KR102278545B1 - Antiviral Composition and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a natural antiviral composition and a method for manufacturing the same. More specifically, the natural antiviral composition includes, as effective ingredients, composite extract containing a gold silverization extract, an eoseongcho extract, a panram root extract, a golden extract, a sulphur extract, a rhubarb extract, an enamel extract, a reinforced lion's balsam extract, a broccoli extract, and a fermented horseradish extract. The natural antiviral composition has antiviral effects against influenza A virus, adenovirus, norovirus, and polyovirus. According to the present invention, the natural antiviral composition is provided, excellent antiviral activity and anti-bacterial activity may be produced against influenza A virus, adenovirus, norovirus, and poliovirus without toxicity or skin irritation. Therefore, the present invention is variously applicable to sanitary products for preventing various viruses and bacteria and preventing viral or bacterial infectious diseases.

Description

Natural antiviral composition with excellent antiviral effect and manufacturing method thereof

The present invention relates to a natural antiviral composition and a method for producing the same, and more particularly, by including a natural complex extract and a fermented wasabi extract as active ingredients, influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus), to a natural antiviral composition excellent in antiviral effect against poliovirus, and a method for producing the same.

Virus means a toxic substance in Latin, and means a group of infectious pathogenic particles that pass through a bacterial filter paper (0.22㎛). Viruses exist around us, and some pathogenic microorganisms and viruses such as bacteria and fungi cause diseases and harm the human body, such as generating bad odors.

Accordingly, interest in hygiene management products to improve the environment for these viruses and microorganisms is increasing. For example, there are various types of viruses that can prevent infection itself with high epidemic viruses such as SARS, Influenza A virus, H1N1 influenza, Ebola virus, MERS-CoV, including COVID-19. Products are being developed.

Influenza virus kills 250,000 to 500,000 people worldwide every year, infects 10-40% of children, and causes a serious global disease that causes national economic loss.

After the Spanish flu pandemic in 1918, influenza pandemics occurred every few decades, such as the Asian flu in 1957 and the Hong Kong flu in 1968. With the advent of the H1N1 influenza virus (2009pdm) in 2009, 270,000 people were infected worldwide, and more than 3,200 people were infected. died Due to the possibility of the outbreak of a new highly contagious mutant in the Southern Hemisphere, even though measures are being taken against the pandemic, the social impact and concerns are not diminishing.

Influenza is an acute respiratory disease accompanied by chills, fever, muscle pain, and cough, and is usually transmitted through human-to-human transmission through aerosol released into the air through coughing or sneezing of an infected person. It can also be transmitted through bird droppings, and compared to the common cold, a cold is a respiratory disease caused by infection with adenovirus, rhinovirus, coxsackie virus, and coronavirus, and usually does not accompany muscle pain or rapid high fever. Influenza infection is caused by an influenza virus belonging to the orthomyxoviridae, and as an RNA virus, antigenic mutation is relatively easy, so repeated reinfection is possible within the infected population.

Influenza viruses that cause human infection are type A and B, and the virus antigen mutation is determined by HA (hemagglutinin) and NA (neuraminidase) on the surface of the virus. According to the genetic combination of the virus surface proteins HA and NA, H1-H16 and N1-N9 subtypes are generated. Among them, the subtypes of human influenza A virus are three types H1, H2, H3, and N1 and N2 are two types of each other. determined by other combinations. The reason influenza B does not cause a pandemic is that, unlike type A, it only infects humans, so there is no gene from an animal virus.

Influenza antiviral agents oseltamivir and amantadine act on the NA and M2 channels, respectively, but the problem of resistant viruses is serious. Although oseltamivir and zanamivir are still effective for 2009pdm H1N1 derived from pigs, type B resistant to H5N1, H1N1, and zanamivir that are resistant to oseltamivir has already been reported. is becoming The life cycle of influenza virus takes approximately 2-3 days in human cells.

Norovirus, a food hazard pathogen, is known to be the cause of more than 90% of nonbacterial enteritis in developed countries, and is a major cause of food poisoning in all age groups around the world. According to a report from the U.S. Centers for Disease Control and Prevention in January 2011, norovirus took the first place with 46% of the causes of food poisoning, and long-term nursing homes had the highest number of norovirus food poisoning facilities, followed by restaurants, ships, and schools. It can be seen that mainly occurs in group catering facilities. Each year in the United States, 48 million people, or 1 in 6 Americans, get food poisoning, 128,000 hospitalizations, and 3,000 deaths. 90% of food poisoning deaths leading to death are caused by the norovirus pathogen (CDC December 15, 2010).

In Korea, the number of food poisoning patients with norovirus continues to increase, accounting for about 30% of the total food poisoning patients, except for 2009, when the swine flu was affected after 2006. is counted as Norovirus is a virus that can survive in winter when the temperature is dry and low. It has a physically and chemically stable structure, so it can survive for a long time at room temperature for 10 days, at 10℃ seawater for 30-40 days, and at minus 20℃ or less. have. It can be transmitted to humans through food and water, and can also be transmitted through direct contact with a patient or through the air. Food poisoning can be induced by ingestion of only 10 particles of norovirus, and it has a strong infectious power that can be transmitted for two weeks even after the symptoms have disappeared. Norovirus is a single-stranded positive RNA virus and has a 7.5 kb RNA genome. Among the three main ORFs, ORF2 and ORF3 have capsid protein genetic information.

Adenovirus (scientific name: Adenoviridae) is a medium-sized virus with a diameter of 90-100 nm and has no envelope. It has an icosahedron shape and has double helix DNA. Adenovirus is responsible for 5-10% of upper respiratory tract infections in children, and adults can also be infected.

Unlike other viruses whose clinical course is relatively better than bacterial pneumonia, adenovirus is known to have a similar pattern to severe bacterial pneumonia, and can cause fatal results or leave sequelae such as permanent lung damage. Viruses belonging to the adenoviridae family can infect several vertebrates, including humans. The adenovirus was first isolated from the human adenoid, hence the name "adenovirus". The most common symptom of adenovirus infection is upper respiratory tract disease. Adenovirus infection is often accompanied by conjunctivitis, tonsillitis, otitis media, laryngitis, and gastroenteritis. In particular, children can get bronchiolitis or pneumonia, which can worsen significantly. Babies may cough as if they have whooping cough. In addition, adenoviruses can rarely cause encephalitis or cystitis.

