous acid, or iodine poisons.

Studies on VHSV vaccines have been steadily carried out, including formalin inactivated vaccines, recombinant protein vaccines, attenuated vaccines, and DNA vaccines, but no chemotherapeutic agents for prevention and treatment have been commercialized in Korea.

In Germany, a toxic vaccine of VHSV has been reported to be limited to rainbow trout. In Japan, a recombinant DNA vaccine using the teleostean interferon regulatory factor-1 (IRF-1) gene, which regulates the fish immune system, There has been research that has induced resistance to VHSV. In addition, studies on recombinant glycoprotein (VHSVg), a subunit vaccine of VHSV, and DNA vaccine (pCMV-VHSg) using this gene have been conducted. Recently, synthetic double-stranded RNA, polyinosinic-polycytidylic acid (poly However, there are no studies that have been conducted to improve the immune function of the flounder and induce resistance to VHSV.

On the other hand, Korea has a rich intellectual property on natural products of Oriental medicine and Oriental medicine accumulated on the basis of long experience of oriental traditional medicine, and it is applied to food, functional products and medicines in various ways. Herbal medicines are eco-friendly materials that have been used to prevent and treat human diseases for a long time. Because they are nature-friendly, they are easy to decompose in nature and have low toxicity, which can help to mitigate toxic effects of chemicals. In particular, there are concerns about environmental impacts and drug residues, and the antibiotics and other chemotherapeutics that have been widely used in livestock and fisheries have faced strong domestic and foreign regulations due to various public health hazards, And herbal medicines may be an important alternative to fish disease prevention research.

One of the plants attracting attention as a medicinal substance derived from such natural products is mandrami. Mandrai is an annual plant in the middle part of the Korean peninsula. It is also called the 'ganghwanhwa' because the flowers resemble chicken crest. The tropical and subtropical regions of Asia, America, and West Indies are native to the country and more than 60 species are known. Celosia cristata , Celosia plumosa , Celosia argentea , and their hybrids are naturally grown or cultivated in Korea. It grows up to 90cm in height. Stems are straight, hard, and sometimes reddish without hair on the whole. Flowers bloom in 7-10 months with red, yellow, or white, and the fruit is ovate with calyxes and sideways.

According to a previous study of the present inventor, it has been found that the extract of Mandraki is effective for the prevention and treatment of viral diseases of humans and livestock, and is effective against influenza A virus, newcastle disease virus, infectious broncheitis virus ), Avian pneumovirus, porcine reproductive and respiratory syndrome virus, coxsakie virus, reticuloendotheliosis virus, aujeszky's disease virus, and Adeno It has been found that there is a suppression of the proliferation of the adenovirus and a virucidal effect.

In Korean Patent No. 10-0881035, in vitro experiments using infected cell lines such as influenza A virus and in vivo experiments using the above-mentioned virus-infected animals have shown that virus replication inhibition and flesh virus , A pharmaceutical composition useful for the prevention and treatment of human diseases caused by the in vivo infection of the virus, and a composition for prevention and treatment of human diseases caused by viruses containing the extract of mandrake that can be used for health functional foods . Korean Patent Laid-Open Publication No. 10-2014-0135488 discloses a method of using a mangalial flower extract containing polyphenols and flavonoids as a natural antioxidant to prevent cell damage caused by oxidative stress, Discloses a composition having an antioxidative activity effect using an extract of Mandrel as an active ingredient, which has an effect of treating and preventing diseases that occur. Korean Patent Laid-Open No. 10-2009-0077303 can provide a method for preventing infection with hemorrhagic septicemia virus (VHSV) of flounder by controlling culture temperature without drug treatment or DNA vaccination, thereby improving the productivity of flounder A method and system for preventing infestation of a flounder's Viral Hemorrhagic Septicemia Virus (VHSV) by controlling the temperature of the flounder culture to be maintained at 18 to 22 ° C. However, the above-mentioned inventions include a mandrami extract, which is produced by hot-water extraction from the stem and root of Celosia cristata and decompression distillation thereof and using root extract of Raphanus sativus as an excipient. And the composition and effect are different from those of the pharmaceutical composition for preventing viral infection of fish.

