KR20070042895A - Composition comprising an extract of pine needle for preventing and treating human disease caused by viruses - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of human diseases caused by viruses containing pine needle extract, pine needle extract according to the invention is influenza A viruses, Newcastle disease virus, chicken infectious Bronchitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus, coxsakie virus, reticuloendotheliosis virus, Ozeski virus In vitro experiments with infected cell lines of aujeszky's disease virus and adeno virus and in vivo experiments with the infected animals resulted in inhibition of virus growth and antiviral effects by killer virus. As it is excellent, the body of the virus The pharmaceutical compositions useful in the prevention and treatment of human disease caused by salt and health can be used for food.

Viruses, pine needles, pharmaceutical compositions, dietary supplements.

Description

Composition comprising an extract of Pine needle for preventing and treating human disease caused by viruses}

1 is a diagram showing the results of the plaque reduction analysis of influenza virus of pine needle 60% ethanol extract,

2 is a diagram showing the intracellular antiviral efficacy of PRRSU of pine needle 60% ethanol extract,

Figure 3 is a view showing the plaque reduction results of pine needle soluble extract non-solvent.

The present invention relates to a composition for the treatment and prevention of human diseases caused by viruses containing pine needle extract.

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and its serotypes are classified into three types, type A, type B and type C. Among them, type B and C have been identified only in humans, and type A has been identified in humans, horses, pigs, other mammals, and various types of poultry and wild birds (Selmons et al ., Avian Dis. , 18 (1), pp119-124, 1974; Turek R et al ., Acta Virol. , 27 (6), pp523-527, 1983; Webster RG et al., Microbiol Rev. , 56 (1), pp152- 179, 1992).

The serotypes of influenza A viruses are classified according to the two types of proteins on the surface of the virus: hemagglutinin (HA) and neuraminidase (NA), and 144 types (HA proteins) 16 species and 9 NA proteins). HA acts as a virus to attach to somatic cells, and NA allows the virus to penetrate into cells. (Alexander DJ, Vet. Microbiol. , 74 (1-2), pp3-13, 2000).

The normal natural host of influenza A virus is known as wild waterfowl such as ducks and seagulls.As a result of epidemiological investigations of influenza infection in wild birds around the world, there are 16 existing HA and 9 NA influenza species. It has been confirmed that viruses are infected in wild birds (Selmons et al ., Avian Dis. , 18 (1), pp119-124, 1974). Avian influenza virus classified as type A is a common infectious disease virus, a non-pathogenic avian influenza that causes mild respiratory symptoms when infected with chickens, and low pathogenicity that causes mortality and spawning degradation of about 1-30%. Low pathogenic avian influenza (LPAI) and high pathogenic avian influenza (HPAI) with over 95% high mortality and also called "bird flu". (Alexander DJ, Vet. Microbiol. , 74 (1-2), pp3-13, 2000). The highly pathogenic avian influenza is classified as Class A by the International Water Office (OIE) and domestically infected with animal species I.

High pathogenic avian influenza (HPAI) has been reported in the United States since 1980 (1983), Australia (1985, 1992, 1994, 1997), Mexico (1994), Pakistan (1994, 2004), Hong Kong (1997, 2001), It has been confirmed worldwide, in Italy (1997, 1999), the Netherlands (2003), Belgium (2003), Germany (2003) and Canada (2004). In particular, in December 2003, serotype A / H5N1 highly pathogenic avian influenza occurred in Southeast Asia and all of Far East Asia including Vietnam, Japan, Thailand, Cambodia, Laos, Laos, Indonesia and China in 2004. . In particular, highly pathogenic avian influenza in Vietnam and Thailand, unlike the traditional high-pathogenic avian influenza, which did not infect humans in Korea and Japan at that time, is a mutant avian influenza that can infect a human body when it comes into contact with infected birds (mutant A / H5N1). HPAI) virus has infected 22 infected birds in Vietnam and killed 15 of them by March 2004. In Thailand, neighboring Thailand, 11 infected infected birds, 8 of them died and the world is concerned. I'm raising my voice. In recent years, the incidence of highly pathogenic avian influenza has increased about 10 times compared to the past, and especially since 2001, it has been taking place worldwide every year. This is required (Song CS et al ., Korean J. Poult. Sci. , 31 (2), pp129-136, 2004).

The first record of human influenza dates back to 412 B.C.E., but the first pandemic influenza record of mankind is estimated to have occurred throughout Europe from 1173 to 1174 (Grmek MD, Les Maladies a L '). aube de la Civilization Accidentale, Payot, Paris, 1893; Hirsch A, Handbook of Geographical and Histological Pathology, New Syndenham Society , London, 1883). Since the 20th century, records of influenza have been more scientifically treated, based on the data that have been left behind, since the 20th century there have been three pandemic influenza. Since the twentieth century, a worldwide pandemic between 1918 and 1920, the first influenza pandemic (aka Spanish flu) has been documented as the greatest damage to humanity, with between 20 and 50 million people dying during that period ( Walter JH, Bull.NY Acad.Med . , 54, pp 855-864, 1978). The causative agent of the Spanish flu virus has been identified as serotype A / H1N1, which is very similar to the swine influenza virus and is still the major epidemic of pandemic flu, which is still prevalent worldwide every year (Taubenberger JK et al ., Science , 275, pp1793-1796, 1997). Since the 20th century, which occurred between 1957 and 1958, the second influenza pandemic began in China and rapidly spread along the coast to nearby Hong Kong, Singapore, Japan and Taiwan in six to seven months. About 40-50% of the world's population was infected during this period, of which 25% had clinical symptoms, mainly infants and middle-aged people who died of infection, and about 1 million people died. (Potter CW, J. appl. Microbiol. , 91, pp572-579, 2001). The causative virus was identified as serotype A / H2N2 influenza virus. In addition, the third pandemic of the influenza pandemic between 1968 and 1969 was confirmed to originate in Hong Kong and spread rapidly to Taiwan, the Philippines, Singapore and Vietnam. The causative virus is serotype A / H3N2, which differs from the previous serotype H2N2 virus and HA, which has been analyzed to be transmitted from birds, and is still the main epidemic of pandemic flu, which is still prevalent worldwide every year. (Oxford JS, Rev. Med. Virol. , 10 (2), pp 119-133, 2000).

Some serotypes of AIV, a major concern in birds, are infected by humans, causing flu and causing death. Several strains of avian-induced avian influenza viruses, including serotype A / H5N1, which has been called "Hong Kong bird flu" to date, are serotype A / H7N7, and serotype A / H9N2. It is estimated that there is a possibility. Therefore, the present study on the mutant viruses derived from these three serotypes is being conducted worldwide (Suarez DL et al ., J. Virol. , 72 (8), pp6678-6688, 1998).

The case of human infection that overcomes the barriers between avian influenza virus (serum-type A / H5N1 avian influenza virus) recently reported in Vietnam is the prelude to the pandemic of the fourth human influenza pandemic since the 20th century. Attention has been paid to the spread of the mutant virus, including direct transmission from person to person. The mutant virus is an avian influenza virus belonging to serotype A / H5N1, and this virus is also the avian influenza virus that has been prevalent only in birds that humans experience for the first time.

Influenza viruses can infect the respiratory tract, cause systemic symptoms, change their appearance periodically, and move to other hosts without killing them before they die. Although it is the virus that causes the greatest economic loss to human beings and preventive vaccines have been developed, they have not caught up with the mutation of the virus, and there is no fundamental virus treatment yet.

The live virus vaccine among the avian influenza vaccines is almost impossible to develop due to the characteristics of the virus that is easily mutated. The vaccines developed to date can be largely classified into a deadly vaccine and a recombinant vaccine. In Italy and Hong Kong in 1999, the high-pathogenic avian influenza outbreak was prolonged and spread throughout the country, and avian influenza vaccine was used as a means of combating high-pathogenic avian influenza in combination with a selective killing policy. It has been positively evaluated that the use of is effective in controlling high pathogenic avian influenza (Capua I et al ., Avian Pathol. , 32, pp47-55, 2003; Capua I and Marangon S, Avian Pathol. , 32, pp335- 343, 2003; Swayne DE et al ., Avian Pathol. , 28, pp 245-255, 1999). Vaccines positively evaluated in Italy (serum type A / H7N1) and Hong Kong (serum type A / H5N1) are all serotoxins and are heterologous serotypes with the same HA type but different NA type (serum type A / H7N3, This is the case of serotype A / H5N2), which attempts to distinguish it from outdoor infection during antibody testing. However, this deadly vaccine cannot be distinguished from vaccine and field infection by the Agar Gel Precipitation (AGP) test, which is the standard A type of avian influenza standard diagnosis. It is pointed out that it is not suitable for inspection. In addition, the now-developed dead venom vaccine can reduce the amount of virus released into feces during high-pathogenic avian influenza infection, but it does not completely prevent the spread of the disease (Capua I et al ., Avian Pathol. , 32, pp 47-55, 2003; Capua I and Marangon S, Avian Pathol. , 32, pp335-343, 2003; Swayne DE et al ., Avian Pathol. , 28, pp245-255, 1999).

In Mexico, when the serotype A / H5N2 low-pathogenic avian influenza developed in late 1993, the virus spread throughout the country became highly pathogenic and abolished pathogenicity. The vaccine is produced and used, and it has been converted to advanced genetic vaccination to distinguish between the vaccination system and the field infection system. The vaccine used so far in Mexico is a cutting-edge technology that effectively prevents chickenpox and highly pathogenic avian influenza at the time of vaccination by inserting the hemagglutin gene, a surface protein of the highly pathogenic avian influenza virus, into fowl pox virus. In the countries where the recombinant viral vector vaccine or chicken pox vaccine is used, including Korea, on the other hand, the effect of viral vector vaccine is halved due to the interference of antibodies induced by the existing poultry vaccine vaccination. have. A recombinant viral vector vaccine has been developed by inserting the hemagglutin gene, a surface protein of the highly pathogenic avian influenza virus, into an infectious laryngotracheitis virus (ILT), but this also includes infectious laryngotracheitis, including Korea. In countries where vaccines are used, the effect is halved, and the use of the vaccine is limited to chickens (Beard CW et al ., Avian Dis., 35, pp356-359, 1991; Webster RG et al . , Avian Dis., 40, pp461-465, 1996; Swayne DE et al ., Avian Dis., 41, pp910-922, 1997; Swayne DE et al ., Avian Dis., 44, pp132-137, 2000).

Amantadine and rimantadine are substances that inhibit the function of M2 ion channel proteins of influenza viruses and are representative antiviral agents that inhibit the growth of influenza viruses in vivo. However, these two antiviral agents were found to be effective only against serotype A influenza virus and not against serotype B influenza virus without M2 protein. In addition, amantadine and rimantadine have been found to have the disadvantage that the emergence of a mutant virus that does not affect the ion channel function of influenza virus M2 protein when used. Zanamivir and oseltamivir, which have been developed to compensate for these disadvantages, are substances that inhibit the function of the neuraminidase protein of influenza viruses and are representative of the suppression of influenza virus proliferation in vivo. Antiviral agents. These two antiviral agents are known to be effective against all 16 serotype A influenza viruses and serotype B influenza viruses. However, zanamivir has the disadvantage of inhalation and intravenous administration, while oseltamivir can be administered orally, but it has been pointed out as a disadvantage due to side effects such as recent reports of resistant virus and vomiting and dizziness upon oral administration (Ward P et. al ., J. antimicrob. Chemother. , 55 (supp1), ppi5-i21, 2005).

Newcastle disease virus is a single-stranded RNA virus belonging to the genus Avulavirus genus of the family Pararamyxoviridae. Newcastle disease viruses have an envelope, which contains the Haemaglutinin-Neuraminidase (HN) protein, which allows the virus to bind to the host cell, and the F (Fusion) protein, which causes the envelope to fuse with the host cell. F and HN proteins are glycoproteins that are distributed on the surface of the envelope (Alexander DJ, Disease of poultry, 10 th edn , pp 541-569, 1997).

Newcastle disease is a lethal, highly contagious livestock epidemic in poultry, with a 100% mortality rate when infected with unvaccinated chickens.If not properly vaccinated, respiratory and digestive symptoms and laying rates in laying hens It is a deadly disease that causes economic damage from degradation. In Korea, the issuer notice is issued every year, but the occurrence continues to increase and occurs nationwide, causing serious damage to poultry farmers. The main symptoms of Newcastle disease begin to slumber at first, which causes respiratory symptoms such as runny nose and cough, and dies of green diarrhea. In addition, if the vaccine is not properly administered, laying hens or breeders with low vaccine antibody titers may have lowered or stopped egg production, and even if they are vaccinated. (Song CS et al., Korean J. Vet. Res., 40 (3) , pp563-573, 2000).

Korea has been studying the Newcastle virus by Japanese researchers since the 1920s. Since the virus from Southeast Asia spreads to the UK and is first known to the West, a worldwide outbreak has been reported. As a result of comparing and analyzing the genes of Newcastle disease virus isolated in Korea, Europe, Japan, Taiwan and China in 1949, 1982-1984, 1988-1997 and 1995-2002, the Korean isolated strain Were identified as belonging to genotypes III, V, VI, and VII, respectively. These results are reported to mean that the genotypes of the viruses isolated during the past five Korean Newcastle disease epidemics have been replaced with the III, V, VI, and VII types in chronological order. Newcastle Disease Virus The genotype V virus among Korean isolates is associated with European epidemic strains in the 1970s, whereas the genotype VI and VII viruses isolated since 1988 have caused Newcastle disease in Far East Asia such as Japan, Taiwan, and China. Was found to be highly relevant. Recently, genotypes VI and VII from Far East Asia have been reported to be the mainstream of Korean isolates (Lee YJ et al., Avian Pathology, 33 (5) , pp 1-10, 2004).

