TWI755725B - Bioactive components of phellinus linteus mycelia and its use in preparation of antiviral composition against enterovirus type 71 - Google Patents

Abstract

The present invention relates to a bioactive component of Phellinus linteus mycelia, which can be produced by inoculating Phellinus linteus mycelia into a solid-state culture, a liquid-state culture, a liquid fermentation culture and an extraction. The extract product of Phellinus linteus mycelia includes the bioactive components against enterovirus type 71 infections, thereby be applied to a use in the preparation of an antiviral composition against enterovirus type 71.

Description

Phellinus linteus mycelium biologically active substance and its use in the preparation of anti-enterovirus type 71 disease resistance Use of toxic compositions

The present invention relates to a fungal mycelium active substance, its preparation method and its use, in particular to a Phellinus linteus mycelium bioactive substance, its preparation method and its use for preparing an antiviral composition.

Virus is a non-cellular form composed of a nucleic acid molecule (DNA or RNA) and protein, which is between living things and non-living things. At present, it is not classified as one of the five kingdoms (prokaryotes, protists, fungi, plants and animals). It is an organic species between living and non-living organisms, which cannot express life phenomena on its own, and must rely on parasitism to survive and reproduce. The viral structure is primarily a stretch of DNA or RNA wrapped in a protective shell. Through the mechanism of infection, these simple organisms can utilize the host's cellular system to replicate themselves, but are unable to grow and replicate independently.

Viruses are made up of two to three components: Viruses all contain genetic material (RNA or DNA); they also have capsids made of proteins that wrap and protect the genetic material within them; in addition, some viruses can reach the cell surface. A lipid envelope is formed around it. Viruses come in a variety of shapes, from simple helical and icosahedral shapes to complex structures. Virus particles are about one-thousandth the size of bacteria.

Most viruses cause disease in an organism, but not all, and many viruses replicate without appreciable harm to an infected organ. A small number of viruses, such as HIV, can coexist with the human body for a long time and remain infectious without being affected by the host's immune system, known as "viral persistence". But under normal circumstances, viral infections trigger an immune response that destroys the invading virus. These immune responses can be produced by injecting vaccines, so that the vaccinated human or animal can be immune to the corresponding virus for life. Different viruses have different pathogenic mechanisms, mainly depending on the type of virus. At the cellular level, the main destructive effect of the virus is to cause cell lysis and thus cell death. In multicellular organisms, once enough cells in the body die, there are consequences for the body's health. Although viruses can cause disease, they can also exist harmlessly in the body. For example, the herpes simplex virus, which causes cold sores, can remain dormant in the body; this state is also known as "latency," which is characteristic of all herpes viruses. dive into The varicella-zoster virus in a swollen state can cause shingles after it "wakes up".

Enterovirus 71 (EV71) is a single-stranded RNA virus and one of the causative agents of hand, foot and mouth disease. EV71 infection is common in Southeast Asia, mostly in summer and early autumn. Outbreaks have occurred in Australia, Mainland China, Malaysia, Singapore, Taiwan, etc. EV71 infection is more common in young children. Symptoms are generally the same as those of hand, foot and mouth disease, including fever, painful sores in the mouth, and a rash with blisters. Symptoms such as fever, loss of appetite, tiredness or sore throat are usually the first symptoms of illness. One to two days after a fever, painful blisters may appear in the mouth. These blisters start as small red spots and then develop into ulcers. Ulcers are usually located on the tongue, gums, and the inside of the cheeks of the mouth. In addition, a rash that is not itchy and sometimes has small blisters can also appear on the palms and soles, and even on the buttocks and/or genitals. HFMD patients may also have no symptoms, or only develop symptoms such as rashes or mouth ulcers. EV71 can cause more serious conditions including viral meningitis, encephalitis, polio-like paralysis and myocarditis. At present, there is no vaccine to prevent EV71 infection, and in terms of drug development, there is currently no, and there is currently no effective drug to treat EV71. Only a few drugs can relieve pain caused by fever and ulcers. Therefore, we are looking for effective drugs to prevent/treat EV71 necessary.

Dengue fever is an acute infectious disease caused by the dengue virus, which is spread by mosquitoes to humans. And it is divided into four serotypes: I, II, III, and IV, and each type has the ability to infect and cause disease. A patient infected with a certain type of dengue virus will have lifelong immunity to that type of virus, but only temporary immunity to other types of dengue virus, and it is still possible to re-infect other serotype viruses. Clinically, repeated infection with different types of dengue virus can cause different degrees of host response, ranging from mild or insignificant symptoms, to typical dengue fever with fever and rash, or warning signs such as lethargy, restlessness, and liver enlargement, and even Severe dengue fever that can cause severe bleeding or severe organ damage. The prone areas of dengue fever in the world are mainly concentrated in tropical and subtropical countries where Aedes aegypti and Aedes albopictus are distributed. , Dengue fever also began to spread to various countries, becoming a serious public health problem. Taiwan is located in a subtropical region, and a slightly hot and humid environment like this is the favorite growth environment for mosquitoes, and it is a high-risk area for dengue fever. Therefore, the demand for safe vaccines in various countries is very urgent. The dengue vaccine has been studied since the 1970s, but the progress has been slow. Until recently, four types of live attenuated vaccines have entered human trials, but many problems have been found in the vaccine trials, including (1) side effects: single The side effects of the type vaccines are mild, but the side effects of the four types of vaccines are more frequent and more serious. (2) Competitive interference phenomenon: The replication speed of type 3 dengue virus was obviously faster than that of type 1 and type 2 dengue viruses when the vaccine was made. The production of antibodies is mainly type 3, and the protective power is insufficient. Therefore, it is necessary to find effective drugs to prevent/treat dengue fever.

