KR20240034975A - Composition comprising extracts of mushroom mixed mycelia for preventing or treating pneumonia - Google Patents

Abstract

The present invention relates to a composition for preventing or treating pneumonia containing complex mushroom mycelium of chaga, reishi, and saenghang mushrooms, and a method for producing the same. The complex mushroom mycelium has excellent efficacy in suppressing edema in lung tissue and suppressing the expression of inflammatory cytokines TNF-α and IL-1β, and chemokines CXCL16, CXCL1, MCP1, and TARC, thereby preventing, treating, and improving pneumonia. It is possible to provide excellent pharmaceutical compositions or health functional foods.

Description

Composition comprising extracts of mushroom mixed mycelia for preventing or treating pneumonia}

The present invention relates to a composition for preventing or treating pneumonia containing the mycelium of three types of mushrooms: Chaga, Reishi, and Phellodendron mushroom.

Pneumonia is an inflammation of the lungs caused by infection with microorganisms such as bacteria, viruses, and fungi. Pneumonia is characterized by pulmonary symptoms that interfere with the normal function of the lungs, such as coughing, phlegm due to excretion of inflammatory substances, and difficulty breathing due to breathing problems, digestive symptoms such as nausea, vomiting, and diarrhea, and headaches, fatigue, muscle pain, and joint pain. It is a disease that causes systemic symptoms throughout the body. Pneumonia must be treated according to the cause, and most methods include using antibiotics. However, if the symptoms of pneumonia are severe, the disease may continue to progress and death may occur even if appropriate antibiotics are used.

Sanghwang mushroom ( Phellinus linteus ) is also called woody mud mushroom, and is recorded in the decoction section under the name Sangmoki (桑木耳) in Donguibogam. It grows naturally on mulberry tree trunks and is yellow except for the surface of the hat. At first, it looks like a lump of mud, but when it grows up, it looks like a tree stump with its tongue sticking out, so it is also called a tree stump. Sanghwang mushrooms have been used since ancient times for uterine bleeding, menstrual irregularities, etc., and have recently been reported to have excellent effects in suppressing tumors, strengthening immunity, and whitening.

Reishi ( Ganoderma lucidum ) grows on the roots of broadleaf trees in summer. It is also known as Qin Shi Huang's herb of immortality, and in the herbal medicine class, this mushroom is ranked among the best medicines along with ginseng. Reishi mushrooms are known to have tonic, anti-inflammatory, and anti-inflammatory effects, and are effective for respiratory diseases, nervous breakdown, heart disease, and high blood pressure. They also lower cholesterol and are known to have anti-cancer effects.

Chaga mushroom ( Inonotus obliquus ) is a sclerotium that grows naturally on birch and alder trees that grow in cold regions such as Russia, Canada, and Hokkaido, Japan. In the United States, chaga mushroom is classified as a special natural substance and is being developed as a future pharmaceutical and health food. In Korea, it was reported that chaga mushroom was used in the private sector for stomach cancer patients and diabetic patients, and that the effect was superior to that of other mushrooms. In addition, chaga mushrooms are reported to be effective in increasing body resistance, inhibiting tumor development, and whitening.

Meanwhile, in order to use the above-mentioned mushrooms as mixed raw materials for medicines and functional foods, mushrooms, which are fruiting bodies, must be collected and used. However, mushrooms themselves do not reproduce well in natural conditions, and overuse of collection causes resource depletion and destruction of the ecosystem. There is a problem. It is not possible to meet the demand for mass production due to the difficulty of requiring large production facilities and costs to supply raw materials sufficient for industrial use. Likewise, although the mushrooms are effective as anticancer agents or immune enhancers, their supply is limited and mass production and rapid production are not easy, so they are not widely used. Therefore, for the above mushrooms, research has recently been actively conducted on mass culturing methods that can produce mycelium that has the same effect as the fruiting body of the mushroom fungus.

Republic of Korea Patent No. 10-922311 discloses a method for complex culturing Chaga, Sanghwang, Reishi, cauliflower, and Cordyceps sinensis mycelium, and Republic of Korea Patent No. 10-1358648 discloses Chaga, Sanghwang, and Reishi mushrooms. The composite culture method of the Republic of Korea Patent No. 10-1652035 discloses a method of cultivating composite mushroom mycelium of Chaga mushroom, Sanghwang mushroom, and cauliflower mushroom. However, complex mushroom mycelium has differences in its own enzyme activity and beta-glucan content depending on the culture conditions, and when used to process food, it can produce different tastes and flavors, and also has different pharmacological efficacy. It flies.

