CN116018144A

CN116018144A - An antiviral pharmaceutical composition containing therapeutic agent derived from Astraeus Asiaticus

Info

Publication number: CN116018144A
Application number: CN202180037605.6A
Authority: CN
Inventors: 布兰特·塔萨尼特利塔普; 金塔纳·法旺; 纳萨吉·努尔凯; 通坤·普里恩普洛姆; 詹提普·萨莱
Original assignee: Mahidol University
Current assignee: Mahidol University
Priority date: 2020-06-15
Filing date: 2021-04-30
Publication date: 2023-04-25
Also published as: WO2021257007A1

Abstract

The present invention relates to an antiviral pharmaceutical composition comprising a polar extract of Astraeus asiaticus; and one or more pharmaceutically acceptable excipients selected from solvents, additives, buffers, preservatives, flavouring agents and stabilisers. The composition further comprises acyclovir. The invention also relates to the use of a compound for the treatment of a disease caused by enterovirus 71 (EV 71), coxsackievirus a16 (CA V16), herpes simplex virus type 1 (HSV-1) and/or herpes simplex virus type 2 (HSV-2), wherein the HSV-2 strain is resistant to acyclovir, wherein the disease comprises hand-foot-and-mouth disease (HFMD). The invention also relates to the use of ergosterol for treating hand-foot-mouth disease.

Description

An antiviral pharmaceutical composition containing therapeutic agent derived from Astraeus Asiaticus

Technical Field

The present disclosure relates to a pharmaceutical composition having antiviral properties. The disclosed compositions comprise at least one active therapeutic agent derived from Astraeus asiaticus or a derivative thereof. Alternatively, the disclosed compositions may have one or more antiviral drugs or agents to synergistically improve their achieved therapeutic effect.

Background

Enterovirus 71 (EV 71) and Coxsackie virus A16 (CA 16) belong to the family picornaviridae, and are causative agents of pandemic disease in a number of ways worldwide [1]. In particular, EV71 and CVA16 are both major causes of hand-foot-and-mouth disease (HFMD) and herpangina [2]. In general, EV71 is an RNA-based virus that is capable of causing human complications associated with the Central Nervous System (CNS), including cerebellar ataxia, acute brainstem encephalitis, and poliomyelitis-like paralysis [3]. More importantly, EV71 primarily infects infants and children under 5 years [4], which are more susceptible to the complications that arise than adults. Therefore, the mortality rate of infants infected with the hand-foot-and-mouth disease is high. Common symptoms of hand-foot-and-mouth disease are blisters on the hands, soles and buttocks or maculopapules on the skin of infected subjects [5]. Hand-foot-and-mouth disease is easily transmitted through the fecal-oral route or by touching surfaces contaminated with oropharyngeal secretions [6]. Unfortunately, there is currently no effective clinical drug available for treating or significantly alleviating the symptoms of hand-foot-and-mouth disease patients [7].

Astraeus is a member of the Diplocystaceae (Astraeaceae) family, a fungus common in tropical countries [8]. As conventional knowledge is widely spread in this area, efforts have been made to investigate the medicinal value of the different Astraeus members of A.sirindhorniae [9], A.asepticus and A.odoratus [10] in southeast Asia. Some early studies reported that the most abundant compounds in Astraeus were triterpenes. In these studies, extracts of A.odoratus containing astraosorol A and B have been found to have great potential against cancer cell proliferation and tuberculosis [11]. Modification of the astraodorol chemical structure also showed antimalarial activity in vero cell lines [12]. In addition, antibacterial and antifungal properties of astraodorol were also observed [13]. Nevertheless, the effect of Astraeus derived compounds and/or extracts on viral activity is still unclear. Thus, it is very valuable to explore the potential use of extracts, crude or purified extracts of a. Aseptic us, especially extracts from different plant parts and any other derivatives obtained thereof, for neutralizing harmful viral activity in subjects suffering from enterovirus infections.

Disclosure of Invention

It is an object of the present disclosure to provide an antiviral composition comprising at least one active therapeutic agent of natural origin. The inventors of the present disclosure found that the active therapeutic agent has little or no cytotoxicity to a subject treated with the disclosed compositions.

