KR102001769B1 - A Composition comprising extracts, fractions or isolated compounds of Liriope platyphylla for inhibition of Hepatitis E virus proliferation - Google Patents

Abstract

Since a Liriope muscari extract, a fraction thereof, or a spicatoside A compound separated from the same inhibits the proliferation of hepatitis E virus, it is possible to be usefully used for preventing or treating diseases caused by infection of the virus. It is confirmed that a composition containing the Liriope muscari extract, the fraction thereof, or the spicatoside A compound separated from the same can inhibit the expression of an ORF2 region.

Description

[0001] The present invention relates to a composition for inhibiting the proliferation of hepatitis E virus, comprising fractions or fractions thereof or a compound isolated therefrom as an active ingredient. [0002] The present invention relates to a composition for inhibiting hepatitis E virus proliferation,

The present invention relates to a composition for inhibiting the proliferation of hepatitis E virus, which comprises Aspergillus oryzae extract, fractions thereof or a compound isolated therefrom as an active ingredient.

Hepatitis E is transmitted through the fecal-oral route, which is orally transmitted by drinking water contaminated with hepatitis E virus (HEV), and its prevalence is determined by the health and hygiene level of each country There is a close relationship with. For this reason, hepatitis E is predominantly present in Asian countries such as India, Nepal, Myanmar, Pakistan and China, Latin America countries such as Sudan, Somalia and Mexico, and Latin America, and in countries where other regions and economic levels are relatively high Reported sporadically.

The hepatitis E virus, which is the cause of hepatitis E virus, is differentiated from other hepatitis viruses. It shows viremia (viremina) for about 2 weeks when infected. In addition to jaundice, it also causes plasma bilirubin elevation, abdominal pain, , Hepatomegalia (hepatomegalia), vomiting, and fever. Symptoms due to HEV infection last for 40 days on average, and most of them are known to heal naturally, but some are chronicized by cirrhosis. In general, the mortality rate is as low as 3%, but it is known to rise to 25-30% in pregnant women and elderly people.

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

The viral genomic RNA of HEV entering the host cell acts as a template for mRNA replication and replication of ORF1. The ORF1 protein is translated in viral genomic RNA, and the largest ORF1 domain, RdRp, synthesizes (-) - sense RNA. The (+) - sense genome and the subgenomic RNA are synthesized by RdRp using (-) - sense RNA as a template. ORF2 and ORF3 are translated from the subgenomic RNA, and the viral genomic RNA is packaged into the virion

McMundong (麦 门冬, Liriope platyphylla) is the pulsation (麥冬), mundong (門冬), jog (寸冬), bulsacho (不死草), second linkage (沿階草) a herbaceous perennial of the lily (Liliaceae), also called the purple flowers are in bloom It is also used for ornamental purposes, and the bulge of roots is used for edible and medicinal purposes. In the oriental medicine, it is said that there is the efficacy that the five moon is relaxed, the eyes are lightened, and the mouth is dried or the cough is stopped in the mukmundong, and the efficacy of the mukmundong known through the existing scientific studies is lipid reduction, Stress reduction, memory improvement effect, and the like are known. Korean Patent Laid-Open No. 2006-0007871 discloses a variety of documents on the use of marsupialis such as the induction of neurite outgrowth of spiccotoside A contained in McDo-Dong and the inhibitory activity on brain ischemic neuronal apoptosis .

Korea Patent Publication No. 2006-0007871

The invention maekmundong (Liriope The present invention provides a pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus (HEV) infection, which comprises as an active ingredient an extract of platyphylla or fractions thereof.

The present invention also provides a health functional food for improving diseases caused by hepatitis E virus infection, which comprises Aspergillus oryzae extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection, comprising a spicatoside A compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health functional food for improving diseases caused by hepatitis E virus infection, which comprises as an active ingredient a spicatosteride A compound or a pharmaceutically acceptable salt thereof.

In order to achieve the above object, the present invention maekmundong (Liriope The present invention provides a pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus (HEV) infection, which comprises as an active ingredient an extract of platyphylla or fractions thereof.

The present invention also provides a health functional food for improving diseases caused by hepatitis E virus infection, which comprises Aspergillus oryzae extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection, comprising a spicatoside A compound or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a health functional food for improving diseases caused by hepatitis E virus infection, which comprises as an active ingredient a spicatosteride A compound or a pharmaceutically acceptable salt thereof.

