A method for detecting fingerprints of Qingwen Hufei granules

Abstract

The present invention provides a method for detecting the fingerprint of Qingwen Hufei Granules. The detection method generates a standard fingerprint and determines a total of 14 common chromatographic peaks. The fingerprint detection method established by the present invention is tested on 14 batches of Qingwen Hufei Granules, and the similarity value is ≥0.943. The present invention also establishes a method for determining the content of 7 index components, namely, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and angloside. The methodological results show that the method has good precision, stability, and sample recovery rate, and can be used for the intrinsic quality detection and identification of the Qingwen Hufei Granules traditional Chinese medicine composition. It has good market prospects in industrial large-scale production applications.

Description

Technical Field

[0001] The present invention belongs to the field of quality detection of chemical components of traditional Chinese medicine, and in particular relates to a method for detecting the fingerprint of Qingwen Hufei granules. Background Art

[0002] Qingwen Hufei Granules are composed of 16 Chinese medicinal herbs including honeysuckle, forsythia, fritillaria thunbergii, isatis indigotica, loblolly bamboo, platycodon, codonopsis pilosula, stir-fried atractylodes macrocephala, poria, scrophularia, ophiopogon japonicus, bitter almond, capillaris, saposhnikovia divaricata, perilla leaf, and licorice. They have the effects of relieving wind-heat, clearing the lungs and relieving cough, and protecting the spleen and stomach. They are suitable for mild cases of new coronavirus infection and acute respiratory infections in winter and spring.

[0003] The applicant has filed two related patent applications for this product, specifically CN111097021A, which discloses a Chinese medicine composition for treating mild cases, suspected cases and acute respiratory infections of COVID-19. The Chinese medicine composition is made of the following Chinese medicine raw materials in parts by weight: 10-20g of honeysuckle, 10-20g of forsythia, 10-20g of isatis indigotica, 10-20g of Scrophularia, 6-15g of stir-fried bitter almonds, 10-20g of thunbergia thunbergii, 6-15g of platycodon grandiflorum, and 10-20g of purpurogenous ginseng. 6-15g of perilla leaves, 6-15g of siler leaves, 10-20g of stir-fried atractylodes macrocephala, 10-20g of poria, 10-20g of capillaris, 10-20g of codonopsis pilosula, 6-15g of bamboo leaves, 10-20g of ophiopogon japonicus, and 4-10g of raw liquorice. The preparation method of stir-fried bitter almonds is to stir-fry bitter almonds; the preparation method of stir-fried atractylodes macrocephala is to heat raw atractylodes macrocephala and then stir-fry it with wheat bran. Take the above-mentioned Chinese medicinal raw materials by weight, decoct in water for 30 minutes, and take 400ml of juice. This medicine has the characteristics of treating both the exterior and interior, clearing and replenishing, and preventing the disease from changing. It has a definite effect on the treatment of respiratory infections in winter and spring. The symptoms improved before and after taking the medicine are runny nose, fever, cough, dry and sore throat, sputum, poor appetite, and shortness of breath.

[0004] Its publication number CN202211478410.8 describes an ultra-high performance liquid chromatography-mass spectrometry detection method for Qingwen Hufei granules. Using ultra-high performance liquid chromatography-quadrupole / electrostatic field orbital trap high-resolution mass spectrometry UHPLC-QExactiveFocusMS / MS technology, a total of 17 terpenoid compounds (including cleaved strychnine, cleaved strychnine and other components) and 28 phenylpropanoid compounds (1-O-caffeoylquinic acid, chlorogenic acid, The detection method of the present invention can quickly separate and identify the chemical components in Qingwen Hufei Granules online, providing a scientific basis for the research on the basic substances of its pharmacological effects.

[0005] However, because Qingwen Hufei Granules is a traditional Chinese medicine compound preparation, the traditional Chinese medicine formula contains a variety of traditional Chinese medicine flavors, and the traditional Chinese medicine active ingredients it contains are very complex. Currently, the only internal control standard is the thin layer identification method, but because thin layer identification is a qualitative identification, it cannot fully control the intrinsic quality of the traditional Chinese medicine compound. Traditional Chinese medicine fingerprints can more comprehensively reflect the overall characteristics of the chemical components of traditional Chinese medicine and are an effective method for evaluating the authenticity, quality, consistency and stability of traditional Chinese medicines. Combining the multi-index component quantification method with the fingerprint spectrum can provide qualitative and quantitative information of traditional Chinese medicines at the same time, and is an effective method for quality control of traditional Chinese medicines. Summary of the Invention

[0006] The present invention provides a method for detecting the fingerprint of Qingwen Hufei Granules. The method generates a standard fingerprint, identifies 14 common chromatographic peaks, and identifies 7 of the peaks, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and angoside as index components. Furthermore, the fingerprint detection method established by the present invention was tested on 14 batches of Qingwen Hufei Granules, and the similarity value was ≥0.943. The present invention also established a method for determining the content of the seven index components, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and angoside. The methodological results show that the method has good precision, stability, and sample recovery rate, and can be used for intrinsic quality detection and identification of Qingwen Hufei Granule traditional Chinese medicine compositions. It has good market prospects in large-scale industrial production applications.

