Method for extracting melanin from inonotus obliquus by deep eutectic solvent

Abstract

The application discloses a method for extracting melanin from inonotus obliquus in deep eutectic solution, and belongs to the field of functional food. The yield of melanin is 13.75%, the PTCA / PDCA of the melanin of the application is 4.17, and the PTCA / PDCA of the melanin of a conventional method is 1.71. Compared with the conventional method, the melanin obtained by the application is more complete. In addition, the melanin obtained by the application has good thermal stability, light stability and alkaline stability, and metal ions have no significant effect on the stability of the melanin. Meanwhile, the melanin extracted from the inonotus obliquus is a biological endogenous substance, is safe and non-toxic, and has great application potential in the treatment of intestinal injury diseases. The in-vivo experiment proves that the melanin extracted from the inonotus obliquus can restore the damaged colon tissue structure, helps to maintain the integrity and function of the intestinal tract of mice, inhibits inflammation, relieves oxidative stress, and improves the level of short-chain fatty acids, and has a significant treatment effect on colitis.

Description

TECHNICAL FIELD

[0001] The application relates to a melanin extraction method, in particular to a method for extracting melanin in Inonotus obliquus in a deep eutectic solution, and belongs to the functional food field. BACKGROUND

[0002] Inonotus obliquus, also known as chaga, is a fungus belonging to the phylum Fungi, subphylum Basidiomycotina, class Hymenomycetes, order non-brown fungi, family polypore, and genus Inonotus obliquus. The sclerotium of the fungus is nodule-shaped, has irregular furrows on the surface, is generally dark brown or black in color, has no stipe, has a diameter of 25-40 cm, has a fertile part of 5 mm thick, has a pore tube of 3-10 mm, and is yellow-brown inside. The spores are widely broad-elliptical or oval, smooth, 9-10 microns x (5.5-6.5) microns, and have bristles. Inonotus obliquus is a wood-decaying fungus growing in cold regions. This fungus is widely distributed in northern European countries such as Norway, Finland and Poland, and also has a certain distribution in Japan and Heilongjiang, China. It mainly grows on the dead trunks of silver birch, white birch and elm trees. The sclerotium can survive on the dead trunks for about 6 years. It is rich in various active substances such as triterpenes, polysaccharides, melanin, saponins and polyphenols needed by the human body, and has high nutritional value. It has obvious effects of anti-cancer, anti-tumor, blood glucose and blood lipid reduction, antioxidant and immune enhancement.

[0003] Melanin is a common biological pigment, which is a biological macromolecule polymerized from indoles and phenols. It is easily combined with proteins, oils and polysaccharides, and is insoluble in water, acidic solution and most organic solvents. Natural melanin has a wide source, and there are related research reports on melanin in animals, plants and microorganisms. Melanin has physiological activities such as anti-tumor, anti-radiation and immune enhancement, so the application value of melanin in medicine, cosmetics and biological materials is self-evident.

[0004] At present, most of the processing methods of Inonotus obliquus are mainly dry processing and other primary processing methods. The deep processing of Inonotus obliquus is mainly concentrated on polysaccharides, and there is still very little research on melanin. Due to the characteristics that melanin is easily soluble in alkali and will form a precipitate under acidic conditions, the alkali dissolution and acid precipitation method is a commonly used method for extracting melanin. However, the traditional alkali dissolution and acid precipitation method needs a long time and has low efficiency, which may cause the structure of melanin to be destroyed in the extraction process due to decarboxylation. In addition, due to the fact that melanin is easily combined with proteins, oils and polysaccharides, and its heterogeneity and non-uniformity, it is difficult to purify melanin. The extraction method of melanin is determined according to the type, source, production site, content and impurity content of melanin. How to extract and purify melanin in Inonotus obliquus to realize the extraction and development of melanin in Inonotus obliquus is a technical problem to be solved at present. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a method for extracting melanin from Inonotus obliquus in a deep eutectic solvent, and aims to solve the technical problem that the traditional extraction method requires a long time, has low efficiency, and causes the structure of melanin to be destroyed during the extraction process and the subsequent purification to be difficult, thereby limiting the purification of melanin from Inonotus obliquus.

[0006] The first technical solution provided by the present application is a method for extracting melanin from Inonotus obliquus in a deep eutectic solvent, comprising the following steps:

[0007] (1) adding Inonotus obliquus powder into a deep eutectic solvent for extraction, and collecting the precipitate after the extraction is completed;

[0008] (2) washing the precipitate in step (1) with water and neutralizing, and then washing the precipitate with chloroform, ethyl acetate, anhydrous ethanol and pure water in sequence until the supernatant is clear and transparent, collecting the precipitate and drying to obtain Inonotus obliquus melanin powder.

