Japanese encephalitis virus (JEV) infection inhibiting combination with sarcodia sp. extracts and usage thereof

Abstract

A Sarcodia sp. extract usage for JEV infection inhibiting combination is disclosed. A Sarcodia extract is extracted from a Sarcodia algae material or a Sarcodia algae. The Nostoc extract has a JEV infection inhibiting ingredient such that the Sarcodia extract is provided to be performed as functions of anti-JEV infection and inhibiting activation of JEV. The Sarcodia extract is non-cytotoxic.

Description

[Technical Field]

[0001] This invention relates to a composition prepared from Sarcodia sp. extract for inhibiting Japanese encephalitis virus (JEV) and its use; particularly to various compositions prepared from Sarcodia sp. extract for inhibiting Japanese encephalitis virus (e.g., health foods or health supplements) and their various uses (e.g., pharmaceuticals, medical supplies or disinfectants). [Previous Technology]

[0002] For example, conventionally used tripeptides that resist Japanese encephalitis virus infection and their applications include, for example, Chinese Patent Publication No. CN-102827248, "A tripeptide that resists Japanese encephalitis virus infection and its application", which discloses a tripeptide that resists Japanese encephalitis virus infection and its application, which belongs to antiviral biological product technology.

[0003] As above, the tripeptide with anti-Japanese encephalitis virus infection and its application mentioned in CN-102827248 are based on the fact that a tripeptide can be screened and identified from the loop peptide of the EDIII domain of Japanese encephalitis virus, and the sequence of the tripeptide is: NSK.

[0004] As above, the tripeptide with anti-Japanese encephalitis virus infection of the aforementioned CN-102827248 showed in vitro experimental results that: pretreatment of BHK-21 cells with the tripeptide can significantly interfere with and inhibit the infection of Japanese encephalitis virus; after administering the tripeptide intraperitoneally or intracerebrally and then attacking BALB / c mice with a lethal dose of Japanese encephalitis virus, the survival rate of mice can be significantly improved.

[0005] Another commonly used ingredient related to anti-Japanese encephalitis virus infection is pomegranate glycoside and its application, for example: Chinese Patent Publication No. CN-113876794, "Potential Application of Pomegranate Glycoside in Anti-Japanese Encephalitis Virus Infection", which discloses a pomegranate glycoside used in anti-Japanese encephalitis virus infection and its application.

[0006] As mentioned above, the pungent glycoside mentioned in CN-113876794 is an inhibitor of the non-structural protein NS2B(H)-NS3pro two-component protease, which is an enzyme that is extremely important for the growth and replication of Japanese encephalitis virus. The inhibitor is pungent glycoside, and the use of pungent glycoside in the treatment of Japanese encephalitis virus infection is a potential drug for the treatment of Japanese encephalitis virus infection.

[0007] As mentioned above, the pungent glycoside described in CN-113876794, used in the treatment of Japanese encephalitis virus infection, has a significant inhibitory effect on the serine protease activity of NS2B(H)-NS3pro in Japanese encephalitis virus. Therefore, this inhibitor can be used to prepare a small molecule inhibitor targeting the two-component protease NS2B(H)-NS3pro in Japanese encephalitis virus.

[0008] Another common application of triptolide and its use in the preparation of drugs against Japanese encephalitis virus is, for example, Chinese Patent Publication No. CN-117159558, "Application of triptolide in the preparation of drugs against Japanese encephalitis virus", which discloses a triptolide and its use in the preparation of drugs against Japanese encephalitis virus.

[0009] As above, the application of triptolide in the preparation of anti-Japanese encephalitis virus drugs as described in CN-117159558 targets the JEV NS2B-NS3 protease in Japanese encephalitis virus and uses triptolide as an inhibitor of the protease.

[0010] As mentioned above, the application of triptolide in the preparation of anti-Japanese encephalitis virus drugs according to CN-117159558 has good inhibitory activity against JEV NS2B-NS3 protease in Japanese encephalitis virus, and triptolide has multiple carriers and administration methods.

