Melanoidin with anti-fatigue effect and application thereof

Abstract

The present invention provides a melanoidin with anti-fatigue effect and its use. The present invention verifies through an animal model that the melanoidin extracted from wine-processed polygonatum can significantly reduce the content of lactic acid, blood urea nitrogen and malondialdehyde in the animal body, improve the activity of antioxidant enzymes SOD, CAT, GSH-Px and glycogen level, and has antioxidant and anti-fatigue effects. The melanoidin in wine-processed polygonatum is prepared into an anti-fatigue drug or health food, which has broad market application prospects.

Description

Technical Field

[0001] The invention relates to melanoidin with anti-fatigue effect and application thereof. Background Art

[0002] Melanoidins are a brown-black mixture formed by various chemical reactions such as condensation, polymerization, degradation, and cyclization in the late stage of the Maillard reaction. There are three main inferences about the structure of melanoidins: ① The skeleton of melanoidins is formed by sugar degradation products and amino compounds through aldol reaction and Michael addition, and the product melanoidins should have a partial structure of the chromophore. ② Melanoidins in food are derived from the cross-linked products of proteins and are low-molecular-weight pigment polymers formed by sugars and amino acids. ③ The conversion of sugars and amino acids into melanoidins is related to the spontaneous polymerization of pyrrole and furan structures, the products of the late stage of the Maillard reaction. It is necessary to conduct in-depth research on the generation of natural melanin from selectively labeled precursors. Due to the complex structure and different types of melanoidins, they are closely related to the reaction environment, such as food processing technical conditions, reaction temperature and time, and pH. At present, the structural research on melanoidins focuses on the use of sugars and amino acids to simulate reactions to separate and purify melanoidins, but the composition of melanoidins obtained after the Maillard reaction of different foods is different, and the unit structure of naturally isolated melanoidins is still unknown.

[0003] Most of the research on melanoidins focuses on food categories such as black garlic, and optimizes the extraction, separation, and purification processes of melanoidins in food. At present, little is known about the Maillard reaction and its product melanoidins that occur during the processing of traditional Chinese medicine. It is of great significance to study and explore the composition of melanoidins, the product of the Maillard reaction during the processing of traditional Chinese medicine, and their effects on human body functions. However, there has been no report on the efficacy of melanoidins in wine-processed Polygonatum odoratum and its effects on human body functions. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a melanoidin with anti-fatigue effect, wherein the melanoidin is derived from wine-processed polygonatum.

[0005] Furthermore, the PY-GC-MS chromatogram of the melanoidin should have chromatographic peaks of propiolic acid, acetone, 2,3-butanedione, 2-methylfuran, acetic acid, hydroxyacetone, 2,5-dimethylfuran, 2-acetic furan, 2-cyclopentene-1-one, 2-methyl-2-cyclopentene-1-one, γ-butyrolactone, 3-methyl-2-cyclopentene-1-one and 2,3-dimethyl-2-cyclopentenone.

[0006] Furthermore, the retention time of the propiolic acid chromatographic peak is 1.56min, and the peak area ratio is 66.14%; the retention time of the acetone chromatographic peak is 1.71min, and the peak area ratio is 10.56%; the retention time of the 2,3-butanedione chromatographic peak is 1.894min, and the peak area ratio is 1.51%; the retention time of the 2-methylfuran chromatographic peak is 1.952min, and the peak area ratio is 1.76%; the retention time of the acetic acid chromatographic peak is 2.014min, and the peak area ratio is 5.99%; the retention time of the hydroxyacetone chromatographic peak is 2.22min, and the peak area ratio is 2.45%; the retention time of the 2,5-dimethylfuran chromatographic peak is 2.469min, and the peak area ratio is 4.5 9%; the retention time of the chromatographic peak of 2-acetic acid furan is 3.275min, and the peak area ratio is 0.76%; the retention time of the chromatographic peak of 2-cyclopenten-1-one is 3.686min, and the peak area ratio is 1.21%; the retention time of the chromatographic peak of 2-methyl-2-cyclopenten-1-one is 4.507min, and the peak area ratio is 0.69%; the retention time of the chromatographic peak of γ-butyrolactone is 4.607min, and the peak area ratio is 0.75%; the retention time of the chromatographic peak of 3-methyl-2-cyclopenten-1-one is 5.205min, and the peak area ratio is 1.75%; the retention time of the chromatographic peak of 2,3-dimethyl-2-cyclopentenone is 5.982min, and the peak area ratio is 0.73%.

[0007] The present invention also provides a use of the aforementioned melanoidin in preparing anti-fatigue medicines and / or health foods.

[0008] Furthermore, the medicine and / or health food is a medicine and / or health food that reduces the content of lactic acid, urea nitrogen and / or malondialdehyde in serum.

[0009] Furthermore, the medicine and / or health food is a medicine and / or health food that increases the content of antioxidant enzymes SOD, CAT, GSH-Px and / or glycogen in serum.

[0010] Furthermore, the medicine and / or health food is a preparation prepared by using melanoidin as an active ingredient and adding pharmaceutically and / or food acceptable excipients or auxiliary ingredients.

