CN108717002B

CN108717002B - Method for detecting galanthamine in lycoris aurea

Info

Publication number: CN108717002B
Application number: CN201810541070.6A
Authority: CN
Inventors: 傅力
Original assignee: Hangzhou Dianzi University
Current assignee: Shanghai Lianxing Pharmaceutical Technology Co.,Ltd.
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2020-09-29
Anticipated expiration: 2038-05-30
Also published as: CN108717002A

Abstract

The invention discloses a method for detecting galanthamine in lycoris aurea. The method relates to plant sample treatment, preparation of a nano-gold printed electrode sensor and electrochemical detection. And (3) fully adsorbing the nano-gold modified printed electrode sensor in the sample extracting solution, and then scanning the nano-gold printed electrode sensor by using a differential pulse voltammetry method to obtain the oxidation peak current value. In the detectable range, the galanthamine content in the lycoris aurea adsorbed by the sensor and the oxidation peak current intensity are in a linear relationship, and the content of the galanthamine in the sample to be detected can be calculated according to the working curve. The nano gold modified printed electrode sensor prepared by the invention combines a nano technology and a screen printing technology, and can obviously improve the sensitivity of the sensor to the detection of the concentration of galanthamine in the lycoris aurea and ensure that the lowest detection limit can reach 0.013 mu M.

Description

Method for detecting galanthamine in lycoris aurea

Technical Field

The invention relates to a method for detecting galanthamine in lycoris herb and flower lycoris neglecta, in particular to a method for detecting galanthamine by using a nano-gold modified printed electrode.

Background

The lycoris aurea is a perennial herb flower of lycoris, mostly grows in shade and in the dark, has warm property and mild and sweet taste, and has the effects of relieving swelling, relieving pain, dissipating toxin, promoting vomiting and the like. Galanthamine is a secondary metabolite alkaloid of lycoris neglecta and is mainly distributed in bulbs of galanthamine, and the Chinese pharmacopoeia records that injection and tablets of galanthamine hydrobromide can be used for treating sensory or motor disorders caused by nervous system diseases, including poliomyelitis sequelae, myasthenia gravis and enteroparalysis, and can be used as an anti-curare medicament and a waking agent after operative anesthesia, and the like. In recent years, galantamine hydrobromide has been used as an acetylcholinesterase inhibitor for the treatment of mild to moderate alzheimer's syndrome at home and abroad.

At present, the most widely applied methods for detecting substances in plants are instrument analysis methods such as chromatography and mass spectrometry, and the like, which can rapidly and accurately quantitatively detect the substances to be detected under the condition of using a small amount of samples, but the detection means needs complex pretreatment processes, expensive professional instruments and skilled operators, and is difficult to meet the real-time and online detection requirements. The electrochemical detection technology is a simple, rapid and high-sensitivity detection method, and an Iranian scholars (Journal of electrochemical Chemistry 785(2017): 220-. The electrochemical detection method based on the printed electrode is a novel rapid detection technology, can detect plant samples subjected to simple pretreatment, and has the advantages of rapidness, simplicity, convenience, low price, portability, easiness in miniaturization and the like. However, due to the limited performance of the printed electrode, the method has the defect of low sensitivity, so that the method cannot be used for direct detection of galanthamine.

The nano-gold is a micro gold particle with the diameter of 1-100nm, has the characteristics of simple preparation method, excellent surface adsorption capacity, strong electric conductivity, stable chemical state and the like, and can improve the electron transfer rate and the electrode response speed by modifying the printing electrode by utilizing the nano-gold.

Disclosure of Invention

The invention aims to provide a method for detecting the content of galanthamine in lycoris herb lycoris neglecta, which is used for a nano gold-modified printed electrode sensor and has the advantages of simple structure, convenience in use, low price, high sensitivity and rapid detection process, aiming at overcoming the defects of the prior art.

The invention relates to plant sample treatment, nano-gold printed electrode sensor preparation and electrochemical detection, and specifically comprises the following steps:

step (1), plant sample treatment: chopping up the bulbodium of smilax glabra, stirring the bulbodium of smilax glabra into paste by using a clean food stirrer, weighing a sample into a centrifuge tube, adding a certain amount of methanol into the centrifuge tube, covering the centrifuge tube with a tube cover, and placing the centrifuge tube into an ultrasonic machine for ultrasonic extraction; filtering the obtained dispersion liquid to obtain a plant sample extracting solution.

Step (2), preparing the nano gold modified printed electrode sensor: and cleaning the surface of the printed electrode. And (3) placing the cleaned printed electrode in a mixed solution of dilute hydrochloric acid and chloroauric acid for constant potential electrodeposition to obtain the nano-gold modified printed electrode sensor.

