Construction method and application of characteristic chromatogram of Chimonanthus praecox (L.) Link medicinal materials, decoction pieces, standard decoctions or formula granules

Abstract

The present invention discloses a method for constructing a characteristic chromatogram of Chimonanthus praecox (L.) Link medicinal materials, decoction pieces, standard decoctions or formula granules, comprising: preparing a reference substance solution, the reference substance solution including a reference substance solution of the control medicinal material and a reference substance solution of the reference substance, and the reference substance solution of the reference substance including quercetin reference substance, kaempferide reference substance and rutin reference substance; preparing a test solution from Chimonanthus praecox (L.) Link medicinal materials, decoction pieces, standard decoctions or formula granules; taking a preset amount of the reference substance solution and the test solution and injecting them into a liquid chromatograph, the liquid chromatograph using octadecylsilane chemically bonded silica as the filler, methanol as mobile phase A, and phosphoric acid solution as mobile phase B for gradient elution to establish a characteristic chromatogram, the characteristic chromatogram having 5 common peaks and 3 identified peaks, namely rutin, quercetin and kaempferide. The present invention can provide a data basis for the quality control of Chimonanthus praecox (L.) Link medicinal materials and effectively ensure the stability and controllability of the quality of Chimonanthus praecox (L.) Link medicinal material products.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and in particular to a method for constructing a characteristic spectrum of wintersweet flower medicinal materials, decoction pieces, standard decoctions or formula granules and its application. Background Art

[0002] Wintersweet flowers, listed in Volume 1 of the 1992 edition of the "Pharmaceutical Standards of the Ministry of Health of the People's Republic of China" (Chinese Medicinal Materials), are the dried flower buds of Chimonantlita praecox (L.) Link, a member of the Calycanthaceae family. Harvested from January to March, they are sun-dried or oven-dried. They are round, rectangular, or obovate, 1 to 1.5 cm long and 0.4 to 0.8 cm wide. The perianth is overlapping, forming a bud-like shape. The lower half is tightly enclosed by numerous membranous scales, which are yellowish-brown, slightly triangular, and slightly hairy. They are sweet and slightly bitter in nature, relieving summer heat and promoting the production of body fluids. They are used to treat feverish thirst, chest tightness, and cough. The main chemical components of wintersweet flowers include volatile compounds such as borneol, eucalyptol, saccharine, saccharin, linoleic acid, and oleic acid. They also contain alkaloids, flavonoids, coumarins, and steroids.

[0003] Wintersweet flowers are easily confused with plum blossoms during use. Both are commonly used traditional Chinese medicines. Zhang Xukui et al. frequently observed the two being confused during testing, so they conducted research on their medicinal properties, microscopic characteristics, and thin-layer chromatography (TLC) identification. Consequently, methods for distinguishing and identifying wintersweet flowers from plum blossoms are limited, and the quality standards for wintersweet flowers urgently need to be standardized and improved. Literature review and patent searches revealed limited research on the composition, fingerprints, or characteristic spectra of wintersweet flowers. Literature studies examining the quality standards for wintersweet flower formula granules have employed TLC identification combined with rutin content determination as a quality control method. The "Jiangsu Province Traditional Chinese Medicine Standards" (2016 edition) uses total flavonoid content as an indicator for evaluating medicinal material quality. This suggests a lack of comprehensive quality control methods for wintersweet flowers. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of wintersweet flower medicinal materials, decoction pieces, standard decoctions or formula granules. The method has good reproducibility, accuracy and reliability, and can provide a data basis for the quality control of large-scale production of wintersweet flower medicinal materials, thereby ensuring the stability and controllability of the quality of wintersweet flower medicinal materials products.

[0005] In order to solve the above technical problems, the present invention provides a method for constructing a characteristic spectrum of wintersweet flower medicinal materials, decoction pieces, standard decoctions or formula granules, comprising the following steps:

[0006] Prepare a reference solution, which includes a reference solution of control crude drug and a reference solution of reference substance. The reference solution of reference substance includes quercetin reference substance, kaempferol reference substance and rutin reference substance;

[0007] Take the Chimonanthus praecox L. crude drug, cut crude drug, standard decoction or formula granule to prepare a test solution;

[0008] Take a preset amount of the reference solution and the test solution, and inject them into a liquid chromatograph. The liquid chromatograph uses octadecylsilane chemically bonded silica gel as the filler, methanol as mobile phase A, and phosphoric acid solution as mobile phase B for gradient elution to establish a characteristic chromatogram.

