Characteristic chromatogram detection method of traditional Chinese medicine preparation or intermediate extract thereof and application of characteristic chromatogram detection method

Abstract

The invention relates to a specific chromatogram detection method and application of a traditional Chinese medicine preparation or an intermediate extract thereof. The detection method comprises the following steps: (1) preparing a test solution of the traditional Chinese medicine preparation or the intermediate extract thereof; the traditional Chinese medicine preparation or an intermediate extract thereof is prepared from the following raw materials: rhizoma pinellinae praeparata, poria cocos, rhizoma anemarrhenae, radix scrophulariae, radix gentianae, vinegar tortoise shell, spina gleditsiae and calcined oyster shell. And (2) sucking the test solution, injecting the test solution into a liquid chromatograph, determining according to a high performance liquid chromatography, and recording a chromatogram, thereby obtaining the target product. The conditions of the high performance liquid chromatography are as follows: a chromatographic column is an octadecyl silane bonded silica gel chromatographic column, acetonitrile is taken as a mobile phase A, an acid solution is taken as a mobile phase B, and gradient elution is performed. According to the method, the characteristic chromatogram of the infantile poria cocos and dragon precocity compound preparation or the intermediate extract thereof is established by utilizing the high performance liquid chromatography, and the characteristic chromatogram is stable in baseline, large in information amount, uniform in chromatographic peak distribution and good in separation effect, and has the advantages of rapidness, simplicity, convenience and good reproducibility and stability.

Description

Technical Field

[0001] The present invention relates to the field of quality control of traditional Chinese medicines, and in particular to a characteristic spectrum detection method for a traditional Chinese medicine preparation or an intermediate extract thereof and an application thereof. Background Art

[0002] Patent application CN111759938A discloses a Chinese medicine composition for treating precocious puberty in children, and the Chinese medicine preparation prepared by the composition (hereinafter referred to as "Xiaoer Linglong Precocious Compound Preparation") mainly treats phlegm and heat, softens and disperses hard masses. Precocious puberty in girls, according to TCM syndrome differentiation, belongs to phlegm-heat internal knot syndrome, with symptoms of breast development or swelling and pain accompanied by hard masses, dark areola color, fat body, red tongue, thin yellow or greasy fur, and slippery pulse. The prescription is composed of eight herbs, including Pinellia ternata, Poria, Rhizoma Anemarrhenae, Scrophulariae, Gentiana, Vinegar Turtle Shell, Gleditsia sinensis, and Calcined Oyster, and is used to treat precocious puberty in girls.

[0003] The main active ingredients of Xiaoer Linglong Zaoshu compound preparation include alkaloids, volatile oils, saponins, organic acids, monoterpenoid glycosides, flavonoids and other substances. Modern research shows that Anemarrhena asphodeloides can improve learning and memory disorders, osteoporosis, Alzheimer's disease and other symptoms, and also has anti-inflammatory, anti-depressant, anti-pathogenic microorganism, hypoglycemic, antipyretic, anti-tumor, anti-platelet aggregation and other pharmacological effects. Anemarrhena asphodeloides contains steroidal saponins, alkaloids, biphenylpyrazone, lignans, polysaccharides, amino acids, volatile oils and other ingredients, among which steroidal saponins and aglycones are its most important active ingredients, accounting for about 6%, and have a wide range of pharmacological activities. The main active ingredients in gentian are iridoid glycosides, including gentiopicroside, swertiamarin, swertiain, loganinic acid, etc., which have multiple biological activities such as antiviral, antitumor, liver protection and choleretic. Gentianine in gentian also has antipyretic effects. The volatile oil and organic acid components have the effects of regulating smooth muscle, anti-oxidation and stimulating the central nervous system. Gleditsia sinensis mainly contains flavonoids, triterpenes, phenolic acids, and coumarins, among which flavonoids are its main active ingredients. Taxifolin is a flavonoid compound with a high content in Gleditsia sinensis, which has antioxidant, anti-inflammatory, antibacterial, anti-tumor, neuroprotective, and improvement of myocardial cell fibrosis. Taurine in calcined oyster is a sulfur-containing amino acid, but it is not a component of protein. It is also called taurine and taurine. Taurine has a wide range of biological functions. Studies have found that taurine has multiple effects such as visual development, neural development, detoxification, calcium flow regulation, bile acid binding, osmotic pressure regulation, cell membrane stabilization, etc., as well as anti-arrhythmic, myocardial protection, lowering blood pressure, and lowering blood sugar in the cardiovascular system. The above-mentioned ingredients are the main active ingredients of Xiaoer Linglong Zaoshu Granules.

[0004] In order to control the quality level of traditional Chinese medicine preparations and ensure the safety and efficacy of clinical medication, it is necessary to conduct quality control on traditional Chinese medicine preparations. Since traditional Chinese medicine and its compound preparations are multi-component complex systems, the current content determination methods are not sufficient to solve this problem.

