CN110687219A

CN110687219A - Detection method of suhuang cough-relieving capsule fingerprint and application thereof

Info

Publication number: CN110687219A
Application number: CN201910784734.6A
Authority: CN
Inventors: 贾毓宁; 谭宁华; 陈东; 巫兴东
Original assignee: Beijing Haiyan Pharmaceutical Industry Co Ltd Yangzijiang Pharmaceutical Ind; Yangtze River Pharmaceutical Group Co Ltd
Current assignee: Beijing Haiyan Pharmaceutical Industry Co Ltd Yangzijiang Pharmaceutical Ind; Yangtze River Pharmaceutical Group Co Ltd
Priority date: 2019-08-23
Filing date: 2019-08-23
Publication date: 2020-01-14
Anticipated expiration: 2039-08-23
Also published as: CN110687219B

Abstract

The invention discloses a detection method of a threo Huang cough relieving capsule fingerprint spectrum and application thereof. The detection method comprises the following steps: (1) preparing a test solution; (2) determining HPLC chromatographic conditions; (3) measuring an HPLC (high performance liquid chromatography) spectrum; (4) and establishing a standard fingerprint spectrum to obtain the suhuang cough relieving capsule fingerprint spectrum. The detection method can be used for quality control of Suhuang cough relieving capsule.

Description

Detection method of suhuang cough-relieving capsule fingerprint and application thereof

Technical Field

The invention relates to but not limited to a drug analysis technology, in particular to but not limited to a detection method of a threo yellow cough-relieving capsule fingerprint spectrum and application thereof.

Background

The prescription of the cough-relieving perilla yellow capsule comprises honey-fried ephedra, perilla leaf, earthworm, fried loquat leaf, perilla seed, cicada slough, radix peucedani, burdock and schisandra fruit. Wherein, the ephedra herb, the earthworm, the cicada slough, the perilla leaf and the whiteflower hogfennel root are used for dispelling wind and dispersing wind and are used for relieving cough, relieving sore throat and relieving itching; the perilla fruit and the Chinese magnoliavine fruit relieve spasm and spasm; the burdock and the loquat leaf can moisten lung and clear heat. The medicines are used together, the medicine has the effects of dispersing lung qi with pungent and warm natured drugs, dispelling wind and relieving spasm, dredging orifices and lowering qi, eliminating phlegm and relieving asthma, dispelling phlegm and eliminating spasm, and lung qi can be dispersed and lowered, and qi movement is smooth, so that the aims of integrally adjusting wind cough and relieving cough and treating both principal and secondary aspect of disease are fulfilled, and the medicine is used for treating cough variant asthma and cough after cold. Clinical tests show that the medicine has good effect and less adverse reaction compared with the current common western medicines.

Chinese patent application CN104007222A discloses a detection method of Suhuang cough-relieving capsules, which is used for quality control of the Suhuang cough-relieving capsules; the method adopts thin-layer chromatography to qualitatively identify herba Ephedrae, folium Perillae, Lumbricus, fructus Arctii, and fructus Schisandrae chinensis in SUHUANG ZHIKE JIAO NANG; the content of ephedrine hydrochloride in the capsule is determined by high performance liquid chromatography (octadecylsilane chemically bonded silica is used as filler, acetonitrile-water-phosphoric acid-triethylamine 1.5:98.3:0.1:0.1 is used as mobile phase, and detection wavelength is 205 nm).

Chinese patent application CN108490083A discloses a quality detection method of a Suhuang cough-relieving capsule, which comprises a content determination method of ephedrine and pseudoephedrine (polar ether is connected with phenyl bonded silica gel as a filling agent, methanol-0.092% phosphoric acid solution (containing 0.125% triethylamine) (1.5: 98.5) as a mobile phase, the detection wavelength is 210nm) and arctiin (octadecyl silane bonded silica gel as a filling agent, methanol-water (1: 1.1) as a mobile phase, the detection wavelength is 280nm), and a quality detection method of ephedrine hydrochloride and pseudoephedrine hydrochloride in the Suhuang cough-relieving capsule.

Although the prior art discloses a quality detection method for ephedrine hydrochloride, pseudoephedrine hydrochloride and arctiin, a detection method for a fingerprint of a suhuang cough-relieving capsule does not exist, and meanwhile, the relationship among effective components is not established yet.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

The application provides a detection method of a threo Huang Zhike capsule fingerprint spectrum and application thereof, which not only establishes a marker for quality control of the threo Huang Zhike capsule, but also can better control the quality of a product.