Most people heal themselves when they become infected, but people with immunodeficiency may die from infection, and in very rare cases, even healthy people can die from infection. Adenovirus is often transmitted by coughing up material, but it can also be transmitted by direct contact with an infected person or by virus particles left on objects such as towels or faucets.

According to a weekly report by the Respiratory and Intestinal Virus Surveillance System (NREVS) in the United States, 3.4% of adenovirus gene (PCR) tests and about 0-1% of virus isolation cultures in patients with respiratory symptoms were positive. Confirmed (US CDC, as of July 2017).

Poliovirus (poliovirus) is a pathogen of polio, a virus belonging to the genus Enterovirus of the Picornaviridae family, has a size of 24 to 30 nm, and is an icosahedral single-stranded RNA genome without an envelope. Polio (poliomyelitis or polio) is an acute infectious disease that invades the central nervous system and is a serious disease. Poliovirus is unique to humans, and the symptoms of infection vary from subclinical infection to mild symptoms, aseptic meningitis or paralysis.

Most poliovirus infections are asymptomatic, but in about 1% of infected people, the virus invades the motor neurons of the anterior spinal cord, causing acute flaccid paralysis (AFP). Viral infection shows various clinical symptoms, from subclinical infection to mild fever symptoms to severe permanent paralysis. The incubation period is usually 7-14 days, and initial clinical symptoms include fever, fatigue, headache, vomiting, constipation, stiff neck, and pain in the extremities. Infected by oral route, primary virus propagation occurs in the throat or gastrointestinal tract, and the virus is already excreted before symptoms appear. Poliovirus proliferates in the tonsils, lymph nodes of the neck, Peyer'patches, and small intestine in humans, invades regional lymph nodes, enters the central nervous system through the bloodstream, and destroys motor neurons, causing polio symptoms. The virus spreads by releasing the virus.

Under this circumstance, recent efforts have been made to overcome the shortcomings of existing antiviral agents overseas and domestically, and as one of the efforts, domestic research on antiviral efficacy of herbal extracts and plant extracts is being actively conducted.

However, when the extract of a single substance was applied, the antiviral effect was mostly insignificant.

KR 10-2016-0079459 A KR 10-2173159 B1 KR 10-1566441 B1 KR 10-0812246 B1

An object of the present invention is to provide a natural antiviral composition having an antiviral effect against influenza A (Influenza A virus), adenovirus, norovirus, and poliovirus, and a method for preparing the same. .

Another object of the present invention is to provide a natural antiviral composition that can ensure stability even when used for a long period of time without toxicity and skin irritation, and a method for preparing the same.

The natural antiviral composition of the present invention for achieving the above object is a complex extract comprising a gold leaf extract, eoseongcho extract, banyan root extract, golden extract, yellow rhubarb extract, rhubarb extract, ephedra extract, fortified sagebrush extract and broccoli extract ; and fermented wasabi extract; as an active ingredient, it is characterized in that it has an antiviral effect against influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus), and poliovirus (Poliovirus).

The fermented wasabi extract is characterized in that it is fermented by inoculating the Lactobacillus sp. strain into the extract of wasabi.

80 to 99.9% by weight of the complex extract and 0.1 to 20% by weight of the wasabi extract are included as active ingredients, and the complex extract is, 0.1 to 20% by weight of the ginseng extract, 0.1 to 20% by weight of the Eosinaceae extract, 0.1 to 20% by weight of the dandelion root extract. 20% by weight, golden extract 0.1 to 20% by weight, yellow rhubarb extract 0.1 to 20% by weight, rhubarb extract 0.1 to 20% by weight, ephedra extract 0.1 to 20% by weight, Ganghwa sajabal mugwort extract 0.1 to 20% by weight, and broccoli extract 0.1 to It is characterized in that it contains 20% by weight.

The method for producing the natural antiviral composition comprises the steps of preparing a complex extract comprising a gold leaf extract, eoseongcho extract, dandelion root extract, golden extract, yellow rhubarb extract, rhubarb extract, ephedra extract, fortified sagebrush extract, and broccoli extract; Comprising the steps of preparing a fermented wasabi extract, mixing and homogenizing the prepared complex extract and fermented wasabi extract, the homogenized composition is influenza A (Influenza A virus), adenovirus (Adenovirus), noro It is characterized in that it has an antiviral effect against viruses (Norovirus) and poliovirus (Poliovirus).

In the step of preparing the complex extract, gold and silver ginseng, eoseongcho, banyan root, gold, yellow rhubarb, rhubarb, ephedra, sagebrush and broccoli are mixed, and this is extracted with a solvent, or ginseng ginseng, eoseongcho, banyan root, gold, yellow rhubarb, After extracting each of rhubarb, ephedra, sagebrush and broccoli with a solvent, the extracts are mixed, and the step of preparing the fermented wasabi extract is extracting wasabi, and inoculating the extract with a Lactobacillus spp. It is characterized by fermentation.

According to the present invention, by being composed of a natural extract, without toxicity or skin irritation, influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus), poliovirus (Poliovirus) excellent antiviral activity and It has antibacterial activity. Accordingly, the present invention has the advantage that it can be applied to various kinds of hygiene products for the prevention of various viruses and bacteria and the prevention of viral or bacterial infectious diseases.

1 to 8 are views showing the results of Test Example 3 according to the present invention.

Hereinafter, the present invention will be described in detail.

The natural antiviral composition of the present invention is a complex extract comprising a gold leaf extract, eoseongcho extract, banyan root extract, golden extract, hwangryeon extract, rhubarb extract, ephedra extract, fortified sagebrush extract and broccoli extract; and fermented wasabi extract; as an active ingredient.

