KR101623500B1 - Use of acacetin and apigenin for improving and treating gamma herpesvirus infection - Google Patents

Abstract

The present invention provides the use of compounds of formula (1), namely acacetin and apigenin, for use in the amelioration and treatment of gamma herpesvirus infections. According to the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is effective for treating a gamma-herpesvirus infectious disease with few side effects and toxicity, and being biocompatible and drug-resistant.

Description

Use of acacetin and apigenin for use in the amelioration and treatment of gamma herpesvirus infections. ≪ Desc / Clms Page number 2 > Use of acacetin and apigenin for improving and treating gamma herpesvirus infection,

The present invention relates to the use of acacetin and apigenin for use in the amelioration and treatment of gamma herpesvirus infections.

Most human herpes viruses are 70-80% (See Table 1. Characteristics of human chronic viruses). It is a typical chronic virus that maintains infection for a lifetime by repeating latent infection and reactivation in the host.

Human herpes virus Kinds Latent infection A major disease HHV-1 Herpes simplex virus-1 Alpha Nervous system Herpes simplex, encephalitis HHV-2 Herpes simplex virus-2 Alpha Nervous system Genital herpes (STD), encephalitis HHV-3 Varicells-Zoster virus Alpha Nervous system Varicella, shingles HHV-4 Epstein-Barr virus gamma B lymphocyte Infectious mononucleosis, bucket lymphoma, coin cancer, B lymphoma, gastric cancer HHV-5 Human cytomegalovirus beta Lymphatic system Infectious mononucleosis, congenital anomaly, retinitis, blindness HHV-6 Human herpesvirus-6 beta T lymphocyte Epidemic, meningitis, infectious mononucleosis, fulminant hepatitis, atypical lymphocyte hyperplasia HHV-7 Human herpesvirus-7 beta T lymphocyte HHV-8 Kaposi's sarcoma-associated herpesvirus gamma B lymphocyte Kaposi's sarcoma, B lymphoma

Herpes viruses are a typical opportunistic infection Reactivation due to decreased immune function or congenital infection causes various diseases and it shows a fatal infection prognosis according to the immune status of the patient. Prevention of Varicells-Zoster Virus (VZV) There are no preventive or therapeutic vaccines against the seven human herpes viruses except the live vaccine.

Control of reactivated herpes virus infection due to immune degradation, such as organ transplant and hematopoietic stem cell transplant, HIV-1 infection, is becoming an important issue.

In addition, the immune decline in the elderly population due to persistent infections of the herpes virus (see review article: Koch et al, Cytomegalovirus infection: a driving force in human T cell immunosenescence, Ann NY Acad Sci 2007 1114: 23-35) It will be an important health and social burden in Korea, which is entering an aging society.

Among herpes viruses, gamma herpesvirus is known to cause latent infection with lymph cells, causing tumors, and lytic infection in epithelial cells and fibroblasts. Gamma-herpes viruses include Epstein-Barr Virus (hereinafter referred to as EBV) and Kaposi's sarcoma associated virus (hereinafter referred to as KSHV).

Normally, the herpes virus is characterized by a high infection rate. Especially, in EBV, more than 90% of the world's population, including Korea, is seropositive. EBV has been implicated in chronic infections caused by latent infections such as nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), Burkitt's lymphoma BL, post-transplant B cell lymphomas (PTBL ), And Hodgkin's disease (HD) (Roizman, 2007).

KSHV is the most recent human infectious herpesvirus found in 1994. Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), a type of B cell lymphoma (PEL) or multicentric casts Castleman's disease) (Alkan et al, 1997; Huang et al, 1995; Liberman et al, 1990; Moore et al, 1994).

Most of the existing anti-herpes virus preparations were developed targeting the viral DNA polymerase (DNA pol). Among them, acyclovir and its derivatives are specifically phosphorylated by thymidine kinase (hereinafter referred to as "TK") of the herpes virus and then interrupted with the viral genome which is replicated by the DNA pol, It is a drug that induces premature termination and has been used most often for the past 40 years after its development. In addition, drugs such as forcarnet, such as a pyrophosphate analogue, are commonly used.

While they are effective in the treatment of alpha-herpes virus infections, their clinical potency is limited for early infections (eg, infectious mononucleosis) or reactivation infections of beta or gamma-herpes viruses. In particular, there is no antiviral agent that can be useful for the control of gamma herpesvirus, and thus it relies mainly on steroid-based drugs for the treatment of hypertension.

