Preparation method of phallus impudicus extract as well as product and application of phallus impudicus extract

Abstract

The invention discloses a preparation method of a phallus impudicus extract as well as a product and application of the phallus impudicus extract, and belongs to the technical field of biology. The phallus impudicus extract is prepared by taking phallus impudicus buds as a raw material and adopting a water extraction process, and the mass percentage of polysaccharide in the extract is not less than 70%. The obtained extract has the biological activity effects of resisting oxidation, resisting inflammation, resisting saccharification, inhibiting elastinase, relieving repair activity or promoting skin repair, and experiments prove that the extract is free of cytotoxicity, sensitization and phototoxicity. The phallus impudicus extract is very suitable for sensitive skin and used as a daily skin care product. Development of traditional fungus phallus impudicus is led to the field of high-tech cosmetic raw materials, a complete technical scheme and solid data support are provided for high value-added utilization of phallus impudicus, and the phallus impudicus is wide in application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a preparation method of Phallus impudicus extract, products and applications thereof. BACKGROUND

[0002] Phallus impudicus, as a kind of large fungi, is widely distributed in the forests of the temperate and subtropical regions of the Northern Hemisphere, and is a typical representative of the phallus family. In traditional folk medicine, Phallus impudicus is often used to treat rheumatism, inflammation and skin ulcers. However, the current systematic research and application of Phallus impudicus is still insufficient. For example, the existing technology focuses on the extraction of single raw material form, i.e. dried fruiting bodies, ignoring the significant influence of raw material growth stage (such as unopened fruiting bodies, commonly known as "mushroom buds") and processing form (fresh mushrooms, dried mushrooms) on the composition and activity of the extract. In fact, dried mushrooms (dried fruiting bodies) are more commonly studied due to their convenience in storage and transportation. However, during the drying process, some heat-sensitive components such as certain terpenes and antioxidants may be lost. Fresh mushrooms (mature fruiting bodies) have a comprehensive composition, but they are difficult to handle due to the abundance of volatile and foul-smelling substances, and the active ingredients are prone to degradation, which greatly limits their application. Research on mushroom buds (unopened stage) is even less. Moreover, the existing extraction processes, such as conventional alcohol extraction or water extraction, have not been optimized for the above-mentioned differences in raw materials, resulting in low extraction efficiency, incomplete retention of active ingredients, lack of outstanding functionality, and lack of systematic comparative research, which has greatly limited the application of Phallus impudicus.

[0003] Based on the above, the present application systematically studies the different forms of Phallus impudicus raw materials and creates a new, industrialized extraction method for Phallus impudicus extract and its products and applications, which can efficiently enrich specific active ingredients and ensure that the obtained extract has stable and significant multiple functions, while meeting the high safety standards of cosmetic raw materials, becoming the current industry's much-needed improvement target. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a Phallus impudicus extract that can efficiently enrich specific active ingredients and ensure that the obtained extract has stable and significant multiple functions, while meeting the high safety standards of cosmetic raw materials, thereby overcoming the shortcomings of existing Phallus impudicus extraction methods.

[0005] To solve the above technical problems, the present application provides a Phallus impudicus extract, which is extracted from Phallus impudicus bodies, and the mass percentage of polysaccharides in the extract is greater than 45%, and the Phallus impudicus bodies are Phallus impudicus buds, dried Phallus impudicus or fresh Phallus impudicus.

[0006] Further, the white ghost pen entity adopts white ghost pen bud, the extract is made of white ghost pen bud as raw material, and is made by water extraction process, and the mass percentage of polysaccharide in the extract is not less than 70%. The present application studies and shows that the extract obtained from white ghost pen bud has the best activity.

[0007] As another improvement of the present application, the present application further provides a preparation method of white ghost pen extract, which comprises the following steps: (1) taking white ghost pen bud as raw material, washing and slicing; (2) refluxing extraction at 85-95℃ for 2-3h with water as solvent, and the ratio of material to liquid is 1:8-1:15 (w / v), and the extraction is carried out for 2-3 times; (3) combining the extract, filtering, concentrating and freeze-drying to obtain the white ghost pen extract. In the preparation method, hot water extraction at 85-95℃ is adopted, which aims to efficiently dissolve the active ingredients such as water-soluble polysaccharides, and at the same time, can avoid excessive degradation caused by high temperature and prevent the loss of active ingredients.

[0008] Further, the thickness of the slice in step (1) is 2-3mm to increase the extraction contact area; the concentration condition in step (3) is vacuum condition at 60℃ and-0.08MPa, and the concentration is carried out to the relative density of 1.10, and the freeze-drying condition is-45℃ and <10Pa, and the freeze-drying is carried out for 24h. The concentration and drying conditions can further prevent the loss of active ingredients in the extract, and form efficient enrichment of active ingredients.

[0009] As another improvement of the present application, the present application further provides a composition comprising the above-mentioned white ghost pen extract and cosmetically or pharmaceutically acceptable adjuvant, wherein the addition amount of the white ghost pen extract is 0.1-5 wt%, and the composition has the biological activity of antioxidant, anti-inflammatory, anti-glycation, inhibition of elastase, soothing sensitive skin or promoting skin repair.

[0010] The cosmetic can be essence, emulsion, cream, cosmetic water or mask, which contains one or more auxiliary ingredients selected from moisturizing agent, anti-irritant, skin barrier repair agent and preservative.

[0011] Further, the composition is soothing repair essence, which contains the following raw materials in the following mass percentage: white ghost pen extract 1.0%, glycerol 5%, 4-tert-butylcyclohexanol 0.5%, panthenol 2%, asiaticoside 0.2%, sodium hyaluronate 0.3%, dipotassium glycyrrhizinate 0.2%, 1,2-hexanediol 1.0%, and the balance is deionized water.

[0012] In addition, the present application further provides the use of the white ghost pen extract in the preparation of drugs or cosmetics with anti-inflammatory soothing activity.

[0013] The present invention also provides the application of the aforementioned white stinkhorn extract in the preparation of antioxidant and anti-skin aging drugs or cosmetics.

[0014] The present invention also provides the application of the aforementioned white stinkhorn extract in the preparation of pharmaceuticals or cosmetics that promote skin repair.

[0015] With this design, the present invention has at least the following advantages:

[0016] 1. Through systematic comparison, this invention has found that the buds of *Phallus scutellariae* are the optimal stage for the enrichment of its active polysaccharides. Combined with a specific water extraction method, it can maximize the dissolution and retention of polysaccharide components with high bioactivity, and standardize the extract. This solves the problems of unclear raw materials and crude processes in the prior art, and provides a core raw material and key extraction method with controllable quality for the development of stable and efficient cosmetics. It is simple, stable and controllable.