It was confirmed that the composition comprising the Liriope platyphylla extract or fractions thereof or the Spicatoside A compound isolated therefrom inhibits the proliferation of the hepatitis E virus and can inhibit the expression of the ORF2 region. Thus, the extracts of McMurong Dong, its fractions or compounds isolated therefrom can be usefully used for the prevention or treatment of diseases caused by hepatitis E virus infection by inhibiting the proliferation of hepatitis E virus.

FIG. 1 shows the results of the HEV inhibitory effect of the extracts and fractions of McMurray.
FIG. 2 shows the effect of inhibiting HEV according to the concentration of the ethyl acetate fraction on the extract of McMurdoong.
Fig. 3 shows the results of inhibiting the replication of HEV by the ethyl acetate fraction of the extracts of mackerel mushroom (A: qRT-PCR, B: immunofluorescence microscopy).
Fig. 4 is a cytotoxicity test result of the ethylacetate fraction of the mungumun dong extract (A: Huh7.5 cells, B: A549 cells).
FIG. 5 shows the results of the fractionation and TLC of the ethyl acetate fraction of McMurray extract.
Fig. 6 shows the results of identification of active ingredients from the extracts of mugwort.
Fig. 7 shows the effect of suppressing the replication of HEV by spicatoside (A: qRT-PCR result, B: immunofluorescence microscopy result).

Hereinafter, the present invention will be described in detail.

The present invention relates to the use of Liriope The present invention provides a pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus (HEV) infection, which comprises as an active ingredient an extract of platyphylla or fractions thereof.

The extract may be, but not limited to, a conventional extraction method known in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction.

The extract may be extracted by using water, C ₁ to C ₄ lower alcohols or a mixed solvent thereof, but the present invention is not limited thereto. When a lower alcohol is used as the extraction solvent, it is more preferably selected from methanol or ethanol.

In addition, the extract of the present invention includes not only the extract obtained by the above-mentioned extraction solvent, but also the extract obtained through other conventional extraction methods, and the extract obtained through purification and fermentation processes. Separation by supercritical extraction method by high temperature, extraction by extraction method by ultrasonic, separation by ultrafiltration membrane having a constant molecular weight cutoff value, separation by various chromatography, separation of natural state or various microorganisms And the active fraction obtained through various purification and extraction methods such as an extract from the fermentation product used are also included in the extract of the present invention.

The root canal may be any one or more selected from the group consisting of roots, stems, leaves and flowers of the rootstock, preferably roots, but is not limited thereto.

The fraction may be one prepared by further extracting marshmallow extract with water, butanol or ethyl acetate, but is not limited thereto. The fraction of the present invention includes not only the fraction obtained by the above-mentioned solvent but also the active fraction obtained through various fractionation methods such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value and separation by various chromatographies.

The composition may be one that inhibits the proliferation of hepatitis E virus. Infection and proliferation of hepatitis E virus causes hepatitis E in the host and causes symptoms such as viremina, jaundice, plasma bilirubin elevation, abdominal pain, anorexia, hepatomegalia, vomiting, fever or cirrhosis .

The composition may inhibit gene expression of the ORF2 region of hepatitis E virus.

The composition of the present invention may further contain one or more active ingredients which exhibit the same or similar functions in addition to the above extracts or fractions. For administration, one or more additional pharmaceutically acceptable carriers may be prepared. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, powders, tablets, capsules, rings, granules or injection solutions. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 19th, 1990), in a suitable manner in the art.

The composition of the present invention can be formulated into tablets, capsules, powders, granules, suspensions, emulsions, syrups and other liquid preparations by conventional methods.

Specifically, the pharmaceutical composition of the present invention can be administered orally or parenterally. Formulations for oral administration can be in the form of tablets, troche, lozenge, aqueous or predominant suspension, prepared powders or granules, emulsions, Hard or soft capsules, syrups, or elixir. Binders such as lactose, saccharose, sorbitol, mannitol, erythritol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, Magnesium stearate, calcium stearate, sodium stearyl fumarate, or polyethylene glycol wax. In the case of capsule formulations, in addition to the above-mentioned materials, liquid carriers such as fatty oils may be included. In addition, for the purpose of formulation as a formulation, it is possible to use, in addition to the above-mentioned components, an acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methylcellulose, methylhydroxybenzoate, Talc, magnesium stearate, and mineral oil, but are not limited thereto.