The application provides a detection method of a threo Huang Zhike capsule fingerprint, which comprises the following steps:

(1) preparation of a test solution:

taking Suhuang cough relieving capsules, removing capsule shells, precisely weighing 0.5g, placing into a conical flask with a plug, precisely adding 30 +/-5 mL of 60 +/-10 volume percent methanol aqueous solution, weighing, ultrasonically treating for 40 +/-10 min, cooling to room temperature, supplementing weight loss, filtering, taking a subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution;

(2) establishment of HPLC chromatographic conditions

A chromatographic column: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, and the specification of the chromatographic column is 4.6 multiplied by 250mm and 5 mu m;

mobile phase: a mixture of mobile phase a and mobile phase B, wherein mobile phase a is 0.1 ± 0.05 vol% aqueous formic acid and mobile phase B is acetonitrile; gradient elution of mobile phase;

column temperature: 30 + -5 deg.C, preferably 28 deg.C;

flow rate: 1 plus or minus 0.5 mL/min^-1Preferably, 1 mL. min^-1；

Detection wavelength: 230 ± 30nm, preferably 254 nm;

sample introduction amount: 10 ± 5 μ L, preferably 10 μ L;

(3) determination of HPLC Profile

Sampling the sample solution according to the HPLC chromatographic condition to obtain an HPLC chromatogram;

(4) establishment of standard fingerprint

Identifying common characteristic peaks in the above HPLC profile, preferably, establishing 22 common characteristic peaks comprising common characteristic peaks of:

adenosine, 1-caffeoylquinic acid, chlorogenic acid, schaftoside, arctiin, schizandrol A, gomisin D, schizandrol B, angeloylgomisin H, praeruptorin A and schisandrin B.

In the embodiments of the present application, adenosine, 1-caffeoylquinic acid, chlorogenic acid, schaftoside, arctiin, schizandrol A, gomisin D, schizandrol B, angeloylgomisin H, praeruptorin A and schizandrin B are 11 common peaks, and their relative retention times are as follows as indicated by the control: relative retention times are respectively based on retention times of chromatographic peaks at 19.6417min, 51.5968min and 103.2624min as reference: 0.3207, 0.7817 with 19.6417min chromatographic peak retention time as reference; 0.6455, 0.9116 and 1.3868 by taking the retention time of a chromatographic peak of 51.5968min as a reference; 1.0000, 1.0374, 1.0663, 1.1281, 1.1975 and 1.3501 by taking the retention time of a chromatographic peak of 103.2624min as a reference.

In an embodiment of the present application, the relative retention times of the 22 common characteristic peaks are: the retention times of chromatographic peaks at three different times (19.6417min, 51.5968min, and 103.2624min) were selected as references to calculate the relative retention times of the remaining 19 common peaks, 22 of which were: 0.3207, 0.3605, 0.4805, 0.5080, 0.7817, 1.0000(19.6417min as reference);

0.5141, 0.6455, 0.7566, 0.9116, 1.0000, 1.3868, 1.4096(51.5968min as reference);

0.9580, 1.0000, 1.0374, 1.0663, 1.1281, 1.1975, 1.3234, 1.3501, 1.3561(103.2624min as reference).

In embodiments of the present application, the mobile phase elution gradient is:

0-15 min, wherein the volume ratio of 0.1 +/-0.05 percent of formic acid water to acetonitrile is 96: 4;

15-40 min, wherein the ratio of 0.1 +/-0.05 volume percent of formic acid water to acetonitrile is 96-85: 4-15;

40-90 min,0.1 +/-0.05 volume percent of formic acid water, acetonitrile 85-65: 15-35;

90-110 min, wherein the ratio of 0.1 +/-0.05 volume percent of formic acid water to acetonitrile is 65-50: 35-50;

110-130 min, 50-40: 50-60 of 0.1 +/-0.05 volume percent of formic acid water and acetonitrile;

130-140 min, 40-10: 60-90% of 0.1 +/-0.05 volume% of formic acid water and acetonitrile;

140-145 min,0.1 + -0.05 volume percent of formic acid water and acetonitrile, 10-5: 90-95.

In a preferred embodiment of the present application, the present application provides a method for detecting a fingerprint of a threo yellow cough-relieving capsule, wherein the HPLC chromatographic conditions are as follows:

mobile phase: a mixture of mobile phase a and mobile phase B, wherein mobile phase a is 0.05 vol% aqueous formic acid and mobile phase B is acetonitrile; gradient elution of mobile phase;

column temperature: 28 ℃;

flow rate: 1 mL. min^-1；

Detection wavelength: 254 nm;

sample introduction amount: 10 μ L.