[0011] Furthermore, the preparation is an oral preparation; the oral preparation is a granule, a solution, a pill, a paste or a tablet.

[0012] Finally, the present invention provides a method for preparing the above-mentioned melanoidin, which comprises the steps of preparing polygonatum with wine, extracting with ethanol, and purifying the extract with macroporous resin.

[0013] Furthermore, the wine-processed polygonatum is extracted with 40% ethanol, the extract is adjusted to pH 2, loaded onto D3520 resin, washed with water until the effluent has no molish reaction, then washed with 60% ethanol at pH 8 until the resin is colorless, and then dried with the 60% ethanol washing solution to obtain the product.

[0014] The invention discloses a use of melanoidin in wine-processed polygonatum in the preparation of anti-fatigue medicine and / or health food. The animal model confirms that melanoidin can be extracted from wine-processed polygonatum. The melanoidin can significantly reduce the content of lactic acid, blood urea nitrogen and malondialdehyde in the animal body, improve the activity of antioxidant enzymes SOD, CAT, GSH-Px and glycogen level, and has anti-oxidation and anti-fatigue effects. The melanoidin in wine-processed polygonatum is prepared into an anti-fatigue medicine or health food, which has broad market application prospects.

[0015] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0016] The following is a further detailed description of the above contents of the present invention through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Absorbance of extraction solutions from different methods

[0018] Figure 2 Absorbance of solutions after adsorption by different resins

[0019] Figure 3 Absorbance of solution after adsorption under different pH conditions

[0020] Figure 4 Thermal cracking product structure

[0021] Figure 5 Infrared spectrum

[0022] Figure 6 MALDI-TOF-MS molecular weight assignment

[0023] Figure 7 Antioxidant capacity results

[0024] Figure 8 Exhaustive swimming time of mice * Compared with the blank group, # Compared with the positive group, p<0.05 is significantly different

[0025] Fig. 9 Serum lactate level* Compared with the blank group, # Compared with the positive group, p<0.05 is significantly different

[0026] Fig.10 Serum blood urea nitrogen level * Compared with the blank group, # Compared with the positive group, p<0.05 is significantly different

[0027] Fig.11 Antioxidant enzymes and oxidation product levels ( * Compared with the blank group, # Compared with the positive group, p<0.05 is significantly different)

[0028] Fig.12 Liver and muscle glycogen levels (* indicates comparison with the blank group, # indicates comparison with the positive group, p<0.05 has significant difference) DETAILED DESCRIPTION

[0029] Example 1 Preparation of Melanin

[0030] 1. Instruments and Materials

[0031] 1.1 Experimental Instruments

[0032] Table 1 Experimental instruments

[0033]

[0034]

[0035] 1.2 Experimental Materials

[0036] The medicinal materials used in this experiment were collected from the standardized planting base of traditional Chinese medicine in Dazhu County, Dazhou City, Sichuan Province. They were identified as fresh rhizomes of Polygonatumcyrtonema Hua of the Liliaceae family by Associate Professor Long Fei of the Pharmacy Teaching and Research Section of Chengdu University of Traditional Chinese Medicine. The fresh rhizomes were washed, the fibrous roots were removed, and dried for use as raw Polygonatum cyrtonema Hua. Take the raw product, mix it with rice wine, and seal it for moistening. Steam it in a 300W electric steamer, let it dry to room temperature, cut it into 4mm thick slices, dry it, and get the wine-made Polygonatum cyrtonema Hua slices, grind it into powder, pass it through a No. 5 sieve, and set it aside.

[0037] Take the raw product for later use, add 20% rice wine of the medicinal material weight to moisten it for 12 hours, steam it in an electric steamer (600W) for 24 hours, let it cool to room temperature, cut into 4mm thick slices, dry them, obtain the processed polygonatum decoction slices, grind them into powder, pass through No. 5 sieve, and set aside.

[0038] Macroporous resin D101, D3520, AB-8, X-5, NKA-9, Zhengzhou Hecheng New Materials Co., Ltd.

[0039] 1.3 Experimental Reagents

[0040] 95% ethanol, sodium hydroxide, concentrated hydrochloric acid, potassium bromide, trifluoroacetic acid, salicylic acid, potassium ferrocyanide, ferric chloride, hydrogen peroxide, and diphenylpicrylhydrazine (DPPH) were all domestically produced analytical grade.

[0041] 2 Methods

[0042] 2.1 Investigation of extraction methods

[0043] Melanoidin extraction is mainly divided into water immersion method and alcohol extraction method. Referring to the existing melanoidin extraction processes, four of the more commonly used methods were selected for investigation, as shown in Table 2.

[0044] Table 2 Melanoid extraction method

[0045]

[0046] 2.2 Investigation of purification methods

[0047] The main method for purifying melanoidins is macroporous resin adsorption purification. The type of macroporous resin, elution pH, and desorption alcohol concentration were investigated.

