CN107515259B

CN107515259B - Method for determining blumea balsamifera element content in blumea balsamifera ketone tablets

Info

Publication number: CN107515259B
Application number: CN201710657468.1A
Authority: CN
Inventors: 谭道鹏; 曾伟珍; 卢禁; 江东龙; 冯汉林; 于琳
Original assignee: Shenzhen Neptunus Pharmaceutical Research Institute Co Ltd
Current assignee: Shenzhen Neptunus Pharmaceutical Research Institute Co Ltd
Priority date: 2017-08-03
Filing date: 2017-08-03
Publication date: 2020-04-21
Anticipated expiration: 2037-08-03
Also published as: CN107515259A

Abstract

The invention provides a method for determining the content of blumea balsamifera element in blumea balsamifera ketone tablets. The method comprises the following steps: preparing a standard substance solution, preparing a test sample solution, carrying out external standard method chromatographic detection, and calculating the content of the blumea balsamifera element in the test sample. The method can specifically detect the blumea balsamifera A, and has the advantages of good chromatographic peak separation, no interference, good linearity, high accuracy, repeatability, recovery rate and stability.

Description

Method for determining blumea balsamifera element content in blumea balsamifera ketone tablets

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a method for determining the content of effective components in an Aixintong tablet.

Background

According to the statistics of a lancet, the coronary heart disease becomes one of the three lethal factors before human diseases, the current clinical medicines for the coronary heart disease are mainly chemical medicines, the current commonly used myocardial ischemia resisting medicines comprise nitrates, β -receptor blockers, calcium antagonists and the like, and angiotensin converting enzyme inhibitors and statins lipid lowering medicines also have indirect myocardial ischemia resisting effects and are mainly used for symptomatic treatment and prognosis improvement of the coronary heart disease.

However, the clinical effectiveness and prognosis of the current commonly used therapeutic drugs for coronary heart disease and angina pectoris have its limitations. For example, nitrate based drugs are only symptomatic relief drugs with no evidence of benefit for outcome, and theoretically, these drugs could reflexively increase heart rate, possibly negatively affecting the long-term outcome of myocardial ischemia. To date, these drugs remain one of the most commonly used drugs in the treatment of coronary heart disease. However, the first generation of calcium antagonists found more than 20 years after clinical use, such drugs increased the risk of myocardial infarction. Clinical tests show that the calcium antagonist has no improvement effect on prognosis of malformed myocardial infarction, variant angina and cardiac insufficiency. Drugs that improve blood flow supply are often associated with headache, increased heart rate reflexes and adverse reactions such as hypotension, peripheral edema, constipation, palpitations, flushing of the face, etc., while platelet inhibitors that improve prognosis are at risk of bleeding. The safe treatment range of the chemical drugs is narrow according to different patient conditions, and uncertain risks exist when the chemical drugs are clinically used and used as secondary preventive drugs. Therefore, the development of new drugs for treating coronary heart disease, which can increase the clinical drug selection range of patients and improve the long-term use safety of the drugs, has remarkable economic and social benefits

The Blumea balsamifera tablets are a novel anti-myocardial ischemia drug developed by the traditional Chinese medicine Blumea balsamifera (L.) DC according to the theory of the traditional Chinese medicine and the modern pharmaceutical technology, are used for treating ischemic heart diseases (coronary heart disease and angina pectoris), and are preparations of the effective parts of the total flavonoids of the Blumea balsamifera. Chinese patent application CN 100563669C discloses a blumea balsamifera total flavone extract and a preparation method thereof, chinese patent application CN 1923191B discloses an application of flavanone compounds in the preparation of drugs for treating cardiovascular diseases, and chinese patent application CN 100420453C discloses a preparation method of blumea balsamifera total flavone extract solid dispersion.

In the traditional Chinese pharmacology, the blumea balsamifera has the functions of warming middle-jiao and promoting blood circulation, regulating menstruation, dispelling wind and removing dampness, and is used for treating diseases such as exogenous wind-cold, diarrhea, abdominal pain, borborborygmus, swelling, irregular menstruation, arthralgia and myalgia and the like. The application of the traditional Chinese medicine blumea balsamifera in the existing prescription preparation is mainly embodied in two aspects, namely, borneol (blumea balsamifera tablets) is extracted and then the latter is used as the medicine; the second one is the blumea balsamifera medicinal material, especially the volatile component of blumea balsamifera. Obviously, the volatile components of blumea balsamifera and the main chemical substance borneol therein should be one of the material bases of blumea balsamifera for warming middle-jiao and promoting blood circulation; therefore, the research on the medicinal value of the traditional Chinese medicine blumea balsamifera from the perspective of total flavonoids is significant. The systematic research shows that the blumea balsamifera total flavonoids have good anti-myocardial ischemia activity and have a certain blood fat reducing effect on hyperlipoidemia animals; the safety research shows that the blumea balsamifera total flavonoids have higher safety.