[0007] The technical solution of the present invention patent application is:

[0008] A fingerprint detection method for Qingwen Hufei granules, the detection method comprising the following steps:

[0009] (1) Preparation of test solution: weigh the Qingwen Hufei granules of the present invention, add methanol to dissolve, ultrasonicate, cool, shake well, filter, evaporate to dryness, fix to volume, filter, and take the filtrate;

[0010] ⑵. Preparation of reference solution: weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-caffeoylquinic acid, and angloside C reference substances, dilute with 40-60% methanol, and make up to volume;

[0011] ⑶、Chromatographic conditions: Chromatographic column is C 18 , mobile phase: 0.1% formic acid water as mobile phase A, acetonitrile as mobile phase B; the present invention adopts a gradient elution method, and the elution order is: 0-14 min, 6%-8% B; 14-15 min, 8%-13% B; 15-17 min, 13%-15% B; 17-20 min, 15%-17% B; 20-23 min, 17%-33% B; 23-27 min, 33% B; 27-31 min, 33%-98% B; flow rate 0.2-0.6 mL / min; column temperature 23-28 ° C, detection wavelength 220-270 nm;

[0012] (4) Establish fingerprint: Determine the test solution in step (1) and the reference solution in step (2) using the chromatographic conditions under step (3), perform detection according to the chromatographic conditions under step (3), inject and analyze multiple batches of Qingwen Hufei Granule test solutions, convert the data file into "AIA" format, and import it into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to generate a control fingerprint.

[0013] As a preferred embodiment of the present invention, in step (1) of the detection method, the ultrasonic frequency is 40-60kHz, the ultrasonic power is 280-320W, and the ultrasonic treatment time is 20-40min.

[0014] As a preferred embodiment of the present invention, the concentration of methanol in step (2) of the detection method is 50%.

[0015] As a preferred embodiment of the present invention, in step (2) of the detection method, the concentration of neochlorogenic acid is: 0.0467-0.280 mg / mL, the concentration of chlorogenic acid is: 0.0375-0.2250 mg / mL, the concentration of cryptochlorogenic acid is: 0.0395-0.2370 mg / mL, the concentration of forsythiaside A is: 0.0448-0.2688 mg / mL, the concentration of 3,5-O-dicaffeoylquinic acid is: 0.00506-0.03036 mg / mL, the concentration of 4,5-caffeoylquinic acid is: 0.00396-0.02376 mg / mL, and the concentration of angloside C is: 0.00576-0.03456 mg / mL.

[0016] As a preferred embodiment of the present invention, in step (2) of the detection method, the concentration of neochlorogenic acid is 0.2802 mg / mL, the concentration of chlorogenic acid is 0.2250 mg / mL, the concentration of cryptochlorogenic acid is 0.2370 mg / mL, the concentration of forsythiaside A is 0.1344 mg / mL, the concentration of 3,5-O-dicaffeoylquinic acid is 0.03036 mg / mL, the concentration of 4,5-caffeoylquinic acid is 0.02376 mg / mL, and the concentration of angloside C is 0.03456 mg / mL.

[0017] As the preferred embodiment of the present invention, the detection method step (3) chromatographic column C 18 Model: Agilent ZORBAX Eclipse Plus, column specifications: 250 mm × 4.6 mm, 5 μm.

[0018] As a preferred embodiment of the present invention, in step (3) of the detection method, the chromatographic column temperature is 25° C., the flow rate is 0.3 mL / min, and the detection wavelength is 254 nm.

[0019] The multiple batches are more than 10 batches.

[0020] As a preferred embodiment of the present invention, the chromatographic conditions of step (3) are used to detect the test solution of the traditional Chinese medicine for clearing away the wen and protecting the lungs, and the generated high performance liquid chromatography peak is compared with the control fingerprint spectrum, and the similarity is calculated to be 0.94 to 1.00.

[0021] As a preferred embodiment of the present invention, the detection method can be used for quality detection and component identification of Qingwen Hufei Granules.

[0022] In order to further illustrate the creativity of the detection method of the fingerprint spectrum of Qingwen Hufei Granules of the present invention, some experimental contents screened by the technical solution of the present invention are summarized as follows.

[0023] During the establishment of the fingerprint method of the present invention, the optimal chromatographic conditions were obtained after repeated experiments. This is mainly because the preparation process of the Qingwen Hufei Granules of the present invention is a traditional water extraction process, with a large prescription volume (containing 16 Chinese medicines), but the proportion of single medicines is relatively small (the total prescription is 206g, and the 10 medicines such as honeysuckle and forsythia are all 15g, which are the medicinal materials with the largest dosage in the prescription, that is, the largest medicinal material accounts for only 7.3%). The above multiple reasons have caused the following problems in the Qingwen Hufei Granules product: (1) The ingredients are complex and the chromatographic peaks are difficult to separate; (2) The content of each component is low and it is difficult to quantify; (3) The peak height is low and the baseline is not easy to stabilize. The method described in this paper can better separate the chromatographic peaks under a large number of chromatographic conditions. In addition, it has the following advantages: (1) Standards are readily available and inexpensive, which reduces the cost for quality control; (2) The detection time is shortened (in the process of exploration, the separation is good at about 130 minutes. Considering the long time, the conditions are optimized and compressed to 100 minutes), which improves the detection efficiency for quality control; (3) The reagents in the detection method of the present invention are all consumables that are readily available, inexpensive, harmless to humans and the environment, and meet environmental protection requirements; (4) The chromatographic conditions are simple and the separation is good, which reduces the detection cost for quality control while ensuring accuracy.

