TWI724890B - Use of lignosus rhinocerus mycelia active substance for manufacturing an antiviral composition - Google Patents

Abstract

A novel use of Lignosus rhinocerusmycelia active substance is provided. The Lignosus rhinocerusmycelia active substance is for manufacturing of anti-enterovirus and/or anti-influenza virus composition and preparation of these composition. The method of prepare Lignosus rhinocerusmycelia active substance comprises following steps: (a)culturing a Lignosus rhinocerusmycelium in a plate media between 15℃ and 30℃ for 1 to 2 weeks; (b)inoculating the mycelium of step(a) to a flask and culturing it between 15℃ and 30℃ with a pH of 2 to 6 for 3 to 14 days; and (c)inoculating the mycelium of step(b) to a fermentation tank and culturing it between 15℃ and 30℃ with a pH of 2 to 6 for 3 to 21 days, so as to obtain a Lignosus rhinocerusmycelium fermentation liquid containing said Lignosus rhinocerusmycelium active substance.

Description

Use of tiger milk ganoderma lucidum mycelium active substance for preparing antiviral composition

The present invention relates to the use of a tiger milk ganoderma lucidum mycelium active substance, in particular to its use in resisting enterovirus and/or influenza virus, and its use in preparing a composition against enterovirus and/or influenza virus.

A virus is a non-cellular form composed of a nucleic acid molecule (DNA or RNA) and protein. It is between living things and non- living things. At present, it is not classified as one of the five realms (prokaryotes, protists, fungi, plants and animals). It is an organic species between living and non-living forms that cannot show life phenomena on its own and must rely on parasitism to survive and multiply. The structure of the virus is mainly a piece of DNA or RNA wrapped in a protective shell. Through the mechanism of infection, these simple organisms can use the host cell system to replicate themselves, but they cannot grow and replicate independently.

Viruses are composed of two to three components: genetic material (RNA or DNA); a capsid formed by protein, which is used to wrap and protect the genetic material; in addition, some viruses can form a lipid envelope around the cell surface when they reach the cell surface. Viruses have different shapes, ranging from simple spirals and icosahedrons to complex structures. Virus particles are about one thousandth the size of bacteria.

Most viruses can cause diseases of the organism, but not all of them. The replication of many viruses does not cause obvious damage to the infected organs. A small number of viruses, such as HIV, can coexist with the human body for a long time and still remain infectious without being affected by the host's immune system, that is, "viral persistence" (viral persistence). But under normal circumstances, a viral infection can trigger an immune response and eliminate the invading virus. These immune responses can be produced by injecting vaccines, so that the vaccinated people or animals can be immune to the corresponding viruses 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, thereby causing cell death. In multicellular organisms, once enough cells in the body die, it will have an impact on the health of the body. Although viruses can cause diseases, they can also exist in the body harmlessly. For example, the herpes simplex virus that can cause cold sores can remain dormant in the human body; this state is also called "latency", which is also a characteristic of all herpes viruses. The varicella-zoster virus that has entered the latent state can cause shingles after it "woke up."

Enterovirus 71 (EV71) is a single-stranded ribonucleic acid virus and one of the pathogens that cause hand, foot and mouth disease. EV71 infection is common in Southeast Asia, mostly in summer and early autumn. There have been outbreaks in Australia, China, Malaysia, Singapore, Taiwan, etc., and EV71 infection is more common in young children. The symptoms are generally the same as hand, foot and mouth disease, mainly including fever, sore mouth sores, and blistered rash. Symptoms such as fever, loss of appetite, fatigue, or sore throat are usually the first symptoms of the disease. One to two days after fever, painful blisters may appear in the mouth. These blisters appear as small red spots at first and then form ulcers. Ulcers are usually located on the tongue, gums, and inside the cheeks of the mouth. In addition, the palms and soles of the feet, and even the buttocks and/or genitals will also have red rashes that are not itchy or have small blisters. Patients with hand, foot and mouth disease may also have no symptoms, or may only have symptoms such as skin rashes or oral ulcers. The more serious conditions of the EV71 type include viral meningitis, encephalitis, polio-like paralysis and myocarditis. Currently, there is no vaccine to prevent EV71 infection. In terms of drug development, there is currently no effective drug to treat EV71. Only a few drugs can relieve the pain caused by fever and ulcers. Therefore, it is necessary to find effective drugs to prevent/treat EV71.