In addition, the composition of the present invention can be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intraperitoneal injection method for parenteral administration. For formulation into a parenteral administration form, the mixture is mixed with water and extracted with water to prepare a suspension, which is then formulated into unit dosage forms of ampoules or vials.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The dose of the active ingredient according to the present invention will consider safety and efficiency when used in the human body, and it is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations in determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman ' s Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin et al., Remington's Pharmaceutical Sciences, 18th ed., (1990), Mack Publishing Co. In the case of oral administration, an adult dose of 0.0001-500 mg of the extract of the present invention per kg of body weight per day And may be administered in an amount of 0.001 to 100 mg per day. However, the dosage may be varied depending on the route of administration, the severity of the disease, sex, weight, age, and the like, and therefore the dose is not limited to the scope of the present invention by any means.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Compositions of the present invention may also include carriers, diluents, excipients, or a combination of two or more thereof, which are commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Sodium chloride, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. If necessary, antioxidants, buffers, And other conventional additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990) in a suitable manner in the art.

The composition of the present invention may further contain one or more active ingredients showing the same or similar functions. The composition of the present invention may contain 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the above compound, based on the total weight of the composition.

The present invention also provides a health functional food for improving diseases caused by hepatitis E virus infection, which comprises Aspergillus oryzae extract or a fraction thereof as an active ingredient.

The health functional food of the present invention may further contain one or more active ingredients showing the same or similar functions. For administration, one or more additional carriers acceptable for food can be prepared.

The health functional food of the present invention is preferably one selected from beverage, ring, tablet, capsule, and powder, but is not limited thereto. The health functional food composition of the present invention may be added as it is, May be prepared together with food or food ingredients, and may be suitably prepared according to conventional methods. Examples of foods to which the health functional food composition of the present invention can be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, , Beverage, tea, drink, alcoholic beverage, and vitamin complex, and includes all the health foods in a conventional sense.

The food may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alkynic acid and its salts, Stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices and vegetable drinks.

The above components can be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention. In addition, the health functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The natural carbohydrates may be selected from the group consisting of glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, Polysaccharides such as cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

Maekmundong In a particular embodiment of the present invention (Liriope platyphylla ) extract or its fractions can inhibit the replication of hepatitis E virus (HEV), so that it is possible to provide a pharmaceutical composition for preventing or treating diseases caused by infection of HEV, And can be usefully used for functional foods (Figs. 1 to 3).

The present invention also relates to a pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection, comprising a spicatoside A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient Lt; / RTI >

The spicatosteride A compound may inhibit the proliferation of hepatitis E virus.

The spicatosteride A compound may inhibit gene expression in the ORF2 region of hepatitis E virus.

The present invention includes all the possible solvates and hydrates, which can be prepared therefrom, as well as the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof.

The compound of the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfo, naphthalene-1-sulfonate Naphthalene-2-sulfonate, or mandelate.

The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the compound of the present invention in an excess amount of an aqueous acid solution, adding the salt to a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI >

By heating the same amount of the compound represented by the formula (1) and the acid or alcohol in water, and then evaporating and drying the mixture, or by filtering the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

In addition, the present invention provides a health functional food for improving diseases caused by hepatitis E virus infection, comprising the spicatosteride A compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The health functional food of the present invention may further contain one or more active ingredients showing the same or similar functions. For administration, one or more additional carriers acceptable for food can be prepared.

The health functional food of the present invention is preferably one selected from beverage, ring, tablet, capsule, and powder, but is not limited thereto. The health functional food composition of the present invention may be added as it is, May be prepared together with food or food ingredients, and may be suitably prepared according to conventional methods. Examples of foods to which the health functional food composition of the present invention can be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, , Beverage, tea, drink, alcoholic beverage, and vitamin complex, and includes all the health foods in a conventional sense.

The food may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and enhancers (cheese, chocolate etc.), pectic acid and its salts, alkynic acid and its salts, Stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices and vegetable drinks.

