WO2006009395A1

WO2006009395A1 - Natural anti- virus and composition comprising thereof

Info

Publication number: WO2006009395A1
Application number: PCT/KR2005/002348
Authority: WO
Inventors: Dong-Il Jang; Jung-Hwa Lee
Original assignee: Cotde, Ltd.
Priority date: 2004-07-20
Filing date: 2005-07-20
Publication date: 2006-01-26
Also published as: CN1997378A; KR20050087742A; CN1997378B; BRPI0513466A; EP1781308A4; CN101843640A; EP1781308A1; CN101843640B; KR100552462B1

Abstract

Disclosed herein are a natural antiviral agent having high inhibitory activity against a broad spectrum of viruses without adverse side effects, obtainable using lactic acid bacteria isolated from kimchi, and a composition comprising the natural antiviral agent. The natural antiviral agent is safe to the body and can be applied to compositions, such as fodder, beverages, food, medicine, and cosmetics, imparting antimicrobial activity thereto.

Description

NATURAL ANTI- VIRUS AND COMPOSITION COMPRISING

THEREOF

Technical Field

[1] The present invention relates to a natural antiviral agent and a composition comprising the same. More particularly, the present invention relates to a natural antiviral agent, prepared using lactic acid bacteria isolated from Kimchi, which exhibits high inhibitory activity against the growth of viruses without side effects and is safe to the body, and a composition including the natural antiviral agent therein, such as a beverage, fodder, food, medicine, cosmetic, etc. Background Art

[2] The influenza virus, causing annual flu epidemics, is a basic single-stranded RNA virus, having an envelope 1/10,000 mm in size surrounding the RNA. According to antigenicity, influenza viruses are classified into types A, B and C. Of them, influenza type A and B viruses cause epidemics. Glycoproteins of two kinds, called haemaglutinin (HA) and neuramidase (NA), protrude from the surface as spikes, and 8 RNA segments exist inside the virus. Both the surface glycoproteins HA and NA of influenza viruses frequently undergo variation within the same subtype, resulting in the emergence of new antigen mutants each year.

[3] Influenza viruses are ejected, along with a violent release of air, from the lungs upon coughing and enter other bodies through the nose or the mouth. Inside the body, influenza viruses, using the viral glycoprotein HA spikes, attach to and penetrate the mucous epithelial cells of the upper airway, and subsequently proliferate therein. Research results accumulated thus far suggest definite mechanisms of virus infection. Generally, viruses bind to receptors composed of sugar chains of glycoproteins on the surface of target cells and undergo endocytosis and subsequent fusion of the viral envelope with endosomes to enter the target cells. Inside cells, viruses are proliferated by the transcription and replication of their genes and the production of progeny virus particles from plasma membranes of their hosts. Infection with influenza viruses is generally accompanied by fever, headache, pain in the joints, general prostration, etc., together with respiratory symptoms including sore throat, runny nose, stuffy nose, etc. The flu is different from colds in symptoms and severity, and features highly notorious infectivity, bringing about explosive pandemics over the world for a short period of time. Because influenza viruses have HA structures that undergo variation every year, antibodies produced in response to previous infections are not effective against ongoing epidemics, which is the reason why flu is epidemic. [4] For the prevention of influenza virus infection, scientists have mapped strategies for inhibiting the attachment onto epithelial cells, penetration into cells, transcription, replication, translation, and progeny release from cells, all of which are the targets of anti-influenza virus agents. Under the strategies, anti-viral agents, such as amantadine, rimantadine, zanamivir, etc., have been developed, but must be applied with extreme caution because they are reported to produce significant adverse side effects, such as hypersensitivity, in the central nervous system, the digestive system and the autonomic nervous system.

[5] As described above, influenza viruses infect the mucous epithelia of the airways and proliferate therein. Vaccination may be useless in preventing the infection and pro¬ liferation of influenza viruses if a virus type of a vaccine is different from the epidemic influenza virus. In fact, there are no therapeutically effective treatments or preventive agents against influenza viruses. Now, frequent tooth brushing, keeping the throat moist, and taking sufficient rest and nutrients are known as the most effective prevention of influenza virus infection.

