Phellinus igniarius polyphenol extract as well as preparation method and application thereof

Abstract

The invention provides a phellinus igniarius polyphenol extract as well as a preparation method and application thereof, and belongs to the technical field of phellinus igniarius extraction. The preparation method of the phellinus igniarius polyphenol extract comprises the following steps: mixing phellinus igniarius powder with an ethanol solution, carrying out ultrasonic treatment, standing overnight, and centrifuging to take supernatant, so as to obtain a phellinus igniarius alcohol extract; drying the phellinus igniarius alcohol extract to obtain a phellinus igniarius polyphenol extract; the volume fraction of ethanol in the ethanol solution is 55%-65%; the mass volume ratio of the phellinus igniarius powder to the ethanol solution is 1g: (45-55) mL; the phellinus igniarius polyphenol extract mainly contains active ingredients such as osmundone, and lipid metabolism disorder, oxidative stress and neurotoxicity caused by microplastics can be improved. The phellinus igniarius polyphenol extract is used for the research of relieving the toxicity of the microplastics, and a theoretical basis is provided for the application of the phellinus igniarius polyphenol extract in drug development.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of Sanghuang extraction, and particularly relates to a Sanghuang polyphenol extract and a preparation method and application thereof. BACKGROUND

[0002] "Microplastics" refers to plastic particles with a diameter of less than 5 mm. These particles are derived from daily chemicals (such as toothpaste, cleaning and care products), textiles and automobile tires, and can be enriched through the food chain: starting from phytoplankton, shellfish and other bottom organisms, as the trophic level increases, eventually, through biological enrichment, microplastics will enter the human food chain, posing a potential threat to human health, and the harm mainly lies in inducing oxidative stress, neurotoxicity and lipid metabolism disorder.

[0003] Sanghuang (Sanghuangporus sanghuang) is a perennial edible fungus with medicinal value. The main active components of Sanghuang are polysaccharides, flavonoids and terpenoids, which endow it with multiple pharmacological activities such as anti-tumor, antioxidant and anti-inflammatory. Due to its significant biological activity and low cytotoxicity characteristics, Sanghuang has become an important research object for new drug development. At present, there is no report on the related research of Sanghuang polyphenol extract on neurotoxicity. SUMMARY

[0004] The application aims to provide a Sanghuang polyphenol extract and a preparation method and application thereof. The Sanghuang polyphenol extract provided by the application can relieve neurotoxicity, and provides a theoretical basis for the application of the Sanghuang polyphenol extract in drug development.

[0005] The application provides a preparation method of a Sanghuang polyphenol extract, which comprises: mixing Sanghuang powder and an ethanol solution, then performing ultrasonic treatment, standing overnight, centrifuging to obtain supernatant, and obtaining a Sanghuang alcohol extract;

[0006] The volume fraction of ethanol in the ethanol solution is 55% to 65%; and the mass-volume ratio of the Sanghuang powder to the ethanol solution is 1g: 45 to 55mL.

[0007] Preferably, the temperature of the ultrasonic treatment is 25 to 35℃, and the time of the ultrasonic treatment is 90 to 120min.

[0008] The application further provides a Sanghuang polyphenol extract prepared by the preparation method.

[0009] Preferably, the active ingredients of the Phellinus baumii polyphenol extract include one or more of aspidin, pinic acid, abietic acid, palmitic acid, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid, 10-hydroxydecanoic acid, phellopterin J, protocatechuic aldehyde, azelaic acid, 9-hydroxy-16-oxohexadecanoic acid, phosphatidyl ethanolamine, L-malic acid, phellopterin C, 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid, caffeic acid, lycorine, L-aspartic acid, 4-(4-hydroxyphenyl)-3-buten-2-one, resorcinol, 16-hydroxyhexadecanoic acid, and phellopterin B.

[0010] The application also provides use of the Phellinus baumii polyphenol extract in the preparation of a product for alleviating toxicity induced by microplastic exposure.

[0011] Preferably, the microplastics are polystyrene; and the Phellinus baumii polyphenol extract alleviates toxicity of nematodes induced by microplastic exposure.

[0012] Preferably, the toxicity is one or more of lipid metabolism disorder, oxidative stress, and neurotoxicity; and the nematodes are Caenorhabditis elegans.

[0013] Preferably, the neurotoxicity includes neurodegenerative disease.

[0014] Preferably, the neurodegenerative disease is Parkinson's disease and / or Huntington's disease.

[0015] Preferably, the product includes a pharmaceutical product.

[0016] The application also provides a method for alleviating toxicity induced by microplastic exposure using a Phellinus baumii polyphenol extract, the method being used for non-therapeutic purposes, and including the following steps:

[0017] Feeding the Phellinus baumii polyphenol extract to nematodes to alleviate toxicity induced by microplastic exposure.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The application provides a preparation method of a Phellinus baumii polyphenol extract, the Phellinus baumii polyphenol extract obtained by the preparation method mainly containing active ingredients such as aspidin and having strong antioxidant capacity, and being capable of improving lipid metabolism disorder, oxidative stress, and neurotoxicity caused by microplastics.

