Pseudo-ginseng-paecilomyces cicadae lysate aqueous extract as well as preparation method and application thereof

Abstract

The invention provides a preparation method of a pseudo-ginseng-paecilomyces cicadae lysate aqueous extract, which comprises the following steps: uniformly mixing pseudo-ginseng root hair powder and water, and sterilizing to obtain a pseudo-ginseng liquid culture medium; inoculating a paecilomyces cicadae seed solution into the culture medium for fermentation treatment; after fermentation, centrifuging to obtain mycelium, drying, and crushing to obtain mycelium powder; and carrying out water extraction treatment on the mycelium powder under a constant-temperature water bath condition, then centrifuging to take supernate, cooling, freeze-drying, crushing and grinding to obtain freeze-dried powder of the pseudo-ginseng-paecilomyces cicadae lysate water extract. The invention also provides the pseudo-ginseng-paecilomyces cicadae lysate aqueous extract prepared by the method and application of the pseudo-ginseng-paecilomyces cicadae lysate aqueous extract in preparation of skin external preparations and hair preparations. In the pseudo-ginseng-paecilomyces cicadae lysate aqueous extract for inducing and enhancing the antioxidant and anti-inflammatory effects based on the mycelium metabolic pathway, the content of beta-glucan and uronic acid is relatively high, the pseudo-ginseng-paecilomyces cicadae lysate aqueous extract has an extremely remarkable inhibition effect on IL-1beta and TNF-alpha, and has a relatively strong inhibition capability on MMP-9.

Description

Technical Field

[0001] The present disclosure belongs to the technical field of cosmetic raw materials, and particularly relates to a water extract of the lysate of Panax notoginseng-Paecilomyces cicadae and its preparation method and application. Background Art

[0002] Paecilomyces cicadae is the asexual stage of the traditional Chinese medicine Cordyceps cicadae, and is an edible and medicinal fungus. Its active substances have effects such as antioxidant, anti-inflammatory, anti-tumor, and immunomodulatory effects, and have high medicinal value and good application and development prospects. Panax notoginseng (Burkill) F.H.Chen ex C.H.Chow is a perennial herb of the genus Panax in the Araliaceae family. Panax notoginseng is a traditional precious Chinese herbal medicine in China, and has effects such as anti-inflammatory, anti-aging, and immunomodulatory effects. At present, there is still an expectation in the market for the advent of a Paecilomyces cicadae extract with better performance. Summary of the Invention

[0003] A brief overview of the present disclosure is given below to provide a basic understanding of certain aspects of the present disclosure. It should be understood that this overview is not an exhaustive overview of the present disclosure. It is not intended to identify the key or important parts of the present disclosure, nor is it intended to limit the scope of the present disclosure. Its purpose is only to present certain concepts in a simplified form as a prelude to the more detailed description discussed later.

[0004] To solve the above technical problems, the technical solution provided by the present disclosure is:

[0005] In a first aspect, the present disclosure provides a preparation method of a water extract of the lysate of Panax notoginseng-Paecilomyces cicadae, comprising:

[0006] Steps for preparing the mycelium of Panax notoginseng-Paecilomyces cicadae: Mix the root and whisker powder of Panax notoginseng and water evenly, sterilize to obtain a liquid medium of Panax notoginseng; inoculate the seed liquid of Paecilomyces cicadae into the liquid medium of Panax notoginseng for fermentation treatment; after fermentation is completed, centrifuge to obtain mycelium, dry it, and pulverize it into mycelium powder.

[0007] Steps for preparing the water extract: Perform water extraction treatment on the mycelium powder under the condition of a constant temperature water bath, then centrifuge to take the supernatant, and after cooling, perform freeze-drying, pulverizing, and grinding treatments to obtain a freeze-dried powder of the water extract of the lysate of Panax notoginseng-Paecilomyces cicadae.

[0008] In the above preparation method of the water extract of the lysate of Panax notoginseng-Paecilomyces cicadae, as a preferred embodiment, in the steps for preparing the mycelium of Panax notoginseng-Paecilomyces cicadae, the root and whisker powder of Panax notoginseng in the liquid medium of Panax notoginseng accounts for 0.5-2 wt%, preferably 1 wt%.

[0009] The preparation method of the aqueous extract of the Cordyceps cicadae-Panax notoginseng lytic product, as a preferred embodiment, in the step of preparing the Cordyceps cicadae-Panax notoginseng mycelium, the sterilization conditions are 118-125°C for 25-35 minutes, preferably 121°C for 30 minutes.

[0010] The preparation method of the aqueous extract of the Cordyceps cicadae-Panax notoginseng lytic product, as a preferred embodiment, in the step of preparing the Cordyceps cicadae-Panax notoginseng mycelium, the Cordyceps cicadae is Cordyceps cicadae CH2347, and its preservation number is CGMCC No. 40399; more preferably, the volume ratio of the Cordyceps cicadae mycelium in the Cordyceps cicadae seed liquid accounts for 50-80% of the entire liquid medium; further preferably, the volume ratio of the Cordyceps cicadae seed liquid to the Panax notoginseng liquid medium is 3-7%, preferably 5%; furthermore, the preparation method of the Cordyceps cicadae seed liquid includes: (1) inoculating the Cordyceps cicadae CH2347 strain growing on a PDA plate into a potato dextrose water medium, and the inoculation ratio is 2-3 solid strains with a diameter of about 0.5 cm: 300 mL of the medium, and culturing at 28°C at 180 rpm for 3 days; (2) after homogenizing the culture in step (1), continue to inoculate it as a seed liquid into a new potato dextrose water medium, and the inoculation volume ratio is 1:15, and culturing at 28°C at 180 rpm for 4 days to obtain the Cordyceps cicadae seed liquid.

[0011] The preparation method of the aqueous extract of the Cordyceps cicadae-Panax notoginseng lytic product, as a preferred embodiment, in the step of preparing the Cordyceps cicadae-Panax notoginseng mycelium, the fermentation conditions are fermentation at 26-30°C for 4-6 days, preferably fermentation at 28°C for 4-5 days; more preferably, the fermentation is carried out on a shaker, and the shaker speed is 160-200 rpm, further preferably 180 rpm.

[0012] The preparation method of the aqueous extract of the Cordyceps cicadae-Panax notoginseng lytic product, as a preferred embodiment, in the step of preparing the Cordyceps cicadae-Panax notoginseng mycelium, the centrifugation conditions are 4500-5000 rpm for 25-35 minutes; preferably 4800 rpm for 30 minutes; and / or, the drying temperature is 35-45°C.

[0013] The preparation method of the aqueous extract of the Cordyceps cicadae-Panax notoginseng lytic product, as a preferred embodiment, in the step of preparing the Cordyceps cicadae-Panax notoginseng mycelium, the particle size of the mycelium powder is below 200 μm, preferably below 177 μm; and / or, the mass ratio of the mycelium powder to distilled water is 0.5-2:10, preferably 1:10.

[0014] The preparation method of the aqueous extract of the Cordyceps cicadae Paecilomyces hepiali cell lysate mentioned above, as a preferred embodiment, in the steps of preparing the aqueous extract, the constant temperature water bath condition is a temperature of 60 - 80°C and an extraction time of 1.5 - 2.5 h, preferably extracting for 2 h in a constant temperature water bath at 70°C; and / or, the centrifugation condition is 3500 - 5000 r / min for 8 - 15 min, preferably 4000 r / min for 10 min.

