CN117860791A - Isaria cicadae mycelium aqueous extract and application thereof in preparing cosmetics for improving acne - Google Patents
Isaria cicadae mycelium aqueous extract and application thereof in preparing cosmetics for improving acne Download PDFInfo
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- CN117860791A CN117860791A CN202311719044.5A CN202311719044A CN117860791A CN 117860791 A CN117860791 A CN 117860791A CN 202311719044 A CN202311719044 A CN 202311719044A CN 117860791 A CN117860791 A CN 117860791A
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- mycelium
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Abstract
The invention discloses an aqueous extract of Isaria cicadae mycelium and application thereof in preparing cosmetics for improving acne. According to the invention, after the freeze-dried mycelium of the corynespora cicadae C1 is crushed, a cellulase synergistic hot water extraction method is adopted to extract the mycelium to obtain the corynespora cicadae water extract, and the obtained water extract contains various active ingredients such as polysaccharide, nucleoside, sugar alcohol, protein and the like, has good effects of resisting inflammation and oxidation, promoting the growth of staphylococcus epidermidis, inhibiting propionibacterium acnes and regulating the microecological balance of skin, and can be used as an anti-acne and anti-inflammatory active raw material in the fields of cosmetics and the like.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to an isaria cicadae mycelium aqueous extract and application thereof in preparing cosmetics for improving acne.
Background
A variety of microorganisms inhabit the surface layers of human skin and hair follicles, and play an important role in maintaining skin health, also known as the microbial barrier of the skin. The skin microbiota, the host skin and the environment form a skin micro-ecological system, and the interaction and the mutual restriction between the skin microbiota, the host skin and the environment keep the coordinated, physiological and dynamic balance. Balanced skin micro-ecology is beneficial to skin health. In daily life, factors such as ultraviolet radiation, frequent use of cosmetics, irregular life pressure and work and rest can cause the stable state of skin flora to be destroyed, so that frequent occurrence of skin problems is caused.
Acne is a common skin disease with chronic inflammation, is frequently found in teenagers, and has incidence rate of more than 90%. Although the disease is not generally life-threatening, the disease is good to the face, and can cause the patient to be damaged or destroyed, so that the psychological influence on the patient is quite large, and the psychological diseases such as anxiety, depression and the like can be caused in serious cases, so that the disease cannot be ignored. The occurrence of acne is closely related to the factors of hypersecretion of sebum, keratosis of the pilosebaceous ducts, infection of pathogenic bacteria, inflammation and the like. The sebaceous glands secrete sebum to be vigorous, so that pores are blocked, and propionibacterium acnes capable of inducing inflammation in hair follicles are promoted to be propagated in large quantities. Studies show that the imbalance of skin flora exists on the skin of acne patients, which is mainly characterized by the increase of the quantity of propionibacterium acnes and the decrease of staphylococcus epidermidis, and the imbalance of the proportion of the propionibacterium acnes and the staphylococcus epidermidis leads to the mass propagation of propionibacterium acnes, the secretion of lipase to hydrolyze triglyceride and the metabolism of more free fatty acid, so that inflammatory reaction is aggravated. At present, the acne is mainly treated by using hormone antagonists, vitamin A acid medicaments, antibiotics and other medicaments, but the long-term administration has the risks of causing allergy, kidney function damage, even toxic and side effects such as deformity and the like. The acne-removing cosmetic raw material with the functions of restoring the flora balance and resisting the inflammation is selected, so that the acne-removing cosmetic raw material is a safe and effective means for preventing and treating acne.
The Cordyceps sobolifera (Cordyceps cicada) is commonly called as Cordyceps cicadae, is a precious fungus for both medicine and food, is one of four Cordyceps sinensis in China, and has long use history in China. Modern medical researches have proved that the cicada fungus and its effective components have the effects of resisting tumor, regulating immunity, improving renal function, reducing blood lipid, blood pressure and cholesterol. Isaria cicadae (Isaria cicadae) is a conidiophore stage, i.e., asexual stage, of Cordyceps sobolifera, and is a fungus that forms Cordyceps sobolifera. The cicada fungus mycelium contains a plurality of secondary metabolites such as polysaccharide, nucleoside, amino acid, hyaluronic acid, micromolecular sugar alcohol (such as cordycepic acid) and the like, has important biological activity functions such as immunoregulation, anti-tumor, nervous system regulation, renal function improvement, antibiosis, antivirus and the like, and has good application prospect in the development of skin external preparations.
The applicant has previously obtained a strain of exopolysaccharide-producing coryneform cicadae (Isaria cicadae) deposited in China general microbiological culture Collection center, deposit number, at 11.05, 2019: CGMCC No.18807, which provides the corynespora cicadae (marked as corynespora cicadae C1) which is a strain with fast growth and high extracellular polysaccharide yield, has narrower molecular weight distribution, good uniformity, good antioxidant capacity, good immunocompetence, moisture retention, apparent viscosity and suspension property, can be used as antioxidant and moisture retention raw materials, thickening agents, suspension stabilizing agents and the like to be applied to foods and cosmetics (see ZH 202110230202.5, a corynespora cicadae strain for producing extracellular polysaccharide, a preparation method and application). On this basis, the applicant has further developed the function of its mycelium in the hope of being used for the treatment of acne.
