A fingerprint spectrum establishment method and fingerprint spectrum of Qingfei mixture

Abstract

The present invention relates to a method for establishing a fingerprint of a Qingfei mixture and its fingerprint, and belongs to the technical field of analysis of traditional Chinese medicine preparations. The method for establishing a fingerprint of the Qingfei mixture of the present invention includes the preparation of a test solution, the determination of HPLC chromatographic conditions, and the production of a fingerprint. There are 18 common peaks in the fingerprint of the Qingfei mixture obtained by the method provided by the present invention, and the method has high stability and good reproducibility, and the obtained characteristic peaks are more, and the separation between the chromatographic peaks is high. By comparing the common peaks in the fingerprint, the quality of the Qingfei mixture can be comprehensively evaluated, the product quality is guaranteed, and the safety and effectiveness of clinical medication are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation analysis, and relates to a fingerprint spectrum establishment method of a lung-clearing mixture and a fingerprint spectrum thereof. Background Art

[0002] Cough is a common symptom, which may be caused by colds, tracheitis, bronchitis, pneumonia and other diseases. In traditional Chinese medicine, cough is usually classified as "cough syndrome". Its cause is different from cough caused by exogenous wind-cold, internal heat cough and asthma, and kidney deficiency. Therefore, the treatment of cough in traditional Chinese medicine should be based on the cause and condition of the disease.

[0003] Maxingshigantang is a decoction. It has a pungent and cool effect, clears the lungs and relieves asthma. It is mainly used to treat lung heat, asthma and cough, even shortness of breath, flaring of the nostrils, sweating or no sweating, persistent fever, thirst, slippery and rapid pulse, and thin yellow fur. It is currently used for pneumonia, chronic bronchitis, bronchial asthma, measles, whooping cough, chronic sinusitis and other diseases caused by pathogenic heat blocking the lungs.

[0004] Qingfei Mixture is a specialty preparation of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. It has been used clinically for more than 20 years. It is based on the ancient prescription "Ma Xing Shi Gan Tang" and has the effects of clearing heat and promoting lung function, removing phlegm, relieving cough and relieving asthma. As a Chinese patent medicine, the efficacy of Qingfei Mixture is the result of the combined action of multiple active ingredients. However, at present, Qingfei Mixture is an in-hospital preparation of the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine. Its quality standard detection is single, and the effective ingredients have not been fully tested. There are certain limitations, which cannot fully reflect the quality level of Qingfei Mixture.

[0005] As a quality control technology, the fingerprint of traditional Chinese medicine can control the quality of medicines more comprehensively. It has the characteristics of systematicity, integrity and stability. At present, there are no relevant patent disclosures and literature reports on the fingerprint of Qingfei Mixture at home and abroad. Summary of the invention

[0006] The main purpose of the present invention is to provide a lung-clearing mixture with the effects of clearing away heat, promoting lung function, removing phlegm, relieving cough and relieving asthma. The lung-clearing mixture can be clinically used to treat lung heat, cough and asthma, bronchitis, pneumonia, etc., with significant therapeutic effect. At the same time, its fingerprint spectrum and establishment method are provided to make up for the shortcomings of the quality control method of the lung-clearing mixture in the prior art.

[0007] The present invention adopts the following technical solutions to achieve the above purpose:

[0008] A lung-clearing mixture is prepared from the following ingredients:

[0009] 10-25 weight parts of Houttuynia cordata, 3-10 weight parts of Ephedra, 5-15 weight parts of Scutellaria baicalensis, 5-20 weight parts of Platycodon grandiflorum, 5-20 weight parts of Bitter Apricot, 5-15 weight parts of Trichosanthes, 5-20 weight parts of Peucedanum, 5-20 weight parts of Lemongrass, 3-10 weight parts of Citrus aurantium, 3-10 weight parts of Belamcanda chinensis, 2-8 weight parts of Indigo Naturalis, and 2-8 weight parts of Licorice.

