Method for detecting antioxidant capacity of traditional Chinese medicine polysaccharide and application of Fe / NC nano-enzyme

Abstract

The invention provides a method for detecting the oxidation resistance of traditional Chinese medicine polysaccharide and application of Fe / NC nano-enzyme, and relates to the technical field of biological detection. The method for detecting the oxidation resistance of the traditional Chinese medicine polysaccharide comprises the following steps: providing Fe / NC nano-enzyme; providing a buffer solution, and adding the Fe / NC nano-enzyme, traditional Chinese medicine polysaccharide, hydrogen peroxide and a chromogenic substrate into the buffer solution to form a first reaction solution; and measuring the absorbance value of the first reaction liquid at a specific wavelength, and detecting the oxidation resistance of the traditional Chinese medicine polysaccharide according to the change of the absorbance value of the first reaction liquid. According to the method for detecting the antioxidant capacity of the traditional Chinese medicine polysaccharide, antioxidant activity evaluation on different traditional Chinese medicine polysaccharides can be realized, so that the application of the nano-enzyme in the traditional Chinese medicine polysaccharide is promoted, and the method has important significance in knowing the pharmacological action of the nano-enzyme and developing novel medicines and functional foods.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a method for detecting the antioxidant capacity of traditional Chinese medicine polysaccharides and the use of Fe / NC nanozymes. Background Art

[0002] Traditional Chinese medicine polysaccharides are an important class of active ingredients in traditional Chinese medicine, with a variety of biological activities, including immunomodulation, anti-tumor, anti-inflammatory, hypoglycemic and other effects. Among them, antioxidant activity is one of the important mechanisms by which traditional Chinese medicine polysaccharides exert their effects. In vivo, the imbalance between the production and removal of free radicals can lead to oxidative stress, which in turn causes cell damage and the occurrence of various diseases. Therefore, the evaluation of the antioxidant activity of traditional Chinese medicine polysaccharides is of great significance for understanding their pharmacological effects and developing new drugs and functional foods. Colorimetric analysis is widely used due to its advantages such as simple operation, low cost and short analysis time. Nanozymes, as a new type of biocatalyst, show great potential in the field of colorimetric detection of the antioxidant capacity of substances due to their unique enzyme-like catalytic performance and stability. Although nanozymes show good sensitivity in the detection of total antioxidant capacity, compared with natural enzymes, the enzyme-like catalytic activity of nanozymes needs to be improved, and the colorimetric sensing system based on nanozymes has not been reported in the evaluation of the antioxidant capacity of traditional Chinese medicine polysaccharides. Summary of the invention

[0003] In view of the above problems existing in the prior art, the present invention provides a method for detecting the antioxidant capacity of Chinese medicine polysaccharides and the use of Fe / NC nanozymes, so as to evaluate the antioxidant activity of different Chinese medicine polysaccharides, thereby promoting the application of nanozymes in Chinese medicine polysaccharides.

[0004] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present invention provides a method for detecting the antioxidant capacity of traditional Chinese medicine polysaccharides, comprising the following steps: providing Fe / NC nanozymes; providing a buffer solution, adding the Fe / NC nanozymes, traditional Chinese medicine polysaccharides, hydrogen peroxide and a chromogenic substrate to the buffer solution to form a first reaction solution; measuring the absorbance value of the first reaction solution at a specific wavelength, and detecting the antioxidant property of the traditional Chinese medicine polysaccharides by the change in the absorbance value of the first reaction solution.

[0005] In one embodiment of the present invention, the detection method also includes a process of exploring the catalytic performance of the Fe / NC nanozyme.

[0006] In one embodiment of the present invention, the process of exploring the catalytic performance of Fe / NC nanozymes includes the following steps: providing the buffer; adding the Fe / NC nanozyme, hydrogen peroxide and the chromogenic substrate to the buffer to form a second reaction solution; The absorbance value of the second reaction liquid at a specific wavelength is measured, and the catalytic performance of the Fe / NC nanozyme is detected by the change in the absorbance value of the second reaction liquid.

[0007] In one embodiment of the present invention, the pH of the second reaction solution is 3.5-4.5.

[0008] In one embodiment of the present invention, the temperature of the second reaction liquid is 25-45°C.