[0009] In some embodiments, in step (1), the Inonotus obliquus powder is Inonotus obliquus water extract powder.

[0010] Further, the preparation method of the Inonotus obliquus water extract powder is as follows: S1, grinding and sieving Inonotus obliquus fruiting bodies to obtain ground powder;

[0011] S2, dissolving the ground powder in S1 in pure water for extraction, and then centrifuging to remove the precipitate to obtain an extraction liquid;

[0012] S3, concentrating and drying the extraction liquid in S2 to obtain Inonotus obliquus water extract powder.

[0013] Further, in S1, the mesh size of the sieving is 100 mesh.

[0014] Further, in S2, the solid-liquid ratio of the ground powder to pure water is 1g:(10-50)mL, the number of extractions is 1-3 times, the extraction time is 1-2h each time, and the extraction temperature is 40-100℃.

[0015] Further, in S3, the extraction liquid is concentrated to 30% of the original volume at 50℃ using a vacuum rotary evaporator, and the drying method is freeze-drying.

[0016] In some embodiments, in step (1), the deep eutectic solvent comprises choline chloride and a hydrogen donor, and the hydrogen donor is selected from any one of malic acid, oxalic acid, formic acid, acetic acid, propionic acid, butyric acid, hexanoic acid and citric acid.

[0017] In some embodiments, in step (1), the deep eutectic solution comprises choline chloride and oxalic acid, and the molar ratio of the choline chloride to the oxalic acid is 1:2-2:1.

[0018] In some embodiments, in step (1), the ratio of the Inonotus obliquus powder to the deep eutectic solution is (10-200) mg / mL.

[0019] In some embodiments, in step (1), the temperature of the extraction is 30-70℃, and the time of the extraction is 2-6 h.

[0020] The second technical solution provided by the present application is a melanin prepared by the method of the first technical solution.

[0021] The third technical solution provided by the present application is a product containing the melanin of the second technical solution.

[0022] In some embodiments, the product is a food or a medicine.

[0023] The fourth technical solution provided by the present application is the use of the method of the first technical solution or the melanin of the second technical solution in the preparation of a medicine for relieving and / or treating intestinal inflammation.

[0024] Further, the medicine comprises the above-mentioned composition and a pharmaceutically acceptable carrier.

[0025] Still further, the carrier comprises one or more of a filler, a binder, a humectant, a disintegrant, a lubricant, and a flavoring agent commonly used in medicine.

[0026] Further, the dosage form of the medicine comprises a granule, a capsule, a tablet, a pill, or an oral liquid.

[0027] Still further, the medicine comprises enteric-coated tablets and capsules, and oral liquids.

[0028] Advantages and effects of the present application:

[0029] The present application obtains the optimal extraction process of Inonotus obliquus melanin through the screening of deep eutectic solvents and single-factor tests. Under the optimal process, the yield of melanin is 13.75%, and the PTCA / PDCA of the melanin obtained by the present application is 4.17, while that of the melanin obtained by the conventional method is 1.71. Therefore, the melanin obtained by the present application is more complete than that obtained by the conventional method. Meanwhile, in the extraction process, the present application adds the water extract of Inonotus obliquus into the deep eutectic solution, and then directly centrifugates and purifies, which can avoid the reduction of hydrogen bond strength, and thus improves the extraction efficiency of melanin. In addition, the Inonotus obliquus melanin obtained by the present application has good thermal stability, light stability, and alkaline stability, and is not affected by metal ions Fe 3+ , Al 3+Mn 2+ Cr 2+ Na + K + Fe 2+ Cu 2+ Ca 2+ Mg 2+ and Zn 2+ The melanin extracted from Inonotus obliquus has no significant effect on the stability of melanin. At the same time, the melanin extracted from Inonotus obliquus is an endogenous substance of living organisms, safe and non-toxic, and has great application potential in the treatment of intestinal injury diseases. The in vivo experiment proves that the melanin extracted from Inonotus obliquus can restore the damaged colon tissue structure, help maintain the integrity and function of the intestinal tract of mice, inhibit inflammation, relieve oxidative stress, and improve the level of short-chain fatty acids, and has a significant therapeutic effect on colitis. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The extraction rate of the melanin extracted from Inonotus obliquus by different extraction solutions of the present application;

[0031] Figure 2 The extraction rate of the melanin extracted from Inonotus obliquus by different proportions in chcl-oa of the present application;

[0032] Figure 3 The extraction rate of the melanin extracted from Inonotus obliquus by different solid-liquid ratios in chcl-oa of the present application;

[0033] Figure 4 The extraction rate of the melanin extracted from Inonotus obliquus by different extraction temperatures in chcl-oa of the present application;

[0034] Figure 5 The extraction rate of the melanin extracted from Inonotus obliquus by different extraction times in chcl-oa of the present application;