[0005] At present, there is no method for identifying the characteristic spectrum of the traditional Chinese medicine flavor of the Linglong Zaoshu compound preparation for children, so it is necessary to establish a detection method that can provide rich identification information and evaluate its quality, so as to achieve effective quality control and ensure the safety and efficacy of clinical medication. Summary of the invention

[0006] One of the purposes of the present invention is to overcome the shortcomings of the prior art and provide a method for detecting a characteristic spectrum of a Chinese medicine preparation or its intermediate extract. The method has a stable spectrum baseline, a large amount of information, uniform distribution of chromatographic peaks and a good separation effect, and is fast, simple, has good reproducibility and stability.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is:

[0008] The present invention provides a method for detecting characteristic spectra of a Chinese medicine preparation or an intermediate extract thereof, comprising the following steps:

[0009] (1) Preparation of test sample solutions of Chinese herbal medicines or their intermediate extracts;

[0010] The Chinese medicine preparation or its intermediate extract is made from the following raw materials: French pinellia, Poria, Anemarrhena, Scrophularia, Gentiana, Vinegar tortoise shell, Gleditsia sinensis, and calcined oyster shell;

[0011] (2) Pipette the test solution, inject it into the liquid chromatograph, measure it according to the high performance liquid chromatography method, and record the chromatogram to obtain;

[0012] The conditions of the high performance liquid chromatography method include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, acetonitrile is used as mobile phase A, and acid solution is used as mobile phase B, and gradient elution is performed.

[0013] The present invention uses high performance liquid chromatography and selects a specific mobile phase to establish a characteristic spectrum of the Xiaoer Linglong Zaoshu compound preparation or its intermediate extract, the spectrum has a stable baseline, a large amount of information, a uniform distribution of chromatographic peaks, a good separation effect, and is fast, simple, and has good reproducibility and stability. The characteristic spectrum obtained by the method of the present invention determines 9 characteristic peaks, identifies the four medicinal flavors of Anemarrhena, Gentiana, Gleditsia sinensis, and Scrophularia ningpoensis in Xiaoer Linglong Zaoshu granules, and identifies the four chemical components of mangiferin, gentiopicroside, harpagoside, and cinnamic acid.

[0014] By using the characteristic spectrum detection method of the present invention, the quality control level of the Linglong Zaoshu compound preparation for children or its intermediate extracts can be further improved, the quality of traditional Chinese medicine can be effectively controlled, and the safety and efficacy of clinical medication can be ensured.

[0015] In the present invention, the intermediate extract refers to the intermediate product obtained by extracting, concentrating and drying the prescription drug flavor, including but not limited to the extract, concentrated solution or dry paste powder.

[0016] In the present invention, the types of the preparation include but are not limited to granules, tablets, capsules, pills, dry suspensions, oral liquids or decoctions.

[0017] Preferably, the Chinese medicine preparation is prepared from the following raw materials in parts by weight: 1-8 parts of Pinellia ternata, 2-10 parts of Poria cocos, 2-10 parts of Anemarrhena asphodeloides, 2-10 parts of Scrophularia ningpoensis, 1-8 parts of Gentiana scabra, 2-10 parts of Vinegar-free Tortoise Shell, 2-10 parts of Gleditsia sinensis, and 2-10 parts of calcined Oyster shell.

[0018] Preferably, the Chinese medicine preparation is prepared from the following raw materials in parts by weight: 2-6 parts of Pinellia ternata, 4-8 parts of Poria cocos, 4-8 parts of Anemarrhena asphodeloides, 4-8 parts of Scrophularia ningpoensis, 2-6 parts of Gentiana scabra, 4-8 parts of Vinegar-free Tortoise Shell, 4-8 parts of Gleditsia sinensis, and 4-8 parts of calcined Oyster shell.

[0019] Preferably, the Chinese medicine preparation is prepared from the following raw materials in parts by weight: 3 parts of Pinellia ternata, 5 parts of Poria cocos, 5 parts of Anemarrhena asphodeloides, 5 parts of Scrophularia ningpoensis, 3 parts of Gentiana scabra, 5 parts of Vinegar-free Tortoise Shell, 5 parts of Gleditsia sinensis, and 5 parts of calcined Oyster shell.

[0020] Preferably, step (1) comprises: mixing the Chinese medicine preparation or its intermediate extract with an extraction solvent, extracting, filtering, and obtaining the test solution.

[0021] Preferably, the extraction solvent comprises ethyl acetate or water-saturated n-butanol, preferably ethyl acetate.

[0022] The present invention preferably uses ethyl acetate as a solvent to dissolve the test sample Xiaoer Linglong Zaoshu Granules or its intermediate extract. Compared with other solvents, the characteristic peak response value is higher.

[0023] Preferably, in step (1), the extraction method comprises any one of ultrasound, extraction or column chromatography separation or a combination of at least two thereof, preferably a combination of ultrasound and extraction.

[0024] The present invention preferably uses a combination of ultrasound and extraction, first subjecting the sample to be tested to ultrasound treatment and then extraction. Compared with the method of ultrasound alone or ultrasound followed by column separation, the obtained spectrum is better and the information presented is richer.

[0025] Preferably, step (1) specifically comprises the following steps: taking the traditional Chinese medicine preparation into a container, adding water, ultrasonic treatment, transferring to a separatory funnel, extracting with ethyl acetate, recovering the solvent with ethyl acetate until dry, adding 70% methanol to dissolve the residue, transferring to a volumetric flask, adding 70% methanol to dilute to the scale, shaking well, filtering, and taking the filtrate to obtain the test solution.

[0026] Preferably, step (1) specifically comprises the following steps: taking the Chinese medicine preparation, placing it in a stoppered conical flask, accurately adding 25 ml of water, ultrasonically treating (power 250 W, frequency 40 kHz) for 30 minutes, transferring it to a separatory funnel, extracting it twice with ethyl acetate, each time with 25 ml, combining the ethyl acetate solution to recover the solvent to dryness, adding 70% methanol to dissolve the residue, and transferring it to a 10 ml volumetric flask, adding 70% methanol to dilute to the scale, shaking well, filtering, and taking the filtrate to obtain the test solution.

