Preparation method for plate-cultured antrodia cinnamomea active distinguishing product and use thereof

Abstract

Provided is a preparation method for a plate-cultured Antrodia cinnamomea active distinguishing product, comprising: carrying out twice extraction and filtration on plate-cultured Antrodia cinnamomea fruiting body powder by using alcohol, combining the two filtrates to obtain a mixed solution, and carrying out drying treatment on part of the mixed solution to obtain an Antrodia cinnamomea extract; and adsorbing the remaining mixed solution with macroporous resin, concentrating under reduced pressure until drying, separating by macroporous resin and a glass tube column, and eluting multiple times by using secondary water, alcohol, and ethyl acetate according to a preset mode to separately obtain first, second, third, and fourth Antrodia cinnamomea active distinguishing products. The Antrodia cinnamomea extract and the Antrodia cinnamomea active distinguishing products can be used for preparing a pharmaceutical composition for treating atopic dermatitis.

Description

Preparation method of petri dish culture antrodia cinnamomea active differentiator and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of microbiological technology, in particular to a preparation method of petri dish culture antrodia cinnamomea active differentiator and use thereof in treating atopic dermatitis, which has better activity than directly using antrodia cinnamomea extract. BACKGROUND

[0002] Antrodia cinnamomea is a perennial polypore fungus, and the fruiting body has various shapes such as plate, bell, horseshoe or tower; it is initially bright red, and gradually changes into light red-brown, light brown or light yellow-brown. Antrodia cinnamomea is a species endemic to Taiwan Province of China, also known as antrodia camphorata, antrodia camphorata, etc., and only grows in the hollow heartwood of the evergreen broad-leaved large tree camphor tree in the natural state. It is only native to Taiwan Province of China in the world, and its traces can be found at an altitude of 200 to 1500 meters. The wood of camphor tree is extremely fragrant and hard, and is also a good carving material. It is currently an endangered and protected plant. In the literature review of Geethangili and Tzeng, it is mentioned that the indigenous people of Taiwan Province of China used to eat antrodia cinnamomea to cure hangover, liver cirrhosis, liver inflammation, detoxification, stomachache, diarrhea, skin itching, cold, high blood sugar, high blood pressure, tumors, improve fatigue, and enhance physical strength, etc.

[0003] The petri dish culture antrodia cinnamomea fruiting body is grown in a specific medium at a specific temperature, and has the advantages of uniform quality, stable yield, shorter production period and reasonable price. It is different from the segment culture antrodia cinnamomea, which grows on the old segment of the camphor tree. The fungus antrodia cinnamomea fruiting body needs to grow in a dark, humid and slightly low temperature environment, and has the problems of slow growth, large quality difference and easy contamination of mixed bacteria.

[0004] Atopic dermatitis (AD) is also known as atopic dermatitis, allergic dermatitis, atopic eczema, and was called four-bending wind in ancient times. Common symptoms include itching, redness, and skin cracking. Clear liquid often flows out of the inflamed area, and the liquid becomes thicker as the inflammation lasts longer. Atopic dermatitis often occurs in childhood, and the severity varies with age; red rashes on children under the age of one often cover the whole body; when they are a little older, the affected area is mainly located in the nasolabial and cubital fossa; adults are prone to occur in the hands and feet. Scratching the lesion will worsen the symptoms and increase the risk of skin infection. Many patients often have symptoms of allergic rhinitis and asthma. SUMMARY

[0005] To solve the risk of skin infection caused by ectopic dermatitis, the present application provides a preparation method of Ganoderma cochlear active fraction, which comprises five steps of first extraction step, second extraction step, mixing step, separation step and layering step. The first extraction step is to extract Ganoderma cochlear fruiting body powder with alcohol and filter to collect first filtrate. The second extraction step is to extract the residue with alcohol again and filter to collect second filtrate. The mixing step is to mix the first filtrate and the second filtrate to form a mixed solution, and to take part of the mixed solution for drying treatment to obtain Ganoderma cochlear extract. The separation step is to adsorb the remaining mixed solution with macroporous resin, to concentrate to dryness under reduced pressure, and to separate with macroporous resin and glass column. The layering step is to perform multiple elution layering with twice distilled water, alcohol and ethyl acetate in a preset manner to obtain first Ganoderma cochlear active fraction, second Ganoderma cochlear active fraction, third Ganoderma cochlear active fraction and fourth Ganoderma cochlear active fraction respectively.