[0035] Figure 6 The temperature stability of the melanin extracted from Inonotus obliquus in chcl-oa of the present application;

[0036] Figure 7 The metal stability of the melanin extracted from Inonotus obliquus in chcl-oa of the present application;

[0037] Figure 8 The redox reagent stability of the melanin extracted from Inonotus obliquus in chcl-oa of the present application;

[0038] Figure 9 The ultraviolet absorption spectrum of the melanin extracted from Inonotus obliquus in chcl-oa of the present application;

[0039] Figure 10The Fourier infrared transform spectrum of the Inonotus obliquus melanin extracted in the chcl-oa of the application;

[0040] Figure 11 The nuclear magnetic resonance spectrum of the Inonotus obliquus melanin extracted in the chcl-oa of the application;

[0041] Figure 12 The standard substance liquid chromatogram of AHPO;

[0042] Figure 13 The liquid chromatogram of the Inonotus obliquus melanin extracted in the chcl-oa of the application;

[0043] Figure 14 The liquid chromatogram of the Inonotus obliquus melanin extracted by the conventional method of the application;

[0044] Figure 15 The DAI score, body weight change rate and colon length of mice after oral administration of the extracted melanin of the application;

[0045] Figure 16 The H&E staining diagram of the pathological changes of the colon of mice after oral administration of the extracted melanin of the application;

[0046] Figure 17 The immunohistochemical staining diagram of the colon of mice after oral administration of the extracted melanin of the application;

[0047] Figure 18 The serum inflammatory factor content of mice after oral administration of the extracted melanin of the application;

[0048] Figure 19 The catalase content of the colon tissue of mice after oral administration of the extracted melanin of the application;

[0049] Figure 20 The glutathione content of the colon tissue of mice after oral administration of the extracted melanin of the application;

[0050] Figure 21 The myeloperoxidase content of the colon tissue of mice after oral administration of the extracted melanin of the application;

[0051] Figure 22 The superoxide dismutase content of the colon tissue of mice after oral administration of the extracted melanin of the application;

[0052] Figure 23 The short-chain fatty acid expression amount of the cecum content of mice after oral administration of the extracted melanin of the application. DETAILED DESCRIPTION

[0053] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to specific examples and with reference to the drawings.

[0054] 1. Inonotus obliquus fruiting bodies were purchased from Dalian Siberia International Trade Co., Ltd. DSS (molecular weight = 36-50 KD) was purchased from MP Biomedicals (Irvine, CA, USA). Commercial kits for glutathione (GSH), catalase (CAT), myeloperoxidase (MPO) and superoxide dismutase (SOD) were purchased from Nanjing Jianshen Biological Engineering Institute (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) kits for determining tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) levels were purchased from Enzyme-Linked Bio-technology Co., Ltd. Short-chain fatty acid (SCFA) standards were purchased from Shanghai Maikelin Biochemical Engineering Co., Ltd. Other chemicals used were analytical grade reagents purchased commercially.

[0055] 2. Melanin detection method: The stability of melanin was identified using thermal stability, metal stability, and redox stability. Melanin structure was identified using ultraviolet-visible spectroscopy, attenuated total reflection Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The integrity of melanin was identified using the alkaline hydrogen peroxide method.

[0056] 3. Calculation method of melanin extraction rate: Melanin extraction rate = weight of melanin freeze-dried powder / weight of Inonotus obliquus fruiting bodies.

[0057] Example 1

[0058] The detailed composition information of the hydrogen donor and the hydrogen acceptor in this example is listed in Table 1. All natural deep eutectic solutions were prepared by mixing the hydrogen donor and the hydrogen acceptor according to Table 1 using microwave heating, i.e. exposing the natural deep eutectic solution in a glass vial to a microwave of 160 W, irradiating for 5 seconds, pausing for 5 seconds, and repeating the operation in a cycle until a clear liquid was obtained, and then vortexing in air for cooling.

[0059] Table 1 Different natural deep eutectic solution combinations

[0060]

[0061]

[0062] Method for preparing Inonotus obliquus water extract powder

[0063] The surface layer of Inonotus obliquus fruiting body was selected, ground, and then passed through a 100-mesh sieve to obtain a uniform powder. 100 g of the powder was extracted with pure water (according to a material-to-water ratio of 1:24, W / V) twice at 90°C for 1.5 h each time, the precipitate was removed, the supernatant was concentrated to 30% of the original volume using a vacuum rotary evaporator at 50°C, and finally freeze-dried to obtain Inonotus obliquus water extract powder.