In a preferred embodiment of the present application, the test solution is prepared by:

taking Suhuang cough relieving capsules, removing capsule shells, precisely weighing 0.5g, placing in a conical flask with a plug, precisely adding 30mL of 60 volume percent methanol aqueous solution, weighing, performing ultrasonic treatment for 40min, complementing the weight loss, filtering, taking a subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution;

the mobile phase elution gradient is:

0-15 min, 96:4 of 0.05 volume percent of formic acid water and acetonitrile;

15-40 min, wherein the ratio of 0.05 volume percent formic acid water to acetonitrile is 96-85: 4-15;

40-90 min,0.05 volume percent of formic acid water, acetonitrile 85-65: 15-35;

90-110 min, wherein the ratio of 0.05 volume percent formic acid water to acetonitrile is 65-50: 35-50;

110-130 min, 50-40: 50-60 of 0.05 volume percent formic acid water and acetonitrile;

130-140 min, 40-10: 60-90% of 0.05 volume% formic acid water and acetonitrile;

140-145 min, and 10-5: 90-95% of 0.05 volume% formic acid water and acetonitrile.

In the embodiment of the present application, after the standard fingerprint is determined, the evaluation is performed by using "traditional Chinese medicine chromatography fingerprint similarity evaluation system software (2012 edition)" of the national pharmacopoeia committee.

On the other hand, the application provides the application of the fingerprint obtained by the detection method in quality control of the suhuang cough relieving capsules.

The established fingerprint spectrum can monitor the whole quality of the perilla yellow cough-relieving capsule systematically, and simultaneously the established fingerprint spectrum identifies that the common components of praeruptorin A, schizandrol A and pseudoephedrine, and arctiin metabolite arctigenin have obvious anti-inflammatory activity. Can control and produce stable Suhuang cough-relieving capsules and ensure the stability of the efficacy of the Suhuang.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.

Fig. 1 is a chromatogram obtained by optimizing the fingerprint chromatogram conditions of the suhuang cough-relieving capsule prepared in example 1;

FIG. 2 is a precision test spectrum of the Suhuang Zhike capsule prepared in example 1;

FIG. 3 is a stability test spectrum of Suhuang cough relieving capsules prepared in example 1;

FIG. 4 is a repeatability test spectrum of Suhuang Zhike capsules prepared in example 1;

FIG. 5 is a comparative fingerprint of the Suhuang cough relieving capsule prepared in example 1;

fig. 6 is the fingerprint of 16 batches of suhuang cough relieving capsules prepared in example 1;

FIG. 7 is an HPLC chromatogram of Suhuang cough relieving capsules of different mobile phase compositions in a comparative example;

FIG. 8 is an HPLC chromatogram of Suhuang Zhike capsules with different mobile phase elution gradients in comparative example;

FIG. 9 is an HPLC chromatogram of Suhuang Zhike capsules of different acids in a comparative example;

FIG. 10 is a HPLC chromatogram of a Suhuang Zhike capsule on different chromatographic columns in a comparative example;

FIG. 11 is an HPLC chromatogram of Suhuang Zhike capsules of different wavelengths in a comparative example;

FIG. 12 is an HPLC chromatogram of Suhuang cough relieving capsules at different flow rates in a comparative example;

FIG. 13 is an HPLC chromatogram of Suhuang Zhike capsules of different column temperatures in the comparative example.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.

The instrument comprises the following steps: waters 1525 (containing Waters 2707 Auto-sampler, Waters 2489 photodiodeArray Detector, Waters 1525 binary HPLC pump) and Waters ACQUITY Arc high performance liquid chromatograph (containing Sample Manager FTN-R, Waters 2998 photodiodeary Detector, QuaternarySolvent Manager-R), column: welch (

plus C18, 4.6X 250mm,5 μm), METTLERTODO XS105DU one tenth of a ten thousand electronic balance (Mettler-Torlado group), SI-234 type electronic analytical balance (Danver instrument)(Beijing) Ltd.); grant XB-22 type ultrasonic extractor.

Material reagent: acetonitrile (analytically pure starfish biochemically available high purity solvent, ltd.); methanol (analytically pure starfish biochemically available high purity solvent, ltd); formic acid (analytically pure Shanghai Shenbo chemical Co., Ltd.). Suhuang Zhike capsules (Yangzhijiang pharmaceutical group Beijing Haiyan pharmaceutical Co., Ltd., batch No. 15121111)

Example 1

Preparation of sample solution: taking Suhuang cough relieving capsules, removing capsule shells, precisely weighing 0.5g, placing the capsules in a conical flask with a plug, precisely adding 30mL of 60 volume percent methanol aqueous solution, weighing, ultrasonically treating for 40min, cooling to room temperature, complementing weight loss, filtering, taking subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution for later use.