[0006] In a preferred embodiment of the present application, the layering step specifically comprises: elution layering with a mixture of twice distilled water and alcohol in a volume ratio of 80:20, drying the eluate to obtain the first Ganoderma cochlear active fraction; elution layering with a mixture of twice distilled water and alcohol in a volume ratio of 60:40, drying the eluate to obtain the second Ganoderma cochlear active fraction; elution layering with a mixture of twice distilled water and alcohol in a volume ratio of 40:60 and elution layering with a mixture of twice distilled water and alcohol in a volume ratio of 20:80, collecting and combining the two eluates, drying the eluate to obtain the third Ganoderma cochlear active fraction; and elution layering with alcohol and then elution layering with ethyl acetate, collecting and combining the two eluates, and drying the eluate to obtain the fourth Ganoderma cochlear active fraction. In a preferred embodiment of the present application, the volume percentage concentration of the alcohol is 90%-95%.

[0007] In the present application, the Ganoderma cochlear extract can effectively inhibit anti-inflammatory chemokine CCL5, and different active fractions have certain inhibitory capacity and show dose dependence. In a more preferred embodiment of the present application, the first Ganoderma cochlear active fraction, the second Ganoderma cochlear active fraction, the third Ganoderma cochlear active fraction and the fourth Ganoderma cochlear active fraction all have specific ultra-high performance liquid chromatography fingerprint.

[0008] In the present application, the first Ganoderma cochlear active fraction, the second Ganoderma cochlear active fraction, the third Ganoderma cochlear active fraction and the fourth Ganoderma cochlear active fraction are superior to Ganoderma cochlear fruiting body extract in terms of CCL5 immune chemotactic factor activity.

[0009] The present application provides the use of the active fraction of Ganoderma cochlear from the above method for preparing a pharmaceutical composition for treating atopic dermatitis.

[0010] The present application provides a pharmaceutical composition, which is prepared by using the active fraction of Ganoderma cochlear from the above method as an active ingredient, and adding a pharmaceutically acceptable excipient or carrier or auxiliary ingredient.

[0011] The present application also provides the use of the above pharmaceutical composition for preparing a drug for treating atopic dermatitis.

[0012] The present application provides a preparation method of the active fraction of Ganoderma cochlear, which is obtained by multiple elution and layering through macroporous resin and a glass column, and the active fraction of Ganoderma cochlear can effectively inhibit anti-inflammatory chemokine CCL5, and different active fractions have certain inhibition capacity and show dose dependence, and the active fraction of Ganoderma cochlear also has a specific UPLC fingerprint, and the activity of CCL5 immune chemokine is better than that of Ganoderma cochlear fruiting body extract. BRIEF DESCRIPTION OF DRAWINGS

[0013] FIG. 1 is a flowchart of the preparation method of the active fraction of Ganoderma cochlear according to the present application;

[0014] FIG. 2 is a column chart drawn by using GraphPad Prism 9 software according to the numerical values of each group according to the present application;

[0015] FIG. 3 is the ultra performance liquid chromatography (UPLC) fingerprint of the active fraction of Ganoderma cochlear ACF003-F02 according to the present application;

[0016] FIG. 4 is a test flowchart of the active fraction of Ganoderma cochlear in the MC903-induced atopic dermatitis mouse model according to the present application;

[0017] FIG. 5 is a test result of the active fraction of Ganoderma cochlear in the MC903-induced atopic dermatitis mouse model according to the present application, wherein the abbreviation (L) represents the left ear, and (R) represents the right ear. DETAILED DESCRIPTION

[0018] The technical solutions of the present application will be further described below in combination with the drawings and examples.

[0019] Referring to FIG. 1, the preparation method of the active fraction of Ganoderma cochlear according to the present application includes the following steps:

[0020] Step S101, extract the Antrodia camphorata fruiting body powder with alcohol and filter to collect a first filtrate; Step S102, extract the residue with alcohol again and filter to collect a second filtrate;

[0021] Step S103, mix the first filtrate and the second filtrate to form a mixed solution, and take out a portion of the mixed solution for drying treatment to obtain an Antrodia camphorata extract;

[0022] Step S104, use a macroporous resin to adsorb the remaining mixed solution, reduce pressure to concentrate to dryness, and use a macroporous resin and a glass column to separate;

[0023] Step S105, use secondary water, alcohol, and ethyl acetate to perform multiple elution fractionations according to a predetermined manner to obtain a first Antrodia camphorata active fraction, a second Antrodia camphorata active fraction, a third Antrodia camphorata active fraction, and a fourth Antrodia camphorata active fraction, respectively.

