Method for detecting specific chromatogram of pine pollen

Abstract

The invention belongs to the field of analysis and detection, and particularly relates to a method for detecting a characteristic spectrum of pine pollen. The method comprises the following steps: preparing a pine pollen test solution by adopting a methanol solution, carrying out gradient elution by using a C18 chromatographic column and an acetonitrile-phosphoric acid aqueous solution system, and respectively determining the characteristic chromatograms of masson pine, pinus yunnanensis, pinus armandii, pinus tabulaeformis, pinus comatus and pinus bungeana. The method for detecting the characteristic spectrum of the pine pollen is determined by screening and optimizing extraction conditions and chromatographic column conditions, and a scientific detection method is provided for quality control of the pine pollen.

Description

Technical Field

[0001] The present invention belongs to the field of analytical detection, and more specifically, the present invention relates to a method for detecting the characteristic fingerprint of pine pollen. Background Art

[0002] Pine pollen, also known as pine yellow, is the dried pollen of Pinus massoniana Lamb., Pinus tabuliformis Carr. or several species of the same genus in the pine family of China. It is one of the two kinds of pollen recorded in ancient Chinese medical classics and is the only pollen variety that can be used as both food and medicine in the treasure house of traditional Chinese medicine. It has the effects of drying dampness and arresting bleeding.

[0003] The main Pinus resources plants from which pine pollen can be harvested in China are: Pinus tabuliformis Carr., Pinus massoniana Lamb., Pinus yunnanensis Franch., Pinus densiflora Sieb. et Zucc., Pinus thunbergii Parl.

[0004] The nutritional components of pine pollen mainly include proteins, amino acids, fats, carbohydrates, organic acids, nucleic acids, vitamins, mineral elements, unsaturated fatty acids, flavonoids, cellulose, polysaccharides, hormones, choline, enzymes and coenzymes, etc., with more than 200 kinds of component types. Pine pollen is a natural nutrient reservoir, containing a variety of nutritional components. It contains 20 kinds of amino acids, including 8 kinds of essential amino acids for the human body, which can provide the basic substances for protein synthesis in the human body; it contains 14 kinds of vitamins, such as vitamin A, B vitamins, vitamin C, vitamin D, vitamin E, etc., and these vitamins play important roles in the growth and development, metabolism, immune regulation, etc. of the human body; it also contains 30 kinds of minerals, such as calcium, iron, zinc, magnesium, phosphorus, etc., which are of great significance for maintaining the normal physiological functions of the human body.

[0005] In recent years, pine pollen has been widely used in the fields of health products, cosmetics and additives, etc. due to its effects of enhancing immunity, anti-aging, anti-fatigue, beauty, etc. Pine pollen is rich in a variety of nutrients essential for the human body and is known as a "miniature nutrient reservoir". Pine pollen is a traditional pollen variety that can be used as both food and medicine in China, with rich nutritional value and biological activity, and has broad prospects both in clinical applications and health product development.

[0006] Traditionally, the quality judgment of pine pollen mainly relies on simple characteristics such as appearance, color, smell, etc. However, it is difficult to accurately identify the authenticity and quality of pine pollen only through these methods, because some substances with similar appearances may be incorporated or confused. For example, pollen typhae, corn flour, etc. are extremely similar to pine pollen in appearance and color, and it is impossible to effectively distinguish them only by visual inspection.

[0007] In the 2011 edition of the Chinese Pharmacopoeia, pine pollen quality is controlled through microscopic identification, moisture content, and total ash content. However, there is a lack of qualitative and quantitative indicators to evaluate pine pollen quality, making it difficult to fully and accurately reflect the intrinsic quality and effective components of pine pollen, making it difficult to meet the needs of modern pine pollen quality control and research. Therefore, the development of more scientific and accurate pine pollen detection methods has become necessary. Furthermore, because pine pollen obtained from different origins and varieties can have subtle differences in efficacy and quality, existing detection methods generate fingerprints of pine pollen medicinal samples with few characteristic peaks, resulting in relatively rough test results and poor specificity. These methods are unable to effectively and accurately identify the differences between pine pollen from different sources, and consequently, are unable to accurately monitor pine pollen quality. Summary of the Invention

[0008] In order to overcome the deficiencies of the prior art, the present invention determines a method for detecting pine pollen characteristic patterns through condition screening and optimization, thereby providing a scientific detection method for pine pollen quality control.

[0009] In a first aspect, the present invention provides a method for detecting a characteristic spectrum of pine pollen, characterized in that the method comprises: (1) Preparation of pine pollen test solution Take 1-3g of pine pollen, accurately weigh it, place it in a stoppered conical flask, accurately add 25%-75% methanol or 100% methanol 25ml, ultrasonically treat for 30 minutes or heat under reflux for 40 minutes, remove it, cool, shake it well, centrifuge it at 12000rpm for 5 minutes, filter it, and take the filtrate to obtain the test solution; (2) Column conditions A C18 column was used with acetonitrile as mobile phase A and aqueous phosphoric acid as mobile phase B for gradient elution; the flow rate was 0.5 ml / min-1.5 ml / min; the column temperature was 25°C-35°C; the detection wavelength was 210 nm, and the injection volume was 0.5-5 μl; (3) Establishment of feature maps The characteristic spectra of Pinus massoniana, Pinus yunnanensis, Pinus armandii, Pinus tabulaeformis, mixed pine and Pinus bungeana were determined respectively.

