CN113281437A

CN113281437A - Method for detecting dissolution rates of terpene lactones and flavonoid components in ginkgo biloba extract dripping pills

Info

Publication number: CN113281437A
Application number: CN202110581401.0A
Authority: CN
Inventors: 李海鹏; 王晓玲; 阎红; 范玲玲; 姜敏
Original assignee: Zhejiang Jiuxu Pharmaceutical Co ltd
Current assignee: Zhejiang Jiuxu Pharmaceutical Co ltd
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2021-08-20
Anticipated expiration: 2041-05-27
Also published as: CN113281437B

Abstract

The invention provides a dissolution rate detection method of terpene lactones and flavonoid ingredients in a ginkgo ketoester dripping pill, which comprises the following steps of 1) preparing beta-glucosidase liquid; 2) preparing a dissolving liquid; 3) preparing a test solution; 4) preparing a self-control solution; 5) preparing a reference substance solution; 6) detecting; 7) and (4) calculating the dissolution rate.

Description

Method for detecting dissolution rates of terpene lactones and flavonoid components in ginkgo biloba extract dripping pills

Technical Field

The invention belongs to the technical field of analysis and detection of traditional Chinese medicine components, and relates to a method for detecting the dissolution rates of terpene lactone components and flavonoid components in a ginkgo biloba extract dripping pill.

Background

In vitro dissolution rate research is one of the important evaluation indexes of the traditional Chinese medicine preparation, and not only can simulate the disintegration and dissolution conditions of the oral solid preparation in the gastrointestinal tract, but also can control the quality of the medicinal preparation. The ginkgo biloba extract dripping pill is a main product produced by Jiuzhouxu pharmaceutical company Limited in Zhejiang, and is widely used for treating cardiovascular diseases such as dizziness, coronary heart disease, angina and the like caused by blood stasis type thoracic obstruction and blood stasis type mild cerebral arteriosclerosis.

The terpene lactones in the ginkgo biloba extract dripping pill are the specific components of ginkgo biloba, and are not found in other plants at present. The ginkgolide component mainly comprises ginkgolide A, B, C and bilobalide, is an important active component in ginkgolide dripping pills, and has effects of antagonizing platelet activation and protecting nervous system.

The content of the ginkgolide component in the ginkgolide dripping pill is low, no conjugated group exists in the chemical structure of the ginkgolide component, the ultraviolet absorption is poor, the detection sensitivity is low, and the ginkgolide component is easily interfered by other components. At present, the dissolution rate of terpene lactone components in the ginkgo biloba extract dripping pills is not detected in national standards, and the in vitro dissolution condition of the ginkgo biloba extract dripping pills cannot be well reflected.

The flavone compounds in the ginkgo ketoester dripping pills are mainly glycosides formed by kaempferol, quercetin, isorhamnetin and various glycosyl groups, wherein the glycosyl groups can be monosaccharide, disaccharide and trisaccharide, and most of the glycosyl groups are glucose and rhamnose. The research shows that the ginkgetin has the pharmacological actions of resisting inflammation, resisting oxidation, dilating blood vessels, inhibiting platelet aggregation and the like. In the national standard, kaempferide, quercetin and isorhamnetin aglycone are obtained by an acid water hydrolysis method, the content of the three aglycones is measured, and the three aglycones are multiplied by a coefficient (2.51) to obtain the content of the total flavonol glycoside.

Liuhui Min and Hui Zhen ren, using UPLC-MS/MS method to determine the dissolution of terpene lactone in 4 kinds of ginkgo leaf preparations. CN109738544A adopts UPLC-MS/MS method to provide a method for detecting the dissolution of terpene lactones and flavonoid glycosides in ginkgo biloba extract tablet, the method does not need complex sample pretreatment, the MS detector has the characteristics of strong specificity and high detection sensitivity, but the equipment is expensive, the requirement for operators is high, and the method is not widely applied at present.

Disclosure of Invention

The high performance liquid chromatography equipped with the evaporative light scattering detector has wide application, and the evaporative light detector responds to both ginkgo flavonoid components and ginkgo terpene lactone components, but has the following technical problems: 1. the ginkgo flavone components in the ginkgo ketone ester dripping pills mostly exist in the form of flavonoid glycoside, and interfere the detection of terpene lactone components; 2. other components in the bilobalide dripping pill interfere with terpene lactone component detection; 3. the sample pretreatment process is too violent, or the steps are too many, so that the components to be detected are easily damaged or lost, and the detection method is low in accuracy.