[0024] The specific process is as follows:

[0025] 1.1 Optimization of chromatographic conditions

[0026] Different mobile phase types (methanol-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, methanol-0.1% formic acid) and ratios, as well as detection wavelengths (220, 230, 250, 270, 290, 310, 330 nm) were investigated, and their effects on the number, area, peak shape, resolution, and baseline stability of chromatographic peaks were analyzed. Finally, an Agilent ZORBAX EclipsePlus-C18 column was selected, with methanol-0.1% phosphoric acid aqueous solution as the mobile phase and detection and separation at a wavelength of 330 nm.

[0027] 1.2 Investigation of test solution preparation method

[0028] Based on the comprehensive evaluation criteria of chromatographic peak information richness, peak shape and relative peak area, the effects of different extraction solvents (methanol with volume fractions of 50%, 70% and 100% respectively), ultrasonic time (30, 45, 60 min) and solvent after evaporation (water, methanol, 50% methanol) on the extraction effect of the measured components were investigated. Finally, it was determined that 100% methanol by volume was used as the solvent and ultrasonic extraction was performed for 30 min.

[0029] 1.3 Optimization of chromatographic mobile phase conditions

[0030] Since the chemical components contained in Qingwen Hufei Granules are complex and the polarity differences are obvious, it is difficult to achieve effective separation and analysis by simply relying on isocratic elution, so gradient elution is adopted to achieve the effect of improving peak shape and increasing separation degree. This application uses methanol (A)-0.1% phosphoric acid solution (B) for elution. In the early stage during 2-15 minutes, only Peak 1 exists. This application shortens the elution time between the two by accelerating the growth ratio of methanol in the mobile phase, thereby reducing the overall time. In about 12-40 minutes, gradient elution is used to separate Peak 2 neochlorogenic acid, Peak 3 chlorogenic acid and Peak 5 cryptochlorogenic acid in Qingwen Hufei Granules. Because Peaks 6-10 are all unknown peaks in Peaks 6-12, and Peak 8 is preceded and followed by interference peaks, the methanol ratio is rapidly increased in 35-38 minutes, and the methanol ratio is slowly increased in 38-80 minutes to separate Peaks 6-12. Rapidly increasing the methanol ratio significantly shortens analysis time, but it's worth noting that too rapid an increase in the ratio can also cause an upward baseline shift, significantly impacting subsequent chromatographic peaks. Therefore, after extensive experimentation, this application identified an appropriate methanol increase ratio. Peak 11, forsythiaside A, and Peak 12, 3,5-O-dicaffeoylquinic acid, are too close in polarity to be separated using an isocratic method. Therefore, a reduced flow rate was employed, changing the overall flow rate from 1 ml / min to 0.8 ml / min, successfully separating peaks 11 and 12. Between 80 and 100 minutes, only two control peaks, 13 and 14, were observed, with an interfering peak adjacent to peak 13. Given the long overall analysis time, the methanol ratio was also varied rapidly. Finally, after a lot of experimental exploration and proportion adjustment, the optimal mobile phase ratio was determined to be 0-12 min, 10%-16% A; 12-35 min, 16%-18% A; 35-38 min, 18%-30% A; 38-80 min, 30%-34% A; 80-95 min, 34%-58% A; 95-100 min, 58%-90% A.

[0031] 1.4 Selection of indicator components

[0032] The fingerprint spectrum was compared with the reference substances and identified 15 peaks, namely neochlorogenic acid (peak 2), chlorogenic acid (peak 3), cryptochlorogenic acid (peak 5), forsythiaside A (peak 11), 3,5-O-dicaffeoylquinic acid (peak 12), 4,5-dicaffeoylquinic acid (peak 13), and angloside C (peak 14).

[0033] After reviewing relevant literature, we found that the organic acid components in honeysuckle can inhibit the overexpression of interferon beta (IFN-β) or the secretion of NO, IL-6, IL-1β, and TNF-α in inflammatory cells, thereby exerting anti-inflammatory effects. Forsythiaside A in forsythia can also reduce the production of cellular inflammatory factors, such as inhibiting STAT3 protein expression by upregulating miR-124, thereby inhibiting the production of IL-6, thereby exerting its anti-inflammatory effects. The aqueous extract of Scrophularia ningpoensis can promote the growth of mouse immune organs and carbon clearance index, and improve the levels of hemolysin and IFN-γ in mouse serum, exerting an immunomodulatory effect. Therefore, these seven components were included in the indicator components for the content determination of Qingwen Hufei Granule.

[0034] In this study, an HPLC method was established to simultaneously determine the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythoside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and angoside C in Qingwen Hufei Granules. This content determination method is accurate, precise, and simple, which helps to improve the quality control level of this preparation and ensure its effectiveness and safety in clinical application.

[0035] The beneficial effects of the patented technical solution of this invention are as follows:

[0036] ⑴. After a large number of experimental trials, the present invention finally found the optimal chromatographic conditions for the fingerprint of Qingwen Hufei Granules. Agilent ZORBAX Eclipse Plus-C18 chromatographic column (250mm×4.6mm, 5μm); mobile phase methanol (A), 0.1% phosphoric acid (B), gradient elution (0-12min, 10%-16% A; 12-35min, 16%-18% A; 35-38min, 18%-30% A; 38-80min, 30%-34% A; 80-95min, 34%-58% A; 95-100min, 58%-90% A); volume flow rate 0.8mL / min; column temperature 30℃; detection wavelength 330nm.

