A method for constructing a characteristic spectrum of pine resin formulation granules, the characteristic spectrum and its application.

Abstract

This invention provides a method for constructing a characteristic spectrum of Pinus tabuliformis granules, the characteristic spectrum, and its application. The construction method includes the following steps: (1) preparing a test solution of Pinus tabuliformis granules; preparing a reference solution of Pinus tabuliformis reference material; and preparing a reference solution of pine syrup monomethyl ether reference standard; (2) performing ultra-high performance liquid chromatography (UHPLC) on the test solution, the reference solution of the reference material, and the reference solution of the reference standard, respectively, and obtaining the characteristic spectrum of the Pinus tabuliformis granules based on the detection. The characteristic spectrum constructed by the method provided by this invention enhances the specific identification of Pinus tabuliformis granules and can effectively identify the source of Pinus tabuliformis medicinal material, providing support for ensuring the single source of the medicinal material source of Pinus tabuliformis granules. The characteristic spectrum constructed by this invention can comprehensively reflect the characteristic peak information of the sample, and the method is stable, highly precise, and has good reproducibility.

Description

Technical Field

[0001] This invention relates to the fields of traditional Chinese medicine testing, quality control, and identification, specifically to a method for constructing a characteristic spectrum of pine resin granules, the characteristic spectrum, and its application. Background Technology

[0002] Pinus tabuliformis Carr. or Pinus massoniana Lamb., belonging to the Pinaceae family, are dried nodular nodes or branch nodes. They are warm in nature, slightly bitter and pungent, and enter the liver and kidney meridians. They dispel wind and dampness, unblock the meridians, and relieve pain. They are used for wind-cold-dampness arthralgia, joint pain due to wind, muscle spasms, and traumatic injuries. The main active ingredient and indicator component in Pinus tabuliformis is α-pinene. It also contains cellulose, lignans, vitamins, amino acids, polyphenols, volatile oil (turpentine oil), resin, fatty acids, and flavonoids.

[0003] The Chinese Pharmacopoeia (2020 edition) sets standards for Pinus tabuliformis medicinal materials based on the volatile oil extracted from the medicinal material, specifying the content of the volatile oil and its volatile component α-pinene. However, Pinus tabuliformis granules are prepared from Pinus tabuliformis slices through extraction, volatile oil encapsulation, concentration, drying, and formulation. Their material basis differs significantly from that of the raw medicinal material. Furthermore, in the field of Pinus tabuliformis granules, there are almost no comprehensive evaluation methods for water-soluble components other than volatile components. Pinus tabuliformis monomethyl ether has strong antibacterial properties and is mainly found in Pinus genus plants of the Pinaceae family. Pinus tabuliformis monomethyl ether also has analgesic and anti-inflammatory effects, and as a water-soluble component, it exhibits good transfer rate in granules. Therefore, establishing a characteristic spectrum of water-soluble substances in Pinus tabuliformis based on pinus tabuliformis monomethyl ether can achieve effective quality control from raw Pinus tabuliformis medicinal materials to granules, providing a more comprehensive and objective evaluation method.

[0004] Patent CN115541792A discloses a method for studying the content and characteristic spectrum of pine resin monomethyl ether in pine resin formulation particles using ultra-high liquid chromatography. However, this method is not only cumbersome in detecting gradient changes, but also yields a small number of characteristic peaks in the obtained characteristic spectrum, showing only 4 characteristic peaks. This method cannot fully represent the material basis of pine resin formulation particles, and these 4 characteristic peaks cannot be used to identify the origin of pine resin.

[0005] The raw materials for pine resin are Pinus tabuliformis and Pinus massoniana. For pine resin formulation granules, a single, fixed raw material is required. Currently, research on the identification of the raw material for pine resin is lacking. To ensure the singularity of the raw material source for pine resin formulation granules, effective identification of the raw material for pine resin is necessary. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for constructing a characteristic spectrum of pine resin formulation particles, the characteristic spectrum itself, and its applications.