H1N1 new influenza is a type A influenza virus. The transmission route is the same as that of seasonal influenza. Both are droplet transmission and contact transmission. The incubation period is estimated to be 1-4 days. The infectious period is from the day before the onset of the disease to 7 days after the onset. The infectious period in children's cases is usually It is longer than adult cases and is also the main source of influenza transmission. The symptoms of the new H1N1 flu are similar to seasonal flu, including fever, cough, sore throat, body aches, headaches, chills and fatigue. In some cases, symptoms of diarrhea and vomiting occur. So far, the epidemiology of the new H1N1 influenza has shown that its severity is still mild, comparable to that of seasonal influenza, with a mortality rate ranging from one to four in a thousand. In some high-risk groups, the probability of complications or death is higher. High-risk groups include children under five, pregnant women, elderly people over 65, people with underlying diseases (such as HIV, diabetes, cardiovascular disease, chronic lung disease), immunosuppressed patients, and obese people. At present, the main prevention and treatment method is the administration of vaccines. However, due to the high specificity of the vaccine and the large variation of the new H1N1 influenza, the influenza vaccine administered each year may not be effective against the H1N1 of the current year. Therefore, it is necessary to find effective drugs to prevent/treat H1N1.

Tiger Milk Ganoderma ( Lignosus rhinocerus ), also known as tiger milk, belongs to the Basidiomycota (Basidiomycota) Polyporaceae (Polyporaceae) plant. Tiger Milk Ganoderma is a traditional medicine used in Malaysia. It is traditionally used for cough, asthma, bronchitis, vomiting, fever, food poisoning and other symptoms. Modern pharmacological research and analysis have found that Tiger Milk Ganoderma has immune regulation, anti-inflammatory, anti-oxidant, anti-microbial, anti-viral, anti-coagulation, and anti-breast cancer effects. However, most of these studies focused on tiger milk ganoderma as a nutrient/health food to enhance the eater's own immunity to resist the above symptoms; or the anti-inflammation was extended to anti-viral inflammation, which actually has no anti-viral effect. There is no evidence in the prior art that when viruses or bacteria invade the body, Tiger Milk Ganoderma lucidum has a clear anti-infection benefit as a medicine. Therefore, the present invention conducts detailed research on the active substance of tiger milk Ganoderma lucidum mycelium and enterovirus/influenza virus itself, and provides a use of the active substance of tiger milk Ganoderma lucidum mycelium to prove that it can effectively resist enterovirus and/or influenza virus infection cell.

According to the present invention, there is provided a use of the active substance of Tiger Milk Ganoderma lucidum mycelium, which is used to prepare a composition against enterovirus and/or influenza virus. The preparation method of the tiger milk ganoderma lucidum mycelium active substance includes the following steps:

(A) Take a tiger milk Ganoderma lucidum mycelia (Lignosus rhinocerus mycelia) on a plate medium and culture it at a temperature of 15 to 30°C for 1 to 2 weeks; (b) Add the tiger milk Ganoderma lucidum after step (a) The silk body is inoculated into a flask and cultured at 15 to 30°C and pH 2 to 6 for 3 to 14 days; (c) the tiger milk ganoderma lucidum mycelium after cultivation in step (b) is inoculated into a fermentation tank, Stir and cultivate for 3 to 21 days at 15 to 30°C and pH 2 to 6 to form a tiger milk ganoderma lucidum mycelium fermentation broth; (d) freeze-dry the tiger milk ganoderma lucidum mycelium fermentation broth and pulverize it. Forming a tiger milk ganoderma lucidum mycelium freeze-dried powder; (e) extracting the tiger milk ganoderma lucidum mycelium freeze-dried powder with at least one solvent to form a tiger milk ganoderma lucidum mycelium extract; and (f) the tiger milk ganoderma lucidum mycelium extract The milk Ganoderma lucidum mycelium extract is dried to obtain the tiger milk Ganoderma lucidum mycelium active substance.

In one embodiment, the active substance of tiger milk ganoderma lucidum mycelium is used to prepare a composition against enterovirus type 71 or H1N1 influenza virus.

In one embodiment, the solvent in step (e) of the method for preparing the active substance of tiger milk ganoderma lucidum mycelium is selected from water, ethanol, ethyl acetate or hexane.

In one embodiment, the flask culture in step (b) of the method for preparing the active substance of Tiger Milk Ganoderma lucidum mycelium is shaking culture, and the rotation speed is 110 to 130 rpm.

In one embodiment, the fermentation tank in step (c) of the above method for preparing active material of tiger milk ganoderma mycelium is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium or a combination thereof, the fermentation tank The tank pressure is 0.5 to 1.0 kg/cm2 and the ventilation rate is 0.1 to 1.5 VVM.

