CN115032316A - Quality evaluation method of Gaodanling tablets - Google Patents

Abstract

A quality evaluation method for Gandouling tablets belongs to the technical field of quality evaluation of compound traditional Chinese medicine preparations. An appropriate amount of Gandouling tablets is crushed, ultrasonically extracted with methanol, and filtered through a filter membrane; a reference substance is precisely weighed and methanol is added to prepare a certain concentration control Product solution; mobile phase selects acetonitrile-potassium dihydrogen phosphate solution, adopts gradient elution; detector selects DAD, chromatographic column selects BEH C ₁₈ ; Measure and obtain 10 batches of samples HPLC chromatograms, and import the obtained HPLC chromatograms into similarity evaluation software , calculate the similarity of the samples, and determine the standard fingerprint; if the similarity is greater than 0.90, it meets the quality requirements of Gandouling tablets, thus completing the quality evaluation of the samples to be tested. The present invention uses ultra-high performance liquid chromatography to construct the fingerprint of Gandouling tablets, analyzes multiple batches of Gandouling tablets to determine its standard fingerprint, and establishes a stable and reliable quality evaluation of Gandouling tablets method.

Description

A kind of quality evaluation method of Gandouling tablets

technical field

The invention belongs to the technical field of quality evaluation of compound traditional Chinese medicine preparations, and particularly relates to a quality evaluation method of Gandouling tablets.

Background technique

At present, traditional Chinese medicine fingerprint technology mainly includes chromatography methods such as thin layer scanning (TLCS), high performance liquid chromatography (HPLC), gas chromatography (GC) and high performance capillary electrophoresis (HPCE), as well as ultraviolet spectroscopy (UV), Spectroscopic methods such as Infrared Spectroscopy (IR), Mass Spectrometry (MS), Nuclear Magnetic Resonance (NMR) and X-ray Diffraction.

Because high performance liquid chromatography (HPLC) has the characteristics of high separation efficiency, high selectivity, high detection sensitivity, fast analysis speed and wide application range, and most of the components of traditional Chinese medicine can be analyzed and detected on high performance liquid chromatography. Therefore, high performance liquid chromatography has become the preferred method of traditional Chinese medicine fingerprint technology.

Gandouling Tablet is a hospital preparation of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine. The prescription of the preparation is mainly composed of traditional Chinese medicines such as Coptis chinensis, Salvia miltiorrhiza, etc., and the main functions are to clear heat and detoxify, remove blood stasis and soften firmness, invigorate gallbladder and expel copper. At present, the main quality control method of the preparation is to use Coptis chinensis in the preparation as a reference medicinal material, and use the HPLC method to determine the content of berberine hydrochloride and aloe-emodin in the preparation. However, at present, this quality control method is relatively single, and cannot comprehensively control and reflect the quality of the preparation.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a method for evaluating the quality of Gandouling tablets, which firstly establishes the fingerprint of Gandouling tablets by using HPLC technology, and then controls the quality of the preparation more comprehensively.

The technical scheme adopted in the present invention is: a method for evaluating the quality of Gandouling tablets, the steps are as follows:

(1) Preparation of Gandouling Tablets

The prescription composition of Gandouling tablet is: 182g of Coptis chinensis, 91g of Salvia miltiorrhiza, 91g of Radix Glycyrrhizae, 82g of Rhubarb, 82g of Curcuma Radix, and 82g of Turmeric;

The production method of Gandouling tablets is as follows:

①Mix rhubarb, curcuma curcuma and turmeric in a baking tray, put them in an oven to dry (≤75°C), pulverize, use a 100-mesh sieve, and fine powder for later use;

②The crude powder was combined with Coptis chinensis and Salvia miltiorrhiza and extracted twice with 75% ethanol. The first time, 10 times the amount of ethanol was added to reflux for 2 hours, and the second time, 8 times the amount of ethanol was added to reflux for 1.5 hours. The alcohol extracts were combined, filtered, and recovered. Ethanol; add 8 times the amount of water to the dregs and decoct for 1 hour, filter, stand for precipitation for 48 hours, absorb the supernatant, combine with the alcohol extract and concentrate under reduced pressure (pressure: -0.02～-0.04MPa) to a relative density of 1.10 (60℃) clear cream;