[0020] The polystyrene microplastic exposure induced neurotoxicity can be relieved by the Phellinus baumii polyphenol extract, the number of head swings and body bends of the Caenorhabditis elegans caused by the polystyrene (PS) microplastic toxicity can be significantly reduced, the accumulation level of ROS and lipofuscin in the Caenorhabditis elegans is reduced, the learning and memory ability and chemotaxis behavior of the Caenorhabditis elegans are improved, and the neurotoxicity suffered by the neurodegenerative disease model Caenorhabditis elegans is reduced. The research of the Phellinus baumii polyphenol extract for relieving PS toxicity provides a theoretical basis for the application of the Phellinus baumii polyphenol extract in drug development. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a mass spectrum of the substance contained in the Phellinus baumii alcohol extract in Example 1.

[0022] Figure 2 It is the in vitro antioxidant activity detection result of the Phellinus baumii polyphenol extract in Example 2, wherein A is the ABTS scavenging capacity determination curve of the Phellinus baumii polyphenol extract, B is the ABTS scavenging capacity determination curve of ascorbic acid, C is the DPPH scavenging capacity determination curve of the Phellinus baumii polyphenol extract, D is the DPPH scavenging capacity determination curve of ascorbic acid, E is the iron ion reduction / antioxidant capacity determination result of the Phellinus baumii polyphenol extract, and F is the iron ion reduction / antioxidant capacity determination result of ascorbic acid.

[0023] Figure 3 It is the influence result of different treatments on the body length and body width of the Caenorhabditis elegans in Example 3, wherein A is the influence result of different treatments on the body length of the Caenorhabditis elegans, and B is the influence result of different treatments on the body width of the Caenorhabditis elegans; control represents a control group fed only with OP50 bacterial liquid, PS represents a PS treatment group fed with OP50 bacterial liquid containing 1 mg / L of PS with a particle size of 0.5 μm, and PS+SH represents a PS+SH treatment group fed with OP50 bacterial liquid containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of the Phellinus baumii polyphenol extract; the same lowercase letters indicate no significant difference, and different lowercase letters indicate significant difference at the level of p<0.05.

[0024] Figure 4 It is the influence result of different treatments on the head swing and body bend of the Caenorhabditis elegans in Example 4, wherein A is the influence result of different treatments on the body bend number of the Caenorhabditis elegans, and B is the influence result of different treatments on the head swing number of the Caenorhabditis elegans; control represents a control group fed only with OP50 bacterial liquid, PS represents a PS treatment group fed with OP50 bacterial liquid containing 1 mg / L of PS with a particle size of 0.5 μm, and PS+SH represents a PS+SH treatment group fed with OP50 bacterial liquid containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of the Phellinus baumii polyphenol extract; different lowercase letters indicate significant difference at the level of p<0.05.

[0025] Figure 5 The results of the influence of different treatments on the learning and memory and chemotaxis of nematodes in Example 5, wherein A is the test method design of learning and memory and chemotaxis, B is the influence of different treatments on the learning and memory of nematodes, C is the influence of different treatments on the chemotaxis of nematodes; Control represents the control group fed only with OP50 bacterial solution, PS represents the PS treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm, PS+SH represents the PS+SH treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract; different lowercase letters indicate significant differences at the level of p<0.05.

[0026] Figure 6 The results of the influence of different treatments on the oxidative stress of nematodes in Example 6, wherein A is the fluorescence intensity result chart of the control-treated nematodes under fluorescence microscopy, B is the fluorescence intensity result chart of the PS-treated nematodes under fluorescence microscopy, C is the fluorescence intensity result chart of the PS+SH group-treated nematodes under fluorescence microscopy, D is the fluorescence intensity result of the influence of different treatment groups on oxidative stress analyzed by Fiji / ImageJ software; Control represents the control group fed only with OP50 bacterial solution, PS represents the PS treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm, PS+SH represents the PS+SH treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract; different lowercase letters indicate significant differences at the level of p<0.05.

[0027] Figure 7 The results of the influence of different treatments on the lipofuscin of nematodes in Example 7, wherein A is the fluorescence intensity result chart of the control-treated nematodes under fluorescence microscopy, B is the fluorescence intensity result chart of the PS-treated nematodes under fluorescence microscopy, C is the fluorescence intensity result chart of the PS+SH group-treated nematodes under fluorescence microscopy, D is the fluorescence intensity result of the influence of different treatment groups on lipofuscin analyzed by Fiji / ImageJ software; Control represents the control group fed only with OP50 bacterial solution, PS represents the PS treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm, PS+SH represents the PS+SH treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract; different lowercase letters indicate significant differences at the level of p<0.05.

