Screening method and activity evaluation method of natural antioxidant

Abstract

The invention relates to a screening method and an activity evaluation method of a natural antioxidant, and the screening method is characterized in that a cerium dioxide-based nano composite material with a specific structure is used as oxidase, a nano oxidase + natural antioxidant reaction system is constructed, and the activity of the natural antioxidant is evaluated through a high performance liquid chromatography-ultraviolet detector-tandem mass spectrometry analysis technology. Screening of the natural antioxidant is realized; according to the activity evaluation method, a cerium dioxide-based nano composite material with a specific structure is used as oxidase, a nano oxidase + natural antioxidant + TMB reaction system is constructed, and the activity evaluation of the natural antioxidant is realized through an oxidized TMB photo-thermal detection technology. According to the method disclosed by the invention, the used reaction system is high in applicability, high in accuracy and good in selectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine analysis, and more specifically, to a screening method and an activity evaluation method for natural antioxidants. Background Art

[0002] Antioxidant activity can be classified into in vitro chemical analysis methods, cell model evaluation, in vivo biomarker determination, and standardized methods according to the evaluation mechanism. Among them, the 2,2'-diphenyl-1-picrylhydrazyl (DPPH·) method and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS· + ) method in in vitro chemical analysis methods are widely used for the activity evaluation of the antioxidant activity of natural products due to their low cost and easy implementation. The DPPH·-HPLC method and ABTS·+-HPLC method developed in combination with high performance liquid chromatography (HPLC) are widely used for screening and analyzing antioxidant active ingredients in natural products. However, studies have found that antioxidants show different electron transfer modes for different free radicals, resulting in different screening results. For example, 26 phenylpropanoid compounds found in Lycium barbarum fruits show ROO· scavenging activity, but only 9 of them have DPPH· scavenging activity. And there are research reports that the correlation between the DPPH· scavenging assay and the ABTS· + scavenging assay is weak, and the ABTS· + scavenging assay can better reflect highly hydrophilic antioxidants. In addition, DPPH· and ABTS· + do not exist in vivo and cannot comprehensively reflect the antioxidant activity of the tested sample in the real in vivo environment. Therefore, discovering antioxidants from a physiological perspective and rationally designing reliable antioxidant (with ROS scavenging activity) evaluation and screening methods are the keys to the research. Summary of the Invention

[0003] Based on the above technical problems existing in the prior art, the present invention provides a screening method for natural antioxidants. This method uses a cerium oxide-based nanocomposite with a specific structure as a peroxidase-like enzyme to construct a nano-peroxidase-like enzyme + natural antioxidant reaction system, and realizes the screening of natural antioxidants through high performance liquid chromatography-ultraviolet detector-tandem mass spectrometry analysis technology. Moreover, this reaction system has strong applicability, high accuracy, and good selectivity.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] A screening method for natural antioxidants, which is: constructing a peroxidase-like enzyme + natural antioxidant reaction system, using a cerium oxide-based nanocomposite as a peroxidase-like enzyme, and catalyzing the absorbed O2 to generate ·O2 at physiological pH - , ·O2 -It can react with natural antioxidants to cause a decrease in HPLC-UV-MS / MS signals, thereby realizing the screening of antioxidants; wherein, the cerium dioxide-based nanocomposite includes nanoceria and adenosine triphosphate coated on the surface of ceria.

[0006] In some embodiments, the method for screening natural antioxidants includes the following steps:

[0007] (1) Prepare citrus extract: Wash the citrus, separate the peel and the edible part, then squeeze the edible part until no obvious liquid is released to obtain the pulp; dry the peel and the pulp separately, grind them into powder, extract by alcohol extraction method, filter, and collect the filtrate; evaporate the ethanol from the filtrate, then extract with ethyl acetate, and collect the ethyl acetate phase to obtain the citrus extract;

[0008] (2) Analyze and identify the citrus extract by HPLC-UV-MS / MS to obtain the first liquid chromatogram;

[0009] Then mix the citrus extract with the above-mentioned cerium dioxide-based nanocomposite and PBS buffer solution, and after reacting in the dark, analyze and identify the resulting reaction solution by HPLC-UV-MS / MS to obtain the second liquid chromatogram. By comparing the first liquid chromatogram and the second liquid chromatogram, and comparing and verifying with the standard products and prior art documents, the antioxidants in the citrus extract are screened out; wherein, the HPLC-UV-MS / MS analysis of the citrus extract before and after the reaction is as follows:

