Composition prepared from sarcodia montagneana extract and used for inhibiting Japanese encephalitis virus and application of composition

Abstract

The invention provides application of a sarcodia montagneana extract in preparation of a composition for inhibiting Japanese encephalitis virus. Wherein the sarcodia montagneana extract is extracted from a sarcodia montagneana frond material or a sarcodia montagneana frond, the sarcodia montagneana extract contains a component for inhibiting the Japanese encephalitis virus, the sarcodia montagneana extract can be applied to resisting infection of the Japanese encephalitis virus and inhibiting the activity of the Japanese encephalitis virus, and the sarcodia montagneana extract does not have cytotoxicity.

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 (e.g., health foods or health supplements) prepared from Sarcodia sp. extract for inhibiting JEV and their various uses (e.g., pharmaceuticals, medical supplies or disinfectants). Background Technology

[0002] For example, tripeptides and their applications that are commonly used to combat Japanese encephalitis virus infection are often mentioned, such as Chinese Patent Publication No. CN-102827248, "A tripeptide with anti-Japanese encephalitis virus infection and its application", which discloses a tripeptide with anti-Japanese encephalitis virus infection and its application, belonging to the field of antiviral biological product technology.

[0003] As mentioned above, the tripeptide with anti-Japanese encephalitis virus infection and its application 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 mentioned above, the tripeptide with anti-Japanese encephalitis virus infection described in CN-102827248 showed in vitro experimental results that: pretreatment of BHK-21 cells with the tripeptide could significantly interfere with and inhibit Japanese encephalitis virus infection; 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 the mice could 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", discloses a pomegranate glycoside used in anti-Japanese encephalitis virus infection and its application.

[0006] As mentioned above, the pungent glycoside described in CN-113876794, used in the treatment of Japanese encephalitis virus infection, is an inhibitor of the non-structural protein NS2B(H)-NS3pro two-component protease, which is an enzyme extremely important for the growth and replication of Japanese encephalitis virus. Moreover, the inhibitor is pungent glycoside, and the use of pungent glycoside in the treatment of Japanese encephalitis virus infection is considered 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, exhibits 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 commonly used example is triptolide and its application in the preparation of drugs against Japanese encephalitis virus, such as Chinese Patent Publication No. CN-117159558, "Application of triptolide in the Preparation of Drugs Against Japanese Encephalitis Virus", which discloses a triptolide and its application in the preparation of drugs against Japanese encephalitis virus.

[0009] As mentioned 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 this protease.

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

[0011] However, the aforementioned Chinese patents, including Chinese Patent No. CN-102827248 concerning a tripeptide and its application against Japanese encephalitis virus infection, Chinese Patent No. CN-113876794 concerning pomegranate glycoside and its application against Japanese encephalitis virus infection, and Chinese Patent No. CN-117159558 concerning triptolide and its application in the preparation of drugs against Japanese encephalitis virus, inevitably have the potential need for further improvement of their products, such as providing other natural substances (or extracts) that can fight Japanese encephalitis virus.

[0012] Another commonly used sea fungus extract, its uses and extraction methods, for example: Taiwan Patent Publication No. TW-I685344, "Sea Fungus Extract and Extraction Method Thereof", discloses a sea fungus extract and its extraction method, and the sea fungus extract contains a sea fungus extract powder.

[0013] As mentioned above, the sea fungus extract powder described in 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 for 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: Taiwan 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 in preparing a composition to enhance fish disease resistance or immunity.

[0016] Continuing above, the preparation method of the sea fungus extract in the aforementioned No. TW-I745896 includes: Step 1, selecting a sea fungus and extracting it in a water bath 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] As mentioned above, the immune capacity described in TW-I745896 refers to phagocytic ability, while the enhancement of fish disease resistance or immunity refers to increasing the expression level of fish immune-related genes. Furthermore, the disease resistance refers to resistance against pathogens, specifically bacteria, and the bacteria are *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: Taiwan 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: Taiwan 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 Taiwan Patent Publications No. TW-I685344, TW-I745896, TW-I780840 and TW-I780841 only pertain to sea fungus extract, its 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 inevitably 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, CN-113876794, and CN-117159558, and Taiwan patent publications No. TW-I685344, TW-I745896, TW-I780840, and TW-I780841 are merely 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 that inhibits 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 commonly used 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 prepared from seaweed extract for inhibiting Japanese encephalitis virus and its use therein. The seaweed extract contains a component that inhibits Japanese encephalitis virus and a polysaccharide active component. The seaweed extract can be used to fight Japanese encephalitis virus infection and inhibit its activity. Furthermore, the seaweed extract is not cytotoxic, thereby achieving the purpose or effect of providing a composition that fights Japanese encephalitis virus infection and inhibits its activity, and enhancing the economic value of seaweed.