The above components can be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention. In addition, the health functional food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient. The natural carbohydrates may be selected from the group consisting of glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, Polysaccharides such as cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In a specific example of the present invention, spicatoside A (FIGS. 5 and 6), a compound isolated from McMurdoe extract and fractions, showed that the replication inhibitory effect of HEV was superior to that of universal antiviral agent (FIG. 7) , And a health functional food for improving diseases caused by infection of HEV.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

EXAMPLES The following Examples and Experiments are for the purpose of illustrating the present invention, but the present invention is not limited by the following Examples and Experimental Examples.

< Example  1> Mukmun dong extract and Fraction  Produce

Liriope, who bought herbal medicines through wholesalers, platyphylla ) was pulverized and extracted with 5 L of 70% ethanol at room temperature for 72 hours, followed by concentration with a rotary evaporator to obtain 72 g of an ethanol extract. 1 g of the ethyl acetate fraction, 1.29 g of the butanol fraction and 68.7 g of the water fraction were obtained by resuspending the obtained ethanol extract in water (ddH ₂ O) and using a liquid-partitioning technique. After lyophilization, And fractions were used in the experiment while being stored at 4 &lt; 0 &gt; C.

< Experimental Example  1> Macromolecular Extract or Fraction HEV  Duplication suppression

A luciferase-reporter assay was performed to measure the effect of the extract of McMundumong and its fractions on the replication of hepatitis E virus (HEV).

The genotype 3-type porcine HEV Renilla luciferase repreicon (pSHEV3-luc) was transfected into hepatoma cell line Huh7.5 cell and the firefly luciferase-pcDNA3 (luc pCDNA3) plasmid was used as a control to compare the degree of transformation. To transform, the pSHEV3-luc plasmid was linearized with XbaI (New England BioLabs) and luc-pcDNA3 with BglII (New England BioLabs) and ligated with mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific) The capped RNA transcript was synthesized and transformed into Huh 7.5 cells according to the manufacturer's protocol with DMRIE-C reagent (Invitrogen). The fractions obtained in Example 1, each of which was fractionated with ethyl acetate (EtOAc), butanol ( n- BuOH) or water (ddH ₂ O), were each treated at 10 μg / ml. Luciferase activity was measured according to the manufacturer's protocol using the Dual-Luciferase Reporter Assay System (Promega).

As a result, after 4 days of transformation, the luciferase activity of pSHEV3-luc increased 234.7-fold in the DMSO-treated group. However, in the LPE - treated cells, the LPE - treated cells showed a low level of about 67%, while the LPEE - treated group showed 33.8%, the butanol fraction (LPE BuOH) treated group showed 47.2% (LPE ddH ₂ O) treated group showed a 40.8% level of luciferase activity lower than that of the control group. Thus, it was confirmed that the extracts or fractions thereof showed inhibition of replication of hepatitis E virus (Fig. 1).

< Experimental Example  2> Fraction HEV  Duplication suppression

Experimental Example  2-1. McMundong Fraction  By concentration HEV  Inhibitory effect

In order to confirm the effective dose of the ethyl acetate fraction of McMun Dong, which was confirmed to have the best effect of inhibiting replication of HEV through Experimental Example 1, the ethyl acetate fraction of McMundong was treated in various amounts and analyzed. Data are presented as means ± SD (SD), and the significance of differences between the means was determined as Student't-test. The P-value <0.05 was considered statistically significant and the half-maximal inhibitory concentration (IC ₅₀ ) was calculated using Graphpad Prism 7 (Graphpad Software).

As a result, it was confirmed that the IC ₅₀ value of the ethyl acetate fraction for inhibiting replication of pSHEV3-luc replicon was 1.78 ± 0.93 ㎍ / ml (FIG. 2).

Experimental Example  2-2. McMundong Fraction HEV  Duplication suppression

The inhibition of HEV replication by ethyl acetate fraction was confirmed by qRT-PCR (Quantitative RT-PCR).