In the present invention, "antivirus" is influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus), poliovirus (Poliovirus), as well as Coronavirus Disease 19 (COVID-19), Newcastle virus, bullous Vesicular stomatitis virus, Cossackievirus, Enterovirus-71, Herpes Simplex Virus, Foot and mouth disease (FMD), Colorado tick fever virus ( Antiviral activity against Colorado tick fever Virus), Reovirus, Human immunodeficiency virus, and Hepatitis B,C viruses, Rotavirus, and Parbovirus It means to have, but does not limit the type of the virus.

The complex extract includes a gold leaf extract, eoseongcho extract, dandelion root extract, golden extract, yellow rhubarb extract, rhubarb extract, ephedra extract, fortified wormwood extract and broccoli extract, gold and silver ginseng, eoseongcho, banyan root, gold, yellow rhubarb, rhubarb , may be extracted from a mixture of ephedra, fortified sagebrush and broccoli, and may be a mixture of extracts extracted individually from these, but is not limited thereto.

The Lonicerae Flos is a bud or a flower just beginning to bloom of Lonicera japonica Thunberg belonging to the Caprifoliaceae family, and is a herbal medicine used in oriental medicine for diuresis, dryness, detoxification, ongjongjeongchang, external persimmon fever, etc. to be. The components of gold and silver coins include chlorogenic acid, iridoid glucosides, loganin, sweroside, secoxyloganin, vogeloside, epivogeloside, and tannin; apigenin, quercetin, ochnaflavone, astragalin and their glycosides have been reported. Recent studies on the gold and silver coins have reported antibacterial, anti-inflammatory, antioxidant, anticancer and immune enhancing effects, and hepatoprotective effects.

The eoseongcho is a perennial herb belonging to the family Saururaceae, called eoseongcho because it smells fishy, and has been widely used as oriental medicine in China. It has been used for whooping cough, bronchitis, pneumonia, and tonsillitis. It has been reported that eoseongcho can be used as a variety of natural preservatives because it has no toxicity and side effects.

The dandelion root (Isatis indigotica Fortune) is a dried root of daechung, a plant of the Brassica family, and has a bitter and cold taste. It is known to have a detoxifying effect and to purify the blood. It has been reported to be used to treat influenza, meningitis, type B encephalitis, pneumonia, solitary, heat-toxic rash, sore throat, mumps, and acute conjunctivitis.

The gold (Scutellariae Radix) is the root of Scutellaria baicalensis Georgi, a perennial herb belonging to the family Lamiaceae, and in oriental medicine, antipyretic, diuretic, small swelling, anti-inflammatory, diarrhea, anesthesia, fever, hypertension, arteriosclerosis, It is used for cholecystitis, jaundice and gastritis. It has been reported that the root contains ingredients such as baicalin, baicalein, wogonin, wogonoside, neobaicalein, and sitosterol. This has been reported

The yellow lily (Coptis chinensis) is a perennial herbaceous plant cultivated in Korea, Japan, China, etc., and as a main component, berberine, palmatin, coptisine, magnoflorine, epiberine ( epiberine), etc. Yellow lotus has been prescribed a lot since ancient times, and it is known to have many pharmacological activities such as antibacterial action, antioxidant action, melanin production inhibitory action, and cancer cell suppression action.

The rhubarb (Eisenia bicyclis) is a marine plant belonging to the brown algae, and is a seaweed belonging to the end of the seaweed family, and the length usually varies depending on the depth of the habitat, but the large one is 1 to 1.5 m or more, and it lives up to 4 years. It is a perennial, and the fluid appears mainly in spring. It is generally distributed in Japan and Korea, especially Eulleungdo, and mainly inhabits the rocky area of the lower intertidal zone. It is mainly used for food, and the concentration of alginic acid, iodine and potassium is high, so it is also used for the production of alginic acid.

The ephedra (Ephedra sinica Stapf.) is an evergreen shrub of the ephedra family, and has been widely applied to haemophilia, bronchial asthma, rhinitis, etc.

The Ganghwa lion wormwood (Artemisia princeps Pampanini) is a perennial herb that grows mainly only in Ganghwa-do, and has been widely used as an anti-inflammatory, analgesic, cardiac, antitussive, and inhalant in oriental medicine, and its antibacterial action is known mainly for its pharmacological action. In addition, the content of phenol, flavonoid, and amino acid is relatively higher than that of other mugwort, so it is expected to contain a lot of active ingredients. And among edible plants, mugwort is consumed a lot in Korea, and many reports have been made on its components and pharmacological effects. In particular, as for the anticancer study of mugwort, anti-mutagenic effects were confirmed in several plants including mugwort (A.lavandulaefolia DC), and water-soluble extracts of mugwort (A. capilaris) were reported to have the effect of activating tumor necrosis factor. In addition, the anticancer activity of phenolic compounds of mugwort was also announced.

The broccoli (Brassica oleracea L. var. italica) is a plant belonging to the cruciferous family, and is widely consumed as a salad or food additive in Western Europe, the United States and Far East Asian countries. Broccoli, a rich source of many types of vitamins and antioxidants, also contains a secondary metabolite called glucosinolate (GSL). As a result of various epidemiological studies, plants of the genus Brassica in general, especially broccoli, have anticancer activity because they are rich in GSL, and their bactericidal activity has been reported in the literature for decades.

In the complex extract, the type of solvent is not particularly limited, and if necessary, various solvents, for example, water, and at least one of C1 to C4 alcohols may be used. In addition, the extraction may be performed by a conventional method in the art, such as cooling, warming, heating, etc. using the solvent. However, it is most preferable to use an extraction method using water as a solvent, since it has the best antiviral activity.

In the present invention, the complex extract exhibits an excellent antiviral effect due to the synergistic effect according to the interaction between the active ingredients derived from each raw material, and the antiviral activity significantly increased compared to the conventional single extract alone did not show sufficient antiviral activity. has

And the fermented wasabi extract also exhibits an excellent antiviral effect through interaction with the complex extract, and is prepared by inoculating a Lactobacillus spp. strain into the extract of wasabi and fermenting. The Lactobacillus sp. strain may be any one selected from Lactobacillus casei, Lactobacillus acido pillars, Lactobacillus bulgaricus, Lactobacillus confusus and Lactobacillus reuteri.