[0017] 2. This invention, through extensive in vitro and in vivo experiments, has demonstrated that the prepared stigmata extract not only possesses powerful antioxidant capabilities but also exhibits significant anti-inflammatory, soothing, skin cell repair-promoting, anti-glycation, and anti-aging effects, comprehensively addressing multiple core needs of modern skincare. Furthermore, it demonstrates high safety; cytotoxicity, sensitization, and phototoxicity tests show that the extract is safe and non-irritating to skin cells at effective concentrations, exhibiting no sensitization or phototoxicity, making it highly suitable for sensitive skin and for use in daily skincare products.

[0018] 3. This invention leads the development of the traditional fungus Strigopteris in the field of high-tech cosmetic raw materials, providing a complete technical solution and solid data support for its high-value-added utilization, with broad application prospects. Attached Figure Description

[0019] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 The images shown are photographs of the actual product of the *Phallus spp.* of this invention, where a is a photograph of the actual product and b is a cross-sectional view.

[0021] Figures 2-4 The results of the cytotoxicity evaluation of the white phalloides extract PI-M015 of this invention are shown.

[0022] Figures 5-6 The results show the effect of the extract PI-M015 of the present invention on the sensitization activity of THP-1 cells.

[0023] Figure 7 The phototoxicity evaluation results of the white stinkhorn extract PI-M015 of this invention are shown.

[0024] Figure 8 The test results of the antioxidant activity of the white ghost pen extract PI-M015.

[0025] Figures 9-11 The test results of the white ghost pen extract PI-M015 in inhibiting the release of NO, IL-6 and TNF-α in the LPS-induced Raw264.7 cell model.

[0026] Figure 12 The test results of the anti-non-enzyme glycosylation of the white ghost pen extract PI-M015.

[0027] Figure 13 The test results of the elastase activity inhibition rate of the white ghost pen extract PI-M015.

[0028] Figure 14 The comparison of the results of the white ghost pen extract PI-M015 on the scratch repair ability of HaCaT cells at 24h and 48h.

[0029] Figure 15 The test results of the antioxidant effect of the white ghost pen extract PI-M015 in the skin care essence.

[0030] Figure 16 The test results of the anti-inflammatory effect of the white ghost pen extract PI-M015 in the skin care essence.

[0031] Figure 17 The test results of the skin soothing and anti-irritation of the white ghost pen extract PI-M015 in the skin care essence. DETAILED DESCRIPTION

[0032] Exemplary embodiments of the present application will be described in greater detail below with reference to the accompanying drawings. While exemplary embodiments of the present application are shown in the drawings, it is to be understood that the present application can be embodied in various forms without being limited by the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the application to those skilled in the art.

[0033] The present inventors have found that the polysaccharide (especially β-glucan) content of white ghost pen buds (unopened umbrella stage) is extremely high, and the volatile odor components are extremely few, which is very suitable for the development of high value-added cosmetics. The specific examples of the present application are as follows:

[0034] Example 1: Preparation of white ghost pen unopened umbrella fruiting body (bud) extract

[0035] 1.1 Raw material pretreatment

[0036] Collect fresh, intact, non-rotten white ghost pen unopened umbrella fruiting body (bud), such as Figure 1 The intact and cut pictures of white ghost pen unopened umbrella fruiting body (bud) are shown, showing its typical morphological characteristics as the preferred raw material.

[0037] Pre-treatment: gently brush with flowing cold water to remove surface soil and impurities, and keep the outer veil intact;

[0038] Use filter paper to absorb surface moisture;

[0039] Use a stainless steel knife to cut it into about 2-3mm thick slices to increase the extraction contact area, ready for use.

[0040] 1.2 Water extraction method

[0041] Feeding: take a portion of the bud slices and deionized water at a ratio of 1:12 (w / v) into the extraction tank.

[0042] Extraction: heat reflux extraction at 92°C for 2.5h.

[0043] Filtering: filter with 200 mesh filter cloth while hot, collect the filtrate. The dregs can be added with 8 times (w / v) deionized water for repeated extraction for 2h, and the two filtrates are combined.

[0044] Concentration: concentrate the combined filtrate to about 1 / 5 of the original volume under the conditions of 60°C, -0.08 MPa vacuum, so that the relative density of the concentrated solution is about 1.10.

[0045] Drying: transfer the concentrated solution to the tray and place it in the freeze dryer under the conditions of -45°C, <10 Pa for 24h to obtain light gray, sponge-like polysaccharide enriched extract powder, which is the bud extract 1.

[0046] Yield: weigh and calculate the yield of the bud extract 1, which is about 4.8% of the weight of the raw material.

[0047] 1.3 Alcohol extraction method

[0048] Feeding: take a portion of the bud slices and 50% (v / v) ethanol solution at a ratio of 1:8 (w / v) into the extraction tank.

[0049] Extraction: heat reflux extraction at 85°C for 2 times, 3h each time. Combine the two extraction solutions.

[0050] Concentration: concentrate the combined extraction solution at 60°C under reduced pressure until there is no alcohol smell, to obtain a syrup-like crude extract.

[0051] Dispersion: add an equal weight (1:1, w / w) of pure water to the crude extract, stir well to form a water dispersion.

[0052] Solvent extraction: The aqueous dispersion was extracted with n-hexane and dichloromethane successively to remove liposoluble impurities and part of pigments.

[0053] n-hexane extraction 2 times: the volume ratio of organic phase to aqueous phase was 1:1 for the first time and 1:2 for the second time.

[0054] dichloromethane extraction 2 times: the volume ratio of organic phase to aqueous phase was 1:1 for the first time and 1:2 for the second time.

[0055] All the aqueous phases after extraction were collected and combined.

[0056] The aqueous phase was concentrated under reduced pressure at 60°C and -0.08 MPa to a paste, and further freeze-dried to obtain the extract powder in the form of brownish yellow powder, which was the budlet extract 2.

[0057] Yield: the yield of the budlet extract 2 was calculated to be about 4.2% of the weight of the raw material.

[0058] 1.4 Gradient extraction method

[0059] Alcohol extraction: a portion of the budlet slices was mixed with 65% (v / v) ethanol solution at a ratio of 1:10 (w / v), and soaked for extraction at room temperature for 24 h in the dark with intermittent stirring. This step was aimed at extracting phenolic acids, flavonoids, terpenes and other alcohol-soluble components.