3. Add water to decoct and extract for three times, the first time add 10 times the amount of water to decoct for 1.5 hours, the second and third times add 8 times the amount of water to decoct for 1 hour, combine the decoctions, filter, and let stand for 48 hours. Aspirate the supernatant and concentrate under reduced pressure (pressure: -0.02～-0.04MPa) to a clear paste with a relative density of 1.10 (60°C);

4. Merge above-mentioned two kinds of clear pastes, add above-mentioned medicine fine powder, starch 90g, simple syrup 20mL granulation, dry (<75 ℃), granulate (10 meshes on the upper sieve, 60 meshes on the lower sieve), add 1.5 mg of magnesium stearate g, mix well, and press into 1000 tablets, namely Gandouling tablets;

(2) Preparation of the test solution:

Take 10 pieces of Gandouling tablets, grind them, take 0.3 g of fine powder, put them in a 10ml volumetric flask, add methanol to the volume, ultrasonicate for 20 minutes, centrifuge at 12000r/min to get the filtrate and filter it with a 0.22μm filter membrane to get the test product solution;

(3) Preparation of reference solution:

Precisely weigh a certain amount of reference substances, epiberberine, coptisine, palmatine, berberine hydrochloride, aloe-emodin, rhein, tanshinone I, and tanshinone IIA, and add methanol to prepare 0.239 mg/ml of epiberberine, Coptisine 0.211mg/ml, Palmatine 0.277mg/ml, Berberine Hydrochloride 0.216mg/ml, Aloe-emodin 0.243mg/ml, Rhein 0.252mg/ml, Tanshinone I 0.224mg/ml, Tanshinone IIA 0.261mg /ml;

(4) Chromatographic conditions:

The mobile phase is acetonitrile (A)-potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) (B), gradient elution is used; elution conditions are: 0-2min, 22% A; 2-9min, 22% A; 9-12min, 25%A; 12-14min, 55%A; 14-22min, 65%A; 22-23min, 100%A; 23-24min, 15%A; 24-25min, 15%A; Stop after 25min;

The detector selects DAD, the detection wavelength is 250nm; the column temperature is 30.0°C; the flow rate is 0.2ml/min; the injection volume is 1ul; the chromatographic column selects ACQUITY UPLC BEH C ₁₈ ;

(5) Establishment of fingerprints:

Prepare 10 batches of Gandouling tablet samples for test solution, inject samples according to the above chromatographic conditions, obtain 10 batches of samples HPLC chromatograms, import them into similarity evaluation software, calculate the similarity of 10 batches of samples, and determine standard fingerprints;

The prepared reference substance solution is injected and analyzed under the same chromatographic conditions, the obtained reference substance spectrum is compared with the standard fingerprint spectrum, and the peak number of the chromatographic peak corresponding to the reference substance in the standard fingerprint spectrum is identified;

(6) Quality evaluation:

After establishing the fingerprint, carry out methodological verification to examine the precision, stability and repeatability, and then use the same chromatographic conditions to detect the samples to be tested, and the test results are compared and evaluated by the similarity evaluation software. requirements, so as to complete the quality evaluation of the Gandouling tablet samples to be tested.

As another object of the present invention, the present invention proposes a composition and production method of Gandouling tablet, wherein the formulation of Gandouling tablet is composed of: 182g of Coptis chinensis, 91g of Salvia miltiorrhiza, 91g of Radix Glycyrrhizae, 82g of Rhubarb , Curcuma 82g, Turmeric 82g; The production method of described Gandouling tablet is:

①Mix rhubarb, curcuma curcuma and turmeric in a baking tray, put them in an oven to dry (≤75°C), pulverize, use a 100-mesh sieve, and fine powder for later use;

②The crude powder was combined with Coptis chinensis and Salvia miltiorrhiza and extracted twice with 75% ethanol. The first time, 10 times the amount of ethanol was added to reflux for 2 hours, and the second time, 8 times the amount of ethanol was added to reflux for 1.5 hours. The alcohol extracts were combined, filtered, and recovered. Ethanol; add 8 times the amount of water to the dregs and decoct for 1 hour, filter, stand for precipitation for 48 hours, absorb the supernatant, combine with the alcohol extract and concentrate under reduced pressure (pressure: -0.02～-0.04MPa) to a relative density of 1.10 (60℃) clear cream;

