Agaric melanin as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological medicines, and particularly relates to auricularia auricula melanin and a preparation method and application thereof, and tests prove that the extracted auricularia auricula melanin can effectively improve the action ability and exploration desire of aged mice and relieve depression behaviors. Besides, the black fungus melanin can also remarkably improve the activity of serum superoxide dismutase and glutathione peroxidase and reduce the content of malondialdehyde, which indicates that the black fungus melanin can effectively relieve oxidative stress injury in vivo by enhancing an antioxidant enzyme system and reducing lipid peroxidation. In addition, the black fungus melanin can reduce hippocampus nerve cell apoptosis in the brain and improve the neurological function; the area of the glomerulus in the kidney can be reduced, glomerular sclerosis is relieved, and the renal function is improved; meanwhile, liver sinus deformation in the liver can be repaired, lipid droplet accumulation and cell necrosis are reduced, liver tissue pathological damage is improved, and the oxidative stress degree is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to auricularia melanin, a preparation method thereof and an application thereof. Background Art

[0003] The essence of aging is the accumulation of multi-dimensional physiological imbalances, and its core mechanisms include oxidative stress, cellular senescence, telomere attrition, and metabolic disorders, etc. Recent studies have revealed that systematic intervention in the common pathways of aging can achieve the synergistic treatment of multiple diseases. For example, metformin reverses the biological age index by 18 years in a primate model by resetting the cellular senescence program; stem cell therapy and traditional Chinese medicine active ingredients (such as auricularia melanin) improve organ and nerve functions by regulating the inflammatory pathway and antioxidant stress. The World Health Organization (WHO) defined aging as a disease in 2018, promoting the transformation of anti-aging research from single-disease treatment to multi-organ collaborative intervention. In addition, the development of precise aging markers (such as DNA methylation clocks) and targeted technologies (such as nano-delivery systems) provides new tools for neuroprotection across the blood-brain barrier and delaying "cliff-like aging". Coping with aging requires the integration of preventive medicine, technological innovation, and social policies. At the medical level, strengthening primary care services (such as general practitioner training) and promoting long-term care insurance can relieve the pressure on medical resources; the application of technology (such as real-time monitoring by wearable devices and AI-assisted diagnosis) improves the management efficiency of chronic diseases. In the field of scientific research, establishing a natural aging population cohort, developing multi-omics biological clock models, and promoting industry-university-research cooperation (such as the research and development of anti-aging vaccines) are the breakthrough directions.

[0004] As a model of medicine and food homology, the anti-aging effect of auricularia has been verified by both traditional medicine and modern science. Traditional Chinese medicine classics "Shennong Ben Cao Jing" and "Ben Cao Gang Mu" both record its effect of "replenishing qi without hunger, lightening the body and strengthening the will", belonging to the lung, spleen, and liver meridians, and laying a theoretical foundation for delaying aging by tonifying qi and blood and regulating the functions of zang-fu organs. Modern research further reveals its core mechanisms:

[0005] Antioxidation and free radical scavenging: Auricularia polysaccharide can significantly enhance the activity of superoxide dismutase (SOD) and reduce the content of lipofuscin (an aging marker). Animal experiments show that it can reduce plasma peroxidized lipids by 30% and scavenge oxidation products such as hydroxyl free radicals, thereby delaying cell damage;

[0006] Multi-organ collaborative protection: The iron content in auricularia is 7 times that of pig liver, which can improve brain oxygen supply and neurotransmitter synthesis, reducing the risk of Alzheimer's disease. Its vitamin K and purine nucleoside components prevent atherosclerosis through an anticoagulant mechanism. Clinical trials show that long-term consumption can reduce the incidence of coronary heart disease by 15%;

[0007] Metabolic Regulation and Anti - inflammation: The glial substance of Auricularia auricula can adsorb heavy metals and metabolic wastes, reducing the burden on the liver and kidneys. Its polysaccharide component can improve insulin sensitivity by activating the AMPK pathway, showing potential for intervening in diabetes - related aging.

