KR102133927B1 - Pharmaceutical composition for inhibiting of Varicella-Zoster Virus and Hepatitis E virus comprising Lysimachia mauritiana extract or fraction thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a magpie swimming extract or a fraction thereof having the effect of inhibiting the proliferation of varicella-zoster virus and hepatitis E virus.
The magpie swimming extract of the present invention or a fraction thereof inhibits the expression of Immediate early (IE) gene, Early (E) gene, and Late (L) gene for replication of chickenpox-herpes zoster virus, and also of hepatitis E virus Since it is possible to suppress the expression of the ORF2 region for replication, the extract and fractions can be useful for the treatment of diseases caused by varicella-zoster virus or hepatitis E virus infection.

Description

Pharmaceutical composition for inhibiting of Varicella-Zoster Virus and Hepatitis E virus comprising Lysimachia mauritiana extract or fraction thereof as an active ingredient}

The present invention relates to a magpie swimming extract or a fraction thereof having the effect of inhibiting the proliferation of varicella-zoster virus and hepatitis E virus.

Varicella-Zoster Virus (VZV) is a human pathogenic virus that is highly contagious and predominantly occurs in children 2 to 8 years of age in winter and spring. Varicella-zoster virus replicates the virus's DNA through sequential expression of Immediate early (IE), Early (E) and Late (L) genes upon infection. The IE62 protein encoded by the ORF (open reading frame) 62 is essential for activation of a gene related to lytic action, and binds transcription factors to induce the expression of E genes. The E genes play a critical role in chickenpox-herpes virus replication, and induce the expression of L genes to promote transcription of the components that make up the virion.

Chickenpox is caused by the primary infection of the varicella-zoster virus, but it can also occur as a herpes zoster when the virus is latent in the ganglion and then reactivated.

Chickenpox or herpes zoster usually progresses well in normal subjects, but can be fatal in patients with hematologic malignancies, patients with solid organ transplants or hematopoietic stem cell transplants, or patients with autoimmune diseases or congenital immunodeficiency disorders.

The vaccine against varicella-zoster virus was developed in Japan in the 1970s, and has been inoculated as a national essential vaccination since 2005 since the introduction of the chickenpox vaccine in 1988 in Korea. Since the introduction of the chickenpox vaccine, the incidence and hospitalization rates of chickenpox and shingles have decreased significantly. In the United States, as the vaccination rate increased, the incidence of chickenpox decreased 71-84% compared to before vaccination, and the hospitalization rate associated with chickenpox decreased 75-88%. In addition, the death rate from chickenpox was reduced by 66% at the age of 50 years or younger, and the mortality rate was reduced by 92% in children 1 to 4 years old.

However, 180,000~240,000 cases have been reported every year even though chickenpox vaccine shows a high vaccination rate exceeding 80% after being included in the national essential immunization. This is because the rate of antibodies produced by chickenpox vaccine when getting a vaccine is 77-82%, which is lower than that of other vaccines. It is presumed to be the cause, which is more at risk for immunocompromised patients or those who have not received the vaccine. Therefore, chickenpox is a disease that has high need for treatment as well as prevention.

The varicella-zoster virus is a virus belonging to the Herpesviridae family, which causes various symptoms such as blisters, skin peeling, encephalitis, pain, sensory abnormalities, and nervous system damage, and is divided into several as shown in Reference 1.

[Reference Figure 1]

Viruses belonging to the Herpes family of viruses are infected with the kidneys and cause cytomegalic symptoms as shown in Table 1 below.Human Herpes Virus-5 (HHV-5), infected with lymphoid tissues, and have lymphocyte proliferation (lymphoproliferative) It is differentiated to have different infection sites and life cycles, such as the causing Epstein-Barr virus (Human Herpes Virus-5; HHV-4). Herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) belonging to the Alpha subfamily are chickenpox-herpes virus and genome as shown in Table 2. Since there is a difference from the stage, even the same herpes virus family needs to develop a treatment for each virus.

Classification of the Herpesviridae designation subfamily
(subfamily) genus
(genus) Life History/
Cytopathological features Infection site HHV-1 Herpes simplex virus-1
(Herpes simplex virus-1) Alpha Simplexvirus Short life cycle/
Cell lysis nerve HHV-2 Herpes simplex virus-2
(Herpes simplex virus-2) Alpha Simplexvirus Short life cycle/
Cell lysis nerve HHV-3 Varicella-zoster virus
(Varicella-zoster virus) Alpha Varicellovirus Short life cycle/
Cell lysis nerve HHV-4 Epstein-Barr virus
(Epstein-Barr virus) Gamma Lymphocryptovirus Various life cycles/
Lymphocyte hyperplasia Lymphatic tissue HHV-5 Cytomegalovirus Beta Cytomegalovirus Long life cycle/
Cell giant Gland, kidney HHV-6 Herpes simplex virus-6
(Herpes simplex virus-6) Beta Roseolavirus Long life cycle/
Lymphocyte hyperplasia Lymphatic tissue HHV-7 Herpes simplex virus-7
(Herpes simplex virus-7) Beta Roseolavirus Long life cycle/
Lymphocyte hyperplasia Lymphatic tissue HHV-8 Kaposi's sarcoma-inducing virus
(Kaposi's sarcoma associated virus) Gamma Rhadinovirus Various life cycles/
Lymphocyte hyperplasia Lymphatic tissue

HSV-1 (HHV-1) and HSV-2 (HHV-2) VZV(HHV-3) genome size ~152 Kbp ~125Kbp GC ratio 67% 47% protein Immediate Early protein ICP0, ICP4, ICP22, ICP27, ICP47 IE4, IE62 and IE63 Cortical protein UL46-49 ORF9-12 The nerve that lurks when infected
HSV-1: Trigeminal ganglion
(Trigeminal ganglia),
HSV-2: cranial ganglion
(Sacral ganglia) Sensory ganglia
(Sensory ganglion)

Drugs acting on DNA polymerases, including acyclovir (ACV), famciclovir, valacyclovir and foscarnet, are currently used to treat varicella-zoster virus. Are used. However, the antiviral agent is known to induce side effects in chronically infected patients with weakened immunity, and a type resistant to the antiviral agent has been shown. There is a need to develop a therapeutic agent that eliminates the side effects.

Hepatitis E is transmitted through a fecal-oral route orally spread by drinking water contaminated with Hepatitis E virus (HEV), the prevalence of which is the health and economic level of each country. It is closely related to. For this reason, it mainly affects Asian countries such as India, Nepal, Myanmar, Pakistan and China, African countries such as Sudan, Somalia, and Latin American countries such as Mexico. have.

