KR20130081479A - A composition comprising the combined extract of ivy leaf extract and coptis chinensis for preventing and treating influenza viral disease - Google Patents

Abstract

PURPOSE: A complex herbal medication extract composed of a Hedera helix leaf and a barberry root is provied to utilize as a medicinal composition for prevention and treatmentof an infectious disease caused by an influenza virus by checking survival rate when injected together with an anti-virus agent. CONSTITUTION: A medicinal composition for prevention and treatment of an infectious disease caused by an influenza virus contains a complex herbal extract with a complex herb medication composed of a Hedera helix leaf and a barberry root as an active component. a complex medicinal extract is a compound in which a dried weight ratio (w/w) of the Hedera helix leaf extract and the barberry root in 1-10:1 are mixed in a mixing ratio. The Hedera helix extract is a crude extract, a polar solvent extract, or a non-polar solvent soluble extract. The barberry extract is water, a low quality alcolhol with carbon number ranged from 1-4 such as methanol, butanol, an extract which is useful in a selected solvent from a mixed solvent or a bubbled butanolo extract. The medicinal composition for prevention and treatment of an infectious disease caused by an influenza virus contains the combination of the complex herbal medication extract composed of the Hedera helix leaf and the barberry root and an anti virus agent. [Reference numerals] (AA) Survival rate (%); (BB) Time (day) after infection; (CC) Ivy leaf; (DD) Ivy leaf + coptis

Description

A composition comprising the combined extract of ivy leaf extract and Coptis chinensis for preventing and treating influenza viral disease}

The present invention provides a composition for the prevention and treatment of influenza virus-related diseases containing a complex herbal extract consisting of ivy leaf and yellow lotus 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.

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

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

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

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

12 Demirci et al., Pharmazie 59, 770-774, 2004:

[Document 13] Korean Patent Publication No. 10-2006-8310

Document 14 Majester-Savornin et al., Planta Med. 57, 260-262, 1991;

Reference 15 Exp. Parasitol. 116, 340-345, 2007,

Mendel et al., J. Ethnopharmacol. 134, 796-802, 2011,

Document 17 Gepdiremen et al., Phytomedicicne 12, 440-444, 2005

[Document 18] One Information Interview, Dohae Hyangjeomsa Dictionary, Younglimsa, p490-493, 1998

[19] Sang-Uk Seo et al., J. Virol. 2010.84 (24): 12713-12722)

Due to the concentration of the population due to the development and urbanization of transportation, respiratory infectious diseases caused by influenza, pneumococci, etc., which are spread to the air, pose a serious threat to the public. Among them, influenza virus belongs to the orthomyxovirus family, and is an RNA virus that is infected through the respiratory mucosa, and glycoproteins HA (Hemagglutinin) and NA (Neuraminidase) exist on the surface and are classified into various subtypes according to the antigenicity of HA and NA. . In addition, Avian influenza A (H5N1), the first human infection reported in 1997, continues to report infections in livestock, including birds, with mortality of more than 60%. It is the object of alert.

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), which are more than 95% highly deadly and also called "bird flu" (Alexander DJ, Vet. Microbiol., 74 (1-2), p. 3-13, 2000). The highly pathogenic algal influenza is classified as Class A by the International Water Services Bureau (OIE) and domestic type 1 infectious disease.

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 20th century, the world-wide pandemic between 1918 and 1920, the pandemic of the first influenza pandemic (aka Spanish flu) has been recorded as the greatest damage to humanity, and between 20 and 50 million people died 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).

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 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).

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 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.

To date, subunit vaccines, inactivated vaccines or attenuated vaccines, etc. have been used to prevent influenza virus infections, but the prevention of the influenza virus is limited due to high antigenic mutations. For example, influenza A (H1N1), first detected in March 2009 in San Diego, California, USA, affected more than 14,000 people in 48 countries, including Korea, in just two months. It is epidemic worldwide and has resulted in more than 130,000 infections and more than 300 deaths ( http://www.who.int/influenza ).

