FormalPara Key Points

The pharmacokinetic characteristics of Salvia miltiorrhiza and Carthamus tinctorius active ingredients were different in normal and cerebral ischemia rats

The reliability of the high performance liquid chromatography with diode array ultraviolet detection method for simultaneous determination of DSS, SAA, SAB, and HSYA in the plasma of normal and cerebral ischemia rats was fully validated by specificity, linear relationship, precision, recovery, and stability tests

1 Introduction

Dan-Hong injection is composed of extracts of Salvia miltiorrhiza and Carthamus tinctorius, which has the effect of promoting blood circulation and reducing blood stasis. The main active ingredients of S. miltiorrhiza (Danshen in Chinese) include fat-soluble and water-soluble ingredients, among which the water-soluble ingredients are danshensu (DSS), salvianolic acid A (SAA), and salvianolic acid B (SAB) (Fig. 1) [1]. The compatibility of these three active ingredients with hydroxysafflor yellow A (HSYA) in C. tinctorius (Honghua in Chinese) has a better therapeutic effect on cerebral ischemia [2,3,4]. SAA can scavenge free oxygen radicals [5, 6] and has a protective effect on cell damage caused by oxidative stress [7, 8]. SAB not only has strong antioxidant effects [9,10,11,12,13], but it can also inhibit apoptosis [14,15,16,17,18]. DSS is an important water-soluble ingredient, which has been pharmacologically shown to improve blood circulation [19] and eliminate blood stasis [20, 21], and can also protect against cerebral ischemia reperfusion injury [22]. HSYA can inhibit platelet aggregation [23, 24], reduce inflammatory response [25,26,27,28] and protect against myocardial ischemia [29, 30].

Fig. 1
figure 1

Chemical structures of the four active ingredients. a Danshensu; b salvianolic acid A; c salvianolic acid B; d hydroxysafflor yellow A

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The active ingredients of both S. miltiorrhiza and C. tinctorius have obvious pharmacological effects, and there are many studies examining the pharmacodynamic combinations of the active ingredients; however, there are few studies examining the pharmacokinetics of the active ingredients of S. miltiorrhiza and C. tinctorius in combination. The aim of this study was to establish a high-performance liquid chromatography with diode array ultraviolet detection (HPLC–DAD) method to examine the pharmacokinetic characteristics of the four above-mentioned active ingredients in both normal and cerebral ischemia rats, and to evaluate whether the pharmacokinetic properties in both normal and cerebral ischemia–reperfusion rats show any significant differences. The dosage of the four ingredients was from the results of our previous experiment [31].

2 Materials and Methods

2.1 Chemicals and Reagents

DSS (99%, purity, SZ201707038DSS), SAA (99%, purity, SZ201706001DA), SAB (99%, purity, SZ201706003DB) and HSYA (99%, purity, SZ201702005QA) were all obtained from Nanjing Shizhou Biology Technology Co., Ltd. (Nanjing, Jiangsu, China). Methanol and acetonitrile (chromatographic purity) were from Tedia Company (OH, USA). Water was of ultra-pure quality while the remainder of the chemicals used were of analytical grade.

2.2 Animal Experiments

Animal experiments were performed using adult male Sprague–Dawley (SD) rats (7–8 weeks, 280–300 g, Certification No. SCXK 2014-0001) which were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. The rats were nurtured under standard conditions of humidity (45 ± 5%) and temperature controlled (26 ± 2 °C) with an alternating 12 h light/12 h dark cycle with freely available drinking water. The animals were nurtured for 7 days before surgery, and then fasted for 12 h with free access to water before surgery. All experimental procedures strictly followed the National Institutions of Health Guide for the Care and Use of Laboratory Animals.

2.3 Instrumentation

The following instruments were used in this study—an Agilent 1200 High Performance Liquid Chromatography System (including a G1311A Quaternary Gradient Pump, a G1316A Column Thermostat, a G1315D Diode Array Detector, a G1315D DAD Detector, a G1322A online degasser, and a chemical workstation; Waldbronn, Germany); a 2-16PK Centrifuge (Sigma, Germany); an XS205DU METTLER Precision Scale (Mettler Toledo); an ND100-1 Nitrogen Purger (Hangzhou Ruicheng Instrument Co., Ltd., Hangzhou, China); a QL-861 Vortex Mixer (Haimen Kylin-Bell Lab Instruments Co., Ltd., Haimen, China); an SK5210HP Ultrasonic Cleaner (Zhangjiagang Deke Ultrasound Co., Ltd., Zhangjiagang, China); a pure water meter (Millipore, Seattle, USA); and an SHB-IIIA Circulating Water Multi-purpose Vacuum Pump (Henan Taikang Teaching Instrument Co., Ltd, Henan, China).

