CN115266954A

CN115266954A - Deafness capsule fingerprint spectrum, construction method thereof and deafness capsule quality detection method

Info

Publication number: CN115266954A
Application number: CN202110473411.2A
Authority: CN
Inventors: 项阳东; 崔兵; 李爱英; 樊慧蓉; 张一昕; 彭小园; 刘兴超
Original assignee: Jingzhongshan Sinopharm Tangshan Co ltd
Current assignee: Jingzhongshan Sinopharm Tangshan Co ltd
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2022-11-01

Abstract

The invention provides a deafness capsule fingerprint, a construction method thereof and a deafness capsule quality detection method, wherein the construction method mainly comprises the steps of detecting a deafness capsule test solution, a mixed reference solution and a negative sample solution, the detection wavelength of high performance liquid chromatography is 270-290nm, the chromatographic column is a C18 silica gel chromatographic column, and the column temperature is 28-38 ℃; the mobile phase A is acetonitrile, the mobile phase B is 0.05 to 0.15 percent phosphoric acid aqueous solution, gradient elution is adopted, and the flow rate is 0.5 to 2ml/min; the deafness capsule fingerprint spectrum is constructed by the method, and 13 common peaks are determined and calibrated. The invention can well carry out qualitative analysis on the quality of the deafness capsule product by constructing the HPLC fingerprint of the deafness capsule, and provides a basis for comprehensively evaluating the quality standard of the deafness capsule.

Description

Deafness capsule fingerprint spectrum, its construction method and deafness capsule quality detection method

Technical Field

The invention belongs to the technical field of medicine detection, and particularly relates to a deafness capsule fingerprint, a construction method thereof and a deafness capsule quality detection method.

Background

The deafness capsule is a Chinese patent medicine prepared by ten Chinese medicinal materials of scutellaria baicalensis, gentiana scabra bunge, akebiaquinata, cape jasmine fruit, liquorice, rehmannia glutinosa, rhizoma alismatis, chinese angelica, irkutsk anemone rhizome and antelope horn according to a production process, is a Chinese medicinal preparation produced by a single company of Jingzhong mountain medicine (Tangshan) Limited company, has the effects of clearing liver, purging fire, promoting diuresis and relieving stuffiness, can improve the hearing of patients and tinnitus symptoms, and is clinically and commonly used for treating otodynia, purulence, dizziness and headache caused by damp-heat in liver and gallbladder. In the recipe, gentian root, bitter in taste and cold in nature, is the monarch drug for purging liver-gallbladder excess fire and promoting diuresis; scutellaria baicalensis and cape jasmine fruit for eliminating dampness and purging fire^[7]The gentian is used as a minister to assist the efficacy of purging fire; the akebia stem is used for assisting in clearing heat and promoting diuresis, and the liquorice is used for regulating the effects of the medicines; the combination of the medicines can play a role in both upper and lower aspects, and has the functions of tonifying, eliminating evil, promoting diuresis, dredging orifices and the like in purgation.

The deafness capsule relates to 10 traditional Chinese medicines, has relatively complex components, the execution standard of the deafness capsule is loaded in national medicine standard (new drug transfer standard) WS3-992 (Z-259) -2007Z of the State food and drug administration, only baicalin is quantified, and the quantitative determination of a single component is difficult to be used as the evaluation standard of the overall quality of the deafness capsule. Based on the above, the research on the HPLC fingerprint of the deafness capsule has important practical significance for the evaluation of various components in the deafness capsule, and the quality standard of the deafness capsule can be comprehensively controlled and evaluated.

Disclosure of Invention

The invention aims to provide a deafness capsule fingerprint, a construction method thereof and a deafness capsule quality detection method, and aims to solve the problem that in the prior art, only baicalin is quantified, and the quantitative determination of a single component is difficult to be used as an evaluation standard of the overall quality of a deafness capsule.