Step (3), drawing a standard curve: and weighing mother liquor prepared from galanthamine solid, adding the mother liquor into phosphate buffer according to the gradient volume, and performing constant volume to obtain a standard solution to be detected with gradient concentration. The nano-gold modified printed electrode sensor is sequentially inserted into standard galanthamine mother liquor with different concentrations, differential pulse voltammetry scanning is carried out within the current range of 0.5-1.5V after full adsorption, a standard working curve is drawn by utilizing the measured oxidation peak current value, and a linear equation is calculated.

Step (4), detection of galanthamine in the smile: and (3) fully adsorbing the nano-gold modified printed electrode sensor in a sample extracting solution, and then scanning the nano-gold modified printed electrode sensor by using a differential pulse voltammetry method to obtain an oxidation peak current value. In the detectable range, the galanthamine content in the lycoris aurea adsorbed by the sensor and the oxidation peak current intensity are in a linear relationship, and the content of the galanthamine in the sample to be detected can be calculated according to the working curve.

Preferably, the amount of the plant sample in step (1) is 1-10g, and the amount of methanol is 5-20 mL.

Preferably, the ultrasonic power in the step (1) is 200-500W, and the ultrasonic frequency is 20 kHz.

Preferably, the filter paper in the step (1) is made of polyether sulfone and has a pore diameter of 200nm-2 μm.

Preferably, the printed electrode is a screen-printed electrode in which the working electrode, the reference electrode and the auxiliary electrode are integrated. The printed electrode is a disposable electrode with low cost and is very suitable for rapid detection.

Preferably, the printed electrode is in transmission connection with the printed electrode through an NDK wild port bus, a hole plate and a DuPont wire.

Preferably, the concentration of the dilute hydrochloric acid in the electrolyte in the step (2) is 0.1M-0.5M.

Preferably, the concentration of chloroauric acid in step (2) is 0.1 wt% to 0.5 wt%.

Preferably, the electrodeposition potential in the step (2) is +0.4 to 0.5V.

Preferably, the electrodeposition time in step (2) is 1 to 5 min.

The invention has the beneficial effects that:

1. the method does not need purification and concentration in the pretreatment process of the sample of the smiling sample, and greatly shortens the time required by sample treatment.

2. The nano gold modified printed electrode sensor prepared by the invention combines a nano technology and a screen printing technology, and can obviously improve the sensitivity of the sensor to the detection of the concentration of galanthamine in the lycoris aurea and ensure that the lowest detection limit can reach 0.013 mu M.

Detailed Description

The present invention is further analyzed with reference to the following specific examples.

Example 1.

A method for detecting galanthamine in lycoris aurea comprises the following operation steps:

(1) sample treatment: chopping the bulbodium caucasiae of the lycoris aurea, stirring the bulbodium caucasia into paste by using a clean food stirrer, weighing 10g to 50mL of a centrifuge tube of the sample, adding 20mL of methanol, covering the tube cover, then arranging the tube cover, and then placing the tube cover in an ultrasonic machine for ultrasonic extraction for 10min, wherein the ultrasonic power is 200W, and the ultrasonic frequency is 20 kHz. Filtering the obtained dispersion liquid by polyethersulfone filter paper with the aperture of 200nm to obtain the extract of the lycoris aurea.

(2) Preparing a nano gold modified printed electrode sensor: respectively cleaning the surface of the printed electrode by using distilled water and ethanol, placing the cleaned printed electrode into a mixed solution of 20mL of dilute hydrochloric acid (0.1M) and chloroauric acid (0.1 wt%), and performing constant potential electrodeposition for 1min under the voltage of +0.4V to obtain the nano-gold modified printed electrode sensor.

(3) Drawing a standard curve: weighing mother liquor prepared from galanthamine solid, diluting with methanol, and diluting to constant volume to obtain mother liquor with concentration of 0.1mM, adding phosphate buffer solution with pH of 0.1mM 7.0 into a certain amount of mother liquor, and diluting to constant volume to obtain standard solutions to be tested with galanthamine gradient concentrations of 0.1 μ M,0.2 μ M, 0.4 μ M, 0.8 μ M and 1.0 μ M in sequence.

(4) Detection of galantamine in laugh: and immersing the nanogold modified printed electrode sensor into a sample extracting solution for full adsorption, and scanning by using a CH1760b electrochemical workstation at 0.5-1.5V through differential pulse voltammetry to detect the current intensity of an oxidation peak. The peak current of the oxidation peak obtained by differential pulse voltammetry scanning and the galanthamine concentration are in a linear relationship within the range of 0.1-1.0 mu M, and the detection limit is 0.05 mu M.

Example 2.

(1) sample treatment: chopping the bulbodium of the lycoris aurea, stirring the bulbodium of the lycoris aurea into paste by using a clean food stirrer, weighing 5g to 50mL of a centrifuge tube for a sample, adding 10mL of methanol, covering the centrifuge tube with a tube cover, then placing the centrifuge tube with the tube cover, placing the centrifuge tube into an ultrasonic machine for ultrasonic extraction for 10min, and filtering the obtained dispersion liquid by using polyether sulfone filter paper with the aperture of 2 mu m to obtain the lycoris aurea extract.