The hepatitis E virus, which is the cause of hepatitis E, is a virus differentiated from other hepatitis viruses and exhibits viremia for about two weeks upon infection. In addition, symptoms such as elevated plasma bilirubin, abdominal pain, anorexia, hepatomegalia, vomiting, and fever are accompanied by major symptoms. Symptoms from hepatitis E virus infection last on average for 40 days, after which most of them heal naturally, but some become chronic with cirrhosis. In general, the mortality rate is as low as 3%, but in pregnant women and the elderly, the mortality rate increases to 25 to 30%.

Hepatitis E virus is a virus belonging to the Hepeviridae family, consisting of a single RNA strand of about 7.2 kb in length, and specifically has a (+)-sense strand RNA genome. The hepatitis E virus genome includes ORF (open reading frame) 1, 2 and 3, where ORF1 is a Met (methyltransferase) domain, Y domain, PCP (papain-like cysteine protease) domain, HVR (hypervariable region), Pro (proline) It encodes a non-structural replicase, a polyprotein with a -rich region, a X-domain, a Helicase (Hel) domain, and a functional domain of RNA dependent RNA polymerase (RdRp). The coding regions of ORF2 and ORF3 overlap and are translated from bicistronic sub-genomic RNA. ORF2 encodes capsid proteins that bind to cellular proteins such as heparin sulfate proteoglycan (HSPG), heat-shock protein 90 (HSP90) and glucose-regulated protein 78 (Grp78), and ORF3 is the hepatitis E virus virion It encodes a multifunctional phosphoprotein, which is important for the release of (virion). ORF3 inhibits the host's innate immune response by degrading TRADD (tumor necrosis factor receptor 1-associated death domain) protein and reducing ubiquitination of RIP1 (receptor interacting protein 1) protein, thereby inhibiting NF-κB activation. Was reported.

The viral genomic RNA of the hepatitis E virus entering the host cell serves as the mRNA for translation of ORF1 and the template for replication. The ORF1 protein is translated from viral genomic RNA, and the largest ORF1 domain, RdRp, synthesizes (-)-sense RNA. The (+)-sense genomic and sub-genomic RNAs are synthesized by RdRp using (-)-sense RNA as a template. ORF2 and ORF3 are translated from subgenomic RNA, and viral genomic RNA is packaged into virions.

Currently, there is no effective treatment for the hepatitis E virus, and in general, hepatitis E virus is used to treat hepatitis E, which exhibits various side effects during long-term administration, although ribavirin and interferon alpha are administered for chronic hepatitis. It is desired to develop a therapeutic agent that is suitable for and eliminates the side effects.

Lysimachia mauritiana is a dicotyledonous plant distributed in the rocky seashores of southern areas including Jeju and Ulleungdo. Young leaves are edible and fruits are used for ornamental purposes. It is used as a strong heart and diuretic in oriental medicine, and is known to be effective in hypertension, diabetes, constipation, edema and bruises. Republic of Korea Patent No. 10-1168567 discloses the anti-fungal activity of saponin-based substances contained in blackcurrant swimming, and is increasingly used in various ways for the use of blackcurrant swimming.

Thus, the present inventors confirmed that the larvae of the Magpie Squirrel (Lysimachia mauritiana extracts; LME) or a fraction thereof has the effect of inhibiting the proliferation of chickenpox-herpes virus and hepatitis E virus, and treatment for chickenpox-herpes virus and hepatitis E virus The present invention has been completed by revealing that it can be usefully used.

Republic of Korea Registered Patent No. 10-1168567

An object of the present invention is to provide a therapeutic use of diseases caused by infection of the varicella-zoster virus and hepatitis E virus containing the Magpie Swim extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by varicella-zoster virus infection containing the Magpie Swim extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food for the improvement of diseases caused by the varicella-zoster virus infection containing the blackcurrant swimming extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by hepatitis E virus infection containing the Magpie Swim extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a health functional food for the improvement of diseases caused by hepatitis E virus infection containing a blackcurrant swimming extract or a fraction thereof.

The magpie swimming extract or fractions thereof of the present invention inhibit the expression of Immediate early (IE) gene, Early (E) gene, Late (L) gene for replication of chickenpox-herpes zoster virus, and also of hepatitis E virus Since it is possible to suppress the expression of the ORF2 region for replication, the extract and fractions can be useful for the treatment of diseases caused by varicella-zoster virus or hepatitis E virus infection.

