A method for detecting leonurine

Abstract

The present invention relates to the detection of leonurine. The present invention provides a method for detecting leonurine. A sample to be tested is dissolved in a 50-90% methanol aqueous solution, and synchronous fluorescence analysis is used. A Stokes shift of 25-35 nm and an emission wavelength of 300-310 nm are set to detect the fluorescence intensity of the test solution to qualitatively detect the leonurine component. The present invention has the following advantages: synchronous fluorescence analysis is used for determination, which is simple to operate, can quickly respond to leonurine, has high sensitivity and strong anti-interference ability, uses optimized measurement parameters for emission wavelength and Stokes shift, improves measurement selectivity, has high linearity of the standard curve equation, has a low detection limit, is fast to identify, and has accurate detection results, and has good development and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of detection, and in particular to the detection of leonurine. Background Art

[0002] Leonurine, scientifically known as 3,5-dimethoxy-4-carboxyl-benzoic acid (4-guanidino)-1-butyl ester (Structural Formula I), is most commonly found as its hydrochloride hydrate. It is extracted from the leaves of the Lamiaceae plant Leonurine, or the whole plant of Leonurine, Artemisia annua, and Leonurine. It has the effects of promoting blood circulation, removing blood stasis, promoting diuresis and reducing swelling. Leonurine is an alkaloid with anti-inflammatory, antioxidant and anti-tumor effects. Studies have shown that Leonurine can dilate peripheral blood vessels, increase blood flow, and has a protective effect on cerebral ischemia and myocardial ischemia; it can also significantly reduce the area of ​​cerebral cortical infarction caused by cerebral ischemia and improve the symptoms of neurological impairment. Leonurine preparations are used to a certain extent in clinical practice.

[0003]

[0004] Currently, methods for detecting the content of leonurine include reverse-phase high-performance liquid chromatography (HPLC) or ultra-high-pressure liquid chromatography (UPLC). Both HPLC and UPLC require pretreatment of the substance to be tested, filtering it through a microporous membrane before injection. Chromatographic conditions must also be established, with mobile phases of varying volume ratios, such as methanol-water, methanol-triethylamine, and methanol-buffered solution, tested. Furthermore, liquid chromatography requires complete separation of leonurine from other substances to detect leonurine.

[0005] Fluorescence analysis uses the fluorescence intensity of alkaloid molecules when excited by specific wavelengths to calculate their content. For example, sodium fluorescein emits green fluorescence under ultraviolet light, which can be used for content determination. Conventional fluorescence analysis of complex mixtures encounters technical challenges such as spectral overlap, requiring complex pre-separation of the sample. Summary of the Invention

[0006] The object of the present invention is to provide a method for detecting leonurine, which can achieve rapid response and quantitative detection of leonurine, rapid identification, accurate detection results, and does not require complex pre-separation of samples.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for detecting leonurine. A sample to be tested is dissolved in a 50-90% methanol aqueous solution as the test solution. Synchronous fluorescence analysis is adopted with a Stokes shift of 25-35 nm and an emission wavelength of 300-310 nm to detect the fluorescence intensity of the test solution and qualitatively detect leonurine.

[0009] This method uses synchronous fluorescence analysis, under the conditions of Stokes shift 25-35nm and emission wavelength in the range of 300-310nm. It can be seen from the wavelength-fluorescence intensity spectrum that the spectrum of leonurine has a higher spectral peak than the spectra of other components of the sample to be tested, that is, it has a higher fluorescence intensity, and leonurine can be qualitatively detected.

[0010] Preferably, a 70% methanol aqueous solution is selected as the detection system. When the methanol concentration is high, the sample to be tested is difficult to dissolve, and when the methanol concentration is low, the fluorescence intensity is too low. The 70% methanol aqueous solution is used for the best measurement results.

[0011] Preferably, the Stokes shift is set to 30 nm.

[0012] Preferably, the emission wavelength is set to 303 nm.

[0013] Under these setting conditions, the peak shape of leonurine is narrower and better, and the anti-interference effect is stronger.

[0014] Preferably, according to the standard curve of leonurine concentration-fluorescence intensity, the fluorescence intensity corresponds to the leonurine content, and a quantitative calculation is performed to obtain the leonurine content of the sample to be tested.

[0015] Preferably, the concentration range of leonurine tested by synchronous fluorescence method is 0 to 20 μM.