[0024] The following application examples are given as follows:

[0025] Example

[0026] Take 10 g of Antrodia camphorata fruiting body dry medicinal material from Taiwan Province, China, and grind it into powder. Use 20 times the weight of the Antrodia camphorata fruiting body powder of 95% volume percentage concentration alcohol to ultrasonic oscillate for 99 minutes, then stand and soak for extraction. After 24 hours of extraction, filter with filter paper to collect a first filtrate. Take the residue and use 20 times the weight of the Antrodia camphorata fruiting body powder of 95% volume percentage concentration alcohol to ultrasonic oscillate for 99 minutes, then stand and soak for extraction. After 24 hours of extraction, collect a second filtrate. Mix the first filtrate and the second filtrate to form a mixed solution, and take out a portion of the mixed solution for drying to obtain an Antrodia camphorata extract ACF003 (about 10 g). The Antrodia camphorata extract ACF003 is used to provide pharmacological experiments.

[0027] The remaining mixture is added to 4 times the weight of ACF003 macroporous resin (HP20) for adsorption, and concentrated under reduced pressure to dryness, and then separated by macroporous resin (HP20) and a glass column, with twice-distilled water, 95% alcohol by volume, and ethyl acetate as the elution solvent, and multiple times of elution and fractionation according to the preset mode, to obtain the first, second, third, and fourth active fraction of T. ceriferum, respectively. The specific process is as follows: twice-distilled water and 95% alcohol by volume are used as the elution solvent, the volume ratio of twice-distilled water to alcohol is 80:20, 70 mL of the elution solvent is collected, and after concentration and drying, the first active fraction of T. ceriferum ACF003-F01 is obtained; twice-distilled water and 95% alcohol by volume are used as the elution solvent, the volume ratio of twice-distilled water to alcohol is 60:40, 70 mL of the elution solvent is collected, and after concentration and drying, the second active fraction of T. ceriferum ACF003-F02 is obtained; twice-distilled water and 95% alcohol by volume are used as the elution solvent, the volume ratio of twice-distilled water to alcohol is 40:60, 70 mL of the elution solvent is collected, and twice-distilled water and 95% alcohol by volume are used as the elution solvent, the volume ratio of twice-distilled water to alcohol is 20:80, 70 mL of the elution solvent is collected, the elution solvents collected twice are combined, and after concentration and drying, the third active fraction of T. ceriferum ACF003-F03 is obtained; 95% alcohol by volume is used for elution and fractionation, and then ethyl acetate is used as the elution solvent for elution and fractionation, 70 mL of the elution solvent is collected, and after concentration and drying, the fourth active fraction of T. ceriferum ACF003-F04 is obtained.

[0028] To test the ability of T. ceriferum extract ACF003 to inhibit the inflammatory factor CCL5, the following tests need to be performed:

[0029] I. Cell activity test

[0030] Method for testing anti-inflammatory activity using macrophage RAW 264.7 (lipopolysaccharide (LPS) activated macrophage RAW 264.7):

[0031] 1) General culture solution preparation: DMEM is added with FBS and P / S to make the final concentration 10% and 1%;

[0032] 2) The macrophage RAW 264.7 cultured to 90% full is collected using a scraper to scrape the bottom of the culture dish;

[0033] 3) The number of cells collected is calculated using a cell counter for experimental plate division.

[0034] or 5 x 10 6 cell / 10cm dish of cells were seeded and incubated in a 37°C, 5% CO2 incubator for 2-3 days until the dish was confluent.

[0035] 1. Incubate the seed cells

[0036] 1) 3 x 10 4 cells / well / 0.1 mL DMEM were incubated in a 96-well plate;

[0037] 2) Incubate in a 37°C, 5% CO2 incubator for 24 hours.

[0038] 2. Add lipopolysaccharide (LPS)

[0039] 1) Remove old media;

[0040] 2) Add 50 μL of LPS at a concentration of 100 ng / mL and incubate in a 37°C, 5% CO2 incubator for 1 hour;

[0041] 3) Add 50 μL of test article and incubate in a 37°C, 5% CO2 incubator for 24 hours;

[0042] Final LPS concentration is 100 ng / mL.