1 is a diagram showing the effect of inhibiting the growth of varicella-zoster virus of the Magpie Swim extract:
VZV-pOka: artificially recombined pOka strain of varicella-zoster virus (VZV);
DMSO: dimethyl sulfoxide treatment control group;
LME: experimental group treated with the Magpie Swim Extract;
BF: Bright field microscopic image;
FL: Fluorescence microscopic image; And
hpi: hours post-infection.
Figure 2 is a diagram showing the effect of inhibiting the growth of varicella-zoster virus according to the concentration of magpie swimming:
VZV-YC01: Varicella-zoster virus (VZV) YC01 strain obtained clinically.
Figure 3 is a diagram showing the cytotoxicity of the magpie swimming extract.
4 is a diagram showing the effect of inhibiting the varicella-zoster virus Immediate early (IE) gene, Early (E) gene, and Late (L) gene of the Magpie Swim extract:
DMSO: dimethyl sulfoxide treatment control group;
LME: experimental group treated with the Magpie Swim Extract;
ORF62: Open reading frame 62;
ORF28: Open reading frame 28;
gB: glycoprotein B; And
hpi: hours post-infection.
Figure 5 is a diagram showing the effect of inhibiting the chicken pox-shingles virus IE62 (Immediate early 62) protein of the magpie swimming:
VZV-pOka: artificially recombined chickenpox-herpes virus (VZV) pOka strain;
DMSO: dimethyl sulfoxide treatment control group;
LME: experimental group treated with the Magpie Swim Extract; And
IE62: Immediate early 62 protein.
6 is a view showing the hepatitis E virus proliferation inhibitory effect of the Magpie Swim extract:
DMSO: dimethyl sulfoxide treatment control group; And
LME: Experimental group for the treatment of Magpie swimming.
Figure 7 is a diagram showing the hepatitis E virus proliferation inhibitory effect and cytotoxicity according to the concentration of Magpie swimming extract:
LME: Experimental group for the treatment of Magpie swimming.
8 is a diagram showing the hepatitis E virus proliferation inhibitory effect of the Magpie Swim extract:
DMSO: dimethyl sulfoxide treatment control group;
LME: experimental group treated with the Magpie Swim Extract;
HEV: Hepatitis E virus;
BF: Bright field microscopic image; And
FL: Fluorescence microscopic image.
9 is a diagram showing the cytotoxicity of the magpie swimming extract:
LME: Experimental group for the treatment of Magpie swimming.
Figure 10 is a schematic diagram showing the process of fractionating the magpie swimming with an organic solvent:
EtOH: Ethanol;
CHCl ₃ : Chloroform;
EtOAc: Ethyl acetate;
n-BuOH: butanol (n-butanol); And
ddH ₂ O: Double distilled water.
11 is a diagram showing the effect of inhibiting the growth of varicella-herpes virus of the magpie swimming fraction:
VZV-pOka: artificially recombined chickenpox-herpes virus (VZV) pOka strain;
DMSO: dimethyl sulfoxide treatment control group;
LM-total: 70% ethanol extract treatment experiment group;
LM-EA: experimental group for treatment of ethyl acetate fraction of Magpie swimming extract;
LM-BuOH: experimental group for the treatment of butanol fractions of Magpie swimming extract;
LM-DW: Experimental group for re-distilled water fraction treatment of Magpie swimming;
LM-CHCl ₃ : Experimental group for treating chloroform fractions of magpie swimming extract; And
LM-Hx: Experimental group for treating hexane fractions of magpie swimming extract.
12 is a diagram showing the cytotoxicity of the magpie swimming fraction:
DMSO: dimethyl sulfoxide treatment control group;
LM-total: 70% ethanol extract treatment experiment group;
LM-EA: experimental group for treatment of ethyl acetate fraction of Magpie swimming extract;
LM-DW: Experimental group for re-distilled water fraction treatment of Magpie swimming;
LM-BuOH: experimental group for the treatment of butanol fractions of Magpie swimming extract;
LM-CHCl ₃ : Experimental group for treating chloroform fractions of magpie swimming extract;
LM-Hx: Experimental group for treating hexane fractions of magpie swimming extract; And
HFF: Human foreskin fibroblast.
13 is a diagram showing the hepatitis E virus proliferation inhibitory effect of the magpie swimming fraction:
pSHEV3-luciferase replicon: porcine hepatitis E virus of type 3 genotype (HEV) Renilla luciferase replicon;
DMSO: dimethyl sulfoxide treatment control group;
LM-total: 70% ethanol extract treatment experiment group;
LM-EA: experimental group for treatment of ethyl acetate fraction of Magpie swimming extract;
LM-BuOH: experimental group for the treatment of butanol fractions of Magpie swimming extract;
LM-DW: Experimental group for re-distilled water fraction treatment of Magpie swimming;
LM-Hx: Experimental group for treating hexane fractions of magpie swimming extract; And
LM-CHCl ₃ : Experimental group for treating chloroform fractions of magpie swimming extract.
14 is a view showing the hepatitis E virus proliferation inhibitory effect of the Magpie swimming fraction:
HEV47832c: Hepatitis E virus (HEV) 47832c strain of type 3 genotype;
DMSO: dimethyl sulfoxide treatment control group;
LM-total: 70% ethanol extract treatment experiment group;
LM-EA: experimental group for treatment of ethyl acetate fraction of Magpie swimming extract;
LM-BuOH: experimental group for the treatment of butanol fractions of Magpie swimming extract;
LM-DW: Experimental group for re-distilled water fraction treatment of Magpie swimming;
LM-Hx: Experimental group for treating hexane fractions of magpie swimming extract; And
LM-CHCl ₃ : Experimental group for treating chloroform fractions of magpie swimming extract.
15 is a diagram showing the cytotoxicity of the magpie swimming fraction.
Huh7.5 cell: Human hepatocellular carcinoma cell;
DMSO: dimethyl sulfoxide treatment control group;
LM-total: 70% ethanol extract treatment experiment group;
LM-EA: experimental group for treatment of ethyl acetate fraction of Magpie swimming extract;
LM-BuOH: experimental group for the treatment of butanol fractions of Magpie swimming extract;
LM-DW: Experimental group for re-distilled water fraction treatment of Magpie swimming;
LM-CHCl ₃ : Experimental group for treating chloroform fractions of magpie swimming extract; And
LM-Hx: Experimental group for treating hexane fractions of magpie swimming extract.
16 is a diagram showing the cytotoxicity of the magpie swimming fraction.
A549 cell: Human adenocarcinoma cell;
DMSO: dimethyl sulfoxide treatment control group;
LM-total: 70% ethanol extract treatment experiment group;
LM-EA: experimental group for treatment of ethyl acetate fraction of Magpie swimming extract;
LM-BuOH: experimental group for the treatment of butanol fractions of Magpie swimming extract;
LM-DW: Experimental group for re-distilled water fraction treatment of Magpie swimming;
LM-CHCl ₃ : Experimental group for treating chloroform fractions of magpie swimming extract; And
LM-Hx: Experimental group for treating hexane fractions of magpie swimming extract.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by varicella-zoster virus infection that contains the Magpie Swim extract or a fraction thereof as an active ingredient.

The magpie swimming can be prepared by a manufacturing method comprising the following steps:

1) preparing an extract by adding an extracting solvent to the magpie swimming;

2) filtering the extract of step 1); And

3) Concentrating the filtered filtrate of step 2) under reduced pressure and drying.

In the above manufacturing method, the magpie swimming may include all parts such as leaves, stems, roots, fruits, and shells of the magpie swimming.

In addition, the extraction solvent may be water, alcohol or a mixture thereof. The alcohol may be a lower alcohol of C1 to C2, specifically, the alcohol may be ethanol or methanol. In one embodiment of the present invention, the extraction solvent may be 70% ethanol. The extraction solvent may be added in an amount of 1.7 ml per 1 g of the magpie swimming used for extraction.

The extraction method may be shaking extraction, Soxhlet extraction or reflux extraction. At this time, the extraction time may be 72 hours.

On the other hand, the reduced pressure concentration of step 3) may use a vacuum decompressor or a vacuum rotary evaporator. In addition, the drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying, and specifically, freeze drying.

The fraction may be further divided into hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction, and hexane and chloroform used in the production of each fraction of magpie swimming according to the present invention. , The concentration and amount of ethyl acetate and butanol, the number of repetitions of shaking and fractionation, and the degree of concentration can be easily changed according to the intended use.

The disease caused by the varicella-zoster virus infection may include, but is not limited to, varicella and herpes zoster.

The composition exhibits an inhibitory effect on the expression of Immediate-early(IE), Early(E) and Late(L) genes for chickenpox-herpes virus replication.

The Immediate-early (IE) gene of the varicella-zoster virus includes, but is not limited to, the ORF62 (IE62) gene.

The Early(E) gene of the varicella-zoster virus includes, but is not limited to, the ORF28 gene.

The Late(L) gene of the varicella-zoster virus includes, but is not limited to, the glycoprotein B (gB) gene.

