Isaria cymbae mycelium water extract and application of the same in preparing a cosmetic for improving acne

Abstract

The application discloses a water extract of Isaria cicadae mycelium and application of the water extract in preparation of cosmetics for improving acne. The water extract of the Isaria cicadae mycelium is obtained by crushing freeze-dried mycelium of Isaria cicadae C1 and then using a cellulase and hot water to perform extraction, the water extract contains various active ingredients such as polysaccharides, nucleosides, sugar alcohols and proteins, has good effects of anti-inflammation, anti-oxidation, promotion of growth of Staphylococcus epidermidis, inhibition of Propionibacterium acnes and regulation of skin microecological balance, and can be used as an anti-acne and anti-inflammatory active material in the fields of cosmetics and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to an Isaria cicadae mycelium water extract and application of the Isaria cicadae mycelium water extract in preparation of a cosmetic for improving acne. BACKGROUND

[0002] There are various microorganisms inhabiting in the surface layer of human skin and hair follicles, which play an important role in maintaining skin health and are also known as the microbial barrier of skin. The skin microbiota, host skin and environment constitute the skin microecosystem, which interact with and restrict each other to maintain a coordinated, physiological and dynamic balance. Balanced skin microecosystem is conducive to skin health. In daily life, factors such as ultraviolet radiation, frequent use of cosmetics, life pressure and irregular work and rest will cause the skin flora homeostasis to be destroyed, thereby causing skin problems to occur frequently.

[0003] Acne is a chronic inflammatory common skin disease, which occurs mainly in adolescents, and the incidence rate is as high as more than 90%. Although the disease generally does not endanger life, it can cause patients to lose or disfigure their appearance due to its predilection for the face, and has a considerable psychological impact on patients, and can cause psychological diseases such as anxiety and depression in severe cases, so it cannot be ignored. The occurrence of acne is closely related to factors such as excessive sebum secretion, keratinization of hair follicle sebaceous duct, pathogenic bacterial infection and inflammation. Excessive secretion of sebaceous glands causes pore blockage, which promotes the proliferation of Propionibacterium acnes in the hair follicle, which can cause inflammation. Studies have shown that there is a disorder of skin flora in the skin of acne patients, mainly manifested as an increase in the number of Propionibacterium acnes and a decrease in the number of Staphylococcus epidermidis, and the imbalance of the two leads to the proliferation of Propionibacterium acnes, which secretes lipase to hydrolyze triglycerides and metabolizes more free fatty acids, thereby aggravating the inflammatory response. At present, acne is mainly treated by using hormone antagonists, retinoid drugs, antibiotics and other drugs, but long-term use of drugs has the risk of causing allergies, kidney function damage and even deformity and other toxic side effects. Selecting acne-removing cosmetic raw materials with components of restoring flora balance and anti-inflammatory is a safe and effective means for preventing and treating acne.

[0004] Cordyceps cicadae, commonly known as golden cicada, is a kind of precious fungus for both medicine and food, and is one of the four major Cordyceps in China, which has a long history of use in China. Modern medical research has confirmed that Cordyceps cicadae and its active ingredients have the effects of anti-tumor, immune regulation, improving kidney function, and reducing blood lipids, blood pressure and cholesterol. Isaria cicadae is the conidial stage of Cordyceps cicadae, which is the asexual stage of Cordyceps cicadae. The mycelium of Isaria cicadae contains many secondary metabolites such as polysaccharides, nucleotides, amino acids, hyaluronic acid, and small molecule sugar alcohols (such as cordycepic acid), which have important biological activity functions such as immune regulation, anti-tumor, regulation of nervous system, improvement of kidney function, antibacterial, antiviral and other functions, and have a good application prospect in the development of skin external preparations.

[0005] The applicant previously obtained a strain of Isaria cicadae producing exopolysaccharide, which was preserved in China General Microbiological Culture Collection Center on November 5, 2019, and the preservation number is CGMCC No. 18807. The Isaria cicadae provided by the applicant (referred to as Isaria cicadae C1) is a strain with fast growth and high yield of exopolysaccharide. The exopolysaccharide has a narrow molecular weight distribution, good uniformity, good antioxidant capacity, good immune activity, and good moisturizing property, apparent viscosity and suspension performance, and can be applied to food, cosmetics as antioxidant and moisturizing raw materials, thickening agents and suspension stabilizers (see: ZH 202110230202.5, a strain of Isaria cicadae producing exopolysaccharide, exopolysaccharide and preparation method and application). On this basis, the applicant further developed the function of the mycelium thereof for the treatment of acne. SUMMARY

[0006] The present application first provides a water extract of Isaria cicadae C1 fermentation mycelium, which has good anti-inflammatory, antioxidant, and balanced effects on Staphylococcus epidermidis and Propionibacterium acnes. As an efficacy additive in skin external preparations, it is natural, safe and can significantly relieve the symptoms of acne skin.