[0009] In one embodiment, the gradient elution is carried out according to the following procedure:

[0010] 0 - 3 min, mobile phase A is 25%, mobile phase B is 75%;

[0011] 3 - 4 min, mobile phase A changes from 25% to 27%, mobile phase B changes from 75% to 73%;

[0012] 4 - 13 min, mobile phase A changes from 27% to 29%, mobile phase B changes from 73% to 71%;

[0013] 13 - 14 min, mobile phase A changes from 29% to 36%, mobile phase B changes from 71% to 64%;

[0014] 14 - 30 min, mobile phase A changes from 36% to 42%, mobile phase B changes from 64% to 58%.

[0015] In one embodiment, the volume concentration of the phosphoric acid solution is 0.1 - 0.25%. Preferably, the volume concentration of the phosphoric acid solution is 0.2%.

[0016] In one embodiment, the preset amount of the reference solution and the test solution is 1 μL - 3 μL;

[0017] The liquid chromatograph uses octadecylsilane chemically bonded silica gel as the filler, the column length is 100 mm, the inner diameter is 2.1 mm, the particle size is 1.6 μm - 1.8 μm, and the column temperature is 32°C - 37°C;

[0018] When performing gradient elution, the flow rate of the mobile phase is 0.28 - 0.32 mL / min;

[0019] The ultraviolet detection wavelength of the liquid chromatograph is 340 nm - 365 nm.

[0020] Preferably, the column temperature is 35°C, the flow rate of the mobile phase is 0.3 mL / min, and the ultraviolet detection wavelength is 355 nm.

[0021] In one embodiment, the test sample solution is prepared by the following method:

[0022] Take Chimonanthus praecox herbs, cut pieces, standard decoctions or formula granules, grind them, and sieve through a 45-mesh to 55-mesh sieve. Then weigh 0.3 g to 1 g and mix with 60% to 75% ethanol. The added amount of ethanol is 40 mL to 60 mL. After heating under reflux for 30 min to 90 min, cool it, shake well, filter, and take the subsequent filtrate to obtain the test sample solution.

[0023] In one embodiment, the reference substance solution of the reference standard is prepared by the following method:

[0024] Take appropriate amounts of quercetin reference standard, kaempferide reference standard, and rutin reference standard, accurately weigh them, and dissolve them in methanol to prepare a mixed solution containing 60 μg of quercetin, 20 μg of kaempferide, and 90 μg of rutin per 1 mL as the reference substance solution of the reference standard;

[0025] The reference substance solution of the control herb is prepared by the following method:

[0026] Take Chimonanthus praecox herbs, grind them, and sieve through a 45-mesh to 55-mesh sieve. Then weigh 0.3 g to 1 g and mix with 60% to 75% ethanol. The added amount of ethanol is 40 ml to 60 mL. After heating under reflux for 30 min to 90 min, cool it, shake well, filter, and take the subsequent filtrate to obtain the reference substance solution of the control herb.

[0027] In one embodiment, the characteristic chromatogram includes the characteristic chromatogram of Chimonanthus praecox herbs, the characteristic chromatogram of Chimonanthus praecox cut pieces, the characteristic chromatogram of Chimonanthus praecox standard decoctions, and the characteristic chromatogram of Chimonanthus praecox formula granules. The characteristic chromatogram includes 5 common peaks; among them, the 2nd peak corresponds to rutin, the 4th peak corresponds to quercetin, and the 5th peak corresponds to kaempferide.

[0028] In one embodiment, in the characteristic chromatogram of Chimonanthus praecox herbs, the peak corresponding to the rutin reference standard peak is the S peak, and the relative peak areas of the common peaks to the S peak meet the following: for the 1st peak, 0.009 to 0.065; for the 2nd peak, 1.000; for the 3rd peak, 0.057 to 0.091; for the 4th peak, 0.262 to 1.743; for the 5th peak, 0.027 to 0.149.

[0029] In one embodiment, in the characteristic chromatogram of Chimonanthus praecox cut pieces, the peak corresponding to the rutin reference standard peak is the S peak, and the relative peak areas of the common peaks to the S peak meet the following: for the 1st peak, 0.010 to 0.067; for the 2nd peak, 1.000; for the 3rd peak, 0.059 to 0.090; for the 4th peak, 0.277 to 1.757; for the 5th peak, 0.032 to 0.173.

[0030] In one embodiment, in the characteristic chromatogram of the standard decoction of Chimonanthus praecox flowers, the peak corresponding to the rutin reference substance peak is the S peak, and the relative peak areas of the common peaks and the S peak meet the following requirements: peak 1 is 0.042 - 0.131, peak 2 is 1.000, peak 3 is 0.050 - 0.082, peak 4 is 0.363 - 1.274, and peak 5 is 0.031 - 0.082.