[0010] Furthermore, the ephedra used in the lung-clearing mixture is honey ephedra, the fried bitter almonds are fried bitter almonds, and the fructus aurantii is fried fructus aurantii with bran.

[0011] Furthermore, the preparation method of the Qingfei mixture is:

[0012] Mix houttuynia cordata, honey ephedra, scutellaria baicalensis, platycodon grandiflorum, stir-fried bitter almond, trichosanthes, peucedanum chinense, scutellaria baicalensis seeds, stir-fried fructus aurantii with bran, belamcanda chinensis, indigo naturalis and liquorice, add water and boil twice, combine the decoctions, filter, concentrate the filtrate into a clear paste, add ethanol to make the alcohol content reach 65%, let it stand overnight, absorb the supernatant to recover ethanol and eliminate the alcohol taste, add stevioside with a mass fraction of 1.2‰, stir evenly, filter, fill, and sterilize by hot pressing at 110℃ for 40 minutes.

[0013] A method for establishing the HPLC fingerprint of the Qingfei mixture obtained above is characterized by comprising the following steps:

[0014] Step a, preparing a test solution and a reference solution of Qingfei mixture;

[0015] Step b, HPLC chromatographic conditions: chromatographic column: Agilent Eclipse XDB-C 18 Column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution; detection wavelength is 210nm; column temperature is 30℃; injection volume is 10μl; flow rate is 1.0ml / min.

[0016] Step c, analyzing and comparing the Qingfei mixture test solution obtained in step a according to the chromatographic conditions in step b, to obtain the fingerprint of the Qingfei mixture.

[0017] Furthermore, the preparation method of the Qingfei mixture test solution in step a is: accurately measure 1 ml of the Qingfei mixture according to claim 3, add 70% methanol, shake well, filter, and obtain the filtrate.

[0018] Furthermore, the preparation method of the reference solution in step a is:

[0019] Accurately weigh naringin, neohesperidin, baicalin, and praeruptorin A reference substances, and add methanol to dissolve them respectively to prepare naringin reference substance solution with a concentration of 407.66 μg / ml, neohesperidin reference substance solution with a concentration of 429.08 μg / ml, baicalin reference substance solution with a concentration of 125.15 μg / ml, and praeruptorin A reference substance solution with a concentration of 529.80 μg / ml.

[0020] Furthermore, the gradient elution procedure in step b is:

[0021]

[0022]

[0023] Furthermore, the characteristic peaks of the fingerprint spectrum of step c are: peak No. 9 is -naringin, peak No. 11 is neohesperidin, peak No. 12 is baicalin, and peak No. 18 is leucoderma.

[0024] The present invention provides a specific application of the method for establishing the HPLC fingerprint of the Qingfei mixture, and the method for establishing the fingerprint can be used for quality detection of Qingfei mixture preparations.

[0025] The present invention provides a quality detection method for Qingfei mixture, comprising the following steps:

[0026] Step 1, establishing the HPLC fingerprint of Qingfei mixture according to the above-mentioned method for establishing the HPLC fingerprint of Qingfei mixture.

[0027] Step 2, performing quality inspection on the obtained Qingfei mixture according to the established Qingfei mixture HPLC fingerprint.

[0028] The present invention has the following beneficial effects:

[0029] 1. Qingfei mixture is a special preparation of our hospital. It is modified based on the ancient prescription "Ma Xing Shi Gan Tang". It has the effects of clearing heat, promoting lung function, removing phlegm, relieving cough and relieving asthma, and has good clinical effect.