The Chinese name of Phellinus linteus belongs to the genus Phellinus of the family Hymenochaetaceae. Phellinus sinensis grows on the trunks of mulberry plants, also known as mulberry or mulberry. Li Shizhen's "Compendium of Materia Medica" in the Ming Dynasty recorded that it is cold in nature, slightly bitter in taste, can benefit the five internal organs, dispel intestinal qi, detoxify qi or stop bleeding, etc. It is commonly used in traditional Chinese medicine for diuresis, stomach strengthening or diarrhea. Modern pharmacological research and analysis have found that Phellinus linteus has the effects of immune regulation, anti-inflammatory, antioxidant, anti-microbial, anti-virus, and anti-cancer. In terms of the antiviral benefits of fungi, most of them are to enhance the consumer's own immunity to resist viruses, and it cannot really represent the benefits of drugs against viral infection when the virus invades the body.

Therefore, one aspect of the present invention is to provide the use of a Phellinus linteus mycelium bioactive substance for preparing an antiviral composition against enterovirus 71, which uses Phellinus Phellinus mycelium bioactive substance as an active ingredient .

Another aspect of the present invention is to provide an oral composition with antiviral efficacy against enterovirus 71, comprising an in vitro effective dose of Phellinus linteus mycelium bioactive substances.

According to the above aspect of the present invention, there is provided the use of a Phellinus linteus mycelium bioactive substance for preparing an antiviral composition against enterovirus 71, wherein the Phellinus Phellinus mycelium bioactive substance is an active ingredient. The aforementioned Phellinus linteus is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China Consortium, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is July 18, 2019, and the deposit number is BCRC 930210).

According to an embodiment of the present invention, the in vitro effective dose of the above-mentioned Phellinus linteus mycelium bioactive substance may be, for example, 250 μg/mL to 1000 μg/mL.

According to an embodiment of the present invention, the above-mentioned Phellinus Phellinus mycelium bioactive substances may include but are not limited to Phellinus Phellinus mycelium fermentation liquid, Phellinus Phellinus mycelium freeze-dried product, Phellinus Phellinus mycelium freeze-dried powder, Phellinus Phellinus mycelium Filament extract, Phellinus linteus mycelium extract and any combination of the above.

According to an embodiment of the present invention, the above-mentioned Phellinus linteus mycelium extract may include, but is not limited to, water extract, ethanol extract, dichloromethane extract, hexane extract and any combination of the above.

According to an embodiment of the present invention, the above-mentioned antiviral composition further includes a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.

According to an embodiment of the present invention, the above-mentioned antiviral composition can be, for example, an oral composition.

According to an embodiment of the present invention, the above-mentioned antiviral composition can be administered to cells in vitro, for example, before, during or after infection of the cells by the virus.

According to an embodiment of the present invention, the above viruses may include but are not limited to enterovirus type 71 (EV71) and dengue virus type 2 (DENV-2).

According to another aspect of the present invention, an oral composition with antiviral efficacy is provided, comprising the above-mentioned bioactive substance from Phellinus linteus mycelium, wherein the in vitro effective dose of the bioactive substance from Phellinus Phellinus mycelium can be, for example, 250 μg/ mL to 1000 μg/mL.

According to an embodiment of the present invention, the above-mentioned Phellinus Phellinus mycelium bioactive substances may include but are not limited to Phellinus Phellinus mycelium extract, Phellinus Phellinus mycelium extract and any combination of the above, and Phellinus Phellinus mycelium extract It may also include, but is not limited to, water extracts, ethanol extracts, dichloromethane extracts, hexane extracts, or any combination of the above.

Using the bioactive substance of Phellinus linteus mycelium of the present invention, it has the effect of anti-viral infection, and can be used as an active ingredient and applied to prepare an anti-viral composition.

In order to make the above-mentioned and other purposes, features, advantages and embodiments of the present invention more obvious and easy to understand, the detailed description of the accompanying drawings is as follows: [Fig. Bioactive substances prevent the viability of cells treated with EV71 virus after treatment of vero cells.

[Fig. 2] shows the cell viability after co-cultivation of Phellinus linteus mycelium bioactive substances, EV71 virus and vero cells according to an embodiment of the present invention.

Fig. 3 shows the cell survival rate of EV71 virus firstly treating vero cells and then adding the bioactive substance of Phellinus linteus according to an embodiment of the present invention.