[0027] Illustratively, in the preparation of the above-mentioned test solution, if the Chinese medicine composition is an intermediate extract, the sampling amount is 1 g, and if it is a granule, the sampling amount is 2 g.

[0028] Preferably, in step (2), the elution gradient of the high performance liquid chromatography is as follows:

[0029] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 7→20 93→80 30～40 20→25 80→75 40～50 25 75 50～60 25→30 75→70 60～75 30→80 70→20 75～95 80→90 20→10。

[0030] In the above elution gradient, “→” represents a change from one ratio to another. For example, in the time period of 0 to 30 minutes, the volume ratio of mobile phase A gradually changes from 7% to 20%, and the volume ratio of mobile phase B gradually changes from 93% to 80%.

[0031] The present invention further preferably adopts the above-mentioned gradient for elution, which can obtain a spectrum with better separation effect and richer chromatographic peak information.

[0032] Preferably, in step (2), the chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0033] Preferably, in step (2), the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid solution with a concentration of 0.1%.

[0034] Preferably, in step (2), the detection wavelength of the high performance liquid chromatography is 190 to 400 nm, for example, 210 nm, 230 nm, 258 nm, 270 nm, 290 nm, 310 nm, etc., preferably 258 nm.

[0035] The present invention further prefers that the detection wavelength is 258 nm, at which the amount of information is greater and the baseline is more stable.

[0036] Preferably, in step (2), the flow rate of the HPLC is 0.8-1.2 ml / min, such as 0.8 ml / min, 1.0 ml / min, 1.2 ml / min, preferably 1.0 ml / min.

[0037] Preferably, in step (2), the column temperature of the HPLC is 28-32°C, such as 28°C, 30°C, 32°C, etc., preferably 30°C.

[0038] Preferably, in step (2), the injection volume of the HPLC method is 10 μL.

[0039] Preferably, in step (2), the conditions of the high performance liquid chromatography include: the chromatographic column is an octadecylsilane bonded silica gel column, the mobile phase A is acetonitrile, the mobile phase B is a phosphoric acid solution with a concentration of 0.08%-0.12%, the detection wavelength is 190-400nm, the flow rate is 0.8-1.2ml / min, the column temperature is 28-32°C, and the elution gradient is as follows:

[0040] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 7→20 93→80 30～40 20→25 80→75 40～50 25 75 50～60 25→30 75→70 60～75 30→80 70→20 75～95 80→90 20→10。

[0041] Preferably, in step (2), the conditions of the high performance liquid chromatography include: the chromatographic column is an octadecylsilane bonded silica gel column, the mobile phase A is acetonitrile, the mobile phase B is a 0.1% phosphoric acid solution, the detection wavelength is 258 nm, the flow rate is 1.0 ml / min, the column temperature is 30° C., and the elution gradient is as follows:

[0042] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 7→20 93→80 30～40 20→25 80→75 40～50 25 75 50～60 25→30 75→70 60～75 30→80 70→20 75～95 80→90 20→10。

[0043] In the preferred technical solution of the present invention, the detection result is optimized by comprehensively considering and analyzing the comprehensive influence of mobile phase gradient elution program, flow rate, column temperature and other conditions on separation and detection.

[0044] Preferably, four substances, namely mangiferin, gentiopicroside, harpaoside and cinnamic acid, can be identified in the chromatogram.

[0045] Preferably, in the chromatogram, mangiferin is used as a reference peak and as the S peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of gentiopicroside is 0.777-0.949, the relative retention time of the characteristic peak of cinnamic acid is 1.942-2.374, and the relative retention time of the characteristic peak of harpagoside is 2.150-2.628.

[0046] Preferably, four medicinal flavors of Anemarrhena asphodeloides, Gentiana scabra, Gleditsia sinensis and Scrophularia ningpoensis can be identified in the chromatogram.

[0047] Preferably, in the chromatogram, mangiferin is used as a reference peak and as the S peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of Anemarrhena asphodeloides includes 0.931-1.138, 1.397-1.708, and 2.856-3.491, and the characteristic peak retention time of Gentiana scabra includes 0.780-0.953, 0.904-1.105, 0.975-1.192, and 1 .005~1.228, 1.242~1.518, the retention time of the characteristic peaks of Gleditsia sinensis were 0.415~0.507, 0.618~0.755, 0.734~0.897, 1.294~1.581, and the retention time of the characteristic peaks of Scrophularia ningpoensis were 0.622~0.760, 1.357~1.659, 1.971~2.409, 2.212~2.703.

[0048] The second object of the present invention is to provide a quality control method for a Chinese medicine preparation or an intermediate extract thereof, the quality control method comprising the characteristic spectrum detection method described in the first object;

[0049] The Chinese medicine preparation or its intermediate extract is made from the following raw materials: French pinellia, tuckahoe, rhizoma anemarrhenae, radix scrophulariae, gentian, tortoise shell with vinegar, thorn of Gleditsia sinensis, and calcined oyster shell.

[0050] Preferably, the quality control method specifically comprises: obtaining a characteristic spectrum by the method of one of the purposes; the characteristic spectrum can identify four substances, mangiferin, gentiopicroside, harpaoside and cinnamic acid, and the characteristic spectrum presents 9 characteristic peaks corresponding to the control characteristic spectrum, and the relative retention time is within ±10% of the specified value, and the specified value is: 0.446 (peak 1), 0.680 (peak 2), 0.865 (peak 3), 1.038 (peak 5), 2.166 (peak 6), 2.401 (peak 7), 3.183 (peak 8), 3.354 (peak 9). If the above conditions are met, it is a qualified product.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] (1) The present invention uses high performance liquid chromatography to establish a characteristic spectrum of the Xiaoer Linglong Zaoshu compound preparation or its intermediate extract, which has a stable baseline, a large amount of information, a uniform distribution of chromatographic peaks, a good separation effect, and is fast, simple, and has good reproducibility and stability. The characteristic spectrum obtained by the method of the present invention determines 9 characteristic peaks, identifies the four herbs of Anemarrhena, Gentiana, Gleditsia sinensis, and Scrophularia ningpoensis in Xiaoer Linglong Zaoshu granules, and identifies the four chemical components of mangiferin, gentiopicroside, harpagoside, and cinnamic acid.