[0060] Filtration: the alcohol extract was collected by filtration with a filter press. The residue was retained.

[0061] Water extraction: the residue was mixed with deionized water at a ratio of 1:12 (w / v), and extracted by hot reflux at 90°C for 2 h, aiming to extract polysaccharides.

[0062] Filtration: the water extract was collected by filtration.

[0063] Combination: the alcohol extract was concentrated under reduced pressure at 50°C to remove ethanol, and concentrated to no alcohol smell, and then combined with the above-mentioned water extract.

[0064] Concentration and drying: the combined solution was concentrated under reduced pressure at 60°C, and then freeze-dried to obtain the full-spectrum extract powder with water-soluble and alcohol-soluble components, which was the budlet extract 3 in the form of dark brown.

[0065] Yield: the yield of the budlet extract 3 was calculated to be about 3.9% of the weight of the raw material.

[0066] Example 2: Preparation of dry fruiting body (dry fungus) extract of white ghost pen

[0067] 2.1 Pretreatment of raw material

[0068] The collected mature fruiting bodies (pods opened) of A. albus were dried completely to constant weight (crumble at a pinch) in a blast drying oven at 50°C.

[0069] The dried material was ground into fine powder of 80 mesh using a universal grinder, ready for use.

[0070] 2.2 Water extraction method

[0071] A portion of the dry mushroom powder was used as raw material, and the extraction, purification and post-treatment were carried out according to the method of "1.2 Water extraction method" in Example 1, to obtain a light brown powder extract, which was the dry mushroom extract 1.

[0072] Yield: The yield of the dry mushroom extract 1 was calculated to be about 4.5% of the weight of the dry mushroom powder.

[0073] 2.3 Alcohol extraction method

[0074] A portion of the dry mushroom powder was used as raw material, and the extraction, purification and post-treatment were carried out according to the method of "1.3 Alcohol extraction method" in Example 1, to obtain a light brown powder extract, which was the dry mushroom extract 1.

[0075] Yield: The yield of the dry mushroom extract 1 was calculated to be about 4.5% of the weight of the dry mushroom powder.

[0076] 2.4 Stepwise extraction method

[0077] A portion of the dry mushroom powder was used as raw material, and the extraction, purification and post-treatment were carried out according to the method of "1.4 Stepwise extraction method" in Example 1, to obtain a light brown powder extract, which was the dry mushroom extract 1.

[0078] Yield: The yield of the dry mushroom extract 1 was calculated to be about 4.5% of the weight of the dry mushroom powder.

[0079] Example 3: Preparation of fresh mature fruiting body (fresh mushroom) extract of A. albus

[0080] 3.1 Raw material pretreatment

[0081] Mature and morphologically intact fresh fruiting bodies of A. albus (pods opened) were collected.

[0082] Deodorization: To reduce the strong odor, the fruiting bodies were placed in a high-speed blast drying oven at 45°C for 3h to promote the volatilization of most volatile sulfur-containing compounds.

[0083] The treated fruiting bodies were cut into small pieces, ready for use.

[0084] 3.2 Water extraction method

[0085] A portion of the fresh mushroom pieces was used as raw material, and the extraction, purification and post-treatment were carried out according to the method of "1.2 Water extraction method" in Example 1, to obtain a light brown powder extract, which was the fresh mushroom extract 1.

[0086] Yield: The yield of the fresh mushroom extract 1 was calculated to be about 2.0% of the weight of the fresh mushroom.

[0087] 3.3 Alcohol extraction method

[0088] A portion of fresh mushroom was taken and subjected to feeding, extraction, purification and post-treatment according to the method of "1.3 Alcohol extraction method" in Example 1 to obtain a brownish yellow powder extract, which was fresh mushroom extract 2.

[0089] Yield: The yield of the fresh mushroom extract 2 was calculated to be about 2.3% of the weight of the fresh mushroom.

[0090] 3.4 Stepwise extraction method

[0091] A portion of fresh mushroom was taken and subjected to stepwise extraction and post-treatment according to the method of "1.4 Stepwise extraction method" in Example 1 to obtain a dark brown powder extract, which was fresh mushroom extract 3.

[0092] Yield: The yield of the fresh mushroom extract 3 was calculated to be about 2.1% of the weight of the fresh mushroom.

[0093] Example 4: Comparison of the activity and polysaccharide content of white ghost pen extracts from different raw material sources

[0094] 4.1 Polysaccharide content detection (phenol-sulfuric acid method)

[0095] Standard curve preparation: The dried glucose standard was accurately weighed and prepared into a series of standard solutions. The standard solutions were accurately pipetted, phenol solution and concentrated sulfuric acid were added, and the absorbance was measured at 490 nm after color development. The standard curve was drawn.

[0096] Sample determination: The bud, dried mushroom and fresh mushroom extract samples obtained above were accurately weighed and dissolved in water. An appropriate amount of sample solution was pipetted and operated according to the same method as above to determine the absorbance value, which was substituted into the standard curve to calculate the polysaccharide content (calculated as glucose) in each sample.

[0097] 4.2 Activity detection method

[0098] The bud extract 1-3, dried mushroom extract 1-3 and fresh mushroom extract 1-3 obtained in Examples 1-3 were taken and subjected to antioxidant activity detection. The antioxidant activity of the nine extract samples was tested using the experimental method described in Example 8 below.

[0099] 4.3 Experimental results

[0100] The results are shown in Table 1 below.

[0101] Table 1 Polysaccharide content and antioxidant activity of extracts obtained from different raw material sources and different extraction methods

[0102]

[0103] 4.4 Experimental results analysis

[0104] As can be seen from Table 1, the polysaccharide content of the mushroom bud extract 1 prepared by using the unopened cap fruiting body (mushroom bud) as the raw material and adopting the water extraction method is the highest (72.5 ± 2.1%), and the DPPH free radical scavenging capacity of the mushroom bud extract 1 is also the strongest (EC 50 = 67.57 μg / mL). The results show that the unopened cap fruiting body of white ghost pen adopted by the water extraction method is the best combination for enriching polysaccharides with high antioxidant activity.

[0105] Moreover, by comparing the same raw material, it is found that the extraction method has a significant effect on the content of active ingredients and antioxidant efficacy. For example, when the mushroom bud is extracted by different methods, the polysaccharide content and antioxidant activity of the mushroom bud follow the order: water extraction method > gradient extraction method > alcohol extraction method. This further shows the unique advantage of water extraction in efficiently dissolving water-soluble active polysaccharides in the mushroom bud.