[0024] To achieve the above objectives, the preferred embodiment of the present invention relates to the use of seaweed extract in the preparation of a composition for inhibiting Japanese encephalitis virus, wherein the composition comprises: A seaweed extract is extracted from a seaweed thallus material or a seaweed thallus. The seaweed extract contains a component that inhibits Japanese encephalitis virus. The seaweed extract can be used to fight Japanese encephalitis virus infection and inhibit the activity of Japanese encephalitis virus. The seaweed extract is not cytotoxic.

[0025] The sea fungus extract of the preferred embodiment of the present invention has a concentration, and the concentration of the sea fungus extract is positively correlated with an antiviral activity.

[0026] To achieve the above objectives, a preferred embodiment of the present invention provides a composition for inhibiting Japanese encephalitis virus prepared from seaweed extract, 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 make a food composition. 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. Moreover, the sea fungus extract does not have cytotoxicity.

[0027] 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.

[0028] To achieve the above objectives, a preferred embodiment of the present invention provides a composition for inhibiting Japanese encephalitis virus prepared from seaweed extract, comprising: At least one health food ingredient, which has a first content; and 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. 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. Moreover, the sea fungus extract does not have cytotoxicity.

[0029] 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, to be made into a nutritional supplement, a dietary supplement, a health food, or a health food.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The sea fungus extract of the preferred embodiment of the present invention contains a polysaccharide active ingredient.

[0034] This invention provides the use of seaweed extract in the preparation of compositions that inhibit Japanese encephalitis virus, which has good application prospects. Attached Figure Description

[0035] Figure 1 : A block diagram illustrating 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.

[0036] Figure 2 : A block diagram illustrating 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.

[0037] Figure 3 : A schematic diagram of a block diagram of a drug for inhibiting Japanese encephalitis virus prepared from a composition made of seaweed extract according to the third preferred embodiment of the present invention.

[0038] 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.

[0039] Figure 5 : A schematic diagram illustrating the relationship between cell survival percentage and concentration in a preferred embodiment of the present invention, which uses seaweed extract to prepare a composition for inhibiting Japanese encephalitis virus, in a test of inhibiting Japanese encephalitis virus.

[0040] 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 a composition prepared from seaweed extract to inhibit Japanese encephalitis virus, in an inhibitory test of Japanese encephalitis virus.

[0041] Figure 7 The figure shows the effect of SSWE against JEV in BALB / c mice according to a preferred embodiment of the present invention, with body weight as the indicator.

[0042] Figure 8The preferred embodiment of the present invention shows the effect of SSWE against JEV in BALB / c mice. The figure shows the change in body weight.

[0043] Figure 9 The preferred embodiment of the present invention shows the effect of SSWE against JEV in BALB / c mice. The figure shows the survival rate.

[0044] Figure 10 : Another preferred embodiment of the present invention shows the relationship between cell viability (pre-infection treatment, in-infection treatment, and post-infection treatment) and concentration in the Japanese encephalitis virus inhibition test of a composition prepared from seaweed extract to inhibit Japanese encephalitis virus.

[0045] In the picture: 1a Food ingredients 1b Health food ingredients 100 processing equipment 2. Seaweed extract 21. Seaweed thallus material 22 Agaric algae 3a Food composition 3b. Composition of health foods 3c Inhibitory Composition for Japanese Encephalitis Virus Detailed Implementation

[0046] To fully illustrate the present invention, the following preferred embodiments will be described in detail with reference to the accompanying drawings, but they are not intended to limit the present invention.