[0008] The anti - aging value of Auricularia auricula is not only reflected at the mechanistic level, but also realized through diverse applications in traditional dietary therapy and modern preparation development:

[0009] Traditional Dietary Therapy Formulas:

[0010] Blood Tonifying and Anti - aging: The Auricularia auricula and red date soup can increase the skin water content by 20% and improve iron - deficiency anemia;

[0011] Organ Protection: The Auricularia auricula, Chinese wolfberry and black - boned chicken soup can relieve numbness in hands and feet and pain in the waist and legs by regulating the metabolism of the liver and kidneys, and is especially suitable for people in postoperative rehabilitation;

[0012] Metabolic Optimization: The stir - fried Auricularia auricula with Chinese yam combines dietary fiber and mucoprotein, promoting the balance of intestinal flora and reducing the re - absorption of toxins;

[0013] Modern Preparation Development: Japan has used its extracts in anti - aging health products. Domestic research focuses on nano - encapsulation technology to enhance the ability of Auricularia auricula polysaccharide to cross the blood - brain barrier and develop targeted products for neurodegenerative diseases;

[0014] Safety and Universality: Compared with synthetic anti - aging drugs, Auricularia auricula has a mild nature, with a per - capita daily consumption cost of less than 2 yuan and few taboos (only those with spleen - stomach deficiency - cold need to consume moderately). Its soaking and cooking methods (such as soaking in warm water below 60°C for 2 - 3 hours) have formed a standardized guide to avoid the risk of contamination by Pseudomonas cocovenenans.

[0015] Through the integration of the three dimensions of "mechanism - application - safety", Auricularia auricula provides a scientific and feasible solution for the anti - aging field and becomes an important resource in the healthy aging strategy.

[0016] Existing research on the specific anti - aging components of Auricularia auricula mostly focuses on large - category components such as Auricularia auricula polysaccharide, but lacks the structural analysis and function verification of single highly active anti - aging substances. For example, although Auricularia auricula polysaccharide has been proven to have antioxidant ability, its molecular weight distribution is complex, and the metabolic pathways and action targets of components with different degrees of polymerization in vivo have not been clarified, resulting in the "black - box" feature of anti - aging mechanism research. This directly restricts the industrial development of the anti - aging function of Auricularia auricula. Therefore, it is urgent to establish an extraction technology system for anti - aging substances in Auricularia auricula, and clarify its molecular mechanism of action and clinical application path to provide a new solution for addressing the global aging challenge. Summary of the Invention

[0017] The object of the present invention is to provide a black fungus melanin, its preparation method and application, which not only clarify the efficient extraction method of black fungus melanin, but also clarify its protective effects on the brain, liver and kidney in D-galactose-induced senile model mice, as well as its improvement effect on neurodegenerative behavior.

[0018] The object of the present invention is achieved by the following technical solutions:

[0019] The present invention provides a preparation method of black fungus melanin, comprising the following steps:

[0020] (1) Mix the powder obtained by crushing the fruiting body of Auricularia auricula with NaOH solution, perform ultrasonic extraction, and take the supernatant after suction filtration;

[0021] (2) Adjust the pH of the supernatant, and remove the proteins and carbohydrates therein by constant temperature water bath to obtain the treated supernatant;

[0022] (3) Wash the precipitate obtained by centrifuging the treated supernatant with deionized water until neutral to obtain crude melanin;

[0023] (4) Redissolve the crude melanin in sodium hydroxide solution after washing and centrifuging, adjust the pH value again and then centrifuge, wash the precipitate with deionized water until neutral, and dry it at room temperature to obtain the purified black fungus melanin.

[0024] Further, in step (1), the solid-liquid ratio of the powder to the NaOH solution is 1:35, the concentration of the NaOH solution is 1.25 mol / L, and the ultrasonic extraction is ultrasonic extraction at 60 °C for 60 min.

[0025] Further, in step (2), adjusting the pH of the supernatant is to adjust the pH value of the supernatant to 1.5 with 7M hydrochloric acid, and the constant temperature water bath is a constant temperature water bath at 80 °C for 10 h.

[0026] Further, in step (3), the centrifugation is centrifugation at 4000 rmp / min for 10 min.

[0027] Further, in step (4), the washing and centrifuging is to continuously wash the crude melanin with dichloromethane, ethyl acetate, absolute ethanol and deionized water 3 times and then centrifuge at 7000 rpm / min for 10 min; the concentration of the sodium hydroxide solution is 2M; adjusting the pH value again and then centrifuging is to adjust the pH value to 1.5 with 7M hydrochloric acid and then centrifuge at 7000 rpm / min for 10 min.

[0028] The present invention also provides a black fungus melanin prepared by using the above preparation method, and the black fungus melanin comprises eumelanin containing indole and phenolic polymers.

