A method for evaluating the quality of Danshen injection based on interface diffusion

Abstract

The present invention aims at the current situation that the existing content determination and fingerprint spectrum lack the analysis of the existence state of the index components in quality evaluation, and based on the fact that the component state is directly related to the stability and effectiveness of the preparation, provides a method for evaluating the quality of Danshen injection based on interface diffusion. The present invention innovatively reflects the difference in the existence state of Danshen medicinal components according to the characteristics of interface distribution, creates a method for evaluating the quality difference of Danshen injection from the interface diffusion at the molecular state level, solves the technical bottleneck that the traditional quality evaluation is difficult to explain the difference in the stability and efficacy of Danshen injection, and helps to improve the standardization and orderliness of the production process of Danshen injection.

Description

Technical Field

[0001] The invention belongs to the technical field of drug quality evaluation, and in particular relates to a method for evaluating the quality of salvia miltiorrhiza injection based on interface diffusion. Background Art

[0002] Danshen injection is a water solution made by extracting Danshen with water, precipitating with alcohol, treating with acid and alkali, and finally sterilizing. It has the functions of promoting blood circulation and removing blood stasis, promoting circulation and nourishing the heart, and is mainly used for the treatment of coronary heart disease, chest tightness, and angina pectoris. The quality control implementation standard is the national drug standard (WS3-B-3766-98-2011). Danshen sodium, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B are used as quality markers. The quality of Danshen injection is controlled by combining content determination and fingerprint / characteristic spectrum.

[0003] In the drug quality standard, the content determination item is to study whether the content of a certain ingredient meets the requirements to determine the quality of the drug. The content determination of Chinese medicine preparations is an important indicator in quality control. By determining the content of active ingredients, toxic ingredients or certain indicator ingredients in the preparations, the stability of the preparation process and the quality of Chinese medicinal materials are measured to ensure the quality of the drug preparations and achieve safe and effective clinical use.

[0004] The composition of traditional Chinese medicine is relatively complex, mainly divided into effective ingredients and toxic ingredients. The effective ingredients are divided into chemical components and bioactive components, which are important biochemical indicators for the quality control of traditional Chinese medicine. Traditional Chinese medicine fingerprint is a comprehensive and quantifiable identification method. It is based on the systematic study of the chemical components of traditional Chinese medicine and is mainly used to evaluate the authenticity, quality and stability of traditional Chinese medicine and semi-finished products of traditional Chinese medicine preparations. Traditional Chinese medicine and its preparations are multi-component complex systems. Therefore, the evaluation of their quality should adopt a detection method that is suitable for them and can provide rich identification information. The establishment of traditional Chinese medicine fingerprint will be able to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then provide an overall description and evaluation of the quality of the medicine. On this basis, if further spectrum efficacy research is carried out, the quality of traditional Chinese medicine can be truly combined with its efficacy, which will help to clarify the mechanism of action of traditional Chinese medicine.

[0005] Content determination and fingerprint can effectively control the content range and material composition characteristics of the index components in Danshen injection, which basically reflects the quality of the drug and can also evaluate the differences in the quality of Danshen injection between different manufacturers and batches. The existence state of Chinese medicine components is diverse in the solution. Danshen phenolic acid in Danshen injection exists in molecular state, ionic state, and associated state, and the component state is directly correlated with stability and efficacy. Danshen phenolic acid B can be converted into sodium danshensu, protocatechuic aldehyde, and rosmarinic acid by heating, alkaline hydrolysis, and light. The stability of danshen phenolic acid in ionic state is better than that in molecular state, and phenolic acid in molecular state is easier to convert, thereby changing the ratio of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and danshen phenolic acid B in Danshen injection during drug storage, thereby causing efficacy and safety risks during clinical use. Summary of the invention

[0006] Based on the above problems, it is particularly urgent to provide a method for systematically characterizing the differences in the presence states of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B in danshen injection. The presence state of the components determines the interfacial diffusion behavior during membrane separation. Therefore, the present invention provides a method for evaluating the quality of danshen injection based on interfacial diffusion, which innovatively reflects the differences in the presence states of danshen medicinal ingredients according to the characteristics of interfacial distribution, and creates a method for evaluating the quality differences of danshen injection from the molecular state level. The present invention is helpful to evaluate the quality differences of traditional Chinese medicine preparations with multiple components coexisting in different production processes, manufacturers, and production batches.