Newcastle disease vaccines are largely divided into live and killed vaccines. Live vaccines can be classified into mesogenic strain, lentogenic strain, and apathogenic strain depending on the residual virulence of the virus used as a vaccine strain (Alexander DJ, Disease of poultry, 10 th). edn , pp 541-569, 1997). Among the poisoning strains, B1 and La Sota (including Clone) are the most widely used live vaccines for Newcastle disease, not only in Korea but also throughout the world. Although varying in degree, it is generally known to cause a vaccination response that is easily detected after vaccination. In order to minimize vaccination response during recent vaccination, various live organisms using V4 strain, Ulster 2c strain, VG / GA strain, and NDV-6 / 10 strain, such as respiratory nonpathogenic (entrepreneurial) vaccine strains, which are mainly grown in the gastrointestinal mucosa, are used. Toxins have been developed. However, these respiratory non-pathogenic vaccines have the advantage of having almost no vaccination response at the time of vaccination, whereas the vaccination efficacy is somewhat lower than that of attenuated strains such as B1 strain, especially visceral virulent belonging to genotype VII, which is currently popular in Korea. It is estimated that there is an ineffective disadvantage in the defense of Newcastle disease virus. Recently, the problem of the live poison vaccine of Newcastle disease is frequently confirmed mainly in winter broiler farms, especially because of the recent epidemic of Newcastle disease despite 2-3 live vaccines (Song CS et al., Korean J. Vet. Res., 40 (3) , pp563-573, 2000).

Newcastle disease poisoning vaccines are divided into gel vaccines and oil vaccines. Recently, multivalent mixed poisoning oil vaccines, which can prevent three or more diseases such as Newcastle disease, chicken infectious bronchitis and spawning hypoplasia, have been developed and used worldwide. The development of multivalent mixed poisoning oil vaccines has prevented three or more diseases at the same time with a single vaccination, which has the effect of reducing labor and labor costs. However, in case of laying farms, cases of Newcastle disease are increasing due to reduced immunity. In particular, in case of winter laying hen farms, the outbreak of New Cattle disease has been increasing regionally, increasing the economic damage caused by the lowering of spawning. Unlike broiler chickens, despite several inoculations of live and dead vaccines, mortality does not occur. Multivalent mixed serotoxin oil vaccine has been pointed out as a disadvantage that the relative ratio of antigen and oil used in the manufacture is less of Newcastle disease virus antigen compared to the New American disease vaccine, resulting in a decrease in immunity and persistence of the vaccine. Therefore, the EU recently tried to increase the antigen content by quantitatively evaluating the viral antigen content of Newcastle disease vaccine by measuring the antibody level produced by inoculating 1/50 of the vaccine recommended dose into the host animal chicken. Doing. In addition, it is pointed out how effectively the Newcastle disease vaccine strain for the production of killed vaccine can protect against the recent genotype VII Newcastle disease virus strain. However, Newcastle disease deadly vaccines that can completely prevent spawning have not yet been developed worldwide (Darrell R. Kapczynski et al., Vaccine, 23 , pp3424-3433, 2005).

Infectious bronchitis (IB) is an acute infectious disease of chickens caused by the IBV and is a major cause of economic damage to the poultry industry as well as domestically. Productivity is one of the diseases. Chickens infected with IBV generally cause three major conditions. The first is a condition that causes respiratory symptoms such as runny nose or cough due to infection with young chicks. Sometimes it causes secondary respiratory bacterial infections and causes mortality with high morbidity. The second is a condition that damages egg laying organs, and lowers egg production and egg quality. The third is a condition that causes severe damage to the kidneys, causing many deaths (King DJ et al., Disease of poultry, 9 th edn , pp 471-484, 1991).

IBV is a virus belonging to the coronavirus and Coronaviridae coronavirus, and it has been experimentally confirmed that mutations occur due to recombination between different IBVs, and there is a possibility that new serotype IBVs will continue to emerge. It is estimated that there will be dozens of species including each of these serotypes, including mutated mutant viruses. In addition, these serotypes do not have a good cross-reaction or cross-immunity, and thus, prevention of variant IBV presents many difficulties (Lambrechts C. et al., Avian Pathology, 22 , pp 577-599, 1993).

The IBV serotypes, which have been seen worldwide, to date, are Massachusetts-type and 793B or 4/91 variant or region-specific viruses. In the United States, Massachusetts-type IBVs became popular since 1950, and Connecticut-type IBVs became popular since 1957. Since then, a wide variety of serotypes of IBV, including Arkansas, Florida, Delaware, and Georgia Mutant strains, have become popular. Currently in the United States, based on Massachusetts-type vaccines that show a wide range of defense against various serotypes, Connecticut and Arkansas-type vaccines that are popular in each region of the United States are being developed and used in parallel. In the UK, eight serotypes of IBV, including UK6 / 82, have been isolated and reported. Before Massachusetts-type IBV vaccines were used in 1991 before the 793 / B type IBV was identified, it is now 793 / B. Type IBV vaccines are being used in parallel. In Australia, a new type of IBV is mainstream that is completely different from the IBV that is popular in the United States and Western European countries. That is, IBV, which is named serotypes A (Vac1), B (Vic S), C, D, E, F, G, H, I, J, K, L, M, O, etc., is in epidemic, and double serum Two vaccines, Vac1 and Vic S, derived from types A and B are used. The Australian-based IBV classifies epidemic IBV in Australia into two genetic groups, Group I (kidney type) and Group II (respiratory type), based on the S1 genetic analysis of the virus. In Korea, the first IB report was confirmed in 1986, and live and dead venom oil vaccines made with Massachusetts type IBV were used to prevent damage. At that time, the damage caused by IBV infection was mainly caused by respiratory symptoms and spawning damage accompanied by malformed eggs. Massachusetts type IB vaccination effectively prevented the damage caused by outdoor IBV infection. However, despite the use of Massachusetts-type IBV vaccines nationwide since 1990, various cases of low laying losses have been reported in laying hens and broilers, and in broiler chicks, about 10% mortality and severe nephritis have been reported in broilers. The accompanying variant IBV has occurred and continues to cause damage. IBV, which is currently in vogue in Korea, can be classified into respiratory type IBV belonging to Korean 1 group and renal type IBV (genotype III) belonging to Korean 2 group (Rhee YO et al., Korean J. Vet. Res., 26 , pp 277-282, 1986; Song CS et al., Avian Pathology, 27 , pp 409-416, 1998; Song CS et al., Korean J. Poult. Sci ., 27 (2) , pp91-98, 2000).

It is estimated that there are more than a dozen serotypes of IBV that are popular all over the world, and cross-protection between serotypes is often not recognized. However, each time a new serotype is found, a vaccine based on the same serotype is developed. It is impossible to do. Among the IBVs that are prevalent in the open air, it is reported that one serotype does not complete the IBV of several other serotypes but may cause significant cross-protection effects. It is mainly used for the development of IB vaccines.

Currently, IB live vaccines that are used all over the world are Massachusetts type (H120, Ma5), which cannot completely prevent the renal IB infection, which is a kind of mutant, but it is analyzed that it is effective in repeated spray vaccination. . Recently, however, the number of damages caused by mutant kidney IB infection has increased from Korea and Asia to Europe. In particular, the damage of respiratory and peritonitis caused by mutant kidney IB infection has been enormously economical in the broiler and laying industry. It is causing problems and is a problem. Existing Massachusetts type IB live vaccines, which are known to have a wide range of immunogenicity to relatively diverse types of IBV, have been pointed out as disadvantages due to problems of respiratory and peritonitis as well as decreased spawning in mutant kidney IBV infection.

Coronavirus was a virus that caused respiratory symptoms when infected with humans, but it was not noticed as a human infectious disease because the symptoms were minimal. However, Severe acute respiratory disease (SARS) in southern China at the end of 2002 has spread worldwide, resulting in many lives and economic losses. On February 11, 2003, the World Health Organization (WHO) Infectious Disease Surveillance and Response (CSR) said, "300 people were diagnosed with SARS, an unknown cause in the province of Guangdong, China. Patients and five deaths have been found and Chinese health authorities are conducting probate and epidemiological investigations.

Following the outbreak in Guangdong Province, China, 20 hospital staff showed similar symptoms while a first-time patient visited Shanghai, Hong Kong, China, and was hospitalized for acute respiratory syndrome. In a public hospital in Hong Kong, 23 out of 50 hospital staff showed acute respiratory symptoms and 8 patients were hospitalized with pneumonia. The SARS corona virus has not been confirmed in humans until now, but the bats in the caves in Hong Kong are attracting attention because of SARS coronavirus and a hereditary virus. Vaccines that can prevent SARS coronavirus are under development and not yet commercialized, and effective treatments are also under development or not commercialized, and the nature of the virus may lead to the emergence of mutant strains resistant to existing antiviral agents. (Henry LY et al., TRENDS in molecular medicine, 9 (8) , pp323-325, 2003; Kathryn VH et al., J. Clin . Invest, 111 , pp 1605-1609, 2003).

Pneumovirus (hereinafter referred to as 'APV') is a single-string RNA virus belonging to the paramyxovirus and pneumovirus. APV infection has been known as ostrich rhinotracheitis (TRT) in Europe in connection with catarrhal inflammation of the upper respiratory tract and foam conjunctivitis and sinusitis. The upper respiratory tract also causes ciliary loss and loss of motility, leading to Swollen head syndrome (SHS) in chickens that show coughing, sneezing or swelling of the head (Cook JKA, The Veterinary Journal, 160 , pp118-125, 2000).

Pneumovirus types A and B are mainly a problem in chickens, and type C causes respiratory diseases mainly in turkeys. Clinical symptoms in chickens are aggravated by secondary infections of bacteria such as Escherichia coli, Mycoplasma and Bodetella, and play an important role in the proliferation of chicken infectious bronchitis in the upper respiratory tract. Along with respiratory diseases, the reproductive organs are affected, resulting in malformations and spawning. Even without clinical symptoms, antibodies can be detected as a result of serological tests, which may be a potential cause of the onset, and are easily affected by airborne transmission (Cook JKA et al., Avian Pathology, 31 , pp 117-132). , 2002).

In recent years, the common infectious disease has emerged as a social problem. Recently, a new virus belonging to the metapneumovirus in the Netherlands was isolated from 28 children and received great social attention. This new virus is called human pneumovirus and is reported to be similar to avian pneumovirus type C. Therefore, the prevailing opinion is that an assessment should be made of the possibility that human viruses may be infected with birds or vice versa (Njenga MK et al., Virus Research, 91 , pp163-169, 2003).

To identify pathogenic mechanisms for respiratory infections, existing respiratory affinity viruses and bacteria such as Newcastle disease, Infectious laryngotracheitis, Fowl pox, Mycoplasmosis and Pasteur Studies on the pathogenesis and differential diagnosis of mixed infections for Pastellallosis and Hemophilus paragallinarum have been conducted.

As a vaccine for disease control, deadly oil vaccine and lively poison vaccine have been developed and various clinical evaluations have been made. Although pneumovirus vaccines have been shown to be effective in preventing productivity degradation, such as clinical symptoms and lowered egg production in turkeys and chickens, questions still remain about the cross-protective ability of pneumovirus serotype vaccines, especially vaccination prevents pneumovirus infection itself. It is not possible to be pointed out as a disadvantage. Vaccines and therapeutics for human pneumoviruses are diseases that have yet to be developed and commercialized, but require urgent countermeasures.

Porcine reproductive and respiratory syndrome (PPRS) is caused by the pig reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-stranded virus of the arteriviriridae. About 10 to 15 years ago, two other PRRSV strains appeared distinctly and independently in the United States and Europe, and the disease now causes reduced fertility and respiratory disease in pigs in many swine producing countries in North America, Europe and Asia. The prevalence of infection in the United States is estimated at 70% (Snijder EJ and Meulenberg JJ, J. Gen. Virol. , 79, pp961-979, 1998). PRRSV is infected by breathing, achievement, bite wounds or needles, and releases the virus in vitro through body fluids, blood, excreta, urine and semen from macrophages in mucous membranes, lungs or local areas. Clinical symptoms in sows are associated with preterm delivery of aborted or weakly born pigs, stillbirth pigs, and autolyzed fetuses. PRRSV occurs worldwide and causes huge economic losses in the livestock industry, but it is urgently needed to take measures due to the lack of appropriate measures (Hampl H et al., J. Virol . , 52 , pp583-590, 1984 ).

PRRS is known to occur in most swine-producing countries, and in 1989, when the disease first developed, the loss was most pronounced during reproduction and delivery, but it was commercialized to induce swine respiratory complex infections or post-weaning neoplastic syndrome. It is also very important as a factor. PRRSV makes it difficult to prevent damage caused by subclinical infections, persistent virus cycles in swine herds, continuous influx of susceptible breeding pigs, and the emergence of mutant strains (Goyal SM et al., J. Vet. Diagn . Invest., 5 , pp 656-664, 1993).

In Korea, PRRSV was isolated in 1993, but serological results indicate that it was introduced into Korea by the rapid growth of pig industry in the mid-1980s and by PRRS-infected sows imported from the United States (Kang JY et al., Korean J). Vet.Res ., 39 (2) , pp 294-300, 1999). At that time, due to the lack of understanding of PRRS, it was estimated that due to the spread of infected breeding pigs, disease spread easily throughout the country. PRRS is persistently infected during viral infection, and it is reported that it is very difficult to stably maintain the population immunity of pigs because the virus can coexist with genetically serological traits in the same farm and spread vertically and horizontally. . In addition, the vaccine currently inoculated does not sustain long-term protective immunity, and does not reduce virus discharge through semen or manure, and thus, defense against diseases dependent on vaccines is not assured (Kwon CH). et al., Korean J. Vet. Res., 34 , pp77-83, 1994).