[0015] In a second aspect, the present disclosure provides an aqueous extract of Cordyceps cicadae Paecilomyces hepiali cell lysate, which is prepared according to the above preparation method.

[0016] In a third aspect, the present disclosure provides the application of the aqueous extract of Cordyceps cicadae Paecilomyces hepiali cell lysate in the preparation of skin topical agents and hair agents; preferably, the aqueous extract of Cordyceps cicadae Paecilomyces hepiali cell lysate is used as at least one of the antioxidant active ingredient and the anti-inflammatory active ingredient in the skin topical agent; preferably, the skin topical agent is at least one of skin lotion, milk, cream, and facial mask.

[0017] Compared with the prior art, the beneficial effects of the present disclosure include but are not limited to:

[0018] 1. The present disclosure provides an aqueous extract of Cordyceps cicadae Paecilomyces hepiali cell lysate and its preparation method for inducing and enhancing antioxidant and anti-inflammatory effects based on the mycelial metabolic pathway;

[0019] 2. The present disclosure explores the effects of Panax notoginseng on the composition function and cosmetic efficacy of Cordyceps cicadae Paecilomyces hepiali cell lysate from three aspects: the content of active ingredients, the evaluation of anti-inflammatory and antioxidant effects, and non-targeted metabolomics analysis. The results show that the contents of total sugar, reducing sugar, and protein in the Cordyceps cicadae Paecilomyces hepiali cell lysate fermented with Panax notoginseng (SQ-CH) are lower, but the contents of β-glucan and uronic acid are higher; in terms of free radical scavenging ability, SQ-CH has a stronger DPPH free radical scavenging ability, showing a concentration dependence. After UVB damage, the contents of inflammatory factors IL-1β and TNF-α in the culture medium of HaCaT cells in the SQ-CH group are significantly reduced, indicating that adding Panax notoginseng to ferment Cordyceps cicadae Paecilomyces hepiali has a very significant inhibitory effect on IL-1β and TNF-α, showing a strong anti-inflammatory effect. After UVA damage, the contents of MMP-1 and MMP-9 in HSF cells in the SQ-CH group are significantly lower than those in the model group, indicating that adding Panax notoginseng to ferment Cordyceps cicadae Paecilomyces hepiali has a strong inhibitory ability on MMP-9, showing a strong antioxidant effect. The mycelium may face the pressure of the external environment, inducing the adjustment of the internal metabolic pathway of the mycelium, reducing carbohydrate metabolism, turning to amino acids as the main energy source or the direction of regulating metabolism, increasing amino acid metabolites, and enhancing antioxidant and anti-inflammatory effects. From the above data, it can be seen that after adding Panax notoginseng, the anti-inflammatory and antioxidant effects of the cell lysate are improved, providing ideas for the development and application of Cordyceps cicadae Paecilomyces hepiali cell lysate in the future. Brief Description of the Drawings

[0020] Figure 1 Shows the test results of the DPPH free radical scavenging ability of the freeze-dried powder of the aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate (SQ-CH) and the aqueous extract of Paecilomyces cicadae lysate (TD-CH) in Example 3;

[0021] Figure 2 Respectively show the effects of SQ-CH (Figure A) and TD-CH (Figure B) on the viability of HaCaT cells, and the effects of SQ-CH (Figure C) and TD-CH (Figure D) on the viability of HSF cells in Example 4;

[0022] Figure 3 Respectively show the effects of SQ-CH and TD-CH on the contents of TNF-α (Figure A) and IL-1β (Figure B) in UVB-induced photo-damaged HaCaT cells in Example 5 (UVB is selected as 30 mJ / cm 2 );

[0023] Figure 4 Respectively show the effects of SQ-CH and TD-CH on the contents of MMP-1 and MMP-9 in UVA-induced photo-damaged HSF cells in Example 6 (UVA is selected as 12 J / cm 2 );

[0024] Figure 5 Respectively show the PCA score plots of SQ-CH and TD-CH in positive ion (Figure A) and negative ion (Figure B) modes in Example 7;

[0025] Figure 6 Respectively show the PLS-DA score plots of SQ-CH and TD-CH in positive ion (Figure A) and negative ion (Figure B) modes in Example 7;

[0026] Figure 7 Respectively show the results of the permutation test of the PLS-DA model of SQ-CH and TD-CH in positive ion (Figure A) and negative ion (Figure B) modes in Example 7;

[0027] Figure 8 Is the Venn diagram of SQ-CH and TD-CH;

[0028] Figure 9 Is the volcano plot of the differential components of SQ-CH and TD-CH in positive ion (Figure A) and negative ion (Figure B) modes;

[0029] Figure 10 Is the clustering heat map of the differential components;

[0030] Figure 11 Is the heat map of the correlation analysis of the differential components;

[0031] Figure 12 is a bar graph of differential component pathway types;

[0032] Figure 13 is a bubble chart of KEGG enrichment analysis;

[0033] Figure 14 is a graph of the differential abundance scores of KEGG pathways. Specific implementation manners

[0034] The following examples further illustrate the content of the present invention, but should not be construed as a limitation to the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention belong to the scope of the present invention.

[0035] In the following, the technical solutions of the present disclosure will be described in conjunction with exemplary embodiments. Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified. The Paecilomyces cicadae used in the examples and comparative examples of this application is Cordyceps cicadae CH2347, and its preservation number is CGMCC No. 40399. This strain has been recorded in CN202410898360.1 and CN202410898355.0; the potato dextrose broth medium is purchased from Haibo Biotechnology Co., Ltd., model HB0233-4.

[0036] Example 1.1 Growth Adaptability of Different Additives - Paecilomyces cicadae Mycelia

[0037] The preparation method of the Paecilomyces cicadae seed liquid is as follows: inoculate the solid strain of Cordyceps cicadae CH2347 (with medium, take 2-3 solid strains with a diameter of about 0.5 cm using a sterilized punch) growing on a PDA plate into 300 mL of potato dextrose broth medium that has been sterilized (115 °C, 20 min), and culture at 28 °C with 180 rpm for 3 days to obtain Paecilomyces cicadae seed liquid 1. At this time, the mycelial balls of Paecilomyces cicadae seed liquid 1 are of different sizes; 2) Homogenize Paecilomyces cicadae seed liquid 1 with a sterile glass homogenizer, and continue to take 20 mL as the seed liquid and inoculate it into 300 mL of potato dextrose broth medium, and culture at 28 °C with 180 rpm for 4 days to obtain Paecilomyces cicadae seed liquid 2. At this time, the volume ratio of the mycelium of Paecilomyces cicadae to the whole liquid medium is about 50-80%.

[0038] The preparation method of different additives - Paecilomyces cicadae mycelium is as follows:

[0039] 1.1.1 Preparation of Paecilomyces cicadae mycelia: Prepare 300 mL of potato dextrose broth, sterilize it at 115 °C for 20 min, inoculate the Paecilomyces cicadae seed liquid into the broth, with an inoculation ratio of 5% (i.e., inoculate 15 mL of seed liquid into 300 mL of broth), place it in a shaker with parameters of 180 rpm and 28 °C, and ferment for 5 days to obtain the Paecilomyces cicadae fermentation broth; put the fermentation broth into a centrifuge with a rotation speed of 4800 rpm for 30 min. After centrifugation, pour out the supernatant, take out the mycelia at the bottom, blot dry the water, weigh it, and record the wet weight as 112.7 g.