[0007] Therefore, the present invention provides a method for evaluating the quality of Danshen injection based on interface diffusion, comprising the following steps:

[0008] (1) Take Danshen injection and use high performance liquid chromatography to detect the concentrations of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B, which are C1, C2, C3, and C4 respectively;

[0009] (2) using a membrane separation device to separate Danshen injection, the membrane components used have molecular weight cutoffs of 10 KDa, 5 KDa, 1 KDa, and 800 Da, respectively;

[0010] (3) closing the retentate end of the membrane separation device, wherein the transmembrane pressure difference of the 10KDa, 5KDa, and 1KDa membrane separation device systems is adjusted to 0.1MPa, 0.2MPa, 0.3MPa, and 0.4MPa, respectively, and the transmembrane pressure difference of the 800Da membrane module is adjusted to 0.2MPa, 0.5MPa, 0.8MPa, and 1.0MPa, respectively, and the membrane flux is calculated, which are J1, J2, J3, and J4, respectively;

[0011] (4) collecting the filtrate of the membrane separation device, and using high performance liquid chromatography to detect the concentrations of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B, which are C1', C2', C3', and C4', respectively;

[0012] (5) respectively calculating the concentration differences of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B in step (1) and step (3), which are △C1, △C2, △C3, and △C4, respectively;

[0013] (6) Using the membrane flux data in step (3) as the horizontal coordinate and the concentration difference in step (5) as the vertical coordinate, a logarithmic function is fitted, wherein the coefficient of the logarithmic function is the membrane interface diffusion coefficient, and the membrane interface diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and tanshinone B are F1, F2, F3, and F4, respectively;

[0014] (7) The diffusion coefficient under the flux of J1 is set as the static diffusion coefficient Fstatic, the average diffusion coefficient under the flux of J2 and J3 is set as the transition diffusion coefficient Ftransition, and the diffusion coefficient under the flux of J4 is set as the concentration polarization diffusion coefficient Fconcentration;

[0015] (8) According to the comprehensive diffusion coefficient Fzong=0.4×Fconcent+0.4×Fstatic+0.2×Ftrans, the comprehensive diffusion coefficient Fzong of sodium danshensu, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B was calculated;

[0016] (9) The average value of the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B obtained from multiple batches of danshen injection was calculated as the reference value. The same method was used to obtain the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B in the batch of danshen injection to be tested, and the comprehensive diffusion coefficients were compared with the reference value to obtain the quality evaluation of the batch of danshen injection to be tested.

[0017] In a preferred embodiment, in step (9), the average value of the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B obtained in more than 10 batches of danshen injection is calculated as a reference value.

[0018] In a preferred embodiment, the multiple batches of Danshen injection are Danshen injections obtained from different batches and different production processes.

[0019] In a preferred embodiment, the separation layer material of the membrane component is one of cellulose, polyethersulfone, and composite polyamide; more preferably, the separation layer material of the membrane component is one of cellulose and polyethersulfone.

[0020] In a preferred embodiment, the area of ​​the membrane module is 0.1-0.5m 2 .

[0021] In a preferred embodiment, the membrane flux is calculated after the membrane module is saturated with sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B.

[0022] In a preferred embodiment, the correlation coefficients of the logarithmic functions are all greater than 0.95.

[0023] In a preferred embodiment, for sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B, F concentrated > F static.

[0024] In a preferred embodiment, the comprehensive diffusion coefficient of the tested Danshen injection exceeds the reference value by ±10%, which is judged as a large quality difference and an obvious quality difference.

[0025] Technical effects:

[0026] (1) The existence state of the components determines the interfacial diffusion behavior during membrane separation. Therefore, the present invention reveals the connotation of the quality difference of Danshen injection from the molecular state level.

[0027] (2) It solved the technical bottleneck that traditional quality evaluation could not explain the differences in stability and efficacy of Danshen injection.