The technical scheme adopted by the invention for realizing the purpose is as follows: a construction method of a deafness capsule fingerprint comprises the following steps:

a. taking the content of several batches of deafness capsules, grinding, extracting with 60-80% ethanol water solution, and filtering to obtain a test solution;

b. dissolving chlorogenic acid, gentiopicrin, baicalin, baicalein, and wogonin reference substances in methanol to obtain mixed reference substance solution;

c. b, preparing negative samples lacking scutellaria baicalensis, negative samples lacking gardenia, angelica sinensis and irkutsk anemone rhizome and negative samples lacking gentiana scabra bunge according to the preparation process of the deaf capsule, grinding the content of each negative sample according to the operation of the step a, extracting the ground content with 60-80% ethanol water solution, and filtering to obtain each negative sample solution;

d. respectively detecting the test solution, the mixed reference solution and the negative sample solution by a high performance liquid chromatograph, wherein the detection wavelength is 270-290nm, the chromatographic column is a C18 silica gel chromatographic column, and the column temperature is 28-38 ℃; the mobile phase A is acetonitrile, the mobile phase B is 0.05-0.15% phosphoric acid water solution, gradient elution is adopted, the flow rate is 0.5-2ml/min, and the gradient elution procedure is as follows: 0-10min, 95% -92% B;10 to 15min,92 to 85% by weight of B; 15-35min, 85% by weight of B; 35-65min, 85% -70% B; 65-85min, 70-50% by weight of B; 85-95min, 50-30 percent of B;

e. respectively recording chromatograms of the test solution, the mixed reference solution and the negative sample solution, and performing attribution positioning on index components in the chromatogram of the test solution by using the chromatograms of the mixed reference solution; and (3) performing attribution positioning on medicinal materials in the chromatogram of the test solution by using the chromatogram of each negative sample solution, and simultaneously processing the chromatogram of the test solution by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to obtain the fingerprint of the deafness capsule.

In the step a, the preparation of the test solution is as follows: weighing the content of the deafness capsule, grinding, placing into a conical flask with a plug, adding 75% ethanol water solution, weighing, refluxing for 25-35min, cooling, supplementing 75% ethanol water solution, shaking, and filtering with microporous membrane; wherein, the ratio of the content of the deafness capsule to the added 75% ethanol water solution is 2 g: 20-30mL.

In the step b, the concentrations of chlorogenic acid, gentiopicrin, baicalin, baicalein and wogonin are respectively 0.02-0.03mg/mL, 0.08-0.09mg/mL, 0.3-0.4mg/mL, 0.05-0.07mg/mL and 0.02-0.03mg/mL.

In step d, the detection wavelength is 280nm, the chromatographic column is a Wondasil C18-WR chromatographic column, the mobile phase B is 0.1% phosphoric acid aqueous solution, the column temperature is 35 ℃, and the sample injection amount is 10 mu L.

In step e, single-component reference substance solutions of chlorogenic acid, gentiopicrin, baicalin, baicalein and wogonin are detected in advance to determine components corresponding to peaks in a chromatogram of the mixed reference substance solution.

A deafness capsule fingerprint is detected and constructed by the construction method.

The fingerprint comprises 13 common peaks, and the relative retention time of each common peak is calculated by taking the No. 5 peak as a reference peak: 7.20-7.25 of No. 1 peak, 10.01-10.06 of No. 2 peak, 22.53-22.63 of No. 3 peak, 23.87-23.95 of No. 4 peak, 62.67-62.75 of No. 5 peak, 66.90-67.00 of No. 6 peak, 69.55-69.63 of No. 7 peak, 72.00-72.60 of No. 8 peak, 77.50-77.60 of No. 9 peak, 78.15-78.30 of No. 10 peak, 79.00-79.15 of No. 11 peak, 88.15-88.30 of No. 12 peak and 90.10-90.20 of No. 13 peak.

A deafness capsule quality detection method comprises the steps of detecting a sample to be detected of a deafness capsule according to the construction method to obtain a high performance liquid chromatography spectrum of the sample to be detected of the deafness capsule, then constructing a fingerprint spectrum of the deafness capsule according to the construction method, and finally comparing the high performance liquid chromatography spectrum of the sample to be detected of the deafness capsule with the fingerprint spectrum of the deafness capsule.