It is a further object of the present disclosure to provide an antiviral pharmaceutical composition that is effective in inhibiting viral replication of EV71, CA16, herpes simplex virus type 1 (HSV-1) and/or herpes simplex virus type 2 (HSV-2) in a subject. Thus, the disclosed compositions are useful as a means of treating conditions associated with or caused by these viruses.

It is another object of the present disclosure to obtain synergistic therapeutic effects by incorporating one or more other antiviral compounds through the disclosed compositions.

Furthermore, another object of the present disclosure relates to the use of ergosterol or any derivative thereof obtained for the treatment of hand-foot-and-mouth disease.

One aspect of the present disclosure relates to an antiviral pharmaceutical composition comprising a polar extract of a. Asepticus; and one or more pharmaceutically acceptable excipients selected from solvents, additives, buffers, preservatives, flavouring agents and stabilisers. Preferably, the composition is effective for viral replication in a host cell infected with the predetermined virus.

Further embodiments of the disclosed compositions also include acyclovir, which has been found to work synergistically with the polar extract of Astraeus asiaticus against herpes simplex virus.

For further embodiments, the weight ratio of extract of a. Asepticus to acyclovir is from 1:1 to 4:1 for the disclosed antiviral composition.

For further embodiments, the concentration of polar extract and acyclovir in the disclosed compositions is from 60 to 500ug/ml and from 0.10 to 100ug/ml, respectively.

In several embodiments, the polar extract used in the disclosed compositions is obtained by heating a. Aseptic us with water for a predetermined time, removing a. Aseptic us from the water, and concentrating the water to produce the polar extract.

For further embodiments, the predetermined virus is any one or combination of enterovirus 71, coxsackievirus a16, herpes simplex virus type 1, herpes simplex virus type 2, anti-acyclovir herpes simplex virus type 1, and anti-acyclovir herpes simplex virus type 2.

In some embodiments, herpes simplex virus type 1 and herpes simplex virus type 2 refer to strains that are resistant to acyclovir when acyclovir alone is used against the resulting infection.

Another aspect of the present disclosure relates to an aqueous extract of a. Asepticus for treating hand-foot-and-mouth disease.

Furthermore, another aspect of the present disclosure relates to ergosterol or any derivative thereof obtained for use in the treatment of hand-foot-and-mouth disease.

Drawings

FIG. 1 is an HPLC chromatogram of an aqueous extract of A. Asepticus showing peaks corresponding to ergosterol;

FIG. 2 is a graph showing the cytotoxicity of aqueous extracts of A. Aseptic us at various concentrations on Vero cells;

FIG. 3 is a graph showing the results of an aqueous extract of A. Aseptic us inhibiting the efficacy of HSV1 and HSV 2 in Vero cells pretreated with different concentrations of extract;

FIG. 4 is a graph showing the results of the efficacy of aqueous extracts of A. Aseptic us as virucide in killing and/or inhibiting HSV1 and HSV 2 infection in Vero cells treated with different concentrations of extracts;

FIG. 5 is a graph showing the results of aqueous extracts of A.aseptic us in inhibiting the efficacy of HSV1 and HSV 2 on infection of Vero cells treated with different concentrations of extracts;

FIG. 6 is a graph showing the results of an aqueous extract of A. Aseptic us in inhibiting viral replication of HSV1 and HSV 2 in infected Vero cells treated with different concentrations of extract; and

FIG. 7 is a diagram illustrating an aqueous extract of A. Aseptic us (H ₂ O[μg/ml]) Graph of results and efficiency of inhibition of enterovirus 71 and coxsackievirus a16 viral replication in Vero cells pretreated with the extract.

Detailed Description

Hereinafter, the present invention will be described according to preferred embodiments with reference to the accompanying description and drawings. It should be understood, however, that the description with respect to the preferred embodiments of the present disclosure and the accompanying drawings is for the purpose of facilitating the discussion of the various disclosed embodiments only, and that various modifications may be envisaged by those skilled in the art without departing from the scope of the appended claims.

The terms "comprising" and "including," and grammatical variants thereof, as used herein, are intended to mean "open" or "inclusive" language such that they include the recited elements but also allow for the inclusion of additional, unrecited elements, unless otherwise indicated.