[0009] In one embodiment of the present invention, the color developing substrate is 3,3',5,5'-tetramethylbenzidine.

[0010] In one embodiment of the present invention, the buffer is acetic acid / sodium acetate buffer.

[0011] In one embodiment of the present invention, the preparation of the Fe / NC nanozyme comprises the following steps: Dissolve Zn(NO3)2•6H2O and 2-methylimidazole in anhydrous methanol to obtain Zn(NO3)2•6H2O solution and 2-methylimidazole solution respectively; The Zn(NO3)2•6H2O solution and the 2-methylimidazole solution are mixed, stirred, allowed to stand, centrifuged, and dried to obtain ZIF-8; The ZIF-8 and iron powder are placed in a nitrogen atmosphere, pyrolyzed at 800-1000° C. for 2-3 h, and naturally cooled to obtain the Fe / NC nanozyme.

[0012] The second aspect of the present invention provides a use of a Fe / NC nanozyme, wherein the Fe / NC nanozyme is used for detecting the antioxidant capacity of polysaccharides in traditional Chinese medicine.

[0013] The present invention utilizes the excellent peroxidase-like catalytic performance of Fe / NC nanozymes to catalyze the substrate H2O2 to produce hydroxyl radicals (·OH), and promote the colorless chromogenic substrate to lose electrons to generate colored products. Chinese medicine polysaccharides can quickly remove free radicals and inhibit the formation of colored products. Therefore, based on the linear relationship between the concentration of Chinese medicine polysaccharides and the degree of inhibition of Fe / NC enzyme activity, a colorimetric sensing system is constructed to achieve the antioxidant activity evaluation of different Chinese medicine polysaccharides, thereby promoting the application of nanozymes in Chinese medicine polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other embodiments can be obtained based on these drawings without paying creative work.

[0015] Figure 1 It is a flow chart of a method for detecting the antioxidant capacity of Chinese medicine polysaccharides in one embodiment of the present invention; Figure 2 This is the morphology characterization diagram of Fe / NC nanozyme; Figure 2 (A) is the scanning electron microscope (SEM) image of Fe / NC nanozyme; Figure 2 (B) is the transmission electron microscopy (TEM) image of Fe / NC nanozyme; Figure 3 X-ray photoelectron spectroscopy (XPS) graphs of Fe / NC nanozyme and NC; Figure 3 (A) is the full XPS spectrum of Fe / NC and NC; Figure 3 (B) is the high-resolution XPS spectrum of Fe 2p of Fe / NC; Figure 3 (C) is the N1s XPS high-resolution spectrum of Fe / NC; Figure 3 (D) is the C 1s XPS high-resolution spectrum of Fe / NC; Figure 3 (E) is the atomic percentage diagram of C, N, O and Fe in Fe / NC; Figure 4 To explore the catalytic performance of Fe / NC nanozymes; Figure 4 (A) shows the peroxidase and oxidase-like catalytic activities of Fe / NC nanozymes; Figure 4 (B) shows the performance comparison between Fe / NC nanozyme and NC peroxidase; Figure 4 (C) shows the performance comparison of Fe / NC nanozyme peroxidases at different concentrations; Figure 4 (D) is the effect of the pH value of the reaction system on the performance of Fe / NC nanozyme peroxidase; Figure 4 (E) is the effect of the temperature of the reaction system on the performance of Fe / NC nanozyme peroxidase; Figure 5 The Michaelis-Menten curve and the corresponding Lineweaver-Burk plot of Fe / NC nanozyme; Figure 5 (A) is the Michaelis-Menten curve of different H2O2 concentrations; Figure 5 (B) in Figure 5 (A) The corresponding double reciprocal Lineweaver-Burk plot; Figure 5 (C) in the figure shows the Michaelis-Menten curve of different TMB concentrations; Figure 5 (D) in Figure 5 (C) The corresponding double reciprocal Lineweaver-Burk plot; Figure 6 Antioxidant activity of polysaccharides from traditional Chinese medicine; Figure 6 (A) is the absorbance comparison of different traditional Chinese medicine polysaccharides; Figure 6 (B) in the figure shows the corresponding relationship between different concentrations of Lycium barbarum polysaccharides (LBP) and the absorbance difference ΔA; Figure 6 The (C) in Figure 6 Linear simulation of (B); Figure 6 (D) in the figure shows the corresponding relationship between different concentrations of Panax notoginseng polysaccharide (PNP) and the absorbance difference ΔA; Figure 6 The (E) in Figure 6 Linear simulation of (D); Figure 6 (F) in the figure shows the corresponding relationship between different concentrations of Salvia miltiorrhiza polysaccharide (SMP) and the absorbance difference ΔA; Figure 6 The (G) in Figure 6 Linear simulation of (F) in; Figure 6 (H) in the figure is the corresponding relationship between different concentrations of Chinese medicine polysaccharides and inhibition rate; Figure 7 Antioxidant activity of Chinese herbal medicines; Figure 7 (A) Comparison of absorbance of different Chinese herbal medicines; Figure 7 (B) in the figure is a comparison of the inhibition rates of different Chinese herbal medicines. DETAILED DESCRIPTION

