Construction method of specific chromatogram of pinus tabulaeformis knot formula granules, specific chromatogram and application of specific chromatogram

Abstract

The invention provides a construction method of a specific chromatogram of pinus tabulaeformis nodule formula granules, the specific chromatogram and application of the specific chromatogram. The construction method comprises the following steps: (1) preparing the pinus tabulaeformis nodule formula granules into a test solution; preparing the pinus tabulaeformis knot reference medicinal material into a reference medicinal material reference solution; preparing a silver pinocembrin monomethyl ether reference substance into a reference substance solution; and (2) respectively carrying out ultra-high performance liquid chromatography detection on the test solution, the reference substance solution of the reference medicinal material and the reference substance solution of the reference substance, and obtaining the specific chromatogram of the pinus tabulaeformis knot formula granules according to detection. According to the specific chromatogram constructed by the method provided by the invention, the specific identification of the pinus tabulaeformis formula granules is enhanced, the pinus tabulaeformis medicinal material base can be effectively identified, and a support is provided for ensuring the singleness of the source of the pinus tabulaeformis formula granules medicinal material base. The specific chromatogram constructed by the invention can comprehensively reflect the characteristic peak information of the sample, and the method is stable, high in precision and better in reproducibility.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine detection, quality control and identification, and in particular to a method for constructing a characteristic spectrum of pine knot formula particles, a characteristic spectrum and an application thereof. Background Art

[0002] Chinese pine knots are dried nodular knots or branch knots of Pinus tabulieformis Carr. or Pinus massoniana Lamb. of the Pinaceae family. It is warm in nature, slightly bitter and pungent, enters the liver and kidney meridians, dispels wind and dampness, dredges the meridians and relieves pain. It is used for wind-cold and dampness arthralgia, rheumatism, cramps, and traumatic injuries. The main active ingredient and index ingredient in Chinese pine knots is α-pinene. In addition, it also contains cellulose, lignans, vitamins, amino acids, polyphenols, volatile oils (turpentine), resins, fatty acids, flavonoids, etc.

[0003] The Chinese Pharmacopoeia (2020 edition) formulates the standard for Chinese pine knot medicinal materials based on the volatile oil extracted from Chinese pine knot medicinal materials as the research object, and stipulates the content of volatile oil and volatile component α-pinene. The Chinese pine knot formula granules are formulated granules obtained by extracting Chinese pine knot slices, encapsulating volatile oils, concentrating, drying, and preparing them. There is a big difference between the material basis of the Chinese pine knot formula granules and the material basis of the medicinal materials. In addition to volatile components, there is almost no comprehensive evaluation method for water-soluble components in the field of Chinese pine knot formula granules. Silver pine monomethyl ether has strong antibacterial properties and is mainly found in plants of the genus Pinus in the Coniferaceae family. Silver pine monomethyl ether has analgesic and anti-inflammatory effects, and as a water-soluble component, it has a good transfer rate in the formula granules. Therefore, the characteristic spectrum of water-soluble substances in Chinese pine knot is established based on silver pine monomethyl ether, which can achieve effective quality control from Chinese pine knot medicinal materials to granules, and the evaluation method is more comprehensive and objective.

[0004] Patent CN115541792A discloses the use of ultra-high liquid phase to study the content and characteristic spectrum of pine pine monomethyl ether in pine knot formula particles. However, this method is not only cumbersome to detect gradient changes, but also the characteristic spectrum obtained has fewer characteristic peaks, showing only 4 characteristic peaks, which cannot fully present the material basis of the pine knot formula particles, and the origin of pine knot cannot be identified by these 4 characteristic peaks.