3. Add water to decoct and extract for three times, the first time add 10 times the amount of water to decoct for 1.5 hours, the second and third times add 8 times the amount of water to decoct for 1 hour, combine the decoctions, filter, and let stand for 48 hours. Aspirate the supernatant and concentrate under reduced pressure (pressure: -0.02～-0.04MPa) to a clear paste with a relative density of 1.10 (60°C);

4. Merge above-mentioned two kinds of clear pastes, add above-mentioned medicine fine powder, starch 90g, simple syrup 20mL granulation, dry (<75 ℃), granulate (10 meshes on the upper sieve, 60 meshes on the lower sieve), add 1.5 mg of magnesium stearate g, mix well, and press into 1000 pieces to obtain Gandouling tablets; properties of Gandouling tablets: khaki-yellow tablets; slightly fragrant and bitter in taste.

The beneficial effects of the present invention are shown in:

(1) Fingerprint technology is a comprehensive identification method with strong specificity, high stability and good repeatability, which can comprehensively evaluate the authenticity and quality of medicinal materials. Therefore, the present invention uses ultra-high performance liquid chromatography to construct the fingerprint of Gandouling tablets, and analyzes multiple batches of Gandouling tablets to determine its standard fingerprint, and establishes a stable and reliable quality of Gandouling tablets. Control Method.

(2) The present invention selects reasonable chromatographic conditions and chromatographic columns, has good separation effect, high reproducibility and excellent identification effect. The method verification showed that the instrument had good precision, good sample stability and good method repeatability, which met the technical requirements of fingerprints, and provided a better evaluation method for the quality control of Gandouling tablets.

(3) The composition and production method of Gandouling Tablets proposed by the present invention have reasonable compatibility, can clear away heat and detoxify, dissolve blood stasis and soften firmness, invigorate gallbladder and expel copper, and have good curative effect on hepatolenticular degeneration .

Description of drawings

Figure 1 is the standard fingerprint of Gandouling tablets.

Figure 2 is an overlay of 10 batches of Gandouling tablets HPLC fingerprints.

Fig. 3 is the contrast spectrum of 8 single reference substances and fingerprints of Gandouling tablets.

Detailed ways

Example 1

The invention provides a method for evaluating the quality of Gandouling tablets, and the specific steps are as follows:

(1) Production of Gandouling tablets:

The prescription composition of Gandouling tablet is: 182g of Coptis chinensis, 91g of Salvia miltiorrhiza, 91g of Radix Glycyrrhizae, 82g of Rhubarb, 82g of Curcuma curcuma, 82g of Turmeric; the production method of Gandouling tablet is:

①Mixed rhubarb, curcuma curcuma and turmeric are spread on a baking tray, dried in an oven (≤75°C), pulverized, sieved with a 100-mesh sieve, and finely powdered for use.

②The crude powder was combined with Coptis chinensis and Salvia miltiorrhiza and extracted twice with 75% ethanol. The first time, 10 times the amount of ethanol was added to reflux for 2 hours, and the second time, 8 times the amount of ethanol was added to reflux for 1.5 hours. The alcohol extracts were combined, filtered, and recovered. Ethanol. Add 8 times the amount of water to the dregs and decoct for 1 hour, filter, stand for precipitation for 48 hours, absorb the supernatant, combine with the alcohol extract and concentrate under reduced pressure (pressure: -0.02～-0.04MPa) to a relative density of 1.10 ( 60°C) clear paste.

3. Add water to decoct and extract for three times, the first time add 10 times the amount of water to decoct for 1.5 hours, the second and third times add 8 times the amount of water to decoct for 1 hour, combine the decoctions, filter, and let stand for 48 hours. The supernatant was aspirated and concentrated under reduced pressure (pressure: -0.02 to -0.04MPa) to a clear paste with a relative density of 1.10 (60°C).