[0031] In one embodiment, in the characteristic chromatogram of the formula granules of Chimonanthus praecox flowers, the peak corresponding to the rutin reference substance peak is the S peak, and the relative peak areas of the common peaks and the S peak meet the following requirements: peak 1 is 0.035 - 0.042, peak 2 is 1.000, peak 3 is 0.080 - 0.083, peak 4 is 0.329 - 0.355, and peak 5 is 0.033 - 0.037.

[0032] Correspondingly, the present invention also provides a characteristic chromatogram of Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules, and the characteristic chromatogram of the Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules is obtained by using the construction method of the characteristic chromatogram of the above-mentioned Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules.

[0033] In addition, the application of the characteristic chromatogram of the Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules in (1) or (2):

[0034] (1) Authenticity identification and component detection of Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules;

[0035] (2) Differentiation between Chimonanthus praecox flower medicinal materials and Prunus mume flower medicinal materials.

[0036] Implementing the present invention has the following beneficial effects:

[0037] Through the construction method of the characteristic chromatogram, the present invention first establishes a characteristic chromatogram of Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules. This characteristic chromatogram can fully display the chemical composition characteristics of Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules. The characteristic peak information is rich, and it can comprehensively reflect the quality information of Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules, so as to achieve the purpose of comprehensively and effectively controlling the quality of Chimonanthus praecox flower medicinal materials, cut pieces, standard decoctions or formula granules. And it can realize the identification and differentiation of Chimonanthus praecox flower medicinal materials and Prunus mume flower medicinal materials, providing a reference basis for the identification of Chimonanthus praecox flower medicinal materials and Prunus mume flower medicinal materials. Description of the Drawings

[0038] Figure 1 It is the characteristic chromatogram of the Chimonanthus praecox flower medicinal materials in Example 1 of the present invention, in which peak 2 is the rutin peak, peak 4 is the quercetin peak, and peak 5 is the kaempferol peak;

[0039] Figure 2It is the characteristic chromatogram of Chimonanthus praecox (L.) Link cut crude drug in Example 2 of the present invention, wherein Peak 2 is the rutin peak, Peak 4 is the quercetin peak, and Peak 5 is the kaempferide peak;

[0040] Figure 3 It is the characteristic chromatogram of the standard decoction of Chimonanthus praecox (L.) Link in Example 3 of the present invention, wherein Peak 2 is the rutin peak, Peak 4 is the quercetin peak, and Peak 5 is the kaempferide peak;

[0041] Figure 4 It is the characteristic chromatogram of the formula granules of Chimonanthus praecox (L.) Link in Example 4 of the present invention, wherein Peak 2 is the rutin peak, Peak 4 is the quercetin peak, and Peak 5 is the kaempferide peak;

[0042] Figure 5 It is the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 5 of the present invention;

[0043] Figure 6 It is the comparative study diagram of the Chimonanthus praecox (L.) Link crude drug and the Prunus mume Sieb. et Zucc. crude drug in Example 5 of the present invention, wherein 1 is the characteristic chromatogram of the Prunus mume Sieb. et Zucc. crude drug, and 2 is the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug;

[0044] Figure 7 It is the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 6 of the present invention;

[0045] Figure 8 It is the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 7 of the present invention;

[0046] Figure 9 It is the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Comparative Example 1 of the present invention;

[0047] Figure 10 It is the specificity investigation diagram of the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 1 of the present invention;

[0048] Figure 11 It is the identification diagram of the rutin peak in the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 1 of the present invention;

[0049] Figure 12 It is the identification diagram of the quercetin peak in the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 1 of the present invention;

[0050] Figure 13 It is the identification diagram of the kaempferide peak in the characteristic chromatogram of the Chimonanthus praecox (L.) Link crude drug in Example 1 of the present invention. Detailed Embodiments

[0051] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] Chimonanthus praecox flowers are included in the first volume of the Chinese Pharmacopoeia of the Ministry of Health of the People's Republic of China (1992 edition), and are the dried flower buds of Chimonanthus praecox (L.) Link of the Calycanthaceae family. In the process of application, Chimonanthus praecox flowers are extremely easy to be confused with Prunus mume flowers. However, in the existing technology, the identification methods for the two are limited, and the quality standards of Chimonanthus praecox flower medicinal materials urgently need to be standardized and improved, lacking means to comprehensively control the quality of Chimonanthus praecox flower medicinal materials, decoction pieces, standard decoctions or formula granules.

