Detection method of macleaya cordata alkaloid
Through the steps of extracting Macleaya cordata alkaloids, drawing a standard curve and determining samples, a small ultraviolet spectrophotometer is used to achieve simple and rapid detection of Macleaya cordata alkaloids, which solves the problems of the complexity and high cost of the existing methods and achieves rapid and accurate detection results.
Patent Information
- Application Number
- CN202510683262.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-26
- Publication Date
- 2025-09-19
AI Technical Summary
Existing Macleaya cordata alkaloid detection methods are complex to operate, costly, slow to detect, and lack simple and rapid detection methods, making it difficult to meet industry needs.
The method adopts the steps of extracting Macleaya cordata alkaloids, drawing a standard curve and determining samples, and uses a small ultraviolet spectrophotometer for detection. The Macleaya cordata alkaloids are extracted with a strong acid-alcohol solution, an absorbance-concentration curve is drawn, and the extraction rate is calculated.
A simple, rapid and low-cost detection of Macleaya cordata alkaloids is achieved, with a detection time of less than half an hour, a wide linear detection range, accurate results, and a cost lower than traditional large-scale instruments.
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Figure CN120668431A_ABST
Abstract
Description
[0001] The invention belongs to the technical field of chemical detection, and particularly relates to a method for detecting Macleaya cordata alkaloids. Background Art
[0002] Macleaya cordata alkaloids are a class of natural nitrogen-containing organic compounds extracted mainly from the Macleaya cordata plant and belong to the isoquinoline alkaloid family. Macleaya cordata alkaloids have certain toxicity and can damage organs such as the heart. Macleaya cordata extracts are used as botanical pesticides in some areas (such as to control vegetable aphids). Testing Macleaya cordata alkaloids can ensure that alkaloid residues in agricultural products (such as vegetables and fruits) do not exceed the standard and prevent them from entering the human body through the food chain. There are also some businesses that may illegally add Macleaya cordata alkaloids to health foods (such as claiming "antibacterial and detoxification" effects). Testing Macleaya cordata alkaloids can identify illegal ingredients and prevent consumers from long-term intake causing cumulative toxicity (such as liver damage and reproductive toxicity).
[0003] Methods for determining the content of Macleaya cordata alkaloids include high-performance liquid chromatography (HPLC) and acid dye colorimetry. The HPLC method uses total alkaloids (sanguinarine, chelerythrine, protopine, and allocryptine) as a quality marker for Macleaya cordata, but suffers from drawbacks such as complex operation, high cost, and slow detection. The acid dye colorimetry method uses papaverine as a standard and requires the preparation of a color developer, buffer, and extractant, resulting in cumbersome operation, poor reproducibility, and low stability.
[0004] Currently, there is a lack of a simple and rapid detection method for Macleaya cordata alkaloids, which can better lower the detection threshold, improve industry efficiency and ensure product quality. Summary of the Invention
[0005] In order to solve the problem of lack of a simple and rapid detection method for Macleaya cordata alkaloids, the present invention aims to provide a detection method for Macleaya cordata alkaloids.
[0006] This is achieved specifically through the following technical solutions:
[0007] The detection of Macleaya cordata alkaloids is achieved through three steps: extraction of Macleaya cordata alkaloids, drawing of standard curve, and sample determination. The three steps are:
[0008] A method for detecting Macleaya cordata alkaloids specifically comprises the following steps:
[0009] (1) Extraction of alkaloids from Macleaya cordata: Accurately weigh a powder sample of Macleaya cordata fruit or Macleaya cordata leaf and place it in a container. Add a strong acid-alcohol solution for extraction, then heat it in a microwave reactor. After heating, take it out and let it cool to room temperature before filtering. The residue is washed three times with a strong acid-alcohol solution. The filtrate and the washing solution are transferred together to a volumetric flask and fixed to volume with a strong acid-alcohol solution to obtain the Macleaya cordata extract.