[0040] 1.1.2 Preparation of Panax notoginseng - Paecilomyces cicadae mycelia: The method is the same as 1.1.1 of this example, the difference is that the potato dextrose broth is replaced with Panax notoginseng liquid medium (the mass ratio of Panax notoginseng rootlet powder to water is 1:99), and the wet weight of the obtained mycelia is 132.24 g.

[0041] 1.1.3 Preparation of Angelica pubescens - Paecilomyces cicadae mycelia: The method is the same as 1.1.1 of this example, the difference is that the potato dextrose broth is replaced with Angelica pubescens liquid medium (the mass ratio of dry Angelica pubescens powder to water is 1:99), and the wet weight of the obtained mycelia is 56.22 g.

[0042] 1.1.4 Preparation of Citrus medica - Paecilomyces cicadae mycelia: The method is the same as 1.1.1 of this example, the difference is that the potato dextrose broth is replaced with Citrus medica liquid medium (the mass ratio of dry Citrus medica powder to water is 1:99), and the wet weight of the obtained mycelia is 108.94 g.

[0043] 1.1.5 Preparation of Gleditsia sinensis - Paecilomyces cicadae mycelia: The method is the same as 1.1.1 of this example, the difference is that the potato dextrose broth is replaced with Gleditsia sinensis liquid medium (the mass ratio of Gleditsia sinensis to water is 1:99), and the wet weight of the obtained mycelia is 60.04 g.

[0044] 1.1.6 Preparation of Rosa davurica - Paecilomyces cicadae mycelia: The method is the same as 1.1.1 of this example, the difference is that the potato dextrose broth is replaced with Rosa davurica liquid medium (the mass ratio of Rosa davurica to water is 1:99), and the wet weight of the obtained mycelia is 104.80 g.

[0045] 1.1.7 Preparation of Bombyx batryticatus - Paecilomyces cicadae mycelia: The method is the same as 1.1.1 of this example, the difference is that the potato dextrose broth is replaced with Bombyx batryticatus liquid medium (the mass ratio of Bombyx batryticatus to water is 1:99), and the wet weight of the obtained mycelia is 75.9 g.

[0046] In summary, the weight of Panax notoginseng - Paecilomyces cicadae mycelia is the largest, followed by potato dextrose broth, Citrus medica - Paecilomyces cicadae mycelia, and Rosa davurica - Paecilomyces cicadae mycelia. It can be seen from this that Panax notoginseng has the effect of promoting the growth of Paecilomyces cicadae.

[0047] Example 1.2 Preparation of Aqueous Extract (SQ-CH) of the Lysate of Panax notoginseng - Paecilomyces cicadae

[0048] 1.2.1 Preparation of Paecilomyces cicadae seed liquid: The same as in Example 1.1.

[0049] 1.2.2 Preparation of Paecilomyces cicadae mycelia: The same as 1.1.1 in Example 1.1, except that: ferment for 4 days to obtain the Paecilomyces cicadae fermentation broth; put the fermentation broth into a centrifuge, with a rotation speed of 4800 rpm for 30 min. After centrifugation, pour out the supernatant, take out the mycelia at the bottom, dry them to constant weight in an oven at 40 °C, pulverize them and pass through an 80-mesh sieve to make mycelia powder.

[0050] 1.2.3 Preparation of Panax notoginseng-Paecilomyces cicadae mycelia: The same as 1.1.2 in Example 1.1, except that: ferment for 4 days; take out after fermentation is completed, put it into a centrifuge, with a rotation speed of 4800 rmp for 30 min. After centrifugation is completed, pour out the supernatant, take out the mycelia at the bottom, dry them to constant weight in an oven at 40 °C, pulverize them with a pulverizer and pass through an 80-mesh sieve to make mycelia powder.

[0051] 1.2.4 Preparation of water extract: Weigh the dry powders of Paecilomyces cicadae mycelia and Panax notoginseng-Paecilomyces cicadae mycelia respectively, add distilled water and mix them according to a mass ratio of 1:10, stir evenly and then put them into a constant temperature water bath at 70 °C for extraction for 2 h. After taking out, centrifuge at 4000 rpm for 10 min, take the supernatant, transfer it to a vacuum freeze dryer for freeze-drying treatment after cooling, and pulverize and grind the freeze-dried product to obtain the freeze-dried powder of the water extract of the lysate of Panax notoginseng-Paecilomyces cicadae (hereinafter referred to as SQ-CH) and the freeze-dried powder of the water extract of the lysate of Paecilomyces cicadae (hereinafter referred to as TD-CH) respectively.

[0052] Example 2 Determination of the Content of Active Substances in the Aqueous Extract (SQ-CH) of the Lysate of Panax notoginseng - Paecilomyces cicadae

[0053] 2.1 Determination of total sugar content by phenol-sulfuric acid method

[0054] Preparation of standard solution: Dissolve 10 mg of D-glucose powder in 10 mL of water to obtain a 1 mg / mL glucose solution, and dilute it with water to 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0 mg / mL for standard curve determination. Reagent preparation: Dissolve 600 μL of phenol solution in 9.4 mL of water to obtain a phenol solution. Preparation of test solution: Dilute the sample so that the measurement result is within the range of the standard curve to prepare the test solution. Experimental procedure: Sequentially add 200 μL of the sample, 100 μL of the phenol solution, and 400 μL of concentrated sulfuric acid solution into a 1.5 mL centrifuge tube, mix well, and place in a boiling water bath for 30 min. Set three groups of samples: The standard curve group is the diluted glucose solution; the sample group is the sample diluted to an appropriate concentration; the blank control group is distilled water. Pipette 200 μL into a 96-well plate, zero with the blank well, measure the absorbance at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and set three replicates for each sample. Draw the standard curve, calculate the content according to the standard curve formula, take the average of three parallel data, and the results are shown in Table 4. The total sugar standard curve is y = 3.2997x + 0.107, R 2 = 0.9988.

[0055] 2.2 Determination of reducing sugar

[0056] Use the Beijing Bairuiji Total Sugar and Reducing Sugar Detection Kit (DNS Microplate Method) to detect the content of reducing sugar. Preparation of standard solution: Dissolve 10 mg of D-glucose powder in 10 mL of water to obtain a 1 mg / mL glucose solution, and dilute it with water to 1, 0.8, 0.6, 0.4, 0.2, 0 mg / mL for standard curve determination. Preparation of test solution: Dilute the sample so that the measurement result is within the range of the standard curve to prepare the test solution. Experimental procedure: Take a 1.5 mL centrifuge tube, set the blank well, standard well, and measurement well according to Table 1. The solutions should be added in sequence, and care should be taken to avoid generating bubbles and mix well carefully. Set three groups of samples: The standard curve group is the diluted glucose solution; the sample group is the sample diluted to an appropriate concentration; the blank control group is distilled water. Sequentially pipette 200 μL and transfer it to the corresponding 96-well plate, zero with the blank well, measure the absorbance at 540 nm using an ELISA reader, and set three replicates for each sample. The results are shown in Table 4.

[0057] Table 1

[0058]

[0059] 2.3 Determination of protein content

[0060] The protein content was detected using the BCA Protein Assay Kit from BRIGHT. Preparation of the standard solution: The 10 mg / mL protein standard was diluted to 1000 μg / mL with PBS and then further diluted into solutions with concentration gradients of 500, 250, 125, 62.5, and 31.2 μg / mL for the determination of the standard curve. Preparation of the reagent: Prepare the BCA protein quantitative detection working solution according to the instructions in the kit. Preparation of the sample solution to be measured: Dilute the sample so that the measurement result is within the range of the standard curve to obtain the sample solution to be measured. Experimental procedure: Use a 96-well plate, add the samples sequentially according to Table 2 below, mix well, incubate at 37 °C for 1 hour, measure the absorbance at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and set three replicates for each sample. Plot the standard curve, calculate the content according to the standard curve formula, take the average of the three parallel data, and the results are shown in Table 4. The protein standard curve is y = 0.0006x + 0.1441, R 2 = 0.997.