1. Comparison of chromatographic conditions

The part is used for inspecting and optimizing optimal chromatographic conditions of influencing factors influencing chromatographic behavior, such as mobile phase composition, mobile phase elution gradient, different acids, different acid concentrations, different chromatographic columns, different detection wavelengths, different flow rates and different column temperatures. Results the chromatographic conditions were determined to be: by Welch (

plus C18,4.6 × 250mm,5 μm) chromatography column, mobile phase acetonitrile (B) -0.05 vol% formic acid water (a), mobile phase elution gradient: 0-15 min, 4% (B); 15-40 min, 4-15% (B); 40-90 min, 15-35% (B); 90-110 min, 35-50% (B); 110-130 min, 50-60% (B); 130-140 min, 60-90%; 140-145 min, 90-95% (B), column temperature 28 ℃, flow rate 1 mL/min^-1The detection wavelength is 254nm, and the sample injection amount is 10 muL. The optimization results are shown in figure 1.

2. Comparison of test article preparation conditions

The part takes 13 chromatographic peak areas of which the peak areas in the spectrogram account for more than 2 percent of the total peak area as evaluation indexes, and optimizes the optimal extraction conditions on the influence factors influencing the extraction of the contents of the Suhuang cough relieving capsule, such as extraction method, extraction solvent, extraction time, extraction times, feed liquid ratio and methanol concentration investigation. As a result, the extraction conditions were determined to be: an ultrasonic extraction mode is adopted, the extraction time is 40min, one time, and the material-liquid ratio (V/m) is 1: 60, the extraction solvent is 60% methanol by volume.

3. Methodology validation

The fingerprint chromatogram takes 22 chromatographic peaks with a single peak area of more than 1 percent and a total peak area of more than 40 percent as reference, and is used for evaluating the feasibility of the establishment method.

3.1 precision test

Preparation of a test solution: removing capsule shell from Suhuang cough relieving capsule, mixing well, precisely weighing 0.5g of Suhuang cough relieving capsule powder, placing in 50mL conical flask with plug, precisely adding 30mL of 60% methanol solution, weighing, ultrasonically extracting for 40min, cooling, supplementing with corresponding 60% methanol solution, reducing weight loss, filtering, collecting subsequent filtrate, and filtering the filtrate with 0.45 μm microporous membrane to obtain test solution. And continuously carrying out sample injection for 6 times, and inspecting the consistency of relative peak areas and relative retention times of main chromatographic peaks in spectrograms of different sample injection times to obtain a precision spectrogram, which is shown in an attached figure 2.

And (3) taking 22 chromatographic peaks with peak areas accounting for more than 1% of the total peak area in the chromatogram as references, and respectively taking the retention time and the peak area of the three chromatographic peaks with the retention time of 19.6417min, 51.5968min and 103.2624min as references to calculate the relative retention time and the relative peak area result of the rest chromatographic peaks, which are shown in tables 1 and 2. TABLE 1 fingerprint precision test relative retention time results

TABLE 2 fingerprint precision test relative peak area results

The relative retention time of the chromatographic peaks and the RSD value of the relative peak area of the chromatographic peaks are both less than 3 percent, which indicates that the precision of the instrument is good.

3.2 stability test

Preparation of a test solution: removing capsule shell from Suhuang cough relieving capsule, mixing well, precisely weighing 0.5g of Suhuang cough relieving capsule powder, placing in 50mL conical flask with plug, precisely adding 30mL of 60% methanol solution, weighing, ultrasonically extracting for 40min, cooling, supplementing with corresponding 60% methanol solution, reducing weight loss, filtering, collecting subsequent filtrate, and filtering the filtrate with 0.45 μm microporous membrane to obtain test solution. The sample is injected when the sample is extracted for 0, 4, 8, 12, 16, 24 and 48 hours respectively, the consistency of the relative peak areas and the relative retention times of the main chromatographic peaks in the spectrogram of the sample in different time periods is inspected, and the result stability spectrogram is shown in figure 3.

And (3) taking 22 chromatographic peaks with peak areas accounting for more than 1% of the total peak area in the chromatogram as references, and respectively taking the retention time and the peak area of the three chromatographic peaks with the retention time of 19.6417min, 51.5968min and 103.2624min as references to calculate the relative retention time and the relative peak area result of the rest chromatographic peaks, which are shown in tables 3 and 4.

TABLE 3 fingerprint stability test relative retention time results

TABLE 4 fingerprint stability test relative peak area results

As a result, the relative retention time of each chromatographic peak and the RSD value of the relative peak area are both less than 3 percent, which indicates that the stability of the test solution is good within 48 h.