[0028] Figure 8Figure 8 shows the results of the effects of different treatments on NL5901 nematodes in Example 8, wherein A is a fluorescence intensity result diagram of NL5901 nematodes under a fluorescence microscope under a control treatment, B is a fluorescence intensity result diagram of NL5901 nematodes under a PS treatment, C is a fluorescence intensity result diagram of NL5901 nematodes under a PS+SH group treatment, and D is the number of α-syn protein aggregates in NL5901 nematodes under different treatment groups; control represents a control group fed only with OP50 bacterial solution, PS represents a PS treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm, and PS+SH represents a PS+SH treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of mulberry polysaccharide extract; different lowercase letters represent significant differences at the level of p<0.05.

[0029] Figure 9 Figure 9 shows the results of the effects of different treatments on AM141 nematodes in Example 9, wherein A is a fluorescence intensity result diagram of AM141 nematodes under a fluorescence microscope under a control treatment, B is a fluorescence intensity result diagram of AM141 nematodes under a PS treatment, C is a fluorescence intensity result diagram of AM141 nematodes under a PS+SH group treatment, and D is the number of polyQ40 focuses in AM141 nematodes under different treatment groups; control represents a control group fed only with OP50 bacterial solution, PS represents a PS treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm, and PS+SH represents a PS+SH treatment group fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of mulberry polysaccharide extract; different lowercase letters represent significant differences at the level of p<0.05. DETAILED DESCRIPTION

[0030] The present application provides a preparation method of mulberry polysaccharide extract, comprising: mixing mulberry powder with an ethanol solution, and then performing ultrasonic treatment, standing overnight, centrifuging to obtain supernatant, and obtaining a mulberry alcohol extract.

[0031] In the preparation method of the mulberry polysaccharide extract, the volume fraction of ethanol in the ethanol solution is preferably 55% to 65%, further preferably 56% to 64%, more preferably any one of 57%, 58%, 59%, 60%, 61%, 62%, or 63%, and most preferably 60%; the mass-volume ratio of the mulberry powder to the ethanol solution is preferably 1 g: 45 to 55 mL, further preferably 1 g: 48 to 52 mL, more preferably any one of 1 g: 49 mL, 1 g: 50 mL, or 1 g: 51 mL, and most preferably 1 g: 50 mL.

[0032] In the preparation method of the Phellinus baumii polyphenol extract, the temperature of the ultrasonic treatment is preferably 25-35℃, further preferably 26-34℃, further preferably any one of 27, 28, 29, 30, 31, 32 or 33, and most preferably 30℃; and the time of the ultrasonic treatment is preferably 90-120min, further preferably 90-110min, further preferably 90-100min or 100-110min, and most preferably 90min.

[0033] As an optional embodiment, 1g of Phellinus baumii is added into 50mL of ethanol solution with a concentration of 60%, stirred uniformly, and then ultrasonically treated at 30℃ for 90min to obtain a mixed solution; the mixed solution is taken out, and left to stand at 4℃ overnight; the mixed solution left to stand overnight is centrifuged at 4000rpm for 30min, and the supernatant is taken; the supernatant is filtered, concentrated by rotary evaporation, and freeze-dried to obtain the Phellinus baumii polyphenol extract. In the present application, the conditions of the filtration, concentration by rotary evaporation and freeze-drying are not limited, and the conventional methods in the art can be used.

[0034] The present application also provides a Phellinus baumii polyphenol extract prepared by the preparation method; preferably, the active ingredients of the Phellinus baumii polyphenol extract include one or more of aspidin, pinic acid, abietic acid, palmitic acid, 9(S), 10(S), 11(R)-trihydroxy-12(Z), 15(Z)-octadecadienoic acid, 10-hydroxydecanoic acid, phellopterin J, protocatechuic aldehyde, azelaic acid, 9-hydroxy-16-oxohexadecanoic acid, phosphatidyl ethanolamine, L-malic acid, phellopterin C, 11-hydroperoxy-12, 13-epoxy-9-octadecenoic acid, caffeic acid, lacciferin, L-aspartic acid, 4-(4-hydroxyphenyl)-3-buten-2-one, resorcinol, 16-hydroxyhexadecanoic acid and phellopterin B, further the active ingredients of the Phellinus baumii polyphenol extract include aspidin, pinic acid, abietic acid, palmitic acid, 9(S), 10(S), 11(R)-trihydroxy-12(Z), 15(Z)-octadecadienoic acid, 10-hydroxydecanoic acid, phellopterin J, protocatechuic aldehyde, azelaic acid, 9-hydroxy-16-oxohexadecanoic acid, phosphatidyl ethanolamine, L-malic acid, phellopterin C, 11-hydroperoxy-12, 13-epoxy-9-octadecenoic acid, caffeic acid, lacciferin, L-aspartic acid, 4-(4-hydroxyphenyl)-3-buten-2-one, resorcinol, 16-hydroxyhexadecanoic acid and phellopterin B.

[0035] The present application also provides the use of the Phellinus baumii polyphenol extract in the preparation of a product for relieving the toxicity induced by microplastic exposure. In the use of the present application, the microplastic is preferably polystyrene.