Accordingly, the present inventors completed the present invention by confirming that the mycelium of three types of mushrooms obtained through a complex culture method and a composition using the same suppressed pulmonary edema and significantly suppressed inflammatory symptoms within lung cells.

Republic of Korea Registered Patent No. 10-1923408 (Title of Invention: Complex culture method of mycelium of Chaga, Reishi and Sanghwang mushrooms, Applicant: Giunchan Co., Ltd., Registration date: November 23, 2018) Republic of Korea Registered Patent No. 10-1652035 (Title of invention: Production method of composite mushroom mycelium of chaga mushroom, Sanghwang mushroom and cauliflower mushroom, Applicant: Giunchan Co., Ltd., Registration date: August 23, 2016) Republic of Korea Patent No. 10-1358648 (Title of Invention: Complex cultivation method of Chaga, Sanghwang, and Reishi mushrooms using mushroom extract, Applicant: Taebong Lee, Registration date: January 28, 2014) Republic of Korea Registered Patent No. 10-1269410 (Title of invention: Complex culture method of chaga mushroom, Sanghwang mushroom, Reishi mushroom, cauliflower mushroom, and Cordyceps sinensis using mushroom medium, Applicant: Taebong Lee, Registration date: May 24, 2013) Republic of Korea Patent No. 10-0922311 (Title of Invention: Method for culturing chaga, Sanghwang, Reishi, cauliflower and Cordyceps sinensis producing substances containing activated sugar-related compounds, Applicant: Taebong Lee, Registration date: 2009 October 12, 2018)

The purpose of the present invention is to provide a composition for preventing or treating pneumonia containing the composite mushroom mycelium of three types of mushrooms: chaga, reishi, and shanghai.

The present invention relates to a pharmaceutical composition for the prevention or treatment of pneumonia containing the complex mushroom mycelium of three types of chaga, reishi, and saenghang mushrooms or extracts thereof.

The composition can also be provided as a health functional food.

The composite mushroom mycelium includes the steps of: (first step) inoculating the fruiting body tissues of chaga, reishi, and sanghwang mushrooms into PDA (Potato Dextrose Agar) and separately cultivating each mushroom into a mycelium state;

(Step 2) Mixing and inoculating mycelium of each of the three types of Chaga, Reishi, and Sanghwang mushrooms cultured in the first step into PDB (Potato Dextrose Broth);

(Step 3) Obtaining complex culture mycelium by liquid culturing the three types of mycelium inoculated in PDB (Potato Dextrose Broth) medium for 4 to 6 weeks;

(Step 4) Inoculating and culturing the complex culture mycelium obtained through the third step of culturing on rice and barley medium; and,

It may have been cultured through .

The mycelium liquid culture in the third step is preferably performed at 25 to 30°C and a relative humidity of 10 to 30%. In addition, it is advisable to cultivate the culture statically for the first 1 to 2 weeks, with agitation for 1 to 5 minutes once or twice a day. Afterwards, shaking culture while stirring at 50 to 150 rpm is good for mycelium culture.

The rice barley medium in the fourth step is made by soaking the rice barley for 4 to 8 hours, dehydrating it, adding 0.5 to 2 parts by weight of calcium carbonate based on 100 parts by weight of the dehydrated rice barley, and then sterilizing it at 120 to 125°C for 30 minutes to 2 hours. It may have been obtained by doing so.

The mycelium liquid culture in the fifth step is preferably carried out at 25-30°C at a relative humidity of 40-60%, and it is advisable to perform stationary culture with stirring for 1-5 minutes once or twice a day.

PDA (Potato Dextrose Agar) used for culturing mushroom mycelium in the present invention is manufactured by sterilizing 3 to 5 g of potato starch, 10 to 30 g of dextrose, and 10 to 30 g of agarose, based on a total volume of 1 liter. You can. At this time, the remaining water can be added to bring the total volume to 1 liter.