In a specific embodiment of the present invention, the magpie swimming extract was found to have an effect of inhibiting the replication of varicella-zoster virus DNA (FIGS. 1 and 2), and did not show cytotoxicity at IC ₅₀ concentrations (FIG. 3). In the cells infected with varicella-zoster virus, the group treated with blackcurrant swimming extract showed that the transcription of the immediate early gene, ORF62, the transcription of the early gene, ORF28, and the transcription of the Late gene, gB (FIG. 4 ), were reduced. The expression of IE62 protein synthesized through transcription and translation was also reduced (Fig. 5). In addition, the butanol fraction of the Magpie swimming showed an effect of inhibiting chickenpox-herpes virus replication without cytotoxicity at a concentration of 5 μg/ml or less (FIGS. 11 and 12 ).

Therefore, the composition containing the Magpie Swim extract or a fraction thereof as an active ingredient can be useful for the prevention or treatment of diseases caused by infection of the varicella-zoster virus.

The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of the Magpie Swim Extract as an active ingredient relative to the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the above-mentioned active ingredients.

The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof, which are commonly used in biological agents. Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., a saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatic agents and the like can be added as necessary.

When formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used may be added.

The composition of the present invention may be formulated as an oral preparation or parenteral preparation. Oral formulations may include solid and liquid formulations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and the solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or a mixture thereof. In addition, the solid formulation may include a lubricant, and examples thereof include magnesium stearate and talc. Meanwhile, the liquid formulation may be a suspending agent, an intravenous solution, an emulsion, or a syrup. At this time, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, preservatives.

The parenteral preparation may include injections, suppositories, respiratory inhalation powders, spray aerosols, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. At this time, as a non-aqueous solvent or a suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally depending on the desired method. Parenteral administration can include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.

The composition can be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, drug activity, patient sensitivity to the drug, administration time, route of administration, duration of treatment, and drugs used simultaneously. However, for a desired effect, the amount of the active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg/kg, specifically 0.001 to 10 mg/kg. The administration can be several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration can be sequential or simultaneous.

In addition, the present invention provides a health functional food for the improvement of diseases caused by the varicella-zoster virus infection containing the blackcurrant swimming extract or a fraction thereof as an active ingredient.

Magpie swimming according to the present invention or fractions thereof are as described above.

In a specific embodiment of the present invention, the magpie swimming extract was found to have an effect of inhibiting the replication of varicella-zoster virus DNA (FIGS. 1 and 2), and did not show cytotoxicity at IC ₅₀ concentrations (FIG. 3). In the cells infected with varicella-zoster virus, the group treated with blackcurrant swimming extract showed that the transcription of the immediate early gene, ORF62, the transcription of the early gene, ORF28, and the transcription of the Late gene, gB (FIG. 4 ), were reduced. The expression of IE62 protein synthesized through transcription and translation was also reduced (Fig. 5). In addition, the butanol fraction of the Magpie swimming showed an effect of inhibiting chickenpox-herpes virus replication without cytotoxicity at a concentration of 5 μg/ml or less (FIGS. 11 and 12 ).

Therefore, the composition containing the Magpie Swim extract or its fraction as an active ingredient can be usefully applied to health functional foods for improving diseases caused by infection of chickenpox-herpes zoster virus.

The composition of the present invention may be added to food as it is, or used with other foods or food ingredients. At this time, the content of the active ingredient to be added may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total food weight.

The form and type of health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The health functional food may include various flavors, sweeteners or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include tau martin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide and sugar alcohol.

The health functional food of the present invention, in addition to the additional ingredients described above, nutrients, vitamins, electrolytes, flavors, colorants, pectans and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives , Glycerin, alcohol, and the like. These ingredients can be used independently or in combination. The proportion of the additive may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention provides a pharmaceutical composition for the prevention or treatment of diseases caused by hepatitis E virus infection containing the Magpie Swim extract or a fraction thereof as an active ingredient.

The magpie swimming can be prepared by a manufacturing method comprising the following steps:

1) preparing an extract by adding an extracting solvent to the magpie swimming;

2) filtering the extract of step 1); And

3) Concentrating the filtered filtrate of step 2) under reduced pressure and drying.

In the above manufacturing method, the magpie swimming may include all parts such as leaves, stems, roots, fruits, and shells of the magpie swimming.

In addition, the extraction solvent may be water, alcohol or a mixture thereof. The alcohol may be a lower alcohol of C1 to C2, specifically, the alcohol may be ethanol or methanol. In one embodiment of the present invention, the extraction solvent may be 70% ethanol. The extraction solvent may be added in an amount of 1.7 ml per 1 g of the magpie swimming used for extraction.

The extraction method may be shaking extraction, Soxhlet extraction or reflux extraction. At this time, the extraction time may be 72 hours.

On the other hand, the reduced pressure concentration of step 3) may use a vacuum decompressor or a vacuum rotary evaporator. In addition, the drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying, and specifically, freeze drying.

The fraction may be further divided into hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction and water fraction, and hexane and chloroform used in the production of each fraction of magpie swimming according to the present invention. , The concentration and amount of ethyl acetate and butanol, the number of repetitions of shaking and fractionation, and the degree of concentration can be easily changed according to the intended use.

The composition exhibits an effect of inhibiting hepatitis E virus proliferation.

The composition shows the hepatitis E virus ORF2 gene expression inhibitory effect.

In a specific embodiment of the present invention, the magpie swimming extract was found to have a hepatitis E virus RNA replication inhibitory effect (FIGS. 6 to 8 ), and did not show cytotoxicity at an IC ₅₀ concentration (FIG. 7B ). In the group infected with hepatitis E virus, the group treated with the magpie swimming showed an effect of reducing ORF2 capsid expression (FIG. 8). In addition, the ethyl acetate fraction of the Magpie swimming showed an effect of inhibiting the replication of varicella-zoster virus without cytotoxicity at a concentration of 10 μg/ml or less (FIGS. 13 to 16 ).

Therefore, the composition containing the Magpie Swim extract or a fraction thereof as an active ingredient can be useful for the prevention or treatment of diseases caused by infection of the hepatitis E virus.

The pharmaceutical composition according to the present invention may contain 10 to 95% by weight of the Magpie Swim Extract as an active ingredient relative to the total weight of the composition. In addition, the pharmaceutical composition of the present invention may further include one or more active ingredients exhibiting the same or similar functions in addition to the above-mentioned active ingredients.

The pharmaceutical composition of the present invention may include carriers, diluents, excipients, or mixtures thereof, which are commonly used in biological agents. Any pharmaceutically acceptable carrier can be used as long as it is suitable for delivering the composition in vivo. Specifically, the carrier is Merck Index, 13th ed., Merck & Co. Inc., a saline solution, sterile water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture thereof. In addition, conventional additives such as antioxidants, buffers, bacteriostatic agents and the like can be added as necessary.

When formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used may be added.