[0047] First, the preferred embodiment of the present invention, which uses seaweed extract to prepare a composition for inhibiting Japanese encephalitis virus and its uses, is 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 it is not intended to limit the scope of application of the present invention.

[0048] Furthermore, the seaweed extract of the preferred embodiment of the present invention, or a 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.

[0049] 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 Culex tritaeniorhynchus mosquito. It can infect mosquitoes, birds, bats, livestock, and even animals such as chickens, cattle, and goats, as well as humans. The main route of transmission for Japanese encephalitis is through mosquito bites, and its main endemic areas are Southeast Asia and the western Pacific region.

[0050] 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 are recorded in historical documents as having medicinal potential. In particular, red algae are rich in essential amino acids, fatty acids, polyphenols, flavonoids, and other components, all of which have been proven to have various biological activities such as antibacterial, anticancer, and anti-inflammatory effects.

[0051] Continuing from the above, this invention uses Sarcodia sp., 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, which not only increases the value of Sarcodia sp. as a functional raw material, but also provides new anti-JEV and treatment methods.

[0052] This invention uses seaweed (Sarcodia suae or Sarcodia sp.) as an edible seaweed, a species endemic to the waters of Taiwan, China. Its thallus is dark red, thick and leathery, with irregular forked branches, and its unfolded length is approximately 5 to 15 centimeters. Originally, seaweed was harvested by residents of the Xiaoliuqiu area from the seashore and eaten directly after blanching. A preferred embodiment of this invention uses artificially cultivated seaweed (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.

[0053] As mentioned above, seaweed has generally been used as a food ingredient or related product material. Previously, seaweed extracts were only found to possess ferrous chelating ion capacity, reducing power, superoxide anion scavenging capacity, DPPH free radical scavenging capacity, or other biological activities (e.g., neuroprotection, promoting bone formation, or treating allergic dermatitis). However, this invention uses seaweed extract to prepare a composition for inhibiting Japanese encephalitis virus, which has the functions of anti-infecting Japanese encephalitis virus and inhibiting its activity (e.g.,...). Figure 5 and 6 As shown in the figure, 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.

[0054] Example 1 Figure 1 This is a schematic diagram illustrating a preferred embodiment of the present invention, showing a seaweed extract used to prepare a composition for inhibiting Japanese encephalitis virus, and a seaweed additive product. Please refer to... Figure 1 As shown, for example, the composition prepared from seaweed extract to inhibit Japanese encephalitis virus in the first preferred embodiment of the present invention can be appropriately formulated into a seaweed additive product, and the seaweed additive product comprises at least one food ingredient 1a and at least one seaweed extract 2, and the food ingredient 1a (first predetermined amount) and seaweed extract 2 (second predetermined amount) are formulated into a food composition 3a.

[0055] Please refer to again Figure 1 As shown, for example, the sea fungus extract 2 can be selected from a low-temperature sea fungus extract, a solvent extract, or other appropriate extraction techniques. The sea fungus extract 2 can be extracted 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), meaning it has components that inhibit Japanese encephalitis virus (such as...). Figure 5 and 6 As shown), and this seaweed extract 2 does not have cytotoxicity (e.g. Figure 4 (As shown).

[0056] Please refer to again Figure 1 As shown, for example, the sea fungus extract 2 can be selected as a sea fungus extract liquid, a sea fungus extract essential oil, a sea fungus extract tablet, a sea fungus extract granules, a sea fungus extract powder, or any combination thereof.

[0057] Please refer to again Figure 1 As shown, for example, the sea fungus extract 2 is added to the food ingredient 1a using a processing device 100 (or other device with similar functions) with appropriate technical means, and 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.

[0058] Please refer to again Figure 1 As shown, 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).

[0059] Example 2 Figure 2This is a schematic diagram illustrating a second preferred embodiment of the present invention, showing a seaweed extract used to prepare a composition for inhibiting Japanese encephalitis virus, resulting in a seaweed additive product. Please refer to... Figure 2 As shown, relative to the first embodiment, the sea fungus additive product of the second preferred embodiment of the present invention includes at least one health food ingredient 1b and at least one sea fungus extract 2, and the health food ingredient 1a (first predetermined amount) and the sea fungus extract 2 (second predetermined amount) are made into a health food composition 3b.