[0011] However, the aforementioned Chinese patents, namely, the tripeptide for combating Japanese encephalitis virus infection and its application, No. CN-102827248, the pungent glycoside for combating Japanese encephalitis virus infection and its application, and No. CN-113876794, the tripterin for combating Japanese encephalitis virus infection and its application, and No. CN-117159558, the tripterin and its application in the preparation of drugs for combating Japanese encephalitis virus, inevitably have the potential need to further improve their products, for example, by providing other natural substances (or extracts) that can combat Japanese encephalitis virus.

[0012] Another commonly used sea fungus extract, its uses and extraction method, for example: Republic of China Patent Announcement No. TW-I685344 "Sea Fungus Extract and Extraction Method Thereof" invention patent, which discloses a sea fungus extract and its extraction method, and the sea fungus extract contains a sea fungus extract powder.

[0013] As above, the sea fungus extract powder of the aforementioned No. TW-I685344 is extracted from a sea fungus algae, and the sea fungus extract powder has the ability to chelate ions, reduce, scavenge superoxide anions and scavenge DPPH free radicals.

[0014] Continuing above, the extraction method of the sea fungus extract in the aforementioned No. TW-I685344 includes: providing a sea fungus thallus; subjecting the sea fungus thallus to a first freeze-drying treatment to obtain a freeze-dried sea fungus thallus; pulverizing the freeze-dried sea fungus thallus to obtain a sea fungus algae powder; subjecting the sea fungus algae powder to hot water extraction to obtain a sea fungus extract; separating the sea fungus extract to obtain a clear extract; and subjecting the clear extract to a second freeze-drying treatment to obtain a sea fungus extract.

[0015] Another commonly used sea fungus extract, its uses and extraction methods, for example: Republic of China Patent Publication No. TW-I745896, "A sea fungus extract for enhancing fish immunity and disease resistance, its preparation method and uses", which discloses the use of a sea fungus extract for preparing a composition to enhance fish disease resistance or immunity.

[0016] As above, the preparation method of the sea fungus extract in the aforementioned No. TW-I745896 includes: Step 1, selecting a sea fungus and performing water bath extraction at a temperature of 30°C to 50°C to obtain a sea fungus extract; Step 2, centrifuging the sea fungus extract and selecting a supernatant to obtain the sea fungus extract from the supernatant.

[0017] Continuing from the above, the immune capacity described in the aforementioned No. TW-I745896 refers to phagocytic capacity, and the enhancement of fish disease resistance or immunity refers to enhancing the expression level of fish immune-related genes. Furthermore, the disease resistance refers to resistance against pathogens, and the pathogen is bacteria, specifically Streptococcus agalatiae. Additionally, the composition has a dosage form, which includes an oral dosage form, an immersion dosage form, a spray dosage form, or an injection dosage form.

[0018] Another commonly used sea fungus extract, its uses and extraction methods, for example: Republic of China Patent Publication No. TW-I780840, "A Sea Fungus Extract and Its Uses", discloses a composition for anti-inflammatory and skin whitening purposes, and the composition contains a sea fungus extract.

[0019] Another commonly used sea fungus extract, its uses and extraction methods, for example: Republic of China Patent Publication No. TW-I780841 "A sea fungus extract and its uses" discloses a composition for use in preparing a medicine that provides neuroprotection, promotes bone formation and treats allergic dermatitis, and the composition contains a sea fungus extract.

[0020] However, the aforementioned Republic of China Patent Publications No. TW-I685344, No. TW-I745896, No. TW-I780840 and No. TW-I780841 are only sea fungus extracts, their compositions (e.g., providing neuroprotection, promoting bone formation and treating allergic dermatitis) and their uses (e.g., ferrous chelating ion capacity, reducing capacity, superoxide anion scavenging capacity and DPPH free radical scavenging capacity; enhancing fish immunity and disease resistance; or, anti-inflammatory and skin whitening). There is necessarily a potential need for further improvement or the provision of other technical means to achieve compositions and uses that can inhibit Japanese encephalitis virus.