[0005] The base materials of Chinese pine knot are Chinese pine and Pinus massoniana. Chinese pine knot formula granules need to have a fixed base material. Currently, there is no research on the identification of Chinese pine knot base materials. In order to ensure the uniqueness of the source of the base materials of Chinese pine knot formula granules, it is necessary to effectively identify the base materials of Chinese pine knot. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for constructing a characteristic spectrum of pine knot formula particles, a characteristic spectrum and applications thereof.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for constructing a characteristic spectrum of pine knot formula particles, the method for constructing the characteristic spectrum comprising the following steps:

[0009] (1) Mixing the pine knot formula particles and methanol aqueous solution, and ultrasonically treating them to prepare a test solution; preparing a reference medicinal material reference solution from the pine knot reference medicinal material; mixing the pine knot monomethyl ether reference substance with methanol to prepare a reference substance reference solution;

[0010] (2) subjecting the test solution, the control medicinal material reference solution and the control substance reference solution to ultra-high performance liquid chromatography detection, respectively, screening out common peaks with consistent retention time, good peak shape and high separation degree as characteristic peaks according to the detection results, and selecting the characteristic peaks with a certain composition and good peak shape as S peaks, and calculating the relative retention times of other characteristic peaks relative to the S peak, thereby obtaining the characteristic spectrum of the pine knot formula particles;

[0011] Wherein, the fluidity of the high performance liquid chromatography in step (2) comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution;

[0012] The ultra-high performance liquid chromatography adopts gradient elution, and the specific process of the gradient elution is as follows:

[0013] In the 0th to 10th minute, the volume fraction of mobile phase A changes uniformly from 4-6% (for example, 4%, 4.5%, 5%, 5.5% or 6%) to 18-22% (for example, 18%, 19%, 20%, 21% or 22%), and the volume fraction of mobile phase B changes uniformly from 94-96% (for example, 94%, 94.5%, 95%, 95.5% or 96%) to 78-82% (for example, 78%, 79%, 80%, 81% or 82%). Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0014] In the 10th to 25th minute, the volume fraction of mobile phase A changes uniformly to 33-37% (for example, 33%, 34%, 35%, 36% or 37%, etc.), and the volume fraction of mobile phase B changes uniformly to 63-67% (for example, 63%, 64%, 65%, 66% or 67%, etc.). Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0015] At 25-32 minutes, the volume fraction of mobile phase A changes uniformly to 63-67% (for example, 63%, 64%, 65%, 66% or 67%, etc.), and the volume fraction of mobile phase B changes uniformly to 33-37% (for example, 33%, 34%, 35%, 36% or 37%, etc.). Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0016] At 32-34 minutes, the volume fraction of mobile phase A changes uniformly to 4-6% (for example, 4%, 4.5%, 5%, 5.5% or 6%, etc.), and the volume fraction of mobile phase B changes uniformly to 94-96% (for example, 94%, 94.5%, 95%, 95.5% or 96%, etc.). Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0017] The present invention aims at the technical defect that the prior art cannot distinguish different base crude pine knots (pine knots and masson pine knots) and provides a new method for constructing a characteristic spectrum of pine knot formula particles. The obtained characteristic spectrum strengthens the specific identification of pine knot formula particles and can effectively identify the base of pine knot medicinal materials, providing support for ensuring the singleness of the source of the base of pine knot formula particles. The characteristic spectrum constructed by the present invention can fully reflect the characteristic peak information of the sample, and the method is stable, with high precision and good reproducibility.

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

[0019] Preferably, the ratio of the pine knot formula particles and 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 point values ​​within this numerical range can be selected, and they will not be described one by one here.

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

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

[0022] The Chinese pine knot reference medicinal material was mixed with methanol, subjected to ultrasonic treatment, and the subsequent filtrate was taken to obtain a reference medicinal material solution.

[0023] Preferably, the ratio of the Chinese pine knot control medicinal 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 point values ​​within this numerical range can be selected, which will not be described one by one here.

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

[0025] Preferably, the volume fraction of the aqueous formic acid solution is 0.05-0.15%, for example, 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 point values ​​within this numerical range can be selected and will not be described here one by one.

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

[0027] Preferably, the detection wavelength of the ultra-high performance liquid chromatography is 320-350 nm (for example, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm or 350 nm, etc.), and other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

[0028] Preferably, the chromatographic column filler of the ultra-high performance liquid chromatography is octadecylsilane bonded silica gel.

