KR102668370B1 - Pharmaceutical composition for treating arthritis containing ganoderma lucidum spore supercritical extract - Google Patents

Abstract

The present invention relates to a composition and health functional food for preventing or treating arthritis containing a supercritical extract of reishi mushroom spores, and a method for preventing or treating arthritis using the composition.

Description

Pharmaceutical composition for treating arthritis comprising supercritical extract of reishi mushroom spores {PHARMACEUTICAL COMPOSITION FOR TREATING ARTHRITIS CONTAINING GANODERMA LUCIDUM SPORE SUPERCRITICAL EXTRACT}

The present invention relates to a composition for preventing or treating arthritis and a health functional food containing a supercritical extract of reishi mushroom spores, a method for preventing or treating arthritis using the composition, and a method for producing the supercritical extract of reishi mushrooms.

Rheumatoid arthritis (RA) is a systemic autoimmune disease that affects 0.5-1.0% of the world's population and causes chronic symptoms such as joint swelling, stiffness, and immune senescence. In particular, immune aging is a major cause of increased risk of disability in elderly people with rheumatoid arthritis. In addition, RA directly or indirectly affects systemic symptoms and inflammation, which may increase the likelihood of complications such as cardiovascular disease and osteoporosis. There are various reasons for the onset of RA, including genetics, infectious diseases, environmental changes, and hormonal influences. Additionally, research has shown that the onset of RA is influenced by the complex interaction and activation of innate and adaptive immune cells. However, the mechanism of RA is still not clearly known.

DMARDs (disease-modifying antirheumatic drugs) are used to prevent joint damage in the early stages of RA. Anti-rheumatic drugs are often used to treat or slow the progression of RA, including methotrexate, hydroxychloroquine, sulfasalazine, and leflunomide. Excessive use of these drugs may cause digestive problems or other side effects, which in some cases may lead to ulcers or perforations. In addition, arthritis treatment drugs may cause liver and kidney damage, causing symptoms such as skin rash, hives, headache, dizziness, and drowsiness, and some patients may also develop high blood pressure and edema. Therefore, the development of effective alternative drugs that can reduce these side effects is urgently needed.

Reishi ( Ganoderma lucidum ; GL) is an annual mushroom that grows in salt water in summer and has a strong bitter taste. It is a medicinal mushroom known as 'Ling zhi' in China and 'Rei shi' in Japan. Reishi mushroom spores (GLS) are released from reishi mushrooms in the form of brown ovals. Reishi mushrooms have been used as an effective medicine for improving heart function, increasing memory, and anti-aging for over 2,000 years. Organic germanium reishi mushrooms are considered useful for relieving cough and asthma, dizziness, insomnia, and breathing difficulties. Physiologically active components of reishi mushroom fruiting bodies include beta-glucans, sterols, triterpenes, and selenium. The spores of reishi mushrooms have higher bioactivity than the fruiting bodies, and are attracting attention as a promising drug that can be used for multiple purposes. The physiologically active components of reishi mushroom spores include polysaccharides, triterpenoids, peptides, amino acids, fatty acids and trace elements. These biologically active ingredients have been studied to have immune-modulating, anti-aging, blood lipid-lowering, antiviral, and anticancer properties. Many studies have proven the anti-cancer, anti-pain, and immune-enhancing effects of reishi mushroom fruits and mycelium. However, the effectiveness of reishi mushroom spore oil in rheumatoid arthritis has not yet been proven.

Korean Patent No. 10-1156775

The inventors of the present invention completed the present invention by producing a reishi mushroom spore extract using supercritical CO ₂ and confirming its effect on improving rheumatoid arthritis in an animal model.

Accordingly, the present invention provides a composition for preventing or treating arthritis and a health functional food containing a supercritical extract of reishi mushroom spores, a method for preventing or treating arthritis using the composition, and a method for producing a supercritical extract of reishi mushroom spores. The purpose is to

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating arthritis containing a supercritical extract of Ganoderma lucidum spore (GLS) as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving arthritis containing a supercritical extract of Ganoderma lucidum spore (GLS) as an active ingredient.

Additionally, the present invention provides a method for preventing or treating arthritis comprising administering the pharmaceutical composition according to the present invention to a patient.