[0048] 2.2.1 Macroporous resin types

[0049] Weigh 5.0g of five different activated macroporous resins into a 50mL stoppered conical flask, add 30mL of melanoidin extract, shake for 3h to allow the resin to fully adsorb the pigment, and take the supernatant after standing. Measure the absorbance at 420nm, the absorbance before adsorption is A0, and the absorbance after adsorption is A1.

[0050]

[0051] 2.2.2 Different pH values

[0052] Weigh 5.0 g of activated D3520 macroporous resin into a 50 mL stoppered conical flask, add 30 mL of melanoidin solution with different pH (2-10), shake at intervals for 3 h to allow the resin to fully adsorb the pigment, measure the absorbance of the supernatant, and calculate the static adsorption rate.

[0053] 2.2.3 Desorbents at different concentrations

[0054] Weigh 5.0 g of saturated macroporous resin, add 30 mL of ethanol solution of different concentrations (mass fraction of 20, 40, 60, 80, 95%), oscillate for 3 h, measure the absorbance of the supernatant, and calculate the static adsorption rate.

[0055] 2.3 Preparation of melanin

[0056] According to the results of the extraction and purification experiments in Items “2.1” and “2.2”, melanoidin is extracted, purified and prepared, and the prepared melanoidin is used for component analysis.

[0057] 2.4 Composition analysis

[0058] Melanoidin samples were dissolved in 60% ethanol solution, and the polysaccharides, mono-oligosaccharides and total saponins therein were determined by the determination method.

[0059] 2.4.1 The polysaccharide content was determined by the phenol-sulfuric acid method.

[0060] Preparation of standard solution Accurately weigh the glucose standard to prepare a 0.175 mg / mL standard solution.

[0061] Preparation of phenol solution Weigh phenol, heat in a water bath to dissolve, and prepare a 4.5% phenol solution. Seal and store in a dark place for later use.

[0062] The sample solution was prepared according to the 2020 edition of the Chinese Pharmacopoeia.

[0063] Plotting the standard curve Take 0.2-0.8mL of the gradient volume standard solution in a 10mL stoppered test tube, add distilled water to 2mL, shake well and place in ice water. Add 1.5mL of phenol solution to the test tube, shake well and add 6.5mL of concentrated sulfuric acid solution as soon as possible and shake well. After cooling, keep warm in a 40℃ water bath for 30min, ice water bath for 5min, take out and put it at room temperature. Set up a blank group and measure the absorbance at a wavelength of 490nm.

[0064] Determination of polysaccharide content in samples: Take 1.0 mL of sample solution and follow the same steps as above. Substitute the standard curve to calculate the sample content.

[0065] 2.4.2 Determination of Mono-oligosaccharide Content

[0066] Preparation of reference solution: Accurately weigh 10.0-15.0 mg of fructose, glucose, sucrose, and melibiose reference substances, record the weighed value, and dilute to 100 mL with 50% methanol to obtain the standard stock solution.

[0067] Preparation of sample solution Weigh 0.5 g of sample, add 50 mL of 25% methanol, seal and weigh. Ultrasonicate for 30 min, take out, cool and make up the weight.

[0068] Chromatographic conditions: Ultimate HILIC-NH2 column (250 mm × 4.6 mm, 5 μm); mobile phase: water (A)-acetonitrile (B), isocratic elution (0-30 min, 79% B); flow rate: 0.8 mL / min; detector: ELSD, column temperature: 30°C, drift tube temperature: 95°C, flow rate: 2.2 mL / min.

[0069] Standard curve drawing: Take the mother solution of standard solution and prepare 0.1, 0.15, 0.2, 0.25, 0.3 mg / mL standard solution. The chromatographic conditions are the same as above, and the standard curve is drawn with the peak area as the ordinate.

[0070] 2.4.3 Determination of total saponin content

[0071] The total saponins were determined by UV-visible spectrophotometry.

[0072] Preparation of reference solution: Accurately weigh 20.0 mg of diosgenin reference substance and prepare a 0.2 mg / mL standard solution in 80% ethanol.

[0073] Preparation of sample solution: Weigh 0.5 g of sample, add 20 mL of 80% ethanol, seal and ultrasonicate for 30 min, twice, and adjust the volume to 50 mL.

[0074] Draw the standard curve Take 0.2-0.6mL of gradient volume standard solution and evaporate the solvent. Add 0.2mL of 5% vanillin-glacial acetic acid solution, ice bath, add 0.8mL of perchloric acid, and shake well. Heat in a 60℃ water bath for 15min, ice bath for 2min, and quickly add 5ml of glacial acetic acid. Seal and let stand for 5min. Set up a blank group, measure the absorbance at a wavelength of 550nm, and draw the standard curve.

[0075] Sample determination: Take 0.2 mL of sample solution and follow the other steps as above.

[0076] 2.5 Melanoid PY-GC-MS Analysis

[0077] PY-GC-MS analysis, namely pyrolysis gas chromatography-mass spectrometry analysis, uses high temperature to break down the macromolecules in the sample into small molecular compounds and fragments, and then separates and purifies them through a gas chromatograph, and finally detects them. It has the advantages of fast analysis speed and high sensitivity. The conditions are as follows:

[0078] Thermal cracking conditions: temperature 550°C, thermal cracking time 10s.

