Alpha-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxide nanosheets

Abstract

The invention discloses an alpha-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxide nanosheets, the alpha-glucosidase inhibitor screening kit comprises an R-CDs (at) CoOOH NCs nano composite material, and a preparation method of the R-CDs (at) CoOOH NCs nano composite material comprises the following steps: preparing an R-CDs solution and a CoOOH NCs solution: mixing the R-CDs solution, the CoOOH NSs solution and ultrapure water according to a volume ratio of 5: 2: 8 to obtain the R-CDs (at) CoOOH NCs nano composite material. The fluorescence sensor is used for detecting alpha-Glu and screening a natural inhibitor in a polygonum cuspidatum extract, is simple to operate, low in cost and high in sensitivity, and is composed of the FRET effect between R-CDs and CoOOH NSs. Through verification, the method can be used for determining the activity of alpha-Glu in vitro, the linear detection range of the alpha-Glu activity under 625nm fluorescence emission is between 0.01 U / mL and 15U / mL, and the detection limit is 0.0037 U / mL. The determination method can also be used for screening anti-diabetic active components from polygonum cuspidatum.

Description

Technical Field

[0001] The invention relates to an inhibitor screening technology, in particular to an alpha-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxyl nanosheets. Background Art

[0002] Diabetes is a chronic metabolic disease accompanied by metabolic endocrine disorders, mainly caused by insufficient insulin secretion or pancreatic islet dysfunction and external factors, which has attracted widespread attention worldwide. Among them, type 2 diabetes (T2D) is considered to be one of the main types of diabetes. α-Glu is a hydrolase that accelerates the release of glucose during digestion and is a very important factor in regulating glucose concentration to the normal range. The screening and use of α-glucosidase inhibitors are currently the key methods for the prevention and treatment of T2D. T2D patients often choose oral α-Glu inhibitors such as acarbose, miglitol and voglibose to balance the glucose level in the blood. However, long-term use of these drugs can cause some side effects such as intestinal obstruction, flatulence, nausea and vomiting. Since natural products are green, low-toxic and effective, screening enzyme inhibitors from natural products is a promising method for disease prevention and treatment. Therefore, the accurate detection of α-Glu and the screening of α-Glu inhibitors are of great significance.

[0003] The p-nitrophenolpyranoglucoside colorimetric method is a classic method for sensing α-Glu, but this method is prone to overlapping absorption, often has high interference and low sensitivity. Subsequently, although some new α-Glu determination and high-throughput screening methods have been developed using nuclear magnetic resonance (NMR), capillary electrophoresis (CE), colorimetry and electrochemistry, there are still some inevitable disadvantages. For example, the long waiting time required for NMR makes it difficult to improve the screening efficiency, CE is highly sensitive to environmental factors, and the sensitivity of colorimetry is relatively low. In addition, electrochemistry also has high interference problems. Fluorescence technology sensing has attracted widespread attention due to its rapid detection, simple operation and high sensitivity.

[0004] In the prior art, a multifunctional CD (mCDs) nanoplatform was assembled using blue fluorescent carbon dots (CDs), β-cyclodextrin and phenylboronic acid for sensitive detection of α-Glu and screening of inhibitors. The high recognition and strong binding ability of the hydrolysis product p-nitrophenol with β-cyclodextrin and phenylboronic acid effectively quenched the fluorescence of mCDs. Although most CDs emit bright blue-green light under ultraviolet light, they have relatively low quantum yields (QY) in the long wavelength range, resulting in low sensitivity and narrow detection range. When screening inhibitors, they are easily interfered by the autofluorescence of some natural sample matrices, resulting in false positive results. Recent research results show that the optimization of synthetic solvents and reaction conditions will red-shift the emission wavelength of carbon dots and greatly improve their quantum yield. Long-wavelength red carbon dots show great application potential and have beneficial effects in the fields of environment and biomedicine. Cobalt oxyhydroxide nanosheets (CoOOH NSs) are 2D sheet-like nanomaterials with large specific surface area.

[0005] Polygonum cuspidatum is the dried rhizome and root of Polygonum cuspidatum Sieb.et Zucc., a plant of the Polygonaceae family. It has the effects of clearing away heat and dampness, removing jaundice and removing blood stasis. It can be used to treat inflammation, diabetes, gout, cancer, viral diseases, bacterial infectious diseases and neurodegenerative diseases. Previous studies have shown that the ethyl acetate and ether extracts of sea cucumbers, sea cucumbers and Bohemian sea cucumbers Chrtek and Chrtkova showed higher anti-glycolytic oxidative activity compared with aminoguanidine. Resveratrol can well reduce blood glucose levels, protect pancreatic B cells and improve insulin resistance. In addition, polydatin also improved the salivary insufficiency caused by diabetes in db / db2 type 2 diabetic model mice. Although resveratrol-4'-OD-glucoside, polydatin and malonyl glucoside resveratrol in Polygonum cuspidatum were found to have good inhibitory effects on α-glucosidase in previous studies, the components that have inhibitory effects on α-glucosidase activity are still not comprehensive. Therefore, a screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets is needed. Summary of the invention

[0006] The purpose of the present invention is to provide an alpha-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxide nanosheets.

[0007] The present invention includes a R-CDs@CoOOH NCs nanocomposite material. The preparation method of the R-CDs@CoOOH NCs nanocomposite material includes: preparing R-CDs and CoOOH NCs solutions: mixing the R-CDs, CoOOH NSs solution and ultrapure water in a volume ratio of 5:2:8 to obtain the R-CDs@CoOOH NCs nanocomposite material.