[0021] In short, the aforementioned Chinese patent applications No. CN-102827248, No. CN-113876794 and No. CN-117159558, and the Republic of China patent announcements No. TW-I685344, No. TW-I745896, No. TW-I780840 and No. TW-I780841 are only for reference and explanation of the technical background of this invention and the current state of technological development, and are not intended to limit the scope of this invention.

[0022] In view of the above, in order to meet the above-mentioned needs, the present invention provides a composition for inhibiting Japanese encephalitis virus prepared from seaweed extract and its use therein, wherein the seaweed extract contains a component for inhibiting Japanese encephalitis virus and a polysaccharide active component, and the seaweed extract can be used for anti-infection with Japanese encephalitis virus and inhibition of Japanese encephalitis virus activity, and the seaweed extract does not have cytotoxicity, thereby improving the technical method of conventional natural substances (or extracts) that can resist Japanese encephalitis virus or drugs used to treat infection with Japanese encephalitis. [Summary of the Invention]

[0023] The main objective of the preferred embodiment of the present invention is to provide a composition for inhibiting Japanese encephalitis virus prepared from sea fungus extract and its use therein. The sea fungus extract contains a component that inhibits Japanese encephalitis virus and a polysaccharide active component. The sea fungus extract can be used to fight Japanese encephalitis virus infection and inhibit the activity of Japanese encephalitis virus. The sea fungus extract does not have cytotoxicity, thereby achieving the purpose or effect of providing a composition for fighting Japanese encephalitis virus infection and inhibiting the activity of Japanese encephalitis virus and improving the economic value of sea fungus.

[0024] To achieve the above objective, a preferred embodiment of the present invention describes the use of seaweed extract to prepare a composition for inhibiting Japanese encephalitis virus, comprising:

[0025] A sea fungus extract is extracted from a sea fungus thallus material or a sea fungus thallus, and the sea fungus extract contains a component that inhibits Japanese encephalitis virus, and the sea fungus extract can be used for anti-infection of Japanese encephalitis virus and inhibition of Japanese encephalitis virus activity, and the sea fungus extract is not cytotoxic.

[0026] In a preferred embodiment of the present invention, the sea fungus extract has a concentration, and the concentration of the sea fungus extract is positively correlated with an antiviral activity.

[0027] To achieve the above objective, a preferred embodiment of the present invention provides a composition for inhibiting Japanese encephalitis virus prepared from seaweed extract, comprising:

[0028] At least one food ingredient having a first content; and

[0029] At least one sea fungus extract, having a second content, wherein the sea fungus extract is extracted from a sea fungus thallus material or a sea fungus thallus, and the sea fungus extract is added to the food ingredient to make a food composition;

[0030] The sea fungus extract contains a component that inhibits Japanese encephalitis virus, and the sea fungus extract can be used to fight Japanese encephalitis virus infection and inhibit the activity of Japanese encephalitis virus, and the sea fungus extract does not have cytotoxicity.

[0031] In a preferred embodiment of the present invention, the food composition is a beverage, a food, a sauce, a seasoning powder, an edible oil, or a dairy product.

[0032] To achieve the above objective, a preferred embodiment of the present invention comprises a composition for inhibiting Japanese encephalitis virus prepared from seaweed extract, comprising:

[0033] At least one health food ingredient, having a first content; and

[0034] At least one sea fungus extract, having a second content, wherein the sea fungus extract is extracted from a sea fungus thallus material or a sea fungus thallus, and the sea fungus extract is added to the health food raw material to make a health food composition.

[0035] The sea fungus extract contains a component that inhibits Japanese encephalitis virus, and the sea fungus extract can be used to fight Japanese encephalitis virus infection and inhibit the activity of Japanese encephalitis virus, and the sea fungus extract does not have cytotoxicity.