The present invention relates to a method for preventing and treating viral hemorrhagic septicemia in a fish which has not yet been fully established for its prevention and treatment, though it has recently caused massive deaths in freshwater and marine fish farms including flounder, And to provide a pharmaceutical composition for such a pharmaceutical composition.

In order to solve the above problems, the present invention relates to a method for preparing a pharmaceutical composition by using a root extract of Raphanus sativus as an excipient and extracting a mandrami extract obtained by hot-water extraction from the stem and root of Celosia cristata and distillation under reduced pressure, The present invention also provides a pharmaceutical composition for preventing viral infection of a fish, which contains a mandrami extract for prevention and treatment of viral hemorrhagic septicemia in fish.

The pharmaceutical composition for the prevention of fish viruses containing the extract of Mandrel according to the present invention is added to the diet of the cultured fish such as flounder using the easy-to-grow and easy-to-obtain mandrel, and the immune response gene Inducing the expression of the virus, and inhibiting the proliferation of the infectious virus. Therefore, it can be easily used for inhibiting and treating infection of fish virus including viral hemorrhagic sepsis virus.

FIG. 1 is a graph showing the effect of Bombyx mori extract on cell activity of an EPC cell line. (B) Cellular activity of EPC cell line in which (A) excipient was added at concentrations of (0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 10.0 mg / Cellular activity of
FIG. 2 is a graph showing the effect of Bombyx mori extract on EPC cell lysis by VHSV. (A) Cell lysis by VHSV of EPC cell line supplemented with excipient (B) Cell lysis by VHSV of EPC cell line supplemented with a mixture of Mandraki extract and excipient
FIG. 3 is a graph showing the effect of Bombyx mori extract on the EPC cell lysis by VHSV. (A) Cell lysis by VHSV of EPC cell line treated with PBS at different concentration (B) Cell lysis by VHSV of EPC cell line treated with concentration of Mandraham extract and excipient mixture
FIG. 4 is a photograph showing the effect of Bombyx mori extract on the CPE (Cytophatic effect) of a VHSV-infected EPC cell line. (A) A CPE (B) EPC cell line treated with a mixture of mandrami extract and excipient first in an EPC cell line and infected with VHSV, followed by treatment with a CPE
FIG. 5 is a photograph of a plaque assay showing the effect of Bombyx mori extract on the growth of VHSV-infected EPC cell lines. (A) Plaque Assay of VHSV-infected EPC cell line treated with 10 μg / ml of Bombyx mori extract and vehicle mixture (B) Plaque Assay of PBS-treated VHSV-infected EPC cell line
FIG. 6 is a graph showing the effect of Bombyx mori extract on the increase curve of VHSV infected with EPC cell line. FIG.
Fig. 7 shows the effect of Bombyx mori extract on the expression of the Mx gene of the EPC cell line. (A) is an electrophoresis image of RT-PCR result of Mx gene in EPC cell line infected with VHSV-infected Bombyx mori extract with different treatment time with PBS control. (B) is a graph showing the relative expression level of the RT-PCR result of the Mx gene in the EPC cell line infected with the VHSV-infected Bombyx mori extract with different treatment time with PBS treatment (Control).
FIG. 8 is a photograph of a flounder to examine the effect of the extract on the immune gene of the flounder. (a) Immunoreactive flounder (b) Obtained liver, kidney and spleen for RT-PCR in flounder of (a)
FIG. 9 shows the effect of Bombyx mori extract on the immune response genes of flounder. (A) is an electrophoresis photograph of RT-PCR result of TLR2 and TLR7 genes of flounder, which was orally administered with a mixture of Bombyx mori extract and excipient. (B) is a graph showing the relative abundance of Mx, TLR7, TLR2, and ISG15 gene in the flounder that was orally administered with a mixture of Bombyx mori extract and excipient.
10 is a graph showing the effect of the pharmaceutical composition containing the extract of Bombyx mori L. on the VHS infection of the flounder according to the present invention.

