CN116500145A - Fifteen-ingredient Sierfighting pill fingerprint detection method - Google Patents

Abstract

The invention discloses a fifteen-ingredient Sierkui pill fingerprint detection method, which comprises the following steps: a. preparation of a control solution: dissolving gallic acid, magnolol, mangiferin, ellagic acid and berberine hydrochloride reference substance in methanol; b. preparation of test solution: extracting fifteen kinds of Sierkuw pills with methanol solution, filtering, and collecting filtrate; c. and respectively sucking the reference substance solution and the test substance solution, and injecting the reference substance solution and the test substance solution into a liquid chromatograph for detection. The method establishes the fingerprint of fifteen-flavor Sierkuchi pills for the first time, provides a simple and accurate method for detecting and identifying fifteen-flavor Sierkuchi pills, ensures the quality of fifteen-flavor Sierkuchi pills, provides scientific basis for clinical application, and has good application prospect.

Description

Fifteen-ingredient Sierfighting pill fingerprint detection method

Technical Field

The invention relates to the field of medicine detection and analysis, in particular to a fifteen-ingredient Sierfighting pill fingerprint detection method.

Background

The shizandra berry pill is a traditional preparation in Tibetan medicine and is used for treating liver and gall heat, cholecystitis, cholelithiasis and choledocholithiasis. Is widely applied to clinic in minority nationality areas. The only current quality standard at present is the national pharmaceutical standard of the national food and pharmaceutical administration, standard number: WS-10738 (ZD-0738) -2002, which has no control over a lot of active ingredients, has relatively simple quality standard, can not well reflect uniformity, stability and controllability of medicine quality, and does not meet the requirements of modern medicine quality.

The fifteen-flavor Sier pill is a compound prepared from fifteen Tibetan medicines such as India swertia, myrobalan, pomegranate fruit, berberis cortex and the like, the compound preparation plays a comprehensive treatment role based on various chemical components of the compound preparation, has the characteristics of integrity and ambiguity, and is difficult to achieve the purpose of quality control and evaluation of a traditional Chinese medicine prescription only by using a traditional Chinese medicine quality evaluation method with single or a plurality of chemical components as indexes, and the quality control of the traditional Chinese medicine preparation can be more accurate and reliable by using more comprehensive chemical components. However, no research on multi-component of the shizandra berry pill exists at present.

Disclosure of Invention

In order to solve the problems, the invention provides a fifteen-ingredient Sierkui pill fingerprint detection method, which is characterized by comprising the following steps of: it comprises the following steps:

a. preparation of a control solution: dissolving gallic acid, magnolol, mangiferin, ellagic acid and berberine hydrochloride reference substance in methanol;

b. preparation of test solution: extracting fifteen kinds of Sierkuw pill powder with methanol solution, filtering, and collecting filtrate;

c. respectively sucking the reference substance solution and the sample solution, and injecting into a liquid chromatograph; the chromatographic conditions were as follows:

chromatographic column: hydrophilic octadecylsilane chemically bonded silica is used as a filler; the gradient elution procedure was as follows with 0.1% phosphoric acid solution as mobile phase a and acetonitrile as mobile phase B:

further, the control solution in the step a contains 10-30 mug gallic acid, 20-50 mug magnolol, 1-10 mug mangiferin, 10-30 mug ellagic acid and 5-25 mug berberine hydrochloride in each 1 mL.

Further, the mass volume ratio of the fifteen-flavor Sierkudo pill to the methanol solution in the step b is 0.2-2 g:50mL.

Still further, the concentration of the methanol solution was 75%, mL/mL.

Further, the extraction in the step b is ultrasonic extraction, the power ratio of ultrasonic extraction is 99%, the frequency is 45kHz, and the time is 45-60 min.

Further, the chromatographic column in the chromatographic condition in the step c is SwellC18,5 μm, 4.6X1250 mm, flow rate 0.6-1.0 mL/min, sample injection amount 2-16. Mu.L, column temperature 25-35 ℃, wavelength 254+ -5 nm.