[0028] (3) Based on the interfacial diffusion behavior of salvianolic acid components in different membrane components, the differences in the states of salvianolic acid components were evaluated. Based on the correlation between the component states and the material transfer between steps in the pharmaceutical process, it was helpful to improve the standardization and orderliness of the production process of salvianolic acid injection. DETAILED DESCRIPTION

[0029] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0030] Example 1

[0031] Danshen injection (manufacturer: Jiangsu Shenlong Pharmaceutical Co., Ltd., batch number: 20240510101) was taken and detected by high performance liquid chromatography. The chromatographic conditions were as follows: Waters e2695 high performance liquid chromatograph, full wavelength ultraviolet detector, Hanbang Hedera ODS-C18 chromatographic column, acetonitrile (A)-0.05% trifluoroacetic acid aqueous solution (B), gradient elution (0min-65min, A2%-30%; 65min-66min, A30%-2%; 66min-80min, A2%), volume flow rate 0.8mL / min, column temperature 40°C, detection wavelength 288nm, and the standard curve method was used to calculate the concentrations of the components in Danshen injection, which were 1.27mg / mL of sodium danshensu, 0.38mg / mL of protocatechuic aldehyde, 0.25mg / mL of rosmarinic acid, and 0.36mg / mL of salvianolic acid B.

[0032] The membrane separation equipment was used to separate the Danshen injection, with a membrane area of ​​0.5m 2 The molecular weight cutoffs of the membrane components used were 10KDa, 5KDa, 1KDa, and 800Da, respectively. The retentate end of the membrane separation device was closed. The transmembrane pressure differences of the 10KDa, 5KDa, and 1KDa membrane separation device systems made of polyethersulfone were adjusted to 0.1MPa, 0.2MPa, 0.3MPa, and 0.4MPa, respectively. The transmembrane pressure differences of the 800Da membrane components made of composite polyamide were adjusted to 0.2MPa, 0.5MPa, 0.8MPa, and 1.0MPa, respectively. The membrane fluxes were calculated as J1, J2, J3, and J4, respectively, in units of L / m 2 h. The results are shown in Table 1.

[0033] Table 1

[0034] Membrane flux 800Da 1KDa 5KDa 10KDa J1 17.8 21.4 72.7 93.6 J2 36.5 49.8 122.3 159.5 J3 46.2 62.5 147.8 209.4 J4 51.7 71.1 179.2 247.5

[0035] The filtrate of the membrane separation equipment was collected, and the concentrations of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B were detected by HPLC, which were C1', C2', C3', and C4', respectively. The concentration differences of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B before and after membrane separation were calculated, which were △C1, △C2, △C3, and △C4, respectively. The results are shown in Table 2.

[0036] Table 2

[0037] 800Da 1KDa 5KDa 10KDa Danshensu Sodium △C1 0.537 0.409 0.123 0.102 Protocatechuic aldehyde △C2 0.358 0.284 0.137 0.099 Rosmarinic acid △C3 0.562 0.397 0.157 0.033 Salvianolic acid B△C4 0.491 0.360 0.123 0.038

[0038] The membrane flux data in Table 1 is used as the horizontal coordinate (x), and the concentration difference in Table 2 is used as the vertical coordinate (Y). Logarithmic function fitting is performed, and the results are shown in Table 3. The correlation coefficients are all greater than 0.95, and the logarithmic function is established. The coefficient of the logarithmic function is the membrane interface diffusion coefficient. The membrane interface diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and tanshinone B are F1, F2, F3, and F4, respectively, as shown in Table 4.

[0039] Table 3

[0040]

[0041] Table 4

[0042] F1 F2 F3 F4 Danshensu Sodium -0.25 -0.302 -0.298 -0.286 Protocatechuic aldehyde -0.143 -0.173 -0.171 -0.164 Rosmarinic acid -0.275 -0.334 -0.332 -0.381 Salvianolic acid B -0.23 -0.278 -0.275 -0.281

[0043] Under the flux of J1, the diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B are higher than those under the fluxes of J2, J3 and J4. This is because with the increase of the transmembrane pressure difference, the diffusion of the four components of danshen injection at the membrane interface transitions from static diffusion to concentration polarized diffusion. Therefore, the diffusion coefficient under the flux of J1 is set as the static diffusion coefficient (Fstatic), the average values ​​of J2 and J3 are set as the transition diffusion coefficient (Ftransition), and the flux of J4 is set as the concentration polarized diffusion coefficient (Fconcent). Among them, the static diffusion is mainly completed by the concentration difference between the solution and the interface layer of the component state, and the concentration polarized diffusion is mainly completed by the concentration difference between the solution and the interface layer generated under the pressure. The dead-end filtration caused by the closure of the interception end strengthens the concentration effect, showing that the absolute value of Fconcent is greater than the absolute value of Fstatic. Among them, due to the molecular competitive diffusion, the absolute value of Fconcent of rosmarinic acid and salvianolic acid B is greater than the absolute value of Ftransition.