[0053] In order to comprehensively reflect the quality information of Chimonanthus praecox flower medicinal materials, decoction pieces, standard decoctions or formula granules, and to achieve comprehensive and effective control of the quality of Chimonanthus praecox flower medicinal materials, decoction pieces, standard decoctions or formula granules, the present invention provides a method for constructing a characteristic chromatogram of Chimonanthus praecox flower medicinal materials, decoction pieces, standard decoctions or formula granules, which is described in detail below: 1 Instruments and reagents

[0054] Instruments: Waters ultra-high performance liquid chromatograph (H-Class, Waters Co., Ltd.), Thermo ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher Scientific (China) Co., Ltd.), Waters BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm), ten-thousandth balance (ME204E, Mettler Toledo Co., Ltd.), millionth balance (XP26, Mettler Toledo Co., Ltd.), ultra-pure water system (Milli-Q Direct, Merck KGaA).

[0055] Reagents: Ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) are both of analytical purity; phosphoric acid (Tianjin Kemiou Chemical Reagent Co., Ltd.), acetonitrile (Merck KGaA), and methanol (Merck KGaA) are of HPLC chromatographic grade, and water is ultra-pure water (prepared in the laboratory).

[0056] Test drugs: Control Chimonanthus praecox flower medicinal materials (from Guangdong Yifang Pharmaceutical Co., Ltd.), rutin reference substance (batch number: 100080 - 202012, content: 91.6%, National Institutes for Food and Drug Control); quercetin reference substance (batch number: 100081 - 201610, content: 99.1%, National Institutes for Food and Drug Control); kaempferol reference substance (batch number: 110861 - 202013, content: 93.2%, National Institutes for Food and Drug Control).

[0057] 17 batches of Chimonanthus praecox flower medicinal materials, 17 batches of Chimonanthus praecox flower decoction pieces, 17 batches of Chimonanthus praecox flower standard decoctions and 3 batches of Chimonanthus praecox flower formula granule samples, and the sources of 17 batches of Chimonanthus praecox flower medicinal materials are as follows:

[0058]

[0059]

[0060] 2 Chromatographic Conditions and Preparation of Reference Substance Solution and Test Solution

[0061] 2.1 Chromatographic Conditions

[0062] Chromatographic conditions and system suitability test: The liquid chromatograph uses octadecylsilane-bonded silica gel as the filler, methanol as mobile phase A, and phosphoric acid solution as mobile phase B for gradient elution according to the gradient elution table shown in Table 1 to establish a characteristic chromatogram.

[0063] In one embodiment, the volume concentration of the phosphoric acid solution is 0.1 - 0.25%. Preferably, the volume concentration of the phosphoric acid solution is 0.2%.

[0064] In one embodiment, the liquid chromatograph uses octadecylsilane-bonded silica gel as the filler, with a column length of 100 mm, an inner diameter of 2.1 mm, a particle size of 1.6 - 1.8 μm, and a column temperature of 32°C - 37°C; when performing gradient elution, the flow rate of the mobile phase is 0.28 - 0.32 mL / min; the ultraviolet detection wavelength of the liquid chromatograph is 340 nm - 365 nm. Preferably, the column temperature is 35°C, the flow rate of the mobile phase is 0.3 mL / min, and the ultraviolet detection wavelength is 355 nm.

[0065] Table 1 Gradient Elution Table

[0066]

[0067] 2.2 Preparation of Reference Substance Solution

[0068] In one embodiment, the reference substance solution of the reference standard is prepared according to the following method:

[0069] Take appropriate amounts of quercetin reference standard, kaempferol reference standard, and rutin reference standard, accurately weigh them, and dissolve them in methanol to prepare a mixed solution containing 60 μg of quercetin, 20 μg of kaempferol, and 90 μg of rutin per 1 mL as the reference substance solution of the reference standard;

[0070] The reference substance solution of the control medicinal material is prepared by the following method:

[0071] Take Chimonanthus praecox (L.) Link medicinal material, grind it and pass through a 45 - 55 mesh sieve, then weigh 0.3 g - 1 g and mix it with 60% - 75% ethanol, with the addition amount of ethanol being 40 mL - 60 mL. After heating under reflux for 30 min - 90 min, let it cool, shake well, filter, and take the continued filtrate to prepare the reference substance solution of the control medicinal material.