In addition, when preparing the wasabi extract, the type of the solvent is not particularly limited, and if necessary, various solvents, for example, water, and at least one of C1 to C4 alcohols may be used. In addition, the extraction may be performed by a conventional method in the art, such as cooling, warming, heating, etc. using the solvent. However, it is most preferable to use an extraction method using water as a solvent, since it has the best antiviral activity.

The wasabi is a perennial plant that grows wild in cold valleys and is also cultivated in cold areas. It is roughly divided into a Japanese product with a scientific name of Eutrema wasabi and a Southeastern European product with a scientific name of Armoracia rusticana and English name Horse radish. In the present invention, it can be used irrespective of its origin. It has been reported that allyisothiocyanate, a major volatile component of wasabi essential oil, exhibits antibacterial action against Escherichia coli, Staphyllococcus aureus, Saccharomyces cerevisiae and Aspergillus oryzae.

On the other hand, the complex extract and the fermented wasabi extract are preferably contained in 80 to 99.9 wt% of the complex extract and 0.1 to 20 wt% of the fermented wasabi extract based on 100 wt% of the natural antiviral composition. This is in consideration of its excellent antiviral activity.

In addition, the complex extract is 0.1 to 20% by weight of ginseng extract, 0.1 to 20% by weight of eoseongcho extract, 0.1 to 20% by weight of dandelion root extract, 0.1 to 20% by weight of golden extract, 0.1 to 20% by weight of rhubarb extract, 0.1% by weight of rhubarb extract It is preferable to include ~20% by weight, 0.1 ~ 20% by weight of ephedra extract, 0.1 ~ 20% by weight of sagebrush extract and 0.1 ~ 20% by weight of broccoli extract, because it exhibits the best antiviral activity in this combination ratio. .

The natural antiviral composition of the present invention as described above, without toxicity or skin irritation, influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus), poliovirus (Poliovirus) excellent antiviral activity and antibacterial activity.

In addition, the natural antiviral composition of the present invention also has an antibacterial effect against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococci and Candida albicans.

Hereinafter, a method for producing a natural antiviral composition according to the present invention will be described.

The method for producing a natural antiviral composition according to the present invention is to prepare a complex extract comprising an extract of gold and silver lilies extract, eoseongcho extract, banyan root extract, golden extract, yellow rhubarb extract, rhubarb extract, ephedra extract, fortified sagebrush extract and broccoli extract It is characterized in that it comprises the steps of preparing a fermented wasabi extract, and mixing and homogenizing the prepared complex extract and fermented wasabi extract.

Preparing a complex extract comprising a gold leaf extract, eoseongcho extract, banyan root extract, golden extract, yellow rhubarb extract, rhubarb extract, ephedra extract, fortified sagebrush extract, and broccoli extract

First, a complex extract is prepared.

Here, as described above, the complex extract is obtained by mixing gold and silver ginseng, eoseongcho, dandelion root, gold, yellow rhubarb, rhubarb, ephedra, sagebrush and broccoli, and extracting it using a solvent, or egeumhwahwa, eoseongcho, dandelion root. , gold, yellow rhubarb, rhubarb, ephedra, sagebrush, and broccoli may be prepared by extracting each using a solvent, and then mixing the extracts, and the method is not limited.

In addition, the type of solvent for the complex extract is not particularly limited, and if necessary, various solvents, for example, water, and at least one of C1 to C4 alcohols may be used. In addition, the extraction can be carried out by a conventional method known in the art, such as cold soaking, warm soaking, heating, etc. using the solvent, and the implementation is not limited. However, it is most preferably extracted using water as a solvent, because the antiviral activity is the best. In addition, the process of filtration, concentration, or drying after extraction may be additionally performed, and this is not limited thereto.

And the mixing ratio of each extract is, 0.1 to 20% by weight of gold extract, 0.1 to 20% by weight of Eoseongcho extract, 0.1 to 20% by weight of dandelion root extract, 0.1 to 20% by weight of golden extract, 0.1 to 20% by weight of rhubarb extract, rhubarb It is preferable that the extract is 0.1 to 20% by weight, ephedra extract 0.1 to 20% by weight, Ganghwa sajabal mugwort extract 0.1 to 20% by weight, and broccoli extract 0.1 to 20% by weight, and when extracting by mixing the raw materials, the mixing ratio is the mixing ratio of the raw materials. meaning is natural.

Steps to Prepare Fermented Wasabi Extract

Next, a fermented wasabi extract is prepared. In the present invention, for ease of explanation, it is described that the fermented wasabi extract is prepared after the preparation of the complex extract, but since this is a separate process, the order is not limited.

The fermented wasabi extract is prepared by first preparing an extract by adding a solvent to one or more of the washed outplants, roots, and leaves of wasabi, and then inoculating the extract with a Lactobacillus spp. strain and fermenting it.

In this case, the type of the solvent is not particularly limited, and if necessary, various solvents, for example, water, and at least one of C1 to C4 alcohols may be used. In addition, the extraction can be carried out by a conventional method known in the art, such as cold soaking, warm soaking, heating, etc. using the solvent, and the implementation is not limited. However, it is most preferably extracted using water as a solvent, because the antiviral activity is the best. In addition, the process of filtration, concentration, or drying after extraction may be additionally performed, but is not limited thereto.

And the fermentation is to inoculate a Lactobacillus sp. strain into an extract of wasabi, and ferment for 60 to 90 hours at 30-40 ℃. The Lactobacillus sp. strain may be any one selected from Lactobacillus casei, Lactobacillus acido pillars, Lactobacillus bulgaricus, Lactobacillus confusus and Lactobacillus reuteri. In addition, after fermentation, the process of filtration, concentration or drying may be additionally performed, but this is not limited.

Mixing and homogenizing the prepared complex extract and fermented wasabi extract

Next, the prepared complex extract and the fermented wasabi extract are mixed. At this time, the mixing ratio is preferably 80 to 99.9% by weight of the complex extract and 0.1 to 20% by weight of the wasabi extract, as described above.

