Quality evaluation method of traditional Chinese medicine

Abstract

A traditional Chinese medicine quality evaluation method is disclosed, the quality evaluation method comprises the following steps: according to the structural characteristics of the active ingredient group, using the same detection mechanism as the active group action mechanism, using molecularly imprinted polymer to simulate antigen-antibody reaction, capturing the active ingredient group, determining the content of the active group in the active ingredient group by an electrochemical sensor, directly reflecting the pharmacological activity of the traditional Chinese medicine, and developing a new method for traditional Chinese medicine quality evaluation based on "active action mechanism as detection principle and active group as detection index", which provides a new idea for precise evaluation and control of traditional Chinese medicine quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a traditional Chinese medicine quality evaluation method. BACKGROUND

[0002] Traditional Chinese medicine has the characteristics of complex and diverse components, unclear effective substances and unclear mechanism of action. The existing quality control of traditional Chinese medicine mostly adopts the combination of traditional experience identification and chemical component analysis. However, this method has the problem of insufficient specificity of control indicators, and cannot accurately identify the authenticity of raw materials and distinguish their quality. At the same time, the evaluation of indicator components only relies on chemical methods, which cannot directly reflect the effectiveness and safety of traditional Chinese medicine, and cannot comprehensively evaluate the quality of traditional Chinese medicine products. Therefore, it is difficult to ensure the safety, effectiveness and controllable quality of traditional Chinese medicine products. In the past decade, with the development of instrument technology and the improvement of analysis methods, the traditional Chinese medicine quality control system has made great progress, and the new quality control modes such as multi-component overall analysis, quality marker (Q-Marker) of traditional Chinese medicine, grade differentiation and characteristic fragment have brought hope for solving the key problems of traditional Chinese medicine quality evaluation. However, these methods are limited by high cost and high difficulty of high-end instruments, cannot reflect the correlation between activity and integrity, are difficult to build evaluation models, and are complicated to operate. Therefore, they have not been collected by pharmacopoeia and related quality standards, and cannot play a role in practical application.

[0003] Artemisia annua shows significant anti-malarial activity due to the presence of peroxides such as artemisinin, artemisinene, artemisinin H, etc. The peroxide bond of artemisinin (ART) and other peroxides reacts with the ferrihematin produced by the decomposition of hemoglobin in host red blood cells through redox reaction, and the peroxide bridge is cracked to produce highly active intermediates (such as oxygen free radicals, carbon free radicals, etc.), which can alkylate susceptible proteins and cellular metabolites in the body of Plasmodium and produce reactive oxygen species (ROS). These free radicals collectively cause metabolic disorders in Plasmodium and lead to death. The peroxide bridge structure of peroxide sesquiterpenes contained in anti-malarial traditional Chinese medicine is considered to be the key group for the occurrence of anti-malarial activity, but it is also the main factor causing the instability of this type of compounds. If the total content of the peroxide bridge bond of the effective component group can be accurately determined, the anti-malarial traditional Chinese medicine can be intuitively, accurately, specifically and sensitively evaluated in a complex matrix. SUMMARY

[0004] In view of the technical problem in the prior art that the internal quality of traditional Chinese medicine is difficult to be scientifically evaluated and controlled, the present application provides a traditional Chinese medicine quality evaluation method.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] A traditional Chinese medicine quality evaluation method, the quality evaluation method comprising the following steps:

[0007] According to the structural characteristics of the active ingredient group, the same detection mechanism as the active group action mechanism is used, the molecularly imprinted polymer is used to simulate the antigen-antibody reaction, the active ingredient group is captured, the content of the active group in the active ingredient group is determined by an electrochemical sensor, and the quality evaluation of the direct pharmacological activity of the traditional Chinese medicine is performed.

[0008] Further, the traditional Chinese medicine includes an anti-malaria traditional Chinese medicine, the structural characteristics include a peroxide sesquiterpene, the action mechanism includes an active oxygen generated by ring opening of a peroxide bridge active group, and the electrochemical sensor is an iron porphyrin as an active center, and the iron porphyrin is a peroxidase as a core.

[0009] Further, the anti-malaria traditional Chinese medicine includes artemisia annua, eurya japonica and nardostachys jatamansi.

[0010] Further, the molecularly imprinted polymer includes MOF-525(Fe)@MWCNT-MIP.

[0011] Further, the preparation steps of the MOF-525(Fe)@MWCNT-MIP include:

[0012] Multi-walled carbon nanotubes MWCNT are used as crystal seeds to prepare a composite material MOF-525(Fe)@MWCNT with the MOF-525(Fe), and the multi-walled carbon nanotubes can enhance the conductivity of the metal organic framework in the electrochemical sensor and improve the detection sensitivity.

[0013] Methacrylic acid MAA is used as a functional monomer, SA is used as a porogen, and ART is used as a template molecule to prepare a composite ART molecularly imprinted polymer microsphere MOF-525(Fe)@MWCNT-MIP with the MOF-525(Fe)@MWCNT.

[0014] Further, the amount of the MAA is 0.16 mmol.

[0015] Further, the amount of the ART is 0.02 mmol.

