KR101035463B1 - A composition comprising the processed root extract of Panax ginseng for the prevention and treatment of pathogenic avian influenza virus - Google Patents

Abstract

The present invention relates to a composition containing an extract extracted from black ginseng root prepared using a high temperature and high pressure manufacturing method as an active ingredient, specifically, the composition of the present invention is excellent against highly pathogenic avian influenza virus (highly pathogenic avian influenza) Since it shows an inhibitory effect, it can be usefully used as a pharmaceutical composition and health functional food for the prevention and treatment of high pathogenic avian influenza virus infection.

Black ginseng, highly pathogenic avian influenza virus

Description

A composition comprising the processed root extract of Panax ginseng for the prevention and treatment of pathogenic avian influenza virus}

It is an object of the present invention to provide an antiviral pharmaceutical composition and health functional food having an inhibitory effect on high pathogenic avian influenza virus containing an extract extracted from black ginseng root prepared using high temperature and high pressure manufacturing method as an active ingredient.

Selmons et al., Avian Dis., 18 (1), p. 119-124, 1974.

Webster RG et al., Microbiol Rev., 56 (1), p. 152-179, 1992

Document 3 Alexander DJ, Vet. Microbiol., 74 (1-2), p.3-13, 2000

[4] Song CS et al., Korean J. Poult. Sci., 31 (2), p.129-136, 2004

Document 5 JH, Bull. NY Acad. Med., 54, p. 855-864, 1978

[6] Taubenberger JK et al., Science, 275, p. 1179-1796, 1997

7 Potter CW, J. appl. Microbiol., 91, p. 572-579, 2001

[Reference 8] Oxford JS, Rev. Med. Virol., 10 (2), p. 119-133, 2000

9 Suarez DL et al., J. Virol., 72 (8), p.6678-6688, 1998

10. Capua I et al., Avian Pathol., 32, p. 47-55, 2003

Document 11 Capua I and Marangon S, Avian Pathol., 32, p. 335-343, 2003

Swayne DE et al., Avian Dis., 41, p. 910-922, 1997

Swayne DE et al., Avian Dis., 44, p. 132-137, 2000

Ward P et al., J. antimicrob. Chemother., 55 (supp1), p.i5-i21, 2005

[Reference 15] Understanding Korean Ginseng, Korean Ginseng Society, p.9, 1995

[Reference 16] Advances in Ginseng Research, Korean Ginseng Society, p. 127-137, 1998

[Document 18] Korean Unexamined Patent Publication No. 2003-0005089

[Document 19] Republic of Korea Patent Registration No. 10-0754253

[Document 20] Korea Patent Registration No. 10-0781571

Influenza virus is an RNA virus belonging to the family Orthomyxoviridae, and serotypes are classified into three types, type A, type B and type C. Among them, hepatitis B and C have been identified only in humans, while hepatitis 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), p. 119-124, 1974; Webster RG et al., Microbiol Rev., 56 (1), p. 152-179, 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 according to the serotype. 16 species and 9 NA types). The HA protein plays a role in attaching the virus to somatic cells, and the NA protein allows the virus to penetrate into the cell (Alexander DJ, Vet. Microbiol., 74 (1-2), p.3-13, 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. The virus was confirmed to be infected in wild birds (Selmons et al., Avian Dis., 18 (1), p. 119-124, 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 more than 95% high mortality and also called "bird flu". Alexander DJ, Vet. Microbiol., 74 (1-2), p.3-13, 2000). The highly pathogenic avian influenza is classified as Class A by the International Water Services Bureau (OIE) and domestic type 1 epidemics.

High pathogenic avian influenza (HPAI) has been reported since 1980 in the United States (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 HPAI occurred almost simultaneously in all of Southeast Asia and Far East Asia, including Vietnam, Japan, Thailand, Cambodia, Laos, Laos, Indonesia, and China. In particular, HPAI, which originated in Vietnam and Thailand, is a variant A / H5N1 HPAI virus that can infect human body when it comes into contact with infected birds, unlike the traditional HPAI that does not infect humans in Korea and Japan at that time. By March 2004, 22 people in contact with infected birds were infected in Vietnam, and 15 of them were killed. In neighboring Thailand, 11 people in contact with infected birds were infected, and eight of them were killed. It is true. In recent years, the incidence of HPAI has increased by 10 times compared to the past, and since 2001, it has been taking place every year worldwide. Therefore, a new concept of preventive measures to effectively control HPAI is required. Song CS et al., Korean J. Poult. Sci., 31 (2), p. 129-136, 2004).

