A method for detecting artepillin c in brazilian propolis using a thin layer scanner

Abstract

The application discloses a method for detecting artepillin C in Brazilian propolis by using a thin layer scanner and belongs to the field of traditional Chinese medicine quality detection. The method specifically comprises the following steps: sucking the pretreated propolis sample solution and spotting the sample solution on a thin layer chromatography silica gel plate; adopting a mixed system of petroleum ether, ethyl acetate and formic acid with a volume ratio of (8-14):(3-6):(0.2-1) as a developing agent, flowing the developing agent through the fixed phase with the propolis sample solution, and drying to obtain a sample to be measured; scanning the chromatographic spots in the sample to be measured by using a thin layer scanner, testing the wavelength of 290-320 nm, the reference wavelength of 400-800 nm, and obtaining the absorbance integral value of the sample to be measured; and converting the absorbance integral value with an artepillin C standard curve to obtain the content of artepillin C in the sample to be measured. The method can simultaneously determine the content of artepillin C in multiple batches of propolis samples, the chromatographic peak of artepillin C is well separated and stable in scanning, and the method has the advantages of fast detection speed and low cost.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality testing, specifically relating to a method for detecting artepillin C in Brazilian propolis using a thin-layer scanner. Background Technology

[0002] Propolis is a sticky substance formed by worker bees collecting secretions such as collagen and plant resins, which are then mixed with secretions from their mandibular and wax glands. Propolis extract is a substance obtained from propolis raw materials through methods such as solvent extraction, critical fluid extraction, ultrasonic-assisted extraction, microwave-assisted extraction, and ultra-high pressure processing extraction.

[0003] Brazilian propolis primarily refers to propolis derived from plants of the genus *Baccharis*. Ateptalin C (3,5-diisopentenyl-4-hydroxycinnamic acid) is the main active phenolic acid in Brazilian propolis, with a much higher content than propolis from other regions. In China, the main plant source for propolis is *Populus* and its hybrids, with flavonoids as the main active ingredient. Propolis quality is judged based on flavonoid content. The 2020 edition of the *Chinese Pharmacopoeia* uses thin-layer chromatography (TLC) with toluene-ethyl acetate-glacial acetic acid (10:3:0.5) as the developing solvent to identify apigenin, galangin, and pinocembrin. The content determination section uses two high-performance liquid chromatography (HPLC) methods to determine the content of apigenin, galangin, and pinocembrin. However, the developing solvents used in these identification methods contain toluene, a precursor chemical for drug production, and experiments have shown that these methods are not suitable for identifying Brazilian propolis or determining the content of ateptalin C in Brazilian propolis.

[0004] The presence or absence of atepsilin C is a key indicator for determining whether a product is Brazilian propolis, and its content is a primary indicator of its quality. Because Brazilian propolis is several times more expensive than Chinese poplar-type propolis, there is a phenomenon in the market of Chinese poplar-type propolis being passed off as Brazilian propolis. Therefore, there is an urgent need for a method suitable for detecting atepsilin C in Brazilian propolis.

[0005] The industry standard uses high-performance liquid chromatography (HPLC) to determine the aspirin C content in propolis (GH / T1114-2015), but this method is time-consuming and cannot simultaneously analyze multiple batches. High-performance thin-layer chromatography (HPLC) can separate components using thin-layer chromatography and quantify them through scanning, allowing for simultaneous analysis of multiple samples. HPLC is currently used to determine caffeine content in tea, saponin content in Panax notoginseng powder, luteolin, apigenin, and farnesin content in Chrysanthemum morifolium, and astragalin content in Astragalus membranaceus. HPLC requires specific selection of the developing solvent and setting of scanning parameters for different components. Currently, there is no suitable thin-layer chromatography method for determining the content of aspirin C in Brazilian propolis, nor are there any reports of HPLC being applied to this method. Based on the identification experiments of thin-layer chromatography, HPLC eliminates the need for color development and, by following quantitative operation requirements, can obtain results comparable to HPLC. Therefore, there is an urgent need to develop a new method for determining the aspirin C content in Brazilian propolis, so as to provide technical support for the quality control of Brazilian propolis. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] This invention provides a method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner, the specific method being as follows:

[0009] Thin-layer chromatography was used. The pretreated propolis test solution was spotted onto a silica gel plate used as the stationary phase, so that the propolis test solution appeared as a strip. A mixture of petroleum ether, ethyl acetate and formic acid was used as the developing solvent. The developing solvent was allowed to flow through the stationary phase on which the propolis test solution was spotted. After development and separation of the components, the sample was dried to obtain the test sample.

[0010] A thin-layer scanner was used to scan the chromatographic spots in the sample to obtain the absorbance integral value of the sample. This value was then converted with the atepspirin C standard curve to obtain the content of atepspirin C in the sample.

[0011] Preferably, the volume ratio of petroleum ether, ethyl acetate and formic acid in the above developing agent is (8-14):(3-6):(0.2-1).

[0012] As a preferred option, the scanning parameters of the thin-layer chromatograph are set as follows: the test wavelength is 290-320 nm, and the reference wavelength is 400-800 nm.

[0013] Preferably, the above pretreatment process is as follows: dissolve the propolis test sample in methanol and sonicate it for 10 to 60 minutes to mix it evenly to obtain a mixture; the concentration range of the propolis test sample in the mixture is 5 to 100 mg / ml; filter the above mixture and take the filtrate as the propolis test sample solution.

[0014] Furthermore, the aforementioned propolis test sample was Brazilian propolis raw material or propolis extract.

[0015] Furthermore, the above mixture is filtered using qualitative filter paper with a particle size of 30–120 μm.

[0016] Preferably, the amount of the propolis test solution taken is 1-10 μl, and the sample is applied using an intelligent spotting instrument.

[0017] Preferably, the aforementioned thin-layer chromatography silica gel plate is of the high-efficiency G type.

[0018] As a preferred method, the above-mentioned atespirin C standard curve is obtained as follows:

[0019] Take atepspirin C standard and add methanol to prepare atepspirin C standard solution; the concentration range of the atepspirin C standard solution is 0.1-0.5 mg / ml; take 1-10 μl of atepspirin C standard solution and spot it on a thin-layer chromatography silica gel plate using an intelligent spotting instrument, so that the atepspirin C standard solution is in strip shape. Use the developing solvent to flow through the thin-layer chromatography silica gel plate spotted with atepspirin C standard solution, develop to achieve the purpose of separating the components, and then dry to obtain atepspirin C standard samples of different masses;

[0020] A thin-layer scanner was used to scan the chromatographic spots in atepspirin C standard samples of different masses to obtain the absorbance integral values ​​of atepspirin C standard samples of different masses; linear regression was performed with the mass of atepspirin C as the abscissa and the absorbance integral value as the ordinate to obtain the atepspirin C standard curve.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) This invention has optimized a method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner through extensive experiments. A mixed system of petroleum ether, ethyl acetate, and formic acid is selected as the developing solvent. The scanning parameters are set to a test wavelength of 290–320 nm and a reference wavelength of 400–800 nm, ensuring good separation and stable scanning of the atepspirin C peak in the thin-layer chromatography of the test sample. Compared with the existing propolis determination method included in the 2020 edition of the Chinese Pharmacopoeia, the developing solvent used in this invention does not contain toluene, a precursor chemical for drug production.

[0023] (2) The present invention can simultaneously separate and analyze multiple propolis samples on the same thin-layer chromatography silica gel plate, and has the advantages of simple operation, fast analysis speed, low solvent consumption and low analysis cost. Attached Figure Description

[0024] Figure 1 The scan absorption chromatograms are for the test sample and the reference sample. Detailed Implementation

[0025] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.