Disclosure of Invention
The invention firstly provides a corynespora cicadae C1 fermentation mycelium aqueous extract which has good anti-inflammatory, antioxidant and acne propionibacterium balance adjusting effects, and the corynespora cicadae C1 fermentation mycelium aqueous extract is used as an efficacy additive in an external skin preparation, so that the external skin preparation is natural, safe and can obviously relieve acne skin symptoms.
In order to achieve the above object, the present invention provides the following technical solutions: a preparation method of a Isaria cicadae mycelium aqueous extract is characterized in that Isaria cicadae C1 (CGMCC No. 18807) is fermented, fermentation liquor is centrifuged, mycelium is collected, and then the mycelium is washed and freeze-dried to obtain freeze-dried mycelium; after the freeze-dried mycelium is crushed, the cellulase and hot water extraction method are adopted for extraction.
Further, the crushing is that freeze-dried mycelium is crushed and passes through a 80-mesh sieve.
Furthermore, the extraction method adopting the cellulase and hot water extraction method comprises the following steps: adding water and cellulase (more than or equal to 8 ten thousand U/g) (food grade) into the crushed freeze-dried mycelium for enzymolysis, wherein the enzymolysis temperature is 40-50 ℃ and the enzymolysis time is 1-2 h; after enzymolysis, the enzyme is quickly heated to deactivate the enzyme; then the extraction is continued by a hot water extraction method, the extraction temperature is 90-100 ℃, the extraction time is 2-4 h, and the extraction times are 2-3 times; filtering the extractive solution while it is hot, mixing filtrates, concentrating, and lyophilizing to obtain water extract. The enzyme dosage is 0.10-0.30% (based on the mass of the freeze-dried mycelium).
The Isaria cicadae mycelium aqueous extract (ICE-01 for short) prepared by the method comprises the following components in percentage by weight: 0.05 to 0.10 percent of uridine, 0.05 to 0.10 percent of adenosine, 0.04 to 0.08 percent of guanosine, 0.01 to 0.015 percent of inosine, 0.01 to 0.02 percent of uracil, 0.50 to 1.00 percent of erythritol, 5.00 to 10.00 percent of cordycepic acid, 1.00 to 1.50 percent of glucose, 0.50 to 1.00 percent of trehalose, 5.00 to 10.00 percent of polysaccharide and 0.15 to 0.20 percent of protein.
The invention also provides the application of the cicada fungus mycelium aqueous extract as an active ingredient in preparing the skin external preparation with the antioxidation effectIs used. Preferably, the concentration of the Isaria cicadae mycelium aqueous extract is 2.00-10.00 mg/mL. The antioxidation effect is as follows: has better antioxidant activity for removing any one or more of DPPH free radical, hydroxyl free radical, ABTS free radical and iron ion reducing capability. When the concentration of the aqueous extract ICE-01 is 10.00mg/mL, the clearance rate of DPPH free radicals can reach 96.24 percent, and the clearance rate of OH can reach 70.82 percent; when the concentration of the ICE-01 of the water extract is 8.00mg/mL, the clearance rate of the ABTS free radicals can reach 90.71 percent; when the ICE-01 sample concentration is 0.05g/mL, the concentration is equivalent to 0.88mM FeSO 4 ·7H 2 O and the iron ion reducing capability is stronger.
The invention also provides application of the cicada fungus mycelium aqueous extract as an active ingredient in preparing a skin external preparation with anti-inflammatory effect. Preferably, the concentration of the Isaria cicadae mycelium aqueous extract is 0.10-10.00 mg/mL. The anti-inflammatory agent comprises the following components: reduces the inflammatory response of macrophage RAW264.7 induced by LPS and reduces the expression of inflammatory factors IL-6, IL-beta and TNF-alpha. The aqueous extract ICE-01 at a concentration of 1.0mg/mL inhibited the release of IL-6, IL-beta and TNF-alpha from RAW264.7 cells (P < 0.05, P < 0.01) relative to the LPS treated model group. It can be seen that medium doses of ICE-01 of the present invention have an inhibitory effect on LPS-induced increases in inflammatory factor levels.
The invention also provides application of the corynespora cicadae mycelium aqueous extract in preparing 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 aqueous extract is 1.25-10.00 mg/mL. When the concentration of the aqueous extract ICE-01 is 7.50mg/mL, the antibacterial rate of the aqueous extract ICE-01 on propionibacterium acnes reaches 50.12%. When the concentration of the aqueous extract ICE-01 is 10.00mg/mL, the growth promotion rate of staphylococcus epidermidis reaches 66.58 percent.
Preferably, the external skin preparation includes a drug having a therapeutic effect on a disease, or a cosmetic having no therapeutic effect on a disease.
Further, the invention provides application of the corynespora cicadae mycelium aqueous extract in preparing cosmetics for improving acne.
