WO2021257007A1

WO2021257007A1 - An antiviral pharmaceutical composition with therapeutic agent originated from astraeus asiaticus

Info

Publication number: WO2021257007A1
Application number: PCT/TH2021/000016
Authority: WO
Inventors: Boonrat TASSANEETRITAP; Chintana PHAWONG; Natsajee NUALKAEW; Thongkoon PRIENGPROM; Jantip SAELEE
Original assignee: Mahidol University
Priority date: 2020-06-15
Filing date: 2021-04-30
Publication date: 2021-12-23
Also published as: CN116018144A

Abstract

The present invention relates to an antiviral pharmaceutical composition comprising a polar extract of Astraeus asiaticus, and one or more pharmaceutically acceptable excipient selected from the group consisting of a solvent, an additive, a buffering agent, a preservative, a flavoring agent, and a stabilizer. The composition may further comprise Acyclovir. The invention further relates to use of the composition for treating diseases caused by Enterovirus 71 (EV71), Coxsackies A16 (CAV16), Herpes Simplex Virus type 1 (HSV-1), Herpes Simplex Virus type 2 (HSV-2), and HSV-2 strain resistant to Acyclovir, including hand, foot and mouth disease (HFMD). The invention also relates to use of ergosterol for treating HFMD.

Description

AN ANTIVIRAL PHARMACEUTICAL COMPOSITION WITH THERAPEUTIC

AGENT ORIGINATED FROM ASTRAEUS ASIATICUS

Technical Field

The present disclosure relates to a pharmaceutical composition equipped with antiviral property. The disclosed composition comprises at least one active therapeutic agent or its derivatives originated from Astraeus asiaticus. Alternatively, the disclosed composition can be equipped with one or more antiviral drug or medicine to synergistically improve therapeutic outcome achieved thereof.

Background

Enterovirus71 (EV71 ) and coxsackievirus A16 (CA16), which belong to the Picomaviridae family, have been the causative agents resulting pandemic of several diseases worldwide [1]. Particularly, both EV71 and CVA16 are the primary cause of hand, foot, and mount disease (HFMD) and herpangina [2]. Generally, EV71 is an RNA-based virus capable of triggering complications in human associated with central nervous system (CNS) including cerebella ataxia, acute brainstem encephalitis, and poliomyelitis-like paralysis [3]. More importantly, EV71 mainly infects infant and child under five years old [4] that these subjects are far more vulnerable to the complications triggered compared to adults. Consequently, mortality rate in infant and child contracted with HFMD is relatively high. Common symptoms of HFMD are blisters found on the hands, soles, and buttocks or maculopapular rash on the skin of the infected subjects [5]. HFMD can be easily transmitted through fecal-oral route or touching on surface contaminated with oropharyngeal secretions [6]. Unfortunately, there is no effective clinical drug for treating or substantially relieving symptoms of HFMD’s patients [7].

Astraeus, a member of the Diplocystaceae (Astraeaceae) family, is a fungus commonly found in tropical countries [8]. Owing to traditional knowledge widespread in the region, effort has been consistently put into researching medicinal value of the different members of Astraeus such as A. sirindhorniae [9], A. asiaticus, and A. odoratus [10] in the southeast Asia. Some of the earlier studies reported that the most abundant compounds in the Astraeus are triterpenoids. It was found in these studies that extraction of A. odoratus containing astraodorol such as astraodoric acids A and B possessing great potential for antiproliferation of cancer cells and antituberculosis [11]. Modification of the chemical structure of the astraodorol also exhibited anti-malarial activity in vero cell line [12]. Moreover, antibacterial and anti-fungi properties of the astraodorol were observed as well [13]. Nonetheless, effects of Astraeus-derived compounds and/or extract towards viral activities are still remined unclear. Therefore, it is well-worth to explore the potential application of the extract, crude or purified, of A. asiaticus especially from different plant parts and any other derivatives acquired thereof in neutralizing harmful viral activities in the subject suffering from Enterovirus infection. Summary

One object of the present disclosure is to provide an antiviral composition which comprises at least one active therapeutic agent of natural origin. It was found by the inventors of the present disclosure that the mentioned active therapeutic agent has no or almost no cytotoxicity towards the subject treated using the disclosed composition.

