Pharmacokinetics comparison of four major bio-active components in normal and blood stasis rats after administration of Naoxintong Capsule by UPLC-TQ/MS


 Background Blood stasis is one major cause of cardiovascular and cerebrovascular diseases. Naoxintong capsule (NXTC), a Chinese patent medicine, has been widely employed in the prevention and treatment against cardiovascular and cerebrovascular diseases. However, the pharmacokinetics comparison of NXTC in normal and blood stasis rats were remain obscured. Methods Acute blood stasis rats were induced by being placed in ice-cold water during the interval between two injections of adrenaline hydrochloride. Normal and blood stasis rats were administrated of NXTC suspension at the dosage of 5 g⋅kg -1 , and blood was collected at different time points after then. Concentrations of four main components including caffeic acid, ferulic acid, formononetin and tanshinone IIA in rat plasma were quantified by UPLC-TQ/MS. The pharmacokinetic parameters were calculated by Phoenix WinNonlin v6.2 software. Results It was found that C max , AUC all , AUC INF_obs , Vz_F_obs and MRT last of ferulic acid, AUC all, Vz_F_obs and MRT last of caffeic acid in blood stasis rats were significantly different ( P < 0.05) from normal rats. Compared with normal rats, C max of ferulic acid and formononetin decreased significantly in the plasma of acute blood stasis rats, AUC all of caffeic acid and ferulic acid decreased notably, AUC INF_obs of ferulic acid decreased remarkably, Vz_F_obs and MRT last of ferulic acid and caffeic acid increased reversely. It is suggested that the absorption of the four components of NXTC in acute blood stasis rats was weakened, and the elimination time was prolonged. Conclusions The significant difference in some different parameters of the 4 NXTC components in normal and blood stasis rats might be caused by increasing of blood viscosity and slowing down of blood flow in acute blood stasis rats. The pharmacokinetic study under pathological condition provided important information for more rational use of NXTC in clinical situations.

National Basic Drug List (2012 edition). Modern researches show that the pharmacological effects of NXTC mainly focus on improvement of blood rheology and blood coagulation function, anti-myocardial ischemia and ischemia/reperfusion injury, anti-atherosclerosis, anti-myocardial brosis, anti-cerebral I/R injury, improvement on learning and memory functions [3]. It is commonly used for the treatment of coronary heart disease, angina pectoris, stroke, secondary prevention of myocardial ischemia, cerebral infarction, transient ischemic attack, vertebro-basilar insu ciency, carotid atherosclerosis, and other cardiovascular and cerebrovascular diseases [4].
Promoting blood circulation and removing blood stasis is the basic pharmacological action of NXTC in the treatment of cardiovascular and cerebrovascular diseases [5]. The active chemical ingredients of NXTC mainly include avonoids, avonoid glycosides, phenanthraquinones, phenolic acids, terpenoids, and polysaccharide, which are related to the pharmacological functions of NXTC on inhibition of platelet aggregation, anti-in ammation, reduction of apoptosis and ROS production, activation of lipid metabolism, promotion of angiogenesis and lesion plaque stability, attenuation of vascular calci cation [1,[6][7][8][9]. Many researchers have been trying to explore and identify the major and bioactive constituents of NXTC, and many chemical constituents in NXTC in vitro were quanti ed [10][11][12].
Nowadays, it is of great signi cance to study the pharmacokinetics and metabolism of TCH components for promoting its modernization. Therefore, some studies were conducted to explore the pharmacokinetics and metabolites of the main components of NXTC. A total of 36 prototype compounds and 52 metabolites of NXTC were identi ed or tentatively characterized in beagle dog urine and feces [13]; the pharmacokinetic pro les of caffeic acid, ferulic acid, formononetin, cryptotanshinone and tanshinone IIA in healthy rats have already been reported [14]. However, it is generally known that when the body is in pathological state, the pharmacokinetic process of compounds will change, and even has a signi cant difference with the normal state [15,16]. Therefore, it is necessary to study the pharmacokinetic parameters changes in pathological state. In addition, the pharmacokinetic data obtained in pathological state will be more bene cial to clinical application than in normal state. Based on this, we employed the pharmacokinetics comparison of the main active components of NXTC between normal and blood stasis model rats, which will provide more valuable data for illuminating the variation mechanism of invigorating qi and activating blood circulation, removing blood stasis and dredging collaterals after oral administration of NXTC, and for better clinical applications of NXTC.
Internal standards including clarithromycin and propione sulphonate were purchased from National Institutes for Food and Drug Control with purity above 98% (Beijing, China). Ultra-pure water was puri ed by Molecular waters puri cation system. Acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany). Formic acid (HPLC grade) was purchased from Dikma Technology Co., Ltd (Tianjin, China). Ultra-pure water was puri ed by using a Milli-Q system (Milford, MA, USA).