The first record of human influenza dates back to 412 B.C.E., but the first pandemic influenza record of humanity is estimated to have occurred throughout Europe from 1173 to 1174. 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, p. 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 a major epidemic of pandemic flu, which is still prevalent worldwide every year (Taubenberger JK et al., Science, 275, p.1793-). 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, p.572-579, 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), p. 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 of the mutant viruses derived from these three serotypes is being conducted worldwide (Suarez DL et al., J. Virol., 72 (8), p.6678-6688, 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, which is also avian influenza virus that has been prevalent only in birds for the first time among humans.

Influenza viruses can infect the respiratory tract, cause systemic symptoms, change their appearance periodically, and move to another host before the host dies, without killing the host. 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 the basic virus treatment is not yet performed.

Among the avian influenza vaccines, live virus vaccines are almost impossible to develop due to the characteristics of viruses that are easily mutated. The vaccines developed to date can be largely classified into a deadly poison vaccine and a recombinant vaccine. As HPAI outbreaks prolonged and spread throughout the country in 1999 and Hong Kong in 2003, avian influenza vaccine was used as a means of combating HPAI in combination with a selective killing policy. Currently, in Italy and Hong Kong, the use of avian influenza vaccine (Capua I et al., Avian Pathol., 32, p.47-55, 2003; Capua I and Marangon S, Avian Pathol., 32, p.335-343, 2003). Vaccines positively evaluated in Italy (serum type A / H7N1) and Hong Kong (serum type A / H5N1) are all serotoxin vaccines and are heterologous serotypes with the same HA type but different NA type (serum type A / H7N3, A serotoxin vaccine with serotype A / H5N2) was used to distinguish it from field infection in antibody testing. However, the deadly venom vaccine cannot be distinguished from vaccine and outdoor infection by the Agar Gel Precipitation (AGP) standard, 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 currently developed dead venom vaccine can reduce the amount of virus released into feces during HPAI infection, but does not completely prevent the spread of disease (Capua I et al., Avian Pathol., 32, pp 47-55, 2003; Capua I and Marangon S, Avian Pathol., 32, p. 335-343, 2003).

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 has been converted to advanced genetic vaccination to date to distinguish between the vaccination system and the field infection system. The vaccines used in Mexico so far include the hemagglutin gene, which is the surface protein of the HPAI virus, into the fowl pox virus, an advanced genetic recombinant vector that can effectively prevent poultry and HPAI during vaccination. Vaccines, and on the other hand, countries that use chicken pox vaccines, including Korea, have been evaluated to have a disadvantage in that the effects of viral vector vaccines are halved due to the interference of antibodies induced by conventional poultry vaccination. In addition, genetically engineered viral vector vaccines have been developed by inserting the Hemagglutin gene, a surface protein of HPAI virus, into infectious laryngotracheitis virus (ILT). In the countries of use, the effect is halved, and it is estimated that the available subject is limited to chickens (Swayne DE et al., Avian Dis., 41, p.910-922, 1997; Swayne DE et al. , Avian Dis., 44, p. 132-137, 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 are developed to compensate for these drawbacks, 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), p.i5-i21, 2005). Therefore, along with vaccines and therapeutics, the development of safe natural materials while increasing human immunity will be an important means of reducing mortality during pandemic.

Ginseng is a perennial plant belonging to the genus Panax, and the genus Panax is a perennial root of the genus Araliaceae. Typical species include Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax japonica, Panax trifolia, Panax pseudoginseng, Panax pseudoginseng, and Panax ginseng. vietnamensis) (Understanding Korean Ginseng, Korean Ginseng Society, p.9, 1995; Advances in Ginseng Research, Korean Ginseng Society, p. 127-137, 1998).

The most important component of these Panax plants is saponin. In particular, the genus Panax, unlike other plants contain a common saponin 1 to 4 sugars are bonded to the dammarane skeleton. In particular, saponins with high content in Korean ginseng are ginsenosides Rb1, Rb2, Rc, Rd, Rg1 and Re. These saponin components show various effects, and the types and strengths of the drugs vary greatly depending on their structure (Understanding Korean Ginseng, Korean Ginseng Society, p.9, 1995).