[0030] 2. In the lung-clearing mixture of the present invention, Houttuynia cordata, Indigo Naturalis, and Scutellaria baicalensis are the main drugs for clearing heat, descending qi, and relieving cough and asthma, and are matched with Belamcanda chinensis and Platycodon grandiflorum, which can clear the throat and relieve phlegm, and Fructus Aurantii Immaturus, which can relieve lung qi, relieve cough and asthma, and relieve phlegm and asthma. Scutellaria baicalensis and Ephedra are matched, which can clear and resolve the stagnant heat in the lung. Fructus Aurantii Immaturus is matched with Platycodon grandiflorum, which comes from Fructus Aurantii Immaturus Decoction in Su Shen's Prescriptions, which has the effect of widening the middle and lowering qi, benefiting lung qi, clearing the pharyngeal septum, widening the middle and regulating qi, relieving depression and removing phlegm, which is the so-called "regulating qi when treating phlegm". The combination of bitter almond and scutellaria baicalensis comes from the bitter scutellaria baicalensis pill in the "Shengji Zonglu". Bitter almond is used to relieve lung qi and asthma, and scutellaria baicalensis is used to relieve lung qi and asthma. The two medicines are used together, one is used to promote qi and the other is used to relieve qi, so that the qi mechanism is unobstructed and asthma is flat. Qianhu treats phlegm-heat blocking the lungs, and cough, asthma, and chest fullness caused by the lungs failing to descend. This recipe combines the essence of many classic ancient recipes to comprehensively treat lung heat, cough, and asthma.

[0031] 3. The HPLC fingerprint detection method for Qingfei mixture provided by the present invention has good precision, stability and repeatability. The quality of Qingfei mixture can be comprehensively evaluated by comparing the presence or absence of common peaks in the obtained standard fingerprint, effectively ensuring the quality of the finished product. Compared with the use of a single indicator for multiple detections in the prior art, it can more comprehensively reflect the quality of Qingfei mixture, and the operation is simple, and multiple components can be detected simultaneously, saving time and cost; at the same time, under the chromatographic conditions of the present invention, each characteristic peak achieves baseline separation and high stability, and there is no interference between the characteristic peaks, which can accurately and comprehensively evaluate the quality of Qingfei mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 : HPLC fingerprints of 15 batches of Qingfei mixture samples in Example 4;

[0033] Figure 2 : The attribution of characteristic peaks in Example 4 is that peak No. 9 is naringin, peak No. 11 is neohesperidin, peak No. 12 is baicalin, and peak No. 18 is leucophyllotoxin;

[0034] Figure 3 : Chromatogram of baicalin reference substance;

[0035] Figure 4 : Chromatogram of naringin reference substance;

[0036] Figure 5 :Chromatogram of neohesperidin reference substance;

[0037] Figure 6 : Chromatogram of leucoderma protozoan reference substance;

[0038] Figure 7 : Chromatogram of the Qingfei mixture solution obtained in Example 1. DETAILED DESCRIPTION

[0039] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent modifications to the present invention by those skilled in the art all fall within the scope of protection of the claims of this application.

[0040] Example 1 Preparation of Qingfei Mixture

[0041] Mix 15g of Houttuynia cordata, 10g of honey ephedra, 15g of Scutellaria baicalensis, 20g of Platycodon grandiflorum, 5g of stir-fried bitter almond, 15g of Trichosanthes kirilowii, 20g of Peucedanum chinense, 20g of Lepidium officinale, 10g of stir-fried Citrus aurantium with bran, 10g of Belamcanda chinensis, 8g of Indigo naturalis, and 8g of Licorice, add water and boil twice, combine the decoctions, filter, concentrate the filtrate into a clear paste, add ethanol to make the alcohol content reach 65%, let it stand overnight, absorb the supernatant, recover ethanol to eliminate the alcohol taste, add stevioside with a mass fraction of 1.2‰, stir evenly, filter, fill, and sterilize by hot pressure at 110℃ for 40 minutes.