[0060] Please refer to again Figure 2 As shown, 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.

[0061] Please refer to again Figure 1 and 2 As shown, for example, the food ingredient 1a or health food ingredient 1b can be selected from a powdered ingredient, a paste ingredient, a liquid ingredient, or any mixture thereof, and the powdered ingredient, paste ingredient, or liquid ingredient can be selected according to different needs.

[0062] Example 3 Figure 3 This is a schematic diagram illustrating a third preferred embodiment of the present invention, showing a composition prepared from seaweed extract to inhibit Japanese encephalitis virus, used to produce a drug for inhibiting Japanese encephalitis virus. Please refer to... Figure 3 As shown, relative to the first embodiment, the Japanese encephalitis virus inhibitory composition of the third preferred embodiment of the present invention comprises at least one seaweed extract 2, and the seaweed extract 2 is made into a Japanese encephalitis virus inhibitory composition 3c (e.g., a human or animal medicine) by appropriate technical means.

[0063] Example 4 Please refer to again Figure 1 , 2 As shown in Figure 3, for example, the preferred embodiment of the present invention uses a sea fungus extraction method: 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.

[0064] Please refer to again Figure 1 , 2As shown in Figure 3, for example, a preferred embodiment of the present invention employs a sea fungus extraction method: 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 bath at a predetermined temperature (e.g., 40°C or other suitable temperatures, such as 30-50°C) 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 for a predetermined time (e.g., 15 minutes or other processing time), and the supernatant is further freeze-dried to obtain at least one sea fungus algae extract, which effectively retains polysaccharide active ingredients, but this is not intended to limit the scope of the present invention.

[0065] Specifically, the preferred embodiment of the present invention uses the following seaweed extraction method: This invention uses *Sarcodia suae* or *Sarcodia sp.*, an edible seaweed endemic to the waters of Taiwan, China. Its thallus is dark red, thick and leathery, with irregular forked branches, and extends approximately 5 to 15 centimeters. Originally, residents of the Xiaoliuqiu area collected *Sarcodia suae* from the seashore and consumed it directly after blanching. A preferred embodiment of this invention uses artificially cultivated *Sarcodia suae* (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.

[0066] The preparation method of this sea fungus extract includes: Step 1: After cleaning the sea fungus thallus sample to remove surface impurities, it is first freeze-dried and then pulverized using a pulverizer for later use, so as to provide sea fungus thallus sample powder. Step 2: Add 1000ml of deionized water to every 100g of the powder prepared in Step 1, and perform water bath extraction at 40℃ for 15 minutes to obtain a sea fungus extract. Step 3: After centrifuging the sea fungus extract at 1200 rpm for 15 minutes, select a supernatant to obtain the sea fungus extract. Step 4: The sea fungus extract obtained above can also be freeze-dried to obtain sea fungus extract powder.

[0067] Please refer to again Figure 1 , 2As shown in Figure 3, for example, in a preferred embodiment of the present invention, the polysaccharide content of the *Auricularia auricula-judae* thallus extract was determined as follows: The polysaccharide content was detected using the phenol-sulfuric acid method. First, glucose standard solutions (concentrations of 0, 25, 50, 75, 100, and 125 mg L⁻¹) were prepared. One mL of the standard solution was thoroughly mixed with 0.5 mL of 5% phenol solution, and then 2.5 mL of concentrated sulfuric acid was added. After mixing thoroughly, the mixture was allowed to stand for 15 minutes. The absorbance at 490 nm was then measured using a spectrophotometer (Metertek SP-830, Taiwan, China), and the data were analyzed using a regression curve. The polysaccharide content of the water extract was determined using the above method. The polysaccharide concentration was 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 was also calculated. Polysaccharide percentage of sample dry weight (%) = (Dry weight of polysaccharides / Dry weight of sample) × 100% As mentioned above, for example, the preferred embodiment of the present invention uses seaweed extract which has polysaccharide active ingredients, but this is not intended to limit the scope of the present invention.