[0106] As can be seen from Table 1, the DPPH free radical scavenging capacity is positively correlated with the polysaccharide content in the extract, that is, the DPPH free radical scavenging capacity of the mushroom bud extract prepared by using the mushroom bud as the raw material is obviously better than that of the other two extracts. The results show that the high polysaccharide content in the white ghost pen extract may be the material basis for its high biological activity.

[0107] Based on the above comparison, it is proved that the mushroom bud as the raw material and the water extraction method for extracting the white ghost pen extract have significant advantages, which provides the best solution for developing high-performance cosmetics or drugs. Based on this, the mushroom bud extract 1 prepared in the above is used for subsequent efficacy experiments, and is named as white ghost pen extract PI-M015.

[0108] Example 5: Cytotoxicity test

[0109] 5.1 Reagents and materials

[0110] High-sugar medium (DMEM), 1640 medium, fetal bovine serum (FBS), DPBS, dimethyl sulfoxide (DMSO), CCK-8.

[0111] 5.2 Instruments

[0112] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader.

[0113] 5.3 Cell lines

[0114] Mouse monocyte macrophage leukemia cells (Raw264.7), human acute mononuclear leukemia cells (THP-1), human immortalized epidermal cells (HaCaT).

[0115] 5.4 Samples to be tested

[0116] Sample group: Phallus tiglium extract PI-M015, tested at concentrations of 300 μg / mL, 100 μg / mL, 30 μg / mL, 10 μg / mL, 3 μg / mL, and 1 μg / mL.

[0117] Solvent control group: DMEM complete medium or 1640 complete medium;

[0118] Positive control group: DMEM complete medium containing 10% DMSO, or 1640 complete medium containing 10% DMSO;

[0119] Zeroing group: DMEM complete medium (cell-free) or 1640 complete medium (cell-free).

[0120] 5.5 Experimental Methods

[0121] Raw264.7 cells, THP-1 cells, or HaCaT cells were cultured at a density of 2.5 × 10⁻⁶ cells / year. 5 100 μL of cell suspension per well was seeded into each well of a 96-well plate and cultured until the cell confluence reached approximately 40%. The supernatant was discarded, and different concentrations of the sample were added sequentially for treatment with bioactive substances. After 24 h of treatment, CCK-8 reagent was added at a 1:10 ratio. After 40 min, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the following formula:

[0122] .

[0123] 5.6 Results

[0124] The CCK-8 assay is a method for detecting cell viability and growth; the measured OD value is directly proportional to cell activity. For example... Figures 2-4 As shown, the results of the test samples on the cytotoxicity of Raw264.7 cells, THP-1 cells and HaCaT cells showed that the cell viability of the white phalloides extract PI-M015 was greater than 90% in the above cells at a concentration of 100 μg / mL and below, and no obvious cytotoxicity was observed, indicating that the white phalloides extract PI-M015 has high safety for immune-related cells and skin cells.

[0125] Example 6: Evaluation of THP-1 cell sensitization

[0126] 6.1 Reagents and Materials

[0127] 1640 medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), β-mercaptoethanol (β-ME), DPBS, bovine serum albumin (BSA), dinitrochlorobenzene (DNCB), lactic acid (LA), 7-AAD, FITC anti-human CD86 Antibody, PE anti-human CD54 Antibody, 5 FITC Mouse IgG1 kappa Isotype Control Antibody, PE Mouse IgG1 kappa Isotype Control Antibody.

[0128] 6.2 Instruments

[0129] CO2 incubator, biosafety cabinet, inverted microscope, flow cytometer.

[0130] 6.3 Cell lines

[0131] Human acute monocytic leukemia cells (THP-1) were purchased from the China Academy of Sciences Cell Bank.

[0132] 6.4 Samples to be tested

[0133] Blank control group: 1640 complete medium;

[0134] Solvent control group: 0.2% DMSO, prepared with 1640 complete medium;

[0135] Positive control group: 3.125 μg / mL DNCB, prepared with 1640 complete medium;

[0136] Negative control group: 1 mg / mL lactic acid, prepared with 1640 complete medium;

[0137] Dosing group: Bai Guibi extract PI-M015, with concentrations of 100, 50, 25, and 12.5 μg / mL, prepared with 1640 complete medium.

[0138] 6.5 Experimental methods

[0139] THP-1 cells in good condition in the exponential growth phase were centrifuged to remove the supernatant and resuspended with culture medium, and the cell density was adjusted to 1.5×10 6 / mL, and the cell suspension was inoculated in a 48-well plate at 200 μL / well. According to the experimental grouping, a 2-fold concentration of the corresponding to-be-tested solution was prepared. In the corresponding well, 200 μL / well of the prepared to-be-tested sample was added, and was placed in a 37°C, 5% CO2 incubator for incubation for 24 h. After the incubation was completed, the cell suspension in each well was collected and was divided into two parts, one part of the cells was used for CD86 and CD54 staining, and one part was used for isotype control staining. After the staining was completed, the cells were fixed using paraformaldehyde, and the mean fluorescence intensity (MFI) of the FITC, PE, and 7-AAD dyes of each group of cells was detected using a flow cytometer. The relative fluorescence intensity value (RFI) of each group of tested substances was calculated using the following formula.

[0140]

[0141] 6.6 Experimental principle and results

[0142] When the sensitizer contacts the skin, the dendritic cells differentiate and mature in the process of moving to the lymphatic organs, and the expression of a series of surface molecules is up-regulated. The use of fluorescent antibody dyes to stain the cell surface molecules CD86 and CD54 and the use of a flow cytometer to detect them can determine whether the to-be-tested substance has sensitization.

[0143] The experimental results are shown in Table 6. Figures 5-6 The positive control group produced positive expression on the cells (RFICD86>150, RFICD54>200) and the cell survival rate was >50%; the negative control group produced negative expression on the cells (RFICD86<150, RFICD54<200) and the cell survival rate was >50%; the solvent control group produced negative expression on the cells (RFICD86<150, RFICD54<200) and the cell survival rate was >90%; indicating that the cells passed the stability test.

[0144] The extract PI-M015 of A. versicolor was negative expression on the CD86 and CD54 of THP-1 cells at all concentrations tested, and the cell survival rate was >90%, indicating that the extract PI-M015 of A. versicolor had no sensitization.

[0145] Example 7: Phototoxicity test

[0146] 7.1 Reagents and materials

[0147] High-sugar medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, neutral red, ice acetic acid, anhydrous ethanol, chlorpromazine hydrochloride (CPZ), HEPES.