[0064] Extraction of Inonotus obliquus melanin

[0065] 100.0 mg of Inonotus obliquus water extract powder was water-bathed with 2 mL of natural deep eutectic solution in a 50°C constant-temperature water bath for 4 h. The precipitate was then separated by centrifugation at 10,000 rpm for 15 min, and then subjected to a purification procedure involving multiple washes with pure water until a neutral pH was reached, and the insoluble precipitate was washed with chloroform, ethyl acetate, ethanol, and pure water in sequence until the supernatant was clear and transparent. The precipitate was then centrifuged and dried in a freeze dryer to obtain Inonotus obliquus melanin, referred to as NADES melanin.

[0066] As shown in Figure 1 , 11 natural deep eutectic solutions all had an extraction effect on Inonotus obliquus melanin, except for the choline chloride-urea combination, the choline chloride-fructose combination, and the choline chloride-glycerol combination, the extraction rates of the other combinations were all higher than 6%. Among them, the choline chloride-oxalic acid combination had the highest extraction efficiency for melanin.

[0067] Example 2

[0068] The natural deep eutectic composition ratio, solid-to-liquid ratio, extraction temperature, and extraction time were selected, and the melanin extraction process flow according to Example 1 was used to analyze the effects of the four factors on the yield of Inonotus obliquus melanin.

[0069] 1. Natural deep eutectic composition ratio

[0070] Under the fixed conditions of a liquid-to-solid ratio of 50:1 mg / mL, an extraction temperature of 50°C, and an extraction time of 4 h, different natural deep eutectic composition ratios (ChCl / Oa, mol / mol) 2:1, 3:2, 1:1, 2:3, and 1:2 were set to investigate the effect of the natural deep eutectic composition ratio on the yield of Inonotus obliquus melanin. As shown in Figure 2 , when the deep eutectic composition ratio (ChCl / Oa, mol / mol) was 3:2, the yield of melanin was the highest, reaching 11.95%

[0071] 2、Under the fixed conditions of natural deep eutectic solvent composition ratio 1:1 (ChCl / Oa, mol / mol), extraction temperature 50℃, and extraction time 4h, different liquid-solid ratios (mg / ml) 10, 50, 100, 150, and 200 were set to investigate the effect of single-factor liquid-solid ratio on the yield of Inonotus melanin. The results are shown in Table 1. Figure 3 As shown in Table 1, when the liquid-solid ratio (mg / ml) was from 10 to 150, the extraction rate of melanin increased with the increase of liquid-solid ratio, and decreased at 200. Therefore, the extraction rate of melanin was the highest when the liquid-solid ratio (mg / ml) was 150, and the highest was 12.79%.

[0072] 3、Extraction temperature

[0073] Under the fixed conditions of natural deep eutectic solvent composition ratio 1:1 (ChCl / Oa, mol / mol), liquid-solid ratio 50:1 mg / mL, and extraction time 4h, different extraction temperatures 30℃, 40℃, 50℃, 60℃, and 70℃ were set to investigate the effect of single-factor extraction temperature on the yield of Inonotus melanin. The results are shown in Table 2. Figure 4 As shown in Table 2, when the extraction temperature was from 30 to 60℃, the extraction rate increased with the increase of temperature, and then the extraction rate did not increase accordingly with the increase of temperature. Therefore, 60℃ was selected as the optimal extraction temperature, and the highest was 11.14%.

[0074] 4、Extraction time

[0075] Under the fixed conditions of natural deep eutectic solvent composition ratio 1:1 (ChCl / Oa, mol / mol), liquid-solid ratio 50:1 mg / mL, and extraction temperature 50℃, different extraction times 2h, 3h, 4h, 5h, and 6h were set to investigate the effect of single-factor extraction time on the yield of Inonotus melanin. The results are shown in Table 3. Figure 5 As shown in Table 3, when the extraction time was from 2 to 5h, the extraction rate increased with the extension of time, and then decreased. Therefore, the extraction rate of melanin was the highest when the extraction time was 5h, and the highest was 11.14%.

[0076] 5、Optimal condition extraction

[0077] Two batches of extraction experiments were carried out under optimal conditions, i.e. natural deep eutectic composition ratio of 3:2 (ChCl / Oa, mol / mol), liquid material ratio of 150:1 mg / mL, extraction temperature of 60°C, and extraction time of 5h. After the first batch of extraction, the precipitate was separated by centrifugation at 10,000 rpm for 15 minutes, and then directly freeze-dried to obtain the crude melanin extract, and the extraction rate of the crude melanin extract was 15.21%. After the second batch of extraction, the precipitate was separated by centrifugation at 10,000 rpm for 15 minutes, and then subjected to a purification procedure, which included multiple washing with pure water until a neutral pH was reached, and the insoluble precipitate was sequentially washed with chloroform, ethyl acetate, ethanol and pure water until the supernatant was clear and transparent. Subsequently, the precipitate was centrifuged and dried in a freeze dryer to obtain the Inonotus melanin, referred to as NADES melanin, and the extraction rate of the NADES melanin was 13.75%.

