CN117487376A - Method for extracting melanin from inonotus obliquus by deep eutectic solution - Google Patents
Method for extracting melanin from inonotus obliquus by deep eutectic solution Download PDFInfo
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- CN117487376A CN117487376A CN202311417898.8A CN202311417898A CN117487376A CN 117487376 A CN117487376 A CN 117487376A CN 202311417898 A CN202311417898 A CN 202311417898A CN 117487376 A CN117487376 A CN 117487376A
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- Prior art keywords
- melanin
- inonotus obliquus
- extraction
- powder
- acid
- Prior art date
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses a method for extracting melanin from inonotus obliquus by using deep eutectic solution, and belongs to the field of functional foods. The melanin yield is 13.75%, the PTCA/PDCA of the melanin is 4.17, and compared with the conventional method, the melanin obtained by the method is more complete, namely 1.71. In addition, the inonotus obliquus melanin obtained by the method has good thermal stability, light stability and alkaline stability, and the stability of the melanin is not obviously influenced by metal ions. Meanwhile, the melanin extracted from the inonotus obliquus is an endogenous substance of organisms, is safe and nontoxic, and has great application potential in the treatment of intestinal injury diseases. In vivo experiments prove that the melanin extracted from the inonotus obliquus can restore the damaged colon tissue structure, is beneficial to maintaining the integrity and the function of the intestinal tract of a mouse, inhibits inflammation, relieves oxidative stress, improves the level of short chain fatty acid, and has remarkable treatment effect on colonitis.
Description
Technical Field
The invention relates to a method for extracting melanin, in particular to a method for extracting melanin from inonotus obliquus by using deep eutectic solution, belonging to the field of functional foods.
Background
Inonotus obliquus, also known as chaga, english name Inonotus obliquus, belongs to the phylum of fungi, basidiomycotina, class Hymenomycetes, order Phlebopulariales, family Polyporaceae and genus Phlebopus, and its sclerotium shows tumor shape, has irregular groove mark on its surface, is generally dark brown or black, has no handle, has diameter of 25 cm-40 cm, has a fertility part of 5 mm thickness, has a fungus tube of 3-10 mm, and has brown inside. The spores are widely in a broad oval shape or an egg shape, smooth, 9-10 micrometers x (5.5-6.5) micrometers and have bristles, and the inonotus obliquus is a wood rot fungus growing in cold areas. The fungus is widely distributed in Nordic countries such as Norway, finland, polish, etc., and also in Japan and China in Heilongjiang. It mainly grows on dead dry trunk of silver birch, white birch, elm, etc., and its sclerotium can survive on the dry trunk for about 6 years. Is rich in triterpene, polysaccharide, melanin, saponins, polyphenol and other active substances required by human body, has rich nutritive value, and has obvious effects of resisting cancer, resisting tumor, reducing blood sugar, reducing blood lipid, resisting oxidation and enhancing human immunity.
Melanin is a common biological pigment, is a biological macromolecule polymerized by indoles and phenols, is easily combined with proteins, grease and polysaccharides, and is insoluble in water, acid solution and most organic solvents. The natural melanin has wide sources, and related research reports on melanin are available in animals, plants and microorganisms. Melanin has physiological activities such as anti-tumor, anti-radiation, immunity improvement and the like, so the application value of the melanin in medicines, cosmetics, biological materials and the like is self-evident.
At present, most of inonotus obliquus processing modes mainly include primary processing modes such as drying and the like. The deep processing of inonotus obliquus has been focused on polysaccharides, and there have been very few studies on melanin. Because melanin is easily soluble in alkali, and can form precipitate under acidic condition, alkali-based acid precipitation is a common method for extracting melanin. However, the conventional alkali-dissolution and acid-precipitation method requires long time and is inefficient, and the decarboxylation reaction occurs, so that the structure of melanin is destroyed in the extraction process. In addition, melanin is difficult to purify due to its tendency to bind to proteins, oils and polysaccharides, as well as its heterogeneity and heterogeneity. The extraction method of melanin depends on the type, source, site of generation, content of melanin and its impurity content. How to extract and purify the melanin in the inonotus obliquus so as to realize the extraction and development of the inonotus obliquus black is a technical problem which needs to be solved at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for extracting melanin in inonotus obliquus by using a deep eutectic solution, which aims to solve the technical problems that the traditional extraction method is long in time and low in efficiency, and the structure of the melanin is damaged in the extraction process and the subsequent purification difficulty is high, so that the purification of the melanin in the inonotus obliquus is limited.