[0148] 7.2 Instruments

[0149] CO2 incubator, biosafety cabinet, inverted microscope, microplate reader.

[0150] 7.3 Cell lines

[0151] Mouse embryonic fibroblast cells (BALB / C 3T3).

[0152] 7.4 Sample to be tested

[0153] Sample group: Bai Guibi extract PI-M015, test concentrations of 1 μg / mL, 3 μg / mL, 10 μg / mL, 30 μg / mL, 100 μg / mL, 300 μg / mL, 1000 μg / mL;

[0154] Blank control group: DMEM medium containing 10% FBS;

[0155] Background group: DMEM medium containing 10% FBS (without cells).

[0156] 7.5 Experimental method

[0157] The cells were inoculated in a 96-well cell culture plate at a density of 2x10 5 cells / mL cell suspension per well 100 μL, and when the cell fusion rate was about 100%, the supernatant was removed. The sample to be tested was added in turn for 1 h of biological activity treatment. Then the sample-containing cell culture plate requiring light treatment was subjected to UVA irradiation; the non-light group was placed in the incubator for continued culture. After UVA irradiation was completed, the culture medium was removed and washed, and after adding DMEM medium, the 96-well plate was placed in a CO2 incubator (37°C, 5% CO2) for continued culture for 18-22 h.

[0158] After the end of the culture, neutral red staining solution was added to the cell culture box for 3 h of incubation. After incubation was complete, the neutral red working solution was completely removed and washed, and neutral red desorption solution (distilled water: ethanol: acetic acid = 49:50:1) was added to completely lyse the cells, and a microplate reader was used to detect the absorbance at 540 nm. The survival rate of the positive control or sample cells was calculated according to the following formula:

[0159]

[0160] The half-maximal inhibitory concentration IC 50 of the cells under UVA irradiation (+Irr) and non-irradiation (-Irr) conditions was calculated, respectively, and the photo-stimulation factor (PIF) was calculated according to the following formula:

[0161] .

[0162] 7.6 Results

[0163] Certain drugs are converted from stable state to excited state after receiving light, and then produce damage to the body. In this test, according to the method of 3T3 neutral red uptake in vitro phototoxicity test of cosmetic raw materials in the Cosmetic Safety Technical Specification (2015 edition), the ability of BALB / C 3T3 fibroblasts to absorb neutral red or the change of cell toxicity after being subjected to the combined action of the test substance and ultraviolet irradiation is determined to determine whether the substance has phototoxicity.

[0164] The typical phototoxic drug CPZ has a light stimulation factor PIF value usually ≥ 5, indicating "phototoxicity". As shown in Table 1, the light stimulation factor PIF value of the sample white ghost pen extract PI-M015 is calculated to be about 1.76≤2, indicating that the white ghost pen extract PI-M015 has no phototoxicity. Figure 7

[0165] Example 8: Evaluation of antioxidant activity

[0166] 8.1 Reagents and materials

[0167] 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH), anhydrous ethanol, 96-well plate.

[0168] 8.2 Instruments

[0169] Microplate reader, hundred-thousandth microbalance.

[0170] 8.3 Sample to be tested

[0171] DPPH working solution: 50 μg / mL, prepared with anhydrous ethanol;

[0172] Sample group: white ghost pen extract PI-M015, test concentration 1.25, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL, prepared with distilled water;

[0173] Positive control group: GSH, concentration 300, 100, 33.3, 11.1, 3.7, 1.25, 0.41 μg / mL.

[0174] 8.4 Experimental method

[0175] ​Take 1.5 mg of DPPH powder in a 50 mL centrifuge tube, wrap it in tin foil to avoid light, add 30 mL of anhydrous ethanol, mix well, ultrasonic to make the powder completely dissolved, obtain 50 μg / mL DPPH working solution. The corresponding concentration of GSH working solution and test sample working solution is prepared by gradient dilution method, 50 μL of test sample working solution, or 50 μL of GSH working solution, or 50 μL of anhydrous ethanol is added to each well, and then 150 μL of DPPH working solution, or 150 μL of anhydrous ethanol is added to each well; after the addition is completed, mix well with 96-well microplate mixer, react at room temperature for 30 min, and read the absorbance at 517 nm with an enzyme-labeled instrument;

[0176] The DPPH clearance rate (%) of the test sample and GSH is calculated by the following formula:

[0177]

[0178] Among them, As represents the absorbance of the mixture of test sample (or GSH) and DPPH, Ac represents the absorbance of the mixture of test sample (or GSH) and EtOH, Ab represents the absorbance of the mixture of EtOH and DPPH, and A0 represents the absorbance of the mixture of EtOH and EtOH.

[0179] After calculating the clearance rate, the GraphPad Prism software is used for plotting, taking the final concentration of the sample in the reaction system as the horizontal coordinate and the clearance rate as the vertical coordinate, to establish a nonlinear fitting equation of the concentration of the solution to be tested and the clearance rate, and to calculate the half clearance amount EC 50 , the antioxidant capacity AO value of the polypeptide or small molecule active substance sample is calculated according to the following formula:

[0180]

[0181] Among them, EC50(S) represents the concentration (μg / mL) corresponding to the half clearance rate of the test sample, and EC50(R) represents the concentration (μg / mL) corresponding to the half clearance rate of GSH.

[0182] 8.5 Results

[0183] When there is a free radical scavenger (reducing agent), DPPH reacts with its single electron pairing, causing the characteristic purple color of DPPH ethanol solution to gradually disappear, turning colorless or light yellow, thereby causing the light absorption of DPPH to weaken. Studies have shown that the degree of fading of DPPH ethanol solution is linearly related to the number of electrons it receives, so the ability of the sample to scavenge free radicals, i.e., the size of the antioxidant activity, can be evaluated by measuring the light absorption of the reaction solution of the sample and DPPH at 517 nm.

[0184] The test results are as follows Figure 8As shown, the white ghost pen extract PI-M015 has the ability to scavenge DPPH, and its EC 50 67.57 pg / mL, and according to the AO value, the AO value of the white ghost pen extract PI-M015 is 2.6, i.e. the scavenging ability of DPPH is strong, close to the positive control GSH, indicating that the extract PI-M015 has strong antioxidant activity.

[0185] Example 9: Evaluation of Anti-inflammatory Activity

[0186] 9.1 Reagents and Materials

[0187] DMEM high-sugar medium, fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, lipopolysaccharide (LPS), dexamethasone (DEX), Mouse TNF-a ELISA detection kit, Mouse IL-6 ELISA detection kit, NO detection kit.