In addition, recently, immunodeficiency patients with organ transplantation and hematopoietic stem cell transplantation and HIV infection are severely toxic to the drug, and more importantly, the mutation of drug resistance is increasing in these patients (Danve-Szatanek C et al. Surveillance network for herpes 단 정비 저항 to antiviral drugs : 3- year follow - up . J Clin Microbiol. 2004 42 (1): 242-9 .; Limaye AP et al. Emergence of ganciclovir -resistant cytomegalovirus disease among recipients of solid - organ transplants . Lancet 2000; 356: 645649; Marfori JE et al. Development of new cytomegalovirus UL97 and DNA polymerase mutations conferring drug resistance after valganciclovir therapy in allogeneic stem cell recipients . J Clin Virol 2007; 38: 120-125).

Most drug-resistant mutations are mutations in TK or viral DNA pols, and it is very urgent to develop antihernospiral drug candidates with novel targets and mechanisms.

It is an object of the present invention to provide a novel component capable of effectively improving or treating gamma-herpesvirus infection without drug toxicity and drug resistance.

To this end, the present invention provides a composition for improving or treating a gamma herpesvirus infection comprising as an active ingredient a compound of formula (I) or a pharmaceutically acceptable salt thereof, a therapeutically effective amount of a compound of formula (I) A method for treating a gamma herpesvirus infection comprising administering a salt to a subject in need thereof, a novel use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of an antiviral agent against gamma herpesvirus.

In this formula,

R ₁ is CH ₃ or H.

In one embodiment, the compound of formula (1) may be acacetin having the structure of formula (2).

Acacetin (5,7-dihydroxy-4'-methoxy-flavone) is an O-methylated flavone component mainly found in Robinia pseudoacacia L. (black locust) , Turnera diffusa (damiana) and the fern Asplenium normale. Acacetin is a secondary metabolite that is synthesized by supplying methyl groups from S-adenosylmethionine by apigenin 4'-O-methyltransferase with apigenin as a backbone. In addition to anti-inflammatory and analgesic effects, acacetin has been reported to have anti-cancer efficacy against a variety of cancer cell lines (Jaganathan and Mandal, 2009). Previous studies have shown that acacetin has no antiviral efficacy against herpesvirus, adenovirus, coronavirus and rotavirus (Debiaggi M1, Tateo F, Pagani L, Luini M, Romero E. 1990 Effects of propolis flavonoids on virus infectivity and replication Microbiologica 13 (3): 207-13.), Inhibiting the activation and replication of HIV stimulated by HIV in latently infected cells (Critchfield et al., Critchfield JW1, Butera ST, Folks TM 1996. Inhibition of HIV activation in latently infected cells by flavonoid compounds 1996, 12 (1): 39-46. ). Recently, it has been reported that a high concentration of acacetin has an antiviral efficacy against influenza virus (1. Wu Q, Yu C, Yan Y, Chen J, Zhang C, Wen X. 2010 Antiviral flavonoids from Mosla scabra. Phytotherapy 81 (5): 429-33).

In another embodiment, the compound of formula (1) may be an apigenin having the structure of formula (3).

Apigenin (5,7,4'-trihydroxyflavone) is not only found in the acacia tree extract but also contains fruits such as oranges, apples, cherries, and grapes, vegetables and tea such as onions, parsley, celery, (3): 297-300., Zhao, J, Dasmahapatra (1991), "Flavonoid distribution in asplenioid ferns" , AK, Khan, SI, and Khan, IA (2008). "Anti-aromatase activity of the constituents from damiana (turnera diffusa) "Journal of Ethnopharmacology 120 ( 3):... 387-393) antioxidant activity, has been reported to exhibit anti-inflammatory and anti-cancer effect (1. Tong X, Pelling JC 2013 Targeting the PI3K / Akt / mTOR axis In addition, apigenin has been shown to have antiviral efficacy against avian influenza virus, hepatitis C virus, HSV-1 and HIV-1 virus (see, for example, Antigenic Agents Med. Chem. 13 (7): 971-8) (Sithisarn P, Michaelis M, Schubert-Zsilavecz M, Cinatl J Jr. 2013 Differential antiviral and anti-inflammatory mechanisms of the flavonoids biochanin and baicalein in H5N1 influenzae virus-infected cells. : 41-8., Manvar D, Mishra M, Kumar S, Pandey V. 2012 Identification and evaluation of anti-hepatitis C virus phytochemicals from Eclipta alba J Ethnopharmacol 144 (3): 545-54).