[0068] The NMR analysis results of the above-mentioned extract powder are as follows: Example 5 Figure 4 This diagram illustrates the relative percentage of cell toxicity in a preferred embodiment of the present invention, using seaweed extract to prepare a composition for inhibiting Japanese encephalitis virus, and performing a cytotoxicity test. Please refer to... Figure 4 As shown, for example, the tissue control (TC) is a cell group whose cells have not been treated with seaweed extract, meaning the TC represents 100% cell viability. In this invention, African green monkey kidney epithelial cells (Vero) were cultured in a medium containing 5% fetal bovine serum and then placed in a 96-well plate (4 × 10⁻⁶ cells / year). 4Cell / well culture was performed overnight. After removing the culture medium, six 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, and 0.15625 mg / mL, extracted according to the specific extraction method in Example 4) and DMEM containing 2% FBS were added. The cells were cultured for two days in an incubator at 37°C and 5% CO2. Cell morphology was observed and photographed under a microscope, and cell viability was determined using the MTT assay, with intracellular mitochondrial activity as the indicator of cell viability. Finally, a spectrophotometer (e.g., 560 nm) was used to measure cell viability. The absorbance value was measured at wavelength and converted into cell viability. A high cell viability rate was considered to indicate low toxicity. The seaweed extract (SSWE) of the preferred embodiment of this invention showed no significant toxicity.

[0069] Figure 5 This diagram illustrates the relationship between cell survival percentage and concentration in a preferred embodiment of the present invention, using a composition prepared from seaweed extract to inhibit Japanese encephalitis virus, and its performance in a Japanese encephalitis virus inhibition test. Please refer to... Figure 5 As shown, for example, the virus control (VC) was 0%, while TC was 100%. The viral activity inhibition assay of this invention also used African green monkey kidney epithelial cells, and the cells were cultured overnight in 96-well plates (4×10⁴ cells / well). After removing the culture medium, six concentrations of algal extracts (5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, and 0.15625 mg / mL, extracted according to the specific extraction method in Example 4) 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 water extract (SSWE) and DMEM containing 2% FBS were added. Infection was observed under a microscope at 3 dpi (day post-infection), and cell viability was measured.

[0070] Please refer to again Figure 1 , 2As shown in 3, 4 and 5, 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.

[0071] Figure 6 This diagram illustrates the relationship between cell viability (pre-infection treatment, during-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 viral activity assay of the present invention also uses African green monkey kidney epithelial cells, and the cells are plated in 96 well plates (4 × 10⁻⁶). 4 Cells were cultured overnight in cells / wells. After removing the culture medium, they were divided into control group (TC and VC), pre-treatment experimental group, co-treatment experimental group, and post-treatment experimental group. The infection status of all four groups was observed under a microscope and cell viability was measured to evaluate the effect of inhibiting viral replication.

[0072] Please refer to again Figure 6 As shown, for example, the method for inhibiting viral activity in this 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, and no seaweed extract was given during the 2-hour infection process or after infection; the mid-infection treatment experimental group was not given seaweed extract before or after infection, and seaweed extract and virus were added simultaneously only during the 2-hour infection period; and the post-infection treatment experimental group was not given seaweed extract before or during infection, and different concentrations of seaweed extract were given only after washing away unattached virus one hour after infection.

[0073] Please refer to again Figure 6 As shown, 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 after infection with Japanese encephalitis virus at various concentrations of seaweed extract (SSWE), which shows that seaweed extract (SSWE) has significant anti-infective properties against JEV at various concentrations.

[0074] Example 6 This example is an experiment related to the effect of SSWE on JEV in BALB / c mice.

[0075] The experimental design is as follows: • Subjects: 15 four-week-old female BALB / c mice (n=5 per group) • Viral dose: 1.28 × 10 4 PFU / 33μL • Group: Pre-: 33 μL SSWE administered the day before for nasal infection (IN). Co-: Administered via nasal injection after mixing with the virus (SSWE+JEV). • Comparison of three treatment groups: VC (virus control), 5 mg / mL, and 2.5 mg / mL SSWE. The results are as follows Figure 7 , 8 As shown in Figure 9: 1. Weight change chart ( Figure 7 The VC group showed significant weight loss, indicating that the infection led to a deterioration in health; the two SSWE groups showed a gradual increase in weight, indicating a protective effect; the 5 mg / mL group showed the best effect, with stable weight recovery.