Among the RNA viruses are relatively small viruses. They combine the word 'pico' which means 'small' with RNA and call it piconavirus, and the viruses belonging to it are collectively called the piconaviridae. Enteroviruses belonging to the family of piconaviruses contain about 70 serotypes that cause various clinical symptoms such as aseptic meningitis, hand and foot disease, herpetic stenosis, dilated myocarditis, and acute febrile conjunctivitis. , Cossackievirus and echoviruses and other enteroviruses. Enteroviruses have a diameter of about 20 to 30 nanometers of viral particles and have a single strand of RNA as a gene. Most of the infections in the digestive organs of vertebrates, as well as the respiratory tract and the central nervous system, but often do not show a clear symptom. Coxsackievirus (CXV) is a human enterovirus belonging to the family of piconaviruses, and is largely divided into A type and B type (Pallansch MA and Roos RP, Fields Virology, 4 th edi , pp 723-775, 2001).

In addition, recently, high-risk enteroviruses and mutant viruses (entrovirus type 71, coxsackie virus A24 mutant strain) have been newly discovered and spread all over the world, and it is required to establish a joint surveillance system among countries to detect them early. Indeed, in 2002, when enterovirus 71 and aseptic meningitis were prevalent in Korea, coxakivirus A24 mutant strains were prevalent in 2002 and 2003, when there was an explosion of ecovirus 13 and acute hemorrhagic conjunctivitis nationwide. We confirmed and publicized the public.

Enteroviruses, including coxsackieviruses, infect the vertebrate digestive system, respiratory tract and central nervous system and cause various clinical symptoms. Therefore, it is urgently required to prepare measures at the national level, but the types and serotypes of viruses are very diverse. As such, no effective commercialized vaccines or therapeutics have been developed.

Reticuloendotheliosis virus (RE) is an RNA virus belonging to the family of retroviridae (retroviridae), which mainly occurs in turkeys, chickens, ducks, etc., and also in pheasants and quails. RE virus (REV), like avian leukosis / sarcoma virus, has RNA-dependent reverse transcriptase but differs antigenically and morphologically. REV includes strain T isolated from turkey, chicken syncytial virus (CSV) isolated from chicken, spleen necrosis virus (SNV) isolated from duck, and duck infectious anemia virus (duck). infectious anemia virus (DIAV), but neutralization tests are known to show no differences between viruses. Of these, T strains have a tumor gene (v-rel) that can cause acute tumors but cannot replicate on their own. It consists of REV associated helper virus (REV-A) (Witter RL and Fadly AM, Disease of poultry, 10 th edn , pp 517-536, 1997).

Reticuloendothelial disease may also result in running disease syndrome, which indicates adenitis, feather abnormalities, lower body weight gain and immunodeficiency in chickens, and may cause chronic tumors similar to tumors or leukemia tumors. In addition, REV-infected chickens have been reported to have reduced immunity and significantly reduce resistance to infectious diseases such as infectious laryngotracheitis, chicken pox, coccidiosis, and Salmonella, and reduce vaccine immunity such as Marek's disease and Newcastle disease. The spread of reticuloendothelial disease in chickens is easily caused by horizontal infection from the infectious system to the cohabitation system, but in this case, the damage caused by the disease is not usually observed, and the damage caused by the disease is vertically transmitted from the infected mother to the later chick. Appears only in early infections (Theilen GH et al., Journal of the National Cancer Institute, 37 , pp731-743, 1966).

REV is composed of single-stranded RNA, but with RNA-dependent DNA polymerase, it can be double-stranded DNA during proliferation. Such double-stranded DNA can be inserted into the chromosomal DNA of a cell and exist as a provirus (Fritsch E). et al., Virology, 83 , pp 313-321, 1977). Due to the nature of the REV infection in the host genome, the live venom vaccine for retinopathy cannot be developed and used. The dead venom vaccine also has a glycoprotein 85 (glycoprotein 85: gp85) that produces neutralizing antibodies to viruses. It is very unstable to manufacture a vaccine.

Ojeskiky's disease virus (ADV) A DNA virus belonging to the herpesviridae's alpha herpesvirus subfamily and is a natural host of pigs in cattle, dogs, cats, rabbits, mink, mice, rats, guinea pigs and birds. It is a virus with a wide range of host regions (Thowley DG et al., J. AM. Vet. Med . Assoc ., 176 , pp1001-1003, 1980). Ozeski's disease is also called psedorabies, and ADV's swine disease does not show the characteristic itch seen in other diseases, severe respiratory symptoms and low growth in piglets, reproductive disorders such as similar acid in pregnant pigs, and high mortality in piglets. It causes a lot of economic damage to the pig industry (Lee JB et al., Korean J. Vet. Res., 28 (1) , pp99-103, 1988).

Vaccination is performed to reduce the economic loss caused by Ozeski's disease, but the clinical symptoms do not appear during infection, but the infection itself cannot be prevented and latent infection is established, which is not effective for eradication of ADV. Therefore, in order to distinguish between outdoor ADV infected pigs and vaccinated pigs, an antibody test kit has been developed and used to differentially diagnose outdoor ADV infection. It remains homework.

Adenovirus is a DNA virus belonging to the family Adenoviridae, which causes a variety of respiratory infections and gastroenteritis in children and immunocompromised patients, epidemic keratoconjunctivitis, hemorrhagic cystitis and meningitis. (Ruuskanen O et al., Clinical Virology, pp525-548, 1997). Adenovirus infection is transmitted by the secretion of infected people by droplets, contact with contaminated objects, etc., and can result in death from adenovirus infections, as well as deaths from patients with infections or immune function caused by certain serotypes. Known.

Respiratory diseases caused by adenoviruses include acute pharyngitis, acute respiratory disease (ARD), and pneumonia, and 4-18% of adenoviruses are in the form of pneumonia. It is known to represent various infections such as upper respiratory tract infection, pharyngitis and laryngitis. Lung damage from respiratory infections of adenoviruses is often a very serious problem, with 60-65% of patients with pneumonia and 10% of patients with upper respiratory infections including a variety of complications including obstructive bronchitis, bronchiectasis and McLoed syndrome. It is known to cause lung injury (Gold R et al., J. Can. Assoc . Radiol ., 20 , pp218-224, 1969).

The incidence of adenovirus infections is high in late winter, spring and early summer, but occurs sporadically throughout the year (Ahn KM et al., Korean Med . Sci ., 14 , pp405-411, 1999). Although it is known to occur mainly in the elderly and patients with reduced immune function, the incidence of cases may be epidemic in group facilities such as schools, hospitals, the military, and day care centers (Esteban RE et al., Rev. Clin . Esp ., 196 , pp82 -86, 1996).

In the case of other viruses such as influenza causing respiratory diseases, various effective vaccines and treatments have been developed and used. On the other hand, effective vaccines against adenoviruses have not been developed yet. There is no specific treatment except symptomatic therapy such as timely hydration, stabilization and antipyretic.

There are about 80-90 species of pine in the world of Pinus, but among them, P. palustris Miller (North America), P. pinaster Aiton (France), Pinus Sylvestris ( P. sylvestris L. (European)), Pinus laricid Poiret (Austria), Pinus longifolia Rocvurgh (India), Pinus densiflora sieb ( P) densiflora Sieb. et Zucc .: Korea, Japan), terpenes from P. thunberii Palatore (Haesong, Japan), etc. are mainly used in industry.

The main components of pine needles are Terpentine oil, Cineole, Salinigrin, Coniferin, P-Cymen, Densipimaric acid, Retten (Retene) and chlorophyll, protein, no fat, phosphorus, iron, enzymes, minerals, fat-soluble vitamin A and vitamin C, etc. The main ingredients are somewhat different depending on the species and the season harvested.

Essential oils obtained from the leaves of Pinus densiflora Sieb. Et Zucc. Include alpha-pinene, beta-pinene, camphene, and pelandene ( phellandrene, borneol, bornyllacetate, caryophyllene, cardinene diterpene, sesquiterpene, sesquiterpenalcohol, seryl alcohol ( cerylalcohol, beeswax contains juniperic acid, sabinic acid, hexadecane-diol, triacontan-1-ol, etc. matsusterin), Pitot Ste Lin (phytosterin), when acid (chinic acid), shikimic acid (shikimic acid), quercetin (quercetine), Kam Ferrol there is contained the like (kaempferol) (赤松金芳low, New Year's Chemistry medicine, pp664 -665, 1980).

Among pines growing in Korea, pine needles are used as a medicine for gonorrhea and syphilis in folk medicine (御 影 雅 幸, 日本 生 藥學 雜誌 , p 336, 1991), but there are no known pharmacological effects of pine needles. And it is known to have the effect of quickly restoring the coagulated skin to the bruises of the skin, and to heal eczema, scabies and sweat bands (Park Jong-gap, Korean Medicine , p134, 1984; Culture Broadcasting, Korean Civil Therapy Exhibition, p21, 1988). It is also mentioned in the appendix that it has the effect of preventing the whitening of hair. The herb wood is said to strengthen swelling, hair improvement, intestines and prolong life. There is also a deodorizing effect, insomnia healing effect and skin cosmetic effect, but none of the literature teaches or disclosed that pine needles have an antiviral effect.

Accordingly, the present inventors have conducted research for minimizing the damage caused by the virus infection, overcoming the shortcomings of existing live vaccines, and developing a safe and effective next-generation natural product virucide which can replace them. The present invention was completed by confirming that the pine needle extract of the present invention had an excellent virus growth inhibitory effect and a virucidal effect.

An object of the present invention is to provide a pharmaceutical composition and health functional food useful for the prevention and treatment of human diseases caused by viruses containing pine needle extract having an excellent antiviral effect as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by viruses containing pine needle extract as an active ingredient.

Extracts as defined herein include crude extracts or nonpolar solvent soluble extracts, wherein the crude extracts are polar solvents selected from water, C ₁ to C ₄ lower alcohols or mixed solvents thereof, preferably 50 to 70% water and C ₁ to a mixed solvent of a lower alcohol, and more preferably include the extract soluble in 60% ethanol, a non-polar solvent soluble extract of the C ₄ are in a non-polar solvent selected from dichloromethane, chloroform or ethyl acetate, preferably dichloromethane Contains extracts available for use.

Pine needles as defined herein include the leaves of Pinus densiflora Sieb. Et Zucc., Pinus thunbergii parlatore or Pinus koraiensis Sieb. Et Zucc.

Viruses as defined herein include RNA-type viruses or DNA-type viruses.

RNA-type viruses as defined herein include influenza virus, newcastle disease virus, infectious broncheitis virus, avian pneumovirus or porcine reproductive and respiratory syndrome viruses (porcine reproductive and respiratory syndrome virus or coxsakie virus and reticuloendotheliosis virus.

Influenza viruses as defined herein are characterized as having serotypes of H1N1, H9N2 or H6N5.

DNA-type viruses, as defined herein, include auzeszky's disease virus and adeno virus.

Diseases caused by viruses as defined herein include orthomyxovirus family viruses, including influenza viruses, paramyxovirus family viruses, including the Newcastle disease virus, and infectious broncheitis viruses. Coronavirus famil virus, including the virus, picorna virus family including the coxsakie virus, retrovirus family including the reticuloendotheliosis virus ) Is caused by an infection in the body of the herpesvirus family virus, which includes the azezky's disease virus, or the adenovirus famoly virus, which includes the adeno virus. It features.

Diseases caused by in vivo infection of the influenza virus family viruses as defined herein include flu, cold, bird flu or variant infectious bird flu.

Diseases caused by in vivo infection of the paramyxo virus and paramyxo virus families as defined herein include Parainfluenza, Mumps, Measles, Nipah virus, Hendra virus virus), human Respiratory syncytial virus or human metapneumovirus.

Diseases caused by in vivo infection of the coronavirus family virus as defined herein include human respiratory coronavirus (HCoV-229E), Severe Acute Respiratory Syndrome (SARS) or human respiratory coronavirus (HCoV-). OC43).

Diseases caused by in vivo infection of the picornavirus family of viruses as defined herein may include Encephalomyocarditis virus, Poliovirus, Coxsackievirus, Echovirus, and rhinovirus. (Rhinovirus), Parecovirus or Hepatitis A virus.

Diseases caused by the infection of the retrovirus family viruses as defined herein include human immunodeficiency virus types 1 and 2 or human foamy virus. do.

Diseases caused by the infection of the herpesvirus family virus as defined herein include human herpes simplex virus, epstein-barr virus, cytomegalovirus, vericellazo Varicella-zoster virus, human herpes virus 6, 6A, 6B, type 7 (human herpesvirus 6, 6A, 6B, 7), kaposi's sarcoma or cercopithecine herpesvirus. Include.

Diseases caused by in vivo infection of the adenovirus family viruses as defined herein cause respiratory infections, gastroenteritis, epidemic keratoconjunctivitis, hemorrhagic cystitis, or meningitis in humans. Person adenovirus infectious virus.

The present invention also provides a health functional food for the prevention and improvement of diseases caused by viruses containing pine needle extract and food supplements food additive.

Pharmaceutical composition for the prevention and treatment of diseases caused by viruses containing pine needle extract of the present invention as an active ingredient, 0.1 to 50% by weight of the extract relative to the total weight of the composition.

Hereinafter, the present invention will be described in detail.

Pine needle extract of the present invention can be obtained as follows.

The pine needles of the present invention are collected, rinsed with water, and then dried and finely pulverized to have a volume of about 1 to 30 times the weight of the pine needle sample, preferably about 1 to 15 times C ₁ to C ₄ such as water, ethanol and methanol. Polar alcohols of lower alcohols or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably 30 to 100% of ethanol at about 10 to 100 ° C. for about 1 hour to 1 day, preferably For example, using an extraction method such as hot water extraction, reflux circulation extraction or pressurized extraction, and ultrasonic extraction for 5 to 20 hours, preferably hydrothermal extraction or reflux circulation extraction under reduced pressure filtration, centrifugation and polar solvent of lower alcohol. Alternatively, the supernatant extracted with a mixed solvent may be placed in a dry heat sterilizer and dried at 50-150 ° C., preferably 60-130 ° C., and then applied to distilled water and then lyophilized to obtain pine needle extract.