[0037] After methodological investigation, the results of the repeatability test showed that the relative standard deviation (RSD) of the relative retention time of each common peak was 0.019% to 0.637%, and the RSD of the relative peak area was 0.049% to 0.596%, indicating that the injection repeatability was good. The results of the repeatability test showed that the RSD of the relative retention time of each common peak was 0.004% to 0.082%, and the RSD of the relative peak area was 0.062% to 2.520%, indicating that the method had good repeatability. The results of the stability test showed that the RSD of the relative retention time of each common peak was 0.006% to 0.142%, and the RD of the relative peak area was 0.210% to 3.066%, indicating that the test sample of the present invention was stable within 24 hours at room temperature.

[0038] Similarity analysis of the fingerprint of the present invention: the similarities of 14 batches of Qingwen Hufei Granules test samples were 0.972, 0.984, 0.992, 0.992, 0.983, 0.981, 0.992, 0.993, 0.943, 0.996, 0.996, 0.997, 0.949 and 0.996, respectively, which also shows that the quality of Qingwen Hufei Granules medicinal materials in different batches is relatively stable.

[0039] (2) The detection method of the present invention generates a standard fingerprint, identifies a total of 14 common chromatographic peaks, and identifies 7 of them as the index components: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and angoside. This detection method can be used to detect the intrinsic quality and identify the components of Qingwen Hufei Granules, providing a new reference.

[0040] (3) The present invention establishes an HPLC method for simultaneously determining the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and angoside C in Qingwen Hufei granules. The results of the methodological validation show that the RSDs of the peak areas of the components were 0.10% for neochlorogenic acid, 0.06% for chlorogenic acid, 0.14% for cryptochlorogenic acid, 0.14% for forsythiaside A, 0.43% for 3,5-O-dicaffeoylquinic acid, 0.03% for 4,5-O-dicaffeoylquinic acid, and 0.08% for angoside C, respectively, indicating good injection repeatability. The results of the stability test showed that the RSDs of the peak areas of the components were 0.35% for neochlorogenic acid, 0.16% for chlorogenic acid, 0.10% for cryptochlorogenic acid, 0.25% forsythiaside A, 1.82% for 3,5-O-dicaffeoylquinic acid, 2.04% for 4,5-O-dicaffeoylquinic acid, and 0.38% for angoside C, indicating good stability within 24 hours. The results of the repeatability test showed that the RSDs of the content of the components were 2.28% for neochlorogenic acid, 2.55% for chlorogenic acid, 2.34% for cryptochlorogenic acid, 1.41% forsythiaside A, 2.14% for 3,5-O-dicaffeoylquinic acid, 2.00% for 4,5-O-dicaffeoylquinic acid, and 1.39% for angoside C, indicating good repeatability of the method. The results of the sample recovery test showed that the average recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and angloside C were 97.67%, 96.31%, 96.46%, 96.94%, 97.39%, 103.84% and 102.45%, respectively, and the RSDs were 1.42%, 0.56%, 1.20%, 1.22%, 0.52%, 0.96% and 0.36%, respectively.

[0041] (4) After multiple batches of content determination, it was found that the content of neochlorogenic acid in the Qingwen Hufei granules of the present invention was 0.0210-0.0459%, chlorogenic acid 0.0269-0.0506%, cryptochlorogenic acid 0.028-0.0461%, 3,5-O-dicaffeoylquinic acid A 0.0444-0.1045%, forsythiaside A 0.0067-0.0156%, 4,5-O-dicaffeoylquinic acid 0.0088-0.0144%, and angoside C 0.0057-0.0101%. The above detection method can be used as an internal quality control standard for this product to further ensure its intrinsic quality.

[0042] ⑸. There are 14 common peaks in the fingerprints of 14 batches of Qingwen Hufei Granules, with a similarity greater than 0.9. Cluster analysis can divide the 14 batches of samples into 3 categories, and principal component analysis obtains 4 principal components, with a cumulative variance contribution rate of 99.529%. The content determination results show that the average sample recovery of the above 7 components is 97.11% to 101.14%, the RSD is 0.60% to 2.65%, and the RSD of precision, stability and repeatability tests is less than 3.0%. The HPLC fingerprint and content determination method established by the present invention are efficient, feasible, and reproducible, and can be used for the quality control of Qingwen Hufei Granules, providing a reference basis. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 , 14 batches of Qingwen Hufei granules high performance liquid chromatography (HPLC) fingerprints;

[0044] Figure 2 , HPLC chromatogram of the mixed reference substance, wherein, chromatographic peak 2-neochlorogenic acid; chromatographic peak 3-chlorogenic acid; chromatographic peak 5-cryptochlorogenic acid; chromatographic peak 11-forsythoside A; chromatographic peak 12-3,5-O-dicaffeoylquinic acid; chromatographic peak 13-4,5-dicaffeoylquinic acid; chromatographic peak 14-angoloside C;

[0045] Figure 3 , the peaks of the HPLC standard fingerprint of Qingwen Hufei Granules, among which, peak 2 is neochlorogenic acid; peak 3-chlorogenic acid; peak 5-cryptochlorogenic acid; peak 11-forsythiaside A; peak 12-3,5-O-dicaffeoylquinic acid; peak 13-4,5-dicaffeoylquinic acid; peak 14-angoloside C; peaks 1, 4, 6 to 10-unknown peaks;

[0046] Figure 4 , Cluster analysis diagram of 14 batches of Qingwen Hufei granule samples;

[0047] Figure 5 , PCA score chart of 14 batches of Qingwen Hufei Granule samples. DETAILED DESCRIPTION

[0048] Unless otherwise defined, the technical or scientific terms used in the patent application specification and claims of the present invention shall have the common meanings understood by persons having ordinary skills in the field to which the invention belongs.