[Fig. 4] is a graph showing the cell viability after BHK-21 cells are treated with the bioactive substance of Phellinus linteus mycelium according to an embodiment of the present invention, and then DENV-2 virus is added.

[Fig. 5] is a graph showing the cell viability after co-culture treatment of Phellinus linteus mycelium, DENV-2 virus and BHK-21 cells according to an embodiment of the present invention.

[Fig. 6] shows the cell viability of BHK-21 cells treated with DENV-2 virus first and then adding the bioactive substance of Phellinus linteus mycelium according to an embodiment of the present invention.

As mentioned above, the present invention provides a biologically active substance of Phellinus linteus mycelium, its manufacturing method and its use for preparing an antiviral composition.

The present invention further provides a method for producing a bioactive substance of Phellinus linteus mycelium, which comprises the steps of culturing the Phellinus linteus mycelium in multiple stages to obtain a fermentation broth, wherein the fermentation broth contains Phellinus linteus Yellow mycelium bioactive substances. The biologically active substances mentioned here refer to the mycelium of Phellinus linteus and/or its derivatives and other inseparable substances.

The present invention provides a bioactive substance of Phellinus linteus mycelium, a method for producing the same, and its use for preparing an antiviral composition. Propositionally, the Phellinus linteus referred to here belongs to the genus Phellinus of the family Hymenochaetaceae , and the Chinese name is Cleft hoof Xyloporus, especially referring to July 18, 2019 The strain is deposited in the Biological Resource Center (BCRC) of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan, and the deposit number is BCRC 930210.

In one embodiment, the manufacturing method of the above-mentioned Phellinus linteus mycelium bioactive substance may include, for example, the following steps. First, step (a) is performed, which is to take Phellinus linteus mycelia for solid-state culture. In an example, the aforementioned solid-state culture can Phellinus mycelium is inoculated on a plate medium, such as a potato dextrose agar (Potato Dextrose Agar, PDA) medium, and the solid-state culture is performed at a temperature of 15°C to 30°C for 1 to 2 weeks. . If the temperature of step (a) is outside the aforementioned range, the growth of mycelium will be inhibited. If the incubation time of step (a) is less than 1 week, the quality of mycelium will be insufficient. In addition, when the culturing time of step (a) reaches 2 weeks, the mycelium has grown completely, so there is no need to cultivate for more than 2 weeks.

Next, step (b) is carried out, which is step (a) solid-state culture The cultured Phellinus linteus mycelium is inoculated into a liquid medium, and the liquid culture is carried out at a temperature of 15°C to 30°C for 3 to 14 days. In an example, step (b) can optionally be supplemented with shaking culture at a rotational speed of 110 rpm to 130 rpm. If the culture temperature of step (b) exceeds the aforementioned range, the growth of mycelium will be inhibited. If the incubation time of step (b) is less than 3 days, the quality of mycelium will be insufficient. If the culturing time of step (b) exceeds 14 days, it will not help or even inhibit the growth of mycelium. In an example, the pH value of the liquid medium is preferably pH 2 to 6. If the pH value of the liquid medium exceeds the aforementioned range, the mycelium will grow poorly.

In one example, the aforementioned liquid culture medium may include, for example, 1% to 3% by weight of a comprehensive carbon and nitrogen source (such as cereals and/or beans), 1% to 4% by weight of carbohydrates (such as monosaccharides and/or disaccharide), 0.1 to 1 wt% yeast extract, 0.1 to 1 wt% protease, and 0.01 to 0.05 wt% inorganic salts such as phosphates and/or sulfates. It should be added that the aforementioned liquid culture medium is not limited to those listed above, and in other examples, the composition and content of the liquid culture medium can also be appropriately adjusted according to requirements.

Afterwards, carry out step (c), it is to inoculate the Phellinus linteus mycelium after the step (b) cultivation in the fermentation medium, carry out liquid fermentation culture with the temperature of 15 ℃ to 30 ℃ and reach 3 days to 21 days, Obtain Phellinus linteus mycelium fermented liquid thereby, and it is containing biological activity quality. In an example, the liquid medium of step (b) can be used as the fermentation medium in step (c), or the composition and content of the fermentation medium can be appropriately adjusted as required.

In an example, step (c) can be carried out in a conventional fermentation tank, wherein the fermentation tank can selectively introduce a gas, such as air, oxygen, carbon dioxide, helium or any combination of the above, the fermentation tank. The tank pressure may be, for example, 0.5 to 1.0 kg/cm ² , and the aeration rate of the fermentation tank may be, for example, 0.01 to 1.5 VVM.

Here, if the culture temperature of step (c) exceeds the aforementioned range, the growth of mycelium will be inhibited. If the incubation time of step (c) is less than 3 days, the quality of mycelium will be insufficient. If the culture time of step (c) exceeds 14 days, the growth of mycelium is not helped or even inhibited. In one example, the pH value of the fermentation medium is preferably pH 2 to 6. If the pH value of the fermentation medium exceeds the above-mentioned range, the mycelium will not grow well.