In view of the above disadvantages of the prior art, the present invention aims to provide a method for simultaneously determining the dissolution rates of terpene lactone components and flavonoid components in ginkgo biloba extract dripping pills. It is characterized in that: 1. the solid phase extraction technology replaces the traditional liquid-liquid extraction, so that the operation is convenient, and the sample purification is realized, so that other components in the ginkgo biloba extract dripping pill do not interfere with the detection of terpene lactone components; 2. by adopting an enzyme hydrolysis method, the terpene lactone component of ginkgo is not damaged while the flavonoid glycoside component is hydrolyzed, so that the problem that the flavonoid glycoside component interferes with terpene lactone detection is solved; 3. the method can simultaneously determine 4 main components (ginkgolide A, B, C and bilobalide) in the ginkgolide and 2 main aglycones (kaempferide and quercetin) in the ginkgetin, and the detection method has high accuracy; 4. the invention adopts an Evaporative Light Scattering Detector (ELSD), and the device has wide application range and is more popular in use.

The method specifically comprises the following steps:

1) preparation of beta-glucosidase solution A proper amount of beta-glucosidase is weighed and diluted by a dissolution medium to prepare the beta-glucosidase solution with a certain concentration.

2) Preparation of dissolution liquid taking 4 pills (taking dosage per time), according to a dissolution determination method, taking 100mL of buffer salt with pH 4.0-8.0 as a dissolution medium, rotating at 100 revolutions per minute, taking 7mL of dissolution liquid after 30 minutes, and filtering.

3) Preparation of test solution

Measuring 0.5-1.0 mL beta-glucosidase solution, transferring to a Chem Elut s (5mL) small column, standing for more than 15 minutes, precisely absorbing 4mL of dissolved solution, transferring to the Chem Elut s (5mL) small column, standing for a period of time under a certain temperature condition, eluting with 15mL of ethyl acetate, evaporating the eluent by using a nitrogen blower, dissolving the residue in methanol to a 1mL measuring flask, diluting to a scale by using methanol, shaking up, and filtering to obtain the sample solution.

4) Preparing self-contrast solution by taking 10 pills of the ginkgo ketoester dripping pill, grinding, precisely weighing 1 pill by weight, placing in a 25mL measuring flask, adding 10mL of 70% ethanol for ultrasonic dissolution, diluting with dissolution medium to scale, taking 7mL, and filtering. Preparing the product by the same method as the step 3).

5) Preparing reference solution, precisely weighing appropriate amount of quercetin and kaempferide, and adding methanol to obtain mixed solution containing 1500 μ g and 1500 μ g per 1ml, and making into stock solution of ginkgetin. Precisely weighing appropriate amount of bilobalide, bilobalide A, bilobalide B and bilobalide C reference substances, respectively, adding methanol to obtain mixed solution containing 2000 μ g, 1000 μ g and 1000 μ g per 1ml, and using as bilobalide reference substance stock solution. Precisely measuring 5ml of ginkgetin reference substance stock solution and 1ml of ginkgolide reference substance stock solution into the same 100ml measuring flask, and diluting with methanol to scale as reference substance solution.

6) Detection of

The chromatographic conditions were gradient elution with octadecylsilane bonded silica gel as a filler (column length 250mm, inner diameter 4.6mm, particle size 5 μm), tetrahydrofuran-acetonitrile-methanol (13:55:32) as mobile phase A, and 0.4% acetic acid solution as mobile phase B, as specified in the following table; the flow rate is 1.0 mL/min; column temperature 30 ℃, detection with evaporative light scattering detector, drift tube temperature: 105 ℃, nitrogen flow rate: 2.5 mL/min.

The determination method comprises precisely sucking 10 μ l and 20 μ l of reference solution, respectively, injecting 15 μ l of test solution and 15 μ l of self-reference solution into liquid chromatograph, determining, and respectively calculating 4 terpene lactone components and 2 flavonoid components by using external standard two-point logarithmic equation.