In one embodiment, the tiger milk ganoderma lucidum mycelium active substance comprises an aqueous extract, an ethanol extract, an ethyl acetate extract, a hexane extract, or a mixture of the tiger milk ganoderma mycelium.

In one embodiment, the composition further comprises pharmaceutically acceptable carriers, excipients, diluents, adjuvants, fillers, absorption enhancers, sweeteners, or combinations thereof.

In one embodiment, the composition includes a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

In one embodiment, the form of the composition includes powder, lozenge, granulation, suppository, microcapsule, ampoule, liquid spray or suppository.

In one embodiment, the tiger milk Ganoderma lucidum mycelium is selected from the tiger milk Ganoderma lucidum mycelium deposited at the Biological Resources Preservation and Research Center with a deposit number of BCRC 930202.

In order to make the above and other aspects of the present invention clearer and easier to understand, the following specific embodiments are described in detail in conjunction with the accompanying drawings.

Example 1: Culture of Tiger Milk Ganoderma Lucidum Mycelium

The tiger milk Ganoderma lucidum mycelium used in this example was purchased from Malaysian wild tiger milk Ganoderma lucidum fruiting bodies. After the surface of the fruiting body is disinfected, a small cut is made to obtain a bacteria-free area by tearing, and a small piece of fruiting body is clamped with tweezers and placed in a plate medium PDA for cultivation. In this way, the strains are separated and after the hyphae grow on the PDA, different types of hyphae are purified, and the strains are identified after confirming that the hyphae types are consistent.

The strains were cultured in liquid form to collect the mycelium, and then the DNA was extracted by grinding and breaking with liquid nitrogen, and the ITS gene fragment was amplified and amplified with ITS-1 and ITS-4 primers (ITS-1: TCCGTAGGTGAACCTGCGG/ ITS-4: TCCTCCGCTTATTGATATGC) .

PCR amplification conditions were 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 52°C for 30 seconds, 72°C for 1 minute and 10 seconds, and finally 72°C for 5 minutes to carry out the polymerase chain reaction. The amplified products are sequenced and analyzed, and the NCBI website resources are used for BLAST search for sequence comparison. The sequence similarity is 99% with that of Lignosus rhinoceros. The above-mentioned Tiger Milk Ganoderma lucidum mycelium is deposited in the Bioresource Conservation and Research Center of the Food Industry Development Institute, and the deposit number is BCRC 930202.

However, the species of tiger milk Ganoderma lucidum that can be used in the present invention is not limited to this single one, and tiger milk Ganoderma lucidum from other sources can also be used, which is not limited by the present invention. The following describes the steps of cultivating tiger milk ganoderma lucidum of the present invention:

(1) Plate culture: Inoculate the tiger milk ganoderma lucidum mycelium on the plate and culture it at 15-30°C for 1 to 2 weeks (in this example, it is cultured at 25°C for 7 days). The composition of the plate medium may include Potato Dextrose Agar (PDA), carbon source and nitrogen source, and there is no particular limitation.

(2) Flask culture: (1) Scrape the mycelium on the plate and inoculate it in a flask, and cultivate it for 3 to 14 days with shaking at 15 to 30°C, pH 2 to 6 and rotation speed of 110 to 130 rpm (this implementation) In the example, culture was shaken for 7 days at 25°C, pH 5, and rotating speed 120 rpm). This shaking culture was cultured in the medium shown in Table 1 below.

Table 1 Medium formula ingredient The content used in this embodiment (wt%) Preferred content range (wt%) Comprehensive carbon and nitrogen source 1 1-3 carbohydrate 2 1-4 Yeast extract 0.6 0.5-1 Egg White 0.6 0.5-1 Inorganic salts 0.05 0.01-0.05

In the above medium formula, the comprehensive carbon and nitrogen sources can be selected from cereals (such as wheat flour) or beans (such as soybean powder, mung bean powder, soybean powder, cinnamon powder, etc.); sugars can be glucose, fructose, maltose, sucrose Etc.; inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, iron sulfate and the like. In particular, the medium formula in Table 1 is only one example, and the ingredients can be adjusted according to requirements during use, or used with commercially available medium, and there is no special restriction.