[0008] Further, the method for preparing R-CDs comprises:

[0009] Anhydrous citric acid and urea were ultrasonically dispersed in N, N-dimethylformamide in a mass ratio of 1:2 and mixed evenly, and the mixture was placed in a polytetrafluoroethylene autoclave, heated at 180°C for 5 hours, and then cooled to room temperature after the reaction was completed;

[0010] Add NaOH solution to the mixture at a mass ratio of 30:1 and stir for 5 minutes. Gradually add concentrated hydrochloric acid to the solution and stir for 10 minutes to adjust the pH to neutral.

[0011] The R-CDs were obtained by dialysis for 4 hours, centrifugation at 10,000 rpm for 20 minutes, and freeze-drying.

[0012] Further, the method for preparing CoOOH NCs includes:

[0013] NaOH was added to CoCl2 at a mol ratio of 3:10 and ultrasonicated for 5 min;

[0014] Centrifuge at 7000 rpm for 20 minutes to obtain a precipitate, and redisperse the precipitate in distilled water to a constant concentration of 50 mL to obtain a precipitate dispersion;

[0015] Slowly drip 0.9 mol / L NaClO solution into the precipitate dispersion at a volume ratio of 20:1 and perform ultrasonic treatment for 20 minutes;

[0016] The precipitate was collected by centrifugation at 7000 rpm for 10 min and washed three times with water;

[0017] The precipitate was reconstituted to 50 mL with deionized water, placed at 37°C for 20 minutes, and reconstituted to 50 mL with deionized water.

[0018] On the other hand, a method for screening α-glucosidase inhibitors is provided, wherein the active components of the α-glucosidase inhibitors are screened out after automatic fraction chromatography analysis and gradient elution of the pretreated Polygonum cuspidatum powder.

[0019] Further, the fraction collection chromatographic conditions are: gradient elution with 0.1% formic acid (A) and acetonitrile (B) as mobile phases; B content is 7-17% within 0-6 min; B content is 17% within 6-14 min; B content is 17-20% within 14-15 min; B content is 20-25% within 15-25 min; B content is 25-28% within 25-28 min; B content is 28-30% within 28-30 min; 30% B at 30-42.5 min; B content is 30-40% within 42.5-43 min; B content is 40-45% within 43-50 min; 1 mL min within 50-60 min -1 45-90% B was added at a flow rate of 10 μL; the injection volume was 10 μL, the column temperature was 25°C; and the DAD detection wavelength was 254 nm.

[0020] Furthermore, the gradient elution was carried out with 0.1% formic acid (A) and acetonitrile (B) as mobile phases, 5-50% B was carried out within 0-20 min; the flow rate was 0.3 mL min -1 , the content of B was 50-92% within 20-30 min; the injection volume was 3 μL, the column temperature was 30 °C; the acquisition parameters were set as follows: drying gas flow rate, 9.0 L min -1 ; Drying gas temperature, 350°C; Nebulizer gas pressure, 40 psig; Fragment or voltage, 120 V; Capillary voltage, 3500 V; Quadrupole RF, 750 V; Skimmer voltage, 65 V.

[0021] On the other hand, R-CDs@CoOOH NCs nanocomposites were applied in detecting α-Glu activity and screening for inhibitors of Polygonum cuspidatum. The target Polygonum cuspidatum assay produced glucose and ascorbic acid to reduce CoOOH NSs to Co 2+ The catalytic activity of R-CDs@CoOOHNCs was then destroyed to restore the red fluorescence emission of R-CDs at 625 nm. The detection range of α-Glu was 0.01 to 25 U / mL, and the detection limit was 0.0037 U / mL.

[0022] Furthermore, 10 U / mL α-Glu was used to screen the active ingredients in the experiment.

[0023] Furthermore, the reaction time of glucose and ascorbic acid with R-CDs@CoOOH NCs was controlled at 40 min.

[0024] The beneficial effects of the present invention are:

[0025] The present invention constructs a fluorescent nanocomposite sensor based on R-CDs and CoOOH NSs for detecting α-Glu and screening natural inhibitors in Polygonum cuspidatum extracts. The fluorescent sensor is simple to operate, low in cost, and highly sensitive, and is composed of the FRET effect between R-CDs and CoOOH NSs. It has been verified that the method can determine α-Glu activity in vitro, and its linear detection range under 625nm fluorescence emission is between 0.01 and 15U / mL, and the detection limit is 0.0037U / mL. The determination method can also be used to screen anti-diabetic active ingredients from Polygonum cuspidatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of the effect of R-CDs on a screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets of the present invention;

[0027] in Figure 1 (a) Fluorescence emission spectrum of R-CDs; (b) UV absorption spectrum; (c) HRTEM spectrum of R-CDs; (d) Size distribution of R-CDs; (e) XPS spectrum of R-CD and CoOOH NSs; (f) FTIR spectrum of R-CD and CoOOH NSs. XPS spectra of C1s (g), N1s (h), and O1s (i) of R-CDs.

[0028] Figure 2 Schematic diagram of the R-CDs conformation of an α-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxide nanosheets;

[0029] in Figure 2 (a) is the ESP distribution diagram of urea; (b) is the ESP distribution diagram of citrate; (c) is the S0 conformation and ESP distribution of R-CDs. (d) is the S1(d) conformation and ESP distribution of R-CDs; (e) is the fluorescence emission mechanism diagram; (f) is the LUMO and HOMO conformation diagram of R-CDs.