As used herein, a phrase in "an embodiment" refers to in some embodiments, but not necessarily in all embodiments.

As used herein, the term "about" or "approximately" in the context of component concentrations, conditions, other measurements, etc., refers to +/-5% of the value, or +/-4% of the value, or +/-3% of the value, or +/-2% of the value, or +/-1% of the value, or +/-0.5% of the value, or +/-0% of the value.

The terms "aqueous extract" and "aqueous extract" are used interchangeably throughout the description provided below and refer to an extract obtained by contacting a. The aqueous extract with water, preferably deionized water, under one or more predetermined conditions to promote dissolution of polar or water-soluble compounds present in a. The aqueous extract, separating the water and dissolved compounds from the a. The aqueous extract, and removing and concentrating the separated water, unless otherwise indicated. Preferably, the A.aseptic us and water for the extraction process are prepared in a ratio of 1:1 to 1:6 (w/v).

The term "antiviral" or "viral inhibition" or "antiviral replication" refers to the disruption of replication of the viral life cycle, including preventing the virus from penetrating into cells and/or directly killing the virus. But should not be construed as having a direct effect on the virus, such as preventing binding of cellular receptors to the virus, inhibiting gene expression in viral replication, etc. By "killing the virus" is meant acting directly on the virus itself, resulting in inactivation of the virus. The antiviral properties mentioned below in relation to an a. Aseptic extract may refer to the ability of the extract to interfere with viral replication, in particular by inhibition of binding between host cell receptors and the virus, or by inhibition of penetration of the host cell, or even by inhibition or killing of the virus after replication.

As used herein, the term "synergistic effect" may refer to a combination of at least two therapeutic agents during a treatment or course of treatment, wherein the combination of at least two therapeutic agents produces a therapeutic effect that is greater than the sum of only each therapeutic agent, e.g., when acyclovir is combined with an extract of a. Asepticus, the resulting therapeutic result is very effective in a synergistic manner for viral replication, particularly for drug resistant strains.

According to one aspect of the present disclosure, an antiviral pharmaceutical composition is disclosed. In essence, the disclosed antiviral composition comprises a polar extract of a. Asepticus; and one or more pharmaceutically acceptable excipients selected from solvents, additives, buffers, preservatives, flavouring agents and stabilisers. The disclosed compositions are effective for viral replication in host cells infected with a predetermined virus. For many embodiments, the disclosed compositions are effective against, but not limited to, enterovirus 71, coxsackievirus a16, herpes simplex virus type 1, and/or herpes simplex virus type 2.

Preferably, the polar extract is obtained by heating clean a. Aseptic us with water or an aqueous phase at a temperature of 95 to 100 ℃. After the heat treatment to leave the desired polar compounds from the a. Aseptic us matrix, the water with dissolved compounds is separated from the used a. Aseptic us by one or more filtration steps to remove any unwanted solid residues from the aqueous phase. The aqueous phase, free of solid residues, is then concentrated by further heating or vacuum drying to precipitate a polar extract for use in the preparation of antiviral pharmaceutical compositions. It will be appreciated by those skilled in the art that other polar solvents such as solvent alcohols, acetonitrile, etc. may also be used to extract by mixing and contacting a. Asepticus with these solvents, with or without heat, to carry out and dissolve the desired polar compounds. The inventors of the present disclosure have discovered that ergosterol can be one of the active therapeutic agents available in polar extracts, imparting antiviral properties to the disclosed compositions.

Furthermore, the type of excipient used with the a. Aseptic us aqueous extract is highly dependent on the formulation being prepared to deliver a given bioactive compound in the extract to achieve the desired therapeutic effect. For example, the disclosed compositions may employ emulsion formulations for oral administration, and similar emulsion formulations may contain one or more buffers and/or stabilizers to preserve the emulsion produced and thereby facilitate absorption of the bioactive compound. Alternatively, the disclosed compositions may be in the form of creams, lotions, ointments, and the like, for topical application to the skin, particularly to the feet and hands of a subject suffering from injury.

