Inositol extract and preparation method and application thereof

Abstract

The application belongs to the technical field of biological fermentation, and particularly discloses a chaga extract with anti-inflammatory and repairing effects, and a preparation method and application thereof. The preparation method of the chaga extract comprises the following steps: (1) strain activation: the frozen chaga is streaked and activated on a solid culture medium; (2) preparation of a seed liquid: the mycelium of the activated chaga is inoculated into a liquid fermentation culture medium to culture a seed liquid; (3) fermentation culture: the seed liquid is inoculated into the liquid fermentation culture medium to culture, and the liquid fermentation culture medium is added with Platycladus orientalis oil to obtain a chaga fermentation liquor; and (4) extraction: the chaga fermentation liquor is extracted to obtain the chaga extract. The chaga extract is prepared by adopting specific culture medium for fermentation treatment and adding specific inducers, so that the content of active ingredients in the extract can be effectively improved, the active effect is good, the chaga extract has good effects in skin repair and anti-inflammation, and can be applied as a raw material in cosmetics or medical products.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and in particular to an extract of Inonotus obliquus with anti-inflammatory and repairing effects, its preparation method, and its application. Background Technology

[0002] Chaga mushroom (Inonotus obliquus), also known as birch polypore, is a rare medicinal fungus that parasitizes the bark or dead trunks of birch, elm, and alder trees. Its fruiting bodies are irregularly shaped lumps, ranging in color from yellowish-brown to black. Chaga mushroom has a complex composition, containing polysaccharides, polyphenols, melanin, steroids, and other bioactive substances, exhibiting pharmacological effects such as anti-tumor activity, immune enhancement, antiviral activity, and enhancement of catalase activity.

[0003] Traditionally, the active substances of Inonotus obliquus are extracted from its fruiting bodies using methods such as hot water extraction, organic solvent extraction, ultrasonic or microwave-assisted extraction, and enzymatic hydrolysis. However, this method of extracting active substances from Inonotus obliquus fruiting bodies has many limitations. First, as a parasitic fungus, Inonotus obliquus typically requires a long growth cycle of more than ten years and has demanding growth environment requirements. Its probability of naturally growing on host trees is extremely low, resulting in extremely scarce wild resources. Although there are currently methods for artificially cultivating Inonotus obliquus fruiting bodies, the composition and content of its active substances are easily affected by differences in growth region, climate conditions, and harvesting time, leading to problems such as inconsistent quality, poor quality stability, and difficulties in standardization between different batches. Second, the fruiting body extraction process suffers from technical bottlenecks such as low extraction efficiency, complex composition, and cumbersome procedures. Extraction of active substances from Inonotus obliquus often requires expensive enzymatic hydrolysis or high-pressure crushing technology. The crude extract obtained is complex in composition and contains impurities such as proteins, pigments and inorganic salts. Therefore, it is necessary to further process it through multiple steps such as precipitation, deproteinization and chromatography purification, which ultimately leads to the loss of active substance content. Moreover, these processes mostly rely on organic solvents such as chloroform, which not only poses the risk of excessive residues, but also increases the cost of hazardous waste treatment.

[0004] Therefore, developing a high-efficiency, simple, and industrially applicable method for preparing Inonotus obliquus extract with high content and activity of active ingredients is of great significance for solving the shortage of Inonotus obliquus resources, developing its active substance products, and realizing its broad application prospects. Summary of the Invention

[0005] In view of the above, it is necessary to provide an extract of Inonotus obliquus with anti-inflammatory and repairing effects, its preparation method, and its application. This invention uses a specific culture medium for fermentation treatment, and adds a specific inducing agent during the fermentation process, which can effectively increase the content of active ingredients such as polysaccharides and triterpenes in the extract, and improve its activity. It has a good effect on skin repair and anti-inflammatory acne treatment, and can be used as a raw material in cosmetics or pharmaceutical products.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing an extract of Inonotus obliquus, comprising the following steps:

[0008] (1) Activation of strain: The frozen Inonotus obliquus was picked up with an inoculation loop and streaked on a solid culture medium to activate the strain. After three generations of continuous activation culture, activated Inonotus obliquus was obtained.

[0009] The solid culture medium comprises the following components in the indicated weight percentages: glucose 3-5%, agar 1.5-2.5%, yeast extract 0.1-0.3%, peptone 0.4-0.6%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.1-0.2%, birch sap 0.5-1%, with the remainder being water;

[0010] (2) Preparation of seed liquid: The mycelium of the activated Inonotus obliquus was inoculated into the liquid fermentation medium using an inoculation loop and cultured at 26-28℃ for 5-6 days to obtain seed liquid;

[0011] The liquid fermentation medium comprises the following components in the indicated weight percentages: glucose 3-5%, yeast extract 0.2-0.4%, peptone 0.4-0.7%, potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.1-0.3%, birch sap 1-2%, kudzu root extract 1-2%, and the remainder being water.

[0012] (3) Fermentation culture: The above seed liquid is inoculated into the liquid fermentation medium at 10-15% (v / v) and cultured at 26-30℃ for 7-9 days. On the 4th day of culture, 2-4 g / L of cypress seed oil is added. After the culture is completed, the fermentation broth of Inonotus obliquus is obtained.

[0013] (4) Extraction: The fermentation broth of Inonotus obliquus was filtered to obtain fermentation filtrate and bacterial cells; the bacterial cells were crushed and mixed with the fermentation filtrate, and then boiled at 90-95℃ for 30-40 min, then cooled to 70-80℃ and ultrasonically treated for 20-25 min, and filtered. The obtained filtrate is the extract of Inonotus obliquus.