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

[0030] In a second aspect, the present invention provides a characteristic spectrum of pine knot formula particles, wherein the characteristic spectrum of the pine knot formula particles is obtained by the method for constructing the characteristic spectrum of pine knot formula particles as described in the first aspect;

[0031] The number of characteristic peaks in the characteristic spectrum is 11, the number of S peaks is 1, and they are sorted from small to large in order of retention time, with peak 11 being the S peak, and 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] The peak 11 component is argentopsis monomethyl ether.

[0033] In the third aspect, the present invention provides a method for constructing a characteristic spectrum of pine knot formula particles as described in the first aspect, and an application of the characteristic spectrum of pine knot formula particles as described in the second aspect in the quality inspection of pine knot related products.

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

[0035] In a fourth aspect, the present invention provides a method for identifying pine knot base, the method comprising the following steps:

[0036] The pine knot medicinal material is detected according to the ultra-high performance liquid chromatography detection method of the first aspect to obtain a characteristic spectrum of the pine knot medicinal material, showing 11 characteristic peaks, and the area ratios of peak 4 / peak 6, peak 5 / peak 6, and peak 7 / peak 6 are calculated;

[0037] Preferably, when the area ratio of Peak 4 / Peak 6 is ≥0.9 (for example, 0.9, 1.0, 1.1, 1.2, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0, etc.), the area ratio of Peak 5 / Peak 6 is ≥0.4 (for example, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3 or 1.4, etc.), and the area ratio of Peak 7 / Peak 6 is ≥5.0 (for example, 5.0, 5.5, 6.0, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, etc.), the origin of the Chinese pine knot medicinal material is Chinese pine, and other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

[0038] Preferably, when the area ratio of Peak 4 / Peak 6 is less than 0.9 (for example, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1, etc.), the area ratio of Peak 5 / Peak 6 is less than 0.4 (for example, 0.3, 0.2 or 0.1, etc.), and the area ratio of Peak 7 / Peak 6 is less than 5.0 (for example, 4.9, 4.7, 4.5, 4.3, 4.1, 3.5, 3.0, 2.5, 2.0, 1.5 or 1.0, etc.), the origin of the Chinese pine knot medicinal material is Pinus massoniana, and other specific point values ​​within the above numerical range can be selected, which will not be described one by one here.

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

[0040] The present invention aims at the technical defect that the prior art cannot distinguish different base crude pine knots (pine knots and masson pine knots) of medicinal materials, and provides a new method for constructing a characteristic spectrum of pine knot formula particles. The obtained characteristic spectrum strengthens the specific identification of pine knot formula particles, and can effectively identify the base of pine knot, providing support for ensuring the single source of the base of the pine knot formula particles. The characteristic spectrum constructed by the present invention can fully reflect the characteristic peak information of the sample, and the method is stable, with high precision and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0043] Figure 3 It is the confirmation diagram of peak No. 11 in Example 1;

[0044] Figure 4 is the pine knot atlas obtained in Application Example 1;

[0045] Figure 5 This is the Masson pine knot map obtained in Application Example 1. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0047] In the following examples, unless otherwise specified, the reagents and consumables used were purchased from conventional reagent manufacturers in the field; unless otherwise specified, the experimental methods and technical means used were conventional methods and means in the field.

[0048] Example 1

[0049] This embodiment provides a method for constructing a characteristic spectrum of pine knot formula particles and its application, and the specific steps are as follows:

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

[0051] Preparation of reference substance solution: Add methanol to the reference substance of argentopsis monomethyl ether to obtain a 0.1 mg / L solution as the reference substance solution.