[0072] 2.3 Preparation of Test Solution

[0073] In one embodiment, the test sample solution is prepared by the following method:

[0074] Take Chimonanthus praecox herbs, cut pieces, standard decoctions or formula granules, grind them and sieve through a 45-mesh to 55-mesh sieve, then weigh 0.3 g to 1 g and mix with 60% to 75% ethanol. The added amount of ethanol is 40 mL to 60 mL. After heating under reflux for 30 min to 90 min, cool it, shake well, filter, and take the subsequent filtrate to prepare the test sample solution.

[0075] 3 Chromatographic determination and establishment of characteristic chromatograms

[0076] 3.1 Determination method

[0077] Precisely pipette a preset amount of the reference substance solution and the test sample solution into the liquid chromatograph for determination. The preset amounts of the reference substance solution and the test sample solution are 1 μL to 3 μL.

[0078] 3.2 Establishment of characteristic chromatograms

[0079] Use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to identify the common peaks in the characteristic chromatograms of Chimonanthus praecox herbs, cut pieces, standard decoctions and Chimonanthus praecox formula granules, and generate a reference chromatogram. The characteristic chromatograms include the characteristic chromatogram of Chimonanthus praecox herbs, the characteristic chromatogram of Chimonanthus praecox cut pieces, the characteristic chromatogram of Chimonanthus praecox standard decoctions and the characteristic chromatogram of Chimonanthus praecox formula granules. Five characteristic peaks should be presented in the test sample chromatogram, and should correspond to the retention times of the five characteristic peaks in the reference chromatogram of the reference medicinal material. Among them, peak 2, peak 4, and peak 5 should respectively correspond to the retention times of the corresponding reference substance peaks. Specifically, peak 2 corresponds to rutin, peak 4 corresponds to quercetin, and peak 5 corresponds to kaempferide.

[0080] In one embodiment, in the characteristic chromatogram of Chimonanthus praecox herbs, the peak corresponding to the rutin reference substance peak is the S peak. The relative peak areas of the common peaks and the S peak are as follows: peak 1 is 0.009 - 0.065, peak 2 is 1.000, peak 3 is 0.057 - 0.091, peak 4 is 0.262 - 1.743, and peak 5 is 0.027 - 0.149.

[0081] In one embodiment, in the characteristic chromatogram of Chimonanthus praecox cut pieces, the peak corresponding to the rutin reference substance peak is the S peak. The relative peak areas of the common peaks and the S peak are as follows: peak 1 is 0.010 - 0.067, peak 2 is 1.000, peak 3 is 0.059 - 0.090, peak 4 is 0.277 - 1.757, and peak 5 is 0.032 - 0.173.

[0082] In one embodiment, in the characteristic chromatogram of the standard decoction of Chimonanthus praecox flowers, the peak corresponding to the rutin reference substance peak is the S peak, and the relative peak areas of the common peaks and the S peak meet the following: peak 1, 0.042 - 0.131; peak 2, 1.000; peak 3, 0.050 - 0.082; peak 4, 0.363 - 1.274; peak 5, 0.031 - 0.082.

[0083] In one embodiment, in the characteristic chromatogram of the formula granules of Chimonanthus praecox flowers, the peak corresponding to the rutin reference substance peak is the S peak, and the relative peak areas of the common peaks and the S peak meet the following: peak 1, 0.035 - 0.042; peak 2, 1.000; peak 3, 0.080 - 0.083; peak 4, 0.329 - 0.355; peak 5, 0.033 - 0.037.

[0084] The present invention will be explained below with reference to specific embodiments:

[0085] Example 1

[0086] A method for constructing the characteristic chromatogram of Chimonanthus praecox flower medicinal materials includes the following steps:

[0087] S1. Prepare the reference substance solution, which includes the reference substance solution of the control medicinal materials and the reference substance solution of the reference substances. The reference substance solution of the reference substances includes quercetin reference substance, kaempferide reference substance, and rutin reference substance;

[0088] Specifically, take 0.5 g of the control medicinal materials of Chimonanthus praecox flowers, place them in a stoppered conical flask, add 50 mL of 70% ethanol, heat under reflux for 1 hour, cool, shake well, filter, and take the subsequent filtrate as the reference substance solution of the control medicinal materials.

[0089] Take appropriate amounts of quercetin reference substance, kaempferide reference substance, and rutin reference substance, weigh them accurately, and prepare a mixed solution containing 60 μg of quercetin, 20 μg of kaempferide, and 90 μg of rutin per 1 mL with methanol as the reference substance solution of the reference substances.