[0007] In order to achieve the above purpose, the present application provides the following technical scheme: a preparation method of water extract of Isaria cicadae mycelium, characterized in that Isaria cicadae C1 (CGMCC No. 18807) is fermented, the fermentation broth is centrifuged, the mycelium is collected, then washed and freeze-dried to obtain freeze-dried mycelium; the freeze-dried mycelium is crushed, and then extracted by cellulase and hot water immersion method.

[0008] Further, the crushing is crushing the freeze-dried mycelium through an 80-mesh sieve.

[0009] Further, the cellulase-assisted hot water extraction method is as follows: the crushed freeze-dried mycelium is added with water and cellulase (≥80,000 U / g) (food grade) for enzymolysis, the enzymolysis temperature is 40-50°C, and the enzymolysis time is 1-2 h; the enzyme activity is rapidly inactivated after the enzymolysis; then the hot water extraction method is continuously used for extraction, the extraction temperature is 90-100°C, the extraction time is 2-4 h, and the extraction times is 2-3 times; the extraction liquid is hot-filtered, the filtrates are combined and concentrated, and then freeze-dried to obtain the water extract. The enzyme dosage is 0.10-0.30% (based on the mass of the freeze-dried mycelium).

[0010] The mycelium water extract (referred to as ICE-01) of Isaria cicadae prepared by the above method contains the following components: the content of uridine is 0.05-0.10%, the content of adenosine is 0.05-0.10%, the content of guanosine is 0.04-0.08%, the content of inosine is 0.01-0.015%, the content of uracil is 0.01-0.02%, the content of erythritol is 0.50-1.00%, the content of cordycepic acid is 5.00-10.00%, the content of glucose is 1.00-1.50%, the content of trehalose is 0.50-1.00%, the content of polysaccharide is 5.00-10.00%, and the content of protein is 0.15-0.20%.

[0011] The application further provides use of the mycelium water extract of Isaria cicadae as an active ingredient in preparation of a skin external agent with antioxidant efficacy. Preferably, the concentration of the mycelium water extract of Isaria cicadae is 2.00-10.00 mg / mL. The above antioxidant efficacy is antioxidant activity with any one or more of the following effects: good DPPH free radical scavenging, hydroxyl radical scavenging, ABTS radical scavenging and iron ion reducing capacity. When the concentration of the water extract ICE-01 is 10.00 mg / mL, the DPPH free radical scavenging rate can reach 96.24%, and the ·OH scavenging rate reaches 70.82%; when the concentration of the water extract ICE-01 is 8.00 mg / mL, the ABTS radical scavenging rate can reach 90.71%; and when the concentration of the ICE-01 sample is 0.05 g / mL, which is equivalent to 0.88 mM FeSO4·7H2O, the iron ion reducing capacity is strong.

[0012] The application also provides use of the Isaria cicadae mycelium water extract as an active ingredient in the preparation of a skin external preparation with anti-inflammatory efficacy. Preferably, the concentration of the Isaria cicadae mycelium water extract is 0.10-10.00 mg / mL. The above-mentioned anti-inflammatory is specifically: reducing the inflammatory response of LPS-induced macrophage RAW264.7, and reducing the expression of inflammatory factors IL-6, IL-β and TNF-α. Compared with the model group treated with LPS, the concentration of 1.0 mg / mL of the water extract ICE-01 can inhibit the release of IL-6, IL-β and TNF-α of RAW264.7 cells (P<0.05, P<0.01). It can be seen that the medium dose of the application ICE-01 has an inhibitory effect on the increase of the level of inflammatory factors induced by LPS.

[0013] The application also provides use of the Isaria cicadae mycelium water extract in the preparation of a skin external preparation for promoting the growth of Staphylococcus epidermidis, inhibiting the growth of Propionibacterium acnes, and regulating the balance of Staphylococcus epidermidis and Propionibacterium acnes. Preferably, the concentration of the Isaria cicadae mycelium water extract is 1.25-10.00 mg / mL. When the concentration of the water extract ICE-01 is 7.50 mg / mL, the antibacterial rate on Propionibacterium acnes reaches 50.12%. When the concentration of the water extract ICE-01 is 10.00 mg / mL, the growth promotion rate on Staphylococcus epidermidis reaches 66.58%.

[0014] Preferably, the above-mentioned skin external preparation includes a medicine with disease treatment use or a cosmetic without disease treatment use.

[0015] Further, the application provides use of the Isaria cicadae mycelium water extract in the preparation of a cosmetic for improving acne.