[0061] Table 2

[0062] Additive (μL) Blank Well Standard Sample Well BCA Protein Quantification Detection Working Solution 200 200 200 PBS 20 — — Standard — 20 — Test Solution — — 20

[0063] 2.4 Determination of β-glucan content

[0064] The content was detected using the β-glucan content assay kit from Suzhou Grees Biotech Co., Ltd. Preparation of the standard solution: Dilute 1 mg of the barley flour (containing 4.1% glucan, moisture content 10.9%) standard to 0.5 mg / mL with PBS. Preparation of the reagent: ① Take 200 μL of the sample solution into an EP tube, add 640 μL of Reagent II, mix well, and incubate in a 95 °C water bath for 3 min (mix once every 1 min), then cool to 50 °C after taking out. ② Add 40 μL of Reagent III, vortex and mix well, then incubate in a constant temperature shaking incubator at 50 °C and 200 rpm for 60 min. ③ Take out and add 1 mL of Reagent IV, mix well, let it stand for 5 - 10 min to cool to room temperature, centrifuge at 1000 g at room temperature for 10 min, and take the supernatant for use. Preparation of the sample solution to be measured: Dilute the sample so that the measurement result is within the range of the standard curve to obtain the sample solution to be measured. Experimental procedure: Use a 96-well plate, add the reagents sequentially according to Table 3, mix well, incubate at 50 °C for 20 min, read the absorbance at 510 nm, set three replicates for each sample, and the results are shown in Table 4.

[0065] Table 3

[0066]

[0067] Calculation: β-glucan content (mg / g dry weight) = (C 标准 × V1) × ΔA ÷ (A 标准品 - A 空白管 ) ÷ (V1 ÷ V × W) × 0.9 × D = 0.846 × ΔA ÷ (A标准品 -A 空白管 ) ÷ W × D, where C 标准 - Standard product concentration, 0.5 mg / mL; V - Volume of sample supernatant, 1.88 mL; V1 - Volume of sample taken during measurement, 0.024 mL; D - Self-dilution factor, 1 if not diluted; W - Dry weight of sample, g; 0.9 - Dehydration conversion factor for glucose to β-glucan.

[0068] 2.5 Determination of uronic acid content:

[0069] Preparation of standard product solution: Dissolve 10 mg of D-glucose powder in 10 ml of water to obtain a 1 mg / mL glucose solution, and dilute it with water to 0.8, 0.6, 0.4, 0.2, 0.1, 0 mg / ml for standard curve determination. Preparation of test solution: Dilute the sample so that the measurement result is within the standard curve range to obtain the test solution. Reagent preparation: (1) Borax-sulfuric acid solution: 0.025 mol / L. Weigh 4.7673 g of Na2B4O7·10H2O and add it to 1 L of concentrated sulfuric acid. Cover and shake irregularly until completely dissolved. (2) Carbazole-ethanol solution: 0.125%. Accurately weigh 0.1254 g of carbazole and dissolve it in 100 g of anhydrous ethanol, and store it in a refrigerator at 4°C in the dark. Experimental steps: Take a 5 mL test tube, add the sample solution, cool it to about 4°C in an ice bath, slowly add 1.25 mL of borax-sulfuric acid solution, stopper it, heat it in a boiling water bath for 15 min (shake it once in the middle), quickly cool it with an ice-water bath, add 50 μL of carbazole anhydrous ethanol solution, shake well, heat it in a boiling water bath for 20 min, shake it once in the middle, and then quickly cool it to room temperature with an ice-water bath. Set up three groups of samples: The standard curve group is the diluted glucose solution; the sample group is the sample diluted to an appropriate concentration; the blank control group is distilled water. Pipette 200 μL into a 96-well plate, zero with the blank well, measure the absorbance at 530 nm with an enzyme-linked immunosorbent assay reader, and set three replicates for each sample. Calculation: Draw a standard curve, calculate the content according to the standard curve formula, take the average of three parallel data, and the results are shown in Table 4. The standard curve of uronic acid is y = 0.8055x + 0.1349, R 2 = 0.9991.

[0070] Table 4

[0071]

[0072] It can be observed from Table 4 that in SQ-CH, the contents of total sugar, reducing sugar, and protein are not very high. Instead, the contents of uronic acid and β-glucan are relatively high, which are significantly better than the water extract of Paecilomyces cicadae lysate without adding Panax notoginseng (TD-CH).

[0073] Example 3 Free Radical Scavenging Ability Test of the Aqueous Extract (SQ-CH) of the Lysate of Panax notoginseng - Paecilomyces cicadae

[0074] Reagent preparation: Dissolve DPPH in absolute ethanol to prepare a DPPH solution with a concentration of 0.2 mM. Prepare it freshly before use and store it in the dark at 0 - 4 °C. Preparation of the test solution: Before measurement, the sample can be diluted to 4, 2, 1, 0.5, 0.25 mg / mL. Experimental steps: (1) Sample group A 测 : Mix equal volumes of the test solution and the 0.2 mM DPPH solution; (2) Blank group A 空 : Mix equal volumes of the test solution and absolute ethanol; (3) Control group A 对 : Mix equal volumes of absolute ethanol and the 0.2 mM DPPH solution. After reacting in the dark for 30 min, pipette 200 μL and add it to a 96-well plate, and measure the absorbance at 517 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Set three replicates for each sample. Calculation: Use the formula DPPH radical scavenging rate = (A 空 + A 对 - A 测 ) / A 空 * 100% to calculate the DPPH radical scavenging rate of the sample. Take the average of three parallel data, and the results are as Figure 1 shown.

[0075] As Figure 1 shown, the DPPH radical scavenging abilities of SQ-CH and TD-CH are concentration-dependent. As the concentration increases, the scavenging ability gradually enhances. At a concentration of 1 mg / mL, the DPPH radical scavenging rate of SQ-CH is significantly higher than that of TD-CH.

[0076] Example 4 Cell Experiment

[0077] 4.1 Cell culture: Human immortalized keratinocytes (HaCaT cells) and human fibroblasts (HSF cells) are cultured in MEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and DMEM medium supplemented with 15% fetal bovine serum and 1% penicillin-streptomycin, respectively. The cells are cultured in an environment of 37 °C and 5% CO2. When the cell confluence reaches 80%, digest with 0.05% trypsin-EDTA and passage at a volume ratio of 1:3.

[0078] 4.2 Detection of cell viability by CCK-8 method: The CCK-8 method is used to detect the cytotoxicity of SQ-CH and TD-CH on HaCaT and HSF. The cells are seeded at a density of 1.5×10 4Inoculate at a density of cells / well in a 96-well plate and culture for 24 h. Discard the original medium and add different concentrations of SQ-CH and TD-CH test solutions diluted with serum-free DMEM medium. After culturing for 24 h, gently wash the cells twice with phosphate buffered saline (PBS), add 100 μL of serum-free DMEM to each well, and then add 10 μL of CCK-8 solution. Measure the absorbance at 450 nm after culturing for 1 h.

