Anti-inflammatory active melanoidin based on black American ginseng as well as preparation method and application of anti-inflammatory active melanoidin

Abstract

The invention belongs to the technical field of extraction and purification of natural active substances, and discloses an anti-inflammatory active melanoidin based on black American ginseng as well as a preparation method and application of the anti-inflammatory active melanoidin, and the black American ginseng melanoidin with anti-inflammatory activity is obtained by performing separation and purification by utilizing the metal chelating property of the melanoidin. The content of the anti-inflammatory active melanoidins is 63.29-73.52 g / 100g, the molecular weight distribution range is 4.6 * 10 < 2 >-2.49 * 10 < 4 > Da, the average molecular weight is 17.46 kDa, and the anti-inflammatory active melanoidins are of uniform rod-shaped and sheet-shaped structures. A skeleton in the black American ginseng melanoidin with anti-inflammatory activity is rich in C = O, C-N and N-H groups, characteristic absorption peaks of amide, carboxyl and the like appear at 1600cm <-1 > of a separated product, the influence of the separated product on LPS-induced Caco-2 colon cancer cells is researched through an in-vitro cell experiment, and a research result shows that the melanoidin isolate has a relatively remarkable anti-inflammatory effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and purification of natural active substances, and in particular relates to an anti-inflammatory active melanoidin based on black American ginseng, and a preparation method and application thereof. Background Art

[0002] Melanoidins are a class of complex high-molecular-weight compounds, mainly formed in the final stage of the Maillard reaction. They are widely present in a variety of foods and medicinal materials, such as coffee, black tea, black garlic, fermented foods, etc., endowing them with unique colors, flavors and biological activities. In recent years, with the in-depth study of Maillard reaction products, melanoidins have received extensive attention in the fields of food science and biomedicine due to their excellent antioxidant, antibacterial, anti-inflammatory and intestinal health regulatory functions.

[0003] Inflammation is a defense mechanism of the body against infection, injury and other stimuli, but chronic inflammation may lead to tissue damage and is closely related to chronic diseases such as diabetes, cardiovascular diseases, neurodegenerative diseases, etc. Research shows that melanoidins can play an anti-inflammatory role in in vitro and in vivo experiments, and its mechanism of action mainly includes: inhibiting the release of pro-inflammatory factors, inhibiting oxidative stress and regulating inflammatory signaling pathways. And inflammatory mediators refer to a class of bioactive molecules released by cells during the inflammatory process, which can regulate immune responses, cell communication and tissue repair. According to their chemical structures and biological functions, inflammatory mediators mainly include cytokines, chemokines, arachidonic acid derivatives (such as prostaglandins and leukotrienes), and nitric oxide (NO), etc. Different types of inflammatory mediators may have pro-inflammatory or anti-inflammatory effects in different pathological environments, affecting the duration and severity of inflammation. In in vitro cell experiments, black garlic melanoidins can significantly inhibit the inflammatory response of macrophages induced by LPS and reduce the expression of TNF-α, IL-6 and IL-1β. In addition, melanoidins can also reduce the transcription and release of inflammatory factors by inhibiting the NF-κB and MAPK signaling pathways, thereby reducing the inflammatory response. Moreover, tea melanoidins can reduce the expression of COX-2 and iNOS, reduce the synthesis of prostaglandin E2 (PGE2), and relieve the symptoms of inflammation-related diseases. The intake of melanoidins can significantly reduce the serum C-reactive protein (CRP) level in an inflammatory mouse model and reduce oxidative damage to the liver and intestine. In addition, the research on melanoidins in arthritis and neuroinflammation also shows potential application value, which makes it an important candidate for natural anti-inflammatory foods or drugs.

[0004] The melanoidin extraction method is often applied before separation and purification. According to the principle that the solubilities of various components in a sample are different in a specific solvent, a suitable organic solvent is selected to separate melanoidins. Currently, relatively common organic solvents include methanol, ethanol, ether, chloroform, acetone, etc. When choosing an organic solvent, special attention should be paid to its toxicity and safety. In addition, the solvent concentration range also needs to be experimentally determined. If the concentration is too low, the extraction efficiency will decrease; if the solvent concentration is too high, the extraction time of melanoidins will be correspondingly prolonged, and the problem of increased leached impurities will occur, and the leached melanoidins will inhibit the substances that have not been leached to a certain extent. The ethanol extraction method is a method of separating and purifying substances using an ethanol solution as a solvent, which is relatively common in the extraction of melanoidins. An ethanol solution with an appropriate volume fraction promotes the extraction of melanoidins, is beneficial to the stability of the structure and function of melanoidins, and can also precipitate some water-soluble proteins and impurities, making the extracted melanoidins purer.

[0005] The separation and purification of melanoidins are mainly carried out according to the molecular weight and polarity. These separation methods mainly include dialysis, ultrafiltration, size exclusion chromatography, macroporous resin separation, high performance liquid chromatography, etc. Among various different separation and purification methods, dialysis and ultrafiltration are the simplest and most efficient methods, and are often used to separate and purify active substances. The principle of the dialysis method is to separate small molecules and large molecules according to the permeability of molecules in a solution with different molecular weights in a semipermeable membrane, which has the advantage of convenient operation. Macroporous resin has the advantages of good selectivity, fast adsorption speed and short time, and gel chromatography is widely used in the purification of substances. It separates according to the size and shape of the substances to be purified. Hydrophilic gel is an inert carrier, without charge, and various micropores of different sizes are distributed on the gel surface. When the sample is injected into the chromatography column, the macromolecules that cannot enter the micropores are eluted first, and the small molecules that enter the micropores are eluted later.

[0006] Therefore, the use of multiple separation and purification technologies can simply and efficiently separate and extract melanoidins from food materials, separate melanoidins with different polarities and molecular weights, improve the yield of food melanoidins, shorten the extraction time of melanoidins, increase the extraction efficiency, reduce the damage to the structure or activity of melanoidins, and be more suitable for large-scale production, which has received wide attention.

[0007] Therefore, the development of an anti-inflammatory active melanoidin based on black American ginseng and its preparation method and application are of great significance for solving many problems existing in technology. Summary of the Invention

[0008] The purpose of the present invention is to overcome the deficiencies in the prior art and provide an anti-inflammatory active melanoidin based on black American ginseng and its preparation method and application.

[0009] The technical solution adopted by the present invention to solve its technical problems is:

[0010] An anti-inflammatory melanoidin based on black American ginseng, which is prepared by metal chelation separation and purification and has the following characteristics:

[0011] (i) The content of anti-inflammatory melanoidin is 63.29 - 73.52 g / 100 g;

[0012] (ii) The molecular weight distribution range of the anti-inflammatory melanoidin is 4.6×10 2 ~2.49×10 4 Da;

[0013] (iii) The anti-inflammatory melanoidin presents a uniform rod-shaped and flaky microstructure;

[0014] (iv) The skeleton in the anti-inflammatory melanoidin is rich in C=O, C-N and N-H groups, and the separated product of the melanoidin shows characteristic absorption peaks of amide and carboxyl groups at 1600 cm -1 .