[0016] Preferably, when the cosmetic is a lotion, the lotion includes the following raw materials by weight: 0.125-1 parts of the Isaria cicadae mycelium water extract, 6.23-16.88 parts of phase A, 9-26 parts of phase B, 0.1-0.4 parts of phase C, and water to make up 100 parts; the phase A includes stearyl polyoxyethylene ether-2, stearyl polyoxyethylene ether-21, cetyl stearyl alcohol, dimethyl silicone oil, octyl / decyl triglyceride, white oil and BHT; the phase B includes propylene glycol, glycerol, carbopol 20 and a preservative, and the preservative includes 1,2-hexanediol and glyceryl monocaprylate; and the phase C includes sodium hydroxide.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The present application takes Isaria cicadae C1 mycelium obtained by fermentation as raw material, and obtains mycelium water extract ICE-01 by combining enzymatic hydrolysis and hot water extraction, and the preparation method is simple and easy to operate, and no organic reagents such as ethanol and methanol are used in the extraction process, and the obtained water extract contains polysaccharides, nucleosides, sugar alcohols, proteins and other active ingredients, and can be used as a new type of raw material for anti-acne and anti-inflammatory cosmetics, which not only has good antioxidant activity, strong inflammatory factor inhibition effect, but also can effectively promote the growth of Staphylococcus epidermidis and inhibit the growth of Propionibacterium acnes, thereby relieving the symptoms of acne skin, and can be applied to the fields of cosmetics, food and medicine, especially in cosmetics. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Nucleoside mixture standard spectrum (peaks 1-12: cytosine, uracil, cytidine, hypoxanthine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine, cordycepin, respectively);

[0020] Figure 2 Nucleoside component determination liquid chromatogram of the water extract ICE-01 of the present application (peaks 1-5: uracil, uridine, inosine, guanosine, adenosine, respectively);

[0021] Figure 3 Sugar alcohol component determination ion chromatogram of the water extract ICE-01 of the present application (peaks 1-4: erythritol, trehalose, mannitol, glucose, respectively);

[0022] Figure 4 Water extract ICE-01 of the present application on RAW264.7 macrophage cytotoxicity test results;

[0023] Figure 5 Effect of different concentrations of ICE-01 (0.50, 1.00, 5.00 mg / mL) on IL-6 level produced by LPS-induced RAW264.7 cells (NT is the untreated control group, LPS is the model group, DEX is the positive group, ICE-01 is the drug group, compared with the control group,####P<0.0001; compared with the model group, *P<0.05, **P<0.01);

[0024] Figure 6 Effect of different concentrations of ICE-01 (0.50, 1.00, 5.00 mg / mL) on IL-β level produced by LPS-induced RAW264.7 cells (NT is the untreated control group, LPS is the model group, DEX is the positive group, ICE-01 is the drug group, compared with the control group,####P<0.0001; compared with the model group, *P<0.05, **P<0.01);

[0025] Figure 7 Effect of different concentrations of ICE-01 (0.50, 1.00, 5.00 mg / mL) on the level of TNF-α produced by LPS-induced RAW264.7 cells (NT is the non-treatment control group, LPS is the model group, DEX is the positive group, and ICE-01 is the drug group. Compared with the control group,####P<0.0001; compared with the model group, *P<0.05, **P<0.01). DETAILED DESCRIPTION

[0026] In order to further illustrate the present application, the application of Isaria cicadae mycelium water extract and preparation of cosmetics for improving acne will be described in detail below in conjunction with examples and drawings, but they cannot be understood as limiting the scope of protection of the present application.

[0027] Example 1: Preparation of Isaria cicadae mycelium water extract ICE-01

[0028] 1) Preparation of Isaria cicadae mycelium

[0029] Strain activation: inoculate the glycerol-preserved Isaria cicadae C1 strain into PDA solid plates and incubate at 28°C for 5-6 days for activation twice;

[0030] Seed culture: under sterile conditions, use an inoculation shovel to transfer fresh mycelium to liquid seed culture medium, place in a 70 mL / 250 mL flask on a shaker at 28°C and 160 r / min for 3 days to obtain first-stage seed liquid; inoculate the first-stage seed liquid into the seed culture medium at a 8% (V / V) inoculation amount, incubate at 28°C and 160 r / min for 2 days to obtain second-stage seed liquid. The seed culture medium contains the following components in the following mass concentrations: potato 200 g / L, sucrose 20 g / L, KH2PO4 2 g / L, MgSO4·7H2O 1 g / L, and the pH is natural. Sterilize at 121°C for 20 min.

[0031] Fermentation culture: add the second-stage seed liquid to the fermentation culture medium at a 6% (V / V) inoculation amount, with a liquid volume of 70 mL / 250 mL, a shaker speed of 180 r / min, and incubate at 28°C for 4 days. The fermentation culture medium contains the following components in the following mass concentrations: glucose 20 g / L, yeast extract 2 g / L, peptone 2 g / L, KH2PO4 2 g / L, MgSO4·7H2O 1 g / L, and the pH is adjusted to 6. Sterilize at 121°C for 20 min.

[0032] Centrifuge the fermentation liquid (8000 r / min, 4°C) for 15 min, and use the supernatant as raw material for extracellular polysaccharides. Collect the C1 mycelium after washing with distilled water and freeze-dry.