[0014] Furthermore, in step (2), the kudzu root extract is a kudzu root ethanol extract.

[0015] Furthermore, in step (2), the mass ratio of the birch sap to the kudzu root extract is 1:1.

[0016] The present invention also provides an extract of Inonotus obliquus prepared by the above preparation method.

[0017] The present invention also provides an application of the above-mentioned Inonotus obliquus extract in the preparation of a product having at least one of the following effects:

[0018] 1) Anti-inflammatory; 2) Repairing.

[0019] The present invention also provides an acne-removing essence, wherein the essence comprises the above-mentioned Inonotus obliquus extract.

[0020] Furthermore, the acne-removing essence, by weight percentage, comprises the following ingredients: 0.40-0.45% thickener, 0.60-0.80% preservative, 4.50-5.50% Inonotus obliquus extract, and the remainder is water.

[0021] Furthermore, the thickener includes xanthan gum and sodium polyacrylamide dimethyl taurate; the preservative includes a blend of phenoxyethanol and ethylhexylglycerin.

[0022] Furthermore, the acne-removing essence, by weight percentage, comprises the following ingredients: 0.15% xanthan gum, 0.28% sodium polyacrylamide dimethyl taurate, 0.70% a blend of phenoxyethanol and ethylhexylglycerin, 5.00% Inonotus obliquus extract, and the remainder being water.

[0023] This invention also provides a method for preparing the aforementioned acne-removing essence, comprising the following steps:

[0024] ① Add the thickener to the water, stir to disperse, heat to 80-82℃, soak and swell, stir to homogenize for 2-3 minutes;

[0025] ② Stir and cool to below 40°C, add the Inonotus obliquus extract, and stir until homogeneous;

[0026] ③ Continue adding the preservative, stir well, and discharge to obtain the essence.

[0027] The present invention has the following beneficial effects:

[0028] 1. Regarding the Birch Inonotus extract: First, this invention utilizes liquid fermentation technology to solve the problem of the scarcity of Birch Inonotus raw materials and avoids the instability of components caused by the depletion of wild resources and fluctuations in the cultivation environment. Second, in terms of process, this invention uses a specific culture medium combined with a specific inducer to effectively increase the polysaccharide and total triterpenoid content of the Birch Inonotus extract, resulting in high content and activity of active ingredients, effectively enhancing its repair and anti-inflammatory acne-reducing effects. Furthermore, there is a certain interaction between the birch sap, kudzu root extract, and the inducer, Platycladus orientalis seed oil, in the culture medium. These three elements mutually reinforce each other, promoting the conversion and release of the active ingredients of Birch Inonotus, thus optimizing the content and activity of the active substances in the Birch Inonotus extract, further enhancing its anti-inflammatory and repairing effects.

[0029] 2. Regarding the serum: First, the specially formulated Inonotus obliquus extract of this invention is used as the active ingredient to exert its anti-inflammatory and repairing effects, thereby achieving good acne-removing results. Second, through screening and optimization of the excipients used in conjunction with the Inonotus obliquus extract, the final serum product ensures the stability of the Inonotus obliquus extract, thus making its efficacy more stable. Furthermore, the resulting serum is gentle, safe, and non-irritating, with a stable pH of around 6.0, providing a pleasant user experience. Attached Figure Description

[0030] Figure 1 A plate diagram showing the activation of Inonotus obliquus, in which, Figure 1 (a) is the back view of the flat panel diagram. Figure 1 (b) is the front view of the flat panel diagram.

[0031] Figure 2 This is a temperature graph of the environment during test four.

[0032] Figure 3 This is a graph showing the ambient humidity during the four testing periods.

[0033] Figure 4 This is a comparison chart of the number of pimples before and after the subjects in Test 4 used the acne-removing essence.

[0034] Figure 5 The test involved four subjects who used an acne-removing serum before and after experiencing skin redness. Value comparison chart.

[0035] Figure 6 This is a graph showing the changes in the red zone of subject number 6 in test 4.

[0036] Figure 7 This is a graph showing the changes in the red zone of subject number 18 in test 4. Detailed Implementation

[0037] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0038] In this invention, unless otherwise specified, all reagents and consumables used are purchased from conventional reagent manufacturers in the field; unless otherwise specified, all experimental methods and techniques used are conventional methods and techniques in the field.

[0039] The Inonotus obliquus (Ach. ex Pers.) Pilát involved in this invention was purchased from the China Forestry Microbiology Preservation and Management Center, with the strain number CFCC6584.

[0040] The preparation method of the cypress seed oil involved in this invention is as follows: after pulverizing the cypress seeds, pass them through a 40-mesh sieve, mix them with ethyl acetate at a material-to-liquid ratio of 1:10 g / mL, place them in an ultrasonic-microwave synergistic extractor, keep the ultrasonic waves running during extraction, extract for 155 seconds at a microwave power of 350W, add anhydrous Na2SO4 to the extract, filter, evaporate and concentrate to obtain the cypress seed oil.

[0041] First, the present invention provides a method for preparing Inonotus obliquus extract, which includes the following steps:

[0042] (1) Activation of the strain: The frozen Inonotus obliquus was picked up with an inoculation loop and streaked on a solid culture medium to activate the strain. After three generations of continuous activation culture, obvious mycelium was formed on the solid culture medium, and the interior was yellow to yellowish-brown, thus obtaining activated Inonotus obliquus; the back and front views of the plate image of the activated Inonotus obliquus are shown in the figure below. Figure 1 (a) and Figure 1 As shown in (b);

[0043] The solid culture medium comprises the following components in the indicated weight percentages: glucose 3-5%, agar 1.5-2.5%, yeast extract 0.1-0.3%, peptone 0.4-0.6%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.1-0.2%, birch sap 0.5-1%, with the remainder being water;

[0044] (2) Preparation of seed liquid: The mycelium of the activated Inonotus obliquus was inoculated into the liquid fermentation medium using an inoculation loop and cultured at 26-28℃ for 5-6 days to obtain seed liquid;

[0045] The liquid fermentation medium comprises the following components in the indicated weight percentages: glucose 3-5%, yeast extract 0.2-0.4%, peptone 0.4-0.7%, potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.1-0.3%, birch sap 1-2%, kudzu root extract 1-2%, and the remainder being water.