The sterilization is preferably performed at 120 to 125°C for 15 to 20 minutes at a pressure of at least 1.3 to 1.7 atmospheres, and can be equally applied to sterilization conditions for all media.

The most preferable manufacturing conditions for PDA (Potato Dextrose Agar) may be sterilization of 4 g of potato starch, 20 g of dextrose, and 15 g of agarose by adding water to a total volume of 1 liter.

In addition, PDB (Potato Dextrose Broth) used for culturing mushroom mycelium in the present invention may be manufactured by adding water instead of agarose under the manufacturing conditions of PDA (Potato Dextrose Agar). The most preferable manufacturing conditions for PDB (Potato Dextrose Broth) may be sterilization of 4 g of potato starch and 20 g of dextrose by adding water to a total volume of 1 liter.

When cultivating each mushroom fruiting body separately in the first step, it is preferable to inoculate each mushroom fruiting body piece on PDA (Potato Dextrose Agar) and then culture it at 25-30°C for 1-3 weeks. The culture period can be appropriately adjusted within 1 to 3 weeks depending on the growth capacity of the mushroom fruiting body. Additionally, the relative humidity during cultivation under these conditions is preferably 10 to 60%. Even if the humidity is less than 10% or more than 60%, mycelium growth can be slow.

However, if the culture period is less than 1 week, the mycelium may not grow well enough to activate subsequent liquid culture, and even if it exceeds 3 weeks, mycelium culture may not work well during subsequent liquid culture. Additionally, even if the culture temperature is less than 25°C or more than 30°C, it is also undesirable because it affects mycelium culture during liquid culture.

When inoculating the three types of mycelium into PDB (Potato Dextrose Broth) in the second step, it is sufficient to inoculate a similar amount of mycelium from each mushroom cultured in the first step, but more preferably, each culture The mycelium can be cut into 0.5~2mm square pieces and inoculated with 3~7 pieces of each mushroom mycelium into PDB (Potato Dextrose Broth).

The mycelium culture in the third step is preferably performed at 25 to 30 ℃. In particular, if the culture temperature is less than 25 ℃, the mycelium may not grow well, and even if it exceeds 30 ℃, the culture of the mycelium may be slow. . In addition, it is advisable to cultivate the culture statically for the first 1 to 2 weeks, with agitation for 1 to 5 minutes once or twice a day. Afterwards, shaking culture while stirring at 50 to 150 rpm is good for mycelium culture. Meanwhile, the relative humidity during cultivation under these conditions is not greatly limited, but is preferably 10 to 30% relative humidity.

The rice barley medium of the fourth step can be inoculated with 1 to 10 ml of mycelium obtained through the third step of culture in the form of a culture solution. At this time, if the mycelium culture is inoculated with less than 1 ml, the amount of mycelium is small and additional culture may not work well, and even if the culture is inoculated with more than 10 ml, the culture is not further activated, which is not desirable.

The rice barley medium in the fourth step is made by soaking the rice barley for 4 to 8 hours, dehydrating it, adding 0.5 to 2 parts by weight of calcium carbonate based on 100 parts by weight of the dehydrated rice barley, and then sterilizing it at 120 to 125°C for 30 minutes to 2 hours. It may have been obtained by doing so. At this time, if the barley is not sufficiently soaked in water and soaked for less than 4 hours, the barley may not be used well as a nutrient source due to the lack of moisture during mycelium culture, and if soaked for more than 8 hours, more moisture will be used. Since it is not absorbed into rice and barley, it has the effect of only delaying the manufacturing time, which is not desirable.

The culturing in the fourth step is preferably performed at 25 to 30°C. If the culture temperature is less than 25℃, mycelium may not grow well, and if it exceeds 30℃, mycelium culture may also be slow. Additionally, it is better that the relative humidity during cultivation under these conditions is 40 to 60%. Mycelium growth may be slow even when humidity is below 40% or above 60%.

The three types of composite mushroom mycelium extract can be extracted from the three types of composite mushroom mycelium using water, C1 to C4 alcohol, or a mixed solution thereof as a solvent, and the C1 to C4 alcohol is methanol, ethanol, propanol, isopropanol, butanol, and It may be selected from the group consisting of isobutanol. More preferably, water can be used as a solvent, and the liquid may be freeze-dried after extraction for 8 to 15 hours by adding 5 to 20 times the weight of hot water at 70 to 75 ° C. compared to the weight of the three types of composite mushroom mycelium.