And the mixture is homogenized to prepare a natural antiviral composition. The homogenization method is not limited, but most preferably MF (Microfluidizer) process is used.

Here, the MF (Microfluidizer) process is a high-efficiency intensifier pump that pressurizes the fluid at high pressure and injects it into a slot of 100 microns or less, and Dispersion, Emulsion, Cell rupture, Deaggloberation, Liposome, etc. It means the process of passing a microfluidizer consisting of an interaction chamber that causes it to occur. In this chamber, submicron particle size (droplet) and narrow distribution can be obtained by the three forces of shear, impact, and cavitation. Since the MF process is already known in the field to which this technology belongs, a detailed description thereof will be omitted.

The homogenized composition, that is, the natural antiviral composition as described above, has an antiviral effect on influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus) and poliovirus (Poliovirus).

Since the natural antiviral composition prepared as described above has antiviral and antibacterial activity, it can be applied to various antiviral and antibacterial products for sterilization or disinfection, illustratively wet wipes, disinfectants, etc., as well as virus infection It can be applied to pharmaceutical compositions, food compositions, cosmetic compositions, etc. for the treatment or prevention of , and the field of use thereof is not limited.

In addition, it is natural that various additives, carriers, etc. known in the art may be further included in the preparation of the above-described antiviral and antibacterial products, pharmaceutical compositions, food compositions, and cosmetic compositions, and are formulated in various forms. It is natural to be angry.

Hereinafter, embodiments of the present invention will be described in detail.

(Example 1): Preparation of natural antiviral composition

Preparation of complex extracts

In 2L of purified water, 100 g of gold and silver lilies, 100 g of Eosongcho, 100 g of dandelion root, 100 g of gold, 100 g of rhubarb, 100 g of rhubarb, 100 g of ephedra, 100 g of Ganghwa sajabal mugwort and 100 g of broccoli were put, and the mixture was heated to 90-95° C. in a hot water bath, and maintained for 5 hours. Then, it was cooled to room temperature and filtered through a 1 μm filter to remove solids, thereby preparing 1 L of a composite extract.

Preparation of Fermented Wasabi Extract

100 g of wasabi root was added to 300 mL of purified water, and the mixture was heated to 90-95° C. in a hot water bath, and maintained for 5 hours. Then, it was cooled to room temperature and filtered through a 1 μm filter to remove solids, thereby preparing a wasabi extract.

Next, the wasabi extract was inoculated with Lactobacillus casei (KCTC 13086) at 0.5 wt%, fermented at 32° C. for 72 hours, and filtered through a 1 μm filter to prepare 150 mL of fermented wasabi extract.

Preparation of natural antiviral compositions

All of the prepared complex extract and fermented wasabi extract were added, stirred homogeneously, and then subjected to a microfluidizer process to prepare a homogeneously dispersed natural antiviral composition. Hereinafter, in showing the test results, it is expressed as a natural antiviral composition (DKV-50AF).

(Test Example 1): Antiviral effect test of natural antiviral composition

^{1×10 6} cells per well of a 12-well plate were inoculated in a Petri dish with a diameter of 6 cm using Minimum essential medium (MEM) supplemented with 10% FBS (Fetal Bovine Serum). And the sample of Example 1 was added by concentration (10%, 1%, 0.1%) and then cultured for 12 hours.

After 12 hours, each of Influenza A virus (H1N1) was inoculated at a concentration of 0.1 mol, the inoculum was removed after 3 minutes, washed 3 times with PBS, and the concentration of the natural antiviral composition (10%, 1%, 0.1%) to confirm the virus titer and reduction rate against Influenza A virus. The results were shown in Tables 1 and 2 below.

Virus titer and reduction rate (log value) Sample natural antiviral composition Virus Virus titer
(log CCID ₅₀ /well) Virus reduction
(log) Name concent Name Concent 3 min 3 min hard water
90% 0% FluA
Mock 10% 5.08
<0.00 <0.00
Natural Antiviral Composition (DKV-50AF) 10% 90% 9.00% FluA
Mock 10% <3.2
0.51 >4.57
Natural Antiviral Composition (DKV-50AF) 1% 90% 0.90% FluA
Mock 10% <3.2
0.51 >4.57
Natural Antiviral Composition (DKV-50AF) 0.1% 90% 0.09% FluA
Mock 10% <3.2
0.51 >4.57

Virus titer and reduction (%) Sample natural antiviral composition Virus Virus titer
(log CCID ₅₀ /well) Virus reduction
(%) Name concent Name Concent 3 min 3 min hard water
90% 0% FluA
Mock 10% 120,000
<1.0 0%
Natural Antiviral Composition (DKV-50AF) 10% 90% 9.00% FluA
Mock 10% <3.2
3.2 >99.97%
Natural Antiviral Composition (DKV-50AF) 1% 90% 0.90% FluA
Mock 10% <3.2
3.2 >99.97%
Natural Antiviral Composition (DKV-50AF) 0.1% 90% 0.09% FluA
Mock 10% <3.2
3.2 >99.97%

As shown in the results of Tables 1 and 2, the natural antiviral composition prepared according to Example 1 of the present invention is an excellent antiviral substance that significantly kills 99.99% of the virus within 3 minutes with respect to the Influenza A virus virus. (Log >4.57, >99.997%).

(Example 2): Preparation of wet tissue containing natural antiviral composition

5.00% by weight of the natural antiviral composition prepared in Example 1 and 95.00% by weight of water were mixed to prepare an impregnation solution for wet tissues. And the nonwoven fabric for wet tissues was impregnated with the impregnation solution to prepare wet tissues.

(Test Example 2): Antiviral effect test of wet tissue containing natural antiviral composition

The antiviral effect test of the wet tissue prepared in Example 2 was conducted by requesting Analytice (43, Route du Polygone 67100 STRASBOURG FRANCE), a French authorized testing institution.

1. Experimental conditions

1) Standard test method: EN 14476 + A2: 07-2019

2) Exam period: August 25, 2020 - October 5, 2020

3) Test temperature: 20℃±1℃

4) Titration method: log DICT ₅₀ and log UFP/mL (Poliovirus).