[0090] S2. Prepare the test solution from the Chimonanthus praecox flower medicinal materials;

[0091] Specifically, take 0.5 g of each of the 17 batches of Chimonanthus praecox flower medicinal materials, grind them, pass through a 50-mesh sieve, place them in a stoppered conical flask, add 50 mL of 70% ethanol, heat under reflux for 60 minutes, cool, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0092] S3. Take 1 μL of the reference solution and the test solution, and inject them into a liquid chromatograph. The liquid chromatograph uses octadecylsilane bonded silica gel as a filler (column length is 100 mm, inner diameter is 2.1 mm, and particle size is 1.7 μm); methanol is used as mobile phase A, and 0.2% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in Table 1; the flow rate is 0.30 mL per minute; the column temperature is 35°C; the ultraviolet detection wavelength is 355 nm, and the number of theoretical plates should be not less than 5000 calculated based on the rutin peak, and a characteristic spectrum of wintersweet flower medicinal materials is established. In the characteristic spectrum of wintersweet flower medicinal materials, peak No. 2 corresponds to rutin, peak No. 4 corresponds to quercetin, and peak No. 5 corresponds to kaempferol. Table 2 shows the relative peak areas of the common peaks of the characteristic spectrum of wintersweet flower medicinal materials. Figure 1 This is an overlay of the characteristic spectra of the medicinal material Chimonanthus jasminoides in Example 1.

[0093] Table 2 Relative peak areas of common peaks in characteristic spectrum of Chimonanthus odoratus

[0094]

[0095]

[0096] Example 2

[0097] A method for constructing a characteristic spectrum of wintersweet flower slices, the construction method is based on Example 1, and the characteristic spectrum of the obtained wintersweet flower slices corresponds to peak 2 corresponding to rutin, peak 4 corresponding to quercetin, and peak 5 corresponding to kaempferol. Table 3 shows the relative peak areas of the common peaks in the characteristic spectrum of the wintersweet flower slices. Figure 2 It is an overlay of characteristic spectra of the Flos Chimonanthus Waxensis slices in Example 2.

[0098] Table 3 Relative peak areas of common peaks in characteristic spectra of Chimonanthus dasyphylla decoction pieces

[0099]

[0100] Example 3

[0101] A method for constructing a characteristic spectrum of a standard decoction of wintersweet flower is described. The construction method is based on Example 1. The characteristic spectrum of the obtained standard decoction of wintersweet flower has peak 2 corresponding to rutin, peak 4 corresponding to quercetin, and peak 5 corresponding to kaempferol. Table 4 shows the relative peak areas of the common peaks of the characteristic spectrum of the standard decoction of wintersweet flower. Figure 3 It is an overlay of characteristic spectra of the standard decoction of wintersweet flower in Example 3.

[0102] Table 4 Common peak relative peak areas of characteristic spectrum of standard decoction of wintersweet flower

[0103]

[0104] Example 4

[0105] A method for constructing the characteristic fingerprint of Chimonanthus praecox formula granules, different in that the sampling amount of Chimonanthus praecox formula granules is 3 batches, and the rest are the same as in Example 1. In the characteristic fingerprint of the obtained Chimonanthus praecox formula granules, the peak corresponding to rutin is Peak 2, the peak corresponding to quercetin is Peak 4, and the peak corresponding to kaempferide is Peak 5. Table 5 shows the relative peak areas of the common peaks in the characteristic fingerprint of Chimonanthus praecox formula granules. Figure 4 It is the superimposed diagram of the characteristic fingerprint of Chimonanthus praecox formula granules in Example 4.

[0106] Table 5 Relative peak areas of the common peaks in the characteristic fingerprint of Chimonanthus praecox formula granules

[0107]

[0108] Example 5

[0109] Application of the characteristic fingerprint of Chimonanthus praecox medicinal materials in the differentiation between Chimonanthus praecox medicinal materials and Prunus mume medicinal materials, including the following steps:

[0110] Take 12 batches of Prunus mume medicinal material samples and determine them with reference to the construction method provided in Example 1; use the "Similarity Evaluation Software for Chromatographic Fingerprints of Traditional Chinese Medicines" to identify the common peaks in the characteristic fingerprints of 17 batches of Chimonanthus praecox medicinal materials in Example 1 and 12 batches of Prunus mume medicinal materials in this example, and generate a control fingerprint for each, and then superimpose them. The result of the superimposed diagram of the characteristic fingerprint of Prunus mume medicinal materials is shown in Figure 5 , and the comparison research diagram of Chimonanthus praecox and Prunus mume is shown in Figure 6 . As can be seen from the figure, Prunus mume medicinal materials have a peak at 2 minutes and two peaks at 12 - 13 minutes, while Chimonanthus praecox medicinal materials do not. Prunus mume medicinal materials lack Peak 5, that is, the kaempferide peak. It can be seen that this fingerprint method can be used to identify Chimonanthus praecox medicinal materials and Prunus mume medicinal materials.