Water, C1 to C4 alcohol, or a mixed solution thereof used in the production of the three types of composite mushroom mycelium extract can be used in an amount 1 to 40 times the volume (1 to 40 liters per 1 kg) based on the weight of composite mushroom mycelium used, preferably 5 to 40 times the volume can be used. Extraction conditions for the three types of composite mushroom mycelium extract may be 1 minute to 48 hours at 20 to 100°C. The above process can be repeated 1 to 4 times.

In addition, as a common method in the art, water, C1 to C4 alcohol, or their mixed solution extracts of the three types of composite mushroom mycelium are dissolved in water and then dissolved in n-hexane, methylene chloride, acetone, chloroform, ethyl acetate, and n-butanol. It can be prepared as a fraction by additional fractionation using one or more solvents selected from the group consisting of.

In another method, water is added to the three types of composite mushroom mycelium extract obtained by extracting and concentrating the three types of composite mushroom mycelium with water, C1 to C4 alcohol, or a mixed solvent thereof, and then suspended, preferably the three types of composite mushrooms. After suspending the mycelium extract by adding 1 to 1000 times, more preferably 1 to 500 times, and most preferably 1 to 50 times the weight of water, the suspension is mixed with hexane, chloroform, ethyl acetate, and butanol. It can be prepared from a composite mushroom mycelium fraction obtained by adding a solvent selected from the group consisting of. The three types of composite mushroom mycelium fraction is preferably obtained by extracting and concentrating the three types of composite mushroom mycelium with water, C1 to C4 alcohol, or a mixed solvent thereof, suspending the three types of composite mushroom mycelium extract in water and then mixing it with hexane. A concentrate of the hexane layer obtained, a concentrate of the chloroform layer obtained by mixing chloroform with the residue (water layer) remaining after removing the hexane layer, or ethyl obtained by mixing ethyl acetate with the residue (water layer) remaining after removing the chloroform layer. It may be a concentrate of the acetate layer, a concentrate of the butanol layer obtained by mixing butanol with the residue (water layer) remaining after removing the ethyl acetate layer, or a concentrate of the residue (water layer) remaining after removing the butanol layer. Meanwhile, other fractionation conditions are not limited, but after preparing a suspension by adding 1 to 50 times the weight of the three types of composite mushroom mycelium extract to the above three types of composite mushroom mycelium extract, the same amount of hexane as the water was added. It can be fractionated by adding a solvent selected from the group consisting of chloroform, ethyl acetate, and butanol. In addition, when chloroform is added to the residue remaining after removing the hexane layer, ethyl acetate is added to the residue remaining after removing the chloroform layer, and butanol is added to the residue remaining after removing the ethyl acetate, even when done in stages, the same amount of the residue as the residue is added. It can be fractionated by sequentially adding each solvent (chloroform, ethyl acetate, or butanol).

The production temperature of the extract or fraction thereof may be 20 to 100° C., but is not limited thereto. The extraction or fractionation time is not particularly limited, but is preferably extracted within 10 minutes to 2 days, and the extraction device may be a conventional extraction device, an ultrasonic grinding extractor, or a fractionator. The extract or fraction prepared in this way can be dried with hot air, dried under reduced pressure, or freeze-dried to remove the solvent. Additionally, the extract or fraction can be purified and used using column chromatography.

The extract may be prepared alone or appropriately using known methods used for separation and extraction of plant components, such as extraction with organic solvents (alcohol, ether, acetone, etc.), distribution of hexane and water, and column chromatography, according to commercial methods. It can be used after fractionation or purification using a combined method.

The chromatography includes silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, and medium pressure liquid chromatography. chromatography), thin layer chromatography (TLC), silica gel vacuum liquid chromatography, and high performance liquid chromatography.

The present invention can provide a pharmaceutical composition for preventing or treating pneumonia comprising three types of complex mushroom mycelium, or extracts thereof, and pharmaceutical excipients.

The pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or the complex mushroom mycelium of the present invention. It is prepared by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection. The complex culture mycelium of the present invention has little toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for preventive purposes.