[0029] The present invention also provides an application of the auricularia melanin as described above in the preparation of a product for preventing and treating anti-aging.

[0030] Furthermore, the anti-aging includes regulating the activities and social behaviors of aging mice, reducing oxidative stress in the body, and improving the functions of the kidney, whole brain, and liver tissues of aging mice.

[0031] Furthermore, the reduction of oxidative stress in the body is to increase the contents of antioxidant factors SOD and GSH-Px and decrease the content of lipid oxidation marker MDA.

[0032] Furthermore, the improvement of the functions of the kidney, whole brain, and liver tissues of aging mice includes reducing the apoptosis of hippocampal neurons in the brain, improving nerve function; reducing the glomerular area in the kidney, alleviating glomerulosclerosis, and improving renal function; repairing the deformation of hepatic sinusoids in the liver, reducing lipid droplet accumulation and cell necrosis, and improving the pathological damage of liver tissues.

[0033] Beneficial effects:

[0034] The present invention provides an efficient extraction and purification method of auricularia melanin based on alkali hydrolysis-acid precipitation-organic solvent washing, optimizes key parameters such as solid-liquid ratio, extraction temperature, and pH adjustment, significantly improves the yield of melanin, the purity reaches more than 95%, and the process has good repeatability and controllable cost, laying a technical foundation for industrial production.

[0035] The present invention is experimentally proved that in a D-galactose-induced aging mouse model, auricularia melanin significantly increases the total moving distance of mice, decreases the stationary time, and increases the central residence time, indicating that it can effectively improve the locomotor ability and exploration desire of aging mice and relieve depressive behavior. In addition, auricularia melanin can also significantly increase the activities of serum superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and decrease the content of malondialdehyde (MDA), indicating that it can effectively reduce oxidative stress damage in the body by enhancing the antioxidant enzyme system and reducing lipid peroxidation. In terms of tissue protection, auricularia melanin can reduce the apoptosis of hippocampal neurons in the brain, improve nerve function, which is consistent with the behavioral results, suggesting that it can delay neurodegenerative diseases; it can also reduce the glomerular area in the kidney, alleviate glomerulosclerosis, and improve renal function; at the same time, it can repair the deformation of hepatic sinusoids in the liver, reduce lipid droplet accumulation and cell necrosis, improve the pathological damage of liver tissues, and reduce the degree of oxidative stress. It can be seen that auricularia melanin can regulate the activities and social behaviors of aging mice, reduce oxidative stress in the body, and improve the functions of the kidney, whole brain, and liver tissues of aging mice. Brief Description of the Drawings

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 The ultraviolet scanning spectrum of the purified melanin from Auricularia auricula prepared in Example 1 of the present invention;

[0038] Figure 2 The graph of the logarithm of the absorbance of the purified melanin from Auricularia auricula prepared in Example 1 of the present invention plotted against the absorption wavelength;

[0039] Figure 3 The Fourier transform infrared scanning spectrum of the purified melanin from Auricularia auricula prepared in Example 1 of the present invention;

[0040] Figure 4 The nuclear magnetic resonance spectrum of the purified melanin from Auricularia auricula prepared in Example 1 of the present invention. Among them, the upper figure is 1 the 1H-NMR spectrum; the lower figure is the 13C-NMR spectrum;

[0041] Figure 5 The graph of the effects of Auricularia auricula melanin on the locomotor and depressive behaviors of mice. Among them, A is the total moving distance in the open field test; B is the stationary time; C is the residence time in the corner; D is the residence time in the center; compared with the adjacent group: ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001;

[0042] Figure 6 The graph of the effects of Auricularia auricula melanin on the changes of oxidative stress in vivo. Among them, A is the effect on SOD; B is the effect on GSH-Px; C is the effect on MDA;

[0043] Figure 7 The graph of the effects of Auricularia auricula melanin on the apoptotic nerve cells in the glomeruli and hippocampi of mice. Among them, A is the control group; B is the aging group; C is the Auricularia auricula melanin group;

[0044] Figure 8 The graph of the effects of Auricularia auricula melanin on the livers of mice. Among them, A is the control group; B is the aging group; C is the Auricularia auricula melanin group. Detailed implementation manners

[0045] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation manners of the present invention.