In addition, the pine needles are collected, washed with water, then dried and finely pulverized to a volume of about 1 to 30 times the weight of the pine needle sample, preferably about 1 to 15 times the volume of C ₁ to C ₄ such as ethanol and methanol. Polar solvents of lower alcohols or mixed solvents having a mixing ratio of about 1: 0.1 to 1:10, preferably 30 to 100% of ethanol at about 10 to 100 ° C. for about 1 hour to 1 day, preferably For 5 to 20 hours using an extraction method such as hot water extraction, reflux circulation extraction or pressurized extraction, ultrasonic extraction, preferably hot water extraction or reflux circulation extraction under reduced pressure filtration, centrifugation and the supernatant taken under reduced pressure The pine needle extract obtained by evaporation to dryness was suspended in water, and then extracted with a solvent in the order of dichloromethane, ethyl acetate, n -butanol, and the pine needle non-polar solvent soluble extract of the present invention, Preferably, ethyl acetate soluble fraction can be obtained, more specifically, dichloromethane fraction by mixing the same amount of dichloromethane: water: methanol in a ratio of 60: 9: 1 in pine needle 60% ethanol extract and A water soluble fraction can be obtained, and again, the same amount of ethyl acetate can be added to the water soluble fraction to obtain an ethyl acetate soluble fraction and a water soluble fraction, and finally the water soluble fraction is extracted with the same amount of butanol soluble Fractions and water soluble fractions can be obtained.

The present invention provides a pharmaceutical composition for the prevention and treatment of diseases caused by viruses containing pine needle extract obtained by the above manufacturing process as an active ingredient.

The pine needle extract according to the present invention showed a constant antiviral effect irrespective of the type of pine needle, antiviral effect according to the extraction solvent is 60% ethanol extract in the polar solvent extract, dichloromethane extract in the non-polar solvent soluble extract The antiviral effect of was excellent.

In addition, the pine needle polar solvent soluble extract according to the present invention is influenza A viruses, newcastle disease virus, chicken infectious bronchitis virus, avian pneumovirus, avian pneumovirus, pig reproduction And MDCK cells infected with porcine reproductive and respiratory syndrome virus, coxsakie virus, reticuloendotheliosis virus, ausjeszky's disease virus and adeno virus, system When treated in vitro antiviral efficacy test using fetal cells, MARK-145 cells, Hep-2 cells and SPF oocytes, the virucidal effect was excellent, and pine needle non-polar solvent soluble extracts were treated with influenza A virus and Newcastle disease virus. Infected MDCK Cells and Fetal Fibroblasts Heomgwan in (in vitro) wherein was a live virus effective excel by treatment with a virus efficacy testing, and were excellent therapeutic effect in accordance with the negative and the gavage administration of a pine needle extract of the present invention against mouse influenza A virus infection, influenza A The SPF chickens infected with the virus and Newcastle disease virus were excellent in the therapeutic effect of administration of the pine needle extract of the present invention, and the treatment effect of the pine needle extract of the present invention was excellent in the ducks infected with influenza A virus. .

Pine needle extract of the present invention has little toxicity and side effects, so can be used with confidence even for long-term administration for the purpose of prevention.

Pharmaceutical compositions comprising pine needle extract of the present invention may further comprise suitable excipients and diluents according to conventional methods.

Carriers, excipients and diluents that may be included in the pharmaceutical composition comprising the pine needle extract of the present invention, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, Alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. .

Pharmaceutical compositions comprising pine needle extract according to the present invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used.

The amount of pine needle extract of the present invention may vary depending on the age, sex, and weight of the patient, but the amount of 0.1 to 100 mg / kg may be administered once to several times a day. In addition, the dosage of the pine needle extract may be increased or decreased depending on the route of administration, degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The present invention also provides a health functional food for the prevention and improvement of diseases caused by viruses containing pine needle extract obtained in the process as an active ingredient.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

The dietary supplement for the prevention and improvement of the disease caused by the virus of the present invention comprises the extract in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.

In addition, for the purpose of preventing and improving diseases caused by viruses, the manufacture and processing of health functional foods in the form of pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or health drinks, etc. It is possible.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for having the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by the following reference examples, examples and experimental examples.

However, the following reference examples, examples and experimental examples are merely to illustrate the invention, the content of the present invention is not limited by the following reference examples, examples and experimental examples.

Reference Example 1. Obtaining Pine Needle Powder

Leaves from (1) red pine ( Pinus denstifora Sieb. Et Zucc.), (2) pine ( Pinus thunbergii parlatore), and (3) Pinus koraiensis Sieb. Et Zucc. After collecting and washing with water, the surface water was removed, and then dried and finely pulverized with a mill (ball mill, mental company, Korea) to dry powder.

Reference Example 2. RNA Virus Preparation

RNA viruses include Influencza A virus, Newcastle disease virus, Infectious broncheitis virus, Avian pneumovirus, Porcine reproductive and respiratory syndrome virus, Coxsakie virus (CXV), and reticuloendotheliosis virus were prepared.

2-1. Influencza A virus

2-1-1. A / PR / 8/34 (H1N1)

The influenza virus A / PR / 8/34 (H1N1), a published virus, was distributed at the University of Hokkaido and used for 48 hours in 10-day-old SPF eggs for in vitro and in vivo experiments and stored at -80 ° C.

2-1-2. A / HS / K5 / 01 (H9N2-1), A / HS / MS96 / 96 (H9N2-2)

A / HS / K5 / 01, a Korean isolate, was isolated from domestic herds with avian influenza symptoms at the College of Veterinary Medicine, Konkuk University, and A / HS / MS96 / 96 were distributed by the National Veterinary Research and Quarantine Service for in vitro and in vivo experiments. 48 days in 10 days old SPF egg breeding was used while storing at -80 ℃.

2-1-3. A / CSM / D2 / 05 (H6N5)

A / CSM / D2 / 05 (H6N5) isolated from migratory birds was isolated from the veterinary college of Konkuk University through migratory fecal samples collected from Cheonsu Bay area, and grown for 48 hours in 10-day-old SPF eggs for in vitro and in vivo experiments. It was used while storing at -80 ℃.

2-2. Newcastle disease virus (NDV)

Domestic isolates Kr-005 / 00 and KJW strain were obtained from the National Veterinary Research and Quarantine Service and used for 48 hours in 10-day-old SPF eggs for in vitro and in vivo experiments and stored at -80 ° C.

2-3. Infectious broncheitis virus (IBV)

M41 strain was obtained from the National Veterinary Research and Quarantine Service, and used for 48 hours in 10-day-old SPF eggs and stored at -80 ℃ for in vitro experiments.

2-4. Pneumonia virus (Avian pneumovirus: APV)

Poulvac® SHS vaccine (Fort Dodge Animal Health, UK) was used.

2-5. Porcine reproductive and respiratory syndrome virus (PRRSV)

The parental strain of Ingelvac® PRRS, a pig reproductive and respiratory syndrome virus live vaccine (ATCC VR-2332), was distributed by the National Veterinary Research and Quarantine Service and used to grow in MARK-145 cells and store at -80 ° C.

2-6. Coxsakie virus (CXV)

Coxsackie virus was distributed by the National Institutes of Health, and was used while proliferating in Hep-2 cells and storing at -80 ° C.

2-7. Reticuloendotheliosis virus (REV)

The reticuloendothelial virus received chicken syncytial virus (CSV) from ATCC, and was used while proliferating in fetal fibroblasts and storing at -80 ° C.

Reference Example 3. DNA Virus Preparation

As the DNA virus, azejeky virus (aujeszky's disease virus), adenovirus (Adeno virus) was prepared.

3-1. Azejezky's disease virus (ADV)

Ozesky virus was distributed by the National Veterinary Research and Quarantine Service, and used to grow in MDCK cells and store at -80 ° C.

3-2. Adeno virus (ADV)

Adenoviruses were distributed by the National Institutes of Health, and were used in proliferating Hep-2 cells and storing at -80 ° C.

Reference Example  4. Cell Culture

4-1. MDCK (Madin Darby Canine Kidney) cells

MDCK cells were purchased from the American Type Culture Collection (Manassas, VA, USA), using Eagle's minimum essential medium (EMEM) medium supplemented with 10% fetal bovine serum (Gibco, USA). Cells were maintained at 37 ° C. and 5% CO ₂ culture conditions. For antiviral efficacy screening, trypsin 5 μg / ml, 0.22% sodium bicarbonate (NaHCO ₃ ) and gentamycin 40 μg / ml were added to MEM medium.

4-2. Hep-2 cells

Hep-2 cells were cultured by the Korea Research Institute of Chemical Technology and cultured at 37 ° C. and 5% CO ₂ using a medium essential medium (MEM) medium supplemented with 5% fetal bovine serum (Gibco, USA). Cells were maintained at the conditions.

4-3. Chicken embryo fibroblast cell

Aseptic internal organs of 10-day-old SPF chicken embryos are aseptically removed and digested with trypsin, M199 medium containing TPB (Tryptose phosphate broth) and 8% calf serum (Gibco, USA) (5% Cells were maintained at 37 ° C. and 5% CO ₂ culture conditions using) + HAM's F10 medium (5%) (Gibco, USA).

4-4. MARK-145 cells

MARK-145 cells were distributed by the National Veterinary Research and Quarantine Service and were subjected to 37 ° C. and 5% CO ₂ using medium essential medium (MEM) medium supplemented with 5% fetal bovine serum (Gibco, USA). Cells were maintained in culture conditions.

Reference Example  5. In vitro  Quantitative suspension test with viruses

The virus disinfection efficacy test was performed in accordance with the guidelines for disinfectant efficacy testing of National Veterinary Research and Quarantine Service Regulation No. 30 (2003.1.27). To test the in vitro virucidal activity of the sample, the sample was diluted 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold, and 32000-fold on the basis of 100-fold dilution with distilled water. 2.5 ml of the virus solution prepared at intervals of 1 minute were added to the test tube containing 2.5 ml of the sample dilution solution at 4 ° C. in the test tube, followed by mixing for 30 minutes at 4 ° C., followed by shaking every 10 minutes. The control group experimented differently using distilled water instead of diluted samples. The diluted solution was mixed with the virus using cell culture medium, and the cells were cultured in a single layer by diluting folds of the stock solution, 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 . It was applied to a 96 well tissue culture plate (greiner, Germany) in 8 well (dilution), inoculated 25 μl of the mixed solution, and then adsorbed for 30 minutes, and the medium was changed. In the case of the influenza virus test, the medium was changed to a medium containing 5 ug / ml trypsin to induce a cytopathic effect (CPE). After inoculation, the cells were incubated for 4 days in a CO ₂ incubator at 37 ° C., and daily cytoplasmic effect was observed under a microscope, and finally, 3-4 days after the inoculation, CPE was read for the presence of viruses. Judging According to the virus used, cells used for inoculation were appropriately replaced. In the case of viruses (H9N2 and IBV) that do not show CPE in the cells, the diluting fold samples and each virus reaction solution were inoculated with 0.2 ml of 10 days old SPF eggs. Three days after the inoculation, the presence of virus was determined by hemagglutination and dot immunoassay (Song CS et al., Avian Diseases, 42 (92) , pp92-100, 1998) . Virus content calculations were calculated using the Karber method (Payment P et al., Methods and Techniques in Virology , p33, 1993).

Reference Example  6. Plaque reduction assay

MDCK cells were placed in a 6-well tissue culture plate at a concentration of 6 X 10 ⁵ cells / well and cultured when a monolayer was formed. The medium was removed and inoculated with 0.1 ml of 400 pfu / ml of virus. The virus was adsorbed in a 37 ° C., 5% CO ₂ incubator for hours. Samples diluted in MEM medium were diluted in the same amount with 1% agarose so as to have concentrations of 1000, 500, 250, 125, 62.5, 31.25 μg / ml, and double-layered onto virus-inoculated cells. After the agarose was hardened for 2-3 days at 37 ° C. and 5% CO ₂ , the number of plaques was measured.

Reference Example  7. Neutral Red  Colorimetric analysis colorimetric  assay)

[Microtiter Neutral Red Assay for Antiviral or Cytotoxicity Activity: Neutral red colorimetric assay]

In order to test the virus growth inhibitory effect of the samples, the samples were each diluted 500 times with distilled water. 2.5 ml of the prepared virus solution was added to a test tube containing 2.5 ml of the sample dilution solution at 4 ° C., followed by reaction at 4 ° C. for exactly 30 minutes, and well shaken every 10 minutes. The control group experimented differently using distilled water instead of diluted samples. The mixed solution of the diluted sample and virus was cultured using the MDCK cell culture medium, and the diluting fold was made into the stock solution, 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , and the MDCK was cultured in a single layer. One 96-well tissue culture plate (greiner, Germany) was applied in 8 wells / dilution multiple times to inoculate 25 μl of the mixed solution, adsorbed for 30 minutes, and the medium was changed. After inoculation, the cells were incubated for 4 days in a CO ₂ incubator at 37 ° C., and each day was observed for cytopathic effect (CPE) under a microscope and a neutral red assay was performed on day 4 of the inoculation. Neutral red assay was performed by adding 0.034% of neutral red to 100ul / well on the plate containing the cells, blocking the light with foil, etc., and reacting for 2 hours at 37 ° C to allow the living cells to absorb the neutral red. The cells are washed twice with PBS. After the cells are completely dried, add 100% ethanol and Sorensen citrate buffer in a solution of equal volume dilution to 200ul / well and absorbance values at the spectrophotometer (SUNRISE, Tecan, Austria) at a wavelength of 540 nm Optical density value: OD value) was measured (Donald FS et al., Antimicrobial Agents and Chemotherapy, 45 (3) , pp743-748, 2001).

