Cistanche deserticola quality evaluation method based on rapid detection of echinacoside

Abstract

The invention discloses a cistanche quality evaluation method based on rapid detection of echinacoside, and relates to the field of quality detection of traditional Chinese medicinal materials, and the method comprises preparation and quality detection of a molecularly imprinted electrochemical sensor MIP-PG / GCE. According to the invention, echinacoside is taken as a template molecule, methacrylic acid is taken as a functional monomer, a molecularly imprinted polymer film is prepared on the surface of a porous graphene modified glassy carbon electrode in a self-assembly manner, and the molecularly imprinted electrochemical sensor MIP-PG / GCE for high-selectivity, high-sensitivity and rapid detection of echinacoside is developed. The sensor shows a relatively low detection limit, a relatively wide linear range and relatively high selectivity on echinacoside. The molecularly imprinted electrochemical sensor is used for quantitatively detecting the content of echinacoside in a cistanche traditional Chinese medicinal material or a compound preparation, and the quality evaluation of cistanche can be quickly realized.

Description

Technical Field

[0001] The invention relates to the field of quality detection of traditional Chinese medicines, and in particular to a quality evaluation method for Cistanche deserticola based on rapid detection of echinacoside. Background Art

[0002] Phenylethanol glycosides (PhGs), represented by echinacoside, are characteristic components of Cistanche deserticola and are also index components for the quality control of Cistanche deserticola in the Chinese Pharmacopoeia. They have many biological effects. Rapid, real-time and accurate analysis of echinacoside content in medicinal materials and preparations, as well as the lowest blood drug concentration in the body, is of great significance for the quality control of medicinal materials and preparations with echinacoside as an evaluation indicator and for the efficacy of echinacoside in preventing and treating related diseases.

[0003] At present, the main methods used for the analysis of echinacosides include liquid chromatography, liquid chromatography-tandem mass spectrometry (LC-MS / MS) coupling technology, dynamic microdialysis sampling, and HPLC-DAD-MS. Although these detection methods have been widely used and generally accepted, most of them require complicated sample pretreatment and professional technicians to operate, and even require the purchase of expensive instruments and equipment, which is not conducive to the popularization and expansion of analytical technology. Molecular imprinting electrochemical sensing technology is a new analytical method that has attracted much attention in recent years. It has been applied in the fields of drug analysis, environmental testing, food safety, and clinical drug monitoring because of its characteristics of no need for complicated sample pretreatment, simple operation, strong selectivity, high sensitivity, and fast analysis speed. Summary of the invention

[0004] The purpose of the present invention is to provide a quality evaluation method for Cistanche deserticola based on rapid detection of echinacoside. By developing a molecular imprinting electrochemical sensor with high selectivity and high sensitivity for rapid detection of echinacoside, rapid detection of echinacoside, an intrinsic microscopic component of Cistanche deserticola in a formula is achieved, the detection steps are simplified, the detection cost is reduced, and the detection cycle is shortened. This plays a vital role in ensuring that the compound preparation containing Cistanche deserticola is of high quality and efficacy and the overall quality is controllable, and also provides a forward-looking exploration for electrochemical analysis technology in the quality evaluation of traditional Chinese medicine and drug safety.

[0005] In order to achieve the above object, the present invention adopts the following technical solution: The present invention provides a method for evaluating the quality of Cistanche deserticola based on rapid detection of echinacoside, comprising the following steps: (1) Preparation of molecular imprinted electrochemical sensor MIP-PG / GCE 2.96 mg of echinacoside (ECH) was added to a solution containing 70 μL of methacrylic acid (MAA) and 0.5 mL of PBS solution with pH = 4. The mixture was sonicated at room temperature for 10 min to obtain a prepolymer solution. Then, 2 μL of the prepolymer solution was pipetted onto the surface of the PG / GCE modified electrode, and it was then placed in an oven at 40 °C and heated and dried for 24 h. Subsequently, it was immersed in a methanol / acetic acid eluent with a volume ratio of 5:5 for 20 min to remove the template molecules. The surface eluent was washed off with distilled water and dried at room temperature to obtain the molecularly imprinted electrochemical sensor MIP-PG / GCE; (2)Quality Detection Using the molecularly imprinted electrochemical sensor MIP-PG / GCE as the working electrode, a platinum wire electrode as the auxiliary electrode, and a saturated calomel electrode as the reference electrode, 10 mL of 0.1 mol / L acetate buffer solution with pH = 4.0 was added to the electrolytic cell. Differential pulse voltammetry was used to detect the sample extract of Cistanche deserticola Chinese herbal medicine or compound preparations to determine the content of echinacoside, and the quality of Cistanche deserticola was evaluated according to the Chinese Pharmacopoeia; among them, when the concentration of echinacoside was in the range of 1.26×10 -7 ~5.26×10 -5 mol / L, its oxidation peak current showed a good linear relationship with the concentration. The linear equation was expressed as Ip = 0.2609C + 0.1187, and the correlation coefficient R 2 = 0.9928. The detection limit was 3.28×10 -8 mol / L, the signal-to-noise ratio S / N = 3, Ip was the oxidation peak current with the unit of μA, and C was the concentration with the unit of μM.