[0036] In the application, the toxicity is one or more of lipid metabolism disorder, oxidative stress and neurotoxicity; and the nematode is Caenorhabditis elegans. The neurotoxicity includes neurodegenerative diseases, preferably Parkinson's disease and / or Huntington's disease.

[0037] The Phellinus baumii polyphenol extract can alleviate the toxicity of microplastic exposure on nematodes.

[0038] In the application, the product includes a pharmaceutical product.

[0039] The application further provides a method for alleviating the toxicity induced by microplastic exposure by using the Phellinus baumii polyphenol extract, which is used for non-therapeutic purposes, and includes the following steps:

[0040] The Phellinus baumii polyphenol extract is fed to the nematodes to alleviate the toxicity induced by microplastic exposure.

[0041] In the method, the nematode is Caenorhabditis elegans, and the Caenorhabditis elegans is fed with standard food Escherichia coli and the Phellinus baumii polyphenol extract under the condition of microplastic exposure; and the addition amount of the Phellinus baumii polyphenol extract is 100 mg / L based on the standard food Escherichia coli OP50 liquid.

[0042] In the application, the Phellinus baumii polyphenol extract can alleviate the decrease in the number of head swings and body bends of nematodes caused by the toxicity of polystyrene (PS) microplastics, reduce the accumulation level of ROS and lipofuscin in the nematodes, improve the learning and memory ability and chemotaxis behavior of the nematodes, and reduce the neurotoxicity of the model nematodes of neurodegenerative diseases, which indicates that the Phellinus baumii polyphenol extract can alleviate the toxicological effect of microplastics on Caenorhabditis elegans.

[0043] In the application, the Phellinus baumii powder can be obtained from a commercial channel, and the specific source is not limited. In the following examples, the Phellinus baumii powder is purchased from Shupin Ci Food Store in Bozhou High-tech Industrial Development Zone, with a production license number of SC11464052101006 and a production date of October 25, 2024.

[0044] In the application, the OP50 liquid refers to Escherichia coli OP50 liquid, which is the standard food Escherichia coli fed to nematodes, and is not specifically limited.

[0045] In the following examples, PS represents polystyrene (PS) microplastics with a particle size of 0.5 μm, which is purchased from Tianjin Junyi Jia Science and Technology Co., Ltd. with a product number of 6-1-0050.

[0046] In the following examples, the N2 nematode is from the C. elegans Genetics Center, University of Minnesota, the NL5901 nematode is from the C. elegans Genetics Center, University of Minnesota, and is used as a model nematode for Parkinson's disease (PD) research, and the AM141 nematode is an AM141 transgenic nematode model from the C. elegans Genetics Center, University of Minnesota, which mimics the protein aggregation pathology of Huntington's disease (HD) by muscle-specific expression of Q40::YFP fusion protein.

[0047] In the following examples, the NGM plate refers to a nematode growth medium plate, and NGM is a medium that can be obtained from commercial channels, and the source thereof is not specifically limited.

[0048] In the following examples, the test methods used are conventional test methods unless otherwise specified, and the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] The technical solutions in the present application will be described clearly and completely below in combination with the examples in the present application.

[0050] Example 1

[0051] Preparation of the Phellinus baumii polyphenol extract

[0052] (1) 30 g of Phellinus baumii powder was weighed out, 1.5 L of 60% ethanol was added at a ratio of 1:50 (m / v), and the mixture was stirred uniformly and then ultrasonically treated at 30°C for 90 min to obtain a mixed solution.

[0053] (2) The mixed solution was taken out and left to stand at 4°C overnight.

[0054] (3) The mixed solution after standing overnight was centrifuged at 4000 rpm for 30 min, and the supernatant was taken out.

[0055] (4) The supernatant was suction-filtered, the filtrate after suction-filtering was concentrated by rotary evaporation, and freeze-drying was performed to obtain the Phellinus baumii polyphenol extract.

[0056] When detecting, the Phellinus baumii polyphenol extract was dissolved in 60% ethanol solution at a ratio of 1:200 (m / v) to obtain a Phellinus baumii alcohol extract, and the types of substances contained in the Phellinus baumii alcohol extract were detected by a rapid high performance liquid chromatography-quadrupole time-of-flight (Q-TOF) mass spectrometer, and the detection results are shown in Table 1. Figure 1 and Table 1.

[0057] Table 1 Types of substances contained in the Phellinus baumii alcohol extract detected by Q-TOF

[0058]

[0059]

[0060] Example 2

[0061] Evaluation of the antioxidant activity of the extract of Phellinus baumii in vitro

[0062] ABTS radical scavenging capacity of the extract of Phellinus baumii was determined.

[0063] ABTS mother liquor was prepared by mixing 10 mL of 7 mmol / L ABTS solution with 179 μL of 140 mmol / L potassium persulfate solution, incubating at room temperature in the dark for 14 h, and diluting 20 times to obtain the working solution; 500 μL of the working solution was mixed with an equal volume of a series of concentration gradient solutions of the extract of Phellinus baumii (dissolved in deionized water, with the final concentration of the extract being 20, 40, 60, 80, and 100 μg / mL, respectively), ascorbic acid solution (positive control), or deionized water (blank), and reacted at room temperature in the dark for 6 min, followed by centrifugation at 7000 g for 10 min, and determination of the absorbance value (A734) of the supernatant at 734 nm.