[0079] The results are as Figure 2 shown. Among them, Figure A and Figure B show the effects of SQ-CH and TD-CH on the viability of HaCaT cells, respectively, and Figure C and Figure D show the effects of SQ-CH and TD-CH on the viability of HSF cells, respectively. In the figure, ***: p < 0.001, indicating extremely significant decrease compared with the blank group; **: p < 0.01; *: p < 0.05; n.s.: indicating no significance compared with the blank group.

[0080] See Figure 2 Figure A in. When the concentrations of the SQ-CH solution are 2 and 4 mg / mL, respectively, compared with the blank control group, the viability of HaCaT cells decreases extremely significantly, inhibiting the proliferation of HaCaT cells. When the concentration is 0.5 mg / mL, there is no obvious effect on the viability of HaCaT cells. When the concentration is below 0.5 mg / mL, the viability of HaCaT cells increases. Among them, the SQ-CH solutions with concentrations of 0.25 and 0.13 mg / mL make the viability of HaCaT cells increase extremely significantly, promoting the proliferation of HaCaT cells. When the concentration of the SQ-CH solution is 1 mg / mL, the survival rate of HaCaT cells is 83.79%, which is suitable for subsequent experiments.

[0081] See Figure 2 Figure B in. When the concentrations of the TD-CH solution are 1, 2, and 4 mg / mL, respectively, compared with the blank control group, the viability of HaCaT cells decreases extremely significantly, inhibiting the proliferation of HaCaT cells. When the concentration of the TD-CH solution is between 0.03 and 0.25 mg / mL, there is no obvious effect on the viability of HaCaT cells. When the concentration of the TD-CH solution is 0.13 mg / mL, it promotes the proliferation of HaCaT cells. When the concentration of the TD-CH solution is 0.5 mg / mL, the survival rate of HaCaT cells is 83.32%, which is suitable for subsequent experiments.

[0082] See Figure 2In Figure C, when the concentrations of the SQ-CH solution were 1, 2, and 4 mg / mL respectively, compared with the blank control group, the viability of HSF cells decreased extremely significantly, inhibiting the proliferation of HSF cells; when the concentrations of the SQ-CH solution were 0.08 and 0.13 mg / mL, there was no obvious effect on the viability of HSF cells; when the concentrations of the SQ-CH solution were 0.25 and 0.5 mg / mL, the viability of HSF cells increased significantly, promoting the proliferation of HSF cells; when the concentration of the SQ-CH solution was 0.04 mg / mL, the survival rate of HSF cells was 89.02%, which was suitable for subsequent experiments.

[0083] See Figure 2 In Figure D, when the concentrations of the TD-CH solution were 1 and 2 mg / mL respectively, compared with the blank control group, the viability of HSF cells decreased extremely significantly, inhibiting the proliferation of HSF cells; when the concentrations of the TD-CH solution were 0.08, 0.13, 0.25, and 0.5 mg / mL, there was no obvious effect on the viability of HSF cells; when the concentrations of the TD-CH solution were 0.02 and 0.04 mg / mL, the survival rates of HSF cells were 84.77% and 84.00% respectively, both of which were suitable for subsequent experiments. The TD-CH solution with a concentration of 0.02 mg / mL with a smaller standard deviation was selected as the sample for subsequent experiments.

[0084] Example 5 Anti-inflammatory Efficacy Determination

[0085] HaCaT cells in the logarithmic growth phase were inoculated into 6-well plates at a density of 3×10 5 cells / well, and 2 mL of complete DMEM medium was added to each well. They were divided into a blank group, a model group, and an experimental group. When cultured to 80% confluence, 2 mL of the SQ-CH solution with a concentration of 1 mg / mL and the TD-CH solution with a concentration of 0.5 mg / mL were added to the experimental group, and 2 mL of serum-free DMEM was added to the blank group and the model group. Then they were continued to be cultured in an incubator for 24 h. The supernatant was removed, and the cells were carefully rinsed twice with PBS, and 1 mL of PBS was added to cover the cells. The model group and the experimental group were irradiated with UVB using an ultraviolet cross-linking agent at 30 mJ / cm 2Cells were stimulated with UVB at a certain dose. The blank group was not irradiated. After irradiation, the cells were washed twice with PBS, and 2 mL of serum-free DMEM was added to each well. After continued culturing for 24 h, the supernatant was collected and centrifuged at 500 g for 10 min for protein content determination. After collecting the supernatant, 200 μL of lysis buffer (prepared by mixing IP lysis buffer and PMSF at a ratio of 100:1) was added to each well. After the cells were completely lysed, the cells were carefully scraped off with a cell scraper and the cell lysate was collected, centrifuged at 10,000 r / min for 10 min, and the supernatant was collected for standby. This was the supernatant of the cell lysate. The contents of TNF-α and IL-1β in the cell culture supernatant of HaCaT cells in the blank group, model group, and sample group were measured using the ELISA kits for human tumor necrosis factor-α (TNF-α) and human interleukin 1β (IL-1β) from BiYunTian. For details, refer to the instruction manual. The protein content in the supernatant of the cell lysate of HaCaT cells in the blank group, model group, and sample group was measured using the BCA protein quantification kit from Bio-Rad for protein labeling and quantification.

[0086] The results are as Figure 3 shown. Figure 3 Figures A and B in it respectively show the effects of SQ-CH and TD-CH on the contents of TNF-α and IL-1β in UVB-induced photo-damaged HaCaT cells (UVB was selected at 30 mJ / cm 2 ). In the figure, , p < 0.001, indicates an extremely significant difference between the Model group and the Control group; **, p < 0.01, indicates a very significant difference between the sample group and the Model group; ***, p < 0.001, indicates an extremely significant difference. It can be seen that compared with the blank control group, the contents of TNF-α and IL-1β in the culture medium of HaCaT cells in the model group increased extremely significantly. Compared with the model group, after treatment with 1 mg / mL of SQ-CH solution, the content of TNF-α in HaCaT cells decreased significantly, and the content of IL-1β decreased extremely significantly; after treatment with 0.5 mg / mL of TD-CH solution, the contents of TNF-α and IL-1β in HaCaT cells both decreased significantly. The results show that both SQ-CH and TD-CH can reduce the contents of the pro-inflammatory cytokines TNF-α and IL-1β in HaCaT cells caused by UVB irradiation, and significantly reduce the inflammatory level in HaCaT cells, indicating that both have relatively strong anti-inflammatory effects. At the same time, the ability of TD-CH to reduce the pro-inflammatory cytokine TNF-α is stronger than that of SQ-CH; the ability of SQ-CH to reduce the pro-inflammatory cytokine IL-1β is significantly stronger than that of TD-CH.