[0016] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the features in the following embodiments and the embodiments can be combined with each other without conflict. It should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods for which specific conditions are not specified in the following examples are usually carried out under conventional conditions or according to the conditions recommended by the manufacturers.

[0017] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention are familiar to those skilled in the art and the description of the present invention, and any method, device and material of the prior art similar or equivalent to the method, device and material in the embodiments of the present invention can also be used to implement the present invention.

[0018] It should be noted that the terms such as "upper", "lower", "left", "right", "middle" and "one" etc. used in this specification are only for the convenience of description and are not intended to limit the scope of implementation of the present invention. Changes or adjustments to their relative relationships, without substantially changing the technical content, should also be regarded as the scope of implementation of the present invention.

[0019] At present, the detection indicators of the in vitro antioxidant capacity of Chinese herbal polysaccharides mainly include scavenging hydroxyl radicals (·OH), scavenging 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH·), and scavenging superoxide anion radicals (O 2- ·), antioxidant activity, reducing power, scavenging nitrite and scavenging alkyl free radicals (R·). DPPH is a stable nitrogen-centered free radical in organic solvents. The solution is purple and has a maximum absorption peak at 517nm. Therefore, the change in absorbance can be used to evaluate the scavenging of free radicals by Chinese herbal polysaccharides, thereby evaluating the antioxidant capacity of Chinese herbal polysaccharides. The colorimetric analysis method based on scavenging DPPH· has the advantages of simple operation, fewer experimental steps and easy implementation. However, since the DPPH method mainly targets the hydrogen atom transfer mechanism, it is impossible to fully evaluate the antioxidant capacity of Chinese herbal polysaccharides. Moreover, the DPPH method cannot accurately detect substances with weak antioxidant activity. In addition, 2,2′-azobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) is also often used to detect the total antioxidant capacity of Chinese herbal polysaccharides. The detection principle of the ABTS method is that ABTS is oxidized to generate green ABTS free radicals under the action of appropriate oxidants, and an absorption peak can be detected at 734nm. When antioxidants are present, the generation of ABTS will be suppressed. The absorbance of ABTS free radicals at 734nm can be measured and the total antioxidant capacity of the sample can be calculated. The ABTS method has higher sensitivity and is suitable for substances with weak antioxidant activity. However, the operation steps of the ABTS method are relatively more, and it is necessary to react with potassium persulfate to prepare ABTS free radicals in advance, which limits the rapid and accurate evaluation of the antioxidant activity of Chinese medicine polysaccharides. Therefore, the present application provides a detection method for the antioxidant capacity of Chinese medicine polysaccharides, which can realize rapid, sensitive and practical detection of the antioxidant capacity of Chinese medicine polysaccharides.

[0020] See also Figure 1 The method for detecting the antioxidant capacity of Chinese medicine polysaccharides of the present invention comprises the following steps: S1, providing Fe / NC nanozymes; S2, providing a buffer solution, adding Fe / NC nanozyme, Chinese medicine polysaccharide, hydrogen peroxide and a color developing substrate into the buffer solution to form a first reaction solution; S3. Measure the absorbance value of the first reaction solution at a specific wavelength, and detect the antioxidant property of the Chinese medicine polysaccharide by the change of the absorbance value of the first reaction solution.