[0033] 2) Preparation of water extract ICE-01

[0034] The freeze-dried mycelium was crushed through an 80-mesh sieve and extracted by cellulase (≥80,000 U / g) (food grade) synergistic hot water extraction method: add an appropriate amount of water, enzyme dosage 0.20% (based on the mass of freeze-dried mycelium), enzymolysis time 1.5 h, and enzymolysis temperature 50°C. After enzymolysis, quickly heat to 90°C for 10 min to inactivate the enzyme. Continue to extract by hot water extraction method, with a solid-liquid ratio of 1:30, extraction temperature of 95°C, and extraction time of 3 h for 2 times. The extraction liquid was hot-filtered, the filtrate was concentrated, and then freeze-dried to obtain the water extract.

[0035] Example 2: Analysis of active ingredients in water extract ICE-01

[0036] (1) Determination of polysaccharide content

[0037] Dissolve the ICE-01 sample described in Example 1 in water, precipitate with 3 times the amount of ethanol, dissolve the precipitate in water, and then dilute to a certain multiple to prepare a polysaccharide solution.

[0038] Determine the polysaccharide content by the sulfuric acid-phenol method: weigh an appropriate amount of glucose standard, dry at 105°C to prepare a standard solution of 100 μg / mL, respectively, take 0, 0.10, 0.20, 0.40, 0.60, 0.80, and 1.00 mL into a glass test tube with a stopper, add distilled water to 1.00 mL, then add 5% phenol solution 0.50 mL, quickly add 2.50 mL of concentrated sulfuric acid, mix the reaction liquid well by vortex shaker, stand for 10 min, then place in a boiling water bath for 15 min, measure OD 490 , parallel three times, and take the average value. The standard curve y = 11.973x - 0.0099 (R 2 = 0.9991) is obtained.

[0039] Take 1.00 mL of ICE-01 solution into a 10 mL test tube, and determine the absorbance value of the sample according to the above method and determination conditions. The polysaccharide content in the water extract ICE-01 of the present application is calculated to be 0.0975 g / g (polysaccharide / water extract).

[0040] (2) Nucleotide component analysis

[0041] The nucleoside components were determined by high performance liquid chromatography. 1.00 g of the sample described in Example 1 was accurately weighed into a 100 mL volumetric flask, dissolved by adding water to the mark, and then filtered through a 0.45 μm water phase filter membrane for liquid chromatography analysis. Each sample was repeated 3 times. The mixed standard of nucleoside standard: containing cytosine, uracil, cytidine, hypoxanthine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine and cordycepin. Chromatographic conditions: Venusil MP C18 column (5 μm, 4.6 mm x 250 mm) (Agilent, USA). Mobile phase: water (A phase) - methanol (B phase); flow rate: 1 mL / min; elution program: 0-5 min: 100% A; 5-10 min: 100% A-95% A, 0% B-5% B; 10-30 min: 95% A-70% A, 5% B-30% B; 30-40 min: 70% A-95% A, 30% B-5% B; 40-45 min: 95% A-100% A. UV detector detection, detection wavelength: 254 nm; column temperature: 40 ℃; injection volume: 10 μL. The nucleoside mixed standard spectrum is shown in Figure 1 , and the detection results are shown in Figure 2 .

[0042] As can be seen from Figure 1 , 2 , the water extract ICE-01 of the present application contains 5 nucleoside components, wherein the content of uridine is 0.7746 mg / g, the content of adenosine is 0.8929 mg / g, the content of guanosine is 0.5683 mg / g, the content of inosine is 0.1296 mg / g, and the content of uracil is 0.1496 mg / g (nucleoside / water extract).

[0043] (3) Sugar alcohol component analysis

[0044] The content of sugar alcohol was determined by high performance ion chromatography. 0.50 g of the Isaria cicadae mycelium water extract sample described in Example 1 was accurately weighed into a 100 mL volumetric flask, dissolved by adding distilled water to the mark, and then diluted to an appropriate concentration with distilled water, filtered through a 0.45 μm microporous filter, and then determined by ion chromatography. Erythritol, cordycepic acid, trehalose, arabitol, mannitol, mannose, glucose and galactose were taken as standard substances, dissolved in deionized water and diluted to different concentrations to prepare a standard solution. Chromatographic conditions: CarboPac MA1 anion exchange column (4 mm x 250 mm) (Dionex, USA), mobile phase: 480 mmol / L NaOH solution, flow rate: 0.40 mL / min, sample size: 25 μL, column temperature: 30 ℃. The specific determination results are shown in Figure 3 .

[0045] As can be seen from Figure 3It can be known that the water extract ICE-01 contains five sugar alcohol components, wherein the erythritol content is 5.0348 mg / g, the cordycepin content is 55.9177 mg / g, the glucose content is 10.5073 mg / g, and the trehalose content is 5.5249 mg / g (sugar alcohol / water extract).