[0016] This synchronous fluorescence analysis method is immune to interference from interfering components in the test solution, demonstrating excellent interference resistance. Within the range of 0 to 20 μM, the concentration of motherwort is linearly correlated with its fluorescence intensity. By measuring the fluorescence intensity of motherwort, the motherwort content in the sample can be determined. This method allows for rapid detection with a low limit of detection.

[0017] Preferably, the solution to be tested is filtered through a 0.1-0.5 μM microporous filter membrane.

[0018] The beneficial effects of the present invention are:

[0019] (1) Synchronous fluorescence analysis is used to detect leonurine, which has good stability. Under the condition of Stokes shift of 25-35nm, the detection wavelength is 300-310, the fluorescence intensity of interfering substances is low, and leonurine can be qualitatively detected. The emission wavelength and Stokes shift are optimized to improve the determination selectivity.

[0020] (2) The present invention quantitatively detects leonurine by synchronous fluorescence, which can achieve a rapid response to leonurine. At the same time, the leonurine concentration at 0-20 μM shows a significant linear correlation with the fluorescence intensity at 303 nm. The standard curve equation has high linearity and low detection limit. The minimum detection limit of leonurine is as low as 0.37 μM. This method achieves high sensitivity, high selectivity, and wide linear range of concentration detection of leonurine.

[0021] (3) The synchronous fluorescence method of the present invention is simple to operate, has high sensitivity and strong anti-interference ability, and can realize rapid qualitative and quantitative detection of leonurine in actual samples. It has good development and application prospects and has broad application prospects in biological sciences, traditional Chinese medicine and clinical medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a graph showing the fluorescence intensity stability of leonurine in a methanol-water solution detection system according to the present invention;

[0023] Figure 2 This is a graph showing the instrument precision test of the synchronous fluorescence method of the present invention;

[0024] Figure 3 For the present invention's leonurine, Na + , K + , Ca 2+ 、Cu 2+ 、Cd 2+ 、Ni 2+ Mg 2+ 、Co 2+ 、Zn 2+ , Pb 2+ 、Al 3+ 、Cl - 、F - 、CO3 2- 、SO4 2- , threonine (Thr), arginine (Arg), lysine (Cys), and rutin (Rutin) spectra in 70% methanol solution detection system;

[0025] Figure 4 A bar graph showing the anti-interference performance of the synchronous fluorescence method of the present invention on leonurine;

[0026] Figure 5 This is a schematic diagram of the change in fluorescence of leonurine according to the present invention as its concentration changes;

[0027] Figure 6 This is a working curve of leonurine concentration-fluorescence intensity for detecting leonurine using the synchronous fluorescence method of the present invention;

[0028] Figure 7 This is the spectrum of the content of leonurine in Yimucao granules of brand A detected by synchronous fluorescence method;

[0029] Figure 8 This is the spectrum of the leonurine content in Brand B Yimucao Granules detected by synchronous fluorescence method;

[0030] Figure 9 This is the working curve of leonurine concentration-fluorescence intensity detected in brand B Yimucao granules using the external standard method. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1 Study on the stability of leonurine

[0033] To test the stability of leonurine in a 70% methanol aqueous solution, the synchronous fluorescence response to 10 μM leonurine was tested over time. The specific process is as follows:

[0034] (1) Prepare a 10 mM leonurine stock solution. The detection system is 70% methanol in water.

[0035] (2) Place 2 mL of 70% methanol aqueous solution in a 3.5 mL four-sided light-transmitting quartz cuvette. Then add 2 μL of leonurine stock solution to obtain a test object with a concentration of 10 μM. Shake well and place in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection wavelength range to 250-400 nm. Set the time to 0, 2, 4, 6, 8, and 10 h, and test the synchronous fluorescence spectrum of the above test solution respectively.

[0036] (3) The synchronous fluorescence spectra of leonurine at different times were investigated using a fluorescence spectrometer. As time went on, the synchronous fluorescence intensity of leonurine remained basically unchanged, with a calculated RSD of 1.6%. The experimental results showed that leonurine had good stability from 0 to 10 hours. Figure 1 As shown in the figure, it can be seen that within 0-10 hours, as time goes by, the synchronous fluorescence intensity of leonurine remains basically unchanged, and the RSD of the calculator is 1.6%. The experimental results show that leonurine has good stability within 0-10 hours.

[0037] Example 2 Precision Test

[0038] To test the precision of the fluorescence spectrometer, the same sample solution was scanned 6 times and the RSD was calculated. The specific steps are as follows:

[0039] (1) Prepare a 10 mM leonurine stock solution. The detection system is 70% methanol in water.