[0030] Figure 3 Schematic diagram of the characterization of CoOOH NS based on a screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets;

[0031] in Figure 3 (a) TEM image of CoOOH NS;

[0032] in Figure 3 (b) Co2pXPS spectrum of CoOOH NSs;

[0033] in Figure 3(c) XPS spectra of O1sd and O1s(c) of CoOOH NSs;

[0034] in Figure 3 (d) TEM image of R-CDs@CoOOHNSs.

[0035] in Figure 3 (e) Zeta potential diagram of R-CDs (1), CoOOH NSs (2) and R-CDs@CoOOH NCs (3);

[0036] in Figure 3 (f) Fluorescence lifetime diagram of R-CDs and R-CDs@CoOOH NCs.

[0037] Figure 4 Schematic diagram of the effect of a META based on red carbon dots and cobalt oxyhydroxide nanosheet α-glucosidase inhibitor screening kit on the mitochondrial respiration oxygen consumption of H9c2 cells under high glucose conditions;

[0038] in Figure 4 (a) Fluorescence intensity of R-CDs, CoOOH NSs, and R-CDs@CoOOH NCs.

[0039] Figure 4 (b) Fluorescence emission spectra of R-CDs incubated with various raw materials;

[0040] Figure 4 (c) Fluorescence spectra of R-CDs@CoOOH NCs incubated with 0-25 μM concentration of AA;

[0041] Figure 4 (d) Linear relationship between R-CDs@CoOOH NCs and AA (0-12.5 μM);

[0042] Figure 5 This is a diagram of the optimized conditions for a screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets;

[0043] Figure 6 A schematic representation of the optimized reaction conditions of a screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets;

[0044] Figure 6 (a) Fluorescence emission spectra of R-CDs@CoOOH NCs after incubation with different concentrations of α-Glu (0-25U / mL);

[0045] Figure 6(b) Linear relationship diagram of R-CDs@CoOOH NCs in the range of 0.01-15U / mLα-Glu;

[0046] Figure 6 (c) is an analysis diagram of the selectivity and anti-interference of the sensor for detecting α-Glu under fluorescence mode;

[0047] The concentrations of inorganic ions, biomolecules and amino acids were 1 mM, and the activity of the enzyme was 6 U / mL.

[0048] Figure 7 A schematic diagram of the characterization of α-Glu activity based on a red carbon dot and cobalt oxyhydroxide nanosheet α-glucosidase inhibitor screening kit;

[0049] in Figure 7 (a) Fluorescence spectra of the inhibition of α-Glu activity by acarbose at different concentrations; (b) The inhibition rate curve and linear relationship of acarbose on α-Glu activity under the fluorescence measurement mode; (c) Fluorescence spectra of the inhibition of α-Glu activity by Polygonum cuspidatum extract at different concentrations; (d) The inhibition rate curve and linear relationship of Polygonum cuspidatum extract on α-Glu activity under the fluorescence measurement mode.

[0050] Figure 8 A schematic diagram of the separation chromatogram characterization of Polygonum cuspidatum based on a screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets;

[0051] Figure 8 (a) Separation chromatogram of Polygonum cuspidatum and schematic diagram of the inhibition rate of different components on α-Glu activity. The black line is a typical chromatogram of PC, and the bar graph is the inhibition rate of 16 fractions on α-Glu activity. (b) Schematic diagram of the inhibitory activity of screening active compounds. The inhibition curves of seven active ingredients on α-Glu, (c) polysaccharides, (d) (-)-epicatechin gallate, (e) rhamnolide-1-O-β-D-glucoside, (f) resveratrol, (g) cassia ketone-8-O-β-D-glucoside, (h) rhamnolide-8-O-β-D-glucoside, (i) rhamnolide;

[0052] Fig. 9 Detailed diagram of molecular docking of acarbose and seven active ingredients with α-Glu;

[0053] Among them: (1) acarbose; (2) polysaccharide; (3) (-)-epicatechin gallate; (4) rhamnolide-1-OD-glucoside; (5) resveratrol; (6) cassia ketone-8-OD-glucoside; (7) rhamnolide-8-OD-glucoside; (8) rhamnolide. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0055] Experimental Section

[0056] Preparation of R-CDs@CoOOHNCs nanocomposites

[0057] Weigh 1.0g of anhydrous citric acid and 2.0g of urea and ultrasonically disperse them in 10mL of N,N-dimethylformamide. After thorough mixing, place the mixture in a 50mL polytetrafluoroethylene autoclave and heat at 180°C for 5 hours. After the reaction is completed, cool to room temperature, add 20mL of NaOH solution (50mg / mL) to the mixture and stir for 5 minutes. Then, gradually add 1.25mL of concentrated hydrochloric acid to the solution and stir for 10 minutes. Dialyze the solution in a dialysis bag for 4 hours and centrifuge at 10000rpm for 20 minutes. Finally, freeze-dry to obtain R-CDs and store them in a refrigerator at 4°C.

[0058] 1.5mL NaOH (1.0mM) was added to 50mL CoCl2 (10mM) solution, supersonicated for 5 minutes, and then the precipitate was redispersed in 50mL distilled water after centrifugation at 7000rpm for 20 minutes. 2.5mL 0.9M NaClO solution was slowly dripped into the above mixture and supersonicated for 20 minutes. Subsequently, 7000rpm was centrifuged for 10 minutes to collect the precipitate, and washed three times with water. Finally, the precipitate was redissolved to 50mL with deionized water to carry out subsequent experiments.

[0059] R-CDs@CoOOH NCs were prepared from 40 μg mL -1 The R-CD aqueous solution, CoOOH NSs aqueous solution and ultrapure water were fully mixed to prepare the solution with a volume ratio of 5:2:8. The mixed solution was placed at 37 °C for 20 min before further experiments.