The composition of the present invention may be formulated as an oral preparation or parenteral preparation. Oral formulations may include solid and liquid formulations. The solid preparation may be a tablet, pill, powder, granule, capsule or troche, and the solid preparation may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin or a mixture thereof. In addition, the solid formulation may include a lubricant, and examples thereof include magnesium stearate and talc. Meanwhile, the liquid formulation may be a suspending agent, an intravenous solution, an emulsion, or a syrup. At this time, the liquid formulation may include excipients such as wetting agents, sweeteners, fragrances, preservatives.

The parenteral preparation may include injections, suppositories, respiratory inhalation powders, spray aerosols, powders and creams. The injection may include a sterilized aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, and the like. At this time, as a non-aqueous solvent or a suspension solvent, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil, or injectable esters such as ethyl oleate may be used.

The composition of the present invention may be administered orally or parenterally depending on the desired method. Parenteral administration can include intraperitoneal, rectal, subcutaneous, intravenous, intramuscular or intrathoracic injection.

The composition can be administered in a pharmaceutically effective amount. This may vary depending on the type of disease, severity, drug activity, patient sensitivity to the drug, administration time, route of administration, duration of treatment, and drugs used simultaneously. However, for the desired effect, the amount of active ingredient contained in the pharmaceutical composition according to the present invention may be 0.0001 to 100 mg/kg, specifically 0.001 to 10 mg/kg. The administration can be several times a day.

The composition of the present invention may be administered alone or in combination with other therapeutic agents. In combination administration, administration can be sequential or simultaneous.

In addition, the present invention provides a health functional food for the improvement of diseases caused by hepatitis E virus infection, containing the Magpie Swim Extract or a fraction thereof as an active ingredient.

Magpie swimming according to the present invention or fractions thereof are as described above.

In a specific embodiment of the present invention, the magpie swimming extract was found to have a hepatitis E virus RNA replication inhibitory effect (FIGS. 6 to 8 ), and did not show cytotoxicity at an IC ₅₀ concentration (FIG. 7B ). In the group infected with hepatitis E virus, the group treated with the magpie swimming showed an effect of reducing ORF2 capsid expression (FIG. 8). In addition, the ethyl acetate fraction of the Magpie swimming showed an effect of inhibiting the replication of varicella-zoster virus without cytotoxicity at a concentration of 10 μg/ml or less (FIGS. 13 to 16 ).

Therefore, the composition containing the Magpie Swim extract or its fraction as an active ingredient can be usefully applied to health functional foods for the improvement of diseases caused by infection of the hepatitis E virus.

The composition of the present invention may be added to food as it is, or used with other foods or food ingredients. At this time, the content of the active ingredient to be added may be determined according to the purpose, and generally may be 0.01 to 90 parts by weight of the total food weight.

The form and type of health functional food are not particularly limited. Specifically, the health functional food may be in the form of tablets, capsules, powders, granules, liquids and pills. The health functional food may include various flavors, sweeteners or natural carbohydrates as additional ingredients. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include tau martin and stevia extract. Meanwhile, examples of synthetic sweeteners include saccharin and aspartame. In addition, the natural carbohydrate may be monosaccharide, disaccharide, polysaccharide, oligosaccharide and sugar alcohol.

The health functional food of the present invention, in addition to the additional ingredients described above, nutrients, vitamins, electrolytes, flavors, colorants, pectans and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives , Glycerin, alcohol, and the like. These ingredients can be used independently or in combination. The proportion of the additive may be selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only for illustrating the present invention, and the present invention is not limited by them.

Example 1 Preparation of Magpie Swim Extract (LME) and its fractions

Lysimachia mauritiana used individuals collected from Jeju Island and Geoje Island, and plant specimens are stored in the Department of Life Science, Sungkyunkwan University and the Department of Pharmacy, Gachon University.

Specifically, 70% ethanol (EtOH) was extracted with 5 L of 70% ethanol at room temperature for 72 hours with a solvent for dried blackcurrant swimming (3 kg), then concentrated with a rotary vacuum concentrator to obtain 285 g of ethanol extract. Did.

After re-suspending the extract in water (ddH ₂ O), 52 g of n-hexane (n-Hexane) fraction, 17 g of chloroform (CHCl ₃ ) fraction, and ethyl acetate (EtOAc) using liquid-partitioning technology 15 g of the fraction, 60 g of the n-butanol (n-BuOH) fraction, and 135 g of the water fraction were obtained, and after lyophilization, the extracts and fractions were stored in 4°C and used in the experiment (FIG. 10).

Experimental Example 1. Inhibition of proliferation of chickenpox-herpes virus (VZV) of blackcurrant extract (LME)

We investigated whether the Magpie Swim extract can inhibit chickenpox-herpetic virus replication.

MRC-5 cells (Korean Cell Line Bank no.10171) in uninfected state are infected with a multiplicity of infection (MOI) of 0.1 with artificially recombined chickenpox-herpes virus pOka strain (Chungbuk National University) expressing green fluorescent protein And treated with a magpie swimming extract at a concentration of 50 μg/ml. At 0, 24, 48 or 72 hours after treatment, analysis was performed using a Nikon TS100-F inverted fluorescence microscope (Tokyo, Japan) equipped with a digital camera and Nikon NIS-Elements microscope imaging software. In addition, at 0, 24, 48 or 72 hours after treatment, the amount of VZV-pOka-GFP DNA was measured by quantitative PCR (qPCR) using the ORF62 primer of Table 3. Specifically, qPCR was separated total DNA using Accuprep Genomic DNA Extraction kit (Bioneer), and amplified and measured according to the manufacturer's protocol using StepOnePlus Real-Time PCR system (Applied Biosystem).

As a result, as shown in FIG. 1A, fluorescence expression of cells treated with the Magpie Squash extract 72 hours after treatment was significantly reduced compared to the DMSO treatment group. In addition, as shown in FIG. 1B, VZV-pOka-GFP DNA was amplified 170000 times and 280000 times after 48 hours and 72 hours, compared to the case where DMSO was treated as a control. However, in the case of cells treated with the Magpie Swim extract, after 48 hours and 72 hours, VZV-pOka-GFP DNA replication decreased by 3.8 and 4.7 times, respectively, compared to the control group. The above results suggest that the Magpie Swim extract can inhibit the replication of chickenpox-herpetic virus.

Primer name order Sequence number VZV ORF62 Forward 5’-TCTTGTCGAGGAGGCTTCTG-3’ One VZV ORF62 Reverse 5’-TGTGTGTCCACCGGATGAT-3’ 2 GAPDH Forward 5’-CATGAGAAGTATGACAACAGCCT-3’ 3 GAPDH Reverse 5’-AGTCCTTCCACGATACCAAAGT-3’ 4

Experimental Example 2. Determination of the concentration of suppression of replication of varicella-zoster virus (VZV) of Magpie swimming

<2-1> IC of the Magpie swimming extract in the activity of inhibiting the replication of varicella-zoster virus ₅₀ decision

It was intended to confirm the IC ₅₀ (concentration that inhibits the growth of VZV by 50%) of the magpie swimming extract in the activity of inhibiting replication of varicella-zoster virus.