[0040] (2) Place 2 mL of 70% methanol aqueous solution in a 3.5 mL four-sided light-transmitting quartz cuvette. Then add 2 μL of the leonurine stock solution to obtain a test object concentration of 10 μM. Shake well and place in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection wavelength range to 250-400 nm. Continuously measure the synchronous fluorescence spectrum of the above test solution 6 times.

[0041] (3) The synchronous fluorescence spectrum of leonurine was investigated using a fluorescence spectrometer. The calculated RSD was 1.6%, and the instrument precision of the experimental results met the requirements. Figure 2 As shown in the figure, it can be seen that the difference in fluorescence intensity of the six times is not obvious, and the calculated RSD is 1.6%. The instrument precision of the experimental results meets the requirements.

[0042] Example 3 Synchronous Fluorescence Spectrum of Leonurine

[0043] In order to test the synchronous fluorescence spectrum of leonurine, the fluorescence responses of different types of anions and cations, amino acids and other interfering substances contained in motherwort were tested. + , K + , Ca 2+ 、Cu 2+ 、Cd 2+ 、Ni 2+ Mg 2+ 、Co 2+ 、Zn 2+ , Pb 2+ 、Al 3+ 、Cl - 、F - 、CO3 2- 、SO4 2- , threonine (Thr), arginine (Arg), lysine (Cys), and rutin (Rutin). The working concentration of the research objects was 10μM, and the concentration of leonurine was also 10μM. The specific operation process is as follows:

[0044] (1) Prepare a 10 mM stock solution of the test object. Weigh the appropriate amount of the substance into a 5 mL volumetric flask, dilute to the mark with deionized water, shake well, and refrigerate until ready for use.

[0045] (2) Place 2 mL of 70% methanol aqueous solution in a 3.5 mL four-sided light-transmitting quartz cuvette. Add 2 μL of stock solutions of various anions, cations, amino acids, and other interfering substances to the cuvette to obtain a series of test solutions with a concentration of 10 μM. Shake well and place in a fluorescence spectrometer. Set the Stokes value to 30 nm and the detection wavelength range to 250-400 nm. Measure the synchronous fluorescence spectra of the above test solutions.

[0046] (3) The fluorescence spectra of different anions and cations, amino acids, other interfering substances contained in Leonurus japonicus, and leonurine were investigated using a fluorescence spectrometer. Under the condition of a Stokes shift of 30 nm, the synchronous fluorescence emission wave of leonurine is located at 303 nm.

[0047] like Figure 3 As shown in the figure, it can be seen that leonurine shows strong fluorescence at 303nm. The experimental results show that under the condition of Stokes shift of 30nm, leonurine can be qualitatively detected at 303nm.

[0048] Example 4 Anti-interference performance of synchronous fluorescence method

[0049] (1) Prepare a 10 mM stock solution of the test object. Weigh the appropriate amount of the substance into a 5 mL volumetric flask, dilute to the mark with deionized water, shake well, and refrigerate until ready for use.

[0050] (2) Prepare 19 four-sided light-transmitting quartz cuvettes and place 2 mL of 70% methanol aqueous solution in 3.5 mL four-sided light-transmitting quartz cuvettes. Add 2 μL of stock solutions of various anions, cations, amino acids, and other interfering substances to the cuvettes to obtain a series of test solutions with a concentration of 10 μM. Shake well and place in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection wavelength range to 250-400 nm. Measure the synchronous fluorescence spectra of the above test solutions.

[0051] (3) Record the fluorescence intensity at the maximum emission point (303 nm) of the synchronous fluorescence spectrum to obtain the synchronous fluorescence emission intensity values ​​(I1) of various interfering substances.

[0052] (4) Add 2 μL of the leonurine stock solution (10 mM) to each of the cuvettes in (2) to obtain a series of leonurine test solutions with various anions, cations, amino acids, and other interfering substances. Test the synchronous fluorescence spectra of the above test solutions.

[0053] (5) Record the fluorescence intensity at the maximum emission point (303 nm) of the synchronous fluorescence spectrum to obtain the synchronous fluorescence emission intensity value (I2) of leonurine under various interference backgrounds.

[0054] (6) Draw a bar graph with the different interfering substances added as the horizontal axis and the fluorescence emission intensity (I1, I2) as the vertical axis. Figure 4 Experiments have shown that leonurine is not interfered with by the above-mentioned other interfering substances in a 70% methanol aqueous solution system and has good anti-interference properties.