[0010] In some embodiments, the C18 chromatographic column is selected from Agilent Extend-C18 RRHD, Agilent Poroshell 120 AQ-C18, Agilent Poroshell 120 EC-C18, Agilent ZORBAX RRHD Extend–C18, Agilent ZORBAX RRHD SB–C18, and Dikma Navigatorsil C18.

[0011] In some embodiments, the concentration of the phosphoric acid aqueous solution is 0.02% - 0.15%.

[0012] In some embodiments, the concentration of the phosphoric acid aqueous solution is 0.05%.

[0013] In some embodiments, the column temperature is 30 °C.

[0014] In some embodiments, the flow rate is 0.5 ml / min or 1 ml / min.

[0015] In some embodiments, the C18 chromatographic column is Agilent ZORBAX RRHD Extend-C18 (3.0×100 mm, 1.8 μm).

[0016] In some embodiments, the concentration of methanol in step (1) is 50%.

[0017] In some embodiments, the ultrasonic treatment conditions in step (1) are a power of 300 W and a frequency of 40 kHz.

[0018] In some embodiments, 2 g of pine pollen is taken in step (1).

[0019] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. Through condition optimization, the present invention obtains the parameter conditions for detecting the general characteristic chromatogram of pine pollen, with good repeatability, and effectively controls the quality of pine pollen. 2. The method of the present invention is simple and reliable, and the reliability of the detection method is verified by comparing the results of multiple batches.

[0020] 3. The method of the present invention can distinguish pine pollen from different origins. Among them, Pinus bungeana has more obvious chromatographic peaks at 12 - 20 minutes than other origins; Pinus armandii lacks peaks 1', 3', and 8', and the peak areas of each chromatographic peak are also smaller, especially the difference in the peak area of peak 23' is the most obvious; Pinus massoniana and mixed pine have two obvious chromatographic peaks after peak 26', and the peak areas of peaks 14' - 22' in the chromatogram of Pinus tabuliformis are significantly larger than those of Pinus yunnanensis. Description of the Drawings [[ID=३५]]

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments.

[0022] Figure 1 Chromatogram of pine pollen sample (extracted with 50% methanol) Figure 2 Chromatogram of pine pollen sample (extracted with methanol) Figure 3Chromatogram of pine pollen sample (Condition 2) Figure 4 Chromatogram of pine pollen sample (Condition 3) Figure 5 Chromatogram of pine pollen sample (Condition 4) Figure 6 Chromatogram of pine pollen sample (Condition 5) Figure 7 Chromatogram of pine pollen sample (Condition 6) Figure 8 Chromatogram of pine pollen sample (Condition 7) Figure 9 Chromatogram of pine pollen sample (Condition 8) Figure 10 Chromatogram of pine pollen sample (Condition 9) Figure 11 Chromatogram of pine pollen sample (Condition 10) Figure 12 Chromatogram of pine pollen sample (Condition 11) Figure 13 Chromatogram of pine pollen sample (Condition 12) Figure 14 Chromatogram of pine pollen sample (Condition 13) Figure 15 Chromatogram of pine pollen sample (Condition 14) Figure 16 Chromatogram at a wavelength of 210 nm Figure 17 Chromatogram at a wavelength of 220 nm Figure 18 Chromatogram at a wavelength of 240 nm Figure 19 Chromatogram at a wavelength of 254 nm Figure 20 Chromatogram at a wavelength of 280 nm Figure 21 Chromatogram at a wavelength of 320 nm Figure 22 Chromatogram of the test sample under the conditions of Agilent Extend–C18 chromatographic column Figure 23 Chromatogram of the test sample under the conditions of Agilent ZORBAX SB–C18 RRHD chromatographic column Figure 24 Chromatogram of the test sample under the conditions of Dikma Navigatorsil C18 chromatographic column Figure 25 Chromatogram for the investigation of water-acetonitrile system Figure 26 Chromatogram for the investigation of 0.1% phosphoric acid aqueous solution-acetonitrile system Figure 27 Chromatogram obtained by examining with 0.02% phosphoric acid - acetonitrile system Figure 28 Chromatogram obtained by examining with 0.05% phosphoric acid - acetonitrile system Figure 29 Chromatogram obtained by examining with 0.1% phosphoric acid - acetonitrile system Figure 30 Chromatogram obtained by examining with 0.15% phosphoric acid - acetonitrile system Figure 31 Chromatogram at column temperature of 25°C Figure 32 Chromatogram at column temperature of 30°C Figure 33 Chromatogram at column temperature of 35°C Figure 34 Chromatogram at flow rate of 0.5 ml / min Figure 35 Chromatogram at flow rate of 0.6 ml / min Figure 36 Chromatogram at flow rate of 0.7 ml / min Figure 37 Chromatogram obtained by examining gradient 1 Figure 38 Chromatogram obtained by examining gradient 2 Figure 39 Chromatogram obtained by examining gradient 3 Figure 40 Chromatogram using water as extraction solvent Figure 41 Chromatogram using 25% methanol as extraction solvent Figure 42 Chromatogram using 50% methanol as extraction solvent Figure 43 Chromatogram using 75% methanol as extraction solvent Figure 44 Chromatogram using methanol as extraction solvent Figure 45 Chromatogram using ultrasonic wave as extraction method Figure 46 Chromatogram using reflux as extraction method Figure 47 Chromatogram obtained by extraction with reflux for 20 min Figure 48 Chromatogram obtained by extraction with reflux for 30 min Figure 49 Chromatogram obtained by extraction with reflux for 40 min Figure 50 Chromatogram obtained by extraction with reflux for 60 min Figure 51 Chromatogram obtained by extraction with sampling amount of 1 g Figure 52 Chromatogram of extraction with 2g sample Figure 53 Chromatogram of extraction with 3g sample Figure 54 Comparison of characteristic spectra of pine pollen from different origins Figure 55 Overlay of characteristic spectra of pine pollen (44 batches) Figure 56 Overlay of characteristic spectra of mixed pine pollen (17 batches) Figure 57 Overlay of characteristic spectra of Pinus tabulaeformis pollen (14 batches) Figure 58 Overlay of characteristic spectra of Masson pine pollen (8 batches) Figure 59 Overlay of characteristic spectra of Yunnan pine pollen (5 batches) Figure 60 Pine Pollen (44 batches) comparison chart Figure 61 Comparative experimental diagrams of the method of the present invention and the prior art method (A: present invention; B: prior art) DETAILED DESCRIPTION

