WO2023239175A1 - Composition for preventing or treating alphaherpesvirus, gammaherpesvirus or norovirus infectious disease comprising extract of tsuga sieboldii, potentilla discolor bunge, potentilla nivea l., hydrocotyle yabei makino, or anthriscus sylvestris - Google Patents

Abstract

The present invention relates to a natural substance extract exhibiting antiviral activity against gammaherpesvirus, alphaherpesvirus, or norovirus. Specifically, the present invention relates to: a composition for treating or preventing a disease caused by gammaherpesvirus infection, comprising an extract of Tsuga sieboldii, Potentilla discolor Bunge, Potentilla nivea L., or Hydrocotyle yabei Makino; a composition for treating or preventing a disease caused by alphaherpesvirus infection, comprising an extract of Potentilla discolor Bunge, Potentilla nivea L., or Anthriscus sylvestris; and a composition for treating or preventing a disease caused by norovirus infection, comprising a Potentilla discolor Bunge or Potentilla nivea L. extract. The composition of the present invention inhibits the activity of viruses, and thus can be effectively used as a therapeutic agent for viral infectious diseases.

Description

Composition for preventing or treating alphaherpesvirus, gammaherpesvirus, or norovirus infectious diseases containing extracts of hemlock, cypress, euphorbia, Jeju mantle, or Jeonho.

The present invention relates to a natural product extract that exhibits antiviral activity against gammaherpesvirus, alphaherpesvirus, or norovirus. Specifically, a composition for treating or preventing diseases caused by gammaherpes virus infection comprising extracts of hemlock, hemlock, chinensis, or Jeju capsicum; A composition for treating or preventing diseases caused by alphaherpesvirus infection, comprising extracts of Euphorbia chinensis, Euphorbia chinensis, or Jeonho; And it relates to a composition for treating or preventing diseases caused by norovirus infection, comprising extracts of Euphorbia chinensis or Euphorbia chinensis.

Viruses are microorganisms that cannot replicate on their own, and thousands of types of viruses currently exist on Earth. Some of these viruses infect humans, and these viral infections are currently on the rise worldwide due to global warming and globalization.

Herpesviridae is an enveloped virus with a relatively large and complex structure whose viral genome is double-stranded DNA of 120 to 230 kbp in size. The above virus family has evolved to be able to infect the host throughout its life by evading the host's immune system and forming a latent infection in addition to lytic infection, in which progeny viruses replicate, and alpha according to differences in biological characteristics. It is classified into three groups: Alpha herpesvirinae , Beta herpesvirinae , and Gamma herpesvirinae .

Most human-infecting herpes viruses have a high infection rate of over 70-90% in Korea. Once infected, they form a latent infection in the human body and are chronic viruses that remain infectious throughout life by repeating latent infection and reactivation in the host, affecting immune function. They are representative opportunistic infection pathogens that cause various diseases through reactivation or congenital infection due to degradation and have a fatal infection prognosis depending on the patient's immune status. Control of reactivated herpes virus infections due to decreased immunity, such as the increase in organ transplants and hematopoietic stem cell transplants and human immunodeficiency virus (HIV-1) infections, is becoming an important issue. The decline in immunity of the elderly population due to persistent infections is acting as an important health and socioeconomic burden in countries that are entering aging societies at the fastest rate.

To date, eight types of human herpesviruses have been discovered: Herpes simplex virus I (HSV-I), Herpes simplex virus II (HSV-II), Varicella zoster virus (VZV), Human cytomegalovirus (HCMV), and Human herpesvirus 6. , Human herpesvirus 7, Epstein-barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV), of which Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) belong to gammaherpesviruses.

Unlike alpha and beta herpesviruses, gammaherpesvirus has distinct cell tropism and has the characteristic of causing persistent infection in the host through latency in lymphocytes. It causes benign or malignant tumors in infected hosts, affecting 90% of the world's population. In chronic infection, EBV causes Burkitt's lymphoma, gastric carcinoma, nasopharyngeal carcinoma, post-transplant B cell lymphomas, and Hodgkin's lymphoma ( KSHV contributes to the formation of tumors such as Hodgkin's disease, and KSHV is the cause of Kaposi's sarcoma, primary effusion lymphoma, and multicentric castleman's disease.

Herpes simplex virus-1 (HSV-1), a type 1 human infectious herpes virus, is an alphaherpesvirus that forms a latent infection in the nervous system. It continues to infect the host for life and is reactivated and proliferates due to decreased immunity, etc., infecting the skin and skin. Herpes that appears around the mouth, lips, eyes, or genitals can cause herpes simplex, cold sores, stomatitis, and herpes simplex keratitis, as well as meningitis and encephalitis. In newborns, it can cause fatal encephalitis.

Varicella-Zoster virus (VZV), a type 3 human infectious herpesvirus, is well known as the causative agent that causes highly contagious chickenpox (Varicella) in the initial infection. After the initial infection, VZV affects the sensory nervous system ( A latent infection forms in the dorsal root ganglia (DRG) and reactivation due to decreased immunity causes shingles, which causes extreme pain. Reactivation of VZV causes shingles that occurs in the chest, optic ganglion, and facial nerve areas, and can also cause herpes zoster (zoster sine herpete), which causes abnormal skin sensation or radiating pain even without skin herpes. In some patients, it can cause ‘postherpetic neuralgia (PHN),’ where pain persists even after shingles is treated. 'Postherpetic neuralgia (PHN)', the most common complication after shingles caused by reactivated VZV, causes varying degrees of pain for each patient, and usually occurs when pain persists for at least 4 months or more after the rash. It is classified as posterior neuralgia. This chronic pain causes not only physical pain but also psychological anxiety, depression, and insomnia in patients, which has a significant negative impact on the patient's quality of life. Therefore, rapid control of VZV reactivation and infection is very important. It is important.

In the case of existing antiherpesvirus drugs, there is no fundamental treatment that can control latent infection, and most of them are nucleoside analogs developed to target viral DNA polymerase (DNA pol). These drugs are highly toxic not only to the elderly but also to patients with organ transplants, hematopoietic stem cell transplants, and immunodeficiency due to HIV infection, and more importantly, the emergence of drug-resistant mutations is increasing in these patients, thereby controlling new infections. Excavation of the material is urgent.

