WO2020083354A1

WO2020083354A1 - Application of chlorogenic acid derivative in drug quality control

Info

Publication number: WO2020083354A1
Application number: PCT/CN2019/113166
Authority: WO
Inventors: 张洁; 张亮; 姬勋; 张飞; 黄望
Original assignee: 四川九章生物科技有限公司
Priority date: 2018-10-25
Filing date: 2019-10-25
Publication date: 2020-04-30
Also published as: CN111103375A; CN111103375B

Abstract

Use of a chlorogenic acid derivative as a reference substance in detection of the quality of preparations comprising chlorogenic acid and mannitol.

Description

Application of a chlorogenic acid derivative in medicine quality control

Technical field

The invention belongs to the field of chemical analysis and detection, and in particular relates to the application of a chlorogenic acid derivative in drug quality control.

Background technique

Chlorogenic acid (Chlorogenic acid) is a phenolic acid produced by Caffeic acid and Quinic acid. It is a phenylpropanoid produced by the shikimic acid pathway during the aerobic respiration of plants. Similar compounds, the structure is as follows:

Chlorogenic acid is widely present in plants, with higher contents in honeysuckle and eucommia. Chlorogenic acid has a wide range of biological activities, such as anti-cancer, antibacterial, anti-viral, liver protection and gall bladder, blood pressure, blood fat, scavenging free radicals and excite the central nervous system, which reflects the extraordinary medicinal and social value . The chlorogenic acid is prepared into a preparation, which is used in clinic and can treat various diseases.

However, the chemical nature of chlorogenic acid is unstable, and it is easily degraded and reacted after it is made into a preparation, and impurities are generated, especially a preparation containing chlorogenic acid and mannitol, which is easily degraded during storage, affecting the quality of the preparation. Therefore, chlorogenic acid preparations are difficult to store, have poor controllability, and it is difficult to accurately control product quality.

In order to more accurately control the quality of chlorogenic acid preparations and ensure the safety of medications, currently the main content of chlorogenic acid preparations is controlled by detecting the total and partial impurity content of chlorogenic acid preparations. However, there are many types of chlorogenic acid impurities. In addition to the known impurities, there are many impurities with unclear components. These impurities are difficult to be effectively detected, which affects the quality control of chlorogenic acid preparations.

In order to make chlorogenic acid preparations safer, controllable, and effective, and to ensure the safety of patients' medication, it is very important to find the exact composition of impurities in chlorogenic acid preparations and find a method to detect the impurities.

Summary of the invention

The object of the present invention is to provide an application of chlorogenic acid derivatives in drug quality control.

The present invention provides the use of the compound of formula I as a reference substance in detecting the quality of a preparation containing chlorogenic acid and mannitol:

Further, the preparation method of the compound includes the following steps:

(1) Dissolve chlorogenic acid and mannitol in an organic solvent, and react under an acidic system to obtain a reaction solution;

(2) The compound reaction solution is prepared by precipitation, adsorption analysis, washing, crystallization and recrystallization, and freeze-drying.

further,

In step (1), the molar ratio of the chlorogenic acid to mannitol is (1: 1) to (5: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 2-24 h;

And / or, in step (2), the chromatography column used for the adsorption analysis is a non-polar or weakly polar macroporous adsorption resin chromatography column;

And / or, in step (2), the washing is washing with an organic solvent;

Preferably,

In step (1), the molar ratio of chlorogenic acid to mannitol is (1: 1) to (3: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or concentrated sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 24 h;

And / or, in step (1), the organic solvent is N, N-dimethylformamide or absolute ethanol;

And / or, in step (2), the model of the macroporous adsorption resin chromatography column is HPD-100, HPD-450 or LS-46;

And / or, in step (2), the chromatography column is eluted with 5% to 80% ethanol after the chromatography column is completed, the eluent is collected, concentrated to dryness, and dissolved in water with saturation;

And / or, in step (2), the organic solvent used for the washing is ethyl acetate;

And / or, in step (2), the crystallization is refrigerated crystallization;

And / or, in step (2), the recrystallization is recrystallization with water.