In addition, the compound of Chemical Formula 1 used as an active ingredient in the present invention may include all of the prodrug forms of the compound of Chemical Formula 1, which is converted into the compound of Chemical Formula 1 in vivo to exhibit antiviral efficacy against gamma herpes virus . For example, such a prodrug may be a glycoside of the compound of formula (I).

In addition, the pharmaceutically or physiologically acceptable salt of the compound of Formula 1 according to the present invention may be an acid addition salt formed using an organic acid or an inorganic acid. The organic acid may be selected from the group consisting of formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, Acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid and methanesulfonic acid-based salts; The inorganic acid includes, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid and boric acid-based salts. The above-mentioned acid addition salts can be prepared from the compound of the formula (1) through methods known in the art.

As can be seen in the following examples, the compound of formula (I) exhibits an antiviral efficacy against gamma herpesvirus.

The compounds of formula (I) may be useful for the amelioration or treatment of infection with gamma-herpes viruses, particularly Epstein-Barr virus (EBV) or Kaposi's sarcoma associated virus (KSHV).

The compounds of formula (I) according to the present invention may be used for the treatment or amelioration of diseases caused by infection with gamma herpesvirus, including, but not limited to, infectious mononucleosis, bucket lymphoma, Lymphoma, stomach cancer, Kaposi sarcoma, and B lymphoma.

The compound of Chemical Formula (1) used in the present invention can be chemically synthesized or isolated or semisynthetic from an extract of a plant containing the compound. For example, the compound of formula (I) may be contained in the composition as a form contained in an extract of a plant or a fraction thereof, or as a compound isolated from an extract of a plant or a fraction thereof.

The composition of the present invention comprising the compound of formula (I) as an active ingredient may be a pharmaceutical composition or a food composition.

In one embodiment, the present invention provides a pharmaceutical composition for treating a gamma herpesvirus infection comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

The medicinal composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.

Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

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 as an appropriate method in the field.

The present invention also provides a method of treating a gamma herpesvirus infection comprising administering a therapeutically effective amount of a compound of formula 1 to a subject in need thereof.

The term "subject" as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

As used herein, the term "therapeutically effective amount " refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, This includes an amount that induces relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective amount and the administration frequency of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the compound of the formula (1) of the present invention in a dose of 0.01 mg / kg to 200 mg / kg once to several times a day.

In the therapeutic method of the present invention, the composition comprising the compound of formula (I) of the present invention as an active ingredient can be administered orally, rectally, intravenously, intraarterially, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, ≪ / RTI > can be administered in a conventional manner.

The present invention also provides a food composition for improving gamma-herpesvirus infection comprising a compound of formula (I) or a physiologically acceptable salt thereof.

The food composition according to the present invention can be formulated in the same manner as the above pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectioneries, diet bars, dairy products, meat, chocolates, pizza, ram noodles, other noodles, gums, ice cream, .

For example, when the food composition of the present invention is prepared as a beverage such as a drink, in addition to the above-mentioned plant extract or fraction thereof containing the compound of Chemical Formula 1 or the compound of Chemical Formula 1, citric acid, Malic acid, persimmon, various plant extracts, and the like.

The present invention provides a health functional food comprising the composition for improving the gamma herpesvirus infection. The health functional food is prepared by adding the extract of the above-exemplified plant or the fraction thereof containing the compound of the formula (1) or the compound of the formula (1) to a food material such as beverage, tea, spices, gum, confectionary, or by encapsulating, Suspension, etc., which means that a certain effect is obtained when the food is consumed. However, unlike the general medicine, there is an advantage that there is no side effect that may be caused when a food is used as a raw material for a long time. The health functional food of the present invention thus obtained is very useful because it can be ingested routinely. The amount of the extract of the above-exemplified plant or the fraction thereof containing the compound of the formula (I) or the compound of the formula (I) in such a health food can not be uniformly determined depending on the kind of the health food as the target, And it is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight based on the target food. In the case of foods in the form of granules, tablets or capsules, they may be added usually in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a drink.