[0053] (2) In the preferred technical solution of the present invention, the present invention obtains a further optimized characteristic spectrum by selecting specific elution gradient, detection wavelength and other conditions.

[0054] (3) In another preferred technical solution of the present invention, a specific sample preparation method is adopted, ethyl acetate is used as a solvent, or an extraction method is adopted, so that the separation degree of the obtained characteristic spectrum is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The HPLC chromatogram of Example 1 is shown.

[0056] Figure 2 The HPLC chromatogram of Comparative Example 1 is shown.

[0057] Figure 3 The HPLC chromatogram of Example 2 is shown.

[0058] Figure 4a It is the HPLC chromatogram at a wavelength of 210 nm in Example 3.

[0059] Figure 4b It is the HPLC chromatogram at a wavelength of 230 nm in Example 3.

[0060] Figure 4c It is the HPLC chromatogram at a wavelength of 270 nm in Example 3.

[0061] Figure 4d It is the HPLC chromatogram at a wavelength of 290 nm in Example 3.

[0062] Figure 4e It is the HPLC chromatogram at a wavelength of 310 nm in Example 3.

[0063] Figure 5 The HPLC chromatogram of Example 4 is shown in FIG.

[0064] Figure 6 The HPLC chromatogram of Example 5 is shown in FIG.

[0065] Figure 7 The HPLC chromatogram of Example 6 is shown in FIG.

[0066] Figure 8 This is an overlay of HPLC comparison spectra for the identification of medicinal flavors.

[0067] Fig. 9 Overlay of HPLC comparison spectra for the location of related components.

[0068] Fig.10 HPLC chromatograms were examined for specificity.

[0069] Fig.11 This is an overlay of the precision results of the characteristic spectrum.

[0070] Fig.12 This is an overlay of the characteristic spectrum stability results.

[0071] Fig.13 This is an overlay of the repeatability results of the characteristic spectrum.

[0072] Fig.14 This is an overlay of the test results at different flow rates.

[0073] Fig.15 This is an overlay of the test results at different column temperatures.

[0074] Fig.16 The following are HPLC chromatograms of different columns.

[0075] Fig.17 This is an overlay chart of test results from instruments from different manufacturers.

[0076] Fig.18 This is the common pattern diagram of the characteristic spectra of 10 batches of test products.

[0077] Fig.19 It is the reference characteristic map. DETAILED DESCRIPTION

[0078] The present invention is further described in detail below by examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0079] The samples tested in the following examples and comparative examples are Xiaoer Linglong Zaoshu Granules or their intermediate extracts, and their prescriptions and preparation methods are as follows:

[0080] (1) Prescription:

[0081] Weigh the following pieces: 333 parts of French Pinellia, 555 parts of Poria, 555 parts of Anemarrhena, 555 parts of Scrophularia, 333 parts of Gentiana, 555 parts of Vinegar Turtle Shell, 555 parts of Gleditsia Spinosa, and 555 parts of Calcined Oyster. The above eight herbs should comply with the corresponding medicinal material standards in the 2020 edition of the Chinese Pharmacopoeia, and be prepared according to the preparation methods of the herbs under each medicinal flavor.

[0082] (2) Preparation of intermediate extract: The above eight ingredients, Pinellia ternata, Vinegar-coated Tortoise Shell, and Calcined Oyster Mushroom are first decocted with 10 times the total amount of water in the prescription for 30 minutes, and then Poria cocos, Scrophularia ningpoensis, and other five ingredients are added and decocted twice, each time for 1 hour, filtered, and the filtrate is concentrated under reduced pressure to a clear paste with a relative density of 1.10-1.15 (60°C), and ethanol is added to make the alcohol content reach 70%, stirred evenly, and allowed to stand for more than 12 hours, filtered, and the filtrate is decompressed to recover the ethanol and concentrated to a thick paste with a relative density of 1.25-1.30 (60°C), and vacuum dried to obtain a dry paste.

[0083] (3) Preparation of compound preparation (granules): The eight ingredients above, Pinellia ternata, Vinegar-scented Tortoise Shell, and Calcined Oyster Mushroom are first decocted with 10 times the total amount of water in the prescription for 30 minutes, and then Poria cocos, Scrophularia ningpoensis, and other five ingredients are added and decocted twice, each time for 1 hour, filtered, and the filtrate is concentrated under reduced pressure to a clear paste with a relative density of 1.10-1.15 (60°C), and ethanol is added to make the alcohol content reach 70%, stirred evenly, and allowed to stand for more than 12 hours, filtered, and the filtrate is decompressed to recover ethanol and concentrated to a thick paste with a relative density of 1.25-1.30 (60°C), vacuum dried to obtain a dry paste, and crushed. Add 8g sucralose, 5g strawberry essence, 10g citric acid, and an appropriate amount of maltodextrin, and granulate to obtain the product.