The first technical scheme provided by the invention is a method for extracting inonotus obliquus melanin by using a deep eutectic solution, which comprises the following steps:
(1) Adding inonotus obliquus powder into deep eutectic solution, extracting, and collecting precipitate after extraction;
(2) Washing the precipitate in the step (1) with water to neutrality, washing the precipitate with chloroform, ethyl acetate, absolute ethyl alcohol and pure water in turn until the supernatant is clear and transparent, collecting the precipitate, and drying to obtain inonotus obliquus melanin powder.
In certain embodiments, in step (1), the inonotus obliquus powder is an inonotus obliquus aqueous extract powder.
Further, the preparation method of the inonotus obliquus water extract powder comprises the following steps: s1, grinding and sieving inonotus obliquus fruiting bodies to obtain ground powder;
s2, dissolving the ground powder in the S1 in pure water for extraction, centrifuging again, and removing sediment to obtain an extract;
and S3, concentrating and drying the extract liquid in the S2 to obtain the inonotus obliquus water extract powder.
Still further, in S1, the number of the sieved mesh is 100 mesh.
Still further, in S2, the feed water ratio of the milled powder to pure water was 1g: (10-50) mL, wherein the extraction times are 1-3 times, the extraction time is 1-2 h each time, and the extraction temperature is 40-100 ℃.
Still further, in S3, the extract was concentrated to 30% of the original volume at 50 ℃ using a vacuum rotary evaporator and dried by freeze drying.
In certain embodiments, in step (1), the deep eutectic solution comprises choline chloride and a hydrogen donor selected from any one of malic acid, oxalic acid, formic acid, acetic acid, propionic acid, butyric acid, caproic acid, and citric acid.
In certain embodiments, in step (1), the deep eutectic solution comprises choline chloride and oxalic acid, wherein the molar ratio of the choline chloride to the oxalic acid is 1:2 to 2:1.
In certain embodiments, in step (1), the ratio of the inonotus obliquus powder to the deep eutectic solution is (10-200) mg/mL.
In certain embodiments, in step (1), the temperature of the extraction is from 30 to 70 ℃ and the time of the extraction is from 2 to 6 hours.
The second technical scheme provided by the invention is melanin prepared by the method of the first technical scheme.
The third technical scheme provided by the invention is a product containing the melanin according to the second technical scheme.
In certain embodiments, the product is a food product or a pharmaceutical product.
The fourth technical scheme provided by the invention is application of the method of the first technical scheme or the second technical scheme in preparation of medicines for relieving and/or treating intestinal inflammation.
Further, the pharmaceutical product comprises the above composition and a pharmaceutically acceptable carrier.
Still further, the carrier includes one or more of fillers, binders, humectants, disintegrants, lubricants, flavoring agents commonly used in medicine.
Further, the dosage forms of the medicine comprise granules, capsules, tablets, pills or oral liquids.
Still further, the medicine comprises tablets, capsules and oral liquid which are coated with enteric coatings through mouth.
The invention has the advantages and effects that:
the invention obtains the optimal extraction process of the inonotus obliquus melanin through screening of the deep eutectic solvent and a single factor test. Under the optimal process, the melanin yield is 13.75%, the PTCA/PDCA of the melanin is 4.17, the conventional method is 1.71, and compared with the conventional method, the melanin obtained by the method is more complete. Meanwhile, in the extraction process, the inonotus obliquus aqueous extract is added into the deep eutectic solution and then is directly subjected to centrifugal purification, so that the reduction of the hydrogen bond strength can be avoided, and the extraction efficiency of melanin is further improved. In addition, the inonotus obliquus melanin obtained by the invention has better thermal stability, light stability and alkaline stability, and metal ion Fe 3+ 、Al 3+ 、Mn 2+ 、Cr 2+ 、Na + 、K + 、Fe 2+ 、Cu 2+ 、Ca 2+ 、Mg 2+ And Zn 2+ Has no significant effect on the stability of melanin. Meanwhile, the melanin extracted from the inonotus obliquus is an endogenous substance of organisms, is safe and nontoxic, and has great application potential in the treatment of intestinal injury diseases. In vivo experiments prove that the melanin extracted from the inonotus obliquus can restore the damaged colon tissue structure, is beneficial to maintaining the integrity and the function of the intestinal tract of a mouse, inhibits inflammation, relieves oxidative stress, improves the level of short chain fatty acid, and has remarkable treatment effect on colonitis.