Further, the flow rate in the chromatographic condition is 0.8mL/min, the column temperature is 30 ℃, and the wavelength is 254nm.

Further, 13 common characteristic peaks are provided in the fingerprint of the fifteen-flavor Sier-bucket pill, specifically shown in figure 1, wherein peak 2 is a gallic acid chromatographic peak, peak 8 is an ellagic acid chromatographic peak, peak 5 is a magnaline chromatographic peak, peak 10 is a berberine hydrochloride chromatographic peak, and peak 6 is a mangiferin chromatographic peak.

According to the fingerprint detection method of the fifteen-flavor Sier pill, the fingerprint of the fifteen-flavor Sier pill is established for the first time through a specific sample processing method and chromatographic conditions, a simple and accurate method is provided for detection and identification of the fifteen-flavor Sier pill, the quality of the fifteen-flavor Sier pill is ensured, a scientific basis is provided for clinical application of the fifteen-flavor Sier pill, and the fingerprint detection method has a good application prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a diagram showing the superimposed fingerprint (S1-S11) and the reference fingerprint (R) of fifteen-ingredient Sierkudo pills

The fifteen herb Sier pill of FIG. 2 has common characteristic peaks (S1. Test article, S2. Indian swertia herb, S3. Indian swertia herb, S4. Sal Nitri, S5. Achillea Wilsoniana, S6. Lagotis herb, S7. Tang Gute aconite, S8. Granati, S9. Engella seed, S10. Berberis bark, S11. Rebaudiana, S12. Barbadeta herb, S13. Black borneol, S14. Murraya root, S15. Chebula, S16. Wilsonia herb, S17. Indian swertia herb, S18. Lecanina herb, S19. Sal Nitri, S20. Hordeum vulgare, S21. Lagotis herb, S22. Tang Gute aconite, S23. Granati, S24. Boussonetia herb, S25. Small bark, S26. Peel seed, S27. Barbadeta herb, S28. Barbadeta herb, S29. Murraya herb, S30. Chen, 33. Chebula) and a blank solution

Detailed Description

Example 1 fifteen-ingredient Sierkui pill fingerprint detection

(1) Preparation of test solution

Taking fifteen kinds of Sierkui pill powder 0.5g, precisely weighing, placing into a conical bottle with a plug, precisely adding 70% methanol solution 50ml, performing ultrasonic treatment (power ratio 99%, frequency 45 kHz) for 45min, cooling, filtering, and collecting the subsequent filtrate, and passing through a 0.22 μm microporous filter membrane;

(2) Preparation of control solution

Precisely weighing 13.66mg of gallic acid reference substance, 7.51mg of magnolol reference substance, 6.42mg of mangiferin reference substance, 5.07mg of ellagic acid reference substance and 8.67mg of berberine hydrochloride reference substance, and respectively adding methanol to a volumetric flask with volume of 10ml to obtain reference substance stock solution; precisely sucking the prepared control stock solutions respectively, diluting with methanol to volume to scale to obtain mixed control solutions of gallic acid, magnaline, mangiferin, ellagic acid and berberine hydrochloride with mass concentrations of 27.32, 45.06, 6.42, 20.28 and 17.34 μg/mL;

(3) Respectively sucking 10 μl of reference solution and test solution, and injecting into liquid chromatograph; the chromatographic conditions were as follows:

chromatographic column: swellC18 column (5 μm, 4.6X1250 mm); column temperature: 30 ℃; flow rate: 0.8ml/min; detection wavelength: 254nm; mobile phase: acetonitrile (B) -0.1% phosphoric acid solution (a), gradient elution procedure:

(4) 13 common characteristic peaks are arranged in the fingerprint of the fifteen-flavor Sier pills, and the specific structure is shown in figure 1, wherein peak 2 is a gallic acid chromatographic peak, peak 8 is an ellagic acid chromatographic peak, peak 5 is a magnolol chromatographic peak, peak 10 is a berberine hydrochloride chromatographic peak, and peak 6 is a mangiferin chromatographic peak.