4. Merge above-mentioned two kinds of clear pastes, add above-mentioned medicine fine powder, starch 90g, simple syrup 20mL granulation, dry (<75 ℃), granulate (10 meshes on the upper sieve, 60 meshes on the lower sieve), add 1.5 mg of magnesium stearate g, mix well, and press into 1000 tablets, namely Gandouling tablets;

(2) Preparation of the test solution:

According to the above method, 10 batches of Gandouling tablets were prepared, and the batch numbers are shown in Table 1.

Table 1. Batch numbers of 10 batches of Gandouling tablets

Take 10 Gandouling tablets, grind them, accurately weigh about 0.3 g into a volumetric flask, add methanol to make up to 10 ml, extract by ultrasonic for 20 min, centrifuge at 12000 r/min, and filter the filtrate with a 0.22 μm filter membrane to obtain Test solution.

(3) Preparation of reference solution:

Precisely weigh a certain amount of reference substances, epiberberine, coptisine, palmatine, berberine hydrochloride, aloe-emodin, rhein, tanshinone I, and tanshinone IIA, and add methanol to prepare 0.239 mg/ml of epiberberine, Coptisine 0.211mg/ml, Palmatine 0.277mg/ml, Berberine Hydrochloride 0.216mg/ml, Aloe-emodin 0.243mg/ml, Rhein 0.252mg/ml, Tanshinone I 0.224mg/ml, Tanshinone IIA 0.261mg /ml.

(4) Chromatographic conditions:

The mobile phase is acetonitrile (A)-potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid) (B), gradient elution is used; elution conditions are: 0-2min, 22% A; 2-9min, 22% A; 9-12min, 25%A; 12-14min, 55%A; 14-22min, 65%A; 22-23min, 100%A; 23-24min, 15%A; 24-25min, 15%A; Stop after 25 minutes.

The detector selected DAD, the detection wavelength was 250 nm; the column temperature was 30.0 °C; the flow rate was 0.2 ml/min; the injection volume was 1 μL; the chromatographic column was ACQUITY UPLC BEH C ₁₈ .

(5) Establishment of fingerprints:

10 batches of Gandouling tablet samples were injected into the test solution according to the above chromatographic conditions, to obtain 10 batches of sample HPLC chromatograms, and 19 common peaks were determined to form a common pattern. Among them, peak No. 11 is the reference peak, and its relative retention time and relative peak area are 1. The relative retention time and relative peak area of other peaks are calculated by comparing with them. The results are shown in Table 2. Thereby, the standard fingerprint of Gandouling tablets was obtained, as shown in Figure 1.

Table 2. Relative peak area and relative retention time of 19 peaks in 10 batches of Gandouling tablets

Note: Peak 11 is the reference peak, (RRT: relative retention time, RPA: relative peak area)

Import the analysis results into the similarity evaluation software (Chinese medicine chromatographic fingerprint similarity evaluation system (2004A version), the same below), calculate the similarity by the mean method, and obtain the similarity data of 10 batches of samples, the results are shown in Table 3, The overlay of HPLC chromatograms of each sample is shown in Figure 2.

Table 3 Similarity data results of 10 batches of samples

The HPLC chromatograms and fingerprints of 8 reference substances—epiberberine, coptisine, palmatine, berberine hydrochloride, aloe-emodin, rhein, tanshinone I, and tanshinone IIA were combined and compared with the fingerprints to identify quercetin. In the 19 common peaks, the corresponding peak numbers are peaks 8, 9, 11, 12, 13, 14, 16 and 19, respectively. The results are shown in Figure 3.

(6) Methodological investigation

①Inspection of precision

The S5 sample was prepared according to the preparation method of the test solution, and the determined optimal chromatographic conditions were repeated 6 times. The RSD values of the relative retention time and relative peak area of each common peak were measured to be 0.117-1.084% and 0.038-1.990. % (as shown in Table 4), indicating that the precision of the instrument is good and meets the technical requirements of fingerprints.

Table 4. Precision experimental results

Note: Peak 11 is the reference peak, (RRT: relative retention time, RPA: relative peak area)

②Stability test

Sample No. S5 was prepared according to the preparation method of the test solution, and the determined optimal chromatographic conditions were injected and analyzed at 0, 4, 8, 12, 16, and 24 h, respectively, and the relative retention time and relative peak area of each common peak were measured. The RSD value of the test solution is between 0.195-2.366% and 0.329-2.752% (as shown in Table 5), indicating that the test solution has good stability within 24h.