[0043] 3. NO assay

[0044] 1) Prepare a standard curve of NaNO2;

[0045] Standard curve concentrations are: 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0 μM (prepared in media);

[0046] 2) Add 80 μL of cell supernatant or NaNO2 to the 96-well plate;

[0047] 3) Add 80 μL of Griess reagent (40 mg / mL);

[0048] 4) Incubate for 10 minutes in the dark and measure the OD550 nm absorbance.

[0049] 4. MTS assay

[0050] 1) Dilute the MTS reagent 1:10 in 5% FBS media;

[0051] 2) Remove the remaining old culture medium, add MTS reagent 100 μL / well, and react for 1 hour in a CO2 incubator at a temperature of 37°C and a CO2 concentration of 5%; 3) Read the absorbance at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC

[0052] 3) Read the absorbance at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC 50 .

[0053] Activity results: ACF003-F02 showed the best performance with an IC 50 value of 32.51 μg / mL under the condition of 90-100% cell survival.

[0054] II. Inhibition of chemokine CCL5 using human skin keratinocytes (HaCaT)

[0055] 1. Reagent preparation

[0056] 1) TNF-α stock solution preparation: Dissolve lyophilized TNF-α in sterile water at a concentration of 200 mg / mL;

[0057] 2) IFN-γ stock solution preparation: Dissolve lyophilized IFN-γ in sterile water at a concentration of 200 mg / mL;

[0058] 3) Treated medium: Dilute the TNF-α and IFN-γ stock solutions with sterile water, and then dilute with serum-free DMEM medium to a final concentration of 20 ng / mL;

[0059] 4) Antrodia cinnamomea extract sample stock solution preparation: Dissolve the Antrodia cinnamomea extract sample in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / mL;

[0060] 5) Antrodia cinnamomea extract sample dilution: Perform serial dilution to prepare working solutions using 100% DMSO at concentrations of 5000 μg / mL, 2500 μg / mL, and 1250 μg / mL, and then dilute with serum containing TNF-α and IFN-γ at a volume ratio of 1:100 to final concentrations of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL, respectively.

[0061] 6) Antrodia cinnamomea extract active fraction sample dilution: Perform serial dilution to prepare working solutions using 100% DMSO at concentrations of 500 μg / mL, 250 μg / mL, and 125 μg / mL, and then dilute with serum containing TNF-α and IFN-γ at a volume ratio of 1:100 to final concentrations of 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL, respectively.

[0062] 2. Cell culture

[0063] HaCaT cells were cultured in DMEM medium containing 10% FBS and 100 U / mL penicillin at 37 °C with 5% CO2. Prior to testing, HaCaT cells (1 x 10 5 cells / well) were cultured in 24-well plates overnight.

[0064] Experimental procedure: HaCaT cells (1 x 10 5 cells / well) were cultured in 24-well plates overnight. After 24 h of incubation in DMEM without FBS, DMEM medium with TNF-a and IFN-g (20 ng / mL each) was used and different concentrations of T. antho extract were added according to groups for 24 h. Enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s instructions.

[0065] Data analysis: Column charts were plotted using GraphPad Prism 9 software according to the values of each group.

[0066] This experiment was to evaluate the inhibitory effect of T. antho extract ACF003 sample on the production of inflammatory factor CCL5. HaCaT cells were used to test the ability of the sample to inhibit the production of inflammatory factor CCL5. The sample used DMSO as a solvent, and the test results are as follows (as shown in Figure 2):

[0067] 1) The positive control group (TNF-a / IFN-g, 20 ng / mL) increased CCL5 by nearly 2.4 times compared to the negative control group (normal ctrl.), while the ACF003 extract group (50-12.5 pg / mL) increased CCL5 by only 1.2-1.5 times compared to the negative control group, showing that the ACF003 sample can effectively inhibit the increase in CCL5 caused by TNF-a and IFN-g, and exhibits a dose-dependent effect.

[0068] 2) Inhibitory effect on CCL5 activity can also be observed in the active fraction of ACF003, in which ACF003-F02 is the most active among all the active fractions, having inhibitory ability in all the three concentrations tested, and showing dose-dependent, in the lowest dose of 1.25 μg / mL tested, the expression of CCL5 has almost the same ratio as the negative control group (close to 1), while ACF003-F01, ACF003-F03, ACF003-F04 have a ratio greater than 2 with the negative control group at 1.25 μg / mL, showing that ACF003-F02 fraction has better activity than the other three fractions, and the order is ACF003-F02 > ACF003-F04 > ACF003-F03 > ACF003-F01.