Accordingly, antiviral agents currently used to treat influenza infections include Oseltamivir (Tamiflu) and Zanamivir (Relenza), which are inhibitors of influenza NA (neuraminidase), or Amantadine and Rimantadine, which are inhibitors of M2 ion channels of influenza A virus. It is used for the purpose of prevention and treatment of influenza, and it has been reported to be effective to administer influenza virus using these antiviral agents within 24-48 hours after infection. In particular, the recent emergence of resistant viruses to these antiviral agents is the urgent situation to develop a new concept of treatment to overcome this.

Ivy ( Hedera helix ) is a plant called English ivy and its leaf extract has an expectorant action and is used for the treatment of bronchitis (Korean Patent Publication No. 10-2004-106201), its main ingredient is reported as saponin component called hederacoside ( Demirci et al., Pharmazie 59, 770-774, 2004: Korean Patent Publication No. 10-2006-8310). In addition to their expectorant action, the extracts of their leaves have been reported to have antibacterial, antifungal, anti-lesmanian, and digestive effects (Majester-Savornin et al., Planta Med. 57, 260-262, 1991; Exp. Parasitol. 116, 340-345, 2007, Mendel et al., J. Ethnopharmacol. 134, 796-802, 2011), several activities of hederacoside C, the active substance of ivy leaf, have been reported. It is. In other words, anti-inflammatory activity, antibacterial activity and antifungal activity have been reported (Gepdiremen et al., Phytomedicicne 12, 440-444, 2005), but there is no known antiviral activity.

Rhubarb refers to the root of perennial herbaceous Coptis chinensis and the same plant belonging to the genus Ranuculaceae, and its components include alkaloid compounds such as berberine, coptisine, and palmatine. And it is known to contain components such as obacunone and obaculactone, antibacterial action, blood pressure lowering effect, organ smooth muscle relaxation action such as uterus is known (information interception 1 person, illustrated flavor medicine) Ambassadors, Younglimsa, p490-493, 1998).

However, none of these documents discloses or teaches the inhibitory activity against the influenza virus of the complex herbal extract consisting of ivy leaf and yellow lotus.

Therefore, the present inventors tested the inhibitory activity against influenza virus against ivy leaf and rhubarb extracts for the purpose of use as a therapeutic agent for influenza virus infection, and showed a strong inhibitory activity, especially when combined with existing antiviral agents. The inventors discovered for the first time the present invention and completed the present invention.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of infection by influenza virus containing a complex herbal extract consisting of ivy leaf and yellow lotus as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of the infection caused by influenza virus containing a complex herbal extract consisting of ivy leaf and yellow lotus as an active ingredient.

Complex herbal extracts defined herein are formulated at a blending ratio of 1 to 10: 1, preferably 1 to 5: 1, and more preferably 1 to 3: 1 dry weight ratio (w / w) of the ivy leaf and sulfur extract. Characterized in that it comprises a formulated formulation.

Ivy leaf extract as defined herein is characterized in that the crude extract, polar solvent soluble extract or non-polar solvent soluble extract.

The yellow lotus extract as defined herein is characterized in that the crude extract, polar solvent soluble extract or nonpolar solvent soluble extract, preferably crude extract.

The crude extract as defined herein is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably 10 to Characterized in that the extract is available in 60% ethanol.

Non-polar solvent soluble extract fractions as defined herein include extract fractions soluble in non-polar solvents purified only from the crude extract of the present invention in hexane, methylene chloride, chloroform, or ethyl acetate, preferably hexane, or chloroform solvent. do.

The polar solvent soluble extract as defined herein is a solvent selected from water, methanol, butanol, or a mixed solvent thereof remaining after removing the nonpolar solvent soluble fractions from the crude extract, preferably water or butanol, more preferably saturated butanol Extract fractions available for use.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of infection by influenza virus containing a combination of a combination herbal extract consisting of ivy leaf and yellow lotus as an active ingredient as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of the infection caused by influenza virus containing a combination of a combination herbal extract consisting of ivy leaf and rhubarb and an existing antiviral agent as an active ingredient.