[0021] In step S1, in one embodiment, Fe / NC nanozyme is prepared by high temperature gas migration method, specifically comprising the following steps: dissolving Zn(NO3)2•6H2O and 2-methylimidazole in anhydrous methanol respectively to obtain Zn(NO3)2•6H2O solution and 2-methylimidazole solution; mixing Zn(NO3)2•6H2O solution and 2-methylimidazole solution, stirring, standing, centrifuging, and drying to obtain ZIF-8; placing ZIF-8 and iron powder in a nitrogen atmosphere, pyrolyzing at 800~1000℃ for 2~3h, and cooling naturally to obtain Fe / NC nanozyme. For example, the pyrolysis temperature can be 800℃, 900℃ or 1000℃, etc., and the pyrolysis time can be 2h, 2.5h or 3h. Exemplarily, 1.116 g of Zn(NO3)2•6H2O was dissolved in 12.5 mL of anhydrous methanol to obtain a Zn(NO3)2•6H2O solution, 2.608 g of 2-methylimidazole was dissolved in 12.5 mL of anhydrous methanol to obtain a 2-methylimidazole solution, the Zn(NO3)2•6H2O solution and the 2-methylimidazole solution were mixed, stirred at 25°C for 2 h, allowed to stand for 24 h, centrifuged, and dried to obtain ZIF-8; 100 mg of iron powder and 100 mg of ZIF-8 were placed at the two ends of a calcined boat, respectively, and pyrolyzed at 900°C for 3 h under a nitrogen atmosphere, and naturally cooled to obtain Fe / NC nanozyme.

[0022] The prepared Fe / NC nanozyme was characterized by morphology. Figure 2 As shown. Figure 2 (A) and Figure 2 As can be seen from (B), the Fe / NC nanozyme is a dodecahedral morphology with an average size of 120 nm. In order to study the composition and chemical state of the Fe / NC nanozyme, the Fe / NC nanozyme was tested by X-ray photoelectron spectroscopy (XPS). The test results are shown in Figure 2. Figure 3 As shown. Figure 3 As can be seen in (A), Fe / NC nanozymes include C, N, O, and Fe elements, proving the presence of iron in Fe / NC nanozymes. Figure 3 As can be seen in (B), the high-resolution XPS of Fe 2P in Fe / NC nanozyme shows two peaks, corresponding to Fe 2p3 / 2 and Fe 2p1 / 2 respectively; Figure 3 As can be seen in (C), the high-resolution XPS of N 1s in Fe / NC nanozyme is well fitted, corresponding to five peaks of graphitic N, pyridinic N, pyrrolic N, oxidized N and Fe-N. Figure 3 In (D), it can be seen that the C 1s spectrum in the Fe / NC nanozyme is divided into three peaks, which are respectively attributed to C=C, C=N and CN. Figure 3In (E), it can be seen that the atomic percentages of C, N, O and Fe in Fe / NC nanozyme are 86.1%, 4.81%, 8.48% and 0.7%, respectively.

[0023] In one embodiment, the detection method further includes a process of exploring the catalytic performance of the Fe / NC nanozyme, and the exploration process includes the following steps: S11, providing buffer; S12, adding Fe / NC nanozyme, hydrogen peroxide and a color-developing substrate to a buffer to form a second buffer; S13. Measure the absorbance value of the second reaction liquid at a specific wavelength, and detect the catalytic performance of the Fe / NC nanozyme by the change in the absorbance value of the second reaction liquid.

[0024] In step S11, the buffer is an acidic buffer, such as glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, citric acid-sodium citrate buffer, acetic acid / sodium acetate buffer, etc. Common acidic buffers in the art. Exemplarily, the buffer is acetic acid / sodium acetate buffer.

[0025] In step S12, the concentration of Fe / NC nanozyme is 25-50 μg / mL, the pH of the second reaction solution is 3.5-4.5, and the temperature of the second reaction solution is 25-45°C. The color developing substrate is selected from 3,3',5,5'-tetramethylbenzidine, 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, dopamine, diaminobenzidine or o-phenylenediamine, which are commonly used color developers in the art. Exemplarily, the color developing substrate is selected from 3,3',5,5'-tetramethylbenzidine (TMB), and the second reaction solution is a Fe / NC-TMB-H2O2 system. TMB is colorless and generates blue oxTMB when oxidized. oxTMB has a strong absorption peak at 652nm.