[0060] Ascorbic acid detection

[0061] First, 150 μL of R-CDs@CoOOH NCs solution was mixed with 250 μL of PBS buffer solution at pH 6.8, and then different concentrations of AA were added. The solution was then quantified to 500 μL with ultrapure water, and the mixed solution was incubated at 37 °C for 40 min, and the fluorescence signal was recorded in the fluorescence spectrum at an emission wavelength of 625 nm.

[0062] Fluorescence assay for α-Glu activity

[0063] 100 μL of α-Glu with different concentrations was mixed with 100 μL of L-ascorbic acid-2-OaD-pyranose (AAG) (10 mM) in PBS (0.1 mM, pH 6.8) buffer for 30 min. Then, 150 μL of L-CDs@CoOOH NCs was added to the mixed solution, and the mixture was incubated at 37 °C for 40 min, and the fluorescence spectrum was finally obtained.

[0064] Sample preparation

[0065] 1g of Polygonum cuspidatum powder was immersed in a 50% methanol solution, ultrasonically treated for 30 minutes, and then filtered through a 0.22μM filter membrane. The filtrate was concentrated, dried, weighed, and completely dissolved in 50% methanol. The solution was then ultrasonically treated for 20 minutes and centrifuged at 13400rpm for 6 minutes. The supernatant was filtered through a 0.22μM pore size filter and placed at 4°C for use.

[0066] Fraction collection chromatographic conditions

[0067] HPLC separated the above filtrate for 60 minutes. The HPLC was connected to an automatic fraction collector to collect peaks according to the time pattern. 16 peaks were filtered at different separation times. The fractions collected after enrichment 4 times were dried with nitrogen and redissolved with methanol to obtain sample fractions. The PC extract was chromatographed by an HPLC system combined with photodiode array detection (DAD). The analytical column was Agilent HPLC Eclipse plus C18 (5μm, 4.6×250mm). Gradient elution was performed with 0.1% formic acid (A) and acetonitrile (B) as the mobile phase. The B content is 7-17% within 0-6min; 17% within 6-14min; 17-20% within 14-15min; 20-25% within 15-25min; 25-28% within 25-28min; 28-30% within 28-30min; 30% B within 30-42.5min; 30-40% within 42.5-43min; 40-45% within 43-50min; 1mL min within 50-60min -1 45-90% B was added at a flow rate of 1.5 %. The injection volume was 10 μL, the column temperature was 25° C. The DAD detection wavelength was 254 nm.

[0068] Screening for α-Glu inhibitors

[0069] First, 100 μL of α-Glu (10 U / mL) was mixed with 140 μL of PBS (0.1 M, pH 6.8), and then 10 μL of different concentrations of acarbose and antidiabetic active ingredients from Polygonum cuspidatum were added. The prepared R-CDs@CoOOH NCs were placed at 37°C and incubated for another 40 minutes before recording the fluorescence emission spectrum. The inhibition rate (%) was calculated as follows: Inhibition rate (%) = 1-(F2-F1) / (F-F0), where F0 is the fluorescence intensity of R-CDs@CoOOH NCs without α-Glu, F is the fluorescence intensity of R-CDs@CoOOH NCs in the presence of α-Glu, F1 is the fluorescence intensity of R-CDs@CoOOH NCs in the presence of the inhibitor at the same concentration, and F2 is the fluorescence intensity of R-CDs@CoOOH NCs in the presence of α-Glu and the inhibitor.

[0070] Characterization of Polygonum cuspidatum extract

[0071] The qualitative analysis of the Polygonum cuspidatum extract was performed using an Agilent 6546LC / Q-TOF mass spectrometer and a 1290UPLC system with an electrospray ionization source. The ratio of the mobile phase and the separation time were simply optimized. The analytical column was an Agilent UPLC Eclipse XDB-C18 (1.8 μm, 2.1×150 mm) with an Agilent Eclipse XDB-C18 (1.8 μm, 2.1×5 mm) guard column. Gradient elution was performed with 0.1% formic acid (A) and acetonitrile (B) as the mobile phase, with 5-50% B in 0-20 min; the flow rate was 0.3 mL min -1 , the content of B was 50-92% within 20-30 min. The injection volume was 3 μL, and the column temperature was 30 °C. In addition, the acquisition parameters of Q-TOF were set as follows: drying gas flow rate, 9.0 L min -1 ; Drying gas temperature, 350 °C; Nebulizer gas pressure, 40 psig; Fragmentor voltage, 120 V; Capillary voltage, 3500 V; Quadrupole RF, 750 V; Skimmer voltage was 65 V. In order to provide comprehensive information for the structural identification of the samples, both positive and negative ion modes were recorded. The collision energy in MS / MS analysis was set to 10 V and 30 V, and the mass range was set to 50–1000. The acquired MS and MS were summarized on the SCIEX OS software. 2 Ion fragments were used to find the molecular formula, relative molecular mass error and characteristic ion fragments of various compounds in Knotweed. The results were compared with published literature, PubMed, PubChem and ChemSpider. The chemical structure information was used to determine the chemical components in Knotweed extracts.

[0072] Molecular docking

[0073] The crystal structure of α-Glu (PDB ID: P3A4A) was collected from the Protein Data Bank and docked into the binding site pocket for molecular docking. Autodock Vina 36 Used as docking engine, Gasteiger charge 37 For electrostatic interaction calculations, set the parameter to 9. The docking space is set to 3. All ligands were docked 100 times for adequate sampling. Finally, docking simulations including binding energy calculations were performed to determine the best conformation with lower energy.