Existing antiviral agents as defined herein are Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel) and Rimantadine, Rivaririn, Taribavirin, and the like, preferably Oseltamivir or Amantadine.

Influenza viruses as defined herein are H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 or H10N2 or H10N7 It is characterized by having the H1N1 serotype.

Infections caused by influenza viruses as defined herein include avian influenza (bird flu), pandemic flu, and flu.

Hereinafter, the present invention will be described in more detail.

The extracts of the present invention can be obtained by the following production methods.

For example, the present invention will be described in detail below.

Ivy leaf and sulfur extract of the present invention can be prepared as follows. After washing and cutting the dried ivy leaf and the yellow lotus, a solvent selected from water, including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably Preferably, 30-100% ethanol is mixed several times, followed by ultrasonic extraction method, hydrothermal extraction method for 30 minutes to 48 hours, preferably 1 hour to 12 hours at a temperature of 30 ° C to 150 ° C, preferably 50 ° C to 100 ° C, The crude extract of the present invention can be obtained by filtration, concentration under reduced pressure, and drying of the extract obtained by repeating room temperature extraction or reflux extraction, preferably ultrasonic extraction, about 1 to 20 times, preferably 2 to 10 times.

In addition, the polar solvent or non-polar solvent soluble extract of the present invention is about 0.0005 to 0.005 times the weight of the crude extract, preferably 30 to 90% ethanol crude extract, preferably 0.05 to 0.5 times the volume (v / w% Non-polar solvent soluble extract fractions which are available in non-polar solvents such as n-hexane, methylene chloride, ethyl acetate by carrying out a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol after addition of water; And polar solvent-soluble extract fractions soluble in polar solvents such as butanol and water can be obtained.

The present inventors have tested the inhibitory activity against the influenza virus in the mixture of the extracts obtained by the above production method, showed a strong inhibitory activity, especially when combined with an existing antiviral agent shows a high virus inhibitory activity By confirming, the composition was found to be useful as a pharmaceutical composition or health functional food for the prevention and treatment of infection by influenza virus.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of infectious diseases caused by influenza virus containing as an active ingredient a complex herbal extract consisting of ivy leaf and yellow lotus obtained by the above method.

The pharmaceutical composition of the present invention comprises 0.1 to 50% by weight of the extract, based on the total weight of the composition.

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

Carriers, excipients and diluents that may be included in the compositions of the present invention 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.

The composition comprising the extract of the present invention is formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. Can be used.

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules and capsules, which may contain at least one excipient such as starch, calcium carbonate, sucrose, ), Lactose, gelatin and the like.

In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. As the non-aqueous solvent and suspending agent, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyloleate may be used. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, and glycerol gelatin can be used.

Preferred dosages of the mixtures of the extracts of the present invention depend 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, but may be appropriately selected by those skilled in the art. However, for the desired effect, the mixture of the extract of the present invention may be administered in 0.0001 ~ 100 mg / kg, preferably in an amount of 0.001 ~ 100 mg / kg divided once to several times daily. Extract of the present invention in the composition may be formulated in an amount of 0.0001 to 50% by weight relative to the total weight of the composition.

The pharmaceutical composition 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 and intracere broventricular injections.

In addition, the present invention provides a health functional food for the prevention and improvement of the infection caused by influenza virus containing a complex herbal extract consisting of ivy leaf and yellow lotus obtained by the above production method as an active ingredient.

Extract of the present invention can be used in various ways, such as drugs, foods and drinks for the prevention and treatment of the desired disease. Foods to which the extract of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes and dietary supplements, and may be used in the form of powders, granules, tablets and capsules or beverages. have.

The mixture of the extract of the present invention may be added to food or beverage for the purpose of preventing and treating the desired disease. At this time, the amount of the extract in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 30 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 10 g.