[0188] 9.2 Instruments

[0189] CO2 incubator, biological safety cabinet, inverted microscope, microplate reader.

[0190] 9.3 Cell Strains

[0191] Mouse monocyte macrophage leukemia cells (Raw264.7) were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences Typical Culture Preservation Committee.

[0192] 9.4 Samples to be Tested

[0193] LPS working solution: 25 ng / mL, prepared using DMEM medium containing 1% FBS;

[0194] Dexamethasone working solution (Dex): 10 pg / mL, prepared using LPS working solution;

[0195] White ghost pen extract: white ghost pen extract PI-M015, test concentration 1, 3, 10, 30, 100 pg / mL, prepared using LPS working solution.

[0196] 9.5 Experimental Methods

[0197] Take well-growing Raw264.7 cells in the exponential growth phase, use a cell scraper to make the cells fall off, and after centrifugation, adjust the cells to a density of 1.5 x 10 5 / mL of cell suspension. The above cell suspension was inoculated in a 48-well plate, 500 μL / well, and incubated in a 37°C, 5% CO2 incubator for 16-24 h. When the cell fusion rate reached 30-50%, the supernatant was removed, and 400 μL of the above sample to be tested was added in turn, except for the control group, and incubated in a 37°C, 5% CO2 incubator for 24 h. After incubation, the supernatant was collected, centrifuged to remove cell debris, and the concentration of NO, IL-6 and TNF-α in the supernatant of each group was detected according to the steps of the kit instructions.

[0198] 9.6 Experimental principle and results

[0199] Lipopolysaccharide can activate cells to synthesize and release various cytokines and inflammatory mediators through the cell signal transduction system. The aggregation of inflammatory factors can cause vasodilation, redness, stinging, itching and other symptoms, thereby causing the state of skin stinging sensitivity. In this experiment, LPS-induced Raw264.7 cells were used to establish an inflammation model to evaluate the anti-inflammatory activity of white ghost pen extract PI-M015.

[0200] The experimental results are shown in Table 1. Figures 9-11 Compared with the control group, the expression levels of NO, TNF-α and IL-6 of Raw264.7 cells were significantly increased after LPS induction, indicating that the inflammation model of Raw264.7 cells was successfully established. White ghost pen extract PI-M015 at a concentration of 1-100 μg / mL can significantly inhibit the release of NO, IL-6 and TNF-α of LPS-induced Raw264.7 cells in a dose-dependent manner. This indicates that the white ghost pen extract PI-M015 of the present application has good anti-inflammatory activity.

[0201] Example 10: Evaluation of non-enzymatic glycation inhibition activity

[0202] 10.1 Reagents and materials

[0203] Bovine serum albumin (BSA), D-anhydrous glucose, DPBS, penicillin / streptomycin (P / S).

[0204] 10.2 Instruments

[0205] Microplate reader, hundred-thousandth microbalance.

[0206] 10.3 Sample to be tested

[0207] BSA working solution: 40 μg / mL, prepared using DPBS containing 1% P / S;

[0208] Glucose working solution: 480 μg / mL, prepared using DPBS containing 1% P / S;

[0209] Test sample: White ghost pen extract PI-M015, concentration of 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL, prepared with DPBS containing 1% P / S.

[0210] 10.4 Experimental method

[0211] According to the following table, add the prepared white ghost pen extract PI-M015 solution of different concentrations, BSA working solution, glucose working solution and DPBS to the 96-well plate respectively, seal with sealing film, and place in a 37°C incubator, avoid light for 7 days. After incubation, use the enzyme marker to detect the fluorescence intensity under the condition of excitation wave 370 nm and emission wave 440 nm.

[0212] Table 2 Composition and proportion of non-enzymatic glycation inhibition activity evaluation experiment of white ghost pen extract PI-M015

[0213]

[0214] According to the following formula, the AGEs inhibition rate (P AGEs ) and half inhibition concentration (IC 50 ) of the test sample are calculated.

[0215]

[0216] Wherein, T, T0, C, C0 represent the fluorescence intensity of sample group, sample background group, glycation reaction group and BSA group under the condition of excitation wave 370 nm and emission wave 440 nm.

[0217] 10.5 Experimental principle and results

[0218] Non-enzymatic glycation (NEG) refers to the non-enzymatic condensation reaction between the carbonyl group on the reducing sugar (such as glucose, fructose, etc.) and the free amino group on the macromolecule such as protein, lipid or nucleic acid. The stable covalent adduct generated by the reaction is called advanced glycation end product (AGEs).

[0219] In this experiment, the ability of each test substance to inhibit the production of AGEs was determined to evaluate its anti-glycation ability. The experimental results are shown in Figure 12 White ghost pen extract PI-M015 has significant non-enzymatic glycation inhibition activity, and its half inhibition concentration (IC 50 ) is 75.45 μg / mL.

[0220] Example 11: Elastase inhibition activity evaluation

[0221] 11.1 Reagents and materials

[0222] Elastase, N-succinyl-Ala-Ala-Ala-pNA (NSAAAP), 1 M Tris-HCl buffer (pH= 8.0), DPBS.

[0223] 11.2 Instruments

[0224] Microplate reader, ten millionth microbalance.

[0225] 11.3 Samples to be tested

[0226] NSAAAP working solution: 0.5 mg / mL, prepared with 50 mM Tris-HCl buffer;

[0227] Test object: white ghost pen extract PI-M015, test concentration 0.41, 1.23, 3.7, 11.1, 33.33, 100, 300 μg / mL, prepared with 50 mM Tris-HCl buffer;

[0228] Elastase working solution: 5 U / mL, prepared with 50 mM Tris-HCl buffer.

[0229] 11.4 Experimental method

[0230] According to the following table, the prepared elastase working solution, white ghost pen extract of different concentrations, 50 mM Tris-HCL buffer were added respectively in the 96-well plate, and incubated at 25°C for 10 min, then NSAAAP working solution or 50 mM Tris-HCL buffer was added and incubated at 25°C for 30 min. After incubation, the absorbance at 410 nm was detected using a microplate reader.

[0231] Table 3 Component proportion of elastase inhibitory activity evaluation experiment of white ghost pen extract

[0232]

[0233] According to the following formula, the elastase activity (P 弹性蛋白酶 ) and half inhibitory concentration IC 50 of the sample to be tested were calculated:

[0234]

[0235] Wherein, T, T0, C, C0 represent the absorbance at 410 nm of sample group, sample background group, reaction group, solvent group respectively.