In addition, the present invention provides a health functional food for preventing or improving pneumonia containing three types of complex mushroom mycelium, or extracts thereof, and various food excipients. In addition, the above health functional food can also be used as a health functional food for preventing or improving dementia. The mycelium can be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes the form of tablets, capsules, pills, or liquid, and foods to which the extract of the present invention can be added include, for example, various drinks, meat, sausages, bread, candy, These include snacks, noodles, ice cream, dairy products, soups, electrolyte beverages, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

The present invention relates to a composition for preventing or treating pneumonia containing complex mushroom mycelium of chaga, reishi, and saenghang mushrooms, and a method for producing the same. The complex mushroom mycelium has excellent efficacy in suppressing edema in lung tissue and suppressing the expression of inflammatory cytokines TNF-α and IL-1β, and chemokines CXCL16, CXCL1, MCP1, and TARC, thereby preventing, treating, and improving pneumonia. It is possible to provide excellent pharmaceutical compositions or health functional foods.

Figure 1 is a schematic diagram showing the process of producing an experimental animal model of the present invention.
Figure 2 shows the body temperature of the experimental animal group after administration of dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 3 shows the pulmonary edema state of experimental animals administered dexamethasone, the composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 4 shows the expression level of TNF-α in the lung tissue of experimental animals administered dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 5 shows the expression level of IL-1β in the lung tissue of experimental animals administered dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 6 shows the expression level of CXCL16 in the lung tissue of experimental animals administered dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 7 shows the expression level of CXCL1 in the lung tissue of experimental animals administered dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 8 shows the expression level of MCP1 in the lung tissue of experimental animals administered dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.
Figure 9 shows the expression level of TARC in the lung tissue of experimental animals administered dexamethasone, composite mushroom mycelium (GMK-Immune) of Example 1, and LPS.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the content introduced here is thorough and complete, and to sufficiently convey the spirit of the present invention to those skilled in the art.

<Example 1. Complex culture of mushroom mycelium>

The fruiting body tissues of Chaga, Reishi, and Sanghwang mushrooms were separated and inoculated into PDA, and the mycelium of each mushroom was cultured at 27-29°C for 2 weeks. After preparing PDB medium divided into 100 ㎖ units, each mushroom mycelium cultured in PDA was cut into 1 mm square pieces using a scalpel, and 5 sections of the three types of mycelium were mixed per bottle of Erlenmeyer flask containing PDB medium. Inoculated.

The medium inoculated with mixed mycelium was cultured in a BOD incubator (Bio-Oxygen Demand incubator, low-temperature incubator) at 27-28°C and 20% humidity for one week, and stirred for about 1 minute every day during incubation. One week later, the complex inoculated and cultured flask was transferred to a shaking incubator and cultured at 27°C and 100 rpm for 4 weeks to prepare three types of complex mushroom mycelium cultures.

Rice barley was soaked for 6 hours, then dehydrated for 8 hours, and 1 g of calcium carbonate based on 100 g of dehydrated rice barley was added, mixed evenly, and sterilized in an autoclave at 121°C for 1 hour to prepare a rice barley medium. After sterilization was completed, 5 ml of complex mushroom mycelium culture cultured for 5 weeks was dispensed and inoculated per 1 kg of rice and barley medium cooled to 25°C. After inoculation, the cells were cultured for 30 days in a culture room maintained at a temperature of 26-28°C and humidity of 45-50%.

The cultured composite mushroom mycelium containing rice barley was dried at 57-60°C for 24 hours using a dryer and pulverized using a pin mill grinder to make powder.

Meanwhile, this method is the same as the method disclosed in Example 1 of Republic of Korea Patent No. 10-1923408.

<Comparative Examples 1 to 8. Cultivation of mushrooms under comparative conditions>

Comparative Example 1

In the method of Example 1, instead of complex inoculating the mycelium of the three types of mushrooms, only the chaga mycelium was singlely inoculated into the PDB, and the subsequent culturing process was the same.

Comparative Example 2

In the method of Example 1, instead of complex inoculation with the mycelium of three types of mushrooms, only reishi mushroom mycelium was singlely inoculated into the PDB, and the subsequent culture process was the same.

Comparative Example 3

In the method of Example 1, instead of complex inoculation with the mycelium of three types of mushrooms, only Sanghwang mushroom mycelium was single-inoculated into PDB, and the subsequent culture process was the same.