[0046] (4) Protein content determination

[0047] A 0.50 mg Coomassie brilliant blue G250 is weighed, 25 mL of 95% ethanol solution and 50 mL of 85% H3PO4 solution are added, and distilled water is added to 500 mL, and it is stored in the dark. 1 mL of 3.13, 6.25, 12.50, 25.00, 50.00, 100.00 mg / mL bovine serum albumin solution is taken, 5.00 mL of Coomassie brilliant blue G250 solution is added, and it is mixed well by a vortex oscillator, and it is placed at room temperature for 20 min, and OD 595 is measured, and the average value is taken for three parallel tests. The standard curve equation is y=0.00334x+0.3259 (R2=0.9992).

[0048] 1 mL of the ICE-01 sample solution diluted to a certain multiple according to the ICE-01 described in Example 1 is taken into a 10 mL test tube, and the absorbance is measured at 595 nm according to the above determination method, and the protein content of the water extract ICE-01 of the application is calculated to be 1.5480 mg / g (protein / water extract).

[0049] Example 3: Determination of the antioxidant performance of the water extract ICE-01

[0050] (1) Determination of DPPH free radical scavenging capacity

[0051] 2 mL of the sample solution diluted to a certain multiple is taken into a 10 mL test tube, 2.00 mL of DPPH solution (0.10 mmol / L) is added, and it is reacted in the dark at room temperature for 30 min. The absorbance is measured at 517 nm as A s , 2.00 mL of water is reacted with 2.00 mL of DPPH solution as above, and the absorbance is recorded as A0, and 2.00 mL of the sample solution is reacted with 2.00 mL of ethanol as above, and the absorbance is recorded as A c . The DPPH free radical scavenging rate is calculated according to the following formula, and the IC 50 concentration is calculated, reflecting the strength of DPPH scavenging.

[0052]

[0053] Table 1. DPPH free radical scavenging rate of the water extract ICE-01

[0054]

[0055] As shown in Table 1, the aqueous extract ICE-01 of this invention exhibits good DPPH free radical scavenging effect, showing a dose-dependent relationship with concentration within the range of 2.00–10.00 mg / mL. The DPPH scavenging rate gradually increases with increasing concentration. When the concentration of the aqueous extract ICE-01 is 10.00 mg / mL, the DPPH free radical scavenging rate reaches 96.24%. Calculations show that its IC50... 50 The value was 2.69 mg / mL.

[0056] (2) Determination of ·OH scavenging ability

[0057] Take 50.00 μL of sample solution diluted to a certain factor and add it to a 96-well plate. Add 50.00 μL of 9 mmol / L FeSO4 solution, 50.00 μL of 9 mmol / L salicylic acid-ethanol solution, and finally add 50.00 μL of 9 mmol / L H2O2 to start the reaction. This solution is denoted as solution A. s Deionized water was used instead of H₂O₂ solution as the background for the sample, denoted as solution A0; deionized water was used instead of the sample solution as the blank control, denoted as solution A. c The above solution was placed in a 37°C incubator in the dark for 30 minutes, and the absorbance value A was measured at 510 nm. s A c A0, each group has 3 replicates, and the experiment is repeated 3 times. Calculate the ·OH radical scavenging rate using the following formula, and calculate the half-life (IC50) concentration. 50 This reflects the strength of the ability to scavenge ·OH free radicals.

[0058]

[0059] Table 2. Scavenging rate of ICE-01 water extract against ·OH radicals

[0060]

[0061] Table 2 shows that, within a certain range, the scavenging rate of ·OH by the aqueous extract ICE-01 increases with increasing concentration. The scavenging effect of the aqueous extract ICE-01 on ·OH reaches its maximum at 10.00 mg / mL, with a scavenging rate of 70.82%. Calculations show that the IC50 concentration... 50 It has a concentration of 2.28 mg / mL and strong antioxidant capacity.

[0062] (3) Determination of ABTS free radical scavenging ability

[0063] ABTS clearance rate was determined by micro method. 200.00 μL of ABTS working solution (determined after 1:1 configuration of ABTS mother liquor and oxidant at room temperature for 12 hours in dark, stable within 2-3 days, and diluted 50 times with PBS to make the absorbance at 734 nm be 0.7±0.05) was added into a 96-well plate, mixed with 10.00 μL of sample with different concentrations, and then placed at room temperature in dark for 5 minutes, and the absorbance was determined at OD 734 t The absorbance was recorded as A0. 200.00 μL of PBS solution was reacted with 10.00 μL of sample to record the absorbance as B. The ABTS free radical clearance rate was calculated according to the following formula, and the half clearance concentration (IC 50 ) was calculated to reflect the ability of clearing ABTS free radicals.

[0064]

[0065] Table 3. ABTS free radical clearance rate of water extract ICE-01

[0066]

[0067] As shown in Table 3, the water extract ICE-01 has good ability of clearing ABTS free radicals, and shows dose-dependent within 2.00-8.00 mg / mL. When the concentration of the water extract ICE-01 is 8.00 mg / mL, the clearance rate can reach 90.71%, and the IC 50 is calculated to be 2.97 mg / mL.