[0026] The following examples and comparative examples use the following reagents, experimental instruments, and specifications:

[0027] 0.0001g electronic balance (XS105DU, Mettler); 0.0001g electronic balance (ME204, Mettler); CNC ultrasonic cleaner (KQ-500, Kunshan Ultrasonic Instrument Co., Ltd.); SP-20Start intelligent spotting instrument (Shanghai Kezhe Biochemical Technology Co., Ltd.); KH-3100 thin-layer chromatography scanner (Shanghai Kezhe Biochemical Technology Co., Ltd.).

[0028] Atespirin C was purchased from Shanghai Yuanye Biotechnology Co., Ltd., batch number: A26GB159041, purity: 97.00%.

[0029] Example 1

[0030] This embodiment provides a method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner. The specific steps are as follows:

[0031] (1) Preparation of the test solution

[0032] Take 0.15g of Brazilian propolis extract powder, add 25ml of methanol, sonicate for 30 minutes, filter, and use the filtrate as the propolis test solution.

[0033] (2) Preparation of Atespirin C standard solution

[0034] Take atepspirin C standard, add methanol, mix well to prepare an atepspirin C standard solution containing 0.188 mg per ml. The concentration of atepspirin C is 0.188 g / L.

[0035] (3) Development of thin-layer chromatography of the test sample

[0036] Thin-layer chromatography (refer to Chinese Pharmacopoeia 2020, Part IV, General Chapter 0502) was used. 4 μl of the propolis test solution prepared in step (1) was precisely pipetted onto a high-performance G thin-layer chromatography silica gel plate using an intelligent spotting instrument, so that the propolis test solution appeared as a strip.

[0037] A mixture of petroleum ether, ethyl acetate, and formic acid was used as the developing solvent. The developing solvent flowed through a stationary phase containing the propolis sample solution. After development and separation of the components, the sample was dried to obtain the test sample. The volume ratio of petroleum ether:ethyl acetate:formic acid in the developing solvent was 12:4:0.5.

[0038] Set the thin-layer chromatograph scanning parameters as follows: test wavelength 310 nm, reference wavelength 500 nm, set the number of sample groups to be scanned and the location of the sample spots, run the scan, and measure the absorbance integral value of the sample to be tested.

[0039] (4) Obtaining the C standard curve of atepspirin

[0040] Accurately pipette 2, 4, 6, 8, and 10 μl of the above-mentioned atespirin C standard solution and spot the different volumes of atespirin C standard solution onto the same high-performance G thin-layer chromatography silica gel plate using an intelligent spotter, forming strips. Perform thin-layer chromatography development and scanning according to the prescribed method. Plot the mass of the atespirin C standard solution on the x-axis and the absorbance integral value on the y-axis to perform linear regression, obtaining the standard curve: y = 31825x + 1024.7, R0. 2 =0.9935, with good linearity in the range of 0.376 to 1.880 μg.

[0041] (5) Conversion of Atespirin C content in propolis test sample

[0042] The absorbance integral value of the sample obtained in step (3) is converted according to the atepspirin C standard curve obtained in step (4) to obtain the content of atepspirin C in the sample. The thin-layer chromatograms of the test sample and the reference sample are shown below. Figure 1 As shown.

[0043] In this embodiment, the content of artepillin C in the five batches of Brazilian propolis extracts was determined to be 4.79%, 4.49%, 3.71%, 5.63%, and 6.42%, respectively.

[0044] To examine the precision, repeatability, stability, and recovery of the thin-layer scanner method for detecting atepsilin C in Brazilian propolis provided in this embodiment, the specific operation is as follows:

[0045] (1) Precision: 4 μl of atepspirin C standard solution was precisely pipetted onto the same high-performance G thin-layer chromatography silica gel plate using an intelligent spotter, forming a strip. The spotting was repeated 6 times, and the determination was performed according to the protocol. The RSD of the Rf value of atepspirin C was 0.27%, and the RSD of the absorbance integral value was 1.16%, indicating that the method has good precision.