Preferably, when the cosmetic is an emulsion, the emulsion comprises the following raw materials in parts by weight: 0.125-1 part of a Isaria cicadae mycelium aqueous extract, 6.23-16.88 parts of A phase, 9-26 parts of B phase, 0.1-0.4 part of C phase and 100 parts of water; the phase A comprises stearyl polyoxyethylene ether-2, stearyl polyoxyethylene ether-21, cetyl-stearyl alcohol, simethicone, octyl/decyl triglyceryl, white oil and BHT; the phase B includes propylene glycol, glycerin, carbopol 20, and a preservative comprising: 1, 2-hexanediol, glycerol monocaprylate; the phase C includes sodium hydroxide.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the cicada fungus mycelium obtained by fermentation of the cicada fungus C1 as a raw material, combines an enzymolysis method and a hot water extraction method to obtain the mycelium aqueous extract ICE-01, has simple and easy preparation method, does not use organic reagents such as ethanol, methanol and the like in the extraction process, contains various active ingredients such as polysaccharide, nucleoside, sugar alcohol, protein and the like, can be used as a novel raw material for removing acnes and resisting inflammation in cosmetics, has good antioxidant activity and stronger inflammatory factor inhibition effect, can effectively promote the growth of staphylococcus epidermidis, inhibits the growth of propionibacterium acnes, further relieves the symptoms of acnes, and can be applied to the fields of cosmetics, foods and medicines, and particularly can be applied to cosmetics.
Drawings
FIG. 1 shows a nucleoside mixed standard substance pattern (peak 1-12: cytosine, uracil, cytidine, hypoxanthine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine, cordycepin in this order);
FIG. 2 is a liquid chromatogram of the determination of nucleoside components in aqueous extract ICE-01 of the present invention (peaks 1-5: uracil, uridine, inosine, guanosine, adenosine, in order);
FIG. 3 shows ion chromatograms of sugar alcohol component determination in aqueous extract ICE-01 of the present invention (peaks 1-4: erythritol (erythrotol), trehalose (trehalose), cordycepic acid (mannitol), glucose (glucose), in this order);
FIG. 4 shows the cytotoxicity test results of the aqueous extract ICE-01 of the present invention on RAW264.7 macrophages;
FIG. 5 shows the effect of different concentrations of ICE-01 (0.50, 1.00, 5.00 mg/mL) on IL-6 levels produced by LPS-induced RAW264.7 cells (NT is no treatment control group, LPS is model group, DEX is positive group, ICE-01 is drug group, # # P < 0.0001 compared to control group, # # P < 0.05, # P < 0.01 compared to model group);
FIG. 6 shows the effect of different concentrations of ICE-01 (0.50, 1.00, 5.00 mg/mL) on LPS-induced production of IL- β by RAW264.7 cells (NT is no treatment control group, LPS is model group, DEX is positive group, ICE-01 is drug group, # # P < 0.0001 compared to control group, # # P < 0.05, # P < 0.01 compared to model group);
FIG. 7 shows the effect of different concentrations of ICE-01 (0.50, 1.00, 5.00 mg/mL) on TNF- α levels produced by LPS-induced RAW264.7 cells (NT is no treatment control, LPS is model, DEX is positive, ICE-01 is drug, and, # # P < 0.0001 compared to control, # # P < 0.05, # P < 0.01 compared to model).
Detailed Description
For further explanation of the present invention, an aqueous extract of Isaria cicadae mycelium and the use of the same for preparing cosmetics for improving acne of the present invention will be described in detail with reference to the examples and drawings, but they should not be construed as limiting the scope of the present invention.
Example 1: preparation of Isaria cicadae mycelium aqueous extract ICE-01
1) Preparation of Isaria cicadae mycelium
Activating strains: inoculating a strain of Isaria cicadae C1 (Isaria cicadae C1) preserved in glycerol to a PDA solid flat plate, culturing for 5-6 days at a constant temperature of 28 ℃, and activating twice;
seed culture: picking fresh mycelium with an inoculating shovel under aseptic condition, transferring to a liquid seed culture medium, placing in a 70mL/250mL triangular flask on a shaking table, and culturing at 28deg.C for 3d at 160r/min to obtain first-stage seed liquid; the primary seed solution is transferred into a seed culture medium with the inoculation amount of 8% (V/V), and is cultured for 2d at 28 ℃ and 160r/min to obtain the secondary seed solution. The seed culture medium contains the following components in mass concentration: 200g/L of potato and 20g/L, KH of sucrose 2 PO 4 2g/L、MgSO 4 ·7H 2 O1 g/L, pH is natural. Sterilizing at 121deg.C for 20min.
Fermentation culture: the secondary seed solution was added to the fermentation medium at an inoculum size of 6% (V/V), a liquid loading amount of 70mL/250mL, a shaking speed of 180r/min, and cultured at 28℃for 4d. The fermentation medium contains the following components in mass concentration: glucose 20g/L, yeast extract 2g/L, peptone 2g/L, KH 2 PO 4 2g/L、MgSO 4 ·7H 2 O1 g/L, adjusting pH to 6, sterilizing at 121deg.C for 20min.
The fermentation broth was centrifuged (8000 r/min,4 ℃) for 15min, the supernatant was used as the other (as the starting material for the extracellular polysaccharide), and the C1 mycelia were collected after washing with distilled water and freeze-dried.