Further, the compound represented by formula I is a degradation product during storage of the formulation containing chlorogenic acid and mannitol; preferably, the compound represented by formula I is a degradation product of chlorogenic acid and mannitol; the formulation It is a freeze-dried powder injection.

Further, during the storage process, the moisture content of the preparation is less than 5%; during the storage process, the temperature is 0 ° C to 40 ° C.

Further, in the formulation, the content of the compound represented by formula I is 0.01% to 5.0% w / w; preferably, the content of the compound represented by formula I is 0.01% to 2.0% w / w.

The present invention also provides a method for detecting a compound represented by Formula I in a formulation containing chlorogenic acid and mannitol, which includes the following steps:

a. Take the compound shown in formula Ⅰ as a reference substance and prepare it as a reference substance solution;

b. Take a sample of the preparation containing chlorogenic acid and mannitol to be tested and prepare a test solution;

c. Detection by high-performance liquid chromatography, the packing of the column is octadecylsilane-bonded silica gel, the mobile phase is the volume ratio of (7.0 ～ 8.0) :( 7.0 ～ 8.0) :( 8.0 ～ 9.0) Acetonitrile: methanol: a mixed solution of phosphoric acid aqueous solution, the detection wavelength is 325-335 nm; the phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a concentration of 0.4-0.6%;

further,

In step a, the solvent for preparing the reference solution is purified water, and the concentration of the reference solution is 10 μg / mL;

In step b, the solvent for preparing the test product solution is purified water, and the concentration of chlorogenic acid in the test product solution is 0.1 to 1.0 mg / mL, preferably 0.5 to 1.0 mg / mL;

In step c, the chromatographic conditions of the high performance liquid chromatography method are:

The column is an Agilent ZORBAX SB-C18 column with a length of 4.6mm × 150mm and an inner diameter of 5μm;

And / or, the mobile phase is a mixed solution of acetonitrile: methanol: 0.5% phosphoric acid aqueous solution with a volume ratio of 7.5: 7.5: 85;

And / or, the column temperature is 30 ° C;

And / or, the flow rate is 1.0 mL / min;

And / or, the injection volume is 10 μL;

And / or, the detection wavelength is 327 nm.

The present invention also provides a formulation comprising chlorogenic acid and mannitol, which contains the compound represented by Formula 1;

Preferably, the preparation is a freeze-dried powder injection.

The present invention finds an impurity generated by degradation of a preparation containing chlorogenic acid and mannitol during storage, and gives the specific chemical structural formula of the impurity. The impurity is a chlorogenic acid derivative, which is used as a reference. It can be used to detect the quality of preparations containing chlorogenic acid and mannitol, to better monitor the quality of preparations containing chlorogenic acid and mannitol, and to ensure the safety of medication.

Obviously, according to the above content of the present invention, in accordance with the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or alteration can be made without departing from the above basic technical idea of the present invention.

The above content of the present invention will be further described in detail below through specific implementations in the form of examples. However, it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples. All technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION

Figure 1 is an HPLC chart of the compound of the present invention.

Figure 2 is the nuclear magnetic resonance spectrum of the compound of the present invention; A: ¹ H-NMR spectrum, B: ¹ H- ¹ H COSY spectrum, C: heavy water exchange hydrogen spectrum, D: ¹³ C-NMR spectrum, E: ¹³ C-DEPT Map, F: ¹ H- ¹³ C HSQC map, G: ¹ H- ¹³ C HMBC map.

detailed description

Example 1, the preparation method of the compound of the present invention

Take 200 g of chlorogenic acid and 100 g of mannitol, dissolve in N, N-dimethylformamide, add 50 mL of p-toluenesulfonic acid, and stir at room temperature for 24 hours to obtain a reaction solution.