[0064] ABTS radical scavenging capacity was calculated using formula (1), and the determination was repeated three times, and a curve was plotted as shown in FIG. A and FIG. B. Figure 2 Figure 2

[0065] ABTS radical scavenging rate (%) = [1-A734(sample) / A734(blank)] x 100 formula (1)

[0066] DPPH radical scavenging capacity of the extract of Phellinus baumii was determined. DPPH was dissolved in 80% ethanol to prepare a working solution with a concentration of 0.2 mmol / L; 500 μL of the working solution was mixed with an equal volume of a series of concentration gradient solutions of the extract of Phellinus baumii (dissolved in deionized water, with the final concentration of the extract being 20, 40, 60, 80, and 100 μg / mL, respectively), ascorbic acid solution (positive control), or deionized water (blank), and reacted at room temperature in the dark for 30 min, followed by centrifugation at 7000 g for 10 min, and determination of the absorbance value (A517) of the supernatant at 517 nm.

[0067] ABTS radical scavenging capacity was calculated using formula (2), and the determination was repeated three times, and a curve was plotted as shown in FIG. C and FIG. D. Figure 2 Figure 2

[0068] DPPH radical scavenging rate (%) = [1-A517(sample) / A517(blank)] x 100 formula (2)

[0069] ​​​​The iron reduction / antioxidant activity (FRAP) of mulberry linterinary polyphenol extract was determined. A FRAP reagent was prepared by mixing 300 mmol / L acetate buffer (pH 3.5), 10 mmol / L LTPTZ solution, and 20 mmol / L FeCl3 solution at a ratio of 10:1:1 (v / v / v). 500 μL of this reagent was mixed with equal volumes of a series of concentration gradient mulberry linterinary polyphenol extract solutions (dissolved in deionized water, with final concentrations of 10, 20, 30, 40, and 50 μg / mL), ascorbic acid solution (positive control), or deionized water (blank). The mixture was incubated at 37°C in the dark for 30 min, then centrifuged at 7000 g for 10 min. The absorbance of the supernatant at 593 nm (A593) was measured.

[0070] Ascorbic acid equivalent is used to express the reducing ability of the mulberry linterinary phenol extract solution, and a curve is drawn, such as Figure 2 E and Figure 2 As shown in F.

[0071] Depend on Figure 2 From the A to F values, it can be seen that the in vitro antioxidant capacity of mulberry ignia polyphenol extract gradually increases with the increase of concentration. The half inhibitory concentration (IC50) of mulberry ignia on the scavenging rate of ABTS and DPPH free radicals was obtained by linear fitting. 50 ) were 28.23 μg / mL and 58.17 μg / mL, and the IC 50 The value was 28.37 μg / mL. Although the in vitro antioxidant capacity of the mulberry ignia polyphenol extract was slightly weaker than that of ascorbic acid at the same mass concentration, the mulberry ignia polyphenol extract still showed strong antioxidant activity.

[0072] Example 3

[0073] Nematode body length and width measurement experiment

[0074] (1) Experimental grouping: The experiment was divided into 3 groups:

[0075] Control group: fed with the same dose of OP50 bacterial solution as the PS group or PS+SH group;

[0076] PS group: fed with the same dose of OP50 bacterial solution containing 1 mg / L PS with a particle size of 0.5 μm as the PS+SH group;

[0077] PS+SH group: fed with OP50 bacterial solution containing 1 mg / L PS with a particle size of 0.5 μm and 100 mg / L mulberry linterus polyphenol extract.

[0078] (2) Body length and width test method: N2 nematodes were cultured in NGM plates containing reagents of each test group at 20°C for 60 h starting from eggs.

[0079] After 60 h, each group of nematodes was rinsed with M9 solution in a centrifuge tube and placed in a 50°C water bath for 5 min to kill and straighten the nematodes. The body length and width of the nematodes were measured under a stereomicroscope at the same magnification. 20-25 nematodes were measured in each experimental group.

[0080] In this example, the effect of mulberry linterinary polyphenol extract on the body length and width of nematodes can be found in Figure 3 .

[0081] Depend on Figure 3 As shown in Figures A and B, compared with the OP50 control group, PS intake increased the body width of the nematodes. This may be because PS interferes with lipid metabolism in the nematodes, causing fat deposition in the nematodes and resulting in "obesity." However, intervention with mulberry linterinary polyphenol extract significantly ameliorated the increase in body width caused by PS intake (p < 0.05).

[0082] Example 4

[0083] Nematode head swing and body bending measurement experiment

[0084] The number of head swings and body bends represents the nematode's locomotion ability.