[0046] (3) Preparation of fermentation broth: The above seed liquid is inoculated into the liquid fermentation medium at 10-15% (v / v) and cultured at 26-30℃ for 7-10 days. On the 4th day of culture, 2-4 g / L of cypress seed oil is added. After the culture is completed, the fermentation broth of Inonotus obliquus is obtained.

[0047] (4) Extraction: The fermentation broth of Inonotus obliquus was filtered to obtain fermentation filtrate and bacterial cells; the bacterial cells were crushed and mixed with the fermentation filtrate, and then boiled at 90-95℃ for 30-40 min, then cooled to 70-80℃ and ultrasonically treated for 20-25 min, and filtered. The obtained filtrate is the extract of Inonotus obliquus.

[0048] For example, in step (1), in the solid culture medium:

[0049] The glucose mass percentage includes, but is not limited to: 3%, 3.2%, 3.5%, 3.7%, 4%, 4.5%, and 5%.

[0050] The mass percentage of the agar includes, but is not limited to: 1.5%, 1.7%, 2.0%, 2.2%, and 2.5%.

[0051] The yeast powder's mass percentage includes, but is not limited to: 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%.

[0052] The peptone mass percentages include, but are not limited to: 0.4%, 0.43%, 0.45%, 0.48%, 0.5%, 0.52%, 0.55%, and 0.6%.

[0053] The mass percentage of potassium dihydrogen phosphate includes, but is not limited to: 0.1%, 0.14%, 0.15%, 0.18%, and 0.2%.

[0054] The mass percentage of the magnesium sulfate includes, but is not limited to: 0.1%, 0.12%, 0.15%, 0.17%, and 0.2%.

[0055] The mass percentage of the birch sap includes, but is not limited to, 0.5%, 0.7%, 0.9%, and 1%.

[0056] For example, in step (2) or (3), in the liquid fermentation medium:

[0057] The glucose mass percentage includes, but is not limited to: 3%, 3.2%, 3.5%, 3.7%, 4%, 4.5%, and 5%.

[0058] The yeast powder's mass percentage includes, but is not limited to: 0.2%, 0.25%, 0.3%, 0.35%, and 0.4%.

[0059] The peptone mass percentages include, but are not limited to: 0.4%, 0.45%, 0.48%, 0.5%, 0.55%, 0.6%, 0.65%, and 0.7%.

[0060] The mass percentage of potassium dihydrogen phosphate includes, but is not limited to: 0.1%, 0.15%, 0.18%, 0.2%, 0.24%, 0.27%, and 0.3%.

[0061] The mass percentage of the magnesium sulfate includes, but is not limited to: 0.1%, 0.12%, 0.15%, 0.17%, 0.2%, 0.25%, and 0.3%.

[0062] The mass percentage of the birch sap includes, but is not limited to: 1%, 1.5%, 1.7%, 1.9%, and 2%.

[0063] The mass percentage of the kudzu root extract includes, but is not limited to, 1%, 1.5%, 1.7%, 1.9%, and 2%.

[0064] Preferably, the kudzu root extract is a kudzu root ethanol extract.

[0065] For example, the preparation method of the kudzu root ethanol extract is as follows: kudzu root is pulverized and passed through a 60-mesh sieve, mixed with 60% ethanol at a material-to-liquid ratio of 1:40 g / mL, and extracted by heating under reflux. This process is repeated three times, with each extraction lasting 1 hour. The filtrates are then filtered, combined, concentrated, and dried to obtain the kudzu root extract.

[0066] Preferably, the mass ratio of the birch sap to the kudzu root extract is 1:1.

[0067] For example, in step (2), the culture temperature can be 26℃, 26.5℃, 27℃, 27.5℃, 28℃, etc., as long as it is within the range of 26-28℃; the culture time can be 5d, 5.5d or 6d.

[0068] For example, in step (3), the inoculation amount can be 10% (v / v), 11% (v / v), 12% (v / v), 13% (v / v), 14% (v / v) or 15% (v / v), as long as it is within the range of 15-20% (v / v); the culture time can be 7 days, 8 days or 9 days; and the amount of cypress seed oil added includes, but is not limited to: 2g / L, 2.5g / L, 2.8g / L, 3g / L, 3.5g / L or 4g / L.

[0069] For example, in step (4), the decocting temperature can be 90℃, 91℃, 92℃, 93℃, 94℃, or 95℃, and the decocting time can be 30min, 31min, 32min, 33min, 35min, 36min, 38min, or 40min; the ultrasonic treatment temperature can be 70℃, 71℃, 72℃, 73℃, 74℃, 78℃, or 80℃, and the ultrasonic treatment time can be 20min, 21min, 22min, 23min, 24min, or 25min.

[0070] The present application will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present application.

[0071] Example 1

[0072] This embodiment provides a Chaga mushroom extract, the preparation method of which includes the following steps:

[0073] (1) Activation of strain: The frozen Inonotus obliquus was picked up with an inoculation loop and streaked on a solid culture medium to activate the strain. After three generations of continuous activation culture, activated Inonotus obliquus was obtained.