Comparative Example 4

In the method of Example 1, the PDB culture step was omitted, and the mycelium of the three types of mushrooms was directly inoculated into the rice and barley medium, and instead, the culture period was additionally performed as the PDB culture step.

Comparative Example 5

After performing the PDB culture step in the method of Example 1, the cells were re-inoculated into new PDB medium and additional liquid culture was performed for 30 days (culturing in rice and barley medium was omitted).

Comparative Example 6

Composite mushroom mycelium of Chaga mushroom, Sanghwang mushroom, and cauliflower mushroom was prepared by the method of Preparation Example 1 and Example 1 of Republic of Korea Patent No. 10-1652035.

Comparative Example 7

As in Comparative Example 2, composite mushroom mycelium was prepared by the method of Preparation Example 1 and Example 1 of Korean Patent No. 10-1652035, except that reishi mushrooms were used instead of cauliflower mushrooms.

Comparative Example 8

Composite mushroom mycelium of Chaga, Reishi, and Sanghwang mushroom mycelium was prepared by the method of Example 1 of Republic of Korea Patent No. 10-1358648.

<Experimental Example 1. Preparation of experimental animals and sample administration>

Five 6-week-old BALB/c mice were prepared per group. Pneumonia was induced by intranasal administration of LPS at 1㎍/50㎕/mouse. The positive control group was orally administered 3 mg/kg of Dexamethasone, an adrenocortical hormone with anti-inflammatory activity, and 5 mg/mouse of complex mushroom mycelium (GMK-Immune) of Example 1. Dexamethasone was administered orally 2 days prior to LPS administration, and composite mushroom mycelium (GMK-Immune) of Example 1 was administered orally 1 day prior to LPS administration. Distilled water was used as a placebo (vehicle) (control). Each mouse was sacrificed at 18 hours of age. The process of inducing pneumonia and administering samples is shown in Figure 1.

<Experimental Example 2. Checking body temperature and pulmonary edema>

The body temperature and pulmonary edema status of each mouse group prepared in Experimental Example 1 were confirmed.

As a result, as shown in FIG. 2, it can be seen that the body temperature raised due to LPS in the group ingesting complex mushroom mycelium (GMK-Immune) in Example 1 had a similar effect in lowering the body temperature as the group ingesting Dexamethasone.

In addition, lung tissue samples were collected, and the wet/dry weight ratio (W/D) was measured to confirm the pulmonary edema state. Referring to FIG. 3, the pulmonary edema increased due to LPS administration was also confirmed in Example 1. It can be seen that the pulmonary edema was almost alleviated in the group that consumed composite mushroom mycelium (GMK-Immune), returning to the level of the normal group.

<Experimental Example 4. Confirmation of inflammatory cytokines in lung tissue>

The content of inflammatory cytokines in the lung tissue of each mouse group prepared in Experimental Example 1 was confirmed using ELISA (abcam).

As a result, as shown in Figures 4 and 5, the complex mushroom mycelium (GMK-Immune) intake group of Example 1 significantly reduced the expression of TNF-α (Tumor Necrosis Factor-α) like the dexamethasone intake group, It can be confirmed that IL-1β (Interleukin 1β) is also inhibited by more than 50%.

<Experimental Example 5. Confirmation of chemokine inhibition efficacy in lung tissue>

The content of chemokines in the lung tissue of each mouse group prepared in Experimental Example 1 was confirmed using ELISA (abcam).

As a result, as shown in Figures 6 to 9, the chemokines CXCL16 (C-X-C Motif Chemokine Ligand 16), CXCL1 (C-X-C Motif Chemokine Ligand 1), MCP1 (Monocyte chemoattractant protein-1), and TARC (Thymus and activation-regulated chemokine) Although it increased with LPS administration, its expression decreased in all groups ingesting complex mushroom mycelium (GMK-Immune) in Example 1.