The HEV genotype 3 strain 47832c (HEV genotype 3 strain 47832c) was transformed into the human lung cancer cell line A549, and the ethylacetate fraction prepared in Example 1 was treated at 10 占 퐂 / ml. On the third day after infection, the ethyl acetate fraction Was reprocessed at 10 ㎍ / ml, and the number of HEV copies on days 1, 3, 7 and 14 of infection was measured by qRT-PCR. RNA was isolated according to the protocol of the manufacturer using TRI Reagent (Molecular Research Center, Inc.), and the RNA was reverse transcribed with cDNA using the TOPscript ™ cDNA synthesis kit (Enzynomics) according to the manufacturer's protocol. Real-time quantitative polymerase chain reaction (PCR) was performed using the ORF1 primer of Table 1 and the 1X HOT FIREPol EvaGreenq PCR Mix Plus (Solis BioDyne) kit.

ORF1 order SEQ ID NO: forward 5'-CCTGTTAGTGACATTTGGGTGT-3 ' One reverse 5'-AGACCTTTGCCCCATCCGGATA-3 ' 2

As a result, the amount of HEV RNA in the control group was increased by 23.8-fold at the 14th day of infection, but the amount of HEV RNA was reduced to 63% of the control group in the group treated with the ethyl acetate fraction (FIG. 3A).

Experimental Example  2-3. McMundong Fraction  By solvent HEV  Inhibitory effect

The expression of ORF2 capsid in HEV was examined by immunofluorescence microscopy to confirm the effect of ethyl acetate fraction on HEV replication.

The transformed A549 cells treated with the HEV genotype 3 type 47832c strain and treated with the ethyl acetate fraction were washed three times with PBS (phosphate-buffered saline) for 5 minutes in the same manner as in Experimental Example 2-2, At -20 占 폚 for 10 minutes, and blocked with 1X PBS containing 0.5% BSA for 30 minutes. The blocked cells were washed with PBS and reacted with mouse-HEV ORF2 antibody (MAB8002, Millipore) at a ratio of 1: 200. After overnight incubation, the cells were washed with PBS and reacted with 1: 200 fluorochrome-conjugated anti-mouse IgG secondary antibody (4408, Cell Signaling) for 1 hour at 37 ° C. Cells were washed and counterstained with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories), then stained with a Nikon TS100-F inverted fluorescence microscope equipped with a digital camera and Nikon NIS-Elements microscopy imaging software , Japan).

As a result, after 14 days of inoculation, ORF2 was strongly expressed, but the expression of ORF2 was significantly reduced in the ethyl acetate fraction treated group as compared with the control group (FIG. 3B). This suggests that the ethyl acetate fraction of McMurdo extract is more effective at inhibiting ORF2 expression than HEV RNA replication inhibition.

< Experimental Example  3> Fraction HEV  Check cytotoxicity

The cytotoxicity of the ethyl acetate fraction of McMurdo extract was confirmed.

Huh7.5 cells and A549 cells were treated with 0, 10, 25, 50 and 100 占 퐂 / ml of the ethyl acetate fraction described in Example 1, and the survival rate of the cells was measured.

As a result, there was no significant cytotoxicity against Huh7.5 cells except for a slight decrease in survival rate of 87% when treated with 100 ㎍ / ml at the highest concentration 4 days after treatment, and A549 cells showed the highest cytotoxicity But did not show cytotoxicity even at a concentration of 100 占 퐂 / ml (Fig. 4). These results suggest that the HEV inhibitory effect of the ethyl acetate fraction of McMurdo extract is not due to the toxicity of the solvent.

< Example  2> Fraction  Active ingredient separation

20 kg of dry root of McMundum dough was pulverized and extracted with 60 L of 95% ethanol at room temperature for 72 hours, followed by concentration with a rotary evaporator to obtain 290 g of ethanol extract. The obtained ethanol extract was resuspended in water (ddH2O) and then 20 g of the ethyl acetate fraction (LPEEA), 30 g of the butanol fraction (LPEBuOH) and 238 g of the water fraction (LPEdd ₂ O) were obtained by liquid-partitioning technique . Each fraction was analyzed by HPLC-ESI-Q-TOF-MS / MS and the Agilent 6530 Q-TOF mass spectrometer (Agilent Technologies) and Agilent series 1290 Infinity HPLC instrument (Agilent Technologies) were used. The ethyl acetate fraction was chromatographed on a silica gel column (500 × 50.0 mm, particle size 35-75 μm, Intertechnologies) column using 0.1% chloroform (solvent A) and methanol (solvent B) The gradient profile was performed by forming A: B from 9: 1 to 0:10.