[0010] Filter with 0.22 μm, the eluent is formic acid solution (5‰, v / v, A) and acetonitrile (B), and the elution program is as follows:

[0011]

[0012] The sample injection volume is 20 μL; the mobile phase flow rate is 0.8 mL / min, the column temperature is 25 °C, and the DAD detection wavelength is 254 nm;

[0013] Quadrupole time-of-flight mass spectrometry / mass spectrometry analysis is carried out in the positive ion mode using an electrospray ionization source, and the mass spectrometry analysis is carried out under the following conditions:

[0014] Capillary voltage: 3500 V; drying gas flow rate: 8.0 L / min; drying temperature: 200 °C; nebulizer pressure: 0.3 bar; ion energy: 5.0 eV; collision energy: 35 eV; end plate offset voltage: 500 V; scanning range (m / z): 100 - 1000.

[0015] In some embodiments, in the reaction system, the concentration of the nanocomposite is 5 - 40 mg / mL.

[0016] In some embodiments, the method for preparing the cerium dioxide-based nanocomposite comprises the following steps:

[0017] Dissolve the soluble cerium salt in water, then add ammonia water to react to form cerium dioxide precipitate; add the cerium dioxide precipitate to water, then add adenosine triphosphate to react to obtain the cerium dioxide-based nanocomposite.

[0018] The present invention also provides a method for evaluating the activity of a natural antioxidant. This method constructs a peroxidase-like enzyme + natural antioxidant + TMB reaction system, uses the cerium dioxide-based nanocomposite as the peroxidase-like enzyme to catalyze the absorbed O2 to generate ·O2 - ·O2 - which catalyzes TMB to generate oxidized oxTMB. Using oxTMB as a photothermal reagent, the detection signal is converted into a thermal signal to achieve the photothermal detection of the antioxidant activity, and the activity of the antioxidant is calculated according to the photothermal detection result.

[0019] In some embodiments, the method for evaluating the activity of the natural antioxidant comprises the following steps:

[0020] (1) Mix the compound to be tested with the cerium dioxide-based nanocomposite, TMB and PBS buffer solution, after reacting in the dark, irradiate with an infrared laser, and monitor the temperature change with an infrared thermal imager and record it as ΔT 样品 ;

[0021] (2) Mix the cerium dioxide-based nanocomposite, TMB and the first buffer solution, after reacting in the dark, irradiate with an infrared laser, and monitor the temperature change with an infrared thermal imager and record it as ΔT 空白 ;

[0022] (3) Calculate the half-inhibition rate IC 50 according to the following inhibition rate calculation formula, where the IC 50 value represents the concentration of the compound to be tested corresponding to an inhibition rate of 50%, and the smaller the IC 50 value, the better the antioxidant activity of the compound to be tested; the inhibition rate calculation formula is as follows:

[0023] Inhibition rate (%) = (1 - ΔT 样品 / ΔT 空白 ) × 100.

[0024] In some embodiments, in the reaction system, the concentration of the nanocomposite is 5 - 50 μg / mL; the pH of the reaction system is 3 - 8; the concentration of TMB is 1 - 5 mM.

[0025] In some embodiments, the wavelength of the infrared laser is 750 - 850 nm.

[0026] In some embodiments, the method for preparing the cerium dioxide-based nanocomposite comprises the following steps:

[0027] Dissolve a soluble cerium salt in water, then add ammonia water for reaction to form a cerium dioxide precipitate; add the cerium dioxide precipitate to water, then add adenosine triphosphate for reaction to obtain the cerium dioxide-based nanocomposite.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The present invention uses a cerium dioxide-based nanocomposite with a specific structure as a peroxidase-mimicking enzyme to construct a peroxidase-mimicking enzyme + natural antioxidant reaction system, and realizes the screening of natural antioxidants through high performance liquid chromatography-ultraviolet detector-tandem mass spectrometry analysis technology; in addition, by constructing a peroxidase-mimicking enzyme + natural antioxidant + TMB reaction system and using the oxidation of TMB photothermal detection technology, the activity evaluation of natural antioxidants is realized; the screening method and activity evaluation method of the present invention have the characteristics of strong applicability, high accuracy and good selectivity.