[0068] (4) Iron ion reduction capacity determination

[0069] FRAP value was determined by micro method. 180.00 μL of FRAP working solution (TPTZ diluent, TPTZ solution, and detection buffer were configured at 10:1:1 to be preheated at 37℃ for standby) was added into a 96-well plate detection hole, and then 5.00 μL of sample diluted to a certain concentration was added, and distilled water was used as a blank control. The absorbance was determined at 593 nm. The corresponding FeSO4·7H2O concentration was obtained on a standard curve (y=0.294x-0.0058, R 2 =0.998) to define as FRAP value, and the greater the value, the stronger the antioxidant activity.

[0070] The detection result is that when the concentration of the ICE-01 sample is 0.05 g / mL, it is equivalent to 0.88 mM of FeSO4·7H2O, and the ABTS free radical clearance ability is strong, which indicates that the antioxidant property of the water extract ICE-01 of the application can be realized by electron transfer.

[0071] ​Example 4: Water extract ICE-01 inhibits inflammatory response caused by LPS-induced RAW264.7 cells (1) Cytotoxicity test

[0072] Macrophage RAW264.7 was normally cultured in high-glucose DMEM medium containing 10% fetal bovine serum under culture conditions of 5% CO2, 37℃. When the cells grew to 80-90% confluence, they were digested with trypsin and inoculated in 96-well plates at a density of 8x10 5 After the cells adhered for 24h, different concentrations of ICE-01 samples were added for 24h, and a no-treatment blank group (NT) and a series of experimental groups with concentrations of 0.10, 0.50, 1.00, 5.00, 10.00 mg / mL were set up, with 3 parallel holes in each group. After washing the cells in the holes once with DPBS, the absorbance was measured at 450nm using a CCK-8 kit, and the relative cell activity was calculated. If the cell activity was less than 90%, it was considered to have cytotoxicity.

[0073]

[0074] In the formula:

[0075] OD0----blank group OD value;

[0076] OD1----experimental group OD value.

[0077] The results are shown in Figure 4 The water extract of the present application had no cytotoxicity to RAW264.7 at an addition concentration of 0.10-10.00 mg / mL, and could also promote the proliferation thereof. Subsequently, 0.50, 1.00, 5.00 mg / mL samples were selected to treat LPS-induced cells for anti-inflammatory efficacy evaluation.

[0078] (2) Measurement of cytokines

[0079] According to the results of the above cell viability, 0.50, 1.00, 5.00 mg / mL ICE-01 samples were selected as sample groups for LPS (1 μg / mL) induced cell inflammation experiments, and a positive group (DEX, dexamethasone 80 μM) was set up. The contents of TNF-α, IL-6, IL-β cytokines were detected by ELISA kit. At the same time, a no-treatment blank group was set up as NT group, and a model group was set up as LPS group.

[0080] The results are shown in Figure 5 , 6, 7It can be seen that the contents of IL-6, IL-β and TNF-α in the supernatant of the model group cells increased significantly, and the difference was extremely significant. At the same time, the secretion of IL-6, IL-β and TNF-α in the positive DEX group was significantly inhibited. Therefore, it can be considered that the test model group and the positive group are running normally, and the test system is effective. Compared with the model group, 1.00 mg / mL ICE-01 can significantly inhibit the release of IL-6, IL-β and TNF-α in RAW264.7 cells (P<0.05, P<0.01). It shows that the water extract has an inhibitory effect on the increase of LPS-induced inflammatory factor levels.

[0081] Example 5: Promoting effect of water extract ICE-01 on the growth of Staphylococcus epidermidis

[0082] The Staphylococcus epidermidis used in this example is Staphylococcus epidermidis CCSM0287, with a preservation number of CCTCC No: M2022779.

[0083] The promoting effect of water extract ICE-01 on the growth of Staphylococcus epidermidis was determined by liquid culture method. The water extract ICE-01 sample was prepared into a solution, and all were sterilized by sterile 0.22 μm filter membrane. Six dilution gradients of half dilution were performed using sterile 96-well plates, and four replicate wells were set for each group. First, 200.00 μL of sample with a concentration of 20.00 mg / mL (prepared with sterile TSB) was added to the first well, 100.00 μL of solution from the first well was added to the second well for gradient dilution, and the above operation was repeated after mixing well to the sixth well. 100.00 μL of sterile TSB medium was added to the sixth well, mixed well, and then 100.00 μL of liquid was discarded. Subsequently, 100.00 μL of 10 6 cfu / mL Staphylococcus epidermidis bacterial suspension was added to the first to sixth wells of the 96-well plate. Sample group (a): 100.00 μL of each half dilution sample + 100.00 μL of 10 6 cfu / mL Staphylococcus epidermidis bacterial suspension; sample control group (b): 100.00 μL of each half dilution sample + 100.00 μL of sterile TSB medium; negative control group (c): 200.00 μL of sterile TSB medium; positive control group (d): 100.00 μL of 10 6 cfu / mL Staphylococcus epidermidis bacterial suspension + 100.00 μL of sterile TSB medium, and placed at 37°C for static culture for 48 h. The turbidity change was observed, and the absorbance value at 600 nm of each well was determined by enzyme marker instrument. Each group of test was repeated for 3 times, and the bacteriostatic rate was calculated according to the following formula:

[0084]

[0085] Table 4. Bacteriostatic rate of water extract ICE-01 on Staphylococcus epidermidis

[0086]

[0087]

[0088] From Table 4, it can be seen that the water extract ICE-01 has obvious promoting effect on the growth of Staphylococcus epidermidis in the range of 0.625-10.00 mg / mL, and the growth rate can reach 66.58% when the concentration of the water extract is 10.00 mg / mL.