Human norovirus is a norovirus belonging to the Caliciviridae family that has positive single-stranded RNA as its genome, and accounts for the highest percentage of non-bacterial gastroenteritis, the main cause of enteritis in winter worldwide. Human norovirus enters the environment through the feces or aerosolized vomit of infected patients, and is contracted by consuming food or drinking water contaminated with the virus. There are reports that shedding of human norovirus can be observed for up to eight weeks even after symptoms such as diarrhea have stopped. In addition, since infection is possible with less than 100 virus particles, it is highly contagious, and a small amount of virus contamination can cause widespread mass enteritis, making it an important pathogen that must be controlled to improve public health. In particular, prevention and treatment for norovirus is urgent because it is easy to spread in facilities where many immunocompromised people live together, such as nursing homes, and symptoms can worsen to severe cases.

The biggest problem in studying human noroviruses is that the culture system that can produce viruses by infecting human noroviruses is very inefficient. As a result, research on the role and life cycle of viral genes of infectious human noroviruses is inevitably very limited. Additionally, the absence of a virus culture system that can confirm viral infection control serves as the most important limiting step in the development of antiviral drugs. Murine noroviruses (MNVs) are being used as a surrogate virus system to overcome these problems. In particular, since mouse norovirus (MNV-1) was discovered as a norovirus that infects mice as a host, it is the only norovirus that is easy to cell culture and has the same infection route as human norovirus. Before the discovery of mouse norovirus, feline calicivirus 9 (FCV-9), which belongs to the caliciviridae family, was once accepted as an alternative virus to human norovirus, but after the discovery of mouse norovirus, the transmission route and pathogenic characteristics changed. It is not used because it is different. While human norovirus and mouse norovirus are transmitted through feces through gastrointestinal infection, feline calicivirus is a virus transmitted through respiratory infection and has many different properties, so mouse norovirus is accepted as a more appropriate alternative virus system. .

Currently, there are no specific vaccines or antiviral drugs approved for human norovirus worldwide, but the unmet medical demand for treatment of norovirus infections is increasing, as the number of group living facilities such as nursing homes increases due to the aging population.

The present inventors studied the effect of natural products on virus activity and completed the present invention by developing natural product extracts that can effectively inhibit infection by norovirus, alphaherpesvirus, and gammaherpesvirus.

The purpose of the present invention is to provide a composition with antiviral activity containing extracts of hemlock, hemlock, euphorbia, or Jeju mantle as an active ingredient, which can treat or prevent diseases caused by gammaherpesvirus infection.

In addition, the purpose of the present invention is to provide a composition with antiviral activity containing extracts of Euphorbia or Euphorbia as an active ingredient, which can prevent or treat diseases caused by norovirus infection.

Additionally, the purpose of the present invention is to provide a composition with antiviral activity containing as an active ingredient extracts of Euphorbia chinensis, Euphorbia chinensis, or Jeonho chinensis, which can prevent or treat diseases caused by alphaherpesvirus infection.

In order to solve the above problems, the present invention provides a composition containing extracts of hemlock, hemlock, euphorbia, or Jeju mantle.

The composition may be a pharmaceutical composition, food composition, or animal feed composition.

In one embodiment, the composition may be used to prevent, treat, or improve gammaherpes virus infectious disease.

In one embodiment, the composition may be for preventing, treating, or improving Kaposi's sarcoma herpes virus infectious disease.

In one embodiment, the composition is capable of reducing the expression of the lytic protein of Kaposi's sarcoma herpes virus.

In one embodiment, the infectious disease may be one or more diseases selected from the group consisting of Kaposi's sarcoma, primary exudative lymphoma, and multicentric Castleman's disease.

In one embodiment, the composition may be for preventing, treating, or ameliorating Epstein-Barr virus infectious disease.

In one embodiment, the composition is capable of reducing expression of the lytic protein of Epstein Barr virus.

In one embodiment, the infectious disease may be one or more diseases selected from the group consisting of Burkitt's lymphoma, gastric carcinoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, and post-transplant B cell lymphoma.

The present invention provides a pharmaceutical composition for the prevention or treatment of norovirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of Prickly pear and Prickly pear extracts.

In one embodiment, the pharmaceutical composition may include one or more extracts selected from the group consisting of Lilium vulgaris and Lilium vulgaris extracts, or may include an extract extracted from a mixture selected from the group consisting of Lilium vulgaris and Lilium vulgaris extracts.

In one embodiment, the norovirus may be a human norovirus.

In one embodiment, the norovirus infectious disease may be non-bacterial gastroenteritis.

In one embodiment, the Euphorbia chinensis extract may be fractionated with one or more solvents selected from the group consisting of hexane, chloroform, butanol, ethyl acetate, and water.

In one embodiment, the Euphorbia chinensis extract may be a hexane fraction or a chloroform fraction.

The present invention provides a food composition for preventing or improving norovirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of Prickly pear and Prickly pear extracts.

In one embodiment, the norovirus infectious disease may be non-bacterial gastroenteritis.

The present invention provides a feed composition for animals for preventing or ameliorating norovirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of Prickly pear and Prickly pear extracts.

In one embodiment, the norovirus infectious disease may be non-bacterial gastroenteritis.

The present invention provides a pharmaceutical composition for the prevention or treatment of alphaherpesvirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of Euphorbia chinensis, Euphorbia chinensis and Jeonho extract.

In one embodiment, the pharmaceutical composition comprises one or more extracts selected from the group consisting of extracts of Corolla, Corolla, and Corolla extracts, or comprises an extract extracted from a mixture selected from the group consisting of extracts of Corolla, Corolla, and Corolla extracts. can do.

In one embodiment, the alphaherpesvirus may be herpes simplex virus or varicella-zoster virus.

In one embodiment, the herpes simplex virus infectious disease may be one or more diseases selected from the group consisting of herpes simplex, cold sores, stomatitis, meningitis, and encephalitis.

In one embodiment, the varicella-zoster virus infectious disease may be one or more diseases selected from the group consisting of varicella-zoster virus shingles and Zoster Sine Herpete.

In one embodiment, the Euphorbia chinensis extract may be fractionated with one or more solvents selected from the group consisting of hexane, chloroform, butanol, ethyl acetate, and water.

In one embodiment, the Euphorbia chinensis extract may be a hexane fraction or a chloroform fraction, and preferably may be a hexane fraction.