[0006] Furthermore, the preparation method of the PG / GCE modified electrode was as follows: The glassy carbon electrode was polished successively with 0.3 μm and 0.05 μm alumina powder, rinsed with distilled water, and then ultrasonically cleaned in methanol and distilled water for 1 min each, and air-dried naturally for later use; 5 μL of the PG dispersion was pipetted onto the surface of the treated glassy carbon electrode and air-dried naturally, denoted as the PG / GCE modified electrode Furthermore, the preparation method of the PG dispersion was as follows: 2 g of Na and 5 mL of ethanol were mixed and heated in a reactor at 220 °C for 48 h, cooled to room temperature. The obtained white product was lysed with deionized water, and the obtained black product was washed repeatedly with deionized water several times and then freeze-dried to obtain the product porous graphene (PG), which was dissolved with isopropanol to prepare a 1 mg / mL PG dispersion; Furthermore, the potential range for detection by differential pulse voltammetry was 0.10 - 0.70 V.

[0007] Compared with the prior art, the beneficial technical effects of the present invention are as follows: The present invention uses echinacoside (ECH) as the template molecule and methacrylic acid (MAA) as the functional monomer, and prepares a molecularly imprinted polymer film on the surface of a glassy carbon electrode modified with porous graphene (PG) in a self-assembled manner, developing a molecularly imprinted electrochemical sensor MIP-PG / GCE for highly selective, highly sensitive and rapid detection of echinacoside. The sensor shows a low detection limit, a wide linear range and high selectivity for echinacoside. By quantitatively detecting the content of echinacoside in Cistanche deserticola Chinese medicinal materials or compound preparations with this molecularly imprinted electrochemical sensor, the quality evaluation of Cistanche deserticola can be quickly realized. Description of the Drawings

[0008] Figure 1 It is a characterization diagram of the physical and chemical structure of the materials in the embodiments of the present invention, where both A and B are scanning electron microscope characterizations of porous graphene (PG), C is the infrared spectrum diagram of graphene oxide (a) and porous graphene (b), and D is the Raman spectrum diagram of graphene oxide (a) and porous graphene (b).

[0009] Figure 2 It is an electrochemical characterization diagram of two electrodes, GCE and PG@GCE, in the embodiments of the present invention. Among them, A is the characterization diagram of cyclic voltammetry test of GCE (a) and PG@GCE (b) in a probe solution containing 0.5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl; B is the characterization diagram of electrochemical impedance spectroscopy test of GCE (a) and PG@GCE (b) in a probe solution containing 0.5 mmol / L [Fe(CN)6] 3- / 4- and 0.1 mol / L KCl; C is the cyclic voltammogram of GCE (a, b) and PG@GCE (c, d) in 0.1 mol / L phosphate buffer solution (a, c) and 0.1 M acetate buffer solution (b, d) containing 2.0×10 −5 mol / L echinacoside; D is the enlarged cyclic voltammogram of GCE in 0.1 mol / L phosphate buffer solution (a) and 0.1 mol / L acetate buffer solution (b) containing 2.0×10 -5 mol / L echinacoside.

[0010] Figure 3 It is an electrochemical characterization diagram of three electrodes, GCE, GO / GCE and PG@GCE, in the embodiments of the present invention. Among them, A is the Q-t curve of GCE (a), GO / GCE (b) and PG@GCE (c) in a 1.0 mol / L KCl solution containing 0.5 mmol / L [Fe(CN)6], and B is the Q-t 3- / 4- curve of GCE (a), GO / GCE (b) and PG@GCE (c) 1 / 2Linear relationship diagram.