[0036] In a preferred embodiment of the present invention, the raw material for the health food is selected from a nutritional supplement raw material, a dietary supplement raw material, or a health food raw material, so as to make a nutritional supplement, a dietary supplement, a health food, or a health food.

[0037] In a preferred embodiment of the present invention, the raw material for the health food is selected from a powdered raw material, a paste-like raw material, or a liquid raw material.

[0038] In a preferred embodiment of the present invention, the sea fungus thallus is selected from an artificially cultured sea fungus thallus or a natural sea fungus thallus.

[0039] In a preferred embodiment of the present invention, the sea fungus extract is a sea fungus extract essence liquid, a sea fungus extract essential oil, a sea fungus extract essence tablet, a sea fungus extract essence granules, a sea fungus extract essence powder, or any combination thereof.

[0040] The sea fungus extract of the preferred embodiment of the present invention contains a polysaccharide active ingredient. [Simplified Explanation of the Diagram]

[0072] Figure 1: A block diagram of a seaweed additive product prepared from a composition for inhibiting Japanese encephalitis virus using seaweed extract according to the first preferred embodiment of the present invention.

[0073] Figure 2: A block diagram of a seaweed additive product prepared from a composition for inhibiting Japanese encephalitis virus using seaweed extract according to the second preferred embodiment of the present invention.

[0074] Figure 3: A block diagram of a drug for inhibiting Japanese encephalitis virus prepared from a composition of seaweed extract to inhibit Japanese encephalitis virus according to the third preferred embodiment of the present invention.

[0075] Figure 4: A schematic diagram showing the relative percentage of survival rate in a cytotoxicity test of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus according to a preferred embodiment of the present invention.

[0076] Figure 5: A schematic diagram showing the relationship between cell survival percentage and concentration in a preferred embodiment of the present invention, which is a composition prepared from seaweed extract to inhibit Japanese encephalitis virus, in a test of inhibiting Japanese encephalitis virus.

[0077] Figure 6: A schematic diagram of the relationship between cell viability (pre-infection treatment, in-infection treatment, and post-infection treatment) and concentration in a preferred embodiment of the present invention, which is prepared from seaweed extract to inhibit Japanese encephalitis virus, in a test of inhibiting Japanese encephalitis virus.

Implementation Method

[0041] In order to fully understand the present invention, preferred embodiments will be described in detail below with reference to the accompanying drawings, and these are not intended to limit the present invention.

[0042] First, the composition prepared from seaweed extract to inhibit Japanese encephalitis virus and its use in the preferred embodiment of the present invention are applicable to various pharmaceutical compositions (e.g., antiviral drugs), various animal pharmaceutical compositions, various medical product compositions, various medical external compositions, or various medical cleaning and disinfection product compositions, but are not intended to limit the scope of application of the present invention.

[0043] Furthermore, the seaweed extract of the preferred embodiment of the present invention or the composition prepared from seaweed extract to inhibit Japanese encephalitis virus (e.g., freeze-dried powder material, extract material or other extract) is added to various processed products, such as beverages, sauces, seasoning powders, health foods, health care products, pharmaceuticals or related ointment products, but it is not intended to limit the scope of the present invention.

[0044] Generally speaking, Japanese encephalitis virus is a mosquito-borne positive single-stranded RNA virus and a zoonotic natural focal disease. It primarily originates from animal hosts, with pigs and migratory birds being the most common. It is mainly transmitted among vertebrate hosts via the mosquito *Culex tritaeniorhynchus*. It can infect mosquitoes, birds, bats, livestock, and even animals such as chickens, cattle, and goats, as well as humans. The main transmission route of Japanese encephalitis is through mosquito bites, and its main endemic areas are Southeast Asia and the western Pacific region.