[0023] 1. Instruments and reagents 1.1 Instruments and Equipment Table 1 Instrument and equipment statistics 1.2 Reagents and test drugs Table 2 Reagent and drug statistics Example 1 Investigation of chromatographic conditions Pine pollen contains complex components that are difficult to separate using isocratic elution, so gradient elution was used. Based on previous research and literature reports on fingerprint chromatograms, the test solution was initially prepared according to the following method, and a preliminary experiment was conducted under the following chromatographic conditions.

[0024] Preparation of the test solution: Take two portions of Yunnan pine powder (DYC220601), 2 g each, accurately weighed, placed in a stoppered conical flask, accurately added 25 ml of 50% methanol or 100% methanol, respectively, and ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes. Remove, cool, shake well, centrifuge at 12000 rpm for 5 minutes, filter, and take the filtrate to obtain the product.

[0025] Preliminary study of chromatographic conditions Condition 1: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent Poroshell 120 AQ-C18 (4.6×150mm, 2.7μm) as the chromatographic column and perform gradient elution according to the regulations in the following table; the flow rate is 1 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 1-2 .

[0026] It can be seen from the results that there are more chromatographic peaks in the low-polarity part of the chromatogram obtained with methanol as the extraction solvent. Therefore, methanol extraction solution is used for method development in the follow-up.

[0027] Condition 2: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent Poroshell 120 AQ-C18 (4.6×150mm, 2.7μm) as the chromatographic column and perform gradient elution according to the regulations in the following table; the flow rate is 1 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results Figure 3 .

[0028] It can be seen from the results that the chromatographic peaks with retention times between 35 and 55 minutes are dense, and the mobile phase gradient needs to be adjusted.

[0029] Condition 3: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent Poroshell 120 AQ-C18 (4.6×150mm, 2.7μm) as the chromatographic column and perform gradient elution according to the regulations in the following table; the flow rate is 1 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 4 .

[0030] It can be seen from the results that the baseline is uneven at about 25 minutes of retention time, and the chromatographic peaks between 42 and 55 minutes are not well separated. Continue to adjust the mobile phase gradient.

[0031] Condition 4: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent Poroshell 120 AQ-C18 (4.6×150mm, 2.7μm) as the chromatographic column, and perform gradient elution according to the regulations in the following table; the flow rate is 1 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 5 .

[0032] It can be seen from the results that the separation effect of some chromatographic peaks is not good, and try to replace the chromatographic column.

[0033] Condition 5: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, and perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 6 .

[0034] It can be seen from the above results that after reducing the column temperature and replacing the chromatographic column with a smaller particle size, the resolution has been improved, and continue to optimize the mobile phase gradient.

[0035] Condition 6: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, and perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 7 .

[0036] It can be seen from the results that there are many chromatographic peaks with retention times between 16 and 20 minutes and the separation is poor. Continue to adjust the mobile phase gradient.

[0037] Condition 7: Use the instrument Agilent 1290, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B. Use Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, and perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown inFigure 8 。

[0038] As can be seen from the results, the separation effect of some chromatographic peaks has been improved, but the mobile phase gradient still needs to be further adjusted.

[0039] Condition 8: Using Agilent 1290 instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 9 。

[0040] As can be seen from the results, the separation effect of some chromatographic peaks has been improved, but the mobile phase gradient still needs to be further adjusted.

[0041] Condition 9: Using Agilent 1290 instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 10 。

[0042] From the above results, it can be seen that the separation of chromatographic peaks with retention times between 33 - 36 minutes is poor, so the mobile phase gradient is further adjusted.

[0043] Condition 10: Using Agilent 1290 instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 13 。

[0044] As can be seen from the results, good separation of chromatographic peaks can be achieved at present. However, due to the overly complex elution gradient, it needs to be simplified. In order to expand the applicability of this chromatographic condition and optimize the separation effect in the middle section, the test solution used was changed from Pinus yunnanensis to Pinus tabuliformis, and subsequently, the final chromatographic condition will be used to confirm the applicability of Pinus yunnanensis.