Further object of the present disclosure aims to offer an antiviral pharmaceutical composition being effective in prohibiting viral replication of EV 71, CA 16, Herpes Simplex Virus type 1 (HSV-1) and/or Herpes Simplex Virus type 2 (HSV-2) in a subject. Hence, the disclosed composition is usable as a mean for treating diseased states associated to or caused by these viruses.

Another object of the present disclosure is directed to attain a synergistical therapeutic outcome through the disclosed composition by incorporating one or more other antiviral compounds.

Still, another object of the present disclosure refers to the application of Ergosterol or any of derivatives acquired thereof for use in the treatment ofHFMD.

One aspect of the present disclosure refers to an antiviral pharmaceutical composition comprising a polar extract of A. asiaticus; and one or more pharmaceutically acceptable excipient selected from the group consisting of a solvent, an additive, a buffering agent, a preservative, a flavoring agent, and a stabilizer. Preferably, the composition is effective against viral replication within a host cell infected with a predetermined virus.

More embodiments of the disclosed composition further comprise Acyclovir, which is found to act against Herpes Simplex Virus synergistically along with the polar extract of Astraeus asiaticus.

For more embodiments, the extract of A. asiaticus and Acyclovir are in a weight ratio of 1 : 1 to 4:1 for the disclosed antiviral composition.

For more embodiments, the polar extract and the Acyclovir in the disclosed composition have a concentration of 60 to 500 ug/ml, and 0.10 to 100 ug/ml, respectively. In several embodiments, the polar extract used in the disclosed composition is acquired by heating A. asiaticus together with water for a predetermined period, removing the A. asiaticus from the water, and concentrating the water to yield the polar extract.

For more embodiments, the predetermined virus is any one or combination of Enterovirus 71, Coxsackies A16, Herpes Simplex Virus type 1, Herpes Simplex Virus type 2, Acyclovir- resistant Herpes Simplex Virus type 1, and Acyclovir- resistant Herpes Simplex Virus type 2.

In some embodiments, the Herpes Simplex Virus type 1 and the Herpes Simplex Virus type 2 refer to the strain which is resistant against Acyclovir when Acyclovir is being used alone against the infection caused.

Another aspect of the present disclosure relates to an aqueous extract of A. asiaticus for use in the treatment ofHFMD.

Also, further aspect of the present disclosure is directed to Ergosterol or any of derivatives acquired thereof for use in the treatment ofHFMD.

Brief Description of Drawings Fig. 1 is a HPLC chromatogram of the aqueous extract of A. asiaticus illustrating a peak corresponding to Ergosterol;

Fig. 2 is a graph presenting cytotoxicity effect of the aqueous extract of A. asiaticus at different concentration towards the Vero cells;

Fig. 3 is a graph showing results about efficiency of the aqueous extract of A. asiaticus in prohibiting entrance of HS V 1 and HSV 2 into the Vero cells pretreated with the extract at different concentrations;

Fig. 4 is a graph showing results about efficiency of the aqueous extract of A. asiaticus in acting as virucide to kill and/or prohibit of HSV 1 and HSV 2 infection towards Vero cells treated with the extract at different concentrations; Fig. 5 is a graph showing results about efficiency of the aqueous extract of A. asiaticus in prohibiting infection of HSV 1 and HSV 2 towards the Vero cells treated with the extract at different concentrations;

Fig. 6 is a graph showing results about efficiency of the aqueous extract of A. asiaticus in prohibiting viral replication of HSV 1 and HSV 2 within the infected Vero cells treated with the extract at different concentrations; and

Fig. 7 is a graph illustrating the results and efficiency of the aqueous extract of A. asiaticus (HzO [μg/ml]) in prohibiting viral replication of Enterovirus 71 and Coxsackie A16 within Vero cells pre-treated with the extract. Detailed Description

Hereinafter, the disclosure shall be described according to the preferred embodiments and by referring to the accompanying description and drawings. However, it is to be understood that referring the description to the preferred embodiments of the disclosure and to the drawings is merely to facilitate discussion of the various disclosed embodiments and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

Unless specified otherwise, the terms "comprising" and "comprise" as used herein, and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, un-recited elements.

As used herein, the phrase in “embodiments” means in some embodiments but not necessarily in all embodiments.

As used herein, the terms “approximately” or “about”, in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.