The present invention provides a food composition for preventing or ameliorating alphaherpesvirus infectious disease, comprising as an active ingredient one or more extracts selected from the group consisting of Euphorbia flower, Euphorbia chinensis and Jeonho extract.

In one embodiment, the alphaherpesvirus may be herpes simplex virus or varicella-zoster virus.

The present invention provides a feed composition for animals for preventing or ameliorating alphaherpesvirus infection disease, which contains as an active ingredient one or more extracts selected from the group consisting of Euphorbia chinensis, Euphorbia chinensis and Jeonho extract.

In one embodiment, the alphaherpesvirus may be herpes simplex virus or varicella-zoster virus.

A composition containing extracts of Hemlock, Hemlock, Euphorbia, or Jeju mantle according to the present invention has an antiviral effect against KSHV and EBV. Therefore, the composition of the present invention can treat or prevent diseases caused by gammaherpesvirus infection.

The Prickly pear and Prickly pear extracts of the present invention have an excellent effect of inducing inhibition of the proliferation of norovirus. Accordingly, the extracts of Prickly pear and Prickly pear of the present invention can be used for the prevention or treatment of norovirus infectious diseases.

In addition, the extracts of the present invention, such as euphorbia vulgaris, euphorbia vulgaris, and jeonho, have an excellent effect of inducing inhibition of the proliferation of alphaherpesvirus. Therefore, the composition of the present invention can be used for the prevention or treatment of alphaherpesvirus infectious diseases.

Figure 1 shows the level of GFP expression when 86 natural product-derived mixtures were treated with the BC-3-G cell line.

Figure 2 shows the level of GFP expression when 86 natural product-derived mixtures were treated with the BC-3-G cell line, with the negative control group designated as 0.

Figure 3 shows GFP positive cells in flow cytometry when BC-3-G cell line was treated with six types of natural product-derived mixtures.

Figure 4 shows the expression levels of RTA and K8 when BC-3-G cell line was treated with six types of natural product-derived mixtures.

Figure 5 shows the expression levels of RTA and K8 when BC-3 cell line was treated with three mixtures derived from natural products at different concentrations.

Figure 6 shows the expression levels of RTA and K8 when BC-3-G cell line was treated with three mixtures derived from natural products at different concentrations.

Figure 7 shows the expression levels of RTA and K8 when BCBL-1 cell line was treated with three mixtures derived from natural products at different concentrations.

Figure 8 shows the expression level of ZTA when Raji cell line was treated with six types of natural product-derived mixtures.

Figure 9 shows the expression levels of EA-D and ZTA when Akata (+) cell line was treated with three types of natural product-derived mixtures.

Figure 10 shows the expression levels of EA-D and ZTA when the Akata (+) cell line was treated with three mixtures derived from natural products at different concentrations.

Figure 11 shows the expression levels of RTA and K8 when nine types of natural product extracts were treated with BC-3-G cell line.

Figure 12 shows the expression levels of RTA and K8 when nine types of natural product extracts were treated with the BCBL-1 cell line.

Figure 13 shows the expression levels of EA-D and ZTA when nine types of natural product extracts were treated with Raji cell line.

Figure 14 shows the expression levels of EA-D and ZTA when nine types of natural product extracts were treated with Akata(+) cell line.

Figure 15 is a schematic diagram showing the process of evaluating antiviral efficacy against norovirus.

Figure 16 is a photograph and a graph showing the results of evaluating the antiviral efficacy of extracts of Euphorbia and Euphorbicus flowers against norovirus.

Figure 17 is a photograph and a graph showing the results of evaluating the antiviral efficacy of five types of noroviruses from the extracts and fractions of Somnambula flower.

Figure 18 is a schematic diagram showing the process of evaluating antiviral efficacy against alphaherpesvirus.

Figure 19 is a photograph and graph showing the results of evaluating the antiviral efficacy of extracts of Lithuanian lily and Lithuanian lily against HSV-1 alphaherpesvirus.

Figure 20 is a photograph and graph showing the change in plaque size in the evaluation of the antiviral efficacy of extracts of Lilium vulgaris and Lilium vulgaris against HSV-1 alphaherpesvirus.

Figure 21 is a photograph and graph showing the results of evaluating the antiviral efficacy of Jeonho extract against HSV-1 alphaherpesvirus.

Figure 22 is a photograph and graph showing the results of evaluating the antiviral efficacy of 5 types of HSV-1 alphaherpesvirus extracts and fractions of Sulfuria chinensis.

Figure 23 is a photograph and a graph showing the results of evaluating the antiviral efficacy of extracts of Lithuanian vulgaris, Liquoria vulgaris, and Chrysanthemum vulgaris against VZV alphaherpesvirus.

Figure 24 is a photograph and graph showing the change in plaque size in the evaluation of the antiviral efficacy of extracts of Prickly pear and Prickly pear extract against VZV alphaherpesvirus.

Figure 25 is a photograph and graph showing the results of evaluating the antiviral efficacy of 5 types of extracts and fractions of Sulfuria chinensis against VZV alphaherpesvirus.

Figure 26 is a photograph and a graph showing the results of evaluating the antiviral efficacy of two types of Prickly pear fractions against VZV alphaherpesvirus.

Figure 27 is a photograph and graph showing the change in plaque size in the evaluation of the antiviral efficacy of the hexane fraction of Solanum vulgaris against VZV alphaherpesvirus.

Hereinafter, with reference to the attached drawings, embodiments and examples of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present application may be implemented in various forms and is not limited to the embodiments and examples described herein.

Throughout the specification of the present application, when a part “includes” a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary.

As used in the present invention, the term “prevention” refers to any action that suppresses or delays a disease, etc. by administration of the composition according to the present invention, and “treatment” refers to symptoms of a suspected disease or diseased individual by administration of the composition. This means any action that improves or changes to benefit. As used herein, the term “improvement” refers to any action that results in at least a reduction in the severity of a parameter, such as a symptom, associated with the condition being treated by administration of a composition comprising an extract of the present invention.

The hemlock tree of the present invention is Tsuga sieboldii Carriere , which belongs to the genus of Hemlock family of the Pinaceae family, is mainly distributed in Ulleungdo and Japan, and is known to be used as an ingredient in the treatment of allergic diseases.

The cotton bellflower of the present invention is Potentilla discolor Bunge , which belongs to the Rosaceae genus and is widely distributed in Korea, Taiwan, Russia, Japan, China, etc., and is known to be used for diabetes, immune diseases, etc.

