Detection method for nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules and application of detection method

Abstract

The invention discloses a detection method for nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules and application of the detection method, and relates to the technical field of traditional Chinese medicine identification. The detection method for nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules comprises the following steps: extracting cuttlebone medicinal materials, decoction pieces, standard decoction or formula granules by adopting an extraction solvent to obtain a test solution; dissolving or extracting a reference substance and / or a cuttlebone reference medicinal material by adopting an extraction solvent to obtain a reference substance solution and / or a reference medicinal material solution; determining the test solution, the reference substance solution and / or the reference medicinal material solution by adopting a liquid chromatograph; wherein a chromatographic column of the liquid chromatograph takes octadecylsilane chemically bonded silica as a stationary phase, methanol as a mobile phase A and water as a mobile phase B for gradient elution, and the reference substances comprise a uracil reference substance, a cytidine reference substance, a hypoxanthine reference substance, a thymine reference substance, a guanosine reference substance and an adenosine reference substance; the extraction solvent is water or a methanol aqueous solution with the concentration of 10-30 vol%. The detection method disclosed by the invention can be used for determining various nucleoside components in the cuttlebone, effectively identifying common counterfeit products and providing a scientific basis for formulating a quality standard of the cuttlebone.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and in particular to a method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules and an application thereof. Background Art

[0002] Cuttlebone is the dried inner shell of Sepia maindroni de Rochebrune or Sepiasculenta Hoyle, both species of the family Sepiidae. It is salty, astringent, and warm in nature, and enters the spleen and kidney meridians. It has anti-acid and analgesic properties, astringent and hemostatic properties, and astringent and anti-leucorrhea properties. It is one of the marine traditional Chinese medicines included in the 2020 edition of the Chinese Pharmacopoeia. Cuttlebone is primarily composed of calcium carbonate, but also contains chitin, proteins, peptides, amino acids, polysaccharides, nucleosides, and some other trace elements. Common counterfeit cuttlebone products are derived from the inner shells of several species of the genus Sepia in the family Sepiidae. These products are similar in appearance, with differences primarily focusing on the overall outline, the morphology of the dorsal longitudinal ribs, and the transverse striations on the ventral surface. This identification requires considerable experience and is highly subjective, making it prone to misjudgment. Therefore, establishing an objective identification method is crucial for quality control of medicinal materials.

[0003] Modern pharmacological research has shown that cuttlebone has clinically proven efficacy in neutralizing gastric acid, combating peptic ulcers, protecting mucosa, reducing phosphorus, stopping bleeding, and debridement. It boasts a proven efficacy and widespread use. Modern research indicates that cuttlebone is associated with angiogenesis, promotes bone induction in the early stages of fracture cartilage formation, and significantly impacts the proliferation and synthetic activity of osteoblasts. As a phosphate binder, cuttlebone effectively reduces serum phosphorus, calcium-phosphate deposition, and phenylthiohydantoin (PTH) levels, while having minimal effects on serum calcium. This provides a basis for better clinical use of phosphate binders. Its nucleosides have physiological activities such as modulating immune function and influencing the nervous and cardiovascular systems, as well as antibacterial and antiviral properties. The diverse pharmacological effects of cuttlebone are closely related to its components, such as nucleosides and amino acids. Various trace elements participate in numerous metabolic processes in the body. This is consistent with the clinical efficacy of cuttlebone: astringent, hemostatic, acid-reducing, analgesic, and dampness-reducing properties. In summary, in recent years, the pharmacology and clinical effects of cuttlebone have made great breakthroughs based on its basic ancient efficacy, and the medicinal value of cuttlebone has been greatly improved.

[0004] Cuttlebone contains a variety of human nucleosides. Modern methods for their determination include ultrafast liquid chromatography-tandem mass spectrometry (UFLC-MS / MS), high-performance liquid chromatography (HPLC), capillary electrophoresis, thin-layer chromatography scanning, and ultraviolet-visible spectrophotometry. Tandem mass spectrometry instruments are expensive and require sophisticated operation, while only three HPLC-based assays are currently available for detecting nucleosides in cuttlebone, making it difficult to fully and effectively characterize the nucleosides present in cuttlebone. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for detecting the nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules and its application, which can realize the determination of nucleoside components in cuttlebone and identify common counterfeits.

[0006] In order to solve the above technical problems, the present invention provides a method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules, which comprises:

[0007] Extracting cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules with an extraction solvent to obtain a test solution;

[0008] Dissolving or extracting the reference substance and / or the cuttlebone reference medicinal material with an extraction solvent to obtain a reference substance solution and / or a reference medicinal material solution;

[0009] The test solution, reference solution and / or reference medicinal material solution are measured by liquid chromatography;

[0010] The chromatographic column of the liquid chromatograph uses octadecylsilane bonded silica gel as the stationary phase, methanol as the mobile phase A, and water as the mobile phase B for gradient elution. The gradient elution curve is:

[0011] 0 min to 11 min, mobile phase A from 1% to 6%, mobile phase B from 99% to 94%;

[0012] 11 min to 30 min, mobile phase A from 6% to 9%, mobile phase B from 94% to 91%;

[0013] 30-35 min, mobile phase A: 9% → 60%, mobile phase B: 91% → 40%;

[0014] Wherein, the reference substances include uracil reference substance, cytidine reference substance, hypoxanthine reference substance, thymine reference substance, guanosine reference substance and adenosine reference substance;

[0015] The extraction solvent is water or a methanol aqueous solution with a concentration of 10 vol% to 30 vol%. Exemplarily, the concentration of the methanol aqueous solution is 12 vol%, 14 vol%, 16 vol%, 18 vol%, 21 vol%, 24 vol%, 27 vol%, or 29 vol%, but is not limited thereto. Preferably, it is 10 vol% to 20 vol%, and more preferably 10 vol%.