[0078] Since its discovery, deep eutectic solution has been highly praised for its excellent extraction performance. In the process of extracting melanin from deep eutectic solution, complex hydrogen bond principles are involved, and hydrogen bonds can dissolve the structural components (chitin, protein) of the fungal cell wall in the solvent. This dissolution promotes the separation of melanin from other substances, thereby precipitating out. As the hydrogen bond strength decreases, the separated melanin again polymerizes with the structural components and re-dissolves in water. In the prior art, in order to improve the flowability of the deep eutectic solution, pure water, ethanol and other solutions are often added at the end of the extraction, but such treatment can reduce the hydrogen bond strength of the system and affect the extraction efficiency of the melanin. In the extraction process, the present application adds Inonotus water extract to the deep eutectic solution, and then directly centrifuges and purifies to obtain melanin. This can avoid the reduction of hydrogen bond strength and greatly improve the extraction efficiency of melanin.

[0079] Comparative Example 1

[0080] Conventional strong acid high-temperature extraction

[0081] 100.0 mg of Inonotus water extract powder was water-bathed with 2 mL of 6M HC1 solution in a 100°C constant temperature water bath for 10h. Subsequently, the precipitate was separated by centrifugation at 10,000 rpm for 15 minutes, and then subjected to a purification procedure, which included multiple washing with pure water until a neutral pH was reached, and the insoluble precipitate was sequentially washed with chloroform, ethyl acetate, ethanol and pure water until the supernatant was clear and transparent. Subsequently, the precipitate was centrifuged and dried in a freeze dryer to obtain the Inonotus melanin, referred to as HC1 melanin. The extraction rate was 6.43%. The conventional extraction method is time-consuming, requires long-term maintenance of high temperature, has high energy consumption, and the use of strong acid pollutes the environment and is not conducive to sustainable development. At the same time, the extraction rate is low and the extraction efficiency is not high.

[0082] Test Example

[0083] I. Performance identification of Inonotus obliquus melanin

[0084] 1. Thermal stability

[0085] 1.0 mg of Inonotus obliquus melanin was dissolved in 10 mL of 0.1 mol / L NaOH solution, and then incubated at 20, 40, 60, 80 and 100°C. Samples were taken every hour, cooled to room temperature, and analyzed for absorbance at 218 nm using a UV-Vis spectrophotometer with a 0.1 mol / L NaOH reference solution. As shown in Figure 6 , the absorbance of Inonotus obliquus melanin did not show a significant decrease, indicating that Inonotus obliquus melanin has good thermal stability.

[0086] 2. Metal ion stability

[0087] To determine how metal ions affect the stability of Inonotus obliquus melanin, 1.0 mg of the compound was thoroughly mixed with 10 mL of 0.1 mol / L NaOH solution containing the following additional 0.01 mol / L metal ions: Fe 3+ , Ca 2 + , Cr 3+ , Zn 2+ , Al 3+ , Mn 2+ , Mg 2+ , Na + , K + , Cu 2+ , Fe 2+ and Ba 2+ . Samples were taken every 12 hours, and the absorbance was measured at 218 nm. As shown in Figure 7 , the absorbance of Inonotus obliquus melanin did not show a significant change over time compared to the blank after the addition of metal ions, so the added metals had no effect on the stability of Inonotus obliquus melanin.

[0088] 3. Redox properties

[0089] Different volumes (0, 1, 2, 3, 4 and 5 mL) of reducing agent (30% Na2SO3) and oxidizing agent (30% H2O2) were added to 5 mL of 0.1 mg / mL Inonotus obliquus melanin solution, respectively, and the total volume of the solution was adjusted to 10 mL with pure water. The solution was thoroughly shaken and left to stand for 30 minutes, and the absorbance was measured at 218 nm. As shown in Figure 8 , Na2SO3 and H2O2 had no effect on the absorbance of Inonotus obliquus melanin, indicating that Inonotus obliquus melanin has good redox properties.

[0090] 4. UV-Vis spectrum

[0091] In 0.1 mol / L NaOH, dissolve the Inonotus obliquus melanin to prepare a solution with a final concentration of 0.1 mg / mL. Using the ultraviolet-visible spectrophotometer, measure the ultraviolet-visible absorption spectrum of the Inonotus obliquus melanin in the range of 200-800 nm, with 0.1 mol / L NaOH solution as the reference. The results are shown in Figure 9 The ultraviolet-visible spectrum of the Inonotus obliquus melanin shows a single absorption peak, with strong ultraviolet absorption, and the maximum absorption wavelength is 218 nm, which is consistent with the ultraviolet absorption characteristics of traditional melanin.