The flower of the present invention is Potentilla nivea L. , which belongs to the Rosaceae genus and is distributed in the northern part of the Korean Peninsula, Russia, Japan, China, Europe, North America, etc.

The Jeju marigold of the present invention is Hydrocotyle yabei Makino , which belongs to the genus Apiaceae and is distributed in Jeju Island and Japan.

The general name of the present invention is Anthriscus sylvestris , which belongs to the genus Apiaceae and is distributed in Europe, Asia, Africa, etc.

The term "norovirus" used in the present invention refers to a virus belonging to the Caliciviridae and Norovirus genus, which has positive single-stranded RNA as its genome.

The term “alphaherpesvirus” used in the present invention refers to a virus belonging to the herpesviridae or alphaherpesvirinae subfamily.

Among them, Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) belong to the gammaherpesviruses.

The term "Epstein Barr virus" used in the present invention refers to the Epstein-Barr virus belonging to the Gammaherpesvirinae and Lymphocryptovirus genus, and is also referred to as "EBV".

The term "Kaposi's sarcoma herpesvirus" used in the present invention refers to Kaposi's sarcoma-associated herpesvirus belonging to the Gammaherpesvirinae and Rhadinovirus genus, and is also referred to as "KSHV".

The term "varicella-zoster virus" used in the present invention refers to Varicella-zoster virus belonging to the Alphaherpesvirinae and Varicellovirus genus, and is referred to as "VZV" or "Human herpesvirus 3" It can also happen.

The term “herpes simplex virus” used in the present invention refers to Herpes simplex virus-1 belonging to the Alphaherpesvirinae and Simplexvirus genus, and is also referred to as “HSV-1.”

The present inventors discovered for the first time that one or more extracts selected from the group consisting of hemlock, hemlock, euphorbia, and Jeju marigold have an antiviral effect against gammaherpesvirus. Specifically, it was found that one or more extracts selected from the group consisting of hemlock, hemlock, euphorbia, and Jeju capsicum inhibit the activity of KSHV and EBV.

The extracts of hemlock, hemlock, euphorbia, jeju capifolia, and jeonho may be extracts of stems, fruits, roots, or leaves, but are not limited thereto.

The term “extract” used in the present invention refers to what is commonly used in the art as a crude extract, but in a broad sense also includes fractions obtained by additional fractionation of the extract. That is, the extract includes not only those obtained using the above-mentioned solvents, but also those obtained by additionally applying a purification process. For example, fractions obtained by passing the extract through an ultrafiltration membrane with a certain molecular weight cut-off value, separation by various chromatographs (designed for separation according to size, charge, hydrophobicity, or affinity), etc. Fractions obtained through purification methods are also included in the extract of the present invention.

The extract used in the present invention may be extracted through hot water extraction, cold needle extraction, reflux cooling extraction, ultrasonic extraction, or conventional extraction methods known in the art.

The extract used in the present invention can be obtained using a conventional extraction solvent known in the art. As the extraction solvent, a polar solvent or a non-polar solvent can be used. Polar solvents include water, C1 to C6 alcohols (e.g., methanol, ethanol, propanol, butanol, n-propanol, iso-propanol, and n-butanol, etc.), acetic acid, or mixtures of the above polar solvents. Nonpolar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, butyl acetate, fluoroalkane, hexane, ether, chloroform, dichloromethane, or mixtures of the above nonpolar solvents.

The food composition of the present invention may be a health functional food, dairy product, fermented product, or food additive.

The above health functional foods refer to foods manufactured and processed using raw materials or ingredients with functional properties useful to the human body. “Functional” refers to foods that are useful for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. It means taking it for the purpose of obtaining effects.

The animal feed composition of the present invention can be ingested by all non-human animals, such as non-human primates, sheep, dogs, cows, horses, etc.

The present invention will be described in more detail through examples below. However, the examples below are for illustrative purposes only and are not intended to limit the scope of the present invention.

[Production Example 1]

Manufacture of Extracts of Euphorbia flower, Euphorbia chinensis and Jeonho extract

Extracts of three types of natural products, including Psyllium vulgaris herbaceous plant, Psyllium spp. leaf, stem, and flower, were purchased from the Korea Plant Extract Bank (Korea Research Institute of Bioscience and Biotechnology, Ochang Branch), and the extracts were prepared by dissolving 100% DMSO as a solvent.

[Production Example 2]

Preparation of the whole plant fraction of the flower

After adding 10 times 99.5% methyl alcohol to the whole plant, it was sonified for 450 minutes and then immersed and extracted for 72 hours. The extract was filtered and then concentrated under reduced pressure at 50°C to prepare an extract of the whole plant extract. Afterwards, the whole plant extract prepared by the above method was dissolved in 10 times the amount of distilled water. An equal volume of nucleic acid was added to the solution, extracted three times by liquid-liquid extraction, and then concentrated under reduced pressure at 50°C to prepare a hexane fraction of the herbaceous plant. An equal volume of chloroform was added to the filtrate, extracted three times using liquid-liquid extraction, and then concentrated under reduced pressure at 50°C to prepare a chloroform fraction from the herbaceous plant. An equal volume of ethyl acetate was added to the filtrate, and the extract was extracted three times using liquid-liquid extraction, and then concentrated under reduced pressure at 50°C to prepare an ethyl acetate fraction. An equal volume of saturated butanol was added to the filtrate, extracted three times using liquid-liquid extraction, and then concentrated under reduced pressure at 50°C to prepare a butanol fraction from the plant. The filtrate was concentrated under reduced pressure at 50°C to prepare a water fraction of Soybean herbaceous plant.

[Example 1]

Screening to select natural extracts showing gammaherpesvirus inhibitory effect

To select a natural product extract showing gammaherpesvirus inhibition efficacy, the BC-3-G cell line, in which dsGFP (Green Fluorescent Protein) was introduced as a reporter gene into BC-3 cells latently infected with KSHV, a gammaherpesvirus, was used. The BC-3-G cell line has the characteristic of expressing GFP due to active infection (lytic replication) of the virus.

A total of 86 mixture samples derived from natural products were prepared for screening, and each mixture sample consisted of three types of natural product extracts.