[0010] (2) Plotting the standard curve: Dissolve the alkaloid standard in a strong acid-alcohol solution, transfer to a volumetric flask, and then dilute to volume to make a 400 μg / mL standard stock solution. Accurately pipette 2.50 mL of the stock solution and transfer to a 25 mL volumetric flask. Then, accurately pipette 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of the diluted solution to a 25 mL volumetric flask. Scan the spectrum using the solvent as a reference and measure the absorbance at the maximum absorption wavelength of 265-285 nm to plot the absorbance-concentration curve.
[0011] (3) Sample determination: Accurately pipette 0.50 mL of the Macleaya cordata extract from (1) into a 25 mL volumetric flask, add the corresponding extractant to the volume, and measure the absorbance at the maximum absorption wavelength using the extractant as a reference. Repeat 3 times. The Macleaya cordata alkaloid extraction rate is calculated as follows:
[0012]
[0013] Wherein: ω1 is the extraction rate (%); c is the concentration of Macleaya cordata alkaloids in the extract (μg / mL); V is the final volume of the extract (mL); N is the dilution multiple; m is the mass of Macleaya cordata powder (g).
[0014] The mass ratio of the powder of Macleaya cordata fruit or Macleaya cordata leaf to the strong acid-alcohol solution is 1:30 to 1:200.
[0015] The heating temperature in the microwave reactor is 60° C. to 80° C., and the heating time is 5 to 15 minutes.
[0016] The strong acid-alcohol solution described in (1) refers to one, two or three of sulfuric acid, hydrochloric acid and nitric acid, and the total acid concentration is 0.05 to 0.2 mol / L in terms of hydrogen ions.
[0017] The strong acid-alcohol solution described in (1) refers to a solution obtained by mixing one or both of methanol and ethanol with water, with the ratio of alcohol to water being 8:2-2:8.
[0018] The alkaloid standard described in (2) is a mixture of one or more of sanguinarine, chelerythrine, protopine, and allocryptine.
[0019] The maximum absorption wavelength mentioned in (3) refers to the wavelength of light used when measuring absorbance in the ultraviolet region in ultraviolet-visible spectrophotometry, which ranges from 265 to 285 nm.
[0020] Beneficial effects
[0021] The detection method of Macleaya cordata alkaloids protected by the present invention has the following beneficial effects:
[0022] 1. Compared with the traditional large-scale instrument detection method, the detection method of the present invention is simpler.
[0023] 2. Compared with the traditional large-scale instrument detection method, the detection method of the present invention is faster, and the whole process takes less than half an hour.
[0024] 3. Compared with the traditional large-scale instrument detection method, the detection method of the present invention is lower in cost and only requires a small instrument, such as a UV spectrophotometer, to complete the detection.
[0025] 4. The detection linear range of the present invention is wide and can accurately detect the total alkaloids of Macleaya cordata. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The absorbance standard curve of sanguinarine was measured at 275 nm;
[0027] Figure 2 The absorbance standard curve of chelerythrine was measured at 272 nm;
[0028] Figure 3 The absorbance of the mixed base was measured at 278 nm to obtain a standard curve;
[0029] Figure 4 The absorbance of the mixed base was measured at 280 nm to obtain a standard curve. DETAILED DESCRIPTION
[0030] Accurately weigh 0.50 g of a Macleaya cordata fruit powder sample and place it in a two-necked round-bottom flask. Add 50 mL of a 0.15 mol / L hydrochloric acid-methanol aqueous solution (60% methanol). Heat the mixture at 70° C. in a microwave reactor for 5 min, remove the mixture, and cool it to room temperature to obtain a Macleaya cordata crude extract. Filter the Macleaya cordata crude extract, wash the residue with an extraction solvent 2 to 3 times, and transfer the filtrate and the washing solution to a 100 mL volumetric flask to a constant volume to obtain a Macleaya cordata extract.