Ginseng has long been known to be effective for fatigue recovery and strength enhancement. In fact, ginseng is known to have increased exploratory activity, decreased spontaneous movement, elevated seizure threshold, increased memory, and analgesic effects (Understanding Korean Ginseng, Korean Ginseng Society, p.9, 1995).

Recently, as a result of efforts to further increase the active ingredient content of ginseng, black ginseng using the principle of gujeungpo, which is the most common method of using water and fire among the processing methods of the herbal medicine, which is one of the steaming methods, has been developed ( Korean Patent Application Publication No. 2003-0005089; Manufacturing method of black ginseng and black rice ginseng) In this laboratory, patent has been registered for a standardized manufacturing method of black ginseng that can be shortened in time and mass-produced compared to this method. No. 10-0754253; Novel black ginseng manufacturing method and composition containing the same, Patent registration No. 10-0781571; Pharmaceutical composition containing the extract of the novel black ginseng). However, there has been no disclosure or study on the inhibitory effect on the HPAI of the extract extracted from the black ginseng root prepared by the above method.

Accordingly, the present inventors have completed the present invention by confirming that the extract extracted from the black ginseng root prepared by high temperature and high pressure shows the excellent inhibitory effect on the high pathogenic avian influenza virus in the era of premiumization of ginseng.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of high pathogenic avian influenza virus containing the extract of the root of Black Panax Ginseng prepared by high temperature and high pressure manufacturing method as an active ingredient.

In addition, the present invention provides a health food for the prevention and improvement of high pathogenic avian influenza virus containing the extract of black ginseng root prepared by the high temperature and high pressure manufacturing method as an active ingredient.

The extract includes crude extracts available from water, including purified water, lower alcohols having 1 to 4 carbon atoms, or a mixed solvent thereof, preferably a solvent selected from water, ethanol or a mixed solvent thereof.

The extract comprises extracts available from water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably butanol.

The extract as defined herein is characterized in that the extract of black ginseng root (root), leaves or buds, preferably black ginseng root (root).

      The highly pathogenic avian influenza virus as defined herein is H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N7, H9N2 , H3N2, Human B, H5N1, H9N2, H7N1 or has a serotype of H7N2.

Hereinafter, the present invention will be described in detail.

Black ginseng root extract of the present invention can be prepared through the following process.

  Black ginseng root of the present invention is 3 to 7 years old, preferably 4 to 6 years old Korean ginseng after washing three times for 10 minutes with an ultrasonic cleaner, 10 to 70 ℃, preferably 12 to 36 hours at a temperature of 30 to 50 ℃ Preferably, the first step of drying for 20 to 28 hours; A second step of putting dried Korean ginseng root into a steamer for 1 to 8 hours, preferably 4 to 6 hours at a temperature of 60 to 120 ° C., preferably 85 to 105 ° C. except for a preheating time; A third step of drying at a temperature of 40-80 ° C., preferably 50-70 ° C. for 6-18 hours, preferably 9-15 hours; 4 to 8 hours, preferably 5 at a temperature of 95 to 155 ° C., preferably at a temperature of 100 to 120 ° C., under a pressure of 0.05 to 0.45 MPa, preferably 0.10 to 0.20 MPa, more preferably 0.10 to 0.14 MPa A fourth step of steaming for 7 to 7 hours; The black ginseng root (hereinafter referred to as "BPG") of the present invention can be obtained by a fifth step of drying in a drier for 8 to 16 hours, preferably 10 to 14 hours.

Approximately 3 to 7 times, preferably 4 to 6 times mass water of black ginseng root prepared by the high temperature and high pressure method was added thereto, and the mixture was left at room temperature for 3 to 7 hours, and then water containing purified water and lower carbon atoms having 1 to 4 carbon atoms. Alcohol or a mixed solvent thereof, preferably 80% ethanol, added 5 to 20 times the mass, preferably 8 to 12 times the mass for 2 to 8 hours, preferably 3 to 4 hours, 1 to The extract obtained by refluxing five times, preferably three times, may be cooled, filtered and concentrated to obtain the black ginseng root crude extract of the present invention (hereinafter referred to as “BPGE”).

