Composition for inhibiting vesicular stomatitis virus by using sarcodia montagneana extract and application of composition

Abstract

The present invention provides a composition for inhibiting vesicular stomatitis virus using a sarcodia montagneana extract and a use thereof, the sarcodia montagneana extract is extracted from a sarcodia montagneana material or a sarcodia montagneana frond, and the sarcodia montagneana extract comprises a vesicular stomatitis virus inhibiting component, the sarcodia montagneana extract can be used for resisting vesicular stomatitis virus infection and inhibiting vesicular stomatitis virus activity, and the sarcodia montagneana extract does not have cytotoxicity.

Description

Technical Field

[0001] The present invention relates to a composition using an extract of Sarcodia sp. as an inhibitor of Vesicular Stomatitis Virus (VSV) and its uses; in particular, it relates to various compositions (such as health foods or functional foods) using an extract of Sarcodia sp. as an inhibitor of Vesicular Stomatitis Virus and various uses (such as pharmaceuticals, medical supplies or disinfectants). Background Art

[0002] In the prior art, there are infectious clones of Vesicular Stomatitis Virus, their preparation methods and their applications. For example, in the invention patent application case of "Infectious Clone of Vesicular Stomatitis Virus Carrying Green Fluorescent Protein and Transferrin and Its Preparation Method and Application" of Chinese Patent Publication No. CN-108277208, it discloses an infectious clone of Vesicular Stomatitis Virus carrying green fluorescent protein and magnetic resonance imaging reporter gene transferrin.

[0003] The sequence of the infectious clone of Vesicular Stomatitis Virus in Chinese Patent No. CN-108277208 is SEQ ID NO.1, and this infectious clone of Vesicular Stomatitis Virus can be used to prepare a recombinant Vesicular Stomatitis Virus carrying enhanced green fluorescent protein (EGFP) and magnetic resonance imaging reporter gene transferrin (nuclear magnetic resonance imaging reporter gene ferritin).

[0004] The recombinant Vesicular Stomatitis Virus carrying green fluorescent protein and magnetic resonance imaging reporter gene transferrin in Chinese Patent No. CN-108277208 can be widely used in neuronal marking, brain structure analysis, magnetic resonance imaging or neurotoxicity analysis of Vesicular Stomatitis Virus.

[0005] Another prior art is fluorescent LAMP primers for detecting Swine Vesicular Disease Virus and its detection method. For example, in the invention patent application case of "Fluorescent LAMP Primers for Detecting Swine Vesicular Disease Virus and Its Detection Method" of Chinese Patent Publication No. CN-106755599, it discloses fluorescent LAMP primers for detecting Swine Vesicular Disease Virus, their synthesis method and their detection method.

[0006] Several sequences of the fluorescent LAMP primers of Chinese Patent No. CN-106755599 include: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0007] The synthesis method of the fluorescent LAMP primers for swine vesicular disease virus of Chinese Patent No. CN-106755599 includes: according to the gene sequence of swine vesicular disease virus in the GenBank database, first select a highly conserved region on its VP1 gene as the amplification target, design LAMP primers using the software LAMP designer 1.14, perform an optimal pairing screening test on the primers, and purify them by HPLC.

[0008] Another prior art is a fluorescent LAMP primer for detecting vesicular stomatitis virus and its detection method. For example, the invention patent application case of "Fluorescent LAMP Primer and Detection Method for Detecting Vesicular Stomatitis Virus" of Chinese Patent Publication No. CN-106591498 discloses a fluorescent LAMP primer for detecting vesicular stomatitis virus, its synthesis method, and its detection method.

[0009] Several sequences of the fluorescent LAMP primers of Chinese Patent No. CN-106591498 include: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6. The final concentrations of the inner primers FIP and BIP of the detection method are 1.6 μmol / L, the final concentrations of the outer primers F3 and B3 are 0.2 μmol / L, the final concentrations of the loop primers LF and LB are 0.8 μmol / L, and the reaction temperature is 63°C.

[0010] The synthesis method of Chinese Patent No. CN-106591498 includes: according to the gene sequence of vesicular stomatitis virus in the GenBank database, first select a highly conserved region on its NP gene as the amplification target, design real-time fluorescent LAMP primers using the software LAMPdesigner 1.14, and purify the primers by HPLC.