[0045] Preparation of test solution: Weigh accurately 2 g of Pinus tabuliformis powder (DYC220634), place it in a stoppered conical flask, accurately add 25 ml of methanol, sonicate (power 300 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, shake well, centrifuge at 12000 rpm for 5 min, filter, and take the subsequent filtrate, that is, obtain the test solution.

[0046] Condition 11: Using Agilent 1290 instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent Extend-C18 RRHD (3.0×100 mm, 1.8 μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30 °C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 12 .

[0047] As can be seen from the above results, the impact on chromatographic peaks after simplification is relatively small, so the mobile phase gradient is continued to be adjusted.

[0048] Condition 12: Using Agilent 1290 instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100 mm, 1.8 μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30 °C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 13 .

[0049] Condition 13: Using Agilent 1290 instrument, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100 mm, 1.8 μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30 °C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 14 .

[0050] It can be seen from the results that the separation effect of some chromatographic peaks has been improved, but the mobile phase gradient still needs to be adjusted continuously.

[0051] Condition 14: Using Agilent 1290 instrument, acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100mm, 1.8μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 1 μl. The results are shown in Figure 15 .

[0052] It can be seen from the results that the current separation effect is good. In order to amplify the response value of the chromatographic peak, the injection volume is increased to 2 μl, and this method is temporarily used as the initial condition for subsequent system optimization.

[0053] Example 2 Determination of the detection wavelength Take the test solution of pine pollen (Pinus yunnanensis DYC220601), inject the test solution into the liquid chromatograph according to the chromatographic conditions of Condition 16 above, perform a full wavelength scan from 210 to 400 nm, and compare the chromatograms at 3 different absorption wavelengths (200 nm, 210 nm (reference wavelength), 220 nm, 240 nm, 254 nm, 280 nm, 320 nm) according to the amount of information in the chromatogram. The results are shown in Appendix Figure 16-21 . Use the number of detected chromatographic peaks, the absorption intensity corresponding to the retention time, and the baseline stability as the evaluation indicators.

[0054] It can be seen from the results that the chromatographic information is rich at 200 - 220 nm, but compared with 210 nm at 220 nm, there is obviously one less chromatographic peak at 40 minutes of retention time; compared with 200 nm at 210 nm, the baseline of the former is more stable. Therefore, 210 nm is used as the detection wavelength for the characteristic chromatogram of pine pollen.

[0055] To sum up, the preliminary chromatographic conditions for the determination of the content of pine pollen are as follows: acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, Agilent Extend-C18 RRHD (3.0×100mm, 1.8μm) as the chromatographic column, perform gradient elution according to the regulations in the following table; the flow rate is 0.6 ml per minute; the column temperature is 30°C; the detection wavelength is 210 nm, and the injection volume is 2 μl.

[0056] Example 3 Optimization of chromatographic conditions 3.1 Investigation of different chromatographic columns Under the above chromatographic conditions, the test solution of pine pollen (Pinus yunnanensis DYC220601) was injected and analyzed using chromatographic columns of three models (Agilent ZORBAX RRHD Extend–C18 1.8μm, 3.0×100mm; Agilent ZORBAX RRHD SB–C18 1.8μm, 3.0×100mm; Dikma Navigatorsil C18 2.7μm, 3.0×100mm), and the chromatograms were recorded. The specific results are shown in the following table and appendix Figure 22-24 .

[0057] Table 3 System suitability parameters of chromatographic peaks under different chromatographic column conditions The results showed that there were co-eluting chromatographic peaks in the chromatograms obtained with the Dikma Navigatorsil C18 and gilent ZORBAX SB–C18 RRHD chromatographic columns, while the peak shape and resolution of the chromatogram obtained with the Agilent Extend–C18 RRHD chromatographic column were better. Therefore, the Agilent Extend–C18 RRHD chromatographic column was used for the subsequent method research.

[0058] 3.2 Investigation on whether to add phosphoric acid to the mobile phase According to the preliminary chromatographic conditions determined above, the effects of different mobile phase additives (without additive, phosphoric acid) on the separation effect of the characteristic chromatogram of pine pollen were investigated. The results are shown in Figure 25-26 .

[0059] It can be seen from the results that the chromatographic information is rich after adding 0.1% phosphoric acid to the mobile phase. Therefore, phosphoric acid was selected as the additive for subsequent condition screening.

[0060] 3.3 Investigation on different phosphoric acid concentrations in the mobile phase According to the preliminary chromatographic conditions determined above, the effects of different phosphoric acid concentrations (0.02%, 0.05%, 0.10%, 0.15%) in the mobile phase on the separation effect of the characteristic chromatogram of pine pollen were investigated. The results are shown in the following table and appendix Figure 27-30 .

[0061] Table 4 System suitability parameters of chromatographic peaks under different phosphoric acid concentrations It can be seen from the results that different phosphoric acid concentrations have a relatively obvious effect on the separation of each chromatographic peak. Among them, the separation effect is the best when the phosphoric acid concentration is 0.05%. Based on this, subsequent condition screening and investigation were carried out.