[0030] Specifically, as Figure 1 shown, the principle of the cerium dioxide-based nanocomposite of the present invention as a peroxidase-mimicking enzyme in the screening and activity evaluation of natural antioxidants is: the cerium dioxide-based composite catalyzes O2 to generate ·O2 - , and the peak area of a compound with potential antioxidant activity will decrease or disappear in the HPLC chromatogram after reacting with the generated ·O2 - , while for a compound without antioxidant activity, the peak area hardly changes. Combining high performance liquid chromatography-ultraviolet detector-tandem mass spectrometry (HPLC-UV-MS / MS) technology to efficiently screen natural antioxidants; at the same time, using ·O2 - can oxidize TMB to obtain oxTMB with a photothermal effect, thereby realizing the photothermal detection of antioxidant activity.

[0031] Through the screening method and activity evaluation method of natural antioxidants provided by the present invention, antioxidants can be accurately and selectively screened from a physiological perspective and the activity of antioxidants can be evaluated, providing a novel idea and a reliable platform for screening antioxidants in natural complex samples and evaluating the activity of antioxidants, greatly making up for the deficiencies of traditional screening methods. At the same time, the detection based on the photothermal method can exclude matrix interference, thereby improving the detection accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram of screening and evaluating the activity of natural antioxidants by the cerium dioxide-based composite of the present invention;

[0033] Figure 2 In it, Figure A is the TEM image of CeO2-A prepared in Example 1; Figure B is the XRD pattern of CeO2-A, Figure C is the Raman spectrum of CeO2-A; Figure D is the XPS pattern of CeO2-A; Figure E is the Ce 3d spectrum of CeO2-A; Figure F is the infrared spectrum of CeO2-A;

[0034] Figure 3 are the comparative dot-line graph and ultraviolet spectrum of the reaction activities of CeO2-A and CeO2 prepared in Example 1 with TMB under different pH conditions;

[0035] Figure 4 is the evaluation result of the peroxidase-like activity of CeO2-A prepared in Example 1;

[0036] Figure 5 is the screening result of potential antioxidants in the mixed standard sample by the CeO2-A method, DPPH· (HAT mechanism) method and ABTS·+ (SET mechanism) method in Example 2;

[0037] Figure 6 are the HPLC chromatograms of lime peel, lime pulp, sweet orange peel, sweet orange pulp and CeO2-A before and after reaction in Example 2; among them, Figure A is the HPLC chromatogram of lime peel before and after reaction with CeO2-A; Figure B is the HPLC chromatogram of lime peel before and after reaction with CeO2-A; Figure C is the HPLC chromatogram of lime peel before and after reaction with CeO2-A; Figure D is the HPLC chromatogram of lime peel before and after reaction with CeO2-A; in the figure, the black line is the HPLC chromatogram before reaction, and the red line is the HPLC chromatogram after reaction;

[0038] Figure 7 are the antioxidant flavonoids identified in lime and sweet orange in Example 2;

[0039] Figure 8 In it, Figure A is the optimization of the reaction concentration of TMB in Example 3, and 1 mM is selected; Figure B is the optimization of the reaction concentration of CeO2-A, and 30 μg / mL is selected; Figure C is the optimization of the incubation temperature, and 37 °C is selected; Figure D is the optimization of the reaction time, and 5 min is selected; Figure E is the optimization of the reaction pH, and pH = 7.4 is selected;

[0040] Figure 9 are the inhibition rates of four standard products, quercetin, rutin, hesperidin and nobiletin, in Example 3 (the accompanying figure is the color change presented with the change of antioxidant concentration under photothermal detection); among them, Figure A is the inhibition curve of quercetin; Figure B is the inhibition curve of rutin; Figure C is the inhibition curve of hesperidin; Figure D is the inhibition curve of nobiletin;

[0041] Figure 10 Inhibitory curves of lime peel, lime pulp, sweet orange peel and sweet orange pulp in Example 3 (the accompanying figure shows the color change with the change of antioxidant concentration under photothermal detection); among them, Figure A is the inhibitory curve of lime peel; Figure B is the inhibitory curve of lime pulp; Figure C is the inhibitory curve of sweet orange peel; Figure D is the inhibitory curve of sweet orange pulp. Detailed implementation manners

[0042] Many specific details are set forth in the following description in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementations disclosed below.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs. The terms used in the description of the present invention herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention.