The terms “water extract” and “aqueous extract” are used interchangeably throughout the description provided hereinafter referring to an extract obtained by way of contacting the A. asiaticus with water, preferably deionized water, trader one or more predetermined conditions facilitating dissolution of polar or water-soluble compounds available in the A. asiaticus into the water, separating the water along with the dissolved compound from A. asiaticus and removing or concentrating the separated water, unless mentioned otherwise. Preferably, the A. asiaticus and water are prepared in a ratio of 1 : 1 to 1 :6 (by w/v) for the extraction process.

The terms “antiviral” or “viral inhibition” or “against viral replication” refers to an interference of the viral life cycle from replication, including preventing the viruses from penetrating into cell and/or directly killing the virus. However, it shall not be interpretated as having direct effect to the virus such as prevention of the binding between the cell receptor and the virus, inhibition the gene expression in viral replication, etc. The term “killing virus” refers to the action directly to the virus itself, resulting inactivation of the virus. The antiviral property referred hereinafter with respect to the A. asiaticus extract may direct to the capacity of the extract to interfere the viral replication, either by binding inhibition between the host cell receptor and the virus, or penetration inhibition to the host cell, or even post-replicating inhibition, or killing virus. The terms “synergistic effect” as used herein, may refer to a combination of at least two therapeutic agents in a treatment or course of treatment that the therapeutic outcome derived thereof is greater than the sum of each therapeutic agent alone, for example when the Acyclovir is combined with the extract of A. asiaticus, the derived therapeutic outcome is highly effective against viral replication, particularly against the drug-resistant strains, in a synergistic manner.

According to one aspect of the present disclosure, an antiviral pharmaceutical composition is disclosed. Essentially, the disclosed antiviral composition comprises a polar extract of A. asiaticus; and one or more pharmaceutically acceptable excipient selected from the group consisting of a solvent, an additive, a buffering agent, a preservative, a flavoring agent, and a stabilizer. The disclosed composition is effective against viral replication within a host cell infected with a predetermined virus. For a number of embodiments, the disclosed composition is effective against, but not limited to, Enterovirus 71, Coxsackies A 16, Herpes Simplex Virus type 1 and/or Herpes Simplex Virus type 2.

Preferably, the polar extract is acquired by way of heating the cleaned A. asiaticus with water or an aqueous phase at a temperature ranging from 95 to 100 °C. After the heat treatment to bring the desired polar compounds out from the matrix of the A. asiaticus, the water having the dissolved compounds is separated from the used A. asiaticus through 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 the polar extract out for being used to prepare the antiviral pharmaceutical composition. One skilled in the field shall appreciate the fact that other polar solvent such as solvent alcohol, acetonitrile, etc. can be employed as well to perform the extraction by mixing and contacting the A. asiaticus to these solvent with or without heating to bring out and dissolved the desired polar compounds. It was discovered by inventors of the present disclosure that Ergosterol may be one of the active therapeutic agents available in the polar extract rendering the disclosed composition with the antiviral property.

Furthermore, the type of excipients used along with the aqueous extract of A. asiaticus largely depends on formulation prepared to deliver the bioactive compounds given in the extract for acquiring the desired therapeutic outcome. For instance, the disclosed composition may adopt an emulsified formulation for being administered orally that the like emulsified formulation may comprise one or more buffering agent and/or stabilizer to preserve the emulsion prepared thus facilitating absorption of the bioactive compounds. Alternatively, the disclosed composition may take the form of cream, lotion, ointment, etc. to be applied topically on the skin especially foot and hands of the subject inflicted with lesions thereto.

Pursuant to more preferred embodiments, the disclosed composition may further comprise Acyclovir. Particularly, Acyclovir is a known antiviral drug for treating symptoms caused by certain types of virus. For instance, it has been applied to treat cold sore associated to Herpes simplex and shingles triggered by Herpes Zoster. Generally, Acyclovir is di- and tri- phosphorylated by the kinases of the infecting viruses and host cells to inhibit HSV DNA polymerase from functioning and result chain termination. Despite being an antiviral drug, Acyclovir is not a cure for the infection caused by the mentioned viruses. It merely reduces the episodes of the outbreak in the infected subject and decrease the severity as well as shortens the length of each outbreak. Still, it has become more worrisome that the cases reported with respect to HSV strain resistant against Acyclovir treatment have been ever increasing. Through the experiments performed, it has come to the attention of the inventors of the present disclosure that the antiviral efficiency against HSV, particularly HSV 1 and HSV 2, is boosted in the presence of Acyclovir. More importantly, the HSV 1 and HSV 2 referred in these embodiments can be of those strains resistant against the treatment or therapeutic effect of Acyclovir. Accordingly, the HSV 1 or the HSV 2, which its replication can be prohibited by the synergistic therapeutic effect generated through the combined usage of the aqueous extract of A. asiaticus and Acyclovir refers to the strain resistant against Acyclovir when Acyclovir is being used alone.