[0016] Further, the concentration of the MOF-525(Fe)@MWCNT is 5 mg / mL.

[0017] Further, the electrochemical sensor includes an ART molecularly imprinted electrochemical sensor, and the elution condition of the template molecule of the ART molecularly imprinted electrochemical sensor is-1 V for 100 s.

[0018] Further, the method comprises using the ART molecular imprinting electrochemical sensor to determine the anti-malaria traditional Chinese medicine, and the specific steps comprise: ultrasonic treatment for 5 minutes before testing the sample, incubating the ART molecular imprinting electrochemical sensor in a solution containing the sample extract, and performing cyclic voltammetry (CV) detection.

[0019] Further, the detection limit (LOD) of the ART molecular imprinting electrochemical sensor for determining the anti-malaria traditional Chinese medicine is 1.738*10 -13 M, and the limit of quantification (LOQ) is 4.764*10 -9 M.

[0020] The present application has the following beneficial effects:

[0021] 1. The reaction mechanism with a peroxide bridge as an active center provides a scientific basis for the quality evaluation method of the anti-malaria traditional Chinese medicine with a peroxide sesquiterpene component, and a new traditional Chinese medicine quality evaluation method with "active action mechanism as the detection principle and active group as the detection index" is developed, which provides a new idea for precise evaluation and control of traditional Chinese medicine quality.

[0022] 2. The quality evaluation method has low detection limit (LOD) and limit of quantification (LOQ), and in particular, the multi-walled carbon nanotube enhances the conductivity of the metal-organic framework in the electrochemical sensor, improves the sensitivity of the detection, and has a wide application range.

[0023] 2. The linear range is wide and suitable for analyzing actual samples.

[0024] 3. The method has good selectivity, specificity, and accuracy, is not easily disturbed by the complex matrix in the medicinal material and decoction piece sample, and reduces the sample pretreatment steps.

[0025] 4. The material has good stability and can be stored for a long time.

[0026] 5. The required instrument is simple, and the operation is simple, which endows the present application with higher conversion application value and commercialization potential. BRIEF DESCRIPTION OF DRAWINGS

[0027] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated in and constitute a part of this specification. The drawings illustrate the embodiments and, together with the description, serve to explain the principles of the present application. Other embodiments and many of the intended advantages of the embodiments will be readily appreciated as the same becomes better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale relative to each other. Like reference numerals designate corresponding similar parts.

[0028] Figure 1 A schematic diagram of the electro-reduction process of the ART molecule on the MOF-525 (Fe) modified electrode GCE according to the present application is shown;

[0029] Figure 2 A schematic diagram showing the preparation process of the ART molecularly imprinted electrochemical sensor according to the present application is shown;

[0030] Figure 3 A schematic diagram showing the working process of the ART molecularly imprinted electrochemical sensor according to the present application is shown;

[0031] Figure 4 A schematic diagram showing the characterization analysis according to one specific embodiment of the present application is shown;

[0032] Figure 5 A plot of voltammetry curves obtained in 0.01 M ART solution for different modified electrodes according to one specific embodiment of the present application is shown;

[0033] Figure 6 A plot of dynamic light scattering particle size analysis (DLS) results according to one specific embodiment of the present application is shown;

[0034] Figure 7 A plot of scanning electron microscope (SEM) results for materials according to one specific embodiment of the present application is shown;

[0035] Figure 8 A plot of response current values in 0.01 M ART solution for MOF-525(Fe)@MWCNT-MIP modified GCEs obtained under different preparation conditions and after different re-adsorption times according to one specific embodiment of the present application is shown;

[0036] Figure 9 A plot of current change curves during elution of MOF-525(Fe)@MWCNT-MIP and MOF-525(Fe)@MWCNT-NIP modified electrodes (GCEs) according to one specific embodiment of the present application is shown;

[0037] Figure 10 A plot of the relationship between CV peak current and the arithmetic square root of scan rate and the linear fitting results according to one specific embodiment of the present application is shown;

[0038] Figure 11 A plot of differential pulse voltammetry (DPV) curves according to one specific embodiment of the present application is shown;

[0039] Figure 12 A plot of the working curve obtained for the sensor in gradient concentration ART solution and its fitting results according to one specific embodiment of the present application is shown;

[0040] Figure 13A column chart showing the current response values obtained in 0.003M ART solution containing ART derivative dihydroartemisinin DHA and artesunate ASE as interfering substances according to one specific embodiment of the present application;

[0041] Figure 14 A column chart showing the current response values obtained in 0.01M ART solution after elution of ART after preparation of MOF-525(Fe)@MWCNT-MIP modified electrode GCE after different time according to one specific embodiment of the present application;

[0042] Figure 15 A chart showing the comparison results of ART content levels of 11 batches of artemisia pieces samples from 4 provinces with different production dates and 5 batches of deteriorated samples according to one specific embodiment of the present application. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely intended for the purpose of explanation and are not intended to limit the present application. In addition, it should be noted that only parts related to the present application are shown in the accompanying drawings for the purpose of description.

[0044] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the accompanying drawings and embodiments.