[0007] To achieve this objective, the present invention employs the following technical solution:

[0008] In a first aspect, the present invention provides a method for constructing a feature map of pine resin formulation particles, the method comprising the following steps:

[0009] (1) Mix the granules of Pinus tabuliformis with an aqueous methanol solution, sonicate them, and prepare a test solution; prepare a reference solution of Pinus tabuliformis reference material; mix the reference standard of Pinus tabuliformis monomethyl ether with methanol to prepare a reference solution of reference standard.

[0010] (2) The test solution, the reference medicinal material solution and the reference standard solution were subjected to ultra-high performance liquid chromatography. Based on the detection results, the common peak with consistent retention time, good peak shape and high resolution was selected as the characteristic peak. Among the characteristic peaks, the one with a definite component and good peak shape was selected as the S peak. The relative retention time of other characteristic peaks relative to the S peak was calculated, and the characteristic spectrum of the pine resin formula granules was obtained.

[0011] In step (2), the high performance liquid chromatography fluidity includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous formic acid solution;

[0012] The ultra-high performance liquid chromatography employs gradient elution, and the specific procedure for gradient elution is as follows:

[0013] From 0 to 10 minutes, the volume fraction of mobile phase A is uniformly changed from 4-6% (e.g., 4%, 4.5%, 5%, 5.5%, or 6%) to 18-22% (e.g., 18%, 19%, 20%, 21%, or 22%), and the volume fraction of mobile phase B is uniformly changed from 94-96% (e.g., 94%, 94.5%, 95%, 95.5%, or 96%) to 78-82% (e.g., 78%, 79%, 80%, 81%, or 82%). Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0014] Between 10 and 25 minutes, the volume fraction of mobile phase A changes uniformly to 33-37% (e.g., 33%, 34%, 35%, 36%, or 37%), and the volume fraction of mobile phase B changes uniformly to 63-67% (e.g., 63%, 64%, 65%, 66%, or 67%). Other specific values ​​within the above range can be selected, and will not be elaborated on here.

[0015] Between 25 and 32 minutes, the volume fraction of mobile phase A changes uniformly to 63-67% (e.g., 63%, 64%, 65%, 66%, or 67%), and the volume fraction of mobile phase B changes uniformly to 33-37% (e.g., 33%, 34%, 35%, 36%, or 37%). Other specific values ​​within the above range can be selected, and will not be elaborated on here.

[0016] Between 32 and 34 minutes, the volume fraction of mobile phase A changes uniformly to 4-6% (e.g., 4%, 4.5%, 5%, 5.5%, or 6%), and the volume fraction of mobile phase B changes uniformly to 94-96% (e.g., 94%, 94.5%, 95%, 95.5%, or 96%). Other specific values ​​within the above range can be selected, and will not be elaborated on here.

[0017] This invention addresses the technical deficiency of existing technologies in distinguishing between different types of pine resin (Pinus tabuliformis and Pinus massoniana) medicinal materials. It provides a novel method for constructing characteristic chromatograms of Pinus tabuliformis formulation granules. The resulting characteristic chromatograms enhance the specificity identification of Pinus tabuliformis formulation granules, effectively identifying the original source of the medicinal material and supporting the guarantee of the singularity of the medicinal material source for Pinus tabuliformis formulation granules. The characteristic chromatograms constructed by this invention comprehensively reflect the characteristic peak information of the sample, and the method is stable, highly precise, and has good reproducibility.

[0018] Preferably, the volume fraction of methanol in the methanol aqueous solution in step (1) is 50%-90%, such as 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0019] Preferably, the ratio of the pine resin formulation particles to the methanol aqueous solution is 1g:(40-60)mL, for example, 1g:40mL, 1g:42mL, 1g:44mL, 1g:46mL, 1g:48mL, 1g:50mL, 1g:52mL, 1g:54mL, 1g:56mL, 1g:58mL, or 1g:60mL, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0020] Preferably, the ultrasonic treatment power is 200-300W (e.g., 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 290W, or 300W, etc.), the frequency is 30-50kHz (30kHz, 32kHz, 34kHz, 36kHz, 38kHz, 40kHz, 42kHz, 44kHz, 46kHz, 48kHz, or 50kHz, etc.), and the time is 20-40min (e.g., 20min, 22min, 24min, 26min, 28min, 30min, 32min, 34min, 36min, 38min, or 40min, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0021] Preferably, the reference solution of the control medicinal material in step (1) is prepared by a method comprising the following steps:

[0022] The pine resin reference material was mixed with methanol, ultrasonically treated, and the filtrate was collected to obtain the reference material solution.