Table 5. Results of stability experiments

Note: Peak 11 is the reference peak, (RRT: relative retention time, RPA: relative peak area)

③ Repeated test

According to the determined preparation method of the test solution, 6 samples of No. S5 were prepared in parallel, and were respectively injected and analyzed under the optimal chromatographic conditions determined above. The RSD values of the relative retention time and relative peak area of each common peak were measured between 0.135 and between 1.490% and 0.433-2.425% (as shown in Table 6), indicating that the method has good repeatability.

Table 6 Repeated experimental results

Note: Peak 11 is the reference peak, (RRT: relative retention time, RPA: relative peak area)

(7) Quality evaluation of Gandouling tablets

Prepare the sample to be tested according to the above-mentioned preparation method of the test solution, and then use the same chromatographic conditions to detect the sample to be tested. The test results are compared and evaluated by the similarity evaluation software. Quality evaluation of Gandouling tablet samples.

The above content is only an example and description of the concept of the present invention. Those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them in a similar manner, as long as they do not deviate from the concept of the invention. Or beyond the scope defined by the claims, all belong to the protection scope of the present invention.

Claims (2)

1. A quality evaluation method of Gaodanling tablets is characterized by comprising the following steps:

(1) preparation of Ganduoling tablet

The prescription of the Gaodaoling tablet comprises the following components: 182g of coptis root, 91g of salvia miltiorrhiza, 91g of suberect spatholobus stem, 82g of rhubarb, 82g of curcuma zedoary and 82g of turmeric;

the production method of the Ganduoling tablet comprises the following steps:

mixing and spreading rhubarb, zedoary and turmeric in a baking pan, putting the mixture into a baking oven for drying (less than or equal to 75 ℃), crushing the mixture, and sieving the crushed mixture with a 100-mesh sieve to obtain fine powder for later use;

② coarse powder, coptis root and salvia miltiorrhiza are mixed and extracted twice by 75 percent ethanol, 10 times of ethanol is added for 2 hours of reflux for the first time, 8 times of ethanol is added for 1.5 hours of reflux for the second time, and the ethanol extract is mixed, filtered and recovered; decocting the dregs of a decoction for 1 hour by adding 8 times of water, filtering, standing and precipitating for 48 hours, sucking supernatant, merging the supernatant with an alcohol extract, and concentrating under reduced pressure (the pressure is-0.02 to-0.04 MPa) to obtain clear paste with the relative density of 1.10(60 ℃);

thirdly, adding water into the suberect spatholobus stem for decocting and extracting for three times, adding 10 times of water for decocting for 1.5 hours for the first time, adding 8 times of water for decocting for 1 hour for the second time and the third time, merging decoction, filtering, standing and precipitating for 48 hours, sucking supernatant fluid, and concentrating under reduced pressure (the pressure is-0.02 to-0.04 MPa) to obtain clear paste with the relative density of 1.10(60 ℃);

mixing the two kinds of clear paste, adding the medicine fine powder, 90g of starch and 20mL of simple syrup, granulating, drying (less than 75 ℃), granulating (10 meshes on an upper sieve and 60 meshes on a lower sieve), adding 1.5g of magnesium stearate, mixing uniformly, and pressing into 1000 tablets to obtain the Gaodaoling tablets;

(2) preparing a test solution:

taking 10 Gaodaoling tablets, grinding, taking 0.3g of fine powder, placing in a 10ml volumetric flask, adding methanol to a constant volume, carrying out ultrasonic treatment for 20 minutes, centrifuging at a rotating speed of 12000r/min, taking filtrate, and filtering with a 0.22 mu m filter membrane to obtain a test solution;

(3) preparation of a control solution:

precisely weighing reference substances including epiberberine, coptisine, palmatine, berberine hydrochloride, aloe-emodin, rhein, tanshinone I and tanshinone IIA, adding methanol to prepare 0.239mg/ml of epiberberine, 0.211mg/ml of coptisine, 0.277mg/ml of palmatine, 0.216mg/ml of berberine hydrochloride, 0.243mg/ml of aloe-emodin, 0.252mg/ml of rhein, 0.224mg/ml of tanshinone I and 0.261mg/ml of tanshinone II;