[0236] 11.5 Experimental principle and results

[0237] Inhibition of elastase activity is beneficial to improve skin aging, in this experiment, NSAAAP is used as substrate, elastase can hydrolyze it to Nsuccinyl-Ala-Ala-Ala and p-nitroaniline (pNA) in 50 mM Tris-HCl buffer at pH 8.0, the absorbance of pNA can be measured at 410 nm in spectrophotometer, the test substance with elastase activity inhibition can reduce the degradation of elastin, thereby reducing the absorbance.

[0238] In this experiment, the change of absorbance is used to evaluate the inhibition of elastase activity of the test substance. The experimental results are shown in Figure 13 , the elastase activity (P 弹性蛋白酶 ) decreases with the increase of the concentration of white ghost pen extract PI-M015, and the half inhibitory concentration IC 50 is 28.89 μg / mL. It shows that the white ghost pen extract PI-M015 of the application has the anti-aging effect of inhibiting elastase.

[0239] Example 12: Evaluation of zebrafish anti-inflammatory soothing activity

[0240] 12.1 Reagents and materials

[0241] Anhydrous copper sulfate, methyl cellulose, indomethacin, dimethyl sulfoxide, embryo culture solution, tricaine.

[0242] 12.2 Instruments

[0243] Biochemical incubator, fluorescent microscope, 100,000th scale balance.

[0244] 12.3 Zebrafish strain

[0245] Transgenic neutrophil red fluorescent strain zebrafish Tg (Lyz: dsRed).

[0246] 12.4 Test sample

[0247] Sample group: white ghost pen extract PI-M015, test concentration is 1 μg / mL, 2 μg / mL;

[0248] Model group: 0.8 μg / mL copper sulfate solution;

[0249] Positive group: 1 μg / mL indomethacin;

[0250] Blank control group: embryo culture solution.

[0251] 12.5 Experimental method

[0252] Select the 3 days (72hpf) after fertilization of the zebrafish development normally in 24-well plate, 12 tails per hole, each group repeated 2 complex holes. The above solution is added to the corresponding group, 2 mL solution per hole, incubated at 28℃ in biochemical incubator for 1h, after incubation, washed with culture solution for 2 times. The zebrafish after washing is anesthetized with 3% tricaine solution for 15 min, and the zebrafish is fixed on the glass slide with 5% methyl cellulose, and the imaging is carried out under microscope with unified parameters and body position. The number of neutrophils in the lateral line area of each fish embryo from the anus is counted, and the inhibition rate is calculated according to the following formula:

[0253]

[0254] 12.6Results

[0255] Neutrophils are the most important immune leukocytes, which function for phagocytosis and clearance of infection or harmful substances in the skin. The number of neutrophil migration in the zebrafish caudal neuropil can reflect the inflammatory condition in vivo, so as to evaluate the anti-inflammatory soothing effect of the raw material.

[0256] The experimental results are shown in Table 4. Compared with the model group, the number of neutrophil migration to the inflammatory site of the zebrafish treated with white ghost pen extract (PI-M015) is significantly reduced, and shows a dose-dependent relationship (2 μg / mL is better than 1 μg / mL). The neutrophil inhibition rates at 1 μg / mL and 2 μg / mL concentrations are 55.3% and 65.2%, respectively.

[0257] Table 4 Inhibition effect of white ghost pen extract on CuSO4 induced neutrophil aggregation in zebrafish tail

[0258]

[0259] The results fully show that the white ghost pen extract PI-M015 of the application can effectively inhibit the acute inflammatory response induced by CuSO4, has significant anti-inflammatory and soothing activity, and can be used for the development of cosmetics for soothing sensitive skin and reducing skin irritation.

[0260] Example 13: HaCat cell scratch repair activity

[0261] 13.1 Reagents and materials

[0262] High-sugar medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (P / S), DPBS, epidermal growth factor (EGF).

[0263] 13.2 Instruments

[0264] CO2 incubator, biosafety cabinet, inverted microscope, microplate reader, 2 Well Culture-lnserts for self-insertion (Ibidi, 80209).

[0265] 13.3 Cell lines

[0266] Human immortalized keratinocytes (HaCaT).

[0267] 13.4 Test sample

[0268] Sample group: white ghost pen extract PI-M015, test concentration 20 μg / mL;

[0269] Model group: 5 ng / mL EGF;

[0270] Blank control group: DMEM medium containing 0.1% FBS.

[0271] 13.5 Experimental method

[0272] HaCaT cells were inoculated in the scratch insert at a density of 6x10 5 cells / mL cell suspension per well 70 μL, and when the cell fusion rate reached 100%, the supernatant was removed. The test sample was added in turn for biological activity treatment. After the administration was completed, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) for culture. At 0h, 8h and 12h after administration, a 5x microscope was used to take photos of the same field and record the scratch gap at the corresponding time. The area of the scratch in the photo was calculated using Image J, and the scratch healing rate was calculated using the following formula:

[0273]

[0274] 13.6 Results

[0275] The differentiation ability of keratinocytes plays a crucial role in the process of skin wound healing, and the scratch test is a commonly used method to detect cell migration ability.

[0276] The experimental results are shown in Figure 14 Compared with the blank control, the sample group PI-M015 showed a significant migration-promoting trend at 24h, and the healing ability of the PI-M015 group remained at a high level when cultured for 48h, indicating that the white ghost pen extract PI-M015 can continuously promote the migration and repair of HaCaT cells. The above results suggest that the white ghost pen extract PI-M015 has significant keratinocyte scratch repair activity in vitro, and its effect may be related to the enhancement of cell migration ability.

[0277] Example 14: Application of white ghost pen extract in soothing repair serum

[0278] 14.1 Formulation design

[0279] Soothing repair serum: white ghost pen extract PI-M015 1.0%, glycerin 5%, 4-tert-butylcyclohexanol 0.5%, panthenol 2%, asiaticoside 0.2%, sodium hyaluronate 0.3%, dipotassium glycyrrhizate 0.2%, 1,2-hexanediol 1.0%, and the balance deionized water.

[0280] Comparative serum: glycerin 5%, 4-tert-butylcyclohexanol 0.5%, panthenol 2%, asiaticoside 0.2%, sodium hyaluronate 0.3%, dipotassium glycyrrhizate 0.2%, 1,2-hexanediol 1.0%, and the balance deionized water.