[0045] According to the structural features of the active ingredient group, the same detection mechanism as the active group action mechanism is used, the antigen-antibody reaction is simulated by using molecularly imprinted polymers to capture the active ingredient group, and the content of the active group in the active ingredient group is determined by an electrochemical sensor to directly reflect the pharmacological activity of the quality evaluation of traditional Chinese medicine.

[0046] The quality evaluation method proposed in the present application is suitable for various natural medicines and their processed products, Figure 1 A schematic diagram showing the electro-reduction process of ART molecules on MOF-525(Fe) modified GCE according to the present application is shown; Figure 2 A schematic diagram showing the preparation process of the ART molecularly imprinted electrochemical sensor according to the present application is shown; Figure 3 A schematic diagram showing the working process of the ART molecularly imprinted electrochemical sensor according to the present application is shown, as Figure 1-3 As shown, the traditional Chinese medicine includes anti-malaria traditional Chinese medicine, and the quality evaluation method includes the following steps:

[0047] According to the structural characteristics of the active ingredient group of the anti-malaria traditional Chinese medicine, a detection mechanism same as the action mechanism of the active group is used, the structural characteristics include peroxide sesquiterpene, the action mechanism includes ring opening of the peroxide bridge active group to produce active oxygen, and a molecular imprinting polymer is used to simulate the antigen-antibody reaction to capture the active ingredient group;

[0048] An electrochemical sensor with iron porphyrin as an active center is used to determine the content of the active group peroxide bridge in the active ingredient group, to determine the effective component in the anti-malaria traditional Chinese medicine matrix, and to directly reflect the pharmacological activity of the anti-malaria traditional Chinese medicine for quality evaluation.

[0049] In a specific embodiment, the quality evaluation method includes placing the sensor in a solution containing an extract of the sample to be tested, and incubating for a period of time, during which ART molecules and substances similar in structure to ART molecules are specifically adsorbed into the imprint cavities formed on the electrode surface in advance, while other non-target impurities can only form weak non-specific adsorption on the electrode surface. After a simple washing process, these non-specifically adsorbed impurities can be effectively removed, so that the electrode surface is finally enriched with ART.

[0050] During the sensor detection process, the molecular imprinting polymer MIPs modification layer selectively screens the compounds present in the extract solution based on spatial structure. Only those molecules with similar spatial shape and functional group structure to ART molecules can be tightly bound in the imprint film and are not easily detached. Among all the adsorbed molecules, only those containing peroxide bridge structural units can form effective complexes with the iron porphyrin units in the MOF-525(Fe) modified on the electrode surface. When an external voltage is applied, these complexes will undergo a reduction reaction to produce a detectable characteristic current signal. In this way, sensitive detection of the concentration of ART and its analogs containing peroxide bridge structures in the sample is achieved. This method not only combines the high selectivity of MIPs in molecular spatial structure recognition, but also utilizes the high selectivity of MOF-525(Fe) in molecular electronic structure recognition, providing an efficient and sensitive analysis method for the detection of ART and its structural analogs in complex samples. Embodiment

[0051] (1) Preparation of MOF-525(Fe)@MWCNT

[0052] The 300 mg ZrOCl2·8H2O (0.93 mmol), 100 mg FeTCPPCl (0.12 mmol), 3.3 g benzoic acid (27 mmol) were dissolved in 100 mL of N, N-dimethyl formamide DEF, 150 mg multi-walled carbon nanotubes MWCNT were added, and ultrasonic dispersion was performed for 10 min. The obtained uniform solution was transferred to a 250 mL blue cap bottle. The blue cap bottle containing the solution was placed in an oven. After the oven temperature was raised to 90°C, it was kept for 5 h. The dark brown precipitate product was collected by centrifugation. The obtained product was washed with N, N-dimethyl formamide DMF, centrifuged at 8000 rpm for 10 min, and repeated three times until the washing liquid was colorless. Then, the product was washed with methanol, centrifuged at 8000 rpm for 10 min, and repeated six times to remove the remaining DMF. The product was placed in a vacuum drying box and vacuum dried at 60°C overnight to obtain the final product MOF-525(Fe)@MWCNT. The obtained product was stored in the refrigerator at -20°C.