[0079] Gas chromatography conditions: DB-5 quartz capillary column (30 mm × 0.25 mm, 1 μm), initial temperature 60°C, increased to 270°C after 5 minutes, maintained for 5 minutes; flow rate 25 mL / min.

[0080] Mass spectrometry conditions: electron bombardment ion source; ion source temperature 220°C; scan time 0.2s in full scan mode; mass scan range 50-400m / z.

[0081] 2.6 FTIR analysis of melanin

[0082] Take 0.5 mg of melanoidin sample and add potassium bromide powder at a ratio of 1:100, grind and press into tablets to make 1 mm tablets, and then perform 400-4000 cm -1 Wavelength scanning was performed and infrared spectrum data were collected using OMNIC software to analyze the functional group information.

[0083] 2.7 Melanoidin MALDI-TOF-MS Analysis

[0084] Ion source (MALDI), time-of-flight mass analyzer (TOF) and mass spectrometer (MS) together constitute matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This technology has fast analysis speed and high sensitivity and is widely used in polymer research to obtain data such as molecular weight distribution and end group structure of polymers.

[43] .

[0085] Acetonitrile and 0.1% trifluoroacetic acid aqueous solution are prepared into acetonitrile acid solvent (CHCA) in a volume ratio of 1:1 for standby use. After the sample is diluted, 0.5 μL of the sample solution is placed on the MALDI target plate, dried naturally, and then 0.5 μL of 4 mg / ml CHCA solution is added dropwise, and dried naturally at room temperature. The MALDI-TOF mass spectrometer is calibrated with horse myoglobin enzymatic peptides for external standard. The mass spectrometry laser source has a wavelength of 355 nm and an acceleration of 20 kV. The sample is scanned in the range of 100-10000 Da, and the data is collected in the positive ion reflection mode and the automatic data acquisition mode. The molecular weight distribution of melanoidin is determined.

[0086] 2.8 In vitro antioxidant activity assay

[0087] 2.8.1 Determination of total reducing power

[0088] Take a gradient concentration of melanoidin solution, add 2.5mL of 0.2mol / L phosphate buffer and 2.5mL of 1% potassium ferricyanide solution. Keep warm in a 50℃ water bath for 20min and then cool in a water bath. Add 2.5mL of 10% trichloroacetic acid to terminate the reaction. Take 2.5mL of supernatant, add 2.5mL of distilled water and 1.0mL of 0.1% ferric chloride, and let stand for 10min. Measure the absorbance at 700nm.

[43] .

[0089] 2.8.2 Hydroxyl free radical scavenging ability assay

[0090] The Fenton reaction was modified. Take 1.0 mL of 9 mmol / L salicylic acid-ethanol solution, 1 mL of 9 mmol / L FeSO4 solution, and 2 mL of distilled water, and finally add 1.0 mL of 8.8 mmol / L hydrogen peroxide, and place in a 37°C water bath for 10 min as the control group, and measure the absorbance A0 at 510 nm. Take a gradient concentration of melanoidin solution, and the rest of the reaction steps are the same as above, as the reaction group, and measure the absorbance A1.

[0091]

[0092] 2.8.3 DPPH free radical scavenging ability determination

[0093] DPPH reaction method, take 2.0mL of gradient concentration sample solution, add 2.0mL of 0.4mmol / L DPPH (diphenylpicrylhydrazyl) anhydrous ethanol solution. Keep it in the dark for 30min as the reaction group. Measure the absorbance value A1 at 517nm. Take the same volume of anhydrous ethanol instead of the sample as the control group, and measure the absorbance value A0. DPPH is purple when dissolved in alcohol, so the same volume of anhydrous ethanol is used instead of the DPPH free radical anhydrous ethanol solution as the background group, and the absorbance value A2 is measured.

[0094]

[0095] 2.9 Data Analysis

[0096] All data were analyzed by Duncan test using SPSS 23.0 software, and data between groups were considered significant different (P<0.05). ORIGIN 2018 was used for drawing graphs.

[0097] 3 Results and analysis

[0098] 3.1 Selection of extraction method

[0099] The absorbance results of the four extraction methods are shown in Figure 1 , selected extraction method B, that is, the solid-liquid ratio was 1:40, the solvent was 40% ethanol solution, refluxed in a water bath at 85°C for 1h, and extracted twice.

[0100] 3.2 Selection of purification method

[0101] 3.2.1 Effect of macroporous resin type

[0102] The performance of different types of resins varies greatly. Five commonly used resins on the market were selected, and the adsorption capacity was shown in Figure 2 The adsorption capacity of D3520 type adsorption resin is the strongest, and the static adsorption rate is 85.79%. Therefore, D3520 type resin is used to purify melanoidin in subsequent experiments.

[0103] 3.2.2 Effect of adsorption solution pH

[0104] The adsorption capacity under different pH conditions is shown in Figure 3 The static adsorption rate is the highest when pH=2, so the pH of the adsorption solution should be controlled at 2 for the best result.