The prescription of the deafness capsule (180 ten thousand capsules) is as follows:

medicinal materials	Dosage/kg
		Angelica sinensis/angelica sinensis decoction pieces	244.6
Antelope horn	11.5
		Radix Gentianae	225.0
Radix Scutellariae	225.0
		Rehmannia root (rehmannia root)	225.0
Rhizoma alismatis	225.0
		Wood block	225.0
Gardenia jasminoides ellis	225.0
		Rhizoma anemones Altaicae	225.0
Licorice root, radix Glycyrrhizae	225.0
		Corn starch	28.0
Silicon dioxide	15.1

Pulverizing radix Angelicae sinensis and cornu Saigae Tataricae into fine powder respectively; extracting the rest eight materials such as radix Gentianae with ethanol under reflux for 2 hr and 1.5 hr, mixing extractive solutions, filtering, recovering ethanol under reduced pressure, and concentrating to obtain soft extract; decocting the dregs in water twice for 1.5 hours for the first time and 1 hour for the second time, mixing the decoctions, filtering, concentrating the filtrate under reduced pressure to obtain clear paste with the relative density of 1.30-1.35 (50 ℃), mixing the clear paste with the thick paste, stirring uniformly, drying in vacuum, crushing into fine powder, adding the fine powder of the angelica and the antelope horn, mixing uniformly, adding a proper amount of corn starch and 2% of superfine silica gel powder, mixing uniformly, and filling into capsules to obtain the traditional Chinese medicine.

The traditional Chinese medicine compound preparation related by the invention has more traditional Chinese medicines and more complex active ingredients, and the quality of the traditional Chinese medicine preparation is difficult to accurately grade only by measuring the content of one or more active ingredients. The invention can well carry out qualitative analysis on the quality of the deafness capsule product by constructing the HPLC fingerprint of the deafness capsule, and provides a basis for comprehensively evaluating the quality standard of the deafness capsule.

Drawings

FIG. 1 shows HPLC finger print (A) of 10 batches of deafness capsules and HPLC finger print (B) of baicalin reference substance.

FIG. 2 is a dendrogram of 10 batches of deafness capsule clustering analysis.

FIG. 3 is the analysis score chart of the main components of the deafness capsule.

Detailed Description

The invention is further illustrated by the following examples, which are given by way of illustration only and are not intended to limit the scope of the invention in any way. In the examples of the present invention, LC-20AT HPLC (Shimadzu corporation, japan); KQ-300DE type numerical control ultrasonic cleaner (ultrasonic instruments, inc. of Kunshan city); a Wondasil C18-WR column (4.6 mm. Times.250mm, 5 μm); DK-98-II electric constant temperature water bath (Tester instruments, tianjin Co., ltd.); an electronic analytical balance model ME204E102 (MettlerToledo, switzerland);

chlorogenic acid (lot No. AF9082001, content mass fraction 98.83%), baicalein (lot No. AF0022325, content mass fraction 98.86%), ferulic acid (lot No. AF20062351, content mass fraction 99.65%), gentiopicroside (lot No. AF9061002, content mass fraction 99.75%), baicalin (lot No. AF20022601, content mass fraction 98.33%), wogonin (lot No. AF0022331, content mass fraction 98.91%) were purchased from Duyage Biotechnology Limited. Deafness capsules 10 batches (batch nos. 190202, 190802, 20030, 201001, 200901, 190502, 200903, 200302, 200902, 201002) were all from the Jingzhong Shang national drug (Tangshan) Co., ltd.; acetonitrile and phosphoric acid are chromatographically pure, methanol and ethanol are analytically pure, and ultrapure water is adopted for testing.

Example 1 construction of HPLC fingerprint of deafness capsule

1. Preparation of test solution

Grinding the content of the deafness capsule, precisely weighing 2g, placing in a conical flask with a plug, precisely adding 25mL of 75% ethanol, weighing, refluxing for 30min, cooling, supplementing 75% ethanol, shaking up, taking the subsequent filtrate, and filtering with a 0.45-micron microporous filter membrane to obtain the deafness capsule.