[6] For these reasons, there is an imperative need for an antiviral agent that has high inhibitory activity against influenza viruses and can be generally utilized without adverse side-effects.

[7] Viral diseases are one of the factors that most greatly threaten and damage the poultry industry. Now, poultry influenza viruses are said to be a threat to all of mankind as well as the poultry industry. Recently, H5N1 highly pathogenic avian influenza (HPAI) outbreaks in poultry in China and Southeast Asia have been frequently reported. In the opinion of specialists from current situations, avian influenza viruses are highly apt to undergo mutation, rather than to be eradiated, bringing about an awful problem for humans.

[8] In Indonesia, it has already been found impossible to take preventive measures against epidemics of bird flu. In China, a variety of H5N1 HPAI viruses are detected, and spread at rates faster than the control rates thereof. Thus, there is no recourse but vaccination. In fact, vaccination is being conducted in many regions.

[9] Vietnam and Thailand are under more severe situations, with an increasing number of reports that bird flu has caused death in humans. What is worse, there is little possibility of controlling influenza epidemics in these countries. Thailand, a repre¬ sentative poultry-exporting nation, adheres to the policy of the slaughter of poultry that is at risk, rather than vaccination, in response to an outbreak of avian influenza. To eradicate avian influenza, the government of Thailand takes the drastic measure of imposing fines on persons who immunize poultry without permission. The reason is that, because vaccination makes it impossible to eradicate poultry influenza throughout the country, Thailand cannot help but give up the export of poultry. [10] Avian infectious bronchitis (IB) is a representative disease that greatly damages poultry farming in almost all nations having a poultry industry. In practice, no countries have terminated IB thus far, even though some countries eradicated Newcastle disease (ND) or avian influenza (AI).

[11] In Korea, the inspection data of the National Veterinary Research and Quarantine

Service show that IB was the most prevalent, comprising 6% in 2001 and 5.2% in 2002 of the total number of occurrences of avian diseases. An IB virus is the most intensely studied corona virus and has an incredibly simple structure. IB viruses, also called mobile viruses, are now undergoing mutation all over the world. The mutation is mainly caused by the gene recombination of the IB viruses that differ in serotype from each other when they simultaneously infect a host. When IB viruses of two or more types infect a host concurrently, they grow in the same cells with the concomitant re¬ combination of their genes, resulting in the emergence of new types of viruses having characteristics different from those of the progenitor viruses. For instance, if gene re¬ combination occurs between an attenuated vaccine virus and a virulent wild type virus, there is an increasing possibility that the advantages of the two viruses, that is, high proliferativity and pathogenicity, will be combined, and thus the resulting progeny virus is highly likely to be a drastic disease-causing agent that rapidly proliferates. For this reason, vaccines, although attenuated, must not be domestically introduced without impunity.

[12] Typical properties of IB viruses include the continuous emergence of various serotypes and variants, the ability to spread faster than any other disease (a latent period of 18-36 hours), and survival ability within poultry flocks (persistence on farms). In addition, the defense of serum antibodies against the viruses and cross im¬ munization between the serotypes are limited. Further, when viruses of two or more serotypes attack poultry flocks simultaneously, it is difficult to defend against the viruses only with vaccines.

[13] In Korea, ND, caused by Newcastle disease viruses, has been designated the first legal communicable disease that arises every year in poultry farms. Various vaccines based on the inactivation of ND viruses have been developed, however, the occurrence of ND has not yet decreased.