2) Preparation of aqueous extract ICE-01
Crushing the freeze-dried mycelium, sieving with an 80-mesh sieve, and extracting by adopting a cellulase (more than or equal to 8 ten thousand U/g) (food grade) synergistic hot water extraction method: adding proper amount of water, and performing enzymolysis at 50deg.C for 1.5 hr with enzyme dosage of 0.20% (based on lyophilized mycelium mass). And after enzymolysis, the mixture is quickly heated to 90 ℃ to inactivate enzyme for 10min. The extraction is continued by a hot water extraction method, wherein the ratio of feed to liquid is 1:30, the extraction temperature is 95 ℃, the extraction times are 2 times, and the extraction time is 3 hours each time. Filtering the extractive solution while it is hot, mixing filtrates, concentrating, and lyophilizing to obtain water extract.
Example 2: analysis of active ingredients in aqueous extracts ICE-01
(1) Polysaccharide content determination
Dissolving the ICE-01 sample in water, precipitating with 3 times of ethanol, dissolving the precipitate in water, volatilizing ethanol, and diluting with a certain multiple to obtain polysaccharide solution.
The content of polysaccharide is determined by sulfuric acid-phenol method: weighing a proper amount of glucose standard substance, drying at 105 ℃ to prepare 100 mug/mL standard substance solution, respectively sucking 0, 0.10, 0.20, 0.40, 0.60, 0.80 and 1.00mL into a glass test tube with a plug, supplementing 1.00mL with distilled water, adding 0.50mL of 5% phenol solution, quickly adding 2.50mL of concentrated sulfuric acid, fully mixing the reaction solution by a vortex oscillator, standing for 10min, then placing in a boiling water bath for reaction for 15min, and measuring OD 490 Leveling out three times in parallelAnd (5) an average value. A standard curve y= 11.973x-0.0099 (R 2 ＝0.9991)。
The polysaccharide content of the ICE-01 water extract is 0.0975g/g by taking 1.00mL ICE-01 solution into a 10mL test tube and measuring the absorbance value of a sample according to the method and the measuring conditions. (polysaccharide/water extract).
(2) Analysis of nucleoside Components
The nucleoside components are determined by high performance liquid chromatography, 1.00g of the sample described in the example 1 is accurately weighed in a 100mL volumetric flask, water is added to fix the volume until the sample is uniformly dissolved by shaking to scale, the solution is filtered through a 0.45 mu m water phase filter membrane for liquid chromatography analysis, and each sample is repeated for 3 times. Mixing and labeling of nucleoside standard substances: comprises cytosine, uracil, cytidine, hypoxanthine, uridine, thymine, adenine, inosine, guanosine, thymidine, adenosine and cordycepin. Chromatographic conditions: column Venusil MP C18 column (5 μm,4.6 mm. Times.250 mm) (Agilent, USA). Mobile phase: water (phase a) -methanol (phase B); flow rate: 1mL/min; the elution procedure is 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: 70-95% of A, 30-5% of B; 40-45 min:95% A to 100% A. UV detector detects, detection wavelength: 254nm; column temperature: 40 ℃; sample injection amount: 10 mu L. The spectrum of the nucleoside mixed standard substance is shown in figure 1, and the detection result is shown in figure 2.
As can be seen from FIGS. 1 and 2, the aqueous extract ICE-01 of the present invention contains 5 nucleoside components, wherein the uridine content is 0.7746mg/g, the adenosine content is 0.8929mg/g, the guanosine content is 0.5683mg/g, the inosine content is 0.1296mg/g, and the uracil content is 0.1496mg/g (nucleoside/aqueous extract).
(3) Sugar alcohol component analysis
And measuring the sugar alcohol content by adopting high-performance ion chromatography. Accurately weighing 0.50g of the Isaria cicadae mycelium aqueous extract sample in the embodiment 1, fixing the volume to a 100mL volumetric flask scale by distilled water, diluting to a proper concentration by distilled water, filtering by a 0.45 mu m microporous filter membrane, and measuring by ion chromatography. Taking erythritol, cordycepic acid, trehalose, arabitol, mannitol, mannose, glucose and galactose as standard substances, dissolving with deionized water, and diluting to different concentrations to prepare a standard substance solution. Chromatographic conditions: carboPac MA1 anion exchange column (4 mm. Times.250 mm) (Dionex Co., U.S.A.), mobile phase 480mmol/L NaOH solution, flow rate 0.40mL/min, loading 25. Mu.L, column temperature 30 ℃. The specific measurement results are shown in FIG. 3.
As can be seen from FIG. 3, the aqueous extract ICE-01 of the present invention contains 5 sugar alcohol components, wherein the erythritol content is 5.0348mg/g, the cordycepic acid content is 55.9177mg/g, the glucose content is 10.5073mg/g, and the trehalose content is 5.5249mg/g (sugar alcohol/aqueous extract).
(4) Protein content determination
0.50mg of Coomassie brilliant blue G250 was weighed and added with 25mL of 95% ethanol solution and 50mL of 85% H, as determined by the Coomassie brilliant blue method 3 PO 4 Dissolving, adding distilled water to volume to 500mL, and preserving in dark. 1mL of 3.13, 6.25, 12.50, 25.00, 50.00 and 100.00mg/mL bovine serum albumin solution is absorbed, 5.00mL of Coomassie brilliant blue G250 solution is added, the mixture is fully mixed by a vortex oscillator, the mixture is placed at room temperature for 20min, and OD is measured 595 Three times in parallel, and taking an average value. The standard curve formula is obtained as follows: y=0.00334 x+0.3259 (r2= 0.9992).