[0089] Example 6: Evaluation of the bacteriostatic activity of water extract ICE-01 on Propionibacterium acnes

[0090] The Propionibacterium acnes used in this example is Propionibacterium acnes ATCC11827, which is a standard strain of skin pathogenic bacteria with strong biofilm production.

[0091] The liquid culture method was used to determine the inhibitory effect of water extract ICE-01 on Propionibacterium acnes. The water extract ICE-01 sample was prepared into a solution, and all were filtered and sterilized with sterile 0.22 μm filter membranes. All processes were completed under sterile conditions, and four replicate wells were set up for each group. 100.00 μL of samples with concentrations of 20.00, 15.00, 10.00, 5.00 and 2.50 mg / mL (prepared with sterile TSB) were added to the well plates according to the following groups. Sample group (a): 100.00 μL of each diluted sample + 100.00 μL of 10 5 cfu / mL of Propionibacterium acnes bacterial suspension; sample control group (b): 100.00 μL of each diluted sample + 100.00 μL of sterile TSB medium; negative control group (c): 100.00 μL of sterile TSB medium + 100.00 μL of sterile water; positive control group (d): 100.00 μL of 10 5 cfu / mL of Propionibacterium acnes bacterial suspension + 100.00 μL of sterile TSB medium, and placed at 37°C for static culture for 48 h. The turbidity change was observed, and the absorbance value at 600 nm of each well was determined by an enzyme marker. Each group of tests was repeated 3 times, and the bacteriostatic rate was calculated according to the following formula:

[0092]

[0093] Table 5. Bacteriostatic rate of water extract ICE-01 on Propionibacterium acnes

[0094]

[0095] As shown in Table 5, the water extract ICE-01 has gradually enhanced bacteriostatic effect on Propionibacterium acnes in the range of 1.25-7.50 mg / mL, and the bacteriostatic rate can reach 50.12% when the concentration of the water extract is 7.50 mg / mL.

[0096] Example 7: Preparation process of emulsion containing Isaria cicadae mycelium water extract

[0097] (1) Basic formula of emulsion containing water extract ICE-01

[0098] The formula of the emulsion is shown in Table 6. The ingredients in phase A are weighed according to the proportion of each formula and added to beaker A and placed in a 90℃ water bath for melting, and kept for standby. An appropriate amount of deionized water is weighed and added to beaker B. The components in phase B are dispersed in the water phase of beaker B in order. When the two phases of A and B reach 90℃ in the 90℃ water bath, the stirring of phase B is not stopped. The melted liquid in A is added to beaker B at a uniform speed, and stirring and dispersion are maintained for 5 min. After that, homogenization is performed, and slow stirring is maintained after homogenization. When the temperature of the system decreases to about 55℃, phase C is added. At this time, stirring is continued for 20-30 min. After the system is uniform, it is sealed with preservative film and stored.

[0099] Table 6: Emulsion formula table

[0100]

[0101] (2) Cosmetic stability evaluation

[0102] According to the experimental methods described in GBT29665-2013 and GB / T16497-2007, the cold and hot stability experiments of the emulsion containing water extract ICE-01 are performed.

[0103] Thermal stability: The sample to be tested is placed in two stoppered test tubes. One of the test tubes is placed in a 40℃ constant temperature incubator for 24 h, and then taken out to recover to room temperature. The other test tube is placed at room temperature for 24 h. Whether the properties of the samples in the two test tubes are consistent and whether the sample is layered after heating is observed.

[0104] Cold stability: The sample to be tested is placed in a-8℃ refrigerator for 24 h, and then taken out to room temperature. The sample is compared with the sample placed at room temperature, and whether the sample is layered is observed.

[0105] Cold and hot alternating stability: The sample to be tested is placed in a-8℃ refrigerator for 24 h, and then placed in a 40℃ constant temperature incubator for 24 h. The sample is compared with the sample placed at room temperature, and whether the sample is layered is observed.

[0106] Table 7: Stability test results of emulsion of water extract ICE-01

[0107]

[0108]

[0109] A1: base emulsion (without adding ICE-01), A2, A3: emulsion with 0.15%, 0.50% ICE-01 added

[0110] The emulsion stability test results of the aqueous extract ICE-01 are shown in Table 7. As can be seen from Table 7, the base emulsion and the emulsion containing different amounts of cicada polysaccharide all show good stability. This shows that the addition of the mycelium water extract of Cordyceps cicada does not adversely affect the stability of the cosmetic formulation.