In the effective part of the total flavone of the blumea balsamifera tablet, the main active ingredient is mainly flavanone compounds, and 3,3 ', 5, 7-tetrahydroxy-4' -methoxy-flavanone (blumeatin A), 3,3 ', 5-trihydroxy-4', 7-dimethoxy-flavanone (blumeatin B) have been identified; 3 ', 4', 5-trihydroxy-7-methoxy-flavanone (blumeatin C), and the like. The flavanone compound components are rarely reported in the existing relevant literature data about the traditional Chinese medicine prescription preparations on the market; they are also very different from the traditional Chinese medicine preparations which are on the market and take flavonoid components as effective components or effective parts for resisting myocardial ischemia.

Preclinical research shows that the blumea balsamifera tablets can effectively improve myocardial ischemia of various experimental model animals, can effectively protect ischemic tissues, and have certain functions of reducing blood fat and diluting blood. Clinical test results show that the treatment of chronic stable coronary heart disease angina by the erigeron ketone has better curative effect and better safety, is obviously superior to a placebo control group in the aspects of improving angina symptoms of patients, reducing angina attack frequency and pain duration, has a treatment window far larger than all chemical drugs of the current standard treatment, does not have common adverse reactions of standard treatment drugs, and can ensure the overall safety of long-term administration. The existing results preliminarily show that the advantages of the Aixintong tablets in improving the life quality of patients can be obviously reduced, the clinical dosage of nitroglycerin can be obviously reduced, and the Aixintong tablets have good clinical benefit value for patients with coronary heart disease and angina pectoris.

Literature "RP-HPLC method for determination of blumea balsamifera element and flavanonol content" in blumea balsamifera [ huang-yong lin, wen-yong xin, zhao-shi, juting-chun-guangxi science, 2007,14 (2): 140-142] reports the content determination method of two chemical components in blumea balsamifera. Chinese patent application CN 104458993B discloses a finger print of Yunnan Gui blumea balsamifera with protocatechuic acid and protocatechuic aldehyde as main components. But no report of the method for measuring the content of the blumea balsamifera element is found. In addition, since the blumea balsamifera ketone tablets are recently available, a rapid and efficient detection method for the content of the effective component blumea balsamifera A is still lacked, which is a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a method for determining the content of blumea balsamifera A in blumea balsamifera ketone tablets.

The content of the blumea balsamifera A in the blumea balsamifera ketone tablets is rapidly and accurately determined by high performance liquid chromatography.

In order to achieve the above object, the method of the present invention comprises:

(1) dissolving a blumea balsamifera A standard substance in a methanol water solution to prepare a standard substance solution, and performing a system applicability test and calibrating a detection system by using the standard substance solution under the following chromatographic conditions;

chromatographic conditions are as follows: the chromatographic column is a C-octadecyl silane bonded silica gel column, and the particle size of the C-octadecyl bonded silica gel is less than or equal to 5 mu m;

gradient elution is carried out by taking 30-90% methanol-acid aqueous solution in volume ratio as a mobile phase, and the detection wavelength is 289 nm;

(2) dissolving the blumea balsamifera ketone tablets in a methanol water solution to prepare a test solution;

(3) according to the chromatographic conditions in the step (1), injecting a standard solution and a test solution which are equal in volume into a high performance liquid chromatograph, measuring the peak areas of the standard solution and the test solution, and calculating to obtain the content of the blumea balsamifera element in the test solution.

Further, the methanol aqueous solution has a concentration of 70% (V/V); the specification of the chromatographic column is 250 multiplied by 4.6 mm; the particle size of the carbon octadecyl bonded silica gel is 5 mu m;

the mobile phase used in the gradient elution is formed by mixing methanol and phosphoric acid solution, and the phosphoric acid solution used for mixing contains 0.4% (V/V) of phosphoric acid.

The invention also carries out a series of methodological verifications on the detection method.