2.4 Focal Cerebral Ischemia Model of Rats

An improved rat model of middle cerebral artery occlusion (MCAO) using the thread embolization method, as reported by Longa et al., was used in this study [31]. Rats were intraperitoneally injected with 10% chloral hydrate (4 mL/kg) as an anesthetic. An incision was then made in the median position of the neck and blunt dissections of the right common carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) were performed. The CCA was ligated at the proximal end and the ECA root using a nylon thread and the ICA was closed using a miniature arterial clip. Another incision was made close to the bifurcation of the CCA and a nylon thread was inserted accordingly. The arterial clip was opened at the ICA end, and the line transited for approximately 20 mm until resistance could be felt before it was fixed. One hour later, the thread was gently extracted to finish the reperfusion. The incision was sutured and the rats returned to their cages. After recovery, Horner syndrome appeared in the right eye of the rats. The neurological symptoms of the rats were evaluated, and those with a score ≥ 2 were selected as cerebral ischemia model animals.

2.5 Grouping and Administration

The rats were divided into a normal group and a model group (n = 5). In the model group, DSS (30 mg/kg), SAA (10 mg/kg), SAB (8 mg/kg), and HSYA (4 mg/kg) were injected into the tail vein immediately after 1 h of cerebral ischemia. The normal group underwent the same injections as the model group.

2.6 Preparation of Reference Solution

The reference standards for SAA, SAB, DSS, and HSYA were precisely weighed, and dissolved in methanol to obtain a 1 g/L mother liquor. In parallel, a precise amount of p-hydroxybenzoic acid was weighed and dissolved in methanol to obtain a 1 g/L p-hydroxybenzoic acid solution as the internal standard mother liquor. The internal standard solution was diluted five-fold (0.2 g/L) prior to use.

2.7 Blood Sample Collection

Before administration, the rats were fasted with free access to water for 12 h. In the normal group, 0.5 mL of blank plasma was added to a test tube which contained 20 μL of sodium heparin. The model group was administered drugs via tail vein injection, and 0.5 mL of blood was collected from the jaw vein [55] at 2, 5, 10, 15, 30, 45, 60, 90, 120, 180, 240, and 360 min after administration. Sodium heparin (20 μL) was added as an anticoagulant. Samples were centrifuged at 3000 rpm (825g) for 12 min, and the supernatant was removed and stored at − 20 °C.

2.8 Plasma Sample Handling Methods

Plasma samples (100 μL) were transferred to a centrifuge tube containing 15 μL of the internal standard solution (0.2 g/L) after which 300 μL of methanol was added. The samples were vortexed for 5 min and then centrifuged at 13,000 rpm (15,493g) for 12 min. The supernatant was removed and then dried under a nitrogen stream. The dried residue was reconstituted with 100 μL of mobile phase and re-centrifuged at 13,000 rpm (15,493g) for 12 min, and the supernatant was used for HPLC analysis.

2.9 Chromatographic Conditions

An Eclipse XDB-C18 chromatographic column (4.6 mm × 250 mm, 5 μm) was used with a binary mobile phase system. Phase B was a 0.2% aqueous solution of phosphoric acid. The gradient elution consisted of 0–16 min, 90% A–72% A; 16–28 min, 72% A–65% A; 28–30 min, 65% A–90% A; A was 100% acetonitrile. The injection volume was 20 μL, the column temperature was 30 °C, the flow rate was 1.0 mL/min, and quantification was carried out by the internal standard peak area method. Chromatography was assessed using dual wavelengths—280 nm for SAA, SAB, and DSS and 403 nm for HSYA.

2.10 Data Analysis

Pharmacokinetic parameters were calculated using a non-compartment model in software Kinetica version 4.4 (Innapase, Massachusetts, USA). Statistical comparisons were made between different groups using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA) by an analysis of variance (ANOVA). All data were expressed as mean ± standard deviation.

3 Results

3.1 Specificity

The treated blank plasma, mixed reference solution, and plasma samples were analyzed according to the above-mentioned chromatographic conditions, as shown in Fig. 2. The results showed that under the selected chromatographic conditions, DSS, SAA, SAB, HSYA and the internal standard p-hydroxybenzoic acid could be completely separated from each other, had good peak shapes, and the endogenous peak impurities did not interfere with each other. These data indicated that the method had good specificity.