[14] Today, poultry farms have three lines of defense for inhibiting bird diseases. The first line of defense is dependent on biosecurity, the second line of defense on vaccination, and the third line of defense on the immune system of the poultry itself. However, the occurrence of IB in a poultry farm conversely demonstrates that IB viruses penetrate the first line of defense of biosecurity, render vaccination useless, and destroy the body of the chicken, completely defeating the immune system. Therefore, there is an imperative need for a therapeutic agent that kills viruses or a multifunctional treatment agent that exceptionally enhances the immune system. Disclosure of Invention

Technical Problem

[15] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a novel natural antiviral agent, which has highly potent inhibitory activity against the infection of various viruses including influenza viruses, avian influenza viruses, avian infectious bronchitis viruses, etc. and is highly safe to the human body without adverse side effects.

[16] In addition, it is another object of the present invention to provide a composition containing the natural antiviral agent as an active ingredient, which can be included in various products. Technical Solution

[17] In accordance with an aspect of the present invention, the objects of the present invention can be accomplished by the provision of a natural antiviral agent, prepared from one selected from a group consisting of a culture solution of lactic acid bacteria isolated from Kimchi during the fermentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof.

[18] In the natural antiviral agent, the lactic acid bacteria may be selected from a group consisting of Leuconostoc spp., Lactobacillus spp., Weissella spp., and combinations thereof.

[19] Preferably, Leuconostoc spp. useful in the natural antiviral agent of the present invention, include Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. Mesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc gellidum, Leuconostoc kimchii, Leuconostoc inhae, Leuconostoc gasicomitatum, and combinations thereof.

[20] Preferably, Lactobacillus spp. useful in the natural antiviral agent of the present invention include Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus kimchii, Lactobacillus para- plantarum, Lactobacillus curvatus subsp. curvatus, Lactobacillus sakei subsp. sakei, and combinations thereof.

[21] Preferably, Weissella spp. useful in the natural antiviral agent of the present invention include Weissella koreens, Weissella hanii, Weissella kimchii, Weissella soli, Weissella confusa and combinations thereof.

[22] In accordance with another aspect of the present invention, the object of the present invention could be accomplished by the provision of a composition that comprises a natural antiviral agent prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from Kimchi during the fermentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof, as an active ingredient.

[23] In this regard, the composition is preferably in the form of one of a beverage, fodder, food, a medicine or a cosmetic. Advantageous Effects

[24] Culture solutions of Kimichi lactic acid bacteria, or their concentrates or dried materials have excellent antimicrobial activity against a wide spectrum of viruses and can endow foods, fodders, beverages, medicines and cosmetics with antiviral activity with safety and efficiency. Best Mode for Carrying Out the Invention

[25] Leading to the present invention, intensive and thorough research, conducted by the present inventors, into the preservation of foods and cosmetics, resulted in the finding that a culture of lactic acid bacteria isolated from Kimchi (hereinafter referred to as Kimchi lactic acid bacteria) can act as a multifunctional natural antiviral agent that has potent inhibitory activity against a broad spectrum of viruses. As well known, kimchi lactic acid bacteria proliferate during the fermentation of kimchi, which is generally prepared by pickling radish, cabbage and cucumber with salt, mixing the pickles with a condiment composition including powdered pepper, crushed garlic, chopped green onions, salted seafood, etc., and fermenting the pickled vegetables. Mode for the Invention

[26] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[27]

[28] EXAMPLES

[29]

[30] Preparation of Culture Solution of Kimchi Lactic Acid Bacteria

[31]

[32] Among Kimchi lactic acid bacteria, Leuconostoc kimchii (KCTC2386),

Leuconostoc mesenteroides subsp. Mesenteroides)(KCTC3530), Leuconostoc citreum ( KCTC3524J, Leuconostoc lactis (KCTC3528), Lactobacillus plantarum (KCCMl 1322), and Weissella koreensis (KCTC3746) were utilized for the preparation of the culture solution.

[33] First, 100 ml of MRS (Difco) broth was autoclaved at 121°C for 15 min, and

Kimchi lactic acid bacteria was inoculated in the broth and cultured at 30°C for 16 hours with agitation. [34] After the centrifugation of the resulting culture at 5000xg for 3 min, the su¬ pernatant was filtered through a membrane filter having a pore size of 0.22 D to afford a bacteria-free solution.