[0023] Preferably, the ratio of the pine resin reference material to methanol is 1g:(20-30)mL, for example, 1g:20mL, 1g:21mL, 1g:22mL, 1g:23mL, 1g:24mL, 1g:25mL, 1g:26mL, 1g:27mL, 1g:28mL, 1g:29mL, or 1g:30mL, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0024] Preferably, the ultrasonic treatment power is 200-300W (e.g., 200W, 210W, 220W, 230W, 240W, 250W, 260W, 270W, 280W, 290W, or 300W, etc.), the frequency is 30-50kHz (30kHz, 32kHz, 34kHz, 36kHz, 38kHz, 40kHz, 42kHz, 44kHz, 46kHz, 48kHz, or 50kHz, etc.), and the time is 15-25min (e.g., 15min, 16min, 17min, 18min, 19min, 20min, 21min, 22min, 23min, 24min, or 25min, etc.). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0025] Preferably, the volume fraction of the formic acid dissolved in water is 0.05-0.15%, such as 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15%, etc. Other specific values ​​within this range can be selected, and will not be elaborated here.

[0026] Preferably, the column temperature of the ultra-high performance liquid chromatography is 33-37℃ (e.g., 33℃, 33.5℃, 34℃, 34.5℃, 35℃, 35.5℃, 36℃, 36.5℃, or 37℃, etc.), and the flow rate of the mobile phase is 0.28-0.32 mL / min (e.g., 0.28 mL / min, 0.29 mL / min, 0.3 mL / min, 0.31 mL / min, or 0.32 mL / min, etc.). Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0027] Preferably, the detection wavelength of the ultra-high performance liquid chromatography is 320-350nm (e.g., 320nm, 325nm, 330nm, 335nm, 340nm, 345nm or 350nm, etc.). Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0028] Preferably, the column packing material for the ultra-high performance liquid chromatography is octadecylsilane-bonded silica gel.

[0029] Preferably, the theoretical plate number of the ultra-high performance liquid chromatography method, calculated based on the peak of pine ether monomethyl ether, is not less than 5000.

[0030] In a second aspect, the present invention provides a feature map of pine resin formulation particles, wherein the feature map of pine resin formulation particles is obtained by the method for constructing a feature map of pine resin formulation particles as described in the first aspect;

[0031] The characteristic spectrum contains 11 characteristic peaks and 1 S-peak. The peaks are sorted by retention time from smallest to largest, with peak 11 being the S-peak. The relative retention times of peaks 1, 2, 3, 4, 5, 7, 8, 9, and 10 relative to the S-peak are 0.17±10%, 0.23±10%, 0.28±10%, 0.41±10%, 0.43±10%, 0.52±10%, 0.68±10%, 0.77±10%, 0.81±10%, and 0.98±10%.

[0032] Peak 11 is composed of pine bark extract monomethyl ether.

[0033] Thirdly, the present invention provides a method for constructing a characteristic spectrum of pine resin formulation particles as described in the first aspect, and the application of the characteristic spectrum of pine resin formulation particles as described in the second aspect in the quality testing of pine resin-related products.

[0034] Preferably, the pine resin-related products include any one or a combination of at least two of the following: pine resin medicinal materials, pine resin formula granules, or pine resin standard decoction.

[0035] Fourthly, the present invention provides a method for identifying the pine resin precursor, the method comprising the following steps:

[0036] The *Pinus tabuliformis* medicinal material was detected according to the ultra-high performance liquid chromatography detection method described in the first aspect, and the characteristic spectrum of the *Pinus tabuliformis* medicinal material was obtained, showing 11 characteristic peaks. The area ratios of peak 4 / peak 6, peak 5 / peak 6, and peak 7 / peak 6 were calculated.