[0044] Therefore, in order to further magnify the differences in interfacial diffusion caused by the concentration and state of the components in Danshen injection, the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B were calculated according to the comprehensive diffusion coefficient = (0.4×Fconcentration+0.4×Fstatic+0.2×Ftransfer). The results are shown in Table 5.

[0045] Table 5

[0046] F F F thick F Danshensu Sodium -0.25 -0.3 -0.286 -0.2744 Protocatechuic aldehyde -0.143 -0.172 -0.164 -0.1572 Rosmarinic acid -0.275 -0.333 -0.381 -0.329 Salvianolic acid B -0.23 -0.2765 -0.281 -0.2597

[0047] Ten batches of Danshen injection (20240310101, 20240310102, 20240310501, 20240310502, 20240402001, 20240402002, 20240402003, 20240510101, 20240510102, and 20240518101) produced by Jiangsu Shenlong Pharmaceutical Co., Ltd. were selected, and the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B were calculated using the same method as above, and the average values ​​were calculated. The results are shown in Table 6.

[0048] Table 6

[0049] batch number Danshensu Sodium Protocatechuic aldehyde Rosmarinic acid Salvianolic acid B 20240310101 -0.2808 -0.1504 -0.3360 -0.2477 20240310102 -0.2763 -0.1519 -0.3315 -0.2568 20240310501 -0.2633 -0.1634 -0.3184 -0.2527 20240310502 -0.2809 -0.1590 -0.3416 -0.2621 20240402001 -0.2823 -0.1581 -0.3217 -0.2580 20240402002 -0.2792 -0.1649 -0.3263 -0.2557 20240402003 -0.2705 -0.1517 -0.3288 -0.2598 20240510101 -0.2744 -0.1572 -0.3290 -0.2597 20240510102 -0.2759 -0.1578 -0.3321 -0.2516 20240518101 -0.2887 -0.1621 -0.3279 -0.2669 average value -0.2772 -0.1576 -0.3293 -0.2571

[0050] Example 2

[0051] The Danshen injections (batch numbers 20211010-01, 20220802-02, 20230109401, 20230501101, and 20231101001) produced by Jiangsu Shenlong Pharmaceutical Co., Ltd. were taken respectively, and the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B were calculated according to the method in Example 1. The results are shown in Table 7.

[0052] Table 7

[0053] batch number Danshensu Sodium Protocatechuic aldehyde Rosmarinic acid Salvianolic acid B 20211010-01 -0.2638 -0.1579 -0.3110 -0.2500 20220802-02 -0.3209 -0.1601 -0.3369 -0.2458 20230109401 -0.2754 -0.1520 -0.3228 -0.2619 20230501101 -0.2726 -0.1557 -0.3252 -0.2524 20231101001 -0.2650 -0.1397 -0.3311 -0.2645

[0054] Among them, the comprehensive diffusion coefficient of sodium danshensu in batch number 20220802-02 exceeded 10% of the reference value of the diffusion coefficient of sodium danshensu in Example 1. Liquid phase detection and analysis of batch 20220802-02 danshen injection found that the concentration of sodium danshensu was 1.75 mg / mL, which was significantly higher than other batches produced. Combined with the increase in the absolute values ​​of the comprehensive diffusion coefficients of protocatechuic aldehyde and rosmarinic acid, and the decrease in the absolute value of the comprehensive diffusion coefficient of salvianolic acid, it can be determined that there are certain differences in the component conversion of salvianolic acid B compared with other batches during the production process.

[0055] The comprehensive diffusion coefficient of protocatechuic aldehyde in batch number 20231101001 exceeds 10% of the reference value of the diffusion coefficient of protocatechuic aldehyde in Example 1. Liquid phase detection and analysis of batch 20231101001 of Danshen injection found that the concentration of protocatechuic aldehyde was 0.39 mg / mL, similar to other batches, indicating that the degree of intermolecular association of protocatechuic aldehyde in Danshen injection increased, causing the absolute value of the comprehensive diffusion coefficient of protocatechuic aldehyde to decrease. After solution pH detection, it was found that it was slightly lower than other batches, indicating that the proportion of protocatechuic aldehyde associates in an acidic environment increased. It is fully proved that the method for evaluating the quality of Danshen injection based on interface diffusion provided by the present invention can effectively and reliably evaluate the stability and efficacy differences of Danshen injection, solving the technical bottleneck.