[0084] The reagents, instruments and reagents used in the following examples and comparative examples are as follows:

[0085] (1) Drug testing:

[0086] name factory Specification batch number Mangiferin China Food and Drug Inspection Institute 20mg / vial 111607-201704 Gentiopicroside China Food and Drug Inspection Institute 20mg / vial 110770-202219 Harpagoside China Food and Drug Inspection Institute 20mg / vial 110730-202110 Cinnamic acid China Food and Drug Inspection Institute 50mg / vial 110773-201915 Glycyrrhetinic acid China Food and Drug Inspection Institute 20mg / vial 110723-201715 Harpagine China Food and Drug Inspection Institute 20mg / vial 110729-201707 Anemarrhena saponin BII China Food and Drug Inspection Institute 20mg / vial 111839-202107 cholesterol Hunan Erkang Pharmaceutical Research Co., Ltd. 100mg / vial 20220901

[0087] (2) Instruments:

[0088]

[0089]

[0090] (3) Reagents:

[0091] name level batch number factory Acetonitrile Chromatographic pure 204128 Concord Phosphoric acid Chromatographic pure 20210201 Tianjin Damao Chemical Reagent Factory Methanol Analytical grade 20210401 Tianjin Damao Chemical Reagent Factory Ethyl acetate Chromatographic pure 20210201 Tianjin Xinbote Chemical Reagent Factory water Purified water 20210201 Wahaha Group Methanol Analytical grade 20230109 Concord

[0092] Example 1

[0093] This embodiment provides a characteristic spectrum determination of Xiaoer Linglong Zaoshu Granules, and the specific steps are as follows:

[0094] (1) Preparation of test sample: Take about 1 g of the intermediate extract of this product, weigh it accurately, place it in a stoppered conical flask, add 25 ml of water accurately, treat it with ultrasound (power 250 W, frequency 40 kHz) for 30 minutes, transfer it to a separatory funnel, extract it twice with water-saturated n-butanol, 25 ml each time, combine the water-saturated n-butanol solutions to recover the solvent until dry, add 70% methanol to dissolve the residue, transfer it to a 10 ml volumetric flask, add 70% methanol to dilute to the scale, shake well, filter, and take the filtrate to obtain the product.

[0095] (2) Determination method: Accurately pipette 10 μl of the test solution, inject it into the high performance liquid chromatograph, measure, and record the chromatogram.

[0096] The chromatographic conditions are as follows:

[0097] Chromatographic column: octadecylsilane bonded silica gel as filler (column length: 250 mm, inner diameter: 4.6 mm, particle size: 5 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid as mobile phase B, flow rate: 1 ml per minute, column temperature: 30°C, detection wavelength: 258 nm, gradient elution according to the table below.

[0098] Gradient elution table

[0099] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 7→20 93→80 30～40 20→25 80→75 40～50 25 75 50～60 25→30 75→70 60～75 30→80 70→20 75～95 80→90 20→10

[0100] Chromatogram see Figure 1 .

[0101] Comparative Example 1

[0102] The only difference from Example 1 is that acetonitrile is replaced by methanol.

[0103] Chromatogram see Figure 2 .

[0104] By comparing Example 1 with Comparative Example 1, it can be seen that the characteristic spectrum detection method provided by the present invention can obtain a characteristic spectrum with good separation effect and rich chromatographic peak information. In Comparative Example 1, the mobile phase A is replaced with methanol, and the spectrum separation effect is significantly worse, and the chromatographic peak information is less.

[0105] Example 2

[0106] The only difference from Example 1 is that the water-saturated n-butanol in step (1) is replaced by ethyl acetate. Figure 3 .

[0107] By comparing Example 1 and Example 2, it can be seen that when ethyl acetate is used as a solvent to dissolve the test sample Xiaoer Linglong Zaoshu compound preparation or its intermediate extract (Example 2), the response value of the chromatographic peak in the obtained spectrum is higher, while when water-saturated n-butanol is used as the extraction solvent (Example 1), the characteristic peak response values ​​at 10 minutes and 76 minutes are lower. Therefore, the present invention further prefers ethyl acetate as the extraction solvent.

[0108] Example 3

[0109] The only difference from Example 1 is that the detection wavelengths are changed to 210 nm, 230 nm, 270 nm, 290 nm, and 310 nm respectively.

[0110] Chromatogram see Figure 4a , 4b , 4c, 4d, 4e.

[0111] By comparing Example 1 and Example 3, it can be seen that the information content is greater at a wavelength of 258 nm ( Figure 1 ), and the baseline is more stable.

[0112] Example 4

[0113] The only difference from Example 1 is that the elution gradient is as follows:

[0114] Gradient elution table

[0115] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～30 7→20 93→7 30～40 20→30 80→70 40～50 30→50 70→50 50～70 50→80 50→20 70～71 80→90 20→10 71～90 90 10

[0116] Chromatogram see Figure 5 .

[0117] By comparing Example 1 and Example 4, it can be seen that when the elution gradient of Example 1 is used for gradient elution, compared with other elution gradients (Example 4), the obtained spectral separation effect is better and the chromatographic peak information is richer.

[0118] Example 5

[0119] The only difference from Example 1 is that step (1) is: take about 1 g of the intermediate extract of the product, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, weigh it again after cooling, make up the lost weight with 70% methanol, filter it, and take the filtrate to obtain.

[0120] Chromatogram see Figure 6 .

[0121] By comparing Example 1 and Example 5, it can be seen that in the test sample preparation method, extraction is performed after ultrasound, and the response value of the characteristic spectrum peak is more moderate. When only ultrasound is used without extraction (Example 5), the characteristic peak responses of gentiopicroside and mangiferin in the obtained spectrum are too high, resulting in the lack of prominence of other characteristic peaks.