[0053] (2) Preparation of MOF-525(Fe)@MWCNT-MIP

[0054] The 5 mg prepared MOF-525(Fe)@MWCNT was added to 1 mL ultrapure water, ultrasonic dispersion was performed for 2 h to obtain a uniform and stable dispersion liquid as an aqueous phase. 5.6 mg ART was added to a mixture of 13.6 μL methacrylic acid MAA (0.16 mmol) and 150 μL ethylene glycol dimethyl acrylate EGDMA (0.8 mmol) in a 5 mL centrifuge tube. The solution was transferred to a measuring cylinder, 1 mL SA was added to adjust the solution volume to 1.2 mL, and the solution was left to stand at room temperature for 1 h. After the solution was stable, the solution was extracted and reinserted into a 5 mL centrifuge tube. 20 mg benzoyl peroxide BPO (0.08 mmol) and 1 mg (0.005 mmol) N, N-diisopropylol p-toluidine DPOPT were added as an oil phase, and a 3 mm x 5 mm small olive-shaped stirrer was put in to help emulsification. After the solid added in the previous step was dissolved, the aqueous phase was quickly added to the oil phase and emulsified vigorously for 3 min to obtain a stable Pickering emulsion. The centrifuge tube containing the obtained emulsion was transferred to a 50°C water bath, and after 5 h of incubation, the water bath power was turned off to slowly cool the reaction system to room temperature with the water bath. A gray-white clumpy powder solid product was obtained. The product was ultrasonically dispersed in methanol, centrifuged at 8000 rpm for 10 min to collect the precipitate, and repeated six times to remove the remaining solvent. The product was placed in a vacuum drying box and vacuum dried at 40°C overnight to obtain the final product MOF-525(Fe)@MWCNT-MIP. The obtained product was stored in the refrigerator at -20°C.

[0055] (4) Preparation of modified electrode

[0056] All the electrodes GCE should be activated in HNO3 solution (volume ratio: concentrated HNO3: H2O = 1:1) for 5 min before use. The GCE was polished with 0.3 μm and 0.05 μm alumina polishing powder on polishing cloth with a proper amount of ultrapure water to draw "8" shape to polish to mirror surface. The residual polishing powder was washed away with ultrapure water and ethanol, and dried under dry nitrogen stream for standby.

[0057] The material was ultrasonically dispersed in 0.5 wt% chitosan 1 vt% acetic acid solution for 2 h before testing, and a stable dispersion solution with a concentration of 3 mg / mL was prepared. 5 μL of the dispersion solution was drawn by a pipette and dropped on the surface of the treated and dried electrode twice, and then dried under an infrared baking lamp to obtain the modified electrode for testing.

[0058] (5) Electrochemical testing and elution of template molecules

[0059] The electrochemical detection of the material was carried out on an eight-channel potentiostat. The reference electrode, the counter electrode and one GCE were connected to channel one of the instrument, and the other two GCEs were connected to the other two channels. The remaining electrodes were empty. The potential values involved in the test, unless otherwise specified, were all relative electrode potentials (vs. SCE) to saturated calomel electrode (SCE).

[0060] The elution of residual ART template molecules in the material was carried out by electro-reduction and electrostatic repulsion. In 1 M potassium chloride solution, a voltage of -1 V was applied to the surface of the modified electrode by chronoamperometry (i-t) for 100 s.

[0061] The ECSA test of the material modified electrode was carried out by probe molecule Fe(CN)6 3+ / 4+ The redox peak current on the cyclic voltammetry (CV) curve was measured. In a solution containing 1 M potassium chloride and 0.005 M K3[Fe(CN)6] and K4[Fe(CN)6]·3H2O prepared with ultrapure water, the scanning was carried out at a scanning rate of 0.025, 0.050, 0.075, 0.100, 0.150 V / s respectively at a potential of -0.2-0.6 V, and the peak current value was recorded. According to Randles-Sevcik formula:

[0062] ,

[0063] I p (A) is the peak current, k is a constant related to temperature (its value is 268600 at 25 °C), n is the number of electron transfer A (1 for this reaction), A (cm 2 ) is the ECSA of the modified electrode, D (cm 2 / s) is the diffusion coefficient of the reactant (Fe(CN)6 3+ / 4+6.5×10 -6 cm 2 / s), c(mol / cm 3 ) is the reactant concentration, ν (V / s) is the scan rate. The slope kn is obtained by plotting the peak current against the arithmetic square root of the scan rate. 3 / 2 AD 1 / 2 c, and the ECSA of the modified electrode can be obtained by reverse calculation.

[0064] (6) CV detection of ART reduction process

[0065] The eluted modified electrode was used in the potential window of -1.5–0.1V with a scanning speed of 50mV / s. The test solution was a mixture of 4mL of a certain concentration of ART acetonitrile solution and 8mL of 10×PBS buffer. The solution should be ultrasonically treated for 5min before testing and added to the electrolytic cell system used in the test. Subsequently, nitrogen was passed through the system for deoxygenation for 10min, and the ventilation needle was lifted to the liquid surface to ensure that nitrogen was still passed through the system during the test to avoid the influence of liquid shaking on the detection current.

[0066] The differential pulse voltammetry (DPV) detection of the ART reduction process using unmodified GCE had a starting potential of -0.2 V, an ending potential of -1.5 V, a step size of 0.01 V, a pulse amplitude of 0.05 V, and a pulse width of 0.06 s. The DPV detection after elution using MOF-525(Fe)@MWCNT-MIP modified GCE had a starting potential of 0 V and an ending potential of -0.8 V. The test system and deoxygenation operation were the same as those of the CV test.

[0067] (7) Characterization of MOF-525(Fe)@MWCNT-MIP

[0068] FT-IR test of MOF-525(Fe)@MWCNT-MIP

[0069] About 5 mg of MOF-525(Fe)@MWCNT-MIP sample and about 200 mg of spectrally pure potassium bromide were placed in an agate mortar and ground evenly. After that, an appropriate amount of the mixture was added to the tableting mold until it covered the bottom of the sample tank. After tableting, the sample was placed in a Fourier transform infrared spectrometer for testing.