2. Preparation of stock solutions of Mixed controls

Precisely weighing appropriate amount of chlorogenic acid, gentiopicrin, baicalin, baicalein, and wogonin as reference substances, adding methanol to obtain mixed reference substance stock solutions containing chlorogenic acid, gentiopicrin, baicalin, baicalein, and wogonin at concentrations of 0.0212mg/mL, 0.0827mg/mL, 0.3797mg/mL, 0.0592mg/mL, and 0.0236mg/mL, respectively, filtering, and collecting filtrate.

3. Negative sample solution preparation

Preparing negative samples of radix Scutellariae (containing baicalin, baicalein, and wogonin), fructus Gardeniae, radix Angelicae sinensis, rhizoma anemones Altaicae (containing chlorogenic acid), and radix Gentianae (containing gentiopicrin) according to the proportion and production process, grinding the contents, collecting 2g, precisely weighing, placing in a conical flask with a plug, precisely adding 25mL of 75% ethanol, weighing, refluxing for 30min, cooling, supplementing 75% ethanol, shaking, collecting the subsequent filtrate, and filtering with 0.45 μm microporous membrane.

4. Chromatographic conditions

A chromatographic column: wondasil C18-WR column (4.6 mm. Times.250mm, 5 μm), column temperature: 35 ℃; mobile phase acetonitrile (A) -0.1% aqueous phosphoric acid (B), gradient elution (0-10min, 95-92% B, 10-15min, 92-85% B, 15-35min, 85-65min, 85-70% B, 85-95min, 70-50% B, 85-95min, 50-30% B; the volume flow is 1.0mL/min; the detection wavelength is 280nm; the sample size was 10. Mu.L.

5. Precision test

Taking the deafness capsule sample solution (batch No. 190202), repeatedly injecting sample for 6 times according to the chromatographic conditions, recording chromatogram, taking baicalin as a reference peak, and calculating to obtain the relative retention time and the relative peak area RSD of each common peak, which are less than 0.27% and 1.52%, thereby indicating that the instrument precision is good.

6. Stability test

Sampling deaf capsule sample solution (batch No. 190202) at 0, 4, 8, 12, 16, 24h respectively according to the chromatographic conditions, recording chromatogram, taking baicalin as reference peak, calculating to obtain relative retention time and relative peak area RSD of each common peak which are less than 0.31% and 1.58%, and indicating that the sample solution has good stability within 24 h.

7. Repeatability test

Taking 6 parts of deafness capsule sample (batch No. 190802), preparing a test solution according to the method in step 1, injecting a sample under the chromatographic conditions, recording a chromatogram, taking baicalin as a reference peak, and calculating to obtain the relative retention time and the relative peak area RSD of each common peak, wherein the relative retention time and the relative peak area RSD are both less than 0.40% and 2.00%, which indicates that the method has good repeatability.

8. HPLC fingerprint map establishment

Taking 10 batches of deafness capsule samples, preparing a test solution according to the method in the step 1, injecting a sample under the chromatographic condition, and recording a chromatogram. And (3) introducing the obtained chromatogram into a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to analyze 10 fingerprints, setting the spectrum of the sample S1 as a reference spectrum, setting the time window width to be 0.1min, adopting multi-point correction full-spectrum peak matching, calibrating 13 common peaks in total, and generating a fingerprint common mode (an average value method, R) shown in figure 1. The similarity between the sample map and the reference map of S1-S10 is 1.000, 0.993, 0.991, 0.993, 0.995, 0.997, 0.990, 0.992 and 0.995 respectively, the similarity is more than 0.99, and the similarity is high. The relative retention time of the common peak and the RSD of the relative peak area of 10 batches of deafness capsule samples are respectively calculated to be 1.82-38.06% and 0.02-0.11% by taking the No. 5 peak (baicalin) as a reference peak, and the results are shown in tables 1 and 2. It can be seen that the deafness gum components of different batches are not very different, and the content of each component is different.

Table 1: relative retention time of common peaks

Table 2: relative peak area of common peak

Example 2 Cluster analysis

Using SPSS 23.0 software, 13 common peak relative peak areas in fingerprint of deafness capsule are used as variables, and the inter-group connection and squared Euclidean distance method are used for drawing, as shown in figure 2. As can be seen from FIG. 2, 10 batches of deafness capsule samples were grouped into 3 types, the first type was S1, the second type was S4, S5, and S7, and the third type was S2, S3, S6, S8, S9, and S10, and the clustering analysis results were not significantly different from the similarity evaluation results.