After infection of the uninfected MRC-5 cells with the varicella-zoster virus YC01 strain obtained from clinical trials (Chungbuk National University, VZV-YC01), the magpie swimming extracts were respectively concentrated at 5, 10, 25, or 50 μg/ml. Treated with. Retreatment was performed at the same concentration on the 3rd day after treatment, and the number of plaques was measured on the 6th day.

As a result, as shown in FIG. 2, the magpie swimming reduced the number of flasks in a concentration-dependent manner, and particularly exhibited excellent antiviral activity at a concentration of 25 to 50 μg/ml. The concentration (IC ₅₀ ) that inhibited the growth of varicella-zoster virus by 50% was found to be 26.09 μg/ml.

<2-2> Cytotoxicity of Giant Magpie Swimming Extract

The purpose of this study was to confirm the presence or absence of cytotoxicity of the magpie swimming pool extract.

MRC-5 cells were treated with blackfish swimming extract at concentrations of 0, 5, 10, 25, and 50 μg/ml, respectively, and CellTiter-Glo luminescent cell viability assay (Promega) was performed at 0, 24, 48, or 72 hours. It was performed according to the manufacturer's protocol.

As a result, as shown in FIG. 3, when treated with the Magpie Swim extract, there was no decrease in cell viability at the time of treatment (after 0 hours) at all concentrations treated, but after 50 hours and 72 hours, 50 µg/ml Magpie Swim When treated with the extract, cell viability was reduced by 33% and 39%, respectively. However, the magpie swimming did not show significant cytotoxicity at the concentration of IC ₅₀ , and this result suggests that the effect of suppressing the varicella-zoster virus of the magpie swimming is not due to cytotoxicity.

Experimental Example 3. Inhibited expression of varicella-zoster virus Immediate Early(IE) gene, Early(E) gene, or Late(L) gene by blackfish swimming extract (LME)

Molecular mechanism of anti-chickenpox virus suppression effect by the Magpie Squash extract, Immediate early(IE) gene, Early(E) gene or Late(L) required for lytic replication of chickenpox-herpes virus We investigated whether gene expression can be controlled.

<3-1> mRNA expression of varicella-zoster virus Immediate Early(IE) gene, Early(E) gene or Late(L) gene

MRC-5 cells in an uninfected state were infected with a multiplicity of infection (MOI) of 0.1 with a varicella-zoster virus YC01 (VZV-YC01) strain obtained clinically, and treated with Magpie Swim extract at a concentration of 25 μg/ml Did. After treatment, the mRNA expression levels of ORF62, IE gene, ORF28, and E gene, gB, of the varicella-zoster virus at 0, 24, 48, or 72 hours were quantified using qRT-PCR (quantitative) using the primers in Table 4, respectively. reverse transcription PCR). Specifically, template cDNA for qRT-PCR was synthesized through reverse transcription using a QuantiTect reverse transcription kit (Qiagen) after separating total RNA using HiGene™ Total RNA Prep kit (BIOFACT). The above process was performed according to the manufacturer's manual, and the synthesized cDNA was quantified after amplification in a StepOnePlus Real-Time PCR system (Applied Biosystems).

Primer name order Sequence number VZV ORF62 Forward 5’-TCTTGTCGAGGAGGCTTCTG-3’ One VZV ORF62 Reverse 5’-TGTGTGTCCACCGGATGAT-3’ 2 ORF28(E) Forward 5’-CGAACACGTTCCCCATCAA-3’ 5 ORF28(E) Reverse 5’-CCCGGCTTTGTTAGTTTTGG-3’ 6 gB(L) Forward 5’-GATGGTGCATACAGAGAACATTCC-3’ 7 gB(L) Reverse 5’-CCGTTAAATGAGGCGTGACTAA-3’ 8 GAPDH Forward 5’-CATGAGAAGTATGACAACAGCCT-3’ 3 GAPDH Reverse 5’-AGTCCTTCCACGATACCAAAGT-3’ 4

As a result, as shown in FIG. 4, even if the blackfish swimming extract was treated, there was no effect on chickenpox-herpes virus lytic gene expression until 24 hours after treatment, but DMSO-treated groups after 48 hours and 72 hours. It has been shown to decrease the transcription of ORF62, ORF28 and gB compared to.

<3-2> Protein expression of varicella-zoster virus Immediate Early (IE) gene

Western blot was performed to measure the protein level encoded by the IE gene ORF62.

The uninfected MRC-5 cells were infected with artificially recombined chickenpox-herpes virus pOka strain (VZV-pOka) with a multiplicity of infection (MOI) of 0.1, and the Magpie swimming extract at a concentration of 25 μg/ml. After treatment, 72 hours later, after obtaining the cells, Kim et al. performed Western blot using the method used in J Microbiol 53: 732-738 in 2015. VZV IE62 (Abcam) and tubulin (Sigma-Aldrich) antibodies were used as primary antibodies, and peroxidase-labeled anti-mouse immunoglobulin G (Amersham Biosciences) antibodies were reacted according to the manufacturer's protocol.

As a result, as shown in Figure 5, compared to the DMSO-treated group, it was found that the expression of IE62 protein was reduced in the group treated with magpie squirrel.

The above results suggest that the Magpie Sprout extract inhibits the expression of IE62 protein and inhibits chickenpox-herpetic virus replication.

Experimental Example 4. Inhibition of varicella-zoster virus (VZV) of the magpie swimming fraction

<4-1> Inhibition of replication of varicella-herpes virus of the magpie swimming fraction

It was investigated whether the fraction of the Magpie Sprout extract prepared in Example 1 can inhibit chickenpox-herpes zoster virus replication.

After infection of an uninfected penile foreskin fibroblast (HFF) with an artificially recombined chickenpox-herpes virus pOka strain (VZV-pOka-luciferase) capable of expressing luciferase, The magpie swimming fractions were treated at a concentration of 5 μg/ml each. Three days after treatment, the effect on chickenpox-herpes virus replication was analyzed by luciferase analysis (Promega).

As a result, as shown in Figure 11, it was confirmed that the butanol fraction can effectively inhibit chickenpox-herpes zoster virus replication.

<4-2> Confirmation of Cytotoxicity of Magpie Swimming Fraction

It was intended to confirm the presence or absence of cytotoxicity of the fraction of the Magpie Sprout extract prepared in Example 1.

The HFF cells were treated with magpie swimming fractions at a concentration of 1, 2 or 5 μg/ml each. On day 3 of treatment, live cells were measured using a CellTiter-Glo luminescent cell viability assay (Promega).