[0070] like Figure 4 As shown in A, the C=O stretching vibration peaks (1604, 1418 cm) characteristic of carboxylate can be observed in the spectrum of MOF-525(Fe). -1 ), the out-of-plane bending vibration peak of =CH of the para-disubstituted benzene ring (801cm -1) and other related absorption peaks in porphyrin compounds (2923, 1700, 1543, 1340, 999, 777, 721 cm -1 The MOF-525(Fe)@MWCNT spectrum also contains the MWCNT characteristic C=C skeleton stretching vibration peak (1630cm -1 ) and the characteristic 1604,801 cm- -1 In addition to the three characteristic peaks mentioned above, the -CH3 and -CH2- stretching vibration peaks of polymethacrylate (PMAA) (2988, 2954 cm) can be observed in the MOF-525(Fe)@MWCNT-MIP spectrum. -1 ), carboxyl C=O stretching vibration peak (1728cm -1 ) and -CH3, -CH2- bending vibration peaks (1471, 1444cm -1 ), the existence of these characteristic peaks proves the successful synthesis of the composite material.

[0071] XRD test of MOF-525(Fe)@MWCNT-MIP

[0072] Take an appropriate amount of MOF-525(Fe)@MWCNT-MIP sample to fill the sample pool, flatten the sample with a glass slide, and then place the sample on the sample holder of the X-ray diffractometer for phase analysis. The instrument X-ray source is Cu-K a1 Wire( =0.15406nm), beam current size 30mA, beam voltage 40kV, scanning range 2θ=5-80°, scanning speed 10° / min.

[0073] like Figure 4As shown in Figure B, the black curve is the X-ray diffraction (XRD) spectrum of MOF-525(Fe), which contains two diffraction peaks at 7.1° and 9.9°, corresponding to the (4 0 0) and (4 -2 1) crystal planes of the material, respectively. The increase in diffraction intensity before 5° should be caused by the interlayer diffraction common in porphyrin MOFs materials. The green curve is the XRD spectrum of MWCNT, on which two characteristic diffraction peaks of MWCNT at 26.6° and 44.6° corresponding to the (0 0 2) and (1 0 1) crystal planes can be observed. The above four diffraction peaks also appear in the XRD spectrum of MOF-525(Fe)@MWCNT (blue curve), proving the successful synthesis of the composite material. However, since the crystallinity of the composite material is lower than that of the two individual materials, the intensity of each peak is reduced, and the peak shape is broadened to varying degrees. In the spectrum of MOF-525(Fe)@MWCNT-MIP (red curve), the characteristic peak of MWCNT has been reduced to the point of being difficult to identify due to the greatly improved dispersion between individual MWCNTs and the random distribution of spatial orientation. However, the characteristic peak of MOF-525(Fe) can still be identified due to the higher crystallinity of its own grains, thus confirming the successful preparation of the composite material MOF-525(Fe)@MWCNT-MIP.

[0074] like Figure 5 As shown in Figure 2, the voltammetric curves of electrodes modified with different materials in 0.01M ART solution were obtained. Figure 5 (A) It can be seen that although a concentration-related reduction peak can be obtained using the unmodified glassy carbon electrode, its potential is relatively negative near -1.0V and cannot be completely separated from another unknown reduction peak, which makes subsequent data processing more difficult and introduces additional error sources. Figure 5 (B) shows that all three materials used can catalyze the reduction of ART to varying degrees, thereby reducing its overpotential on the electrode surface. The MOF-525(Fe)-modified electrode can completely reduce the reduction potential of ART to around -0.5 V, but its inherent poor conductivity results in a low peak current. MWCNTs exhibit a certain degree of catalytic activity, but the peak current at -1.0 V is not completely eliminated. The composite material of the two completely reduces the reduction potential to around -0.5 V while maintaining a high peak current. Furthermore, after the final imprinting polymerization, the poor conductivity of MIPs hinders the transfer of electrons from the electrode to the material-solution surface, resulting in a significant decrease in peak current. However, the peak near -0.5 V is still discernible, and the composite material also exhibits no reduction peak at -1.0 V, indicating that MOF-525(Fe)@MWCNT-MIP has the same catalytic ability as MOF-525(Fe)@MWCNT.

[0075] DLS particle size test

[0076] The obtained MOF-525(Fe)@MWCNT-MIP sample 1 mg was put into 10 mL of methanol, and after ultrasonic dispersion for 1 h to obtain a stable dispersion, 2 mL of the dispersion was added to a polystyrene Malvern particle size sample cell, and the sample cell was placed into a particle size instrument for testing to obtain the particle size distribution of the sample.

[0077] As shown in Figure 6 , the particle size distribution of the obtained MIP microspheres and its statistical analysis in the aqueous phase containing 1, 2, 5, 10, 12 mg / mL MOF-525(Fe)@MWCNT can be found that the microspheres obtained by selecting 5 mg / mL of material concentration have the smallest particle size, which means that the finally obtained microspheres have the largest specific surface area. Relatively speaking, when the amount of emulsifier is too small, the emulsification of the oil phase is weaker, resulting in larger micelle diameter in the emulsion, and thus larger particle size of the polymer is obtained; and when the amount of emulsifier is too large, MOF-525(Fe)@MWCNT itself is easy to agglomerate into larger structures, and thus larger particle size of the product is also obtained.