7) And (3) calculating the dissolution rate: respectively calculating the dissolution rates of the terpene lactones and the flavonoid components in the ginkgo biloba extract dripping pill according to a formula I by using the contents of the terpene lactones and the flavonoid components in the test solution and the self control solution, wherein the formula I is as follows: dissolution rate [ (% Ai × V1)/(Bi × V2 × m1/m2) × 100%; wherein, Ai: the content of each component in the test solution is mu g/mL; bi: the content of each component in the self control solution is mu g/mL; v1: volume of dissolution medium, mL; v2: volume of self-control solution, mL; m 1: adding ginkgetin ester dripping pill in mg into the control solution; m 2: the average weight of each pill of the ginkgo biloba extract dripping pill is mg.

Drawings

FIG. 1 chromatogram of control solution

FIG. 2 chromatogram of test solution

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention.

The reagents and equipment used in the following examples are as follows:

1. the reagent bilobalide B (China institute for food and drug testing); bilobalide a (chinese institute for food and drug testing); bilobalide C (chinese institute for food and drug testing); bilobalide (national institute for food and drug testing); quercetin (chinese institute for food and drug testing); kaempferide (chinese institute for food and drug testing); ginkgo Biloba ester dripping pill (batch No. 20200603, Zhejiang Uyao Jiuzhi pharmaceutical Co., Ltd.). Beta-glucosidase (Sigma Aldrich trade Co., Ltd.)

2. Instrument for measuring the position of a moving object

Agilent 1260 high performance liquid chromatograph (Agilent corporation); a column (Shim-pack VP-ODS, 4.6 mm. times.250 mm, 5 μm, Shimadzu (Shanghai) laboratory instruments Co., Ltd.); dissolution tester model Agilenggt 708-DS (Agilent Corp.); DELTA 320PH meter (Mettler-Torledo Corp.); MS105DU analytical balance (mettler-toledo corporation); chem Elut s (5ml) cartridge (Agilent Corp.).

Example 1

1) Preparation of beta-glucosidase solution A proper amount of beta-glucosidase is weighed and diluted with acetate with pH4.5 to prepare beta-glucosidase solution with 10 mug/ml.

2) Preparation of the eluate 4 pills (dosage per time) are prepared by taking 100mL acetate buffer salt with pH4.5 as dissolution medium at 100 rpm according to dissolution determination method, taking 7mL of eluate after 30 min, and filtering.

3) Preparation of test solution

Measuring 0.5-1.0 mL beta-glucosidase solution, transferring to a Chem Elut s (5mL) column, standing for more than 15 minutes, precisely absorbing 4mL of dissolution liquid, transferring to the Chem Elut s (5mL) column, transferring to a 37 ℃ incubator, taking out after 16 hours, then eluting with 15mL ethyl acetate, evaporating the eluent by using a nitrogen blower, dissolving the residue in methanol to be placed in a 1mL measuring flask, diluting to scale by using methanol, shaking up, and filtering to obtain the sample solution.

6) Detection of

The determination method comprises precisely sucking 10 μ l and 20 μ l of reference solution, respectively, injecting 15 μ l of test solution and 15 μ l of self-reference solution into liquid chromatograph, determining, and respectively calculating the contents of 4 terpene lactones and 2 flavonoids by external standard two-point logarithmic equation

Example 2

1) Preparation of beta-glucosidase solution A proper amount of beta-glucosidase is weighed and diluted with acetate with pH6.8 to prepare 10 mug/ml beta-glucosidase solution.

2) Preparation of dissolution liquid taking 4 pills (dosage per time), according to dissolution determination method, 100mL phosphate buffer salt with pH6.8 as dissolution medium, rotating speed of 100 r/min, taking 7mL dissolution liquid after 30 min, and filtering.

3) Preparation of test solution

6) Detection of

Example 3

6 parts of the sample solution are prepared in parallel according to the method for preparing the sample solution in the step 3) of the example 1, peak areas are measured according to the chromatographic conditions in the step 6 of the example 1, and the repeatability of each component is calculated, so that the repeatability RSD values of each component are respectively 1.5% of ginkgolide B, 1.5% of ginkgolide A, 2.3% of ginkgolide C, 1.1% of bilobalide, 0.8% of quercetin and 0.9% of kaempferide. The repeatability RSD value of each component is less than 5 percent, which shows that the experiment repeatability is better.