[0015] Furthermore, in the basic composition of the anti-inflammatory melanoidin, the content of total sugar is 13.21% - 15.55%, the content of protein is 0.89% - 1.03%, the content of lipid is 1.24% - 1.95%, and the content of ash is 3.26% - 3.68%.

[0016] Use of the anti-inflammatory melanoidin as described above as an active component in the preparation of anti-inflammatory drugs. The in vitro anti-inflammatory activity uses a colon cancer Caco-2 cell model.

[0017] Use of the anti-inflammatory melanoidin as described above as a functional ingredient in the preparation of functional foods and anti-inflammatory drugs.

[0018] The preparation method of the anti-inflammatory melanoidin as described above includes the following steps:

[0019] (1) Put American ginseng into a fluidized bed air classifier for pulverization, steam the pulverized American ginseng powder for 3 - 4 h, dry it in the sun, repeat nine times, and finally vacuum dry it at 50 - 60 °C to obtain black American ginseng powder;

[0020] (2) Add the black American ginseng powder to an ethanol solution, mix evenly, place it in a constant temperature water bath and stir magnetically for 30 - 70 min, centrifuge, repeat 2 - 5 times, combine the supernatant, concentrate it under reduced pressure, and freeze-dry to obtain a crude extract of melanoidin;

[0021] (3) Dissolve the obtained melanoidin crude extract in distilled water to obtain an extract solution, add metal chloride, stir evenly, adjust the pH value with 2M NaOH, perform magnetic stirring at room temperature and then let it stand for 12 - 14 h to form a reddish-brown precipitate, and centrifuge to collect the precipitate;

[0022] (4) Treat the obtained precipitate with a metal chelating agent, perform magnetic stirring in a 50°C water bath for 3 - 4 h, remove the added metal ions, then perform dialysis, protein removal, and vacuum concentration, and then lyophilize to obtain melanoidin;

[0023] (5) Macroporous resin purification: Dissolve the lyophilized melanoidin in ultrapure water, filter through a 0.45 μm filter membrane, adsorb it with macroporous resin, set the concentration gradient of ethanol solutions as 0, 20%, 40%, 60% for gradient elution, collect the eluate, detect the absorbance value at a wavelength of 420 nm, collect the eluate and concentrate to remove the solvent, and freeze-dry;

[0024] (6) Sephadex gel column chromatography purification: Prepare the lyophilized product obtained in step (5) into a melanoidin aqueous solution, use ultrapure water as the eluent to balance the Sephadex gel column chromatography column, load the sample on the top of the chromatography column for gradient elution, control the elution flow rate, automatically collect the eluate, measure the absorbance value of the eluate at 420 nm, and perform determination by semi-preparative high performance liquid chromatography, combine the eluates of the same fractions, perform vacuum concentration, and obtain the anti-inflammatory active melanoidin based on black American ginseng after lyophilization.

[0025] Further, in step (1), the gas flow rate of the fluidized bed is 300 - 500 m / s, the gas pressure is 0.6 - 1.2 MPa, and the particle size of the black American ginseng powder is 60 - 100 μm;

[0026] Or, in step (2), the volume concentration of the ethanol solution is 0 - 50% (the value is not 0), the material-liquid ratio of the black American ginseng powder to the ethanol solution is 1:5 - 1:25, and the water bath temperature of the constant temperature water bath is 30 - 70°C.

[0027] Further, in steps (2) and (4), the pressure for vacuum concentration is 0.05 - 0.1 MPa, and the temperature is 40 - 50°C;

[0028] Or, in step (3), the material-liquid ratio of the melanoidin crude extract to distilled water is 1:20 - 1:25; the addition amount of metal chloride in every 200 mL of the extract solution is 5 - 7 g; adjust the pH value to 3.8 - 4.2; the magnetic stirring time is 15 - 20 min.

[0029] Further, the metal chloride in step (3) includes FeCl3, CoCl2 or NiCl2.

[0030] Further, in step (4), the metal chelator is 7.5% (w / v, mass concentration) disodium ethylenediaminetetraacetate (EDTA-2Na) or tetrasodium ethylenediaminetetraacetate; the metal chelator and the addition amount are 200 mL of the metal chelator added to every 8 g of the crude melanoidin extract.

[0031] Further, in step (4), a dialysis bag with a molecular weight cut-off of 1000 Da is selected for dialysis;

[0032] Alternatively, in step (5), the macroporous resin is D101, the elution flow rate is 1 - 2.5 mL / min, and the sample loading concentration is 15 - 25 mg / mL;

[0033] Alternatively, in step (6), the Sephadex gel chromatography column is Sephadex G-50, the elution flow rate is 1 - 2 mL / min, and the sample loading concentration is 80 - 120 mg / mL.

[0034] The advantages and positive effects achieved by the present invention are as follows:

[0035] 1. The present invention uses fresh American ginseng as the raw material, pulverizes it through the fluidized bed air jet milling technology, and obtains black American ginseng powder through repeated steaming and sunning. The melanoidin is extracted by the water extraction method, and the melanoidin is separated and purified by utilizing the metal chelating property of the melanoidin. After adopting the fluidized bed air jet milling technology, the content of melanoidin in black American ginseng is significantly increased. Compared with the unseparated and purified melanoidin, the melanoidin content is increased from 42.27 g / 100 g to 73.52 g / 100 g, which is 1.7 times that of the crude melanoidin content.

[0036] 2. The present invention separates and purifies by utilizing the metal chelating property of the melanoidin. The average molecular weight of the separated product is 17.46 kDa, showing the enrichment effect of the separation process on high molecular weight components, and the molecular weight distribution range is 4.6×10 2 ~2.49×10 4 Da. In terms of the microstructure, the melanoidin before separation is transformed from an irregular granular structure into a uniform rod-like and flake-like structure, and characteristic absorption peaks such as amide and carboxyl groups appear at 1600 cm -1 for the separated product, revealing the structural characteristics of the separated product.

[0037] 3. In the present invention, melanoidins prepared by separating and purifying black American ginseng as raw materials have good anti-inflammatory activity and can significantly inhibit the secretion of inflammatory factors such as TNF-α, IL-6, and IL-1β in LPS-induced Caco-2 cells. Compared with LPS, the concentrations of TNF-α, IL-6, and IL-1β in Caco-2 cells decreased from 10.89, 7.38, and 13.77 pg / mL to 8.28, 4.83, and 6.03 pg / mL respectively, and the decrease in IL-1β was more than twice. In addition, RT-qPCR analysis further demonstrated that black American ginseng melanoidins decreased the mRNA expression levels of inflammation-related genes. Among them, the mRNA expression level of iNOS decreased from 1.65 to 0.85, nearly twice the decrease. The melanoidins obtained by metal chelation separation and purification in the present invention are used as a functional ingredient to inhibit the inflammatory response. On the one hand, it effectively increases the content of melanoidins. On the other hand, it provides a scientific basis and practical guidance for the application of melanoidins in functional foods and drugs.