In addition, the present invention includes the steps of extracting powder of reishi mushroom spores using supercritical carbon dioxide under conditions of 30 to 50 MPa and 45 to 65°C; A step in which the supercritical extract of the extracted reishi mushroom spores and carbon dioxide are circulated within two separators and kinetic extraction is performed; and collecting the supercritical extract of the extracted reishi mushroom spores in a first separator.

The supercritical extract of reishi mushroom spores of the present invention is a natural product, is biocompatible, has no side effects, and can effectively alleviate various symptoms of rheumatoid arthritis, making it effective in the treatment of arthritis.

1 is (a) a schematic diagram of a supercritical extraction device. (b) GC-MS chromatogram of supercritical CO ₂ extracted GLS oil.
Figure 2 shows the effect of GLS oil on clinical scores in the CIA model: (a) Schematic diagram showing the experimental plan and animal group classification. Clinical score (0-4) (b) and paw thickness (c) were measured every 3 days during the experimental period. (d) This is a photograph showing the difference in foot swelling between experimental groups.
Figure 3 shows that GLS oil relieves pain in the CIA model. The von Frey test (a) and hot plate test (b) for pain response were performed once a week for 7 weeks after immunization.
Figure 4 shows that GLS oil alleviates cartilage degeneration, synovitis, and pannus formation in the knee of a CIA model. Cartilage degeneration and erosion were analyzed by OARSI staining (a), and quantitative results were shown (b). In inflammation, knee synovitis was analyzed by hematoxylin and eosin staining (c), and quantitative results were shown (d). Pannus formation indicating passage of immune cells into the knee is shown (e), and quantitative results are shown (f). Abbreviation: OARSI, Osteoarthritis Research Society International.
Figure 5 shows that GLS oil alleviates ankle cartilage degeneration, synovitis, and pannus formation in a CIA model. Cartilage damage in the ankle was visualized by OARSI staining (a), with quantitative results shown (b). The degree of inflammation in the ankle synovium is shown (c), and quantitative results are shown (d). The degree of inflammation infiltration into bone is visualized (e), and quantitative results are shown (f). Abbreviation: OARSI, Osteoarthritis Research Society International.
Figure 6 shows that GLS oil alleviates plantar cartilage destruction, synovitis, and pannus formation in the CIA model. The degree of cartilage degeneration and damage was visualized with OARSI staining (a), and the OARSI grade was quantified (b). The degree of inflammation (c) and synovitis scoring (d) in the synovial membrane of the foot were shown. The degree of immune cell infiltration into bone (e) and the results of the pannus formation score (f) are shown.
Figure 7 shows that GLS oil attenuates immune cell activation in the CIA model. Eosinophilic leukocyte infiltration in the knee and ankle was analyzed by Congo red staining (a). Quantification of eosinophilic leukocyte infiltration in the knee and ankle is shown in (b) and (c), respectively. Additionally, neutrophil infiltration in the knee and ankle was shown in (d) and (e) with CDr15 staining. Quantification of infiltration of eosinophilic leukocytes in the knee (f) and ankle (g) is shown. Finally, the number and activity of mast cells in the knee and ankle were analyzed by toluidine blue staining (h). Total mast cell counts and degranulated mast cells in the knee and ankle are shown in (i) and (j), respectively.

Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of techniques well known to those skilled in the art may be omitted. Additionally, when describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

The present invention relates to a pharmaceutical composition for preventing or treating arthritis and a health functional food containing a supercritical extract of Ganoderma lucidum spore (GLS) as an active ingredient.

The supercritical extract of reishi mushroom spores is extracted in a supercritical extraction system having one extractor and two separators, and carbon dioxide can be used as the supercritical fluid, but is not limited thereto. The pressure and temperature of the extractor may be 30 to 50 MPa and 45 to 65°C, respectively, for example, 40 MPa and 55°C, but are not limited thereto. The temperature and pressure of the first of the two separators may be 40 to 60°C and 10 to 16 MPa, for example, 50°C and 13 MPa, and the temperature and pressure of the second separator may be 30 to 50°C and 3 to 13 MPa. 7 MPa, for example, may be, but is not limited to, 40° C. and 5 MPa.

The supercritical extract of the reishi mushroom spores contains oleic acid, linoleic acid, palmitate acid, elaidic acid, palmitoleate acid, and stearate acid. It may include, but is not limited to, acid, palmitic acid, fatty acid methyl ester, stearic acid, and pentadecanoic acid.

The arthritis may be osteoarthritis or rheumatoid arthritis, but is not limited thereto.