[0074] Synthesis and characterization of R-CDs@CoOOHNCs

[0075] Citric acid and urea as R-CDs precursors had no fluorescence emission signals, but after heat treatment at 180 °C, they emitted red fluorescence under UV light and then purified to form a purple solution, which indicated that Figure 1 R-CDs appeared in the upper right corner of a. In addition, it can be seen that the excitation and emission of the fluorescence spectrum of the purified CD are unique compared with the unpurified CD in Figure S1, revealing the uniformity of the R-CDs. The fluorescence excitation wavelength was finally determined to be 560 nm. Figure 1 In a, the maximum emission peak appears at 625 nm. R-CDs exhibit a broad UV-Vis absorption band in the range of 300 to 700 nm, with a maximum absorption peak at 370 nm ( Figure 1 b) TEM images of R-CDs show that they are well monodispersed with diameters ranging from 1 to 3.2 nm ( Figure 1 c), the carbon dots with a particle size of about 2.0 nm accounted for the largest proportion ( Figure 1 d). High-resolution HRTEM image shows the spherical shape of R-CD, with a lattice spacing of 0.21 nm ( Figure 1 c), which is consistent with previous reports 38 Then, the elemental composition of R-CDs was studied by X-ray spectrometer in this technical solution. Figure 1 The full XPS spectrum in C shows three strong signals at 284.78, 399.1, and 531.69 eV, verifying that R-CDs are mainly composed of C, N, and O elements ( Figure 1 e). Fourier transform infrared spectroscopy (FTIR) spectroscopy analysis ( Figure 1 f) shows that the R-CD sample has a peak at 3400 cm -1 A characteristic band is shown at 1634 cm, which is considered to be the stretching vibration of OH / NH. -1 and 1576cm -1The other characteristic peaks at 1091 cm-1 represent the stretching vibrations of C=O and C=C. The stretching vibrations of CN and COO- are shown at 1091 cm-1. -1 The XRD pattern in Figure S1 shows three typical peaks at 27.35°, 31.68°, 45.42°, and 56.43°, which are in line with the standards on the JCPDS card (PDF number: 01-074-2138), indicating the successful synthesis of R-CDs. Figure 1 As shown in Fig. gi, the C1s peak deconvoluted into five peaks at 284.78, 285.06, 286.85, 288.31, and 290.11 eV, which were attributed to CC / C=C, CN, CO, C=O, and COOH bonds, respectively. The deconvolution of the N1s peak at 397.06 eV, 399.09 eV, and 400.02 eV produced three peaks in Fig. E, corresponding to pyridinic CNC, pyrrolic CNC, and graphitic CN=C groups, respectively. The high-resolution O1s peak included three peaks at 531.69, 532.72, and 534.54 eV, which were attributed to C=O, CO, and C-OH bonds, respectively. These results implied the formation of pyridinic or pyrrolic conjugated cores in R-CDs, which contributed to the long-wavelength fluorescence emission properties. Combining XPS, FTIR and XRD data, the results show that there are abundant hydrophilic groups on the surface of R-CDs, such as amino and carboxyl groups, which ensure their excellent solubility in water. In addition, density functional theory (DET) of Gaussian software is also used in this technical solution to perform molecular simulation on the red carbon dots synthesized from citric acid and urea. First, the ESP distribution map is used in this technical solution to analyze the formation process of red carbon dots. Figure 2 a and 2b show the areas in the ESP distribution diagrams of citric acid and urea that are most prone to hydrogen bonding, and quantify the ESP on the surfaces of the two raw materials. The local maximum and minimum values ​​of ESP are represented by red and blue, respectively. The blue area is negatively charged and nucleophilic, and can act as a hydrogen bond donor, thus having the ability to attack electrophilic atoms. In contrast, the red area is electrophilic and can act as a hydrogen bond acceptor. For example, the N element in urea is distributed in the red area, and the observable local maximum value of ESP is 49.36 kcal / mol, which can be regarded as a hydrogen bond acceptor. As a hydrogen bond donor, the hydrogen atoms of the carboxyl group on citric acid are distributed in the blue area, and its maximum ESP is -32.29 kcal / mol. Molecular simulation results show that hydrogen bond addition exists during the synthesis of R-CD. Afterwards, the molecular state and ESP distribution state of the red carbon dots in the ground state and the first excited state were simulated in this technical scheme ( Figure 2 cd), which is related to the emission mechanism of fluorescence ( Figure 2 e) In order to further explore its luminescence characteristics, such as Figure 2f, the electronic cloud change of carbon dots and the energy transfer of HOMO and LUMO molecular orbitals (ΔE = 1.340 eV) are also calculated in this technical solution. The results show that R-CDs have been successfully synthesized and have photoluminescence properties.

[0076] CoOOH nanosheets are a typical two-dimensional sheet-like compound with several wrinkles and frills, and an average diameter of about 84 nm ( Figure 3 a). The strong signals at 285eV, 532eV, 775 and 779eV in the XPS analysis of CoOOH NSs indicate that the elements C, O and Co exist in the prepared nanosheets. Figure 1 e). The peaks at 775.0 eV and 779.9 eV are attributed to Co2p1 / 2 and Co2p2 / 3, respectively ( Figure 3 b). Figure 3 The two binding energies centered at 530.4 eV and 529.3 eV in c correspond to the Co-OH and Co-O bonds. Figure 1 f, the FTIR spectrum shows a peak at 3423 cm -1 、1623cm -1 and 562cm -1 The characteristic peaks centered at are derived from the absorption of OH, Co-O, and Co-O2 bonds, respectively. These data confirm the successful preparation of CoOOH NSs. The XRD pattern in Figure S3 shows three typical peaks at 23.46°, 45.46°, and 59.45°, which are consistent with the standards on the JCPDS card (No.07-0169), further indicating the successful synthesis of CoOOH crystals.