According to the present invention, the compound of formula (I) or a pharmaceutically acceptable salt thereof is effective for treating a gamma-herpesvirus infectious disease with few side effects and toxicity, and being biocompatible and drug-resistant.

FIG. 1 is a graph showing the results of verifying the antiviral efficacy of the compound of formula (1) against mouse gamma herpesvirus.
Fig. 2 is a graph showing the effect of FIG. 4 is a graph showing the results of analysis of inhibition of viral gene expression against EBV.
FIG. 3 is a graph showing the results of analysis of the antiviral activity of KSHV of the compound of Chemical Formula 1. FIG.
4 is a graph showing the results of western blot analysis for inhibiting RTA protein expression of the compound of formula (1).
FIG. 5 is a diagram showing the cytotoxicity analysis results of acacetin and azepinin on the Bordeaux cell line. FIG.
FIG. 6 is a graph showing the results of analysis of the antiviral efficacy of acacetin and the effect of BZLF1 and BMRF1 gene on the expression of B95.8 and Raji cell lines, EBV-infected cell lines.
FIG. 7 is a graph showing the results of analysis of the effect of acacetin on antiviral efficacy and ERK phosphorylation in KSHV-infected cell lines BC-3G and BC-3.
Fig. 8 is a graph showing the results of analysis of the antiviral efficacy of apigenin and the effect of BZLF1 and BMRF1 gene on the expression of B95.8 and Raji cell lines, EBV-infected cell lines.
FIG. 9 is a graph showing the results of analyzing the antiviral potency of apigenin and the effect of phosphorylation of ERK on KSHV-infected cell lines BC-3G and BC-3.
10 is a graph showing the results of measurement of IC50 values of acacetin and apigenin.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

Example  1. Mouse Gamma Herpes virus  Antiviral efficacy against

Robinia pseudoacacia L .; antiviral efficacy against the following seven substances including acacetin and apigenin found in the black locust tree was tested.

In the present embodiment and drawings, these compounds are also represented by the abbreviations of R1 to R7, which are listed together with the above-mentioned compound.

The anti-viral efficacy of seven monocot bean-bearing single substances was tested by plaque assay using mouse gamma-herpes virus MHV-68. First, samples of R1 to R7 were pretreated with Vero cell line at concentrations of 20, 50, and 100 uM for 3 hours, and then MHV-68 virus was infected with Vero cell line at 100 pfu / well. After the removal of the virus, the cells were cultured for 5 days in the medium to which the sample was added at the same concentration as the pretreatment, and the number of formed platelets was measured. As a result, as shown in FIG. 1, R2, R3, R4, R5 and R7 did not show the effect of inhibiting the replication of MHV-68, while R1 and R6 showed remarkable antiviral efficacy at low concentrations of 20 uM appear.

Example  2. Human Gamma-1 Herpes virus , EBV Antiviral efficacy against

In order to confirm whether the anti-viral effect of the compound of formula (1) found in the model of the mouse gamma-herpes virus showed the same effect on the similar human gamma-herpes viruses, the gamma- Efficacy was confirmed.

(10, 20, 50 uM or 2, 5, 10, 20, and 50 uM) were preliminarily treated for 1 hour in the B95.8 cell line infected with EBV and induced viral reactivation with TPA (20 ng / ml). Cells were harvested 24 hours after induction of reactivation and the effect of samples 1 to R5 on viral replication was compared with the control group treated with DMSO to confirm the expression level of EA-D, a lytic gene representative of EBV And examined via western blot. Then, the intensity of the protein of EA-D was divided by the intensity of the alpha-tubulin used as the control group to quantitatively analyze the expression amount of the virus protein. As a result, as shown in FIG. 2, dose-dependent antiviral efficacy was shown in all sample treatment groups, among which R1, R3 and R6 showed excellent antiviral efficacy.

Example  3. Human Gamma-2 Herpes virus , KSHV Antiviral efficacy against

In order to confirm the antiviral efficacy against human gamma-herpes virus, KSHV, a gamma-2 herpes virus, KSHV confirmed the antiviral efficacy against BC-3G cell line which is a latently infected cell.