[0111] Example 6

[0112] A method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, different from Example 1 in that the gradient elution table shown in Tables 6 and 7 is used instead of the gradient elution table shown in Table 1, and the rest are the same as in Example 1. Figure 7 It is the characteristic fingerprint of Chimonanthus praecox medicinal materials in Example 6. By comparison, it can be known that the chromatogram obtained by the method provided in Example 1 has a more uniform distribution, good peak shapes of each chromatographic peak, and a stable baseline.

[0113] Table 6

[0114]

[0115] Table 7

[0116]

[0117] Example 7

[0118] A method for constructing a characteristic chromatogram of Chimonanthus praecox medicinal materials, which is different from Example 1 in that the ultraviolet detection wavelengths used in Example 7 are 325 nm, 300 nm, and 254 nm, and the rest are the same as in Example 1. Figure 8 It is the characteristic chromatogram of Chimonanthus praecox medicinal materials in Example 7. By comparison, it can be seen that the baseline of the chromatogram obtained by the method provided in Example 1 is relatively stable, and the responses of Peak 1 and Peak 2 are better.

[0119] Comparative Example 1

[0120] A method for constructing a characteristic chromatogram of Chimonanthus praecox medicinal materials, which is different from Example 1 in that in Comparative Example 1, acetonitrile is used as mobile phase A and 0.2% phosphoric acid is used as mobile phase B, and the rest are the same as in Example 1. Figure 9 It is the characteristic chromatogram of Chimonanthus praecox medicinal materials in Comparative Example 1. By comparison, it can be seen that the chromatographic peaks of the chromatogram obtained by the method provided in Example 1 are evenly distributed and well separated.

[0121] In summary, the present invention has established for the first time the characteristic chromatograms of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules. These characteristic chromatograms can fully display the chemical composition characteristics of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules. The characteristic peaks have rich information and can comprehensively reflect the quality information of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules, so as to achieve the purpose of comprehensively and effectively controlling the quality of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules. And it can realize the identification and distinction between Chimonanthus praecox medicinal materials and Prunus mume medicinal materials, providing a reference basis for the identification of Chimonanthus praecox medicinal materials and Prunus mume medicinal materials.

[0122] The above is the preferred embodiment of the invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also regarded as the protection scope of the present invention.

Claims

1. A method for constructing a characteristic fingerprint of Chimonanthus praecox medicinal materials, decoction pieces, standard decoctions or formula granules, characterized in that, It includes the following steps: Prepare a reference solution, which includes a reference solution of the control crude drug and a reference solution of the reference substance. The reference solution of the reference substance includes quercetin reference substance, kaempferol reference substance and rutin reference substance; Take the Flos Chimonanthi Praecocis crude drug, decoction pieces, standard decoction or formula granules to prepare a test solution; The test solution is prepared by the following method: Take the Flos Chimonanthi Praecocis crude drug, decoction pieces, standard decoction or formula granules, grind them and pass through a 45-mesh to 55-mesh sieve, then weigh 0.3 g to 1 g and mix with 60% to 75% ethanol. The addition amount of the ethanol is 40 mL to 60 mL. After heating under reflux for 30 min to 90 min, cool, shake well, filter, and take the continuous filtrate to prepare the test solution; Take a preset amount of the reference solution and the test solution, inject them into a liquid chromatograph. The liquid chromatograph uses octadecylsilane chemically bonded silica gel as the filler, uses methanol as mobile phase A, and uses a phosphoric acid solution as mobile phase B for gradient elution to establish a characteristic chromatogram; The volume concentration of the phosphoric acid solution is 0.1% to 0.25%; The preset amount of the reference solution and the test solution is 1 μL to 3 μL; The liquid chromatograph uses octadecylsilane chemically bonded silica gel as the filler, with a column length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm to 1.8 μm; The ultraviolet detection wavelength of the liquid chromatograph is 340 nm to 365 nm; The gradient elution is carried out according to the following procedure: From 0 to 3 min, mobile phase A is 25% and mobile phase B is 75%; From 3 to 4 min, mobile phase A changes from 25% to 27% and mobile phase B changes from 75% to 73%; From 4 to 13 min, mobile phase A changes from 27% to 29% and mobile phase B changes from 73% to 71%; From 13 to 14 min, mobile phase A changes from 29% to 36% and mobile phase B changes from 71% to 64%; From 14 to 30 min, mobile phase A changes from 36% to 42% and mobile phase B changes from 64% to 58%.