[0122] Example 6

[0123] The only difference from Example 1 is that step (1) is: take about 1 g of the intermediate extract of the product, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, let it cool and weigh it again, make up the lost weight with 70% methanol, put it on an activated macroporous adsorption resin column, wait for the sample solvent to flow out, elute it with 10 ml of methanol twice, collect the fractions, filter, take the filtrate, and inject it.

[0124] Chromatogram see Figure 7 .

[0125] By comparing Example 1 and Example 6, it can be seen that the number of chromatographic peaks in the sample obtained by passing through the macroporous adsorption resin column is small, and the information presented is limited.

[0126] Analysis of HPLC test results:

[0127] The following studies were all carried out according to the test sample preparation method and chromatographic conditions of Example 2.

[0128] (1) Attribution of drug flavor peaks

[0129] The single herbal extracts of Rhizoma Anemarrhenae, Gentiana, Scrophularia, Gleditsia sinensis, Pinellia ternata, Poria, Vinegar-coated Tortoise Shell and Calcined Oyster were prepared and tested according to the test sample preparation method and chromatographic conditions in Example 2 to determine the attribution of each herbal peak in the characteristic spectrum of the compound preparation. The results are shown in FIG. Figure 8 and Table 1.

[0130] Table 1 Result of attribution of drug flavor peaks

[0131] Characteristic peak number Attributed to medicinal taste Peak 1 Gleditsia sinensis Peak 2 Gleditsia sinensis, Scrophularia ningpoensis Peak 3 Gentian Peak 4 Anemarrhena Peak 5 Anemarrhena Peak 6 Scrophularia Peak 7 Scrophularia Peak 8 Gentian Peak 9 Anemarrhena

[0132] from Fig. 9 (a. Rhizoma Anemarrhenae slices, b. Poria cocos slices, c. Gleditsia sinensis slices, d. Gentiana scabra slices, e. Vinegar-coated tortoise shell slices, f. Radix Pinelliae tuber slices, g. Scrophularia ningpoensis slices, h. Calcined Oyster slices, i. Test sample slices) and the results in Table 1 show that the four herbs of Rhizoma Anemarrhenae, Gentiana scabra, Gleditsia sinensis and Scrophularia ningpoensis in Xiaoer Linglong Zaoshu Granules can all be identified in the characteristic spectrum disclosed in the present invention, indicating that the characteristic spectrum disclosed in the present invention can be used for the quality control of Xiaoer Linglong Zaoshu Granules.

[0133] (2) Chromatographic peak identification

[0134] According to the test index ingredients under each medicinal taste item in the 2020 edition of the Chinese Pharmacopoeia, the possible components that may be present were identified by reference substances through known reference substances. The results are shown in Fig. 9 and Table 2.

[0135] Table 2 Chromatographic peak identification results

[0136]

[0137]

[0138] from Fig. 9 It can be seen from the results in Table 2 that the characteristic spectrum disclosed in the present invention has 9 identifiable characteristic peaks, among which gentiopicroside, mangiferin, cinnamic acid and harpaoside have been identified, thereby achieving the effect of improving the accuracy and effectiveness of the characteristic spectrum evaluation.

[0139] (3) Selection of reference objects

[0140] Reference Fig. 9 In the chromatogram, the absorption intensity of the mangiferin chromatographic peak is moderate and the response is stable, so mangiferin is selected as the reference peak and marked as peak S. At the same time, the mangiferin reference substance is used as a reference substance.

[0141] (4) Specificity test and overall investigation

[0142] Test solution: Take an appropriate amount of the product, grind it into powder, take about 2g, weigh it accurately, put it in a stoppered conical flask, accurately add 25ml of water, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, transfer it to a separating funnel, extract it twice with ethyl acetate, 25ml each time, combine the ethyl acetate solution and recover the solvent to dryness, add 70% methanol to dissolve the residue, transfer it to a 10ml volumetric flask, add 70% methanol to dilute to the scale, shake well, filter, and take the filtrate to obtain.

[0143] Reference solution: Take appropriate amount of mangiferin and gentiopicroside reference substances, weigh accurately, add 50% methanol to make a mixed solution containing 20 μg of mangiferin and 0.12 mg of gentiopicroside per 1 ml.

[0144] Blank solution: test sample solvent water

[0145] Excipient solution: weigh sucralose, maltodextrin, strawberry flavor, and citric acid according to the prescription ratio and prepare the solution in the same way as the test sample.

[0146] The test sample solution, reference solution, blank solvent, and excipient solution were respectively injected into the liquid chromatograph, the chromatogram was recorded, and the integrity of the test sample was examined.

[0147] The chromatographic conditions for the specificity investigation are as follows: octadecylsilane bonded silica gel is used as the filler (column length is 250 mm, inner diameter is 4.6 mm, particle size is 5 μm); acetonitrile is used as mobile phase A, 0.01% phosphoric acid solution is used as mobile phase B, gradient elution is performed as specified in the table below, and the detection wavelength is 258 nm. The theoretical plate number calculated based on the mangiferin peak should not be less than 6000.

[0148]

[0149] The chromatographic conditions for the overall investigation are as follows: octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.01% phosphoric acid solution as mobile phase B, gradient elution as specified in the table below, detection wavelength 258 nm. The theoretical plate number calculated based on the mangiferin peak should be no less than 6000.

[0150]

[0151] Results Fig.10 (a test solution b excipient solution c blank solution d mangiferin and gentiopicroside mixed reference solution).