The advantageous effects of the present invention are described below by way of test examples.

Test example 1 study of characteristic Spectrum of Shiwuwei Sier Jiu pills

1 Experimental materials

1.1 instruments

Table 1 instrument

1.2 fifteen-ingredient Sierfighting pill

The specification of the shizandra Sier pill is 0.5 g/pill, which is provided by Qinghai Jiumei Tibetan medicine industry Co., ltd, and the batch number is shown in the table below. Fifteen medicinal materials of the fifteen-five-flavor Sierkuai pill are also provided by Qinghai Jiumei Tibetan medicine Co.

Table 2 eleven batches of fifteen-ingredient siraitia pills

1.3 control

Table 3 control

1.4 reagents and materials

The water used in the experiment is ultrapure water.

Table 4 reagent

1.5, raw medicinal materials

To ensure the quality of the medicinal materials, fifteen raw medicinal materials provided by Qinghai Jiumei Tibetan medicine industry Co., ltd. Were tested according to the current standard regulation project, and the results are shown in Table 5.

TABLE 5 results of testing of medicinal materials

2. Feature map study

2.1 chromatographic conditions

Chromatographic column: swellC18 column (5 μm, 4.6X1250 mm); column temperature: 30 ℃; flow rate: 0.8ml/min; detection wavelength: 254nm; mobile phase: acetonitrile (B) -0.1% phosphoric acid solution (a), gradient elution procedure:

2.2 preparation method of sample solution

About 0.5g of fifteen-ingredient Sierkui pill powder is taken, precisely weighed, placed in a conical bottle with a plug, precisely added with 50ml of 70% methanol solution, subjected to ultrasonic treatment (power ratio 99% and frequency 45 kHz) for 45min, cooled, filtered, and the subsequent filtrate is taken to pass through a microporous filter membrane of 0.22 mu m, thus obtaining the sample solution.

2.3 preparation of control solution

Precisely weighing 13.66mg of gallic acid reference substance, 7.51mg of magnolol reference substance, 6.42mg of mangiferin reference substance, 5.07mg of ellagic acid reference substance and 8.67mg of berberine hydrochloride reference substance, and respectively adding methanol to a volumetric flask with volume of 10ml to obtain reference substance stock solution; respectively precisely sucking the prepared control stock solutions in the same volumetric flask, diluting with methanol to constant volume to scale to obtain mixed control solutions of gallic acid, magnaline, mangiferin, ellagic acid and berberine hydrochloride with mass concentrations of 27.32, 45.06, 6.42, 20.28 and 17.34 μg/mL.

2.4 preparation of sample solution of Single medicinal Material

Weighing the single medicinal materials according to the prescription amount, and preparing the test solution of the single medicinal materials according to the preparation method under the item of 2.2.

2.5 preparation of negative control solution

The preparation method comprises the steps of weighing each medicinal material according to the prescription proportion of the shizandra berry pill, and preparing negative control solutions of berberis thunbergii skin, aurin deficiency, tamarind deficiency, inula root, black borneol, fennel, aconitum tanguticum, pomegranate seed, herba lagotis, herba leonuri, mirabilite, swertia pseudochinensis, myrobalan, fructus glehniae, and costus root according to the method below item 2.2.

2.6 methodological investigation

2.6.1 precision test

Taking the same batch of sample solution (batch eleven) under the item "2.2", continuously injecting the sample solution for 6 times according to the chromatographic condition under the item "2.1", recording the retention time and the peak area of each common characteristic peak, taking ellagic acid (peak No. 8) as a reference peak, and calculating the relative retention time and the relative peak area of the characteristic peak (the retention time and the peak area of the ellagic acid, namely the reference peak, are calculated as 1). As a result, it was found that the relative retention time of each of the common peaks had an RSD value of less than 0.26%. The RSD values of the relative peak areas of the common peaks, where the unimodal area occupies more than 2% of the total peak area, are all less than 3.44%. Chemometric evaluation is carried out by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation system 2004A version, and the similarity is 0.99, which indicates that the instrument precision is good.