[0111] (3) Cosmetic patch test

[0112] According to the "Cosmetic Safety Technical Specification 2015 Edition", 30 subjects were selected to conduct a patch test on the emulsion containing the aqueous extract ICE-01 to investigate the safety of the sample on the human body. The patch applicator containing the sample was placed on the flexor of the volunteer's forearm, and the palm was pressed lightly to make it evenly adhere to the skin. After 24 hours of continuous application, the patch applicator was removed for 30 minutes, and the test site of the volunteer was observed for positive reactions. The experimental results are judged according to Table 8.

[0113] Table 8 Skin Adverse Reaction Grading Standard

[0114]

[0115] Table 9 Patch Test Results of Emulsion

[0116]

[0117] The patch test results are shown in Table 9. The results show that 25 volunteers did not have an allergic reaction within 24 hours of using the emulsion containing the aqueous extract ICE-01 and the emulsion without adding. The experimental results confirm that the addition of the aqueous extract ICE-01 does not affect the safety of the cosmetic, is mild and low in irritation, and is safe and harmless to human skin.

[0118] (3) Bacteriostatic test

[0119] The emulsion (A2, A3) prepared by the above operation was subjected to hygiene inspection according to the "Cosmetic Safety Technical Specification" (2015 edition), and the test results are shown in Table 10. It can be seen that the microbial indicators meet the requirements.

[0120] Table 10 Test Results

[0121]

[0122] (4) Acne-removing effect test

[0123] Test subjects: 60 patients aged 15-30 years old with acne, papules, acne, comedones and the like on their faces, which were divided into two groups, 30 people in each group, and men and women were evenly divided in each group.

[0124] Test method: The faces of the test personnel were washed clean with water, and (1.00±0.10) mg / cm 2 The emulsion containing ICE-01 in Embodiment 1 was applied to the affected area, and the face was allowed to absorb, and the control group was also evenly applied with an equal amount of general skin care emulsion (the formula does not contain water extract ICE-10) to the affected area. Each was used once a day in the morning and evening, and the test period was 28 days. The acne removal test results are shown in Table 11.

[0125] Effect evaluation standard:

[0126] (1) Significant effect: the symptoms improved significantly, and almost all acne disappeared;

[0127] (2) Improvement: the symptoms improved, and part of the acne disappeared;

[0128] (3) Ineffective: the symptoms did not improve, and the acne did not change.

[0129] Table 11 Acne removal test results

[0130]

[0131] From the results of Table 11 above, it can be seen that the acne removal emulsion using the water extract ICE-01 of Cordyceps cicadae mycelium has good acne removal effect.

[0132] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. Application of aqueous extract of *Bryum cicadae* mycelium in the preparation of a topical skin agent that inhibits the growth of *Propionibacterium acnes*; the preparation method of the aqueous extract of *Bryum cicadae* mycelium is as follows: fermenting *Bryum cicadae* C1, centrifuging the fermentation broth, collecting the mycelium, and then washing and freeze-drying to obtain freeze-dried mycelium; after pulverizing the freeze-dried mycelium, extracting it using a cellulase-assisted hot water extraction method.

2. The application as described in claim 1, characterized in that, The extraction method using cellulase-assisted hot water extraction is as follows: the pulverized freeze-dried mycelium is added to water and cellulase for enzymatic hydrolysis at a temperature of 40-50℃ for 1-2 hours; after hydrolysis, the enzyme activity is rapidly inactivated by heating; then, hot water extraction is continued at a temperature of 90-100℃ for 2-4 hours, and the extraction is repeated 2-3 times; the extract is filtered while hot, the filtrates are combined and concentrated, and then freeze-dried to obtain the aqueous extract of *Cladosporium cicadae* mycelium.

3. Application of aqueous extract of *Cladosporium cicadae* mycelium in the preparation of cosmetics for improving acne; the preparation method of the aqueous extract of *Cladosporium cicadae* mycelium is as follows: fermenting *Cladosporium cicadae* C1, centrifuging the fermentation broth, collecting the mycelium, and then washing and freeze-drying to obtain freeze-dried mycelium; after pulverizing the freeze-dried mycelium, extracting it using a cellulase-assisted hot water extraction method.

4. The application as described in claim 3, characterized in that, The extraction method using cellulase-assisted hot water extraction is as follows: the pulverized freeze-dried mycelium is added to water and cellulase for enzymatic hydrolysis at a temperature of 40-50℃ for 1-2 hours; after hydrolysis, the enzyme activity is rapidly inactivated by heating; then, hot water extraction is continued at a temperature of 90-100℃ for 2-4 hours, and the extraction is repeated 2-3 times; the extract is filtered while hot, the filtrates are combined and concentrated, and then freeze-dried to obtain the aqueous extract of *Cladosporium cicadae* mycelium.

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