[0074] The solid culture medium comprises the following components in the indicated weight percentages: glucose 3%, agar 1.5%, yeast extract 0.1%, peptone 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, birch sap 0.5%, with the remainder being water;

[0075] (2) Preparation of seed liquid: The mycelium of the activated Inonotus obliquus was inoculated into the liquid fermentation medium using an inoculation loop and cultured at 26°C for 5 days to obtain seed liquid;

[0076] The liquid fermentation medium comprises the following components in the indicated weight percentages: glucose 3%, yeast extract 0.2%, peptone 0.4%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.1%, birch sap 1%, kudzu root ethanol extract 1%, and the remainder is water.

[0077] (3) Preparation of fermentation broth: The above seed liquid was inoculated into the liquid fermentation medium at 10% (v / v) and cultured at 26°C for 7 days. On the 4th day of culture, 2 g / L of cypress seed oil was added. After the culture was completed, the fermentation broth of Inonotus obliquus was obtained.

[0078] (4) Extraction: The fermentation broth of Inonotus obliquus was filtered to obtain fermentation filtrate and bacterial cells; the bacterial cells were crushed and mixed with the fermentation filtrate, and then boiled at 95°C for 40 min, then cooled to 80°C and ultrasonically treated for 25 min, and filtered. The obtained filtrate is the extract of Inonotus obliquus.

[0079] Example 2

[0080] This embodiment provides a Chaga mushroom extract, the preparation method of which includes the following steps:

[0081] (1) Activation of strain: The frozen Inonotus obliquus was picked up with an inoculation loop and streaked on a solid culture medium to activate the strain. After three generations of continuous activation culture, activated Inonotus obliquus was obtained.

[0082] The solid culture medium comprises the following components in the indicated weight percentages: glucose 4%, agar 2%, yeast extract 0.2%, peptone 0.5%, potassium dihydrogen phosphate 0.15%, magnesium sulfate 0.15%, birch sap 0.8%, with the remainder being water;

[0083] (2) Preparation of seed liquid: The mycelium of the activated Inonotus obliquus was inoculated into the liquid fermentation medium using an inoculation loop and cultured at 27°C for 5.5 days to obtain seed liquid;

[0084] The liquid fermentation medium comprises the following components in the indicated weight percentages: glucose 4%, yeast extract 0.3%, peptone 0.5%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.2%, birch sap 1.5%, kudzu root ethanol extract 1.5%, and the remainder is water.

[0085] (3) Preparation of fermentation broth: The above seed liquid was inoculated into the liquid fermentation medium at 12% (v / v) and cultured at 28°C for 8 days. On the 4th day of culture, 3 g / L of cypress seed oil was added. After the culture was completed, the fermentation broth of Inonotus obliquus was obtained.

[0086] (4) Extraction: The fermentation broth of Inonotus obliquus was filtered to obtain fermentation filtrate and bacterial cells; the bacterial cells were crushed and mixed with the fermentation filtrate, and then boiled at 90°C for 30 min, then cooled to 70°C, sonicated for 20 min, and filtered. The obtained filtrate is the extract of Inonotus obliquus.

[0087] Example 3

[0088] This embodiment provides a Chaga mushroom extract, the preparation method of which includes the following steps:

[0089] (1) Activation of strain: The frozen Inonotus obliquus was picked up with an inoculation loop and streaked on a solid culture medium to activate the strain. After three generations of continuous activation culture, activated Inonotus obliquus was obtained.

[0090] The solid culture medium comprises the following components in the indicated weight percentages: glucose 5%, agar 2.5%, yeast extract 0.3%, peptone 0.6%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.2%, birch sap 1%, and the remainder being water.

[0091] (2) Preparation of seed liquid: The mycelium of the activated Inonotus obliquus was inoculated into the liquid fermentation medium using an inoculation loop and cultured at 28°C for 6 days to obtain seed liquid;

[0092] The liquid fermentation medium comprises the following components in the indicated weight percentages: glucose 5%, yeast extract 0.4%, peptone 0.7%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.3%, birch sap 2%, kudzu root ethanol extract 2%, and the remainder is water.

[0093] (3) Preparation of fermentation broth: The above seed liquid was inoculated into the liquid fermentation medium at 15% (v / v) and cultured at 30°C for 9 days. On the 4th day of culture, 4 g / L of cypress seed oil was added. After the culture was completed, the fermentation broth of Inonotus obliquus was obtained.

[0094] (4) Extraction: The fermentation broth of Inonotus obliquus was filtered to obtain fermentation filtrate and bacterial cells; the bacterial cells were crushed and mixed with the fermentation filtrate, and then boiled at 92°C for 35 min, then cooled to 75°C, sonicated for 23 min, and filtered. The obtained filtrate is the extract of Inonotus obliquus.

[0095] Comparative Example 1

[0096] This comparative example provides a Chaga mushroom extract. The preparation method of the Chaga mushroom extract is the same as that of Example 1, with steps (1) and (2) being the same, except that cypress seed oil is not added in step (3).

[0097] Comparative Example 2

[0098] This comparative example provides a Chaga mushroom extract. The preparation method of the Chaga mushroom extract is the same as that of Example 1, with steps (1) and (2) being the same, except that in step (3), the cypress seed oil is replaced with coix seed oil.

[0099] Comparative Example 3

[0100] This comparative example provides a Chaga mushroom extract. The preparation method of the Chaga mushroom extract differs from that of Example 2 only in the liquid fermentation medium used. In this comparative example, the kudzu root ethanol extract in the liquid fermentation medium is replaced with Polygonum multiflorum extract.