5) Test time: 10 minutes, 5 minutes, 1 minute

6) Final concentration: 97% and 80%

7) Solution diluent used in the test: distilled water.

8) Virus:

① Adenovirus type 5, adenoid 75 strain, cultured in HEp-2 cells.

② Norovirus, strain S99, _{cultivated in 5% CO 2} condition in RAW 264.7 cells.

③ Poliovirus type 1 strain LSc-2a, cultured in .VERO cells.

2. Virus Titer

rber방법에 따라 계산) : Titration by cytopathic effect of test virus suspension (Spearman-K

Calculated according to the rber method):

1) adenovirus = 6.875 log DICT ₅₀

2) Poliovirus = 6.680 log UFP/mL

3) norovirus = 6.750 log DICT ₅₀

3. Cytotoxicity

1) In the case of adenovirus, as a result of exposing HEp-2 cells to a wet tissue containing a natural antiviral composition, cytotoxicity was not observed at a concentration ^{of 10 −1 in the wet tissue containing the natural antiviral composition.}

2) In the case of poliovirus, as a result of exposing VERO cells to wet wipes containing a natural antiviral composition, cytotoxicity was not observed at a concentration ^{of 10 -1 of the wet tissue containing the natural antiviral composition.}

3) In the case of norovirus, as a result of exposing RAW cells to a wet tissue containing a natural antiviral composition, cytotoxicity was not observed at a concentration ^{of 10 -1 of the wet tissue containing the natural antiviral composition.}

4. Sensitivity of cells to viruses

Comparative titration of virus was performed on cells treated with or without product for each suspension of adenovirus, poliovirus, and norovirus used during this antiviral effect test.

Viral titers of untreated and treated cells of adenovirus ADENOVIRUS Virus titer (log DICT ₅₀ ) sample dilution Virus Suspension for Untreated Cells Virus suspension of treated cells Virus titer difference (log DICT ₅₀ ) Wet wipes with natural antiviral composition (DKV-50AF) 10 ^-2 6.875 6.250 0.625

The products tested at the concentrations indicated above had no significant effect on the adenovirus titration method (difference <1 log).

Viral Titers of Untreated and Treated Cells of Poliovirus POLIOVIRUS Virus titer (log UFP/mL) sample dilution Virus Suspension for Untreated Cells Virus suspension of treated cells Virus titer difference (log UFP/mL) Natural Antiviral Composition (DKV-50AF)
^{10 -2} wipes containing this 6.705 6.278 0.427

Products tested at the concentrations indicated above did not significantly affect the poliovirus titration method (difference <1 log).

Viral titers of untreated and treated cells of norovirus NOROVIRUS Virus titer (log DICT ₅₀ ) sample dilution Virus Suspension for Untreated Cells of treated cells
virus suspension Virus titer difference
(log DICT ₅₀ ) Wet wipes with natural antiviral composition (DKV-50AF) 10 ^-2 6.750 6.250 0.500

Products tested at the concentrations indicated above did not significantly affect the norovirus titration method (difference <1 log).

5. Baseline Inactivation Test

1) Adenovirus

Adenovirus inactivation test results 0.7% formaldehyde Virus titer (log DICT ₅₀ ) decrease in virus titer
(log DICT ₅₀ ) Virus Suspension Control 6.875 5 min inactivation test 6.250 0.625 15 minutes of inactivation test 5.875 1.000 30 min inactivation test 5.375 1.500

If the decrease in virus titer between the control suspension and the suspension applied to formaldehyde after 30 minutes is -0.5 to -2.5 log, it is judged that the validation of the test is effective. In Table 6, the decrease in virus titer is 1.500 log after 30 minutes . Therefore, it was confirmed that the conditions of the standard were satisfied.

2) Poliovirus

Poliovirus Inactivation Test Results 0.7% formaldehyde virus titer
(log UFP/mL) decrease in virus titer
(log UFP/mL) Virus Suspension Control 6.680 5 min inactivation test 6.465 0.215 15 minutes of inactivation test 5.311 1.369 30 min inactivation test 3.256 3.424

If the decrease in virus titer between the control suspension and the suspension applied to formaldehyde after 30 minutes is -0.5 to -4.5 log, the validation of the test is considered effective. In Table 7 above, the decrease in virus titer is 3.424 log after 30 minutes . Therefore, it was confirmed that the conditions of the standard were satisfied.

3) Norovirus

Norovirus Inactivation Test Results 0.7% formaldehyde Virus titer (log DICT ₅₀ ) decrease in virus titer
(log DICT ₅₀ ) Virus Suspension Control 6.750 5 min inactivation test 6.125 0.625 15 minutes of inactivation test 5.625 1.125 30 min inactivation test 5.375 1.375

If the decrease in virus titer between the control suspension and the suspension applied to formaldehyde after 30 minutes is -0.5 to -2.5 log, the test is considered effective. In Table 8, the decrease in virus titer is 1.375 log after 30 minutes . Therefore, it was confirmed that the conditions of the standard were satisfied.

6. Antiviral activity test

1) Adenovirus

Adenovirus primary test (concentration of control virus suspension is 6.875 log TCID ₅₀ ) Sample name density test time test temperature after the exam
(log DICT ₅₀₎ Decrease in virus titer Wet wipes with natural antiviral composition (DKV-50AF) 80% 10 minutes 20℃ 2.500 4.375 5 minutes 2.750 4.125

Adenovirus secondary test (concentration of control virus suspension is 6.875 log TCID ₅₀ ) Sample name density test time test temperature after the exam
(log DICT ₅₀ ) Decrease in virus titer Wet wipes with natural antiviral composition (DKV-50AF) 80% 10 minutes 20℃ 2.375 4.500 5 minutes 2.625 4.250

If the decrease in virus titer with respect to the concentration of the test sample is greater than or equal to 4.0 log, it has an antiviral effect (reduction in virus titer≥4.0 log). As shown in Tables 9 and 10 above, the decrease in virus titer through the wet tissue virus activity test containing the antiviral composition over two cases: after 5 minutes of the first test: 4.125 log, after 10 minutes: 4.375 log, after 5 minutes of the second test : 4.250 log, after 10 minutes: showed a decreasing effect of 4.500 log, indicating that the antiviral effect against adenovirus is excellent.