VHS-onset fish show symptoms such as body color blackening, abdominal distension, and hernia, and lead to massive deaths when they show bleeding from protuberance, gills, eyeballs, and pectoral fin. However, unlike other viral diseases, there is no decrease in food intake during the onset, and food intake is very good. Therefore, it can be approached as a preventive and therapeutic method of VHS by adding drugs to feed. Based on the existing studies of the inventors of the present invention, the present invention was completed while confirming the effect of Celosia crista extract, which is expected to have an effect on viral pathology, on VHSV. Hereinafter, the present invention will be described in detail with reference to specific examples.

A. Cell Stability and In Vitro Activity of Compositions Containing Mandrake Extract

A.1. Cytotoxicity studies of compositions containing mandrami extract

A.1.1. Obtain an extract of Mandraki and excipient

The in vitro and in The extracts from the natural products used for the vivo experiment were collected from collected Mandraki ( Celosia crista ) were shredded and shredded, followed by hot water extraction at 95 ° C for 8 hours, followed by vacuum distillation and lyophilization, and solidified samples were used. In the present invention no (Raphanus sativus ) was used as an excipient and the extract of radish root was also extracted by the same method.

A.1.2. Preparation of EPC cell line

The EPC ( Epithelioma papulse cyprini cell line) used in the present invention was distributed in a laboratory of parasitology at Pukyong National University. The culture medium was prepared by adding Sigma-Aldrich L-15 medium (Leibovitz), Gibco FBS (fetal bovine serum) Strep (Penicillin Streptomycin) was used. 10% FBS was used for cell culture. Cells were cultured at 28 ° C in an SPL cell culture flask at 25 cm ² at 2 × 10 ⁶ cells / flask. For cell separation, 0.25% trysin-EDTA (1x) from Gibco was used.

A.1.3. Determination of cytotoxicity of Echinosophora koreana extract to EPC cell line

The WST-1 cell proliferation assay system was used to study cell cytotoxicity in the EPC cell treatment of environmentally-friendly eukaryotic preparations for the prevention of VHSV and the inhibition of infectious diseases that cause massive deaths of aquaculture fish. Cell Viability, Proliferation & Cytotoxicity Assay Kit (EZ-Cytox) from DOGEN was used for cell viability assay.

First, cells were cultured in a 96-well plate so that the final culture was 100 μl / well. At this time, the cell concentration was 1 × 10 ⁵ cells / well and cultured at 28 ° C. for 24 hours. After the cell culture was completed, the remaining culture was removed using an aspirator, and the mixture of Mandraham extract and excipient was adjusted to a concentration of 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 10.0 mg / / well. After incubation at 28 ° C for 24 hours, the culture medium was removed, and 10 μl of Premix WST-1 was added to 100 μl of L-15 medium per well. After reacting at 28 ° C for 3 to 4 hours, the absorbance of the sample was measured using an ELISA reader at 460 nm, and then the relative cell activity was calculated for the control without the extract of Mandrake extract.

FIG. 1 is a graph showing the effect of Bombyx mori extract on cell activity of an EPC cell line. As shown in FIG. 1, when the excipient or a mixture of Bombyx mori extract and excipient was added to the EPC cell line at concentrations of 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 and 10.0 mg / (B) Mixture of SR22: diuting agent did not show half - lethal dose to 5 ㎎ / ㎖. In addition, it was found that the cell viability was better in a mixture of Mandraki extract (SR22) and excipient than the vehicle treated with excipient alone (Fig. 1).