[0052] Preparation of the test solution: Take pine knot formula granules, grind them into powder, accurately weigh 0.5 g, put them in a stoppered conical flask, accurately add 25 mL of 70% methanol, stopper it, weigh it, and treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 min. Take it out and let it cool, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0053] Chromatographic conditions and system suitability test: Determine according to ultra-performance liquid chromatography, use octadecylsilane bonded silica gel as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.7 μm), acetonitrile is mobile phase A, and 0.1% formic acid solution by volume is mobile phase B. Gradient elution is performed as specified in the following table. The flow rate is 0.3 mL / min, the column temperature is 35°C, the detection wavelength is 335 nm, and the theoretical plate number calculated based on the monomethyl ether peak of argentopsis pine is not 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 spectrum: Take 15 batches of pine knot formula granules (as shown in Table 1), prepare the test solution according to the above method, accurately pipette 1 μL of control medicinal material reference solution, reference substance reference solution, and test solution into ultra-high performance liquid chromatography, and measure according to the above chromatographic conditions to obtain the characteristic spectrum.

[0056] The “Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System” (2012 version) was used to analyze and obtain the superimposed spectra of 15 batches of test samples and generate the reference characteristic spectra. The superimposed color spectra of 15 batches of test sample solutions are shown in Figure 1 , where S2(11)-S16(11) refer to 15 batches of samples respectively, S1(11) is the fitting spectrum, and the generated characteristic spectrum is shown in Figure 2 The numbers 1-11 in the spectra 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 Origin Similarity with 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 Wen 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 Li 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 West 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 spectra of 15 batches of pine knot formula particles and the control characteristic spectra is 0.984-1.000, indicating that the differences between the spectra of the batches are small and the generated control characteristic spectra are representative.

[0060] Table 2

[0061]

[0062]

[0063] It can be seen from the characteristic spectra of 15 batches of pine knot test samples that 15 batches of pine knot formula granules have 11 characteristic peaks in the UPLC characteristic spectra. The relative retention time of each characteristic peak is slightly different, all within 1.0%, which meets the quality control requirements. The average value of the relative retention time is selected as the measured value. Combined with the subsequent durability test results, peak 11 is selected as the reference peak, labeled S, and the relative retention time of characteristic peaks 1-10 is calculated. The relative retention time should be within ±10% of the specified value, and the specified value is 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 in the figure, the retention time of peak 11 in the sample spectrum is consistent with that of the reference sample spectrum of argentopsis monomethyl ether. Therefore, it can be confirmed that peak 11 is argentopsis monomethyl ether.

[0065] Test example:

[0066] Methodological investigation:

[0067] Repeatability:

[0068] Take 6 portions of pine knot formula particles (batch number: K445CP15), and measure them according to the method of Example 1 to obtain their characteristic spectrum. Take peak 11 as the reference peak, calculate its relative peak area and relative retention time, and calculate RSD, and the results are shown in Table 3-4. According to the repeatability investigation results, the relative retention time RSD of each characteristic peak is in the range of 0.05-0.92%, and the relative peak area RSD is in the range of 0.30-1.92%, indicating that the repeatability of the characteristic spectrum is good.

[0069] Table 3

[0070]

[0071] Table 4

[0072]

[0073]

[0074] Precision:

[0075] Take 1 portion of pine knot formula particles (batch number: K445CP15), measure according to the method in Example 1, inject 6 injections continuously, and obtain its characteristic spectrum. Take peak 11 as the reference peak, calculate its relative peak area and relative retention time, and calculate RSD, the results are shown in Table 5-6. According to the precision investigation results, the relative retention time RSD of each characteristic peak is in the range of 0.03% to 0.78%, and the relative peak area RSD is in the range of 0.40% to 1.59%, indicating that the precision of the instrument is good.

[0076] Table 5

[0077]

[0078] Table 6

[0079]

[0080]

[0081] Durability:

[0082] Take 3 portions of pine knot formula particles (batch number: K445CP15), prepare the test solution according to the method in Example 1, and measure under the conditions of different concentrations of formic acid solution (0.08%, 0.10%, 0.12%) (other conditions are consistent with Example 1). Take peak 11 as the reference peak, calculate its relative peak area and relative retention time, and calculate RSD, the results are shown in Table 7-8. The experimental results of different acid concentrations show that the relative retention time of each characteristic peak is within the range of ±10% of the specified value, and the RSD of the relative peak area is within the range of 0.35-1.68%, which meets the system applicability requirements, indicating that the method has good durability for different acid concentrations.