1mL of the ICE-01 sample solution described in example 1 diluted to a certain multiple was taken into a 10mL test tube, absorbance was measured at 595nm according to the above-described measurement method, and the protein content of the aqueous extract ICE-01 of the present invention was calculated to be 1.5480mg/g (protein/aqueous extract).
Example 3: measurement of antioxidant property of ICE-01 of aqueous extract
(1) Determination of DPPH radical scavenging ability
2mL of the diluted sample solution was taken into a 10mL test tube, 2.00mL of DPPH solution (0.10 mmol/L) was added, and the reaction was carried out at room temperature in the absence of light for 30min. The absorbance at 517nm was A s Taking 2.00mL of water and 2.00mL of DPPH solution to react with each other, and recording the absorbance as A 0 Taking 2.00mL of sample solution and reacting with 2.00mL of ethanol, and recording the absorbance as A c . The DPPH radical scavenging rate was calculated according to the following formula, and IC was calculated 50 Concentration, reflecting the intensity of DPPH removal.
TABLE 1 scavenging of DPPH free radical by aqueous extract ICE-01
As shown in Table 1, the aqueous extract ICE-01 of the invention has better DPPH free radical scavenging effect and has a certain dosage relationship with concentration in the range of 2.00-10.00 mg/mL. As the concentration increases, the clearance rate to DPPH tends to increase gradually. When the concentration of ICE-01 of the water extract is 10.00mg/mL, the clearance rate of DPPH free radical can reach 96.24 percent, and the IC is obtained through calculation 50 The value was 2.69mg/mL.
(2) Determination of OH-scavenging Capacity
50.00 mu L of the sample solution diluted to a certain multiple is taken in a 96-well plate, and 50.00 mu L of 9mmol/L FeSO is added 4 Solution, 50.00. Mu.L 9mmol/L salicylic acid-ethanol, and finally 50.00. Mu.L 9 mmol/LH 2 O 2 The reaction was started and designated as solution A s The method comprises the steps of carrying out a first treatment on the surface of the Substitution of deionized water for H 2 O 2 The solution was used as background for the sample and was designated solution A 0 The method comprises the steps of carrying out a first treatment on the surface of the Deionized water was used as a blank to replace the sample solution, designated solution A c . Placing above solution in a 37 deg.C incubator for 30min in dark place, and measuring absorbance value A at 510nm s ，A c ，A 0 3 duplicate wells were set up for each group and 3 replicates were measured. The radical scavenging rate of OH was calculated according to the following formula, and the half-scavenging rate concentration (IC 50 ) Reflecting the strength of the ability to scavenge OH free radicals.
TABLE 2 scavenging of OH free radical by aqueous extracts ICE-01
As is clear from Table 2, the clearance of ICE-01 from OH increases with increasing concentration. The clearance of ICE-01 to OH reaches the maximum at 10.00mg/mL, the clearance rate reaches 70.82%, and the IC is obtained through calculation 50 2.28mg/mL, has strong antioxidation capability.
(3) Determination of ABTS free radical scavenging Capacity
The clearance of the ABTS is measured by a micro method, 200.00 mu L of ABTS working solution (measured after the ABTS mother solution and oxidant are prepared and stored for 12 hours at room temperature and protected from light at 1:1) is added into a 96-well plate, the stability is ensured in 2-3 days, when the solution is used, PBS is diluted 50 times to ensure that the absorbance at 734nm is 0.7+/-0.05), and the solution is evenly mixed with 10.00 mu L of samples with different concentrations and placed for 5 minutes at the OD at room temperature and protected from light 734 The absorbance was measured and designated A t Distilled water was used as a blank control and was designated as A 0 The absorbance was recorded as B by reacting 200.00 μl of PBS solution with 10.00 μl of sample. The ABTS radical scavenging rate was calculated according to the following formula, and the half-scavenging rate concentration (IC 50 ) Reflecting the strength of the ability to scavenge ABTS free radicals.
TABLE 3 scavenging of aqueous extract ICE-01 against ABTS free radicals
As can be seen from Table 3, aqueous extract ICE-01 has a better ability to scavenge ABTS free radicals, exhibiting dose escalation within 2.00-8.00 mg/mL. When the concentration of the aqueous extract ICE-01 is 8.00mg/mL, the clearance rate can reach 90.71 percent, and the IC is obtained through calculation 50 2.97mg/mL.
(4) Determination of iron ion reducing ability
Determining FRAP value by micro method, adding 180.00 μl FRAP working solution (TPTZ diluent, TPTZ solution, detection buffer) into 96-well plate detection well10:1:1, pre-heating at 37 ℃ for standby after configuration), adding 5.00 mu L of a sample diluted to a certain concentration, taking distilled water as a blank control, and measuring the absorbance at 593 nm. In FeSO 4 ·7H 2 O configuration series controls, in standard curve (y=0.294 x-0.0058, r 2 =0.998) to obtain the corresponding FeSO 4 ·7H 2 O concentration, defined as FRAP value, is greater the antioxidant activity.