2) Poliovirus

Poliovirus primary test (concentration of control virus suspension is 6.680 log UFP/mL.) Sample name density test time test temperature after the exam
(log UFP/mL) Decrease in virus titer Wet wipes with natural antiviral composition (DKV-50AF) 97% 10 minutes 20℃ 2.612 4.068

Poliovirus secondary test (concentration of control virus suspension is 6.680 log UFP/mL.) Sample name density test time test temperature after the exam
(log UFP/mL) Decrease in virus titer Wet wipes with natural antiviral composition (DKV-50AF) 97% 10 minutes 20℃ 2.494 4.186

If the decrease in virus titer with respect to the concentration of the test sample is greater than or equal to 4.0 log, it has an antiviral effect (reduction in virus titer≥4.0 log).

As shown in Tables 11 and 12 above, the reduction in virus titer after 10 minutes of the first test: 4.068 log, after 10 minutes of the second test: a reduction effect of 4.186 log through the wet tissue virus activity test containing the natural antiviral composition over two cases showed excellent antiviral effect against poliovirus.

3) Norovirus

Norovirus primary test (concentration of control virus suspension is 6.750 log TCID ₅₀ ). Sample name density test time test temperature after the exam
(log DICT ₅₀ ) Decrease in virus titer Wet wipes with natural antiviral composition (DKV-50AF) 80% 10 minutes 20℃ 2.375 4.375 5 minutes 2.625 4.125

Norovirus secondary test (concentration of control virus suspension is 6.750 log TCID ₅₀ ). Sample name density test time test temperature after the exam
(log DICT ₅₀ ) Decrease in virus titer Wet wipes with natural antiviral composition (DKV-50AF) 80% 10 minutes 20℃ 2.375 4.375 5 minutes 2.500 4.250

If the decrease in virus titer with respect to the concentration of the test sample is greater than or equal to 4.0 log, it has an antiviral effect (reduction in virus titer≥4.0 log). As shown in Tables 13 and 14 above, the reduction in virus titer through the wet tissue virus activity test containing the antiviral composition over two cases: after 5 minutes of the first test: 4.125 log, after 10 minutes: 4.375 log, after 5 minutes of the second test :4.250 log, after 10 minutes: showed a decreasing effect of 4.375 log, indicating excellent antiviral effect against norovirus.

Therefore, as a result of testing the wet tissue of Example 2 through the European standard test method EN 14476 + A2: 07-2019 at Analytice, a French authorized testing institution, it was proved that the antiviral effect was 97% to 99%.

(Test Example 3): Antibacterial effect test of wet tissue containing natural antiviral composition

The antibacterial effect test of the wet tissue according to Example 2 of the present invention was conducted by requesting a French authorized testing institution Analytice (43, Route du Polygone 67100 STRASBOURG FRANCE).

1. Experimental conditions

1) Standard test method: EN 16615: 05-2015.

2) Exam period: August 10, 2020 - September 25, 2020

3) Test temperature: 20℃

4) How to use: dilution and neutralization

5) Test time: 1 minute, 2 minutes, 5 minutes

6) Interfering substance: Contaminated state: BSA (3g / L) + 3mL / L

7) Suspension and test diluent: sterile tryptone salt solution.

8) Strains used:

① Staphylococcus aureus subsp. cocci CIP 4.83 batch 15713-1d (ATCC 6538) (Staphylococcus aureus)

② Pseudomonas aeruginosa DSM 939 batch 0413 (ATCC15442) (Pseudomonas aeruginosa)

③ Enterococcus hirae DSM 3320 batch 0511 (ATCC 10541) (enterococcus)

④ Candida albicans CIP 48.72, batch 265.09 (ATCC 10231) (Candida albicans)

9) Bacteria culture conditions: TSA (Tryptone Soy Agar Agar), 37℃ ±1℃

10) Fungal culture conditions: GEM (Malt Extract Agar), 30℃ ±1℃

2. Antibacterial activity test result

The test results for Staphylococcus aureus are shown in FIGS. 1 (first test) and 2 (second test) below, and the test results for P. aeruginosa are shown in FIGS. 3 (first test) and 4 (second test) below. The test results for enterococci are shown in Figures 5 (first test) and 6 (second test) below, and the test results for Candida albicans are shown in Figures 7 (first test), 8 (secondary test) below. test).

In the case of bacteria > 5 log or more, it is judged that there is an antibacterial activity effect, and in the case of yeast > 4 log or more, it is judged that there is an anti-yeast activity effect. The average of the results of the 1st and 2nd repeated tests is as follows.

* Contaminated state (average of 1st and 2nd tests) - After 5 minutes of testing

- Pseudomonas aeruginoas : R = 5.63

- Staphylococcus aureus : R = 5.67

- Enterococcus hirae: R = 5.86

- Candida albicans: R = 4.64

The test results are as a result of carrying out according to EN 16615: 05-2015 standard, and the wet wipes of Example 2 were Pseudomonas aeruginoas (Pseudomonas aeruginosa), Staphylococcus aureus (Staphylococcus aureus), Enterococcus hirae (Enterococcus) within 5 minutes at 20 °C under contaminated conditions. ), was proven to have an antibacterial effect of 99.9% against Candida albicans (Candida albicans) strains.

(Test Example 4)

Skin irritation test of wet tissue containing natural antiviral composition

A clinical test was conducted on how the wet tissue according to Example 2 can stimulate human skin.

The clinical trial was conducted on 35 healthy women between the ages of 20 and 60 who were suitable for the test subject. The use of other products such as steroids or antibiotics was not allowed from 2 weeks before the start of the experiment until the end of the experiment.

After cleaning the upper part of the back with alcohol to all test subjects, the ingredients of the wet tissue containing the natural antiviral composition according to Example 2 of the present invention were applied at a rate of about 3 mg/cm 2 and the skin condition after 24 hours (erythema, edema, papule, etc.) was observed, and the results were qualitatively evaluated through the evaluation table shown in Table 15 below, and the results were shown in Table 16 below.