[0011] However, there must be a potential need to further improve the products of the infectious clone of vesicular stomatitis virus carrying green fluorescent protein and transferrin of magnetic resonance imaging reporter gene, i.e., the infectious clone of vesicular stomatitis virus carrying green fluorescent protein and transferrin of magnetic resonance imaging reporter gene disclosed in the aforementioned Chinese Patent Publication No. CN-108277208, the fluorescent LAMP primers for detecting vesicular stomatitis virus and their detection methods disclosed in Chinese Patent Publication No. CN-106755599, and the fluorescent LAMP primers for detecting vesicular stomatitis virus and their detection methods disclosed in Chinese Patent Publication No. CN-106591498.

[0012] Another prior art relates to drugs, their uses and preparation methods for treating infections with vesicular stomatitis virus. For example, the invention patent application case of "Use of RXR in the preparation of drugs for treating infections with vesicular stomatitis virus" disclosed in Chinese Patent Publication No. CN-105521498 discloses a use of RXR (Retinoid X receptor) in screening drugs for treating infections with vesicular stomatitis virus, its uses, and a method for constructing a cell model of vesicular stomatitis virus infection caused by over-expression of RXR.

[0013] The use of RXR in screening drugs for treating infections with vesicular stomatitis virus, its uses and the construction method disclosed in Chinese Patent No. CN-105521498 utilize the COX (cyclooxygenase) sensing pathway in an RXR-dependent manner to play a key role in host defense against vesicular stomatitis virus, and further utilize the use of MHV68 in screening drugs for treating infections with vesicular stomatitis virus.

[0014] However, the aforementioned Chinese Patent Publication No. CN-105521498 only discloses the use of RXR in screening drugs for treating infections with vesicular stomatitis virus and its uses. There must be a potential need to further improve or provide other technical means to achieve compositions and their uses that can inhibit vesicular stomatitis virus.

[0015] Another prior art relates to Sargassum fusiforme extract, its uses and extraction methods. For example, the invention patent of "Sargassum fusiforme extract and its extraction method" disclosed in Taiwan Patent Publication No. TW-I685344 discloses a Sargassum fusiforme extract and its extraction method, and the Sargassum fusiforme extract contains a Sargassum fusiforme extraction powder.

[0016] The Sargassum fusiforme extraction powder disclosed in Taiwan Patent No. TW-I685344 is extracted from a Sargassum fusiforme thallus, and the Sargassum fusiforme extraction powder has the abilities of ferrous ion chelation, reduction, superoxide anion scavenging and DPPH free radical scavenging.

[0017] The extraction method of the Gracilaria verrucosa extract of Taiwan Patent No. TW-I685344 in China includes: providing a Gracilaria verrucosa thallus; performing a first freeze-drying treatment on the Gracilaria verrucosa thallus to obtain a freeze-dried Gracilaria verrucosa thallus; performing a pulverization treatment on the freeze-dried Gracilaria verrucosa thallus to obtain a Gracilaria verrucosa powder; performing a hot water extraction treatment on the Gracilaria verrucosa powder to obtain a Gracilaria verrucosa extract; performing a separation treatment on the Gracilaria verrucosa extract to obtain a clear extract; performing a second freeze-drying treatment on the clear extract to obtain a Gracilaria verrucosa extract.

[0018] Another prior art Gracilaria verrucosa extract, its uses and its extraction method, for example: the invention patent of "A Gracilaria verrucosa extract for enhancing the immunity and disease resistance of fish, its preparation method and uses" in Taiwan Patent Publication No. TW-I745896 in China, which discloses the use of a Gracilaria verrucosa extract for preparing a composition for enhancing the disease resistance or immunity of fish.

[0019] The preparation method of the Gracilaria verrucosa extract of Taiwan Patent No. TW-I745896 in China includes: Step 1, selecting a Gracilaria verrucosa and performing a water bath extraction at a temperature of 30°C to 50°C to obtain a Gracilaria verrucosa extract; Step 2, after centrifuging the Gracilaria verrucosa extract, selecting a supernatant to obtain the Gracilaria verrucosa extract from the supernatant.

[0020] The immunity in Taiwan Patent No. TW-I745896 in China is the phagocytosis ability, and the enhancement of the disease resistance or immunity of fish means enhancing the expression level of fish immune-related genes, and the disease resistance is against pathogens, and the pathogen is a bacterium, and the bacterium is Streptococcus agalatiae. In addition, the composition has a dosage form, and the dosage form includes an oral dosage form, a soaking dosage form, a spraying dosage form or an injection dosage form.

[0021] Another prior art Gracilaria verrucosa extract, its uses and its extraction method, for example: the invention patent of "A Gracilaria verrucosa extract and its uses" in Taiwan Patent Publication No. TW-I780840 in China, which discloses a composition, and the composition is used for anti-inflammatory and skin whitening purposes, and the composition includes a Gracilaria verrucosa extract.