[0105] 3.2.3 Effect of desorption solution concentration

[0106] Melanoidin was eluted with ethanol of different concentrations, and the eluate was collected to measure the absorbance, and the results are shown in Table 3. When the ethanol concentration was 60%, the absorbance of the eluate was the highest and the desorption capacity was the strongest, so 60% ethanol solution was selected for desorption.

[0107] Table 3 Absorbance under different concentrations of ethanol

[0108]

[0109] 3.3 Preparation of melanoidin

[0110] (1) Extraction

[0111] Take wine-processed Polygonatum sibiricum at a solid-liquid ratio of 1:40, add 40% ethanol solution, reflux in a water bath at 85°C for 1 hour, extract twice, filter, collect the filtrate, concentrate it into an extract, and preserve it.

[0112] (2) Purification

[0113] Take the extract, dilute it with distilled water until the solution is clear, add 1 mol / L dilute hydrochloric acid solution, adjust to pH = 2, load it on D3520 resin, open the column valve, flow rate 1.5 mL / min, collect the effluent, load it on the column twice, close the column valve for the last time, let it stand for 6 to 12 hours, rinse with distilled water until the effluent has no molish reaction, then rinse with 60% ethanol at pH = 8 until the resin is colorless, collect the 60% ethanol rinse, freeze-dry, and store at -4°C.

[0114] Molish reaction: prepare 5% α-naphthol ethanol solution, take 5 ml of the eluate into a test tube, add 2-3 drops of 5% α-naphthol ethanol solution and shake well, add 1-2 ml of concentrated sulfuric acid along the wall of the tube, let it stand, and observe whether a purple ring-shaped substance is produced at the junction of the two liquids. If there is no molish reaction, there is no sugar.

[0115] After extraction and purification of 500.0 g of wine-processed polygonatum, 49.0 g of melanoidin sample was obtained, with a yield of 9.8%.

[0116] 3.4 Ingredients

[0117] No polysaccharides or monosaccharides could be detected, and the total saponin content was 0.16±0.01%.

[0118] 3.5 Melanin thermal decomposition products

[0119] The PY-GC-MS matching results are shown in Table 4. There are 13 kinds of wine-processed Polygonatum melanin cracking products with a matching degree of ≥80%, including 2 acids with a relative content of 72.13%; 7 ketones with a relative content of 18.90%; 3 furans with a relative content of 7.11%; and 1 ester with a relative content of 0.75%. The products obtained by thermal cracking are similar to those reported in the literature, but the relative contents of the obtained product types are different, which may be related to the different matching degrees screened in the reports and the processing time. The relative content of organic acids in the products is the highest, which is speculated to be related to the sour and astringent taste of Polygonatum after processing.

[0120] Table 4 Gas analysis results of thermal cracking products

[0121]

[0122] Furan products are commonly found in the Maillard reaction, showing a caramelized taste. These substances are mainly obtained by the reaction of dicarbonyl compounds. The products are mostly unsaturated carbonyl and dicarbonyl compounds, which conforms to the Maillard intermediate stage reaction principle. The relative content of acetic acid, diacetyl and acetone in the cracking products is not low, and there are no furfural compounds in the products ( Figure 4 ). Combined with the Maillard reaction principle, it can be inferred that in the second stage, the Amadori intermediate is more likely to be in alkaline conditions for the next reaction.

[0123] 3.6 FTIR analysis of melanin

[0124] FTIR chart Figure 5 ,from Figure 5 Visible: 3405cm -1 The large and blunt strong absorption peak at 2935cm is generally considered to be the -OH functional group in the sample. Combined with the results of thermal pyrolysis gas chromatography-mass spectrometry, the sample contains furan heterocycles, which may be considered to have -NH stretching vibration. -1 and 2881cm -1 The absorption peaks near 1625cm are attributed to the asymmetric and symmetric stretching vibrations of -CH3, -CH2-, aliphatic hydrocarbons or CH in cycloalkanes. -1 and 1412cm -1 The peak at 1080cm is the characteristic peak of the benzene ring or aromatic group, corresponding to the stretching vibration of C=C or C=O on the aromatic ring. -1 The absorption peaks near 875, 790 and 610 cm correspond to the stretching vibration of COC in the sample. -1 The absorption peak at is attributed to the out-of-plane deformation vibration of CH in aromatic compounds.

[0125] 3.7 Melanin molecular weight distribution

[0126] Mass spectra such as Figure 6The molecular weight m / z of melanoidins is concentrated in the range of 200-500Da, which are all small molecule compounds, confirming that polysaccharides have been completely removed during the previous macroporous resin purification process. In addition, literature studies have shown that the Maillard reaction generates oligomers, and the size of polymer molecules may be related to the reaction time and temperature. There is no fixed difference between the molecular arrangements, indicating that the mixture is not polymerized from the same units.

[0127] 3.8 In vitro antioxidant activity

[0128] 3.8.1 Total reduction capacity

[0129] Total reducing power is a common indicator for evaluating antioxidant activity. Antioxidant substances provide electrons to react with oxidants such as Fe 3+ Reaction to Fe 2+ , preventing free radicals from further active reactions. Measure the absorbance, which is related to the Fe 2+ The quantity is positively correlated, that is, the greater the absorbance, the stronger the ability to bind with the oxidant and the better the reducing property.