In addition to containing the composition as an essential ingredient in the indicated ratio, the health beverage composition of the present invention has no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose, polysaccharides such as maltose, sucrose and the like, such as dextrin, cyclodextrin and the like , Xylitol, sorbitol and erythritol. As flavoring agents other than those described 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 said 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 includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and 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 drinks, and the like. In addition, the extract of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. 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 composition of the present invention.

As mentioned above, the composite herbal extract consisting of the ivy leaf and the yellow lotus of the present invention showed a significant increase in survival as a result of measuring the survival rate of mice in the influenza virus infection animal model of the extracts, in particular, By confirming the high survival rate increase when combined with the existing antiviral agent, the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of infection by influenza virus.

1 is a diagram showing the survival rate of mice infected with influenza virus by simultaneous administration of 30% ivy leaf and yellow lotus extract.

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 merely illustrative of the present invention, and the contents of the present invention are not limited by the following Examples, Reference Examples and Experimental Examples.

Example   One. Ivy leaves  Preparation of extract

1-1. Ivy leaves Crude extract  Produce

600 g of dried leaves of English Ivy leaf, identified under Prof. Kwon Yong-soo (Natural Products, www.kangwon.ac.kr) of Kangwon National University, were mixed with various concentrations of water and ethanol, ie 30% and 200 ml of 70% and 95% (v / v) ethanol solvents were added and extracted three times at 60 ° C. with an ultrasonic extractor (8510 DHT; CT, USA) for 3 hours, followed by vacuum filtration to recover the supernatant. This process was repeated four times, and the supernatant was collected, and then dried under reduced pressure and lyophilizer (FD5512, Ilshin, Seoul, Korea) to extract 30% and 70% (v / v) ethanol mixed solvent extracts of the ivy leaf, respectively. g and 80.2 g were obtained respectively.

In addition, 30% and 70% ethanol extract of the ivy leaf was confirmed to be the same when compared with the extract purchased from Europe imported Sinpung Pharmaceuticals.

Example   2. goldthread  Preparation of extract

600 g of dried yellow lotus was identified under the influence of Professor Kwon Yong-soo (Natural Products, www.kangwon.ac.kr) of Kangwon National University, and 200 ml of saturated butanol solvent and various concentrations of water and ethanol mixed solvent were added at 60 ℃. The supernatant was recovered by extraction with an ultrasonic extractor (8510 DHT; CT, USA) three times for an hour and then vacuum filtered. This process was repeated four times to collect the supernatant, and then dried under reduced pressure and lyophilizer (FD5512, Ilshin, Seoul, Korea), of which 91.2 g of saturated saturated butanol extract was obtained.

Example   3. Ivy leaves  And goldthread  Preparation of Extract Combination

The composite herbal extract was prepared by mixing 3: 1 or 1: 1 with respect to the weight of the ivy leaf 30% EtOH extract and the rhubarb saturated butanol extract of Example 1, and was used in the following experimental example. )

Experimental Example  1. Inhibitory activity against influenza virus

In order to confirm the inhibitory activity against the influenza virus of the samples obtained in the above example, the experiment was performed by applying the method described in the literature (Sang-Uk Seo et al., J. Virol. 2010. 84 (24) 1271-12722)

1-1. Influenza virus  culture

In order to obtain influenza virus A / PR / 8/34 (PR8, H1N1) from eggs, SPF fertilized eggs (orientbio) were grown for 11 days in a 37 ° C incubation period and then labeled 2/3 site to the lateral vesicle of the fertilized egg. . The A / PR / 3/34 virus parent strain was diluted 10 ⁴ to 10 ⁶ times by a step dilution method using PBS (Phosphate-Buffered Saline) to infect 100 ul using a syringe. The infected area was closed with a paraffin to prevent infection by other bacteria or viruses. Infected fertilized eggs were grown in a 37 ° C. incubator for 3 days and then stored at 4 ° C. for 12 hours. Urinary fluid was collected after cutting the bladder area with surgical scissors. The ureter was passed through a filter and then dispensed and stored at -80 ° C. For virus titer, 10 ⁶ MDCK cells (Canine kidney epithelial, ATCC) were passaged in each well of a 6 well plate. The next day, virus samples were diluted by step dilution using MEM medium (Invitrogen) and 500 ul of each well was added. Infection was performed on a rocking plate at room temperature for 30 minutes, and after infection, cells were washed with MEM medium and then cultured for 2 days by adding 5 ug / ml of trypsin (Invitrogen) to MEM medium containing 1% agarose. . After 2 days, the number of plaques was measured in a well where 50-200 plaques were observed, and the titer of the virus sample was determined by inverting the dilution factor.