[0031] Example 1
[0032] Dissolve the sanguinarine standard in methanol-water (50:50) containing 0.1 mol / L hydrochloric acid, transfer to a 50 mL volumetric flask, dilute to volume with purified water and shake well to prepare a 400 μg / mL standard stock solution. Accurately pipette 2.50 mL of the stock solution and transfer to a 25 mL volumetric flask, then accurately pipette 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of the diluted solution to a 25 mL volumetric flask, and measure the absorbance at the maximum absorption wavelength of 270 nm after scanning the spectrum with the solvent as a reference. Draw an absorbance-concentration curve, as shown in the following figure: Figure 1 As shown, the regression equation is y = 0.101747x + 0.000155 (R 2=0.999990), the limit of detection (LOD) was 21.7 ng / mL (calculated based on the standard deviation of the regression residuals), and the reproducibility and selectivity were good.
[0033] Accurately pipette 0.50 mL of Macleaya cordata extract into a 25 mL volumetric flask, add the appropriate extractant to the volume, and measure the absorbance at 275 nm using the extractant as a reference. Repeat three times. The Macleaya cordata alkaloid extraction rate is calculated as follows:
[0034]
[0035] Wherein: ω1 is the extraction rate (%); c is the concentration of Macleaya cordata alkaloids in the extract (μg / mL); V is the final volume of the extract (mL); N is the dilution multiple; m is the mass of Macleaya cordata powder (g).
[0036] The above method was used to determine the total alkaloid content in 5 Macleaya cordata samples, and HPLC was used to detect the contents of sanguinarine, chelerythrine, protopine, allocryptine and the total content of the four alkaloids.
[0037] Table 1 Results of determination of total alkaloid content in 5 Macleaya cordata samples by sanguinarine method and standard method
[0038]
[0039] It can be seen from the data in Table 1 that the detection method of Macleaya cordata alkaloids using sanguinarine as a reference proposed in the present invention can be used for the determination of Macleaya cordata alkaloids, and the detection effect is the same as that of the standard method.
[0040] Example 2
[0041] Dissolve the chelerythrine standard in ethanol-water (50:50) containing 0.1 mol / L sulfuric acid, transfer to a 50 mL volumetric flask, dilute to volume with purified water, and shake well to prepare a 200 μg / mL standard solution. Then accurately pipette 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of the standard solution into a 10 mL volumetric flask, and measure the absorbance at the maximum absorption wavelength of 272 nm after scanning the spectrum using the solvent as a reference. Draw an absorbance-concentration curve, as shown in the following figure: Figure 2 As shown, the regression equation y=0.102648x+0.000732(R 2 =0.999999), the limit of detection (LOD) was 15.05 ng / mL (calculated based on the standard deviation of the regression residuals), and the reproducibility and selectivity were good.
[0042] Accurately transfer 0.50 mL of Macleaya cordata extract to a 25 mL volumetric flask, add the corresponding extractant to the volume, measure the absorbance at 272 nm using the extractant as a reference, and repeat 3 times. The Macleaya cordata alkaloid extraction rate is calculated as follows:
[0043]
[0044] Wherein: ω1 is the extraction rate (%); c is the concentration of Macleaya cordata alkaloids in the extract (μg / mL); V is the final volume of the extract (mL); N is the dilution multiple; m is the mass of Macleaya cordata powder (g).
[0045] The above method was used to determine the total alkaloid content in 5 Macleaya cordata samples, and HPLC was used to detect the contents of sanguinarine, chelerythrine, protopine, allocryptine and the total content of the four alkaloids.
[0046] Table 2 Results of determination of total alkaloid content in 5 Macleaya cordata samples by chelerythrine and standard method
[0047]
[0048]
[0049] It can be seen from the data in Table 2 that the detection method of Macleaya cordata alkaloids using chelerythrine as a reference proposed in the present invention can be used for the determination of Macleaya cordata alkaloids, and the detection effect is the same as that of the standard method.