Example 1. Preparation of pine needle polar solvent extract

1-1. Preparation of Pine Needle Extract

80 g of each pine needle powder obtained above was suspended in 800 ml of distilled water, and then put in a hot water extractor, extracted at 90 ° C. for 4 hours, filtered, and centrifuged at 10,000 × g for 30 minutes, followed by lyophilization ( ModulyoD, ThermoSavant, USA) and dried to obtain 5g of red pine leaf hot water extract powder, 6 g of sea pine leaf hot water extract powder, 8 g of pine leaf hot water extract powder, and stored in a 4 ℃ refrigerator used for the experiment.

1-2. Preparation of Pine Needle Ethanol Extract

80 g of each pine needle powder obtained above was suspended in 800 ml of 30%, 60% and 100% ethanol, and then filtered by refluxing at room temperature for 18 hours, followed by centrifugation at 10,000 × g for 30 minutes to remove precipitates. The supernatant was taken, the supernatant was taken and evaporated to dryness under reduced pressure (Evaporate, BUCHI, Switzerland) to completely remove ethanol, and 200 ml of distilled water was applied to each sample, followed by drying with a freeze dryer (ModulyoD, ThermoSavant, USA). Leaf Ethanol Extract Powder (30% 9g, 60% 20g, 100% 20g), Seaweed Leaf Ethanol Extract Powder (30% 9g, 60% 21g, 100% 21g), Pine Tree Ethanol Extract Powder (30 % 9 g, 60% 22 g, 100% 22 g) were obtained and used in the experiment while kept in a 4 ° C refrigerator.

1-3. Preparation of Pine Needle Methanol Extract

80 g of each pine needle powder obtained above was suspended in 800 ml of 100% methanol, filtered under reflux for 18 hours at room temperature, filtered, centrifuged at 10,000 x g for 30 minutes to remove precipitates, and the supernatant was taken under reduced pressure. Methanol was completely removed by evaporation (Evaporate, BUCHI, Switzerland), 200 ml of distilled water was applied to each sample, and then dried by lyophilizer (ModulyoD, ThermoSavant, USA) and red pine leaf methanol extract powder (20 g), transported Leaf methanol extract powder (21 g) and pine needle methanol extract powder (22 g) were obtained and used in the experiment while kept in a 4 ° C. refrigerator.

The recovery rate of the pine needle extract prepared in the above Example was the highest recovery rate when extracted with 60% and 100% ethanol, as shown in Table 1 based on the red pine leaf.

menstruum Pine Needle Powder Used (g) Recovered amount after extraction (g) Recovery rate (%) water 80 5 6.25 30% ethanol 80 9 11.25 60% ethanol 80 20 25 100% ethanol 80 20 25 100% methanol 80 20 25

Example  2. Pine needles Nonpolar Solvent  Preparation of Extract

2-1. Preparation of Dichloromethane Soluble Extract

800 g of the red pine leaf powder obtained above was suspended in 8 L of 60% ethanol, filtered under reflux at 42 ° C., filtered, and centrifuged at 10,000 × g for 30 minutes to remove precipitates, and the supernatant was taken up and evaporated to dryness under reduced pressure. 800 ml of dichloromethane (CH ₂ Cl ₂ ) was added to the red pine leaf extract prepared by evaporate or BUCHI (Switzerland) and then suspended in 800 ml of distilled water. After 800 ml, this dichloromethane soluble fraction was evaporated to dryness under reduced pressure to give 4.97 g of a dichloromethane soluble extract.

2-2. Preparation of pine needle ethyl acetate soluble extract

       800 ml of ethyl acetate (EtOAc) was added to 800 ml of the water-soluble fraction layer obtained in Example 2, mixed, and fractionated to obtain 800 ml of the water-soluble fraction layer and 800 ml of the ethyl acetate soluble fraction. Was evaporated to dryness under reduced pressure to give 7.534 g of a dichloromethane soluble extract.

2-3. Preparation of Pine Needle Soluble Extract

       800 ml of butanol (n-BuOH) was added to 800 ml of the water-soluble fraction layer obtained in Example 3, followed by mixing to obtain 800 ml of the water-soluble fraction layer and 800 ml of the butanol-soluble fraction. The water-soluble fraction was evaporated to dryness under reduced pressure to give 17.23 g of butanol soluble extract and 47.1 g of water-soluble extract.

Experimental Example 1. Screening in vitro virucidal efficacy of extracts by different types of pine needle

In order to test the in vitro virucidal efficacy of the extracts of different types of pine needles, the 60% pine needle ethanol extracts prepared in Example 1-2 were each diluted 500-fold with distilled water. 2.5 ml of the prepared influenza H1N1 virus solution was placed in a test tube containing the same amount of the diluted pine needle extract at 4 ° C., followed by reaction at 4 ° C. for exactly 30 minutes, and well shaken every 10 minutes. The control group experimented differently using distilled water instead of diluted pine needle extract. The diluted pine needle extract and the mixed solution of the virus was mixed using the MDCK cell culture medium, and the dilution factor was 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ . In a well-diluted 96 well tissue culture plate (greiner, Germany) incubated with MDCK in 8 wells / dilution drainage, 25 μl of the mixed solution was inoculated, and the medium was changed after 30 minutes of adsorption. After inoculation, the cells were incubated in a CO ₂ incubator at 37 ° C. for 3-4 days, and the cell denaturation effect (CPE, cytophatic effect) was observed daily under a microscope, and finally, the presence of virus was determined by the degree of observed CPE. According to Reference Example 4, the used cells were changed according to the used virus, and in the case of viruses that did not show CPE (H9N2 influenza virus and Infectious broncheitis virus) in the cells, the pine needle extract and each virus reaction solution by dilution factor were 10 days old SPF eggs 0.2 ml of each was inoculated at 3 days after inoculation to determine the presence of virus through hemagglutination and dot immunoassay. Virus content calculations are shown in Table 2 below using the Kärber method (Payment P et al., Methods and Techniques in Virology , p33, 1993).

 division  In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Influenza virus H1N1 (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 2, 500-fold dilution (500 ㎍ / ㎖) a red pine leaves, black pine leaf, a result of the pine leaf extract each of the virus for 2 hours 10 ^5.6 TCID ₅₀ / ㎖ of influenza virus H1N1 10 ^2, 10 ⁴ and completely reduced, indicating that pine needle extracts from Korean native pine, Red pine, Haesong, and pine tree, all have in vitro virucidal efficacy.

 division  In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Newcastle disease virus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 3 above, the virus was reacted with 500-fold dilution (500 μg / ml) of red pine, sea pine, and pine leaf extracts for 2 hours. 10 ^5.6 TCID ₅₀ / ml of Newcastle disease virus 10 ² , 10 ⁴ and completely reduced, indicating that pine needle extracts from Korean native pine, Red pine, Haesong, and pine nuts, all have in vitro virucidal efficacy.

 division  In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml) 500-fold dilution (500 μg / ml) 500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml) 500-fold dilution (500 μg / ml) 500-fold dilution (500 μg / ml)  Pine Leaf Extract 500-fold dilution (500 μg / ml) 500-fold dilution (500 μg / ml) 500-fold dilution (500 μg / ml) * Treated virus: Infectious bronchitis virus (10 ^5.6 TCID ₅₀ / mL) * Treated extract and treatment condition: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 4 above, the virus was reacted with 500-fold dilutions (500 μg / ml) of red pine, sea pine, and pine leaf extracts for 2 hours. 10 ^5.6 TCID ₅₀ / ml of chicken infectious bronchitis virus was 10 ² , 10 ^4, and totally reduced, indicating that pine needle extracts from Korean pine, Red pine, Haesong, and pine tree, all have in vitro virucidal efficacy.

 division  In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Avian pneumovirus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 5 above, the virus was reacted with 500-fold dilutions (500 μg / mL) of red pine, sea pine, and pine needle extracts for 2 hours. 10 ^5.6 TCID ₅₀ / mL of pneumovirus 10 ² , 10 ⁴ and completely reduced, indicating that pine needle extracts from Korean native pine, Red pine, Haesong, and pine nuts, all have in vitro virucidal efficacy.

 division  In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: PRRS virus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 6 above, the virus was reacted with 500-fold dilutions (500 μg / mL) of red pine, sea pine, and pine needle extracts for 2 hours. 10 ^5.6 TCID ₅₀ / mL of pig respiratory syndrome 10 ² , 10 ⁴ and totally reduced, indicating that pine needle extracts from Korean pine, Red pine, Haesong, and pine nuts are all effective in vitro virucidal.

 division  In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Coxsakie virus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 7 above, the virus was reacted with 500 fold dilutions (500 μg / ml) of red pine, sea pine, and pine leaf extracts for 2 hours, resulting in 10 ^5.6 TCID ₅₀ / ml of Coxsaki virus 10 ² ,. 10 ⁴ and completely reduced, indicating that pine needle extracts from Korean native pine, Red pine, Haesong, and pine tree, all have in vitro virucidal efficacy.

 division In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Reticuloendotheliosis virus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 8, 500-fold dilution (500 ㎍ / ㎖) a red pine leaves, black pine leaf, a result of the pine leaf Virus 2 hours to extract each reaction 10 ^5.6 TCID ₅₀ / ㎖ the reticuloendothelial increased virus 10 ² , 10 ^4, and totally reduced, indicating that pine needle extracts from Korean pine, Red pine, Haesong, and pine tree, all have in vitro virucidal efficacy.

 division In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Aujeszky's disease virus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 9 above, the virus was reacted with 500-fold dilutions (500 μg / ml) of red pine, sea pine, and pine leaf extracts for 2 hours. 10 ^5.6 TCID ₅₀ / ml of Ozeski virus 10 ² , 10 ⁴ and completely reduced, indicating that pine needle extracts from Korean native pine, Red pine, Haesong, and pine nuts, all have in vitro virucidal efficacy.

 division In Vitro Viral Efficacy  Reduction 10 to ⁴ reduction 10 ² reduction  Red Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Seaweed Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  Pine Leaf Extract  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml)  500-fold dilution (500 μg / ml) Treatment virus: Adeno virus (10 ^5.6 TCID ₅₀ / mL) Treatment extract and treatment conditions: 60% ethanol extract, 4 ℃, 30 minutes

As shown in Table 10 above, the virus was reacted with 500-fold dilutions (500 μg / ml) of red pine, sea pine, and pine leaf extracts for 2 hours. 10 ^5.6 TCID ₅₀ / mL of adenovirus 10 ² , 10 ⁴ and completely reduced, indicating that pine needle extracts from Korean native pine, Red pine, Haesong, and pine nuts, all have in vitro virucidal efficacy.

Experimental Example 2 Measurement of In Vitro Viral Efficacy of Pine Needle Extract by Different Extraction Methods

In order to test the in vitro virucidal efficacy of the pine needle extract by the extraction method 1000 times, based on 100 times dilution of red pine leaf hot water, 30%, 60% and 100% ethanol extract prepared in Example 1 with distilled water, Dilution of 2000 times, 4000 times, 8000 times, 16000 times, and 32000 times was carried out to determine the effect of virus proliferation by the method of Reference Example 4. 2.5 ml of the virus solution prepared at 1 minute intervals were added to the test tube containing 2.5 ml of the diluent of the pine needle extract at 4 ° C. in each test tube, followed by mixing for 30 minutes at 4 ° C., and shaking well every 10 minutes. The control group experimented differently using distilled water instead of diluted pine needle extract. The diluted pine needle extract and the mixed solution of the virus was mixed using the MDCK cell culture medium, and the dilution factor was 10 ⁻¹ , 10 ⁻² , 10 ⁻³ , 10 ⁻⁴ , 10 ⁻⁵ , 10 ⁻⁶ . MDCK was applied to a 96-well tissue culture plate (96 well tissue culture plate) cultured in a single layer in 8 wells / dilution multiple times to inoculate 25 μl of the mixed solution, followed by adsorption for 30 minutes, and then the medium was changed. In the case of the influenza virus test, the medium was changed to a medium containing 5 ug / ml trypsin to induce a cytopathic effect (CPE). After inoculation, the cells were incubated for 4 days in a CO ₂ incubator at 37 ° C., and daily cytoplasmic effect was observed under a microscope, and finally, 3-4 days after the inoculation, CPE was read for the presence of viruses. Judging According to the virus used, cells used for inoculation were appropriately replaced. In the case of viruses (H9N2 and IBV) that do not show CPE in the cells, the diluting fold samples and each virus reaction solution were inoculated with 0.2 ml of 10 days old SPF eggs. Three days after the inoculation, the presence of virus was determined by hemagglutination and dot immunoassay (Song CS et al., Avian Diseases, 42 (92) , pp92-100, 1998). Virus content calculations were calculated using the Karber method (Payment P et al., Methods and Techniques in Virology , p33, 1993).

 division In Vitro Viral Efficacy  Reduction 10 ² reduction  Extraction by Solvent  100% Ethanol Extract 60% Ethanol Extract 30% Ethanol Extract Hydrothermal extract 100% Ethanol Extract 60% Ethanol Extract 30% Ethanol Extract Hydrothermal extract  Extract treatment concentration  2000-fold dilution (500 μg)  16000-fold dilution (62.5 ㎍)  4000-fold dilution (250 μg)  1000-fold dilution (1 mg)  8000 fold dilution (125 ㎍)  32000-fold dilution (31.25 μg)  16000-fold dilution (62.5 ㎍)  2000-fold dilution (500 μg) Treatment virus: Influenza virus H1N1 (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 11, 2000-fold diluted pine needle 100% ethanol extract (500 μg), 16000-fold diluted 60% ethanol extract (62.5 μg), 4000-fold diluted pine needle 30% ethanol extract (250 μg) and 1000 30 min reaction of virus to pear-diluted pine needle water extract (1 mg) resulted in a complete reduction of 10 ^5.6 TCID ₅₀ / mL of influenza virus A / H1N1, and 100% ethanol extract of 8000-fold diluted pine needle (125㎍), 32000 The virus was reacted 30 times with 60% ethanol extract (31.25 ㎍) diluted, 16,000 fold diluted pine needles 30% ethanol extract (62.5 ㎍) and 2000 times diluted pine needle water extract (500 ㎍) 10 ^5.6 TCID ₅₀ / Ml of influenza virus A / H1N1 was reduced by 10 ² . The virucidal effect of the 60% ethanol extract was the most excellent.