[0038] 4. The present invention uses the metal chelation property of melanoidins for separation and purification to obtain black American ginseng melanoidins with anti-inflammatory activity. The content of the anti-inflammatory active melanoidins is 63.29 - 73.52 g / 100 g, the molecular weight distribution range is 0.66 - 9682.78 kDa, and the average molecular weight is 17.46 kDa, and it presents a uniform rod-like and flake-like structure; the skeleton in the anti-inflammatory active black American ginseng melanoidins is rich in C=O, C-N, and N-H groups, and characteristic absorption peaks such as amide and carboxyl groups appear at 1600 cm -1 -1 in the separated product, and the influence on LPS-induced Caco-2 colon cancer cells was explored through in vitro cell experiments. The research results show that the melanoidin isolate has a relatively significant anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the high-performance liquid chromatography chart of the purified melanoidins prepared in Example 1 of the present invention;

[0040] Figure 2 is the scanning electron microscope comparison chart of the crude melanoidins prepared in Comparative Example 1 and the purified melanoidins prepared in Example 1 of the present invention; among them, (a) and (b) represent the scanning electron microscope images of Comparative Example 1 at a magnification of 1000×, and (c) and (d) represent the scanning electron microscope images of Example 1 at a magnification of 2000×.

[0041] Figure 3 is the Fourier transform infrared spectrum comparison chart of the crude melanoidins prepared in Comparative Example 1 and the purified melanoidins prepared in Example 1 of the present invention;

[0042] Figure 4The graphs show the effects of the purified melanoidin prepared in Example 1 of the present invention on the cell viability of Caco-2 cells after culturing for 24 hours (a), 36 hours (b) and 48 hours (c);

[0043] Figure 5 The graphs show the effects of the purified melanoidin prepared in Example 1 of the present invention on TNF-α secretion (a), IL-6 secretion (b), IL-1β secretion (c), COX-2 secretion (d) and iNOS secretion (e) in LPS-induced Caco-2 cells;

[0044] Figure 6 The figure shows the effect of the purified melanoidin prepared in Example 1 of the present invention on CAT secretion (a), SOD secretion (b) and MDA secretion (c) in LPS-induced Caco-2 cells;

[0045] Figure 7 This is a graph showing the effect of the purified melanoidin prepared in Example 1 of the present invention on the mRNA expression level of related inflammatory genes in LPS-induced Caco-2 cells. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0047] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0048] An anti-inflammatory active melanoidin based on black American ginseng, wherein the anti-inflammatory active melanoidin is prepared by metal chelation separation and purification, and has the following characteristics:

[0049] (i) the content of anti-inflammatory active melanoidin is 63.29-73.52 g / 100 g;

[0050] (ii) The molecular weight distribution range of anti-inflammatory melanoidin is 4.6×10 2 ~2.49×10 4 Da;

[0051] (iii) anti-inflammatory activity melanoidins present a uniform rod- and flake-like microstructure;

[0052] (iv) Anti-inflammatory activity The backbone of melanoidin is rich in C=O, CN and NH groups. The product of isolated melanoidin has a peak at 1600 cm -1 The characteristic absorption peaks of amide and carboxyl groups appear at

[0053] Preferably, in the basic composition of the anti-inflammatory active melanoidins, the content of total sugar is 13.21% - 15.55%, the content of protein is 0.89% - 1.03%, the content of lipid is 1.24% - 1.95%, and the content of ash is 3.26% - 3.68%.

[0054] The application of the anti-inflammatory active melanoidins as described above as an active component in the preparation of anti-inflammatory drugs. The in vitro anti-inflammatory activity uses a colon cancer Caco-2 cell model.

[0055] The application of the anti-inflammatory active melanoidins as described above as a functional ingredient in the preparation of functional foods and anti-inflammatory drugs.

[0056] The preparation method of the anti-inflammatory active melanoidins as described above includes the following steps:

[0057] (1) Place American ginseng in a fluidized bed air classifier for pulverization. Steam the pulverized American ginseng powder for 3 - 4 h, dry it in the sun, repeat nine times, and finally place it in a vacuum dryer at 50 - 60 °C to obtain black American ginseng powder;

[0058] (2) Add the black American ginseng powder to an ethanol solution, mix evenly, place it in a constant temperature water bath and stir magnetically for 30 - 70 min, centrifuge, repeat 2 - 5 times, combine the supernatant and concentrate it under reduced pressure, and freeze-dry to obtain a crude melanoidin extract;

[0059] (3) Dissolve the obtained crude melanoidin extract in distilled water to obtain an extract, add metal chloride, stir evenly, adjust the pH value with 2M NaOH, stir magnetically at room temperature and then let it stand for 12 - 14 h to form a reddish-brown precipitate, and centrifuge to collect the precipitate;

[0060] (4) Treat the obtained precipitate with a metal chelating agent, stir magnetically in a water bath at 50 °C for 3 - 4 h, remove the added metal ions, then perform dialysis, protein removal, concentration under reduced pressure, and then lyophilize to obtain melanoidins;

[0061] (5) Macroporous resin purification: Dissolve the lyophilized melanoidins in ultrapure water, filter through a 0.45 μm filter membrane, adsorb with macroporous resin, set the concentration gradient of ethanol solutions to 0, 20%, 40%, 60% for gradient elution, collect the eluate, detect the absorbance value at a wavelength of 420 nm, collect the eluate and concentrate to remove the solvent, and freeze-dry;

[0062] (6) Purification by Sephadex column chromatography: The freeze-dried product obtained in step (5) was formulated into a melanoidin aqueous solution. The Sephadex column chromatography column was equilibrated with ultrapure water as the eluent. The sample was loaded onto the top of the chromatography column for gradient elution. The elution flow rate was controlled, and the eluate was automatically collected. The absorbance value of the eluate was measured at 420 nm, and it was also determined by semi-preparative high performance liquid chromatography. The eluates of the same fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain the anti-inflammatory active melanoidin based on black American ginseng.

[0063] Preferably, in step (1), the air flow rate of the fluidized bed is 300 - 500 m / s, the air pressure is 0.6 - 1.2 MPa, and the particle size of the black American ginseng powder is 60 - 100 μm;

[0064] Alternatively, in step (2), the volume concentration of the ethanol solution is 0 - 50%, and the value is not 0. The material-liquid ratio of the black American ginseng powder to the ethanol solution is 1:5 - 1:25, and the water bath temperature of the constant temperature water bath is 30 - 70 °C.