In one embodiment, the manufacturing method of the above-mentioned Phellinus Phellinus mycelium biologically active substance can optionally include step (d), which is to freeze-dry the Phellinus Phellinus mycelium fermentation liquid in step (c) and then grind it. into powder to obtain Phellinus linteus mycelium freeze-dried powder, wherein the Phellinus linteus mycelium freeze-dried powder also contains biologically active substances.

In one embodiment, the manufacturing method of the above-mentioned Phellinus Phellinus mycelium biologically active substance can optionally include step (e), which is to extract the Phellinus Phellinus mycelium freeze-dried powder of step (d) with at least one solvent, by The Phellinus Phellinus mycelium extract is obtained, wherein the Phellinus Phellinus mycelium extract also contains biologically active substances. In one example, suitable solvents for step (e) may include, but are not limited to, water, lower alcohols (eg, methanol, ethanol, propanol, isopropanol, etc.), dichloromethane, and/or hexane, for example. In one embodiment, the step (e) can be performed by a conventional extraction method, such as, but not limited to, solvent extraction method, and optionally combined with heating or shaking, but the present invention is not limited to the above.

In one embodiment, the manufacturing method of the above-mentioned Phellinus Phellinus mycelium bioactive substance can optionally include step (f), which is to dry the Phellinus Phellinus mycelium extract in step (e) to obtain biological activity. substance. The aforementioned drying methods can be carried out by conventional methods, such as freeze drying method, vacuum drying method or spray drying method, etc., but are not limited to those mentioned here.

The present invention also provides a kind of Phellinus Phellinus mycelium biologically active substance, which is prepared by the above method, which can include but not limited to Phellinus Phellinus mycelium fermented liquid (containing mycelium and clarified liquid), Phellinus Phellinus mycelium Lyophilisate, lyophilized powder of Phellinus linteus mycelium, Phellinus linteus mycelium extract (eg, water extract, ethanol extract, dichloromethane extract, hexane extract) and any combination of the above. After the above-mentioned Phellinus Phellinus mycelium fermented liquid, Phellinus Phellinus mycelium freeze-dried product and/or Phellinus Phellinus mycelium freeze-dried powder can increase the concentration of Phellinus Phellinus mycelium biologically active substances.

In one embodiment, the above-obtained Phellinus linteus mycelium bioactive substance is confirmed to have antiviral efficacy after evaluation by in vitro tests, and can be further applied to prepare antiviral compositions of various dosage forms. exist In one example, the above-mentioned composition can be, for example, but not limited to, a pharmaceutical composition or a food additive. In this example, the above-mentioned composition may optionally further comprise pharmaceutically or food acceptable carriers, excipients, diluents, adjuvants and/or additives, such as but not limited to solvents, emulsifiers, suspending agents , disintegrants, binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants and/or absorption delaying agents, etc.

The dosage form of the composition of the present invention is not particularly limited. In one embodiment, the dosage form of the composition can be, for example, but not limited to, an aqueous solution, a suspension, a dispersion, an emulsion (single-phase or multi-phase dispersion system, single- or multi-lamellar liposomes), hydrocolloids, gels, solid lipids Nanoparticles, lozenges, granules, powders and/or capsules, etc.

The aforementioned food composition can be, for example, but not limited to, cereal products, fruit products, vegetable products, meat products, fish products, egg products, dairy products, beverages, health food, health food, functional food, nutritional supplements food or special nutritional food.

It is supplemented that, compared with conventional western medicine or synthetic compound composition, the present invention uses the Phellinus linteus mycelium obtained by liquid fermentation to be more natural, safer and more convenient, and the obtained Phellinus linteus mycelium is fermented and cultivated. The liquid contains natural and safe biologically active substances, which have been confirmed by in vitro tests to be effective against virus-infected cells, especially against enterovirus type 71 (EV71) and dengue virus type 2 (dengue virus type 2, DENV-2).

Several embodiments are used below to illustrate the application of the present invention, but they are not intended to limit the present invention. Those with ordinary knowledge in the technical field of the present invention can make various changes and modifications without departing from the spirit and scope of the present invention. retouch.

Example 1: Preparation of Phellinus linteus mycelium freeze-dried powder and biologically active substances 1. Preparation of Phellinus linden mycelium lyophilized powder

First, the mycelium of Phellinus linteus (deposited at the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute of the Republic of China Foundation, No. 331, Food Road, Hsinchu, Taiwan, the deposit date is July 18, 2019, and the deposit number is BCRC 930210). It was inoculated on potato dextrose agar (Potato Dextrose Agar, PDA) plate medium and cultured at 25°C for 7 days. Then, take part of Phellinus linteus mycelium and inoculate it in liquid medium (containing 1% by weight of comprehensive carbon and nitrogen sources, 1.5% by weight of carbohydrates, 0.3% by weight of yeast extract, 0.3% by weight of protein gluten and 0.05% by weight of % inorganic salts, pH 5) in liquid culture at 25°C and 120 rpm for 7 days. The above-mentioned comprehensive carbon and nitrogen sources are cereals (wheat flour and/or bran flour) and/or beans (soybean flour, mung bean flour, soybean flour and/or cinnamon flour). The above sugars are monosaccharides (glucose and/or fructose) and/or disaccharides (maltose and/or sucrose). The above-mentioned inorganic salts are phosphates (dipotassium hydrogen phosphate, potassium dihydrogen phosphate) and/or sulfates (magnesium sulfate and/or iron sulfate).