(3) Fermentation tank culture: Inoculate the mycelium cultured in the flask in (2) into the fermentation tank at 15 to 30 ℃, tank pressure 0.5 to 1.0 kg/cm ² , pH 2 to 6 and stirring speed 50 to 150 Cultivate for 3 to 21 days at a ventilation rate of 0.1 to 1.5 VVM under rpm conditions to form a tiger milk Ganoderma lucidum mycelium fermentation broth. The gas types introduced can include: air, oxygen, carbon dioxide, helium or a combination thereof ( In this example, it was cultured for 14 days under the conditions of 25°C, tank pressure 0.5 kg/cm ² , pH 5, stirring speed 80 rpm and 1.0 VVM (air) to obtain tiger milk ganoderma lucidum mycelium fermentation broth . The culture medium used for the fermentation tank culture can be the same as the culture medium used for the flask culture in step (2) (the method of this embodiment), and an appropriate medium can also be configured in addition. The tiger's milk ganoderma lucidum mycelium fermentation broth can be further prepared as a freeze-dried powder of the tiger's milk ganoderma lucidum mycelium fermentation broth through a freeze-drying step. Freeze-drying methods are: (a) freezing the sample to -20 to -40°C for about 4 hours; (b) vacuuming the sample to directly sublime the water; and (c) heating to 20 to 40°C for 4-8 hours. carry out. In this example, 100 L of Tiger Milk Ganoderma lucidum mycelium fermentation broth can produce about 2 kg of freeze-dried powder.

(4) Extraction preparation: Divide the lyophilized powder of tiger milk ganoderma lucidum mycelium fermentation broth into 4 equal parts, and add 20 times the weight of distilled water/ethanol/ethyl acetate/hexane for extraction. The sample with water as the solvent was heated at a temperature of 100°C for 30 minutes. After cooling, the supernatant was centrifuged to obtain the supernatant, and dried by the freeze-drying method (the procedure can be the same as in the previous paragraph) to obtain the tiger milk ganoderma mycelium water extract. Samples extracted with ethanol/ethyl acetate/hexane as solvents were shaken with ultrasonic for 1 hour, centrifuged and the supernatant was taken, and the supernatant was concentrated under reduced pressure to obtain ethanol extracts of tiger milk Ganoderma lucidum mycelium. Ethyl acetate extract/hexane extract.

Through the extraction step, water extracts and ethanol extracts/ethyl acetate extracts/hexane extracts containing a higher concentration of active substances from tiger milk ganoderma lucidum mycelium can be obtained. The form of active substances from Tiger Milk Ganoderma lucidum mycelium includes Tiger Milk Ganoderma lucidum mycelium fermentation broth (mycelium and clarified liquid), fermentation broth freeze-dried powder, water extract, ethanol extract, ethyl acetate extract, Alkyl extract and its mixture or other dosage forms. In the following example 2 and example 3, water extract and ethanol extract/ethyl acetate extract/hexane extract are used as the active substance of Tiger Milk Ganoderma lucidum mycelium to treat enterovirus 71 and influenza virus. H1N1 experiment. Effect according to the second Tiger Milk mycelia active material for EV71 embodiment infection

Virus amplify

Inoculate the virus liquid in Vero cells (vero cells) containing 2% FBS RPMI-1640 culture medium. Observe the cell morphology with an inverted microscope. When more than 80% cytopathic effect (CPE) occurs, collect the The virus culture solution is quantified by plaque assay and stored in the refrigerator at -80°C for later use.

Cell source and culture

Vero cells (ATCC CCL-81, African green monkey kidney cells): Green monkey kidney cells, originally purchased from American Type Culture Collection (ATCC, Manassas, USA).

When the cells are eighth full, subculture, first aspirate the old cell culture medium, wash away the residual serum and metabolites with phosphate buffered saline PBS, add 1 mL trypsin typsin-EDTA, and place Act for 3-5 minutes at 37℃, tap the side of the cell disc to promote the detachment of the cells attached to the bottom of the disc. After microscopic examination confirms that the cells are cut off, 0.5 mL of fetal bovine serum FBS is used to stop the trypsin effect, and use RPMI-1640 Wash the cells with the medium and collect them in a 50 mL centrifuge tube for centrifugation (conditions are set to 500 xg, 5 minutes, 4°C), then aspirate the supernatant, and then re-dissolve the precipitated cells with RPMI-1640 medium and count them. Adjust the cell concentration to 3 × 10 ⁵ cells/plate, finally plant in a cell culture dish, add 10 mL of RPMI-1640 medium containing 10% FBS, and culture in an incubator at 37°C and 5% CO ₂ .