Drawings
FIG. 1 shows the extraction yields of Inonotus obliquus melanin extracted by different extraction solutions of the invention;
FIG. 2 shows the extraction yield of Inonotus obliquus melanin obtained in the chcl-oa according to the invention in different proportions;
FIG. 3 shows the extraction yield of Inonotus obliquus melanin obtained by different feed liquid ratios in chcl-oa;
FIG. 4 shows the extraction yield of Inonotus obliquus melanin obtained by different extraction temperatures in chcl-oa according to the invention;
FIG. 5 shows the extraction yields of Inonotus obliquus melanin obtained at different extraction times in chcl-oa according to the present invention;
FIG. 6 shows the temperature stability of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 7 shows the metal stability of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 8 shows the redox stability of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 9 is an ultraviolet absorption spectrum of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 10 is a Fourier infrared transform spectrum of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 11 is a nuclear magnetic resonance spectrum of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 12 is a standard substance liquid spectrum of AHPO;
FIG. 13 is a liquid phase diagram of Inonotus obliquus melanin extracted from chcl-oa of the present invention;
FIG. 14 is a liquid-phase diagram of Inonotus obliquus melanin extracted by the conventional method of the present invention;
FIG. 15 shows the DAI score, the rate of change of body weight and the colon length of mice colonitis after oral administration of melanin extraction in accordance with the present invention;
FIG. 16 is a graph of H & E staining of pathological changes in the colon of mice following oral administration of extracted melanin in accordance with the present invention;
FIG. 17 is a chart of immunohistochemical staining of the colon of mice after oral administration of extracted melanin in accordance with the present invention;
FIG. 18 shows serum inflammatory factor content of mice after oral administration of melanin extraction in accordance with the present invention;
FIG. 19 is a graph showing colon tissue catalase content of mice after oral administration of extracted melanin in accordance with the present invention;
FIG. 20 is a graph showing glutathione content of colon tissue of mice after oral administration of melanin extraction in accordance with the present invention;
FIG. 21 is a graph showing the myeloperoxidase content of colon tissue of mice after oral administration of extracted melanin in accordance with the present invention;
FIG. 22 shows superoxide dismutase content of colon tissue of mice after oral administration of extracted melanin in accordance with the present invention;
FIG. 23 shows the expression level of short chain fatty acids in cecum contents of mice after oral administration of melanin extraction in accordance with the present invention.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
1. Inonotus obliquus fruiting bodies were purchased from Dalian Siberian International trade company Limited. DSS (molecular weight = 36-50 KD) was purchased from MP Biomedicals (Irvine, CA, USA). Commercial kits of Glutathione (GSH), catalase (CAT), myeloperoxidase (MPO), and superoxide dismutase (SOD) were purchased from the institute of biotechnology (south ky, china). An enzyme-linked immunosorbent assay (ELISA) kit for determining tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) levels is available from the limited company, enzyme-linked biotechnology Co. Short Chain Fatty Acid (SCFA) standards were purchased from Shanghai microphone biochemistry limited, china. Other chemicals used were commercially available analytical grade reagents.
2. The detection method of melanin comprises the following steps: the stability of melanin was identified by thermal stability, metal stability, redox stability. And carrying out melanin structure identification by utilizing an ultraviolet-visible light spectrum, an attenuated total reflection Fourier transform infrared spectrum and a nuclear magnetic resonance spectrum. The integrity of melanin was identified using alkaline hydrogen peroxide.
3. The method for calculating the extraction rate of melanin comprises the following steps: melanin extraction = weight of melanin lyophilized powder/weight of inonotus obliquus fruiting body.
Example 1
The detailed composition information of the hydrogen donor and hydrogen acceptor in this example is shown in Table 1. All natural deep eutectic solutions were specifically prepared by mixing hydrogen donors and hydrogen acceptors according to table 1 and heating using microwaves, i.e. exposing the natural deep eutectic solution in a glass vial to 160W microwaves for 5 seconds, suspending for 5 seconds, and repeating the operation in a cycle until a clear liquid was obtained, followed by vortexing in air for cooling.
TABLE 1 different natural deep eutectic solution combinations
Preparation method of Inonotus obliquus water extract powder
Selecting the surface layer of inonotus obliquus fruiting body, grinding, and sieving with 100 mesh sieve to obtain uniform powder. Extracting 100g of powder with pure water (at a water-to-material ratio of 1:24, W/V) at 90deg.C for 1.5 hr twice, removing precipitate, concentrating the supernatant to 30% of the original volume with a vacuum rotary evaporator at 50deg.C, and freeze drying to obtain Inonotus obliquus water extract powder.