Suspending the crude black ginseng root crude extract obtained by suspending water, and then adding a nonpolar solvent such as ether or dichloromethane to remove the nonpolar material; Black ginseng root butanol soluble extract of the present invention containing a large amount of ginseng saponin through a final step of extracting saponin present in ginseng 2 to 6 times and depressurizingly concentrating by adding saturated butanol to the remaining aqueous layer (hereinafter referred to as "BPGBE") ) Can be obtained.

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Black ginseng root extract prepared by the manufacturing method is dried through a drying method common in the art, such as shade, freeze-drying, and then pulverized and powdered into fine particles, preferably a particle size of about 50 to 200 ㎛ Through the use of excipients or carriers well known in the art can be prepared in compositions such as pills, capsules, tablets.

The black ginseng root prepared by the manufacturing method may be used as a raw material of various preparations by grinding into fine powder, preferably about 50 to 200 ㎛ size powder using a herbal medicine grinder.

Black ginseng root prepared by the manufacturing method is characterized in that the content of the active ingredient that is present in the trace ginseng, white ginseng, or red ginseng is present in a very small amount.

In addition, black ginseng root extract prepared by the production method of the present invention is characterized by exhibiting a strong inhibitory effect on high pathogenic avian influenza virus.

The present invention provides a pharmaceutical composition for the prevention and treatment of high pathogenic avian influenza virus containing the black ginseng root extract prepared from the production method as an active ingredient.

Pharmaceutical composition for the prevention and treatment of high pathogenic avian influenza virus of the present invention, the extract comprises 0.1 to 50% by weight based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.2 to 200 mg / kg, preferably 2 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as rats, mice, livestock, humans and the like in various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Black ginseng root extract of the present invention can be used as a raw material that is more potent than the conventional ginseng has been used, that is, a variety of pharmaceutical preparations, Chinese medicine, health food, food, tea, cosmetics and the like.

In addition, the extract of the present invention has little toxicity and side effects, so can be used with confidence even for long-term use for the purpose of prevention or treatment.

The present invention provides a health functional food for the prevention and improvement of high pathogenic avian influenza virus containing the black ginseng root extract obtained by the above production method as an active ingredient.

For the purpose of preventing and improving the highly pathogenic avian influenza virus of the present invention it is possible to manufacture and process as a health functional food in the form of tablets, capsules, powders, granules, liquids, pills and the like.

It may also be added to food or beverages for the purpose of preventing the prophylactic avian influenza virus. At this time, the amount of the black ginseng root extract in the food or beverage may be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except the black ginseng root extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. 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, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrginine, 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 extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh 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 in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

An object of the present invention to provide a pharmaceutical composition and health functional food for the prevention and treatment of high pathogenic avian influenza virus showing an inhibitory effect on the high pathogenic avian influenza virus containing an extract extracted from black ginseng root prepared using high temperature and high pressure manufacturing method It is.

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

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited by the following Experimental Examples.

Example 1 Preparation of Black Ginseng Roots

After purchasing five-year-old ginseng produced and distributed in Geumsan, 1kg of ginseng root was put in a screw washer (Jungwoo Machinery, Korea) and washed three times for 10 minutes. At this time, care was taken not to peel during the cleaning process.

When steamed steamed ginseng without drying, ginseng roots are often bursting, so to prevent this, first dried at 40 ℃ for 24 hours. Ginseng roots dried at low temperature for 24 hours were put into a steamer and steamed for 1 hour at 95 ° C except for the preheating time. The preheating time is the time from the steamer to water vapor, which normally took about 30 minutes. After keeping the internal temperature of the dryer with a heating wire at 60 ℃, spin the pen and dry the second ginseng evenly for 12 hours, then steam it again at 115 ℃ for 6 hours under the pressure of 0.12MPa 250 g of black ginseng root of the present invention was prepared by drying in a third time (hereinafter, referred to as “BPG”).