[0130] The total reducing capacity of melanoidin at different concentrations is shown in Figure 7 When the concentration of melanoidin was 0.4 mg / mL, the absorbance was 0.2567, and the growth rate was the largest; the significant difference in absorbance between different concentrations was p<0.05. Melanoidin has a certain antioxidant capacity, and the antioxidant capacity is positively correlated with the concentration.

[0131] 3.8.2 DPPH scavenging ability

[0132] DPPH free radical scavenging method is a common method for determining antioxidant capacity. The principle is that 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals are purple-red after being dissolved in ethanol. When antioxidants are added to react, the antioxidants give electrons to combine with free radicals, the solution becomes lighter, and the absorbance decreases. The scavenging rate is proportional to the antioxidant capacity.

[0133] The DPPH clearance rate of melanoidin at different concentrations is shown in Figure 7 In the concentration range of 0.2mg / mL-1.0mg / mL, the DPPH clearance rate is proportional to the concentration, and melanoidin has a certain antioxidant capacity. At 0.4mg / mL, the clearance rate growth rate is the largest, and at 1.0mg / mL, the maximum clearance rate is 66.97%.

[0134] 3.8.3 Hydroxyl radical scavenging ability

[0135] ·OH radicals were detected by Fenton reaction. 2+ Reacts with H2O2 to generate Fe 3+And ·OH free radicals, ·OH selectively reacts with salicylic acid to develop color. After adding antioxidants, it reacts with free radicals, the solution becomes lighter, and the absorbance decreases.

[0136] The OH clearance rate of different concentrations of melanoidin is shown in Figure 7 As the concentration of melanoidin increases, the ·OH scavenging ability is significantly enhanced. When the concentration of melanoidin reaches 1.0 mg / mL, the scavenging rate is the highest. Melanoidin has a certain antioxidant capacity.

[0137] 4 Summary

[0138] There is no research on melanoidin in processed Polygonatum sibiricum. This section conducts a preliminary study on the purification method, physicochemical properties and structural characterization of melanoidin.

[0139] (1) In the early stage of the present invention, macroporous resin was used to adsorb and remove the pigment in the processed polygonatum extract to purify the polysaccharide. A preliminary investigation was conducted on the melanoidin adsorption process, which improved the yield of polygonatum polysaccharide and established a melanoidin purification method. The content of polysaccharides, monosaccharides and saponins in the crude melanoidin was very low.

[0140] (2) The results of the antioxidant experiment showed that melanoidin can scavenge free radicals, that is, it has a certain in vitro antioxidant function.

[0141] (3) According to the MALDI-TOF-MS analysis, the molecular weight of melanoidins is concentrated in 200-500Da, and there is no polymerization pattern. The results of FTIR and PY-GC-MS analysis show that melanoidins may contain hydroxyl groups, conjugated structures, heterocyclic structures, aromatic heterocyclic structures, such as furan and lactone. PY-GC-MS data show that the small molecules after thermal cracking contain characteristics such as odor, suggesting that the aroma changes after processing of Polygonatum odoratum may be related to melanoidins. The thermal cracking products contain a lot of acetone, diacetyl, and acetic acid, which suggests that the intermediate stage of the Maillard reaction is under alkaline conditions.

[0142] Test Example 2: Investigation of the anti-fatigue effect of melanin

[0143] 1 Experimental instruments and materials

[0144] 1.1 Experimental Instruments

[0145] Table 5 Experimental instruments

[0146]

[0147]

[0148] 1.2 Experimental Reagents

[0149] Lactic acid (LA) assay kit, urea nitrogen (BUN) assay kit, catalase (CAT) enzyme assay kit, glutathione peroxidase (GSH-Px) assay kit, total superoxide dismutase (SOD) assay kit, malondialdehyde (MDA) assay kit, Wuhan Enzyme Immunity Biological Co., Ltd.; liver glycogen test kit, muscle glycogen test kit, Nanjing Jiancheng Bioengineering Institute; Minghehuo brand Rhodiola rosea and American ginseng oral liquid, Chengdu Yikantang Pharmaceutical Co., Ltd.

[0150] 1.3 Experimental animals

[0151] Sixty 4-week-old SPF-grade KM male mice, weighing 20±2 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd., license number: SCXK(Beijing)2019-0010. The breeding temperature was 20-25°C, the humidity was 40-60%, and the light and dark cycle was 12 h.

[0152] 2 Methods

[0153] 2.1 Grouping and Dosing

[0154] The mice were given basic feed, and the experiment began after 7 days of adaptive feeding. The experiment was divided into 5 groups: blank control group (BC), positive control group (PC), low-dose melanoidin group (LHJ-L), medium-dose melanoidin group (LHJ-M), and high-dose melanoidin group (LHJ-H). There were 12 mice in each group, for a total of 60 mice. The positive control group was gavaged with American ginseng and Rhodiola rosea oral liquid. According to the recommended dosage in the instructions, adults (60kg) were 30mL / d, which was converted to a dosage of 10μL / d for mice (20g). The corresponding dilution was performed and gavage was 1mL / d; the blank control group was gavaged with 1mL of distilled water; according to the results of the previous preliminary experiment, the maximum amount of melanoidin that mice could withstand was 400mg / kg, that is, 400mg / kg in the high-dose group, 200mg / kg in the medium-dose group, and 100mg / kg in the low-dose group, and the gavage was continuous for 28d.