Extraction of Inonotus obliquus melanin
100.0mg of Inonotus obliquus water extract powder and 2mL of natural deep eutectic solution are subjected to water bath in a constant temperature water bath kettle at 50 ℃ for 4 hours. The precipitate was then separated by centrifugation at 10,000rpm for 15 minutes and then subjected to a purification procedure comprising multiple washes with pure water until a neutral pH was reached, and the insoluble precipitate was washed sequentially with chloroform, ethyl acetate, ethanol and pure water until the supernatant was clear and transparent. And centrifuging the precipitate and drying in a freeze dryer to obtain Inonotus obliquus melanin, called NADES melanin for short.
As shown in FIG. 1, 11 natural deep eutectic solutions have extraction effects on inonotus obliquus melanin, and the extraction rate of other combinations on melanin is higher than 6% except for choline chloride-urea combination, choline chloride-fructose combination and choline chloride-glycerol combination. Among them, the combination of choline chloride-oxalic acid has the highest extraction efficiency for melanin.
Example 2
The natural deep eutectic composition ratio, the feed liquid ratio, the extraction temperature and the extraction time were selected, and the influence of 4 factors on the yield of inonotus obliquus melanin was analyzed according to the extraction method melanin extraction process flow of example 1.
1. Natural deep eutectic composition ratio
Setting different natural deep eutectic composition ratios (ChCl/Oa, mol/mol) of 2:1, 3:2, 1:1, 2:3 and 1:2 under the fixed conditions of 50:1mg/mL liquid-material ratio, 50 ℃ extraction temperature and 4h extraction time, and examining the influence of the natural deep eutectic composition ratios on the yield of inonotus obliquus melanin. As shown in FIG. 2, the ratio of deep eutectic composition (ChCl/Oa, mol/mol) was 3:2, and the yield of melanin was highest and 11.95% at the highest
2. The feed liquid ratio is set to 10, 50, 100, 150 and 200 of different feed liquid ratios (mg/ml) under the fixed conditions that the natural deep eutectic composition ratio is 1:1 (ChCl/Oa, mol/mol), the extraction temperature is 50 ℃ and the extraction time is 4 hours, and the influence of the single factor feed liquid ratio on the melanin yield of the inonotus obliquus is examined. As a result, as shown in FIG. 3, the extraction rate of melanin increased with increasing feed liquid ratio (mg/ml) from 10 to 150, and decreased at 200, so that the extraction rate of melanin was maximum at 150 feed liquid ratio (mg/ml) and at most 12.79%.
3. Extraction temperature
Under the fixed conditions of natural deep eutectic composition ratio of 1:1 (ChCl/Oa, mol/mol), liquid-material ratio of 50:1mg/mL and extraction time of 4 hours, different extraction temperatures of 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ are set, and the influence of single-factor extraction temperature on the melanin yield of the inonotus obliquus is examined. As a result, as shown in FIG. 4, the extraction rate increased with an increase in temperature at 30 to 600℃and then the extraction rate did not increase with an increase in temperature, so 60℃was selected as the optimum extraction temperature, and the maximum was 11.14%.
4. Extraction time
Under the fixed conditions of natural deep eutectic composition ratio of 1:1 (ChCl/Oa, mol/mol), liquid-material ratio of 50:1mg/mL and extraction temperature of 50 ℃, different extraction times of 2h, 3h, 4h, 5h and 6h are set, and the influence of single factor extraction time on the yield of inonotus obliquus melanin is examined. As shown in fig. 5, the extraction rate was prolonged with the lapse of time and then decreased in 2 to 5 hours, so that the melanin extraction rate was highest and 11.14% at 5 hours.
5. Optimum condition extraction
Two extraction tests were performed under optimal conditions, i.e. a natural deep eutectic composition ratio of 3:2 (ChCl/Oa, mol/mol), a liquid-to-material ratio of 150:1mg/mL, an extraction temperature of 60℃and an extraction time of 5 h. The first batch was centrifuged at 10,000rpm for 15 minutes after the completion of the extraction to separate the precipitate, and freeze-dried directly to obtain a crude melanin extract with an extraction yield of 15.21%, and the second batch was centrifuged at 10,000rpm for 15 minutes after the completion of the extraction to separate the precipitate, and then subjected to a purification procedure comprising washing with pure water a plurality of times until a neutral pH was reached, and washing the insoluble precipitate with chloroform, ethyl acetate, ethanol and pure water in this order until the supernatant was clear and transparent. And centrifuging the precipitate and drying in a freeze dryer to obtain Inonotus obliquus melanin, called NADES melanin for short, with NADES melanin extraction rate of 13.75%.