The BC-3G obtained in this laboratory is a reporter cell line expressing GFP by the viral active replication (lytic replication). TPA (20 ng / ml), which is a substance that induces reactivation of viruses, was treated with R1-R7 samples (10, 20, 50 and 100 uM) As shown in FIG. 3, the number of EGFP (+) cells was analyzed by repeating the procedure of R4 (Taxifolin) and R5 (Myricetin) showed dose-dependent antiviral efficacy and showed cytotoxicity at high concentration of R1 (Acacetin) and R6 (Apigenin) but showed strong antiviral efficacy at low concentration (10 uM) Showed dose-dependent antiviral efficacy, but was found to be highly cytotoxic.

To confirm this by molecular biology, the difference in the expression level of RTA protein, the extreme early gene of active virus replication of KSHV of BC-3G, a KSHV latent infected cell line, was confirmed by western blot. A sample (20 uM) of R1 to R7 was treated with BC-3G and treated with TPA (20 ng / ml) which is a substance inducing reactivation of virus after 1 hour. After 24 hours, the cells were harvested and Western blot was performed.

As a result, as shown in FIG. 4, it was confirmed that the amount of RTA expressed in the R1, R3 and R6 treated groups was higher than the amount of protein expressed in the DMSO treated group treated with TPA. That is, when treated with 20 uM, R1, R3, and R6 showed antiviral efficacy to reduce viral proteins.

Example  4. Cytotoxicity of the compound of formula 1

MTT assays were performed to determine if the compounds of formula (1) exhibiting antiviral effects were toxic to the cells. The samples were treated with the concentration of the Vero cell line used in the MHV-68 infection experiment, and the amount of living cells was measured after 24 hours. The relative cell viability of each experimental group was determined after the measurement of the untreated control group was set at 100%. As a result, as shown in Fig. 5, it was confirmed that both acacetin and apigenin have a CC50 in the vicinity of 100 uM.

Example  5. Acacetin  Antiviral efficacy and mechanism studies

1) Antiviral efficacy and mechanism of acacetin against EBV

The antiviral efficacy of acacetin in the EBV infected cell lines B95.8 and Raji cell lines was examined and the mechanism of antiviral efficacy was examined. Western blotting was performed to confirm the degree of protein expression of EA-D in the same manner as in Example 2. [ B95.8 was treated with TPA (20 ng / ml) and Raji was treated with TPA (20 ng / ml) and sodium butyrate (NaB) (3 mM) to induce virus reactivation. In the experiment using B95.8 according to Example 2, acacetin was treated with 10 μM of acacetin to lower the concentration of EBV virus protein EA-D. As can be seen from FIG. 6, the B95.8 and Raji showed a decrease in the viral protein even at the lowest treatment concentration of 2 uM and showed dose-dependent antiviral efficacy (FIGS. 6A and 6D).

The antiviral efficacy of acacetin was confirmed at the mRNA level by extracting RNA from cells after acacetin treatment and then performing RT-Q-PCR using cDNA primers specific for viral genes (Cho et al, Antiviral activity of angelicin against gammahepesviruses, Antiviral Res. 2013; 100 (1): 75-83). All RT-Q-PCR was performed with the mRNA level of the cell gene beta-actin corrected. BZLF1 is an immediate early gene that coexists with the onset of EBV replication and BMRF1 is an early gene that expresses in its lower stages. As a result, acacetin showed potent antiviral potency to inhibit viral transcripts BZLF1, BMRF1 in a dose-dependent manner from 2uM in B95.8 and Raji cell lines (FIGS. 6B, 6C, 6E and 6F).

2) Antiviral efficacy and mechanism of acacetin on KSHV

The antiviral efficacy of acacetin and the mechanism of antiviral efficacy were examined in BC-3G and BC-3 cells infected with KSHV.

The antiviral efficacy was confirmed in the same manner as in Example 3. Acacetin was treated at a concentration of 2 uM, 5 uM, 10 uM, 20 uM, and 50 uM as previously treated with EBV infected cell line in Example 4-1. As a result, as shown in FIG. 7 (left drawing), acacetin inhibited the active infection of the virus in a dose-dependent manner by confirming the ratio of GFP positive cells and the decrease of virus protein.