Take the reaction solution, dilute it with water to 1 to 10 times, then apply the HPD-100 macroporous adsorption resin chromatography column. After the column is completed, wash with water until the chlorogenic acid and mannitol cannot be detected in the washing solution. Use 80% ethanol Elute, collect 80% ethanol eluate, and concentrate to dryness. Take the dried product, dissolve it in water, wash it with ethyl acetate, refrigerate and crystallize it, filter it, take the crystallized product, recrystallize it with water, and freeze-dry it to obtain 180g, the content is 98.2%.

The HPLC chart of the compound of the present invention is shown in FIG. 1. The nuclear magnetic resonance spectrum of the compound of the present invention is shown in FIG. 2. ¹ H-NMR spectrum (FIG. 2A) clearly shows that the compound of the present invention is a compound in which chlorogenic acid and mannitol are reacted to form an ester. Due to the symmetry of the molecular structure of free mannitol, the chemical shift of C-17 and C-22 to H is basically the same. After mannitol forms an ester with chlorogenic acid, the group connected to C-17 changes from a hydroxyl group to an ester group. The H on C-17 is affected by the ester group, and the chemical shift moves to a lower field, and the chemical shift increases significantly. It is still connected to the hydroxyl group, and the chemical shift of H on C-22 is not greatly affected. The chemical shift of H (δ (4.19, 3.99)) on all compounds of mannitol and chlorogenic acid C-17 is significantly higher than that on C-22 Chemical shift δ (3.60, 3.37). ¹ H- ¹³ C HMBC (Figure 2G) can also clearly indicate that the compound is a compound of chlorogenic acid and mannitol to form an ester, δ173.35 is the C-1 chemical shift in chlorogenic acid, when chlorogenic acid and mannitol react After ester formation, J _CH coupling of C-1 and C-17 can form a triple bond, showing a strong correlation at ¹ H- ¹³ C HMBC.

The C spectrum of the compound of the present invention shows that there are 21 kinds of C in the molecule, and the DEPT spectrum (Figure 2E) shows that there are 4 secondary carbons, 11 tertiary carbons, and 6 quaternary carbons in the molecule, while the molecular formula has 22 Cs, indicating that there are 1 The tertiary carbons with the same chemical shift are analyzed as two tertiary carbons, C-4 and C-18.

The ¹ H-NMR, ¹ H- ¹ HCOSY and heavy water exchange hydrogen spectrum measurement data are listed in Table 1, combined with ¹³ C-NMR, ¹³ C-DEPT-135, ¹³ C-DEPT-90, ¹ H- ¹³ C HSQC , ¹ H- ¹³ C HMBC data, to determine the attribution of each H.

The ¹³ C-NMR spectrum data are listed in the order of C chemical shift from large to small in Table 2. DEPT spectrum data is used to determine the type of C, HSQC spectrum is used to determine the attribution of directly connected CH, and HMBC spectrum is used to target C Attribution of H on atoms with similar atoms, HSQC and HMBC data and analysis are also listed in Table 2.

Table 1 The data attribution of ¹ H-NMR, ¹ H-1H COSY and heavy water exchange hydrogen spectrum of the compound of the present invention

Table 2 ¹³ C-NMR, ¹³ C-DEPT, ¹ H- ¹³ C HSQC, ¹ H- ¹³ C HMBC measurement data of the 3-caffeoylquinic acid derivatives of the present invention

After examination, the structural formula of the compound of the present invention is as follows: its molecular formula is C ₂₂ H ₃₀ O ₁₄ and its molecular weight is 518.47.

Example 2. Preparation method of the compound of the present invention

Take 500 g of chlorogenic acid and 100 g of mannitol, heat and dissolve in absolute ethanol, add 100 mL of concentrated sulfuric acid, and stir at 40 ° C. for 24 hours to obtain a reaction solution.