Fig. 2
figure 2

HPLC chromatograms of rat plasma samples. a Blank plasma 280 nm; b blank plasma 403 nm; c blank plasma + internal standard + DSS + SAA + SAB 280 nm; d blank plasma + internal standard + HSYA + SAA 403 nm; e plasma samples from rats 10 min after drug administration + internal standard 280 nm; f plasma samples from rats 10 min after drug administration + internal standard 403 nm. HPLC high-performance liquid chromatography, DSS danshensu, SAA salvianolic acid A, SAB salvianolic acid B, HSYA hydroxysafflor yellow A

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3.2 Linear Range and Detection Limit

The mixed reference solution was diluted with blank plasma to provide the following concentrations—DSS 100, 50, 25, 12.5, 4, and 0.5 µg/mL; SAA 200, 100, 50, 25, 6.25, and 0.5 µg/mL; SAB 120, 60, 30, 15, 5, and 0.5 µg/mL; HSYA 60, 30, 15, 7.5, 4, and 0.5 µg/mL. The above-mentioned mixed reference plasma solutions were treated according to the above-mentioned plasma sample handling methods and tested against the above-mentioned chromatographic conditions, and chromatograms and peak areas were recorded. The mass concentration of each drug in the plasma was plotted on the abscissa (X), and the ratio of the peak area of the drug to the internal standard was plotted on the ordinate (Y). The regression equations and correlation coefficients for the four ingredients were then obtained, as shown in Table 1. Based on a signal peak-to-noise ratio = 3, the minimum detection limits were DSS 0.05 µg/mL, SAA 0.04 µg/mL, SAB 0.01 µg/mL, and HSYA 0.02 µg/mL. The calibration curve for DSS, SAA, SAB and HSYA were Y = 0.034X + 0.0301 with R2 = 0.9995 in the range of 0.5–100.0 μg/mL, Y = 0.123X + 0.1142 with R2 = 0.9999 in the range of 0.5–200.0 μg/mL, Y = 0.0115X + 0.0694 with R2 = 0.9974 in the range of 0.5–120.0 μg/mL, and Y = 0.034X + 0.0301 with R2 = 0.9999 in the range of 0.5–60.0 μg/mL, respectively.

Table 1 Linear relationships for each of the four ingredients
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3.3 Precision and Recovery

A low, medium, and high concentration of each ingredient that lay in the linear range of the standard curve was selected, and the corresponding standard plasma samples were prepared using blank plasma, treated according to the above-mentioned plasma sample handling methods and tested against the above-mentioned chromatographic conditions, and the chromatograms were obtained. The intraday precision was calculated over five consecutive runs in a single day. The interday precision was calculated over five consecutive days in a single week. The results are shown in Table 2. The intraday relative standard deviation (RSD) was < 3% (n = 5) and the interday RSD was < 6% (n = 5) using this analytical method, showing that the assay meets the requirements for biological sample analysis.

Table 2 Precision, recovery, and stability of the four active ingredients in rat plasma
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The plasma samples containing low, medium, and high concentrations of the ingredients were prepared in blank plasma, and parallelized five parts for each mass concentration, and then subjected to the same treatment and test as those in the above-mentioned plasma sample handing methods and chromatographic conditions, respectively. The ratio of the peak area and internal standard for each measured ingredient was substituted into the regression equation to calculate the corresponding concentration in order to determine the recovery rate by comparing the average experimentally determined value with the theoretical value. The results are shown in Table 2.

3.4 Stability

Standard plasma samples were prepared containing the following concentrations of DSS (50, 25, 125 µg/mL), SAA (100, 50, 25 µg/mL), SAB (60, 30, 15 µg/mL), and HSYA (30, 15, 7.5 µg/mL). The plasma samples containing the different concentrations of drug were repeatedly frozen and thawed three times at − 20 °C. The plasma samples were treated and tested with the same methods as in the above-mentioned plasma sample handling methods and chromatographic conditions. The RSDs for each ingredient sample after freeze thawing were DSS 0.25%, 0.33%, and 0.37%; SAA 0.53%, 0.25%, and 0.08%; SAB 3.62%, 1.78%, and 1.59%; HSYA 0.27%, 0.33%, and 0.45%, for the respective concentrations listed above. These data indicated that sample stability after freeze-thawing was very good.