[0049] Example 1 Construction of the fingerprint spectrum of Qingwen Hufei granules of the present invention

[0050] 1. Materials

[0051] 1.1 Instruments: Agilent 1260 high performance liquid chromatograph equipped with a VWD detector and an Agilent ChemStation workstation (Agilent Corporation, USA); KQ-100 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BS210S (precision 0.1 mg) and BT25S (precision 0.01 mg) electronic analytical balances (Beijing Sartorius Balance Co., Ltd.).

[0052] 1.2 Reagents and drugs

[0053] 14 batches of Qingwen Hufei Granules (Shaanxi Provincial Hospital of Traditional Chinese Medicine, 10g×10 bags / box, batch numbers 20201101, 20210926, 20220101, 20220701, 20221202, 20210715, 20221228, 20210724, 20210929, 20211005, 20211013, 20211226, 20220418, 20220428, 20220506, numbers S1 to S14). Neochlorogenic acid (CHB220328, purity ≥98%), chlorogenic acid (CHB231010, purity ≥98%), cryptochlorogenic acid (CHB230830, purity ≥98%), forsythiaside A (CHB231017, purity ≥98%), 3,5-O-dicaffeoylquinic acid (CBH231227, purity ≥98%), 4,5-dicaffeoylquinic acid (CHB240104, purity ≥98%), and angloside C (CHB240217, purity ≥98%), as well as reference substances, were purchased from Chengdu Kroma Biotechnology Co., Ltd. Methanol was chromatographically pure (Thermo Fisher Scientific, USA); all other reagents were analytically pure; water was Wahaha purified water.

[0054] 1.2 Methods and Results

[0055] 1.2.1 Study on the fingerprint of Qingwen Hufei Granules

[0056] 1.2.1.1 Chromatographic conditions

[0057] An Agilent ZORBAX Eclipse Plus-C18 column (250 mm × 4.6 mm, 5 μm) was used as the mobile phase: methanol (A), 0.1% phosphoric acid (B); gradient elution (0–12 min, 10%–16% A; 12–35 min, 16%–18% A; 35–38 min, 18%–30% A; 38–80 min, 30%–34% A; 80–95 min, 34%–58% A; 95–100 min, 58%–90% A); flow rate: 0.8 mL / min; column temperature: 30°C; detection wavelength: 330 nm; injection volume: 10 μL.

[0058] 1.2.1.2 Preparation of reference solution

[0059] Accurately weigh an appropriate amount of each reference substance, place it in a 10 mL volumetric flask, dilute with 50% methanol and make up to volume to obtain (neochlorogenic acid 0.2802 mg / mL, chlorogenic acid 0.2250 mg / mL, cryptochlorogenic acid 0.2370 mg / mL, forsythiaside A 0.1344 mg / mL, 3,5-O-dicaffeoylquinic acid 0.03036 mg / mL, 4,5-caffeoylquinic acid 0.02376 mg / mL, and angloside C 0.03456 mg / mL).

[0060] 1.2.1.3 Preparation of test solution

[0061] Grind the contents of this product finely, accurately weigh 2.0 g, place in a 50 mL stoppered conical flask, dissolve in 30 mL of methanol, weigh the mass, and ultrasonically treat (frequency 50 kHz, power 300 W) for 30 min. Cool to room temperature, shake well, filter, evaporate to dryness, and dilute to a 10 mL volumetric flask. Filter through a 0.45 μm microporous membrane and collect the filtrate to obtain the product.

[0062] 1.2.1.4 Injection repeatability test

[0063] The same test solution (batch number 20221228) was taken and injected continuously 6 times according to the chromatographic conditions under "1.2.1.1". Forsythiaside A (peak 11) was used as the reference peak. The relative standard deviation (RSD) of the relative retention time of each common peak was calculated to be 0.019%~0.637%, and the RSD of the relative peak area was 0.049%~0.596%, indicating good injection repeatability.

[0064] 1.2.1.5 Repeatability test

[0065] Take the same batch of Qingwen Hufei Granules (S7) and prepare 6 test solutions in parallel using the method under "2.1.3". The samples were injected separately according to the chromatographic conditions under "2.1.1". Forsythiaside A (peak 11) was used as the reference peak. The RSDs of the relative retention times of the common peaks were calculated to be 0.004% to 0.082%, and the RSDs of the relative peak areas were 0.062% to 2.520%, indicating that the method has good repeatability.

[0066] 1.2.1.6 Stability test

[0067] The same sample solution (S7) was taken and measured according to the chromatographic conditions under "1.2.1.1" at room temperature for 0, 2, 4, 8, 12 and 24 hours, respectively. Forsythiaside A (peak 11) was used as the reference peak. The RSDs of the relative retention times of the common peaks were calculated to be 0.006% to 0.142%, and the RDs of the relative peak areas were 0.210% to 3.066%, indicating that the sample was stable at room temperature within 24 hours.