3.3 repeatability test

Preparation of a test solution: removing capsule shells of the Suhuang cough-relieving capsules, fully and uniformly mixing, precisely weighing 6 parts of 0.5g of Suhuang cough-relieving capsule powder, respectively placing the powder into 50mL conical bottles with stoppers, precisely adding 30mL of 60% methanol solution, weighing, ultrasonically extracting for 40min, cooling, complementing the weight loss by using corresponding 60% methanol solution, filtering, taking subsequent filtrate, and filtering the filtrate by using a 0.45 mu m microporous filter membrane to obtain a test solution. And respectively injecting samples, and inspecting the consistency of the relative peak areas and the relative retention times of the main chromatographic peaks in the spectrogram of the repeated sample to obtain a result stability spectrogram, which is shown in an attached figure 4.

And (3) taking 22 chromatographic peaks with peak areas accounting for more than 1% of the total peak area in the chromatogram as references, and respectively taking the retention time and the peak area of the chromatographic peaks with the retention time of 19.6417min, 51.5968min and 103.2624min as references to calculate the relative retention time and the relative peak area result of the rest chromatographic peaks, which are shown in tables 5 and 6.

TABLE 5 fingerprint repeatability test relative retention time results

TABLE 6 relative peak area results of fingerprint repeatability tests

As a result, the relative retention time of each chromatographic peak and the relative peak area RSD value are both less than 3%, indicating that the method has good reproducibility.

The result of methodology verification shows that 22 chromatographic peaks with peak areas in the chromatogram accounting for more than 1% of the total peak area are taken as references, the retention time and the peak area of three chromatographic peaks with the retention time of 19.6417min, 51.5968min and 103.2624min are taken as references for calculation, and the relative retention time and the relative peak area RSD value of precision, stability and reproducibility all accord with the related regulations of 2015 edition 'Chinese pharmacopoeia' on establishment of methodology verification of fingerprint spectrum methods.

4. Establishment of fingerprint spectrums of different batches of perilla yellow cough-relieving capsules

Preparing a test solution according to the chromatographic conditions under 1 item and the method under 2 items, establishing 16 batches of perilla yellow cough-relieving capsule fingerprints, evaluating by using Chinese medicine chromatographic fingerprint similarity evaluation system software (2012 version) of the national pharmacopoeia committee, setting a sample S8 fingerprint as a reference fingerprint with a time window of 0.10min, and performing similarity calculation by using an average method to generate the perilla yellow cough-relieving capsule reference fingerprint, which is shown in figure 5. A total of 11 peaks were identified by comparison with the control, which showed retention times as shown in Table 7. The 16 batches of suhuang cough relieving capsules have 22 common peaks, as shown in figure 6, which account for 40% of the total peak area in each batch of samples. The sample similarity values for each batch are shown in table 8.

TABLE 711 Total peak component retention times (min) assigned

Similarity of Suhuang cough relieving capsule samples of Table 816 batches

The similarity analysis result shows that the similarity of 16 different batches of the perilla yellow cough relieving capsules is more than 0.9, which indicates that the perilla yellow cough relieving capsules have stable quality within two-year shelf life.

Example 2

Suhuang cough-relieving capsule and research on anti-inflammatory activity of main component thereof

1. Materials and methods

1.1 materials

Mouse monocyte macrophage line RAW264.7 (shanghai academy of sciences cell bank); DMEM medium (Biological Industries, USA, 010521 ACS); fetal bovine serum (Biological Industries, 040011ACS, USA); lipopolysaccharide (Sigma-Aldrich, USA, L2630). Synergy H1 multifunctional microplate detector (BioTek, usa).

1.2 methods

1.2.1 cell culture

RAW246.7 cells were cultured in DMEM medium (containing 10% FBS and 1% penicillin-streptomycin) at 37 deg.C and 5% CO₂Culturing in an incubator, replacing culture solution according to growth conditions, slightly blowing off cells by using the culture solution, collecting cell suspension, centrifuging for 2min at 1000r/min, discarding supernatant, adding 3mL of cell culture solution to re-suspend cells, and inoculating the cells into a new culture dish according to the volume ratio of 1: 3 for culturing.

1.2.2 lipopolysaccharide-induced RAW246.7 cell inflammation model establishment and screening

Taking cells in logarithmic growth phase according to 2 × 10⁴The number of cells/well was inoculated into a 96-well plate and incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 24 h. Observe cell state, set up different experimental groups: (1) blank control group, without any treatment; (2) LPS-treated group with 1. mu.g/mL^-1The LPS continuously stimulates the cells for 12 h; (3) treating with LPS, adding 1 μ g/mL after treating RAW264.7 cells with SUHUANGZHIKE Capsule extract, dexamethasone, arctigenin with different concentrations, schizandrol A, praeruptorin A, ephedrine and pseudoephedrine for 24h^-1The LPS was further treated for 24h and cell culture supernatants were collected. Each group was provided with 6 multiple wells. And adding 100 mu L of Griess reagent into 100 mu L of cell supernatant, reacting for 15min in a dark place at room temperature, and measuring the absorbance value by using a microplate reader at 540 nm. Calculating the inhibition rate, and determining half Inhibition Concentration (IC)₅₀) The value is obtained.