[0026] In order to test the oxidase-like catalytic activity of Fe / NC nanozymes, in one embodiment, a Fe / NC-TMB system is also provided. Exemplarily, Fe / NC nanozymes and 3,3',5,5'-tetramethylbenzidine are added to acetic acid / sodium acetate buffer to form a Fe / NC-TMB system.

[0027] In one embodiment, a NC-TMB-H2O2 system is also provided to compare the catalytic activity of Fe / NC nanozymes. Exemplarily, the preparation method of NC is as follows: 1.116g Zn(NO3)2•6H2O is dissolved in 12.5mL anhydrous methanol to obtain a Zn(NO3)2•6H2O solution, 2.608g 2-methylimidazole is dissolved in 12.5mL anhydrous methanol to obtain a 2-methylimidazole solution, the Zn(NO3)2•6H2O solution and the 2-methylimidazole solution are mixed, stirred at 25°C for 2h, allowed to stand for 24h, centrifuged, and dried to obtain ZIF-8; 100mg ZIF-8 is placed in a calcined boat, pyrolyzed at 900°C for 3h under a nitrogen atmosphere, and naturally cooled to obtain NC. The preparation method of the NC-TMB-H2O2 system is as follows: NC, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine are added to acetic acid / sodium acetate buffer to form a NC-TMB-H2O2 system. In order to study the composition and chemical state of NC, X-ray photoelectron spectroscopy (XPS) test was performed on NC. The test results are shown in Figure 2. Figure 3 As shown in (A), the full XPS spectrum of NC shows that there is no iron element in NC.

[0028] In step S13, the absorbance of the Fe / NC-TMB-H2O2 system, the Fe / NC-TMB system and the NC-TMB-H2O2 system are tested respectively. The test results are as follows: Figure 4 As shown. Figure 4 It can be seen from (A) that when H2O2 exists in the system, the Fe / NC-TMB-H2O2 system has an obvious absorption peak at 652nm compared with the Fe / NC-TMB system, indicating that the Fe / NC nanozyme has excellent peroxidase-like catalytic activity. Figure 4 As can be seen from (B), compared with the NC-TMB-H2O2 system, the Fe / NC-TMB-H2O2 system has an obvious absorption peak at 652 nm, indicating that the iron atoms in the Fe / NC nanozyme participate in the catalytic reaction. Figure 4 As can be seen in (C), the peroxidase-like catalytic activity of Fe / NC nanozyme depends on the concentration. When the concentration of Fe / NC nanozyme is between 25 and 50 μg mL -1 When Fe / NC nanozymes are added, they have good peroxidase-like catalytic activity. Figure 4 (D) and Figure 4 As can be seen from (E) in the figure, the Fe / NC-TMB-H2O2 system has good peroxidase activity when the pH is 3.5~4.5 and the system temperature is 25~45℃.

[0029] The enzyme kinetics of Fe / NC nanozyme in Fe / NC-TMB-H2O2 system were studied under the conditions of pH 3.5 and temperature 25°C, and the typical Michaelis–Menten curve and the corresponding Lineweaver–Burk plot were obtained. Figure 5 The Km values ​​of H2O2 and TMB were 19.37 mM and 0.4229 mM, respectively, while the calculated Vmax values ​​were 3.762*10 -8 Ms -1 and 3.783*10 -8 Ms -1 H2O2 and TMB have lower Km values, indicating a stronger affinity between the enzyme and the substrate. Since Fe / NC nanozyme has a lower Km value, Fe / NC nanozyme can be used as a colorimetric assay.