Pine needles showed the most effective antiviral activity against influenza virus when 60% ethanol extract was applied. In addition, the pine needle extract extracted by 60% ethanol extraction method was used for the efficacy test of virucidal virus against adenovirus.

Experimental Example 3. In Vitro Viral Efficacy Screening of Pine Needle Extract

3-1. RNA virus

3-1-1. Flesh A / PR / 8/34 (H1N1) Influenza Virus Effects in MDCK Cells

To determine the effect of in vitro A / PR / 8/34 (H1N1) influenza virus on MDCK cells, 1000, 2000 and 4000 times, Diluting 8000-fold, 16000-fold, and 32000-fold, the in vitro virucidal efficacy was measured by the method of Reference Example 4, and the results are shown in Table 4 below.

In addition, by using the plaque reduction assay of Reference Example 5 to measure the intracellular virus proliferation inhibitory effect against the A / PR / 8/34 (H1N1) influenza virus, the results are shown in Figure 1 below.

division In Vitro Viral Efficacy   60% Ethanol Pine Needle Extract Concentration   Reduction 10 ² reduction   16000-fold dilution (62.5㎍)  32000-fold dilution (31.25㎍) Treatment virus: Influenza virus H1N1 (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 12, the virus was reacted with 60% ethanol extract (62.5 μg) of pine needles diluted 16000 times for 30 minutes, resulting in 10 ^5.6 TCID ₅₀ / mL of influenza virus A / PR / 8/34 (H1N1). The virus was reacted with 32,000-fold diluted pine needles 60% ethanol extract (31.25 μg) for 30 minutes, resulting in a 10 ² decrease of 10 ^5.6 TCID ₅₀ / ml of influenza virus A / PR / 8/34 (H1N1).

In addition, as shown in Figure 1, the results of the plaque reduction analysis showed that the effective concentration (50% Inhibitory concentration: IC ₅₀ ) that inhibits the virus of the pine needle extract of the present invention by 50% is 31.25 µg, which is consistent with the results of the tube experiment. Indicated.

As a result of the experiment, 60% ethanol extract of pine needles was excellent in in vitro virucidal activity and intracellular virus proliferation inhibitory effect against A / PR / 8/34 (H1N1) influenza virus.

3-1-2. In Vitro A / HS / MS96 / 96 (H9N2) Influenza Virus Efficacy in SPF Eggs

In vitro flesh A / HS / MS96 / 96 (H9N2) influenza virus efficacy in SPF oocytes 1000, 2000 and 4000 times, based on 100-fold dilution of 60% ethanol extracts with distilled water, Diluting 8000-fold, 16000-fold, and 32000-fold, the in vitro virucidal efficacy was measured by the method of Reference Example 4, and the results are shown in Table 13 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 16000-fold dilution (62.5㎍) 32000-fold dilution (31.25㎍) Treatment virus: Avian Influenza virus H9N2 (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 13, the virus was reacted with 16000-fold diluted pine needles 60% ethanol extract (62.5 μg) for 30 minutes. As a result, 10 ^5.6 EID ₅₀ / mL of avian influenza virus A / HS / MS96 / 96 (H9N2) A 30 minute reaction of the virus to 32000-fold diluted pine needles 60% ethanol extract (31.25 μg) resulted in a 10 ² reduction of 10 ^5.6 EID ₅₀ / mL of influenza virus A / HS / MS96 / 96 (H9N2). As a result of the experiment, the pine needle 60% ethanol extract was excellent in vitro virucidal efficacy against the A / HS / MS96 / 96 (H9N2) influenza virus.

3-1-3. Efficacy of In Vitro Newcastle Disease Virus in Fetal Fibroblasts

1000, 2000, 4000, 8000, 16000 times, based on 100-fold dilution of 60% ethanol extract of red pine leaf in vitro to determine the efficacy of in vitro flesh Newcastle disease virus (NDV) in fibroblasts. , 32,000-fold dilution measured the in vitro virucidal efficacy using a calibration strain (KJW strain) of one type of NDV by the method of Reference Example 4 and the results are shown in Table 14 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 4000-fold dilution (250 µg) 8000 dilutions (125 µg) Treatment virus: Newcastle disease virus KJW strain (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 14, the virus was reacted with 4000 times diluted pine needles 60% ethanol extract (250 μg) for 30 minutes, resulting in a complete reduction of 10 ^5.6 TCID ₅₀ / ml of Newcastle Disease Virus Correction strains. After 30 minutes of virus reaction with% ethanol extract (125 μg), 10 ^5.6 TCID ₅₀ / ml of Newcastle Disease Virus Correction strain was reduced by 10 ² . As a result of the experiment, pine needle 60% ethanol extract was excellent in vitro virucidal efficacy against Newcastle disease virus.

3-1-4. Efficacy of In Vitro Chicken Infectious Bronchitis Virus in SPF Eggs

In order to measure the efficacy of in vitro infectious bronchitis virus (IBV) in SPF eggs, 1000, 2000, 4000, 8000, 16000, The in vitro virucidal efficacy was measured by the method of Reference Example 4 by diluting 32000-fold and the results are shown in Table 15 below.

Antiviral Effectiveness Reduction 10 ² reduction 60% Ethanol Pine Needle Extract Concentration 4000-fold dilution (250 µg) - Treatment virus: IBV M41 strain (10 ^5.6 EID ₅₀ / mL) Treatment conditions: 4 ℃, 30 minutes

As shown in Table 15, the virus was reacted with 4000 times diluted pine needle 60% ethanol extract (250 μg) for 30 minutes, which completely reduced the infectious bronchitis virus M41 strain of 10 ^5.6 EID ₅₀ / ml. As a result of the experiment, the pine needle 60% ethanol extract was excellent in vitro virucidal effect against the infectious bronchitis virus.

3-1-5. Efficacy of In Vitro Avian Pneumovirus in Fetal Fibroblasts

To determine the efficacy of in vitro avian pneumovirus (APV) efficacy in fetal fibroblasts 1000, 2000, 4000, 8000, 16000 The in vitro virucidal efficacy was measured by the method of Reference Example 4 by diluting fold and 32000-fold, and the results are shown in Table 16 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 500-fold dilution (2 mg) 2000-fold dilution (500 µg) Treatment virus: Avian pneumovirus (10 ^5.6 TCID ₅₀ / ml) Treatment conditions: 4 ℃, 30 minutes

As shown in Table 16, the virus was reacted with 500-fold diluted pine needles 60% ethanol extract (2 mg) for 30 minutes, resulting in a complete reduction of 10 ^5.6 EID ₅₀ / ml of algal pneumonia virus, and 2000-fold diluted pine needles 60. A 30 min reaction of the virus to% ethanol extract (500 μg) resulted in a 10 ² decrease of 10 ^5.6 TCID ₅₀ / mL avian pneumonia virus. As a result of the experiment, the pine needle 60% ethanol extract was excellent in vitro virus virus efficacy against avian pneumonia virus.

3-1-6. Anti-Swine Genital Respiratory Syndrome Virus Efficacy in MARK-145 Cells

1000, 2000, 4000, 8000 based on 100-fold dilution of 60% ethanol extract of red pine leaf in distilled water to determine the efficacy of in vitro anti-pigment genital and respiratory syndrome virus (PRRSV) in MARK-145 cells. Diluting pears, 16000-fold, and 32000-fold dilutions, the virus growth inhibition effect was measured using NR analysis according to the method of Reference Example 6, and the results are shown in FIG. 2.

As shown in FIG. 2, the EC ₅₀ (50% effective concentration: 50% Effective concentration) against PRRSV of pine needle 60% ethanol extract was 121.25 μg. As a result of the experiment, pine needle 60% ethanol extract was excellent in intracellular antiviral efficacy against PRRSV.

3-2-7. In vitro Salcoxaki Virus Efficacy in Hep-2 Cells

1000, 2000, 4000, 8000, 16000, 60% ethanol red pine leaf extracts were diluted 100-fold with distilled water to determine the efficacy of in vitro Salcoxaki virus (CXV) in Hep-2 cells. In vitro, 32,000-fold dilution measured in vitro virucidal efficacy by the method of Reference Example 4 and the results are shown in Table 17 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 500-fold dilution (2mg / ml) 2000-fold dilution (500 µg / ml) Treatment virus: Coxsakie virus (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 17 above, the virus was reacted with 500-fold diluted pine needles 60% ethanol extract (2 mg) for 30 minutes, resulting in a complete reduction of 10 ^5.6 TCID ₅₀ / mL CXV, and 2000-fold diluted pine needles 60% ethanol. After 30 minutes of virus reaction with the extract (500 μg), CSV of 10 ^5.6 TCID ₅₀ / ml was reduced by 10 ² . As a result of the experiment, pine needle 60% ethanol extract was excellent in vitro virucidal efficacy against CXV.

3-1-8. Efficacy of In Vitro Resistant Retinopathy Virus in Ciliated Fibroblasts

1000, 2000, 4000, 8000, on the basis of 100-fold dilution of 60% ethanol red pine leaf extract with distilled water to determine the efficacy of in vitro flesh retinopathy virus (REV) in Hep-2 cells. 16000-fold, 32000-fold dilution to measure the in vitro killer virus efficacy by the method of Reference Example 4 and the results are shown in Table 18 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 4000-fold dilution (250 µg / ml) 8000 fold dilution (125 µg / ml) Treatment virus: Reticuloendotheliosis virus (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 18, the virus was reacted with 4000 times diluted pine needles 60% ethanol extract (250 μg) for 30 minutes, resulting in a complete reduction of 10 ^5.6 TCID ₅₀ / mL of REV, and 8000-fold diluted pine needles 60% ethanol. The reaction of REV virus for 30 minutes in the extract (125 ㎍) resulted in a 10 ² reduction of 10 ^5.6 TCID ₅₀ / mL REV. As a result of the experiment, pine needle 60% ethanol extract was excellent in vitro virucidal efficacy against REV.

3-2. DNA virus

3-2-1. Efficacy of In Vitro Ozeski Virus in MDCK Cells

1000, 2000, 4000, 8000, 16000 times based on 100-fold dilution of 60% ethanol red pine leaf extract with distilled water to determine the efficacy of in vitro Salvage virus (ADV) in Hep-2 cells. , 32,000-fold dilution to measure the in vitro virucidal efficacy by the method of Reference Example 4 is shown in Table 19 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 4000-fold dilution (250 µg / ml) 8000 fold dilution (125 µg / ml) Treatment virus: Aujeszky's disease virus (10 ^5.6 TCID ₅₀ / ml) Treatment conditions: 4 ℃, 30 minutes

As shown in Table 19 above, the virus was reacted with 4000 times diluted pine needles 60% ethanol extract (250 μg) for 30 minutes, resulting in a complete reduction of 10 ^5.6 TCID ₅₀ / mL of ADV, and 8000-fold diluted pine needles 60% ethanol. 30 min reaction of ADV virus to the extract (125 [mu] g) resulted in a 10 ² reduction in REV of 10 ^5.6 TCID ₅₀ / ml. As a result of the experiment, pine needle 60% ethanol extract was excellent in vitro virucidal efficacy against ADV.

3-2-2. In Vitro Adenovirus Efficacy in Hep-2 Cells

To determine the efficacy of in vitro live adenovirus (ADV) in Hep-2 cells, 1000, 2000, 4000, 8000, and 16000 times 60% ethanol red pine leaf extract was diluted 100-fold with distilled water. , 32,000-fold dilution to measure the in vitro virucidal efficacy by the method of Reference Example 4 and the results are shown in Table 20 below.

division In Vitro Viral Efficacy 60% Ethanol Pine Needle Extract Concentration Reduction 10 ² reduction 100-fold dilution (10mg / ml) - * Treated virus: Adeno virus (10 ^5.6 TCID ₅₀ / mL) * Treated condition: 4 ℃, 30 minutes

As shown in Table 20, the virus was completely reacted with 100-fold diluted pine needles 60% ethanol extract (10 mg / ml) for 30 minutes, resulting in a complete reduction of 10 ^5.6 TCID ₅₀ / ml of adenovirus.

Experimental Example 4 In Vitro Viral Efficacy of Pine Needle Soluble Extract in Vitro

4-1. In Vitro A / PR / 8/34 (H1N1) Influenza Virus Efficacy in MDCK Cells

In order to measure the efficacy of in vitro killing influenza virus in MDCK cells, the pine needle non-polar solvent soluble extracts of Example 2 were 1000-fold, 2000-fold, 4000-fold, 8000-fold, 16000-fold, based on 100-fold dilution with distilled water. The in vitro virucidal efficacy was measured by the method of Reference Example 4 by diluting 32000-fold and the results are shown in Table 21 below.

In addition, using the plaque reduction assay of Reference Example 5 to measure the intracellular virus proliferation inhibitory effect of pine needle non-polar solvent soluble extracts against A / PR / 8/34 (H1N1) influenza virus is shown in Figure 3 below. .

Pine Needle Extract Fraction In Vitro Viral Efficacy  Reduction 10 ² reduction Dichloromethane (CH ₂ Cl ₂ ) Soluble extract  16000-fold dilution (62.5 μg / ml)  32000-fold dilution (31.25 µg / ml)  Ethyl Acetate (EtOAc) Soluble Extract  1000-fold dilution (1mg / ml)  2000-fold dilution (500 µg / ml)  Butanol (n-BuOH) Soluble Extract  8000 fold dilution (125 µg / ml) - Water (H ₂ O) Soluble Extract  32000-fold dilution (31.25 µg / ml) -  Pine Needle Powder 60% Ethanol Extract  16000-fold dilution (62.5 μg / ml)  32000-fold dilution (31.25 µg / ml) Treatment virus: Influenza virus H1N1 (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 21, the dichloromethane soluble extract 62.5 g / ㎖, ethyl acetate soluble extract 1 mg / ㎖, butanol soluble extract 125 ㎍ / ㎖, water soluble extract 31.25 ㎍ / ㎖ concentration was completely reduced.