Influence of Tiger Milk Ganoderma lucidum Mycelium Active Substances on Enterovirus 71 Infection

First dissolve the tiger milk Ganoderma lucidum mycelium extract with DMSO to an appropriate concentration, culture the VERO cells in a 96-well plate, and then culture them overnight after the cells are attached, and then use the MTS assay to determine the various tiger milk Ganoderma lucidum mycelium extracts. VERO cell survival rate at the time point of EV71 infection.

Pre-treatment

Before the virus adsorption stage, add different forms (water extract, ethanol extract, ethyl acetate extract and hexane extract respectively), different concentrations of tiger milk ganoderma lucidum mycelium active substances (each form The active substances are 1000, 500, and 250 μg/mL respectively) in the incubator for 1 hour. After aspirating the culture medium, add the EV71 solution with moi = 0.25 (Multiplicity of infection, MOI) to the cells. The incubator acts for 1 hour. Remove the culture medium and add fresh RPMI-1640 culture medium containing 2% FBS, _{incubate for 48 hours in a 37°C, 5% CO 2} incubator, and finally measure the cell viability by MTS assay. The results are shown in Figure 1.

The +/- value of the EV71 column in Figure 1 indicates whether the sample has EV71 added with enterovirus. DMSO is the control/control group without tiger milk ganoderma lucidum mycelium. Lr H ₂ O, Lr EtOH, Lr EA and Lr Hex are active substances added with tiger milk ganoderma lucidum mycelium (water extract, ethanol/ethyl acetate, respectively) Ester/hexane extract is added to the experimental group. 1000, 500, and 250 are the concentration of active substance added with tiger milk ganoderma lucidum mycelium (μg/mL). The vertical axis is the cell survival rate (100% for the non-added enterovirus EV71 group). The asterisk * indicates that the value of this group is significantly different from the virus control group (*=p ＜0.05, **=p ＜0.01, ***=p ＜0.001). The prevention test is mainly to test whether the active substance of Tiger Milk Ganoderma lucidum mycelium can inhibit the adsorption of viruses to cells. Compared with the EV71 pair with only enterovirus added, the concentration of 1000μg/ml water extract, ethyl acetate extract and In the group treated with hexane extract for preventive treatment, the cell survival rate increased by 26.370% (p<0.001), 12.7% (p<0.001), and 13.9% (p<0.001), respectively, and cell survival at a concentration of 250μg/mL The rate still has a significant increase.

Co-cultivation test (Co-treatment)

Co-cultivate the active substances from Tiger Milk Ganoderma lucidum mycelium in different forms and different concentrations with EV71 with moi = 0.25 for 1 hour, and then add the active substances from Tiger Milk Ganoderma lucidum mycelium in various concentrations and forms to the cells for cultivation. Incubate for 1 hour, remove the original culture medium and add fresh RPMI-1640 culture medium containing 2% FBS, _{incubate for 48 hours in a 37°C, 5% CO 2} incubator, and finally measure the cell survival rate by MTS assay. The result is shown in Figure 2, and the description of the horizontal/vertical axis of the drawing is the same as that in Figure 1. The co-cultivation test is also to test whether the active substance of Tiger Milk Ganoderma lucidum mycelium can inhibit the adsorption of the virus to the cell, or whether the active substance can preferentially bind to the virus to inactivate it. Under the co-cultivation conditions, the cell survival rate of 1000μg/mL Tiger Milk Ganoderma lucidum water extract increased by 51.67% (p<0.001), and there was still a significant difference at a concentration of 250μg/mL; while 1000μg/mL Tiger Milk Ganoderma acetic acid The cell viability of ethyl ester extract and tiger milk ganoderma lucidum hexane extract increased by 35.53% (p<0.001) and 28.64% (p<0.001) respectively; and the water extract, ethyl acetate extract, and hexane extract increased There is still a significant difference at a concentration of 250μg/mL.

Post-treatment

Add EV71 with moi = 0.25 to the cells and place it in the incubator for 1 hour. After sucking off the culture solution, add different concentrations of Tiger Milk Ganoderma lucidum mycelium active substances and then incubate for 1 hour in the incubator to remove the original culture. Add fresh RPMI-1640 culture medium containing 2% FBS and _{incubate for 48 hours in a 37°C, 5% CO 2} incubator. Finally, the cell survival rate was measured by MTS assay. The results are shown in Figure 3. The treatment test mainly tests whether the active substance of Tiger Milk Ganoderma lucidum mycelium can improve the ability of cells to kill intracellular viruses. Under treatment conditions, the cell survival rate of 1000μg/mL Tiger Milk Ganoderma lucidum water extract increased by 47.37% (p<0.001), and there was still a significant difference at a concentration of 250μg/mL; while 1000μg/mL Tiger Milk Ganoderma lucidum ethanol extraction The cell survival rate of the tiger's milk ganoderma lucidum ethyl acetate extract and the tiger's milk ganoderma hexane extract increased by 7.86% (p<0.001), 40.84% (p<0.001) and 30.56% (p<0.001) respectively, and There is still a significant difference at a concentration of 250μg/mL.