[0065] Preferably, in steps (2) and (4), the pressure for concentration under reduced pressure is 0.05 - 0.1 MPa, and the temperature is 40 - 50 °C;

[0066] Alternatively, in step (3), the material-liquid ratio of the melanoidin crude extract to distilled water is 1:20 - 1:25; the addition amount of metal chloride in every 200 mL of the extract is 5 - 7 g; the pH value is adjusted to 3.8 - 4.2; the magnetic stirring time is 15 - 20 min.

[0067] Preferably, the metal chloride in step (3) includes FeCl3, CoCl2 or NiCl2.

[0068] Preferably, in step (4), the metal chelating agent is 7.5% (w / v, mass concentration) disodium ethylenediaminetetraacetate (EDTA-2Na) or tetrasodium ethylenediaminetetraacetate; the addition amount of the metal chelating agent is 200 mL of the metal chelating agent added to every 8 g of the melanoidin crude extract.

[0069] Preferably, in step (4), a dialysis bag with a molecular weight cut-off of 1000 Da is selected for dialysis;

[0070] Alternatively, in step (5), the macroporous resin selected is D101, the elution flow rate is 1 - 2.5 mL / min, and the sample loading concentration is 15 - 25 mg / mL;

[0071] Alternatively, in step (6), the Sephadex chromatography column selected is Sephadex G-50, the elution flow rate is 1 - 2 mL / min, and the sample loading concentration is 80 - 120 mg / mL.

[0072] Specifically, the related preparation and detection are as follows:

[0073] Comparative Example 1

[0074] In this comparative example, crude melanoidins without metal chelation separation and purification were used for comparison. The specific preparation steps were as follows:

[0075] 1) Fresh American ginseng was placed in a fluidized bed air classifier mill. The air flow rate was set at 400 m / s and the air pressure was 0.8 MPa to grind the American ginseng to 60 - 70 μm. The ground American ginseng powder was steamed for 3 h, dried in the sun, and this process was repeated nine times. Finally, it was vacuum dried at 60 °C to obtain black American ginseng powder;

[0076] 2) The black American ginseng powder was mixed evenly with a 10% (v / v) ethanol solution at a solid - liquid ratio of 1:15, placed in a 55 °C water bath and magnetically stirred for 50 min, centrifuged, and this process was repeated 4 times. The supernatant was combined, concentrated under reduced pressure, and freeze - dried to obtain crude melanoidins (CMLD).

[0077] Comparative Example 2

[0078] In this comparative example, crude melanoidins prepared without fluidized bed grinding were used for comparison. The specific preparation steps were as follows:

[0079] 1) Fresh American ginseng was taken and dried at 60 °C, ground through a 60 - mesh sieve. The ground American ginseng powder was steamed for 3 h, dried in the sun, and this process was repeated nine times. Finally, it was vacuum dried at 60 °C to obtain black American ginseng powder;

[0080] 2) The black American ginseng powder was mixed evenly with a 10% (v / v) ethanol solution at a solid - liquid ratio of 1:15, placed in a 55 °C water bath and magnetically stirred for 40 min, centrifuged, and this process was repeated 2 times. The supernatant was combined, concentrated under reduced pressure to obtain a crude melanoidin extract;

[0081] 3) 8 g of the crude melanoidin extract was dissolved in 200 mL of distilled water, and 6 g of FeCl3 was added and stirred evenly. The pH value was adjusted to 4.0 with 2 M NaOH, magnetically stirred at room temperature for 15 min, and then left standing for 12 h to form a reddish - brown precipitate, which was collected by centrifugation.

[0082] 4) The obtained precipitate was treated with 200 mL of a 7.5% (w / v) ethylenediaminetetraacetic acid disodium salt (EDTA - Na) metal chelating agent, magnetically stirred at 50 °C for 3 h to remove the added iron ions, then dialyzed, deproteinized, concentrated under reduced pressure, and finally freeze - dried to obtain melanoidins.

[0083] 5) Macroporous resin purification: The melanoidins after lyophilization were redissolved in ultrapure water to prepare a melanoidin aqueous solution, filtered through a 0.45 μm filter membrane, adsorbed with macroporous resin D101, the loading concentration was 20 mg / mL, and gradient elution was carried out with ethanol solutions at concentration gradients of 0, 20%, 40%, and 60%, the elution rate was 1.5 mL / min, the eluate was collected, the absorbance value was detected at a wavelength of 420 nm, the eluate was collected and concentrated to remove the solvent, and then freeze-dried.

[0084] 6) Sephadex gel column chromatography purification: The chromatography column was equilibrated with ultrapure water as the eluent, 100 mg / mL of the melanoidins obtained in step (5) was prepared, loaded onto the top of the chromatography column for gradient elution, the elution flow rate was controlled, the eluate was automatically collected, the absorbance value of the eluate was measured at 420 nm, and semi-preparative high performance liquid chromatography was used for determination. The eluates of the same fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain the melanoidin isolate.

[0085] Example 1

[0086] This example provides a preparation method of anti-inflammatory active melanoidins based on black American ginseng. Specifically, the preparation steps are as follows:

[0087] 1) Fresh American ginseng was placed in a fluidized bed air flow mill, the air flow rate was set to 400 m / s, the air pressure was 0.8 MPa, and the American ginseng was ground to 60 - 70 μm. The ground American ginseng powder was steamed for 3 h, dried in the sun, repeated nine times, and finally vacuum dried at 60 °C to obtain black American ginseng powder;

[0088] 2) The black American ginseng powder was mixed evenly with a 10% ethanol solution by volume at a solid-liquid ratio of 1:15, placed in a 55 °C water bath and magnetically stirred for 40 min, centrifuged, repeated 2 times, the supernatant was combined and concentrated under reduced pressure, and freeze-dried to obtain the crude melanoidin extract;

[0089] 3) 8 g of the crude melanoidin extract was dissolved in 200 mL of distilled water, 6 g of FeCl3 was added, stirred evenly, the pH value was adjusted to 4.0 with 2 M NaOH, magnetically stirred at room temperature for 15 min and then left standing for 12 h to form a reddish-brown precipitate, and the precipitate was collected by centrifugation.

[0090] 4) The obtained precipitate was treated with 200 mL of 7.5% (w / v, mass concentration) ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) metal chelating agent, magnetically stirred at 50 °C for 3.5 h to remove the added iron ions, then dialyzed, deproteinized, concentrated under reduced pressure, and then freeze-dried to obtain melanoidins.

[0091] 5) Macroporous resin purification: The melanoidins after freeze-drying were redissolved with ultrapure water to prepare a melanoidin aqueous solution, filtered through a 0.45 μm filter membrane, adsorbed with macroporous resin D101, the loading concentration was 20 mg / mL, and ethanol solutions with concentration gradients of 0, 20%, 40%, and 60% were used for gradient elution, the elution rate was 1.5 mL / min, the eluate was collected, the absorbance value was detected at a wavelength of 420 nm, the eluate was collected and concentrated to remove the solvent, and then freeze-dried.