[0062] 3.4 Investigation on different column temperatures According to the preliminary chromatographic conditions determined above, the effects of different column temperatures (25°C, 30°C, 35°C) on the separation effect of the characteristic chromatogram of pine pollen were investigated. The results are shown in the following table and appendix Figure 31-33 .

[0063] Table 5 System suitability parameters of chromatographic peaks at different column temperatures It can be seen from the results that increasing or decreasing the column temperature will affect the separation effect. To ensure the separation effect, a column temperature of 30°C was selected for the subsequent screening and investigation of conditions.

[0064] 3.5 Investigation of different flow rates According to the preliminary chromatographic conditions determined above, the effects of different flow rates (0.5 ml / min, 0.6 ml / min, 0.7 ml / min) on the separation effect of the characteristic chromatogram of pine pollen were investigated. The results are shown in the following table and appendix Figure 34-36 .

[0065] Table 6 System suitability parameters of chromatographic peaks under different flow rate conditions It can be seen from the results that the separation effect is significantly better when the flow rate is 0.5 ml / min. Therefore, it was selected for the subsequent condition investigation.

[0066] 3.6 Investigation of different elution gradients Using acetonitrile as mobile phase A, 0.05% phosphoric acid aqueous solution as mobile phase B, Agilent Extend-C18 RRHD (3.0×100 mm, 1.8 μm) as the chromatographic column, the flow rate was 0.5 ml per minute; the column temperature was 30°C; the detection wavelength was 210 nm, and the injection volume was 2 μl.

[0067] Gradient elution was carried out according to the regulations in the following table respectively, and the effects of different gradients on each chromatographic peak were investigated. The results are shown in the following table and appendix Figure 37-39 .

[0068] Table 7 Gradient 1 Table 8 Gradient 2 Table 9 Gradient 3 Table 10 System suitability parameters of chromatographic peaks under different gradient conditions It can be seen from the results that the separation degree of gradient 2 is better and the retention times of each chromatographic peak are more average. Therefore, it was selected as the elution gradient of pine pollen.

[0069] 3.6 Summary of Chromatographic Conditions Chromatographic conditions and system suitability test: Acetonitrile was used as mobile phase A, 0.05% phosphoric acid solution was used as mobile phase B, Agilent ZORBAX RRHD Extend-C18 (3.0×100mm, 1.8μm) was used as the chromatographic column, and the flow rate was 0.5 ml per minute; the column temperature was 30°C; the detection wavelength was 210nm, the injection volume was 2 μl, and gradient elution was carried out according to the regulations in the following table.

[0070] Example 4 Investigation on the Preparation Method of Test Solution 4.1 Investigation on Extraction Solvents Therefore, the extraction effects of water, 25% methanol, 50% methanol, 75% methanol, and methanol on pine pollen (Pinus yunnanensis DYC220601) were investigated. The results are shown in Figure 40-44 .

[0071] The results showed that the chromatographic information in the chromatogram with methanol as the extraction solvent was the most abundant. Therefore, methanol was used as the extraction solvent for subsequent investigations.

[0072] 4.2 Investigation on Extraction Methods According to the results of the extraction solvent determined above, the effects of different extraction methods on pine pollen (Pinus yunnanensis DYC220601) were investigated: ultrasonic treatment (power 300W, frequency 40kHz) for 30 minutes and heating under reflux for 30 minutes. The results are shown in the following table and appendix Figure 45-46 .

[0073] Table 11 System Suitability Parameters of Chromatographic Peaks with Different Extraction Methods It can be seen from the results that a distinct characteristic peak (peak 13’) appeared at 18 min during reflux extraction, and there were no obvious differences in the other chromatographic peaks. Therefore, reflux was selected as the extraction method for subsequent investigations.

[0074] 4.3 Investigation on Extraction Time According to the extraction solvent and extraction method determined above, the reflux time was further investigated: 20 minutes, 30 minutes, 40 minutes, and 60 minutes for their effects on the extraction of pine pollen (Pinus yunnanensis DYC220601). The information content of chromatographic peaks and system suitability parameters were used as the main investigation indicators. The results are shown in the following table and appendix Figure 47-50 .

[0075] Table 12 System Suitability Parameters of Chromatographic Peaks with Different Extraction Times The results showed that as the extraction time prolonged, the peak area of the chromatographic peak at 18 min (peak 13’) increased significantly, and the peak area tended to be flat from 40 to 60 min. For the convenience of operation, 40 minutes of reflux was selected as the extraction time for subsequent investigation.

[0076] 4.4 Investigation of Sampling Quantity According to the extraction method, extraction solvent and extraction time determined above, the influence of sampling quantities of 1 g, 2 g, and 3 g on the extraction effect of pine pollen (Pinus yunnanensis DYC220601) was further investigated. The information content of chromatographic peaks and system suitability parameters were used as the main investigation indexes. The results are shown in the following table and appendix Figure 51-53 。

[0077] Table 13 System Suitability Parameters of Chromatographic Peaks with Different Sampling Quantities The results showed that as the sampling quantity increased, the peak area of the chromatographic peak increased by a corresponding multiple. Considering the saving of test samples, 2 g was selected as the sampling quantity for this experiment.