[0087] Example 6 Antioxidant Efficacy Determination

[0088] HSF cells in the logarithmic growth phase were seeded into 6-well plates at a density of 3×10 5 cells / well. 2 mL of complete DMEM medium was added to each well, and the cells were divided into a blank group, a model group, and an experimental group. When the confluence reached 80%, 2 mL of SQ-CH solution at a concentration of 0.04 mg / mL and TD-CH solution at a concentration of 0.02 mg / mL were added to the experimental group, while 2 mL of serum-free DMEM was added to the blank group and the model group. Then the cells were continued to be cultured in an incubator for 24 h. The supernatant was removed, and the cells were carefully rinsed twice with PBS, and then 1 mL of PBS was added to cover the cells. The model group and the experimental group were irradiated with UVA using an ultraviolet cross-linker, and the cells were stimulated with UVA at a dose of 12 J / cm 2 . The blank group was not irradiated. After irradiation, the cells were washed twice with PBS, and 2 mL of serum-free DMEM was added to each well. After continued culture for 24 h, the supernatant was collected and centrifuged at 500 g for 10 min for protein content determination. After collecting the supernatant, 200 μL of lysis buffer (prepared by mixing IP lysis buffer and PMSF at a ratio of 100:1) was added to each well. After the cells were completely lysed, the cells were carefully scraped off with a cell scraper and the cell lysate was collected. Then it was centrifuged at 10000 r / min for 10 min, and the supernatant was collected for standby. This was the supernatant of the cell lysate. The contents of MMP-1 and MMP-9 in the cell culture supernatant of HSF cells in the blank group, the model group, and the sample group were determined using the Biyuntian human matrix metalloproteinase 1 (MMP-1) enzyme-linked immunosorbent assay kit and the human matrix metalloproteinase 9 (MMP-9) enzyme-linked immunosorbent assay kit. For details, please refer to the instruction manual. The protein content in the supernatant of the cell lysate of HSF cells in the blank group, the model group, and the sample group was determined using the Beryex BCA protein quantification detection kit for protein labeling and quantification.

[0089] The results are as Figure 4 shown. Figure 4 The effects of SQ-CH and TD-CH on the contents of MMP-1 and MMP-9 in UVA-induced photo-damaged HSF cells are respectively shown (UVA was selected at 12 J / cm 2), In the figure, , p < 0.001 indicates an extremely significant difference between the Model group and the Control group; **, p < 0.01 indicates a very significant difference between the Sample group and the Model group; ***, p < 0.001 indicates an extremely significant difference. It can be seen that compared with the blank control group, the contents of MMP-1 and MMP-9 in the culture medium of HSF cells in the model group increased extremely significantly. Compared with the model group, after treatment with 0.04 mg / mL of SQ-CH solution, the content of MMP-1 in HSF cells decreased extremely significantly, and the content of MMP-9 decreased significantly; after treatment with 0.02 mg / mL of TD-CH solution, the contents of MMP-1 and MMP-9 in HSF cells decreased extremely significantly. The results show that both SQ-CH and TD-CH can reduce the contents of MMP-1 and MMP-9 in HSF cells caused by UVA irradiation, and prevent the destruction of the extracellular matrix structure of HSF cells, indicating that both have relatively strong antioxidant effects. At the same time, the ability of TD-CH to reduce the content of MMP-1 is slightly stronger than that of SQ-CH; the ability of SQ-CH to reduce MMP-9 is slightly stronger than that of TD-CH.

[0090] Example 7 Metabolomics Analysis

[0091] 7.1 Principal Component Analysis (PCA)

[0092] To preliminarily analyze the differences in the metabolite accumulation of the lytic products of Paecilomyces cicadae added with Panax notoginseng, principal component analysis (PCA) was performed on the metabolites of the lytic products. From the degree of dispersion of each sample in the principal component analysis graph, we can see the similarity of the metabolic components between each sample. The closer the distance between samples with higher similarity, and vice versa. In PCA, R under positive and negative ion modes 2 were 0.545 and 0.592 respectively, both greater than 0.5, indicating that the PCA model established by the experiment was stable and could be used for metabolic difference analysis. Figure 5 Figure A in it is the PCA score graph of SQ-CH and TD-CH in the positive ion mode, and Figure B is the PCA score graph of SQ-CH and TD-CH in the negative ion mode. As Figure 5 shown, the abscissa PC1 and the ordinate PC2 in the figure represent the scores of the first and second principal components respectively. The samples of the SQ-CH group and the TD-CH group were in a discrete state, and there were significant differences between the two groups, indicating that there were significant differences in the metabolic components between the two groups, providing a premise for the subsequent screening of differential metabolites.

[0093] 7.2 Orthogonal Partial Least Squares-Discriminant Analysis

[0094] Although the principal component analysis method can effectively extract the main information, it is insensitive to variables with low correlations. The partial least squares discriminant analysis can solve this problem. PLS-DA (Partial Least Squares Discriminant Analysis), namely partial least squares discriminant analysis, is a supervised discriminant analysis method and a multivariate statistical analysis method. Figure 6 Figure A in Figure 6 is the PLS-DA score plot of SQ-CH and TD-CH in positive ion mode, and Figure B is the PLS-DA score plot of SQ-CH and TD-CH in negative ion mode. The ordinate represents the within-group difference, and the abscissa represents the between-group difference. It can be seen from the figure that there are significant differences between the two groups, and differential components can be screened subsequently. And the Q 2 (model predictability) values are 0.548 and 0.547 respectively, indicating that the model is stable and reliable. Such results are consistent with those obtained by PCA analysis.

[0095] After permutation test, by randomly changing the permutation order of the classification variable Y and establishing corresponding PLS-DA models multiple times (n = 200) to obtain the R2 and Q2 values of the random models, it plays an important role in avoiding overfitting of the test model and evaluating the statistical significance of the model. Figure 7 Figure A and Figure B in Figure 7 respectively show the results of the permutation test of the PLS-DA model of SQ-CH and TD-CH in positive and negative ion modes. The abscissa in the figure represents the permutation retention degree of the permutation test, and the ordinate represents the value of R2 (blue dots) or Q2 (red triangles). The two dashed lines respectively represent the regression lines of R2 and Q2. It can be seen from the figure that the intercept of the Q2 regression line with the Y-axis is less than 0.05, indicating that the model is robust and reliable and has not overfitted.

[0096] 7.3 Differential component analysis

[0097] 7.3.1 Screening of differential components

[0098] According to Figure 8 the Venn diagram shown, there are 174 unique metabolites in the SQ-CH group and 18 unique metabolites in the TD-CH group.

[0099] Taking VIP>1 and P<0.05 as the criteria to screen differential metabolites and performing visual analysis to draw a volcano plot. Figure 9Figure A in it is the volcano plot of differential components of SQ-CH and TD-CH in positive ion mode, and Figure B is the volcano plot of differential components of SQ-CH and TD-CH in negative ion mode, respectively. Each point represents a component. The red ones are significantly up-regulated components, the blue ones are significantly down-regulated components, and the components with non-significant differences are gray. The size of each point represents the VIP value of the component. As can be seen from Figure 9 Figure A in it, the number of ion peaks that all meet the differential screening conditions in the positive mode is 1,256. As can be seen from Figure 9 Figure B in it, the number of ion peaks that all meet the differential screening conditions in the negative mode is 1,623. The screened differential metabolites were identified, and a total of 457 differential metabolites were screened out, including 326 significantly up-regulated and 131 significantly down-regulated. The differential components mainly include amino acids, sugars, hormones and mediators, nucleic acids, lipids and phospholipids, etc., as shown in Table 5-1 and Table 5-2 specifically. A total of 9 kinds were screened out among all differential amino acid components, including the contents of L-serine, L-asparagine, levodopa, L-tyrosine, L-tryptophan, L-valine, L-glutamic acid, argininosuccinic acid and L-aspartic acid increased significantly after fermentation with Panax notoginseng powder added. These up-regulated amino acids (such as L-serine, L-asparagine, L-tryptophan, etc.) usually participate in multiple metabolic pathways and jointly participate in amino acid biosynthesis, cyanoamino acid metabolism and aminoacyl-tRNA biosynthesis. L-serine, L-valine, L-glutamic acid, argininosuccinic acid and L-aspartic acid participate in the biosynthesis of various antibiotics, and levodopa and L-tyrosine participate in the tyrosine metabolism process. A total of 9 kinds were screened out among all differential sugar components, among which four monosaccharides (glucosamine, 2-deoxy-D-glucose, D-sorbitol and 2-deoxy-D-ribose) were significantly up-regulated and participated in amino sugar and nucleotide sugar metabolism, fructose and galactose metabolism; five oligosaccharides (cellobiose, trehalose, turanose, sucrose and xylobiose) were significantly down-regulated and participated in the ABC transport pathway.