[0152] from Fig.10The results show that the chromatographic peaks have been basically collected at 95 minutes, and the chromatographic conditions have reached the maximum information collection, so it is reasonable to set the collection time as 95 minutes.

[0153] (5) Precision test

[0154] Take an appropriate amount of Xiaoer Linglong Zaoshu Granules, grind them into powder, take about 2g, accurately weigh, and place in a stoppered conical flask. According to the preparation and determination method of the test solution with ethyl acetate as the extraction solvent in Example 2, inject the sample 6 times continuously, and calculate its relative retention time. The specific determination results are shown in Fig.11 (a precision 1 b precision 2 c precision 3 d precision 4 e precision 5 f precision 6), Table 3.

[0155] Table 3 Precision test relative retention time results (n = 6)

[0156]

[0157] The test results show that the relative retention times are basically consistent, the RSDs are all less than 2.0%, and the precision is good.

[0158] (6) Stability test

[0159] Take an appropriate amount of Xiaoer Linglong Zaoshu Granules, grind them into powder, take about 2g, weigh accurately, and operate according to the preparation and determination method of the test solution with ethyl acetate as the extraction solvent in Example 2. Samples are injected and measured at 0, 2, 4, 8, 12, 24, 36, and 48 hours respectively. The relative retention time is calculated with S peak as reference. The specific measurement results are shown in Fig.12 (a0h b2h c4h d8h e12h f24h g36hh48h), Table 4.

[0160] Table 4 Relative retention time results of stability test

[0161]

[0162] The test results show that the relative retention times are basically consistent, the RSDs are all less than 2.0%, and the test solutions have good stability within 48 hours.

[0163] (7) Repeatability test

[0164] Take an appropriate amount of Xiaoer Linglong Zaoshu Granules, grind them into powder, take about 2g, weigh accurately, and operate according to the preparation and determination method of the test solution with ethyl acetate as the extraction solvent in Example 2 to prepare 6 copies. Take the S peak as a reference to calculate the relative retention time. The specific determination results are shown in Fig.13 (a Repeatability 1 b Repeatability 2 c Repeatability d Repeatability 4 e Repeatability 5 f Repeatability 6), Table 5.

[0165] Table 5 Relative retention time results of repeatability test

[0166]

[0167] The test results show that the relative retention times are basically consistent, and the RSDs are all less than 2.0%, indicating that the repeatability of this method is good.

[0168] (8) Durability test

[0169] Based on the method of Example 2, three different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min), three different column temperatures (28°C, 30°C, 32°C), three different chromatographic columns Welch Ultimate LP C18 (4.6*250 mm, 5 μm) / Welch Ultimate AQ C18 (4.6*250 mm, 5 μm) / Phenomenex Luna C18 (4.6*250 mm, 5 μm), and the durability of instruments from two different manufacturers (Agilent and Waters) for the present chromatographic conditions were investigated. The relative retention times of the remaining peaks were calculated with reference to the S peak. The specific measurement results are shown in Tables 6 to 9. Fig.14 (a 0.8ml / min, b 1.0ml / min, c 1.2ml / min), Fig.15 (a 28℃b 30℃c 32℃), Fig.16 (a Welch μultimate LP C18 4.6*250mm, 5μm) (b Welch μltimate AQ C18 4.6*250mm, 5μm) (c Phenomenex Luna C18 4.6*250mm, 5μm) and Fig.17 (a Shimadzu LC-20AD; b Agilent 1260).

[0170] Table 6 Relative retention time results of different flow rate tests

[0171]

[0172] Table 7 Relative retention time results of different column temperature tests

[0173]

[0174] Table 8 Relative retention time results of different chromatographic column tests

[0175]

[0176] Table 9 Relative retention time results of different instrument tests

[0177]

[0178] The results showed that the measurement results under different flow rates, different column temperatures, different chromatographic columns and different instrument conditions were basically consistent, and the main common chromatographic peaks in the chromatogram were sharp, symmetrical and well separated; this showed that the relative retention times of different column temperatures, different flow rates, different chromatographic columns and different instruments were all within a reasonable range, indicating that this method had good durability.

[0179] (7) Establishment of feature maps

[0180] A. The characteristic spectra of 10 batches of Xiaoer Linglong Zaoshu Granules were measured by the method of Example 2, and the spectra were recorded. The obtained characteristic spectra were analyzed, and the chromatographic peaks with good stability and appropriate response values ​​in the characteristic spectra of the 10 batches of samples were selected as common peaks. A total of 9 common peaks were calibrated. The results are shown in Fig.18 (a 220919 b 220920 c 220921 d 221201 e 230208-01 f230208-02 g 230208-03 h 230201 i 230202 j 230203).

[0181] B. Using the "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation Software System 2012 Edition" issued by the National Pharmacopoeia Committee, 10 batches of Xiaoer Linglong Zaoshu Granules HPLC chromatographic peaks were automatically matched to form a common pattern map and establish a reference map. The results are shown in Fig.19 .

[0182] C. The retention time of peak 4 among the common peaks of 10 batches of samples is consistent with that of its corresponding reference substance. As a result, the retention times of the 10 batches of samples are basically consistent with the retention times of their corresponding reference peaks. The results are shown in Tables 10 and 11.

[0183] Table 10 Retention time results of 10 batches of Xiaoer Linglong Zaoshu Granules

[0184]

[0185] D. Taking the peak corresponding to the mangiferin reference as the S peak, the relative retention times of peaks 1 to 9 in 10 batches of samples were calculated. The results showed that the relative retention times of the 10 batches of samples were all within ±10% of the mean value. The results are shown in Table 11. Therefore, the peak corresponding to the mangiferin reference as the S peak, the relative retention time of peaks 1 to 9 should be within ±10% of the specified value.