[0155] 2.2 Exhaustive swimming time measurement of mice

[0156] 30 minutes after the last intragastric administration, the tail of the mouse was loaded with a lead ball weighing 5% of its body weight, and the mouse was placed in a swimming box at 30°C and 30 cm high. The edge of the swimming box was tapped to stimulate the mouse. The exhaustive swimming time of the mouse was taken as the time when the mouse's mouth and nose sank into the water for 6 seconds and did not float to the surface.

[0157] 2.3 Sample collection

[0158] After the mice were exhausted, they were taken out and dried. Blood was collected from the eyeballs and stored in EP tubes. The blood was allowed to coagulate. The upper serum was collected by centrifugation and stored at -80°C. After the blood was collected, the chest cavity was opened, the liver and hind leg muscles of the mice were taken, and they were quickly placed in liquid nitrogen and stored at -80°C.

[0159] 2.4 Index detection

[0160] Elisa method was used to detect serum lactic acid (LA), urea nitrogen (BUN), glutathione peroxidase (GSH-PX), hydrogen peroxide (CAT), malondialdehyde (MDA), and total superoxide dismutase (SOD) levels in serum according to the instructions of the kit. Liver and hind leg muscle were prepared into homogenate according to the instructions to detect glycogen content.

[0161] 2.5 Data Analysis

[0162] SPSS 23.0 statistical software was used for data analysis. One-way analysis of variance was used for data between multiple groups, and LSD test was used for pairwise comparison between groups. * Compared with the blank group, # Compared with the positive group, p < 0.05 is statistically significant

[0163] 3 Results and analysis

[0164] 3.1 Survival status and weight of mice During the experiment, the survival status of mice in each group, such as drinking water, eating, and moving, was normal, and no obvious toxic reactions occurred. The weight changes are shown in Table 6. There was no significant change in the weight of mice between groups, p>0.05, which was not statistically significant. American ginseng and rhodiola rosea oral liquid and melanoidin had no significant effect on the weight of mice.

[0165] Table 6 Changes in mouse body weight

[0166]

[0167] 3.2 Exhaustive swimming time of mice

[0168] The decline in exercise endurance is an obvious manifestation of fatigue. Figure 8 Compared with the blank group, the weight-loaded swimming exhaustion time of mice in the positive control group and the mice in the low-, medium-, and high-dose melanoidin groups was significantly increased. * P<0.05). Compared with the positive control group, the low- and medium-dose melanoidin groups were inferior to the positive control group, and the exhaustive swimming time of mice in the high-dose group was improved ( # P<0.05), and the anti-fatigue ability was similar to that of positive drugs.

[0169] 3.3 Results of serum index improvement

[0170] Lactic acid (LA) is an important metabolite. When the body is exercising vigorously, lack of oxygen causes the glycolysis reaction to accelerate, and a large amount of lactic acid accumulates in the body. The pH value of muscles and blood decreases, thereby inhibiting muscle contraction and inducing fatigue. Therefore, the lactic acid content in muscles is the main cause of fatigue. Fig. 9The results showed that the lactic acid in the serum of mice in the blank control group accumulated after swimming, causing fatigue. Compared with the blank group, the lactic acid content in the other groups was significantly reduced ( * P<0.05). Compared with the positive control group, the low- and high-dose melanoidin groups significantly increased ( # P<0.05), and the improvement effect of the medium-dose group was almost the same as that of the positive group.

[0171] Blood urea nitrogen (BUN) content Fig.10 , this indicator can reflect the level of blood urea. Under normal physiological conditions, the body's blood urea metabolism is in balance. During strenuous exercise, the body cannot obtain enough energy and the blood urea level increases significantly. Studies have shown that serum urea nitrogen content is significantly negatively correlated with the body's exercise endurance. The blank control group had the highest blood urea nitrogen content in mice. Compared with the blank group, the blood urea nitrogen content in the positive control group and each dose group of melanoidin was significantly reduced (*P<0.05). Compared with the positive control group, there were significant differences in the low, medium, and high dose groups of melanoidin ( # P<0.05), melanoidin had a certain ability to clear BUN but was less than that of the positive control group.

[0172] SOD, CAT, and GSH-Px are the three major antioxidant enzymes in the body and are positively correlated with antioxidant capacity; MDA is a peroxidation product and is negatively correlated with antioxidant capacity. Fig.11 It can be seen that compared with the blank group, the contents of the indicators in the other groups were significantly different (*P<0.05), the levels of SOD, CAT, and GSH-Px increased, and the level of MDA decreased. Compared with the positive control group, the low, medium, and high dose groups of melanoidin were significantly different (#P<0.05), and melanoidin had a certain antioxidant capacity but not as good as the positive control group.