[0100] Table 5-1 Significantly up-regulated differential metabolites

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] Table 5-2 Significantly down-regulated differential metabolites

[0110]

[0111]

[0112]

[0113]

[0114] Note: Metabolite: Name of differential metabolite; Name: Chinese name of the substance; Formula: Molecular formula; Pvalue: p-value obtained from student's t-test; VIP: Variable projection importance obtained from PLS-DA model.

[0115] 7.3.2 Cluster heat map of differential metabolites

[0116] Cluster analysis was performed on the top 50 significantly differential metabolites screened. The cluster heat map can intuitively show the up-regulation or down-regulation of metabolites in each group according to color changes. The results are as Figure 10 shown. Horizontally are different sample groups, on the left are the sub-clusters of metabolites, and on the right are the metabolite names. The 50 differential metabolites are divided into ten sub-clusters. It can be seen from the figure that the sub-clusters with the largest proportions are sub-cluster 8, sub-cluster 1, and sub-cluster 2 in turn. Sub-cluster 8 contains sugars such as maltotetraose, trehalose, raffinose, lactulose, galactinol, and 5-hydroxymethyl-2-furaldehyde; sub-cluster 2 contains various amino acids such as L-valine, L-glutamic acid, L-aspartic acid, L-tryptophan, and ginsenoside Rg3. Sub-cluster 1 contains various metabolites, including asparagine, serine, alanine, β-D-ribosylnicotinate, ustusorin C, 4-(4-methylcyclohexyl)-4-oxobutanoic acid, 4-α-methyl-5-α-cholest-7-en-3-β-ol, D-sorbitol, and succinic acid, etc., which are involved in many metabolic pathways. In the SQ-CH group, among the top 50 differential metabolites, the metabolites significantly up-regulated are those in sub-cluster 1 and sub-cluster 2, and the sugars in sub-cluster 8 all decreased significantly.

[0117] 7.3.3 Correlation analysis of differential metabolites

[0118] The purpose of differential component correlation analysis is to study the consistency of the changing trends between components. Conducting differential metabolite correlation analysis is conducive to further understanding the mutual regulatory relationship between metabolites during the change of biological state. Metabolite correlation often reveals the synergy of changes between metabolites: if the change trend is the same as that of a certain type of metabolite, it is a positive correlation; if the change trend is opposite to that of a certain type of metabolite, it is a negative correlation. This experiment calculates the Pearson correlation coefficient between components to analyze the correlation between components. Figure 11 As shown, blue represents two components with a correlation coefficient less than 0, which means that the two components are negatively correlated, and the closer the coefficient is to -1, the stronger the linear relationship between the two; conversely, red represents two components with a positive correlation, and the closer the coefficient is to 1, the stronger the relationship between the two; white represents no significant correlation between the two. Figure 11 From the overall layout of the graph, the graph shows obvious zoning characteristics, with clear red and blue boundaries, indicating that there are strong and weak groupings of associations between metabolites. This grouping pattern may correspond to specific biological functional modules or metabolic pathways. For example, areas with dense red may represent synergistic metabolic networks, while areas with concentrated blue may imply antagonistic or oppositely regulated metabolic relationships.

[0119] 7.4 Differential component metabolite pathway analysis

[0120] 7.4.1 KEGG metabolic pathway annotation and type analysis

[0121] Pathway enrichment analysis of differential metabolites was performed using the KEGG database, such as Figure 12 As shown, a total of 11 metabolic pathways were detected, which were mainly divided into three categories: metabolism (9 pathways), genetic information processing pathways (1 pathway) and environmental information processing pathways (1 pathway).

[0122] 7.4.2 KEGG pathway enrichment analysis

[0123] Through the KEGG database, the metabolites measured in the experiment are mapped to specific metabolic pathways to identify which metabolic pathways have changed significantly between the experimental group and the control group. Through enrichment analysis, it is possible to determine which metabolic pathways are significantly enriched under different experimental conditions, and changes in these pathways may be a sign of the key biological phenomena of the study. In this study, 70 pathways were enriched, of which 11 were downregulated and 59 were upregulated. By calculating the impact factor and p-value of the enrichment analysis, the pathways containing differential components are screened, and the top 20 pathways are screened according to the p-value from small to large, and a bubble chart and a KEGG pathway difference abundance score chart are made. Figure 13Among them, the abscissa is the enrichment rate. The larger the enrichment rate, the more significant the enrichment level of the differential components in this pathway and the more reference value it has. The ordinate is the names of the top 20 metabolic pathways, and the bubble color is the p-value. The smaller the p-value, the more significant the enrichment. The bubble size is the number of differential components in this pathway. The larger the bubble, the more metabolites there are. The KEGG pathway enrichment analysis results show that adding Panax notoginseng has a significant impact on multiple metabolic pathways during the fermentation process of Paecilomyces cicadae. The main metabolic pathways enriched with differential metabolites include amino acid metabolism (alanine, aspartate and glutamate metabolism, histidine metabolism, phenylalanine metabolism, arginine biosynthesis, glycine, serine and threonine metabolism, tyrosine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis), other amino acid metabolism (cyanoamino acid metabolism), translation (aminoacyl-tRNA biosynthesis), cofactor and vitamin metabolism (niacin and nicotinamide metabolism), lipid metabolism (glycerophospholipid metabolism), energy metabolism (sulfur metabolism), nucleotide metabolism (pyrimidine metabolism, purine metabolism), carbohydrate metabolism (galactose metabolism), biosynthesis of various plant secondary metabolites, membrane transport (ABC transporter pathway) and biosynthesis of other various secondary metabolites.

[0124] Figure 14 The abscissa represents the differential abundance score (DA Score), and the ordinate represents the names of the KEGG metabolic pathways. The DA Score reflects the overall changes of all metabolites in the metabolic pathway. A score of 1 indicates that the expression trends of all annotated differential metabolites in this pathway are up-regulated, and -1 indicates that the expression trends of all annotated differential metabolites in this pathway are down-regulated. The length of the line segment represents the absolute value of the DA Score. The size of the dot represents the number of differential metabolites annotated in this pathway. The larger the dot, the more differential metabolites there are in this pathway. If the dots are distributed on the right side of the central axis and the line segment is longer, it indicates that the overall expression of this pathway tends to be up-regulated; if the dots are distributed on the left side of the central axis and the line segment is longer, it indicates that the overall expression of this pathway tends to be down-regulated. Therefore, it can be seen that the expression trends of the differential metabolites annotated in the galactose metabolism and ABC transporter pathways are overall down-regulated, and the rest are overall up-regulated.

[0125] In the pathways related to amino acid metabolism, L-glutamic acid, L-aspartic acid, and succinic acid are three frequently occurring metabolites. Glutamic acid is a precursor for the synthesis of glutathione, which is an important antioxidant that can scavenge free radicals in the body and reduce oxidative stress. As an amino acid, L-aspartic acid can reduce oxidative damage by regulating the synthesis of antioxidants such as glutathione. Succinic acid participates in mitochondrial energy metabolism and, under certain conditions, activates antioxidant enzymes through the Nrf2 pathway to inhibit oxidative stress. We speculate that certain components in Panax notoginseng powder can regulate amino acid metabolism, modulate the processes of amino acid synthesis and transport, thereby leading to the accumulation of aspartic acid, glutamic acid, and succinic acid, and enhancing the intracellular antioxidant capacity.