Since discovery, the deep eutectic solution is praised for its superior extraction performance, and in the process of extracting melanin from the deep eutectic solution, a complex hydrogen bonding principle is involved, and the hydrogen bonding can enable structural components (chitin and protein) of fungal cell walls to be dissolved in a solvent. This dissolution promotes separation of melanin from other materials and thus precipitates out. With the decrease in hydrogen bond strength, the separated melanin is polymerized again with the structural component and is dissolved in water again. In the prior art, in order to improve the fluidity of the deep eutectic solution, solutions such as pure water and ethanol are often added at the end of extraction, but such treatment can reduce the hydrogen bond strength of the system and affect the extraction efficiency of melanin. In the extraction process, the invention adds the inonotus obliquus water extract into the deep eutectic solution, and then directly carries out centrifugal purification to obtain melanin. Therefore, the reduction of the strength of hydrogen bonds can be avoided, and the extraction efficiency of melanin is greatly improved.
Comparative example 1
Conventional high-temperature extraction with strong acid
100.0mg of Inonotus obliquus water extract powder was water-bathed with 2mL of 6M HCl solution in a water bath at 100deg.C for 10h. The precipitate was then isolated by centrifugation at 10,000rpm for 15 minutes, and then subjected to a purification procedure including washing with purified water multiple times until a neutral pH was reached, and insoluble precipitate was washed sequentially with chloroform, ethyl acetate, ethanol and purified water until the supernatant was clear and transparent. And centrifuging the precipitate and drying in a freeze dryer to obtain Inonotus obliquus melanin, called HCl melanin for short. The extraction rate was 6.43%. The conventional extraction method has long time consumption, needs to maintain high temperature for a long time, has large energy consumption, causes pollution to the environment due to the application of strong acid, and is not beneficial to sustainable development. Meanwhile, the extraction rate is low, and the extraction efficiency is low.
Test case
1. Performance identification of Inonotus obliquus melanin
1. Thermal stability
1.0mg of Inonotus obliquus melanin is dissolved in 10mL of 0.1mol/L NaOH solution and then incubated at 20, 40, 60, 80 and 100 ℃. Samples were taken once an hour, cooled to room temperature, and analyzed for absorbance at 218nm using an ultraviolet-visible spectrophotometer containing 0.1mol/L NaOH reference solution. As shown in FIG. 6, the absorbance of the Inonotus obliquus melanin did not significantly decrease, indicating that the Inonotus obliquus melanin has good thermal stability.
2. Stability of metal ions
To determine how metal ions affect stability of inonotus obliquus melanin, 1.0mg of the power compound was thoroughly mixed with 10mL of a 0.1mol/L NaOH solution containing the following additional 0.01mol/L metal ions: fe (Fe) 3+ ，Ca 2 + ,Cr 3+ ,Zn 2+ ,Al 3+ ,Mn 2+ ,Mg 2+ ,Na + ,K + ,Cu 2+ ,Fe 2+ And Ba (beta) 2+ . Samples were taken every 12 hours and absorbance was measured at 218 nm. As shown in FIG. 7, the absorbance of Inonotus obliquus melanin was not associated with the absorbance of the metal ion-added Inonotus obliquus melanin, as compared with the blankThe time is changed obviously, so that the added metal has no influence on the stability of the inonotus obliquus melanin.
3. Redox attribute
Different volumes (0, 1, 2, 3, 4 and 5 mL) of reducing agent (30% Na) were added to 5mL of a solution of Inonotus obliquus melanin at 0.1mg/mL, respectively 2 SO 3 ) And an oxidant (30% H) 2 O 2 ) The total volume of the solution was adjusted to 10ml with pure water. The solution was thoroughly shaken and allowed to stand for 30 minutes, and absorbance was measured at 218 nm. As a result, as shown in FIG. 8, na 2 SO 3 And H 2 O 2 The absorbance of the inonotus obliquus melanin is not influenced, which indicates that the inonotus obliquus melanin has good oxidation-reduction performance.
4. Ultraviolet-visible spectrum
Inonotus obliquus melanin is dissolved in 0.1mol/L NaOH to prepare a solution with the final concentration of 0.1 mg/mL. The ultraviolet-visible absorption spectrum of Inonotus obliquus melanin in the range of 200-800nm was measured using an ultraviolet-visible spectrophotometer based on 0.1mol/L NaOH solution. As shown in FIG. 9, the ultraviolet-visible spectrum of Inonotus obliquus melanin shows a single absorption peak, has strong ultraviolet absorption, has a maximum absorption wavelength of 218nm, and accords with the ultraviolet absorption characteristics of the traditional melanin.