[0085] (1) Experimental grouping: The experiment was divided into 3 groups:

[0086] Control group: fed with the same dose of OP50 bacterial solution as the PS group or PS+SH group;

[0087] PS group: fed with the same dose of OP50 bacterial solution containing 1 mg / L PS with a particle size of 0.5 μm as the PS+SH group;

[0088] PS+SH group: fed with OP50 bacterial solution containing 1 mg / L PS with a particle size of 0.5 μm and 100 mg / L mulberry linterus polyphenol extract.

[0089] (2) Head bobbing and body bending test method: N2 nematodes were cultured in NGM plates containing reagents of each test group at 20°C for 60 h from the time of eggs.

[0090] After 60 hours, the nematodes were transferred to NGM culture dishes without OP50 and dark-acclimated at 20°C for 30 minutes. The number of head oscillations within 20 seconds and body bends within 30 seconds were counted under a stereomicroscope. Each side-to-side head oscillation was counted as a head oscillation, while a vertical sinusoidal movement in the forward direction was counted as a body oscillation. Twenty nematodes were counted for each group.

[0091] In this example, the effect of mulberry linterinary polyphenol extract on the body bending and head swing of nematodes can be found in Figure 4 A and Figure 4 B.

[0092] Compared with the OP50 control group, PS intake significantly reduced the number of head swings and body bends of nematodes (p<0.05), greatly damaging the movement ability of nematodes, while the movement ability of nematodes was significantly improved under the treatment of Phellinus baumii polyphenol extract (p<0.05).

[0093] Example 5

[0094] Nematode learning and memory, chemotaxis assay

[0095] The chemotaxis of nematodes to sodium chloride and the chemical odor agent decanal reflects the learning and memory ability and sensory perception ability. Since the movement ability and chemical tendency of nematodes are directly regulated by the nervous system, abnormal changes in these behaviors can reflect the neurotoxicity induced by PS microplastic exposure.

[0096] (1) Experimental grouping: The experiment was divided into 3 groups:

[0097] Control group: fed with the same dose of OP50 bacterial liquid as the PS group or the PS+SH group;

[0098] PS group: fed with the same dose of OP50 bacterial liquid containing 1 mg / L of PS with a particle size of 0.5 μm as the PS+SH group;

[0099] PS+SH group: fed with OP50 bacterial liquid containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract.

[0100] (2) Test method of learning and memory and chemotaxis: N2 nematodes were cultured in NGM plates containing reagents of each test group for 60 h at 20°C from eggs.

[0101] A 9 cm diameter sodium chloride-free NGM culture dish was used, and N point and C point (A in the figure) 4 cm apart were marked on the back of the culture dish. Figure 5 A 1 cm diameter NGM agar block containing sodium chloride was placed at the N point, and the C point was not treated, and a sodium chloride concentration gradient was formed by standing overnight at 4°C. The agar block was removed 15 min before the experiment. The nematodes treated for 60 h were washed with M9 buffer and placed at S point 3 cm from the center point of N-C line. After 2 h, the number of nematodes in the area with N, C point as the center and 1.5 cm as the radius was counted, the chemotaxis index (CI) was calculated, and 3 repeated tests were performed.

[0102] The calculation method of chemotaxis index (CI) is:

[0103] CI = (N-C) / (N+C) (N: the number of nematodes in the range of 1.5 cm radius from the center of the sodium chloride gradient, C: the number of nematodes in the range of 1.5 cm radius from the control point).

[0104] Chemotaxis experiment of chemical odor used decanal 10 times diluted. After the nematodes treated for 60h were washed with M9 buffer, they were placed in the center of a 9cm diameter NGM culture dish, 3cm from the center at symmetrical points Figure 5 5μL of 10% decanal solution and 5μL of absolute ethanol were added respectively, and the seal film was sealed and treated at 20°C for 2h in the dark.

[0105] The number of nematodes on the decanal side and the ethanol side was counted, and three repeats were performed.

[0106] The chemotaxis index (CI) was calculated as:

[0107] CI=(G-E) / (G+E) (G: number of nematodes on the decanal side, E: number of nematodes on the ethanol side).

[0108] The effects of the extract of Phellinus baumii on the learning and memory and chemotaxis of nematodes in this example are shown in B and C of Figure 5 The nematodes have a tendency for sodium chloride in a culture dish without sodium chloride, which may be related to taste and osmoreceptor. The results show that PS weakens the chemotaxis of nematodes to sodium chloride, and the intervention of the extract of Phellinus baumii significantly improves this abnormality (p<0.05). Under normal circumstances, Caenorhabditis elegans is sensitive to certain odors, and decanal has a repelling effect on nematodes, which is an olfactory perception behavior. PS can significantly weaken the chemotaxis of decanal to nematodes, and the intervention of the extract of Phellinus baumii can significantly alleviate this weakening effect (p<0.05), indicating that exposure to 1mg / L of PS with a particle size of 0.5μm can affect the sensory perception behavior of nematodes, and the extract of Phellinus baumii has a certain alleviating effect on the neurological behavior abnormalities caused by PS.