[0101] Comparative Example 4

[0102] This comparative example provides a Chaga mushroom extract. The preparation method of the Chaga mushroom extract differs from that of Example 2 only in the liquid fermentation medium used. The liquid fermentation medium of this comparative example lacks kudzu root ethanol extract.

[0103] Comparative Example 5

[0104] This comparative example provides a birch extract. The preparation method of the birch extract differs from that of Example 2 only in the liquid fermentation medium used. The liquid fermentation medium of this comparative example lacks birch sap.

[0105] Comparative Example 6

[0106] This comparative example provides a birch extract. The preparation method of the birch extract differs from that of Example 2 only in the liquid fermentation medium used. The liquid fermentation medium of this comparative example contains 1% by mass of birch sap and 1.5% by mass of kudzu ethanol extract.

[0107] Comparative Example 7

[0108] This comparative example provides a birch extract. The preparation method of the birch extract differs from that of Example 2 in that the liquid fermentation culture medium used lacks birch sap and kudzu ethanol extract; and cypress seed oil is not added in step (3).

[0109] Test 1

[0110] This test determined the polysaccharide content and total triterpenoid content of the *Inonotus obliquus* extracts prepared in Examples 1-3 and Comparative Examples 1-7. The results are shown in Table 1.

[0111] Polysaccharide content detection method: phenol-sulfuric acid method;

[0112] Total triterpenoid content detection method: spectrophotometry.

[0113] Table 1. Results of polysaccharide and triterpenoid content determination of Inonotus obliquus extracts in each group

[0114]

[0115] As shown in Table 1, the polysaccharide and triterpenoid contents of the *Inonotus obliquus* extracts in Examples 1-3 are significantly better than those in Comparative Examples 1-7. This indicates that the various components in the liquid fermentation medium of the present invention interact with each other, enabling *Inonotus obliquus* to effectively synthesize secondary metabolites during its growth period, thereby increasing the accumulation and release of effective components, especially polysaccharides and triterpenoids. At the same time, using *Platycladus orientalis* seed oil as an inducer can further promote the accumulation of *Inonotus obliquus* metabolites, thereby further increasing the content of effective components in the *Inonotus obliquus* extract.

[0116] Test 2

[0117] This test determined the repair effects of the Inonotus obliquus extracts prepared in Examples 1-3 and Comparative Examples 1-7, and the results are shown in Table 3.

[0118] (1) Measurement principle

[0119] Repair refers to actions that help maintain the skin's normal state, keep it hydrated, and reduce the irritation and damage caused by external factors. Damaged skin barriers increase the skin's sensitivity to external stimuli, a major cause of erythema, dryness, peeling, and itching. In daily life, various factors such as sunlight and chemical irritants can damage the skin barrier, leading to increased skin permeability. Therefore, repairing the skin barrier and reducing external irritation is a crucial way to maintain normal skin condition. Using non-specific fluorescent staining reagents, changes in zebrafish skin barrier permeability can be efficiently observed, showing a positive correlation between the fluorescent area of ​​zebrafish embryonic skin and skin barrier permeability. Therefore, this experiment aims to construct a zebrafish embryonic skin barrier damage model, use fluorescent staining reagents to label skin permeability, and evaluate the activity of the test samples in inhibiting skin permeability by testing the red fluorescent area of ​​zebrafish skin, thereby reflecting the skin barrier repair efficacy of the test samples.

[0120] (2) Measurement method

[0121] Zebrafish 48 hpf embryos were collected. 6 mL of the corresponding concentration of sample was added to each well in all groups. 6 mL of gentian violet (staining agent) was added to each well in the blank group, and 6 mL of a mixture of gentian violet (staining agent) and lactic acid (LA) (modeling agent) was added to each well in the other groups. After placing the embryos in a rotating shaker for 20 min, they were aspirated and placed in 6-well plates. The plates were washed with embryo buffer until the solution was colorless. After processing, the fluorescent staining area of ​​each zebrafish was observed using an upright fluorescence microscope, and photographs were taken. The tail of each zebrafish was marked using analysis software, and then "Measure Area" was selected in the "Measurement" column. The "Area" in the test results was selected as the degree of photodamage. The skin barrier permeability inhibition rate was calculated based on the fluorescent staining area.

[0122] Skin barrier permeability inhibition rate = (Fluorescent area of ​​model control group - Fluorescent area of ​​test substance) / Fluorescent area of ​​model control group

[0123] The specific test groups are shown in Table 2 below:

[0124] Table 2 Repair Effect Test Groups

[0125]

[0126] Table 3. Zebrafish Repair Efficacy Table

[0127]

[0128] Table 3 shows that the skin barrier repair rates of test groups 1-10 were all greater than 0, indicating that the test group samples had an improving effect on skin barrier damage caused by lactic acid, that is, the *Inonotus obliquus* extract has a repairing effect. The skin barrier repair rates of test groups 1-3 were significantly higher than those of test groups 4-10, indicating that the *Inonotus obliquus* extract prepared using the specific process of this invention has high activity and significant repairing effect. Changing the composition of the culture medium or the inducing agent will affect the repairing effect of the *Inonotus obliquus* extract. Furthermore, combining the results of test groups 2 and 11-12, it can be seen that the skin barrier repair rate increases with the increase of the concentration of *Inonotus obliquus* extract. Therefore, the preferred concentration of *Inonotus obliquus* extract is 50-100 g / L.

[0129] Test 3

[0130] This test determined the anti-inflammatory effects of the Inonotus obliquus extracts prepared in Examples 1-3 and Comparative Examples 1-7, and the results are shown in Table 5.