Ethical statement
Female Sprague-Dawley (SD) rats (6-8 weeks old, weighing 220 ± 20 g) were purchased by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Before the experiment, the rats were fed adaptively for one week, keeping the circadian rhythm for 12 hours and feeding and drinking freely. The temperature and humidity meet the requirements of animal feeding. Animal care and experimental protocols were performed based on 'Detailed Rules and Regulations for Administration and Implementation of Biomedical Animal Experiments' (No. 1998-55, Ministry of Public Health, China). The study protocol was approved by the Ethical Committee of the First A liated Hospital of Henan University of Chinese Medicine.

NXTC Solution Preparation
The content of NXTC was dispersed in 0.5% CMC-Na aqueous solution to produce the NXT suspension with the concentration of 0.33 g/mL.
Preparation of reference substance reserve solution, internal standard reserve solution and quality control sample Caffeic acid, ferulic acid, anthocyanin and tanshinone II A were weighed accurately and then serially dissolved in methanol to obtain the reference stock solutions with different concentrations of 30.5, 30, 38 and 21.5 mg/mL respectively. Six series of mixed reference solutions were gained with appropriate individual reference stock solution mixed and diluted with methanol in volumetric ask: 0.5, 1, 5, 10, 50 and 100 ng/mL caffeic acid, 1, 5, 10, 50, 100 and 500 ng/mL ferulic acid, 0.05, 0.1, 0.5, 1, 5 and 10 ng/mL anthocyanin and 0.5, 1, 5, 10, 50 and 100 ng/mL tanshinone II A.
As internal standard substances, clarithromycin and probenecid were weighed precisely and dissolved with methanol to obtain the internal standard reserve solutions with concentrations of 532 and 536 mg/mL respectively. Appropriate amount of internal standard reserve solutions was diluted with methanol to provide the mixed internal standard reserve solution with clarithromycin of 106.4 ng/mL and probenecid of 107.2 ng/mL. Quality control (QC) samples of caffeic acid with concentrations of 30, 50, 500 ng/mL, ferulic acid with concentrations of 10, 20, 200 ng/mL, anthocyanin with concentrations of 0.5, 1, 10 ng/mL and tanshinone II A with concentrations of 1, 5, 50 ng/mL were prepared with blank plasma.
The ESI source was set in both positive and negative ionization mode. The parameters in the source were set in the following manner: positive ion scanning voltage, 3500 V; negative ion scanning voltage, 2500 V; sheath gas velocity, 25 arb; auxiliary air velocity, 7 arb; collision gas pressure, 1.5 mTorr; ion transmission tube temperature, 325 °C; ion source temperature, 400 °C. The scanning mode was set multiple reaction monitoring (MRM) mode and the selected monitor ion were m/z 178.96®135.04 for caffeic acid, m/z 192.95®134.00 for ferulic acid, m/z 267.00®251.97 for anthocyanin, m/z 295.05®277.20 for tanshinone IIA, m/z 748.35®590.29 for clarithromycin, and m/z 284.00®240.05 for propafenone. The collision energy and RF-lens were optimized for precursor/product ion pairs of each analyte and the selected values are presented in Table 1.

Modeling, drug administration and preparation of plasma samples
In this experiment, twenty rats were randomly divided into blank administration group and model administration group with 10 rats in each group respectively. After 7 days of acclimation, the rats in the model administration group were given adrenaline hydrochloride injection (0.8 mg/kg) subcutaneously.
After 2 h, the rats were placed in 0°C ~ 2°C ice water to swim for 4 minutes, the rst time 4 hours after the administration of epinephrine hydrochloride injection, subcutaneous injection of epinephrine hydrochloride injection 0.8 mg/kg was performed again, resulting in a model of acute blood stasis in rats. All rats fasted for 12 hours and drank freely. NXTC suspension was administrated by gavage at a dose of 5g/kg. At 0.083, 0.167, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 10 and 12 hours after administration, blood samples were collected from the eye orbital venous plexus of rats and placed in a heparinized centrifuge tube. Plasma was separated by 3000 r/min centrifugation for 10 min and stored at -80 °C for determination.
Plasma Sample Preparation 100 mL of plasma sample was precisely pipetted and 20 mL of 10% formic acid water, 10 mL of internal standard solution were added, the mixture was vortexed for 3 min, then 300 mL of methanol was added and vortexed for 5 min additionally. Subsequently, the sample was centrifugated at 14 000 r/min for 10 min. The supernatant was centrifugally concentrated to dryness under vacuum condition. The residue was re-dissolved in 100 mL methanol, agitated on a vortexer for 3 min and centrifugated at 14000 r/min for 10 min, then the supernatant was obtained for analysis.