[0011] Figure 4 These are the CV and EIS curves of two kinds of electrodes, NIP-PG@GCE and MIP-PG@GCE, in the embodiments of the present invention. Among them, A is the CV curve of NIP-PG@GCE (a), MIP-PG@GCE before elution (b), MIP-PG@GCE after rebinding (c), and MIP-PG@GC after elution (d), and B is the EIS curve of NIP-PG@GCE (a), MIP-PG@GCE before elution (b), MIP-PG@GC after elution (c), and MIP-PG@GCE after rebinding (d).

[0012] Figure 5 These are the DPV detection results of MIP-PG@GCE in the embodiments of the present invention. Among them, A is the DPV curve of acteoside at different concentrations on MIP-PG@GCE in the probe solution of 0.1 M KCl and 2.0 mM [Fe(CN)6] 3- / 4- and B is the relationship between the peak current of the prepared sensor and the concentration of acteoside.

[0013] Figure 6 These are the interference effects of different substances when using MIP-PG@GCE to determine acteoside by DPV in the embodiments of the present invention. Among them, 1: quercetin, 2: formononetin, 3: glycine, 4: glutamic acid, 5: rhamnose, 6: sucrose, 7: interfering ions [Fe 3+ , Cu 2+ , Mg 2+ , Ca 2+ , Cl 2- , SO4 2- , 8: acteoside, 9: quercetin + acteoside, 10: formononetin + acteoside, 11: glycine + acteoside, 12: glutamic acid + acteoside, 13: rhamnose + acteoside, 14: sucrose + acteoside, 15: interfering ions + acteoside. Detailed implementation manners

[0014] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0015] In the present invention, unless otherwise specified, the raw materials involved are all well-known commercially available products in the art.

[0016] Embodiment The present embodiment provides a method for evaluating the quality of Cistanche deserticola based on rapid detection of echinacoside, which comprises preparing porous graphene by high-temperature synthesis and water splitting methods, using the porous graphene as a glassy carbon electrode (GCE) modification material, echinacoside as a template molecule, and methyl methacrylate (MAA) as a functional monomer, preparing a molecular imprinting polymer film by self-assembly on the surface of the porous graphene-modified glassy carbon electrode, and developing a molecular imprinting electrochemical sensor with high selectivity, high sensitivity and rapid detection of echinacoside, an indicator component for quality evaluation of Cistanche deserticola.

[0017] Preparation of PG dispersion: 2 g of sodium (Na) and 5 mL of ethanol were mixed in a molar ratio of 1:1, heated in a stainless steel reactor at 220 °C for 48 h, and cooled to room temperature to obtain a white product. The white product was cracked with deionized water, and the white product turned black. The black product was porous graphene (PG). It was then washed repeatedly with deionized water several times and finally freeze-dried to obtain the final product PG. It was dissolved with isopropanol to prepare a 1 mg / mL PG dispersion for later use.

[0018] At a high temperature of 220°C, metallic sodium reacts violently with ethanol. Sodium captures the protons in the ethanol hydroxyl group to generate sodium ethoxide. The high temperature and high pressure environment accelerates the reaction. The release of hydrogen may form micro-nano bubbles, providing a template for the subsequent porous structure. Sodium ethoxide is further dehydrogenated at high temperature to generate ethoxy free radicals. The free radicals polymerize through coupling reactions to form long-chain polyether structures. The polyether chains are carbonized at high temperatures and form conjugated π-electron systems through aromatization reactions. The intercalation of sodium promotes the exfoliation of the carbon layer. At the same time, the released gases (H2, CO2, etc.) escape to form pores. The intercalation of sodium also induces defects in the carbon layer, promoting the exfoliation and porosity of graphene sheets. The cooled white product (containing sodium ethoxide and carbonization products) is cracked by deionized water. Sodium ethoxide is hydrolyzed to generate ethanol and sodium hydroxide (NaOH). The black carbon product (porous graphene) is repeatedly washed to remove inorganic salts such as NaOH, and freeze-dried to avoid pore collapse.

[0019] Preparation of PG / GCE modified electrode: The glassy carbon electrode was polished with 0.3μm and 0.05μm alumina powders in turn, rinsed with distilled water, and then the treated glassy carbon electrode was ultrasonically cleaned in methanol and distilled water for 1 min in turn, dried naturally and set aside; 5μL PG dispersion was transferred and drop-coated on the treated surface of the glassy carbon electrode, dried naturally, and recorded as PG / GCE modified electrode.