Inhibition ratio (%) ═ a₁-A₂)/(A₁-A₀)×100

Wherein A is₁As absorbance value of model group, A₂For the absorbance values of the dosing groups, A₀Blank absorbance values.

1.3 results

The activity test results of the tested medicaments show that the IC of the praeruptorin A is₅₀The value is 21.35 +/-5.87 mu M, and the value is compared with the positive control group dexamethasone IC₅₀At a value of 19.07. + -. 5.65. mu.M, no comparison was madeThe significant difference and the activity are equivalent, while the activity of the perilla yellow cough-relieving capsule extract, arctigenin, pseudoephedrine and schizandrol A is weaker, but the NO generation activity generated by RAW264.7 inflammatory cells induced by LPS can be inhibited to a certain extent.

TABLE 9 screening of the components IC for NO-producing Activity₅₀

Comparative example

1. Comparative flow ratio investigation

The mobile phase composition has a greater impact on the chromatographic behavior of the sample, so better mobile phases are optimized by changing different mobile phase compositions while ensuring other chromatographic conditions are consistent. The application mainly inspects the influence of organic chromatographic solvents (methanol and acetonitrile) commonly used in laboratories on the separation condition of samples. The results are shown in FIG. 7. Wherein: s1 was acetonitrile-water, S2 was acetonitrile-0.05% formic acid water, S3 was methanol-0.05% formic acid water. The result shows that when the organic phase is acetonitrile, the sample has better separation effect than methanol, and can be separated in a shorter time.

The separation condition of the chromatographic peak of the sample and the analysis time of the sample are directly influenced by the elution gradient of the mobile phase, so that the separation effect of different elution gradients on the extract of the suhuang cough relieving capsule is examined under the condition of controlling the preparation method of the sample and other chromatographic conditions to be consistent. Wherein the elution gradient of the mobile phase is respectively as follows: acetonitrile (a) -0.05% formic acid (B), mobile phase elution gradient: (1) 0-5 min, 5% (A); 5-25 min, 5-26% (A); 25-45 min, 26-40% (A); 45-55 min, 40-54% (A); 55-85 min, 54-66% (A); 85-95 min, 66-71% (A); 95-105 min, 71-86% (A); 105-115 min, 86-92% (A); 115-135 min, 92-95% (A); 135-140 min, 95-100% (A). (2) 0-15 min, 4% (A); 15-40 min, 4-15% (A); 40-90 min, 15-35% (A); 90-110 min, 35-50% (A); 110-130 min, 50-60% (A); 130-140 min, 60-80% (A); 140-145 min, 80-95% (A). (3) 0-15 min, 4% (A); 15-40 min, 4-15% (A); 40-90 min, 15-35% (A); 90-110 min, 35-50% (A); 110-130 min, 50-60% (A); 130-140 min, 60-90%; 140-145 min, 90-95% (A). The results are shown in FIG. 8. By optimizing different elution gradients, the result is that when the sample is run according to the elution gradient (3), the obtained spectrum base line is stable, and the chromatographic peaks are well separated.

The pH of the mobile phase can be adjusted by adding acid to the mobile phase, thereby improving the peak profile. The experiment examined the effect of the addition of the commonly used acids, acetic acid (0.02 and 0.05% by volume), formic acid (0.02 and 0.05% by volume) and phosphoric acid (0.02 and 0.05% by volume) to the laboratory mobile phase on the separation of the suhuang cough capsule samples. The results are shown in FIG. 9. As shown in the figure, when no acid is added in the mobile phase (S1), the separation of the chromatographic peaks of the most polar part is poor, while when different concentrations of acetic acid are added in the water phase, the separation of the chromatographic peaks (S2 and S3) is not good as that of formic acid, when 0.05% formic acid water is added in the water phase (S5) and 0.02% phosphoric acid (S6) are good, but the salting-out phenomenon is easy to occur in the inorganic acid chromatographic system of phosphoric acid. Therefore, 0.05% by volume formic acid was added to the aqueous phase.