According to a more preferred embodiment, the disclosed composition may further comprise acyclovir. In particular, acyclovir is a known antiviral drug for the treatment of symptoms caused by certain types of viruses. For example, it has been used to treat herpes labialis associated with herpes simplex and shingles-induced shingles. In general, acyclovir is biphosphorylated and triphosphorylated by kinases of the infected virus and host cells to inhibit operation of HSV DNA polymerase and cause chain termination. Although acyclovir is an antiviral drug, it does not cure the infection caused by the above viruses. It simply reduces the number of outbreaks in the infected subject, reduces the severity, and shortens the duration of each outbreak. However, there is a growing concern about the reported cases of HSV strains that are resistant to acyclovir treatment. Through experiments conducted, the inventors of the present disclosure have noted that antiviral efficacy against HSV, particularly HSV1 and HSV 2, is improved in the presence of acyclovir. More importantly, the HSV1 and HSV 2 mentioned in these examples may be those strains that are resistant to the treatment or therapeutic effect of acyclovir. Thus, replication of HSV1 or HSV 2 may be prevented by the synergistic therapeutic effect produced by the combination of an aqueous extract of A. Aseptic us and acyclovir, HSV1 or HSV 2 referring to a strain resistant to acyclovir when acyclovir is used alone.

For further examples, the weight ratio of extract of a. Aseptic us to acyclovir is from 1:1 to 4:1 to achieve synergistic therapeutic effect. Preferably, the ratio can be adjusted according to the formulation employed in the disclosed compositions to provide therapeutic results. More importantly, in addition to acyclovir, other antiviral drugs can be incorporated into the disclosed compositions to cover a better broad spectrum against different viruses.

According to further embodiments, the concentration of the polar or aqueous extract may be from 60 to 500ug/ml, with the desired result following administration of the disclosed compositions to a subject infected with the predetermined virus. Also, the concentration or amount of acyclovir used in the disclosed compositions is from about 0.20 to 12.50ug/ml.

Another aspect of the present disclosure relates to an aqueous extract of a. Asepticus for use in treating hand-foot-and-mouth disease (HFMD). Similar aspects of the present disclosure may include an aqueous extract of a. Asetics for treating herpes simplex infections caused by HSV1 and/or HSV 2. For further embodiments, HSV1 and HSV 2 may be those strains resistant to treatment with acyclovir. Notably, the treatment described herein should include alleviation of symptoms arising in subjects infected with enterovirus 71, coxsackievirus a16, HSV1 or HSV 2. Treatment may include improving lesion healing, reducing the time of outbreak in the subject caused by the virus, and/or reducing the time of occurrence.

Further aspects of the disclosure relate to ergosterol or any derivative thereof obtained for use in the treatment of hand-foot-and-mouth disease. The ergosterol used in these examples may be obtained from one or more natural sources, such as a. Asepticus or any other organism capable of spontaneously synthesizing it. Alternatively, the ergosterol used may be produced by chemical synthesis using chemical reactions known in the art.

The following examples are provided to more clearly illustrate the invention, but are not intended to limit the scope of the invention.

Example 1

500g of fresh fruit bodies of both mushrooms were washed, then boiled in 1500mL of deionized water (1 g fresh weight: 3mL water ratio) for 15min, and filtered with Whatman No.1 filter paper. The filtrate was lyophilized to-20 ℃ for further use. The yields of the A.aseptic us were 0.42% and 1.48%, respectively.

Example 2

Vero cell line CO at 37℃in 96 well plates ₂ Culturing in an incubator. After overnight, the cultured cells were further incubated with different concentrations of a.asepticus aqueous extract for 24 hours while DMSO was used as a control. MTT solution was then added to the cells, incubated with the extract for an additional 4 hours, then formazan crystals were dissolved using DMSO and passed over OD ₅₆₀ Absorbance at the site was used to measure cell viability. From the results obtained, experiments showed that in the dosage range of 7.80. Mu.g/ml to 2000. Mu.g/ml, both aqueous extracts of A. Asepticus inhibited the proliferation of Vero cells, which appeared to have no detectable toxic effect on the Vero cell line even at doses up to 2000. Mu.g/ml (FIG. 2).