[0069] 3) If ACF003-F02 is compared with ACF003, ACF003-F02 is close to the negative control group at a dose of 1.25 μg / mL (ratio close to 1), while the ACF003 extract is close to the negative control group at a dose of 25 μg / mL (ratio close to 1), so the activity of ACF003-F02 is better than that of ACF003 extract.

[0070] III. Ultra Performance Liquid Chromatography (UPLC) Fingerprint of ACF003-F02

[0071] UPLC analysis conditions: Ultra Performance Liquid Chromatography (UPLC) is Waters ACQUITY; column (ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 50 mm); column temperature is 35°C; flow rate is 0.3 mL / min; detection wavelength is UV 254 nm.

[0072] Mobile phase system conditions: mobile phase A is water (0.1% formic acid), mobile phase B is acetonitrile (0.1% formic acid), linear gradient is 0-3 min (90% A-90% A), 3-13 min (90% A-65% A), 13-15 min (65% A-63% A).

[0073] As shown in Figure 3, under certain UPLC analysis conditions, five main absorption peaks can be seen in the ACF003-F02 spectrum, which are: 1) retention time (RT): 2.28 min; 2) RT: 4.18 min; 3) RT: 6.63 min; 4) RT: 6.77 min; 5) RT: 7.47 min. This indicates that the main components in the ACF003-F02 fraction appear at different positions in the UPLC spectrum.

[0074] IV. Effect evaluation of the ACF003-F02 fraction in the MC903-induced atopic dermatitis model

[0075] Animals: 6-9 week old C57BL / 6 mice.

[0076] Test substances: 2 batches of A. cinnabarinus active fraction test substances (first batch API (API-I), second batch API (API-II), both with a content of 4% in cream preparation) + disease control, API being ACF003-F02+F03+F04.

[0077] Model inducing substance: MC903.

[0078] Test (dosing) period: 9 days (day 0-day 8).

[0079] Dosing method: right ear local administration (10 uL once a day).

[0080] Total live observation period: 10 days.

[0081] Number of animals: 15 (control group: 5; API-I: 5; API-II: 5).

[0082] The test procedure is shown in Figure 4.

[0083] Test evaluation:

[0084] (A) Evaluation during observation:

[0085] 1. Observation: observed once a day.

[0086] 2. Body weight: recorded on day 0 and day 9.

[0087] (B) Ear thickness measurement:

[0088] On day 0 (before test substance administration), day 1, day 3, day 5, day 7 and day 9, scoring and photographing were performed, ear thickness was measured, and photographs were taken; the percentage change in ear thickness on each evaluation day was calculated.

[0089] As shown in Figure 5, the negative control group (Control blank) and the vehicle control group (Vehicle control, administered with cream without drug) are normal ear thickness.

[0090] The drug control group-I (API-I Blank (L)) and the drug control group-II (API-II Blank (L)) are the right ear of the mouse coated with the test substance (cream containing API), and the left ear is not coated with any substance (i.e. Blank), which is also normal thickness. The disease control group (MC903 (R), administered with 100 μM of MC903) is the most severe inflammation and swelling, followed by the cream control group (cream blank, administered with 100 μM of MC903 + cream without drug), which also has some protective effect due to the moisturizing coverage of the cream on the skin. Test group-1 (administered with 100 μM of MC903 + cream containing the first batch of API) and test group-2 (administered with 100 μM of MC903 + cream containing the second batch of API) are the two batches of Petri dish cultured Antrodia cinnamomea active substances with the best effect, and the mouse ears are closest to normal thickness.

[0091] The Petri dish cultured Antrodia cinnamomea active substance prepared by the method of the embodiment of the present application can be used to prepare a medicine for treating atopic dermatitis, which can effectively inhibit anti-inflammatory chemokine CCL5, and different active substances have certain inhibitory capacity and present dose-dependent.

[0092] The embodiment of the present application provides a pharmaceutical composition, which is prepared by taking the Petri dish cultured Antrodia cinnamomea active substance prepared by the above method as an active ingredient, and adding pharmaceutically acceptable adjuvants or auxiliary ingredients. The pharmaceutical composition can effectively inhibit anti-inflammatory chemokine CCL5 and can effectively treat atopic dermatitis.