[0092] 6) Sephadex gel column chromatography purification: The chromatography column was equilibrated with ultrapure water as the eluent, the melanoidins obtained in step (5) were prepared at 100 mg / mL, loaded onto the top of the chromatography column for gradient elution, the elution flow rate was controlled, the eluate was automatically collected, the absorbance value of the eluate was measured at 420 nm, and semi-preparative high performance liquid chromatography was used for determination. The eluates of the same fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain the anti-inflammatory active melanoidins based on black American ginseng (Panax quinquefolius L. melanoidins, PMLD).

[0093] Example 2

[0094] This example provides a preparation method of anti-inflammatory active melanoidins based on black American ginseng. Different from the extraction conditions of the crude melanoidins in Example 1, the specific preparation steps are as follows:

[0095] 1) Fresh American ginseng was placed in a fluidized bed air flow pulverizer, the air flow speed was set at 300 m / s, the air flow pressure was 0.8 MPa, and the American ginseng was pulverized to 70 - 90 μm. The pulverized American ginseng powder was steamed for 3 h, dried in the sun, repeated nine times, and finally vacuum dried at 60 °C to obtain black American ginseng powder;

[0096] 2) The black American ginseng powder was mixed evenly with a 20% ethanol solution by volume at a solid-liquid ratio of 1:15, placed in a 50 °C water bath and magnetically stirred for 30 min, centrifuged, repeated 2 times, the supernatant was combined and concentrated under reduced pressure, and then freeze-dried to obtain the crude melanoidin extract;

[0097] 3) 8 g of the crude melanoidin extract was dissolved in 200 mL of distilled water, 6 g of FeCl3 was added, stirred evenly, the pH value was adjusted to 4.0 with 2 M NaOH, magnetically stirred at room temperature for 15 min and then left to stand for 14 h to form a red-brown precipitate, and the precipitate was collected by centrifugation.

[0098] 4) The obtained precipitate was treated with 200 mL of 7.5% (w / v, mass concentration) ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) metal chelating agent, magnetically stirred at 50 °C for 3.5 h to remove the added iron ions, then dialyzed, deproteinized, concentrated under reduced pressure, and then freeze-dried to obtain the melanoidin isolate.

[0099] 5) Macroporous resin purification: The melanoidins after lyophilization were redissolved in ultrapure water to prepare a melanoidin aqueous solution, filtered through a 0.45 μm filter membrane, adsorbed with macroporous resin D101, with a loading concentration of 20 mg / mL, and gradient elution was performed with ethanol solutions at concentration gradients of 0, 20%, 40%, and 60%, with an elution rate of 1.5 mL / min. The eluate was collected, the absorbance was measured at a wavelength of 420 nm, the eluate was collected and concentrated to remove the solvent, and then freeze-dried.

[0100] 6) Sephadex gel column chromatography purification: The chromatography column was equilibrated with ultrapure water as the eluent. The melanoidins obtained in step (5) were prepared at a concentration of 100 mg / mL, loaded onto the top of the chromatography column for gradient elution, the elution flow rate was controlled, the eluate was automatically collected, the absorbance of the eluate was measured at 420 nm, and semi-preparative high-performance liquid chromatography was used for determination. The eluates of the same fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain the melanoidin isolate.

[0101] Example 3

[0102] This example provides a preparation method of anti-inflammatory active melanoidins based on black American ginseng, which is different from the melanoidin separation and purification conditions in Example 1. Specifically, the preparation steps are as follows:

[0103] 1) Fresh American ginseng was placed in a fluidized bed air mill, with the air flow rate set at 400 m / s and the air pressure at 0.8 MPa, so that the American ginseng was ground to 60 - 70 μm. The ground American ginseng powder was steamed for 3 h, dried in the sun, repeated nine times, and finally vacuum dried at 60 °C to obtain black American ginseng powder;

[0104] 2) The black American ginseng powder was mixed evenly with a 10% ethanol solution by volume at a solid-liquid ratio of 1:15, placed in a 55 °C water bath and magnetically stirred for 50 min, centrifuged, repeated 4 times, the supernatant was combined and concentrated under reduced pressure, and then freeze-dried to obtain the crude melanoidin extract;

[0105] 3) 8 g of the crude melanoidin extract was dissolved in 160 mL of distilled water, 7 g of FeCl3 was added, stirred evenly, the pH value was adjusted to 3.8 with 2M NaOH, magnetically stirred at room temperature for 20 min and then left to stand for 12 h to form a reddish-brown precipitate, and the precipitate was collected by centrifugation.

[0106] 4) The obtained precipitate was treated with 200 mL of 7.5% (w / v, mass concentration) ethylenediaminetetraacetic acid disodium salt (EDTA-2Na) metal chelating agent, magnetically stirred at 50 °C in a water bath for 3 h to remove the added iron ions, then dialyzed, deproteinized, concentrated under reduced pressure, and then freeze-dried to obtain melanoidins.

[0107] 5) Macroporous resin purification: The melanoidins after freeze-drying were redissolved with ultrapure water to prepare a melanoidin aqueous solution, filtered through a 0.45 μm filter membrane, adsorbed with macroporous resin D101, the loading concentration was 20 mg / mL, and gradient elution was carried out with ethanol solutions at concentration gradients of 0, 20%, 40%, and 60%, the elution rate was 1.5 mL / min, the eluate was collected, the absorbance value was detected at a wavelength of 420 nm, the eluate was collected and concentrated to remove the solvent, and then freeze-dried.

[0108] 6) Sephadex gel column chromatography purification: The chromatography column was equilibrated with ultrapure water as the eluent, the melanoidins obtained in step (5) were prepared at 100 mg / mL, loaded onto the top of the chromatography column for gradient elution, the elution flow rate was controlled, the eluate was automatically collected, the absorbance value of the eluate was measured at 420 nm, and semi-preparative high performance liquid chromatography was used for determination. The eluates of the same fractions were combined, concentrated under reduced pressure, and freeze-dried to obtain the melanoidin isolate.

[0109] The following analysis was carried out on Examples 1 to 3, Comparative Example 1, and Comparative Example 2:

[0110] Table 1

[0111] Sample Name Melanoidin Content (g / 100g) Comparative Example 1 42.27±0.23 Comparative Example 2 48.75±0.35 Example 1 73.52±0.12 Example 2 65.28±0.63 Example 3 63.29±0.27

[0112] Note: The data are expressed as mean ± standard deviation.