3.5 Pharmacokinetics Study

The plasma was treated as in the above-mentioned plasma sample handling methods and tested as in the above-mentioned chromatographic conditions. Using the standard curve, the plasma concentration of the different drugs was determined at different time points. To elucidate the compatibility of the four active ingredients, their concentration–time curves were obtained by plotting the plasma drug concentration on the ordinate and time on the abscissa (Fig. 3). Following this, the pharmacokinetic parameters were calculated (Table 3).

Fig. 3
figure 3

The concentration (µg/mL) and time curves of the four active ingredients in compatibility in normal and cerebral ischemia rats. a Danshensu (DSS); b salvianolic acid A (SAA); c salvianolic acid B (SAB); d hydroxysafflor yellow A (HSYA). C concentration, MCAO  middle cerebral artery occlusion

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Table 3 Pharmacokinetic parameters of four active ingredients in compatibility in normal and cerebral ischemia rats (mean ± standard deviation, n = 5)
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Based on the concentration–time curves and the pharmacokinetic parameters, the pharmacokinetics of the four ingredients in the normal and cerebral ischemia rats were consistent with a two-compartment model. The compatibility of four different ingredients may influence the plasma concentrations and pharmacokinetic parameters.

The four ingredients showed statistically significant differences in absorption and distribution phases.

For DSS, compared with normal rats, there was a statistically significant difference (P < 0.01) in cerebral ischemia rats in the distribution phase-elimination half-life (t1/2α), the distribution volume of central compartment (V1), the central compartment clearance (CL1), the peripheral compartment clearance (CL2), the primary clearance rate (K10), and the central compartment transfer rate (K12).

For SAA, compared with normal rats, there was an extremely significant difference (P < 0.01) in cerebral ischemia rats in t1/2α, the area under the concentration–time curve (AUC(0–t)), and K10; and a significant difference (P < 0.05) in K12, and the peripheral compartment transfer rate (K21).

For SAB, compared with normal rats, there was a statistically significant difference (P < 0.01) in cerebral ischemia rats in t1/2α, V1, the distribution volume of peripheral compartment (V2), CL1, CL2, AUC(0–t), and K12.

For HSYA, compared with normal rats there was significant difference (P < 0.01) in cerebral ischemia rats in t1/2α, V1, K10, and K12.

The above pharmacokinetic models and parameters were selected according to the maximum R2 value and the minimum Akaike’s information criterion (AIC) value. The pharmacokinetic data were matched to one compartment, two compartments, and three compartments where the maximum R2 value and the minimum AIC value were generated. Finally, a two-compartment pharmacokinetic model was selected. The results are shown in Table 4.

Table 4 Two-compartment model parameters for the four active ingredients in compatibility in normal and cerebral ischemia rats
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4 Discussion

The effect of treatment with Chinese medicines is often based on the interactions between multiple drug ingredients [32, 33]. These ingredients interact with each other in multiple pathways, interacting with different targets [34, 35], providing fewer side-effects [36,37,38]. Traditional Chinese medicines can therefore provide new approaches and solutions in the treatment of diseases such as cerebrovascular disease. In recent years, the clinical application of traditional Chinese medicines has increased significantly [39,40,41]. One aspect of the use of such medicines that requires close attention is how to achieve synergy and greater effects but at low doses. Pharmacokinetics can play a key role in the evaluation of drug effects [42], drug-dosing intervals, etc., and is considered an efficient means of elucidating the mechanism of herbal formulas [43].

A Dan-Hong injection is composed of extracts of S. miltiorrhiza and C. tinctorius, which has the effect of promoting blood circulation and reducing blood stasis. It is often used for the treatment of cardio-cerebral-vascular disease caused by blood stasis [30, 44, 45]. Existing studies have shown that DSS, SAA, SAB, and HSYA have the ability to scavenge free oxygen radicals, inhibit apoptosis, and prevent inflammation [5,6,7,8,9,10,11,12,13,14,15,16,17,18, 25,26,27,28], thereby protecting against cerebral ischemic injury. Over the past few years, the Dan-Hong injection and its ingredients have been widely studied in clinical and basic research [30, 46,47,48]. However, there are few studies evaluating the pharmacokinetic parameters of the active ingredients in the Dan-Hong injection.