[0016] It should be noted that, in the present invention, only one of the control medicinal material solution or the reference substance solution can be used together with the test solution for determination, or the control medicinal material solution, the reference substance solution and the test solution can be used together for determination.

[0017] Specifically, in some embodiments of the present invention, the column length of the chromatographic column is 100 mm to 300 mm, exemplarily 100 mm, 150 mm, 200 mm, or 250 mm, but not limited thereto. The column diameter of the chromatographic column is 2.1 mm to 5 mm, exemplarily 3.0 mm, 3.9 mm, or 4.6 mm, but not limited thereto. The particle size of the stationary phase is 3 μm to 10 μm, exemplarily 3 μm, 5 μm, or 10 μm, but not limited thereto.

[0018] Preferably, in some embodiments of the present invention, the chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a particle size of 5 μm of the stationary phase, exemplified by, but not limited to, a Shim-pack VP ODS chromatographic column.

[0019] In some embodiments of the present invention, the column temperature of the chromatographic column is 28°C to 32°C, exemplified by 29.5°C, 30°C, 30.5°C, 31°C or 31.5°C, but not limited thereto.

[0020] The flow rate of the liquid chromatograph is 0.9 mL / min to 1.1 mL / min, exemplified by 0.92 mL / min, 0.95 mL / min, 0.98 mL / min, 1.03 mL / min or 1.08 mL / min, but not limited thereto.

[0021] The detection wavelength of the liquid chromatograph is 250 nm to 260 nm, exemplified by 252 nm, 254 nm, 256 nm or 258 nm, but not limited thereto.

[0022] The injection volume of the test solution is 5 μL to 15 μL, exemplified by 5.5 μL, 6 μL, 6.5 μL, 8 μL, 9.5 μL, 11 μL, or 13.5 μL, but not limited thereto. The injection volume of the reference solution is 5 μL to 15 μL, exemplified by 5.5 μL, 6 μL, 6.5 μL, 8 μL, 9.5 μL, 11 μL, or 13.5 μL, but not limited thereto. The injection volume of the control medicinal material solution is 5 to 15 μL, exemplified by 5.5 μL, 6 μL, 6.5 μL, 8 μL, 9.5 μL, 11 μL, or 13.5 μL, but not limited thereto.

[0023] Preferably, in some embodiments of the present invention, the column temperature of the chromatographic column is 30°C;

[0024] The flow rate of the liquid chromatograph was 1.0 mL / min, the detection wavelength was 254 nm, the injection volume of the test solution was 10 μL, the injection volume of the reference solution was 10 μL, and the injection volume of the reference medicinal material solution was 10 μL.

[0025] Specifically, in some embodiments of the present invention, in the step of extracting the cuttlebone medicinal material, decoction piece, standard decoction or formula granules with an extraction solvent to obtain a test solution, the cuttlebone standard decoction or formula granules is mixed with water and ultrasonically treated for 10 minutes to 60 minutes; wherein the ratio of the cuttlebone standard decoction or formula granules to water is 1g to 3g:20mL to 30mL; exemplary ratios are 1.2g:21mL, 1.4g:23mL, 1.7g:25mL, 2.2g:27mL or 2.7g:29mL, but not limited thereto. Preferably, it is 1.5g to 2g:22mL to 28mL, more preferably, it is 1.8g to 2g:24mL to 28mL, and even more preferably, it is 2g:25mL. The ultrasonic power is 200W to 400W, exemplified by, but not limited to, 220W, 250W, 280W, 300W, 340W, 350W, or 380W. Preferably, it is 250W to 350W. The ultrasonic frequency is 20kHz to 50kHz, exemplified by, but not limited to, 24kHz, 28kHz, 32kHz, 36kHz, 41kHz, or 46kHz. Preferably, it is 30kHz to 40kHz. The ultrasonic treatment duration is exemplified by, but not limited to, 15min, 30min, 40min, 50min, or 55min. Preferably, it is 15min to 40min, more preferably 20min to 35min, and even more preferably 30min.