[0113] The melanoidin contents in the melanoidin products obtained in Examples 1 to 3 of the present invention and the extracts obtained by dialysis using the traditional method as Comparative Group 2 were compared. The results are shown in Table 1. The melanoidin contents in the products obtained in Examples 1 to 3 were significantly higher than those in Comparative Example 2. And compared with the samples that were not subjected to fluidized bed air pulverization, the melanoidin contents in the black American ginseng in Examples 1 to 3 were significantly higher than those in Comparative Example 1.

[0114] Molecular weight distribution determination:

[0115] The molecular weight of melanoidins was determined by high performance liquid chromatography (HPLC). A TSK-gel G6000 PW(XL) (300 mm × 7.8 mm) chromatographic column was used, 50 mmol / L phosphate buffer solution (pH = 7.2) was used as the mobile phase with a flow rate of 0.8 mL / min, and a RID-10A differential refractive index detector was used. The column temperature was 30 °C, the pressure was 1.2 MPa, and the injection volume was 20 μL. 10 mg of melanoidins were dissolved in 1 mL of phosphate buffer solution, and after passing through a microporous filter membrane (0.22 μm), it was used for analysis by the detector. Five molecular weight standard substances were selected as standards for determination: γ-globulin (158 kDa), bovine serum albumin (66 kDa), ovalbumin (44287 Da), myoglobin (16591 Da), and VB12 (1355.37 Da) as standards.

[0116] Figure 1 This is the molecular weight distribution diagram of the purified melanoidin with anti-inflammatory activity prepared in Example 1. The results show that there is one peak for the melanoidin products before and after separation. The average molecular weight of the separated product is 17.46 kDa, and the molecular weight distribution range of the anti-inflammatory active melanoidin is 4.6×10 2 ~2.49×10 4 Da.

[0117] Scanning electron microscope analysis:

[0118] At room temperature, take an appropriate amount of powdered melanoidin sample, evenly smear it on the conductive adhesive, blow off the excess floating sample with an ear balloon, and perform gold sputtering coating treatment. Put the treated sample into the vacuum environment of the scanning electron microscope to lock the scanning area, adjust the beam spot size, and adjust the focal length until it is clear. The surface morphology of the sample is obtained using a scanning electron microscope at an acceleration voltage of 10 kV at magnifications of 1000× and 2000×.

[0119] Figure 2 SEM images of the crude melanoidin extract and the melanoidin isolate at magnifications of 1000 times and 2000 times. From the results, it can be seen that there are irregular polygonal blocks and granular structures in the crude product (Crude MLDs). However, in the separated product (PMLDs isolates), the coexistence of rod-shaped and flake-shaped structures was found. The microstructure of the separated product has changed, and the morphology is clearer. This morphological change indicates that the separation process makes the microstructure of melanoidin more consistent and removes some impurities, thereby improving the purity of melanoidin.

[0120] Fourier transform infrared spectroscopy analysis:

[0121] At room temperature, quickly mix 1 mg of dry melanoidin powder and 150 mg of potassium bromide powder, grind them, and then press (pressure = 7 MPa) for about one minute to form a disc for FTIR instrument measurement. The number of scans is 16 times, and the frequency range is 4000 to 400 cm -1 .

[0122] Figure 3 Fourier transform infrared spectra of the crude melanoidin extract and the isolate. As can be seen from the figure, large irregular polygonal blocks and a small amount of granular structures are observed in the crude melanoidin, and more small flake-shaped and block-shaped structures are included in the purified melanoidin, indicating that there is a change in the microstructure after purification of the crude melanoidin. When functional groups interact at the molecular level, changes in the FTIR spectrum can be seen, such as the appearance of new absorption peaks and changes in the intensity or position of absorption peaks. The infrared spectra of the products before and after separation are shown in the figure. At 3410 cm -1The characteristic absorption bands that appear are related to the stretching vibration of O-H. At 2930 cm -1 and 1420 cm -1 The absorption bands observed correspond to the stretching and bending vibrations of CH3 and CH2 respectively. These characteristics also appear in the infrared spectrum of coffee melanoidins. The absorption peak at 1260 cm -1 is a sign of the C-N stretching vibration in the amide III band. The absorption band at approximately 1020 cm -1 is associated with the bending vibration of C-O. The absorption band at 599 cm -1 corresponds to the skeletal mode of the pyranose ring. The crude extract (Crude MLDs) shows an absorption band at approximately 1630 cm -1 which may come from the bending vibration of O-H or the asymmetric stretching vibration of C=O, showing the characteristics of glucose. However, in the isolated product (PMLDsisolates), the absorption band at 1630 cm -1 no longer exists, and instead, an absorption band at 1600 cm -1 appears, which is attributed to the coupling deformation of the stretching of C=O and N-H (i.e., amide I and II bands). The melanoidin skeleton in the isolated product is rich in C=O, C-N, and N-H groups. Generally speaking, both the crude extract and the isolated product contain protein and carbohydrate structures, which are similar to the model melanoidin structures described in the literature.

[0123] In vitro anti-inflammatory test

[0124] In the determination of test indicators, Caco-2 cells were randomly divided into five groups:

[0125] There were 2 groups in the control group, namely: the normal control group was labeled as Control; the inflammatory cell model group was labeled as LPS;

[0126] There were 3 groups in the drug group, namely: the low-concentration melanoidin group (25 μg / mL) was labeled as L-PMLD; the medium-concentration melanoidin group (50 μg / mL) was labeled as M-PMLD; the high-concentration melanoidin group (100 μg / mL) was labeled as H-PMLD.

[0127] Treat Caco-2 cells according to the test requirements.

[0128] Control: Caco-2 cells were cultured directly in the cell culture plate without treatment.

[0129] LPS: Caco-2 cells were cultured in DMEM complete medium containing 20% fetal bovine serum (FBS) and 1% double antibody (a mixture of penicillin-streptomycin), and placed in a constant temperature cell incubator with 5% CO2 content and 37 °C for 24 h. Then, 1 mL of cell-grade 1×PBS was added to each well for 3 h, and then the cells were treated with 1 μg / mL LPS for 24 h.

[0130] L-PMLD / M-PMLD / H-PMLD: Caco-2 cells were cultured in a cell culture plate for 24 h, the culture medium was aspirated, and different concentrations of PMLD were added to incubate Caco-2 cells for 3 h, and then the cells were treated with LPS for 24 h.

[0131] After all experiments were performed at least 3 times independently, the data were expressed as mean ± standard deviation. One-way analysis of variance was performed using SPSS 21.0 software for data analysis, and then Duncan's test was used. The results are shown below.