[0046] It should be understood that the terms used in the present invention are merely for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0049] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0050] Example 1

[0051] 1. Melanin extraction

[0052] 1.1 Extraction and purification of melanin from Auricularia auricula

[0053] (1) The fruiting bodies of Auricularia auricula (30 g) were crushed, and the powder was poured into 1.25 mol / L NaOH solution at a solid-liquid ratio of 1:35, and ultrasonically extracted at 60 °C for 60 min, then filtered by suction, and the supernatant was taken.

[0054] (2) The pH value of the supernatant was adjusted to 1.5 with 7 M hydrochloric acid, and the mixture was kept in a constant temperature water bath at 80 °C for 10 h to remove the proteins and carbohydrates therein.

[0055] (3) Then, it was centrifuged at 4000 rmp / min for 10 min, and the precipitate was washed with deionized water until neutral to obtain crude melanin.

[0056] (4) The crude melanin was continuously washed 3 times with dichloromethane, ethyl acetate, absolute ethanol and deionized water, and centrifuged at 7000 rpm / min for 10 min.

[0057] (5) The precipitate was redissolved in 2 M sodium hydroxide solution, and the pH value was adjusted to 1.5 with 7 M hydrochloric acid. Then it was centrifuged at 7000 rpm for 10 min, and the precipitate was rinsed with deionized water until neutral and air-dried at room temperature to obtain purified melanin.

[0058] 1.2 UV-Visible Spectral Determination of Auricularia auricula Melanin

[0059] Weigh 1 mg of the purified melanin and add it to the solution prepared with 1 M NaOH. After complete dissolution, centrifuge it at 3000 r / min for 5 min. Take the supernatant and measure the absorbance value in the wavelength range of 200 - 500 nm on a UV-visible spectrophotometer. Use 1 mol / L NaOH solution as the blank control and draw the UV full absorption spectrum.

[0060] 1.3 Infrared Spectral Determination of Auricularia auricula Melanin

[0061] Put 1 mg of the purified and dried melanin and an appropriate amount of dried potassium bromide powder into an agate mortar and grind them until evenly mixed. Then put them into a mold to press tablets. When the mixture is pressed into a thin round tablet, scan it with a Fourier transform infrared spectrometer in the wavelength range of 4000 - 400 cm -1 range.

[0062] 1.4 Nuclear Magnetic Resonance Hydrogen Spectrum and Carbon Spectrum Determination of Auricularia auricula Melanin

[0063] Dissolve the Auricularia auricula melanin powder in deuterated-DMSO, use tetramethylsilane (TMS) as the internal standard, and use ppm as the chemical shift to conduct the determination on a 400 MHz nuclear magnetic resonance spectrometer.

[0064] 1.5 UV-Visible Absorption Spectrum of Auricularia auricula Melanin

[0065] The UV-visible absorption spectrum of the purified melanin at 200 - 600 nm is as Figure 1 shown. It was observed that it has a strong absorption in the UV region and has a maximum absorption peak at 220 nm, which is the same as the maximum absorption wavelength of melanin from other different sources. After 220 nm, the absorption value of melanin gradually decreases with the increase of wavelength, which may be due to the presence of aromatic rings or other complex conjugated double bond systems in its structure. There is a small shoulder peak near 280 nm in the melanin of Auricularia auricula fruiting body, which may be caused by the absorption of aromatic amino acid residues (tyrosine, tryptophan, phenylalanine) in the melanin-binding protein.

[0066] When the logarithm of the absorbance is plotted against the wavelength, a straight line with a negative slope will be obtained, and the slope of this straight line is usually used as an important criterion for the identification and characterization of melanin. As Figure 2As shown, the logarithm of the absorbance of melanin shows a linear relationship with the ultraviolet absorption wavelength, and the calculated slope is -0.0029, which is consistent with the slope of melanin from Auricularia auricula-judae reported previously.