[0092] 5. Attenuated total reflection Fourier transform infrared spectroscopy

[0093] A Fourier transform infrared spectrometer with a diamond crystal ATR accessory and a pressure applicator was used to obtain the spectrum of the Inonotus obliquus melanin. Before measurement, the ATR surface was cleaned with water and isopropyl alcohol, and a background measurement was collected using an empty ATR cell. The ATR-FTIR spectrum was recorded between 4000 and 650 cm -1 The results are shown in Figure 10 There is a strong and wide characteristic absorption peak at 3377 cm -1 , which corresponds to the N-H group connected to the O-H on the indole ring group; the absorption peak at 2939 cm -1 belongs to CH2 and CH3 stretching; the characteristic absorption at 1695 is related to C=O stretching or aromatic C=C stretching; the absorption peaks near 1591 cm -1 and 1501 cm -1 are related to N-H bending vibration; the peak at 1415 cm -1 is attributed to C-N stretching, indicating the presence of the typical indole structure of melanin. The peak at 1213 cm -1 is related to the COH stretching vibration of phenolic aldehyde, and the peak at 1119 cm -1 is related to the asymmetric stretching of COC. The weak absorption band at 771 cm -1 indicates that the aromatic ring has become a conjugated system. The above characteristics show that the infrared spectrum of the Inonotus obliquus melanin extracted and purified is consistent with the structural characteristics of traditional melanin.

[0094] 6. Nuclear magnetic resonance spectroscopy 1 (H NMR)

[0095] 1 The H NMR spectrum was recorded on a Bruker AVANCE III 400. The Inonotus obliquus melanin (20.0 mg) was power dissolved in 500.0 μl of deuterium oxide (D2O) and 5.0 μl of sodium deuterium oxide (NaOD), with ppm as the chemical shift. The results are shown in Figure 11As shown, the Inonotus melanin exhibited five different clear peak groups. The solvent absorption peaks were located at 0 and 4.75 ppm, while the peaks between 1 and 2.5 ppm usually corresponded to the C-H stretching vibration signals of the alkyl chain segment. The peaks between 6.8 and 8.5 ppm in the spectrum were the aromatic hydrogens on the indole or pyrrole ring, and the region between 3.5 and 4.2 ppm could be attributed to the protons on the carbon atoms coupled to nitrogen or oxygen atoms. 1 The H-NMR spectrum was similar to that of the melanin extracted from several other organisms.

[0096] 7. Alkaline hydrogen peroxide degradation test (AHPO)

[0097] The alkaline hydrogen peroxide (AHPO) method of melanin followed the method described by Ito (S. Ito, Del Bino, Hirobe, & Wakamatsu, 2020). Briefly, 100.0 ul of sample water suspension (0.1 mg HCl melanin, 0.1 mg NADES melanin) was heated with 375 ul of 1 mol / L K2CO3 and 25 ul of 30% H2O2 in a 25°C water bath for 20 h, stopped with 50 ul of 10% Na2SO3 solution, and then acidified with 140 ul of 6 mol / L HCl. After centrifugation (8000 g, 10 min), the supernatant obtained was immediately analyzed using a Waters e2695 liquid chromatography system, a Hming C18 column, and a Waters ultraviolet detector at 269 nm. 0.05 mol / l potassium phosphate buffer (pH 2.1) / methanol, 99:1 (v / v) was used as the mobile phase. The analysis temperature was 30°C, and the flow rate was 0.6 ml / min.

[0098] The standard substance liquid chromatogram is shown in Figure 12 As shown, the HCl melanin liquid chromatogram is shown in Figure 13 As shown, the NADES melanin liquid chromatogram is shown in Figure 14 According to the results, it can be concluded that the PTCA concentration of the NADES melanin group is the highest, which is 1.49 ug / ml, while the concentration of the HCl melanin group is 0.59 ug / ml. The PTCA / PDCA ratio of the NADES melanin group is 4.17, which is much higher than that of the HCl melanin group, proving that the melanin structure of the NADES melanin group is more complete.

[0099] II. Animal experiments

[0100] C57BL6 / J mice (19 ± 1 g, 6 weeks, male) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed at the Experimental Animal Center of Jiangnan University (License No. SYXK(Su)2021-0056, Wuxi, China) under room temperature (20-26 °C), constant humidity (40-70%) and light-dark cycle (12 h / 12 h) (Permit No. SYXK(Su)2021-0056, Wuxi, China). All animal treatment protocols were approved by the Experimental Animal Management and Animal Welfare Ethics Committee of Jiangnan University (JN.No20230215c0700402

[011] ).