[0044] Example 1

[0045] Preparation of a cerium dioxide-based nanocomposite (CeO2-A), comprising the following steps:

[0046] S1. Prepare a 0.1M cerium sulfate solution with distilled water to obtain a clear yellow solution, vigorously stir it on a magnetic stirring table for 15 min, and then dropwise add 4 mL of 50% ammonia water. The solution immediately turns yellow, forming a precipitate to obtain a suspension;

[0047] S2. Further stir the suspension for 1 h, centrifuge it at 5000 rpm for 5 min, wash it with absolute ethanol, and air-dry it at room temperature to obtain pale yellow CeO2;

[0048] S3. Take 1 g of the dried CeO2, add 10 mL of ultrapure water and dissolve it with ultrasonic treatment, then add 2 g of ATP (adenosine triphosphate) powder, keep it on a vortex mixer for 12 h, centrifuge and wash it at 5000 rpm, and air-dry it at room temperature to obtain a pale yellow cerium dioxide-based nanocomposite CeO2-A.

[0049] The structure of CeO2-A obtained in this example was characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR). The characterization results are as Figure 2 shown.

[0050] As Figure 2, The TEM image shows that the synthesized CeO2-A consists of dispersed nanoparticles, and the characteristic interplanar spacing (0.25 nm) of the lattice fringes corresponds to its (110) crystal plane. According to the XRD pattern, three prominent peaks are observed at 28.48°, 46.76°, and 56.43°, corresponding to the face-centered cubic structures of (111), (220), and (311). The Raman spectrum shows that CeO2-A contains a characteristic peak at 448 cm -1 which corresponds to the F 2g vibration mode of the fluorite-phase CeO2; the characteristic peak at 600 cm -1 is related to the defects associated with the presence of oxygen vacancies.

[0051] To investigate the elemental composition and surface electronic structure of the CeO2-A material, XPS analysis was performed, as shown in Figure 2 Figure D. In the full XPS spectrum of CeO2-A, elements including Ce, O, C, and P were detected, indicating the successful synthesis of CeO2-A; meanwhile, the mixed valence state of CeO2-A (Ce 3+ and Ce 4+ ) was also shown. Since the ratio of Ce 3+ / Ce 4+ directly determines the enzyme-mimicking activity, a thorough XPS analysis of CeO2-A was carried out to evaluate the Ce 3+ and Ce 4+ ions present on the material surface. The XPS spectrum of Ce 3d ( Figure 2 Figure E) can be divided into Ce 3+ and Ce 4+ . The peaks at 881.2 eV (V0), 884.9 eV (V′), 899.4 eV (U0), and 903.3 eV (U′) can be attributed to Ce 3+ , while the peaks at 882.5 eV (V), 888.8 eV (V′), 898.1 eV (V”'), 901.1 eV (U), 907.6 eV (U″), and 916.7 eV (U”') can be attributed to Ce 4+ . By calculation, the Ce 3+ / Ce 4+ ratio of CeO2-A was found to be 35.23%, further demonstrating that CeO2-A has peroxidase-like activity. Finally, as shown in Figure 2 Figure F, surface functional group analysis was carried out in combination with FT-IR, proving the successful synthesis of the CeO2-A material.

[0052] The evaluation of the peroxidase-like activity of CeO2-A was carried out as follows:

[0053] As shown in Figure 3, CeO2 has the strongest peroxidase-like activity at pH = 3 and almost no activity under neutral conditions. However, CeO2-A not only exhibits peroxidase-like activity under acidic conditions but also has strong peroxidase-like activity under neutral conditions. Therefore, the optimal pH = 7.4 was selected.

[0054] CeO2 and CeO2-A (30 μg / mL, 200 μL) were respectively mixed with TMB (1 mM, 200 μL) and PBS buffer solution (10 mM, pH 7.4, 1.6 mL) and reacted at 37 °C for 5 min, and then analyzed using a UV-visible spectrophotometer. For steady-state kinetic analysis, different concentrations of TMB (3,3',5,5'-tetramethylbenzidine) were selected in the reaction system, and the absorbance at 652 nm was measured. According to the Michaelis-Menten equation, by fitting the reaction rate values and TMB concentrations, the kinetic constants (K m , mM; V max , M s –1 ) were obtained, and the results are as Figure 4 shown.