Analysis of the components in the Dan-Hong injection and their pharmacodynamic effects has revealed that DSS, SAA, SAB, and HSYA are the main active ingredients [30, 46, 49,50,51]. Therefore, a pharmacokinetic study of the these four active ingredients appeared to be reasonable and necessary. In this study, we successfully created an HPLC method to determine the pharmacokinetic parameters for these four active ingredients and verified its sensitivity, specificity, and selectivity. All analytes were found not to have any interference with the endogenous matrix. The plasma sample preparation process is simple and fast, simply requiring the precipitation of proteins with methanol, making it suitable for the analysis of mixed samples. Here, the HPLC method was successful in determining plasma pharmacokinetic parameters for DSS, SAA, SAB, and HSYA following time-course changes in both normal and cerebral ischemia rats [52,53,54,55,56]. We hope that this pharmacokinetic study can be used to fully elucidate the behavior of interaction after multicomponent compatibility, and provide a reference for clinical drug application.

In the cerebral ischemia model group, due to severe damage of the vascular endothelial cells, vascular permeability increases, fluid leakage leads to blood stasis, blood flow is relatively obstructed, and plasma protein content may be lower than in normal rats, which affects the plasma protein rate of SAB [57] which, in turn, leads to an apparent distribution volume (V1 and V2) in the central and peripheral chambers of the model group, which is larger than the normal group.

Metabolomics suggests that MCAO-induced cerebral ischemia is mainly caused by energy metabolism disorders, lipid metabolism disorders, inflammatory responses, and oxidative stress. The neuroprotective effects of SAB are associated with improved energy metabolism, lipid metabolism, inflammatory responses, and oxidative stress [58]. Therefore, we speculate that the clearance rate (CL1, CL2) of the central and peripheral chambers of the model SAB group is increased on the basis of accelerating the metabolic reaction in the body when it produces the above improvement and neuroprotection. Meanwhile, the increase in the elimination phase half-life (t1/2β) indicates that the elimination time and the drug action time are both prolonged in the body.

A large amount of SAA enters the central nervous system of rats with ischemia–reperfusion and promotes its protection and regulation of disorder metabolism [59]. We speculate that the t1/2α of the model SAA group is reduced because SAA promotes its distribution in the body when it enters the central nervous system to protect and regulate.

In addition, the blood barrier of cerebral ischemia rats will be damaged, and some drug components can have a small amount of permeability. Among then, SAB has been proved to improve mitochondrial function and play a neuroprotective role [60]. SAA can also participate in the protection and repair of the blood–brain barrier (BBB) [61]. Therefore, the CL1 and CL2 value of SAB in the plasma of the model group increased and the t1/2α value of SAA decreased compared with the normal group. HSYA can also repair BBB function [62]; HSYA could be detected in cerebrospinal fluid of cerebral ischemic model rats. Therefore, it was confirmed that HSYA can act on the brain through BBB, which explains the significant differences of HSYA V1 and t1/2α in the model group and normal group; the pharmacological action would be likely to promote the distribution and elimination of HSYA, and explains why the V1 and t1/2α of HSYA in the model group and normal group have significant differences.

A further review of the literature shows that cerebral ischemia reperfusion can affect renal function [63] and may have resulted in decreased renal reabsorption function, making the drug more easily excreted, thus leading to accelerated clearance rate. We speculated that the t1/2α of DSS in the rats in the model group and the significant difference in clearance rate of CL1 and CL2 between rats in the normal group and the model group might be related to this.

Our study provides an innovative theory and good research ideas for the research of other proprietary Chinese medicines by detecting the pharmacokinetic characteristics of the active components of Dan-Hong injection in normal and cerebral ischemia rats and evaluating whether there are significant differences in pharmacokinetic properties in vivo. Simultaneously, the formula in our study plays an important role in the research on the creation of new drugs for the treatment of cerebral ischemic diseases, the improvement of dosage forms and the mechanism of prescription mechanism.

5 Conclusion

A HPLC–DAD method was established for simultaneous determination of DSS, SAA, SAB, and HSYA in the plasma of normal and cerebral ischemia rats. The reliability of this method was fully verified by specificity, linear relationship, precision, recovery, and stability tests. The main active ingredients of Danshen and Honghua in the plasma of normal and cerebral ischemia rats were quantitatively analyzed by this method. The pharmacokinetics of the tested ingredients in normal and cerebral ischemia rats were determined. The pharmacokinetics and characteristics of the tested ingredients were quite different in normal and cerebral ischemia rats. This study had certain reference value for studying the pharmacokinetics and characteristics of the main active substances of traditional Chinese medicine under pathological conditions.