[0078] Example 5 Determination of Characteristic Chromatograms of Pine Pollen with Different Origins 5.1 Confirmation of Preparation Method of Test Solution The tentative preparation method of the test solution for pine pollen was as follows: Take about 2 g of pine pollen, weigh it precisely, place it in a stoppered conical flask, precisely add 25 ml of 100% methanol, weigh it, heat under reflux for 40 minutes, take it out, let it cool, weigh it again, make up the lost weight with 50% methanol, shake well, filter, and take the subsequent filtrate to obtain.

[0079] Determination method: Precisely pipette 2 μl of the test solution and inject it into the liquid chromatograph, and record the chromatogram for 50 minutes.

[0080] 5.2 Tentative Detection Method Chromatographic conditions and system suitability test: Use acetonitrile as mobile phase A, 0.05% phosphoric acid solution as mobile phase B, Agilent ZORBAX RRHD Extend–C18 (3.0×100 mm, 1.8 μm) as the chromatographic column, the flow rate is 0.5 ml per minute; the column temperature is 30 °C; the detection wavelength is 210 nm, and gradient elution is carried out according to the regulations in the following table.

[0081] Preparation of test solution: Take about 2 g of this product, weigh it precisely, place it in a stoppered conical flask, precisely add 25 ml of methanol, weigh it, heat under reflux for 40 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain.

[0082] Assay:Precisely pipette 2 μl of the test solution, inject it into the liquid chromatograph, and perform the determination to obtain the result.

[0083] 5.3 Determination of characteristic fingerprints of pine pollen from different origins Use the above method to determine the characteristic fingerprints of Pinus massoniana, Pinus yunnanensis, Pinus armandii, Pinus tabuliformis, mixed pine and Pinus bungeana respectively. The results are shown in the appendix Figure 54 . Figure 54 Among them, 2’: phenylalanine; 5’: protocatechuic acid; 6’: tryptophan; 10’: taxifolin; 18’: 15-hydroxydehydroabietic acid; 23’: dehydroabietic acid; S1: Pinus armandii (DYC220612); S2: Pinus bungeana (ZX9735-3002-056); S3: Pinus massoniana (DYC220501); S4: Pinus yunnanensis (DYC220601); S5: mixed pine (DYC220801); S6: Pinus tabuliformis (DYC220630).

[0084] It can be seen from the results that: 1) This method can distinguish pine pollen from different origins. Among them, Pinus bungeana has more obvious chromatographic peaks at 12-20 minutes than other origins; Pinus armandii lacks peaks 1’, 3’, 8’, and the peak areas of each chromatographic peak are also smaller, especially the difference in the peak area of peak 23’ is the most obvious; Pinus massoniana and mixed pine have two obvious chromatographic peaks after peak 26’, and the peak areas of peaks 14’-22’ in the chromatogram of Pinus tabuliformis are significantly larger than those of Pinus yunnanensis. 2) There are significant differences in the characteristic fingerprints between Pinus armandii, Pinus bungeana and Pinus tabuliformis, Pinus yunnanensis, Pinus massoniana, mixed pine. The differences in the characteristic fingerprints of Pinus tabuliformis, Pinus yunnanensis, Pinus massoniana, mixed pine, and mixed pine are smaller, and the number of common peaks is larger. The characteristic fingerprints can be established according to multiple batches of samples.

[0085] 5.4 Determination of characteristic fingerprints of multiple batches of pine pollen (Pinus massoniana, Pinus tabuliformis, Pinus yunnanensis, mixed pine) Use the above method to determine the characteristic fingerprints of Pinus massoniana, Pinus yunnanensis, Pinus tabuliformis and mixed pine respectively. The results are shown in the appendix Figure 55 .

[0086] Figure 55Among them, 2: phenylalanine; 3: tryptophan; 6: taxifolin; 10: 15-hydroxydehydroabietic acid; 12: dehydroabietic acid; S1: mixed pine (DYC220801); S2: mixed pine (DYC220802); S3: mixed pine (DYC220803); S4: mixed pine (DYC220810); S5: mixed pine (DYC220811); S6: mixed pine (DYC220812); S7: mixed pine (DYC220813); S8 mixed pine (DYC220814); S9: mixed pine (DYC220815); S10: mixed pine (DYC220816); S11: mixed pine (DYC220817); S12: mixed pine (DYC220818); S13: mixed pine (DYC230301); S14: mixed pine (DYC230401); S15: mixed pine (DYC230402); S16: mixed pine (DYC230611); S17: mixed pine (DYC230612); S18: Chinese pine (DYC220701); S19: Chinese pine (DYC220702); S20: Chinese pine (DYC220703); S21: Chinese pine (DYC220704); S22: Chinese pine (DYC220705); S23: Chinese pine (DYC230404); S24: Chinese pine (DYC220630); S25: Chinese pine (DYC220631); S26: Chinese pine (DYC220632); S27: Chinese pine (DYC220633); S28: Chinese pine (DYC220634); S29: Chinese pine (DYC230601); S30: Chinese pine (DYC230602); S31: Chinese pine (DYC230701); S32: masson pine (DYC220501); S33: masson pine (DYC220502); S34: masson pine (DYC220503); S35: masson pine (DYC220504); S36: masson pine (DYC220505); S37: masson pine (DYC230302); S38: masson pine (DYC230303); S39: masson pine (DYC230304); S40: Pinus yunnanensis (DYC220601); S41: Pinus yunnanensis (DYC220602); S42: Pinus yunnanensis (DYC220603); S43: Pinus yunnanensis (DYC220604); S44: Pinus yunnanensis (DYC220605).