[0077] After fully mixing R-CDs and CoOOH NCs for 20 min, R-CDs@CoOOH NCs can be easily obtained. The TEM images of R-CDs@CoOOH NCs revealed that R-CDs were well attached to the surface of CoOOH NSs ( Figure 3 d) In Figure 3 The potential image of e shows that R-CDs are negatively charged, CoOOH NSs are positively charged, and R-CDs@CoOOH NCs are negatively charged as a whole, with Zeta potentials of -25.13 eV, 4.36 eV, and -21.37 eV, respectively. The above results indicate that R-CDs are attached to the surface of CoOOH NSs through electrostatic interactions. Figure 1As shown in ab, the UV absorption spectrum of CoOOH NSs and the excitation spectrum of R-CDs are prerequisites for the quenching mechanism. The fluorescence lifetime of R-CDs and R-CDs@CoOOH NCs was also studied in this technical scheme. The fluorescence lifetime of R-CDs changed from 3.64ns to 3.02ns after incubation with CoOOH NSs, which once again clarified the fluorescence quenching mechanism of FRET.

[0078] The fluorescence of R-CDs was quenched after the addition of CoOOH NSs, but gradually recovered after the addition of AA ( Figure 4 a) This is because AA has a strong reducing ability and can reduce CoOOH NCs to Co 2+39 As a substrate of α-Glu, AAG can be enzymatically hydrolyzed to produce AA, thus destroying R-CDs@CoOOH NCs. Figure 4 In b, it was found in this technical scheme that AA, AAG and α-Glu did not interfere with the fluorescence emission of R-CDs, but in the absence of AA or AAG and α-Glu, the fluorescence of R-CDs@CoOOH NCs remained unchanged. This result provides a favorable prerequisite for further detection of α-Glu. Based on the ability of AA to restore the fluorescence of R-CDs, a fluorescence detection strategy for detecting α-Glu was proposed in Scheme 1. The amount of AA affects the catalytic activity of CoOOH, resulting in changes in fluorescence intensity. Figure 4 c and Figure 4 d, when the AA concentration ranged from 0 to 12.5 μM, the linear regression equation was Y = 0.0048C AA +0.9829(R 2 =0.9982).

[0079] Optimization conditions of fluorescence sensor

[0080] In order to determine the optimal conditions for the fluorescence sensing system, the reaction efficiency between R-CDs, AA, and CoOOH at different ratios and times was studied. Figure 5 As shown in a, under the conditions of reaction with the same volume of R-CDs, the fluorescence quenching efficiency varies with the volume of CoOOH NSs solution. Then, 20 μL of CoOOH NSs solution significantly eliminated 50 μL of R-CDs (40 ng / mL), and there was no significant difference with 25 μL of CoOOH NSs, which was attributed to the excellent photobleaching ability, strong quenching ability and large specific surface area of ​​CoOOH NSs. Figure 5In b, when R-CDs encounter CoOOH NSs, the fluorescence decays considerably, and the fluorescence intensity decreases with the reaction time, while the fluorescence of R-CDs@CoOOH remains almost unchanged after 20 minutes of reaction. Therefore, the optimal reaction conditions for R-CDs@CoOOHNCs were determined to be R-CD:CoOOH (v / v; 50μL:20μL) and the reaction time was 20 minutes. Figure 5 c, When the reaction time of AA and R-CDs@CoOOH NCs was extended to 40 min, the fluorescence hardly recovered. Therefore, 40 min was considered to be the optimal reaction time.

[0081] Fluorescence quantitative detection of α-Glu

[0082] Under optimal conditions, the "switch" fluorescence mode is used in this technical solution to measure α-Glu. The fluorescence intensity in the system will increase as the amount of AA produced by AAG catalyzed by α-Glu increases. Figure 6 a and Figure 6 As shown in b, within the range of 0 to 25 U / mL, as the concentration of α-Glu increases, the fluorescence intensity at 625 nm gradually increases. Linear regression equation Y = 0.2706C α-Glu +0.9745(R 2 =0.9926), the range is 0.01-15U / mL, and the detection limit of α-Glu is 0.0037U / mL (based on LOD=3σ / k rule). Compared with the previously reported detection, when the α-Glu activity is close to 10U / mL, the fluorescence intensity recovery effect has reached an excellent state. Therefore, 10U / mL α-Glu was selected to screen the active ingredients of the experiment.