The determination method of the invention not only can be used for determining the content of the blumea balsamifera element in the blumea balsamifera ketone tablet preparation, but also can be used for detecting blumea balsamifera medicinal materials, congeneric plants, extracts and other preparations containing the blumea balsamifera element.

Has the advantages that: the method can specifically determine the content of the blumea balsamifera A, and has good chromatographic peak separation and no interference; good linearity, high accuracy, repeatability, recovery rate and stability.

Drawings

FIG. 1 is an HPLC chromatogram of a blumea balsamifera A standard substance and a blumea balsamifera ketone tablet sample;

a is blumea balsamifera A standard substance and B is blumea balsamifera A tablet sample

FIG. 2 shows the structural formula of blumea balsamifera A.

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the scope of the claims.

The Aixintong tablets used in the invention are prepared according to the method of Chinese patent ZL 200410070463.1; the blumea balsamifera element is prepared according to the method of Chinese patent ZL 200510093643.6.

Example 1 measurement of the content of Blumeatin in Eisenone tablets (Shenzhen Haiwang pharmaceutical science and technology research institute, Ltd., batch No.: 20160401)

1) Chromatographic conditions and systematic adaptability test:

octadecylsilane chemically bonded silica was used as a filler (250X 4.6mm,5 μm); methanol-phosphoric acid solution was used as the mobile phase, and gradient elution was set as shown in table 1.

Table 1:

the mobile phase is formed by mixing methanol and a phosphoric acid solution, and the phosphoric acid solution contains 0.4% (V/V) of phosphoric acid; the detection wavelength is 289nm, and the theoretical plate number is not less than 3000 calculated according to the blumea balsamifera A peak.

2) Preparation of a test solution: taking 20 tablets of the Aixintong tablets, grinding, precisely weighing 230mg, placing in a 50ml measuring flask, adding 40ml of 70% (V/V) methanol aqueous solution, performing ultrasonic dissolution, cooling, adding 70% methanol aqueous solution for dilution to scale, shaking up, filtering, and taking the subsequent filtrate as a sample solution.

3) Preparation of standard solution: a proper amount of blumea balsamifera A standard substance (Shenzhen Haiwang pharmaceutical and technology research institute, Limited, batch No.: 20160101, purity is calculated by 100%) is precisely weighed, and 70% methanol aqueous solution is added for dissolution to prepare 0.12mg of standard substance solution in each 1 ml.

4) Content determination: respectively and precisely sucking 10 μ l of standard solution and sample solution, injecting into high performance liquid chromatograph, measuring peak areas of the standard solution and the sample solution, and calculating according to external standard method. The detection result shows that the content of the blumea balsamifera element in the blumea balsamifera ketone tablet sample is as follows: 4.03 mg/tablet.

Example 2 verification of the determination methodology of blumea balsamifera A in blumea balsamifera A tablet (Shenzhen Haiwang pharmaceutical science and technology research institute, Limited, batch No.: 20160401)

The detection method described in example 1 was validated.

① specialization

As shown in figure 1, under the preferable chromatographic condition, the chromatographic peak of the blumea balsamifera A in the blumea balsamifera ketone tablets is well separated and has no interference, which indicates that the method has better specificity.

② linearity

Precisely preparing standard solution of blumea balsamifera A with different concentrations. And accurately sucking 10 mu L of the standard substance solution for determination, drawing a standard curve by taking the injection concentration (X) of each reference substance as an abscissa and the peak area integral value (Y) as an ordinate, and obtaining a linear regression equation and a linear range of the blumea balsamifera element, wherein Y is 32.496X +0.0726, and R is 0.9998. Wherein Y is the peak area and X is the concentration (. mu.g/ml). As shown in Table 2, the test results show that the peak area and the concentration of the blumea balsamifera A have a good linear relationship in the concentration range of 0-215.8 mug/ml.

TABLE 2 Linear Range test results of Blumectin A assay

③ precision

Precision in the day (n is 5)

Accurately sucking 10 μ l of the same test solution, continuously injecting sample for 5 times, measuring the peak area of blumea balsamifera A, and calculating the relative standard deviation to be 0.15%.

Precision in the day (n 15)

Accurately sucking 10 μ l of the same test solution, continuously measuring for three days, measuring for 5 times per day, measuring the peak area of blumea balsamifera A, and calculating the relative standard deviation to be 0.63%.

④ repeatability

6 parts of the same sample (batch: 20160401) were taken, and the measurement was carried out under the "preparation of test solution", and the peak areas of the blumea balsamifera A were measured, and the relative standard deviations were calculated to be 1.84%, respectively.