[0173] The body's exercise endurance and load capacity depend on the level of energy sources, including glycogen storage. When the body exercises vigorously, liver glycogen and muscle glycogen are consumed quickly. Insufficient glycogen supply leads to hypoglycemia and fatigue. Therefore, glycogen levels can reflect the degree of fatigue and are positively correlated. Fig.12 It can be seen that compared with the blank group, the glycogen content in the other groups increased ( * P<0.05). Compared with the positive control group, the melanoidin groups showed significant reduction ( # P<0.05), melanoidin had a certain effect in increasing serum glycogen level but not as much as the positive control group.

[0174] 4 Summary

[0175] The present invention mainly studies the anti-fatigue effect of melanoidin through the mouse exhaustive swimming model, and preliminarily explores its anti-fatigue mechanism, and the conclusions obtained are as follows:

[0176] (1) When mice were given melanoidin for 28 days, the three doses did not affect the weight changes and normal growth of the mice, and there was no obvious toxic effect.

[0177] (2) Melanoidin can significantly prolong the exhaustive swimming time of mice. The exhaustive swimming time of mice is positively correlated with the dose of melanoidin.

[0178] (3) After exhaustive swimming, various serum indicators of mice changed to varying degrees. In the blank control group, the levels of lactic acid, blood urea nitrogen, and malondialdehyde were high, and the glycogen level and the activities of antioxidant enzymes SOD, CAT, and GSH-Px were low. After administration of melanoidin, the levels of lactic acid, blood urea nitrogen, and malondialdehyde in mice decreased, and the activities of antioxidant enzymes SOD, CAT, and GSH-Px and the glycogen level increased, reaching a significant difference ( * P<0.05), the results showed that melanoidin can reduce serum lactic acid, blood urea nitrogen and malondialdehyde, and increase the activity of antioxidant enzymes SOD, CAT, GSH-Px and glycogen level to achieve anti-fatigue effect.

Claims

1. Use of melanoidin in the preparation of anti-fatigue drugs and / or health foods, characterized in that: The melanoidin is derived from wine-processed polygonatum; the preparation method of the melanoidin is as follows: the wine-processed polygonatum is extracted with 40% ethanol, refluxed in a water bath at 85°C for 1 hour, extracted twice, the extract is adjusted to pH 2, loaded onto D3520 resin, rinsed with water until the effluent has no molish reaction, then rinsed with 60% ethanol at pH 8 until the resin is colorless, and then dried with the 60% ethanol rinse to obtain the melanoidin; the solid-liquid ratio of the wine-processed polygonatum to 40% ethanol is 1:

40.

2. The use according to claim 1, characterized in that The PY-GC-MS chromatogram of the melanoidin should have chromatographic peaks of propiolic acid, acetone, 2,3-butanedione, 2-methylfuran, acetic acid, hydroxyacetone, 2,5-dimethylfuran, 2-acetic furan, 2-cyclopentene-1-one, 2-methyl-2-cyclopentene-1-one, γ-butyrolactone, 3-methyl-2-cyclopentene-1-one and 2,3-dimethyl-2-cyclopentenone.

3. The use according to claim 2, characterized in that: The retention time of the propiolic acid chromatographic peak is 1.56min, and the peak area ratio is 66.14%; the retention time of the acetone chromatographic peak is 1.71min, and the peak area ratio is 10.56%; the retention time of the 2,3-butanedione chromatographic peak is 1.894min, and the peak area ratio is 1.51%; the retention time of the 2-methylfuran chromatographic peak is 1.952min, and the peak area ratio is 1.76%; the retention time of the acetic acid chromatographic peak is 2.014min, and the peak area ratio is 5.99%; The retention time of the chromatographic peak of hydroxyacetone was 2.22min, and the peak area ratio was 2.45%; the retention time of the chromatographic peak of 2,5-dimethylfuran was 2.469min, and the peak area ratio was 4.59%; the retention time of the chromatographic peak of 2-acetic acid furan was 3.275min, and the peak area ratio was 0.76%; the retention time of the chromatographic peak of 2-cyclopenten-1-one was 3.686min, and the peak area ratio was 1.21%; the retention time of the chromatographic peak of 2-methyl-2-cyclopenten-1-one was 4.507min, and the peak area ratio was 0.69%; the retention time of the chromatographic peak of γ-butyrolactone was 4.607min, and the peak area ratio was 0.75%; the retention time of the chromatographic peak of 3-methyl-2-cyclopenten-1-one was 5.205min, and the peak area ratio was 1.75%; the retention time of the chromatographic peak of 2,3-dimethyl-2-cyclopentenone was 5.982min, and the peak area ratio was 0.73%.

4. The use according to claim 1, characterized in that The medicine and / or health food is a preparation prepared by taking melanoidin as an active ingredient and adding pharmaceutically and / or food acceptable auxiliary materials.

5. The use according to claim 4, characterized in that The preparation is an oral preparation; the oral preparation is a granule, a solution, a pill, a paste or a tablet.

Images