A total of 258 natural product samples were distributed from the Korea Plant Extract Bank (Korea Research Institute of Bioscience and Biotechnology, Ochang Branch) and prepared at a concentration of 50 mg/ml using 100% DMSO as a solvent. Each natural product sample was divided into 3 alphabets by country name (or product name). The 86 natural product-derived mixture samples were prepared by mixing 40 μl each in a 1:1:1 ratio, and the types of natural product samples are shown in Table 1 below.

number	Country name/product name
One	Thin cucumber grass, thorn strawberry, thorn raspberry
2	Prickly pear, prickly pear, prickly pear
3	Day lily, mane lily, potato orchid
4	Gamjeoldae, frog stalk, rhubarb rhubarb
5	Gaemacmundong, Gaebaek tree, and development greens
6	Dog cherry tree, dog apricot tree, dog hornbeam tree
7	Gaesosiranggabi, Gagiho, hazel tree
8	Larch tree, Larch tree, Willow tree
9	Gaehoe tree, Gaetgan bow, Gaetgan jujube tree
10	Pussy willow, Pussy willow, Geomun strawberry
11	Geoje strawberry, beggar strawberry, black strawberry
12	Winter strawberries, gomari, bear strawberries
13	Coniferous pop tree, googol tree, copper pole
14	Korean fir tree, noodle tree, Gunggungi
15	Euphorbia tree, golden persimmon, Geumgang lily
16	Geum orchid, Geum mokseo, Geum saeun orchid
17	Long-leafed spirea, long-leafed spirea, crow's pillow
18	Magpie birch, spirea, flowering apple tree
19	Flower lily, peduncle, pheasant's leg flower
20	Sticky fish, Nado noodle tree, Nado brisket flower
21	Nae willow, broadleaf willow, snowy willow
22	Weeping willow, weeping willow, maple leaf weed
23	Chicken orchid, carrot, poplar tree
24	Danki willow, vine buckwheat, German spruce
25	Stone thorn tree, Stone pear tree, Stone thorn tree
26	Dried plum blossoms, crane blossoms, plum chives
27	Round leaves, wild buckthorn, wild strawberries
28	Prickly pear tree, Prickly pear tree, Prickly pear tree
29	Rigida pine, mountain ash, perennial blue
30	Macdo strawberry, croaker, buckwheat
31	Daughter-in-law's belly button, Myeong-a-ja-yekwi, Mok-seo
32	Water parsley, mukbat screech, seal hazelnut tree
33	Water birch, water birch, teal alder
34	Ash tree, American mountain lion, loach fishing
35	Cottonwood tree, loach fishing, wheat sprouts
36	Body namul, birch tree, birch tree
37	Chickadee, white line, white pine
38	Baekun oil plant, Baekun pear tree, willow tree
39	Cherry tree, chickweed tree, peach tree
40	willow, willow tree, red perennial orchid
41	Red firefly, Red knotweed, Vizzaru
42	Sabang duck, cypress tree, sandol pear
43	Wild strawberries, wild bok choy, wild chives
44	Mountain yeokyui, mountain cucumber grass, mountain outside
45	Sanjo pop tree, Sanpa, Sanhwang tree
46	Prickly pear, Sangdong leaf privet, Sangsan
47	Lively quince tree, hornbeam, Seoul quince tree
48	Seonmilnamul, Seonmilnamul, Japanese cherry tree
49	Island ash tree, island raspberry, island strawberry
50	Summal lily, Sum body, Sum willow
51	Island cherry tree, island cherry tree, pine tree
52	Hemlock, hemlock, and ash tree
53	Suri strawberry, watermelon, sorghum flower stalk
54	sycamore tree, sycamore tree, strobe pine tree
55	Singamchae, Monk fir tree, Agpear tree
56	Daylily, cherry tree, glow-in-the-dark tree
57	Medicinal orchid, prickly pear, ragweed
58	Yeo-ro, fox willow, Yeonyeongcho
59	Cucumber, cherry tree, willow tree
60	Chinese sycamore, Japanese knotweed, Japanese apricot
61	Japanese cherry tree, Yongdung bridle, Yongdung willow
62	Yunpan tree, Yunpan greens, Yunpan sprouts
63	Silver orchid, silver tree, lily of the valley
64	Silver aspen, silver aspen, silver aspen flower
65	Isak-yeo-kwi, Italian poplar, artificial wooden pop tree
66	Japanese larch, Japanese spirea, Ilwolbibichu
67	Plum tree, common cucumber plant, fine leaf body
68	Cherry blossom tree, pine tree, raspberry
69	Rose, Longevity Flower, Fir Tree
70	Jeonho, Jeju marigold, spirea tree
71	Zombie beetle, mothball duck, moth pop tree
72	Spruce tree, Swallow orchid, bamboo flower
73	Strawberry, rattail grass, geoliter grass
74	Jinhwangjeong, Prickly pear tree, Quercus columbine
75	Hazelnut tree, Quercus herb, Cham firefly
76	Chamsan Chives, Chamsorijaengi, Participation
77	Chaejin tree, drooping cherry tree, pear tree
78	Large lily, large columbine, large daylily
79	Large orchid, tall orchid, quiver
80	Turtle grass, Buckthorn tree, Buckthorn tree
81	Prickly pear tree, Prickly pear tree, Prickly pear tree
82	Red bean pear tree, Teda pine tree, Tungdung Gulre
83	Paddeuknamul, red bean pear tree, grass floss stalk
84	Pyracanda, sky lily, swallowtail
85	Japanese knotweed, red thorn tree, red daylily
86	White flowering plant, white-bodied herb, white flowering plant

The KSHV latently infected cells were treated with 20 ng/ml of TPA (12-O-Tetradecanoylphorbol-13-acetate), which induces reactivation of the virus, and 50 ㎍/ml of the mixture sample derived from the 86 natural products, and after 24 hours, the natural product Flow cytometry analysis was performed to confirm the KSHV inhibitory effect of the derived mixture sample, and the degree of KSHV reactivation was measured by analyzing the number of GFP-expressing cells among living cells.

At this time, DMSO (Dimethyl Sulfoxide), which was used as a solvent for the TPA and natural product-derived mixture samples, was used as a negative control.

Figure 1 shows the level of GFP expression when processing the natural product-derived mixture sample based on the negative control group treated with the BC-3-G cell line, and Figure 2 shows the degree of KSHV inhibition of 86 natural product-derived mixture samples for easy analysis. To this end, the level of GFP expression in the negative control group was set to 0 and was schematized.