2.6.2 stability test

Taking the same batch of sample solution (batch eleven) under the item "2.2", respectively carrying out sample injection under chromatographic conditions under the item "2.1" at 0, 2, 4, 8, 12 and 24 hours, and calculating the relative retention time and the relative peak area of the characteristic peak. As a result, the relative retention time of each of the common peaks had an RSD value of less than 0.41%. The RSD values of the relative peak areas of the common peaks with the single peak area accounting for more than 2% of the total peak area are all less than 3.21%, the similarity is evaluated as 0.99, and the sample solution is considered to have good stability within 24 hours.

2.6.3 repeatability test

6 parts of the sample solution to be tested (batch eleven) in the same batch are prepared according to the same method of '2.2', and are respectively sampled and measured according to the chromatographic conditions under the item of '2.1', and the relative retention time and the relative peak area of the characteristic peak are calculated. The RSD values for the relative retention times of the individual peaks in common are all less than 0.11%. The RSD values of the relative peak areas of the common peaks with the single peak area accounting for more than 2% of the total peak area are all less than 4.86%, the similarity is evaluated as 0.99, and the method is considered to have better repeatability.

3. Establishment of characteristic spectrum of shizandra berry pill

According to the chromatographic condition under the item of 2.1, eleven batches of fifteen test sample solutions of the Sierkudo pills are sequentially injected, chromatograms are recorded, data matching is carried out by using a 2004A version of a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and peaks with better separation degree and higher relative retention time matching degree in eleven batches of test samples are selected as common characteristic peaks. In the chromatogram of the fifteen-ingredient Sierkudo pill sample, peak 8 (ellagic acid) with a large peak area and a moderate and relatively stable retention time was selected as the reference peak (S peak). And generating a comparison map by a median method, and calculating matching data and similarity. The eleven batches of samples obtained finally were calibrated for a total of 13 common peaks, the results are shown in FIG. 1. By comparing the chromatograms of the 5 mixed reference solutions and combining the retention time of the 5 components, partial peaks can be identified, wherein the peak No. 2 is gallic acid, the peak No. 5 is magnolol, the peak No. 6 is mangiferin, the peak No. 8 is ellagic acid, and the peak No. 10 is berberine hydrochloride. The similarity between eleven batches of fifteen Sierkuei pill samples and a control map is respectively 0.963, 0.957, 0.971, 0.976, 0.993, 0.994, 0.967, 0.980, 0.898, 0.948 and 0.905 and is more than 0.8. The eleven batches of samples are good in similarity, and different batches have good consistency and stability.

4. Peak spectrum assignment analysis

Sample injection measurement is carried out on the sample solution of each single medicinal material under the item "2.4" and each negative control solution under the item "2.5" according to the chromatographic condition under the item "2.1", chromatograms under 254nm wavelength are recorded, each chromatogram is led into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the attribution of 13 common characteristic peaks is analyzed. As a result, as shown in FIG. 2, it was found that the peak No. 1 was probably a solvent peak, which was present in all of the single drug test solution, the negative control solution, the mixed control solution and the blank solution. Peak No. 2 (gallic acid), peak No. 7, peak No. 8 (ellagic acid), peak No. 9 are derived from myrobalan, peak No. 3, peak No. 4, peak No. 5 (magnaline), peak No. 10 (berberine hydrochloride) are derived from berberis skin, peak No. 6 (mangiferin), peak No. 12 are derived from swertia mussel, peak No. 8 (ellagic acid) is derived from pomegranate seed, peak No. 11 is derived from chrysosporium, peak No. 13 is derived from cantaloupe seed, ellagic acid is a common component of myrobalan and pomegranate seed. Each characteristic spectrum peak can correspond to the chromatographic peak of a single medicinal material, and can provide a reference basis for the quality control research of fifteen-ingredient Sierkudo pills.