In addition, as shown in Figure 3, when the concentration of each of the pine needle non-polar solvent soluble extract of the present invention as a result of plaque reduction analysis, the inhibition of the virus was 47.5% dichloromethane soluble extract, ethyl acetate soluble extract 13 %, Butanol soluble extract 28.4% and water soluble extract 86.5% showed a tendency to agree with the results of the tube experiment.

As a result of the experiment, dichloromethane soluble extract and water soluble extract among pine needle non-polar solvent soluble extracts showed excellent in vitro viral activity and antiviral activity against influenza virus.

4-2. Efficacy of In Vitro Newcastle Disease Virus in Fetal Fibroblasts

1000, 2000, 4000, 8000 based on 100-fold dilution of pine needle non-polar solvent soluble extracts of Example 2 to determine the efficacy of in vitro flesh Newcastle disease virus (NDV) in fetal fibroblasts. Diluting pears, 16000-fold, and 32000-fold, the in vitro virucidal efficacy was measured by the method of Reference Example 4, and the results are shown in Table 22 below.

Pine Needle Extract Fraction In Vitro Viral Efficacy  Reduction 10 ² reduction Dichloromethane (CH ₂ Cl ₂ ) Soluble extract  500-fold dilution (2mg / ml)  4000-fold dilution (250 µg / ml)  Ethyl Acetate (EtOAc) Soluble Extract  No effect  No effect  Butanol (n-BuOH) Soluble Extract  No effect  100 times (10mg / ml) Water (H ₂ O) Soluble Extract  2000-fold dilution (500 µg / ml)  4000-fold dilution (250 µg / ml)  Pine Needle Powder 60% Ethanol Extract  4000-fold dilution (250 µg / ml)  8000 dilutions (125 µg / ml) Treatment virus: Newcastle disease virus KJW strain (10 ^5.6 TCID ₅₀ / mL) Treatment condition: 4 ℃, 30 minutes

As shown in Table 22 above, complete reduction in concentrations of 2 mg / ml dichloromethane soluble extract and 500 μg / ml water soluble extract showed excellent virucidal efficacy in vitro.

Experimental Example 5. Antiviral effect experiment of pine needle extract through animal experiment

5-1. Antiviral Effect of Pine Needle Extract in Mice

5-1-1. Therapeutic Effect by Negative Administration of Pine Needle Extract against Influenza Virus Inoculation

      In order to test the therapeutic effect of pine needle extract on influenza virus-infected mice, the red pine leaf 60% ethanol extract prepared above was diluted 5000 times with distilled water to prepare a concentration of pine needle extract 0.2 mg / ml. The test group was divided into three groups: the experimental group treated with pine needle extract after virus challenge, the positive control group without treatment after virus challenge, and the negative control group without virus challenge.

Mice used 6-week-old female SPF ICR mice (Orient, Korea), and challenge virus was influenza virus A / PR / 8/34 (H1N1), a human-derived mouse adaptation that causes distinct clinical symptoms and high mortality in mice upon infection. ) Was used. Mice were inoculated intranasally with 50 μl / mouse of virus solution with a titer of 10 ^8.0 TCID ₅₀ / ml after inhalation anesthesia. After challenge, the inoculated animals measured feed consumption and drinking water daily for two weeks, and also observed the clinical symptoms and the number of deaths every day to calculate the pathogenic index (PI). If more than 80% of the animals used for the negative control did not survive, a retest was performed. The mouse treatment effect according to the negative administration is shown in Table 23, the daily feed consumption and the negative consumption measurement results are shown in Table 24, respectively.

 Fauna  Inoculation head  Body weight (g) (mean ± SD) Vaccination Result ^A) PI ₁₄ ^{F )} Clinical symptoms ^B) Our ^C)  Before vaccination  After inoculation  Sick MTO ^{D )}  Dead MDT ^{E )}  Pine needle extract treatment group 5000 times diluted (0.2 mg / ml)  10  29.533 ± 1.9802  28.54 ± 3.4852  7/10  4.85  4/10   5.5  1.1  Positive control  10  29.523 ± 1.6428  31.14 ± 4.5208  10/10   4.7  7/10  5.9  1.7  Negative control  10  29.418 ± 1.9398  28.09 ± 4.4156  0/10 - 0/10 - 0 A) Vaccine strain and inoculation: Influenza virus, IN = 10 ^6.5 TCID ₅₀ / dose / 50 ㎕ B) Clinical head count / head shot C) Head count / Head shot D) Mean time of onset clinical signs (day) E) Mean death time (day) F) Pathgenicity index: the mean score per mouse per observation over a 14 day period when each day, mouse are scored 0 if normal, 1 if sick (fever, eyelids, hair, breathing, bent posture, Atrophy), 2 if dead

          DPI processor One 2 3 4 5 6 7 8 9 10 11 12 13 14 Pine needle extract treatment group 5000 times (0.2mg / mL) feed 2.66 1.12 1.33 1.44 1.38 1.13 1.02 1.83 2.67 3.5 3.50 4.83 4.67 3.25 negative 4.54 1.52 3.33 1.67 2.5 2.5 3.33 5.21 5.34 5.33 5.83 6.67 6.67 6.67 Positive control group feed 2.25 0.95 0.72 0.88 0.63 0.67 1.08 1.33 2.23 3.67 4.30 5.33 4.67 4.30 negative 2.53 1.00 1.00 0.63 0.63 0.17 0.33 2.67 3.33 3.33 6.67 6.67 6.67 6.67 Negative Control feed 3,56 3.65 3.21 3.78 2.89 4.55 3.82 3.91 3.69 4.31 3.42 3.61 3.78 3.99 negative 5.21 5.43 5.12 5.56 5.67 5.32 5.65 5.43 5.87 5.78 5.89 5.45 5.65 5.92

As shown in Table 23 and Table 24, the application of the 5000-fold diluted 60% ethanol pine needle extract (0.2 mg / ml) to the test group resulted in 2 to 10 days after virus challenge compared to the positive control group that did not receive the extract. There was a significant difference in the amount of drinking water and feed consumption, and the pine needle extract treatment group showed a significantly lower clinical symptoms, mortality and lesion index after virus challenge vaccination than the positive control group.

5-1-2. Therapeutic Effect by Gastrointestinal (Zonde) Administration of Pine Needle Extract against Influenza Virus Inoculation

In order to test the therapeutic effect of pine needle extract on influenza virus-infected mice, the pine needle extract was prepared by diluting the red pine leaf 60% ethanol extract prepared above with distilled water. The test group was divided into two groups: the experimental group treated with pine needle extract after the virus challenge and the negative control group not treated after the virus challenge.

Mice were used with magnetic SPF BALB / c mice (Orient, Korea), weighing 18-21 g in weight. The challenge virus was influenza virus A / PR /, a human-derived mouse adaptation that causes obvious clinical symptoms and high mortality in mice upon infection. 34 (H1N1) was used. Mice were inoculated intranasally with 50 μl / mouse of virus solution with a titer of 10 ^3.0 TCID ₅₀ / ml after inhalation anesthesia. The treatment was started 4 hours before challenge and treated twice a day for 5 days by gavage so that the pine needle extract had a dose of 10 mg / kg / day. In addition, the negative control group was inserted with physiological saline twice a day instead of a therapeutic agent. After challenge, the inoculated animals measured feed consumption and drinking water daily for 2 weeks and observed clinical symptoms and mortality every day to calculate the treatment effect. In order to determine the virus reduction effect of pine needle extract treatment, lungs were extracted from 5 mice in each group at 3 and 6 days after challenge, and the degree of pulmonary sclerosis, lung weight and virus content in lungs were measured (Sidwell RW et. al., Antiviral Research, 51 , pp 179-187, 2001). The therapeutic effect of mice by gavage administration is shown in Tables 25 and 26 below.

 Dosage  Composition (mg / kg / day) Vaccination titer (CCID ₅₀ / ml)  Body weight (g) (mean ± SD) Vaccination Result Survival rate ^A Before vaccination After inoculation Death toll / total head MDT ^B ± SD Pine needle extract 10 10 ^3.0 21.1 ± 1.4 13.1 ± 1.0 3/10 * 8.7 ± 0.6 Saline - 20.7 ± 0.8 14.6 ± 1.2 14/20 11.8 ± 3.0 A survival / vaccinated head B Mean death time (day) * P <0.05 by Fisher's exact test

  Dosage and Composition (mg / kg / day) Vaccination titer (CCID ₅₀ / ml)  Mean lung parameters Day 3 Day 6  Hardness A ± S.D Weight (mg ± S.D.) Virus titer (log10 / g ± S.D.)  Hardness A ± S.D  Weight (mg ± S.D.) Virus titer (log10 / g ± S.D.) Pine needle extract  10 10 ^3.0  0.1 ± 0.1 140 ± 9 * 4.7 ± 0.5 *** 1.0 ± 0.0 ** 209 ± 0 4.4 ± 0.8 * Saline  - 0.0 ± 0.0  159 ± 7  6.8 ± 0.2  2.5 ± 0.5  212 ± 0  5.6 ± 0.4 A consolidation; 0 (normal)-4 (dark purple) * P <0.05; ** P <0.01; *** P <0.001 by Fisher's exact test

As shown in Table 25, the results of treatment with pine needle extract in the test group for treatment purposes were statistically significant (P <0.05) in influenza virus 10 ^3.0 TCID ₅₀ / ml challenge compared to the negative control without pine needle extract. The effect of reducing mortality was apparent. In addition, as shown in Table 26, the test group treated with pine needle extract after 3 and 6 days after influenza virus challenge was statistically significantly decreased in the lung virus content compared to the negative control group (P <0.05 or more). Compared to the negative control group, the pine needle extract treatment group showed statistically significant lowering in the degree of menopause (after 6 days) and lung weight (after 3 days), respectively.

5-2. Antiviral Effects of Pine Needle Extracts in SPF Chickens

5-2-1. Anti-avian Influenza Virus Effects of Pine Needle Extract

      In order to test the therapeutic effect of pine needle extract on SPF chickens infected with avian influenza virus, the red pine leaf 60% ethanol extract powder prepared above was mixed with chicken feed to 1%, 0.4% and 0.1% (w / w), respectively. Prepared. The experimental group was treated with pine needle extract after virus challenge and the experimental group was treated with pine needle extract after virus challenge and the negative control group without negative treatment after virus challenge after virus challenge. The test group was divided into three groups, and the experimental group was divided into three groups to evaluate the effect of each concentration.

The experimental animals were 6-week-old SPF chickens (Namdeok Senitek, Korea), and the challenge virus was low-pathogenic Avian Influenza (LPAI) virus that caused mortality and spawning degradation of 1-30% in chickens. A / HS / K5 / 01 (H9N2), a domestic isolate, was used. During the vaccination of the virus, a titer of 10 ⁷ EID ₅₀ / ml was inoculated into the nasal cavity by 100 ul. The treatment was started 4 hours before challenge vaccination was supplied with a mixture of pine needle extract for each dose concentration, and was administered as a feed for a total of five consecutive days in a way to freely feed the test group freely. Five days after challenge, the airway and cecum tonsils were collected from both experimental and negative control groups, and the virus was re-isolated and titered by inoculating the emulsion into 10-day-old fetuses. Table 27 shows the therapeutic effects of the virus re-isolation rate and virus titer according to the virus challenge.

 Dosage  Concentration in feed (%, w / w) Change after challenge  Virus Re-Isolation Rate (positive and inoculated) Virus titer (log ₁₀ EID ₅₀ / g ± SD)  Prayer  Cecum one-way  Prayer  Cecum one-way  Pine needle extract  1.0 1/8 ^** 1/8 ^**  2.1 ± 0.4 **  3.9 ± 0.3 **  Pine needle extract  0.4  3/8 * 3/8 ^*  2.6 ± 0.4 **  4.6 ± 0.7 **  Pine needle extract  0.1  4/8  5/8  3.2 ± 1.1  5.0 ± 0.9  Distilled water -  8/8  8/8  4.1 ± 0.5  7.5 ± 1.1 ^* P <0.05 by Fisher's exact test ^** P <0.01 by Fisher's exact test

As shown in Table 27, the virus re-separation rate and virus titer in airway and cecal tonsils after 5 days of challenge were significantly decreased in the pine needle extract 1% and 0.4% treatment groups compared to the negative control group (P <0.05). More than).

5-2-2. Anti-Newcastle Disease Virus Effects of Pine Needle Extract

In order to examine the therapeutic effect of pine needle extract on Newcastle disease virus-infected SPF chickens, the red pine leaf 60% ethanol extract prepared above was diluted with distilled water to prepare 500 ug / ml, 250 ug / ml and 125 ug / ml, respectively. . The test group was divided into experimental group treated with pine needle extract after virus challenge and negative control group not treated after virus challenge.

Inoculated with 0.2 ml of Newcastle Disease Virus poisoning oil vaccine to 13-week-old SPF chickens and collected 7 days later to measure the antibody titer of Newcastle Disease Virus, and then inoculated with Kr-005 / 00 week at 10 ^5.5 EID ₅₀ / 30ul. It was. The treatment was started 4 hours before challenge inoculated with distilled water mixed with pine needle extract at the concentration of each dose, and was administered in a negative manner for a total of 5 days in a way that the test group was free to feed freely. Six days after challenge inoculation, the surface of the oral cavity and the total excretory cavity were scraped with a cotton swab to measure virus release in fibroblasts.