[0022] Another prior art Gracilaria verrucosa extract, its uses and its extraction method, for example: the invention patent of "A Gracilaria verrucosa extract and its uses" in Taiwan Patent Publication No. TW-I780841 in China, which discloses a composition, and the composition is used for preparing drugs for providing neuroprotection, promoting bone formation and treating allergic dermatitis, and the composition includes a Gracilaria verrucosa extract.

[0023] However, the aforementioned Taiwan, China Patent Publications Nos. TW-I685344, TW-I745896, TW-I780840, and TW-I780841 are only about Sarcodia montagnei extract, its compositions (such as drugs for providing neuroprotection, promoting osteogenesis, and treating allergic dermatitis), and its uses (such as ferrous chelating ion ability, reducing ability, superoxide anion scavenging ability, and DPPH free radical scavenging ability; enhancing the immune and disease resistance of fish; or, anti-inflammatory and skin whitening). There must be a potential need to further improve or provide other technical means to achieve a composition for inhibiting vesicular stomatitis virus and its uses.

[0024] In short, the aforementioned patent applications of China Patent Publications Nos. CN-108277208, CN-106755599, CN-106591498, and CN-105521498 and Taiwan, China Patent Publications Nos. TW-I685344, TW-I745896, TW-I780840, and TW-I780841 are only for reference of the technical background of the present invention and for explaining the current state of technological development, and they are not used to limit the scope of the present invention.

[0025] In view of this, in order to meet the above needs, the present invention provides a composition using Sarcodia montagnei extract as an inhibitor of vesicular stomatitis virus and its uses. The Sarcodia montagnei extract contains an ingredient for inhibiting vesicular stomatitis virus and a polysaccharide active ingredient. The Sarcodia montagnei extract can be used for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the Sarcodia montagnei extract has no cytotoxicity, so as to improve the conventional fluorescent LAMP primers and their detection methods for detecting vesicular stomatitis virus or the technical method of RXR in screening drugs for treating vesicular stomatitis virus infection. Summary of the Invention

[0026] The main object of the present invention is to provide a composition using Sarcodia montagnei extract as an inhibitor of vesicular stomatitis virus and its uses. The Sarcodia montagnei extract contains an ingredient for inhibiting vesicular stomatitis virus and a polysaccharide active ingredient. The Sarcodia montagnei extract can be used for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the Sarcodia montagnei extract has no cytotoxicity, so as to achieve the purpose or effect of providing a composition for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus and enhancing the economic value of Sarcodia montagnei.

[0027] In order to achieve the above object, the use of the composition using Sarcodia montagnei extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention is for preparing a composition that can inhibit vesicular stomatitis virus, and it includes:

[0028] The Sargassum fusiforme extract is extracted from Sargassum fusiforme materials or a Sargassum fusiforme thallus, and the Sargassum fusiforme extract contains components that inhibit vesicular stomatitis virus, and the Sargassum fusiforme extract can be used for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the Sargassum fusiforme extract is not cytotoxic.

[0029] The Sargassum fusiforme extract of a preferred embodiment of the present invention has a concentration, and the concentration of the Sargassum fusiforme extract is positively correlated with the antiviral activity.

[0030] In order to achieve the above object, the composition using the Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention comprises:

[0031] At least one food raw material, which has a first content; and

[0032] At least one Sargassum fusiforme extract, which has a second content, and the Sargassum fusiforme extract is extracted from Sargassum fusiforme materials or a Sargassum fusiforme thallus, and the Sargassum fusiforme extract is added to the food raw material to form a food composition;

[0033] Wherein the Sargassum fusiforme extract contains components that inhibit vesicular stomatitis virus, and the Sargassum fusiforme extract can be used for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the Sargassum fusiforme extract is not cytotoxic.

[0034] The food composition of a preferred embodiment of the present invention is a beverage, food, sauce, seasoning powder, edible oil or dairy product.

[0035] In order to achieve the above object, the composition using the Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention comprises:

[0036] At least one health food raw material, which has a first content; and

[0037] At least one Sargassum fusiforme extract, which has a second content, and the Sargassum fusiforme extract is extracted from Sargassum fusiforme materials or a Sargassum fusiforme thallus, and the Sargassum fusiforme extract is added to the health food raw material to form a health food composition;

[0038] Wherein the Sargassum fusiforme extract contains components that inhibit vesicular stomatitis virus, and the Sargassum fusiforme extract can be used for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the Sargassum fusiforme extract is not cytotoxic.

[0039] In a preferred embodiment of the present invention, the health food raw material is selected from nutritional supplement raw materials, dietary supplement raw materials or health food raw materials to form nutritional supplements, dietary supplements, health foods or health foods.