Then, get the Phellinus linteus mycelium of part liquid culture and inoculate in the fermenter tank containing fermented medium (composition is identical with liquid medium, pH 5), with 25 ℃, 0.5kg/cm ² air pressure, 1.0VVM air The aeration rate and the rotational speed of 80 rpm were carried out for liquid fermentation for 14 days, thereby obtaining Phellinus linteus fermented liquid.

After that, take Phellinus linteus fermented liquid and carry out freeze-drying to obtain Phellinus linteus mycelium freeze-dried product.

2. Preparation of Phellinus Phellinus Mycelium Bioactive Substances

The lyophilized powder of Phellinus linteus mycelium in Example 1 was divided into two parts, and 20 times the weight of distilled water/ethanol/dichloromethane/hexane was added for extraction. Regarding the extract of Phellinus linteus mycelium freeze-dried powder with water as solvent, it was heated at 100°C for 30 minutes, and after cooling, the supernatant was taken by centrifugation, and dried by freeze-drying to obtain Phellinus linteus yeast mycelium water. Extract.

About the extract of Phellinus linteus mycelium freeze-dried powder with ethanol/dichloromethane/hexane as solvent, then ultrasonically shake for 1 hour, take the supernatant after centrifugation, and concentrate the supernatant under reduced pressure to obtain Phellinus linteus mycelium ethanol extract (EtOH)/dichloromethane extract (PI DCM)/hexane extract (PI Hex).

The following Example 2 and Example 3 evaluate the water extract (PI H2O), ethanol extract (EtOH), dichloromethane extract (PI DCM), hexane extract (PI) of the bioactive substances of Phellinus linteus mycelium Hex) antiviral efficacy.

Example 2: Evaluating the effect of the Phellinus linteus mycelium bioactive substance of Example 1 against enterovirus 71 infection 1. Virus amplification

The virus liquid of enterovirus 71 was inoculated into vero cells in RPMI-1640 medium containing 2% FBS, and the cell morphology was observed with an inverted microscope. When more than 80% cytopathic effect (CPE) occurred, the culture medium containing virus was collected, the virus titer was quantified by plaque assay, and stored in a -80°C refrigerator for later use.

2. Cell Source and Culture

The aforementioned vero cells (ATCC CCL-81, African green monkey kidney cells; green monkey kidney cells) were purchased from the American Type Culture Collection (ATCC), Manassas, USA].

Subculture was performed when the cells were eighth full. First, the old cell culture medium was removed by suction, the residual serum and metabolites were washed away with phosphate buffered saline (PBS), 1 mL of trypsin typsin-EDTA was added, and placed at 37°C for 3 to 5 minutes, and the cell plate was tapped lightly. The cells adhered to the bottom of the dish were detached. After microscopic examination confirmed the detachment of the cells, the action of trypsin was stopped with 0.5 mL of fetal bovine serum (FBS), and the cells were washed down with RPMI-1640 medium and collected in a 50 mL centrifuge tube. Centrifuge (conditions set at 500 xg, 5 min, 4°C). Then, the supernatant was aspirated, and the precipitated cells were re-dissolved with RPMI-1640 medium and counted, and the cell concentration was adjusted to 3×10 ⁵ cells/dish, and then seeded in a cell culture dish and added with 10% FBS A total of 10 mL of RPMI-1640 medium was cultured in an incubator at 37°C and 5% CO ₂ .

3. Assess the effect of Phellinus linteus mycelium bioactive substance of Example 1 on enterovirus 71 infection

First, the bioactive substance of Phellinus linteus mycelium of Example 1 was dissolved to an appropriate concentration with DMSO, vero cells were cultured in a 96-well plate, and after overnight culture until the cells were attached, a commercially available MTS analysis kit was used to measure Example 1 The effect of Phellinus linteus mycelium bioactive substances on the survival rate of EV71 in vero cells at different infection time points.

3.Pre-treatment

Pre-treatment means that cells are pre-treated or pre-treated with Phellinus linteus mycelium bioactive substances before virus treatment, and then the bioactive substances of Phellinus Phellinus mycelium are evaluated to protect cells and avoid being infected by viruses. Effect.

Before the virus adsorption stage, different concentrations of Phellinus linteus mycelium bioactive substances of Example 1 were added to the vero cells to act in an incubator for 1 hour, the culture medium was sucked off, and then the virus infection dose (Multiplicity of infection, moi) was added. The EV71 solution of = 0.25 and the cells were reacted in the incubator for 1 hour, the culture medium was removed and fresh RPMI-1640 medium containing 2% FBS was added, and incubated at 37°C, 5% CO ₂ incubator for 48 hours, and then MTS ( 3-[4,5,dimethylthiazol-2-yl]-5-[3-carboxymethoxy-phenyl]-2-[4-sulfophenyl]-2H-tetrazolium, inner salt) fraction analysis method to measure the cell viability, the results are as follows Figure 1.

