KR101431877B1 - Antiviral composition against fish pathogenic viruses comprising of flavonoid compounds from Rhus verniciflua Stokes - Google Patents

Abstract

The present invention relates to a method for isolating and purifying a fish pathogenic antiviral active flavonoid compound from a methanol extract of quercus and various organic solvent extract fractions thereof and a method for isolating and purifying a fish pathogenic antiviral activity of a flavonoid compound isolated from the above- .

Description

TECHNICAL FIELD The present invention relates to an antiviral composition for fish pathogenic viruses containing a flavonoid compound derived from an extract of Chrysanthemum morifolium (Rhus verniciflua Stokes)

The present invention relates to an antiviral active composition comprising a flavonoid compound as an active ingredient, and to an antiviral composition for preventing, treating or inhibiting a viral disease of fish.

In the case of aquatic aquaculture disease, which is a global problem in the aquaculture industry, the incidence rate in the first half of the 1990s was only less than 5%, and it increased to around 15% in the late 1990s. The most common diseases are in the year-round trend. Among them, the damage of cultured fish due to the incidence of intractable viral infectious diseases is increasing, and the therapeutic effect is lowered due to the increase of the occurrence of resistant bacteria by abuse of medicines. Especially, recently, the increase of imports of cultivated seeds from overseas has increased the risk of introduction of new outbreak infectious diseases, and the spread of diseases due to the movement of infectious seedlings is concerned. In particular, the damage caused by infection of infectious hematopoietic necrosis virus (IHNV) and infectious pacreatic necrosis virus (IPNV) in salmon, trout and fish has been continuing since the 1970s, The development of appropriate drugs that can be used has not occurred at home or abroad.

Among the viruses that cause lethal and industrially important diseases in fish are the infectious hematopoietic necrosis disease (IHNV) caused by the infectious hematopoietic necrosis virus (IHNV) of salmon and fish. (IPND) and viral hemorrhagic septicemia virus (VHSV), which are caused by infectious pac- tic necrosis virus (IPNV) (VHSD) is the most prevalent disease in the world and its damage is also known to be the most serious disease worldwide.

The IHNV was first reported in 1969 by Amend et al. In rainbow trout. It was first reported in 1992 in Korea in the breeding rainbow trout. The major features of the kidneys and spleen, particularly in the kidney hematopoietic tissues, of the pathogenesis are virus-like shell-like morphology. The average size of the virus is between 160 and 180 nm and between 80 and 90 nm in diameter. (Van Regenmortel et al., 2000). The death rate from IHNV is 70 to 80%. Rainbow trout, mountain fish, trout damage is large. This disease was thought to have an endemic nature of red sea salmon and chinook salmon on the west coast of the United States at the time of its discovery, but since the occurrence of rainbow trout was confirmed, , And expanded throughout Europe in the 1990s.

VHS has traditionally been referred to as the Nierenschwelung and Infectiose Nierenschwald und Leberdegeneration (Schu, 1954; Heutschmann, 1952) in rainbow trout. It was reported in the 1962 Fisheries Symposium of the Office International des Epizooties (OIE) (Jensen, 1963) and the causative virus was defined as the viral hemorrhagic septicemia virus (VHSV). VHS is very prevalent in Europe, North America and Asia, and it is widely used in fisheries such as salmon and fish such as rainbow trout, silver salmon, river trout, brown trout, steelhead trout, And also in marine aquaculture fish such as flounder, and the damage is very serious. Causes Chain V efectiviruses are of the shell type with a length of 160-180 nm and a diameter of 70-80 nm and are classified as ss-RNA viruses in the novidepod virus (van Regenmortel et al., 2000). Mortality rates vary widely depending on conditions, but generally range from 20 to 80%.

Studies on the development of vaccines have been conducted as part of the infection control method of AICENV, IPENV, and VITECHV, the main pathogens of salmon and fish, and active research such as inactivated vaccine, live attenuated vaccine, (Gilmore et al., 1988; LaPatra et al., 2002) and some have been applied as prophylactic vaccines (Fryer et al., 1976; Fukuda et al.

In order to prevent the spread of viruses, disinfection of facilities, securing of virus free water, and disinfection of eggs have been studied and applied (Amend and Pietsch 1972; Yoshitsu, Kasai, 2002). However, there have been no reports on the development of drugs that can be applied for the treatment of infectious diseases.

Regarding the chemotherapy of Aiechen V and Viechs V, sodium benzimidazole, an antiviral agent, was treated at 40 ug / ml or higher to inhibit the virus in Fathead Minnow (FHM) cells (Amend, 1976), but in vivo experiments have not been performed yet. In addition, the anti-iethenzyme activity was found in culture media, herbs and herbal medicines of bacteria isolated from sheep or river, and the effect was recognized in vivo (Direkusarakom, 1996). In 1999, Fabregas et al. Reported the possibility of using viable microalgae culture concentrates to investigate the proliferation inhibition of V. aeruginosa. However, the results of this study have not been published yet.