[0055] The interference object is Na + , K + , Ca 2+ 、Cu 2+ 、Cd 2+ 、Ni 2+ Mg 2+ 、Co 2+ 、Zn 2+ , Pb 2+ 、Al 3+ 、Cl - 、F - 、CO3 2- 、SO4 2- , threonine (Thr), arginine (Arg), lysine (Cys), and rutin (Rutin).

[0056] Gray columns represent the addition of other interfering substances to the detection system, while black columns represent the addition of other interfering substances and leonurine to the monitoring system. As can be seen from the figure, leonurine is not affected by the above-mentioned other interfering substances in the 70% methanol-water system, showing good anti-interference properties.

[0057] Example 5 Study on the changes of synchronous fluorescence intensity under different concentrations of leonurine

[0058] In order to test the synchronous fluorescence response characteristics of leonurine at different concentrations, the experimental process was described in detail using 70% methanol aqueous solution as an example.

[0059] (1) Place 2 mL of 70% methanol aqueous solution in a 3.5 mL four-color transparent quartz cuvette. Gradually add leonurine at concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 μM to the cuvette, shake well, and place in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection wavelength range to 250-400 nm to test the synchronous fluorescence response of the above different concentrations of leonurine.

[0060] (2) The fluorescence spectrometer was used to investigate the synchronous fluorescence response of different concentrations of leonurine. The experimental results showed that as the concentration of leonurine increased, the fluorescence intensity at 303 nm gradually increased. Figure 5As shown in the figure, the fluorescence of leonurine increases with increasing leonurine concentration. Within the detection range of 0 to 20 μM, the fluorescence intensity is linearly related to leonurine concentration, with a detection limit of 0.37 μM. These results demonstrate that the synchronous fluorescence intensity varies with leonurine concentration, enabling quantitative detection.

[0061] Example 6 Establishment of a standard curve of leonurine concentration-fluorescence intensity and investigation of detection limit

[0062] (1) A good detection limit is one of the criteria for testing whether a probe molecule has application value.

[0063] (2) Place 2 mL of 70% methanol aqueous solution in a 3.5 mL four-color transparent quartz cuvette. Gradually add leonurine at concentrations of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 μM to the cuvette, shake well, and place in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection wavelength range to 250-400 nm to test the synchronous fluorescence response of the above different concentrations of leonurine.

[0064] (3) Record the fluorescence intensity at the maximum emission point (303 nm) of the synchronous fluorescence spectrum to obtain a series of synchronous fluorescence emission intensity values ​​related to the concentration of leonurine. Make a scatter plot of the synchronous fluorescence intensity value versus the concentration of leonurine, as shown in Figure 2. Figure 6 shown.

[0065] (4) Yes Figure 6 The linear range (0-20 μM) was fitted, and the detection limit of leonurine was calculated according to the formula (3σ / k).

[0066] (5) According to the calculation, the linear regression equation is: Y = 1552.24411x + 3442.56048, R 2 =0.99753, the unit of C is μmol / L, and the detection limit of leonurine is 0.37 μM according to calculation (3σ / k), which shows a good linear relationship.

[0067] Example 7 Synchronous Fluorescence Detection of Leonurine in Different Brands of Leonurine Granules

[0068] The content of leonurine in brand A and brand B Yi Mu Cao granules was determined by synchronous fluorescence method. The specific steps are as follows:

[0069] (1) Accurately weigh 40 mg of brand A and brand B motherwort granules, respectively, dissolve them in 10 mL of 70% methanol aqueous solution, shake well, and filter through a 0.22 μm filter membrane to prepare a 4 mg / ml actual sample (motherwort granule) solution.

[0070] (2) Place 2 mL of the same sample solution in a 3.5 mL four-color transparent quartz cuvette, shake well, and place in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection range to 250-400 nm. Repeat 6 times.

[0071] (3) Record the fluorescence intensity at 303 nm. The synchronous fluorescence intensities of brand A at 303 nm were 12453.4941, 12833.9834, 12536.0088, 12767.0488, 12823.291, and 13009.7793, respectively. Substituting them into the fluorescence linear equation, the concentrations were calculated to be 5.86, 6.05, 5.86, 6.01, 6.04, and 6.16 μM, respectively. The average concentration was 6.0 μM, and the RSD was 1.9% (e.g. Figure 7 As shown in the figure, it can be seen that leonurine can be qualitatively detected at 303 nm, and the corresponding fluorescence intensity is substituted into the linear regression equation to calculate the leonurine concentration in Brand A Yi Mu Cao Granules to be 6.0 μM, with an RSD of 1.9%.