[0131] 1) Measurement Principle

[0132] Neutrophils in zebrafish embryos are highly similar to human neutrophils in morphology, biochemistry, and physiological function. Neutrophils are the first white blood cells to appear at sites of injury or pathogen invasion, playing a role in clearing infection or harmful substances. A model of neutrophil aggregation induced by CuSO4-induced damage to neurothalamic cells in the lateral line region of zebrafish embryos was tested. The changes in the number of neutrophils in the lateral line region of the embryos in the test substance treatment group and the model control group were compared, and the neutrophil inhibition rate was calculated to evaluate the acne-fighting efficacy of the raw material, formulation, or product.

[0133] 2) Measurement method

[0134] Wild-type zebrafish embryos at 72 hpf were collected and grouped into groups of 24 embryos each. The test required setting up a model control group (CuSO4 working solution), a positive control group (dexamethasone working solution), and a test sample group (the test samples were Inonotus obliquus extracts from Examples 1-3 and Comparative Examples 1-7, respectively).

[0135] (1) Model control group setup

[0136] Twenty-four fish embryos were randomly selected and transferred to a six-well plate. 6 mL of fish embryo culture medium was added, and the six-well plate was placed in a constant temperature incubator at 28℃±1℃. The plate was first cultured with the drug for 2 h, and then cultured with 6 mL of copper sulfate containing 10 μM CuSO4 for 2 h.

[0137] (2) Setting up a positive control group

[0138] Twenty-four fish embryos were randomly selected and transferred to a six-well plate. 6 mL of fish embryo culture medium containing 40 μM dexamethasone was added. The six-well plate was placed in a constant temperature incubator at 28℃±1℃. The culture was first administered and cultured for 2 h, then replaced with 6 mL of copper sulfate containing 10 μM CuSO4 and cultured for another 2 h.

[0139] (3) Subject treatment

[0140] Nine groups of 24 fish embryos each were randomly selected and transferred to six-well plates. 6 mL of fish embryo culture medium containing the test samples (the test samples were *Inonotus obliquus* extracts from Examples 1-3 and Comparative Examples 1-7, respectively) was added. The six-well plates were placed in a 28℃±1℃ incubator for 2 h of incubation after drug administration, followed by incubation with 6 mL of 10 μM copper sulfate (CuSO4) for another 2 h.

[0141] The above-mentioned groups of fish embryos were fixed and stained. After processing, the neutrophils of each group of zebrafish were observed using an upright fluorescence microscope, and photographs were taken and recorded. The number of neutrophils in the three-quarters of the tail lateral line region from the anus of each fish embryo was counted, and the neutrophil aggregation inhibition rate was calculated.

[0142] Inhibition rate = (MT) / M 100%

[0143] In the formula:

[0144] T—The average number of neutrophils in the fish embryos of the tested sample treatment group;

[0145] M—The average number of neutrophils in the embryos of fish in the model control group;

[0146] Two-tailed t-tests were performed to determine the number of neutrophils in the embryos of fish in the test substance treatment group and the number of neutrophils in the model control group, and the p-values ​​were obtained.

[0147] The specific test groups are shown in Table 4 below:

[0148] Table 4 Anti-inflammatory effect test grouping

[0149]

[0150] Table 5 Results of neutrophil inhibition rate in zebrafish

[0151]

[0152] Table 5 shows that the neutrophil inhibition rate of test groups 1-10 was greater than 0, indicating that the test group samples had an inhibitory effect on neutrophil migration in zebrafish compared with the model group, suggesting that the samples had a certain ameliorative effect on the inflammatory response induced by copper sulfate, that is, the Inonotus obliquus extract has anti-inflammatory and acne-reducing effects. The skin barrier repair rate of test groups 1-3 was significantly higher than that of test groups 4-10, indicating that the Inonotus obliquus extract prepared using the specific process of this invention has higher activity and more significant anti-inflammatory and acne-reducing effects. Changing the composition of the culture medium, the type of inducer, or the time of addition will affect the anti-inflammatory and acne-reducing effects of the Inonotus obliquus extract. Furthermore, combining the data from test groups 2 and 11-12, it can be seen that the neutrophil inhibition rate is not positively correlated with the concentration of the Inonotus obliquus extract. Considering the effect data in Tables 3 and 5, the preferred concentration of the Inonotus obliquus extract is 50-100 g / L, and more preferably 50 g / L.

[0153] Secondly, the present invention also provides an application of the above-mentioned Inonotus obliquus extract in the preparation of products having at least one of the following effects: 1) anti-inflammatory; 2) repair.

[0154] For example, the product is a topical skin composition. Specifically, it can be a cosmetic and / or medical product.

[0155] It is understood that the products of the present invention can be used for topical or transdermal application and can be produced in any solid, liquid, or semi-solid formulation. Such formulations include, but are not limited to, creams, emulsions (e.g., multiple emulsions, such as oil-in-water and / or polysiloxane-in-water emulsions, water-in-oil and / or polysiloxane-in-water emulsions, water / oil / water or water / polysiloxane / water emulsions, or oil / water / oil or polysiloxane / water / polysiloxane emulsions), liquid crystals, anhydrous compositions, aqueous dispersions, oils, emulsions, balms, aqueous or oily gels, creams, solutions, hydroalcoholic solutions, aqueous glycol solutions, hydrogels, liniments, conditioning agents, masks, or serums.