Please refer to FIG. 1 , which shows the cell survival rate of vero cells treated with Phellinus linteus mycelium bioactive substances for preventive treatment and then adding EV71 virus according to an embodiment of the present invention. In Figure 1, Phellinus linteus mycelium bioactive substances were tested at concentrations ranging from 250 μg/mL to 1000 μg/mL. Compared with the EV71 group, the cell viability of the prophylactic treatment with 1000 μg/mL ethanol extract (PI EtOH) It was increased by 15.1% ( p<0.001 ) and was still significantly different at 500 μg/mL concentration. However, prophylactic treatment with water extract (PI H2O) and hexane extract (PI Hex) at 250 μg/mL increased cell viability by 11.8% ( p<0.001 ) and 5.8% ( p<0.05 ), respectively, compared with EV71 Compared with the control group, the cell survival rate was significantly improved, indicating that pre-treatment of cells with the bioactive substances of Phellinus linteus mycelium did have the effect of preventing enterovirus infection.

4. Co-treatment

The co-cultivation treatment means that the cells are co-cultured with the Phellinus linteus mycelium bioactive substance and virus at the same time, and then the effect of the Phellinus Phellinus mycelium bioactive substance to protect the cells and avoid being infected by the virus is evaluated.

Phellinus linteus mycelium bioactive substances with different concentrations of Example 1 were co-cultured with EV71 with moi=0.25 for 1 hour, and then the mixed solutions of each concentration were added to vero cells for 1 hour in an incubator, and the original culture solution was removed. Fresh RPMI-1640 medium containing 2% FBS was added, and the cells were incubated at 37°C in a 5% CO ₂ incubator for 48 hours, and then the cell viability was measured by MTS assay. The results are shown in Figure 2.

Please refer to FIG. 2 , which shows the cell viability after co-culturing the Phellinus linteus mycelium bioactive substance, EV71 virus and vero cells according to an embodiment of the present invention. In Figure 2, after co-culturing with virus and cells with ethanol extract of Phellinus linteus mycelium bioactive substance (PI EtOH) at a concentration of 1000 μg/mL, the cell viability increased by 40% ( p<0.001 ), And there is still a significant difference at the concentration of 250 μg/mL; the dichloromethane extract (PIDCM) and the hexane extract (PI Hex) of the bioactive substances of Phellinus linteus mycelium with a concentration of 500 μg/mL were used for co-cultivation treatment. Cell viability was increased by 39.1% ( p<0.001 ) and 30.2% ( p<0.001 ), respectively, and was still significantly different at a concentration of 250 μg/mL. It is shown that the bioactive substances of Phellinus linteus mycelium and enterovirus still have the effect of preventing enterovirus infection after co-treatment.

5. Post-treatment

Therapeutic treatment means that after the cells are treated with viruses, post-treatment or treatment treatment is performed with the bioactive substances of Phellinus linteus mycelium to evaluate the effect of the bioactive substances of Phellinus Phellinus mycelium to protect cells and avoid being infected by viruses.

Add EV71 with moi=0.25 to vero cells and put them into an incubator for 1 hour. After removing the culture medium, add different concentrations of Phellinus linteus mycelium bioactive substances in Example 1 and then act in an incubator for 1 hour. , remove the original culture medium and add fresh RPMI-1640 medium containing 2% FBS, incubate at 37°C, 5% CO ₂ incubator for 48 hours, and then measure the cell viability by MTS assay, the results are shown in Figure 3 Show.

Please refer to FIG. 3 , which shows the cell survival rate of EV71 virus firstly treating vero cells and then adding the bioactive substance of Phellinus linteus mycelium according to an embodiment of the present invention. In Figure 3, under the treatment conditions, the ethanol extract (PI EtOH) and hexane extract (PI Hex) of Phellinus linteus mycelium bioactive substances with a concentration of 1000 μg/mL were treated, and the cell viability was respectively The increase was 38.5% ( p<0.001 ) and 16.2% ( p<0.001 ), and there was still a significant difference at the concentration of 250 μg/mL; while the concentration of 500 μg/mL of Phellinus lindenus mycelium bioactive substance was the dichloromethane extract (PIDCM) increased cell viability by 26.7% ( p<0.001 ) and was still significantly different at the 250 μg/mL concentration. It was shown that the bioactive substances of Phellinus linteus mycelium did have the effect of slowing down enterovirus infection in the case of enterovirus-infected cells.

Example 3: Evaluating the effect of the Phellinus linteus mycelium bioactive substance of Example 1 against dengue virus type 2 infection 1. Virus amplification

The virus production method is to use the suckling mice within about a week of birth, inject about 10 μl, 100 pfu (plaque forming unit) virus from the fontanelle into the brain of the mice to infect the dengue virus type 2, and observe. After 3 to 5 days for half of the mice to die, the remaining mice were sacrificed with _CO . The mouse brain was taken out and ground with the same amount of Hank's balanced buffer as the mouse brain, and then the virus concentrate was purified and stored at -80°C for later use.