[0030] In step S2, the buffer is selected from an acidic buffer, such as a glycine-hydrochloric acid buffer, a phthalic acid-hydrochloric acid buffer, a citric acid-sodium hydroxide-hydrochloric acid buffer, a citric acid-sodium citrate buffer, an acetic acid / sodium acetate buffer, and other acidic buffers commonly found in the art. Exemplarily, the buffer is selected from an acetic acid / sodium acetate buffer. In one embodiment, the pH of the first reaction solution is 3.5-4.5, and the temperature of the first reaction solution is 25-45° C. Exemplarily, Chinese medicine polysaccharides are selected from Chinese medicine polysaccharides commonly found in the art, such as three representative Chinese medicine polysaccharides, namely, Chinese wolfberry Chinese medicine polysaccharides (LBP), Panax notoginseng Chinese medicine polysaccharides (PNP), and Salvia miltiorrhiza Chinese medicine polysaccharides (SMP), and the antioxidant activity of the above-mentioned Chinese medicine polysaccharides is detected based on Fe / NC nanozymes. Fe / NC nanozymes, Chinese medicine polysaccharides, hydrogen peroxide, and a chromogenic substrate are added to the buffer to form a first reaction solution, and TMB is used as a chromogenic substrate. In the first reaction solution, the volume of acetic acid / sodium acetate buffer was 100 µL, the concentration of the buffer was 0.2 mol / L, the amount of H2O2 added was 100 mM, the pH of the first reaction solution was 3.5, the temperature of the first reaction solution was 25°C, and the concentrations of the Chinese medicine polysaccharides were 3.91 µg mL -1 , 7.81 µg mL -1 , 15.62 µg mL -1 , 31.25 µg mL -1 , 62.5 µg mL -1 , 125 µgmL -1 , 250 µg mL -1 and 500 µg mL -1 .

[0031] In step S3, after the mixed solution in step S2 is incubated at room temperature for 5 minutes, the absorbance of the first reaction solution is measured using an ELISA instrument. The test result is as follows: Figure 6 As shown. Figure 6 As shown in (A), with the addition of LBP / PNP / SMP, the characteristic absorption peak of oxTMB at 500~800nm ​​gradually decreased, proving the feasibility of the colorimetric sensing system. The absorbance difference ΔA was calculated by ΔA=A0−A (A0 represents the absorbance value of the Fe / NC-TMB-H2O2 system at 652nm when no Chinese medicine polysaccharide is added, and A represents the absorbance value of the Fe / NC-TMB-H2O2 system at 652nm when Chinese medicine polysaccharide is added). Figure 6 (B) and Figure 6 As can be seen in (C), ΔA and LBP varied from 3.91 to 125 µg mL -1 The concentration range showed a good linear relationship (R 2= 0.9789); from Figure 6 (D) and Figure 6 As can be seen in (E), for PNP, as the concentration increases from 15.62 to 125 µg mL -1 As the value of ΔA increases, ΔA also increases linearly (R 2 =0.9559); from Figure 6 (F) and Figure 6 As can be seen in (G), for SMP, as the concentration increases from 7.81 to 125 µg mL -1 As the value of ΔA increases, ΔA also increases linearly (R 2 =0.9779). In the Fe / NC-TMB-H2O2 system, the antioxidant activity (TAC) of LBP / PNP / SMP was characterized by the inhibition rate (%) of LBP / PNP / SMP on the system, and the inhibition rate was calculated as follows: inhibition rate (%) = ΔA / A. Figure 6 As shown in (H), the antioxidant capacity of LBP / PNP / SMP gradually increased with the increase of the concentration of Chinese medicine polysaccharides. Compared with LBP and PNP, SMP showed higher antioxidant activity. -1 At the concentrations of , the antioxidant activities of Chinese medicine polysaccharides are: SMP>LBP>PNP.

[0032] In one embodiment, in order to further detect the antioxidant activity of Chinese herbal medicines containing Chinese herbal polysaccharides, three Chinese herbal medicines, namely, wolfberry extract (LFE), Panax notoginseng extract (PNRE) and Salvia miltiorrhiza extract (SME), were used as examples. The total antioxidant capacity of Chinese herbal medicines containing Chinese herbal polysaccharides was detected by the Fe / NC nanozyme-based colorimetric sensing system. The total antioxidant capacity of Chinese herbal medicines was determined in the presence of 10 µg mL -1Fe / NC, 100 mM H2O2, 500 µM TMB, and 500 µgmL -1 The results were obtained in a mixed solution of LFE / SME / PNRE and 100 µL acetic acid / sodium acetate buffer (0.2 mol / L, pH=3.5). After incubation at room temperature for 20 minutes, the absorbance at 500-800 nm was measured, and the inhibition rates of LFE, PNRE, and SNE at 625 nm were calculated to characterize their total antioxidant capacity. Figure 7 As shown. Figure 7 It can be seen that at 500 µg mL -1 At the same concentration, the antioxidant activities of traditional Chinese medicine are: SME>LFE>PNRE, which is consistent with the antioxidant activity of traditional Chinese medicine polysaccharides.