[0156] It is understood that the products of the present invention may also contain excipients, adjuvants and / or ingredients acceptable in skin care and / or dermatology, such as humectants (e.g., glycerol, propylene glycol, butylene glycol, pentylene glycol, capryloyl glycol, lactic acid, urea, sodium hyaluronate), emollients or skin conditioning agents (e.g., dimethicone, glyceryl stearate, caprylic / capric triglyceride, cetearyl alcohol, lecithin, C12-15 alkyl benzoate, squalane, lanolin, behenol, tocopherol acetate, panthenol, shea butter, retinyl palmitate, retinol), surfactants (e.g., xanthan gum, sodium lauryl ether sulfate, stearic acid, polysorbate 20, polysorbate 80, stearyl alcohol, cetyl alcohol, stearyl alcohol polyether-2, cetearyl alcohol ether-20, cocamidopropyl betaine), etc.

[0157] Finally, the present invention provides an acne-removing essence, wherein the acne-removing essence comprises the above-mentioned Inonotus obliquus extract.

[0158] Preferably, the acne-removing essence comprises, by weight percentage, the following ingredients: 0.40-0.45% thickener, 0.60-0.80% preservative, 4.50-5.50% Inonotus obliquus extract, and the remainder being water.

[0159] For example, the mass percentage of the thickener may be 0.40%, 0.42%, 0.44%, or 0.45%, etc.; the mass percentage of the preservative may be 0.60%, 0.65%, 0.70%, 0.75%, or 0.80%, etc.; and the mass percentage of the Inonotus obliquus extract may be 4.50%, 4.85%, 5.00%, 5.20%, or 5.50%, etc.

[0160] For example, the thickener includes xanthan gum and sodium polyacrylamide dimethyl taurate; the preservative includes a blend of phenoxyethanol and ethylhexylglycerin.

[0161] Preferably, the acne-removing essence comprises, by weight percentage, the following ingredients: 0.15% xanthan gum, 0.28% sodium polyacrylamide dimethyl taurate, 0.70% a blend of phenoxyethanol and ethylhexylglycerin, 5.00% Inonotus obliquus extract, and the remainder being water.

[0162] The present invention also provides a method for preparing the above-mentioned acne-removing essence, comprising the following steps:

[0163] ① Add the thickener to the water, stir to disperse, heat to 80-82℃, soak and swell, stir to homogenize for 2-3 minutes;

[0164] ② Stir and cool to below 40°C, add the Inonotus obliquus extract, and stir until homogeneous;

[0165] ③ Continue adding the preservative, stir well, and discharge to obtain the essence.

[0166] For example, in step ①, the heating temperature can be 80℃, 81℃ or 82℃, etc.; the stirring and homogenization time can be 2min or 3min, etc.

[0167] The above-mentioned acne-removing essence will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application.

[0168] Example 4

[0169] An acne-removing serum has the following ingredient composition as shown in Table 6 below.

[0170] Table 6. Raw material ratio of acne treatment serum

[0171]

[0172] Weigh the above raw materials according to the corresponding proportions, and prepare them according to the following preparation method:

[0173] ① Add phase B and phase C to phase A in sequence, stir to disperse, heat to 80℃, soak and swell, stir to homogenize for 3 minutes;

[0174] ② Stir and cool to 38℃, add phase D, and stir until homogeneous;

[0175] ③ Continue adding phase E, stir well, and discharge to obtain the essence.

[0176] Upon visual inspection, the prepared acne-removing essence is a light yellow, slightly bluish, viscous liquid with a measured pH of 6.0.

[0177] Test 4

[0178] This test measures the acne-removing effect of the acne-removing essence in Example 4 above, as detailed below.

[0179] 1. Subjects

[0180] Participants were screened based on factors such as facial appearance (a certain number of acne spots (whiteheads + blackheads)) and other individual health conditions. 33 participants were selected, and all 33 completed the study (0 dropouts). Among them, 25 were female and 8 were male, with a mean age of 30.8 ± 7.2 years, meeting the voluntary inclusion criteria. Detailed participant information is shown in Table 7 below.

[0181] Table 7 Subject Information

[0182]

[0183] 2. Experimental Design

[0184] After cleansing, subjects should apply an appropriate amount of this product evenly to their facial skin and gently massage until absorbed. The product should be applied twice daily for 28 days, from July 28, 2025 to August 25, 2025.

[0185] 3. Testing

[0186] (1) Testing basis: Based on the "Test Method for Acne Removal Efficacy of Cosmetics" T / TDCA 004-2021, the effect was evaluated by comparing the subject's own face before and after.

[0187] (2) Testing instruments: VISIA facial image analyzer and analytical balance. All performance indicators meet the requirements of the testing specifications.

[0188] Testing Environment: The instrument testing process was conducted in an environment with a temperature of 21±1℃ and a relative humidity of 50±10% RH. Subjects were required to acclimatize to these conditions for 30 minutes before evaluation and testing. The ambient temperature and humidity during the testing period were as follows: Figure 2 and Figure 3 As shown.

[0189] (3) Testing process

[0190] ① Cleaning: Before the test, cleanse your face with a cleansing product (such as Cetaphil facial cleanser) and dry it with a lint-free and fluorescent agent-free tissue; repeat the cleaning process for each follow-up visit.

[0191] ②Test: First, the assessing physician counts the number of pimples on the subject's face. Then, technicians take VISIA images. Finally, IPP analysis software is used to analyze the skin of the test area in the VISIA facial images. The values ​​were analyzed. The same tests were repeated after 2 and 4 weeks of sample use.

[0192] (4) Statistical methods

[0193] SPSS software was used for statistical analysis of the data. Quantitative data were expressed as mean ± standard deviation and subjected to a normality test. If the data met the normality requirement, a paired t-test was used for comparisons of the data before and after the same data point; otherwise, a rank-sum test of two related samples was used.