[0109] Example 6

[0110] Nematode ROS determination experiment

[0111] The toxic mechanism of microplastics involves oxidative stress, and the characteristic biomarker is reactive oxygen species (ROS). ROS can induce inflammatory response, immune regulation abnormalities and nerve function damage by attacking cell membrane and organelle structure, and is closely related to the occurrence and development of chronic diseases.

[0112] (1) Experimental grouping: the experiment was divided into 3 groups:

[0113] Control group: fed with the same dose of OP50 bacterial solution as the PS group or the PS+SH group;

[0114] PS group: fed with the same dose of OP50 bacterial solution containing 1mg / L of PS with a particle size of 0.5μm as the PS+SH group;

[0115] PS+SH group: fed with OP50 bacteria solution containing 1 mg / L PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract.

[0116] (2) ROS testing method: N2 nematodes were cultured in NGM plates containing reagents of each test group for 60 h at 20°C from eggs.

[0117] After 60 h, the nematodes were collected, washed with M9 buffer, mixed with 10 μM DCF dye, and incubated in the dark for 2 h. After washing with PBS for 3 times, the nematodes were anesthetized with 5 μM levamisole, observed under a fluorescence microscope, and the fluorescence intensity was analyzed using Fiji / ImageJ software, with at least 10 nematodes analyzed in each group.

[0118] The effect of Phellinus baumii polyphenol extract on oxidative stress in nematodes in this example is shown in Figure 6 A-D.

[0119] The fluorescence intensity of the PS group was significantly higher than that of the control group and the treatment group. After PS treatment, a large amount of ROS was produced in the nematodes. Compared with the PS group, the ROS level of the PS+SH group was significantly decreased (p<0.05), indicating that the intervention of Phellinus baumii polyphenol extract effectively limited the oxidative stress response induced by PS.

[0120] Example 7

[0121] Nematode lipofuscin determination experiment

[0122] Oxidative stress can induce aging-related metabolic changes, and its characteristic performance is the accumulation of lipofuscin in intestinal cells. As a lysosomal degradation product, this autofluorescent material is a biomarker of cell aging. Its formation mechanism involves free radical-mediated lipid peroxidation and lysosomal dysfunction.

[0123] (1) Experimental grouping: the experiment was divided into 3 groups:

[0124] Control group: fed with the same dose of OP50 bacteria solution as the PS group or the PS+SH group;

[0125] PS group: fed with the same dose of OP50 bacteria solution containing 1 mg / L PS with a particle size of 0.5 μm as the PS+SH group;

[0126] PS+SH group: fed with OP50 bacteria solution containing 1 mg / L PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract.

[0127] (2) Lipofuscin testing method: N2 nematodes were cultured in NGM plates containing reagents of each test group for 60 h at 20°C from eggs.

[0128] After 60h, the nematodes were anesthetized with a 0.5M levamisole hydrochloride solution, and a proper amount of nematodes were randomly selected and placed on a glass slide, observed under a fluorescence microscope with ultraviolet light, and pictures were collected. The fluorescence intensity was analyzed using Fiji / ImageJ software.

[0129] The effect of the Phellinus baumii polyphenol extract on nematode lipofuscin in this example is shown in Figs. A-D. Compared with the control group, the fluorescence intensity of the nematodes after PS treatment was significantly enhanced within the same treatment time, and the PS increased the lipofuscin content in the nematodes, while the lipofuscin level in the nematodes after treatment with the Phellinus baumii polyphenol extract decreased significantly, and there was a significant difference in the fluorescence intensity between the nematodes in the PS treatment group (p<0.05). Therefore, the Phellinus baumii polyphenol extract can alleviate the oxidative stress induced by PS. Figure 7

[0130] Example 8

[0131] Fluorescence analysis experiment of PD model nematodes

[0132] Parkinson's disease (PD) is a typical age-related neurodegenerative disease, which is characterized by motor dysfunction and cognitive decline. NL5901 nematodes, as a PD research model, express α-synuclein (α-syn) in muscle cells, and abnormal aggregation of this protein can induce PD characteristic pathological changes, including mitochondrial dysfunction, increased reactive oxygen species (ROS) levels, and neuroinflammatory responses. By quantitatively analyzing the fluorescence intensity of α-syn, the degree of neurotoxicity of PD model nematodes can be evaluated.

[0133] (1) Experimental grouping: The experiment was divided into 3 groups:

[0134] Control group: fed with OP50 bacterial solution;

[0135] PS group: fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm;

[0136] PS+SH group: fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of Phellinus baumii polyphenol extract.

[0137] (2) Method for determining the fluorescence intensity of α-syn: NL5901 nematodes were cultured in NGM plates containing reagents of each test group at 20°C for 60h from eggs.

[0138] After 60h, the treated nematodes were collected and anesthetized, and the fluorescence intensity of α-syn::YFP protein was observed under a fluorescence microscope. The specific fluorescence values were analyzed using ImageJ software.