[0093] The preparation method of the Petri dish cultured Antrodia cinnamomea active substance provided by the embodiment of the present application is performed by multiple elution and layering through macroporous resin and a glass column, so as to obtain a plurality of different Antrodia cinnamomea active substances. The Antrodia cinnamomea active substance can effectively inhibit anti-inflammatory chemokine CCL5, and different active substances have certain inhibitory capacity and present dose-dependent. The Antrodia cinnamomea active substance also has a specific UPLC fingerprint, and the activity of CCL5 immune chemotactic factor is better than that of Antrodia cinnamomea fruiting body extract.

[0094] The effect evaluation of the atopic dermatitis mouse model of the Petri dish cultured Antrodia cinnamomea active substance API provided by the embodiment of the present application has animal experiment activity of atopic dermatitis.

[0095] The above-described specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described specific embodiments are merely examples of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A preparation method of a petri dish cultured Antrodia camphorata extract, characterized by, comprising: a first extraction step of extracting and filtering the Ganoderma cochlear powder using an alcohol extraction dish, and collecting a first filtrate; a second extraction step of extracting and filtering the residue after the previous extraction and filtration using alcohol again, and collecting a second filtrate; and a mixing step of mixing the first filtrate and the second filtrate to form a mixed solution, and drying the mixed solution to obtain a Ganoderma cochlear extract.

2. A preparation method of a Ganoderma subamans active fraction, characterized in that, comprising: a first extraction step of extracting and filtering the Ganoderma cochlear powder using an alcohol extraction dish, and collecting a first filtrate; a second extraction step of extracting and filtering the residue after the previous extraction and filtration using alcohol again, and collecting a second filtrate; a mixing step of mixing the first filtrate and the second filtrate to form a mixed solution; a separation step of adsorbing the mixed solution using a macroporous resin, concentrating under reduced pressure to dryness, and separating using a macroporous resin and a glass column; and a separation step of eluting and separating using a solution, and obtaining a Ganoderma cochlear active fraction after concentration and drying, wherein the solution comprises a mixture of distilled water and alcohol, alcohol, or ethyl acetate.

3. The preparation method according to claim 2, characterized in that The ratio of the mixture of distilled water and alcohol is 80-20:20-80.

4. The preparation method according to claim 2, characterized in that The separation step comprises eluting and separating using a mixture of distilled water and alcohol at a volume ratio of 80:20, and obtaining a first Ganoderma cochlear active fraction after concentrating and drying the eluate.

5. The preparation method according to claim 2, characterized in that The separation step comprises eluting and separating using a mixture of distilled water and alcohol at a volume ratio of 60:40, and obtaining a second Ganoderma cochlear active fraction after concentrating and drying the eluate.

6. The preparation method according to claim 2, characterized in that The separation step comprises eluting and separating using a mixture of distilled water and alcohol at a volume ratio of 40:60, and eluting and separating using a mixture of distilled water and alcohol at a volume ratio of 20:80, collecting and combining the two eluates, and obtaining a third Ganoderma cochlear active fraction after concentrating and drying the eluate.

7. The preparation method according to claim 2, characterized in that The separation step comprises eluting and separating using alcohol, and then eluting and separating using ethyl acetate, collecting and combining the two eluates, and obtaining a fourth Ganoderma cochlear active fraction after concentrating and drying.

8. The preparation method according to claim 5, characterized in that The second Ganoderma cochlear active fraction has a specific ultra-high performance liquid chromatography fingerprint, wherein the fingerprint has five main absorption peaks, respectively: 1) retention time: 2.28 minutes; 2) retention time: 4.18 minutes; 3) retention time: 6.63 minutes; 4) retention time: 6.77 minutes; and 5) retention time: 7.47 minutes.

9. Use of the Ganoderma cochlear extract according to claim 1 for preparing a pharmaceutical composition for treating atopic dermatitis.

10. Use of the Ganoderma cochlear active fraction according to any one of claims 2-8 for preparing a pharmaceutical composition for treating atopic dermatitis.

11. A pharmaceutical composition for treating atopic dermatitis, comprising the Ganoderma cochlear extract according to claim 1 as an active ingredient and a pharmaceutically acceptable excipient, carrier, or auxiliary ingredient.

12. A pharmaceutical composition for treating atopic dermatitis, comprising the active fraction of Antrodia cinnamomea as claimed in any one of claims 2-8 as an active ingredient and a pharmaceutically acceptable excipient, carrier or auxiliary ingredient.

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