[0068] 1.2.1.7 Fingerprint establishment and similarity analysis

[0069] According to the chromatographic conditions under "1.2.1.1", 14 batches of Qingwen Hufei granules were sampled and analyzed. The data files were converted into "AIA" format and imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". With S1 as the reference spectrum and the time window width set to 0.3, multi-point calibration and Mark peak matching were performed, and the median method was used to generate the reference fingerprint spectrum. The HPLC fingerprint spectrum overlay of 14 batches of Qingwen Hufei granules and the reference spectrum are shown in Figure 1 The similarities between the fingerprints of samples S1 to S14 and the control were calculated to be 0.972, 0.984, 0.992, 0.992, 0.983, 0.981, 0.992, 0.993, 0.943, 0.996, 0.996, 0.997, 0.949, and 0.996, respectively, indicating that the quality of Qingwen Hufei Granules in different batches was relatively stable.

[0070] 1.3 Cluster Analysis (CA):

[0071] The peak areas of 14 common peaks in 14 batches of Qingwen Hufei granules were imported into SPSS27 software, and plotted using intergroup linkage and squared Euclidean distance method. The results are shown in the attached figure. Figure 4 The 14 batches of Qingwen Hufei Granule samples can be divided into three categories: the first category includes samples S1-2, S5-6, S10-S12, and S14; the second category includes samples S9 and S13; and the third category includes samples S3-4 and S7-8.

[0072] 1.4 Principal Component Analysis (PCA):

[0073] The 14 common peak areas in 14 batches of Qingwen Hufei Granule samples were imported into SPSS27 software, and principal component analysis was performed after standardization to obtain a 14-order × 14-order data matrix. Four principal components with eigenvalues ​​> 1 were extracted, and the cumulative variance contribution rate was 99.529%, which can reflect the main information of the fingerprint of Qingwen Hufei Granule samples, as shown in Table 1. The chromatographic peaks 2, 3, 5, 10, 12, and 13 have a higher contribution rate in the first principal component, the chromatographic peak 4 has a higher contribution rate in the second principal component, the chromatographic peak 6 has a higher contribution rate in the third principal component, and the chromatographic peaks 4 and 7 have a higher contribution rate in the fourth principal component, as shown in Table 2. The PCA score diagram was drawn using Origin software, see Appendix. Figure 5 , 14 batches of Qingwen Hufei granules samples can be divided into 3 categories. The first category is samples S1-2, S5-6, S10-S12, and S14. The second category is samples S9 and S13. The third category is samples S3-4 and S7-8. The third category is sample S3, which is consistent with the results of cluster analysis.

[0074] Table 1 PCA eigenvalues ​​and variance contribution rates of Qingwen Hufei Granules

[0075]

[0076] Table 2 PCA factor loading matrix of Qingwen Hufei Granules

[0077]

[0078]

[0079] Example 2 Determination of the contents of seven index components in Qingwen Hufei granules

[0080] 2.1.1 Chromatographic conditions

[0081] An Agilent ZORBAX Eclipse Plus-C18 column (250 mm × 4.6 mm, 5 μm) was used as the mobile phase: methanol (A), 0.1% phosphoric acid (B); gradient elution (0–12 min, 10%–16% A; 12–35 min, 16%–18% A; 35–38 min, 18%–30% A; 38–80 min, 30%–34% A; 80–95 min, 34%–58% A; 95–100 min, 58%–90% A); flow rate: 0.8 mL / min; column temperature: 30°C; detection wavelength: 330 nm; injection volume: 10 μL.

[0082] 2.2.2 Preparation of reference solution

[0083] Accurately weigh an appropriate amount of each reference substance, place it in a 10 mL volumetric flask, dilute with 50% methanol and make up to volume to obtain (neochlorogenic acid 0.2802 mg / mL, chlorogenic acid 0.2250 mg / mL, cryptochlorogenic acid 0.2370 mg / mL, forsythiaside A 0.1344 mg / mL, 3,5-O-dicaffeoylquinic acid 0.03036 mg / mL, 4,5-caffeoylquinic acid 0.02376 mg / mL, and angloside C 0.03456 mg / mL).

[0084] 2.2.3 Preparation of test solution

[0085] Grind the contents of this product (Qingwen Hufei Granules) finely, accurately weigh 2.0g, place in a 50mL stoppered conical flask, dissolve in 30mL of methanol, weigh the mass, and ultrasonicate (frequency 50kHz, power 300W) for 30min. Cool to room temperature, shake well, filter, evaporate to dryness, and dilute to a 10mL volumetric flask. Filter through a 0.45μm microporous membrane, and collect the filtrate to obtain the product.

[0086] 2.2.4 Linear Relationship

[0087] The reference solution prepared in "2.1.2" was diluted stepwise with 75% methanol to six different appropriate concentrations. The sample was injected and analyzed under the chromatographic conditions described in "2.1.1." The chromatogram and peak area were recorded. A standard curve was constructed using the mass concentration of the index component as the abscissa (X) and the peak area as the ordinate (Y). The regression equation was calculated. The linear relationship of the four index components is shown in Table 3.

[0088] Table 3 Linear regression analysis of 7 index components in Qingwen Hufei granules

[0089]

[0090]

[0091] From the determination results in Table 3, it can be seen that the linear concentration range of neochlorogenic acid is 46.7-280.2 μg·mL -1 The linear concentration range of chlorogenic acid is 37.5~225μg·mL -1 The linear concentration range of cryptochlorogenic acid was 39.5-237 μg·mL -1 The linear concentration range of forsythiaside was A44.8~268.8μg·mL -1 The linear concentration range of 3,5-O-dicaffeoylquinic acid was 5.06-30.36 μg·mL -1 The linear concentration range of 4,5-caffeoylquinic acid was 3.96-23.76 μg·mL -1The linear concentration range of angloside C was 5.76-34.56 μg·mL -1 , and the linear correlation coefficients of the above 7 linear indicator components are ≥0.9996.