[0046] (2) Repeatability: The same batch of propolis extract samples were tested 6 times according to the protocol. The RSD of the Rf value of atepsilin C in propolis extract was 2.72%, and the RSD of the absorbance integral value was 3.11%, indicating that the method has good repeatability.

[0047] (3) Stability: The propolis extract test solution was accurately pipetted and developed according to the protocol, with thin-layer scanning performed at 0, 30, 60, 90, 120, and 150 min. The RSD value of the integral of the absorbance of aspirin C in the propolis extract was 1.86%, indicating that the method has good stability.

[0048] (4) Spiking recovery: Accurately weigh 0.075 g of propolis extract, accurately add an equal amount of atepspirin C standard to the test sample, prepare according to the protocol, perform 6 parallel determinations, and calculate the recovery rate (recovery rate = (measured amount - amount of analyte in the sample) / amount of added standard × 100%). The results showed that the average recovery rate of atepspirin C was 102.12%, and the RSD value was 4.51%, indicating that the method had good accuracy.

[0049] Example 2

[0050] This embodiment provides a method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner. The specific steps are as follows:

[0051] (1) Preparation of the test solution

[0052] Take 0.05g of Brazilian propolis extract powder, add 50ml of methanol, sonicate for 10 minutes, filter, and use the filtrate as the propolis test solution.

[0053] (2) Preparation of Atespirin C standard solution

[0054] Take atepspirin C standard, add methanol, mix well to prepare a standard solution containing 0.103 mg of atepspirin C per ml. The concentration of atepspirin C is 0.103 g / L.

[0055] (3) Development of thin-layer chromatography of the test sample

[0056] Thin-layer chromatography (refer to Chinese Pharmacopoeia 2020, Part IV, General Chapter 0502) was used. 10 μl of the propolis test solution prepared in step (1) was precisely pipetted onto a high-performance G thin-layer chromatography silica gel plate using an intelligent spotting instrument, so that the propolis test solution appeared as a strip.

[0057] A mixture of petroleum ether, ethyl acetate, and formic acid was used as the developing solvent. The developing solvent flowed through a stationary phase containing the propolis sample solution. After development to achieve separation of the components, the sample was dried to obtain the test sample. The volume ratio of petroleum ether:ethyl acetate:formic acid in the developing solvent was 8:3:1.

[0058] Set the thin-layer chromatograph scanning parameters as follows: test wavelength 320nm, reference wavelength 800nm, set the number of sample groups to be scanned and the location of the sample spots, run the scan, and measure the absorbance integral value of the sample to be tested.

[0059] (4) Obtaining the C standard curve of atepspirin

[0060] Accurately pipette 1, 3, 6, and 9 μl of the above-mentioned atespirin C standard solution and spot the different volumes of atespirin C standard solution onto the same high-performance G thin-layer chromatography silica gel plate using an intelligent spotter, forming strips. Perform thin-layer chromatography development and scanning according to the prescribed method. Plot the mass of atespirin C standard solution on the x-axis and the absorbance integral value on the y-axis to perform linear regression, obtaining the standard curve: y = 32154x + 342.65, R0. 2 =0.9955, with good linearity in the range of 0.103 to 0.927 μg.

[0061] (5) Conversion of Atespirin C content in propolis test sample

[0062] The absorbance integral value of the sample obtained in step (3) is converted according to the atepspirin C standard curve obtained in step (4) to obtain the content of atepspirin C in the sample.

[0063] In this example, the content of artepillin C in the five batches of Brazilian propolis extracts was determined to be 4.88%, 4.42%, 3.87%, 5.51%, and 6.69%, respectively.