As a result, as shown in Figure 2, the degree of KSHV inhibition of mixture samples 22, 28, 29, 51, 52, 57, 63, 64, and 70 was found to be more than 40% higher than that of the negative control group. However, cytotoxicity was confirmed in the three natural product-derived mixtures Nos. 29, 57, and 63, and a total of six mixtures Nos. 22, 28, 51, 52, 64, and 70, excluding this, were tested for KSHV antiviral efficacy. It was selected as a mixture derived from natural products.

[Example 2]

Evaluation of KSHV antiviral efficacy of selected natural product-derived mixtures

To evaluate the KSHV antiviral efficacy of the six natural product-derived mixtures selected through Example 1, BC-3-G cells were treated with TPA, and each of the six natural product-derived mixtures was treated with 50 μg/ml. , flow cytometry and western blot analysis were performed using ganciclovir as a positive control.

As shown in Figure 3, flow cytometry results showed that the number of GFP-expressing cells was lower when processing mixture samples 52, 64, and 70 compared to the control group, and the Western blot results in Figure 4 also showed that the lytic protein of KSHV ), it was confirmed that the protein band thickness of RTA and K8 decreased.

In addition, to confirm whether the above three mixture samples inhibit KSHV reactivation in a concentration-dependent manner, TPA and 10, 25, and 50 respectively were added to KSHV latently infected cell lines BC-3, BC-3-G, and BCBL-1. A mixture sample at a concentration of ㎍/ml was processed, and the expression levels of lytic proteins RTA and K8 were confirmed through Western blot analysis.

As a result, as can be seen in Figures 5 to 7, the three mixture samples inhibited the reactivation of KSHV in a concentration-dependent manner.

As a result, it was confirmed that mixture samples 52, 64, and 70 among the six natural product-derived mixtures selected through Example 1 had an antiviral effect against KSHV.

[Example 3]

Evaluation of EBV antiviral efficacy of selected natural product-derived mixtures

To evaluate the antiviral efficacy against EBV among the gammaherpesviruses of the six natural product-derived mixtures selected through Example 1, Raji cell line and Akata(+) cell line latently infected with EBV were used.

The Raji cell line was treated with TPA to induce virus reactivation, and the Akata(+) cell line was treated with IgG, and the Raji cell line and the Akata(+) cell line were treated with 50 μg/ml of each of the six natural product-derived mixtures, The expression levels of ZTA and EA-D, which are lytic proteins of EBV, were confirmed through Western blot analysis.

As shown in Figure 8, when the mixture samples 52, 64, and 70 were treated, the expression level of ZTA was confirmed to decrease in the Raji cell line. As shown in Figure 9, the mixture samples 52, 64, and 70 were confirmed to be When treated, it was confirmed that the protein band thickness of ZTA and EA-D decreased in Akata(+) cell line.

In addition, as in Example 2, in order to confirm whether the selected natural product-derived mixture sample was effective in suppressing EBV reactivation in a concentration-dependent manner, IgG and IgG at concentrations of 10, 25, and 50 μg/ml were applied to the Akata (+) cell line. Mixture samples No. 52, 64, and 70 were processed, and the expression levels of lytic proteins EA-D and ZTA were confirmed by Western blot analysis.

As a result, as shown in FIG. 10, it was confirmed that mixture samples Nos. 52, 64, and 70 had a concentration-dependent antiviral effect against EBV.

As described above, it was confirmed that mixture samples 52, 64, and 70 among the mixtures derived from natural products were effective not only for KSHV but also for EBV. Based on the results, in the examples below, the mixtures contained in the three types of mixtures derived from natural products were tested. An experiment on antiviral efficacy was conducted on a total of 9 types of natural product extracts.

[Example 4]

Evaluation of KSHV antiviral efficacy of extracts of hemlock, cypress, euphorbia, and Jeju mantle extracts

The types of individual natural product extracts included in the three natural product-derived mixtures that showed antiviral efficacy are shown in Table 2 below, and an experiment was conducted to evaluate the antiviral efficacy of each natural product extract.

A	hemlock tree
B	cotton brisket flower
C	ash tree
D	silver white sheep
E	silver aspen tree
F	silver lily flower
G	phone
H	Jeju cover
I	spirea tree

To evaluate whether the nine types of natural product extracts have KSHV antiviral efficacy, 20ng/ml of TPA, which induces virus reactivation, was treated with KSHV latently infected cell lines BC-3-G and BCBL-1, and the natural product extracts were 50 μg/ml of each was treated, and after 24 hours, the expression levels of RTA and K8, the lytic proteins of KSHV, were confirmed through Western blot analysis.

As a result, as shown in Figures 11 and 12, KSHV antiviral efficacy was confirmed in hemlock (A), hemlock (B), euphorbia (F), and jeonho (G), especially in hemlock (B) and Prickly pear (F) showed strong antiviral efficacy in both BC-3-G and BCBL-1 cell lines.

[Example 5]

Evaluation of EBV antiviral efficacy of extracts of hemlock, cypress, euphorbia, and Jeju mantle.

To evaluate the EBV antiviral efficacy of the natural product extract in Table 2, the Raji cell line, an EBV latently infected cell line, was treated with 20ng/ml TPA and 3mM NaB (sodium butyrate), which induce reactivation of the virus, and the Akata(+) cell line. was treated with 10㎍/㎖ of IgG, and 50㎍/㎖ of 9 natural product extracts were treated each on Raji cell line and Akate(+) cell line, and after 24 hours, Western blot analysis revealed the lytic protein of EBV. ), the expression levels of EA-D and ZTA were confirmed.

As a result, as shown in FIGS. 13 and 14, Prickly pear (B), Prickly pear (F), and Jeju capsicum (H) showed antiviral efficacy against Raji cell lines, and Prickly pear (B) and Prickly pear (B) ( F), Jeonho (G), and Jeju Capricorn (H) showed antiviral efficacy against Akata (+) cell line.

Accordingly, through Examples 4 and 5, it was confirmed that Eunuchia, Eunbi and Jeonho have antiviral effects against both KSHV and EBV, Hemlock is specific for Kaposi's sarcoma herpes virus (KSHV), and Jeju capifolia is specific for Kaposi's sarcoma herpes virus (KSHV). It was confirmed that it has a specific antiviral effect against Epstein-Barr virus (EBV).