[0045] In recent years, marine organisms have been widely regarded as an important source for the development of functional bioactive compounds. Among them, algae, as ancient eukaryotic organisms in the ocean, have been consumed by humans for thousands of years and have been recorded in historical documents as having the potential for medicinal use. In particular, red algae are rich in essential amino acids, fatty acids, polyphenols, flavonoids and other components, which have been proven to have various biological activities such as antibacterial, anticancer and anti-inflammatory effects.

[0046] As above, the present invention uses Sarcodia sp., which is a type of red algae. After extraction using a special technique, its extract shows no cytotoxicity and can significantly improve the survival rate of cells infected with Japanese encephalitis virus. It also has good practicality and application. It not only increases the value of Sarcodia sp. as a functional raw material, but also provides new anti-JEV and treatment methods.

[0047] This invention uses Sarcodia suae or Sarcodia sp. as an edible seaweed, a species unique to the waters of Taiwan. Its thallus is dark red, thick and leathery, with irregular forked branches, and its unfolded length is approximately 5 to 15 cm. Originally, residents of the Xiaoliuqiu area collected Sarcodia suae from the seashore, blanched it, and then consumed directly. In a preferred embodiment of this invention, Sarcodia suae is artificially cultivated (e.g., water temperature 25±3℃, seawater salinity 32±3 ppt, or other suitable temperature and salinity), and its extract is added to various processed products.

[0048] As mentioned above, sea fungus has generally been used as food or related product material. Sea fungus extract has previously only been found to have ferrous chelating ion ability, reducing ability, superoxide anion scavenging ability, DPPH free radical scavenging ability or other biological activities (e.g., neuroprotection, promoting bone formation or treating allergic dermatitis). In this invention, the sea fungus extract is used to prepare a composition for inhibiting Japanese encephalitis virus, which has the function of anti-infecting Japanese encephalitis virus and inhibiting the activity of Japanese encephalitis virus (as shown in Figures 5 and 6). That is, its polysaccharide active ingredients effectively enhance the survival ability of cells in vitro and significantly reduce the replication rate of Japanese encephalitis virus in cells.

[0049] Figure 1 shows a block diagram illustrating a seaweed-added product prepared from a composition for inhibiting Japanese encephalitis virus using seaweed extract according to a first preferred embodiment of the present invention. Referring to Figure 1, for example, the composition for inhibiting Japanese encephalitis virus prepared from seaweed extract according to a first preferred embodiment of the present invention can be appropriately prepared into a seaweed-added product, and the seaweed-added product comprises at least one food ingredient 1a and at least one seaweed extract 2, and the food ingredient 1a (first predetermined amount) and the seaweed extract 2 (second predetermined amount) are prepared into a food composition 3a.

[0050] Please refer to Figure 1 again. For example, the sea fungus extract 2 can be selected from a low-temperature sea fungus extract, a solvent extract, or other extracts obtained by appropriate technical means. The sea fungus extract 2 can be selected from a sea fungus thallus material 21 or a sea fungus thallus 22. The sea fungus extract 2 contains a polysaccharide active ingredient or other effective bioactive ingredients (e.g., rich in protein or lipids), that is, it has components that inhibit Japanese encephalitis virus (as shown in Figures 5 and 6). The sea fungus extract 2 does not have cytotoxicity (as shown in Figure 4).

[0051] Please refer to Figure 1 again. For example, the sea fungus extract 2 can be selected as a sea fungus extract essence liquid, a sea fungus extract essential oil, a sea fungus extract essence tablet, a sea fungus extract essence granules, a sea fungus extract essence powder, or any combination thereof.

[0052] Please refer to Figure 1 again. For example, the sea fungus extract 2 is added to the food ingredient 1a by means of appropriate technical means using a processing device 100 (or other device with similar functions). The sea fungus extract 2 may be extracted from a sea fungus body, a sea fungus fragment, a sea fungus powder or any combination thereof.

[0053] Please refer to Figure 1 again. For example, the food component 3a may be selected as a beverage, a food, a sauce, a seasoning powder, an edible oil, a dairy product, other food products (e.g., various snack products, various bread products or various pastry products) or other processed food products (e.g., various meat products).