A.2. Confirmation of the suppression of VHSV infection of mandrami extract

Experiments were conducted to investigate the effects and proper concentration of Bombyx mori extract for inhibiting VHSV infection. The VHS virus was distributed at the Pukyong National University Fish Pathology and Parasitology Lab. The VHS virus strain used in the experiment was Wt VHSV KJ2008 and the virus stock was 4 × 10 ⁷ pfu / ㎖. EPC cells were cultured on a ^6- well plate at a density of 1 × 10 ⁶ cells / well. After the cell culture was completed, the culture medium was removed and the mixture of the excipient or the extract of Mandrake and the excipient was cultured at 28 ° C for 4 days to a final concentration of 2 ml / well at a concentration of 5, 10, and 15 μg / ml. After incubation at 15 ° C for 14-24 hours before virus inoculation, the culture medium was removed and 2 ml of 2% FBS medium was added. Wt VHSV KJ2008 was diluted in 2% FBS medium to an MOI of 0.1 and then inoculated at 100 μl / well. It was incubated at 15 ° C and observed under a microscope.

FIG. 2 is a graph showing the effect of Bombyx mori extract on EPC cell lysis by VHSV. Cell lysis was observed by VHSV in the EPC cell line supplemented with excipient. However, the cell lysis by VHSV was remarkably suppressed in the case of the EPC cell line supplemented with the mixture of Mandraki extract and excipient.

FIG. 3 is a graph showing the effect of Bombyx mori extract on the EPC cell lysis by VHSV. (A) shows the cell lysis by VHSV of the EPC cell line treated with PBS at different concentrations, and (B) shows the cell lysis by VHSV of the EPC cell line treated with the mixture of Mandraham extract and excipient. As shown in FIG. 3, the cell lysis rate was significantly higher in the treatment group treated with the mixture of mandarin extract (SR22) and excipient, compared with the control (A) treated only with PBS, and the highest cell lysis rate was observed at the concentration of 10 ㎍ / Cell viability was confirmed (FIG. 3).

A.3. Identification of the action of V. minstreve extract to inhibit VHSV infection

Experiments were conducted to investigate the working path of mandrami extract inhibiting VHSV infection. In experiment A, cultured EPC cells were treated with 10 ㎍ / ㎖ of mandrami extract and excipient, and VHSV was inoculated at a MOI of 0.01 and cultured at 15 ℃ (Extracts first). In Experiment B, 1 × 10 ⁶ cells / well of EPC cells were cultured in a ^6- well plate at 28 ° C., VHSV was inoculated at a MOI of 0.01 and incubated at 15 ° C. for 2 hours. The mixture of Mandraki extract and excipient Ug / ml (Virus first) and then observed under a microscope.

FIG. 4 is a photograph showing the effect of a mixture of Bombyx mori extract and an excipient on CPE (Cytophatic effect) of a VHSV-infected EPC cell line. CPE could not be confirmed in the experimental group (A) in which a mixture of Mandraham extract and excipient was first treated in an EPC cell line and infected with VHSV. On the other hand, CPE could be confirmed by treating the mixture of Mandraham extract and excipient after treating the same concentration of VHSV.

A.4. Identification of the effect of mandrami extract on growth curves of VHSV-infected cells

In order to confirm the inhibition of viruses by using Bombyx mori extract, 1 × 10 ⁶ cells / well of EPC cells were cultured on a ^6- well plate at 28 ° C., and then 10 μg / ml of a mixture of Mandrel extract (SR22) and excipient was added. Treated with PBS as a negative control. After 3 days, the medium was replaced with 2% FBS medium, Wt VHSV was treated at a MOI of 0.1, and the supernatant was taken after 1 to 5 days of infection to perform a plaque assay. The supernatant diluted in decanterial dilution method was added to the EPC cell for 2 hours, and the medium (0.7% agar) was dispensed for 7 days at 15 ° C. The PFU was confirmed by Crystal violet staining.