2. The method for constructing the characteristic spectrum of Chimonanthus praecox medicinal materials, decoction pieces, standard decoctions or formula granules as described in claim 1, characterized in that, The volume concentration of the phosphoric acid solution is 0.2%; 3. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, cut pieces, standard decoction or formula granules as described in claim 1, characterized in that, The column temperature is 32°C to 37°C; When carrying out the gradient elution, the flow rate of the mobile phase is 0.28 to 0.32 mL / min.

4. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, decoction pieces, standard decoctions or formula granules according to claim 3, characterized in that, The column temperature is 35°C, the flow rate of the mobile phase is 0.3 mL / min, and the ultraviolet detection wavelength is 355 nm.

5. The method for constructing a characteristic spectrum of the Flos Chimonanthus Wintersweet medicinal material, decoction pieces, standard decoction or formula granules according to claim 1, wherein: The reference solution of the reference substance is prepared by the following method: Take appropriate amounts of quercetin reference substance, kaempferol reference substance and rutin reference substance, accurately weigh them, and dissolve them in methanol to prepare a mixed solution containing 60 μg of quercetin, 20 μg of kaempferol and 90 μg of rutin per 1 mL as the reference solution of the reference substance; The reference solution of the control crude drug is prepared by the following method: Take the Flos Chimonanthi Praecocis crude drug, grind it and pass through a 45-mesh to 55-mesh sieve, then weigh 0.3 g to 1 g and mix with 60% to 75% ethanol. The addition amount of the ethanol is 40 ml to 60 mL. After heating under reflux for 30 min to 90 min, cool, shake well, filter, and take the continuous filtrate to prepare the reference solution of the control crude drug.

6. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules as described in claim 1, characterized in that, The characteristic chromatograms include the characteristic chromatogram of Chimonanthus praecox medicinal materials, the characteristic chromatogram of Chimonanthus praecox cut pieces, the characteristic chromatogram of Chimonanthus praecox standard decoction, and the characteristic chromatogram of Chimonanthus praecox formula granules. The characteristic chromatograms include 5 common peaks; among them, the 2nd peak corresponds to rutin, the 4th peak corresponds to quercetin, and the 5th peak corresponds to kaempferide.

7. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, cut pieces, standard decoction or formula granules as described in claim 6, characterized in that, In the characteristic chromatogram of the Chimonanthus praecox medicinal materials, the peak corresponding to the rutin reference substance peak is the S peak. The relative peak areas of the common peaks and the S peak are as follows: the 1st peak is 0.009 - 0.065, the 2nd peak is 1.000, the 3rd peak is 0.057 - 0.091, the 4th peak is 0.262 - 1.743, and the 5th peak is 0.027 - 0.

149.

8. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules as described in claim 6, characterized in that, In the characteristic chromatogram of the Chimonanthus praecox cut pieces, the peak corresponding to the rutin reference substance peak is the S peak. The relative peak areas of the common peaks and the S peak are as follows: the 1st peak is 0.010 - 0.067, the 2nd peak is 1.000, the 3rd peak is 0.059 - 0.090, the 4th peak is 0.277 - 1.757, and the 5th peak is 0.032 - 0.

173.

9. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, cut pieces, standard decoction or formula granules according to claim 6, wherein, In the characteristic chromatogram of the Chimonanthus praecox standard decoction, the peak corresponding to the rutin reference substance peak is the S peak. The relative peak areas of the common peaks and the S peak are as follows: the 1st peak is 0.042 - 0.131, the 2nd peak is 1.000, the 3rd peak is 0.050 - 0.082, the 4th peak is 0.363 - 1.274, and the 5th peak is 0.031 - 0.

082.

10. The method for constructing the characteristic fingerprint of Chimonanthus praecox medicinal materials, cut pieces, standard decoctions or formula granules as described in claim 6, characterized in that, In the characteristic chromatogram of the Chimonanthus praecox formula granules, the peak corresponding to the rutin reference substance peak is the S peak. The relative peak areas of the common peaks and the S peak are as follows: the 1st peak is 0.035 - 0.042, the 2nd peak is 1.000, the 3rd peak is 0.080 - 0.083, the 4th peak is 0.329 - 0.355, and the 5th peak is 0.033 - 0.

037.

11. Application of the characteristic chromatograms of Chimonanthus praecox medicinal materials, cut pieces, standard decoction or formula granules constructed by the construction method according to any one of claims 1 - 10 in differentiating Chimonanthus praecox medicinal materials from Prunus mume medicinal materials.

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