For more embodiments, the extract of A. asiaticus and Acyclovir are in a weight ratio of 1 : 1 to 4:1 in order to attain the synergistic therapeutic outcome. Preferably, the ratio is adjustable according to the formulation adopted by the disclosed composition to deliver the therapeutic outcome. More importantly, it is possible to incorporate other antiviral drug into the disclosed composition in addition to Acyclovir for covering a better spectrum against different viruses.

Pursuant to more embodiments, the polar or aqueous extract may have a concentration of 60 to 500 ug/ml give the desired result upon administering the disclosed composition to the subject infected by the predetermined virus. Likewise, the concentration or amount of the Acyclovir used in the disclosed composition is about 0.20 to 12.50 ug/ml.

Further aspect of the present disclosure is directed to an aqueous extract of A. asiaticus for use in the treatment of Hand, Foot and Mouth Disease (HFMD). The like aspect of the present disclosure may include an aqueous extract of A. asiaticus for use in the treatment of Herpes Simplex Infection caused by HSV 1 and/or HSV 2. For more embodiments, the HSV 1 and HSV 2 can be of those strains resistant against treatment using Acyclovir. It is crucial to note that the treatment described herein shall include relieving the symptoms present on the subject infected by Enterovirus 71, Coxsackies A 16, HSV 1 or HSV 2. Possibly, the treatment may refer to improve healing of the lesions, reduce the episode of the outbreak on the subject caused by the viruses, and/or shorten the occurred episode.

More aspect of the present disclosure relates to Ergosterol or any of derivatives acquired thereof for use in the treatment of HFMD. The Ergosterol used in these embodiments can be obtained from one or more natural sources such as A. asiaticus or any other organisms capable of synthesized it spontaneously. Alternatively, the Ergosterol employed can be produced through chemical synthesis using chemical reactions known in the field.

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

Example 1

The fresh fruiting bodies of both mushrooms 500 g were washed, then they were boiled in 1500 mL deionized water (the ratio 1 g fresh weight: 3 mL water) for 15 min and filtered through the filter paper Whatman No.l. The filtrates were lyophilized to dryness and were kept at -20 °C until used. The yields of A. asiaticus were 0.42% and 1.48 %, respectively.

Example 2

Vero cell lines were cultured in 96-well plate at 37 °C in a CO₂ incubator. After overnight, the cultured cells were further incubated with aqueous extract of A. asiaticus at various concentrations for 24 hours, while using DMSO as a control group. MTT solution was then added into the cells incubated with the extraction for another 4 hours followed by dissolving formazan crystal using DMSO and detecting the cells viability through absorbance at OD₅₆₀. Based on the results acquired, the experiments demonstrated that aqueous extract of both A. asiaticus prohibited proliferation of Vero cell variably around the dosage of 7.80 μg/ml to 2000 μg/ml, which seemed to have no detectable cytotoxicity effect towards Vero cells line even the dosage was as high as 2000 μg/ml (Fig. 2).

Example 3

Vero cells were cultured using 24-well culture plates in Earle's minimal essential medium (EMEM) with 10% fetal bovine serum (FBS), Penicillin and Streptomycin at 37°C with 5% CO₂ for 24 hours. The medium in each well was then replaced and incubated respectively with a fresh medium incorporated with the extract at different concentrations of 62.5, 125, 250 and 500 μg/ml at 37 °C for 1 hour. Again, the medium incorporated with the extract was replaced with a fresh medium followed by incubating the cells along with Herpes Simplex Virus type 1 (HSV 1) and Herpes Simplex Virus type 2 (HSV 2), isolated from patient samples, at 37 °C for 1 hour. Subsequently, the non-attached viruses were removed, and Vero cells were washed. The washed Vero cells were placed to a medium containing 1.2% of the microcrystalline cellulose (Avicel®) for culturing 3 days at 37 °C with 5% COz. The manner which the extract prohibits the attachment of the Herpes Simplex Virus was investigated using a plaque reduction assay.