[0037] Preferably, when the area ratio of peak 4 to peak 6 is ≥0.9 (e.g., 0.9, 1.0, 1.1, 1.2, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0), the area ratio of peak 5 to peak 6 is ≥0.4 (e.g., 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, or 1.4), and the area ratio of peak 7 to peak 6 is ≥5.0 (e.g., 5.0, 5.5, 6.0, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11), the origin of the Pinus tabuliformis medicinal material is Pinus tabuliformis. Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0038] Preferably, when the area ratio of peak 4 to peak 6 is <0.9 (e.g., 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1), the area ratio of peak 5 to peak 6 is <0.4 (e.g., 0.3, 0.2, or 0.1), and the area ratio of peak 7 to peak 6 is <5.0 (e.g., 4.9, 4.7, 4.5, 4.3, 4.1, 3.5, 3.0, 2.5, 2.0, 1.5, or 1.0), the origin of the Pinus tabuliformis medicinal material is Pinus massoniana. Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] This invention addresses the technical deficiency of existing technologies in distinguishing between different types of pine resin (Pinus tabuliformis and Pinus massoniana). It provides a novel method for constructing characteristic chromatograms of Pinus tabuliformis formulation granules. The resulting chromatograms enhance the specificity identification of Pinus tabuliformis formulation granules, effectively identifying the original source of Pinus tabuliformis and supporting the guarantee of the singularity of the original source of the medicinal materials in Pinus tabuliformis formulation granules. The characteristic chromatograms constructed by this invention comprehensively reflect the characteristic peak information of the samples, and the method is stable, highly precise, and has good reproducibility. Attached Figure Description

[0041] Figure 1 It is a superimposed chromatogram of the test solution in Example 1;

[0042] Figure 2 This is the characteristic spectrum of the pine resin formulation particles obtained in Example 1;

[0043] Figure 3 This is a confirmation diagram of peak 11 in Example 1;

[0044] Figure 4 This is the pine knot diagram obtained from Example 1;

[0045] Figure 5 This is the pine knot diagram of Masson pine obtained from Example 1. Detailed Implementation

[0046] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0047] Unless otherwise specified, the reagents and consumables used in the following embodiments were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used were conventional methods and techniques in the art.

[0048] Example 1

[0049] This embodiment provides a method for constructing a characteristic map of pine resin formulation particles and its application. The specific steps are as follows:

[0050] Preparation of reference solution for reference medicinal material: Take 1.0 g of pine resin reference material, place it in a stoppered conical flask, accurately add 25 mL of methanol, seal tightly, and sonicate (power 250 W, frequency 40 kHz) for 20 min. Take the filtrate as the reference solution for reference medicinal material.

[0051] Preparation of reference solution: Add pine ether monomethyl ether reference standard to methanol to obtain a 0.1 mg / L solution, which is used as the reference solution.

[0052] Preparation of the test solution: Take the turpentine formulation granules, grind them finely, accurately weigh 0.5g, place them in a stoppered conical flask, accurately add 25mL of 70% methanol, stopper tightly, weigh, sonicate (power 250W, frequency 40kHz) for 30min, remove and cool, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution.

[0053] Chromatographic conditions and system suitability test: The determination was performed according to ultra-high performance liquid chromatography, using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm), acetonitrile as mobile phase A, and 0.1% formic acid solution as mobile phase B. Gradient elution was performed according to the specifications in the table below, with a flow rate of 0.3 mL / min, a column temperature of 35 ℃, a detection wavelength of 335 nm, and a theoretical plate number calculated based on the pine ether monomethyl ether peak of pine ether should not be less than 5000.

[0054] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-10 5→20 95→80 10-25 20→35 80→65 25-32 35→65 65→35 32-34 65→5 35→95

[0055] Establishment of characteristic chromatograms: Take 15 batches of pine resin granules (as shown in Table 1), prepare test solutions according to the above method, and accurately inject 1 μL of the reference solution of the reference medicinal material, the reference solution of the reference substance, and the test solution into the ultra-high performance liquid chromatograph. Measure the chromatograms according to the above chromatographic conditions to obtain characteristic chromatograms.