[0050] Example 3
[0051] Dissolve a 1:1 mixture of sanguinarine and chelerythrine in methanol-water (60:40) containing 0.15 mol / L sulfuric acid, transfer to a 50 mL volumetric flask, and dilute to volume with purified water to make a 200 μg / mL standard solution. Accurately pipette 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of the standard solution into a 10 mL volumetric flask, and measure the absorbance at the maximum absorption wavelength of 278 nm after scanning the spectrum using the solvent as a reference. Draw an absorbance-concentration curve, as shown in the following example. Figure 3 As shown, the regression equation y=0.11892x+0.001025(R 2 =0.999997), the limit of detection (LOD) was 14.6 ng / mL (calculated based on the standard deviation of the regression residuals), and the reproducibility and selectivity were good.
[0052] Accurately pipette 0.50 mL of the extract into a 25 mL volumetric flask, add the appropriate extractant to the volume, and measure the absorbance at 278 nm using the extractant as a reference. Repeat this three times. The extraction rate of Macleaya cordata alkaloids is calculated as follows:
[0053]
[0054] Wherein: ω1 is the extraction rate (%); c is the concentration of Macleaya cordata alkaloids in the extract (μg / mL); V is the final volume of the extract (mL); N is the dilution multiple; m is the mass of Macleaya cordata powder (g).
[0055] The above method was used to determine the total alkaloid content in 5 Macleaya cordata samples, and HPLC was used to detect the contents of sanguinarine, chelerythrine, protopine, allocryptine and the total content of the four alkaloids.
[0056] Table 3 Results of determination of total alkaloid content in 5 Macleaya samples by sanguinarine and chelerythrine method and standard method
[0057]
[0058] It can be seen from the data in Table 3 that the detection method of Macleaya cordata alkaloids proposed in the present invention using the sanguinarine method and the chelerythrine method as reference can be used for the determination of Macleaya cordata alkaloids, and the detection effect is the same as that of the standard method.
[0059] Example 4
[0060] Dissolve the mixture of sanguinarine, chelerythrine, and ortho-opioid in methanol-water (50:50) containing 0.15 mol / L sulfuric acid, transfer to a 50 mL volumetric flask, dilute to volume with purified water, and shake well to prepare a 200 μg / mL standard solution. Accurately pipette 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of the standard solution into a 10 mL volumetric flask, and measure the absorbance at the maximum absorption wavelength of 280 nm after scanning the spectrum with the solvent as a reference. Draw an absorbance-concentration curve, as shown in the following figure: Figure 4 As shown, the regression equation y=0.119065x+0.000939(R 2 =0.999996), the limit of detection (LOD) was 2.5 ng / mL (calculated based on the standard deviation of the regression residuals), and the reproducibility and selectivity were good.
[0061] Accurately pipette 0.50 mL of the extract into a 25 mL volumetric flask, add the appropriate extractant to the volume, and measure the absorbance at 280 nm using the extractant as a reference. Repeat this three times. The extraction rate of Macleaya cordata alkaloids is calculated as follows:
[0062]
[0063] Wherein: ω1 is the extraction rate (%); c is the concentration of Macleaya cordata alkaloids in the extract (μg / mL); V is the final volume of the extract (mL); N is the dilution multiple; m is the mass of Macleaya cordata powder (g).
[0064] The above method was used to determine the total alkaloid content in 5 Macleaya cordata samples, and HPLC was used to detect the contents of sanguinarine, chelerythrine, protopine, allocryptine and the total content of the four alkaloids.
[0065] Table 4 Results of determination of total alkaloid content in 5 Macleaya cordata samples by sanguinarine, chelerythrine and protopine methods and standard method
[0066]
[0067] From the data in Table 4, it can be seen that the detection method of Macleaya cordata alkaloids using sanguinarine, chelerythrine and protopine as references proposed in the present invention can be used for the determination of Macleaya cordata alkaloids, and the detection effect is the same as that of the standard method.