[0092] 2.2.5 Injection repeatability test

[0093] Take an appropriate amount of the test sample solution and inject it for determination 6 times under the chromatographic conditions in “2.1.1”. The determination results are as follows.

[0094] Table 4 Injection repeatability test results

[0095]

[0096] From the experimental results in Table 4, it can be seen that the RSDs of the peak areas of each component were 0.09% for neochlorogenic acid, 0.08% for chlorogenic acid, 0.07% forsythiaside A, 0.07% forsythiaside A, 0.17% for 3,5-O-dicaffeoylquinic acid, 0.07% for 4,5-O-dicaffeoylquinic acid, and 0.23% forsythiaside C, respectively, indicating good injection repeatability.

[0097] 2.2.6 Stability test

[0098] Take the same portion of this product (S7) and prepare the test solution according to the method under "2.1.3". At room temperature at 0, 2, 4, 8, 12, and 24 hours, use the chromatographic conditions of "2.1.1" for injection and determination. The results are shown in Table 5.

[0099] Table 5 Stability test results

[0100]

[0101] As can be seen from the results in Table 3, the RSDs of the peak areas of each component were 0.22% for neochlorogenic acid, 0.15% for chlorogenic acid, 0.10% for cryptochlorogenic acid, 0.25% forsythiaside A, 1.82% for 3,5-O-dicaffeoylquinic acid, 2.04% for 4,5-O-dicaffeoylquinic acid, and 0.38% forsythiaside C, respectively, indicating that the solution had good stability within 24 h.

[0102] 2.2.7 Repeatability test

[0103] Take the same portion of this product (S7) and prepare 6 test solutions in parallel according to the method under "2.1.3". Inject and measure under the chromatographic conditions of "2.1.1". The results are shown in Table 6.

[0104] Table 6 Repeatability test results

[0105]

[0106]

[0107] As shown in Table 6, the RSDs of the measured contents of each component were 2.28% for neochlorogenic acid, 2.55% for chlorogenic acid, 2.34% forsythiaside A, 1.41% forsythiaside A, 2.14% for 3,5-O-dicaffeoylquinic acid, 2.00% for 4,5-O-dicaffeoylquinic acid, and 1.39% forsythiaside C, indicating that the method had good repeatability.

[0108] 2.2.8 Sample recovery test

[0109] Accurately weigh 6 portions of the product (S7) with known content of each component, 1.0 g each, and accurately add the reference substance at a level of 100%. Prepare the test solution according to the method under "2.1.3". Inject the sample and measure under the chromatographic conditions under "2.1.1" and calculate the recovery.

[0110] Table 7 Sample recovery test results

[0111]

[0112]

[0113] The results are shown in Table 7. From the sample recovery experiment results, it can be seen that the average sample recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid and angloside C are 97.67%, 96.31%, 96.46%, 96.94%, 97.39%, 103.84% and 102.45%, respectively, and the RSDs are 2.98%, 1.15%, 2.44%, 2.55%, 1.05%, 1.87% and 0.70%, respectively.

[0114] 2.2.9 Sample content determination

[0115] Fourteen batches of samples of the Chinese medicine composition of the present invention were taken, and test solutions were prepared according to the method under "2.1.3". The samples were injected and measured under the chromatographic conditions of "2.1.1". The content of each index component was calculated. The results are shown in Table 8.

[0116] Table 8 Content determination results of 14 batches of Qingwen Hufei granules

[0117]

[0118]

[0119] As shown in Table 8, after multiple batches of content determination, the content of neochlorogenic acid in the traditional Chinese medicine composition (Qingwen Hufei Granules) of the present invention is 0.0210-0.0459%, chlorogenic acid 0.0269-0.0506%, cryptochlorogenic acid 0.028-0.0461%, 3,5-O-dicaffeoylquinic acid A 0.0444-0.1045%, forsythiaside A 0.0067-0.0156%, 4,5-O-dicaffeoylquinic acid 0.0088-0.0144%, and angloside C 0.0057-0.0101%. The upper and lower limits of the content of the above active ingredients are maintained within a certain range, thereby placing higher requirements on parameter control during the production process, further ensuring the stability of the inherent quality of the product.