[0101] 1. Experimental design

[0102] All mice were randomly divided into four groups (n = 8): control group (Con), model group (Mod), positive control group (Con) and Inonotus obliquus melanin group (IO-Mel). All mice were given sterile water and standard diet (Irradiated Laboratory Rat and Mouse Growth and Reproduction Diet-CRO) for one week to adapt. From the 8th to the 21st day, all mice except the control group were induced to develop colitis by drinking a 2.5% (w / v) aqueous solution of dextran sodium sulfate (DSS). At the same time, all mice received an additional 0.2 ml of tube-feeding solution, the control and model groups were given sterile normal saline, the positive control group was given mesalazine (0.52 g / kg body weight), and the extract Inonotus obliquus melanin (0.15 g / kg body weight) was administered to the Inonotus obliquus melanin group. On the 21st day of the experiment, the mice’s feces were collected, and the mice were sacrificed after a 12-hour fast. All mice were anesthetized with isoflurane before blood collection and sacrificed by cervical dislocation after blood collection. After dissection, photographs of the entire colon were taken, and the distal 1 cm of the colon was placed in a paraformaldehyde solution at room temperature. The colon tissue and intestinal contents were rapidly frozen in liquid nitrogen for subsequent analysis.

[0103] 2. Disease activity index score (DAI) and histological analysis

[0104] During the entire experimental period, the body weight, fecal consistency and severity of occult blood were observed and recorded daily. The score of disease activity index was based on Table 2. In addition, the distal colon was stained with hematoxylin and eosin, which stained the chromatin within the nucleus and the nucleic acids within the cytoplasm purple-blue, and the components in the cytoplasm and extracellular matrix red, so that the tissue could be observed for pathological analysis. The histopathological score was calculated according to Table 3.

[0105] Table 2 Disease activity index evaluation criteria table

[0106]

[0107] Table 3 Pathology score criteria

[0108]

[0109] Results as shown in Figure 12 Fig. 3, from the 3rd day of modeling, the DAI index of mice receiving DSS increased compared with the blank group mice without receiving DSS, on the 7th day, the DAI index of melanin group was significantly lower than that of the model group, and the weight change rate showed that melanin group significantly inhibited the weight loss caused by DSS, and the colon length also showed that melanin could significantly restore the shortening of colon caused by DSS, which indicated that melanin had the effect of relieving colitis in mice.

[0110] The results of tissue section are shown in Figure 13 Fig. 4, compared with the control group, the mucosal epithelium of the model group was denatured, necrotic and exfoliated, the intestinal crypt structure disappeared, the goblet cells basically disappeared, and there was obvious inflammatory cell infiltration and edema in the mucosa layer, serosa layer, submucosa layer and muscle layer; the mucosal structure of melanin group was complete, the crypt structure was clear, there was no inflammatory cell infiltration and no edema. The pathological score results showed that the control group had no lesions; compared with the control group, the score of the model group was significantly increased; compared with the model group, the score of the melanin group was significantly reduced. It showed that melanin could restore the structure of colon tissue, thereby relieving colitis in mice.

[0111] 3. Immunohistochemical analysis

[0112] Claudin-1, Occludin, ZO-1 antigens were used for immunohistochemical staining. The specific method is as follows: paraffin sections are deparaffinized to water; antigen repair is carried out in citric acid antigen repair buffer with a microwave oven, and after natural cooling, phosphate buffer solution is washed for 3 times, 5 min each time; the sections are placed in 3% hydrogen peroxide, incubated at room temperature for 25 min, and washed for 3 times, 5 min each time; 3% bovine serum albumin is added in the groupization circle to evenly cover the tissue, and incubated at room temperature for 30 min; the blocking solution is gently shaken off, and the primary antibody is added on the section, and the section is placed in a wet box for incubation at 4°C overnight; wash for 3 times, 5 min each time, and after the section is slightly shaken dry, the secondary antibody is added in the circle to cover the tissue, and incubated at room temperature for 50 min; wash for 3 times, 5 min each time, and after the section is slightly shaken dry, fresh DAB developing solution is added in the circle, and the developing time is controlled under a microscope, the positive is brownish yellow, and the section is washed with tap water to stop the developing; the cell nucleus is stained with hematoxylin; dehydrated and mounted; observed under a microscope, and the images were collected for analysis. The results are shown in Figure 14 Fig. 5, the decrease of Claudin-1, Occludin and ZO-1 in the colon tissue of melanin group was significantly inhibited, which was helpful to maintain the integrity and function of the mouse intestine, thereby relieving colitis in mice.

[0113] 4. Inflammatory cytokines

[0114] Blood samples collected from the eyes were immediately centrifuged (3000g, 5°C, 10 min) to obtain serum. Serum inflammatory cytokines (TNF-a, IL-1 b and IL-6) were then measured using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer's procedure. Results are shown in Figures 2-4. Figure 15 As shown in Figures 2-4, DSS caused an increase in serum inflammatory levels in mice, and melanin alleviated this inflammation, having an anti-inflammatory effect.