FIG. 5 is a photograph of a plaque assay showing the effect of Bombyx mori extract on the growth of VHSV-infected EPC cell lines. (A) is a photograph showing a Plaque Assay of a VHSV-infected EPC cell line treated with a mixture of Bombyx mori extract and an excipient mixture (10 μg / ml), and (B) a PBS showing a Plaque Assay of a VHSV-infected EPC cell line. As shown in FIG. 4, the formation of plaques was significantly reduced in the treatment group treated with 10 μg / ml of a mixture of Mandraham extract and excipient.

6 is a graph showing the effect of the EPC cell line on the increase curve of VHSV infected. As shown in the graph, the virus titer was 6.3 × 10 ⁶ pfu / ㎖ after 3 days of infection in the VHSV-infected cells in the treatment group treated with the mixture of mandrami extract (SR22) and excipient, while the virus titer of the control was 8.6 × 10 ⁶ pfu / Ml. In addition, the virus titer was 5.8 × 10 ⁶ pfu / ㎖ in the treatment with the mixture of Mandrake extract (SR22) and excipient 4 days after infection, but in the control without the mixture of Mandrake extract (SR22) and excipient, the virus titer was 2.1 × 10 < ⁷ > pfu / ml. After 5 days of infection, the virus titer of the control and control groups were 4.7 × 10 ⁶ pfu / ㎖ and 2.7 × 10 ⁷ pfu / ㎖, respectively. These results indicated that the treatment of Mandrel extract (SR22) and excipient mixture significantly inhibited the replication of virus compared to the untreated control.

A.5. Effect of Bombyx mori Extract on the Expression of Immune Response in EPC Cell Lines

In order to investigate the mechanism of inhibition of VHSV of mandrami extract, the incubation time of mandrami extract (SR22) and excipient mixture was varied in cultured EPC cell, and VHSV was infected to confirm immune related gene expression level in EPC cell.

To this end, 10 μg / ml of a mixture of mandrami extract (SR22) and excipient was added to the cultured EPC cell, and the cells were cultured for 1 day, 2 days, 3 days, 4 days and 5 days and then infected with VHSV 0.1. After incubation, mRNA was extracted from EPC cells and RT-PCR was performed. The mRNA of EPC cell line was extracted using Dynabeads mRNA DIRECTtmKIT (Ambion), and cDNA was synthesized using SuperScript® VILO ™ cDNA Synthesis Kit (Invitrogen). The cDNA was amplified by PCR using Mx gene expression.

PCR was performed using Prime Taq DNA Polymerase (GeNet Bio) and Mx Primer was constructed using GeneBank number EF635410.1. The sequence was 5'-gtccaagccctggaaaaaga-3 '(sense), 5'-cgggaagctcctttcctcta-3' -sense). β-actin was produced by 5'-caattactgggcagccttca-3 '(sense) and 5'-caattactggggagccttca-3' (anti-sense) based on GenBank no M24113.1 as an internal control. PCR was carried out at 95 ° C for 2 minutes, followed by 30 cycles at 55 ° C for 30 seconds and 72 ° C for 1 minute, followed by reaction at 72 ° C for 10 minutes.

Fig. 7 shows the effect of Bombyx mori extract on the expression of the Mx gene of the EPC cell line. (A) is the electrophoresis of the RT-PCR result of the Mx gene in the EPC cell line with VHSV-infected Bombyx mori extract with different treatment time, and (B) FIG.

The expression of Mx gene in EPC cell line treated with a mixture of Bombyx mori extract and excipient was significantly increased as the treatment time was increased than that of PBS alone treatment. In addition, the highest Mx gene expression was observed after 4 days of the treatment with the Bombyx mori extract and the excipient mixture.

B. Immunoglobulin Expression and Viral Disease Inhibitory Effect of Bombyx mori Extract on Flounder

B.1. Effect of Bombyx mori Extract on Immune Gene Expression in Flounder

To investigate the effect of Bombyx mori extract on the viral diseases of fish, immunoglobulin expression was examined by treatment with 30 g of flounder at different concentrations and different times.