As a result, as shown in FIG. 12, some cytotoxicity was confirmed in the butanol fraction at a concentration of 5 µg/ml, but the butanol fraction at a concentration of 5 µg/ml or less could exhibit chickenpox-herpes virus replication inhibition effect without cytotoxicity. Was confirmed.

Experimental Example 5. Inhibition of hepatitis E virus (HEV) replication of Magpie swimming extract (LME)

The purpose of this study was to determine whether the Magpie Swim extract can inhibit hepatitis E virus replication.

Porcine HEV Renilla luciferase replicon of type 3 genotype in Korean Cell Line Bank, Human hepatocellular carcinoma cell (Huh7.5) (Virginia Polytechnic Institute and State University, Blacksburg, VA, USA) , pSHEV3-luc) was transfected, and the Magpie swimming extract was treated at a concentration of 10 μg/ml. On the third day of treatment, after re-treatment of the Magpie Swim extract and DMSO to the same concentration, the plasmid capable of expressing firefly luciferase was used as a control for comparing the degree of transformation, and the Renilla luciferase activity was measured. Did. Renilla luciferase activity was measured according to the manufacturer's protocol with the Dual-Luciferase Reporter Assay (Promega).

As a result, as shown in FIG. 6, in the DMSO-treated group, the activity of pSHEV3-luc on the 4th day after transformation was increased by 357.2 times compared to the 1st day after transformation. However, on the 3rd and 4th days of transformation, the activity of Renilla luciferase was very low at 4.7% and 5.2%, respectively, of the DMSO-treated group in the Magpie swimming treated group. These results suggest that the Magpie Swim extract inhibits the replication of the hepatitis E virus.

Experimental Example 6. Confirmation of the inhibitory concentration of hepatitis E virus (HEV) in the blackcurrant extract (LME)

<6-1> IC of Magpie Squirrel Extract in Hepatitis E Virus Replication Inhibitory Activity ₅₀ decision

In order to confirm the hepatitis E virus replication inhibitory activity, IC ₅₀ (concentration that inhibits the proliferation of HEV by 50%) of the magpie swimming extract was attempted.

The human hepatocarcinoma cell line (Huh7.5) was transformed with porcine HEV Renilla luciferase replicon (pSHEV3-luc) of genotype 3, and the Magpie swimming extracts were each 0, 1, 2.5, 5 or 10 μg/ml. After transformation, retreatment was performed at the same concentration on the 3rd day, and Renilla luciferase activity was measured on the 4th day from the retreatment day. Data are expressed as mean±standard deviation (SD) and half-maximal inhibitory concentration (IC ₅₀ ) was calculated using Graphpad Prism 7 (Graphpad Software).

As a result, as shown in FIG. 7A, the IC ₅₀ value of the Magpie swimming extract for inhibiting replication of pSHEV3-luc replicon was 2.141±0.242 μg/ml.

<6-2> Cytotoxicity of Giant Magpie Swimming Extract

The purpose of this study was to confirm the presence or absence of cytotoxicity of the magpie swimming pool extract.

The human hepatocarcinoma cell line (Huh7.5) was treated with a blackfish swimming extract at a concentration of 0, 1, 2.5, 5, 10 or 20 µg/ml, respectively, and then retreated at the same concentration on the third day. CellTiter-Glo luminescent cell viability assay (Promega) was performed on the 1st, 2nd, 3rd, and 4th day after re-treatment in the same manner as described in <Experimental Example 2-2>.

As a result, as shown in Fig. 7B, the Magpie Swim extract did not show significant cytotoxicity against the human hepatocarcinoma cell line (Huh7.5), and this result shows that the hepatitis E virus inhibitory effect of the Magpie Swim extract cell It is suggested that it is not caused by toxicity.

Experimental Example 7. Inhibition of hepatitis E virus (HEV) proliferation of magpie swimming extract (LME)

<7-1> Hepatitis E virus replication inhibition

The anti-hepatitis E virus activity was analyzed through qRT-PCR based on the fact that the magpie squirrel extract can inhibit the renilla luciferase activity of pSHEV3-luc without showing cytotoxicity.

The hepatitis E virus 47832c strain (HEV47832c) of type 3 genotype was transformed into a human lung carcinoma cell line (Korean Cell Line Bank, Human adenocarcinoma cell; A549), and the Magpie swimming extract was treated at a concentration of 10 μg/ml. On the 1st, 3rd, 7th and 14th days after transformation, the number of copies of the hepatitis E virus was confirmed by qRT-PCR (quantitative reverse transcription PCR) in the same manner as in <Experimental Example 1> using the ORF2 primer of Table 5. .

Primer name order Sequence number HEV ORF2 Forward 5’-TATCGGGTTGTCCGAGCTAC-3’ 9 HEV ORF2 Reverse 5’-TGCCGGGTTGAACTAGAATC-3’ 10 GAPDH Forward 5’-CATGAGAAGTATGACAACAGCCT-3’ 3 GAPDH Reverse 5’-AGTCCTTCCACGATACCAAAGT-3’ 4

As a result, as shown in FIG. 8A, hepatitis E virus RNA on the 7th and 14th days after transformation was increased to 4.5 times and 6.7 times, respectively. However, in the case of cells treated with the Magpie Swim extract, hepatitis E virus RNA on the 7th and 14th days after transformation was reduced to 4.4% and 11.9%, respectively, compared to the control group. The above results suggest that the magpie swimming can inhibit replication of the hepatitis E virus.

<7-2> Inhibitory Expression of Hepatitis E Virus Gene by Magpie Swim Extract

As a molecular mechanism of the anti-hepatitis E virus inhibitory effect by the Magpie Swim extract, it was investigated whether the expression of the hepatitis E virus ORF2 capsid can be controlled.

The human lung carcinoma cell line (A549) was transformed with a hepatitis E virus (HEV) 47832c strain (Federal Institute for Risk Assessment, Berlin, Germany, HEV47832c) of the genotype 3, and the Magpie Swim extract concentration of 10 μg/ml Treated with. After obtaining the cells on the 14th day after transformation, Huang et al. in J. Gen. Virol. Using the method used in 86, 2585-2593, immunofluorescence assay was performed. As a primary antibody, a mouse-HEV ORF2 antibody (MAB8002, Millipore) was used as a secondary antibody, and a fluorochrome-binding anti-mouse IgG (4408, Cell Signaling) antibody as a secondary antibody was reacted according to the manufacturer's protocol. Analysis was performed using a Nikon TS100-F inverted fluorescence microscope (Tokyo, Japan) equipped with NIS-Elements microscope imaging software.

As a result, as shown in FIG. 8B, it was found that the expression of ORF2 capsid was reduced in the group treated with magpie squirrel extract compared to the DMSO treated group.

<7-3> Cytotoxicity of Giant Magpie Swimming Extract

The purpose of this study was to confirm the presence or absence of cytotoxicity of the magpie swimming pool extract.