According to the above experiments, it can be known that the water extract of Tiger Milk Ganoderma lucidum mycelium and the ethanol/ethyl acetate/hexane extract have antiviral effects such as prevention, inhibition of adsorption, elimination and/or treatment of EV71 virus, and can improve cell survival rate . Example 3 The effect of active substance from Tiger Milk Ganoderma lucidum mycelium on influenza virus H1N1 infection

Virus amplify

After injecting 100 μL (approximately 100 TCID50) of influenza A virus (H1N1) into the allantoic membrane of SPF (Specific pathogen free) chicken embryo eggs, seal the hole with wax for reproduction, and place it in an incubator for 1-2 Day, put it in 4℃ again to wait for coagulation, and finally extract the liquid from the allantoic membrane and place it at -80℃ for storage.

Cell line source and culture

MDCK cells (Madin-Darby Canine Kidney cells): Canine kidney epithelial cells, purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research Institute of Taiwan, the cell line number is BCRC 60004. When the cells grow to eighth full, subculture is performed. First, aspirate the old cell culture medium, wash away the residual serum and metabolites with phosphate buffered saline, PBS, and add 1 mL trypsin-EDTA. Incubate at 37°C for 3-5 minutes, tap the side of the cell disc to promote the detachment of the cells attached to the bottom of the disc. After microscopic examination confirms that the cells are cut off, use 0.5 mL of fetal bovine serum FBS to stop the trypsin effect, and use DMEM medium Wash down the cells and collect them in a 50 mL centrifuge tube for centrifugation (conditions are set to 500 xg, 5 minutes, 4°C), then aspirate the supernatant, then re-dissolve the precipitated cells with DMEM medium and count them to determine the cell concentration Adjusted to 3 × 10 ⁵ cells/plate, finally planted in a cell culture dish, and added 10 mL of DMEM medium containing 10% FBS to the incubator at 37°C and 5% CO ₂ .

Effect of Tiger Milk Ganoderma Mycelium Extract on H1N1 Virus Infection

First dissolve the tiger milk Ganoderma lucidum mycelium extracts with PBS and DMSO to appropriate concentrations (water extracts are dissolved in PBS, and other extracts are dissolved in DMSO), and MDCK is cultured in a 96-well plate, and cultured overnight until the cells are attached Then, MTS assay was used to determine the survival rate of MDCK cells of Tiger Milk Ganoderma lucidum mycelium extract at different H1N1 infection time points.

Pre-treatment

Before the virus adsorption stage, add different forms (water extract, ethanol extract, ethyl acetate extract, and hexane extract) and different concentrations of tiger milk ganoderma lucidum mycelium active substances (respectively 1000 , 500, 250 μg/mL) in the incubator for 1 hour. After aspirating the culture medium, add H1N1 solution with moi = 0.05 (Multiplicity of infection, MOI), and react with the cells in the incubator for 1 hour. Remove the culture medium and add fresh DMEM culture medium containing 2% FBS, _{incubate for 48 hours in a 37°C, 5% CO 2} incubator, and finally measure the cell survival rate by MTS assay. The results are shown in Figure 4. Except that the virus type is changed from EV71 to H1N1, the description of the horizontal/vertical axis of the graph in Figure 4 is the same as that in Figure 1. Compared with the H1N1 virus control group, the results showed that in the group treated with ethyl acetate extract and hexane extract at a concentration of 1000μg/ml, the cell survival rate increased by 6.35% (p<0.05) and 24.7% respectively ( p<0.01), and there is still a significant difference between the hexane extract at a concentration of 250μg/mL; the group of preventive treatment with the water extract and ethanol extract at a concentration of 500μg/ml, the cell survival rate increased by 5.14% (p <0.05) and 7.47% (p<0.05), and the ethanol extract still has a significant difference at a concentration of 250μg/mL.