[0040] In a preferred embodiment of the present invention, the raw materials of the health food are selected from powdery raw materials, paste-like raw materials or liquid raw materials.

[0041] In a preferred embodiment of the present invention, the Sargassum fusiforme thallus is selected from artificially cultured Sargassum fusiforme thallus or natural Sargassum fusiforme thallus.

[0042] In a preferred embodiment of the present invention, the Sargassum fusiforme extract is Sargassum fusiforme extraction essence liquid, Sargassum fusiforme extraction essential oil, Sargassum fusiforme extraction essence tablets, Sargassum fusiforme extraction essence granules, Sargassum fusiforme extraction essence powder or any combination thereof.

[0043] In a preferred embodiment of the present invention, the Sargassum fusiforme extract contains polysaccharide active ingredients. Brief Description of the Drawings

[0044] Figure 1 It is a schematic flow chart of a Sargassum fusiforme-added product made from a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the first preferred embodiment of the present invention.

[0045] Figure 2 It is a schematic flow chart of a Sargassum fusiforme-added product made from a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the second preferred embodiment of the present invention.

[0046] Figure 3 It is a schematic flow chart of a drug for inhibiting vesicular stomatitis virus made from a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the third preferred embodiment of the present invention.

[0047] Figure 4 It is a schematic diagram of the relative percentage of survival rate in the cytotoxicity test of a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the preferred embodiment of the present invention.

[0048] Figure 5 It is a schematic diagram of the relationship between the cell survival percentage and the concentration in the test of inhibiting vesicular stomatitis virus of a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the preferred embodiment of the present invention.

[0049] Figure 6 It is a schematic diagram of the relationship between the cell survival rate (percentage of fluorescence measured by spectrobrightness) and the concentration in the test of inhibiting vesicular stomatitis virus of a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the preferred embodiment of the present invention.

[0050] Figure 7 It is a schematic diagram of the relationship between the cell survival rate (fluorescence value measured by flow cytometer) and the concentration in the test of inhibiting vesicular stomatitis virus of a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus according to the preferred embodiment of the present invention.

[0051] Figure 8Schematic diagram of a fluorescence microscope image showing the infection status of a composition using Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention for testing the inhibition of vesicular stomatitis virus.

[0052] Figure 9 Schematic diagram of an image of an antiviral plaque assay using a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention.

[0053] Figure 10 Schematic diagram of the results of absorbance values of a cytotoxicity test using Sargassum fusiforme extract.

[0054] Figure 11 Results of the relative percentage of survival rate of a cytotoxicity test using Sargassum fusiforme extract.

[0055] Figure 12 Schematic diagram of the results of absorbance values of a cytotoxicity test using a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus.

[0056] Figure 13 Schematic diagram of the relative percentage of survival rate of a cytotoxicity test using a composition with Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention. Detailed Description of the Invention

[0057] To fully understand the present invention, the following preferred embodiments will be enumerated and described in detail in conjunction with the accompanying drawings, but it is not intended to limit the present invention.

[0058] First, in a preferred embodiment of the present invention, a composition using Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus and its uses can be applied to various pharmaceutical compositions (e.g., antiviral drugs), various animal pharmaceutical compositions, various medical supply compositions, various medical topical compositions, or various medical cleaning and disinfection supply compositions, but it is not intended to limit the application scope of the present invention.

[0059] Furthermore, in a preferred embodiment of the present invention, Sargassum fusiforme extract or a composition using Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus (e.g., freeze-dried powder materials, extract liquid materials, or other extracts) is added to various processed products, such as beverages, condiment sauces, seasoning powders, health foods, health care products, drugs, or related ointment products, but it is not intended to limit the scope of the present invention, but it is not intended to limit the scope of the present invention.

[0060] Generally speaking, vesicular stomatitis virus is a zoonotic disease that can not only infect even-toed ungulates, but also fish or other aquatic animals, thus causing significant economic losses to the livestock and aquaculture industries. Currently, there is no vaccine or treatment for vesicular stomatitis virus on the market, so the only means of prevention are usually isolation measures or sacrificing sick animals, and environmental disinfection.

[0061] As mentioned above, since there is no effective vaccine or therapeutic drug available on the market, this has a certain degree of negative impact on industries such as intensive livestock breeding and farming, and poses a certain degree of occupational risk to related practitioners. Therefore, the present invention provides methods for preventing or treating animals or related practitioners infected with vesicular stomatitis virus.