[0055] As Figure 4 shown in Figure A, a blue solution was produced after CeO2-A was incubated with TMB, which is a typical characteristic of TMB oxidation, and there was a typical absorption peak at 652 nm. In an argon-saturated system, the oxidation of TMB was significantly weakened. Therefore, CeO2-A can catalyze the oxidation of TMB with the help of dissolved oxygen, which confirms that CeO2-A has peroxidase-like activity rather than acting as an oxidant.

[0056] In addition, the kinetic parameters were analyzed, and the Michaelis constant (K Figure 4 , 0.33 mM) and the maximum initial velocity (V m , 2.45×10 max M s -6 ) values were obtained according to the Lineweaver-Burk plots (such as -1 shown in Figures B and C).

[0057] Reactive oxygen species (ROS) scavengers were introduced, including terephthalic acid (TPA) for scavenging hydroxyl radicals (·OH), p-benzoquinone (p-BQ) for scavenging superoxide anion radicals (·O2 - ) and L-tryptophan (L-TRP) for scavenging singlet oxygen ( 1 O2) to identify the generated ROS. As Figure 4As shown in Figures D and E, in the presence of TPA, there is almost no change in its fluorescence spectrum. In the presence of L-TRP, there is also almost no change in the absorbance of TMB at 652 nm. Only in the presence of p-BQ, the absorbance drops sharply, indicating that CeO2-A activates the surface-adsorbed O2 to ·O2 - . To verify the generation of ·O2 - during the catalytic process of CeO2-A, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used as the spin trap of ·O2 - to conduct an electron paramagnetic resonance (EPR) experiment. As shown in Figure F of Figure 4 , the peak intensity ratios of the ·O2 - spin adduct are 1:1:1:1 respectively. The results show that ·O2 - with potential oxidation ability is generated during the peroxidase-like catalytic process of CeO2-A,

[0058] Example 2

[0059] The antioxidant flavonoids in the peels and pulps of lime and sweet orange were screened using CeO2-A prepared in Example 1 as follows:

[0060] (1) Extraction of citrus extracts: The lime was from Loudi, Hunan, and the sweet orange was from Ganzhou, Jiangxi. The lime and sweet orange were thoroughly washed with tap water, peeled with a knife, and the peels and edible parts were separated. Then the edible parts were manually squeezed until no obvious liquid was released to produce the pulp. The peels and pulps were dried overnight at 60 °C, ground into powder, and passed through a 500-mesh sieve. Subsequently, the samples (peel, 100 g; pulp, 100 g) were extracted with 75% ethanol (v / v, 750 mL) at 85 °C using the heating reflux method (3 times, 2 hours each). The filtered extracts were combined, evaporated under vacuum at 50 °C to remove ethanol, and then liquid-liquid separation was carried out with ethyl acetate (100 mL, 3 times). Finally, ethyl acetate extracts (lime peel extract, lime pulp extract, sweet orange peel extract, sweet orange pulp extract) were obtained, and the extracts were stored at 4 °C for further experiments;

[0061] (2) Screening of potential antioxidants in the mixed standard samples (quercetin, rutin, hesperidin, nobiletin) based on the scavenging methods of CeO2-A, DPPH· (HAT mechanism), and ABTS·+ (SET mechanism) in Example 1 to verify the successful establishment of the method.

[0062] The detection results are as shown in Figure 5 .

[0063] As shown in Figure 5As shown in Figure A, with the increase in the concentration of CeO2-A, the HPLC peak areas of the four flavonoids gradually decreased until they disappeared, indicating that these compounds reacted with ·O2 generated by CeO2-A catalysis. - When the concentration of CeO2-A was 5 mg / mL, the HPLC peak of quercetin completely disappeared, while the HPLC peak areas of rutin, hesperidin, and nobiletin decreased to varying degrees. When the concentration of CeO2-A was 40 mg / mL, the peak areas of all four standards decreased significantly, and the reaction basically reached equilibrium. As Figure 5 shown in Figure B, the peak area reduction rates decreased in the order of quercetin (100%) > rutin (94.59%) > hesperidin (73.85%) > nobiletin (15.89%), indicating that the peak area reduction rate of quercetin was consistent in the three screening methods, while the results of hesperidin and nobiletin had weaker consistency. As Figure 5 shown in Figure C, the four flavonoids were detected by the DPPH· method and the ABTS·+ method. Hesperidin and nobiletin showed extremely weak activities. This indicates that there are differences in the action mechanisms of different methods with active intermediates, resulting in deviations in the screening results. The method of the present invention can effectively screen and evaluate the activities of antioxidants.