2. Comparative investigation of chromatographic columns

Because the difference of the packing composition of different producers has larger influence on the separation of samples, the experiment of the part is supposed to optimize the chromatographic columns of different producers and models, and preferably optimize the chromatographic columns. Wherein the optimized chromatographic columns are respectively A: agilent (ZORBAX SB-C18, 4.6X 250mm,5 μm, PN: 880957-902); b: agilent (ZORBAX, SB-C18, 4.6X 150mm,5 μm, PN: 883975-902); c: waters (

C18, 4.6X 250mm,5 μm, PN:186000496), D: waters (XTerra C18, 4.6X 150mm,5 μm, PN: 186002559) E is Welch (R) ((R))

plusC18, 4.6 × 250mm,5 μm, PN: 0026-31043). The results are shown in FIG. 10. As shown in the figure, different chromatographic columns have great influence on the separation condition of the perilla yellow cough relieving capsule extract. Wherein S1 is the result of column A separation, S2 is the columnThe separation result of B, S3, S4, and S5 are the separation results of column C, column D, and column E, respectively. As a result, when a Welch column was used, the separation of the peaks was good and the peak pattern was good.

3. Detection wavelength contrast investigation

The components of the suhuang cough relieving capsule are complex, and comprise alkaloid, lignan, coumarin, flavone, triterpenes and other components, and the maximum absorption wavelengths of different components are different. The experiment examines the influence of six different wavelengths 230, 248, 254, 270, 300 and 330nm on the detection condition of the sample. The results are shown in FIG. 11. As a result, as shown in the figure, the baseline shift was severe when the detection wavelength was 230nm (S1), and the small-polarity portion chromatographic peak response was low when the detection wavelength exceeded 270nm (S4). The chromatographic peak information is more selected to be the detection wavelength 254nm (S3).

4. Comparative investigation of flow rates

Different flow rates can affect the time to peak the chromatographic peak and thus affect the sample separation. The experiment was conducted by examining the different flow rates (0.6, 0.8, 1.0, 1.2, 1.4 mL. min.)^-1) Influence on the separation condition of a Suhuang cough relieving capsule sample, thereby optimizing better chromatographic conditions. The results are shown in FIG. 12. The results show that the flow rate is 1 mL/min^-1(S3) the sample separation time was shorter and the chromatographic peak separation was better.

5. Comparative investigation of column temperature

The column temperature can affect not only the sample separation conditions but also the sample run time. The experiment examines the influence of different column temperatures (20, 26, 28 and 30 ℃) on the separation condition of the Suhuang cough relieving capsule sample. The results are shown in FIG. 13. As a result, as shown in the figure, the sample separation was better when the column temperature was selected to be 28 deg.C (S3).

Although the embodiments disclosed in the present application are described above, the descriptions are only for the convenience of understanding the present application, and are not intended to limit the present application. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the appended claims.

Claims

1. A detection method of a Suhuang cough relieving capsule fingerprint spectrum comprises the following steps:

(1) preparation of a test solution:

taking Suhuang cough relieving capsules, removing capsule shells, precisely weighing 0.5g, placing the capsules in a conical flask with a plug, precisely adding 30 +/-5 mL of 60 +/-10 volume percent methanol aqueous solution, weighing the weight, performing ultrasonic treatment for 40 +/-10 min, taking out the capsules, cooling the capsules to room temperature, complementing weight loss, filtering, taking a subsequent filtrate, and filtering the filtrate through a 0.45 mu m microporous filter membrane to obtain a test solution;

(2) determination of HPLC chromatographic conditions

A chromatographic column: chromatographic column adopting octadecylsilane chemically bonded silica as filler

column temperature: 30 +/-5 ℃;

flow rate: 1 plus or minus 0.5 mL/min^-1；

Detection wavelength: 230 +/-30 nm;

sample introduction amount: 10 +/-5 mu L;

(3) determination of HPLC Profile

(4) establishment of standard fingerprint

Determining common characteristic peaks in the HPLC chromatogram, wherein the common characteristic peaks comprise common characteristic peaks of the following components:

2. The assay of claim 1, wherein the adenosine, 1-caffeoylquinic acid, chlorogenic acid, schaftoside, arctiin, schizandrin A, gomisin D, schizandrin B, angeloylgomisin H, praeruptorin A and schizandrin B are 11 common peaks and their relative retention times, as indicated by the control, are as follows: relative retention times are respectively based on retention times of chromatographic peaks at 19.6417min, 51.5968min and 103.2624min as reference: 0.3207, 0.7817 with 19.6417min chromatographic peak retention time as reference; 0.6455, 0.9116 and 1.3868 by taking the retention time of a chromatographic peak of 51.5968min as a reference; 1.0000, 1.0374, 1.0663, 1.1281, 1.1975 and 1.3501 by taking the retention time of a chromatographic peak of 103.2624min as a reference.