[0132] Cytotoxicity analysis:

[0133] Caco-2 cells were prepared into a uniform cell suspension of 1×10 5 / mL, and transferred to a 96-well plate at a density of 5000 cells / well, and the cell suspension in each well was 100 μL. The cells were cultured in an incubator at 37 °C (5% CO2) for 12 hours. When the cells were almost completely adherent, the culture medium was aspirated, and complete medium (DMEM high-glucose medium containing 20% fetal bovine serum and 1% double antibody) containing different melanoidin concentrations (0 - 200 μg / mL) was added for continued culture (melanoidin concentrations: 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL). Six replicate wells were set for each concentration, and cell control wells (containing Caco-2 cells and complete medium) and blank culture medium control wells (containing only complete medium) were also set. After culturing in an incubator at 37 °C (5% CO2) for 12 h, 24 h, and 48 h respectively, 10 μL of CCK-8 reagent was added to each well and cultured for another 1.5 h. Then, an enzyme-linked immunosorbent assay (ELISA) reader was used to measure the OD value at a wavelength of 450 nm, and the maximum non-toxic concentration of melanoidin-treated Caco-2 cells was found as the high-dose drug group for the study.

[0134] Under the conditions of the present invention, the CCK-8 kit was used to detect the changes in cells treated with different concentrations of melanoidin (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL) for 24 h, 36 h, and 48 h. As Figure 4As shown, melanoidins showed a dose-dependent effect on the viability of Caco-2 cells under the treatment conditions of 24 h, 36 h, and 48 h, and the longer the treatment time, the stronger the toxic effect on Caco-2 cells. Compared with the control group (0 μg / mL), when Caco-2 cells were cultured with 25, 50, and 100 μg / mL for 24 h, the cell viability decreased to 98.61%, 97.92%, and 96.10% respectively, with no significant effect on cell toxicity. However, when the concentration reached 200 μg / mL, the cell viability decreased to 78.92%. Therefore, three concentrations of 25, 50, and 100 μg / mL were finally selected for subsequent experiments.

[0135] Enzyme-linked immunosorbent assay (ELISA):

[0136] After Caco-2 cells were cultured until they reached 70% - 80% confluence, the culture medium was removed, and the cells were pre-protected with a medium containing melanoidins at different concentrations (25, 50, 100 μg / mL) for 3 h. After removal, 1 μg / mL LPS was added to stimulate the cells. The supernatant was taken, and the contents of TNF-α, IL-6, IL-1β, COX-2, iNOS, MDA, SOD, and CAT in different groups were detected by ELISA. The specific detection steps were carried out according to the kit instructions.

[0137] As Figure 5 and 6 shown, a cell inflammation model was established by treating Caco-2 cells with 1 μg / mL LPS, and different doses of melanoidins (25, 50, 100 μg / mL) were given simultaneously. TNF-α, IL-6, and IL-1β are important pro-inflammatory factors in the body, which participate in regulating the occurrence and development of the inflammatory response. By detecting the relative expression levels of TNF-α, IL-6, and IL-1β in LPS-induced Caco-2 cells by melanoidins, the changing trend of the degree of inflammation can be reflected. As shown in the figure, compared with the normal control group, in the cell inflammation model induced by LPS, the levels of inflammatory factors and oxidative stress parameters (TNF-α, IL-6, IL-1β, COX-2, iNOS, MDA, SOD, and CAT) increased significantly (p < 0.05), indicating that LPS successfully induced an inflammatory response. In summary, these inflammatory factors play an important role in regulating the body's immune system, enhancing the chemotaxis of immune cells, and promoting inflammatory signal transduction. Excessive release may exacerbate tissue damage and lead to persistent inflammation (p < 0.05).

[0138] Figure 5 and 6It was shown that after the melanoidin pretreatment, the expression levels of inflammatory factors were significantly decreased, indicating that melanoidin could alleviate the LPS-induced inflammatory response to a certain extent. In particular, high-dose melanoidin (H-PMLD) had the best effect in inhibiting the expression of TNF-α, IL-6, and IL-1β (p<0.05). Compared with the LPS group, the concentrations of TNF-α, IL-6, and IL-1β in H-PMLD decreased from 10.89, 7.38, and 13.77 pg / mL to 8.28, 4.83, and 6.03 pg / mL. This might be related to its stronger antioxidant capacity and inhibitory effect on the inflammatory signaling pathway. In addition, the expression levels of oxidative stress factors COX-2 and iNOS also decreased with the increase in the concentration of melanoidin. Compared with LPS, the concentrations of COX-2 and iNOS in Caco-2 containing H-PMLD decreased from 11.58 and 0.99 ng / mL to 1.08 and 0.17 ng / mL, a decrease of nearly tenfold. It was shown that melanoidin might play an anti-inflammatory role by downregulating prostaglandin synthesis and nitric oxide production and reducing the release of pro-inflammatory mediators. At the same time, the level of the oxidative stress factor MDA in H-PMLD decreased, while the contents of SOD and CAT increased, from 4.82 ng / mL and 15.58 pg / mL to 11.98 ng / mL and 62.24 pg / mL, an increase of more than 2 times and 4 times respectively. This further verified the antioxidant effect of melanoidin, which might help reduce the damage of oxidative stress to cells and indirectly inhibit the inflammatory response.

[0139] RT-qPCR analysis:

[0140] The total RNA in the inflammatory model cells was extracted using the Steady Pure Universal RNA Extraction Kit II. The extracted cellular RNA was operated according to the Evo M-MLV Reverse Transcription Premix Kit, and cDNA samples were synthesized by RNA reverse transcription. Subsequently, a real-time PCR reaction system was prepared using the SYBR Green Pro Taq HS Premix qPCR Kit for quantitative analysis. The reaction mixture was synthesized in a mixed system of 2 μL of a five-fold diluted sample, 1 μL of the upstream primer, 1 μL of the downstream primer, 6 μL of ddH2O, and 10 μL of the mixture. The primers used in RT-qPCR are listed in Table 2. Real-time detection was performed using CFX96, and the quantitative RNA expression level was measured by the TM real-time system. The thermal cycling conditions were carried out for 40 cycles according to the manufacturer's recommendations. The cycle threshold (Ct) was recorded. The data were normalized using GAPDH, and the 2-ΔΔCT method was used.

[0141] Table 2 Primer sequences for RT-qPCR analysis

[0142]

[0143]

[0144] To observe the effects of melanoidins at different concentrations on inflammatory factors in LPS-induced Caco-2 cells, we used RT-qPCR technology to detect the changes in mRNA expression levels of inflammation-related genes TNF-α, IL-6, IL-1β, COX-2, and iNOS. As Figure 7 shown, the results showed that compared with the normal control group, the mRNA levels of inflammation-related genes in Caco-2 cells in the cell model treated with LPS were significantly upregulated (p<0.05), indicating that LPS successfully induced an inflammatory response and activated the inflammatory signaling pathway.