The composition comprising the supercritical extract of reishi mushroom spores according to the present invention as an active ingredient may contain one or more pharmaceutically acceptable carriers, excipients, or diluents in addition to a pharmaceutically effective amount of the supercritical extract of reishi mushroom spores. .

In the above, “pharmaceutically effective amount” refers to an amount sufficient to exhibit the desired physiological or pharmacological activity when the bioactive ingredient is administered to animals or humans. However, the pharmaceutically effective amount may vary appropriately depending on the severity of symptoms, the patient's age, weight, health status, gender, administration route, and treatment period.

In addition, “pharmacologically acceptable” as used herein refers to something that is physiologically acceptable and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions when administered to humans. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Examples include polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

Additionally, the composition of the present invention can be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal, and can be formulated in a variety of ways for oral or parenteral administration. It can be formulated in any form. Dosage forms may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, or sterile powders.

The composition according to the present invention can be administered through several routes, including orally, transdermally, subcutaneously, intravenously, or intramuscularly, and the dosage of the active ingredient is determined by various factors such as the route of administration, the patient's age, gender, weight, and patient's severity. It can be appropriately selected depending on. Additionally, the composition of the present invention can be administered in combination with known compounds that can enhance the desired effect.

Furthermore, the composition according to the present invention can not only be used as a pharmaceutical composition as described above, but can also be used as a health functional food. For example, it can be easily used as a main ingredient, secondary ingredient, food additive, functional food or beverage.

The term “food” refers to a natural product or processed product containing one or more nutrients, preferably in a state that can be eaten directly after a certain degree of processing. In the usual sense, it means food. , which includes all food additives, functional foods, and beverages.

Foods to which the food composition can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, functional foods, etc. Additionally, special nutritional foods (e.g., milk formula, infant and baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g., ramen, noodles, etc.), breads, health supplements, seasoned foods (e.g., soy sauce) , soybean paste, red pepper paste, mixed paste, etc.), sauces, confectionery (e.g., snacks), candy, chocolate, gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi) , pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented drinks, etc.), and natural seasonings (e.g., ramen soup, etc.), but are not limited thereto. The food, beverage or food additive can be manufactured by conventional manufacturing methods.

In addition, the above-mentioned “functional food” or “health functional food” refers to a food group or food composition that has added value to the food by using physical, biochemical, bioengineering methods, etc. to function and express the function of the food for a specific purpose. It refers to food designed and processed to fully express the body's regulatory functions related to defense rhythm control, disease prevention and recovery, etc., and specifically, it may be a health functional food. The functional food may include food auxiliary additives that are foodologically acceptable, and may further include appropriate carriers, excipients, and diluents commonly used in the production of functional foods.

The type of health supplement is not limited thereto, but may be in the form of powder, granule, tablet, capsule, or beverage.

Additionally, the present invention provides a method for preventing or treating arthritis comprising administering the pharmaceutical composition according to the present invention to a patient.

The treatment method of the present invention includes administering the pharmaceutical composition to a subject in a therapeutically effective amount. The specific therapeutically effective amount for a specific subject will depend on the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the subject's age, weight, general health, gender and diet, and time of administration. It is desirable to apply it differently depending on various factors including the route of administration, secretion rate of the composition, treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. Therefore, it is desirable to determine the effective amount of the composition suitable for the purpose of the present invention by considering the above-mentioned matters.

The composition is applicable to any mammal, including humans and primates, as well as domestic animals such as cattle, pigs, sheep, horses, dogs and cats.

In addition, the present invention includes the steps of extracting powder of reishi mushroom spores using supercritical carbon dioxide under conditions of 30 to 50 MPa and 45 to 65°C; A step in which the supercritical extract of the extracted reishi mushroom spores and carbon dioxide are circulated within two separators and kinetic extraction is performed; and collecting the supercritical extract of the extracted reishi mushroom spores in a first separator.

The pressure and temperature of the extractor may be, for example, 40 MPa and 55° C., but are not limited thereto. The temperature and pressure of the first of the two separators may be 40 to 60°C and 10 to 16 MPa, for example, 50°C and 13 MPa, and the temperature and pressure of the second separator may be 30 to 50°C and 3 to 13 MPa. 7 MPa, for example, may be, but is not limited to, 40° C. and 5 MPa.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrating the present invention in detail, and the scope of the present invention is not limited to these examples.