[0139] ​The effect of the mulberry mushroom polyphenol extract on NL5901 nematodes in this example is shown in Figure 8 Compared with the control group, the fluorescence intensity in the body wall muscle of the PS treatment group increased significantly (p<0.05), indicating that the expression of a-syn in the nematodes increased, and PS induced the occurrence of Parkinson's disease in the nematodes. The fluorescent parts of the nematodes were mainly concentrated in the head region, indicating that the damage of ZnO-NPs to the nerves of the nematodes may mainly act on the head. After treatment with the mulberry mushroom polyphenol extract, the a-syn protein aggregation in NL5901 nematodes decreased significantly, indicating that the mulberry mushroom polyphenol extract may have a certain alleviating and protective effect on PS-induced neurodegenerative disease PD.

[0140] Example 9

[0141] Fluorescence analysis experiment of HD model nematodes

[0142] Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal extension of polyglutamine (polyQ) (>36 repeats) due to mutation of the huntingtin gene, and the pathological core is the neurotoxicity mediated by mutant huntingtin protein (mHTT) aggregation. The AM141 transgenic nematode model expresses Q40::YFP fusion protein in muscle-specifically, simulating the abnormal protein aggregation pathological process of HD.

[0143] (1) Experimental grouping: the experiment was divided into 3 groups:

[0144] Control group: fed with OP50 bacterial solution;

[0145] PS group: fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm;

[0146] PS+SH group: fed with OP50 bacterial solution containing 1 mg / L of PS with a particle size of 0.5 μm and 100 mg / L of mulberry mushroom polyphenol extract.

[0147] (2) polyQ40 focusing number determination method: AM141 nematodes were cultured in NGM plates containing reagents of each test group at 20°C from eggs for 60 h.

[0148] After 60 h, the treated nematodes were collected, anesthetized, and observed under a fluorescence microscope for polyQ40 focusing number.

[0149] The effect of the mulberry mushroom polyphenol extract on AM141 nematodes in this example is shown in Figure 9 A~D of Example 8.

[0150] Compared with the untreated control group, PS significantly increased the number of polyQ40::YFP aggregates, while the number of polyQ40::YFP aggregates decreased after treatment with the polyphenol extract of Phellinus baumii, and there was a significant difference between the PS treatment group of nematodes (p<0.05). It is proved that the polyphenol extract of Phellinus baumii may have certain remission and protection effect on PS-induced neurodegenerative disease HD.

[0151] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A method for preparing a mulberry linterinary polyphenol extract, characterized in that: include: The mulberry linter powder was mixed with the ethanol solution and then ultrasonicated, allowed to stand overnight, and the supernatant was collected by centrifugation to obtain the mulberry linter ethanol extract; drying the mulberry ignia alcohol extract to obtain a mulberry ignia polyphenol extract; The volume fraction of ethanol in the ethanol solution is 55% to 65%; the mass volume ratio of the phellinus linteus powder to the ethanol solution is 1g:45 to 55mL.

2. The preparation method according to claim 1, characterized in that The temperature of the ultrasonic treatment is 25-35° C., and the time of the ultrasonic treatment is 90-120 min.

3. A mulberry linterus polyphenol extract, characterized in that The preparation method according to claim 1 or 2 is used for preparation.

4. The mulberry linterinary polyphenol extract according to claim 3, characterized in that The active ingredients of the mulberry polyphenol extract include one or more of porphyrin, pinic acid, rosin acid, palmitic acid, 9(S), 10(S), 11(R)-trihydroxy-12(Z), 15(Z)-octadecadienoic acid, 10-hydroxydecanoic acid, mulberry J, protocatechuic aldehyde, azelaic acid, 9-hydroxy-16-oxohexadecanoic acid, phosphatidylethanolamine, L-malic acid, mulberry C, 11-hydroperoxy-12,13-epoxy-9-octadecenoic acid, caffeic acid, milk tree alkaloid, L-aspartic acid, 4-(4-hydroxyphenyl)-3-butene-2-one, resorcinol, 16-hydroxyhexadecanoic acid and mulberry B.

5. Use of the mulberry ignia polyphenol extract according to claim 3 or 4 in the preparation of products that alleviate toxicity induced by microplastic exposure.

6. The application according to claim 5, characterized in that The microplastic is polystyrene; the mulberry ignia polyphenol extract alleviates the toxicity of microplastic exposure-induced nematodes.

7. The use according to claim 5, characterized in that The toxicity is one or more of lipid metabolism disorder, oxidative stress and neurotoxicity; and the nematode is Caenorhabditis elegans.

8. The application according to claim 7, characterized in that: Such neurotoxicity includes neurodegenerative diseases.

9. The application according to claim 8, characterized in that: The neurodegenerative disease is Parkinson's disease and / or Huntington's disease.

10. A method for alleviating toxicity induced by microplastic exposure using mulberry linterinary polyphenol extract, characterized in that: The method is used for non-therapeutic purposes and comprises the following steps: Feeding nematodes with the mulberry ignia polyphenol extract described in claim 3 or 4 alleviates the toxicity induced by microplastic exposure.

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