[0020] Preparation of Molecularly Imprinted Electrochemical Sensor MIP-PG / GCE: Weigh 2.96 mg of echinacoside (ECH) and add it to a solution containing 70 μL of methacrylic acid (MAA) and 0.5 mL of PBS with pH = 4. Sonicate at room temperature for 10 min to obtain a prepolymer solution. Pipette 2 μL of the prepolymer solution onto the surface of the composite electrode PG / GCE, then place it in an oven at 40 °C and heat-dry for 24 h. Next, soak it in a methanol / acetic acid eluent with a volume ratio of 5:5 for 20 min to remove the template molecules. Wash the surface eluent with distilled water and dry at room temperature to obtain the molecularly imprinted electrochemical sensor MIP-PG / GCE.

[0021] Without adding the template molecules, the non-imprinted composite electrode NIP-PG / GCE was prepared according to the above steps in the example.

[0022] As Figure 1 shown, after constructing the MIP-PG / GCE modified electrode, infrared spectroscopy, Raman spectroscopy, and SEM electron microscopy characterization were performed on it. Figure 1 The test results show that the PG dispersion, the PG / GCE modified electrode, and the molecularly imprinted composite electrode MIP-PG / GCE were all successfully prepared.

[0023] Subsequently, electrochemical characterization was carried out to characterize the preparation and performance of the electrode through electrochemical test methods.

[0024] Electrochemical Characterization of the Base Electrode: Different modified electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in a solution containing 5 mmol [Fe(CN)6] 3- / 4- and 0.1 mol KCl. The measurement conditions for CV were: potential range from -0.2 V to 0.8 V, scan rate of 100 mV / s. The measurement conditions for EIS were: amplitude of 0.005 V, voltage of 0.2 V, and frequency range from 0.1 to 105 Hz.

[0025] Add 10 mL of 0.1 mol / L acetate buffer solution (pH = 4.0) to the electrolytic cell. Using MIP-PG / GCE and NIP-PG / GCE as the working electrodes, a platinum wire electrode as the auxiliary electrode, and a saturated calomel electrode as the reference electrode, scan in the potential range of -0.2 to 0.8 V at a scanning rate of 100 mV / s until the curve is stable. Then add standard solutions of acteoside with different concentrations to the system and keep the system in a homogeneous state. Then enrich for 300 s at the open circuit potential and perform cyclic voltammetry scanning to record its cyclic voltammogram. To maintain the reproducibility and stability of the modified electrode, the electrode must be re-modified after each scan. The potential range of differential pulse voltammetry is 0.10 to 0.70 V. Before the electrochemical experiment, the solution needs to be purged with N2 for 10 min, and the electrochemical experiment is carried out in an N2 atmosphere and at room temperature (20 °C).

[0026] Figure 2 and 3 The test results of Figure 3 show that the PG / GCE modified electrode was successfully prepared, and according to 2 .

[0027] The prepared MIPs were also electrochemically characterized by CV and EIS methods, and the detection was carried out using a bottom solution containing 2.0 mM potassium ferricyanide / potassium ferrocyanide. As shown in Figure 4 A, the peak current intensity of NIP-PG@GCE (a) has a small difference compared with the peak current intensity of MIP-PG@GCE before elution (b), and the peak current intensity of MIP-PG@GCE after elution (d) increases. This can be attributed to the addition of ECH during the synthesis of MIPs, so the formation and filling of imprinted cavities will occur during the elution and rebinding processes, resulting in changes in the peak current; as shown in Figure 4 B, it not only strongly proves that MIPs have been successfully prepared, but also there are changes in the peak current when the imprinted cavities are formed or filled, which also verifies the feasibility of the experiment.

[0028] Under the optimized conditions, differential pulse voltammetry detection of gradient concentration samples was carried out, and the detection results are as shown in Figure 5 . Figure 5 In A, the concentrations from a to f are 0.126, 3.289, 6.577, 13.153, 26.306, 52.612 μmol / L respectively, Figure 5 B shows that the concentration of acteoside is in the range of 1.26×10 -7 ~5.26×10 -5When it is within the range of mol / L, its oxidation peak current shows a good linear relationship with the concentration, and the linear equation is expressed as I p (μA)=0.2609C(μM)+0.1187, and the correlation coefficient R 2 =0.9928, and the detection limit is 3.28×10 -8 mol / L, and the signal-to-noise ratio S / N = 3.