[0064] (3) Subsequently, in a PBS buffer solution, CeO2-A and the test samples (i.e., extracts of bitter orange peel, extracts of bitter orange pulp, extracts of sweet orange peel, extracts of sweet orange pulp) were mixed to construct a peroxidase-like enzyme-natural antioxidant reaction system, and then the reaction was carried out in the dark at 37 °C for 5 min to obtain the reaction solution of the experimental group; among them, in the reaction system, the concentration of CeO2-A was 40 mg / mL; the concentration of the PBS buffer solution was 10 mM, pH 7.4;

[0065] (4) The PBS buffer solution was used instead, mixed with the test samples (i.e., extracts of bitter orange peel, extracts of bitter orange pulp, extracts of sweet orange peel, extracts of sweet orange pulp), and then the reaction was carried out in the dark at 37 °C for 5 min to obtain the reaction solution of the blank group;

[0066] (5) The reaction solutions of the experimental group and the blank group were analyzed by HPLC-UV-MS / MS as follows:

[0067] The reaction solution was filtered through 0.22 μm, and the eluent was formic acid solution (5‰, v / v, A) and acetonitrile (B). The elution program was as follows:

[0068]

[0069]

[0070] The sample injection volume was 20 μL; the mobile phase flow rate was 0.8 mL / min, the column temperature was 25 °C, and the DAD detection wavelength was 254 nm;

[0071] Quadrupole time-of-flight mass spectrometry / mass spectrometry analysis was performed in the positive ion mode using an electrospray ionization source, and the mass spectrometry analysis was carried out under the following conditions:

[0072] Capillary voltage: 3500 V; drying gas flow rate: 8.0 L / min; drying temperature: 200 °C; nebulizer pressure: 0.3 bar; ion energy: 5.0 eV; collision energy: 35 eV; endplate offset voltage: 500 V; scan range (m / z): 100 - 1000.

[0073] Bruker Compass Date Analysis (version 4.4) was installed for MS data acquisition and processing.

[0074] The screening results are as Figure 6 shown.

[0075] By comparing the HPLC chromatograms before and after the reaction with CeO2-A, the peak areas of antioxidant flavones 1 - 15 decreased. In addition, their structures were identified by HPLC-UV-MS / MS analysis and compared and verified with standards and reported literature.

[0076] Table 1 and Figure 7 lists the HPLC-UV-MS / MS data and structural information.

[0077] In the positive ion mode, flavone O-glycosides can produce fragment ions by losing the sugar moiety (e.g., rutin or neohesperidin is 308 Da), and flavone C-glycosides will produce fragment ions by consecutive loss of H2O and diagnostic sugar cleavage ions [M + H–120] + ( 0,2 X) and [M + H–150] + ( 0,1 X) to form fragment ions [M + H–n·CH3] + , while polymethoxyflavones are characterized by fragment ions [M + H–n·CH3] generated by the loss of methyl radicals + . By comparison with standards, the identified compounds 1 - 15 were naringin rutinoside, naringenin, hesperidin, apigenin-7-O-neohesperidoside, isosakuranetin, sinensetin, luteolin tetramethyl ether, 3,5,6,7,3’,4’-hexamethoxyflavone, nobiletin, 4',5,6,7-tetramethoxyflavone, 3',4',3,5,6,7,8-heptamethoxyflavone, naringenin chalcone, tangeretin, vitexin, demethylnobiletin in sequence.