5. Feature map of shizandra berry pill

Through early experiments, fifteen Sierkuai pills have rich chromatographic peaks under 254nm in liquid chromatography, so 254nm is selected as the detection wavelength of the characteristic spectrum of the Sierkuai pills. The composition of the shizandra berry pill is complex, common characteristic peaks are subjected to source analysis, and other 12 peaks except for the 1 # peak with high probability of being a solvent peak can be attributed to corresponding medicinal materials, and meanwhile chemical compositions corresponding to 5 chromatographic peaks can be identified. Therefore, the HPLC method is adopted to establish the characteristic spectrum of the shizandra berry pill under 254nm detection wavelength, which is accurate and feasible.

In conclusion, the fingerprint of fifteen-flavor Sier pills is established for the first time through a specific sample processing method and chromatographic conditions, a simple and accurate method is provided for detection and identification of fifteen-flavor Sier pills, the quality of fifteen-flavor Sier pills is ensured, scientific basis is provided for clinical application of fifteen-flavor Sier pills, and the method has a good application prospect.

Claims (8)

1. A fifteen-ingredient Sierkui pill fingerprint detection method is characterized by comprising the following steps of: it comprises the following steps:

a. preparation of a control solution: dissolving gallic acid, magnolol, mangiferin, ellagic acid and berberine hydrochloride reference substance in methanol;

b. preparation of test solution: extracting fifteen kinds of Sierkuw pills with methanol solution, filtering, and collecting filtrate;

c. respectively sucking the reference substance solution and the sample solution, and injecting into a liquid chromatograph; the chromatographic conditions were as follows:

chromatographic column: hydrophilic octadecylsilane chemically bonded silica is used as a filler; the gradient elution procedure was as follows with 0.1% phosphoric acid solution as mobile phase a and acetonitrile as mobile phase B:

2. the content measuring method according to claim 1, wherein: the reference substance solution in the step a contains 10-30 mug of gallic acid, 20-50 mug of magnolol, 1-10 mug of mangiferin, 10-30 mug of ellagic acid and 5-25 mug of berberine hydrochloride in each 1 mL.

3. The content measuring method according to claim 1, wherein: the mass volume ratio of the fifteen-flavor Sierkui pill to the methanol solution in the step b is 0.2-2 g:50mL.

4. A content determining method according to claim 3, wherein: the concentration of the methanol solution is 75% and mL/mL.

5. The content measuring method according to claim 1, wherein: the extraction in the step b is ultrasonic extraction, the power ratio of ultrasonic extraction is 99%, the frequency is 45kHz, and the time is 45-60 min.

6. The content measuring method according to claim 1, wherein: step c, wherein the chromatographic column is Swell under the chromatographic conditionC18,5 μm, 4.6X1250 mm, flow rate 0.6-1.0 mL/min, sample injection amount 2-16. Mu.L, column temperature 25-35 ℃, wavelength 254+ -5 nm.

7. The method of detection according to claim 6, wherein: the flow rate in the chromatographic condition is 0.8mL/min, the column temperature is 30 ℃, and the wavelength is 254nm.

8. The detection method according to claim 1 to 7, characterized in that: 13 common characteristic peaks are arranged in the fingerprint of the fifteen-flavor Sierfight pill, and the specific characteristic peaks are shown in figure 1, wherein peak 2 is a gallic acid chromatographic peak, peak 8 is an ellagic acid chromatographic peak, peak 5 is a magnaline chromatographic peak, peak 10 is a berberine hydrochloride chromatographic peak, and peak 6 is a mangiferin chromatographic peak.