5. Attenuated total reflection Fourier transform infrared spectra
Fourier transform infrared spectrometer with diamond crystal ATR attachment and pressure applicator was used to obtain the spectrum of inonotus obliquus melanin. The ATR surface was cleaned with water and isopropanol prior to measurement and background measurements were collected using an empty ATR cell. ATR-FTIR spectra were recorded at 4000 to 650cm -1 Between them. As a result, as shown in FIG. 10, at 3377cm -1 The position has a strong and wide characteristic absorption peak, and corresponds to an N-H group connected with O-H on an indole ring group; 2939cm -1 Absorption peaks at the sites belong to the extension of CH2 and CH 3; the feature at 1695 is related to c=o expansion or aromatic c=c expansion; 1591cm -1 ，1501cm -1 The nearby absorption peak N-H bending vibration is related; 1415cm -1 The peak at this point was due to C-N stretch, indicating the presence of typical indoles of melaninAn indole structure. 1213cm -1 The peak at the position is related to the expansion and contraction vibration of the phenolic COH, 1119cm -1 The peak at which is related to asymmetric extension of COC. 771cm -1 The weak absorption band at this point indicates that the aromatic ring has become the conjugated system. The characteristics show that the infrared spectrum of the inonotus obliquus melanin obtained by extraction and purification accords with the structural characteristics of the traditional melanin.
6. Nuclear magnetic resonance spectrum [ ] 1 H NMR)
1 H NMR spectra were recorded on bruck AVANCE III 400. Inonotus obliquus melanin (20.0 mg) was power-dissolved in 500.0 μl deuterium oxide (D2O) and 5.0 μl sodium deuterium oxide (NaOD) with ppm as chemical shift. The results are shown in FIG. 11, in which Inonotus obliquus melanin exhibits five distinct peak groups. The solvent absorption peaks are at 0 and 4.75ppm, while peaks between 1 and 2.5ppm generally correspond to the peaks of the C-H tensile vibration signal of the alkyl segment. The peak between 6.8 and 8.5ppm in the spectrum is aromatic hydrogen on the indole or pyrrole ring, and the region between 3.5 and 4.2ppm is attributable to protons on carbon atoms coupled with nitrogen or oxygen atoms. Melanin pigment 1 The H-NMR spectrum is similar to that of melanin extracted from several other organisms.
7. Alkaline peroxide degradation test (AHPO)
The Alkaline Hydrogen Peroxide (AHPO) method of melanin follows the protocol of Ito (s.ito, del Bino, hirobe,&an amp; wakamatsu, 2020). Briefly, 100.0ul of the aqueous sample suspension (0.1 mg HCl melanin, 0.1mg NADES melanin) was combined with 375ul of 1mol/L K 2 CO 3 And 25ul 30% H 2 O 2 Heating in 25 deg.C water bath for 20 hr, stopping 50ul of 10% Na 2 SO 3 The solution was then acidified with 140ul of 6mol/L HCl. Immediately after centrifugation (8000 g,10 min) the supernatant was subjected to a Waters e2695 liquid chromatography system, a Hming C18 column and a Waters UV detector at 269 nm. As mobile phase, 0.05mol/l potassium phosphate buffer (pH 2.1)/methanol, 99:1 (v/v) was used. The analysis temperature was 30℃and the flow rate was 0.6ml/min.
The liquid phase spectrum of the standard substance is shown in FIG. 12, the liquid phase spectrum of HCl melanin is shown in FIG. 13, and the liquid phase spectrum of NADES melanin is shown in FIG. 14. From the results, it can be seen that the PTCA concentration of the NADES melanin group was highest, 1.49ug/ml, and that of the HCl melanin group was 0.59ug/ml. The NADES melanin group had a PTCA/PDCA ratio of 4.17, which was much higher than that of the HCl melanin group, demonstrating that the melanin structure of the NADES melanin group was more complete.
2. Animal experiment
C57BL6/J mice (19.+ -.1 g,6 weeks, male) were purchased from Experimental animal technologies Inc. of Leishuhua, beijing. They were kept at the laboratory animal center of university of Jiangnan (license number: SYXK (Su) 2021-0056, tin-free, china) at room temperature (20-26 ℃), constant humidity (40-70%) and light and dark cycles (12 h/12 h). All animal treatment protocols were approved by the university of Jiangnan laboratory animal management and animal welfare Committee (JN.No20230215 c0700402[011 ]).