2. Cell line source and culture

Baby hamster kidney cells BHK-21 (baby hamster kidney cells, ATCC CCL-10) were purchased from the American Type Culture Collection (ATCC). When the cells grow to 80% full, the subculture is carried out. First, the old cell culture medium is removed, and the residual serum and metabolites are washed with phosphate-buffered saline PBS. After adding 1 mL of trypsin (trypsin-EDTA), Place it at 37°C for 3-5 minutes, tap the side of the cell plate to promote the cells attached to the bottom of the plate to fall off, and confirm that the cells have been excised by microscopy, then stop the action of trypsin with 0.5mL of fetal bovine serum FBS, and use RPMI -1640 medium to wash down the cells and collect them in a 50mL centrifuge tube for centrifugation (500xg, 5 minutes, 4°C), then aspirate the supernatant, and then re-dissolve the precipitated cells with RPMI-1640 medium and count them , the cell concentration was adjusted to 3×10 ⁵ cells/dish, then seeded in a cell culture dish, and a total of 10 mL of RPMI-1640 medium containing 10% FBS was added, and cultured in an incubator at 37°C and 5% CO ₂ .

3. Evaluation of the effect of the Phellinus linteus mycelium bioactive substance of Example 1 on DENV-2 virus infection

First, the water extract and ethanol extract of Phellinus linteus mycelium bioactive substances of Example 1 were dissolved to appropriate concentrations with PBS and DMSO, respectively, and BHK-21 was cultured in a 96-well plate, and the cells were cultured overnight until the cells were attached. , MTS assay was used to determine the effect of Phellinus linteus mycelium bioactive substances on the survival rate of DENV-2 in BHK-21 at different infection time points.

4.Pre-treatment

Before the virus adsorption stage, add the bioactive substances of Phellinus Phellinus mycelium with different concentrations of Example 1 to the cells to act in the incubator for 1 hour, and then add the moi=0.1 (the multiplicity of infection, multiplicity of infection, MOI) DENV-2 solution and cells were reacted in the incubator for 1 hour, the culture medium was removed and fresh RPMI-1640 medium containing 2% FBS was added, and incubated at 37°C, 5% CO ₂ incubator for 48 hours, and then The cell viability was measured by MTS assay, and the results are shown in Figure 4.

Please refer to FIG. 4 , which shows the cell viability after BHK-21 cells are first treated with the bioactive substance of Phellinus linteus mycelium according to an embodiment of the present invention, and then DENV-2 virus is added. In Figure 4, Phellinus linteus mycelium bioactive substances were tested at concentrations ranging from 250 μg/mL to 1000 μg/mL. Compared with the DENV-2 group, the results showed that the water extract (PI H2O) with a concentration of 1000 μg/mL was prevented. ) and the ethanol extract (PI EtOH) were increased by 8.5% ( p<0.05 ) and 8.4% ( p<0.05 ), respectively, and the ethanol extract (EtOH) was still significantly different at the concentration of 250 μg/mL, showing that Pre-treatment of cells with the bioactive substance of Phellinus linteus mycelium of Example 1 has the effect of preventing dengue virus infection.

5. Co-treatment

The bioactive substances of Phellinus Phellinus mycelium with different concentrations of Example 1 and DENV-2 with moi=0.1 were co-cultured for 1 hour, and then the mixed solutions of each concentration were added to the cells and acted in an incubator for 1 hour, and the original culture was removed. Then, fresh RPMI-1640 medium containing 2% FBS was added, and the cells were cultured at 37°C in a 5% CO ₂ incubator for 48 hours, and then the cell viability was measured by MTS assay. The results are shown in Figure 5.

Please refer to FIG. 5 , which shows the cell viability after co-culture of Phellinus linteus mycelium, DENV-2 virus and BHK-21 cells according to an embodiment of the present invention. In Fig. 5, under the co-cultivation treatment conditions, the cell viability of the water extract (PI H2O) and the ethanol extract (PI EtOH) of the Phellinus linteus mycelium biologically active substance of Example 1 with a concentration of 1000 μg/mL were respectively The increase was 28% ( p<0.05 ), 78.2% ( p<0.05 ), and the ethanol extract (PI EtOH) was still significantly different at the concentration of 250 μg/mL, indicating that the bioactive substances of Phellinus linteus mycelium and dengue virus are common Under treatment, it still has the effect of preventing dengue virus infection.

6. Post-treatment

Add DENV-2 with moi=0.1 to the cells and put them into the incubator for 1 hour. After aspirating the culture medium, add different concentrations of Phellinus linteus mycelium bioactive substances and then in the incubator for 1 hour, remove the The original culture medium was added with fresh RPMI-1640 medium containing 2% FBS, and cultured at 37°C in a 5% CO ₂ incubator for 48 hours, after which the cell viability was measured by MTS assay. The results are shown in Figure 6.