Example 2. Preparation of Black Ginseng Root Extract

      20 g of BPG prepared by the method of Example 1 was taken and pulverized, 100 ml of water was added thereto, and the mixture was left at room temperature for 5 hours, followed by 1 liter of 80% ethanol (20% water). It was. The extracted solution was cooled to room temperature, filtered and the ethanol solvent was removed using a vacuum condenser to obtain 7.4 g of black ethanol soluble extract of black ginseng root (hereinafter referred to as “BPGE”).

Example 3. Preparation of Black Ginseng Root Butanol Soluble Extract

200 ml of water was suspended in 3.2 g of BPGE obtained in Example 2, and 200 ml of ether was added to remove the nonpolar material. 200 ml of saturated butanol was added to the remaining aqueous layer to extract saponins present in ginseng, and then the solvent was removed under a reduced pressure concentrator to obtain 0.7 g of black ginseng root butanol soluble extract containing a large amount of ginseng saponin (hereinafter referred to as “BPGBE”). Business card).

      The BPGBE was used as a sample for component analysis and high pathogenic avian influenza virus experiments.

Example  4. Analysis of Black Ginseng Saponin Components

In order to analyze the components of ginseng saponin present in the black ginseng root extract, 20 mg of BPGBE obtained in Example 3 was dissolved in 1 ml of methanol, and the filtrate was filtered through a 0.45 μm filter. It was. As a result of component analysis, Rb1 2.3 mg, Rb2 2.1 mg, Rc 1.3 mg, Rd 1.4 mg, Re 0.5 mg, Rg1 1.2 mg, Rk1 3.7 mg, Rg5 4.7 mg, Rg3 per 1 g of BPGBE. It was confirmed that (S) was 5.4 mg and Rg3 (R) was 4.5 mg.

Column: Shiseido C18 column (5㎛, 1504.6mm) Flow rate 1 ml / min Detector ELSD-LT detector Solvent A; CH ₃ CN: H ₂ O: 5% CH ₃ COOH = 15: 80: 5, B; CH ₃ CN: H ₂ O = 80:20 0 min (0% B), 0-10 min (30% B), 10-25 min (50% B), 25-40 (100% B), 40- 50 minutes (100% B), 50-55 minutes (0% B), 55-58 minutes (0% B) Temperature 40 degrees

Experimental Example  1. Measurement of antiviral activity

In order to measure the virus inhibitory effect of the black ginseng root extract prepared in the above example was performed as follows.

In this experiment, H5N1 (NIBRG-14; UK NIBSC), a highly pathogenic avian influenza virus, was used. Each experimental group was used in the experiment similarly four BALB / c mice as a group. Three-week old BALB / c mice (males) purchased from Samta Co., Ltd. were administered with 30 mg of black ginseng root extract at a concentration of 10 mg / Kg / day for 30 days, and then 100 mouse lethal doses 50 / ml of 0.1 ml H5N1 vaccine strain (NIBRG-14; NIBSC, UK) was inoculated with the nose. Mortality of the mice was observed for 14 days after infection. The control group is a group infected with the virus without administration of the black ginseng root extract sample of the present invention.

sample Dose (mg / kg / day) Lethality (%) BPGE 10 25 BPGBE 10 0 Control group - 100

As a result of the experiment, as shown in Table 2, the control group showed 100% mortality after 2 weeks of virus infection. On the other hand, the black ginseng root ethanol extract administration group (BPGE) showed 25% mortality after one of the four mice used in the experiment died two weeks after the virus infection. On the other hand, two weeks after the virus infection of the black ginseng root butanol extract administration group (BPGBE) all four mice used in the experiment did not die, the mortality rate was 0%. In other words, black ginseng was found to have excellent virus inhibitory effect. Especially, black ginseng root butanol extract showed better virus inhibitory effect than black ginseng root ethanol extract.

Experimental Example  2. Acute Toxicity Test

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats from the Korea Experimental Supply Center.

After the oral administration of the black ginseng root crude extract of Example 2 at a dose of 1 g / kg to two animals in each group, the mortality, clinical symptoms, and weight changes of the animals were observed. An autopsy was performed, and autopsy was performed to visually observe the abnormalities of the organs and thoracic organs.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxic changes were observed in weight change, blood test, blood biochemical test and autopsy findings. As a result, the extract of the present invention did not show a change in toxicity even in rats up to 1 g / kg, respectively, it was confirmed that the minimum lethal dose (LD ₅₀ ) is determined to be a safe substance more than 1 g / kg.