[0026] Specifically, in some embodiments of the present invention, in the step of extracting the cuttlebone medicinal material, decoction piece, standard decoction or formula granule with an extraction solvent to obtain a test solution, the cuttlebone medicinal material or decoction piece is mixed with a methanol aqueous solution having a concentration of 10 vol% to 30 vol%, heated under reflux for 40 min to 80 min, and the solid phase obtained after solid-liquid separation is dissolved in water;

[0027] The ratio of cuttlebone medicinal materials or slices to methanol aqueous solution is 1g to 3g: 10mL to 50mL, exemplified by 1.2g: 13mL, 1.5g: 19mL, 1.8g: 25mL, 2.1g: 32mL or 2.5g: 43mL, but not limited thereto. Preferably, it is 1g to 2g: 20mL to 40mL, more preferably 1.5g to 2g: 24mL to 28mL, and more preferably 2g: 25mL. Exemplarily, the heating reflux time is 45min, 50min, 60min, 65min, 70min or 75min, but not limited thereto. Preferably, it is 50min to 70min, and more preferably 60min. The ratio of cuttlebone medicinal materials or decoction pieces to water is 1g-3g:1mL-5mL, exemplified by 1.2g:1.3mL, 1.5g:1.7mL, 1.5g:2.7mL, 2.2g:3.9mL, 2.5g:4.1mL or 2.7g:4.7mL, but not limited thereto, preferably 1g-3g:1.3mL-2.5mL, more preferably 1.4g-2.5g:1.5mL-2.5mL.

[0028] Specifically, in some embodiments of the present invention, in the step of dissolving or extracting the reference substance and / or cuttlebone control medicinal material with an extraction solvent to obtain a reference substance solution and / or a control medicinal material solution, the cuttlebone control medicinal material is mixed with a methanol aqueous solution having a concentration of 10 vol% to 30 vol%, heated under reflux for 40 min to 80 min, and the solid phase obtained after solid-liquid separation is dissolved in water to obtain the control medicinal material solution;

[0029] Among them, the ratio of cuttlebone control medicinal materials to methanol aqueous solution is 1g~3g:10mL~50mL, and the ratio of cuttlebone control medicinal materials to water is 1g~3g:1mL~5mL.

[0030] Specifically, in some embodiments of the present invention, in the step of dissolving or extracting the reference substance and / or the cuttlebone control medicinal material with an extraction solvent to obtain a reference substance solution and / or a control medicinal material solution, a uracil reference substance, a cytidine reference substance, a hypoxanthine reference substance, a thymine reference substance, a guanosine reference substance, and an adenosine reference substance are mixed with water to prepare a mixed solution containing 20 μg to 40 μg of uracil, 20 μg to 40 μg of cytidine, 20 μg to 40 μg of hypoxanthine, 5 μg to 20 μg of thymine, 10 μg to 20 μg of guanosine, and 5 μg to 20 μg of adenosine per 1 mL, thereby obtaining a reference substance solution.

[0031] Specifically, in some embodiments of the present invention, the cuttlebone slices may be cleaned cuttlebone (see page 307 of Part I of the 2020 edition of the Chinese Pharmacopoeia), or fried cuttlebone, or bran-processed cuttlebone, or vinegar-processed cuttlebone, but not limited thereto. Accordingly, the cuttlebone standard decoction refers to the preparation obtained by decocting, concentrating and freeze-drying the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules". The cuttlebone formula granules refer to the preparation obtained by extracting, separating, concentrating, drying and granulating the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules", but not limited thereto.

[0032] Specifically, in some embodiments of the present invention, the chromatogram obtained by the determination includes 9 common peaks, wherein peak 2 is uracil, peak 3 is cytidine, peak 4 is hypoxanthine, peak 5 is thymine, peak 8 is guanosine, and peak 9 is adenosine;

[0033] Taking peak 5 as the S peak, the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, and peak 9 were calculated. The relative retention time of each peak was within ±10% of the specified value. Among them, the specified values ​​of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, and peak 9 were 0.42, 0.48, 0.64, 0.72, 1.12, 1.37, 1.46, and 2.70, respectively.

[0034] Correspondingly, the present invention also discloses the use of the above-mentioned method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules in identifying cuttlebone and its counterfeits.

[0035] Accordingly, the present invention also discloses a cuttlebone identification method for distinguishing cuttlebone from its counterfeits, comprising:

[0036] Provide the substance to be identified;

[0037] Detecting the substance to be identified using the above-mentioned method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules to obtain a chromatogram;

[0038] If the chromatogram shows peaks 1 to 9 at the same time, the substance to be identified includes or is cuttlebone; otherwise, it is a counterfeit.

[0039] The implementation of the present invention has the following beneficial effects:

[0040] The present invention establishes a method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules. The method can be simultaneously applied to the determination of different types of nucleoside components in traditional Chinese medicine preparations such as cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules. It not only provides a control means for determining nucleoside components for quality control of cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules, thereby greatly saving the cost of quality standard research and future sample testing, but also provides a reference for the determination of nucleoside components in other mineral-based traditional Chinese medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 1 is a chromatogram of the cuttlebone standard decoction when different mobile phase systems are used in Example 1;

[0042] Figure 2 1 is a chromatogram of the cuttlebone standard decoction using different gradient conditions in Example 1;

[0043] Figure 3 1 is a chromatogram of the cuttlebone standard decoction using different detection wavelengths in Example 1;

[0044] Figure 4 1 is a chromatogram of the cuttlebone standard decoction at different column temperatures in Example 1;

[0045] Figure 5 1 is a chromatogram of the cuttlebone standard decoction at different flow rates in Example 1;

[0046] Figure 6 1 is a chromatogram of the cuttlebone standard decoction when different extraction solvents are used in Example 1;

[0047] Figure 7 1 is a chromatogram of the cuttlebone standard decoction when different extraction methods are used in Example 1;

[0048] Figure 8 1 is a chromatogram of the cuttlebone standard decoction at different extraction times in Example 1;