[0033] In summary, due to the excellent peroxidase-like catalytic performance of Fe / NC nanozyme, it can catalyze the substrate H2O2 to produce hydroxyl radicals (·OH), and cause the colorless chromogenic substrate TMB to lose electrons and oxidize to produce blue oxTMB. Traditional Chinese medicine polysaccharides can quickly scavenge free radicals and inhibit the formation of oxTMB. Fe / NC nanozyme has a low Km value, so Fe / NC nanozyme can be used as a colorimetric assay. Fe / NC nanozyme can be used to achieve rapid, sensitive and practical detection of the antioxidant capacity of traditional Chinese medicine polysaccharides. Therefore, Fe / NC nanozyme is used for the detection of the antioxidant capacity of traditional Chinese medicine polysaccharides.

[0034] The present invention utilizes the excellent peroxidase-like catalytic performance of Fe / NC nanozymes to catalyze the substrate H2O2 to produce hydroxyl radicals (·OH), and promotes the colorless chromogenic substrate to lose electrons to generate colored products. Chinese medicine polysaccharides can quickly remove free radicals and inhibit the formation of colored products. Therefore, based on the linear relationship between the concentration of Chinese medicine polysaccharides and the degree of inhibition of Fe / NC enzyme activity, a colorimetric sensing system is constructed to achieve antioxidant activity evaluation of different Chinese medicine polysaccharides, thereby promoting the application of nanozymes in Chinese medicine polysaccharides. Therefore, the present invention effectively overcomes some practical problems in the prior art and has high utilization value and use significance.

[0035] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for detecting the antioxidant capacity of traditional Chinese medicine polysaccharides, characterized in that: The steps include: Provide Fe / NC nanozymes; Providing a buffer solution, and adding the Fe / NC nanozyme, traditional Chinese medicine polysaccharide, hydrogen peroxide and a color developing substrate into the buffer solution to form a first reaction solution; The absorbance value of the first reaction liquid at a specific wavelength is measured, and the antioxidant property of the traditional Chinese medicine polysaccharide is detected by the change of the absorbance value of the first reaction liquid.

2. The detection method according to claim 1, characterized in that: The detection method also includes a process of exploring the catalytic performance of the Fe / NC nanozyme.

3. The detection method according to claim 2, characterized in that: The process of exploring the catalytic performance of Fe / NC nanozymes includes the following steps: providing the buffer; adding the Fe / NC nanozyme, hydrogen peroxide and the chromogenic substrate to the buffer to form a second reaction solution; The absorbance value of the second reaction liquid at a specific wavelength is measured, and the catalytic performance of the Fe / NC nanozyme is detected by the change in the absorbance value of the second reaction liquid.

4. The detection method according to claim 3, characterized in that: The pH of the second reaction solution is 3.5-4.

5.

5. The detection method according to claim 3, characterized in that: The temperature of the second reaction liquid is 25-45°C.

6. The detection method according to claim 3, characterized in that: The color developing substrate is 3,3',5,5'-tetramethylbenzidine.

7. The detection method according to claim 4, characterized in that: The buffer solution is acetic acid / sodium acetate buffer solution.

8. The detection method according to claim 1, characterized in that: The preparation of the Fe / NC nanozyme comprises the following steps: Dissolve Zn(NO3)2•6H2O and 2-methylimidazole in anhydrous methanol to obtain Zn(NO3)2•6H2O solution and 2-methylimidazole solution respectively; The Zn(NO3)2•6H2O solution and the 2-methylimidazole solution are mixed, stirred, allowed to stand, centrifuged, and dried to obtain ZIF-8; The ZIF-8 and iron powder are placed in a nitrogen atmosphere, pyrolyzed at 800-1000° C. for 2-3 h, and naturally cooled to obtain the Fe / NC nanozyme.

9. A use of Fe / NC nanozyme, characterized in that: Fe / NC nanozyme is used to detect the antioxidant capacity of traditional Chinese medicine polysaccharides.

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