FIG. 8 is a photograph of a flounder infected with VHSV in order to examine the effect of Bombyx mori extract on immune gene expression of VHSV-infected flounder. Total RNA was isolated from the flounder after 24, 48, and 72 hours by oral administration of Mandrake Extract (SR22) and excipients 2.5, 5 and 10 ㎍ / fish.

TRIzol (Invitrogen) was added to the crushed tissues, followed by addition of chloroform, followed by centrifugation at 12,000 g for 15 minutes. After transferring the supernatant to a new tube, isopropyl alcohol was added, centrifuged at 12,000 g for 10 minutes, the pellet was washed with 75% ethanol, centrifuged at 10,000 g for 5 minutes, and 20 μl of RNase-free water To dissolve the pellet. The extracted RNA samples were synthesized with SuperScript® VILO ™ cDNA Synthesis Kit (Invitrogen) and subjected to PCR. The PCR primers used for identification of the immune genes were prepared as shown in Table 1. The expression of the type 1 IFN response (Mx, ISG15) and the innate immune response (TLR2, TLR7) in the treatment of the mixture of mandrami extract (SR22) Respectively.

PCR primers used for immunogenetic identification Primer GeneBank Acc.No Product length Sense primer Antisense primer Tm TLR7 HQ845984a 97 cctgggaaatctggaagaac tttgagggaggagaaactgc 62 TLR2 AB109393 120 gctacatctgcgactctcct cacagggacacgaacaaatc 58 MX AB110446.1 399 aacagccaaggcaaagattg aatgtccagctcctccttca 60 ISG15 AB519717.1 295 gaagcagctcatccagcaga cctctgctgactctccgtca 55 β-actin HQ386788.1 199 tttccctccattgttggtcg tacaacgagctgagagtcgc 55

FIG. 9 shows the effect of Bombyx mori extract on the immune response genes of flounder. (A) is an electrophoresis image of RT-PCR result of TLR2 and TLR7 gene of oral flounder orally administered mixture of Bombyx mori extract and excipient. (B) shows the results of MW, TLR7, TLR2 And ISG 15 gene). Mx and ISG 15, which are virus specific immune response markers, were increased in liver (L), kidney (K), and spleen (S) of flounder treated with Bombyx mori extract and excipient mixture. TLR2 And TLR7, respectively.

In addition, the increase of both Mandrake extract and excipient mixture was observed at 24, 48, and 72 hours, and the highest increase of TLR2 and TLR7 was observed at 2.5 ㎍ / fish.

B.2. Inhibitory effect of Bombyx mori extract on VHS development in flounder

Experiments were carried out to investigate the inhibitory effect of VHMV on the treatment of the mixture of Bombyx mori extract and excipient using flounder with an average weight of 9 g. As shown in Table 2, the experiment was repeated three times in three groups of 10 rats.

The inhibitory effect of the onset of the experimental group Group oral administration VHSV One 100 [mu] l of SR22: Dilutant Mixture 50ul of L-15 2 100 [mu] l of PBS 50ul of VHSV 3 100 [mu] l of SR22: Dilutant Mixture 50ul of VHSV

First, L-15 medium was injected intramuscularly 48 hours after oral administration of 100 쨉 l of 2.5 쨉 g / fish mixture of Mandrel extract and excipient. Virus (10 ² pfu / ml) was injected 48 hours after oral administration of 100 袖 l PBS, Finally, virus (10 ² pfu / ml) was injected 48 hours after oral administration of 100 μl of 2.5 ㎍ / fish mixture of Mandrake extract and excipient. Survival was measured 2 weeks after infection.

In order to confirm the inhibition of viral hemorrhagic septicemia caused by the treatment of Bombyx mori extract and excipient mixture in flounder, the experiment was divided into three groups. Group 1 did not infect VHSV after treatment with a mixture of mandrami extract and excipient, Group 2 infected VHSV without treatment of mandrel extract and excipient mixture, Group 3 infected VHSV after treatment with a mixture of mandrami extract and excipient .