The human lung carcinoma cell line (A549) was treated with blackfish swimming extract at a concentration of 0, 1, 2.5, 5, 10 or 20 µg/ml, respectively, and retreated every 3 days. On days 1, 3, 7 and 14, CellTiter-Glo luminescent cell viability assay (Promega) was performed according to the manufacturer's protocol.

As a result, as shown in FIG. 9, some cytotoxicity was observed in the magpie swimming extract at a concentration of 20 μg/ml, but the magpie swimming extract at a concentration below 10 μg/ml did not show significant cytotoxicity against A549 cells. Did.

The above results suggest that there is a bioactive component that has an inhibitory effect on hepatitis E virus in the magpie swimming extract.

Experimental Example 8. Inhibition of hepatitis E virus (HEV) of the magpie swimming fraction

<8-1> Hepatitis E virus replication inhibition of Magpie swimming fraction

It was investigated whether the fraction of the Magpie Sprout extract prepared in Example 1 can inhibit hepatitis E virus replication.

The human hepatocarcinoma cell line (Huh7.5) was transformed into porcine HEV Renilla luciferase replicon (pSHEV3-luc) of genotype 3, and then 10 μg/ml of Magpie Swim extract Concentration. The effect on hepatitis E virus replication was analyzed through Dual-Luciferase Reporter Assay (Promega) 4 days after transformation.

As a result, as shown in Figure 13, it was confirmed that the ethyl acetate fraction can effectively inhibit hepatitis E virus replication.

<8-2> Hepatitis E Virus Replication Inhibition of Magpie Swimming Fraction

It was investigated whether the fraction of the Magpie Sprout extract prepared in Example 1 can inhibit hepatitis E virus replication.

The human lung carcinoma cell line (A549) was transformed with the hepatitis E virus 47832c strain (HEV47832c) of genotype 3, and then treated with a magpie swimming extract at a concentration of 10 μg/ml. The number of copies of hepatitis E virus on day 14 after transformation was confirmed by qRT-PCR ((quantitative reverse transcription PCR) using the ORF2 primer of Table 5 above.

As a result, as shown in FIG. 14, it was confirmed that the ethyl acetate fraction can effectively inhibit hepatitis E virus replication.

<8-3> Confirmation of Cytotoxicity of Magpie Swimming Fraction

It was intended to confirm the presence or absence of cytotoxicity of the fraction of the Magpie Sprout extract prepared in Example 1.

The human liver carcinoma cell line (Huh7.5) was treated with magpie swimming fractions at a concentration of 0, 1, 2, 5, or 10 μg/ml, respectively. On the 4th day after treatment, live cells were measured using a CellTiter-Glo luminescent cell viability assay (Promega).

As a result, as shown in FIG. 15, it was confirmed that the ethyl acetate fraction at a concentration of 10 μg/ml or less can exhibit the hepatitis E virus replication inhibitory effect without cytotoxicity.

<8-4> Confirmation of Cytotoxicity of Magpie Swimming Fraction

It was intended to confirm the presence or absence of cytotoxicity of the fraction of the Magpie Sprout extract prepared in Example 1.

Human lung carcinoma cell line (A549) cells were treated with magpie swimming fractions at concentrations of 0, 1, 2, 5, or 10 μg/ml, respectively. On the 7th day after treatment, live cells were measured using a CellTiter-Glo luminescent cell viability assay (Promega).

As a result, as shown in FIG. 16, it was confirmed that the ethyl acetate fraction at a concentration of 10 μg/ml or less can exhibit hepatitis E virus replication inhibitory effect without cytotoxicity.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Pharmaceutical composition for inhibiting of Varicella-Zoster Virus and Hepatitis E virus comprising Lysimachia mauritiana extract or fraction thereof as an active ingredient <130> 2018P-03-017 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VZV ORF62 Forward <400> 1 tcttgtcgag gaggcttctg 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> VZV ORF62 Reverse <400> 2 tgtgtgtcca ccggatgat 19 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Forward <400> 3 catgagaagt atgacaacag cct 23 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH Reverse <400> 4 agtccttcca cgataccaaa gt 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ORF28(E) Forward <400> 5 cgaacacgtt ccccatcaa 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ORF28(E) Reverse <400> 6 cccggctttg ttagttttgg 20 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> gB(L) Forward <400> 7 gatggtgcat acagagaaca ttcc 24 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> gB(L) Reverse <400> 8 ccgttaaatg aggcgtgact aa 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 Forward <400> 9 tatcgggttg tccgagctac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> HEV ORF2 Reverse <400> 10 tgccgggttg aactagaatc 20

Claims (11)

Pharmaceutical composition for the prevention or treatment of diseases caused by Varicella-Zoster Virus (VZV) infection, which contains the extract of Lysimachia mauritiana or its fraction as an active ingredient. According to claim 1, The extract is water, C1 to C2 lower alcohol or a pharmaceutical composition for the prevention or treatment of diseases caused by varicella-zoster virus infection extracted with a mixed solvent thereof. According to claim 1, wherein the extract is a leaf, stem or root extract of the Magpie Swim, a pharmaceutical composition for the prevention or treatment of diseases caused by varicella-zoster virus infection. According to claim 1, The fraction is n-hexane fraction obtained by sequentially dividing the magpie swimming extract with n-hexane, chloroform, ethyl acetate, n-butanol and water, chloroform fraction, ethyl acetate fraction, n-butanol fraction or A pharmaceutical composition for preventing or treating diseases caused by varicella-zoster virus infection, which is a water fraction. According to claim 1, wherein the disease caused by the varicella-herpes virus infection is chickenpox (Varicella) or herpes (Herpes Zoster) pharmaceutical composition for the prevention or treatment of diseases caused by varicella-herpes virus infection. Health functional food for the improvement of diseases caused by varicella-zoster virus infection that contains the Magpie Squirrel extract or its fraction as an active ingredient. A pharmaceutical composition for the prevention or treatment of diseases caused by Hepatitis E virus (HEV) infection, which contains an extract of Lysimachia mauritiana or a fraction thereof. The pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection extracted with water, C1 to C2 lower alcohol or a mixed solvent thereof. The pharmaceutical composition for the prevention or treatment of diseases caused by hepatitis E virus infection according to claim 7, wherein the extract is a leaf, stem or root extract of a magpie swimming. According to claim 7, The fraction is n-hexane fraction obtained by sequentially dividing the magpie swimming extract with n-hexane, chloroform, ethyl acetate, n-butanol and water, chloroform fraction, ethyl acetate fraction, n-butanol fraction or A pharmaceutical composition for the prevention or treatment of diseases caused by hepatitis E virus infection, which is a water fraction. Health functional food for the improvement of diseases caused by hepatitis E virus infection, which contains the blackfish swimming extract or its fraction as an active ingredient.

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