[0064] Example 3

[0065] This embodiment provides a method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner. The specific steps are as follows:

[0066] (1) Preparation of the test solution

[0067] Take 1g of Brazilian propolis raw material powder, add 10ml of methanol, sonicate for 60 minutes, filter, and take the filtrate as the propolis test solution.

[0068] (2) Preparation of Atespirin C standard solution

[0069] Take atepspirin C standard, add methanol, mix well to prepare an atepspirin C standard solution containing 0.485 mg per ml. The concentration of atepspirin C is 0.485 g / L.

[0070] (3) Development of thin-layer chromatography of the test sample

[0071] Thin-layer chromatography (refer to Chinese Pharmacopoeia 2020, Part IV, General Chapter 0502) was used. 1 μl of the propolis test solution prepared in step (1) was precisely pipetted onto a high-performance G thin-layer chromatography silica gel plate using an intelligent spotting instrument, so that the propolis test solution appeared as a strip.

[0072] A mixture of petroleum ether, ethyl acetate, and formic acid was used as the developing solvent. The developing solvent flowed through a stationary phase containing the propolis sample solution. After development to achieve separation of the components, the sample was dried to obtain the test sample. The volume ratio of petroleum ether:ethyl acetate:formic acid in the developing solvent was 14:6:0.2.

[0073] Set the thin-layer chromatography scanning parameters to: test wavelength 290 nm, reference wavelength 400 nm, set the number of sample groups to be scanned and the location of the sample spots, run the scan, and measure the absorbance integral value of the sample to be tested.

[0074] (4) Obtaining the C standard curve of atepspirin

[0075] Accurately pipette 1, 2, 3, 4, and 5 μl of the above-mentioned atespirin C standard solution and spot the different volumes of atespirin C standard solution onto the same high-performance G thin-layer chromatography silica gel plate using an intelligent spotter, forming strips. Perform thin-layer chromatography development and scanning according to the procedure. Plot the mass of atespirin C standard solution on the x-axis and the absorbance integral value on the y-axis to perform linear regression, obtaining the standard curve: y = 30722x + 937.5, R0. 2 =0.9978, with good linearity in the range of 0.485 to 2.425 μg.

[0076] (5) Conversion of Atespirin C content in propolis test sample

[0077] The absorbance integral value of the sample obtained in step (3) is converted according to the atepspirin C standard curve obtained in step (4) to obtain the content of atepspirin C in the sample.

[0078] In this example, the content of artepillin C in the three batches of Brazilian propolis raw materials was determined to be 2.37%, 1.94%, and 2.76%, respectively.

[0079] Comparative Example 1

[0080] Compared to Example 1, this comparative example provides a method for determining the content of atepspirin C in Brazilian propolis using high performance liquid chromatography (HPLC), referring to "GH / T 1114-2015 Determination of Atepspirin C in Propolis by High Performance Liquid Chromatography" as follows:

[0081] (1) Preparation of Atespirin C standard solution

[0082] Accurately weigh an appropriate amount of atepspirin C standard and add methanol to prepare a standard solution containing 0.2 mg atepspirin C per ml.

[0083] (2) Preparation of the test solution

[0084] Accurately weigh 0.15g of Brazilian propolis extract powder and place it in a 25ml volumetric flask. Add 20ml of methanol, sonicate for 30 minutes, cool, and then add methanol to the mark to make up to volume. Filter and take the filtrate as the propolis test solution.

[0085] (3) Determination by high performance liquid chromatography

[0086] Accurately pipette 5 μl each of the atepspirin C standard solution and the propolis test solution into the liquid chromatograph. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel (4.6 mm × 250 mm, 5 μm); 1% acetic acid solution as mobile phase A and methanol as mobile phase B, with gradient elution: 0–30 min, 25–55% B; 30–60 min, 55–80% B; 60–70 min, 80–95% B; 70–80 min, 95–25% B. The detection wavelength is 310 nm, the column temperature is 30 °C, and the flow rate is 1 mL / min.