[0078] Table 1 Identification results of antioxidant active components in citrus extracts

[0079]

[0080] Example 3

[0081] Application of CeO2-A in the activity evaluation of natural antioxidants is as follows:

[0082] Firstly, the concentrations of TMB, CeO2-A, reaction time, reaction temperature and pH were optimized. Finally, the TMB concentration was selected as 1 mM, the CeO2-A concentration was 30 μg / mL, the optimal reaction time was 5 min, the reaction temperature was selected as 37 °C, and the pH was selected as 7.4. The specific results are as Figure 8 shown.

[0083] Mix 200 μL of antioxidant samples with different concentrations (quercetin, rutin, hesperidin, nobiletin) with CeO2-A (30 μg / mL, 200 μL), TMB (1 mM, 200 μL) and PBS buffer solution (10 mM, pH 7.4, 1.4 mL). After reacting in the dark at 37 °C for 30 min, irradiate with near-infrared laser (808 nm, 2.0 W / cm2) for 6 min, and use an infrared thermal imager to detect the temperature change and record it as ΔT 样品 ; for the blank group, use PBS buffer solution (i.e., mix the cerium oxide-based nanocomposite with TMB and PBS buffer solution), and the temperature change is recorded as ΔT 空白 ; calculate the half-inhibition rate IC 50 according to the measured data and the inhibition rate calculation formula, where the IC 50 value represents the concentration of the test compound corresponding to an inhibition rate of 50%. The smaller the IC 50 value, the better the antioxidant activity of the test compound; the inhibition rate calculation formula is as follows:

[0084] Inhibition rate (%) = (1 - ΔT 样品 / ΔT 空白 ) × 100.

[0085] The activity evaluation results of the standards (quercetin, rutin, hesperidin and nobiletin) and actual samples (lime peel, lime pulp, sweet orange peel and sweet orange pulp) are as Figure 9 and Figure 10 shown.

[0086] As Figure 9 shown, as the concentration of the antioxidant increases, the purple color gradually weakens under the photothermal state, indicating that the oxidation of TMB is inhibited, ΔT gradually decreases, and its inhibition rate is calculated. The inhibition rate shows an excellent linear relationship with the antioxidant concentration. Calculate the IC of quercetin, rutin, hesperidin and nobiletin according to the linear equation50 The values were 1.08±0.06 μg / mL, 2.23±0.04 μg / mL, 40.08±2.44 μg / mL, and 972.93±41.99 μg / mL respectively. It can be seen that the order of antioxidant activity is quercetin > rutin > hesperidin > nobiletin, which is consistent with the liquid phase results, further proving the feasibility of the method.

[0087] Finally, lime and sweet orange were used as application examples for antioxidant activity evaluation. According to the linear equation, the IC 50 value of lime peel was calculated to be 44.64±4.09 μg / mL, and the IC 50 value of lime pulp was 601.33±3.46 μg / mL. The IC 50 value of sweet orange peel was 65.11±1.36 μg / mL, and the IC 50 value of sweet orange pulp was 109.87±0.45 μg / mL. The inhibition curves are as Figure 10 shown. The IC 50 values of lime and sweet orange peels were significantly lower than those of the pulps, indicating that the antioxidant activity of the peels is stronger than that of the pulps. This may be due to the higher content of flavonoids in the peels. The research results provide a broad prospect for the application of photothermal detection in the evaluation of antioxidant activity in actual samples.

[0088] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0089] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A screening method for natural antioxidants, characterized in that, Construct an oxidase-like + natural antioxidant reaction system, using cerium-based nanocomposites as oxidase-like enzymes to catalyze the absorbed O2 to generate ·O2 at physiological pH - , ·O2 - can react with natural antioxidants to cause a decrease in HPLC-UV-MS / MS signals, realizing the screening of antioxidants; wherein, the cerium-based nanocomposites include nano-ceria and adenosine triphosphate coated on the surface of ceria.