⑤ recovery rate of sample

Taking 20 blumea balsamifera core ketone tablets (batch number: 20160401) with determined blumea balsamifera A content, grinding, precisely weighing 6 parts of the blumea balsamifera A, each part of the blumea balsamifera A115 mg, putting the blumea balsamifera A in a 50ml volumetric flask, adding 70% methanol aqueous solution into each part of the blumea balsamifera A standard substance with the same amount to scale, shaking up, filtering, operating according to the method under the item of content determination, determining the area of each spectrum peak, and calculating the sample adding recovery rate as follows: 101.19%, the relative standard deviations are: 1.02 percent.

⑥ stability

Accurately sucking 10 mu l of the same test solution, measuring at 0h, 1h, 2h, 4h, 8h, 12h, 16h, 24h and 48h, observing by the peak area of the blumea balsamifera A, and calculating the relative standard deviation to be 1.71%, which indicates that the sample has good stability within 48 h.

The results of the methodology study show that: the method for measuring the content of the blumea balsamifera element in the blumea balsamifera ketone tablet is rapid, accurate and reliable, and can be used for measuring the content of the blumea balsamifera element in the blumea balsamifera ketone tablet preparation.

Claims

1. A method for determining the content of blumea balsamifera element in blumea balsamifera ketone tablets comprises the following steps:

chromatographic conditions are as follows: the stationary phase of the chromatographic column is carbon octadecyl silane bonded silica gel, and the particle size of the carbon octadecyl silane bonded silica gel is less than or equal to 5 mu m; the detection wavelength is 289 nm;

the gradient elution is: methanol is taken as a mobile phase A, and phosphoric acid aqueous solution with the volume percentage of 0.4 percent is taken as a mobile phase B;

the gradient elution includes: the time gradient is 0min → 30min → 60 min; corresponding to the gradient eluent, the gradient of the volume percentage of the mobile phase a is 45% → 60% → 60%, and the gradient of the volume percentage of the mobile phase B is 55% → 40% → 40%.

(2) Dissolving the blumea balsamifera tablets to be tested in a methanol water solution to prepare a test solution;

(3) according to the chromatographic conditions in the step (1), injecting a standard solution and a test solution which are equal in volume into a high performance liquid chromatograph, measuring the peak areas of the standard solution and the test solution, and calculating to obtain the content of the blumea balsamifera element in the test.

2. The method according to claim 1, wherein the aqueous methanol solution contains 70% by volume of methanol.

3. The method according to claim 1, characterized in that the particle size of the carbooctadecylsilane bonded silica is 5 μ ι η,4 μ ι η or 3.5 μ ι η.

4. The method according to claim 3, wherein the particle size of the carbooctadecylsilane bonded silica is 5 μm.

5. The method according to claim 1, wherein the theoretical plate number of the chromatographic column is not less than 3000 calculated as the blumea balsamifera A peak.

6. A method for determining the content of blumea balsamifera element in blumea balsamifera ketone tablets comprises the following steps:

1) taking a blumea balsamifera element standard substance, and preparing 1ml of a standard solution containing 0.12mg of blumea balsamifera element by using 70% methanol aqueous solution by volume percentage; the system suitability test, calibration of the detection system was carried out under the following chromatographic conditions:

chromatographic conditions are as follows: the stationary phase is carbon octadecyl silane bonded silica gel, the specification of the chromatographic column is 250 multiplied by 4.6mm, and the particle size of the carbon octadecyl silane bonded silica gel is 5 mu m; methanol is taken as a mobile phase A, and phosphoric acid aqueous solution with the volume ratio of 0.4 percent is taken as a mobile phase B; the gradient elution procedure was: time gradient 0min → 30min → 60 min; corresponding gradient eluent, the volume percent gradient of mobile phase A is 45% → 60% → 60%, and the volume percent gradient of mobile phase B is 55% → 40% → 40%; the detection wavelength is 289nm, and the theoretical plate number is not less than 3000 calculated according to the blumea balsamifera A peak;

2) preparing a sample solution from an elaeostereum piece to be detected by using a methanol water solution with the volume percentage of 70%;

3) injecting the standard solution and the test solution with the same volume into a high performance liquid chromatograph according to the chromatographic conditions of the step 1), measuring the peak areas of the standard solution and the test solution, and calculating the content of the blumea balsamifera element in the blumea balsamifera ketone tablets according to an external standard method.