[Example 6]

Evaluation of the antiviral efficacy of the whole plant extract and fractions of the flowering plant and the flowering plant against norovirus

The antiviral efficacy of five types of fractions from Prickly pear (B), Prickly pear (F), and Prickly pear flowers was evaluated against murine norovirus 1 (MNV-1, CW3 strain), a mouse norovirus.

As shown in Figure 15, the Raw264.7 cell line, a murine macrophage cell line, was seeded at 3x10^5 cells/well in a 6-well plate one day before infection, and the next day, MNV-1 was added at an MOI (Multiplicity of infection) of 0.01. After infection with , the extracts of the natural products Somnorrhea (B) and Euphorbia (F) were treated with 50 ug/ml. At 24 hours after infection, the supernatant was collected, serially diluted 10 times, and the diluted solution was infected with BV-2, a mouse microglial cell line, and cultured for 3 days to measure virus titer by plaque assay.

The sample concentration and treatment concentration of the fractions of the five species of Solanum vulgaris are shown in Table 3 below.

sample	Sample concentration (mg/ml)	Treatment concentration (μg/ml)
hexane fraction	7.97	7.97
Chloroform fraction	11.87	11.87
Ethyl acetate fraction	1.92	1.92
Butanol fraction	3.7	3.7
water fraction	24.6	24.6

The antiviral efficacy of the five fractions of the Prickly pear extract (B) was evaluated in the same manner.

The results of the experiment on extracts of Lilium chinensis (B) and Lilium chinensis (F) are shown in Figure 16, and as shown in Figure 2, norovirus proliferation was significantly inhibited when treated with Lilium chinensis (B) and Lilium chinensis (F). In particular, when treated with the Euphorbia flower (F) extract, a 100% inhibitory effect was observed, confirming excellent antiviral efficacy.

The experimental results of the five types of fractions of the extract of Solanum vulgaris (B) are shown in Figure 17, and as shown in Fig. 3, plaque formation was significantly inhibited in the experimental group treated with the hexane fraction and the chloroform fraction of Lilium vulgaris (B). It was confirmed to have excellent antiviral efficacy.

Therefore, the whole plant extracts of Prickly pear (B) and Prickly pear (F), and the hexane and chloroform fractions of Prickly pear (B) have excellent antiviral efficacy and contain powerful antiviral substances that inhibit MNV-1 proliferation. You can see that

[Example 7]

Evaluation of the antiviral efficacy of Elephant chinensis whole plant extract, Elephant perilla alla extract, Fraction of Elephant herbacea, and Jeonho extract against alphaherpesvirus.

A plaque reduction assay was performed to evaluate the antiviral efficacy against HSV-1 and VZV, which are alphaherpesviruses that infect the nervous system.

As shown in Figure 18, Vero cells or 12-well plates were seeded one day before infection, and on the day of the experiment, each well was infected with >100 PFU of virus for 1 hour, and then overlay media mixed with the extract or fraction was added to each well. After filling and culturing HSV-1 for 3 days, the number of plaques formed was counted.

VZV was infected in MeWo cells for 2 hours, the extract or fraction was treated, and the number of plaques was counted after culturing for 4-6 days. The relative rate of viral plaque formation (%) in extract or fraction treated samples was analyzed based on the number of plaques in vehicle treated samples.

2-1 HSV-1 antiviral efficacy evaluation results

To evaluate antiviral efficacy against HSV-1, a plaque reduction assay corresponding to the gold standard assay was performed. Vero cells, a cell line derived from the African Green Monkey kidney, were infected with >100 PFU of HSV-1 per well for 1 hour, then treated with overlay media treated with 10, 25, and 50 ㎍/ml extract, and cultured for 3 days. The results are shown in Figure 19.

As shown in Figure 19, Gancyclovir (GCV), a nucleoside analog used as a positive control, was treated at a concentration of 40 μg/ml, and it was confirmed that plaque formation was inhibited. In addition, when extracts of Prickly pear (B) and Prickly pear (F) were treated at concentrations of 10, 25, and 50 ㎍/㎖, very high antiviral efficacy was shown at high concentrations.

In the plaque reduction assay, the number of plaques formed and the size of the plaques are also indicators of antiviral efficacy. When treated with extracts of Prickly pear (B) and Prickly pear (F), it was confirmed that the size of plaques decreased in a concentration-dependent manner at 10 and 25 μg/ml, as shown in Figure 20.

Additionally, a plaque reduction assay was performed on the Jeonho (G) extract, and the results are shown in Figure 21. As shown in Figure 21, when the Jeonho (G) extract was treated at concentrations of 10, 25, and 50 ㎍/㎖, no plaques were formed at all concentrations, showing excellent antiviral efficacy. In addition, it was confirmed that the formation of plaques was completely inhibited even when the concentration was further lowered to 2 and 5 ㎍/㎖.

In the end, it can be seen that the extracts of Prickly pear (B), Prickly pear (F), and Prickly pear (G) have excellent antiviral efficacy against HSV-1.

Additionally, a plaque reduction assay was performed on the antiviral efficacy of five types of fractions of Prickly pear (B) at the concentrations shown in Table 3 above, and the results are shown in Figure 22. As shown in Figure 22, it was confirmed that the hexane fraction and chloroform fraction of Prickly pear (B) exhibited excellent antiviral efficacy against HSV-1.

2-2 VZV antiviral efficacy evaluation results

MeWo cells, a cell line derived from human malignant melanoma, were infected with >100 PFU of VZV for 2 hours, and media treated with 10, 25, and 50 μg/ml extract were cultured for 4-6 days.

As shown in Figure 23, the number of VZV plaques decreased in a concentration-dependent manner when treated with extracts of Prickly pear (B) and Prickly pear (F) extracts.

In addition, as shown in Figure 24, it was confirmed that the size of VZV plaques decreased in a concentration-dependent manner when treated with extracts of Euphorbia flower (B) and Euphorbia chinensis (F).

Plaque reduction assay was performed on five types of fractions of Prickly pear (B) that showed concentration-dependent inhibitory efficacy against VZV infection at the concentrations shown in Table 3 above, and the results are shown in Figures 25 to 27. As shown in Figure 25, it was confirmed that among the five fractions (hexane, chloroform, ethyl acetate, butanol, and water fractions) of the extract of Prickly pear (B), the butanol, ethyl acetate, and water fractions did not show antiviral efficacy. .