Example 3

Vero cells at 37℃and 5% CO ₂ Under the conditions, 24-well culture plates were used for 24 hours in Earle minimal medium (EMEM) containing 10% Fetal Bovine Serum (FBS), penicillin and streptomycin. The medium in each well was then changed and spiked with 62.5. Mu.g/ml, 125. Mu.g/ml, 250, respectivelyFresh medium of various concentrations of extracts, μg/ml and 500 μg/ml, was incubated for 1 hour at 37 ℃. Again, the medium combined with the extract was replaced with fresh medium, and then the cells were incubated with the cells with herpes simplex virus type 1 (HSV 1) and herpes simplex virus type 2 (HSV 2) isolated from the patient samples at 37 ℃ for 1 hour. Subsequently, the unattached virus was removed and the Vero cells were washed. Placing the washed Vero cells in a solution containing 1.2% microcrystalline cellulose

In the medium of (C) at 37℃and 5% CO ₂ Culturing for 3 days. The way in which the extract prevented herpes simplex virus attachment was investigated using a plaque reduction assay.

The extract of A.aseptic us was found to indeed prevent the attachment of herpes simplex virus type 1 and herpes simplex virus type 2 to the Vero cells tested, with an efficiency of 50 to 100% which corresponds to an extract concentration of less than or equal to 62.50 μg/ml to 500 μg/ml, as shown in FIG. 3. The present disclosure has demonstrated that a polar or aqueous extract of a. Aseptic us is capable of preventing herpes simplex virus from attaching to host cells, thereby preventing the virus from entering and infecting the cells by means of attachment.

Example 4

Vero cells were cultured and seeded in 24-well tissue culture plates in EMEM containing FBS, penicillin and streptomycin at 37 ℃ and 5% carbon dioxide for 1 day. The medium was removed and Vero cells were then washed with cold PBS and contacted with different solvent mixtures containing extracts incubated with HSV1 or HSV 2, respectively. In particular, the solvent mixture is prepared by mixing aqueous extracts of A.asepticus at concentrations of 62.5. Mu.g/ml, 125. Mu.g/ml, 250. Mu.g/ml and 500. Mu.g/ml with HSV1 and HSV 2, respectively, isolated from the patient, and incubating for 1 hour at 4℃and then contacting it with Vero cells. Vero cells and solvent mixtures were incubated at 4 ℃ for 1 hour. Subsequently, the unattached virus was removed and Vero cells were washed with cold Phosphate Buffered Saline (PBS). Placing the washed Vero cells in another cell containing 1.2% microcrystalline cellulose

To a medium of 37℃and 5% CO ₂ The following further incubation for 3 days and the effect of the aqueous extract was studied using a plaque reduction assay.

The present disclosure found that aqueous extracts of a. Aseptic us, when used at concentrations of 62.50 μg/ml to 500 μg/ml, were able to kill 50-100% of HSV1 and HSV 2 isolated from patient samples in all samples, as shown in figure 4. Thus, the conclusion of the present disclosure is that aqueous extracts of a. Aseptic are capable of killing the enveloped viruses HSV1 and HSV 2 to prevent potential infection by these viruses.

Example 5

Vero cells were cultured and seeded in 24-well tissue culture plates in EMEM containing FBS, penicillin and streptomycin at 37 ℃ and 5% carbon dioxide for 1 day. The medium was then removed and the Vero cells were washed with cold PBS. HSV1 and HSV 2 isolated from patient samples were then added to the cultured cells and incubated for 1 hour at 4 ℃. Unattached virus was removed and Vero cells were washed with cold PBS solution. A. Asepticus aqueous extract at concentrations of 200. Mu.g/ml, 300. Mu.g/ml, 400. Mu.g/ml and 500. Mu.g/ml was added to Vero cells and incubated for 1 hour at 4 ℃. The medium was then removed again and the Vero cells were washed with cold PBS solution. Finally, the washed Vero cells were placed in another cell containing 1.2% microcrystalline cellulose

In fresh medium at 37℃and 5% CO ₂ Incubate for 3 days and study using plaque reduction assay.

Based on the results obtained, the present inventors found that the aqueous extract of a. Aseptic us was able to inhibit the entry of HSV1 and HSV 2 into host cells with an efficiency of about 50 to 100%, which corresponds to a concentration range of 62.50 μg/ml to 500 μg/ml, as shown in fig. 5.