[35] Subsequently, this bacteria-free solution was passed through a membrane having a cut-off size of 10,000 Da. The resulting filtrate was concentrated using a membrane having a cut-off size of 1,000 Da. The concentrate thus obtained was again filtered through a membrane filter having a pore size of 0.22 D so as to maintain the bacteria- free state. The final culture solution was adjusted to a pH of 7 in order to exclude the pH-dependent effect of antimicrobial activity.

[36] [37] -Assay of Culture Solution of Kimchi Lactic Acid bacteria for Antiviral Activity against Avian Influenza Virus-

[38] [39] An animal cell line (MDCK (Madin-Darby canine Kidney) or CEF (Chicken embryo fibroblast)) that had grown in a mono-layer was infected with the avian influenza virus HK/220, and then cultured for 3 days. Plaque formation proved viral infection.

[40] To the animal cell line MDCK or CEF which was grown in a monolayer on a plate to confluency of 80% or more, a mixture of an avian influenza virus that was diluted with PBS (pH 7.4) by a factor of 10^"3 and the culture solution of Kimchi lactic acid bacteria that was diluted with the same buffered saline was added, followed by culturing the cell line for 3 days. The infected cells were fixed and stained with neutral red to observe the formation of plaques therein. The results are given in Table 1, below.

[41] [42] Table 1

[43] As is apparent from the data of Table 1, while the control that did not contain the culture solution of kimchi lactic acid bacteria allowed as many as 1.3-1.6x10 plaques to form, the addition of the culture solution of the kimchi lactic acid bacteria remarkably reduced the number of plaques formed. Thus, these results imply that the culture solution of kimchi lactic acid bacteria has very potent antiviral activity against avian influenza viruses.

[44] [45] -Assay of Culture Solution of Kimchi Lactic Acid bacteria for Antiviral Activity against Influenza Virus-

[46] [47] The same procedure as in the assay for antiviral activity against the avian influenza virus was repeated, with the exception that the influenza virus A1H3N2 was used instead of the avian influenza virus HK/220. The results are given in Table 2, below.

[48] [49] Table 2

[50] Whereas the control in which the virus dilution was used alone without the culture solution of Kimchi lactic acid bacteria had 1.3~1.6xlO² plaques per plate, as seen in Table 2, the infection of the influenza virus in the presence of the culture solution of Kimchi lactic acid bacteria developed a significantly reduced number of plaques, implying that the culture solution of Kimchi lactic acid bacteria has very potent antiviral activity against influenza viruses.

[51] [52] -Animal Test of Culture Solution of Kimchi Lactic Acid Bacteria- [53] [54] 1,100 laying hens experiencing significantly decreased egg production due to the concurrent occurrence of avian influenza and Newcastle disease were randomly divided into 10 groups comprising 110 chickens per group. The culture solution of Kimchi lactic acid bacteria prepared above was 10-fold diluted in the hens' drinking water, and observation was made of the changes in egg production over 17 days. The results are given in Table 3, below.

[55] In Table 3, Sample 1 was prepared on the basis of the culture solution of Leuconostoc kimchii (KCTC2386), Sample 2 on the basis of the culture solution of Leuconostoc mesenteroides subsp. Mesenteroides (KCTC3530), Sample 3 on the basis of the culture solution of Leuconostoc citreum (KCTC3524), Sample 4 on the basis of the culture solution of Leuconostoc lactis (KCTC3528), Sample 5 on the basis of the culture solution of Lactobacillus plantarum (KCCMl 1322), Sample 6 on the basis of the culture solution of Weissella koreensis (KCTC3746), Sample 7 on the basis of a mixture of the culture solution of Leuconostoc mesenteroides subsp. Mesenteroides (KCTC3530) and the culture solution of Lactobacillus plantarum (KCCMl 1322), Sample 8 on the basis of a mixture of the culture solution of Leuconostoc kimchii (KCTC2386) and the culture solution of Weissella koreensis (KCTC3746), Sample 9 on the basis of a mixture of the culture solution of Weissella koreensis (KCTC3746) and the culture solution of Lactobacillus plantarum (KCCMl 1322).