[0087] Results Analysis of 44 Batches of Pine Pollen As can be seen from the results, there are 16 common peaks in 44 batches of pine pollen (including 17 batches of mixed pine, 14 batches of Chinese pine, 8 batches of masson pine, and 5 batches of Pinus yunnanensis).

[0088] 5.5 Results Analysis of 17 Batches of Mixed Pine Pollen Figure 56 Among them, 2: phenylalanine; 3: tryptophan; 6: taxifolin; 10: 15-hydroxydehydroabietic acid; 12: dehydroabietic acid; S1: mixed pine (DYC220801); S2: mixed pine (DYC220802); S3: mixed pine (DYC220803); S4: mixed pine (DYC220810); S5: mixed pine (DYC220811); S6: mixed pine (DYC220812); S7: mixed pine (DYC220813); S8 mixed pine (DYC220814); S9: mixed pine (DYC220815); S10: mixed pine (DYC220816); S11: mixed pine (DYC220817); S12: mixed pine (DYC220818); S13: mixed pine (DYC230301); S14: mixed pine (DYC230401); S15: mixed pine (DYC230402); S16: mixed pine (DYC230611); S17: mixed pine (DYC230612).

[0089] From the attached Figure 56 results, it can be seen that the differences among the 17 batches of mixed pine pollen are relatively small. In addition to the 16 common peaks shown in the figure, there are also more unique common peaks among different batches of mixed pine.

[0090] 5.6 Results Analysis of 14 Batches of Chinese Pine Pollen Figure 57 Among them, 2: phenylalanine; 3: tryptophan; 6: taxifolin; 10: 15-hydroxydehydroabietic acid; 12: dehydroabietic acid; S1: Chinese pine (DYC220701); S2: Chinese pine (DYC220702); S3: Chinese pine (DYC220703); S4: Chinese pine (DYC220704); S5: Chinese pine (DYC220705); S6: Chinese pine (DYC230404); S7: Chinese pine (DYC220630); S8: Chinese pine (DYC220631); S9: Chinese pine (DYC220632); S10: Chinese pine (DYC220633); S11: Chinese pine (DYC220634); S12: Chinese pine (DYC230601); S13: Chinese pine (DYC230602); S14: Chinese pine (DYC230701).

[0091] From the attached Figure 57It can be seen from the results that there are certain differences among batches of Pinus tabuliformis pollen. Among them, the chromatographic peaks of S7-S14 (the origin of S7-S11 is Chifeng, Inner Mongolia, and the origin of S13-S4 is Longde, Ningxia) are significantly richer than those of S1-S6 (the origin of S1-S6 is Jinzhou, Liaoning, and the origin of S6 is Lingshan, Shanxi). It is speculated that this may be related to the origin, but all contain the 16 common peaks in the figure.

[0092] 5.7 Results analysis of 8 batches of Pinus massoniana pollen Figure 58 Among them, 2: phenylalanine; 3: tryptophan; 6: taxifolin; 10: 15-hydroxydehydroabietic acid; 12: dehydroabietic acid; S1: Pinus massoniana (DYC220501); S2: Pinus massoniana (DYC220502); S3: Pinus massoniana (DYC220503); S4: Pinus massoniana (DYC220504); S5: Pinus massoniana (DYC220505); S6: Pinus massoniana (DYC230302); S7: Pinus massoniana (DYC230303); S8: Pinus massoniana (DYC230304).

[0093] From the appendix Figure 58 It can be seen from the results that the peak areas of the chromatographic peaks of Pinus massoniana pollen of S1-S5 (originated from Chuzhou, Anhui, harvested in 2022) between peak 2 - peak 3 and after peak 16 are slightly larger than those of S6-S8 (originated from Chuzhou, Anhui, harvested in 2023). It is speculated that this may be related to the harvest, but all contain the 16 common peaks in the figure.

[0094] 5.8 Results analysis of 5 batches of Pinus yunnanensis pollen Figure 59 Among them, 2: phenylalanine; 3: tryptophan; 6: taxifolin; 10: 15-hydroxydehydroabietic acid; 12: dehydroabietic acid; S1: Pinus yunnanensis (DYC220601); S2: Pinus yunnanensis (DYC220602); S3: Pinus yunnanensis (DYC220603); S4: Pinus yunnanensis (DYC220604); S5: Pinus yunnanensis (DYC220605).

[0095] From the appendix Figure 59 It can be seen from the results that the differences among batches of 5 batches of Pinus yunnanensis pollen are relatively small. In addition to containing the 16 common peaks in the figure, there are more common peaks.

[0096] Figure 60 Among them, 2: phenylalanine; 3: tryptophan; 6: taxifolin; Peak 9: naringenin and naringenin chalcone; 10: 15-hydroxydehydroabietic acid; 12: dehydroabietic acid.