Example 6

Vero cells were cultured and inoculated in 24-well tissue culture plates in EMEM containing FBS, penicillin and streptomycin at 37 ℃ and 5% carbon dioxide for 1 day. After removal of the medium, vero cells were washed with PBS solution. HSV1, HSV 2, enterovirus 71 and Coxsackie A16 isolated from patient samples were contacted with the cells and then incubated at 37℃for 1 hour. Unattached virus was removed and Vero cells were washed with PBS solution. Finally, vero cells were placed in a solution containing 1.2% microcrystalline cellulose

To the fresh medium of (C) was added aqueous extracts of A.asepticus at concentrations of 200. Mu.g/ml, 300. Mu.g/ml, 400. Mu.g/ml and 500. Mu.g/ml, and incubated at 37℃for 3 days and 5 days, respectively. The efficacy of the aqueous extract in preventing viral replication in infected cells was studied using a plaque reduction assay.

In view of the results obtained, the present disclosure found that the aqueous extract of a. Aseptic us was able to inhibit viral replication of HSV1 and HSV 2 isolated from patient samples with an efficiency of about 50 to 100%, which corresponds to a concentration of aqueous extract of a. Aseptic us ranging from 400 μg/ml to 500 μg/ml, as shown in fig. 6. Furthermore, the aqueous extract of A. Asepticus exhibits the ability to inhibit replication of enterovirus 71 and Coxsackie virus A16 virus at 50% to 100% efficiency, which corresponds to a concentration range of 100 μg/ml to 400 μg/ml, as shown in FIG. 7.

Example 7

Vero cells were cultured and inoculated in 24-well tissue culture plates in EMEM containing FBS, penicillin and streptomycin at 37 ℃ and 5% carbon dioxide for 1 day. The medium was then removed and the cultured cells were washed with PBS solution. Subsequently, the washed cells were contacted with HSV1 and HSV 2 isolated from the patient samples for further incubation at 37 ℃ for 1 hour. Unattached virus was removed and Vero cells were then washed with PBS solution. The washed Vero cells were then placed into different solution sets: (a) a set of extracts, (b) a set of acyclovir and (c) a set of extracts and acyclovir. For the relevant group, water extract concentrations of 500. Mu.g/ml, 400. Mu.g/ml, 300. Mu.g/ml, 200. Mu.g/ml and 100. Mu.g/ml were used, whereas acyclovir concentrations of 0.20. Mu.g/ml to 12.5. Mu.g/ml were used. Placing the cultured cells together with the corresponding solution into a solution containing 1.2% microcrystalsCellulose

And cultured at 37℃and 5% carbon dioxide for 3 days. The extracts and the efficacy of the combination with acyclovir in preventing replication of HSV1 and HSV 2 were then studied using a plaque reduction assay.

Based on the results obtained, the present disclosure found that the aqueous extract of a. Aseptic and acyclovir were able to act synergistically to inhibit viral replication of HSV1 and HSV 2 in infected cells. In particular, a combination index value (CI value) of less than 1 indicates that the synergy of both the aqueous extract and acyclovir is shown in Table 1. The results further demonstrate that HSV1 and HSV 2 are resistant to treatment with acyclovir alone in all the samples tested.

Table 1 shows the synergistic results of water extracted A.aseptic us extract and acyclovir against herpes simplex virus from isolated patient samples in Thailand.

Remarks: combination Index (CI) <1 = synergy, 1 = additive, 1> antagonism

It should be understood that the present invention may be embodied in other specific forms and should not be limited to the only embodiments described above. However, modifications and equivalents of the disclosed concepts such as those readily apparent to those skilled in the art are intended to be included within the scope of the following claims.

Reference to the literature

1.Wang H,Li K,Ma L,Wu S,Hu J,Yan H,et al.Berberine inhibits enterovirus 71 replication by downregulating the MEK/ERK signaling pathway and autophagy.Virol J.2017；14(1):2.

2.Lee JT,Yen TY,Shih WL,Lu CY,Liu DP,Huang YC,et al.Enterovirus 71 seroepidemiology in Taiwan in 2017 and comparison of those rates in 1997,1999 and 2007.PLoS One.2019；14(10):e0224110.