[0186] The characteristic spectrum of the test sample should present 9 characteristic peaks corresponding to the characteristic spectrum of the control, of which 2 peaks should have the same retention time as the corresponding reference peaks; the peak corresponding to the mangiferin reference is the S peak, and the relative retention time of characteristic peaks 1 to 9 should be calculated within ±10% of the specified values, which are 0.446 (peak 1), 0.680 (peak 2), 0.865 (peak 3), 1.000 [peak 4 (S peak)], 1.038 (peak 5), 2.166 (peak 6), 2.401 (peak 7), 3.183 (peak 8), and 3.354 (peak 9).

[0187] Table 11 Relative retention time results of 10 batches of Xiaoer Linglong Zaoshu Granules

[0188]

[0189] In summary, the characteristic spectrum detection method provided by the present invention can obtain a characteristic spectrum with a stable baseline, a large amount of information, a uniform distribution of chromatographic peaks and a good separation effect, and is fast, simple, has good reproducibility and stability, and can achieve more effective quality control of the pediatric Linglong Zaoshu compound preparation or its intermediate extracts.

[0190] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method 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.

Claims

1. A method for detecting characteristic spectra of a Chinese medicine preparation or its intermediate extract, characterized in that: The steps include: (1) Preparation of test sample solutions of Chinese herbal medicines or their intermediate extracts; The Chinese medicine preparation or its intermediate extract is made from the following raw materials: French pinellia, Poria, Anemarrhena, Scrophularia, Gentiana, Vinegar tortoise shell, Gleditsia sinensis, and calcined oyster shell; (2) Pipette the test solution, inject it into the liquid chromatograph, measure it according to the high performance liquid chromatography method, and record the chromatogram to obtain; The conditions of the high performance liquid chromatography method include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, acetonitrile is used as mobile phase A, and acid solution is used as mobile phase B, and gradient elution is performed.

2. The detection method according to claim 1, characterized in that: Step (1) comprises: mixing the Chinese medicine preparation or its intermediate extract with an extraction solvent, extracting, filtering, and obtaining the test solution; Preferably, the extraction solvent comprises ethyl acetate or water-saturated n-butanol, preferably ethyl acetate; Preferably, in step (1), the extraction method comprises any one of ultrasound, extraction or column chromatography separation or a combination of at least two thereof, preferably a combination of ultrasound and extraction.

3. The detection method according to claim 1, characterized in that: In step (2), the mobile phase A is acetonitrile, and the mobile phase B is a phosphoric acid solution with a concentration of 0.1%.

4. The detection method according to claim 1, characterized in that: In step (2), the elution gradient of the high performance liquid chromatography is as follows:

5. The detection method according to claim 1, characterized in that: In step (2), the detection wavelength of the high performance liquid chromatography is 190 to 400 nm, preferably 258 nm.

6. The detection method according to claim 1, characterized in that: In step (2), the column temperature of the HPLC is 28-32°C, preferably 30°C.

7. The detection method according to claim 1, characterized in that: Four substances, mangiferin, gentiopicroside, harpagoside, and cinnamic acid, can be identified in the chromatogram; Preferably, in the chromatogram, mangiferin is used as a reference peak and as the S peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of gentiopicroside is 0.777-0.949, the relative retention time of the characteristic peak of cinnamic acid is 1.942-2.374, and the relative retention time of the characteristic peak of harpagoside is 2.150-2.

628.

8. The detection method according to claim 1, characterized in that: In the chromatogram, four medicinal flavors can be identified: Rhizoma Anemarrhenae, Gentiana Scabra, Gleditsia sinensis, and Scrophularia ningpoensis. Preferably, in the chromatogram, mangiferin is used as a reference peak and as the S peak, and the relative retention time of each peak is calculated using the reference peak. The relative retention time of the characteristic peak of Anemarrhena asphodeloides includes 0.931-1.138, 1.397-1.708, and 2.856-3.491, and the characteristic peak retention time of Gentiana scabra includes 0.780-0.953, 0.904-1.105, 0.975-1.192, and 1 .005~1.228, 1.242~1.518, the retention time of the characteristic peaks of Gleditsia sinensis were 0.415~0.507, 0.618~0.755, 0.734~0.897, 1.294~1.581, and the retention time of the characteristic peaks of Scrophularia ningpoensis were 0.622~0.760, 1.357~1.659, 1.971~2.409, 2.212~2.

703.

9. A method for quality control of a Chinese medicine preparation or its intermediate extract, characterized in that: The quality control method comprises the characteristic spectrum detection method described in any one of claims 1 to 8; The Chinese medicine preparation or its intermediate extract is made from the following raw materials: French pinellia, tuckahoe, rhizoma anemarrhenae, radix scrophulariae, gentian, tortoise shell with vinegar, thorn of Gleditsia sinensis, and calcined oyster shell.

10. The quality control method according to claim 9, characterized in that: The quality control method specifically includes: obtaining a characteristic spectrum by the characteristic spectrum detection method described in any one of claims 1 to 8; the characteristic spectrum can identify four substances, mangiferin, gentiopicroside, harpaoside, and cinnamic acid, and the characteristic spectrum presents 9 characteristic peaks corresponding to the control characteristic spectrum, and the relative retention time is within ±10% of the specified value, and the specified value is: 0.446, 0.680, 0.865, 1.038, 2.166, 2.401, 3.183, 3.

354. A qualified product is one that meets the above conditions.

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