Please refer to FIG. 6 , which shows the cell viability of BHK-21 cells treated with DENV-2 virus first and then adding the bioactive substance of Phellinus linteus mycelium according to an embodiment of the present invention. In Figure 6, under the treatment conditions, the cell viability of the group treated with Phellinus linteus mycelium bioactive substances was not statistically significantly different from that of the DENV2 group, but in the dengue virus-infected cells However, the water extract (PI H2O) and the ethanol extract (PI EtOH) still had the tendency to slow down the dengue virus infection at the concentration of 250 μg/mL.

To sum up, it is confirmed from the above-mentioned several examples that the bioactive substance of Phellinus linteus mycelium prepared by the present invention can reduce or slow down the efficiency of cells infected by enterovirus type 71 and dengue virus type 2 after pre-treating cells. , can be used in the preparation of antiviral compositions.

It should be added that although the present invention takes a specific manufacturing process or a specific analysis method as an example to describe the bioactive substance of Phellinus linteus mycelium, its manufacturing method and its use for preparing an antiviral composition of the present invention, this Any person with ordinary knowledge in the technical field to which the invention belongs will know that the present invention is not limited to this. Without departing from the spirit and scope of the present invention, the Phellinus linteus mycelium bioactive substance of the present invention and its manufacturing method can also use other manufacturing processes. or other analytical methods law to proceed.

As can be seen from the above-described embodiments, the bioactive substance of Phellinus linteus mycelium of the present invention, its manufacturing method and its use for preparing an antiviral composition have the advantage that the Phellinus linteus mycelium is subjected to solid-state culture, liquid culture and liquid culture. After fermented cultivation, the obtained Phellinus linteus mycelium fermented liquid contains biologically active substances, has the effect of antiviral infection, and can be used for the preparation of antiviral compositions.

Although the present invention has been disclosed above with several embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field to which the present invention belongs, without departing from the spirit and scope of the present invention, can make various Therefore, the scope of protection of the present invention should be determined by the scope of the appended patent application.

【Biological Material Deposit】

Phellinus linteus is deposited at the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is July 18, 2019, and the deposit number is BCRC 930210.

Claims (10)

A kind of Phellinus Phellinus mycelium bioactive substance is used for preparing the purposes of the antiviral composition of anti-enterovirus type 71 (EV71), wherein the Phellinus Phellinus mycelium bioactive substance is an active ingredient, the Phellinus Phellinus ( Phellinus linteus ) is deposited in the Biological Resource Conservation and Research Center of the Food Industry Development Research Institute, No. 331, Food Road, Hsinchu, Taiwan. The deposit date is July 18, 2019, and the deposit number is BCRC 930210. The Phellinus linteus mycelium The biologically active substance is selected from the group consisting of the water extract, ethanol extract, dichloromethane extract, hexane extract and any combination of the above mentioned Phellinus linteus mycelium. The use of the bioactive substance of Phellinus linteus mycelium according to claim 1 for preparing an antiviral composition against enterovirus 71, wherein one of the in vitro effective doses of the water extract of Phellinus linteus mycelium is 250 μg/ mL. The use of the bioactive substance of Phellinus linteus mycelium as claimed in claim 1 for preparing an antiviral composition against enterovirus 71, wherein the in vitro effective dose of the ethanolic extract of Phellinus linteus mycelium is 250 μg/ mL to 1000 μg/mL. The use of the Phellinus linteus mycelium bioactive substance according to claim 1 for preparing an antiviral composition against enterovirus 71, wherein the in vitro effective dose of the dichloromethane extract is 250 μg/mL to 500 μg/mL mL. The use of the bioactive substance of Phellinus linteus mycelium according to claim 1 for preparing an antiviral composition against enterovirus 71, wherein the in vitro effective dose of the hexane extract is 250 μg/mL to 1000 μg/mL . The use of the Phellinus linteus mycelium biologically active substance according to claim 1 for preparing an antiviral composition against enterovirus 71, wherein the antiviral composition is an oral composition. The use of the bioactive substance of Phellinus linteus mycelium according to claim 6 for preparing an antiviral composition against enterovirus 71, wherein the antiviral composition is before a virus infects a cell and infects the cell Or after infection of the cells, the cells are administered in vitro. Use of the Phellinus linteus mycelium biologically active substance as claimed in claim 1 for preparing an antiviral composition against enterovirus 71, wherein the antiviral composition further comprises a pharmaceutically acceptable carrier, a excipient, a diluent and/or an adjuvant. An oral composition with antiviral efficacy against enterovirus 71, comprising the Phellinus linteus mycelium bioactive substance as described in any one of claims 1 to 7, wherein the Phellinus Phellinus mycelium bioactive substance It is selected from the ethanol extract, dichloromethane extract, hexane extract of Phellinus linteus mycelium. A group consisting of the extracted material and any combination of the above. The oral composition with anti-enterovirus type 71 antiviral efficacy as claimed in claim 9, wherein the in vitro effective dose of the water extract of the Phellinus linteus mycelium is 250 μg/mL, one of the ethanol extract The effective in vitro dose is 250 μg/mL to 1000 μg/mL, the effective in vitro dose of the dichloromethane extract is 250 μg/mL to 500 μg/mL, and the effective in vitro dose of the hexane extract is 250 μg/mL to 1000 μg/mL mL.

Images