10 is a graph showing the effect of the pharmaceutical composition containing the extract of Bombyx mori L. on the VHS infection of the flounder according to the present invention. After 13 days of infection, the motrality of Group 1 was found to be decreased compared with that of Group 2, which had not been treated with the extract of Mandrake and the excipient mixture. After 17 days of infection, 100% of the dead animals were identified in Group 2, which had not been treated with a mixture of Mandrake extract and excipient, and 60% of the dead animals were identified in Group 3. Therefore, the inhibition of the onset of viral hemorrhagic sepsis in flounder could be confirmed by treating a mixture of Mandrel extract and excipient.

C. Preparation of medicines for the prevention of infection using the composition

The composition of the present invention may be prepared in the form of tablets, capsules, soft capsules, granules, or liquid form, and may be used as a feed additive to facilitate the administration of the composition of the present invention. The composition may be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, transdermal preparations, suppositories or sterile injectable solutions so as to contain the composition in an amount of 0.01 to 99.9% by weight.

In the case of a sterile injectable solution, the pharmaceutical composition may be prepared to contain 0.01 to 99.9% by weight of the pharmaceutical composition and 99.9 to 0.01% by weight of purified water, glucose solution or saline solution. In the case of capsules, the pharmaceutical composition may be lyophilized to be contained in an amount of 0.01 to 99.9% by weight, and the vitamin and calcium preparations may be mixed in an amount of 99.9 to 0.01% by weight.

The daily dose of the pharmaceutical composition prepared is such that the extract contains 10 to 1000 mg / kg body weight. It is also possible to prepare feed additives containing 0.01 to 99.9% by weight of the above pharmaceutical composition.

As described above, it was confirmed that the mixture of mandrami extract and excipient was significantly inhibited the proliferation of VHSV after the treatment with VHSV after treatment with EPC cell line, and it was most effective when the concentration of the mandrami extract and excipient mixture was 10 / . At this time, the mixture of Mandrel extract and excipient had a better effect than VHSV infection treatment.

In addition, Mx, an immune gene, was strongly expressed in cells treated with a mixture of Bombyx mori extract and excipient. In the flounder application experiment, expression of Mx, TLR2 and TLR7 genes was significantly increased in liver, kidney and spleen. It was confirmed that the extract of Bombyx mori according to the present invention is effective for the prevention of infection of VHSV.

The pharmaceutical composition for preventing viral hemorrhagic septicemia of a fish containing mandrami extract according to the present invention is useful for preventing viral hemorrhagic septicemia, which causes massive mortality by using an extract of mandrami which is easy to cultivate and easy to obtain in Korea, It is effective for prevention of sepsis and economic loss can be reduced, which is industrially applicable.

Claims

Celosia The present invention relates to a pharmaceutical composition for preventing and treating a viral hemorrhagic sepsis virus infection in a fish containing an extract of cristata as an active ingredient

[Claim 2] The method according to claim 1, wherein the extract is hydrothermally extracted from stem and root of mandrami and subjected to reduced pressure distillation. The pharmaceutical composition for prevention and treatment of viral hemorrhagic sepsis virus infection

The method of claim 1, wherein the mandrami extract is mixed with an excipient and the excipient is selected from the group consisting of Raphanus The present invention relates to a pharmaceutical composition for prevention and treatment of viral hemorrhagic sepsis virus infection in fish,

The pharmaceutical composition according to claim 1, wherein the composition is formulated as a granule, a tablet, a suspension, an emulsion, a syrup, a transdermal agent, or a sterilized solution for injection, and a pharmaceutical composition for prevention and treatment of viral hemorrhagic sepsis virus infection

A pharmaceutical composition for the prevention and treatment of viral hemorrhagic sepsis infection in fish characterized in that the composition of any one of claims 1 to 4 is contained in an amount of 2.5 to 10 占 퐂 / fish