Take the reaction solution, place it below 0 ° C to allow sufficient precipitation, filter, and collect the filtrate; after the filtrate is concentrated and recovered into ethanol, it will be loaded onto a macroporous adsorption resin chromatography column of model HPD-450. Chlorogenic acid and mannitol were eluted with 20% ethanol. The 20% ethanol eluent was collected and concentrated to dryness. Take the dried product, dissolve it in water, wash it with ethyl acetate, refrigerate and crystallize it, filter it, take the crystallized product, recrystallize it with water, and freeze-dry it to obtain 120g, the content is 98.6%.

The compound has been tested and has the same structure as in Example 1.

Example 3. Preparation method of the compound of the present invention

Take 300 g of chlorogenic acid and 100 g of mannitol, dissolve in absolute ethanol, add 150 ml of hydrochloric acid, and stir at 60 ° C for 24 hours to obtain a reaction solution.

Take the reaction solution, place it below 0 ° C to allow sufficient precipitation, filter, and collect the filtrate; after the filtrate is concentrated and recovered into ethanol, a large pore adsorption resin chromatography column of model LS-46 is used. After the column is completed, it is washed with water until it is not detected in the washing solution. Chlorogenic acid and mannitol were eluted with 5% ethanol. The 5% ethanol eluate was collected and concentrated to dryness. Take the dried product, dissolve it in water, wash it with ethyl acetate, refrigerate and crystallize it, filter it, take the crystallized product, recrystallize it with water, and freeze-dry it to obtain 140g, 98.3% content.

The compound has been tested and has the same structure as in Example 1.

Example 4. The compound of the present invention is used as a reference substance to detect the quality of a preparation containing chlorogenic acid and mannitol

(1) Chromatographic conditions of high performance liquid chromatography:

The column is an Agilent ZORBAX SB-C18 column with a length of 4.6mm × 150mm, an inner diameter of 5μm, a filler of octadecylsilane-bonded silica gel, and a column temperature of 30 ° C.

The mobile phase was an acetonitrile: methanol: 0.5% phosphoric acid aqueous solution mixed solution with a volume ratio of 7.5: 7.5: 85, and the flow rate was 1.0 mL / min.

The injection volume is 10 μL; the detection wavelength is 327 nm; the number of theoretical plates should not be less than 3000 based on the calculation of the main peak.

(2) Detection steps

Take an appropriate amount of the compound of the present invention as a reference substance, accurately weigh it, and dissolve it in purified water to prepare a 10 μg / mL reference substance solution.

Weigh the lyophilized powder injection sample containing chlorogenic acid and mannitol preparation prescription, accurately weigh it, add purified water to make a solution containing about 0.5 mg of chlorogenic acid per 1 mL, as the test solution.

Accurately measure 10μL of the above reference solution and test solution into the liquid chromatograph, record the chromatogram and calculate according to the external standard method.

Example 5. The compound of the present invention was used as a reference substance to test the quality of the preparation containing chlorogenic acid and mannitol

(1) Chromatographic conditions of high performance liquid chromatography:

The column is an Agilent Eclipse-C18 column with a length of 4.6mm × 100mm, an internal diameter of 3.5μm, a filler of octadecylsilane-bonded silica gel, and a column temperature of 30 ° C.

(2) Detection steps

Take an appropriate amount of the compound as a reference substance, accurately weigh it, and dissolve it in purified water to prepare a 10 μg / mL reference substance solution.

Weigh an appropriate amount of lyophilized powder injection sample containing chlorogenic acid and mannitol preparation prescription, accurately weigh it, add purified water to make a solution containing about 1.0 mg of chlorogenic acid per 1 mL, as a test solution.

Example 6. Changes of the compound of the present invention under different storage conditions of powder injection containing chlorogenic acid and mannitol

(1) Test plan

Weigh out chlorogenic acid and mannitol according to the mass ratio of 10: 1 ～ 1: 10 (see Table 1 for specific mass ratio), dissolve with water for injection, filter with microporous filter membrane, sub-pack, freeze-dry to obtain chlorogenic acid and mann Alcohol powder injection.