The chili pepper was classified into Rhus ( Rhus ) belonging to the Anacardiaceae verniciflua Stokes) bark. The lacquer tree is a deciduous broad-leaved arboreous tree that grows widely in Northeast Asia such as China and Japan. The phenolic compound such as chalcone, flavonoids and the like has been isolated and reported from the bark of bark, Recently, there have been only a few reports of anticancer, anti-inflammatory, antioxidant, hepatocyte protective activity, anti-apoptosis, and immunomodulating activity of the extracts of the present invention or the phenolic compounds separated therefrom.

To date, the anti-infectivity of fish extracts and their isolated compounds against fish pathogens has been reported in Kang's (J. Fish Pathol., 18 (3): 227-237, 2005; , 2007) and the like in In vitro antimicrobial activity and its in vivo immune enhancing activity have only been reported. No studies on the activity of the fish against pathogenic viruses have been reported so far.

Accordingly, the object of the present invention is to verify and confirm the fish pathogenic viral activity of the extract of horseradish and the fraction isolated therefrom.

The above object of the present invention can be achieved by a method for producing an organic solvent extract, Suspending the horsetail extract in distilled water and obtaining a non-polar solvent fraction; Verifying the fish antiviral activity by said fraction; Ethyl acetate fractions were subjected to spectral analysis to isolate and identify flavonoid crab compounds and to measure and evaluate fish antiviral activity.

The present invention has an effect of producing a fish antiviral active flavonoid compound derived from chili pepper, and using this, it has an excellent effect that the stability of fishery can be expected by preventing and treating fish refractory viral diseases at a proper time and eliminating them.

FIG. 1 is a process diagram showing an organic solvent extraction process for separating and purifying flavonoid compounds from the extract of the present invention.
FIG. 2 is a process diagram showing a process for obtaining an active fraction from the ethyl acetate fraction of the present invention (see FIG. 1).
Fig. 3 shows the compounds of the active ingredient of the small fraction obtained by final purification and purification of the flavonoid compound of the present invention.

≪ Example 1 > Preparation of chili pepper extract

6.75 kg of dried and chopped chili pepper were subjected to ultrasonic extraction three times for 99 minutes with 80% methanol (MeOH), and then 33 L of the extract was collected, concentrated under reduced pressure using a vacuum evaporator, lyophilized, And used as a sample (Fig. 1).

Example 2: Preparation of a non-polar solvent fraction of paper

After the 80% MeOH extract (97.6g) extracted in Example 1 was suspended in distilled water, hexane (n -hexane), chloroform (chloroform, CHCl _3), ethyl acetate (ethylacetate, EtOAc), and saturated butanol (n -butanol, and n- BuOH) in the order of increasing the polarity of the organic solvent, followed by sequential fractionation to obtain a total of five fractions (FIG. 1). As a result of screening the antiviral activities against fish pathogenic viruses IHNV and VHSV in the total of 5 fractions obtained above, the ethyl acetate fraction showed the best activity for IHNV and the hexane (Table 1). ≪ tb >< TABLE >

Example 3 Isolation of Active Compound from Ethyl Acetate Fraction

In Example 2, active compounds were separated from the ethyl acetate fraction showing excellent antiviral activity against both viruses (Fig. 2). The ethyl acetate fraction was subjected to MPLC (solvent: water: MeOH 95: 5, 90:10, 85:15, 80:20, 75:25, 30:70, 20:80, 0: 100 / column: 250 g RediSep reversed phase C-18 column Teledyne Iso, USA) and divided into 9 subfractions centered on the major spot according to the TLC results. The compounds were separated from each of the fractions in the following manner.

① Small fraction II

100: 0, 98: 2, 96: 4, 94: 6, 92: 8, 90:10, 88:12, 0: 100 / Column: 40 × 120 mm), and Compound 1 (230 mg) was isolated by MPLC (solvent: 100% water / column: 130 g RediSep ^R reversedphase C-18 column) 3).

② Small fraction Ⅲ

The small fraction (3.55 g) was subjected to Sephadex LH-20 column chromatography (solvent: MeOH: water = 95: 5 / column: 15 × 1300 mm) column chromatography (solvent: 100% MeOH) to obtain six small fractions. Compound 2 (48.5 mg) was isolated by performing MPLC (solvent: water: Acetonitrile = 90: 10 / column: 43 g RediSep ^R reversedphase C-18 column) with small fraction No. 3 (FIGS. 2 and 3).

③ Small fraction Ⅴ

The fraction (V) (4.24 g) was subjected to Sephadex LH-20 column chromatography (solvent: 100% MeOH / column: 15 × 1300 mm) to obtain Compound 3 (285.5 mg). The other major fractions were combined, : water: MeOH = 90:10, 80:20, 70:30, 60:40, 40:60, 30:70, 20:80, 0: 100 / column: 43 g RediSep ^R reversedphase C-18 column) 4 (62.1 mg) was obtained (Fig. 2 and Fig. 3).