Co-cultivation test (Co-treatment)

Co-cultivation of Tiger Milk Ganoderma lucidum mycelium extracts of different forms and concentrations with H1N1 with moi = 0.05 for 1 hour, and then the active substances of Tiger Milk Ganoderma lucidum mycelium of various concentrations and forms were added to the cells for culture Incubate for 1 hour, remove the original culture medium and add fresh DMEM culture medium containing 2% FBS, _{incubate for 48 hours in a 37°C, 5% CO 2} incubator, and finally measure the cell survival rate by MTS assay. The result is as shown in section 5. As shown in the figure. Under the co-cultivation treatment conditions, the cell survival rate of high-concentration 1000μg/mL water extract, ethanol extract, ethyl acetate extract and hexane extract increased by 25.5% (p<0.01) and 12.1% (p<0.01). ), 18.3% (p<0.05) and 15.5% (p<0.001), and ethanol extracts still have significant differences at a concentration of 250μg/mL.

Post-treatment

Add H1N1 with moi = 0.05 to the cells and place them in the incubator for 1 hour. After sucking off the culture solution, add different concentrations and different forms of Tiger Milk Ganoderma lucidum mycelium extracts and incubate for 1 hour in the incubator. The original culture medium was removed and fresh DMEM culture medium containing 2% FBS was added, and _{the cells were cultured in a 37°C, 5% CO 2} incubator for 48 hours. Finally, the cell survival rate was measured by MTS assay. The results are shown in Figure 6, under the treatment conditions, the cell survival rate of high-concentration 1000μg/mL water extract, ethanol extract and hexane extract increased by 22.1% (p<0.01) and 11.3% (p<0.001). ) And 23.9% (p<0.01), and there is still a significant difference between the ethanol extract and the hexane extract at a concentration of 250μg/mL.

According to the above experiments, it can be known that the water extract, ethanol extract and hexane extract of Tiger Milk Ganoderma lucidum mycelium have antiviral effects such as prevention, inhibition of adsorption, elimination and/or treatment of H1N1 virus, and can improve cell survival rate. Example 4 Preparation of composition

The tiger milk ganoderma lucidum mycelium active substance of the present invention can be further prepared as a food or pharmaceutical composition. This composition further contains additives. In a preferred embodiment, the additives may be excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, or combinations thereof. The excipient can be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose or a combination thereof. The preservative can extend the shelf life of the pharmaceutical composition, such as benzyl alcohol and parabens. The diluent may be selected from water, ethanol, propylene glycol, glycerin, or a combination thereof. The filler may be selected from lactose, nougat, high molecular weight ethylene glycol, or a combination thereof. The absorption enhancer may be selected from dimethyl sulfide (DMSO), azone, propylene glycol, glycerin, polyethylene glycol, or a combination thereof. The sweetener can be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame, or a combination thereof. In addition to the additives listed above, other suitable additives can be selected according to requirements, provided that the medicinal effect of the active substance of Tiger Milk Ganoderma lucidum mycelium is not affected.

The composition can be developed into different commodities in the field of medicine. In a preferred embodiment, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

The composition can adopt different forms according to the needs of the recipient. In a preferred embodiment, the composition is in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule (ampoule/ampule), liquid spray or suppository.

If it is applied to food use, the aspect of composition 1 is taken as an illustrative example.

Composition 1: Take the active substance of tiger milk ganoderma mycelium (in the form of water extract, ethanol/ethyl acetate/hexane extract or a combination thereof, 20 wt%), and benzyl alcohol (8 wt%) as a preservative ), glycerin (7 wt%) as a diluent is mixed thoroughly, dissolved in pure water (65 wt%), and stored at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

If the tiger milk ganoderma mycelium active substance of the present invention is applied in a liquid dosage form for medical purposes, the aspect of composition 2 is taken as an illustrative example.

Composition 2: Take the active substance of tiger milk ganoderma lucidum mycelium (in the form of water extract, ethanol/ethyl acetate/hexane extract or a combination thereof, 20 wt%), and benzyl alcohol (8 wt%) as a preservative %), glycerol (7 wt%) as a diluent, and sucrose (10 wt%) as a diluent, mix thoroughly, dissolve in pure water (55 wt%), and store at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

In summary, through the above experiments, it can be proved that the active substance of tiger milk ganoderma lucidum mycelium disclosed in the present invention has the resistance effect of preventing, eradicating and/or curing, in particular, it is effective in preventing, eradicating and/or curing, etc. The purpose of resisting enterovirus and/or influenza virus is more preferably the purpose of resisting enterovirus 71 and/or influenza H1N1 virus.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Those of ordinary knowledge in the field, after considering the above teachings, can make appropriate modifications to the content of the above embodiments, and still achieve the effects claimed in this case. Therefore, the scope of protection of the present invention should be subject to the scope of the patent application appended thereafter.