[0083] In this technical solution, the fluorescence intensity of the system is normalized in the presence of interfering elements and enzymes with similar structures ( Figure 6 c), where F0 is the fluorescence intensity of R-CDs@CoOOH NCs when incubated with PBS and AAG, F is the fluorescence intensity of R-CDs@CoOOH NCs in the presence of α-Glu and AAG, and F1 is the fluorescence intensity of R-CDs@CoOOH NCs in the presence of interfering materials and AAG. The fluorescence ratio bar graph shows that only α-Glu significantly restores the fluorescence intensity in the system, indicating that it is highly selective for the detection of α-Glu. Inhibitory effect of acarbose and PC extract

[0084] In order to evaluate whether the proposed method can be extended to the application of screening α-Glu inhibitors, acarbose was selected as a positive drug in this technical scheme. Acarbose is a good α-Glu inhibitor that has been frequently reported in the past and is also one of the commonly used oral drugs for the treatment of T2D. Figure 7a shows the fluorescence spectra of R-CDs@CoOOH NCs after adding different concentrations of acarbose in the range of 0–20 μM. Figure 7 b shows that the inhibitory effect of acarbose on α-Glu is concentration-dependent. Figure 7 As shown in b-7c, the half inhibitory concentration (IC 50 ) was 0.1062 μM, indicating that the sensing platform can be successfully applied to screen α-Glu inhibitors. Then, this method was used in the present technical scheme to measure the inhibitory effect of Polygonum cuspidatum extract on α-Glu. Increasing the concentration of Polygonum cuspidatum extract will increase the inhibitory effect on α-Glu, and the amount of AA produced in the system will decrease, eventually leading to a slowdown or termination of the fluorescence recovery process ( Figure 7 c and Figure 7 d) IC of Polygonum cuspidatum extract 50 The highest value was 36.71 μg / mL.

[0085] Screening and identification of 85 antidiabetic active ingredients from Polygonum cuspidatum

[0086] Based on the mixedness and complexity of Chinese herbal medicine extracts, HPLC is often used for separation in this technical scheme. The tandem automatic fraction collector greatly improves the collection efficiency and accuracy. Due to the anti-diabetic activity of Polygonum cuspidatum, UPLC-Q-TOF-MS / MS was applied in positive and negative ion modes to accurately identify the potential hypoglycemic active ingredients of Polygonum cuspidatum. Then, 16 fractions were collected after the Polygonum cuspidatum extract was injected into HPLC-FC for 60 minutes in this technical scheme. Each component was tested for anti-diabetic activity, and the highly active components were selected for UPLC-Q-TOF-MS / MS identification. The UPLC-Q / TOF-MS / MS system separated the active ingredients in the Polygonum cuspidatum extract for 30 minutes and analyzed them to obtain structural ion information. The results extracted from the positive and negative ion chromatograms showed that 85 compounds were identified, including 81 compounds in the negative ion and 4 compounds in the positive ion. The compound identification process mainly comes from two aspects. On the one hand, the ion fragmentation comparison and molecular fragmentation rules are derived from previous literature, and on the other hand, the information obtained from UPLC-Q-TOF-MS / MS.

[0087] Comparison of antidiabetic activity 16 fractions were collected according to the peak time by combining HPLC and fraction collector. The collected fractions were then incubated with α-Glu to calculate the inhibitory contribution of the 16 fractions. Figure 8The analysis results shown in the figure, considering their availability and inhibition degree, a total of 7 active components with inhibition rates greater than 50% were screened out. The final active ingredients have been identified, including (7) polydatin (8) (-)-epicatechin gallate (11) rhamnolide-1-OD-glucoside (13) resveratrol (13) cassia ketone-8-OD-glucoside (14) rhamnolide-8-OD-glucoside (16) rhamnolide. The chromatograms and chemical structures of the seven compounds are shown in Figures S5 and S6. The fluorescence intensity of α-Glu activity decreased with the increase of the dose of the seven standard products, indicating that all seven components have a certain inhibitory effect on α-Glu. Figure 8 As shown in c-8i and Table 1, (-)-epicatechin gallate showed the highest inhibitory effect, IC 50 was 0.8725 μM, followed by rhamnosin-1-OD-glucoside, rhamnosin, rhamnosin-8-OD-glucoside, resveratrol, polydatin and cassia ketone-8-OD-glucoside and, IC 50 The results showed that the fluorescence sensor was reliable.

[0088] Molecular docking

[0089] As a typical T2D inhibitor, acarbose can significantly reduce the activity of α-Glu, thereby balancing blood sugar in the body. In order to find out the binding relationship between these candidate compounds and α-Glu, the binding energy and binding site of acarbose and components with α-Glu-protein were compared by molecular docking in this technical scheme. Fig. 9 As shown, the binding sites of the seven ingredients are not only similar to those of acarbose, but they also share the same binding amino acids. The binding energy between all compounds and α-Glu protein was calculated in this technical scheme. The seven compounds screened from Polygonum cuspidatum showed higher affinity. This technical scheme speculates that the conjugated structure of the candidate components will affect additional interactions. Table 2 shows in detail the binding energy, number of hydrogen bonds and binding pockets of α-Glu with acarbose and active compounds. These characteristics indicate that the selected seven ingredients may also be good inhibitors and further verify the practicality of this method.

[0090] The present invention establishes a novel, low-cost, sensitive and efficient fluorescence detection strategy based on red carbon dots (R-CDs) and cobalt oxyhydroxide nanosheets (CoOOH NSs) for detecting α-Glu activity and screening inhibitors. As a switchable fluorescence source, the fluorescence of R-CDs can be quenched by CoOOH NSs through the fluorescence resonance energy transfer effect (FRET) and assembled into non-fluorescent R-CDs@CoOOH composite nanomaterials (R-CDs@CoOOH NCs). α-Glu can hydrolyze L-ascorbic acid-2-O-α-D-pyranose glucose (AAG) to produce glucose and ascorbic acid (AA). AA further reduces CoOOH NSs to Co 2+ The catalytic activity of R-CDs@CoOOH NCs was then destroyed to restore the red fluorescence emission of R-CDs at 625 nm. The results of this fluorescence detection method showed that the detection range of α-Glu was 0.01 to 25 U / mL, and the detection limit was 0.0037 U / mL.