[0194] If, compared before and after product use, the number of acne (whiteheads + blackheads) and skin redness index of the subjects are... If any parameter shows a significant improvement (P < 0.05), it indicates that the sample has acne-removing effects; otherwise, the sample is considered to have no acne-removing effects.

[0195] (4) Test results

[0196] ① The number of pimples and skin redness index of the subjects before and after using the acne-removing serum The value changes are shown in Table 7 below. In the table, D0 represents before use, D14 represents after 14 days of sample use, and D28 represents after 28 days of sample use.

[0197] Table 7. Number of pimples and skin redness index of the subjects Value changes

[0198]

[0199] ② Comparison chart of the number of pimples and skin redness index before and after the subjects used the acne-removing essence. The value comparison charts are as follows: Figure 4 and Figure 5 As shown in the figure. The significance level is indicated by the following methods: "ns" indicates no statistically significant difference (P>0.05); " "Indicates a significant difference, 0.01 ≤ P < 0.05;" "P" indicates a significant difference, 0.001 ≤ P < 0.01; "" indicates a significant difference, P<0.001).

[0200] ③ Results of tests on the number of pimples and skin redness index before and after the subjects used the acne-removing serum. The statistical analysis of the test results is shown in Tables 8 and 9, respectively.

[0201] Table 8 Statistical Analysis of Acne Count Test Results

[0202]

[0203] Table 9 Skin Redness Index Statistical analysis of value test results

[0204]

[0205] ④ After using the acne-removing serum for 28 days, the changes in the red areas of subjects 6 and 18 were as follows: Figure 6 and 7 As shown.

[0206] Combined with Table 7-9 and Figure 4-7 It can be seen that after the subjects used the acne-removing essence for 28 days, the number of pimples (whiteheads + blackheads) and the skin redness index in the test area were [data missing]. All values ​​showed significant improvement (P < 0.05). In conclusion, under the conditions of this experiment, the acne-removing essence of the present invention has good acne-removing efficacy. Furthermore, during this experiment, 0 out of 33 subjects experienced adverse reactions related to the test product, indicating that the acne-removing essence of the present invention is gentle and safe.

Claims

1. A method for preparing an extract of Inonotus obliquus, characterized by, Comprising the following steps: (1) Strain activation: the frozen Inonotus obliquus is streaked on solid culture medium for strain activation. After three generations of continuous activation culture, the activated Inonotus obliquus is obtained; The solid culture medium comprises the following components in mass percentage: glucose 3-5%, agar 1.5-2.5%, yeast powder 0.1-0.3%, peptone 0.4-0.6%, potassium dihydrogen phosphate 0.1-0.2%, magnesium sulfate 0.1-0.2%, white birch juice 0.5-1%, and the balance is water; (2) Preparation of seed liquid: the mycelium of the above activated Inonotus obliquus is inoculated into a liquid fermentation medium, and cultured at 26-28℃ for 5-6d to obtain a seed liquid; The liquid fermentation medium comprises the following components in mass percentage: glucose 3-5%, yeast powder 0.2-0.4%, peptone 0.4-0.7%, potassium dihydrogen phosphate 0.1-0.3%, magnesium sulfate 0.1-0.3%, white birch juice 1-2%, and pueraria extract 1-2%, and the balance is water; and the mass ratio of the white birch juice and the pueraria extract is 1:1, and the pueraria extract is pueraria ethanol extract; (3) Fermentation culture: the above seed liquid is inoculated into the liquid fermentation medium at 10-15% (v / v), and cultured at 26-30℃ for 7-9d, and 2-4g / L of Platycladus orientalis oil is added on the 4th day of culture, and the Inonotus obliquus fermentation broth is obtained after culture; (4) Extraction: the Inonotus obliquus fermentation broth is filtered to obtain a fermentation filtrate and a mycelium; the mycelium is crushed and mixed with the fermentation filtrate, and then subjected to decoction treatment at 90-95℃ for 30-40min, and then cooled to 70-80℃, and then subjected to ultrasonic treatment for 20-25min, and then filtered, and the obtained filtrate is the Inonotus obliquus extract.

2. The Inonotus obliquus extract prepared by the preparation method of claim 1.

3. The application of the Inonotus obliquus extract of claim 2 in the preparation of cosmetics with at least one of the following effects: 1) anti-inflammatory; 2) repair.

4. An acne serum, characterized by, The serum comprises the Inonotus obliquus extract of claim 2.

5. The acne treatment serum of claim 4, wherein the serum is characterized by, The serum comprises the following raw materials in mass percentage: thickening agent 0.40-0.45%, preservative 0.60-0.80%, Inonotus obliquus extract 4.50-5.50%, and the balance is water.

6. The acne treatment serum of claim 5, wherein, The thickening agent comprises xanthan gum and polyacryloyl sodium dimethyl taurate; and the preservative comprises phenoxyethanol and ethylhexylglycerin blend.

7. The acne treatment serum of claim 6, wherein, The serum comprises the following raw materials in mass percentage: xanthan gum 0.15%, polyacryloyl sodium dimethyl taurate 0.28%, phenoxyethanol and ethylhexylglycerin blend 0.70%, Inonotus obliquus extract 5.00%, and the balance is water.

8. The method for preparing an acne-removing essence according to claim 5, characterized in that, Comprising the following steps: ① The thickening agent is added to the water, stirred and dispersed, heated to 80-82℃, soaked and swelled, and stirred and homogenized for 2-3min; ② Stirring and cooling to below 40℃, adding the Inonotus obliquus extract, and stirring uniformly; ③ Continue to add the preservative, stir evenly, discharge, the essence liquid is obtained.

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