[0087] The results of determining the atepspirin C content in five batches of Brazilian propolis extract using high performance liquid chromatography in this comparative example are shown in Table 1. The data obtained using the high performance thin-layer chromatography method are the same as those obtained in Example 1.

[0088] Table 1 Comparison of the results of atepspirin C content determination in Comparative Example 1 and Example 1

[0089]

[0090] As shown in Table 1, the results obtained by high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (TLC) are largely consistent, with relative errors between the two methods ranging from -2.46% to 8.93%. It is evident that the TLC method provided in Example 1 yields more accurate results and is faster and more solvent-efficient than the HPLC method used in Comparative Example 1.

[0091] This invention provides a method for detecting artepillin C in Brazilian propolis using a high-efficiency thin-layer scanner. This method can simultaneously and accurately detect the content of artepillin C in multiple batches of propolis raw materials or propolis extracts. It features fast analysis speed, high precision, repeatability, stability, and accuracy, and is of great significance for objectively and accurately evaluating the quality of Brazilian propolis.

[0092] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.

Claims

1. A method for detecting atepspirin C in Brazilian propolis using a thin-layer scanner, characterized in that, The specific method is as follows: Thin-layer chromatography was used. The pretreated propolis test solution was spotted onto a silica gel plate used as the stationary phase, so that the propolis test solution appeared as a strip. A mixture of petroleum ether, ethyl acetate and formic acid was used as the developing solvent. The developing solvent was allowed to flow through the stationary phase on which the propolis test solution was spotted. After development and separation of the components, the sample was dried to obtain the test sample. The chromatographic spots in the sample to be tested were scanned using a thin-layer scanner to obtain the absorbance integral value of the sample to be tested. The absorbance integral value of the sample was then converted with the atepspirin C standard curve to obtain the content of atepspirin C in the sample to be tested. The volume ratio of petroleum ether, ethyl acetate, and formic acid in the developing solvent is (8~14):(3~6):(0.2~1). The pretreatment process is as follows: dissolve the propolis test sample in methanol and sonicate it for 10-60 minutes to mix it evenly to obtain a mixture; the concentration of the propolis test sample in the mixture is in the range of 5-100 mg / ml; filter the above mixture and take the filtrate as the propolis test sample solution.

2. The method according to claim 1, characterized in that, The thin-layer chromatograph scanning parameters are set as follows: the test wavelength is 290~320nm, and the reference wavelength is 400~800nm.

3. The method according to claim 1, characterized in that, The propolis sample was Brazilian propolis raw material or propolis extract.

4. The method according to claim 1, characterized in that, The mixture is filtered using qualitative filter paper with a particle size of 30~120μm.

5. The method according to claim 1, characterized in that, The amount of propolis test solution taken is 1~10μl, and the sample is applied using an intelligent spotting instrument.

6. The method according to claim 1, characterized in that, The thin-layer chromatography silica gel plate is of the high-efficiency G type.

7. The method according to claim 1, characterized in that, The method for obtaining the atespirin C standard curve is as follows: Take atepspirin C standard and add methanol to prepare atepspirin C standard solution; the concentration range of the atepspirin C standard solution is 0.1~0.5mg / ml; take 1~10μl of atepspirin C standard solution and spot it on a thin-layer chromatography silica gel plate using an intelligent spotting instrument, so that the atepspirin C standard solution is in strip shape. Use the developing solvent to flow through the thin-layer chromatography silica gel plate spotted with atepspirin C standard solution, develop to achieve the purpose of separating each component, and then dry to obtain atepspirin C standard samples of different masses; The chromatographic spots in atepspirin C standard samples of different masses were scanned using a thin-layer scanner to obtain the absorbance integral values ​​of atepspirin C standard samples of different masses. The standard curve of atepspirin C was obtained by performing linear regression with the mass of atepspirin C as the x-axis and the absorbance integral as the y-axis.

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