[0115] 5. Antioxidant parameters

[0116] To measure catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and myeloperoxidase (MPO) levels in the colon tissue of mice. The colon tissue was homogenized (colon tissue / PBS = 1 / 9, w / v) and centrifuged (5000g, 5 min) to obtain the supernatant. Protein concentration was then measured using a protein assay kit, and CAT, GSH, SOD and MPO levels in the colon were measured with commercial kits.

[0117] CAT results are shown in Figure 5. Figure 16 As shown in Figure 5, the levels of the antioxidant mediator CAT in the colon tissue of mice with DSS-induced colitis were relatively low compared to the blank group, and the CAT concentration after melanin administration was significantly different from the model group, and the CAT concentration returned to the level of the blank group.

[0118] GSH results are shown in Figure 6. Figure 17 As shown in Figure 6, the levels of the antioxidant mediator GSH in the colon tissue of mice with DSS-induced colitis were relatively low compared to the blank group, and the GSH content after melanin administration was significantly different from the model group, and the GSH content returned to the level of the blank group.

[0119] MPO results are shown in Figure 7. Figure 18 As shown in Figure 7, the levels of the oxidative mediator MPO in the colon tissue of mice with DSS-induced colitis were relatively high compared to the blank group, and the MPO concentration after melanin administration was significantly lower than the model group.

[0120] SOD results are shown in Figure 8. Figure 18 As shown in Figure 8, the levels of the oxidative mediator SOD in the colon tissue of mice with DSS-induced colitis were relatively high compared to the blank group, and the SOD concentration after melanin administration was significantly higher than the model group.

[0121] These research results show that Inonotus melanin can reduce oxidative stress and thus reduce the extensive tissue damage caused by DSS.

[0122] 6. Quantification of short-chain fatty acids (SCFAs)

[0123] The GC 2010 pro was used to determine the levels of short-chain fatty acids in the gut, including acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid. Prior to analysis, fecal samples were homogenized with saturated sodium chloride solution, acidified with 10% (v / v) H2SO4, spiked with 2-ethylbutyric acid (as an internal standard for calibration data), and extracted with diethyl ether. The samples were filtered using a 0.22 pm membrane filter. Results are shown in Table 1. Figure 20 As shown in Table 1, short-chain fatty acids are one of the secondary metabolites produced by potentially beneficial gut bacteria, which have been reported to play a stabilizing role on colonic homeostasis. The levels of all six SCFAs decreased with the administration of DSS. In contrast, the melanoidin diet increased the levels of short-chain fatty acids in colitic mice (except isobutyric acid), and the total short-chain fatty acid content levels in the melanoidin group returned to the levels of the blank group. Melanoidin successfully increased the production of short-chain fatty acids, which helped to alleviate colitis.

[0124] The above merely preferred embodiments of the present application, not the limitation of the present application, the present application is not limited to the above examples, the person skilled in the art, within the scope of the present application, the changes, modifications, additions or substitutions, also should belong to the protection scope of the present application.

Claims

1. A method for extracting chaga melanin from a deep eutectic solution, characterized by, Comprising the following steps: (1) adding Inonotus obliquus powder into deep eutectic solution for extraction, collecting the precipitate after the extraction is completed; (2) washing the precipitate in step (1) with water in neutral condition, and then washing the precipitate with chloroform, ethyl acetate, anhydrous ethanol and pure water in sequence until the supernatant is clear and transparent, collecting the precipitate and drying to obtain Inonotus obliquus melanin powder; In step (1), the deep eutectic solution comprises choline chloride and oxalic acid, and the molar ratio of the choline chloride to the oxalic acid is 3:2; the ratio of the Inonotus obliquus powder to the deep eutectic solution is 150 mg / mL; The Inonotus obliquus powder is Inonotus obliquus water extract powder, and the preparation method of the Inonotus obliquus water extract powder is as follows: S1, grinding and sieving Inonotus obliquus fruiting bodies to obtain ground powder; S2, dissolving the ground powder in S1 in pure water for extraction, and then centrifuging and removing the precipitate to obtain an extract; S3, concentrating and drying the extract in S2 to obtain Inonotus obliquus water extract powder.

2. The method of claim 1, wherein, In S2, the solid-liquid ratio of the ground powder to pure water is 1 g: (10-50) mL, the extraction is performed for 1-3 times, the extraction time is 1-2 h each time, and the extraction temperature is 40-100℃.

3. The method of claim 1, wherein, In step (1), the extraction temperature is 30-70℃, and the extraction time is 2-6 h.

4. Inonotus obliquus-derived melanin prepared by the method of any one of claims 1-3.

5. Use of the method of any one of claims 1-3 or the Inonotus obliquus-derived melanin of claim 4 in the preparation of a drug for relieving and / or treating intestinal inflammation.

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