It was found that extract of A. asiaticus indeed prohibited attachment of Herpes Simplex Virus type 1 and Herpes Simplex Virus type 2 onto the tested Vero cells with efficiency ranging from 50 to 100% corresponding to extract concentrations less than or equal to 62.50 μg/ml to 500 μg/ml, as shown in Figure 3. The present disclosure has demonstrated that polar or aqueous extract of A. asiaticus is able to prevent Herpes Simplex Virus to attach onto a host cell thus prohibiting the viruses from entering into and infecting the cells by way of attachment.

Example 4

Vero cells were cultured and plated in a 24-well tissue-culture plate in EMEM with FBS, Penicillin and Streptomycin at 37 °C with 5% carbon dioxide for 1 day. The medium was removed followed by washing the Vero cells with cold PBS and bringing into contact respectively with different solvent mixture containing the extract incubated with either HSV 1 or HSV 2. Particularly, the solvent mixtures were prepared by mixing the aqueous extract of A. asiaticus of the concentration 62.5, 125, 250 and 500μg/ml respectively with HSV 1 and HSV 2 isolated from the patient and were incubated at 4 ° C for 1 hour before being brought into contact with the Vero cells. The Vero cells and the solvent mixture were incubated at 4 °C for 1 hour. Subsequently, the non-attached viruses were removed, and the Vero cells were washed with cool Phosphate Buffered Saline (PBS). The washed Vero cells were placed into another medium containing 1.2% microcrystalline cellulose (Avicel®) for further culturing at 37 °C with 5% CO₂ for 3 days and the effect of the aqueous extract was investigated using a plaque reduction assay.

The present disclosure found that the aqueous extract of A. asiaticus is able to kill HSV 1 and HSV 2 isolated from the patient sample from 50 to 100 % in all samples when it was used at the concentration ranging from 62.50 μg/ml to 500 μg/ml, as shown in Figure 4. Therefore, the present disclosure concluded that the aqueous extract of A. asiaticus is able to kill the enveloped viruses, HSV 1 and HSV 2, to prevent potential infection initiated by these viruses. Example 5

Vero cells were cultured and plated in a 24-well tissue-culture plate in EMEM with FBS, Penicillin and Streptomycin at 37 °C with 5% carbon dioxide for 1 day. The medium was then removed and the Vero cells were washed with cold PBS. HSV 1 and HSV 2 isolated from the patient sample were then added to the cultured cells for incubation at 4 °C for 1 hour. The non- attached viruses were removed and Vero cells were washed with cool PBS solution. Aqueous extract of A. asiaticus of the concentration 200, 300, 400 and 500 μg/ml were added to the Vero cells for incubation at 4 °C for 1 hour. Again, the medium was subsequently removed and Vero cells were washed with cool PBS solution. Finally, the washed Vero cells were placed into another fresh medium containing 1.2% microcrystalline cellulose (Avicel ®) for culturing 3 days at 37 °C with 5% CO₂ and investigated using a plaque reduction assay.

Based upon the results obtained, the present disclosure found that the aqueous extract of A. asiaticus is able to inhibit the entrance of HSV 1 and HSV 2 into the host cells at an efficiency around 50 to 100 % corresponding to the concentration ranging from 62.50 μg/ml to 500 μg/ml, as shown in Figure 5.

Example 6

Vero cells were cultured and plated in a 24-well tissue-culture plate in EMEM with FBS, Penicillin and Streptomycin at 37 °C with 5% carbon dioxide for 1 day. The Vero cells were washed with PBS solution after removing the medium. HSV 1, HSV 2, Enterovirus 71 and Coxsackievirus A 16, isolated from patient samples, were brought into contact with the cells followed by incubation at 37 °C for 1 hour. The non-attached viruses were removed and Vero cells were washed with PBS solution. Finally, the Vero cells were placed respectively into a fresh medium containing 1.2% microcrystalline cellulose (Avicel®) added with the aqueous extract of A. asiaticus in the concentrations of 200, 300, 400 and 500 μg/ml for incubation at 37 °C for 3 days and 5 days respectively. The efficiency of the aqueous extract in prohibiting viral replication in the infected cells are investigated using a plaque reduction assay.