[0083] Table 7

[0084]

[0085] Table 8

[0086]

[0087]

[0088] Application Example 1

[0089] This application example is used to identify the medicinal materials of Pinus tabulaeformis pine knot and Pinus massoniana pine knot.

[0090] (1) Accurately weigh 1.0 g of pine pine powder from 15 batches respectively, place in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, and ultrasonically treat (power 250 W, frequency 40 kHz) for 20 min. Take the filtrate to obtain the pine pine test solution.

[0091] (2) 1.0 g of pine knot powder from 10 batches of Masson pine was accurately weighed and placed in a stoppered conical flask. 25 mL of methanol was accurately added, the flask was sealed, and ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 20 min. The filtrate was collected to obtain the Masson pine knot test solution.

[0092] (3) Accurately pipette 1 μL of the above different original pine knot medicinal material test sample solutions, inject into the ultra-high performance liquid chromatograph, and measure according to the chromatographic conditions in Example 1.

[0093] The results are as follows Figure 4-5 As shown, both the Pinus tabulaeformis spectrum and the Masson pine spectrum present 11 characteristic peaks, and the ratios of Peak 4 / Peak 6, Peak 5 / Peak 6, and Peak 7 / Peak 6 are calculated. The results are shown in Table 9.

[0094] Table 9

[0095]

[0096]

[0097] From the above results, it can be seen that the ratios of Peak 4 / Peak 6, Peak 5 / Peak 6, and Peak 7 / Peak 6 of Masson pine are significantly smaller than those of Pinus tabulaeformis. Therefore, the peak area ratio of Peak 4, Peak 5, Peak 7 and Peak 6 can be used as a means to identify two different base crude oil pine knots.

[0098] By comparing the characteristic spectra of multiple batches of Pinus tabulaeformis and Pinus massoniana, it was found that the relative peak area between peak 4 and peak 6 was not less than 0.9, the relative peak area between peak 5 and peak 6 was not less than 0.4, and the relative peak area between peak 7 and peak 6 was not less than 5.0, all of which were Pinus tabulaeformis, and vice versa, they were Pinus massoniana. The 0.9, 0.4 and 5.0 were obtained by multiplying the measured minimum value of Pinus tabulaeformis by 0.7 and then being accurate to the tenth place.

[0099] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0100] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

Claims

1. A method for constructing a characteristic spectrum of pine knot formula particles, characterized in that: The method for constructing the characteristic map comprises the following steps: (1) Mixing the pine knot formula particles and methanol aqueous solution, and ultrasonically treating them to prepare a test solution; preparing a reference medicinal material reference solution from the pine knot reference medicinal material; mixing the pine knot monomethyl ether reference substance with methanol to prepare a reference substance reference solution; (2) subjecting the test solution, the control medicinal material reference solution and the control substance reference solution to ultra-high performance liquid chromatography detection, respectively, screening out common peaks with consistent retention time, good peak shape and high separation degree as characteristic peaks according to the detection results, and selecting the characteristic peaks with a certain composition and good peak shape as S peaks, and calculating the relative retention times of other characteristic peaks relative to the S peak, thereby obtaining the characteristic spectrum of the pine knot formula particles; Wherein, the fluidity of the high performance liquid chromatography in step (2) comprises mobile phase A and mobile phase B, the mobile phase A is acetonitrile, and the mobile phase B is a formic acid aqueous solution; The ultra-high performance liquid chromatography method adopts gradient elution, and the specific process of the gradient elution is as follows: From 0 to 10 minutes, the volume fraction of mobile phase A changes from 4-6% to 18-22% at a constant rate, and the volume fraction of mobile phase B changes from 94-96% to 78-82% at a constant rate; From 10 to 25 minutes, the volume fraction of mobile phase A changes to 33-37% at a constant rate, and the volume fraction of mobile phase B changes to 63-67% at a constant rate; From 25 to 32 minutes, the volume fraction of mobile phase A changes uniformly to 63-67%, and the volume fraction of mobile phase B changes uniformly to 33-37%; At 32-34 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%.