[0126] Amino acid synthesis and metabolism are not only the basis for cell growth but also closely related to the stress response of cells. The significantly upregulated differential metabolites in the SQ-CH group (L-serine, L-asparagine, L-tyrosine, L-tryptophan, L-valine, L-glutamic acid, and L-aspartic acid) are involved in genetic information processing, environmental information processing, and metabolic pathways, indicating that they not only participate in metabolic processes but may also play a key role in maintaining the adaptability of mycelial growth, regulating gene expression, and responding to environmental changes. Analysis of three metabolic pathways shows that in addition to ginsenosides and notoginsenosides being the main metabolites in the SQ-CH group, amino acids are also one of their sources, further indicating that SQ-CH has certain activity and pharmacodynamic properties.

[0127] In the galactose metabolism pathway of the SQ-CH group, mannotriose, raffinose, sucrose, and galactinol are downregulated. Similarly, among the differential metabolites involved in the ABC transporter pathway, cellobiose, maltotriose, trehalose, raffinose, sucrose, xylobiose, taurine, and glycerol 4-phosphate are significantly downregulated, while L-serine, L-valine, L-glutamic acid, L-aspartic acid, 2'-deoxyuridine, and D-sorbitol are significantly upregulated. It is speculated that this may be due to the adaptation to environmental stress, manifested as reprogramming of the carbohydrate metabolism pathway.

[0128] Combined with the evaluation of the content of active ingredients, anti-inflammatory efficacy, and antioxidant efficacy, it can be speculated that in the intracellular products of Paecilomyces cicadae fermented with Panax notoginseng, the contents of total sugar, reducing sugar, and protein are relatively low, which is related to the ABC transporter pathway analyzed by non-targeted metabolomics. The carbohydrate substances in the differential metabolites of the SQ-CH group are significantly downregulated, and the amino acid substances are significantly upregulated. It is speculated that this may be because the mycelium may face external environmental pressures (such as nutrient limitation, pH change, oxygen deficiency, etc.), inducing adjustments in the internal metabolic pathways of the mycelium, reducing carbohydrate metabolism, and turning to amino acids as the main energy source or the direction of regulating metabolism. Consequently, the amino acid metabolites increase, enhancing the intracellular antioxidant and anti-inflammatory capabilities, which has also been verified in cell experiments.

[0129] Finally, it should also be noted that, in the present disclosure, if any, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or device comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the presence of additional identical elements in the process, method, article or device comprising the element.

[0130] Although the present disclosure has been disclosed above through the description of specific embodiments of the present disclosure, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present disclosure within the spirit and scope of the appended claims. These modifications, improvements or equivalents should also be considered to be included within the scope claimed by the present disclosure.

Claims

1. A method for preparing a water extract of Panax notoginseng-Paecilomyces cicadae lysate, characterized in that: include: Preparation steps of Panax notoginseng-Paecilomyces cicadae mycelium: mixing Panax notoginseng root powder and water evenly, sterilizing, and obtaining Panax notoginseng liquid culture medium; inoculating Paecilomyces cicadae seed liquid into the Panax notoginseng liquid culture medium for fermentation; after the fermentation is completed, centrifuging to obtain mycelium, drying, and crushing to obtain mycelium powder; The water extract preparation step is as follows: the mycelium powder is subjected to water extraction treatment in a constant temperature water bath, and then the supernatant is obtained by centrifugation, and after cooling, the supernatant is subjected to freeze-drying, crushing and grinding to obtain freeze-dried powder of the water extract of Panax notoginseng-Paecilomyces cicadae lysate.

2. The method for preparing the Panax notoginseng-Paecilomyces cicadae lysate aqueous extract according to claim 1, characterized in that: In the step of preparing the Panax notoginseng-Paecilomyces cicadae mycelium, the Panax notoginseng root powder in the Panax notoginseng liquid culture medium accounts for 0.5-2wt%, preferably 1wt%; and / or, The sterilization conditions are 118-125° C., 25-35 min, preferably 121° C., 30 min.

3. The method for preparing the Panax notoginseng-Paecilomyces cicadae lysate aqueous extract according to claim 1 or 2, characterized in that: In the step of preparing the mycelium of Panax notoginseng-Paecilomyces cicadae, the Paecilomyces cicadae is Cordyceps cicadae CH2347, and its deposit number is CGMCC No.40399; Preferably, the volume of the mycelium of Paecilomyces cicadae in the Paecilomyces cicadae seed liquid accounts for 50-80% of the entire liquid culture medium; More preferably, the volume ratio of the Paecilomyces cicadae seed solution to the Panax notoginseng liquid culture medium is 3-7%, and further preferably 5%; Furthermore, the preparation method of the Paecilomyces cicadae seed liquid comprises: (1) inoculating the cicada fungus CH2347 grown in a PDA plate into a potato glucose water culture medium, with an inoculation ratio of 2 to 3 solid bacteria with a diameter of about 0.5 cm: 300 mL of culture medium, and culturing at 28° C. and 180 rpm for 3 days; (2) homogenizing the culture of step (1), and continuing to inoculate it as a seed liquid into a new potato glucose water culture medium, with an inoculation volume ratio of 1:15, and culturing at 28° C. and 180 rpm for 4 days to obtain the Paecilomyces cicadae seed liquid.

4. The method for preparing the aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate according to any one of claims 1 to 3, characterized in that: In the step of preparing the Panax notoginseng-Cicada pseudopaecilomyces mycelium, the fermentation conditions are fermentation at 26-30°C for 4-6 days, preferably fermentation at 28°C for 4-5 days; more preferably, the fermentation is carried out on a shaker, and the shaker speed is 160-200rpm, further preferably 180rpm.

5. The method for preparing the aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate according to any one of claims 1 to 4, characterized in that: In the step of preparing the mycelium of Panax notoginseng-Paecilomyces cicadae, the centrifugation condition is 4500-5000 rpm, 25-35 min; preferably 4800 rpm, 30 min; and / or, The drying temperature is 35-45°C.

6. The method for preparing the aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate according to any one of claims 1 to 5, characterized in that: In the step of preparing Panax notoginseng-Paecilomyces cicadae mycelium, the particle size of the mycelium powder is less than 200 μm, preferably less than 177 μm.

7. The method for preparing the aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate according to any one of claims 1 to 6, characterized in that: In the step of preparing the water extract, the mass ratio of the mycelium powder to distilled water is 0.5-2:10, preferably 1:10; and / or, The constant temperature water bath conditions are 60-80°C and extraction time 1.5-2.5h; preferably, extraction is carried out in a constant temperature water bath at 70°C for 2h.

8. The method for preparing the Panax notoginseng-Paecilomyces cicadae lysate aqueous extract according to claim 1, characterized in that: In the water extract preparation step, the centrifugal conditions are 3500-5000 r / min, 8-15 min; preferably 4000 r / min, 10 min.

9. An aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate, prepared according to the preparation method according to any one of claims 1 to 8.

10. An application of an aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate in the preparation of external skin preparations and hair preparations; preferably, the aqueous extract of Panax notoginseng-Paecilomyces cicadae lysate is used as at least one of the antioxidant active ingredients and anti-inflammatory active ingredients in the external skin preparation; preferably, the external skin preparation is at least one of a skin care lotion, a lotion, a face cream and a face mask.

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