(For the silica gel column we would request a specific type of substrate such as Hydrosphere C18 (150 x 3.0 mm, particle size 3 μm, YMC Co. Ltd.).)

LPEEA-10, which exhibited excellent activity among the additional small fractions of the ethyl acetate fraction (LPEEA), was selected and concentration gradient conditions were established with increasing the concentration of methanol to 50% to 100%, and Sephadex ^TM LH-20 ), The LPEEA-10-2 having excellent activity was selected among the small fractions obtained by fractionation by the size exclusion chromatography (Fig. 5). The LPEEA-10-2-1 fraction, which was further fractionated into the Sephadex column by increasing the concentration of methanol to 60% to 100%, was added to the Sephadex column and analyzed by a 600 MHz spectrophotometer using AVANCE (Bruker) ¹ H-NMR and ¹³ C-NMR analyzes were carried out to confirm that spicatoside A of formula (1) was contained (FIG. 6).

[Chemical Formula 1]

< Experimental Example  4> Spicato side  A ( Spicatoside  A) HEV  Inhibitory effect

Experimental Example  4-1. Luciferase  Activity Spicato side  Of A HEV  Inhibitory effect

Spicatoside A, an active ingredient isolated in Example 2, was examined for its ability to inhibit replication of HEV.

As described in Experimental Example 1, pSHEV3-luc was transformed into Huh7.5 cells, treated with a concentration of 10 占 퐂 / ml of spiccotid A, and after 4 days of infection, luciferase activity was measured using Dual-Luciferase Reporter Assay System Promega) according to the manufacturer's protocol.

As a result, it was confirmed that the luciferase activity of pSHEV3-luc was hardly observed in the group treated with spicatosteride A, and it was found that it inhibited the replication of HEV (Fig. 7A).

Experimental Example  4-2. Using immunofluorescence technique Spicato side  Of A HEV  Duplication suppression

Spikatoside A was treated with A549 infected with the HEV genotype 3 type 47832c strain as described in Experimental Example 2-3, and the control group was compared with the DMSO treatment group, the treatment group of the antiviral agent Ribavirin, and the noninflammatory group.

As a result, in the 7th day and 14th day of infection, the spiccotoside A-treated group showed a similar level of HEV inhibitory activity to ribavirin (Fig. 7B).

<110> Gachon University of Industry-Academic cooperation Foundation <120> A Composition of extracts, fractions or isolated          compounds of Liriope platyphylla for inhibition of Hepatitis E          virus proliferation <130> 2017P-11-054 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ORF1-F <400> 1 cctgttagtg acatttgggt gt 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> ORF1-R <400> 2 agacctttgc cccatccgga ta 22

Claims (11)

Liriope A pharmaceutical composition for preventing or treating a disease caused by hepatitis E virus (HEV) infection, comprising as an active ingredient an extract of Platyphylla or a fraction thereof.
The pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection according to claim 1, wherein the extract is extracted using water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof .
The pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection according to any one of claims 1 to 3, wherein the mussel dong is at least one selected from the group consisting of root, stem, leaf and flower of mussel.
2. The pharmaceutical composition according to claim 1, wherein the fraction is prepared by further extracting marshmallow extract with water, butanol or ethyl acetate.
The pharmaceutical composition according to claim 1, wherein the composition inhibits the proliferation of hepatitis E virus.
The pharmaceutical composition according to claim 1, wherein the composition inhibits gene expression in the ORF2 region of hepatitis E virus.
A health functional food for the improvement of diseases caused by hepatitis E virus infection, which comprises the extract of McMundum or its fractions as an active ingredient.
A pharmaceutical composition for preventing or treating diseases caused by hepatitis E virus infection, comprising as an active ingredient, a spicatoside A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

9. The pharmaceutical composition according to claim 8, wherein the spicatosteride A compound inhibits the proliferation of hepatitis E virus.
9. The pharmaceutical composition according to claim 8, wherein the spicatosteride A compound inhibits gene expression in the ORF2 region of hepatitis E virus.
A health functional food for improving diseases caused by hepatitis E virus infection, comprising as an active ingredient, a spicatosteride A compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

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