1. Test design
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 (irradiation laboratory rats and mice growth and reproduction diet-CRO) to accommodate one week. On days 8-21, all mice except the control group induced colitis by drinking 2.5% (w/v) aqueous sodium dextran sulfate (DSS). At the same time, all mice received an additional 0.2ml of tube feeding solution, the control and model groups were given sterile saline, the positive control group was given mesalamine (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 day 21 of the experiment, mouse faeces were collected and mice were sacrificed 12 hours after fasting. All mice were anesthetized with isoflurane prior to blood collection and sacrificed by cervical dislocation after blood collection. After dissection, a photograph of the entire colon was taken and the distal end of the colon was placed in paraformaldehyde solution at room temperature for 1 cm. Colon tissue and intestinal contents were flash frozen in liquid nitrogen for subsequent analysis.
2. Disease Activity index score (DAI) and histological analysis
Throughout the experiment, the weight, fecal consistency and severity of occult blood were observed and recorded daily. The score for the disease activity index is based on table 2. In addition, the distal colon is stained with hematoxylin and eosin, the chromatin in the nucleus and nucleic acids in the cytoplasm are bluish-colored, and the components in the cytoplasm and extracellular matrix are reddish, so that the tissues can be observed for pathological analysis. Histopathological scores were calculated according to table 3.
TABLE 2 disease Activity index evaluation criteria Table
TABLE 3 pathology scoring criteria
The results are shown in fig. 12, and from the modeling of 3d, the DAI index of mice receiving DSS increased compared to the mice in the blank group not receiving DSS, and at 7d, the DAI index of the melanin group decreased significantly compared to the model group, and at the same time, the body weight decrease caused by DSS was significantly inhibited by the melanin group, and the colon length also showed that melanin was able to significantly restore the colon shortening caused by DSS, which indicated that melanin had the efficacy of relieving colitis in mice.
The results of the tissue section are shown in fig. 13, the colon mucosa structure and the crypt structure of the blank group are complete, a large number of cup-shaped cells exist, and the inflammatory cell infiltration phenomenon is avoided; compared with the control group, the mucosa epithelium of the model group is denatured, necrotized and shed, the intestinal crypt structure is disappeared, the goblet cells are basically disappeared, and the mucosa layer, serosa layer, submucosa and myolayer have obvious inflammatory cell infiltration and edema; the melanotic mucosa has complete structure, clear crypt structure, no inflammatory cell infiltration and no edema. The pathological scoring result shows that the control group has no pathological changes; the model component number was significantly increased compared to the control group; the melanin fraction was significantly reduced compared to the model group. It is shown that melanin is able to restore colonic tissue architecture, thereby alleviating colitis in mice.
3. Immunohistochemical analysis
Immunohistochemical staining was performed using Claudin-1, occludin, ZO-1 antigen. The specific method is that paraffin sections are dewaxed to water; in a citric acid antigen retrieval buffer solution, carrying out antigen retrieval by a microwave oven, naturally cooling, and washing for 3 times by a phosphate buffer solution for 5min each time; placing the slices into 3% hydrogen peroxide, incubating for 25min at room temperature in a dark place, and washing for 3 times for 5min each time; dripping 3% bovine serum albumin into the organized ring to uniformly cover tissues, and sealing at room temperature for 30min; gently throwing away the sealing liquid, dripping the primary antibody on the slice, and placing the slice in a wet box for incubation at 4 ℃ for overnight; washing for 3 times, each time for 5min, dripping secondary antibody into the ring to cover the tissue after slicing and spin-drying, and incubating for 50min at room temperature; washing for 3 times, each time for 5min, dripping freshly prepared DAB color development liquid into the ring after the slices are slightly dried, controlling the color development time under a microscope, and washing the slices with tap water to terminate the color development, wherein the positive color is brown; counterstaining the nuclei with hematoxylin; removing the water sealing sheet; microscopic examination, image acquisition and analysis. As shown in FIG. 14, the decrease in Claudin-1, occidin and ZO-1 in the colon tissue of the melanoset was significantly inhibited, thus helping to maintain the integrity and function of the intestinal tract of the mice, thereby alleviating colitis in the mice.
4. Inflammatory cytokines
Immediately, blood samples collected from the eyes were centrifuged (3000 g,5 ℃,10 minutes) to obtain serum. Serum inflammatory cytokines (TNF- α, IL-1β and IL-6) were then measured using enzyme-linked immunosorbent (ELISA) assay kits following the manufacturer's procedure. As shown in FIG. 15, DSS resulted in elevated serum inflammatory levels in mice, and melanin was able to alleviate such inflammation, with anti-inflammatory effects.
5. Antioxidant parameter
To measure Catalase (CAT), glutathione (GSH), superoxide dismutase (SOD) and Myeloperoxidase (MPO) levels in mouse colon tissue. Colonic tissue was homogenized (colonic tissue/pbs=1/9,w/v) and centrifuged (5000 g,5 min) to obtain a supernatant. Protein concentration was then measured using a protein assay kit and CAT, GSH, SOD and MPO levels in the colon were measured using commercial kits.