[0067] 1.6 Infrared absorption spectrum of melanin from Auricularia auricula-judae

[0068] Melanin is a macromolecular polymer with a complex structure. A series of strong and broad characteristic absorption peaks are obtained by Fourier transform infrared spectroscopy, which can be used to judge the information of main functional groups. As Figure 3 shown, a group of strong and broad characteristic absorption peaks can be observed at 3294 cm -1 -1, which may be caused by the stretching vibration of hydrogen bonds on indole amino (–NH) or hydroxyl (-OH) groups; there is a small and sharp characteristic peak at 2870 - 2960 cm -1 -1, indicating the presence of aliphatic C-H groups; the absorption peak at 1653 cm -1 -1 may be caused by the stretching vibration of carbon-carbon double bonds (C=C) in aromatic rings or the stretching vibration of carbon-oxygen double bonds (C=O) in carbonyl groups; the peak of melanin around 1527 cm -1 -1 may be related to the bending vibration of amino (N-H) and the stretching vibration of C-N (secondary amine), indicating that the molecule may have an indole structure; the absorption peak at 1231 cm -1 -1 is caused by the stretching vibration of phenolic hydroxyl (-OH), and there is a group of weak absorption bands between 1160 - 1000 cm -1 -1, which may be related to the symmetric contraction vibration of ether bonds (C-O-C). The absorption peak at 623 cm -1 -1 is usually for alkane C-H bonds and aromatic C-H bonds, and is also related to the in-plane deformation of carbon-hydrogen bonds substitution or conjugated systems in alkenes.

[0069] 1.7 1H nuclear magnetic resonance spectrum of melanin from Auricularia auricula-judae

[0070] The 1H NMR spectrum of purified melanin is as Figure 4 shown. The peaks at 0.5 - 2.2 ppm come from the alkyl fragments (methyl and methylene) of residual proteins. The signal peak at 2.5 ppm is the solvent peak. The peaks at 3.5 - 4.5 ppm come from methyl or methylene attached to oxygen or nitrogen atoms. The peaks at 6.0 - 8.0 ppm come from aromatic hydrogens in pyrrole or pyrrole ring structures on the melanin polymer chain. The single signal at 8.29 (s) ppm indicates the presence of carboxyl substitution, which is consistent with the reported H spectrum of eumelanin.

[0071] Its 13C-NMR spectrum is as Figure 4As shown, the peaks in the range of 10 - 40 ppm are the carbon atom resonance peaks of methyl and methylene groups. Peaks related to aromatic carbon appear in the range of 110 - 160 ppm, which may be related to the pyrrole or indole system. The peaks in the range of 160 - 200 ppm are the carbonyl carbon peaks corresponding to quinones, amides or carboxylic esters.

[0072] Based on the above data, it can be known that the melanin in Auricularia auricula is eumelanin containing indole and phenolic polymers in its structure.

[0073] Study on the anti - aging activity of Auricularia auricula melanin in Example 2

[0074] 1. Experimental animals and drugs

[0075] Male C57BL / 6J mice (6 - week - old) were purchased from Beijing Speedy Biotechnology Co., Ltd. (Beijing, China). The mice were placed in a room with a 12 - hour light - dark cycle, with a relatively constant temperature of 22 ± 1°C and a relatively constant humidity of 50 - 60%. All mice had free access to food. This experiment was approved by the Animal Ethics Committee of Northwest A&F University; Auricularia auricula melanin was purchased from Sichuan Victy Biological Technology Co., Ltd. (Chengdu, Sichuan).

[0076] 2. Experimental methods

[0077] 2.1 Animal model construction

[0078] 2.1.1 Experimental grouping and treatment:

[0079] Control group: Gavage with deionized water (10 mL / kg) + intraperitoneal injection of normal saline (10 mL / kg) daily

[0080] D - gal Model group: Gavage with deionized water + intraperitoneal injection of D - galactose (150 mg / kg, Sigma - Aldrich, D0501) daily

[0081] Intervention group (D - gal + ABM): Gavage with a solution containing 2.5 mg / kg Auricularia auricula melanin (ABM) + intraperitoneal injection of D - galactose (same as the model group) daily

[0082] 2.1.2 Cycle design

[0083] Adaptive feeding: 7 days, environmental conditions were temperature 22 ± 2°C, humidity 50 ± 10%, 12 / 12 - hour light - dark cycle, and free access to standard feed (indicating the brand and nutritional components such as protein ≥ 18%, fat ≥ 5%). Record the body weight daily and exclude individuals deviating from the mean ± 2SD.

[0084] Modeling period: Continuous intervention for 8 weeks. The unified dose of D-galactose is 150 mg / kg / d (intraperitoneal injection), and the drug is administered daily from 9:00 to 11:00 in the morning.