Chlorogenic acid and mannitol powder injection were placed under high temperature (60 ℃), high humidity (90% relative humidity, 25 ℃), strong light (4500LX) conditions for 10 days, according to "Example 4" test The method was used to detect the percentage of the compound of the present invention in the mass of chlorogenic acid on

days

0 and 10;

The chlorogenic acid and mannitol powder injections were placed under the conditions of 40 ° C and 75% relative humidity, and kept in the dark for 6 months, and the invention was tested in January, March, and June according to the detection method of “Example 4”. The compound accounts for the percentage of chlorogenic acid mass;

The chlorogenic acid and mannitol powder injections were placed under the conditions of 25 ° C and 60% relative humidity, and kept in the dark for 24 months. The percentage of the chlorogenic acid in the compound of the present invention was determined according to the detection method of “Example 4”.

(2) Test results

Table 3 The content of the compound of the present invention in the powder injection under the conditions of high temperature, high humidity and strong light

Note: “——” means not detected.

As a result, high temperature factors can promote the formation of the compound of the present invention in chlorogenic acid and mannitol formulations, while the strong light factor and high humidity factor have no obvious effect; different chlorogenic acid and mannitol mass ratios have different effects on the present invention There was no significant difference in compound formation.

Table 4 The content of the compound of the present invention in powder injection at 40 ° C and 75% relative humidity

Note: “——” means not detected.

As a result, the content of the compound of the present invention gradually increased with time at 40 ° C and 75% relative humidity, and the percentage of the compound of the present invention in the mass of chlorogenic acid could reach 2.11% after 6 months of storage; The alcohol mass ratio has no significant difference in the production of the compound of the present invention.

Table 5 The content of the compound of the present invention in powder injection at 25 ° C and 60% relative humidity

Note: “——” means not detected.

As a result, the compound of the present invention gradually increased with time at 25 ° C and 60% relative humidity, and the percentage of the mass of chlorogenic acid reached up to 1.03% after 24 months of storage, compared with 40 ° C and 75% relative humidity. The low content of 6 months shows that high temperature helps the production of the compound of the present invention in the preparation of chlorogenic acid and mannitol; different mass ratios of chlorogenic acid and mannitol have no significant difference in the production of the compound of the present invention.

(3) Test conclusion

The high temperature factor can promote the generation of the compound of the present invention in the preparation of chlorogenic acid and mannitol, while the strong light factor and high humidity factor have no obvious effect; in the range of the mass ratio of chlorogenic acid to mannitol of 10: 1 to 1:10, Different mass ratios of chlorogenic acid and mannitol have no significant difference in the formation of the compound of the present invention. It indicates that the preparation containing chlorogenic acid and mannitol should be stored in a low temperature environment.

In summary, the present invention finds an impurity generated by degradation of a preparation containing chlorogenic acid and mannitol during storage, and gives the specific chemical structural formula of the impurity. The impurity is a chlorogenic acid derivative, which is used as The reference substance can be used to detect the quality of the preparation containing chlorogenic acid and mannitol, to better monitor the quality of the preparation containing chlorogenic acid and mannitol, and to ensure the safety of medication.