3. The detection method according to claim 1 or 2, wherein 22 common characteristic peaks are determined in the HPLC profile, and the relative retention time of the 22 common characteristic peaks is: relative retention times of the remaining 19 common peaks were calculated with respect to retention times of chromatographic peaks at 19.6417min, 51.5968min and 103.2624min, respectively, wherein the retention times of 22 common peaks are: 0.3207, 0.3605, 0.4805, 0.5080, 0.7817 and 1.0000 by taking 19.6417min chromatographic peak retention time as reference; 0.5141, 0.6455, 0.7566, 0.9116, 1.0000, 1.3868 and 1.4096 by taking the retention time of a chromatographic peak of 51.5968min as a reference; 0.9580, 1.0000, 1.0374, 1.0663, 1.1281, 1.1975, 1.3234, 1.3501 and 1.3561 by taking the retention time of a chromatographic peak of 103.2624min as a reference.

4. The detection method of claim 1, wherein the mobile phase elution gradient is:

5. The detection method according to claim 1, wherein the HPLC chromatographic conditions are:

column temperature: 28 ℃;

flow rate: 1 mL. min^-1；

Detection wavelength: 254 nm;

sample introduction amount: 10 μ L.

6. The test method according to claim 1, wherein the test solution is prepared by:

taking Suhuang cough relieving capsules, removing capsule shells, precisely weighing 0.5g, placing in a conical flask with a plug, precisely adding 30mL of 60 volume percent methanol aqueous solution, weighing, ultrasonically treating for 40min, cooling to room temperature, complementing weight loss, filtering, taking a subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution.

7. The detection method according to claim 1, wherein the detection method comprises the steps of:

(1) preparation of a test solution:

taking Suhuang cough relieving capsules, removing capsule shells, precisely weighing 0.5g, placing in a conical flask with a plug, precisely adding 30mL of 60 volume percent methanol aqueous solution, weighing, ultrasonically treating for 40min, cooling to room temperature, complementing weight loss, filtering, taking a subsequent filtrate, and filtering through a 0.45 mu m microporous filter membrane to obtain a test solution;

(2) determination of HPLC chromatographic conditions

column temperature: 28 ℃;

flow rate: 1 mL. min^-1；

Detection wavelength: 254 nm;

sample introduction amount: 10 mu L of the solution;

(3) determination of HPLC Profile

(4) establishment of standard fingerprint

Identifying 22 common characteristic peaks in the HPLC profile, wherein the common characteristic peaks comprise common characteristic peaks of the following components:

adenosine, 1-caffeoylquinic acid, chlorogenic acid, schaftoside, arctiin, schizandrol A, gomisin D, schizandrol B, angeloylgomisin H, praeruptorin A and schisandrin B;

the mobile phase elution gradient is:

0-15 min, 96:4 of 0.05 volume percent of formic acid water and acetonitrile;

40-90 min,0.05 volume percent of formic acid water, acetonitrile 85-65: 15-35;

130-140 min, 40-10: 60-90% of 0.05 volume% formic acid water and acetonitrile;

8. The assay of claim 7, wherein the adenosine, 1-caffeoylquinic acid, chlorogenic acid, schaftoside, arctiin, schizandrin A, gomisin D, schizandrin B, angeloylgomisin H, praeruptorin A and schizandrin B are 11 common peaks and their relative retention times, as indicated by the control, are as follows: relative retention times are respectively based on retention times of chromatographic peaks at 19.6417min, 51.5968min and 103.2624min as reference: 0.3207, 0.7817 with 19.6417min chromatographic peak retention time as reference; 0.6455, 0.9116 and 1.3868 by taking the retention time of a chromatographic peak of 51.5968min as a reference; 1.0000, 1.0374, 1.0663, 1.1281, 1.1975 and 1.3501 by taking the retention time of a chromatographic peak of 103.2624min as a reference;

determining 22 common characteristic peaks in the HPLC chromatogram, wherein the relative retention time of the 22 common characteristic peaks is as follows: relative retention times of the remaining 19 common peaks were calculated with respect to retention times of chromatographic peaks at 19.6417min, 51.5968min and 103.2624min, respectively, wherein the relative retention times of the 22 common peaks are: 0.3207, 0.3605, 0.4805, 0.5008, 0.7817 and 1.0000 by taking 19.6417min chromatographic peak retention time as reference;

0.5141, 0.6455, 0.7566, 0.9116, 1.0000, 1.3868 and 1.4096 by taking the retention time of a chromatographic peak of 51.5968min as a reference;

0.9580, 1.0000, 1.0374, 1.0663, 1.1281, 1.1975, 1.3234, 1.3501 and 1.3561 by taking the retention time of a chromatographic peak of 103.2624min as a reference.

9. The application of the fingerprint obtained by the detection method of any one of claims 1-8 in quality control of Suhuang cough-relieving capsules.