<Manufacturing Example 1>

Sample preparation and materials

Chemicals and Falcon Labware are owned by Sigma Chemical Co. (St. Louis, MO, USA). Experimental equipment was purchased from Becton-Dickinson (Franklin Lakes, NJ, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and recombinant human IL-6 (PHC0064) were purchased from Thermo Fisher Scientific (Grand Island, NY, USA).

Supercritical CO ₂ Preparation of GLS oil extract by

GLS oil was extracted using a supercritical CO ₂ extraction method. Figure 1a shows Nantong Wisdom Supercritical Science and Technology Development Co., Ltd. This is a schematic diagram of the extraction equipment manufactured in (China). The system includes a 5 L extractor, two separators (separator 1: 3 L, separator 2: 2 L), a cooler, and a high pressure CO ₂ pump to transfer CO ₂ from the CO ₂ cylinder to the system. All vessels were heated with a heating jacket. In a typical experiment, 2 kg of GLS powder was placed in an extractor and a specified amount of CO ₂ was pumped into the system. Then, valve 9 (V9) was opened to empty the system. Removal of CO ₂ was repeated three times and replaced with air in the system. After removal, all valves except V1 and V2 were closed and a certain amount of CO ₂ was pumped into the extractor. When the pressure and temperature of the extractor reached 40 MPa and 55°C, V5, V8, and V11 were opened for CO ₂ circulation and kinetic extraction. The temperature and pressure of separators 1 and 2 were set to 50°C, 13 MPa, and 40°C, 5 MPa, respectively. The CO ₂ mass flow rate was maintained at 35 kg/h and the extraction process continued for 2 hours. After extraction was complete, GLS oil was collected through V7 and stored for further experiments.

<Example 1>

Analysis of GLS oil by gas chromatography-mass spectrometry (GC-MS)

Samples were purified to the corresponding Converted to fatty acid methyl ester (FAMEs). Total fatty acids and fatty acid content of oil samples were measured simultaneously in two steps: (1) preparation of FAME (2) and chromatographic analysis. 100 μl of GLS was added to a test tube with a stopper and 4 mL of potassium hydroxide/methanol solution (0.5 M) was added. The mixture was mixed and evaporated for 15 minutes using a rotary evaporator. Then 1.6 mL of methanol:hydrochloric acid was added and the sample was mixed again for 10 minutes. Then, 6 mL of n-hexane was added to the sample and mixed until the two layers separated. To recover the methyl ester in the organic phase, n-hexane in the upper layer was extracted into a new extraction tube. A certain amount of anhydrous sodium sulfate was added to n-hexane to dehydrate it, and the mixture was left to stand. Then, the supernatant was subjected to GC-MS analysis using a Shimadzu QP2010 SE gas chromatography-mass spectrometer (Shimadzu, Kyoto, Japan) equipped with a DB-FFAP (25 m × 0.32 mm × 0.5 μm) column (Agilent, United States). The injector temperature was set at 250°C and the column temperature was set at 50-240°C. The initial column temperature was maintained at 50°C for 2 minutes, then raised to 180°C at a rate of 10°C/min and held for 5 minutes, and then finally raised to 240°C at a rate of 5°C/min and maintained for 25 minutes. Fatty acid methyl ester was separated and the peak was confirmed by comparing the mass spectrum with the mass spectral library database.

Preparation of experimental RA model and von Frey and hot-plate assays

All experiments were organized under the approval of Jeju National University's Animal Experiment Ethics Committee (2022-0002). Seven-week-old DBA/1 J mice were used as an experimental RA model. Equal volumes of type 2 collagen and 4 mg/mL of intact Freund's adjuvant were mixed and injected into the tail skin of mice anesthetized with isoprofen.

Seven days after arthritis induction, the von Frey test was performed weekly. First, the sole of the mouse's foot was pierced with a thick filament and the time when the mouse took a step was recorded. Each stab was performed 5 times, and the filament size when the paw was stabbed more than 3 times was recorded. If the mouse did not respond, the filament was replaced with a lower filament and further analysis was performed. Hot-plate assays were performed using the Plantar Test for Thermal Stimulation-Hargreaves Apparatus (ugo basile®, 37570, Gemonio, Italy). The experimental mouse was placed in five different locations, and the time (seconds) until the mouse left the base plate was recorded. When the mouse licked or jumped, the reaction time was recorded for accurate measurement. Each leg was measured 5 times, and the average value was recorded.