[0042] Example 2 Preparation of Qingfei Mixture

[0043] Take 10g of Houttuynia cordata, 3g of Ephedra, 5g of Scutellaria baicalensis, 5g of Platycodon grandiflorum, 20g of Bitter Apricot, 5g of Trichosanthes, 5g of Peucedanum chinense, 5g of Lepidium officinale, 3g of Citrus aurantium, 3g of Belamcanda chinensis, 2g of Indigo naturalis, and 2g of Licorice, mix them, add water and boil them twice, combine the decoctions, filter, concentrate the filtrate into a clear paste, add ethanol to make the alcohol content reach 65%, let it stand overnight, absorb the supernatant, recover the ethanol and eliminate the alcohol taste, add stevioside with a mass fraction of 1.2‰, stir well, filter, fill, and sterilize by hot pressure at 110℃ for 40 minutes.

[0044] Example 3 Preparation of Qingfei Mixture

[0045] Take 20g of Houttuynia cordata, 8g of honey ephedra, 10g of Scutellaria baicalensis, 10g of Platycodon grandiflorum, 12g of stir-fried bitter almond, 10g of Trichosanthes kirilowii, 15g of Peucedanum peucedanum, 13g of Lepidium officinale, 5g of stir-fried Citrus aurantium with bran, 6g of Belamcanda chinensis, 5g of Indigo naturalis, and 5g of Licorice, mix them, add water and boil them twice, combine the decoctions, filter, concentrate the filtrate into a clear paste, add ethanol to make the alcohol content reach 65%, let it stand overnight, absorb the supernatant to recover ethanol and eliminate the alcohol taste, add stevioside with a mass fraction of 1.2‰, stir well, filter, fill, and sterilize by hot pressure at 110℃ for 40 minutes.

[0046] Example 4 Fingerprint establishment

[0047] 1. Materials and Instruments

[0048] Agilent 1260 high performance liquid chromatograph; acetonitrile and phosphoric acid for liquid analysis were of chromatographic grade; methanol for test solution and reference solution was of analytical grade; water was Wahaha purified water; naringin, neohesperidin, baicalin, and praeruptorin A reference substances were purchased from China Food and Drug Inspection Institutes.

[0049] 2. Methods and Results

[0050] 2.1 Chromatographic conditions: Agilent Eclipse XDB-C 18 Chromatographic column (4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to Table 1 below; detection wavelength 200-400nm, monitoring wavelength 210nm; column temperature 30°C; injection volume 10μl; flow rate 1.0ml / min.

[0051] Table 1 Gradient elution program

[0052] Time (min) A (acetonitrile, %) B (0.1% phosphoric acid solution, %) 0～15 5→13 95→87 15～18 13 87 18～19 13→18 87→82 19～40 18→22 82→78 40～55 22→35 78→65 55～70 35→55 65→45 70～80 55→95 45→5 80～90 95 5

[0053] 2.2 Preparation of test and reference solutions

[0054] 2.2.1 Reference solution: Accurately weigh naringin, neohesperidin, baicalin, and praeruptorin A reference substances, and add methanol to dissolve them respectively to prepare naringin reference solution with a concentration of 407.66 μg / ml, neohesperidin reference solution with a concentration of 429.08 μg / ml, baicalin reference solution with a concentration of 125.15 μg / ml, and praeruptorin reference solution with a concentration of 529.80 μg / ml.

[0055] 2.2.2 Test solution:

[0056] Fifteen batches of Qingfei mixture were prepared according to the preparation method described in Example 1. The sample batch numbers are shown in Table 2 below.

[0057] Accurately measure 1 ml of the above 15 batches of Qingfei mixture respectively, place it in a 50 mL volumetric flask, add 70% methanol to dilute to the scale, shake well, filter through a 0.22 μm filter membrane, and take the filtrate to obtain 15 batches of Qingfei mixture test solution.

[0058] Table 2 Sample batch

[0059]

[0060]

[0061] 2.3 Fingerprint establishment

[0062] 2.3.1 Common peak calibration, select peak 12 as the reference peak, calculate the relative retention time of each chromatographic peak, the chromatogram is shown in Figure 2 , among which 18 peaks are common peaks of 15 batches of samples.