Skin Irritation Scorecard experimental item subjective score degree of stimulation a: No irritation or change in skin condition.
b: There is no change in skin condition, but there is slight irritation.
c: The skin becomes slightly red (erythema) and there is irritation.
d: The skin is slightly red (erythema) and there is a little swelling.
e: The skin becomes very red (erythema) and the swelling is severe.

Skin irritation evaluation result Evaluation items (skin irritation) Evaluation results No irritation or change in skin condition. 35 people There is no change in skin condition, but there is slight irritation. 0 people The skin becomes slightly red (erythema) and there is irritation. 0 people The skin is slightly red (erythema) and slightly swollen. 0 people The skin becomes very red (erythema) and the swelling is severe. 0 people

As can be seen from the evaluation results in Table 16, the composition according to Example 1 of the present invention was proven to be a safe substance without irritation to the skin such as erythema.

(Example 3)

It was carried out in the same manner as in Example 1, except that 99.9% by weight of the complex extract and 0.1% by weight of the fermented wasabi extract were mixed.

(Example 4)

It was carried out in the same manner as in Example 1, except that 80% by weight of the complex extract and 20% by weight of the fermented wasabi extract were mixed.

(Test Example 5): Antiviral effect test

The antiviral effect was tested in the same manner as in Test Example 1 using Examples 3 and 4, and the results are shown in Table 17 below.

Test Example 5 Results Sample natural antiviral composition Virus Virus reduction
(log) Name concent Name Concent 3 min hard water
90% 0% FluA
Mock 10% <0.00
Example 3 Composition 10% 90% 9.00% FluA
Mock 10% >4.57
Example 3 Composition 1% 90% 0.90% FluA
Mock 10% >4.57
Example 3 Composition 0.1% 90% 0.09% FluA
MoFluA
Mockck 10% >4.57
Example 4 Composition 10% 90% 9.00% FluA
Mock 10% >4.57
Example 4 Composition 1% 90% 0.90% FluA
Mock 10% >4.57
Example 4 Composition 0.1% 90% 0.09% FluA
Mock 10% >4.57

As shown in Table 17, Examples 3 and 4 were confirmed to be excellent antiviral substances that significantly kill 99.99% of the virus within 3 minutes against the Influenza A virus (Log >4.57, >99.997%).

(Examples 5 to 10)

It was carried out in the same manner as in Example 1, except that 900 g of each raw material was used in the same ratio as in Table 18 below in the preparation of the complex extract.

Component ratio (% by weight) of the composite extract of Examples 5 to 10 division Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 gold coin 20 20 20 20 0.1 0.1 Eoseongcho 20 20 20 0.1 0.1 0.1 plaza 20 20 20 0.1 0.1 0.1 Gold 20 20 0.1 0.1 0.1 20 goldthread 19.6 0.1 0.1 0.1 20 20 rhubarb 0.1 19.6 0.1 20 20 0.1 ephedra 0.1 0.1 20 20 19.6 19.6 Ganghwa lion wormwood 0.1 0.1 19.6 20 20 20 broccoli 0.1 0.1 0.1 19.6 20 20

(Test Example 6): Antiviral effect test

The antiviral effect was tested in the same manner as in Test Example 1 using Examples 5 to 10, and the results are shown in Table 19 below.

Test Example 6 Results Sample natural antiviral composition Virus Virus reduction
(log) Name concent Name Concent 3 min hard water
90% 0% FluA
Mock 10% <0.00
Example 5 Composition 1% 90% 0.90% FluA
Mock 10% >4.57
Example 6 Composition 1% 90% 0.90% FluA
Mock 10% >4.57
Example 7 Composition 1% 90% 0.90% FluA
MoFluA
Mockck 10% >4.57
Example 8 Composition 1% 90% 0.90% FluA
Mock 10% >4.57
Example 9 Composition 1% 90% 0.90% FluA
Mock 10% >4.57
Example 10 Composition 1% 90% 0.90% FluA
Mock 10% >4.57

As shown in Table 19, Examples 5 to 10 were confirmed to be excellent antiviral substances that significantly kill 99.99% of the virus within 3 minutes against the Influenza A virus (Log >4.57, >99.997%).

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (5)

0.1 to 20% by weight of ginseng extract, 0.1 to 20% by weight of Eoseongcho extract, 0.1 to 20% by weight of dandelion root extract, 0.1 to 20% by weight of golden extract, 0.1 to 20% by weight of rhubarb extract, 0.1 to 20% by weight of rhubarb extract, ephedra 0.1 to 20% by weight of the extract, 0.1 to 20% by weight of the Ganghwa sagebrush extract and 0.1 to 20% by weight of the broccoli extract 80 to 99.9% by weight of the complex extract; and
Contains 0.1 to 20% by weight of fermented wasabi extract as an active ingredient,
The fermented wasabi extract,
It is fermented by inoculating a Lactobacillus spp. strain into an extract of wasabi,
Natural antiviral composition, characterized in that it has antiviral activity against influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus) and poliovirus (Poliovirus).
delete delete 0.1 to 20% by weight of ginseng extract, 0.1 to 20% by weight of Eoseongcho extract, 0.1 to 20% by weight of dandelion root extract, 0.1 to 20% by weight of golden extract, 0.1 to 20% by weight of rhubarb extract, 0.1 to 20% by weight of rhubarb extract, ephedra A step of preparing a complex extract comprising 0.1 to 20% by weight of extract, 0.1 to 20% by weight of extract, and 0.1 to 20% by weight of broccoli extract;
preparing a fermented wasabi extract;
It comprises the step of homogenizing by mixing 80 to 99.9% by weight of the prepared complex extract and 0.1 to 20% by weight of the fermented wasabi extract,
The homogenized composition has antiviral activity against influenza A (Influenza A virus), adenovirus (Adenovirus), norovirus (Norovirus) and poliovirus (Poliovirus),
The step of preparing the fermented wasabi extract,
A method for producing a natural antiviral composition, characterized in that extracting horseradish, inoculating the extract with a Lactobacillus sp. strain and fermenting the extract. delete

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