[0029] To verify the practicability of the sensor in the examples, the MIP-PG@GCE sensor was tested for reproducibility, selectivity and stability. Five modified electrodes were prepared in parallel, and the same concentration of echinacoside sample solution (2.0×10 -5 mol / L) was measured. The relative standard deviation between the measured values was 4.40%, indicating that the constructed sensor has good reproducibility. After the same modified electrode was stored in a 4°C refrigerator for one week, when measuring a 0.1 mol / L acetic acid buffer solution containing 2.0×10 -5 mol / L echinacoside, its electrochemical response signal was 95.70% of the original detection signal, indicating that the sensor has acceptable stability. Finally, the selectivity of the MIP-PG@GCE sensor was tested. Using differential pulse voltammetry, in a 0.1 mol / L acetic acid buffer solution (pH = 4.0) containing 2.0×10 -5 mol / L echinacoside, quercetin and formononetin were added respectively to make their concentrations 2.0×10 -4 mol / L; glycine, glutamic acid, L-rhamnose monohydrate and sucrose were added to make their concentrations 1.4×10 -3 mol / L; Fe 3+ , Cu 2+ , Mg 2+ , Ca 2+ , Cl 2- , SO4 2- and other interfering ions (Traceelement) were added to make their concentrations 2.0×10 -3 mol / L, and the experimental results are as Figure 6 . It can be seen from the experimental results that when there are high concentrations of interfering substances, when the constructed electrochemical sensor is used for echinacoside analysis, the MIP-PG@GCE sensor shows excellent selectivity.

[0030] The sample extract of Cistanche deserticola Chinese herbal medicine or compound preparation was prepared by the conventional ethanol ultrasonic extraction method. The Chinese Pharmacopoeia stipulates that the content of echinacoside in Cistanche deserticola should not be less than 0.3% mg / g.

[0031] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the technical solutions and concepts of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for evaluating the quality of Cistanche deserticola based on the rapid detection of echinacoside, characterized in that, It includes the following steps: (1) Preparation of the molecularly imprinted electrochemical sensor MIP-PG / GCE Add 2.96 mg of echinacoside to 70 μL of methacrylic acid and 0.5 mL of PBS solution with pH = 4, ultrasonically treat for 10 min at room temperature to obtain a prepolymer solution, transfer 2 μL of the prepolymer solution to the surface of the PG / GCE modified electrode, then place it in an oven at 40 °C to heat and dry for 24 h, then soak it in a methanol / acetic acid eluent with a volume ratio of 5:5 for 20 min to remove the template molecules, wash the surface eluent with distilled water, dry at room temperature to obtain the molecularly imprinted electrochemical sensor MIP-PG / GCE; (2) Quality inspection Using the molecularly imprinted electrochemical sensor MIP-PG / GCE as the working electrode, a platinum wire electrode as the auxiliary electrode, and a saturated calomel electrode as the reference electrode, add 10 mL of 0.1 mol / L acetate buffer solution with pH = 4.0 to the electrolytic cell, and use differential pulse voltammetry to detect the sample extract of Cistanche deserticola Chinese herbal medicine or compound preparation to determine the content of acteoside, and evaluate the quality of Cistanche deserticola according to the Chinese Pharmacopoeia; among them, when the concentration of acteoside is in the range of 1.26×10 -7 ~5.26×10 -5 mol / L, its oxidation peak current shows a good linear relationship with the concentration, and the linear equation is expressed as Ip = 0.2609C + 0.1187, and the correlation coefficient R 2 = 0.9928, the detection limit is 3.28×10 -8 mol / L, the signal-to-noise ratio S / N = 3, Ip is the oxidation peak current, the unit is μA, and C is the concentration, the unit is μM.

2. A method for evaluating the quality of Cistanche deserticola based on the rapid detection of acteoside according to claim 1, characterized in that, The preparation method of the PG / GCE modified electrode is as follows: polish the glassy carbon electrode successively with 0.3 μm and 0.05 μm alumina powder, rinse with distilled water, then ultrasonically clean in methanol and distilled water for 1 min successively, and air dry naturally for standby; transfer 5 μL of the PG dispersion, drop-coat it on the surface of the treated glassy carbon electrode, and air dry naturally, denoted as the PG / GCE modified electrode.

3. A method for evaluating the quality of Cistanche deserticola based on the rapid detection of acteoside according to claim 2, characterized in that, The preparation method of the PG dispersion is as follows: mix 2 g of Na and 5 mL of ethanol, heat in a reactor at 220 °C for 48 h, cool to room temperature, pyrolyze the obtained white product with deionized water, wash the obtained black product with deionized water repeatedly for several times, then freeze-dry to obtain the product porous graphene, dissolve it with isopropanol to make a 1 mg / mL PG dispersion.

4. A method for evaluating the quality of Cistanche deserticola based on the rapid detection of acteoside according to claim 1, characterized in that: The potential range for detection by differential pulse voltammetry is 0.10 - 0.70 V.

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