1-2. Influenza virus Dosage and observation of symptoms

The mice used in the experiment (C57BL / 6, female, 6 weeks, purchased from Orient-Bio) were intraperitoneally mixed with ketamine (100 mg / kg, Yuhan) and lump (20 mg / kg, BAYER) before infection. Anesthetized by administration. The nasal cavity of anesthetized mice was infected three times with virus diluted 1 × 10 ⁵ pfu at 30 ul. Survival of mice assessed resistance to influenza virus.

In order to verify the anti-influenza effect of the medicinal herb extract consisting of ivy lobe and nasturtium in the influenza infection model, 10 ⁵ pfu / 30 ul of PR8 influenza virus was administered to the nasal cavity and 2 days after the influenza administration for 5 days And the survival rate of the mice was observed after oral administration of the herbal extract consisting of rhubarb (30 mg / Kg).

1-3. Ivy leaves  extract, Fraction goldthread  Antiviral Effects of Combination Extracts

As a result of the experiment, when the ivy leaf extract and the rhubarb extract at the same time orally administered compared with the control mice and ivy leaf extract administration group was observed a significant increase in survival (Fig. 1). Therefore, it was confirmed that the anti-influenza effect of the mouse significantly increased by the administration of 30% ivy leaf extract containing sulfur.

In a similar manner, the influenza treatment effect of the mixed herbal extract consisting of 30% or 70% of the ivy leaf and the yellow lotus extract is shown in Table 1.

Antiviral Effects of Ivy Leaf Extracts, Fractions and Mixtures Group Dose (mg / Kg) n Survival rate after 15 days of infection (%) PR8-treated - 20 0 PR8 + Ivy Leaf 30% EtOH Extract (30mg / Kg) + Nasturtium Saturated Butanol Extract (10mg / Kg) 40 10 20 PR8 + Ivy Leaf 70% EtOH Extract (30mg / Kg) + Nasturtium Saturated Butanol Extract (10mg / Kg) 40 5 10 PR8 + Ivy Leaf 30% EtOH Extract (30mg / Kg) + Nasturtium Saturated Butanol Extract (30mg / Kg) 60 10 60 PR8 + Ivy Leaf 70% EtOH Extract (30mg / Kg) + Nasturtium Saturated Butanol Extract (30mg / Kg) 60 5 20

1-4. Antiviral effect by coadministration with existing antiviral agents

In the case of antiviral agents currently used for influenza viruses, the emergence of antiviral resistant influenza has been reported, and therefore measures to overcome them are urgently needed. Therefore, we tried to increase the effectiveness of anti-influenza agent when used in combination with anti-influenza agent, which is composed of ivy leaf and rhubarb, to prevent the emergence of resistant virus and to check whether it can be used in combination with influenza treatment.

Table 2 shows the anti-influenza effect of the combined herbal extract consisting of the ivy leaf extract and the antibacterial agent and antiviral agent isolated in the Example.