Example 3 principal component analysis

The SPSS software is used to introduce 13 index components of 10 batches of deafness capsules to carry out main component analysis, 2 main components with main component characteristic values larger than 1 are obtained by extraction, the cumulative variance contribution rate reaches 83.468%, and the table shows that the

chromatographic peaks

9, 10, 11 (baicalein), 12 (wogonin) and 13 have larger contribution rate to the main component 1 and the chromatographic peaks 5 (baicalin), 6, 7 and 8 have larger contribution rate to the main component 2. The PCA analysis of 13 index components of 10 batches of deafness capsule samples was performed by SIMCA software to obtain 3D graphs of PCA scores of 10 batches of deafness capsule samples, as shown in FIG. 3. As can be seen from FIG. 3, 10 batches of samples are distributed differently, the distance between the samples represents the similarity between the samples, the closer the distance is, the greater the similarity degree is, and the 10 batches of deafness capsules are mainly classified into three categories, which are consistent with the classification result of the cluster analysis.

Table 3: deafness capsule sample principal component characteristic value and variance contribution rate

Table 4: deafness capsule sample principal component factor load matrix

Example 4 index Components and internal standards selection

According to the main components and pharmacological action of 10 medicinal materials of the deafness capsule, baicalin, gentiopicrin, 23-acetyl alisol B, geniposide, ferulic acid, baicalein, eugenol, ligustilide, glycyrrhetinic acid, chlorogenic acid, wogonin and verbascoside are investigated in the experiment. Wherein the contents of verbascoside, geniposide, eugenol and ligustilide are too low, and the 23-acetyl alisol B, ferulic acid and glycyrrhetinic acid are not easy to extract and analyze, so gentiopicroside in monarch drug gentian, baicalin, baicalein and wogonin in ministerial drug baical skullcap root and chlorogenic acid in gardenia are selected as index components. In addition, baicalin with the largest peak area and better peak shape is taken as an internal standard.

Example 5 extraction method and chromatographic Condition optimization

The test was conducted according to the method of preparing the test solution of example 1, except that the solvents were extracted with 25%, 50%, 75%, 100% methanol and 25%, 50%, 75%, 100% ethanol, respectively, and as a result, it was found that there was no significant difference in the extraction rates of methanol in different proportions, and that they were all smaller than the extraction contents of ethanol in different proportions, the extraction amount of ethanol in 75% was the highest, and the contents of each component extracted were about twice as much as that of methanol, so that 75% ethanol was selected as the extraction solvent in the most preferable example.

Experiments were performed according to the method for preparing the test solution in example 1, except that reflux extraction and ultrasonic extraction were used for extraction, respectively, and as a result, it was found that the content of reflux extraction was high, so reflux extraction was used in the most preferred embodiment.

The experiment was carried out under the chromatographic conditions of example 1, except that the detection was carried out at detection wavelengths of 254nm, 280nm and 327nm, respectively, and it was confirmed from the obtained chromatograms that gentiopicroside, baicalin, baicalein and wogonin have maximum absorbances at the detection wavelength of 280nm, chlorogenic acid has maximum absorbances at the detection wavelength of 327, and gentiopicroside has no absorption, so that 280nm was selected as the detection wavelength in the most preferable embodiment.

In the process of establishing the HPLC fingerprints, comparing 10 batches of the HPLC fingerprints of the deafness capsules, determining 13 common peaks, wherein the similarity is more than 0.990, which indicates that the production process of the deafness capsules is stable, the components are not greatly different, and the quality standard of the deafness capsules can be well controlled; however, the content of each component of the deafness capsules in different batches has certain difference, so that the deafness capsule samples are analyzed by adopting cluster analysis and main component analysis, the results show that the deafness capsules are divided into 3 types, and the two main components obtained by analyzing and extracting the main components can represent 83.468 percent of common peak information, which indicates that the deafness capsule samples in different batches have certain difference, possibly due to the quality difference of the original medicinal materials, the fine difference exists, and the manufacturer is recommended to strictly control the factors such as the original medicinal material production area, the processing technology, the storage condition and the like.