④ Small fraction Ⅵ

The small fraction 5 (5.29 g) was subjected to Sephadex ™ LH-20 column chromatography (solvent: 100% MeOH / column: 15 × 1300 mm) : 100% MeOH / column: 15 x 1300 mm) to obtain Compound 5 (85 mg). The fraction 7 was further subjected to Sephadex LH-20 column chromatography (solvent: 100% MeOH / column: 15 x 1300 mm) to obtain Compound 6 (41.3 mg) (FIGS. 2 and 3).

Example 4 Analysis and Identification of Chemical Structures of Compounds

In order to analyze the chemical structure of the compound of the present invention, spectroscopic analysis was performed using EIMS / FABMS, ¹ H-NMR and ¹³ C-NMR. As a result, the compound 1-6 was identified as methyl gallate (1), fustin ( 2), fisetin (3), butin (4), sulfuretin (5) and butein (6) as compared with respective reference values (FIG. 3).

≪ Experimental Example 1 > Antiviral activity of extracts and fractions of horseradish

Coin cell culture

Fish coin cells for virus cultures were obtained by seeding Chinook Salmon Embryo-214 (CHSE-214) and Flounder Spleen (FSP) cells in Eagle's minimum essential medium (MEM) Ml of streptomycin and 10% of Fetal Bovine Serum (FBS) was added to each flask at a concentration of 2.5 x 10 < ⁵ > / ml And incubated at 20 ° C for 6 hours.

Virus culture

The viral strains used in the experiments were Iechniv (IHNV, RtPy91), and VeeVIS (VHSV, Wando 2005). The virus was inoculated into the seeds of the E. elegans -214 cell line for MOI of 0.01 and then cultured at 20 ° C for 5 days to observe the cytopathic effect (CPE) The supernatant was dispensed into microtube and stored at -80 ° C until use in the experiment.

Cytotoxicity investigation

Cells were cultured in 96-well cell culture plates, cultured for 48 hours, and cytotoxicity was measured by neutral red uptake assay. CC ₅₀ (50% cytotoxic concentration) was determined by plotting cytotoxicity at each concentration. In addition, the maximum cytotoxic concentration of MNCC (maximal non-cytotoxic concentration), which does not show toxicity by microscopic observation, also showed the cytotoxicity of the sample. The antiviral activity of each sample was measured in a concentration range that did not show any cytotoxicity.

Antiviral activity measurement

The antiviral activity of the sample was measured using a CPE (cytopathic effect) reduction assay. Briefly, cells incubated on a 96-well plate were mixed with virus diluent and sample 1: 1 and incubated at 15 ° C with quadruplicate. After 5 to 6 days, when virus denaturation (CPE) was observed in all wells, the culture was stopped and the CPE of each sample was observed under a microscope and scored compared to the CPE of the virus control. Concentration (EC50), which reduces CPE by 50%, was obtained by plotting CPE reduction (%) for each concentration on the graph.

The antioxidant activities of the extracts of Arnoldschrift and its fractions were 15.0 and 19.1 for IHNV and VHSV, respectively. The antiviral activity against IHNV was the highest in the ethyl acetate fraction of the lower fraction and the hexane fraction had a lower SI value due to the relatively higher toxicity than the EC50 value. For VHSV, the antiviral activity was exhibited in the remaining four fractions except for the hexane fraction. In the chloroform fraction and the ethyl acetate fraction, the EC50 values were 1.8 and 3.6 μg / mL, respectively, and the SI values were 31.7 and 21.9, respectively (Table 1).

<Experimental Example 2> Antiviral activity of the compounds isolated from the ethyl acetate fraction

The antiviral activity of the separated phenol compounds was measured in the same manner as in Experimental Example 1.

As shown in Table 2, fisetin (3) showed the highest antiviral activity against both IHNV and VHSV. In addition, fustin (2) and sulfuretin (5) showed excellent antiviral activity against both viruses. Of the six isolated compounds, the four compounds except methyl gallate and butein showed 50% or less toxicity even at 500 to 1000 μM, and no cytotoxicity test was conducted due to the solubility problem. Currently, ribavirin, which is used as a human antiviral drug, is tested with a positive control. As a result, the EC50 value of the flavonoids proposed in the present invention is significantly higher than that of ribavirin, but the high cytotoxicity of ribavirin is found to be the same in fish cell lines, and a novel structure of antiviral It is considered that the flavonoid-based compound as a substance can also contribute as a candidate substance to the basic skeleton.

The present invention is an extremely useful invention in biological pesticides and technology-intensive fishing because it has an excellent effect of providing a novel fish pathogenic antiviral composition comprising an extract of chrysanthemum methanol or an ethyl acetate fraction thereof as an active ingredient.

Claims (4)

A composition for fish pathogenic antiviral comprising an extract obtained by ultrasonically extracting chili pepper with 80% methanol, or a fraction obtained by adding an organic solvent to the above extract again as an active ingredient
A composition for fish pathogenic antiviral comprising any one of the flavonoid compounds selected from the following structural formulas [1] to [6] as an active ingredient.

The composition according to claim 1, wherein the organic solvent fraction is selected from the group consisting of hexane, chloroform, ethyl acetate, and butanol fractions.


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