<Experimental Example 6. Experiment to suppress inflammatory response related to lung cells>

Inflammatory responses were confirmed in Calu-3 lung cells and PC-14 lung cancer cells. The two types of cells were placed in a 96 well plate at 1x10 ⁴ /well and then induced by treatment with 100 ng/ml LPS (Lipopolysaccharide) for 24 hours. The anti-inflammatory activity of the three types of composite mushroom mycelium extracts was measured by pre-treating these samples with 10 μg/ml for 12 hours before treating each cell with LPS. Anti-inflammatory activity is performed using the NO production kit and ELISA (abcam) kit to measure inflammatory mediators secreted in cell culture, such as Nitric Oxide (NO), TNF-α (Tumor Necrosis Factor-α), and IL-6 (Interleukin 6). It was determined by quantification and is shown in Table 1. The results of the untreated group were so close to 0 that they were not indicated.

each mycelium
extract Inhibitory effect on Calu-3 Inhibition effect in PC-14 NO
(μM) TNF-α
(pg/ml) IL-6
(pg/ml) NO
(μM) TNF-α
(pg/ml) IL-6
(pg/ml) LPS 4.5 394.0 1623.5 4.2 353.1 1423.4 LPS + Example 1 1.2 78.6 532.2 2.5 124.3 789.2 LPS + Comparative Example 1 3.1 225.5 51.0 3.1 211.0 1292.9 LPS + Comparative Example 2 3.2 352.2 12.2 3.0 252.3 1096.0 LPS + Comparative Example 3 3.5 211.0 94.3 3.4 225.5 902.3 LPS + Comparative Example 4 3.2 310.1 91.5 3.1 192.1 1156.6 LPS + Comparative Example 5 3.2 251.3 82.1 3.0 250.2 1222.3 LPS + Comparative Example 6 2.3 280.5 41.0 3.1 313.0 1230.2 LPS + Comparative Example 7 3.1 342.2 22.3 3.2 321.1 1313.4 LPS + Comparative Example 8 3.2 291.3 24.2 3.3 232.2 910.6

Regarding the inflammatory response induced by LPS, the three types of composite mushroom mycelium significantly inhibited the production of inflammatory mediators such as NO, TNF-α, and IL-6 (Table 1). These results confirm that the pneumonia treatment effect of the three types of composite mushroom mycelium extract is effective in both animals and cells.

<Formulation example 1. Pharmaceutical formulation>

Formulation Example 1-1. manufacture of tablets

200 g of the composite mushroom mycelium of Example 1 of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. A 10% gelatin solution was added to this mixture, then pulverized and passed through a 14 mesh sieve. This was dried and 160g of potato starch, 50g of talc, and 5g of magnesium stearate were added to make the resulting mixture into tablets.

<Formulation example 2. Food production>

Formulation Example 2-1. Manufacturing of cooking seasoning

A cooking seasoning for health promotion was prepared by adding 1% by weight of the composite mushroom mycelium of Example 1 of the present invention to the cooking seasoning.

Formulation Example 2-2. Manufacturing of flour foods

The composite mushroom mycelium of Example 1 of the present invention was added to flour at 0.1% by weight, and this mixture was used to prepare bread, cakes, cookies, crackers, and noodles to prepare food for health promotion.

Formulation Example 2-3. Preparation of soups and gravies

The complex mushroom mycelium of Example 1 of the present invention was added at 0.1% by weight to soup and gravy to prepare soup and gravy for health promotion.

Formulation Example 2-4. Manufacturing of dairy products

The composite mushroom mycelium of Example 1 of the present invention was added to milk at 0.1% by weight, and various dairy products such as butter and ice cream were manufactured using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

Vegetable juice for health promotion was prepared by adding 0.5 g of the composite mushroom mycelium of Example 1 of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Fruit juice manufacturing

Health-promoting fruit juice was prepared by adding 0.1 g of the composite mushroom mycelium of Example 1 of the present invention to 1,000 ml of apple juice or grape juice.