[0062] The present invention selects Sarcodia suae or Sarcodia sp. as edible seaweed, which is a species unique to Taiwan's waters. Its algae is dark red, thick and leathery, and has irregular forked branches. Its algae is about 5 to 15 centimeters when unfolded. The residents of Xiaoliuqiu originally collected Sarcodia suae at the seaside and blanched it for direct consumption. The preferred embodiment of the present invention uses artificial cultivation of Sarcodia suae (for example: water temperature 25±3°C, seawater salinity 32±3ppt or other appropriate temperature and salinity). The artificially cultivated algae will present a radially symmetrical spherical shape, and its extract is added to various processed products.

[0063] As mentioned above, generally speaking, sea fungus has been mostly used as food or materials for related products in the past, and sea fungus extract materials have only been found to have ferrous ion chelating ability, reducing ability, superoxide anion scavenging ability, DPPH free radical scavenging ability or other biological activities (for example: neuroprotection, promotion of bone formation or treatment of allergic dermatitis). The present invention uses sea fungus extract as a composition for inhibiting vesicular stomatitis virus, which has the function of resisting infection with vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus (as shown in Figures 5 to 9).

[0064] Embodiment 1 Sea fungus added product

[0065] Figure 1 This is a flow chart of using the Auricularia auricula extract as a composition for inhibiting vesicular stomatitis virus to prepare an Auricularia auricula-added product according to the first preferred embodiment of the present invention. Figure 1 As shown, for example, the first preferred embodiment of the present invention uses the sea fungus extract as a composition for inhibiting vesicular stomatitis virus and can be appropriately prepared into a sea fungus added product, and the sea fungus added product includes at least one food raw material 1a and at least one sea fungus extract 2, and the food raw material 1a (first predetermined amount) and the sea fungus extract 2 (second predetermined amount) are processed into a food composition 3a through processing equipment 100.

[0066] As Figure 1 shown, for example, the Sargassum fusiforme extract 2 can be selected from a low-temperature Sargassum fusiforme extract, a solvent extract, or an extract obtained by other appropriate technical means. The Sargassum fusiforme extract 2 can be selectively extracted from a Sargassum fusiforme material 21 or a Sargassum fusiforme thallus 22, and the Sargassum fusiforme extract 2 contains a polysaccharide active ingredient or other effective bioactive ingredients (for example, rich in protein or lipid), that is, it has components that inhibit vesicular stomatitis virus (as shown in Figures 5 to 9), and the Sargassum fusiforme extract 2 has no cytotoxicity (as shown in Figure 4).

[0067] As Figure 1 shown, for example, the Sargassum fusiforme extract 2 can be selected as a Sargassum fusiforme extraction essence, a Sargassum fusiforme extraction essential oil, a Sargassum fusiforme extraction essence tablet, a Sargassum fusiforme extraction essence granule, a Sargassum fusiforme extraction essence powder, or any combination thereof.

[0068] As Figure 1 shown, for example, using a processing device 100 (or other devices with similar functions) to add the Sargassum fusiforme extract 2 to the food raw material 1a by appropriate technical means, and the Sargassum fusiforme extract 2 can be selectively extracted from a Sargassum fusiforme body, Sargassum fusiforme fragments, Sargassum fusiforme powder, or any combination thereof.

[0069] As Figure 1 shown, for example, the food composition 3a can be selected as a beverage, a food, a sauce, a seasoning powder, an edible oil product, a dairy product, other food products (for example: various snack products, various bread products, or various western pastry products), or other food processing products (for example: various meat products).

[0070] Preparation of the Sargassum fusiforme - added product in Example 2

[0071] Figure 2 This is the second preferred embodiment of the present invention, which is a flowchart of making a Sargassum fusiforme - added product with the Sargassum fusiforme extract as a composition for inhibiting vesicular stomatitis virus. As Figure 2 shown, compared with the first embodiment, the Sargassum fusiforme - added product of the second preferred embodiment of the present invention contains at least one health food raw material 1b and at least one Sargassum fusiforme extract 2, and the health food raw material 1a (the first predetermined amount) and the Sargassum fusiforme extract 2 (the second predetermined amount) are made into a health food composition 3b.

[0072] As Figure 2 shown, for example, the health food raw material 1b can be selected from a nutritional supplement raw material, a dietary supplement raw material, or a health food raw material to make a nutritional supplement, a dietary supplement, a health food, a health food, or other products.

[0073] As Figure 1 and2 As shown in the figure, for example, the food raw material 1a or the health food raw material 1b can be selected from a powdery raw material, a paste raw material, a liquid raw material or any mixture thereof, and the powdery raw material, the paste raw material or the liquid raw material can be selected according to different requirements.