In addition, as shown in Figures 26 and 27, when testing the concentration-dependent antiviral efficacy of the hexane fraction (1.59, 3.99, 7.97 μg/ml) and the chloroform fraction (2.37, 5.94, 11.87 μg/ml), the hexane fraction was It was confirmed that VZV plaque formation was reduced in a concentration-dependent manner and the size of plaques was reduced, showing antiviral efficacy.

Therefore, the extracts of Lilium vulgaris and Lilium vulgaris have antiviral effects that effectively inhibit infection by varicella-zoster virus (VZV), one of the alphaherpesviruses that infect the nervous system. In particular, the hexane fraction of Lilium vulgaris has a concentration-dependent antiviral effect. Able to know.

Claims (10)

1. A pharmaceutical composition for the prevention or treatment of gammaherpesvirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of hemlock, hemlock, euphorbia, and Jeju mantle.
2. The pharmaceutical composition according to claim 1, wherein the gammaherpesvirus is Kaposi's sarcoma-associated herpesvirus (KSHV).
3. The pharmaceutical composition according to claim 2, wherein the composition reduces the expression of the lytic protein of Kaposi's sarcoma herpes virus.
4. The pharmaceutical composition according to claim 2, wherein the gammaherpesvirus infectious disease is one or more selected from the group consisting of Kaposi's sarcoma, primary exudative lymphoma, and multiple Castleman's disease.
5. The pharmaceutical composition according to claim 1, wherein the gammaherpesvirus is Epstein-Barr virus (EBV).
6. The pharmaceutical composition according to claim 5, wherein the composition reduces the expression of the lytic protein of the Epstein-Barr virus.
7. The pharmaceutical composition according to claim 5, wherein the gammaherpesvirus infectious disease is one or more selected from the group consisting of Burkitt's lymphoma, gastric carcinoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, and post-transplant B cell lymphoma.
8. A food composition for preventing or improving gammaherpes virus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of hemlock, cypress, euphorbia, and Jeju eucalyptus.
9. A feed composition for animals for preventing or improving gammaherpesvirus infection disease, comprising as an active ingredient one or more extracts selected from the group consisting of hemlock, hemlock, eucalyptus, and Jeju marigold.
10. The method of claim 1, comprising one or more extracts selected from the group consisting of hemlock, hemlock, euphorbia, and Jeju euphorbia, or extracted from a mixture selected from the group consisting of hemlock, euphorbia, euphorbia, and Jeju eucalyptus. A pharmaceutical composition comprising an extract.

    [Claim 11]

    A pharmaceutical composition for the prevention or treatment of norovirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of Euphorbia flower and Euphorbia chinensis extract.

    [Claim 12]

    The pharmaceutical composition for preventing or treating norovirus infectious diseases according to claim 11, wherein the norovirus is a human norovirus.

    [Claim 13]

    The pharmaceutical composition according to claim 11, wherein the norovirus infectious disease is non-bacterial gastroenteritis.

    [Claim 14]

    12. The pharmaceutical composition according to claim 11, wherein the Euphorbia chinensis extract is fractionated with one or more solvents selected from the group consisting of hexane, chloroform, butanol, ethyl acetate, and water.

    [Claim 15]

    The pharmaceutical composition according to claim 11, wherein the Euphorbia flower extract is a hexane fraction or a chloroform fraction.

    [Claim 16]

    A food composition for preventing or improving norovirus infectious diseases, comprising as an active ingredient one or more extracts selected from the group consisting of Prickly pear and Prickly pear extracts

    [Claim 17]

    A feed composition for animals for preventing or improving norovirus infection disease, comprising as an active ingredient one or more extracts selected from the group consisting of Prickly pear and Prickly pear extracts

    [Claim 18]

    The pharmaceutical composition according to claim 11, comprising one or more extracts selected from the group consisting of extracts of Lilium chinensis and Lilium chinensis extract, or comprising an extract extracted from a mixture selected from the group consisting of extracts of Lilium chinensis and Lilium chinensis extract. Composition.

    [Claim 19]

    A pharmaceutical composition for the prevention or treatment of alphaherpesvirus infectious disease, comprising as an active ingredient one or more extracts selected from the group consisting of Achyranthesia flower, Achyranthesia flower, and Jeonho extract.

    [Claim 20]

    The pharmaceutical composition according to claim 19, wherein the alphaherpesvirus is Herpes simplex virus-1 (HSV-1) or Varicella-Zoster virus (VZV).

    [Claim 21]

    The pharmaceutical composition according to claim 20, wherein the herpes simplex virus infectious disease is at least one selected from the group consisting of herpes simplex, cold sores, stomatitis, meningitis, and encephalitis.

    [Claim 22]

    The pharmaceutical composition of claim 20, wherein the varicella-zoster virus infectious disease is at least one selected from the group consisting of varicella-zoster virus herpes zoster and Zoster Sine Herpete.

    [Claim 23]

    20. The pharmaceutical composition according to claim 19, wherein the Euphorbia chinensis extract is fractionated with one or more solvents selected from the group consisting of hexane, chloroform, butanol, ethyl acetate, and water.

    [Claim 24]

    The pharmaceutical composition according to claim 19, wherein the Euphorbia flower extract is a hexane fraction and a chloroform fraction.

    [Claim 25]

    25. The pharmaceutical composition according to claim 24, wherein the Euphorbia chinensis extract is a hexane fraction.

    [Claim 26]

    A food composition for preventing or improving alphaherpesvirus infection disease, comprising as an active ingredient one or more extracts selected from the group consisting of Euphorbia flower, Euphorbia flower, and Jeonho extract.

    [Claim 27]

    Animal feed composition for preventing or ameliorating alphaherpesvirus infection disease, comprising as an active ingredient one or more extracts selected from the group consisting of Euphorbia flower, Euphorbia flower, and Jeonho extract.

    [Claim 28]

    20. The method of claim 19, comprising one or more extracts selected from the group consisting of Lilac flower, Euphorbia flower, and Jeonho extract, or comprising an extract extracted from a mixture selected from one or more of the group consisting of extracts of Orchid flower, Euphorbia flower, and Jeonho extract. A pharmaceutical composition, characterized in that.

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