[0049] Figure 9 This is a graph showing the results of the specific property investigation in Example 1;

[0050] Figure 10 This is an overlay of chromatograms of 17 batches of cuttlebone medicinal materials in Example 1;

[0051] Figure 11 It is an overlay of chromatograms of 17 batches of cuttlebone slices in Example 1;

[0052] Figure 12 This is an overlay of chromatograms of 17 batches of cuttlebone standard decoction in Example 1;

[0053] Figure 13This is an overlay of chromatograms of three batches of cuttlebone formula granules in Example 1;

[0054] Figure 14 This is a comparison of the chromatograms of cuttlebone and two unknown counterfeits in Example 2;

[0055] Among them, peak 2 is uracil, peak 3 is cytidine, peak 4 is hypoxanthine, peak 5 is thymine, peak 8 is guanosine, and peak 9 is adenosine. DETAILED DESCRIPTION

[0056] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0057] Example 1 Establishment of a method for detecting nucleoside components in cuttlebone

[0058] 1 Preparation of test solution

[0059] Medicinal materials / decoction pieces: Take 2g of fine powder of this product, weigh accurately, place in a stoppered conical flask, add 25mL of 10% methanol, heat and reflux for 60 minutes, cool, filter, take the supernatant, evaporate to dryness, dissolve the residue in water and make up to 2mL, shake well, filter, and take the filtrate.

[0060] Standard decoction: Take an appropriate amount of this product, grind it into powder, take about 1g, place it in a stoppered conical flask, add 25mL of water, ultrasonically treat it for 30 minutes, cool it, shake it well, filter it, and take the filtrate to obtain the standard decoction.

[0061] Formula granules: Take an appropriate amount of this product, grind it into powder, take about 2g, place it in a stoppered conical flask, add 25mL of water, ultrasonically treat for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0062] 2 Preparation of reference solution

[0063] Reference substance solution: Take appropriate amounts of uracil reference substance, cytidine reference substance, hypoxanthine reference substance, thymine reference substance, guanosine reference substance, and adenosine reference substance, add water to make a mixed solution containing 30 μg of uracil, 30 μg of cytidine, 30 μg of hypoxanthine, 10 μg of thymine, 16 μg of guanosine, and 10 μg of adenosine per 1 mL, which is used as the reference substance solution.

[0064] Reference solution of control medicinal material: Take 2 g of cuttlebone control medicinal material, place it in a stoppered conical flask, add 25 mL of 10% methanol, heat and reflux for 60 minutes, cool, filter, take the supernatant, evaporate to dryness, dissolve the residue in water and make up to 2 mL, shake well, filter, and take the filtrate as the reference solution of control medicinal material.

[0065] 3 Chromatographic conditions

[0066] Chromatographic column: Shim-pack VP ODS (column length, 250 mm, inner diameter, 4.6 mm, particle size, 5 μm); gradient elution was performed using methanol as mobile phase A and water as mobile phase B, as specified in Table 1; flow rate, 1.0 mL / min; column temperature, 30°C; detection wavelength, 254 nm. The number of theoretical plates, calculated based on thymidine, should be no less than 5000.

[0067] Table 1 Gradient elution program conditions

[0068]

[0069] 4 Determination

[0070] Accurately aspirate 10 μL of reference substance solution, 10 μL of control medicinal material reference substance solution, and 10 μL of test solution respectively, inject them into liquid chromatograph, and measure to obtain the result.

[0071] 5 Methodological Investigation

[0072] 5.1 Investigation of mobile phase system

[0073] This section examines the effect of the mobile phase system on the detection method. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then the test was performed according to the following chromatographic conditions.

[0074] Chromatographic conditions: Shim-pack VP ODS column (4.6 mm × 250 mm, 5 μm); methanol and acetonitrile as mobile phase A, respectively; water as mobile phase B, at a flow rate of 1.0 mL / min; gradient elution according to the specifications in Table 1; column temperature, 30°C; detection wavelength, 254 nm; injection volume, 10 μL. Gradient conditions are shown in Table 2.

[0075] Table 2 Gradient elution program conditions

[0076]

[0077] The experimental results are as follows Figure 1 As shown, the results show that when the mobile phase system is methanol-water, the chromatographic peaks of the obtained chromatogram are evenly distributed and have good peak shapes. Therefore, methanol-water is selected as the mobile phase system for the chromatogram of the cuttlebone standard soup.

[0078] 5.2 Gradient elution procedure investigation

[0079] This section examines the effect of the gradient elution procedure on the detection method. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then the test was performed under the following chromatographic conditions.

[0080] Chromatographic conditions: Shim-pack VP ODS (4.6 mm × 250 mm, 5 μm) column; methanol as mobile phase A, water as mobile phase B, flow rate of 1.0 mL / min; gradient elution according to the specifications in Table 3; column temperature, 30°C; detection wavelength, 254 nm; injection volume, 10 μL.

[0081] Table 3 Gradient elution program

[0082]

[0083] The experimental results are as follows Figure 2 As shown, the results show that when gradient condition 3 is adopted, the separation effect of each chromatographic peak is better, so gradient condition 3 is adopted.

[0084] 5.3 Wavelength Investigation

[0085] This section examines the effect of wavelength on the detection method. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then tested under the following chromatographic conditions.