[0056] Analysis was performed using the "Similarity Evaluation System for Chromatographic Characteristic Magnetism of Traditional Chinese Medicine" (2012 version). Overlay chromatograms of 15 batches of test samples were obtained, and control characteristic chromatograms were generated. The overlay chromatograms of the 15 batches of test sample solutions are shown below. Figure 1 Where S2(11)-S16(11) refer to 15 batches of samples respectively, and S1(11) is the fitted spectrum. The generated feature spectrum is shown in [reference needed]. Figure 2 In the spectrum, numbers 1-11 represent characteristic peaks 1-11. The similarity evaluation results are shown in Table 1, and the relative retention time calculation results are shown in Table 2.

[0057] Table 1

[0058] Serial Number batch number Place of origin Similarity to the control feature map S1 R 1.000 S2 2021052603 Danfeng County, Shangluo City, Shaanxi Province 0.998 S3 2021050504 Huoshan County, Lu'an City, Anhui Province 0.998 S4 2021052605 Wenxian County, Longnan City, Gansu Province 0.984 S5 2021050506 Huoshan County, Lu'an City, Anhui Province 0.996 S6 2021050507 Yingzhou District, Fuyang City, Anhui Province 0.999 S7 2021050508 Taihe County, Fuyang City, Anhui Province 0.992 S8 2021050509 Funan County, Fuyang City, Anhui Province 0.998 S9 2021052609 Xiangning County, Linfen City, Shanxi Province 0.985 S10 2021050510 Huangshan District, Huangshan City, Anhui Province 0.997 S11 2021052610 Lixian County, Longnan City, Gansu Province 0.987 S12 2021050511 Nanfen District, Benxi City, Liaoning Province 0.999 S13 2021050512 Zhanqian District, Yingkou City, Liaoning Province 1.000 S14 2021050513 Xishi District, Yingkou City, Liaoning Province 0.999 S15 2021050514 Laobian District, Yingkou City, Liaoning Province 0.999 S16 2021050515 Gaizhou City, Yingkou City, Liaoning Province 0.986

[0059] In Table 1, the similarity between the characteristic chromatograms of 15 batches of pine resin formulation granules and the control characteristic chromatograms ranged from 0.984 to 1.000, indicating that the differences between the batches were small and the generated control characteristic chromatograms were representative.

[0060] Table 2

[0061]

[0062]

[0063] The characteristic chromatograms of 15 batches of pine resin samples showed that all 15 batches of pine resin formulation granules exhibited 11 characteristic peaks in their UPLC characteristic chromatograms. The relative retention times of each characteristic peak were relatively small, all within 1.0%, which met the quality control requirements. The average relative retention time was selected as the measured value. Combined with the results of subsequent durability tests, peak number 11 was selected as the reference peak, labeled S. The relative retention times of characteristic peaks 1-10 were calculated. Their relative retention times should be within ±10% of the specified values, which are 0.17 (peak 1), 0.23 (peak 2), 0.28 (peak 3), 0.41 (peak 4), 0.43 (peak 5), 0.52 (peak 6), 0.68 (peak 7), 0.77 (peak 8), 0.81 (peak 9), and 0.98 (peak 10).

[0064] like Figure 3 As shown, the retention time of peak 11 in the spectrum of the test sample is consistent with that of the reference standard chromatin monomethyl ether. Therefore, peak 11 can be confirmed as chromatin monomethyl ether.

[0065] Test example:

[0066] Methodological examination:

[0067] Repeatability:

[0068] Six portions of turpentine granules (batch number: K445CP15) were taken and their characteristic spectra were obtained according to the method in Example 1. Using peak 11 as the reference peak, the relative peak area and relative retention time were calculated, and the RSD was also calculated. The results are shown in Table 3-4. Based on the repeatability test results, the RSD of the relative retention time of each characteristic peak was in the range of 0.05-0.92%, and the RSD of the relative peak area was in the range of 0.30-1.92%, indicating that the repeatability of this characteristic spectrum is good.