GLS oil injection

GLS oil was injected 1 week after CIA induction. GLS oil was mixed with polyethylene glycol 400 (PEG-400) and injected intraperitoneally (200 μL) twice a week for 7 weeks. The control group was injected with 200 μL PEG-400.

Clinical score and foot thickness

From the first day of intraperitoneal GLS injection, both hind paws of the mice were inspected every 3-4 days and the severity was determined by the amount of swelling. The severity of arthritis (0-4) was graded using the CIA scoring index developed by Hooke Laboratories as follows: non-inflamed, normally functioning foot; Inflammation and swelling of one or both toes, but no visible swelling of the foot or ankle; Two or more toes become inflamed and swollen, but the entire foot is not or only slightly swollen; Three or four toes are severely swollen and the mouse is unable to grasp the top of the cage. The most severe swelling in the mouse's ankle was measured every 3-4 days after intraperitoneal GLS injection. After 7 weeks, the experimental mice were anesthetized with CO ₂ , and knee, ankle, and footpad tissues were collected for tissue analysis.

biopsy

The collected tissues were fixed with 4% paraformaldehyde for 24 hours and then decalcified in 0.5 M ethylenediaminetetraacetic acid (pH 7.4) for 2 weeks. After dehydrating the tissue, it was cut into 4 μm thick pieces to prepare slides. Blocks were created by fixation in paraffin. Harris hematoxylin, Fast Green, and Safranin O (Sigma-Aldrich, St. Louis, MO, USA) were used for tissue analysis. Histological grade of inflammation in RA was divided into three categories: OARSI (Osteoarthritis Research Society International) grade (0-6), synovitis score (0-3), and Panus score (0-4). Five observers evaluated OARSI, synovitis, and Panus scores. Scoring results were calculated by averaging the observers' scores for each mouse. Among the cut slides, representative images of Safranin-O with the most representative etiology were selected.

Immune cell infiltration assay

Congo red (Sigma C6277, St. Louis, MO, USA) and Toluidine blue O (Sigma Cat.no, St. Louis, MO, USA) were used to stain eosinoleukocytes and mast cells, respectively. The infiltration of eosinophils and mast cells into the synovial membrane around joints was analyzed. CD15 was used to trap neutrophils extracellularly, and quantification of neutrophils was analyzed using ImageJ.

statistical analysis

Statistical analysis was performed using IBM SPSS Statistics 24 (IBM Corp., Armonk, NY, USA). The biopsy results were analyzed using the Mann-Whitney U test. Statistical significance was set at p < 0.05.

Example 2

Extraction and identification of GLS components

From 2 kg of GLS powder, 600 g of GLS oil was obtained. The GC-MS analysis results of GLS oil are shown in Figure 1b and Table 1. GLS oil contains oleic acid (52.12%), linoleic acid (16.77%), palmitate acid (13.67%), ellidic acid (9.16%), palmitoleate acid (2.95%), and stearate acid. (stearate acid) (1.58%), palmitic acid (1.35%), fatty acid methyl ester (1.01%), stearic acid (0.87%), and pentadecanoic acid (0.52%).

Effect of GLS oil on clinical score, paw thickness and pain in RA model

The group treated with GLS oil had significantly lower severity levels compared to the group treated with PEG alone (control group). Regarding clinical RA score, the GLS oil-infused group showed significantly lower severity compared to the control-infused group (p = 0.0384; Figure 2b). Moreover, CIA-induced paw swelling was reduced by GLS oil treatment (Figures 2c and d).

Additionally, at 35 days after immunization, the von Frey assay revealed that GLS oil had a pain-relieving effect (Figure 3a). At 45 days after vaccination, it was observed that GLS oil had a significant analgesic effect (p = 0.0248). Additionally, the hot plate test indicated that the CIA group showed significantly reduced procrastination compared to the control group (p = 0.03). However, GLS oil restored the CIA-induced decrease in latency (p = 0.0309; Figure 3b).

Effect of GLS oil on cartilage degeneration of the knee, ankle, and sole in a CIA-induced RA model.

GLS oil treatment reduced CIA-induced synovitis, pannus formation, and cartilage degeneration. Safranin-O staining revealed the extent of cartilage degeneration, synovial inflammation, and inflammation-mediated cell passage into bone. GLS oil treatment reduced inflammation of the synovium of the knee (Figures 4a and b) and cartilage degeneration (Figures 4c and d) induced by CIA. Furthermore, GLS oil treatment reduced the development of CIA-induced pannus in the knee (Figures 4e and f).