Claims

1. A method for evaluating the quality of Danshen injection based on interface diffusion, comprising the following steps: (1) Take Danshen injection and use HPLC to detect the concentrations of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B, which are C1, C2, C3, and C4 respectively; (2) The Danshen injection was separated using a membrane separation device, and the molecular weight cutoffs of the membrane components used were 10 KDa, 5 KDa, 1 KDa, and 800 Da, respectively; (3) Closing the retentate end of the membrane separation device, wherein: The transmembrane pressure differences of 10 KDa, 5 KDa, and 1 KDa membrane separation equipment systems were adjusted to 0.1 MPa, 0.2 MPa, 0.3 MPa, and 0.4 MPa, respectively, and the transmembrane pressure differences of 800 Da membrane modules were adjusted to 0.2 MPa, 0.5 MPa, 0.8 MPa, and 1.0 MPa, respectively, and the membrane fluxes were calculated to be J1, J2, J3, and J4, respectively; (4) Collect the filtrate from the membrane separation device and use high performance liquid chromatography to detect the concentrations of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and tanshinone B, which are C1', C2', C3', and C4', respectively; (5) Calculate the concentration differences of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B in step (1) and step (3), which are △C1, △C2, △C3, and △C4, respectively; (6) Using the membrane flux data in step (3) as the horizontal coordinate and the concentration difference in step (5) as the vertical coordinate, a logarithmic function is fitted, wherein the logarithmic function is in the form of a linear logarithm, that is, y=aln(x)+b, and the coefficient a of the logarithmic function is the membrane interface diffusion coefficient, and the membrane interface diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and tanshinone B are F1, F2, F3, and F4, respectively; (7) The diffusion coefficient under the flux of J1 is set as the static diffusion coefficient Fstatic, the average diffusion coefficient under the flux of J2 and J3 is set as the transition diffusion coefficient Ftransition, and the diffusion coefficient under the flux of J4 is set as the concentration polarization diffusion coefficient Fconcent; (8) According to the comprehensive diffusion coefficient Fzong=0.4×Fconc+0.4×Fjing+0.2×Fdu, calculate the comprehensive diffusion coefficient Fzong of sodium danshensu, protocatechuic aldehyde, rosmarinic acid and salvianolic acid B; (9) The average value of the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B obtained from multiple batches of Danshen injection was calculated as the reference value. The same method was used to obtain the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B in the batch of Danshen injection to be tested. The comprehensive diffusion coefficients were compared with the reference value to obtain the quality evaluation of the batch of Danshen injection to be tested.

2. The method according to claim 1, wherein: In step (9), the average value of the comprehensive diffusion coefficients of sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and salvianolic acid B obtained from more than 10 batches of danshen injection is calculated as a reference value.

3. The method according to claim 1, wherein: The multiple batches of Danshen injection are Danshen injections obtained from different batches and different production processes.

4. The method according to claim 1, wherein: The separation layer material of the membrane component is one of cellulose, polyethersulfone and composite polyamide.

5. The method according to claim 1, wherein: The separation layer material of the membrane component is one of cellulose and polyethersulfone.

6. The method according to claim 1, wherein: The area of ​​the membrane assembly is 0.1-0.5 m 2 .

7. The method according to claim 1, wherein: The membrane flux is calculated after the membrane assembly is saturated with the adsorption of sodium danshensu, protocatechuic aldehyde, rosmarinic acid and tanshinone B.

8. The method according to claim 1, wherein: The correlation coefficients of the logarithmic functions are all greater than 0.

95.

9. The method according to claim 1, wherein: For sodium danshensu, protocatechuic aldehyde, rosmarinic acid, and tanshinone B, F concentrated > F static.

10. The method according to any one of claims 1 to 9, wherein: The comprehensive diffusion coefficient of the tested Danshen injection exceeded the reference value by ±10%, and was judged to be of large quality difference and obvious quality difference.

Images