[0074] Preparation of the drug for inhibiting vesicular stomatitis virus in Example 3

[0075] Figure 3 The flowchart shows the preparation of the drug for inhibiting vesicular stomatitis virus using the extract of Sargassum fusiforme as a composition for inhibiting vesicular stomatitis virus in the third preferred embodiment of the present invention. As Figure 3 shown, compared with Example 1, the composition for inhibiting vesicular stomatitis virus in the third preferred embodiment of the present invention contains at least one extract of Sargassum fusiforme 2, and the extract of Sargassum fusiforme 2 is made into a composition 3c for inhibiting vesicular stomatitis virus (VSV) (such as: drugs for human use or drugs for animal use) by appropriate technical means.

[0076] Extraction of Sargassum fusiforme in Example 4

[0077] As Figure 1 、 2 and 3 shown, for example, the preferred embodiment of the present invention adopts the extraction method of Sargassum fusiforme: the thallus of Sargassum fusiforme and deionized water are configured in a container (such as: conical flask) at a ratio of 1:20, and water bath is carried out at 40 °C or other appropriate temperature for a predetermined time (such as: 3 hours), and shaken once every predetermined time (such as: 15 to 30 minutes) to obtain a preliminary extract of Sargassum fusiforme; then, the preliminary extract of Sargassum fusiforme is centrifuged at 4 °C or other appropriate temperature for a predetermined time (such as: 10 minutes) to obtain a centrifuged extract; then, a supernatant is taken out from the centrifuged extract, and the supernatant is freeze-dried.

[0078] The extract of Sargassum fusiforme adopted in the preferred embodiment of the present invention has a total carbohydrate content of about 81.4%, its monosaccharides are about 20.9% and polysaccharides are 60.5%, and the polysaccharides have 83.2% galactose and 16.8% xylose. The extract of Sargassum fusiforme adopted in the preferred embodiment of the present invention also has other substances (such as: 5.0% sulfate), but it does not limit the scope of the present invention.

[0079] Cytotoxicity and inhibition of vesicular stomatitis virus test of the extract of Sargassum fusiforme in Example 5

[0080] Figure 4Schematic diagram of the relative percentage of cell viability in the cytotoxicity test of the composition using Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention. Referring to Figure 4, the abscissa in the figure is the concentration of the Sargassum fusiforme extract, and the ordinate is the percentage of cell viability. For example, the tissue control (TC) is the cells not treated with the Sargassum fusiforme extract, that is, the TC is 100% cell viability. After the Mv-1-Lu cells (American mink lung epithelial cells) were treated with the Sargassum fusiforme extract for two days, the cell viability was measured by MTT assay at 560 nm using a spectrophotometer, and it showed that the Sargassum fusiforme extract had no obvious toxicity to the cells.

[0081] Figure 5 Schematic diagram of the relationship between cell survival percentage and concentration in the test of inhibiting vesicular stomatitis virus of the composition using Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention. As Figure 5 shown, the abscissa in the figure is the concentration of the Sargassum fusiforme extract, and the ordinate is the percentage of cell viability. The viral control (VC, only cells + virus are added, so all cells are infected) has a survival rate of 0%, while the TC is 100%. The Mv1Lu cells were treated with the Sargassum fusiforme extract (SSWE) one hour before and after infection, and the cell viability after infection was tested by MTT assay two days later. It showed that it had the activity against VSV infection, and the antiviral activity was significantly improved with the increase in the concentration of the extract, showing a dose-dependent manner, that is, the higher the concentration, the more effective it was in inhibiting VSV infection, and it still had a cell viability of more than 50% at a concentration of 0.625 mg / mL or above.

[0082] As Figure 1 、 2 、3, 4 and 5 shown, for example, the Sargassum fusiforme extract 2 can be selected to have a predetermined concentration, and the predetermined concentration of the Sargassum fusiforme extract 2 is positively correlated with an antiviral activity, and the Sargassum fusiforme extract 2 not only has no cytotoxicity, but can also significantly improve the cell viability of cells infected with vesicular stomatitis virus.

[0083] Figure 6 Schematic diagram of the relationship between cell viability (percentage of fluorescence measured by spectrophotometer) and concentration in the test of inhibiting vesicular stomatitis virus of the composition using Sargassum fusiforme extract as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention. As Figure 6As shown, for example, after Mv1Lu cells were infected with VSV, the fluorescence value of green fluorescent protein (GFP) in the cells was quantified using a spectrophotometer, and the absorbance value of GFP in the virus control group (VC) at a wavelength of 450 nm was set as 100%, and a relative fluorescence percentage graph was shown. Figure 6 In the figure, the abscissa is the concentration of the extract containing Sargassum fusiforme, and the ordinate is the percentage of green fluorescent protein. It shows that the extract of Sargassum fusiforme (SSWE) has significant anti-VSV infection characteristics at a concentration of 1 mg / mL or more, and its fluorescence is very low.