Claims

Use of the compound of formula I as a reference substance in the detection of the quality of a preparation containing chlorogenic acid and mannitol:
The use according to claim 1, characterized in that the preparation method of the compound comprises the following steps:

(1) Dissolve chlorogenic acid and mannitol in an organic solvent, and react under an acidic system to obtain a reaction solution;

(2) The compound reaction solution is prepared by precipitation, adsorption analysis, washing, crystallization and recrystallization, and freeze-drying.
The use according to claim 2, characterized in that:

In step (1), the molar ratio of the chlorogenic acid to mannitol is (1: 1) to (5: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 2-24 h;

And / or, in step (2), the chromatography column used for the adsorption analysis is a non-polar or weakly polar macroporous adsorption resin chromatography column;

And / or, in step (2), the washing is washing with an organic solvent;

Preferably,

In step (1), the molar ratio of chlorogenic acid to mannitol is (1: 1) to (3: 1);

And / or, in step (1), the acidic system is a p-toluenesulfonic acid, hydrochloric acid or concentrated sulfuric acid system;

And / or, in step (1), the reaction temperature is 20-60 ° C., and the reaction time is 24 h;

And / or, in step (1), the organic solvent is N, N-dimethylformamide or absolute ethanol;

And / or, in step (2), the model of the macroporous adsorption resin chromatography column is HPD-100, HPD-450 or LS-46;

And / or, in step (2), the chromatography column is eluted with 5% to 80% ethanol after the chromatography column is completed, the eluent is collected, concentrated to dryness, and dissolved in water with saturation;

And / or, in step (2), the organic solvent used for the washing is ethyl acetate;

And / or, in step (2), the crystallization is refrigerated crystallization;

And / or, in step (2), the recrystallization is recrystallization with water.
The use according to any one of claims 1 to 3, characterized in that the compound represented by formula I is a degradation product during storage of the formulation containing chlorogenic acid and mannitol; preferably, the compound represented by formula I The compound is a degradation product of chlorogenic acid and mannitol; the formulation is a lyophilized powder injection.
The use according to claim 4, characterized in that: during the storage process, the moisture content of the preparation is less than 5%; the temperature during the storage process is 0 ° C to 40 ° C.
The use according to claim 4, characterized in that: in the preparation, the content of the compound represented by formula I is 0.01% to 5.0% w / w; preferably, the content of the compound represented by formula I is 0.01% to 2.0 % W / w.
A method for detecting a compound represented by formula I in a preparation containing chlorogenic acid and mannitol, characterized in that it includes the following steps:

a. Take the compound shown in formula Ⅰ as a reference substance and prepare it as a reference substance solution;

b. Take a sample of the preparation containing chlorogenic acid and mannitol to be tested and prepare a test solution;

c. Detection by high-performance liquid chromatography, the packing of the column is octadecylsilane-bonded silica gel, the mobile phase is the volume ratio of (7.0 ～ 8.0) :( 7.0 ～ 8.0) :( 8.0 ～ 9.0) Acetonitrile: methanol: a mixed solution of phosphoric acid aqueous solution, the detection wavelength is 325-335 nm; the phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a concentration of 0.4-0.6%;
The method according to claim 7, characterized in that:

In step a, the solvent for preparing the reference solution is purified water, and the concentration of the reference solution is 10 μg / mL;

In step b, the solvent for preparing the test product solution is purified water, and the concentration of chlorogenic acid in the test product solution is 0.1 to 1.0 mg / mL, preferably 0.5 to 1.0 mg / mL;

In step c, the chromatographic conditions of the high performance liquid chromatography method are:

The column is an Agilent ZORBAX SB-C18 column with a length of 4.6mm × 150mm and an inner diameter of 5μm;

And / or, the mobile phase is a mixed solution of acetonitrile: methanol: 0.5% phosphoric acid aqueous solution with a volume ratio of 7.5: 7.5: 85;

And / or, the column temperature is 30 ° C;

And / or, the flow rate is 1.0 mL / min;

And / or, the injection volume is 10 μL;

And / or, the detection wavelength is 327 nm.
A preparation containing chlorogenic acid and mannitol, characterized in that it contains a compound represented by formula 1;

Preferably, the preparation is a freeze-dried powder injection.
The preparation according to claim 9, characterized in that, in the preparation, the content of the compound represented by formula I is 0.01% to 5.0% w / w; preferably, the content of the compound represented by formula I is 0.01% to 2.0 % W / w.