Example 4

6 parts of the sample solution are parallelly operated according to the preparation method of the sample solution in the step 3) of the example 2, peak areas are measured according to the chromatographic conditions in the step 6 of the example 2, and the repeatability of each component is calculated, so that the repeatability RSD values of each component are respectively 1.6% of ginkgolide B, 1.3% of ginkgolide A, 2.1% of ginkgolide C, 1.2% of bilobalide, 0.6% of quercetin and 1.0% of kaempferide. The repeatability RSD value of each component is less than 5 percent, which shows that the experiment repeatability is better.

Example 5

Taking 6 parts of the eluate obtained in the step 2) of the example 1, respectively and precisely adding the reference substance solution, uniformly mixing, preparing the sample solution according to the step 3) of the example 1, and carrying out sample injection according to the chromatographic conditions in the step 6) of the example 1 to determine the sample recovery rate of each component. As a result, the sample recovery rates of the respective components were 96.1. + -. 2.5% for ginkgolide B, 95.4. + -. 1.3% for ginkgolide A, 97.2. + -. 2.5% for ginkgolide C, 91.8. + -. 2.7% for bilobalide, 92.5. + -. 2.1% for quercetin, and 92.8. + -. 3.2% for kaempferide. The RSD values of the recovery rates of the components are respectively 2.0 percent of ginkgolide B, 0.9 percent of ginkgolide A, 2.0 percent of ginkgolide C, 2.3 percent of bilobalide, 2.3 percent of quercetin and 2.5 percent of kaempferide. The recovery rate of each component is between 90% and 105%, and the RSD value is less than 5%, which shows that the experimental method has good accuracy.

Example 6

Taking 6 parts of the eluate obtained in the step 2) of the example 2, respectively and precisely adding the reference substance solution, uniformly mixing, preparing the sample solution according to the step 3) of the example 2, and carrying out sample injection according to the chromatographic conditions in the step 6) of the example 2 to determine the sample recovery rate of each component. As a result, the sample recovery rates of the respective components were 95.2. + -. 2.3% for ginkgolide B, 95.2. + -. 2.3% for ginkgolide A, 96.2. + -. 2.7% for ginkgolide C, 93.8. + -. 3.7% for bilobalide, 93.5. + -. 2.5% for quercetin, and 93.8. + -. 2.2% for kaempferide. The RSD values of the recovery rates of the components are 1.9 percent of ginkgolide B, 2.0 percent of ginkgolide A, 2.3 percent of ginkgolide C, 3.1 percent of bilobalide, 2.2 percent of quercetin and 2.0 percent of kaempferide respectively. The recovery rate of each component is between 90% and 105%, and the RSD value is less than 5%, which shows that the experimental method has good accuracy.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. A method for detecting the dissolution rate of terpene lactones and flavonoids in a ginkgo biloba extract dripping pill comprises the following steps:

1) preparing beta-glucosidase liquid, weighing a proper amount of beta-glucosidase, and diluting the beta-glucosidase liquid with a dissolution medium to prepare beta-glucosidase liquid with a certain concentration;

2) preparation of dissolution liquid taking 4 pills of ginkgo biloba extract dripping pill (dosage per time), determining by dissolution method, taking 100mL buffer salt as dissolution medium, performing slurry method at rotation speed of 100 r/min, taking 7mL dissolution liquid after 30 min, and filtering;

3) taking 0.5-1.0 mL of beta-glucosidase solution for preparing a test solution, transferring the beta-glucosidase solution to a Chem Elut s (5mL) small column, standing for more than 15 minutes, precisely absorbing 4mL of dissolution liquid, transferring the dissolution liquid to the Chem Elut s (5mL) small column, standing for a period of time under a certain temperature condition, eluting by 15mL of ethyl acetate, evaporating the eluent by a nitrogen blower, dissolving residues by adding methanol into a 1mL measuring flask, diluting the solution to a scale by using methanol, shaking uniformly, and filtering to obtain the test solution;

4) preparing self-contrast solution, collecting 10 pills of semen Ginkgo ketone ester dripping pill, grinding, precisely weighing 1 pill, placing into 25mL measuring flask, adding 70% ethanol 10mL, ultrasonic dissolving, diluting with dissolution medium to scale, collecting 7mL, and filtering; preparing by the same method as the step 3);