CAT results as shown in fig. 16, the levels of antioxidant mediator CAT in colon tissue were relatively low in DSS-induced colitis mice compared to the blank, CAT concentration was significantly different from that in the model group after melanin administration, and CAT concentration was restored to the level of the blank group.
GSH results as shown in fig. 17, the levels of antioxidant mediator GSH in colon tissue were relatively low in DSS-induced colitis mice compared to the blank, the GSH levels were significantly different from the model group after melanin administration, and the GSH levels were restored to the levels of the blank group.
MPO results as shown in fig. 18, the levels of oxidative mediator MPO in colon tissue were relatively high in DSS-induced colitis mice compared to the blank group, and MPO concentration was significantly reduced after melanin administration compared to the model group.
SOD results as shown in figure 18, the oxidative mediator SOD levels in colon tissue were relatively high in DSS-induced colitis mice compared to the blank group, and SOD concentrations were significantly elevated after melanin administration compared to the model group.
These findings indicate that inonotus obliquus melanin can reduce oxidative stress and thus reduce extensive tissue damage caused by DSS.
6. Quantification of Short Chain Fatty Acids (SCFAs)
GC 2010pro is 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. Before analysis, the fecal sample was homogenized with saturated sodium chloride solution, with 10% (v/v) H 2 SO 4 Acidify, add 2-ethylbutyric acid (as an internal standard for calibration data) and extract with diethyl ether. The sample was filtered using a 0.22 μm membrane filter. As a result, as shown in fig. 20, short chain fatty acids are one of the secondary metabolites produced by potentially beneficial intestinal bacteria, and are reported to stabilize colonic homeostasis. The levels of all six SCFAs decreased with DSS administration. In contrast, the melanin diet increased the short chain fatty acid levels in the mice with gastroenteritis (except isobutyric acid) and the total short chain fatty acid content level in the melanin group was restored to the blank group level. Melanin successfully increases the yield of short chain fatty acids, which helps to alleviate colitis.
The above preferred embodiments of the present invention are not limited to the above examples, and the present invention is not limited to the above examples, but can be modified, added or replaced by those skilled in the art within the spirit and scope of the present invention.
Claims (10)
1. A method for extracting inonotus obliquus melanin by using a deep eutectic solution, which is characterized by comprising the following steps of:
(1) Adding inonotus obliquus powder into deep eutectic solution, extracting, and collecting precipitate after extraction;
(2) Washing the precipitate in the step (1) with water to neutrality, washing the precipitate with chloroform, ethyl acetate, absolute ethyl alcohol and pure water in turn until the supernatant is clear and transparent, collecting the precipitate, and drying to obtain inonotus obliquus melanin powder.
2. The method of claim 1, wherein in step (1), the inonotus obliquus powder is an inonotus obliquus aqueous extract powder.
3. The method according to claim 2, wherein the method for preparing the inonotus obliquus aqueous extract powder comprises the following steps:
s1, grinding and sieving inonotus obliquus fruiting bodies to obtain ground powder;
s2, dissolving the ground powder in the S1 in pure water for extraction, centrifuging again, and removing sediment to obtain an extract;
and S3, concentrating and drying the extract liquid in the S2 to obtain the inonotus obliquus water extract powder.
4. A method according to claim 3, wherein in S2 the ratio of ground powder to pure water is 1g: (10-50) mL, wherein the extraction times are 1-3 times, the extraction time is 1-2 h each time, and the extraction temperature is 40-100 ℃.
5. The method of claim 1, wherein in step (1), the deep eutectic solution comprises choline chloride and a hydrogen donor selected from any one of malic acid, oxalic acid, formic acid, acetic acid, propionic acid, butyric acid, caproic acid, and citric acid.
6. The method of claim 1 or 5, wherein in step (1), the deep eutectic solution comprises choline chloride and oxalic acid, wherein the molar ratio of choline chloride to oxalic acid is 1:2-2:1; the ratio of the inonotus obliquus powder to the deep eutectic solution is (10-200) mg/mL.
7. The method according to claim 1, wherein in the step (1), the extraction temperature is 30 to 70 ℃ and the extraction time is 2 to 6 hours.
8. Melanin prepared by the method of any one of claims 1-7.
9. A product comprising the melanin of claim 8.
10. Use of a method according to any one of claims 1 to 7 or a melanin according to claim 8 for the preparation of a medicament for the alleviation and/or treatment of intestinal inflammation.
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