[0145] Melanoidin pretreatment significantly reduced the mRNA expression levels of inflammation-related genes, indicating that it can effectively inhibit the gene transcription of inflammatory factors and reduce the transmission of pro-inflammatory signals. Among them, the inhibitory effect of high-dose melanoidin (H-PMLD) on the mRNA levels of TNF-α, IL-6, IL-1β, COX-2, and iNOS was the most obvious (p<0.05). Compared with the LPS treatment group, the mRNA expression levels of TNF-α, IL-6, and IL-1β decreased from 2.72, 4.31, and 3.01 to 1.04, 1.44, and 1.37. It may reduce the transcriptional activity of pro-inflammatory genes by regulating the NF-κB signaling pathway. In addition, with the decrease in the levels of COX-2 and iNOS, the mRNA expression levels of H-PMLD decreased from 1.81 and 1.65 to 0.92 and 0.85, indicating that melanoidin may interfere with the arachidonic acid metabolism and NO synthesis pathways, reduce the production of prostaglandin (PGE2) and nitric oxide (NO), and thus inhibit the inflammatory response.

[0146] At the same time, by comparing Comparative Example 1, Comparative Example 2, and Examples 1-3, especially Example 1, it can be seen that there is a synergistic effect between the two steps of metal chelation separation and purification and fluidized bed grinding in the present invention, which can synergistically improve the relevant properties of the anti-inflammatory active melanoidin based on black American ginseng prepared.

[0147] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art can understand that: without departing from the spirit and scope of the present invention and the appended claims, various substitutions, changes, and modifications are possible. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. An anti-inflammatory active melanoidin based on black American ginseng, characterized in that: The anti-inflammatory active melanoidins are prepared by metal chelation separation and purification and have the following characteristics: (i) The content of anti-inflammatory active melanoidins is 63.29 - 73.52 g / 100 g; (ii) The molecular weight distribution range of anti-inflammatory active melanoidins is 4.6×10 2 ~2.49×10 4 Da; (iii) The anti-inflammatory active melanoidins exhibit a uniform rod-like and flaky microstructure; (iv) Anti-inflammatory activity: The skeleton in melanoidins is rich in C=O, C-N, and N-H groups. The separated product of melanoidins shows characteristic absorption peaks of amide and carboxyl groups at 1600 cm -1 -1.

2. The melanoidin with anti-inflammatory activity based on American ginseng according to claim 1, characterized in that: In the basic composition of the anti-inflammatory active melanoidins, the content of total sugar is 13.21% - 15.55%, the content of protein is 0.89% - 1.03%, the content of lipid is 1.24% - 1.95%, and the content of ash is 3.26% - 3.68%.

3. Use of the anti-inflammatory active melanoidins as claimed in claim 1 or 2 as an active component in the preparation of anti-inflammatory drugs.

4. Use of the anti-inflammatory active melanoidins as claimed in claim 1 or 2 as a functional ingredient in the preparation of functional foods and anti-inflammatory drugs.

5. The preparation method of the anti-inflammatory active melanoidin according to claim 1 or 2, characterized in that: Comprising the following steps: (1) Place American ginseng in a fluidized bed air classifier for pulverization, steam the pulverized American ginseng powder for 3 - 4 h, dry it in the sun, repeat nine times, and finally place it in a vacuum dryer at 50 - 60 °C to obtain black American ginseng powder; (2) Add the black American ginseng powder to an ethanol solution, mix evenly, place it in a constant temperature water bath and stir magnetically for 30 - 70 min, centrifuge, repeat 2 - 5 times, combine the supernatant and concentrate it under reduced pressure, and freeze-dry to obtain a crude melanoidin extract; (3) Dissolve the obtained crude melanoidin extract in distilled water to obtain an extract, add metal chloride, stir evenly, adjust the pH value with 2M NaOH, stir magnetically at room temperature and then let it stand for 12 - 14 h to form a reddish-brown precipitate, and centrifuge to collect the precipitate; (4) Treat the obtained precipitate with a metal chelating agent, stir magnetically in a water bath at 50 °C for 3 - 4 h, remove the added metal ions, then perform dialysis, protein removal, concentration under reduced pressure, and then freeze-dry to obtain melanoidins; (5) Macroporous resin purification: Dissolve the freeze-dried melanoidins in ultrapure water, filter through a 0.45 μm filter membrane, adsorb with macroporous resin, set the concentration gradient of ethanol solutions to 0, 20%, 40%, 60% for gradient elution, collect the eluate, detect the absorbance value at a wavelength of 420 nm, collect the eluate and concentrate to remove the solvent, and freeze-dry; (6) Sephadex gel column chromatography purification: Prepare the freeze-dried product obtained in step (5) into a melanoidin aqueous solution, use ultrapure water as the eluent to balance the Sephadex gel column chromatography column, load the sample on the top of the chromatography column for gradient elution, control the elution flow rate, automatically collect the eluate, measure the absorbance value of the eluate at 420 nm, and use semi-preparative high performance liquid chromatography for determination, combine the eluates of the same fractions, concentrate under reduced pressure, and freeze-dry to obtain the anti-inflammatory active melanoidins based on black American ginseng.

6. The preparation method according to claim 5, characterized in that: In step (1), the air flow rate of the fluidized bed is 300 - 500 m / s, the air pressure is 0.6 - 1.2 MPa, and the particle size of the black American ginseng powder is 60 - 100 μm; Alternatively, in step (2), the volume concentration of the ethanol solution is 0 - 50% (the value is not 0), the material-liquid ratio of the black American ginseng powder to the ethanol solution is 1:5 - 1:25, and the water bath temperature of the constant temperature water bath is 30 - 70 °C.

7. The preparation method according to claim 5, characterized in that: In steps (2) and (4), the pressure for decompression concentration is 0.05 - 0.1 MPa, and the temperature is 40 - 50 °C; Alternatively, in step (3), the material - liquid ratio of melanoidin crude extract to distilled water is 1:20 - 1:25; the addition amount of metal chloride in every 200 mL of extraction solution is 5 - 7 g; adjust the pH value to 3.8 - 4.2; the magnetic stirring time is 15 - 20 min.

8. The preparation method according to claim 5, characterized in that: The metal chloride in step (3) includes FeCl3, CoCl2 or NiCl2.

9. The preparation method of melanoidins with anti-inflammatory activity based on black American ginseng according to claim 5, characterized in that: In step (4), the metal chelating agent is 7.5% (w / v, mass concentration) disodium ethylenediaminetetraacetate (EDTA - 2Na) or tetrasodium ethylenediaminetetraacetate; the addition amount of the metal chelating agent is 200 mL of the metal chelating agent added to every 8 g of melanoidin crude extract.

10. The preparation method according to any one of claims 5 to 9, characterized in that: In step (4), a dialysis bag with a molecular weight cut - off of 1000 Da is selected for dialysis; Alternatively, in step (5), the macroporous resin is D101, the elution flow rate is 1 - 2.5 mL / min, and the sample loading concentration is 15 - 25 mg / mL; Alternatively, in step (6), the Sephadex gel chromatography column is Sephadex G - 50, the elution flow rate is 1 - 2 mL / min, and the sample loading concentration is 80 - 120 mg / mL.

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