(2) Preparing a nano gold modified printed electrode sensor: respectively cleaning the surface of the printed electrode by using distilled water and ethanol, placing the cleaned printed electrode into a mixed solution of 20mL of dilute hydrochloric acid (0.2M) and chloroauric acid (0.5 wt%), and performing constant potential electrodeposition for 5min under the voltage of +0.5V to obtain the nano-gold modified printed electrode sensor.

(3) Drawing a standard curve: weighing mother liquor prepared from galanthamine solid, diluting with methanol, and diluting to constant volume to obtain mother liquor with concentration of 0.1mM, adding phosphate buffer solution with pH of 0.1mM 7.0 into a certain amount of mother liquor, and diluting to constant volume to obtain standard solutions to be tested with galanthamine gradient concentrations of 0.05 μ M,0.1 μ M, 0.5 μ M, 1.0 μ M and 2.0 μ M in sequence.

(4) Detection of galantamine in laugh: and immersing the nanogold modified printed electrode sensor into a sample extracting solution for full adsorption, and scanning by using a CH1760b electrochemical workstation in a current range of 0.5-1.5V by using differential pulse voltammetry to detect the current intensity of an oxidation peak. The peak current of the oxidation peak obtained by differential pulse voltammetry scanning and the galanthamine concentration are in a linear relation within the range of 0.05-2.0 mu M, and the detection limit is 0.013 mu M.

The foregoing embodiments are described for the purpose of illustration and example, and are not to be construed as limiting the invention in any way, except as to the extent that the present invention is set forth in the following claims.

Claims

1. A method for detecting galantamine in laughing gas, characterized in that the method comprises the steps of:

step (1), plant sample treatment:

chopping up the bulbodium of smilax glabra, stirring the bulbodium of smilax glabra into paste by using a clean food stirrer, weighing a sample into a centrifuge tube, adding a certain amount of methanol into the centrifuge tube, covering the centrifuge tube with a tube cover, and placing the centrifuge tube into an ultrasonic machine for ultrasonic extraction; filtering the obtained dispersion liquid to obtain a plant sample extracting solution;

step (2), preparing the nano gold modified printed electrode sensor:

cleaning the surface of the printing electrode; placing the cleaned printed electrode in a mixed solution of dilute hydrochloric acid and chloroauric acid for constant potential electrodeposition to prepare a nano gold modified printed electrode sensor;

step (3), drawing a standard curve:

weighing mother liquor prepared from galanthamine solid, adding into phosphate buffer according to gradient capacity, and performing constant volume to obtain galanthamine standard mother liquor with gradient concentration; sequentially inserting the nano-gold modified printed electrode sensor into standard galanthamine mother liquor with different concentrations, fully adsorbing, then scanning by a differential pulse voltammetry within a current range of 0.5-1.5V, drawing a standard working curve by using the measured oxidation peak current value, and calculating a linear equation;

step (4), detection of galanthamine in the smile:

fully adsorbing the nano-gold modified printed electrode sensor in a sample extracting solution, and then scanning the nano-gold modified printed electrode sensor by using a differential pulse voltammetry method to obtain an oxidation peak current value; in the detectable range, the galanthamine content in the lycoris aurea adsorbed by the sensor and the oxidation peak current intensity are in a linear relationship, and the content of the galanthamine in the sample to be detected can be calculated according to the working curve.

2. The method for detecting galantamine in lycoris neglecta according to claim 1, wherein the amount of the plant sample in step (1) is 1-10g, and the amount of methanol is 5-20 mL.

3. The method for detecting galantamine in lycoris aurea as claimed in claim 1, wherein the ultrasonic power in step (1) is 200-500W, and the ultrasonic frequency is 20 kHz.

4. The method for detecting galantamine in lycoris aurea according to claim 1, wherein the filter paper used in the filtering in the step (1) is polyether sulfone and has a pore size of 200nm-2 μm.

5. The method for detecting galantamine in laughs according to claim 1, wherein the printed electrodes are in transmission connection with the printed electrodes through an NDK portal bus, a cave plate and a dupont line.

6. The method for detecting galantamine in lycoris aurea according to claim 1, wherein the concentration of the dilute hydrochloric acid of the electrolyte in the step (2) is 0.1M to 0.5M.

7. The method for detecting galantamine in lycoris aurea according to claim 1, wherein the concentration of chloroauric acid in step (2) is 0.1 wt% to 0.5 wt%.

8. The method for detecting galantamine in lycoris aurea according to claim 1, wherein the electrodeposition potential in the step (2) is +0.4 to 0.5V.

9. The method for detecting galantamine in lycoris neglecta according to claim 1, wherein the electrodeposition time in step (2) is 1-5 min.