[0086] Chromatographic conditions: Shim-pack VP ODS (4.6 mm × 250 mm, 5 μm) column; methanol as mobile phase A, water as mobile phase B, flow rate of 1.0 mL / min; gradient elution according to the specifications in Table 1; column temperature, 30°C; detection wavelengths at 254 nm and 260 nm, respectively; injection volume, 10 μL.

[0087] The experimental results are as follows Figure 3 As shown in the figure, it can be seen that when the detection wavelength is 254nm and 260nm, the chromatograms have stable baselines, low interference, and the chromatographic peaks are well separated. Furthermore, when the detection wavelength is 254nm, the response value of each chromatographic peak is relatively large. Therefore, 254nm is optimally selected as the detection wavelength.

[0088] 5.4 Column temperature investigation

[0089] This section examines the effect of column temperature on the detection method. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then the test was performed according to the following chromatographic conditions.

[0090] Chromatographic conditions: Shim-pack VP ODS (4.6 mm × 250 mm, 5 μm) column; methanol as mobile phase A, water as mobile phase B, flow rate of 1.0 mL / min; gradient elution according to the specifications in Table 1; column temperatures of 28°C, 30°C, and 32°C, respectively; detection wavelength of 254 nm; injection volume of 10 μL.

[0091] The experimental results are as follows Figure 4 As shown in the figure, when the column temperature is 28℃~32℃, the separation effect of each chromatographic peak is basically the same. When the column temperature is 30℃, the response value of each chromatographic peak is relatively higher, so 30℃ is the optimal column temperature.

[0092] 5.5 Flow rate investigation

[0093] This section examines the effect of flow rate on the detection method. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then the test was performed according to the following chromatographic conditions.

[0094] Chromatographic conditions: Shim-pack VP ODS (4.6 mm × 250 mm, 5 μm) column; methanol as mobile phase A, water as mobile phase B, with flow rates of 0.9 mL / min, 1.0 mL / min, and 1.1 mL / min, respectively; gradient elution according to the specifications in Table 1; column temperature, 30°C; detection wavelength, 254 nm; injection volume, 10 μL.

[0095] The experimental results are as follows Figure 5 As shown in the figure, when the flow rate is 0.9mL / min to 1.1mL / min, the separation effect of each chromatographic peak is basically the same. When the flow rate is 1.0mL / min, the response value of each chromatographic peak is relatively higher, so 1.0mL / min is the optimal flow rate.

[0096] 5.6 Instrument Inspection

[0097] This section examines the effects of different instruments on the detection method. Specifically, a standard decoction of cuttlebone was prepared into a test solution according to the method under "1", and then tested under the following chromatographic conditions.

[0098] Chromatographic conditions: Shim-pack VP ODS column (4.6 mm × 250 mm, 5 μm); methanol as mobile phase A, water as mobile phase B, flow rate 1.0 mL / min; gradient elution according to Table 1; column temperature 30°C; detection wavelength 254 nm; injection volume 10 μL. Determinations were performed on Waters and Shimadzu instruments.

[0099] The experimental results show that there is basically no difference in the separation effect of each chromatographic peak among different instruments, and the determination method has good reproducibility.

[0100] 5.7 Investigation of test sample preparation methods

[0101] (1) Investigation of extraction solvent

[0102] This section investigates the effects of different extraction solvents on the detection method. By observing the peak shape and separation effect of the tentative 9 chromatographic peaks and calculating the "total peak area / weighed sample amount" of the 9 chromatographic peaks, the effects of different extraction solvents on the detection method are compared to select the best extraction solvent. Specifically, take an appropriate amount of standard cuttlebone decoction, grind it into powder, take about 1g, accurately weigh it, and place it in 5 parallel groups, 2 portions each, in a stoppered conical flask, accurately add water, 10% methanol, 30% methanol, 10% ethanol, 30% ethanol 25mL respectively, weigh the weight, ultrasonically treat (power 300W, frequency 40kHz) for 30 minutes, cool, shake well, filter, take the filtrate, inject and analyze, record the peak area of ​​each chromatographic peak, and calculate the "total peak area / weighed sample amount". The experimental results are shown in Table 4 and Figure 6 .

[0103] Table 4 Results of investigation on different extraction solvents

[0104]

[0105] The above results show that the peak shapes of the chromatographic peaks are poor when 10% and 30% ethanol are used as the extraction solvents. However, the peaks are well resolved and have good peak shapes when 10% methanol, 30% methanol, and water are used as the extraction solvents. In particular, the "total peak area / sample weight" ratio of the nine peaks is large when water is used as the extraction solvent, making water the optimal extraction solvent.

[0106] (2) Investigation of extraction methods

[0107] This section examines the effects of different extraction methods on the detection method. Ultrasonic treatment and heating reflux are selected as the extraction methods. By observing the peak shape and separation effect of the nine tentative chromatographic peaks and calculating the "total peak area / sample weight" of the nine chromatographic peaks, the effects of different extraction methods are compared and the optimal extraction method is selected.

[0108] Specifically, take an appropriate amount of cuttlebone standard decoction, grind it into powder, take about 1g, accurately weigh it, and place it in two parallel groups, with 2 portions in each group, in a stoppered conical flask, accurately add 25mL of water, weigh it, heat it under reflux, and ultrasonically treat it (power 300W, frequency 40kHz) for 30 minutes, let it cool, shake it well, filter it, take the filtrate, inject it into the sample for analysis, record the peak area of ​​each chromatographic peak, and calculate "total peak area / sample amount". The experimental results are shown in Table 5 and Figure 7 .