[56] [57] Table 3

[58] As seen in Table 3, laying hens that decreased in egg production, had diarrhea, and showed abnormal respiratory symptoms as a result of infection with the viruses recovered to a normal state 10 days after the water containing the culture solution according to the present invention was fed thereto, as identified by the recovery of egg production to the level of normal hens. Therefore, the culture solution of Kimchi lactic acid bacteria according to the present invention has excellent antiviral activity in vivo, too.

[59] In addition, since the natural antiviral agent of the present invention is prepared from a culture solution of kimchi lactic acid bacteria that flourishes during the fer¬ mentation of the Korean traditional food kimchi, there is no doubt about the safety of the natural antiviral product to the body, even if additional safety tests are not conducted.

[60] As in the drinking water for laying hens, the antiviral activity of the culture solution of the present invention can be embodied in food, fodder, beverages, cosmetics, and medicines, even though no concrete tests were conducted.

[61] When the culture solution of the present invention is applied to cosmetics, beverages, food, fodder, medicines, etc., those skilled in the art will appreciate that various modifications, additions and substitutions in the culture solution are possible according to the properties of the products and the embodiments of preparation, dis¬ tribution, storage and use of the products, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Industrial Applicability

[62] Taken together, the data obtained in the Examples demonstrate that culture solutio ns of kimichi lactic acid bacteria have excellent antiviral activity against a wide spectrum of microbes and can endow food, fodder, beverages, medicine and cosmetics with antiviral activity safely and efficiently.

Claims

[1] A natural antiviral agent, prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from kimchi during the fer¬ mentation of Kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof.

[2] The natural antiviral agent as set forth in claim 1, wherein the lactic acid bacteria is selected from among Leuconostoc spp., Lactobacillus spp., Weissella spp., and combinations thereof.

[3] The natural antiviral agent as set forth in claim 2, wherein Leuconostoc spp. includes Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. Mesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc gellidum, Leuconostoc kimchii, Leuconostoc inhae, Leuconostoc gasicomitatum and com¬ binations thereof.

[4] The natural antiviral agent as set forth in claim 2, wherein Lactobacillus spp. include Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus kimchii, Lactobacillus para- plantarum, Lactobacillus curvatus subsp. curvatus, Lactobacillus sakei subsp. sakei and combinations thereof.

[5] The natural antiviral as set forth in claim 2, wherein Weissella spp. include

Weissella koreensi, Weissella hanii, Weissella kimchii, Weissella soli, Weissella confusa and combinations thereof.

[6] A composition, comprising a natural antiviral agent prepared from one selected from a group consisting of a culture solution of the lactic acid bacteria isolated from kimchi during the fermentation of kimchi, a concentrate of the culture solution, a dehydrate of the culture solution, and combinations thereof, as an active ingredient.

[7] The composition as set forth in claim 6, wherein the lactic acid bacteria is selected from among Leuconostoc spp., Lactobacillus spp., Weissella spp., and combinations thereof.

[8] The composition as set forth in claim 7, wherein Leuconostoc spp. include

Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc mesenteroides subsp. Mesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc gellidum, Leuconostoc kimchii, Leuconostoc inhae, Leuconostoc gasicomitatum and combinations thereof.

[9] The composition as set forth in claim 7, wherein the Lactobacillus spp. include Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lac¬ tobacillus plantarum, Lactobacillus kimchii, Lactobacillus paraplantarum, Lac¬ tobacillus curvatus subsp. curvatus, Lactobacillus sakei subsp. sakei and com¬ binations thereof.

[10] The composition as set forth in claim 7, wherein the Weissella spp. include

[11] The composition as set forth in any one of claims 6 to 10, being selected from a group consisting of beverages, fodder, food, medicine and cosmetics.