2. The method for constructing a characteristic spectrum of pine knot formula particles according to claim 1, characterized in that: The volume fraction of methanol in the methanol aqueous solution in step (1) is 50%-90%; Preferably, the ratio of the pine knot formula particles to the methanol aqueous solution is 1 g: (40-60) mL.

3. The method for constructing a characteristic spectrum of pine knot formula particles according to claim 1 or 2, characterized in that: The power of the ultrasonic treatment is 200-300W, the frequency is 30-50kHz, and the time is 20-40min.

4. The method for constructing a characteristic spectrum of turpentine formula particles according to any one of claims 1 to 3, characterized in that: The control medicinal material reference solution in step (1) is prepared by a method comprising the following steps: The Chinese pine knot reference medicinal material was mixed with methanol, subjected to ultrasonic treatment, and the subsequent filtrate was taken to obtain a reference medicinal material solution.

5. The method for constructing a characteristic spectrum of pine knot formula particles according to claim 4, characterized in that: The ratio of the Chinese pine knot reference medicinal material to methanol is 1 g: (20-30) mL; Preferably, the ultrasonic treatment has a power of 200-300 W, a frequency of 30-50 kHz, and a time of 15-25 min.

6. The method for constructing a characteristic spectrum of pine knot formula particles according to any one of claims 1 to 5, characterized in that: The volume fraction of the formic acid aqueous solution is 0.05-0.15%.

7. The method for constructing a characteristic spectrum of pine knot formula particles according to any one of claims 1 to 6, characterized in that: The column temperature of the ultra-high performance liquid chromatography is 33-37°C; Preferably, the flow rate of the ultra-high performance liquid chromatography mobile phase is 0.28-0.32 mL / min; Preferably, the detection wavelength of the ultra-high performance liquid chromatography is 320-350 nm; Preferably, the chromatographic column filler of the ultra-high performance liquid chromatography is octadecylsilane bonded silica gel; Preferably, the theoretical plate number of the ultra-high performance liquid chromatography is not less than 5000, calculated based on the argentopsis monomethyl ether peak.

8. A characteristic spectrum of pine knot formula particles, characterized in that: The characteristic spectrum of the pine knot formula particles is obtained by the method for constructing the characteristic spectrum of the pine knot formula particles according to any one of claims 1 to 7; The number of characteristic peaks in the characteristic spectrum is 11, the number of S peaks is 1, and they are sorted from small to large in order of retention time, with peak 11 being the S peak, and 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%; The peak 11 component is argentopsis monomethyl ether.

9. A method for constructing a characteristic spectrum of pine knot formula particles according to any one of claims 1 to 7, and application of the characteristic spectrum of pine knot formula particles according to claim 8 in quality inspection of pine knot related products; Preferably, the pine knot related products include any one of pine knot medicinal materials, pine knot formula granules or pine knot standard decoction, or a combination of at least two of them.

10. A method for identifying pine knot root, characterized in that: The identification method of the pine knot base comprises the following steps: Detecting the Chinese pine knot medicinal material according to the ultra-high performance liquid chromatography detection method described in any one of claims 1 to 7, obtaining a characteristic spectrum of the Chinese pine knot medicinal material, presenting 11 characteristic peaks, and calculating the area ratios of peak 4 / peak 6, peak 5 / peak 6, and peak 7 / peak 6; Preferably, when the peak 4 / peak 6 area ratio is ≥0.9, the peak 5 / peak 6 area ratio is ≥0.4, and the peak 7 / peak 6 area ratio is ≥5.0, the origin of the Chinese pine knot medicinal material is Chinese pine; Preferably, when the area ratio of Peak 4 / Peak 6 is less than 0.9, the area ratio of Peak 5 / Peak 6 is less than 0.4, and the area ratio of Peak 7 / Peak 6 is less than 5.0, the origin of the Chinese pine knot medicinal material is Pinus massoniana.

Images