[0091] Fluorescence mode was used to detect α-Glu and screen inhibitors from Polygonum cuspidatum. Under appropriate conditions, citric acid and urea were thermally synthesized into R-CDs in one pot, which emitted strong red light at 625nm after purification. CoOOH NSs quenched the fluorescence of R-CDs through the FRET effect and self-assembled with R-CDs to form a composite nanomaterial R-CDs@CoOOH NCs. AA released after α-Glu hydrolyzed the substrate AAG effectively inhibited the quenching ability of CoOOH NSs and finally induced fluorescence recovery. This method was further applied to the screening of α-Glu inhibitors in Polygonum cuspidatum.

[0092] After separation and preparation of Polygonum cuspidatum extract by HPLC-FC, the inhibitory efficiency of seven potential antidiabetic active components was monitored simultaneously.

[0093] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A screening kit for α-glucosidase inhibitors based on red carbon dots and cobalt oxyhydroxide nanosheets, characterized in that: The invention comprises a R-CDs@CoOOH NCs nanocomposite material, wherein the preparation method of the R-CDs@CoOOH NCs nanocomposite material comprises: preparing R-CDs and CoOOH NCs solutions: mixing the R-CDs, CoOOH NSs solution and ultrapure water in a volume ratio of 5:2:8 to obtain the R-CDs@CoOOH NCs nanocomposite material.

2. The α-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxide nanosheets according to claim 1, characterized in that: The method for preparing R-CDs comprises: Anhydrous citric acid and urea were ultrasonically dispersed in N, N-dimethylformamide in a mass ratio of 1:2 and mixed evenly, and the mixture was placed in a polytetrafluoroethylene autoclave, heated at 180°C for 5 hours, and then cooled to room temperature after the reaction was completed; Add NaOH solution to the mixture at a mass ratio of 30:1 and stir for 5 minutes. Gradually add concentrated hydrochloric acid to the solution and stir for 10 minutes to adjust the pH to neutral. The R-CDs were obtained by dialysis for 4 hours, centrifugation at 10,000 rpm for 20 minutes, and freeze-drying.

3. The α-glucosidase inhibitor screening kit based on red carbon dots and cobalt oxyhydroxide nanosheets according to claim 1, characterized in that: The method for preparing CoOOH NCs includes: NaOH was added to CoCl2 at a mol ratio of 3:10 and ultrasonicated for 5 min; Centrifuge at 7000 rpm for 20 minutes to obtain a precipitate, and redisperse the precipitate in distilled water to a constant concentration of 50 mL to obtain a precipitate dispersion; Slowly drip 0.9 mol / L NaClO solution into the precipitate dispersion at a volume ratio of 20:1 and perform ultrasonic treatment for 20 minutes; The precipitate was collected by centrifugation at 7000 rpm for 10 min and washed three times with water; The precipitate was reconstituted to 50 mL with deionized water, placed at 37°C for 20 minutes, and reconstituted to 50 mL with deionized water.

4. A method for screening α-glucosidase inhibitors, characterized in that: The pretreated Polygonum cuspidatum powder is subjected to automatic fraction chromatography analysis and gradient elution, and the screening kit as described in claims 1 to 3 is used to screen out the active ingredient of the α-glucosidase inhibitor.

5. The method for screening α-glucosidase inhibitors according to claim 4, characterized in that: The fraction collection chromatographic conditions are as follows: gradient elution with 0.1% formic acid (A) and acetonitrile (B) as mobile phases; 7-17% B content within 0-6 min; 17% B content within 6-14 min; 17-20% B content within 14-15 min; 20-25% B content within 15-25 min; 25-28% B content within 25-28 min; 28-30% B content within 28-30 min; 30% B within 30-42.5 min; 30-40% B content within 42.5-43 min; 40-45% B content within 43-50 min; 1 mL min within 50-60 min -1 45-90% B was added at a flow rate of 10 μL; the injection volume was 10 μL, the column temperature was 25°C; and the DAD detection wavelength was 254 nm.

6. The method for screening α-glucosidase inhibitors according to claim 4, characterized in that: The gradient elution was carried out with 0.1% formic acid (A) and acetonitrile (B) as mobile phases, 5-50% B was carried out within 0-20 min; the flow rate was 0.3 mL min -1 , the content of B was 50-92% within 20-30 min; the injection volume was 3 μL, the column temperature was 30 °C; the acquisition parameters were set as follows: drying gas flow rate, 9.0 L min -1 ; Drying gas temperature, 350°C; Nebulizer gas pressure, 40 psig; Fragmentor voltage, 120V; capillary voltage, 3500V; quadrupole RF, 750V; skimmer voltage, 65V.

7. Application of R-CDs@CoOOH NCs nanocomposite materials in detecting α-Glu activity and screening Polygonum cuspidatum inhibitors, characterized in that: Target Polygonum cuspidatum assay produces glucose and ascorbic acid to reduce CoOOH NSs to Co 2+ The catalytic activity of R-CDs@CoOOHNCs was then destroyed to restore the red fluorescence emission of R-CDs at 625 nm. The detection range of α-Glu was 0.01 to 25 U / mL, and the detection limit was 0.0037 U / mL.

8. The use of the R-CDs@CoOOH NCs nanocomposite material according to claim 7 in detecting α-Glu activity and screening for knotweed inhibitors, characterized in that: 10U / mL α-Glu was used to screen the active ingredients in the experiment.

9. The use of the R-CDs@CoOOH NCs nanocomposite material in detecting α-Glu activity and screening for knotweed inhibitors according to claim 7, characterized in that: The reaction time of glucose, ascorbic acid and R-CDs@CoOOH NCs was controlled at 40 min.

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