[0054] Figure 2 shows a block diagram illustrating a seaweed-added product prepared from a composition for inhibiting Japanese encephalitis virus using seaweed extract according to a second preferred embodiment of the present invention. Referring to Figure 2, compared with the first embodiment, the seaweed-added product of the second preferred embodiment of the present invention comprises at least one health food ingredient 1b and at least one seaweed extract 2, and the health food ingredient 1b (first predetermined amount) and the seaweed extract 2 (second predetermined amount) are formulated into a health food composition 3b.

[0055] Please refer to Figure 2 again. For example, the health food ingredient 1b can be selected from a nutritional supplement ingredient, a dietary supplement ingredient, or a health food ingredient to make a nutritional supplement, a dietary supplement, a health food, a health food, or other products.

[0056] Please refer to Figures 1 and 2 again. For example, the food ingredient 1a or health food ingredient 1b can be selected from a powdered ingredient (power material), a paste ingredient (paste material), a liquid ingredient (liquid material), or any mixture thereof, and the powdered ingredient, paste ingredient, or liquid ingredient can be selected according to different needs.

[0057] Figure 3 illustrates a block diagram of a composition for inhibiting Japanese encephalitis virus prepared from seaweed extract according to a third preferred embodiment of the present invention. Referring to Figure 3, compared to the first embodiment, the composition for inhibiting Japanese encephalitis virus in the third preferred embodiment of the present invention comprises at least one seaweed extract 2, and the seaweed extract 2 is prepared into a composition 3c for inhibiting Japanese encephalitis virus (e.g., a human or animal medicine) using appropriate technical means.

[0058] Please refer to Figures 1, 2 and 3 again. For example, in a preferred embodiment of the present invention, a sea fungus extraction method is used: First, a sea fungus algae sample is washed to remove surface impurities, then freeze-dried, and then pulverized using a pulverizer for later use, so as to provide a sea fungus algae sample powder.

[0059] Please refer again to Figures 1, 2 and 3. For example, in a preferred embodiment of the present invention, a sea fungus extraction method is used: Next, at least one solvent (e.g., ethanol) or an aqueous solution (e.g., deionized water) is added to the sea fungus algae sample powder and extracted in a water tank at a predetermined temperature (e.g., 40°C or other suitable temperature) using appropriate technical means (e.g., shaking, constant temperature or other treatment techniques) to obtain at least one preliminary extract. The preliminary extract is then centrifuged in a centrifuge for a predetermined time (e.g., 15 minutes or other treatment time), and the supernatant is further freeze-dried to obtain at least one sea fungus algae extract, which effectively retains polysaccharide active ingredients, but is not intended to limit the scope of the present invention.

[0060] Please refer again to Figures 1, 2, and 3. For example, in a preferred embodiment of the present invention, the polysaccharide content of the *Auricularia auricula-judae* thallus extract is determined as follows: The polysaccharide content is detected using the phenol-sulfuric acid method. First, glucose standard solutions are prepared (concentrations of 0, 25, 50, 75, 100, and 125 mg L⁻¹). 1 mL of the standard solution is thoroughly mixed with 0.5 mL of 5% phenol solution, and then 2.5 mL of concentrated sulfuric acid is added. After mixing thoroughly, the mixture is allowed to stand for 15 minutes. The absorbance at 490 nm is then measured using a spectrophotometer (Metertek SP-830, Taiwan), and the data are analyzed using a regression curve. The polysaccharide content of the water extract is determined using the above method. The polysaccharide concentration is calculated based on the regression curve corresponding to the absorbance value, and the ratio of the polysaccharide content to the dry weight of the sample is calculated.

[0061] Percentage of polysaccharides in sample dry weight [%) = Dry weight of polysaccharides / Dry weight of sample × 100%

[0062] Continuing from the above, for example, the preferred embodiment of the present invention uses sea fungus extract which has effective polysaccharide active ingredients, but it is not intended to limit the scope of the present invention.