[0078] SEM test

[0079] The obtained MOF-525(Fe)@MWCNT-MIP sample 1 mg was put into 10 mL of methanol, and after ultrasonic dispersion for 1 h to obtain a stable dispersion, 10 μL was taken by a pipette and added on a silicon wafer, and after the liquid was naturally air-dried, the silicon wafer was adhered to the SEM sample table with conductive carbon glue, and was sent into the electron microscope sample chamber. The surface morphology of the sample was observed under the electron microscope SE2 probe at an acceleration voltage of 3.0 kV and a magnification of 30K.

[0080] As shown in Figure 7 , are SEM photos of the synthesized MOF-525(Fe)@MWCNT Figure 7 A) and MOF-525(Fe)@MWCNT-MIP materials Figure 7 B-D) obtained by using MOF-525(Fe)@MWCNT in an amount of 1, 5, 12 mg / mL. In Figure 7 A, the interlaced linear structure formed by carbon nanotubes and the MOF material attached thereto can be observed. In Figure 7 B and Figure 7 D, it can be found that the product material mainly presents a disordered connected string bead shape at low concentration, and the potential cavity structure cannot be observed; at high concentration, large particle products are mainly obtained, and there are a large number of exposed MOF-525(Fe)@MWCNT on the surface of the product, which may further lead to a decrease in the selectivity of the material. Relatively speaking, Figure 7The material in C exhibits a multi-core capsule structure consistent with the literature reports of Pickering emulsion polymerization method for preparing MIPs microspheres, so it is reasonable to select 5 mg / mL as the optimal MOF-525(Fe)@MWCNT concentration.

[0081] (8) Comparative experiment

[0082] The MAA amount, ART concentration, and oil phase volume in the preparation of MOF-525(Fe)@MWCNT-MIP were optimized and screened, and the re-adsorption time necessary for electrochemical testing was also optimized and explored. The specific experimental formulations and parameters are shown in Table 1 below:

[0083]

[0084] The current response peak values of the synthesized 17 groups of materials (except for samples 1, 2, 4, and 5 used for comparison of particle size) to 0.01 M ART solution at -0.5 V were tested and recorded, and the results are shown in Figure 8 .

[0085] As can be seen from Figure 8 (A), the current response value of the material increases with the increase of the functional monomer MAA, and there is no significant increase after 0.16 mmol, indicating that before this amount, the adsorption capacity of the microspheres to ART increases with the increase of the amount of MAA. As shown in Figure 8 (B), the response signal of the material reaches the strongest when using 0.02 mmol of the feed amount, and then slowly decreases with the increase of the feed amount, which may be because when the template molecule concentration is low, the effective imprinting sites formed are less, and when the template molecule concentration is high, there is competition between the template molecule and the functional monomer, and thus enough imprinting sites cannot be formed. Figure 8 (C) reflects the effect of the amount of porogen on the adsorption effect of the imprinted microspheres. It can be found that before the oil phase volume reaches 1.2 mL, the response current increases with the increase of the amount of porogen SA, and after 1.2 mL, the response current decreases with the increase of the amount of SA. It is analyzed that when the amount of porogen is low, the porosity of the material is low, and thus the adsorption capacity is weak, but when the amount of porogen is too high, part of the analyte will pass through the material to the electrode surface to react without undergoing the corresponding electrocatalytic process, thus also leading to the decrease of the characteristic peak current. Finally, according to Figure 8 (D), it can be found that the re-adsorption time of the material is set to 5, 7.5, 10, 15, and 30 minutes, and the response current obtained has no obvious difference, indicating that the material has reached adsorption equilibrium before 5 min.

[0086] As shown in Figure 9As shown, the current changes of MOF-525(Fe)@MWCNT-MIP and MOF-525(Fe)@MWCNT-NIP modified GCE during the elution process relying on electrical reduction and electrostatic repulsion. It can be found that at 90s, the current of MOF-525(Fe)@MWCNT-NIP modified GCE quickly reached equilibrium and no longer decreased, while the current curve of MOF-525(Fe)@MWCNT-MIP modified GCE started at a higher position and decreased more slowly, indicating that the ART molecules continued to be reduced and detached from the electrode surface under the action of electrostatic repulsion. After 90s, the two curves reached the same equilibrium current, indicating that the ART molecules had been completely eluted. Therefore, it is reasonable to set the elution condition to -1V for 100s.