The detection result is as follows: when the ICE-01 sample concentration is 0.05g/mL, the concentration is equivalent to 0.88mM FeSO 4 ·7H 2 O and ABTS have strong free radical scavenging capability, which indicates that the oxidation resistance of the aqueous extract ICE-01 can be realized through electron transfer.
Example 4: aqueous extract ICE-01 inhibition of inflammatory response induced by LPS-induced RAW264.7 cells (1) cytotoxicity assay
Macrophage RAW264.7 at 5% CO 2 Culturing at 37deg.C with high sugar DMEM medium containing 10% foetal calf serum. When the cells grow to 80-90% confluence, they are digested with pancreatin and are then treated as 8X 10 5 cells/ml density was seeded in 96-well plates. After 24h of cell attachment, ICE-01 samples of different concentrations were added and incubated for 24h, and a treatment-free blank (NT) and a series of concentration (0.10, 0.50, 1.00, 5.00, 10.00 mg/mL) of experimental groups were set, each group being provided with 3 parallel wells. After washing the cells in 1 well with DPBS, the relative cell activity was calculated by measuring absorbance at 450nm using CCK-8 kit. Cytotoxicity is considered if the cell activity is less than 90%.
Wherein:
OD 0 -blank OD value;
OD 1 -experimental group OD value.
As shown in FIG. 4, the aqueous extract of the present invention was added at a concentration of 0.10 to 10.00mg/mL, and was not cytotoxic to RAW264.7, and also promoted proliferation. Samples of 0.50, 1.00, 5.00mg/mL were subsequently selected for treatment of LPS-induced cells for anti-inflammatory efficacy assessment.
(2) Cytokine determination
Based on the results of the above cell viability, 0.50, 1.00, 5.00mg/mL ICE-01 samples were selected as sample groups for LPS (1. Mu.g/mL) induced cell inflammation experiments, while positive groups (DEX, dexamethasone 80. Mu.M) were established. ELISA kit is used to detect the content of TNF-alpha, IL-6 and IL-beta cytokines. Meanwhile, a blank group without treatment is set as an NT group, and a model group is set as an LPS group.
As can be seen from fig. 5, 6 and 7, the amounts of IL-6, IL- β and TNF- α in the cell supernatants of the model group were greatly increased, the differences were extremely remarkable, and the secretion of IL-6, IL- β and TNF- α was remarkably inhibited in the positive DEX group, so that the test model group and the positive group were considered to be operating normally, and the test system was effective. Compared with the model group, 1.00mg/mL ICE-01 can significantly inhibit the release of IL-6, IL-beta and TNF-alpha (P < 0.05, P < 0.01) of RAW264.7 cells. The aqueous extract was shown to have an inhibitory effect on the LPS-induced increase in inflammatory factor levels.
Example 5: promoting effect of aqueous extract ICE-01 on growth of staphylococcus epidermidis
The staphylococcus epidermidis used in this example was: staphylococcus epidermidis CCSM0287 with a preservation number of CCTCC No: m2022779.
The promoting effect of the aqueous extract ICE-01 on the growth of staphylococcus epidermidis was measured by a liquid culture method. The aqueous extract ICE-01 samples were prepared as solutions, and were all sterilized by filtration through sterile 0.22 μm filters. 6 dilution gradients are diluted in half-times by using a sterile 96-well plate, and 4 compound wells are respectively arranged in each group; firstly, 200.00 mu L of sample (prepared by using sterile TSB) with concentration of 20.00mg/mL is added into a1 st hole, 100.00 mu L of solution in the 1 st hole is added into a2 nd hole for gradient dilution, the operation is repeated to 6 holes after full mixing, 100.00 mu L of sterile TSB culture medium is added into the 6 th hole, 100.00 mu L of liquid is removed after uniform mixing, and then 100.00 mu L of 10 is respectively added into the 1 st hole to the 6 th hole of a 96-well plate 6 cfu/mL staphylococcus epidermidis suspension. Sample group (a): 100.00. Mu.L of each half-diluted sample+100.00. Mu.L of 10 6 cfu/mL of staphylococcus epidermidis suspension; sample control group (b): 100.00 mu L of each half-diluted sample+100.00 μl sterile TSB medium; negative control group (c): 200.00. Mu.L of sterile TSB medium; positive control group (d): 100.00 mu L10 6 cfu/mL of staphylococcus epidermidis suspension +100.00 mu L of sterile TSB culture medium is placed at 37 ℃ for static culture for 48 hours, taken out for observing turbidity change, and absorbance value of each hole at 600nm is measured by an enzyme-labeling instrument. The operation was repeated 3 times for each group of experiments, and the bacteriostasis rate was calculated as follows:
TABLE 4 antibacterial Rate of aqueous extract ICE-01 against Staphylococcus epidermidis
As shown in Table 4, the aqueous extract ICE-01 of the invention has obvious effect of promoting the growth of staphylococcus epidermidis within the range of 0.625-10.00 mg/mL, and the growth rate can reach 66.58% when the concentration of the aqueous extract is 10.00mg/mL.
Example 6: evaluation of bacteriostatic Activity of aqueous extract ICE-01 on Propionibacterium acnes
The propionibacterium acnes used in this example is propionibacterium acnes ATCC11827, which is a standard strain of skin pathogenic bacteria with stronger biofilm production.