[0120] 3.1 Results Analysis

[0121] The fingerprint of Qingwen Hufei Granules was compared with the reference substance and identified a total of 14 peaks, namely neochlorogenic acid (peak 2), chlorogenic acid (peak 3), cryptochlorogenic acid (peak 5), forsythiaside A (peak 11), 3,5-O-dicaffeoylquinic acid (peak 12), 4,5-dicaffeoylquinic acid (peak 13), and angoside C (peak 14). Studies have shown that these compounds have anti-inflammatory, antibacterial, and antiviral activities. Forsythiaside A can significantly inhibit the production of LPS-induced inflammatory mediators TNF-α, IL-1β, NO, and PGE2. It can also inhibit the activation of NF-κB and the Nrf2 / HO-2 signaling pathway, thereby inhibiting the LPS-induced inflammatory response of BV1 microglia and primary microglia. Chlorogenic acid inhibits the in vitro growth of influenza A virus, syncytial virus, Coxsackie B type 3 virus, Coxsackie B type 5 virus, influenza virus subtype 3, and parainfluenza virus type IV, exhibiting antiviral effects and effectively alleviating lung inflammation, providing 60% and 50% protection against mortality caused by H1N1 and H3N2, respectively. Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, 3,5-O-dicaffeoylquinic acid, angloside C, and 4,5-dicaffeoylquinic acid can reduce inflammation and alleviate lung injury by inhibiting nitric oxide (NO) release and lowering levels of IL-1β, IL-6, IL-8, and TNF-α. This suggests that these seven components may be key ingredients in Qingwen Hufei Granules' ability to dispel wind and heat, clear the lungs, and relieve cough.

[0122] The HPLC fingerprint similarities of all 14 batches of Qingwen Hufei Granule samples were greater than 0.9, indicating good consistency in component composition and a stable production process. Cluster analysis and principal component analysis showed that the 14 batches of Qingwen Hufei Granule samples could be divided into three categories, indicating that there were certain differences in the content of ingredients in different batches of Qingwen Hufei Granule.

[0123] In summary, the present invention established an HPLC fingerprint of Qingwen Hufei Granules and simultaneously determined the contents of seven components. This method is accurate, precise, and simple, which helps to improve the quality control level of this traditional Chinese medicine preparation and provide a reference for the formulation of its quality standards.

Claims

1. A fingerprint detection method for Qingwen Hufei granules, characterized in that: The detection method comprises the following steps: (1) Preparation of test solution: weigh the Qingwen Hufei granules of the present invention, add methanol to dissolve, ultrasonicate, cool, shake well, filter, evaporate to dryness, fix to volume, filter, and take the filtrate; ⑵. Preparation of reference solution: weigh neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, forsythiaside A, 3,5-O-dicaffeoylquinic acid, 4,5-caffeoylquinic acid, and angloside C reference substances, dilute with 40-60% methanol, and make up to volume; ⑶、Chromatographic conditions: Chromatographic column is C 18 , mobile phase: 0.1% formic acid water as mobile phase A, acetonitrile as mobile phase B; the present invention adopts a gradient elution method, and the elution order is: 0-14 min, 6%-8% B; 14-15 min, 8%-13% B; 15-17 min, 13%-15% B; 17-20 min, 15%-17% B; 20-23 min, 17%-33% B; 23-27 min, 33% B; 27-31 min, 33%-98% B; flow rate 0.2-0.6 mL / min; column temperature 23-28 ° C, detection wavelength 220-270 nm; (4) Establish fingerprint: Determine the test solution in step (1) and the reference solution in step (2) using the chromatographic conditions under step (3), perform detection according to the chromatographic conditions under step (3), inject and analyze multiple batches of Qingwen Hufei Granule test solutions, convert the data file into "AIA" format, and import it into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to generate a reference fingerprint.

2. The detection method according to claim 1, wherein The detection method step (1) comprises the following steps: the ultrasonic frequency is 40-60 kHz, the ultrasonic power is 280-320 W, and the ultrasonic treatment time is 20-40 min.

3. The detection method according to claim 1, wherein The concentration of methanol in step (2) of the detection method is 50%.

4. The detection method according to claim 1, wherein The concentration of neochlorogenic acid in step (2) of the detection method is: 0.0467-0.280 mg / mL, and the concentration of chlorogenic acid is: The concentration of the extract was 0.0375-0.2250 mg / mL, the concentration of cryptochlorogenic acid was 0.0395-0.2370 mg / mL, the concentration of forsythiaside A was 0.0448-0.2688 mg / mL, the concentration of 3,5-O-dicaffeoylquinic acid was 0.00506-0.03036 mg / mL, the concentration of 4,5-caffeoylquinic acid was 0.00396-0.02376 mg / mL, and the concentration of angloside C was 0.00576-0.03456 mg / mL.

5. The detection method according to claim 4, wherein: In step ⑵ of the detection method, the concentration of neochlorogenic acid is 0.2802 mg / mL, the concentration of chlorogenic acid is 0.2250 mg / mL, the concentration of cryptochlorogenic acid is 0.2370 mg / mL, the concentration of forsythiaside A is 0.1344 mg / mL, the concentration of 3,5-O-dicaffeoylquinic acid is 0.03036 mg / mL, the concentration of 4,5-caffeoylquinic acid is 0.02376 mg / mL, and the concentration of angloside C is 0.03456 mg / mL.

6. The detection method according to claim 1, wherein The detection method step (3) chromatographic column C 18 Model: Agilent ZORBAX Eclipse Plus, column specifications: 250 mm × 4.6 mm, 5 μm.

7. The detection method according to claim 1, wherein In step (3) of the detection method, the column temperature was 25°C, the flow rate was 0.3 mL / min, and the detection wavelength was 254 nm.

8. The fingerprint detection method according to claim 1, wherein: The chromatographic conditions of step (3) are used to detect the test solution of the traditional Chinese medicine for clearing away wen and protecting the lungs, and the generated high performance liquid chromatography peak is compared with the control fingerprint spectrum, and the similarity is calculated to be 0.94 to 1.

00.

9. The detection method according to claims 1 to 8, wherein The detection method can be used in the quality detection and component identification of Qingwen Hufei Granules Chinese medicine.

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