2. The screening method of the natural antioxidant according to claim 1, characterized in that, It includes the following steps: (1) Prepare citrus extract: Wash the citrus, separate the peel and the edible part, then squeeze the edible part until no obvious liquid is released to obtain pulp; dry the peel and pulp separately, grind them into powder, extract by alcohol extraction method, filter, and collect the filtrate; evaporate the ethanol from the filtrate, then extract with ethyl acetate, collect the ethyl acetate phase to obtain the citrus extract; (2) Analyze and identify the citrus extract by HPLC-UV-MS / MS to obtain the first liquid chromatogram; Then mix the citrus extract with the cerium dioxide-based nanocomposite and PBS buffer solution, react in the dark, and analyze and identify the resulting reaction solution by HPLC-UV-MS / MS to obtain the second liquid chromatogram. By comparing the first liquid chromatogram and the second liquid chromatogram, and comparing and verifying with the standard product and the prior art literature, the antioxidants in the citrus extract are screened out; wherein, The analysis of the citrus extract by HPLC-UV-MS / MS before and after the reaction is as follows: Filter with 0.22 μm, the eluent is formic acid solution (5‰, v / v, A) and acetonitrile (B), and the elution program is as follows: 0 - 10 min 5 - 25% B 10 - 16 min 30% B 16 - 25 min 40 - 45% B 25 - 45 min 45 - 100% B 45 - 50 min 100% B The sample injection volume is 20 μL; the flow rate of the mobile phase is 0.8 mL / min, the column temperature is 25 °C, and the DAD detection wavelength is 254 nm; Quadrupole time-of-flight mass spectrometry / mass spectrometry analysis is carried out in the positive ion mode using an electrospray ionization source, and the mass spectrometry analysis is carried out under the following conditions: Capillary voltage: 3500 V; drying gas flow rate: 8.0 L / min; drying temperature: 200 °C; nebulizer pressure: 0.3 bar; ion energy: 5.0 eV; collision energy: 35 eV; end plate offset voltage: 500 V; scanning range (m / z): 100 - 1000.

3. The screening method of the natural antioxidant according to claim 2, wherein In the reaction system, the concentration of the nanocomposite is 5 - 40 mg / mL.

4. The screening method of the natural antioxidant according to any one of claims 1-3, characterized in that, The preparation method of the cerium dioxide-based nanocomposite includes the following steps: Dissolve the soluble cerium salt in water, then add ammonia water to react to form cerium dioxide precipitate; add the cerium dioxide precipitate to water, then add adenosine triphosphate to react to obtain the cerium dioxide-based nanocomposite.

5. A method for evaluating the activity of a natural antioxidant, characterized in that, Construct a peroxidase-like + natural antioxidant + TMB reaction system, using cerium dioxide-based nanocomposites as peroxidase-like enzymes to catalyze the absorbed O2 to generate ·O2 - , ·O2 - catalyzes TMB to produce oxidized oxTMB. Using oxTMB as a photothermal reagent, the detection signal is converted into a thermal signal to achieve photothermal detection of the antioxidant activity, and the activity of the natural antioxidant is calculated according to the photothermal detection result.

6. The method for evaluating the activity of the natural antioxidant according to claim 5, wherein It includes the following steps: (1) Mix the compound to be tested with a cerium dioxide-based nanocomposite, TMB, and a PBS buffer solution. After reacting in the dark and irradiating with an infrared laser, monitor the temperature change with an infrared thermal imager and record it as ΔT 样品 ; (2) Mix the cerium dioxide-based nanocomposite, TMB, and PBS buffer solution. After reacting in the dark and irradiating with an infrared laser, monitor the temperature change with an infrared thermal imager and record it as ΔT 空白 ; (3) Calculate the half-inhibition rate IC according to the following inhibition rate calculation formula 50 , where IC 50 value represents the concentration of the test compound corresponding to an inhibition rate of 50%. The smaller the IC 50 value, the better the antioxidant activity of the test compound. The inhibition rate calculation formula is as follows: Inhibition rate (%) = (1 - ΔT 样品 / ΔT 空白 ) × 100.

7. The method for evaluating the activity of the natural antioxidant according to claim 6, characterized in that In the reaction system, the concentration of the nanocomposite is 5 - 50 μg / mL; the pH of the reaction system is 3 - 8; the concentration of TMB is 1 - 5 mM.

8. The method for evaluating the activity of the natural antioxidant according to claim 6, wherein The wavelength of the infrared laser is 750 - 850 nm.

9. The method for evaluating the activity of the natural antioxidant according to any one of claims 5-8, characterized in that, The preparation method of the cerium dioxide-based nanocomposite includes the following steps: Dissolve the soluble cerium salt in water, then add ammonia water to react to form cerium dioxide precipitate; add the cerium dioxide precipitate to water, then add adenosine triphosphate to react to obtain the cerium dioxide-based nanocomposite.

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