[0068] From the results of Examples 1 to 4, it can be seen that sanguinarine, chelerythrine, protopine, and allocryptine have the same absorbance at 265-285 nm. The total alkaloid content measured by using any one of the alkaloids as a reference can be regarded as the sum of the contents of the four alkaloids.
[0069] Although the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Any researcher in this field may, without departing from the spirit and scope of the present invention, adopt the design parameters and contents of the above-disclosed embodiments to change and modify the research scheme of the present invention. Therefore, any simple modifications, parameter changes, and modifications made to the above-disclosed embodiments based on the research essence of the present invention without departing from the content of the present invention are within the scope of protection of the present invention.
Claims
1. A method for detecting Macleaya cordata alkaloids, characterized in that: The method consists of three steps: extracting Macleaya cordata alkaloids, drawing a standard curve, and determining samples, and specifically includes the following steps: (1) Extraction of Macleaya cordata alkaloids: Accurately weigh a powder sample of Macleaya cordata fruit or Macleaya cordata leaf and place it in a container. Add a strong acid-alcohol solution for extraction, then heat it in a microwave reactor. After heating, take it out and let it cool to room temperature before filtering. The residue is washed three times with a strong acid-alcohol solution. The filtrate and the washing solution are transferred to a volumetric flask and fixed to volume with a strong acid-alcohol solution to obtain a Macleaya cordata extract. (2) Drawing of the standard curve: Dissolve the alkaloid standard in a strong acid-alcohol solution as the solvent, transfer to a volumetric flask, and then dilute to volume and shake well to prepare a 400 μg / mL standard stock solution. Accurately pipette 2.50 mL of the stock solution and transfer to a 25 mL volumetric flask. Then accurately pipette 0.50, 1.00, 2.00, 3.00, 4.00, and 5.00 mL of the diluted solution to a 25 mL volumetric flask, respectively. Scan the spectrum using the solvent as a reference and measure the absorbance at the maximum absorption wavelength of 265-285 nm to draw the absorbance-concentration curve. (3) Sample determination: Accurately pipette 0.50 mL of the Macleaya cordata extract from (1) into a 25 mL volumetric flask, add the corresponding extractant to the volume, and measure the absorbance at the maximum absorption wavelength using the extractant as a reference. Repeat 3 times. The Macleaya cordata alkaloid extraction rate is calculated as follows: Wherein: ω1 is the extraction rate (%); c is the concentration of Macleaya cordata alkaloids in the extract (μg / mL); V is the final volume of the extract (mL); N is the dilution multiple; m is the mass of Macleaya cordata powder (g).
2. The method for detecting Macleaya cordata alkaloids according to claim 1, wherein The mass ratio of the powder of Macleaya cordata fruit or Macleaya cordata leaf to the strong acid-alcohol solution is 1:30 to 1:
200.
3. The method for detecting Macleaya cordata alkaloids according to claim 1, wherein The heating temperature in the microwave reactor is 60° C. to 80° C., and the heating time is 5 to 15 minutes.
4. The method for detecting Macleaya cordata alkaloids according to claim 1, wherein The strong acid-alcohol solution described in (1) refers to one, two or three of sulfuric acid, hydrochloric acid and nitric acid, and the total acid concentration is 0.05 to 0.2 mol / L in terms of hydrogen ions.
5. The method for detecting Macleaya cordata alkaloids according to claim 1, wherein The strong acid-alcohol solution described in (1) refers to a solution obtained by mixing one or both of methanol and ethanol with water, with the ratio of alcohol to water being 8:2-2:
8.
6. The method for detecting Macleaya cordata alkaloids according to claim 1, wherein The alkaloid standard described in (2) is a mixture of one or more of sanguinarine, chelerythrine, protopine, and allocryptine.
7. The method for detecting Macleaya cordata alkaloids according to claim 1, wherein The maximum absorption wavelength mentioned in (3) refers to the wavelength of light used when measuring absorbance in the ultraviolet region in ultraviolet-visible spectrophotometry, which ranges from 265 to 285 nm.