Safranin-O staining of the ankle showed that erosion of the ankle cartilage was restored by GLS oil treatment (Figure 5a). According to the ankle OARSI scale, GLS oil significantly reduced cartilage damage compared to the CIA group (Figure 5b, p = 0.0187). Additionally, GLS oil also significantly reduced ankle synovitis (p = 0.0028) and synovial inflammation (Figures 5c and d). Finally, GLS oil prevented immune cells from infiltrating bone (Figures 5e and f).

In the soles, GLS oil-treated mice had less cartilage degeneration compared to PEG-treated mice (Figures 6a and b). The CIA group showed increased synovial inflammation, whereas the GLS oil-treated group showed reduced inflammation (Figures 6c and d). Pannus formation increased in the CIA group but decreased significantly in the GLS oil-treated group (Figures 6e and f).

GLS oil attenuates immune cell infiltration in a CIA-induced RA model

Congo red staining revealed eosinophilic leukocyte infiltration of the synovium of the knee and ankle in the CIA group (Figure 7a). Knee eosinophilic leukocyte count was significantly reduced in the GLS oil-treated group (p = 0.0056). CDr15 immunostaining showed that neutrophils were infiltrated in the CIA group; However, infiltrated neutrophils were significantly attenuated in both the knee (p = 0.0006) and ankle (p = 0.0023; Figure 7d-g) of the GLS oil-treated group. The total number of mast cells and degranulated mast cells were also increased in the CIA group (Figure 7h-j). Although the number of mast cells decreased in the GLS oil group, the difference was not statistically significant.

So far, the present invention has been examined focusing on its preferred embodiments. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Claims (11)

It contains as an active ingredient a supercritical extract of Ganoderma lucidum spore (GLS) extracted in a supercritical extraction system with one extractor and two separators,
The supercritical extract of the reishi mushroom spores contains oleic acid, linoleic acid, palmitate acid, elaidic acid, palmitoleate acid, and stearate acid. acid, palmitic acid, fatty acid methyl ester, stearic acid, and pentadecanoic acid,
The pressure and temperature of the extractor are 30 to 50 MPa and 45 to 65 ℃, respectively, the temperature and pressure of the first of the two separators are 40 to 60 ℃ and 10 to 16 MPa, and the temperature and pressure of the second separator A pharmaceutical composition for preventing or treating arthritis, wherein the temperature is 30 to 50°C and 3 to 7 MPa. delete The composition according to claim 1, wherein the supercritical extract of reishi mushroom spores is extracted using carbon dioxide as a supercritical fluid. The composition according to claim 1, wherein the arthritis is osteoarthritis or rheumatoid arthritis. delete delete delete It contains as an active ingredient a supercritical extract of Ganoderma lucidum spore (GLS) extracted in a supercritical extraction system with one extractor and two separators,
The supercritical extract of the reishi mushroom spores contains oleic acid, linoleic acid, palmitate acid, elaidic acid, palmitoleate acid, and stearate acid. acid, palmitic acid, fatty acid methyl ester, stearic acid, and pentadecanoic acid,
The pressure and temperature of the extractor are 30 to 50 MPa and 45 to 65 ℃, respectively, the temperature and pressure of the first of the two separators are 40 to 60 ℃ and 10 to 16 MPa, and the temperature and pressure of the second separator A health functional food for preventing or improving arthritis, wherein the temperature is 30 to 50°C and 3 to 7 MPa. A method for preventing or treating arthritis, comprising administering the pharmaceutical composition of any one of claims 1, 3, and 4 to a patient other than a human. Extracting the powder of reishi mushroom spores using supercritical carbon dioxide at 30 to 50 MPa and 45 to 65°C;
A step in which the supercritical extract of the extracted reishi mushroom spores and carbon dioxide are circulated within two separators and kinetic extraction is performed; and
Collecting the supercritical extract of the extracted reishi mushroom spores in a first separator;
Includes,
Extraction of reishi mushroom spores, wherein the temperature and pressure of the first of the two separators are 40 to 60°C and 10 to 16 MPa, and the temperature and pressure of the second separator are 30 to 50°C and 3 to 7 MPa. method. delete