The inhibition of Propionibacterium acnes by aqueous extract ICE-01 was determined by liquid culture. The aqueous extract ICE-01 samples were prepared as solutions, and were all sterilized by filtration through sterile 0.22 μm filters. All the processes are completed under the aseptic condition, and 4 compound holes are respectively arranged in each group; 100.00. Mu.L of samples (formulated with sterile TSB) at concentrations of 20.00, 15.00, 10.00, 5.00, 2.50mg/mL were each added to the well plates according to the following groups. Sample group (a): 100.00. Mu.L of each diluted sample+100.00. Mu.L of 10 5 cfu/mL propionibacterium acnes suspension; sample control group (b)): 100.00. Mu.L of each diluted sample+100.00. Mu.L of sterile TSB medium; negative control group (c): 100.00. Mu.L of sterile TSB medium+100.00. Mu.L of sterile water; positive control group (d): 100.00 mu L10 5 cfu/mL propionibacterium acnes suspension +100.00 mu L sterile TSB medium, standing at 37 ℃ for 48 hours, taking out for observing turbidity change, and measuring absorbance value of each hole at 600nm by using an enzyme label instrument. The operation was repeated 3 times for each group of experiments, and the bacteriostasis rate was calculated as follows:
TABLE 5 antibacterial Rate of aqueous extract ICE-01 against Propionibacterium acnes
As shown in Table 5, the water extract ICE-01 of the invention has the antibacterial effect on Propionibacterium acnes gradually enhanced within the range of 1.25-7.50 mg/mL, and the antibacterial rate can reach 50.12% when the concentration of the water extract is 7.50 mg/mL.
Example 7: preparation process of emulsion containing Isaria cicadae mycelium aqueous extract
(1) Aqueous extract ICE-01 emulsion basic formula
The formulation of the emulsion is shown in Table 6, the components in phase A are weighed according to the ratio of each formulation, added into a beaker A, and placed in a water bath at 90 ℃ to be melted, and left for standby, a proper amount of deionized water is weighed and added into a beaker B, and each component in phase B is dispersed in the water phase of the beaker B in sequence. Heating in a water bath at 90 ℃ while stirring, when A, B two phases reach 90 ℃, stirring and shaking of the B phase are not stopped, adding the melted feed liquid in the A into the B beaker at a constant speed, maintaining stirring and dispersing for 5min, homogenizing, maintaining slow stirring after homogenizing is finished, adding the C phase when the temperature of the system is reduced to about 55 ℃, continuing stirring for 20-30 min at the moment, sealing the preservative film after the system is uniform, and preserving.
Table 6 emulsion formulation table
(2) Evaluation of cosmetic stability
Cold and hot stability experiments were performed on emulsions of aqueous extracts ICE-01 according to the experimental methods described in GBT29665-2013 and GB/T16497-2007.
Thermal stability: and placing the sample to be tested into two plugged test tubes, placing one test tube into a constant temperature incubator at 40 ℃ for 24 hours, taking out, recovering the room temperature, and placing the other test tube at the room temperature for 24 hours. And observing whether the properties of the samples in the two test tubes are consistent or not, and whether the samples are layered or not after being heated or not.
Cold stability: and placing the sample to be tested in a refrigerator at the temperature of minus 8 ℃ for 24 hours, taking out, then placing the sample to the room temperature, comparing the sample with the sample placed at the room temperature, and observing whether the sample is layered.
Cold and hot alternating stability: placing the sample to be tested in a refrigerator at the temperature of minus 8 ℃ for 24 hours, then placing the sample in a constant temperature incubator at the temperature of 40 ℃ for 24 hours, taking out the sample, comparing the sample with the sample placed at the room temperature, and observing whether the sample is layered.
TABLE 7 emulsion stability test results for aqueous extract ICE-01
Wherein A1 is represented as basic emulsion (ICE-01 is not added), and A2 and A3 are respectively emulsions added with 0.15% and 0.50% of ICE-01
The emulsion stability test results for aqueous extract ICE-01 are set forth in Table 7. As can be seen from Table 7, the base emulsion and the emulsion containing the cordyceps sobolifera polysaccharide in different addition amounts all show good stability. The addition of the Isaria cicadae mycelium aqueous extract did not adversely affect the stability of the cosmetic formulation.
(3) Cosmetic spot-stick test
According to cosmetic safety technical Specification 2015, 30 subjects were selected to carry out a plaque test on an emulsion of aqueous extract ICE-01 to investigate the safety of the test sample to the human body. The patch containing the sample is placed on the forearm of a volunteer, the patch is lightly pressed by the palm to be uniformly applied on the skin, the patch is continuously applied for 24 hours, and after the patch is removed for 30 minutes, whether a positive reaction occurs at the tested part of the volunteer is observed. The experimental results were determined with reference to table 8.
TABLE 8 skin adverse reaction grading criteria
Table 9 results of plaque assay of emulsion
The results of the plaque assay are shown in table 9. The results showed that 25 volunteers did not develop allergic reactions within 24 hours after the use of aqueous extract ICE-01 and no added emulsion, and the experimental results confirmed that: the addition of the aqueous extract ICE-01 has no influence on the safety of cosmetics, is mild, has low irritation, and is safe and harmless to human skin.