[0087] (9) ESCA determination of electrochemically active area of ​​modified electrodes

[0088] The probe molecule Fe(CN)6 3+ / 4+ The results obtained by plotting the redox peak current on the CV curve against the arithmetic square root of the scan rate are as follows: Figure 10 As shown, the obtained curves were linearly fitted to obtain the slopes of the corresponding curves before and after elution of MOF-525(Fe)@MWCNT-MIP modified GCE and before and after elution of MOF-525(Fe)@MWCNT-NIP modified GCE, which were 7.870, 7.912, 6.857, and 7.086, respectively. According to the Randles-Sevcik formula, the ESCA corresponding to each modified GCE were 0.01035, 0.01155, 0.01001, and 0.01034 cm 2 By comparison, the ESCA of MOF-525(Fe)@MWCNT-MIP and MOF-525(Fe)@MWCNT-NIP modified electrodes before elution were similar. However, after elution, the MIP modified electrode exposed many open pores as the template molecules were removed, resulting in an 11.59% increase in ESCA. Although the ESCA of the NIP modified electrode also increased after elution (3.30%), it was obviously smaller than the change of the MIP modified electrode. It is speculated that this change of NIP may be due to the shedding of polymers at some weak points in the structure, which led to the exposure of MWCNT. At the same time, it can be observed that in any case, the ESCA of the modified electrode is smaller than the geometric area of ​​GCE (0.07065 cm). 2 This may be because although materials that improve conductivity are added, PMAA itself is still an insulating material, and most of the volume of the MOF-525(Fe)@MWCNT-MIP finally prepared in this project is occupied by PMAA, which leads to a significant decrease in the ESCA of the modified electrode.

[0089] (10) Determination of sensor working curve

[0090] MOF-525(Fe)@MWCNT-MIP modified GCE at 10 -2 -10 -10 The DPV curve obtained in the MART solution is shown as follows after subtracting the current response value of the blank solution. Figure 11 As shown in the figure, it can be found that there is only one reduction peak at -0.5 V, which decreases with the decrease of ART concentration. Therefore, it can be concluded that the prepared sensor has a good quantitative detection ability for ART.

[0091] like Figure 12 As shown by Figure 11 The sensor working curve and fitting results obtained by the peak current value show that the logarithm of the sensor response value is 10 -2 -10 -5 The relationship between the logarithm of ART concentration and the concentration of ART is linear, and the equation is expressed as lgy=0.6146×lgx+2.148. -5 -10 -9 The relationship between the logarithm of ART concentration and the concentration of ART is linear, and the equation is y = 0.0136 × lgx + 0.1994, R 2 The values ​​were all greater than 0.99, indicating good linearity.

[0092] The sensor current response values ​​obtained by continuous parallel measurement 20 times in a blank solution without ART are shown in Table 2 below. By calculating the standard deviation (SD), the system noise of the sensor can be obtained to be 8.622×10 -9 The relative standard deviation of μA was 3.72%, which also showed that the method had good intermediate precision.

[0093]

[0094] Combined with the calibration curve obtained above, the 3-fold noise and 10-fold noise values ​​were substituted into the obtained working curve to determine the limit of detection (LOD) and limit of quantitation (LOQ) of the method: LOD = 1.738 × 10 -13 M, LOQ = 4.764 × 10 -9 Considering that the content of ART in the actual sample is necessarily higher than the detection limit and quantification limit of the method, it can be considered that the sensor of the present application is fully capable of realizing the accurate quantification of the ART content in the Artemisia annua decoction piece sample and the sample quality evaluation.

[0095] like Figure 13are the peak current values of MOF-525(Fe)@MWCNT-MIP and MOF-525(Fe)@MWCNT-NIP modified electrodes tested in 0.003M ART solution in the presence of 10-fold and 100-fold DHA and ASE, apparently the MOF-525(Fe)@MWCNT-MIP obtained in this work has good selectivity for ART molecules, and can accurately quantify the concentration of ART in the sample in the presence of 100-fold interferents, so it can be fully considered that the method developed in this topic has the potential to be applied to actual samples.

[0096] The synthesized MOF-525(Fe)@MWCNT-MIP was stored in a refrigerator at -20°C, and the same batch of synthesized MOF-525(Fe)@MWCNT-MIP material was taken out at 1, 3, 7, 15, 30, 60, and 90 days, respectively, and modified on the GCE, and the current response values obtained by measuring in 0.01M ART solution after eluting the template ART are shown in Figure 14 It was found that the response level of the prepared MOF-525(Fe)@MWCNT-MIP changed little within 3 months, which shows that the material prepared by this method can be stored for at least three months without losing performance, and also implies that MIPs can maintain performance for a longer period of time if stored without eluting the template molecules.

[0097] Example 2

[0098] Another key point for the sensor method to realize effective evaluation of the quality of artemisia leaf pieces is whether the sensor can be not or as little as possible interfered by other complex components in the extract of natural plant samples. As shown in Table 3, the results of the sensor in the spiked recovery experiment in the acetonitrile extract of the deteriorated artemisia leaf pieces are shown, it can be found that the sensor has good recovery response at high concentration (0.01M), medium concentration (10 -5 M) and low concentration (10 -8 M), which proves that the sensor has the ability to quantitatively detect ART molecules from the complex matrix background of the extract of leaf pieces, it is worth mentioning that the recovery rate and relative standard deviation RSD value obtained by the two calibration curve equations at the turning point of the medium concentration are greatly different, the RSD value obtained by the calibration curve at a higher concentration is lower, showing higher method precision, therefore in actual application, the sample concentration should be ensured to fall within the concentration range of 10 -2 -10 -5 M as much as possible through concentration.