[0069] Table 3

[0070]

[0071] Table 4

[0072]

[0073]

[0074] Precision:

[0075] One sample of turpentine granules (batch number: K445CP15) was taken and analyzed according to the method in Example 1. Six consecutive injections were performed to obtain its characteristic spectrum. Using peak 11 as the reference peak, its relative peak area and relative retention time were calculated, and the RSD was also calculated. The results are shown in Table 5-6. Based on the precision test results, the RSD of the relative retention time of each characteristic peak was in the range of 0.03% to 0.78%, and the RSD of the relative peak area was in the range of 0.40% to 1.59%, indicating that the instrument has good precision.

[0076] Table 5

[0077]

[0078] Table 6

[0079]

[0080]

[0081] Durability:

[0082] Three portions of turpentine granules (batch number: K445CP15) were prepared into test solutions according to the method in Example 1. The solutions were then measured using different concentrations of formic acid solution (0.08%, 0.10%, and 0.12%) (all other conditions were the same as in Example 1). Peak 11 was used as a reference peak, and its relative peak area and relative retention time were calculated, along with the RSD. The results are shown in Tables 7-8. The experimental results at different acid concentrations show that the relative retention times of each characteristic peak are within ±10% of the specified value, and the RSD of the relative peak area is within the range of 0.35–1.68%, meeting the system suitability requirements and indicating that the method has good robustness to different acid concentrations.

[0083] Table 7

[0084]

[0085] Table 8

[0086]

[0087]

[0088] Application Example 1

[0089] This application example distinguishes between the resin of Pinus tabuliformis and the resin of Pinus massoniana.

[0090] (1) Weigh 1.0g of each of 15 batches of Pinus tabuliformis powder, place them in a stoppered conical flask, add 25mL of methanol, seal tightly, and sonicate (power 250W, frequency 40kHz) for 20min. Take the filtrate to obtain the Pinus tabuliformis test solution.

[0091] (2) Weigh 1.0g of each of 10 batches of pine resin powder, place them in a stoppered conical flask, add 25mL of methanol, seal tightly, and sonicate (power 250W, frequency 40kHz) for 20min. Take the filtrate to obtain the pine resin test solution.

[0092] (3) Accurately pipette 1 μL of the above-mentioned turpentine medicinal material test solution of different origins and inject it into the ultra-high performance liquid chromatograph. Determine the chromatographic conditions according to Example 1.

[0093] The results are as follows Figure 4-5 As shown, both the pine knot spectrum of Pinus tabuliformis and the spectrum of Pinus massoniana exhibit 11 characteristic peaks. The ratios of peak 4 / peak 6, peak 5 / peak 6, and peak 7 / peak 6 were calculated, and the results are shown in Table 9.

[0094] Table 9

[0095]

[0096]

[0097] The results above show that the ratios of peak 4 / peak 6, peak 5 / peak 6, and peak 7 / peak 6 of Masson pine are all significantly smaller than those of Pinus tabuliformis. Therefore, the ratio of the peak area of ​​peak 4, peak 5, peak 7 to peak 6 can be used as a means to distinguish the resins of two different base Pinus tabuliformis species.

[0098] By comparing the characteristic spectra of multiple batches of *Pinus tabuliformis* and *Pinus massoniana* medicinal materials, it was found that *Pinus tabuliformis* pine knots with a relative peak area of ​​at least 0.9 between peaks 4 and 6, at least 0.4 between peaks 5 and 6, and at least 5.0 between peaks 7 and 6 were all *Pinus tabuliformis* pine knots; otherwise, they were *Pinus massoniana* pine knots. The values ​​of 0.9, 0.4, and 5.0 were obtained by multiplying the minimum measured value of *Pinus tabuliformis* by 0.7 and rounding to the nearest tenth.