[0281] 14.2 Preparation method

[0282] Aqueous phase preparation: Dissolve glycerin, sodium hyaluronate, panthenol, and dipotassium glycyrrhizate in deionized water, and heat to 75°C and stir until completely dissolved.

[0283] Cooling mixing: After cooling to 40°C, add asiaticoside, white ghost pen extract PI-M015 (not added in the comparative serum), 4-tert-butylcyclohexanol, and 1,2-hexanediol in sequence, and homogenously stir (500 rpm x 10 minutes).

[0284] Filtering and filling: Filter through a 0.22 μm microporous filter membrane, and fill into a light-proof sterile container to obtain a transparent serum with a slight color.

[0285] 14.3 Efficacy test

[0286] 14.3.1 Evaluation of in vitro antioxidant capacity

[0287] Method: DPPH free radical scavenging method. Dilute the serum samples to concentrations of 0.5%, 1%, 2%, and 5% (v / v), and use vitamin C (Vc) as the positive control. Measure the absorbance at 517 nm, and calculate the clearance rate.

[0288] Results: The experimental results are shown in Table 1. Figure 15 Compared with the comparative serum, the serum containing 1% PI-M015 showed a concentration-dependent DPPH free radical scavenging ability. At a test concentration of 5%, its clearance rate was significantly higher than that of the comparative sample (p < 0.01), reaching 85.2% ± 3.1%, which confirmed the excellent in vitro antioxidant activity of the formula, which was mainly contributed by PI-M015.

[0289] 14.3.2 Evaluation of in vitro anti-inflammatory effect

[0290] Methods: Establish the inflammation model of mouse macrophage Raw264.7 induced by lipopolysaccharide (LPS). Pretreat with the serum containing PI-M015 at a dilution of 5% (v / v), and detect the contents of key inflammatory factors TNF-α and PGE2 in the cell supernatant by ELISA.

[0291] Results: The experimental results are shown in Table 1. Figure 16 As compared with the LPS model group, the serum containing PI-M015 can significantly inhibit the release of TNF-α and PGE2, and the inhibition rates of TNF-α and PGE2 are 68.4% and 55.7%, respectively, which is significantly better than the control serum without PI-M015 (p < 0.001), indicating that the white ghost pen extract PI-M015 endows the serum with excellent anti-inflammatory efficacy.

[0292] 14.3.3 Human efficacy evaluation (skin soothing and anti-irritation test)

[0293] Subjects: 30 healthy volunteers (aged 25-50 years) were recruited, all of whom were self-evaluated as sensitive skin or had a history of cosmetic intolerance.

[0294] Methods: Lactic acid stinging test was used. 5% lactic acid solution was applied to both sides of the nasolabial folds of the subjects, and the stinging score (0-5 points) was recorded as the baseline irritation value. Then, the serum containing PI-M015 was applied to one side, and the control serum was applied to the other side. The change in stinging score was determined at 5 min and 30 min after application, and the erythema subsidence was quantitatively evaluated using a skin redness value (a* value) meter.

[0295] Results: The experimental results are shown in Table 2. Figure 17 As shown in Table 2, the stinging score of the side applied with the serum containing PI-M015 was significantly reduced at 30 min, and the subsidence rate of the skin redness value (a* value) was also significantly faster than that of the side applied with the control serum. This indicates that the serum containing the white ghost pen extract PI-M015 can quickly soothe the skin irritation reaction, effectively relieve stinging and redness, and is suitable for daily repair and stabilization of sensitive skin.

[0296] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Those skilled in the art can make some simple modifications, equivalent changes or modifications by using the disclosed technical content, which are all within the protection scope of the present application.

Claims

1. An extract of Whitebark pine, characterized in that, The extract is extracted from white ghost pen entity, the mass percentage of polysaccharide in the extract is greater than 45%, and the white ghost pen entity adopts white ghost pen bud, white ghost pen dry fungus or white ghost pen fresh fungus.

2. The extract of A. anomala according to claim 1, characterized in that, The white ghost pen entity adopts white ghost pen bud, the extract is made of white ghost pen bud as raw material by water extraction process, and the mass percentage of polysaccharide in the extract is not less than 70%.

3. The extract of A. anomala according to claim 2, characterized in that, The preparation method of the extract is: (1) taking white ghost pen bud raw material, washing, slicing; (2) using water as solvent, refluxing extraction at 85-95 DEG C for 2-3h, the ratio of material to liquid is 1:8-1:15 (w / v), extracting 2-3 times; (3) combining the extract, filtering, concentrating, freeze-drying, thus obtaining the white ghost pen extract.

4. A method for preparing an extract of Whitebark pine, characterized by, The method comprises: (1) taking white ghost pen bud raw material, washing, slicing; (2) using water as solvent, refluxing extraction at 85-95 DEG C for 2-3h, the ratio of material to liquid is 1:8-1:15 (w / v), extracting 2-3 times; (3) combining the extract, filtering, concentrating, freeze-drying, thus obtaining the white ghost pen extract.

5. The method for preparing the extract of *Phallus spp.* according to claim 4, characterized in that, The thickness of slicing in step (1) is 2-3mm; the concentration condition in step (3) is 60 DEG C, -0.08 MPa vacuum condition, and the relative density is 1.10, the freeze-drying condition is -45 DEG C, <10 Pa, and freeze-drying for 24h.

6. A composition characterized in that, The composition comprises the white ghost pen extract of any one of claims 1 to 3, and cosmetically or pharmaceutically acceptable adjuvant, wherein the addition amount of the white ghost pen extract is 0.1-5 wt%, and the composition has the biological activity of antioxidant, anti-inflammatory, anti-glycation, elastase inhibition, soothing sensitive skin or promoting skin repair.

7. The composition of claim 6, wherein, The composition comprises the following raw materials in mass percentage: White ghost pen extract 1.0%, glycerol 5%, 4-tert-butylcyclohexanol 0.5%, panthenol 2%, asiaticoside 0.2%, sodium hyaluronate 0.3%, glycyrrhizic acid dipotassium 0.2%, 1,2-hexanediol 1.0%, and the rest is deionized water.

8. The use of the white ghost pen extract of any one of claims 1 to 3 in the preparation of a medicine or cosmetic with anti-inflammatory soothing activity.

9. The use of the white ghost pen extract of any one of claims 1 to 3 in the preparation of an antioxidant, anti-skin aging medicine or cosmetic.

10. The use of the white ghost pen extract of any one of claims 1 to 3 in the preparation of a medicine or cosmetic for promoting skin repair.

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