5) preparing reference solution by precisely weighing appropriate amount of quercetin and kaempferide, and adding methanol to obtain stock solution of ginkgetin; precisely weighing appropriate amount of reference substances including bilobalide, bilobalide A, bilobalide B and bilobalide C, and adding methanol to obtain reference substance stock solution of bilobalide; respectively taking the reference substance stock solution and the ginkgolide reference substance stock solution into the same measuring flask, and diluting with methanol to scale to obtain reference substance solution;

6) respectively measuring a reference solution, a self-reference solution and a test solution by using an HPLC-ELSD method, determining 4 terpene lactone components and 2 flavonoid components in the self-reference solution and the test solution according to retention time, and calculating the contents of the 4 terpene lactone components and the 2 flavonoid components by using an external standard two-point method logarithmic equation;

7) calculating the dissolution rate of the terpene lactones and the flavonoid components in the test solution and the self-contrast solution, and respectively calculating the dissolution rate of the terpene lactones and the flavonoid components in the ginkgo biloba extract dripping pill according to a formula I: dissolution rate [ (% Ai × V1)/(Bi × V2 × m1/m2) × 100%; wherein, Ai: the content of each component in the test solution is mu g/mL; bi: the content of each component in the self control solution is mu g/mL; v1: volume of dissolution medium, mL; v2: volume of self-control solution, mL; m 1: adding ginkgetin ester dripping pill in mg into the control solution; m 2: the average weight of each pill of the ginkgo biloba extract dripping pill is mg.

2. The method of claim 1, wherein the 4 terpene lactones are bilobalide A, bilobalide B, bilobalide C, bilobalide, and 2 flavonoid compounds are quercetin and kaempferide.

3. The method for detecting the dissolution rate of terpene lactones and flavonoids in ginkgo biloba extract dripping pill according to claim 1, wherein the preparation concentration of the β -glucosidase solution in step 1) is 1-20 μ g/mL, preferably 8-12 μ g/mL, and more preferably 10 μ g/mL.

4. The method for detecting the dissolution rate of terpene lactones and flavonoids in ginkgo biloba extract dripping pill according to claim 1, wherein in the step 2), the dissolution medium is a buffer salt with pH of 4.0-8.0, preferably an acetate buffer solution with pH of 4.5 or a phosphate buffer solution with pH of 6.8.

5. The method for detecting the dissolution rate of terpene lactones and flavonoids in ginkgo biloba extract dripping pill according to claim 1, wherein in step 3), the temperature of the placing is 30-40 ℃, the time of the placing is 4-24 hours, preferably the time of the placing is 6-24 hours at 37 ℃.

6. The method for detecting the dissolution rates of terpene lactones and flavonoids in ginkgo biloba extract dripping pill as claimed in claim 1, wherein in step 5), the concentrations of quercetin and kaempferide in the stock solution of ginkgo biloba extract reference substance are 1500 μ g and 1500 μ g per 1ml respectively; the bilobalide, bilobalide A, bilobalide B and bilobalide C in the reference stock solution of ginkgolide are respectively 2000 μ g, 1000 μ g and 1000 μ g per 1 ml; precisely measuring 5ml of ginkgetin reference substance stock solution and 1ml of ginkgolide reference substance stock solution into the same 100ml measuring flask, and diluting with methanol to scale as reference substance solution.

7. The method for detecting the dissolution rate of terpene lactones and flavonoids in ginkgo biloba extract dripping pill according to claim 1, wherein the chromatographic conditions in step 6) are as follows:

gradient elution was carried out using octadecylsilane chemically bonded silica as a filler (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm), tetrahydrofuran-acetonitrile-methanol (13:55:32) as a mobile phase A, and 0.4% acetic acid solution as a mobile phase B as specified in the following table; the flow rate is 1.0 mL/min; column temperature 30 ℃, detection with evaporative light scattering detector, drift tube temperature: 105 ℃, nitrogen flow rate: 2.5 mL/min:

。

8. the method for detecting the dissolution rate of terpene lactones and flavonoids in ginkgo biloba extract dripping pill according to claim 1, wherein in step 6), the determination method is as follows:

respectively and precisely sucking 10 mul and 20 mul of control solution, 15 mul of test solution and 15 mul of self control solution into a liquid chromatograph, measuring, and respectively calculating the content of 4 terpene lactone components and the content of 2 flavonoid components by using an external standard two-point method logarithmic equation.