[0085] 2.2 Behavioral test - Open field test

[0086] The open field test was conducted in the last week of drug administration for the mice. It is usually used to evaluate the locomotor ability and social willingness of mice. The experimental site consists of a 90 cm × 90 cm square, and the open field is divided into 9 quadrants. The middle quadrant is the "center", the quadrants at the four corners are the "corners", and the quadrants on the remaining four sides are the "periphery". The mice were placed in the same corner with their backs to the experimenter and allowed to move freely for 5 minutes, and the video recording of the mice's movement trajectories was started. Before taking out the mice and putting in the next one, 75% alcohol was sprayed to remove odor interference, and it was ensured that the ethanol was completely evaporated. There were no other people, sounds or light interferences in the room during the whole process. The movement trajectory data of the mice were recorded and analyzed by VisuTrack animal behavior analysis software (Shanghai Xinruan Information Technology Co., Ltd.).

[0087] 2.3 Detection experiment process of serum MDA, SOD, GSH-Px

[0088] 2.3.1 Sample collection and pretreatment

[0089] Blood collection and centrifugation: After the experimental animals (such as mice) were fasted for 12 hours, whole blood was collected by orbital blood collection or cardiac puncture into a non-anticoagulant centrifuge tube, left to stand at room temperature for 30 minutes. After the blood coagulated, it was centrifuged at 3000 rpm for 10 minutes to separate the upper layer of serum, which was aliquoted and stored at -80 °C for later use.

[0090] Quality control: Avoid repeated freezing and thawing of samples, and the operation was carried out on ice throughout the process to reduce the risk of enzyme activity degradation.

[0091] 2.3.2 Malondialdehyde (MDA) detection

[0092] Principle: Based on the reaction of MDA with thiobarbituric acid (TBA) to form a red complex (maximum absorption peak at 532 nm) under high-temperature acidic conditions.

[0093] Operation steps: Take 100 μL of serum sample, add 0.2 mL of 0.6% thiobarbituric acid solution and 0.2 mL of 8.1% sodium dodecyl sulfate (SDS), and mix well. React in a boiling water bath for 15 minutes, cool in an ice bath, and then centrifuge at 3000 rpm for 10 minutes. Take the supernatant and measure the absorbance (OD value) at a wavelength of 532 nm, and calculate the MDA content (unit: nmol / mL) according to the standard curve.

[0094] 2.3.3 Superoxide dismutase (SOD) activity detection

[0095] Principle: Detect the ability of SOD to inhibit superoxide anion radicals by the WST-8 method (measured at a wavelength of 450 nm).

[0096] Operating steps: Prepare the reaction solution (containing WST-8, xanthine oxidase, and substrate) according to the kit instructions. Take 20 μL of serum sample and mix it with 200 μL of the reaction solution, and incubate it in the dark at 37 °C for 30 minutes. Immediately measure the OD value at 450 nm, and calculate the SOD activity based on the inhibition rate (unit: U / mL).

[0097] 2.3.4 Detection of glutathione peroxidase (GSH-Px) activity

[0098] Principle: GSH-Px catalyzes the reaction of GSH with hydrogen peroxide to generate GSSG, and the DTNB colorimetric method is used to detect the consumption of GSH (measured at a wavelength of 412 nm).

[0099] Operating steps:

[0100] Prepare the reaction system (containing GSH, hydrogen peroxide, glutathione reductase, and NADPH).

[0101] Add 50 μL of serum sample, react at 37 °C for 5 minutes, and immediately add the DTNB color reagent to terminate the reaction.

[0102] Measure the OD value at 412 nm, and calculate the GSH-Px activity based on the consumption of GSH per unit time (unit: U / L).

[0103] 2.3.5 Data analysis and verification

[0104] Standard curve: Set gradient concentration standards (such as 0 - 100 μM MDA, 0 - 200 U / mL SOD) for each batch of experiments, draw the standard curve and calculate the R 2 value (required R 2 ≥ 0.99).

[0105] Statistical analysis: Data are expressed as mean ± standard deviation, and one-way ANOVA is performed using GraphPad Prism 9.0. The significance standard for differences between groups is *p < 0.05.

[0106] Quality control index: Synchronously detect the positive control (such as the vitamin E treatment group) and the negative control (the oxidative stress model group) to verify the sensitivity and specificity of the detection system.