It is merely to illustrate the preparation of the pharmaceutical composition comprising the black ginseng root extract of the present invention as follows, it is not intended to limit the present invention by this only intended to explain in detail.

Formulation example  One. Powder  Produce

BPGE (Example 2) 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation example  2. Preparation of Tablets

BPGBE (Example 3) 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3. Manufacture of capsule

BPGE (Example 2) 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

BPGBE (Example 3) 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5. Liquid  Produce

BPGE (Example 2) 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method for preparing a liquid, each component is added and dissolved in purified water, lemon flavor is added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by adding purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of health food

BPGBE (Example 3) 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

0.13 mg of vitamin B ₁

0.15 mg of vitamin B ₂

0.5 mg of vitamin B ₆

Vitamin B ₁₂ 0.2 g

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

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 mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation example  7. Manufacture of health drinks

BPGE (Example 2) 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

Vitamin B ₁ 0.25 g

Vitamin B ₂ 0.3 g

Water quantification

After mixing the above components in accordance with the 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 refrigerated Used to prepare the healthy beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (7)

A first step of washing 4 to 6 years old Korean ginseng roots three times for 10 minutes with an ultrasonic cleaner and then drying for 22 to 26 hours at a temperature of 35 to 45 ° C .; Putting the dried Korean ginseng root into a steamer and steaming for 4 to 6 hours at a temperature of 85 to 105 ° C except for a preheating time; A third step of drying at a temperature of 55 to 65 ° C. for 10 to 14 hours; A fourth step of steaming for 5 to 7 hours at a temperature of 110 to 120 ° C. under a pressure of 0.1 to 0.20 MPa; Obtaining a black ginseng root formed by performing a fifth step of drying for 10 to 14 hours in a dryer once; After adding 4-6 times the mass of black ginseng root and leaving it at room temperature for 3-7 hours, the mixture of water and ethanol was added 8-12 times the mass for 3 hours to 4 hours, 3 times, reflux extraction. A pharmaceutical composition for the prevention and treatment of highly pathogenic avian influenza virus infection with H5N1 serotype, which contains black ginseng root water and ethanol mixed solvent soluble extract obtained through cooling, filtration and concentration steps as an active ingredient. Suspending the black ginseng root crude extract of claim 1 by adding water and then removing the non-polar substance by adding ether or dichloromethane; High pathogenicity with H5N1 serotype containing black ginseng root butanol soluble extract containing ginseng saponin as an active ingredient through the final step of extracting saponin present in ginseng 2 to 6 times under reduced concentration by adding saturated butanol to the remaining water layer Pharmaceutical composition for the prevention and treatment of avian influenza virus infection. delete delete A first step of washing 4 to 6 years old Korean ginseng roots three times for 10 minutes with an ultrasonic cleaner and then drying for 22 to 26 hours at a temperature of 35 to 45 ° C .; Putting the dried Korean ginseng root into a steamer and steaming for 4 to 6 hours at a temperature of 85 to 105 ° C except for a preheating time; A third step of drying at a temperature of 55 to 65 ° C. for 10 to 14 hours; A fourth step of steaming for 5 to 7 hours at a temperature of 110 to 120 ° C. under a pressure of 0.1 to 0.20 MPa; Obtaining a black ginseng root formed by performing a fifth step of drying the dryer for 10 to 14 hours once; After adding 4-6 times the mass of black ginseng root and leaving it at room temperature for 3-7 hours, the mixture of water and ethanol was added 8-12 times the mass for 3 hours to 4 hours, 3 times, reflux extraction. A health functional food for the prevention and improvement of high pathogenic avian influenza virus infection with H5N1 serotype, which contains black ginseng root water and ethanol mixed solvent soluble extract obtained through cooling, filtration and concentration steps as an active ingredient. Suspending the black ginseng root crude extract of claim 5 in water and then adding ether or dichloromethane to remove the nonpolar material; High pathogenicity with H5N1 serotype containing black ginseng root butanol soluble extract containing ginseng saponin as an active ingredient through the final step of extracting saponin present in ginseng 2 to 6 times under reduced concentration by adding saturated butanol to the remaining water layer Health functional food for the prevention and improvement of avian influenza virus infection. delete