Figure 1 shows the viability test results of Vero cells with different concentrations/extraction solutions of Tiger Milk Ganoderma lucidum mycelium active substances after first treating Vero cells and then adding EV71 virus.

Figure 2 shows the results of Vero cell viability test after treating Vero cells with Tiger Milk Ganoderma lucidum mycelium active substances in different concentrations/extraction solutions together with EV71 virus.

Figure 3 shows the results of the Vero cell viability test after EV71 virus first treats Vero cells, and then adds tiger milk Ganoderma lucidum mycelium active substances in different concentrations/extraction solutions.

Figure 4 shows the survival rate test results of MDCK cells with different concentrations/extraction solutions of Tiger Milk Ganoderma lucidum mycelium active substances after first treating MDCK cells and then adding H1N1 virus.

Figure 5 shows the results of the MDCK cell viability test after treating MDCK cells with Tiger Milk Ganoderma lucidum mycelium active substances in different concentrations/extraction solutions together with H1N1 virus.

Figure 6 shows the MDCK cell viability test results after the H1N1 virus first treats MDCK cells, and then adds tiger milk Ganoderma lucidum mycelium active substances with different concentrations/extraction solutions.

TW Republic of China Food Industry Development Research Institute Biological Resources Conservation and Research Center 2018/12/24 930202

Claims (10)

A use of tiger milk ganoderma lucidum mycelium active substance is used to prepare a composition against enterovirus and/or influenza virus. The preparation method of tiger milk ganoderma lucidum mycelium active substance includes the following steps: (a) take one Tiger milk Ganoderma lucidum mycelia ( Lignosus rhinocerus mycelia) was cultured on a plate medium at a temperature of 15 to 30°C for 1 to 2 weeks; (b) The tiger milk Ganoderma lucidum mycelia after cultivation in step (a) was inoculated to one In the flask, cultivate for 3 to 14 days at a temperature of 15 to 30°C and pH 2 to 6; (c) Inoculate the tiger milk ganoderma lucidum mycelium after cultivation in step (b) in a fermentation tank at 15 to 30°C , Stir and cultivate for 3 to 21 days under a pH environment of 2 to 6, to form a tiger milk ganoderma lucidum mycelium fermentation broth; (d) freeze-dry the tiger milk ganoderma lucidum mycelium fermentation broth and pulverize to form a tiger milk ganoderma lucidum Mycelium freeze-dried powder; (e) extract the tiger milk ganoderma lucidum mycelium freeze-dried powder with at least one solvent to form a tiger milk ganoderma lucidum mycelium extract; and (f) the tiger milk ganoderma lucidum mycelium The extract is dried to obtain the active substance of the tiger milk ganoderma lucidum mycelium. The use as described in item 1 of the scope of patent application, wherein the enterovirus is type 71, or the influenza virus is type H1N1. The use described in item 1 of the scope of patent application, wherein in step (e), the solvent is selected from water, ethanol, ethyl acetate or hexane. The use described in item 1 of the scope of patent application, wherein the flask culture in step (b) is shaking culture, and the rotation speed is 110 to 130 rpm. The use described in item 1 of the scope of patent application, wherein in step (c), the fermentation tank is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof, and the tank pressure of the fermentation tank is 0.5 To 1.0 kg/cm ² and the ventilation rate is 0.1 to 1.5 VVM. The use as described in item 1 of the scope of patent application, wherein the tiger milk ganoderma lucidum mycelium active material comprises an aqueous extract, an ethanol extract, an ethyl acetate extract, a hexane extract of the tiger milk ganoderma lucidum mycelium or It's mixed. The use according to item 1 of the patent application, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, filler, absorption enhancer, sweetener or a combination thereof. The use described in item 1 of the scope of patent application, wherein the composition includes a medicine, feed, beverage, nutritional supplement, dairy product, food or health food. The use described in item 1 of the scope of patent application, wherein the form of the composition includes powder, lozenge, granulation, suppository, microcapsule, ampoule, liquid spray or suppository. The use as described in item 1 of the scope of patent application, wherein the tiger milk Ganoderma lucidum mycelium is selected from the tiger milk Ganoderma lucidum mycelium deposited at the Biological Resources Conservation and Research Center, and the deposit number is BCRC 930202.

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