[0099]

[0100] Example 3

[0101] The results of the detection of 11 batches of purchased artemisinin decoction pieces (Anhui, Sichuan, Hubei and Shandong) and the deteriorated samples of 5 batches of decoction pieces with higher content after detection are shown in Table 4 and Figure 15

[0102]

[0103] The number is set according to the Chinese initial letter abbreviation of the place of origin (AH: Anhui, SC: Sichuan, HB: Hubei, SD: Shandong), and six digits representing the production date (year / month / day). The batch number of the deteriorated sample is set by DETER (Deteriorated) plus the original untreated sample number. According to the artemisinin quality standards stipulated in the WHO monograph (artemisinin content: leaves: at least 0.7%), only 1, 4 and 10 batches of decoction pieces can be considered as qualified as the extraction source of ART, and other decoction piece samples are not valuable as the extraction raw material of ART due to the serious decline of ART content caused by storage time exceeding one year or other environmental factors during the logistics transportation.

[0104] As shown in Figure 15 , the ART content in the same sample is significantly different, but overall, the results are consistent with expectations, that is, the higher the ART content level of the medicinal materials collected in the native producing area of artemisinin (Sichuan), the higher the ART content level. In addition, the longer the storage time of the sample, the lower the ART content. In addition, even if collected in Sichuan, the ART level can be significantly reduced after 1 year of storage. And the ART content detected in the deteriorated sample is also significantly reduced, which is consistent with the characteristic that ART is easily decomposed by heat.

[0105] The specific embodiments of the present application are described above, but the protection scope of the present application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

[0106] ​In the description of the application, it needs to be understood that the terms "upper", "lower", "inner", "outer" and the like indicate the orientation or positional relationship shown in the drawings based on the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the application. The word 'comprising' does not exclude the existence of elements or steps not listed in the claims. The word 'a' or 'an' in front of an element does not exclude the existence of multiple such elements. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that the combination of these measures cannot be used to improve. Any reference signs in the claims should not be interpreted as limiting the scope.

Claims

1. A method for evaluating the quality of traditional Chinese medicines, characterized in that, The quality evaluation method comprises the following steps: According to the structural characteristics of the active ingredient group, the same detection mechanism as the active group action mechanism is used, the molecular imprinting polymer is used to simulate the antigen-antibody reaction, the active ingredient group is captured, the content of the active group in the active ingredient group is determined by an electrochemical sensor, the quality of the traditional Chinese medicine is directly reflected, the pharmacological activity of the traditional Chinese medicine is evaluated, the traditional Chinese medicine includes anti-malaria traditional Chinese medicine, the structural characteristics include peroxide sesquiterpene, the action mechanism includes peroxide bridge active group ring-opening to produce active oxygen, the electrochemical sensor is an iron porphyrin active center, and the molecular imprinting polymer includes MOF-525(Fe)@MWCNT-MIP; The preparation steps of the MOF-525(Fe)@MWCNT-MIP include: Multi-walled carbon nanotubes MWCNT are used as crystal seeds to prepare a composite material MOF-525(Fe)@MWCNT with MOF-525(Fe); Methyl methacrylate MAA is used as a functional monomer, SA is used as a porogen, and ART is used as a template molecule to prepare a composite ART molecular imprinting polymer microsphere MOF-525(Fe)@MWCNT-MIP with the MOF-525(Fe)@MWCNT.

2. The method for evaluating the quality of traditional Chinese medicine according to claim 1, characterized in that, The anti-malaria traditional Chinese medicine includes artemisia annua, alstonia scholaris, and nardostachys jatamansi.

3. The method for evaluating the quality of traditional Chinese medicine according to claim 1, characterized in that, The amount of MAA is 0.16 mmol, and the amount of ART is 0.02 mmol.

4. The method for evaluating the quality of traditional Chinese medicine according to claim 1, characterized in that, The concentration of the MOF-525(Fe)@MWCNT is 5 mg / mL.

5. The method for evaluating the quality of traditional Chinese medicine according to claim 1, characterized in that, The electrochemical sensor includes an ART molecular imprinting electrochemical sensor, and the elution conditions of the ART molecular imprinting electrochemical sensor for the template molecule are -1 V for 100 s.

6. The method for evaluating the quality of traditional Chinese medicine according to claim 5, characterized in that, The method includes using the ART molecular imprinting electrochemical sensor to determine the anti-malaria traditional Chinese medicine, and the specific steps include: ultrasonic treatment of the sample to be tested for 5 min, incubation of the ART molecular imprinting electrochemical sensor in a solution containing the extract of the sample to be tested, and CV detection.

7. The method for evaluating the quality of traditional Chinese medicine according to claim 6, characterized in that, The ART molecularly imprinted electrochemical sensor has a detection limit LOD of 1.738 x 10 -13 M for the anti-malaria traditional Chinese medicine. -9 M for the anti-malaria traditional Chinese medicine.

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