[0084] Figure 7 This is a schematic diagram showing the relationship between the cell viability (fluorescence value measured by flow cytometry) and the concentration of the composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus in the inhibition test of vesicular stomatitis virus in a preferred embodiment of the present invention. As Figure 7 shown, for example, after Mv1Lu cells were infected with VSV (MOI = 0.1), flow cytometry was used to analyze the cells in the cell population that were judged to be infected when they carried GFP fluorescence, and different concentrations of the extract of Sargassum fusiforme were analyzed after treatment. It shows that the extract of Sargassum fusiforme (SSWE) has the ability to inhibit VSV infection, and the effect is more obvious with the increase of concentration (dose dependent). Under the treatment of the highest concentration of 10 mg / mL of the extract of Sargassum fusiforme (SSWE) (two peaks shown in Figure 7), VSV infection can be effectively inhibited, and most cells are not infected (no fluorescence), and nearly 60% of the infection can be significantly improved, and the infection rate can be reduced by about 60%.

[0085] Figure 8 This is a schematic diagram of a fluorescence microscope image showing the infection status of the composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention. As Figure 8 shown, for example, after Mv1Lu cells were infected with VSV (MOI = 0.1), the virus control group (VC) was not treated with the extract of Sargassum fusiforme, and its fluorescence was the strongest. Under the treatment of the extract of Sargassum fusiforme (SSWE) at a concentration of 10 mg / mL, VSV infection can be effectively prevented (almost no fluorescence signal), and its fluorescence is the lowest; under the treatment of 5 mg / mL, about half of the cells were infected; although the infection area is larger at 2.5 mg / mL, its fluorescence level is relatively lower than that below 1 mg / mL, indicating that the infection is lower than that at a concentration below 1 mg / mL, and the infection degree is similar to that of the virus control group (VC) below 1 mg / mL.

[0086] Figure 9 This is a schematic diagram of the image of the antiviral plaque test of the composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus in a preferred embodiment of the present invention. AsFigure 9 As shown, for example, after Mv1Lu cells were infected with VSV (MOI = 0.1) and treated with Sargassum fusiforme extract (SSWE), plaque assay analysis showed that the ability of Sargassum fusiforme extract to inhibit new virus particles was also dose-dependent. It not only inhibited RNA replication of VSV in cells but also inhibited the production of virus particles, that is, the assembly of viral proteins.

[0087] Example 6

[0088] The inventors' research found that when Sargassum fusiforme was extracted with 100°C hot water, the resulting extract contained a high amount of galactose, was not easily soluble in water after rehydration, presented a sticky paste, could not be sterilized and diluted, and could not be formulated into an aqueous solution with a higher concentration, such as 10 mg / mL.

[0089] When Sargassum fusiforme was extracted with 40°C water, the resulting extract contained a lower amount of galactose, was soluble in water after rehydration, could be sterilized by membrane filtration and diluted, and could be formulated into an aqueous solution with a higher concentration.

[0090] SSWE 100C refers to the Sargassum fusiforme extract extracted with 100°C hot water, that is, a water bath was carried out at 100°C, and other operations were the same as in Example 4.

[0091] Such as Figure 10 and Figure 11 It means that Mv-1-Lu cells were cultured with the Sargassum fusiforme extract SSWE 100C extracted with 100°C hot water. In the figure, the abscissa is the concentration of the Sargassum fusiforme extract, and the ordinate is the absorbance at 560 nm and the percentage of cell viability. The cell control group (tissue control, TC) was Mv-1-Lu cells without treatment with the Sargassum fusiforme extract, that is, the cell viability of TC was 100%. After Mv-1-Lu cells were treated with the Sargassum fusiforme extract for two days, the cell viability was measured by MTT assay at 560 nm with a spectrophotometer, and it showed that the Sargassum fusiforme extract-containing had no obvious toxicity to cells.

