Identification of pharmacokinetic markers for safflower injection using a combination of system pharmacology, multicomponent pharmacokinetics, and quantitative proteomics study

Abstract

Safflower injection (SI), a water-extract preparation from safflower (Carthamus tinctorius L.), has been widely used for the treatment of cardio-cerebrovascular diseases. This work aims to develop an approach for identifying PK markers of cardiovascular herbal medicines using SI as a case study. Firstly, qualitative and quantitative analyses were performed to reveal ingredients of the preparation via HPLC-MS. Subsequently, multiple PK ingredients and integrated PK investigations were carried out to ascertain ingredients with favorable PK properties (e.g., easily detected at conventional PK time points and high system exposure) for the whole preparation. Next, ingredients against cardiovascular diseases (CVDs) in the preparation were predicted with target fishing and system pharmacology studies. Finally, ingredients with favorable PK properties, satisfactory PK representativeness for the preparation, and high relevance to CVDs were considered as potential PK markers. Their therapeutic effect was further evaluated using the H₂O₂-induced H9c2 cardiomyocyte-injured model and a proteomics study to identify objective PK markers. As results, it disclosed that SI mainly contains 11 ingredients. Among them, five ingredients, namely, hydroxysafflor yellow A (HSYA), syringin (SYR), p-coumaric acid (p-CA), scutellarin (SCU), and p-hydroxybenzaldehyde (p-HBA), showed favorable PK properties. HSYA, SYR, and rutin (RU) were predicted to show high relevance to CVDs and screened as potential PK markers. However, only HSYA and SYR were confirmed as therapeutic ingredients against CVDs. Combined with these findings, only HSYA demonstrated satisfactory representativeness on PK properties and therapeutic effects of multiple ingredients of the preparation, thereby indicating that HSYA is a potential PK marker for the SI. The results of this study can provide a reference for the characterization of PK markers for traditional Chinese medicines.

Keywords: PK marker; hydroxysafflor yellow A; multicomponent pharmacokinetics; quantitative proteomics; safflower injection; system pharmacology.

SCHEME 1

The proposed strategy for screening and identifying the PK markers of SI.

FIGURE 1

The heatmap for content determination results of multiple ingredients in SI. S1-1 to S1-5, S2-1 to S2-5, and S3-1 to S3-5 represent the five SI samples from the three batches of preparations (Nos. 19050511, 18113011, and 18101511, respectively); the number in each box represents the content of the relative ingredient in the corresponding sample with the unit of μg/ml.

FIGURE 2

The mean plasma drug concentration–time curves and integrated plasma drug concentration-time curves of six ingredients after i.v., administration of SI with high (4 ml/kg, Mean ± SD, n = 3), medium (2 ml/kg, Mean ± SD, n = 3), and low (1 ml/kg, Mean ± SD, n = 4) dosages.

FIGURE 3

DOSE analysis plots for the five ingredients, (A) HSYA, (B) SYR, (C) RU, (D) SCU and (E) p-CA with favorable PK properties. The top 20 diseases relative to the corresponding ingredient and potential targets are shown in the graphs.

FIGURE 4

Molecular docking combinations of (A) HSYA, (B) SYR and (C) RU with their receptor targets, LTA-4H, SDH, and CTSB.

FIGURE 5

(A) Cell viability and (B) protection rate for the treatment of HSYA and SYR on cardiomyocytes injured by H₂O₂. ^* p < 0.05 and ^** p < 0.01 vs. model group (mean ± SD, n = 4); ^# p < 0.05 and ^## p < 0.01 vs. NAC group (mean ± SD, n = 3).

FIGURE 6

(A) Represents the SDS-PAGE analysis for the total proteins of C, M, HSYA and SYR group. (B,C) show the PCA analysis score plot and the difference proteins numbers, respectively for M vs. C, HSYA vs. M and SYR vs. M groups. (D–F) show the difference expression proteins location in the comparisons for M vs. C, HSYA vs. M and SYR vs. M groups, respectively. (G–I) show the GO annotation results of the difference expression proteins for M vs. C, HSYA vs. M and SYR vs. M groups, respectively. (J–L) show the downregulation and upregulation pathways for M vs. C, HSYA vs. M and SYR vs. M groups, respectively.

FIGURE 7

(A) Venn diagram of PK marker identification for SI and (B) the integrated drug plasma concentration-time curves by the effect weighting integrated method with the plasma concentration of two ingredients HSYA and SYR after i.v., administration of SI with high (4 ml/kg, Mean ± SD, n = 3), medium (2 ml/kg, Mean ± SD, n = 3), and low (1 ml/kg, Mean ± SD, n = 4) dosages.