3.Chong P,Liu CC,Chow YH,Chou AH,Klein M.Review of enterovirus 71 vaccines.Clin Infect Dis.2015；60(5):797-803.

4.Yi EJ,Shin YJ,Kim JH,Kim TG,Chang SY.Enterovirus 71 infection and vaccines. Clin Exp Vaccine Res.2017；6(1):4-14.

5.Lee MS,Chiang PS,Luo ST,Huang ML,Liou GY,Tsao KC,et al.Incidence rates of enterovirus 71 infections in young children during a nationwide epidemic in Taiwan,2008-09.PLoS Negl Trop Dis.2012；6(2):e1476.

6.Chang LY,Tsao KC,Hsia SH,Shih SR,Huang CG,Chan WK,et al.Transmission and clinical features of enterovirus 71 infections in household contacts in Taiwan. JAMA.2004；291(2):222-7.

7.Wang SM,Liu CC.Enterovirus 71:epidemiology,pathogenesis and management. Expert Rev Anti Infect Ther.2009；7(6):735-42.

8.Wilson AW,Binder M,Hibbett DS.Diversity and evolution of ectomycorrhizal host associations in the Sclerodermatineae(Boletales,Basidiomycota).New Phytol. 2012；194(4):1079-95.

9.Phosri C,Watling R,Suwannasai N,Wilson A,Martin MP.A new representative of star-shaped fungi:Astraeus sirindhorniae sp.nov.from Thailand.PLoS One. 2014；9(5):e71160.

10.Phosri C,Martin MP,Sihanonth P,Whalley AJ,Watling R.Molecular study of the genus Astraeus.Mycol Res.2007；111(Pt 3):275-86.

11.Arpha K,Phosri C,Suwannasai N,Mongkolthanaruk W,Sodngam S.Astraodoric acids A-D:new lanostane triterpenes from edible mushroom Astraeus odoratus and their anti-Mycobacterium tuberculosis H37Ra and cytotoxic activity.J Agric Food Chem.2012；60(39):9834-41.

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13.Kope HH,Tsantrizos YS,Fortin JA,Ogilvie KK.p-Hydroxybenzoylformic acid and(R)-(-)-p-hydroxymandelic acid,two antifungal compounds isolated from the liquid culture of the ectomycorrhizal fungus Pisolithus arhizus.Can J Microbiol. 1991；37(4):258-64.

Claims

1. An antiviral pharmaceutical composition comprising:

a polar extract of Astraeus asiaticus; and

one or more pharmaceutically acceptable excipients selected from the group consisting of solvents, additives, buffers, preservatives, flavoring agents and stabilizers, wherein the composition is effective against viral replication in a host cell infected with the predetermined virus.

2. The pharmaceutical composition of claim 1, further comprising acyclovir.

3. The pharmaceutical composition of claim 2, wherein the weight ratio of polar extract to acyclovir of Astraeus asiaticus is from 1:1 to 4:1.

4. The pharmaceutical composition according to claim 1, wherein the concentration of the polar extract of Astraeus asiaticus is from 60 to 500ug/ml.

5. The pharmaceutical composition according to claim 2, wherein the concentration of acyclovir is from 0.20ug/ml to 12.50ug/ml.

6. The pharmaceutical composition of claim 1, wherein the polar extract is obtained by heating Astraeus asiaticus with water for a predetermined time, removing Astraeus asiaticus from the water, and concentrating the water to produce a polar extract.

7. The pharmaceutical composition of claim 2, wherein the predetermined virus is selected from the group consisting of enterovirus 71, coxsackievirus a16, herpes simplex virus type 1, herpes simplex virus type 2, anti-acyclovir herpes simplex virus type 1, and anti-acyclovir herpes simplex virus type 2.

8. The pharmaceutical composition of claim 7, wherein the herpes simplex virus type 1 and the herpes simplex virus type 2 are strains resistant to acyclovir.

9. A Astraeus asiaticus aqueous extract for use in the treatment of hand-foot-and-mouth disease (HFMD).

10. The aqueous extract of claim Astraeus asiaticus, wherein the extract of Astraeus asiaticus has a concentration of 60ug/ml to 500ug/ml.

11. Ergosterol or any derivative thereof obtained for use in the treatment of hand-foot-and-mouth disease (HFMD).