In view of the results acquired, the present disclosure found that the aqueous extract of A. asiaticus is able to inhibit the viral replication of HSV 1 and HSV 2 isolated from the patient sample at an efficiency around 50 to 100 % corresponding to the concentrations of the aqueous extract of A. asiaticus ranging from 400 μg/ml to 500 μg/ml, as shown in Figure 6. Also, the aqueous extract of A. asiaticus water-exhibit the capability to inhibit viral replication of Enterovirus 71 and Coxsackievirus A16 at an efficiency of around 50 to 100% corresponding to a concentration ranging from 100 μg/ml to 400 μg/ml, as shown in Figure 7. Example 7

Vero cells were cultured and plated in a 24-well tissue-culture plate in EMEM with FBS, Penicillin and Streptomycin at 37 °C with 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 brought into contact with HSV 1 and HSV 2 isolated from the patient sample for further incubation at 37 °C for 1 hour. The non-attached viruses were removed then Vero cells were washed with PBS solution. The washed Vero cells were then placed into different groups of solutions: (a) a group with the extract, (b) a group with Acyclovir and (c) a group with the extract and Acyclovir. For the relevant groups, the concentration of aqueous extract used was 500, 400, 300, 200, and 100 μg/ml while the concentration of the Acyclovir used was 0.20 to 12.5 μg/ml. The cultured cells along with the corresponding solution were placed into the medium containing 1.2% microcrystalline cellulose (Avicel ®) and incubated at 37 °C with 5% carbon dioxide for 3 day. The efficiency of the extract and the combination with Acyclovir in prevent replication of HSV 1 and HSV 2 were then investigated using a plaque reduction assay.

From the results acquired, the present disclosure discovered that the aqueous extract of A. asiaticus and Acyclovir were able to work synergistically to inhibit viral replication of HSV 1 and HSV 2 in the infected cells. Particularly, the combination index value (Cl value) is less than 1 indicating the synergism of both the aqueous extract and Acyclovir as shown in Table 1. The results further revealed that HSV 1 and HSV 2 are resistant against the treatment using sole Acyclovir in all the tested samples sample.

Table 1 shows synergistic results for aqueous extracted A. asiaticus extract and Acyclovir against Herpes Simplex Virus from a patient sample isolated in Thailand.

Remark: Combination Index (Cl) <1 = synergism, 1= additive, and 1> antagonistic

It is to be understood that the present disclosure may be embodied in other specific forms and is not limited to the sole embodiment described above. However, modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto.

References

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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 Trap Dis. 2012;6(2):el476. 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.

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9. Phosri C, Watting R, Suwannasai N, Wilson A, Martin MP. A new representative of star-shaped fungi: Astraeus sirindhomiae 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;lll(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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Claims

1. An antiviral pharmaceutical composition comprising: a polar extract of Astraeus asiaticus; and one or more pharmaceutically acceptable excipient selected from the group consisting of a solvent, an additive, a buffering agent, a preservative, a flavoring agent, and a stabilizer, wherein the composition is effective against viral replication within a host cell infected with a predetermined virus.

2. The pharmaceutical composition of claim 1 further comprising Acyclovir.

3. The pharmaceutical composition of claim 2, wherein the polar extract of Astraeus asiaticus and Acyclovir are in a weight ratio of 1 : 1 to 4: 1.

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

5. The pharmaceutical composition of claim 2, wherein the Acyclovir has a concentration of 0.20 to 12.50 ug/ml.

6. The pharmaceutical composition of claim 1, wherein the polar extract is acquired by heating

Astraeus asiaticus together with water for a predetermined period, removing the Astraeus asiaticus from the water, and concentrating the water to yield the polar extract.

7. The pharmaceutical composition of claim 2, wherein the predetermined virus is selected from Enterovirus 71, Coxsackies A16, Herpes Simplex Virus type 1, Herpes Simplex Virus type 2, Acyclovir- resistant Herpes Simplex Virus type 1, and Acyclovir- resistant Herpes Simplex Virus type 2.

8. The pharmaceutical composition of claim 7, wherein the Herpes Simplex Virus type 1 and Herpes Simplex Virus type 2 are strain resistant against Acyclovir.

9. An aqueous extract of Astraeus asiaticus for use in the treatment of Hand, Foot and Mouth Disease (HFMD).

10. The aqueous extract of Astraeus asiaticus of claim9, wherein the extract of Astraeus asiaticus has a concentration of 60 to 500 ug/ml.

11. Ergosterol or any of derivatives acquired thereof for use in the treatment of Hand, Foot and Mouth Disease (HFMD).