[0072] The fluorescence intensities of brand B measured at 303 nm were 23074.0723, 24496.5, 23249.0664, 23583.8457, 23626.25, and 23811.7695, respectively. Substituting them into the fluorescence linear equation, the concentrations were calculated to be 12.65, 13.56, 12.76, 12.96, 13.00, and 13.12 μM, respectively. The average concentration was 13.0 μM, and the RSD was 2.4% (e.g. Figure 8 shown).

[0073] As can be seen from the figure, leonurine can be qualitatively detected at 303 nm, and the corresponding fluorescence intensity is substituted into the linear regression equation to calculate the leonurine concentration in Brand B Leonurine Granules to be 13 μM, with an RSD of 2.4%.

[0074] 3) Synchronous fluorescence was used to measure the leonurine content in different brands of Yi Mu Cao granules. The results showed that Brand A contained 6.0 μM leonurine with an RSD of 1.9%, while Brand B contained 13.0 μM leonurine with an RSD of 2.4%. The results were accurate with reasonable tolerances.

[0075] In order to verify the accuracy of this method, we selected brand B Yi Mu Cao granules and used the spike recovery method to verify its accuracy. The specific steps are as follows:

[0076] (1) Accurately weigh 10 mg of brand B motherwort granules, dissolve in 10 mL of 70% methanol aqueous solution, shake well, and filter through a 0.22 μm filter membrane to prepare a 1 mg / ml actual sample (motherwort granules) solution.

[0077] (2) Place 2 mL of the actual sample (Leonurus granules) solution in a 3.5 mL four-sided light-transmitting quartz cuvette and place the cuvette in a fluorescence spectrometer. Set the Stokes shift to 30 nm and the detection wavelength to 250–400 nm. Obtain the initial fluorescence spectrum of leonurusine in the actual sample.

[0078] (3) Add 2, 4, 6, 8, 10, 12, and 14 μL of pure leonurine stock solution (10 mM) to the cuvette, respectively, to obtain test solutions containing 10, 20, 30, 40, 50, 60, and 70 μM leonurine. Measure the fluorescence spectra of the test solutions.

[0079] (4) Record the fluorescence intensity at the maximum emission point (303 nm) of the above fluorescence spectrum to obtain a series of fluorescence emission intensity values ​​related to the concentration of leonurine. Make a scatter plot of the fluorescence intensity versus the concentration of leonurine, as shown below: Figure 9 shown.

[0080] (5) Yes Figure 9 Linear fitting was performed in the linear range (0-70 μM) to obtain the standard curve equation.

[0081] (6) Standard solutions of known concentrations of leonurine (15 μM, 25 μM, and 35 μM) were added to the actual sample (Leonurine Granules). The fluorescence intensity at 303 nm was recorded and the leonurine content in the actual sample (Leonurine Granules) was calculated using the linear equation. This step was repeated three times. The test results are shown in Table 1 below.

[0082] Table 1 Detection of leonurine content in brand B by synchronous fluorescence method

[0083]

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting leonurine, characterized in that: The sample to be tested is dissolved in a 50-90% methanol aqueous solution as the test solution. Synchronous fluorescence analysis is used with a Stokes shift of 25-35 nm and an emission wavelength of 300-310 nm to detect the fluorescence intensity of the test solution for qualitative detection of leonurine. The sample to be tested is Leonurine granules.

2. The method for detecting leonurine according to claim 1, wherein: 70% methanol aqueous solution was selected as the detection system.

3. The method for detecting leonurine according to claim 1, wherein: Set the Stokes shift to 30 nm.

4. The method for detecting leonurine according to claim 1, wherein: Set the emission wavelength to 303 nm.

5. The method for detecting leonurine according to claim 1, wherein: According to the standard curve of leonurine concentration-fluorescence intensity, the fluorescence intensity corresponds to the leonurine content, and quantitative calculation is performed to obtain the content of leonurine in the test sample. The leonurine concentration range of the synchronous fluorescence quantitative test is 0~20 μM.

6. A method for detecting leonurine according to any one of claims 1 to 5, characterized in that: The solution to be tested was filtered through a 0.1-0.5 μM microporous membrane.

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