Combined Administration of Ivy Leaf Extracts and Mixtures with Antiviral Agents Group Dose (mg / Kg) n % Survival 15 days after infection PR8-treated - 9 0 PR8 + Oseltamivir (5mg / Kg) 30 10 60 PR8 + Oseltamivir (5mg / Kg) + Ivy Lobe 30% EtOH Extract (30mg / Kg) + Barberry Saturated Butanol Extract (10mg / Kg) 40 5 100 PR8 + Oseltamivir (5mg / Kg) + Ivy Lobe 30% EtOH Extract (30mg / Kg) + Barberry Saturated Butanol Extract (30mg / Kg) 60 5 100 PR8 + 1-adamatylamine (10mg / Kg) 5 20 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Lobe 30% EtOH Extract (30mg / Kg) 30 5 20 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Leaf 70% EtOH Extract (30mg / Kg) 30 5 40 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Lobe 30% EtOH Extract (30mg / Kg) + Barberry Saturated Butanol Extract (10mg / Kg) 40 5 50 PR8 + 1-adamatylamine (10mg / Kg) + Ivy Lobe 30% EtOH Extract (30mg / Kg) + Barberry Saturated Butanol Extract (30mg / Kg) 60 5 50

Experimental Example  2. Toxicity Test

Acute toxicity was tested in ICR mice (male, 6 weeks, purchased from Aruet) according to KFDA specifications. As a result, the complex herbal extract consisting of 30% ivy leaf extract, 70% ivy leaf extract, ivy leaf extract and rhubarb did not show acute toxicity until oral administration of 2 g / kg.

As a result of the above experiments, the complex herbal extract consisting of the ivy leaf extract and the yellow lotus can be safely used for influenza virus infection, and especially when combined with an antiviral agent, the activity was high. Therefore, the extract of the ivy leaf may be used alone, or may be used together with other herbal extracts (for example, sulfur extract) as necessary.

One example of the formulation of the pharmaceutical composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail.

Formulation example  One. Sanje  Produce

HCE 300 mg

Lactose 100 mg

10 mg talc

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

HCE 300 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

HCE 300 mg

3 mg of microcrystalline cellulose

Lactose 14.8 mg

0.2 mg magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of Injectables

HCE 300 mg

Di-mannitol 180 mg

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 12H ₂ O 26 mg

(2 ml) per ampoule in accordance with the usual injection method.

Formulation example  5. Liquid  Produce

HCE 300 mg

10 g of isomerized sugar

5 g of di-mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

HCE 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

vitamin 0.15 mg

vitamin 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

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

HCE 300 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

0.3 g of vitamin B ₂

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 It is used to prepare a healthy beverage composition of the present 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 (9)

A pharmaceutical composition for the prevention and treatment of infectious diseases caused by influenza virus, which contains a complex herbal extract consisting of ivy leaf and yellow lotus as an active ingredient. The method of claim 1,
The composite herbal extract is a pharmaceutical composition, characterized in that the blending ratio of the dry weight ratio (w / w) of the ivy leaf and sulfur extract of 1 to 10: 1. The method of claim 1,
The ivy leaf extract is a pharmaceutical composition, characterized in that crude extract, polar solvent soluble extract or non-polar solvent soluble extract. The method of claim 3, wherein
The crude extract is a pharmaceutical composition, characterized in that the extract is soluble in a solvent selected from lower alcohols having 1 to 4 carbon atoms, such as water, methanol, ethanol, butanol, or a mixed solvent thereof. The method of claim 1,
The sulfur extract is a pharmaceutical composition, characterized in that the extract or a saturated butanol extract available in a solvent selected from a lower alcohol having 1 to 4 carbon atoms, such as water, methanol, ethanol, butanol, or a mixed solvent thereof. A pharmaceutical composition for the prevention and treatment of infectious diseases caused by influenza virus, which contains a combination herbal extract consisting of ivy leaf and yellow lotus as an active ingredient. 7. The method according to claim 1 or 6,
The influenza virus is H1N1, H1N2, H2N2, Human B, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H9N2 or H10N7. 7. The method according to claim 1 or 6,
The infection caused by the influenza virus is a bird flu (bird flu), pandemic flu, or a pharmaceutical composition, characterized in that the flu. The method according to claim 6,
The existing antiviral agents are Oseltamivir (Tamiflu), Zanamivir (Relenza), Peramivir, Amantadine (Symmetrel). Pharmaceutical composition, which is Rimantadine, Rivaririn, or Taribavirin.

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