 Virus sampling area  Pine needle extract dose  Distilled water administration group  500ug / ml  250 ug / ml   125ug / ml Virus titer released (log ₁₀ TCID ₅₀ / ml ± SD)  Blowjob  0  0  0  0  Total excretion steel  2.56 ± 1.7  3.26 ± 0.9  3.04 ± 1.5  4.05 ± 1.9

^* P <0.05 by Fisher's exact test

^** P <0.01 by Fisher's exact test

Consecutive negative doses of pine needle extract at concentrations of 500, 250, and 125 ug / ml resulted in a reduction of Newcastle disease virus release of more than about 10 ¹ TCID ₅₀ / ml. Particularly, in the group administered 500 ug / ml and 250 ug / ml, 10 ^2.34 TCID ₅₀ / ml and 10 ^2.98 TCID ₅₀ / ml of the virus were discharged, respectively, compared to 10 ^4.17 TCID ₅₀ / ml of the control group. Significantly ( P <0.01 or more) viral emissions were reduced.

5-3. Anti-influenza Virus Effects of Pine Needle Extracts in Ducks

In order to test the therapeutic effect of pine needle extract against influenza virus-infected ducks, the red pine leaf 60% ethanol extract powder prepared above was prepared by mixing 1%, 0.4% and 0.1% (w / w) in duck feed, respectively. The test group was divided into three test groups: the experimental group treated with pine needle extract after the virus challenge and the negative control group not treated after the virus challenge. The experimental group was divided into three groups to evaluate the effect of each concentration. It was.

The experimental animals were three-week-old ducks (positive farms, Korea), and the challenge virus was A / CSM / D2 / 05 (H6N5), a low-pathogenic avian influenza virus isolated from domestic wild ducks. When challenged with the virus, a titer of 10 ^7.7 EID ₅₀ / ml was inoculated into the airways and nasal passages of 50 ul each to inoculate 0.1 ml of the virus. The treatment was started 4 hours before challenge vaccination was supplied with a mixture of pine needle extract for each dose concentration, and was administered as a feed for a total of five consecutive days in a way to freely feed the test group freely. The virus was re-isolated and titers were obtained by inoculating the airway and cecum tonsils from both experimental and negative control groups for 2 days 3 and 6 days after challenge and inoculating the emulsion into 10-day-old fetuses. Table 29 shows the therapeutic effects of the virus re-isolation rate and virus titer according to the virus challenge.

 Dosage Concentration in feed (%, w / w)  3 days after challenge  6 days after challenge  Virus Re-Isolation Rate (positive and inoculated) Virus titer (log ₁₀ EID ₅₀ / g)  Virus Re-Isolation Rate (positive and inoculated) Virus titer (log ₁₀ EID ₅₀ / g)  Prayer  Cecum one-way  Prayer  Cecum one-way  Prayer  Cecum one-way  Prayer  Cecum one-way  Pine needle extract  1.0  2/8 *  0/8  2.3 **  0  0/8  1/8 **  0  2.1 **  Pine needle extract  0.4  4/8  0/8  3.3 *  0  0/8  2/8 *  0  2.1 **  Pine needle extract  0.1  4/8  0/8  3.8  0  0/8  2/8 *  0  2.7 *  Distilled water -  7/8  0/8  5.6  0  0/8  7/8  0  4.1 ^* P <0.05 by Fisher's exact test ^** P <0.01 by Fisher's exact test

As shown in Table 29, the virus re-separation rate after 3 days of challenge was statistically significant compared to the negative control in pine needle extract in 1% treatment group and the airway virus titer in pine needle extract in 1% and 0.4% treatment groups. Decrease (P <0.05). After 6 days of challenge, virus re-separation rate and cecal tonsil virus titer were significantly decreased in pine needle extract 1%, 0.4% and 0.1% compared to the negative control group (P <0.05).

Pine needle extract of the present invention can be administered in the following formulation, the formulation examples below are merely to illustrate the invention, whereby the content of the present invention is not limited.

Formulation example  1. Preparation of Tablets

Pine needle 60% ethanol extract ............ 200mg

Lactose 100 mg

Starch .............................. 100 mg

Magnesium Stearate ...............

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 2 Preparation of Capsule

Pine needle 60% ethanol extract ........................ 100 mg

Lactose 50 mg

Starch ........................................ 50 mg

Talc ........................................ 2mg

Magnesium Stearate ...............

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 3 Preparation of Liquid

Pine needle 60% ethanol extract ......, ............ 1000mg

Sugar ........................................ 20g

Isomerized sugar ......................................... 20g

Lemon Flavor ......................

Purified water was added to make a total of 1000 ml.

According to the conventional method for preparing a liquid, the above components are mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation Example 4 Preparation of Healthy Food

Pine Needle 60% Ethanol Extract ....

Vitamin Blend ...............

Vitamin A Acetate ......... 70 μg

Vitamin E ............ 1.0 mg

Vitamin B1 ........................ 0.13 mg

Vitamin B2 ........................ 0.15 mg

Vitamin B6 ............... 0.5 mg

Vitamin B12 ............... 0.2 μg

Vitamin C ............ 10 mg

Biotin .............................. 10 ㎍

Nicotinamide ......... 1.7 mg

Folic acid ......................... 50 μg

Calcium Pantothenate ......... 0.5 mg

Inorganic mixtures ...............

Ferrous Sulfate ............... 1.75 mg

Zinc Oxide ............... 0.82 mg

Magnesium Carbonate ............... 25.3 mg

Potassium monophosphate ......................................... 15 mg

Dibasic calcium phosphate ............... 55 mg

Potassium Citrate ............... 90 mg

Calcium Carbonate ... 100 mg

Magnesium Chloride ........................... 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 5 Preparation of Healthy Drink

Pine Needle 60% Ethanol Extract ...

Citric Acid .................................... 1000 mg

Oligosaccharide ......................................... 100 g

Plum concentrate ........................... 2 g

Taurine ......................................... 1 g

Purified water is added to the whole ..... 900 ㎖

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

Pine needle extract of the present invention is influenza A viruses, newcastle disease virus, chicken infectious bronchitis virus, avian pneumovirus, porcine reproductive and respiratory syndrome virus (porcine reproductive) cells infected with and respiratory syndrome virus, coxsakie virus, reticuloendotheliosis virus, azejezky's disease virus, and adeno virus and coxsakie virus. in vivo (in vivo) so experiment the antiviral effect of the virus growth inhibition and live virus prominent in the prophylaxis and treatment of diseases caused by the body, infected with the virus using in (in vitro) test and the virus infected animals in vitro For pharmaceutical compositions and nutraceuticals There.

Claims (37)

Pharmaceutical composition for the prevention and treatment of diseases caused by viruses containing pine needle extract as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is a crude extract. The pharmaceutical composition according to claim 2, wherein the crude extract is an extract available in a polar solvent selected from water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof. The pharmaceutical composition of claim 1, wherein the extract is a nonpolar solvent soluble extract.         The pharmaceutical composition according to claim 4, wherein the nonpolar solvent soluble extract is an extract soluble in a nonpolar solvent selected from dichloromethane or ethyl acetate. The pharmaceutical composition of claim 1, wherein the pine needles are leaves of Pinus densiflora Sieb. Et Zucc., Pinus thunbergii parlatore or Pinus koraiensis Sieb. Et Zucc. The pharmaceutical composition of claim 1, wherein the virus is an RNA virus or a DNA virus. The method of claim 7, wherein the RNA-type virus is influenza virus, Newcastle disease virus, chicken infectious broncheitis virus, avian pneumovirus or pig genital and respiratory syndrome virus pharmaceutical composition which is (porcine reproductive and respiratory syndrome virus), coxsakie virus, reticuloendotheliosis virus. The pharmaceutical composition of claim 8, wherein the influenza virus has a serotype of H1N1, H9N2 or H6N5. 8. The pharmaceutical composition according to claim 7, wherein the DNA-type virus is auzeszky's disease virus or adeno virus or coxsakie virus. The disease caused by the virus of claim 1, wherein the disease caused by the virus is orthomyxovirus family virus including influenza virus, paramyxovirus family virus including newcastle disease virus, chicken infectious bronchitis virus ( Coronavirus famil virus containing infectious broncheitis virus, picorna virus family virus including coxsakie virus, retrovirus including reticuloendotheliosis virus It is caused by the infection of the body, the herpesvirus family, which includes the azezky's disease virus, or the adenovirus famoly, which includes the adeno virus. The pharmaceutical composition according to. The pharmaceutical composition according to claim 11, wherein the disease caused by the infection of the influenza virus family virus is flu, cold, bird flu or mutant infectious bird flu. The method of claim 11, wherein the disease caused by the infection of the paramyxo virus and the virus is paraparafluenza, mumps, measles, nipavirus, hendra. Pharmaceutical composition, which is a virus (Hendra virus), human Respiratory syncytial virus or human metapneumovirus (Human metapneumovirus). The method of claim 11, wherein the disease caused by the infection of the coronavirus family virus is human respiratory coronavirus (HCoV-229E), Severe Acute Respiratory Syndrome (SARS) or human respiratory coronavirus (HCoV-OC43). 12. The method of claim 11, wherein the disease caused by the infection of the picornavirus family virus is Encephalomyocarditis virus, Poliovirus, Coxsackievirus, Echovirus. Pharmaceutical composition, which is rhinovirus, Parecovirus or Hepatitis A virus. 12. The method of claim 11, wherein the disease caused by the infection of the retrovirus family virus is human immunodeficiency virus type 1 and 2 or human foamy virus. Pharmaceutical composition. The method of claim 11, wherein the disease caused by the infection of the herpesvirus family virus is human herpes simplex virus, Epstein-barr virus, cytomegalovirus, Varicella-zoster virus, human herpes virus 6, 6A, 6B, human herpesvirus 6, 6A, 6B, 7, kaposi's sarcoma or cercopithecin herpes virus (cercopithecine herpesvirus). The method of claim 11, wherein the disease caused by the infection of the adenovirus family virus is respiratory infection, gastroenteritis, epidemic keratoconjunctivitis, hemorrhagic cystitis or meningitis in humans. Pharmaceutical composition which is a human adenovirus infectious virus. The pharmaceutical composition of claim 1, wherein the extract comprises 0.1 to 50% by weight, based on the total weight of the composition. Health functional foods for the prevention and improvement of diseases caused by viruses containing pine needle extract of claim 1 and food supplements that can be added foodologically. 21. The dietary supplement of claim 20, wherein the extract is a crude extract. 22. The dietary supplement of claim 21, wherein the crude extract is an extract available in a polar solvent selected from water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof. 21. The dietary supplement of claim 20, wherein the extract is a non-polar solvent soluble extract.         24. The dietary supplement of claim 23, wherein the nonpolar solvent soluble extract is an extract soluble in a nonpolar solvent selected from dichloromethane or ethyl acetate. 21. The dietary supplement of claim 20, wherein the pine needles are leaves of Pinus densiflora Sieb. Et Zucc., Pinus thunbergii parlatore or Pinus koraiensis Sieb. Et Zucc. The dietary supplement according to claim 20, wherein the virus is an RNA virus or a DNA virus. The method of claim 26, wherein the RNA-type virus is influenza virus, Newcastle disease virus, infectious broncheitis virus, avian pneumovirus or swine genital and respiratory syndrome virus ( A dietary supplement that is porcine reproductive and respiratory syndrome virus, coxsakie virus or reticuloendotheliosis virus. 28. The dietary supplement of claim 27, wherein the influenza virus has a serotype of H1N1, H9N2 or H6N5. 27. The dietary supplement of claim 26, wherein the DNA-type virus is auzeszky's disease virus or adeno virus. The disease of claim 20, wherein the disease caused by the virus is orthomyxovirus family virus, including influenza virus, paramyxovirus family virus, including newcastle disease virus, chicken infectious bronchitis virus ( Coronavirus famil virus containing infectious broncheitis virus, picorna virus family virus including coxsakie virus, retrovirus including reticuloendotheliosis virus family virus, the herpesvirus family including auzeszky's disease virus, or the adenovirus famoly virus, including adeno virus. Health foods of Jing. 31. The health functional food according to claim 30, wherein the disease caused by the infection of the influenza virus family virus is flu, cold, bird flu or mutant infectious bird flu. 31. The method of claim 30, wherein the disease caused by the infection of the paramyxo virus and the virus in the body is parainfluenza, mumps, measles, nipavirus, hendra. A dietary supplement that is a virus (Hendra virus), human Respiratory syncytial virus or human metapneumovirus (Human metapneumovirus). 31. The method of claim 30, wherein the disease caused by the infection of the coronavirus family virus is human respiratory coronavirus (HCoV-229E), Severe Acute Respiratory Syndrome (SARS) or human respiratory coronavirus (SAR). Health functional food (HCoV-OC43). 31. The method of claim 30, wherein the disease caused by infection of the picornavirus family virus is Encephalomyocarditis virus, Poliovirus, Coxsackievirus, Echovirus. , Nutraceutical which is rhinovirus, Parecovirus or Hepatitis A virus. 31. The method of claim 30, wherein the disease caused by the infection of the retrovirus family of viruses is human immunodeficiency virus type 1 and 2 or human foamy virus. Health functional food. 31. The method of claim 30, wherein the disease caused by infection of the herpesvirus family of viruses is human herpes simplex virus, Epstein-barr virus, cytomegalovirus, Varicella-zoster virus, human herpes virus 6, 6A, 6B, human herpesvirus 6, 6A, 6B, 7, kaposi's sarcoma or cercopithecin herpes virus (cercopithecine herpesvirus). 31. The method of claim 30, wherein the disease caused by infection of the adenovirus family virus is respiratory infection, gastroenteritis, epidemic keratoconjunctivitis, hemorrhagic cystitis, or meningitis in humans. A dietary supplement that is a human adenovirus infection virus.

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