[0107] 2.4 H&E staining

[0108] After weighing the mice, they were anesthetized with ether. When their movements became unsteady, their eyeballs were removed to collect blood, and the mice were euthanized by carbon dioxide inhalation. After collecting the whole brains, livers, and kidneys of the mice, they were placed in centrifuge tubes containing 4% paraformaldehyde and fixed for 24 hours. After fixation, the tissues were stained with hematoxylin-eosin (H&E staining), and this process was completed on behalf by Wuhan Sevier Biotechnology Co., Ltd. (Wuhan, Hubei). When the tissue sections were loaded onto the PANNORAMIC panoramic slide scanner, the sections would move gradually under the lens of the scanner, imaging while moving, and thus all the tissue information on the tissue sections was scanned and imaged to form a folder, which contained all the tissue information on the tissue sections. After the folder was opened with CaseViewer2.4 software, it could be magnified arbitrarily from 1 to 400 times for observation. The CaseViewer2.4 scanning and browsing software was used to select the target area of the brain tissue for imaging at 400 times magnification. When imaging, try to make the tissue fill the entire field of view and ensure that the background light of each photo is consistent. Finally, the stained images of the tissue sections were obtained.

[0109] The Image-Pro analysis software was used to count the number of apoptotic neurons in the hippocampus of the brain sections respectively, and measure the corresponding field of view area. Calculate the number of apoptotic neurons per unit field of view = number of apoptotic neurons / average value of field of view area.

[0110] The Image-Pro analysis software was used to measure the areas of 5 randomly selected glomeruli in the kidney sections respectively, and calculate the average value.

[0111] Determination of oxidative stress, serum MDA, SOD, GSH-Px levels

[0112] 3. Experimental results

[0113] 3.1 Effects of Auricularia melanin on the activities and social willingness of senescent mice

[0114] Although the open field test is a classic behavioral experiment, the experiment is easily affected by many factors, such as genetic variation, experimental environment, estrous cycle, circadian rhythm, etc. Compared with Kunming and BALB / C mice, the open field results of C57BL / 6J mice used in this example do not vary due to circadian rhythm and are relatively stable whether tested in the morning or afternoon. At the same time, compared with other strains of mice, C57BL / 6J mice show more obvious anxiety behaviors when facing a new environment. And all-male mice can dilute the tissue metabolism effects of D-gal and Auricularia melanin on gender.

[0115] D-gal is a drug commonly used to establish an aging model. Compared with the control group of mice, the total moving distance of the aging group of mice decreased significantly (33.8%), and the stationary time increased (49.8%). This indicates that the moving speed and locomotor ability of the aging group of mice decreased. Moreover, the aging group of mice spent more time in the corners (5.6%), the residence time in the center decreased (80.7%), and the stationary time increased significantly, suggesting that the willingness of mice to explore a new environment decreased after aging and depressive behaviors occurred. The total moving distance (42.9%), stationary time (38.2%), residence time in the corners (9.3%), and residence time in the center (180%) of auricularia melanin were reversed compared with those of the aging group of mice. The occurrence of this phenomenon indicates that auricularia melanin can improve the locomotor ability and exploration desire of aging mice, and improve the decreased locomotor ability and depressive behaviors associated with aging, as shown in Table 1 and Figure 5 as follows.

[0116] Table 1 Open field activity of mice

[0117]

[0118] Note: Total moving distance (mm): Aging group vs control group P = 0.0007, Auricularia melanin group vs aging group, P = 0.0031;

[0119] Stationary time (s): Aging group vs control group P < 0.0001, Auricularia melanin group vs aging group P < 0.0001;

[0120] Residence time in the corners (s): Aging group vs control group P = 0.0981, Auricularia melanin group vs aging group P = 0.0091;

[0121] Residence time in the center (s): Aging group vs control group P = 0.0132, Auricularia melanin group vs aging group P = 0.5172.

[0122] 3.2 Improvement of auricularia melanin against oxidative stress caused by aging

[0123] As Figure 6 shown, compared with the control group, the antioxidant factors SOD and GSH-Px in the model group decreased significantly, while the content of the lipid oxidation marker MDA increased, and the melanin intervention group effectively reversed this change.

[0124] 3.3 Improvement effect of auricularia melanin on tissues of aging mice

[0125] Generally, the aging process will lead to a decline in renal function, as shown in Table 2 and Figure 7As shown. H&E staining of mouse kidneys showed that the glomerular area of mice in the aging group increased (28.6%), indicating that their kidney function had decreased. However, the glomerular area of mice treated with Auricularia melanin decreased (5.88%), suggesting that the aging of the mouse kidneys had improved and the kidney function had been restored. At the same time, brain sections showed an increase in apoptosis of hippocampal neurons in mice in the aging group, with an increase in apoptotic cells per unit field of view in the aging group (28.6%), while the number of apoptotic cells in the Auricularia melanin group decreased (175.5%). This corresponded to the results of the previous open field experiment, indicating that the brain aging damage in mice could be alleviated by Auricularia melanin.