[0063] Figure 4 illustrates a schematic diagram of the relative percentage of cell survival rate in a cytotoxicity test of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus according to a preferred embodiment of the present invention. Referring to Figure 4, for example, the tissue control group (TC) represents cells that have not been treated with seaweed extract, i.e., TC represents 100% cell survival rate. This invention selects African green monkey kidney epithelial cells (Vero) and cultures them in a medium containing 5% fetal bovine serum. The cells are then cultured overnight in 96-well plates (4 × 10⁴ cells / well). After removing the culture medium, eight different concentrations of *Auricularia auricula-judae* extract (5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL) and DMEM containing 2% FBS are added. The cells are cultured for two days in an incubator at 37°C and 5% CO₂. Cell morphology is observed and photographed under a microscope, and cell viability is determined. The MTT assay is used to assess cell viability based on the level of mitochondrial activity. Finally, a spectrophotometer (e.g., [specifically, the polysaccharide active component]) is used to measure cell viability; a high viability rate is considered to indicate low toxicity. The *Auricularia auricula-judae* extract (SSWE) of the preferred embodiment of this invention showed no significant toxicity.

[0064] Figure 5 illustrates the relationship between cell survival percentage and concentration in a preferred embodiment of the present invention, showing the composition prepared from seaweed extract for inhibiting Japanese encephalitis virus, in a test of inhibiting Japanese encephalitis virus. Referring to Figure 5, for example, the virus control group (VC) is 0%, while TC is 100%. In this invention, the viral activity inhibition assay also used African green monkey kidney epithelial cells. The cells were cultured overnight in 96 well plates (4×10⁴ cells / well). After removing the culture medium, eight concentrations of algal extracts (5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL) and DMEM containing 2% FBS (20 μL / well) were added and cultured for 2 hours. After removal, Japanese encephalitis virus was added for infection for 2 hours. After removing the virus solution, the cells were washed once with PBS, and then seaweed extract (SSWE) and DMEM containing 2% FBS were added. The infection was observed under a microscope after 2 days post-infection (dpi), and cell viability was measured.

[0065] Please refer to Figures 1, 2, 3, 4 and 5 again. For example, the sea fungus extract 2 can be selected to have a predetermined concentration, and the predetermined concentration of the sea fungus extract 2 is positively correlated with an antiviral activity. Moreover, the sea fungus extract 2 not only does not have cytotoxicity, but can also significantly improve the cell survival rate of Japanese encephalitis virus infection.

[0066] Figure 6 illustrates the relationship between cell viability (pre-infection treatment, co-infection treatment, and post-infection treatment) and concentration in a preferred embodiment of the present invention, using seaweed extract to prepare a composition for inhibiting Japanese encephalitis virus. Referring to Figure 6, for example, the virus inhibition activity assay of the present invention also uses African green monkey kidney epithelial cells, and the cells are cultured overnight in 96 well plates (4 × 10⁴ cells / well). After removing the culture medium, the cells are divided into a control group (TC and VC), a pre-infection treatment experimental group, a co-infection treatment experimental group, and a post-infection treatment experimental group. The infection status of all four groups is observed under a microscope, and cell viability is measured as an evaluation of the effect of inhibiting viral replication.

[0067] Please refer to Figure 6 again. For example, the method for inhibiting viral activity of the present invention is as follows: the control group was not given any seaweed extract; the pre-infection treatment experimental group was given seaweed extract only 2 hours before infection with the virus, and no seaweed extract was given during the 2-hour infection process and after infection; the mid-infection treatment experimental group was not given seaweed extract before or after infection with the virus, and seaweed extract and virus were added simultaneously only during the 2 hours of infection; and the post-infection treatment experimental group was not given seaweed extract before or during infection, and seaweed extract of different concentrations was given after washing away the unattached virus one hour after infection.