(4) chromatographic conditions are as follows:

acetonitrile (A) -potassium dihydrogen phosphate solution (pH value is adjusted to 3.0 by phosphoric acid) (B) is selected as a mobile phase, and gradient elution is adopted; the elution conditions were: 0-2min, 22% A; 2-9min, 22% A; 9-12min, 25% A; 12-14min, 55% A; 14-22min, 65% A; 22-23min, 100% A; 23-24min, 15% A; 24-25min, 15% A; stopping after 25 min;

DAD is selected as a detector, and the detection wavelength is 250 nm; the column temperature was 30.0 ℃; the flow rate is 0.2 ml/min; the sample amount is 1 ul; selection of chromatographic column with ACQUITY UPLC BEH C ₁₈ ；

(5) Establishing a fingerprint spectrum:

preparing 10 batches of liver bean tablet sample test solution, respectively injecting samples according to the chromatographic conditions to obtain 10 batches of sample HPLC (high performance liquid chromatography) spectrums, introducing the samples into similarity evaluation software, calculating the similarity of 10 batches of samples, and determining standard fingerprint spectrums;

injecting the prepared reference substance solution under the same chromatographic condition for analysis, comparing the obtained reference substance spectrum with the standard fingerprint spectrum, and identifying the chromatographic peak number corresponding to the reference substance in the standard fingerprint spectrum;

(6) and (3) quality evaluation:

after the fingerprint is established, methodology verification is carried out to investigate precision, stability and repeatability, the sample to be detected is detected under the same chromatographic conditions, the detection result is compared and evaluated through similarity evaluation software, and if the similarity is more than 0.90, the quality requirement of the liver bean tablet to be detected is met, so that the quality evaluation of the liver bean tablet sample to be detected is completed.

2. The production method of the liver bean tablet is characterized in that the prescription of the liver bean tablet comprises the following components: 182g of coptis root, 91g of salvia miltiorrhiza, 91g of suberect spatholobus stem, 82g of rhubarb, 82g of curcuma zedoary and 82g of turmeric; the production method of the liver bean tablet comprises the following steps:

mixing and spreading rhubarb, zedoary and turmeric in a baking pan, putting the mixture into the baking pan for drying (less than or equal to 75 ℃), crushing the mixture, and sieving the crushed mixture with a 100-mesh sieve to obtain fine powder for later use;

mixing coarse powder with coptis root and salvia miltiorrhiza, performing reflux extraction twice by using 75% ethanol, adding 10 times of ethanol for refluxing for 2 hours for the first time, adding 8 times of ethanol for refluxing for 1.5 hours for the second time, mixing ethanol extract, filtering, and recovering ethanol; decocting the dregs of a decoction for 1 hour by adding 8 times of water, filtering, standing and precipitating for 48 hours, sucking supernatant, merging the supernatant with an alcohol extract, and concentrating under reduced pressure (the pressure is-0.02 to-0.04 MPa) to obtain clear paste with the relative density of 1.10(60 ℃);

thirdly, adding water into the suberect spatholobus stem for decocting and extracting for three times, adding 10 times of water for decocting for 1.5 hours for the first time, adding 8 times of water for decocting for 1 hour for the second time and the third time, merging decoction, filtering, standing and precipitating for 48 hours, sucking supernatant fluid, and concentrating under reduced pressure (the pressure is-0.02 to-0.04 MPa) to obtain clear paste with the relative density of 1.10(60 ℃);

mixing the two clear pastes, adding the fine powder of the medicine, 90g of starch and 20mL of simple syrup, granulating, drying (below 75 ℃), granulating (sieving with an upper sieve of 10 meshes and sieving with a lower sieve of 60 meshes), adding 1.5g of magnesium stearate, mixing uniformly, and pressing into 1000 tablets to obtain Gaodaoling tablets; the characteristics of the GaoDouling tablet are as follows: a yellowish brown flake; slightly fragrant smell, bitter and astringent taste.

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