[0109] Table 5 Results of investigation on different extraction methods

[0110]

[0111] The results showed that there was no significant difference in the peak shape, separation effect, and "total peak area / sample weight" of each chromatographic peak using different extraction methods.

[0112] (3) Extraction time investigation

[0113] This section examines the effects of different extraction times on the detection method. By observing the peak shapes and separation effects of the nine tentative chromatographic peaks and calculating the "total peak area / sample weight" of the nine chromatographic peaks, the effects of different extraction times are compared and the optimal extraction time is selected.

[0114] Take an appropriate amount of standard cuttlebone decoction, grind it into powder, take about 1g, accurately weigh it, and place it in 4 parallel groups, 2 portions in each group, place it in a stoppered conical flask, accurately add 25mL of water, weigh it, and ultrasonically treat it (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, 45 minutes, and 60 minutes respectively. Let it cool, shake it well, filter it, take the filtrate, inject it into the sample for analysis, record the peak area of ​​each chromatographic peak, and calculate "total peak area / sample amount". The experimental results are shown in Table 6 and Figure 8 .

[0115] Table 6 Results of investigation at different extraction times

[0116]

[0117] The results showed that there was no significant difference in the "total peak area / sample weight" of the 9 chromatographic peaks when different extraction times were used.

[0118] 6 Methodological Validation

[0119] 6.1 Specificity Investigation

[0120] Take the cuttlebone standard decoction and blank solvent according to the preparation method under "1" to prepare the test solution and blank solvent solution, weigh the appropriate amount of hypoxanthine reference substance, thymine reference substance, guanosine reference substance, and adenosine reference substance, add water to make a mixed solution containing 30μg of hypoxanthine, 10μg of thymine, 16μg of guanosine, and 10μg of adenosine per 1mL, as the mixed standard solution, take the uracil reference substance, add water to make a uracil reference solution containing 30μg of uracil per 1mL, accurately draw 10μL of each mixed standard solution, uracil reference solution, test solution, and blank solvent solution of the above solutions, inject them into the liquid chromatograph, and determine according to the determined chromatographic conditions. The results are shown in Figure 9 .

[0121] The results showed that the test sample chromatogram had the same peaks at the corresponding retention times as the reference sample chromatogram, and there was no interference from the blank solvent, indicating that the method had good specificity. Peak 2 was uracil, peak 4 was hypoxanthine, peak 5 (S) was thymine, peak 8 was guanosine, and peak 9 was adenosine.

[0122] 6.2 Precision investigation

[0123] Take an appropriate amount of cuttlebone standard decoction, grind it into powder, take about 1 g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under "1", repeat the injection 6 times according to the determined chromatographic conditions, and use the peak 5 thymine chromatographic peak as the reference peak S. The relative retention time RSD values ​​of each chromatographic peak and the S peak are in the range of 0.04% to 0.15%, and the relative peak area RSD values ​​are in the range of 0.06% to 1.51%, all of which are less than 3.0%, indicating that the instrument precision is good.

[0124] 6.3 Repeatability Study

[0125] Take an appropriate amount of the same batch of cuttlebone standard decoction, grind it into powder, take about 1 g, accurately weigh it, and make 6 parallel portions. Prepare 6 test solutions according to the test solution preparation method determined under item "1". According to the determined chromatographic conditions, the samples were injected and analyzed. The results showed that the same batch of samples were measured 6 times. Taking the thymine chromatographic peak as the reference peak S, the relative retention time RSD values ​​of each chromatographic peak and the S peak were in the range of 0.02% to 0.08%, and the relative peak area RSD values ​​were in the range of 0.19% to 3.44%, both less than 5.0%, indicating that the method has good repeatability.

[0126] 6.4 Stability investigation

[0127] Take an appropriate amount of cuttlebone standard decoction, grind it into powder, take about 1 g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under item "1", and inject and analyze the samples at 0, 2, 4, 8, 10, and 16 hours according to the determined chromatographic conditions. The results showed that the same test solution was analyzed at 0, 2, 4, 8, 10, and 16 hours, respectively. With the thymine peak as the reference peak S, the relative retention time RSD values ​​of each chromatographic peak and the S peak were in the range of 0.04% to 0.16%, and the relative peak area RSD values ​​were in the range of 0.19% to 1.60%, indicating that the test solution was relatively stable within 16 hours.

[0128] 7 Establishment of cuttlebone chromatogram

[0129] According to the above-established construction method, cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules were measured respectively. The sample information is shown in Table 7. The thymine chromatographic peak was used as the reference peak S. The relative retention time and relative peak area of ​​each chromatographic peak and the S peak were calculated, and the RSD value was calculated. The "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" was used to identify the common peaks in the chromatograms of cuttlebone medicinal materials, decoction pieces, standard decoctions, and formula granules.