(3) Bacteriostasis test
The emulsions (A2 and A3) obtained by the above procedure were used for sanitary inspection of samples according to the cosmetic safety Specification (2015 edition), and the results are shown in Table 10 below, which shows that the microbial indicators thereof meet the requirements.
TABLE 10 detection results
(4) Acne removal effect test
The human subject: 60 patients with acne, pimple, acne, comedo, etc. on the 15-30 year old face were divided into 2 groups of 30 persons each, and each group was equally divided.
The testing method comprises the following steps: the face of the test person was cleaned with water, and the test group was (1.00.+ -. 0.10) mg/cm 2 The emulsion containing ICE-01 in the embodiment 1 is smeared on an affected part to be absorbed by the face, and the control group is uniformly smeared on the affected part by using the same amount of common skin care emulsion (the formula does not contain aqueous extract ICE-10). The test period is 28d and the use is carried out once a day in the morning and evening. The acne removal test results are shown in table 11.
Effect evaluation criteria:
(1) The effect is shown: the symptoms are obviously improved, and the acnes almost completely disappear;
(2) Improvement: the symptoms are improved, and the acne part disappears;
(3) Invalidation: the symptoms are not improved, and the acne is not changed.
Table 11 acne removal test results
From the results in Table 11, it is clear that the acne-removing emulsion using the Isaria cicadae mycelium aqueous extract ICE-01 has a good acne-removing effect.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; while the invention has been described in detail with reference to the foregoing embodiments, it will be appreciated by those skilled in the art that variations may be made in the techniques described in the foregoing embodiments, or equivalents may be substituted for in part or in whole; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.
Claims (10)
1. A preparation method of a Isaria cicadae mycelium aqueous extract is characterized in that Isaria cicadae C1 is fermented, fermentation liquor is centrifuged, mycelium is collected, and then freeze-dried mycelium is obtained through washing and freeze drying; after the freeze-dried mycelium is crushed, the cellulase and hot water extraction method are adopted for extraction.
2. The method for preparing the isaria cicadae mycelium aqueous extract according to claim 1, wherein the extraction by adopting a cellulase and hot water extraction method is as follows: adding water and cellulase into the crushed freeze-dried mycelium for enzymolysis, wherein the enzymolysis temperature is 40-50 ℃ and the enzymolysis time is 1-2 h; after enzymolysis, the enzyme is quickly heated to deactivate the enzyme; then the extraction is continued by a hot water extraction method, the extraction temperature is 90-100 ℃, the extraction time is 2-4 h, and the extraction times are 2-3 times; filtering the extractive solution while it is hot, mixing filtrates, concentrating, and lyophilizing to obtain Isaria cicadae mycelium water extract.
3. The water extract of the aschersonia cicadae mycelium prepared by the preparation method of claim 1 or 2, which is characterized by comprising the following components in percentage by mass: 0.05 to 0.10 percent of uridine, 0.05 to 0.10 percent of adenosine, 0.04 to 0.08 percent of guanosine, 0.01 to 0.015 percent of inosine, 0.01 to 0.02 percent of uracil, 0.5 to 1.0 percent of erythritol, 5 to 10 percent of cordycepic acid, 1.0 to 1.5 percent of glucose, 0.5 to 1.0 percent of trehalose, 5 to 10 percent of polysaccharide and 0.15 to 0.2 percent of protein.
4. Use of the aqueous extract of mycelium of Isaria cicadae as claimed in claim 3 as an active ingredient for the preparation of an external preparation for skin having an antioxidant effect.
5. The use according to claim 4, wherein the antioxidant efficacy is: antioxidant activity of scavenging any one or more of DPPH radicals, hydroxy radicals, ABTS radicals and iron ion reducing ability.
6. Use of the aqueous extract of mycelium of Isaria cicadae as claimed in claim 3 as an active ingredient for the preparation of an external preparation for skin having anti-inflammatory effect.
7. The use according to claim 6, wherein the anti-inflammatory efficacy is in particular: reduces lipopolysaccharide-induced inflammatory response of macrophage RAW264.7, and reduces expression of inflammatory factors IL-6, IL-beta and TNF-alpha.
8. Use of an aqueous extract of mycelium of corynespora cicadae according to claim 3 for the preparation of an external preparation for skin which promotes the growth of staphylococcus epidermidis, inhibits the growth of propionibacterium acnes, regulates the balance of staphylococcus epidermidis and propionibacterium acnes.
9. Use of an aqueous extract of mycelium of the group of cicada fungus according to claim 3 for the preparation of a cosmetic for improving acne.
10. The cosmetic for improving acne is characterized in that when the cosmetic is an emulsion, the emulsion comprises the following raw materials in parts by weight: the aqueous extract of mycelium of Isaria cicadae of claim 3, wherein the water is 100 parts, and the aqueous extract comprises 0.125-1 part of mycelium of Isaria cicadae, 6.23-16.88 parts of A phase, 9-26 parts of B phase and 0.1-0.4 part of C phase; the phase A is stearyl alcohol polyoxyethylene ether-2, stearyl alcohol polyoxyethylene ether-21, cetyl-stearyl alcohol, simethicone, octyl/decyl triglyceryl, white oil and BHT; the phase B is propylene glycol, glycerol, carbomer 20 and preservative; the C phase is sodium hydroxide.
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