Claims (12)

A pharmaceutical composition for the prevention or treatment of pneumonia, characterized in that it contains complex mushroom mycelium of chaga mushroom, reishi mushroom, and sanghwang mushroom. According to paragraph 1,
The composition is a pharmaceutical composition for preventing or treating pneumonia, characterized in that it has the effect of reducing the expression of one or more inflammatory cytokines among Tumor Necrosis Factor-α (TNF-α) and Interleukin 1β (IL-1β). According to paragraph 1,
The composition contains one or more chemokines selected from the group consisting of CXCL16 (CXC Motif Chemokine Ligand 16), CXCL1 (CXC Motif Chemokine Ligand 1), MCP1 (Monocyte chemoattractant protein-1), and TARC (Thymus and activation-regulated chemokine). A pharmaceutical composition for the prevention or treatment of pneumonia, characterized in that it has the effect of reducing expression. A health functional food for preventing or improving pneumonia, characterized by containing complex mushroom mycelium of chaga mushroom, reishi mushroom, and sanghwang mushroom. According to paragraph 4,
The health functional food is a health functional food for preventing or improving pneumonia, characterized in that it has the effect of reducing the expression of one or more inflammatory cytokines among TNF-α (Tumor Necrosis Factor-α) and IL-1β (Interleukin 1β). According to clause 4,
The health functional food is one or more types selected from the group consisting of CXCL16 (CXC Motif Chemokine Ligand 16), CXCL1 (CXC Motif Chemokine Ligand 1), MCP1 (Monocyte chemoattractant protein-1), and TARC (Thymus and activation-regulated chemokine). A health functional food for preventing or improving pneumonia, characterized by the effect of reducing the expression of chemokines. (Step 1) Inoculating the fruiting body tissues of Chaga, Reishi, and Sanghwang mushrooms on PDA (Potato Dextrose Agar) and separately cultivating each mushroom into a mycelium state;
(Step 2) Mixing and inoculating mycelium of each of the three types of Chaga, Reishi, and Sanghwang mushrooms cultured in the first step into PDB (Potato Dextrose Broth);
(Step 3) Obtaining complex culture mycelium by liquid culturing the three types of mycelium inoculated in PDB (Potato Dextrose Broth) medium for 4 to 6 weeks;
(Step 4) Inoculating and culturing the complex culture mycelium obtained through the third step of culturing on rice and barley medium; and,
(Step 5) Obtaining composite mushroom mycelium by further liquid culturing the mycelium cultured in rice and barley medium for 4 to 7 weeks;
A pharmaceutical composition for the prevention or treatment of pneumonia, characterized in that it contains cultured composite mushroom mycelium. In clause 7,
The composition is a pharmaceutical composition for preventing or treating pneumonia, characterized in that it has the effect of reducing the expression of one or more inflammatory cytokines among Tumor Necrosis Factor-α (TNF-α) and Interleukin 1β (IL-1β). In clause 7,
The composition contains one or more chemokines selected from the group consisting of CXCL16 (CXC Motif Chemokine Ligand 16), CXCL1 (CXC Motif Chemokine Ligand 1), MCP1 (Monocyte chemoattractant protein-1), and TARC (Thymus and activation-regulated chemokine). A pharmaceutical composition for the prevention or treatment of pneumonia, characterized in that it has the effect of reducing expression. (Step 1) Inoculating the fruiting body tissues of Chaga, Reishi, and Sanghwang mushrooms on PDA (Potato Dextrose Agar) and separately cultivating each mushroom into a mycelium state;
(Second step) Mixing and inoculating the three types of mycelium, Chaga, Reishi, and Phellodendron mushrooms cultured in the first step, into PDB (Potato Dextrose Broth);
(Step 3) Obtaining complex culture mycelium by liquid culturing the three types of mycelium inoculated in PDB (Potato Dextrose Broth) medium for 4 to 6 weeks;
(Step 4) Inoculating and culturing the complex culture mycelium obtained through the third step of culturing on rice and barley medium; and,
(Step 5) Obtaining composite mushroom mycelium by further liquid culturing the mycelium cultured in rice and barley medium for 4 to 7 weeks;
A health functional food for preventing or improving pneumonia, characterized in that it contains cultured complex mushroom mycelium. According to clause 10,
The health functional food is a health functional food for preventing or improving pneumonia, characterized in that it has the effect of reducing the expression of one or more inflammatory cytokines among TNF-α (Tumor Necrosis Factor-α) and IL-1β (Interleukin 1β). According to clause 10,
The health functional food is one or more types selected from the group consisting of CXCL16 (CXC Motif Chemokine Ligand 16), CXCL1 (CXC Motif Chemokine Ligand 1), MCP1 (Monocyte chemoattractant protein-1), and TARC (Thymus and activation-regulated chemokine). A health functional food for preventing or improving pneumonia, characterized by the effect of reducing the expression of chemokines.