[0099] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0100] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0101] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for constructing a characteristic map of pine resin formulation particles, characterized in that, The method for constructing the feature map includes the following steps: (1) Mix the granules of Pinus tabuliformis with an aqueous methanol solution, sonicate them, and prepare a test solution; prepare a reference solution of Pinus tabuliformis reference material; mix the reference standard of Pinus tabuliformis monomethyl ether with methanol to prepare a reference solution of reference standard. (2) The test solution, the reference medicinal material solution and the reference standard solution were subjected to ultra-high performance liquid chromatography detection. Based on the detection results, the common peak with consistent retention time, good peak shape and high resolution was selected as the characteristic peak. Among the characteristic peaks, the one with a definite component and good peak shape was selected as the S peak. The relative retention time of other characteristic peaks relative to the S peak was calculated, and the characteristic spectrum of the pine resin formula granules was obtained. In step (2), the mobile phase of the high performance liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of formic acid with a volume fraction of 0.05-0.15%. The ultra-high performance liquid chromatography method employs gradient elution, and the specific procedure for gradient elution is as follows: From 0 to 10 min, the volume fraction of mobile phase A changed uniformly from 4-6% to 18-22%, and the volume fraction of mobile phase B changed uniformly from 94-96% to 78-82%. Between 10 and 25 minutes, the volume fraction of mobile phase A changes uniformly to 33-37%, and the volume fraction of mobile phase B changes uniformly to 63-67%. Between 25 and 32 minutes, the volume fraction of mobile phase A changed uniformly to 63-67%, and the volume fraction of mobile phase B changed uniformly to 33-37%. Between 32 and 34 minutes, the volume fraction of mobile phase A changes uniformly to 4-6%, and the volume fraction of mobile phase B changes uniformly to 94-96%. The detection wavelength of the ultra-high performance liquid chromatography method is 320-350 nm; The ultra-high performance liquid chromatography column used is packed with octadecylsilane-bonded silica gel, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.7 μm.

2. The method for constructing the characteristic map of pine resin formulation particles according to claim 1, characterized in that, The volume fraction of methanol in the methanol aqueous solution in step (1) is 50%-90%.

3. The method for constructing the characteristic spectrum of pine resin formulation particles according to claim 1, characterized in that, The ratio of the pine resin formulation granules to the methanol aqueous solution is 1 g:(40-60) mL.

4. The method for constructing the characteristic map of pine resin formulation particles according to claim 1, characterized in that, The ultrasonic treatment has a power of 200-300 W, a frequency of 30-50 kHz, and a duration of 20-40 min.

5. The method for constructing the characteristic spectrum of pine resin formulation particles according to claim 1, characterized in that, The reference solution of the control medicinal material in step (1) is prepared by a method including the following steps: The pine resin reference material was mixed with methanol, ultrasonically treated, and the filtrate was collected to obtain the reference material solution.

6. The method for constructing the characteristic spectrum of pine resin formulation particles according to claim 5, characterized in that, The ratio of the pine resin reference material to methanol was 1 g:(20-30) mL.

7. The method for constructing the characteristic spectrum of pine resin formulation particles according to claim 5, characterized in that, The ultrasonic treatment has a power of 200-300 W, a frequency of 30-50 kHz, and a duration of 15-25 min.

8. The method for constructing the characteristic spectrum of pine resin formulation particles according to claim 1, characterized in that, The column temperature for the ultra-high performance liquid chromatography method is 33-37℃.

9. The method for constructing the characteristic spectrum of pine resin formulation particles according to claim 1, characterized in that, The flow rate of the mobile phase in the ultra-high performance liquid chromatography method is 0.28-0.32 mL / min.

10. The method for constructing the characteristic map of pine resin formulation particles according to claim 1, characterized in that, The theoretical plate number of the ultra-high performance liquid chromatography method, calculated based on the peak of pine pine monomethyl ether, shall not be less than 5000.

11. The application of a method for constructing a characteristic spectrum of pine resin formulation particles as described in any one of claims 1-10 in the quality inspection of pine resin-related products; The products related to Pinus tabuliformis include Pinus tabuliformis medicinal materials, Pinus tabuliformis formula granules, or Pinus tabuliformis standard decoctions.

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