[0076] 2. Change in body weight ( Figure 8 ):and Figure 7 Similarly, VC resulted in weight loss and significant improvement in SSWE; higher concentrations yielded better results.

[0077] 3. Survival rate ( Figure 9 ): All patients in the VC group died after 5 days; the survival rate of the 2.5 mg / mL group was about 80%; and all patients in the 5 mg / mL group survived, showing excellent protective effect.

[0078] Figure 10 A schematic diagram illustrating the relationship between cell viability (pre-infection treatment, during-infection treatment, and post-infection treatment) and concentration in a Japanese encephalitis virus inhibition test using a composition prepared from seaweed extract according to another preferred embodiment of the present invention, and its control group. Figure 6 Please refer to Figure 10 As shown, for example, in another preferred embodiment of the present invention, the viral activity inhibition assay also selected African green monkey kidney epithelial cells, and the cells were plated in 96 well plates (4×10⁻⁶). 4 Cells were cultured overnight in cell / well culture medium. After the medium was removed, they were divided into control group (TC and VC), pre-infection treatment experimental group, mid-infection treatment experimental group, and post-infection treatment experimental group. The infection status of all four groups was observed under a microscope and cell viability was measured to evaluate the effect of inhibiting viral replication.

[0079] Please refer to again Figure 10As shown, for example, another preferred embodiment of the present invention describes a method for testing the inhibition of viral activity: the control group received no seaweed extract; the pre-infection treatment experimental group received seaweed extract only 2 hours before viral infection, and no seaweed extract was given during the 2-hour infection process or after infection; the mid-infection treatment experimental group received no seaweed extract before or after viral infection, and seaweed extract and virus were added simultaneously only during the 2-hour infection period; and the post-infection treatment experimental group received no seaweed extract before or during infection, and different concentrations of seaweed extract were given only after washing away unattached virus one hour after infection.

[0080] Please refer to again Figure 10 As shown, 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 rates after infection with Japanese encephalitis virus at various concentrations of seaweed extract (SSWE), which shows that seaweed extract (SSWE) at various concentrations has significant anti-infective properties against JEV and can prevent the virus from infecting host cells.

[0081] The foregoing preferred embodiments are merely illustrative of the present invention and its technical features. The technology of these embodiments can still be implemented by 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.

Claims

1. Use of seaweed extract in the preparation of a composition for inhibiting Japanese encephalitis virus, wherein the composition comprises: A seaweed extract is extracted from seaweed thallus material or seaweed thallus, and 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.

2. The use according to claim 1, characterized in that, The sea fungus extract has a concentration that is positively correlated with an antiviral activity.

3. The use of a seaweed extract in the preparation of a composition 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 make a food composition. 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. Moreover, the sea fungus extract does not have cytotoxicity.

4. The extraction method according to claim 3, characterized in that, The food component is a beverage, a food, a sauce, a seasoning powder, an edible oil, or a dairy product.

5. The use of a seaweed extract in the preparation of a composition for inhibiting Japanese encephalitis virus, comprising: At least one health food ingredient, which has a first content; and 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. 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. Moreover, the sea fungus extract does not have cytotoxicity.

6. The extraction method according to claim 5, characterized in that, The raw materials for this health food are selected from a nutritional supplement raw material, a dietary supplement raw material, or a health food raw material, to be made into a nutritional supplement, a dietary supplement, a health food, or a health food.

7. The extraction method according to claim 5, characterized in that, The raw materials for this health food are selected from a powdered raw material, a paste-like raw material, or a liquid raw material.

8. The use according to any one of claims 1, 3, or 5, characterized in that, The sea auricularia thallus is selected from either an artificially cultured sea auricularia thallus or a natural sea auricularia thallus.

9. The use according to any one of claims 1, 3, or 5, characterized in that, The sea fungus extract is sea fungus extract essence liquid, sea fungus extract essential oil, sea fungus extract essence tablets, sea fungus extract essence granules, sea fungus extract essence powder, or any combination thereof.

10. The extraction method according to claim 5, characterized in that, The sea fungus extract contains a polysaccharide active ingredient.

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