[0063] 2.3.2 Similarity evaluation The common peaks were analyzed by the software "Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" and the fingerprint was established by the median method. The results are shown in Figure 1 The similarities between the fingerprints of the 15 batches of samples and the control fingerprints were all above 0.99. The results are shown in Table 3.

[0064] Table 3 Similarity analysis results

[0065] serial number Similarity serial number Similarity S1 1 S9 0.990 S2 0.998 S10 0.990 S3 1 S11 1 S4 1 S12 0.996 S5 1 S13 0.996 S6 1 S14 0.996 S7 1 S15 0.999 S8 0.994 — —

[0066] 2.3.3 Common peak identification

[0067] Take the test solution and reference solution under "2.2" and inject according to the chromatographic conditions under "2.1". The chromatogram is shown in Figure 3-Figure 7 The common peaks were identified by the reference substances: Peak No. 9 was naringin, Peak No. 11 was neohesperidin, Peak No. 12 was baicalin, Peak No. 18 was leucophyllotoxin, and a total of 4 common peaks were identified.

[0068] 2.4 Methodological Review

[0069] 2.3.1 Precision test

[0070] Take a sample (batch number: 231102-XM2024-0270), prepare the test solution according to the method under "2.2", and inject the sample 6 times continuously according to the chromatographic conditions under "2.1". Take peak 12 as the reference peak, calculate the relative retention time and relative peak area of ​​common peaks 9, 11, and 18, and their RSD values ​​are all less than 3%. At the same time, the similarity of each chromatographic fingerprint calculated using the "Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" software is greater than 0.99, indicating that the instrument has good precision.

[0071] 2.3.2 Stability test

[0072] Take sample (batch number: 231102-XM2024-0270), prepare the test solution according to the method under "2.2", and inject the sample at 0h, 2h, 4h, 6h, 8h, 12h, 18h, and 24h according to the chromatographic conditions under "2.1", and calculate the relative retention time and relative peak area of ​​common peaks 9, 11, and 18 with peak 12 as the reference peak. The relative retention time RSD value is less than 2%, and the relative peak area RSD value is less than 3%; at the same time, the similarity of each chromatographic fingerprint calculated by the "Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" software is greater than 0.99, indicating that the test solution is stable within 24h.

[0073] 2.3.3 Reproducibility test

[0074] Take sample (batch number: 231102-XM2024-0270), accurately weigh 6 portions respectively, prepare test solution according to the method under "2.2", and inject according to the chromatographic conditions under "2.1". Take peak 12 as the reference peak, calculate the relative retention time and relative peak area of ​​common peaks 9, 11, and 18, and their RSD values ​​are all less than 3%. At the same time, the similarity of each chromatographic fingerprint calculated using the "Chinese Medicine Fingerprint Similarity Evaluation System 2012 Edition" software is greater than 0.99, indicating that the method has good reproducibility.

[0075] 3. Results and Analysis

[0076] In the process of establishing the fingerprint spectrum of Qingfei mixture, the effect of using methanol as a solvent for sample treatment is better. The baseline of the methanol-treated sample is relatively stable, and there are more soluble effective ingredients. During chromatographic analysis, the number of peaks is relatively large. During chromatographic analysis, it was found that using acetonitrile-0.1% phosphoric acid solution as an elution solvent can detect more components and the separation of each chromatographic peak is better. The method for establishing the fingerprint spectrum of Qingfei mixture in the present invention has good repeatability, stability, precision, strong characteristic, and can control multiple components as a whole. When used for the quality detection of Qingfei mixture, it can fully reflect the quality level of Qingfei mixture.

Claims

1. A lung-clearing mixture, characterized in that: The lung-clearing mixture is prepared from the following ingredients: 10-25 weight parts of Houttuynia cordata, 3-10 weight parts of Ephedra, 5-15 weight parts of Scutellaria baicalensis, 5-20 weight parts of Platycodon grandiflorum, 5-20 weight parts of Bitter Apricot, 5-15 weight parts of Trichosanthes, 5-20 weight parts of Peucedanum, 5-20 weight parts of Lemongrass, 3-10 weight parts of Citrus aurantium, 3-10 weight parts of Belamcanda chinensis, 2-8 weight parts of Indigo Naturalis, and 2-8 weight parts of Licorice.