[0068] Please refer to Figure 6 again. For example, the pre-infection treatment experimental group, the mid-infection treatment experimental group, and the post-infection treatment experimental group of African green monkey kidney epithelial cells all showed significant cell survival rate of Japanese encephalitis virus infection at various concentrations of sea fungus extract [SSWE], which shows that sea fungus extract [SSWE] has significant anti-infective JEV properties at various concentrations.

[0069] The above experimental data are preliminary experimental results obtained under specific conditions, and are only used to facilitate understanding or reference of the technical content of the present invention. Further related experiments are required. The experimental data and results are not intended to limit the scope of the present invention.

[0070] The foregoing preferred embodiments are merely illustrative of the present invention and its technical features. The technology of these embodiments can still be implemented with various substantially equivalent modifications and / or substitutions. Therefore, the scope of the present invention shall be determined by the scope defined in the appended claims. The copyright of this case is limited to the use of the patent application in the Republic of China.

Claims

1. Use of a composition prepared from a seaweed extract for inhibiting Japanese encephalitis virus, comprising: extracting a seaweed extract from a seaweed thallus material or a seaweed thallus, wherein the seaweed extract contains a component that inhibits Japanese encephalitis virus, and the seaweed extract can be used for anti-infection and inhibition of Japanese encephalitis virus activity, wherein the seaweed extract is non-cytotoxic, and the seaweed thallus material or seaweed thallus is selected from *Auricularia auricula-judae*.

2. The use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claim 1, wherein the seaweed extract has a concentration that is positively correlated with an antiviral activity.

3. Use of a composition prepared from seaweed extract for inhibiting Japanese encephalitis virus, comprising: at least one food ingredient having a first content; and at least one seaweed extract having a second content, wherein the seaweed extract is extracted from a seaweed thallus material or a seaweed thallus, and the seaweed extract is added to the food ingredient to prepare a food composition, wherein the seaweed thallus material or seaweed thallus is selected from *Auricularia auricula-judae*; wherein the seaweed extract contains a component that inhibits Japanese encephalitis virus, and the seaweed extract can be used for anti-infection of Japanese encephalitis virus and inhibition of Japanese encephalitis virus activity, and the seaweed extract is not cytotoxic.

4. The use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claim 3, wherein the food composition is a beverage, a food, a sauce, a seasoning powder, an edible oil, or a dairy product.

5. Use of a composition prepared from seaweed extract for inhibiting Japanese encephalitis virus, comprising: at least one health food ingredient having a first content; and at least one seaweed extract having a second content, wherein the seaweed extract is extracted from a seaweed thallus material or a seaweed thallus, and the seaweed extract is added to the health food ingredient to prepare a health food composition, wherein the seaweed thallus material or seaweed thallus is selected from *Auricularia auricula-judae*; wherein the seaweed extract contains a component that inhibits Japanese encephalitis virus, and the seaweed extract can be used for anti-infection of Japanese encephalitis virus and inhibition of Japanese encephalitis virus activity, and the seaweed extract is not cytotoxic.

6. The use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claim 5, wherein the health food ingredient is selected from a nutritional supplement ingredient, a dietary supplement ingredient, or a health food ingredient to produce a nutritional supplement, a dietary supplement, a health food, or a health food.

7. The use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claim 5, wherein the health food ingredient is selected from a powdered ingredient, a paste-like ingredient, or a liquid ingredient.

8. Use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claims 1, 3 or 5 of the patent application, wherein the seaweed thallus is selected from an artificially cultured seaweed thallus or a natural seaweed thallus.

9. The use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claims 1, 3 or 5 of the patent application, wherein the seaweed extract is a seaweed extract liquid, a seaweed extract essential oil, a seaweed extract tablet, a seaweed extract granules, a seaweed extract powder or any combination thereof.

10. The use of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus as described in claim 5, wherein the seaweed extract contains a polysaccharide active ingredient.

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