[0130] Table 7 Sample information table

[0131]

[0132] 7.1 Test results of cuttlebone medicinal materials

[0133] The chromatogram overlay of 17 batches of cuttlebone medicinal materials is shown in the figure below. Figure 10 As shown in the figure, the chromatogram of cuttlebone medicinal material has nine common peaks, which are consistent with the common peaks in the chromatogram of the standard cuttlebone decoction. Using the thymine peak as the reference peak S, the relative retention time RSD values ​​of the chromatograms of the 17 batches of cuttlebone medicinal material range from 0.05% to 0.89%, and the relative peak area RSD values ​​range from 37.32% to 128.46%. This indicates that the relative retention times of the chromatographic peaks are relatively stable, and the relative peak areas vary significantly, indicating that the proportions of the chemical components represented by the chromatographic peaks vary between batches of cuttlebone medicinal material. This difference may be related to various factors such as production area. The nine peaks of nucleoside components are consistently detected in all batches of cuttlebone medicinal material.

[0134] 7.2 Test results of cuttlebone slices

[0135] The chromatogram overlay of 17 batches of cuttlebone slices is shown in the figure below. Figure 11 As shown in the figure, the chromatograms of cuttlebone slices have nine common peaks. Using the thymine peak as the reference peak (S), the relative retention times and peak areas of each chromatographic peak were calculated. The RSD values ​​for the relative retention times of the chromatographic peaks in the 17 batches of cuttlebone slices ranged from 0.13% to 0.39%, and the RSD values ​​for the relative peak areas ranged from 26.41% to 97.17%, indicating that the relative retention times of the chromatographic peaks were relatively stable. The nine peaks of nucleoside components were stably detected in all batches of cuttlebone slices.

[0136] 7.3 Test results of cuttlebone standard decoction

[0137] The chromatogram overlay of 17 batches of cuttlebone standard decoction is shown in the figure below. Figure 12 As shown in the figure, the chromatogram of the cuttlebone standard decoction displays nine common peaks. Using peak 5 (thymine) as the reference peak (S), the relative standard deviations (RSDs) of the retention times of each chromatographic peak relative to the S peak across all 17 batches of cuttlebone standard decoction range from 0.19% to 0.60%, indicating relatively stable relative retention times. The relative standard deviations of the peak areas relative to the S peak range from 7.19% to 60.57%. The nine peaks of nucleoside components were consistently detected across all batches of cuttlebone standard decoction.

[0138] 7.4 Test results of cuttlebone formula granules

[0139] The chromatogram overlay of 3 batches of cuttlebone formula granules is shown in the figure below. Figure 13As shown in the figure, the chromatogram of the cuttlebone formula granules displays nine common peaks. Using Peak 5 (thymine) as the reference peak (S), the relative retention time (RSD) values ​​of each chromatographic peak relative to the S peak across all 17 batches of cuttlebone (golden cuttlebone) formula granules ranged from 0.10% to 0.57%, indicating relatively stable relative retention times. The relative peak area (RSD) values ​​of each chromatographic peak relative to the S peak ranged from 0.45% to 9.39%. The nine peaks of nucleoside components were consistently detected across all batches of cuttlebone (golden cuttlebone) formula granules.

[0140] 7.5 Establishment of Cuttlebone Chromatographic Standards and Peak Identification

[0141] Based on the determination results of the above-mentioned types of samples, it was determined that the chromatogram of cuttlebone should meet the following standards: there are 9 chromatographic peaks in the sample chromatogram, among which peaks 2, 4, 5, 8, and 9 should correspond to the retention times of the corresponding reference peaks, respectively. The peak corresponding to the thymine reference peak (peak 5) is the reference peak S. The relative retention times of peaks 1, 3, 6, and 7 to the S peak are calculated, and their relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.42 (peak 1), 0.48 (peak 2), 0.64 (peak 3), 0.72 (peak 4), 1.12 (peak 6), 1.37 (peak 7), 1.46 (peak 8), and 2.70 (peak 9). The nine chromatographic peaks in the cuttlebone sample were identified, and it was confirmed that peak 2 was uracil, peak 3 was cytidine, peak 4 was hypoxanthine, peak 5 was thymine, peak 8 was guanosine, and peak 9 was adenosine.

[0142] Example 2 Application of the detection method

[0143] This embodiment provides a method for identifying cuttlebone. Specifically, cuttlebone and two unknown counterfeits with similar unknown characteristics are prepared into test solution according to the conditions under item "1" in Example 1, and the sample is injected and tested according to the chromatographic conditions under item "3". The results are as follows. Figure 14 shown.

[0144] The results showed that two batches of counterfeit squid with similar characteristics to cuttlebone showed missing chromatographic peaks, one batch missing peak 9, and one batch missing peaks 5 and 6. This detection method can distinguish cuttlebone from other similar counterfeit cuttlebone.

[0145] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions, or formula granules, comprising: Extracting cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules with an extraction solvent to obtain a test solution; Dissolving or extracting the reference substance and / or the cuttlebone reference medicinal material with an extraction solvent to obtain a reference substance solution and / or a reference medicinal material solution; The test solution, reference solution and / or reference medicinal material solution are measured by liquid chromatography; The chromatographic column of the liquid chromatograph uses octadecylsilane bonded silica gel as the stationary phase, methanol as the mobile phase A, and water as the mobile phase B for gradient elution. The gradient elution curve is: 0 min to 11 min, mobile phase A from 1% to 6%, mobile phase B from 99% to 94%; 11 min to 30 min, mobile phase A from 6% to 9%, mobile phase B from 94% to 91%; 30-35 min, mobile phase A: 9% → 60%, mobile phase B: 91% → 40%; Wherein, the reference substances include uracil reference substance, cytidine reference substance, hypoxanthine reference substance, thymine reference substance, guanosine reference substance and adenosine reference substance; Wherein, the extraction solvent is water or a methanol aqueous solution with a concentration of 10 vol% to 30 vol%.

2. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, wherein: The column length of the chromatographic column is 100 mm to 300 mm, the column diameter is 2.1 mm to 5 mm, and the particle size of the stationary phase is 3 μm to 10 μm.

3. The method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1 or 2, characterized in that: The column length of the chromatographic column is 250 mm, the column diameter is 4.6 mm, and the particle size of the stationary phase is 5 μm.

4. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, wherein: The column temperature of the chromatographic column is 28°C to 32°C; The flow rate of the liquid chromatograph is 0.9 mL / min to 1.1 mL / min, and the detection wavelength is 250 nm to 260 nm; the injection volume of the test solution is 5 μL to 15 μL, the injection volume of the reference solution is 5 μL to 15 μL, and the injection volume of the reference medicinal material solution is 5 μL to 15 μL.

5. The method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1 or 4, characterized in that: The column temperature of the chromatographic column is 30°C; The flow rate of the liquid chromatograph was 1.0 mL / min, the detection wavelength was 254 nm, the injection volume of the test solution was 10 μL, the injection volume of the reference solution was 10 μL, and the injection volume of the reference medicinal material solution was 10 μL.

6. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, wherein: In the step of extracting the cuttlebone medicinal material, decoction piece, standard decoction or formula granules with an extraction solvent to obtain a test solution, the cuttlebone standard decoction or formula granules is mixed with water and ultrasonically treated for 10 minutes to 60 minutes; Among them, the ratio of standard cuttlebone decoction or formula granules to water is 1g~3g:20mL~30mL; the ultrasonic power is 200W~400W, and the ultrasonic frequency is 20kHz~50kHz.

7. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: In the step of extracting the cuttlebone medicinal material, decoction pieces, standard decoction or formula granules with an extraction solvent to obtain a test solution, the cuttlebone medicinal material or decoction pieces is mixed with a methanol aqueous solution having a concentration of 10 vol% to 30 vol%, heated under reflux for 40 min to 80 min, and the solid phase obtained after solid-liquid separation is dissolved in water; Among them, the ratio of cuttlebone medicinal materials or decoction pieces to methanol aqueous solution is 1g~3g:10mL~50mL, and the ratio of cuttlebone medicinal materials or decoction pieces to water is 1g~3g:1mL~5mL.

8. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: In the step of dissolving or extracting the reference substance and / or cuttlebone control medicinal material with an extraction solvent to obtain a reference substance solution and / or a control medicinal material solution, the cuttlebone control medicinal material is mixed with a methanol aqueous solution having a concentration of 10 vol% to 30 vol%, heated under reflux for 40 min to 80 min, and the solid phase obtained after solid-liquid separation is dissolved in water to obtain the control medicinal material solution; Among them, the ratio of cuttlebone control medicinal materials to methanol aqueous solution is 1g~3g:10mL~50mL, and the ratio of cuttlebone control medicinal materials to water is 1g~3g:1mL~5mL.

9. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: In the step of dissolving or extracting the reference substance and / or the cuttlebone reference medicinal material with an extraction solvent to obtain the reference substance solution and / or the control medicinal material solution, the uracil reference substance, the cytidine reference substance, the hypoxanthine reference substance, the thymine reference substance, the guanosine reference substance, and the adenosine reference substance are mixed with water to prepare a mixed solution containing 20 μg to 40 μg of uracil, 20 μg to 40 μg of cytidine, 20 μg to 40 μg of hypoxanthine, 5 μg to 20 μg of thymine, 10 μg to 20 μg of guanosine, and 5 μg to 20 μg of adenosine per 1 mL, thereby obtaining the reference substance solution.

10. The method for detecting nucleoside components in cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 1, characterized in that: The chromatogram obtained by the determination includes 9 common peaks, among which peak 2 is uracil, peak 3 is cytidine, peak 4 is hypoxanthine, peak 5 is thymine, peak 8 is guanosine, and peak 9 is adenosine; Taking peak 5 as the S peak, the relative retention times of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, and peak 9 were calculated. The relative retention time of each peak was within ±10% of the specified value. Among them, the specified values ​​of peak 1, peak 2, peak 3, peak 4, peak 6, peak 7, peak 8, and peak 9 were 0.42, 0.48, 0.64, 0.72, 1.12, 1.37, 1.46, and 2.70, respectively.

11. Use of the method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to any one of claims 1 to 10 in identifying cuttlebone and its counterfeits.

12. A method for identifying cuttlebone, for distinguishing cuttlebone from its counterfeit products, characterized in that: include: Provide the substance to be identified; Detecting the substance to be identified using the method for detecting nucleoside components of cuttlebone medicinal materials, decoction pieces, standard decoctions or formula granules according to any one of claims 1 to 10 to obtain a chromatogram; If the chromatogram shows peaks 1 to 9 at the same time, the substance to be identified includes or is cuttlebone; otherwise, it is a counterfeit.

Images