[0126] Table 2 Glomerular area of mice and the number of apoptotic neurons in the hippocampus

[0127]

[0128] It can be seen from Figure 8 that H&E staining sections of mouse livers showed that the hepatic sinusoids in the model group were deformed and irregular, the hepatocytes beside the hepatic sinusoids were necrotic, the lipid droplets increased, and the cell margins were blurred, indicating that the liver tissue had developed lesions and the degree of oxidative stress was high. However, the hepatic sinusoids in the administration group were less deformed, the lipid droplet accumulation decreased, and the cells were arranged neatly, indicating that the liver tissue of the mice had been improved.

[0129] In summary, the present invention developed a rapid extraction and purification process for Auricularia melanin and confirmed the anti-aging effect of Auricularia melanin on D-gal-induced aging mice. Through behavioral assays and histological section staining, it was explored that Auricularia melanin could regulate the activities and social behaviors of aging mice, reduce oxidative stress in vivo, and improve the functions of the kidneys, whole brain, and liver tissues of aging mice.

[0130] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for preparing auricularia melanin, characterized in that, It includes the following steps: (1) Mix the powder obtained by crushing the fruiting body of Auricularia auricula with NaOH solution, perform ultrasonic extraction, and take the supernatant after filtration by suction; (2) Adjust the pH of the supernatant, and remove the proteins and carbohydrates therein by constant temperature water bath to obtain the treated supernatant; (3) Wash the precipitate obtained after centrifuging the treated supernatant with deionized water until neutral to obtain crude melanin; (4) Re-dissolve the crude melanin in sodium hydroxide solution after washing and centrifuging, adjust the pH value again and then centrifuge, wash the precipitate with deionized water until neutral, and dry it at room temperature to obtain purified Auricularia auricula melanin.

2. The preparation method according to claim 1, wherein In step (1), the solid-liquid ratio of the powder to the NaOH solution is 1:35, the concentration of the NaOH solution is 1.25 mol / L, and the ultrasonic extraction is ultrasonic extraction at 60 °C for 60 min.

3. The preparation method according to claim 1, characterized in that, In step (2), adjusting the pH of the supernatant is to adjust the pH value of the supernatant to 1.5 with 7M hydrochloric acid, and the constant temperature water bath is a constant temperature water bath at 80 °C for 10 h.

4. The preparation method according to claim 1, characterized in that, In step (3), the centrifugation is centrifugation at 4000 rmp / min for 10 min.

5. The preparation method according to claim 1, characterized in that In step (4), the washing and centrifugation is to continuously wash the crude melanin 3 times with dichloromethane, ethyl acetate, absolute ethanol and deionized water and then centrifuge at 7000 rpm / min for 10 min; the concentration of the sodium hydroxide solution is 2M; the centrifugation after adjusting the pH value again is to adjust the pH value to 1.5 with 7M hydrochloric acid and then centrifuge at 7000 rpm / min for 10 min.

6. Auricularia melanin prepared by the preparation method according to any one of claims 1-5, characterized in that, The Auricularia auricula melanin includes eumelanin containing indole and phenolic polymers.

7. Use of the Auricularia auricula melanin according to claim 6 in the preparation of products for preventing and treating anti-aging.

8. The application according to claim 7, characterized in that, The anti-aging includes regulating the activities and social behaviors of aging mice, reducing oxidative stress in the body and improving the tissue functions of the kidneys, whole brains and livers of aging mice.

9. The application according to claim 8, wherein The reduction of oxidative stress in the body is to increase the contents of antioxidant factors SOD and GSH-Px and reduce the content of lipid oxidation marker MDA.

10. The application according to claim 8, wherein, The improvement of the tissue functions of the kidneys, whole brains and livers of aging mice includes reducing the apoptosis of hippocampal nerve cells in the brain, improving nerve function; reducing the glomerular area in the kidneys, alleviating glomerulosclerosis and improving renal function; repairing the deformation of hepatic sinusoids in the liver, reducing lipid droplet accumulation and cell necrosis, and improving the pathological damage of liver tissue.

Images