2. The lung-clearing mixture according to claim 1, characterized in that: The ephedra is honey ephedra, the bitter almond is fried bitter almond, and the fructus aurantii is fried fructus aurantii with bran.

3. The lung-clearing mixture according to claim 2, characterized in that: The preparation method of the lung-clearing mixture is as follows: Mix houttuynia cordata, honey ephedra, scutellaria baicalensis, platycodon grandiflorum, stir-fried bitter almond, trichosanthes root, peucedanum chinense, scutellaria baicalensis seed, stir-fried fructus aurantii with bran, belamcanda chinensis, indigo naturalis and liquorice, add water and boil twice, combine the decoctions, filter, concentrate the filtrate into a clear paste, add ethanol to make the alcohol content reach 65%, let it stand overnight, absorb the supernatant to recover ethanol and eliminate the alcohol taste, add stevioside with a mass fraction of 1.2‰, stir evenly, filter, fill, and sterilize by hot pressing at 110℃ for 40 minutes.

4. A method for establishing the HPLC fingerprint of Qingfei mixture as claimed in claim 3, characterized in that: The following steps are involved: Step a, preparing a test solution and a reference solution of Qingfei mixture; Step b, HPLC chromatographic conditions: chromatographic column: Agilent Eclipse XDB-C 18 Column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution; detection wavelength 210nm; column temperature 30℃; injection volume 10μl; flow rate 1.0ml / min; Step c, analyzing and comparing the test solution and reference solution of the Qingfei mixture obtained in step a according to the chromatographic conditions in step b, to obtain the fingerprint of the Qingfei mixture.

5. The establishment method according to claim 4, characterized in that: The preparation method of the Qingfei mixture test solution in step a is: accurately measure 1 ml of the Qingfei mixture according to claim 3, add 70% methanol, shake well, filter, and obtain the filtrate.

6. The method of establishing as claimed in claim 4, characterized in that: The preparation method of the reference substance solution in step a is: Accurately weigh naringin, neohesperidin, baicalin, and praeruptorin A reference substances, and add methanol to dissolve them respectively to prepare naringin reference substance solution with a concentration of 407.66 μg / ml, neohesperidin reference substance solution with a concentration of 429.08 μg / ml, baicalin reference substance solution with a concentration of 125.15 μg / ml, and praeruptorin A reference substance solution with a concentration of 529.80 μg / ml.

7. The establishment method according to claim 4, characterized in that: The gradient elution procedure in step b is: 0-15min, 5%-13%A, 95%-87%B; 15-18min, 13%A, 87%B; 18-19min, 13%-18%A, 87%-82%B; 19-40min, 18%-22%A, 82%-78%B; 40-55min, 22%-35%A, 78%-65%B; 55-70min, 35%-55%A, 65%-45%B; 70-80min, 55%-95%A, 45%-5%B; 80-90,min, 95%A, 5%B.

8. The establishment method according to claim 4, characterized in that: The characteristic peaks of the fingerprint spectrum in step c are: peak No. 9 is naringin, peak No. 11 is neohesperidin, peak No. 12 is baicalin, and peak No. 18 is leucophyllotoxin.

9. Application of the Qingfei mixture HPLC fingerprint establishment method as described in any one of claims 4 to 8 in the quality inspection of Qingfei mixture preparations.

10. A quality inspection method for Qingfei mixture, characterized in that: The following steps are involved: Step 1, establishing the HPLC fingerprint of Qingfei mixture according to the method for establishing the HPLC fingerprint of Qingfei mixture according to any one of claims 4 to 8. Step 2, performing quality inspection on the Qingfei mixture described in any one of claims 1 to 3 according to the established Qingfei mixture HPLC fingerprint.

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