Claims

1. A construction method of a deafness capsule fingerprint is characterized by comprising the following steps:

d. respectively detecting the test solution, the mixed reference solution and the negative sample solution by using a high performance liquid chromatograph, wherein the detection wavelength is 270-290nm, the chromatographic column is a C18 silica gel chromatographic column, and the column temperature is 28-38 ℃; the mobile phase A is acetonitrile, the mobile phase B is 0.05 to 0.15 percent phosphoric acid aqueous solution, gradient elution is adopted, the flow rate is 0.5 to 2ml/min, and the gradient elution procedure is as follows: 0-10min, 95% -92% B;10 to 15min,92 to 85% by weight of B; 15-35min, 85 percent B; 35-65min, 85% -70% B; 65-85min, 70-50% by weight of B; 85-95min, 50-30 percent of C;

e. respectively recording chromatograms of the test solution, the mixed reference solution and the negative sample solution, and performing attribution positioning on index components in the chromatograms of the test solution by using the chromatograms of the mixed reference solution; and performing attribution positioning on medicinal materials in the chromatogram of the test solution by using the chromatogram of each negative sample solution, and simultaneously processing the chromatogram of the test solution by using a traditional Chinese medicine chromatogram fingerprint similarity evaluation system to obtain the fingerprint of the deafness capsule.

2. The constructing method according to claim 1, wherein in step a, the sample solution is prepared by: weighing the content of the deafness capsule, grinding, placing into a conical flask with a plug, adding 75% ethanol water solution, weighing, refluxing for 25-35min, cooling, supplementing 75% ethanol water solution, shaking, and filtering with microporous membrane; wherein, the ratio of the content of the deafness capsule to the added 75% ethanol water solution is 2 g: 20-30mL.

3. The method according to claim 1, wherein the concentrations of chlorogenic acid, gentiopicroside, baicalin, baicalein, and wogonin in step b are 0.02-0.03mg/mL, 0.08-0.09mg/mL, 0.3-0.4mg/mL, 0.05-0.07mg/mL, and 0.02-0.03mg/mL, respectively.

4. The method of claim 1, wherein in step d, the detection wavelength is 280nm, the chromatographic column is a Wondasil C18-WR chromatographic column, the mobile phase B is a 0.1% phosphoric acid aqueous solution, the column temperature is 35 ℃, and the sample injection amount is 10 μ L.

5. The method according to claim 1, wherein in step e, the single-component control solution of chlorogenic acid, gentiopicrin, baicalin, baicalein, and wogonin is detected in advance to determine the components corresponding to each peak in the chromatogram of the mixed control solution.

6. A deafness capsule fingerprint spectrum, which is characterized by being detected and constructed by the construction method of any one of claims 1 to 5.

7. The deafness capsule fingerprint of claim 6, wherein the fingerprint comprises 13 common peaks, and the relative retention time of each common peak is calculated as follows, taking peak No. 5 as a reference peak: 7.20-7.25 of No. 1 peak, 10.01-10.06 of No. 2 peak, 22.53-22.63 of No. 3 peak, 23.87-23.95 of No. 4 peak, 62.67-62.75 of No. 5 peak, 66.90-67.00 of No. 6 peak, 69.55-69.63 of No. 7 peak, 72.00-72.60 of No. 8 peak, 77.50-77.60 of No. 9 peak, 78.15-78.30 of No. 10 peak, 79.00-79.15 of No. 11 peak, 88.15-88.30 of No. 12 peak and 90.10-90.20 of No. 13 peak.

8. A deafness capsule quality detection method is characterized in that a sample to be detected of a deafness capsule is detected according to any one of the construction methods of claims 1 to 5 to obtain a high performance liquid chromatography spectrum of the sample to be detected of the deafness capsule, a deafness capsule fingerprint is constructed according to any one of the construction methods of claims 1 to 5, and the high performance liquid chromatography spectrum of the sample to be detected of the deafness capsule is compared with the deafness capsule fingerprint.