To investigate the mechanism of this antiretroviral effect of acacetin, we confirmed the phosphorylation of ERK, which is known to be an important signal for KSHV reactivation. KSHV-infected BC-3 cell line was treated with TPA or TPA and sodium butyrate (NaB) to reactivate the virus, while acacetin was treated with concentration, and the cells were harvested to collect anti-RTA and anti- Western blotting was performed using an antibody capable of detecting peroxynitrite and PHR-EKR. As shown in FIG. 7 (right drawing), the phosphorylated ERK (pERK) did not decrease even when acacetin was treated as compared with the control group treated with DMSO.

Example  6. Apigenin  Antiviral efficacy and mechanism studies

1) Antiviral efficacy and mechanism of apigenin against EBV

The antiviral efficacy and mechanism of apigenin against EBV were investigated in the same manner as in Example 5. As can be seen from FIG. 8, when 2 μM of apigenin was treated with B95.8 and Raji, the virus protein was markedly reduced and showed dose-dependent antiviral efficacy (FIGS. 8A and 8D). In addition, treatment with apigenin significantly reduced the amount of mRNA of BZLF1, which is an extremely early gene of EBV, and showed a dose-dependent decrease in the amount of BMRF1 transferred at its lower stage (FIGS. 8B, 8C, 8E and 8F). It was anticipated that apigenin would act early in viral replication to have antiviral efficacy.

2) Antiviral efficacy and mechanism of azepinin against KSHV

The antiviral efficacy and mechanism of apigenin against KSHV were investigated in the same manner as in Example 5. As can be seen from FIG. 9, apigenin inhibited the active infection of the virus in a dose-dependent manner by confirming the ratio of GFP positive cells and the decrease of virus protein (left-hand side of FIG. 9). In addition, it was confirmed that when apigenin was treated, the phosphorylation of ERK was inhibited in a dose-dependent manner (the right side of Fig. 9). Thus, apigenin was expected to inhibit the active replication of the virus through a mechanism that inhibits ERK phosphorylation.

Example  7. Acacetin  And Apigenin IC50  Measure value

Acacetin was added at a concentration of 0.05 uM, 0.1 uM, 0.2 uM, 0.4 uM, 0.4 uM, 0.8 uM, 1.6 uM, 3.2 uM, 6.4 uM, 12.8 uM, 25.6 uM, The cells were treated with MHV-68 at a concentration of 0.8 uM, 1.6 uM, 3.2 uM, 4.8 uM, 6.4 uM, 12.8 uM, 25.6 uM and 30.0 uM, respectively. The IC ₅₀ value of apigenin was measured. Three samples were used for each concentration and a relative decrease in the number of plagues was measured relative to the sample treated with DMSO.

As a result, as can be seen from FIG. 10, the IC ₅₀ of acacetin was 3.888 uM, and the IC ₅₀ of apigenin was 2.670 uM, indicating high antiviral activity.

As described above, acacetin and apigenin according to the present invention can be usefully used for the treatment and improvement of gamma-herpesvirus infections in which a clear therapeutic agent is not present at present.

Claims (10)

A pharmaceutical composition for treating infection with Kaposi's sarcoma associated herpesvirus (KSHV) comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

In this formula,
R ₁ is CH ₃ or H.
The method according to claim 1,
The compound of Chemical Formula (1) is acacetin, which is a pharmaceutical composition for the treatment of Kaposi's sarcoma herpesvirus infection.
The method according to claim 1,
The compound of Chemical Formula (1) is apigenin, which is a pharmaceutical composition for the treatment of Kaposi's sarcoma herpesvirus infection.
delete 4. The method according to any one of claims 1 to 3,
Wherein said composition is for the treatment of a disease selected from the group consisting of Kaposi sarcoma, B lymphoma, and multicentric Castleman's disease due to Kaposi's sarcoma herpesvirus infection.
A composition for improving Kaposi's sarcoma herpesvirus infection comprising a compound of formula (I) or a physiologically acceptable salt thereof.
[Chemical Formula 1]

In this formula,
R ₁ is CH ₃ or H.
The method according to claim 6,
The composition of claim 1, wherein the compound is acacetin.
The method according to claim 6,
The composition of claim 1, wherein the compound of formula (1) is apigenin.
delete 9. The method according to any one of claims 6 to 8,
Wherein the composition is for the improvement of a disease selected from the group consisting of Kaposi sarcoma, B lymphoma, and multicentric Castleman's disease due to Kaposi's sarcoma herpesvirus infection.

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