[0097] To sum up, 17 batches of mixed pine and 14 batches of Pinus tabuliformis (the results are shown in the appendix Figure 60), There are 16 common peaks in 8 batches of Pinus massoniana and 5 batches of Pinus yunnanensis pine pollen. Thus, we have found the common peaks of pine pollen from different sources, as well as the unique peaks of each different variety, and effectively established a general fingerprint quality control method for pine pollen from different sources. This method is reliable and easy to operate, and it has been confirmed that it can be applied to quality control in actual production to meet the needs of medicinal material identification and quality control.

[0098] Example 6 Comparison of the method of the present invention with the methods of the prior art Test samples: The selected samples are: DYC230304 (Pinus massoniana), DYC230404 (Pinus tabuliformis), DYC230612 (mixed pine), DYC240409 (Pinus yunnanensis), DYC240504 (mixed Pinus tabuliformis), 5 batches.

[0099] Preparation of test samples 1 Refer to 5.2 of the present invention to formulate the detection method Preparation of test samples 2 For the preparation methods and chromatographic conditions of different pine pollens, refer to Patent CN200710040134.6: Weigh 1.0 g of pine pollen, add 100 ml of 80% methanol, reflux for 2 h, filter, evaporate the filtrate to dryness, dissolve in 50% methanol to a volume of 25 ml, and filter through a 0.45 μm microporous membrane to obtain the test sample. Chromatographic conditions: column temperature: 30 °C, detection wavelength 260 nm, injection volume 20 μl, chromatographic column: alltima (250 mm × 4.6 mm, 5 μm); elution conditions: 0 min, 5% acetonitrile (A) - 95% 0.2% formic acid aqueous solution (B); 30 min - 40 min, 60% (A) - 40% (B), flow rate 1.000 ml / min.

[0100] The method of the present invention is compared with the method described in the prior art (Patent CN200710040134.6), as shown in the following table: The experimental chromatogram results obtained by the methods of the present invention and the prior art are respectively as Figure 61A and 61B shown. In this experiment, 5 batches of pine pollen were used, namely Anhui Pinus massoniana, Shanxi Pinus tabuliformis, Anhui mixed pine, Yunnan Pinus yunnanensis, and Inner Mongolia Pinus tabuliformis. The experimental chromatogram results show that the patented method of the prior art identifies fewer small-polarity components. A fingerprint is an aggregate of chemical components, aiming to characterize chemical components to the greatest extent. The method of the present invention identifies amino acids, flavonoids, and organic acid components, and can more comprehensively and truly characterize pine pollen.

[0101] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and not for limiting the scope of the present invention. Those skilled in the art should also understand that various modifications can be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A method for detecting the characteristic chromatogram of pine pollen, characterized in that, The method includes: (1) Preparation of the test solution of pine pollen Take 1 - 3 g of pine pollen, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 25% - 75% methanol or 100% methanol respectively, ultrasonically treat for 30 minutes or heat under reflux for 40 minutes, take it out, let it cool, shake well, centrifuge at 12000 rpm for 5 min, filter, and take the subsequent filtrate to obtain the test solution; (2) Chromatographic column conditions Use a C18 chromatographic column, with acetonitrile as mobile phase A and phosphoric acid aqueous solution as mobile phase B for gradient elution; the flow rate is 0.5 ml / min - 1.5 ml / min; the column temperature is 25°C - 35°C; the detection wavelength is 210 nm, and the injection volume is 0.5 - 5 μl; (3) Establishment of the characteristic chromatogram Determine the characteristic chromatograms of Pinus massoniana, Pinus yunnanensis, Pinus armandii, Pinus tabuliformis, mixed pine, and Pinus bungeana respectively.

2. The method for detecting the characteristic chromatogram of pine pollen according to claim 1, characterized in that The C18 chromatographic column is selected from Agilent Extend-C18 RRHD, Agilent Poroshell 120 AQ-C18, Agilent Poroshell 120EC-C18, Agilent ZORBAX RRHD Extend–C18, Agilent ZORBAX RRHD SB–C18, Dikma Navigatorsil C18.

3. The method for detecting pine pollen characteristic pattern according to claim 1, wherein: The concentration of the phosphoric acid aqueous solution is 0.02% - 0.15%.

4. The method for detecting the characteristic chromatogram of pine pollen according to claim 3, wherein The concentration of the phosphoric acid aqueous solution is 0.05%.

5. The method for detecting the characteristic chromatogram of pine pollen according to claim 1, wherein The column temperature is 30°C.

6. The method for detecting the characteristic fingerprint of pine pollen according to claim 1, wherein The flow rate is 0.5 ml / min or 1 ml / min.

7. The method for detecting the characteristic fingerprint of pine pollen according to claim 2, wherein The C18 chromatographic column is Agilent ZORBAX RRHD Extend-C18 (3.0×100 mm, 1.8 μm).

8. The method for detecting the characteristic chromatogram of pine pollen according to claim 1, wherein The concentration of methanol in step (1) is 50%.

9. The method for detecting the characteristic chromatogram of pine pollen according to claim 1, wherein The ultrasonic treatment conditions in step (1) are a power of 300 W and a frequency of 40 kHz.

10. The method for detecting the characteristic chromatogram of pine pollen according to claim 1, wherein Take 2 g of pine pollen in step (1).

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