[0092] Figure 12 and Figure 13 It is a schematic diagram showing the relationship between the absorbance value at 560 nm and the percentage of cell survival and the concentration of the Sargassum fusiforme extract SSWE 100C extracted with 100°C hot water as a composition for inhibiting vesicular stomatitis virus in the test for inhibiting vesicular stomatitis virus. Such as Figure 12 As shown, the abscissa in the figure is the concentration of the Sargassum fusiforme extract, and the ordinate is the absorbance value at 560 nm. Such as Figure 13As shown, the abscissa in the figure is the concentration of the extract of Sargassum fusiforme, and the ordinate is the percentage of cell survival rate. The virus control group (only the virus is added, no cells, so there is no cell infection) is 0%, while the TC is the tissue control group (tissue control, TC), where the cells are not treated with the extract of Sargassum fusiforme and not treated with the virus, that is, the TC has a cell survival rate of 100%. The Mv1Lu cells were treated with the extract of Sargassum fusiforme SSWE 100C extracted with hot water at 100 °C one hour before and after infection, and the cell survival rate after infection was tested by the MTT assay two days later, which showed that it had no activity against VSV infection. Figure 13 Among them, only the concentration of 10 mg / mL had a cell survival rate of less than 10%, and the cells at the other concentrations did not survive.

[0093] The above experimental data are the preliminary experimental results obtained under specific conditions, which are only used to facilitate the understanding or reference of the technical content of the present invention, and other relevant experiments still need to be carried out. The experimental data and its results are not used to limit the scope of the rights of the present invention.

[0094] The foregoing preferred embodiments only illustrate the present invention and its technical features, and the technology of the embodiments can still be appropriately implemented by various substantially equivalent modifications and / or replacement methods; therefore, the scope of the rights of the present invention shall be subject to the scope defined by the claims of this application.

Claims

1. Use of an extract of Sargassum fusiforme as a composition for inhibiting vesicular stomatitis virus, for preparing a composition capable of inhibiting vesicular stomatitis virus, comprising: The extract of Sargassum fusiforme is extracted from Sargassum fusiforme material or thallus, and the extract of Sargassum fusiforme contains components for inhibiting vesicular stomatitis virus, and the extract of Sargassum fusiforme is used as an application for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the extract of Sargassum fusiforme is not cytotoxic.

2. The composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus according to claim 1, wherein the extract of Sargassum fusiforme has a certain concentration, and the concentration of the extract of Sargassum fusiforme is positively correlated with an antiviral activity.

3. A composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus, comprising: At least one food raw material, having a first content; and At least one extract of Sargassum fusiforme, having a second content, and the extract of Sargassum fusiforme is extracted from Sargassum fusiforme material or thallus, and the extract of Sargassum fusiforme is added to the food raw material to make a food composition; Wherein the extract of Sargassum fusiforme contains components for inhibiting vesicular stomatitis virus, and the extract of Sargassum fusiforme can be used as an application for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the extract of Sargassum fusiforme is not cytotoxic.

4. The composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus according to claim 3, wherein the food composition is a beverage, food, sauce, seasoning powder, edible oil or dairy product.

5. A composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus, comprising: At least one health food raw material, having a first content; and At least one extract of Sargassum fusiforme, having a second content, and the extract of Sargassum fusiforme is extracted from Sargassum fusiforme material or thallus, and the extract of Sargassum fusiforme is added to the health food raw material to make a health food composition; Wherein the extract of Sargassum fusiforme contains components for inhibiting vesicular stomatitis virus, and the extract of Sargassum fusiforme can be used as an application for anti-infecting vesicular stomatitis virus and inhibiting the activity of vesicular stomatitis virus, and the extract of Sargassum fusiforme is not cytotoxic.

6. The composition using the extract of Sargassum fusiforme as an inhibitor of vesicular stomatitis virus according to claim 5, wherein the health food raw material is selected from raw materials for nutritional supplements, dietary supplements or health food raw materials to make nutritional supplements, dietary supplements, health foods or health foods.

7. The composition for inhibiting vesicular stomatitis virus using Sargassum fusiforme extract according to claim 5, wherein the health food raw material is selected from powdery raw materials, paste-like raw materials or liquid raw materials.

8. The composition for inhibiting vesicular stomatitis virus using Sargassum fusiforme extract according to claim 1, 3 or 5, wherein the Sargassum fusiforme thallus is selected from artificially cultured Sargassum fusiforme thalli or natural Sargassum fusiforme thalli.

9. The composition for inhibiting vesicular stomatitis virus using Sargassum fusiforme extract according to claim 1, 3 or 5, wherein the Sargassum fusiforme extract is Sargassum fusiforme extraction essence, Sargassum fusiforme extraction essential oil, Sargassum fusiforme extraction essence tablets, Sargassum fusiforme extraction essence granules, Sargassum fusiforme extraction essence powder or any combination thereof.

10. The composition for inhibiting vesicular stomatitis virus using Sargassum fusiforme extract according to claim 5, wherein the Sargassum fusiforme extract contains polysaccharide active ingredients.

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