Speci city
The blank plasma of rats, blank plasma of rats added with reference solution and internal standard solution, and the plasma samples obtained from rats of model group were taken 100 mL respectively, the samples were processed applying the sample processing procedure in item 2.5. The ion chromatograms of the ingredients were acquired abiding by the corresponding chromatographic and mass spectrometric methods in item 2.3.
linearity, limit of detection (LOD) and lower limit of quanti cation (LLOQ) Six duplicates of 100 mL blank rat plasma were added with a series of mixed reference solutions with different concentrations. The samples were processed according to the sample processing procedure in item 2.5 and conducted LC-MS/MS analysis. Taking the concentration of each substance to be measured as the abscissa (x), the ratio of the peak area of the substance against the internal standard as the ordinate (y), and the reciprocal of the concentration (1/x) as the weighting coe cient for linear regression, the regression equations of the calibration curves of each ingredient in rat plasma were established. By comparing the results of samples with known analyte concentration and those of blank samples, the signal-to-noise ratio (S/N) of 3:1 was determined to be the LOD, the S/N of 10:1 was determined to be the LLOQ, which is the lowest concentration point of the standard curve.

Precision and accuracy
Certain duplicates of 100 mL blank plasma were respectively added with different concentrations of mixed reference solution and internal standard solution to prepare the lower limit of quanti cation sample and quality control samples with low, medium and high concentrations. Six samples were prepared in parallel with each concentration, which were processed according to the procedure displayed in item 2.5. The measured concentration of QC sample was were calculated according to the accompanying standard curve. Measurements for 3 consecutive days were implemented to investigate the accuracy and precision of the established analysis method.

Recovery of extraction and matrix effect
Quality control samples of low, medium and high concentrations prepared from 100 mL blank plasma were processed according to the sample processing method above. The peak area of each component was recorded as A1. Other duplicates of 100 mL blank plasma were also processed according to the method above. The residues were respectively re-dissolved in reference solutions of low, medium and high concentration containing internal standard. The peak area of each component was recorded as A2. The low, medium and high concentration reference solutions containing internal standard were also respectively injected into the analysis system. The peak area of each component was recorded as A3 here. Each sample was prepared in 6 duplicates. The recovery (%) was the ratio of A1 to A3, and the matrix effect (%) was the ratio of A2 to A3.

Stability
The quality control samples with low, medium and high concentration were prepared and placed at 4 °C for 0, 2, 4, 8, 12 and 24 h for analysis. The quality control samples were also frozen at -80 °C and thawed at room temperature for 3 cycles with 6 duplicates for each concentration respectively. According to the established calibration curve, the measured concentration of QC sample was calculated, and compared with the labeled concentration to investigate the stability of each component in rat plasma.

Data processing and analysis
The data collection and sample analysis were controlled by Trace nder 4.1 software and the data were processed with Microsoft Excel. The content of each compound was expressed as x ± s. The obtained data of content were processed with Phoenix Winnonlin (version 6.2, Pharsight Corporation, USA) pharmacokinetic software, and the pharmacokinetic parameters were calculated applying noncompartment model.

Contents of four components in Naoxintong Capsule
The LC-MS/MS analysis of the sample solution revealed that the contents of caffeic acid, ferulic acid, anthocyanin and tanshinone IIA in Naoxintong capsule were determined as 0.011, 0.099, 0.060 and 0.148 mg×g -1 respectively. The chemical structure formulas of the four compounds are shown in Figure 1.

Methodological investigation
Speci city test The endogenous substances in blank plasma did not interfere with the determination of caffeic acid, ferulic acid, anthocyanin, tanshinone IIA, clarithromycin and probenecid. The extracted ion ow chromatograms of each component are shown in Figure 2.
Linearity, LOD and LLOQ The calibration curve and lower limit of quanti cation of the four components are displayed in Table 2.
The linearity of each component is good within the corresponding concentration range, and the correlation coe cient is greater than 0.99, meeting the requirements of in vivo drug analysis method.

Precision and accuracy
Three QC samples of different mass concentrations with six duplicates for each were analyzed for three consecutive days. The results showed that the RSD of intra-day precision, intra-day precision and accuracy of each sample could meet the requirements (Table 3).

Recovery and matrix effect
The results of matrix effect and extraction recovery of each component could meet the requirements of biological sample detection (Table 4).

Stability
The stability of the plasma sample is good after 24 hours of cold storage (4 °C) or 3 cycles of freeze-thaw (Table 5).

Comparative analysis of blood concentration-time curves for four blood components
The blood concentration-time curves of caffeic acid, ferulic acid, formonetin and tanshinone IIA in NXTC in normal and acute blood stasis model rats are shown in Figure 3.

Comparative analysis of pharmacokinetic parameters
The statistical analysis results of main pharmacokinetic parameters of caffeic acid, ferulic acid, formononetin and tanshinone IIA in normal and acute blood stasis model rats intragastrically administrated with NXTC are shown in Table 6. As the pharmacokinetic parameters of caffeic acid, ferulic acid, formononetin and tanshinone IIA exhibited above, the peak time (T max ) of the four components was 0.12 ± 0.05, 0.11 ± 0.04, 0.08 ± 0.00 and 0.13 ± 0.05 h, respectively, which indicated that the above four components could be absorbed into the blood rapidly. Concluded from the results of AUC all and AUC INF_obs and the peak concentrations (C max ) of the four components being 5.01±0.74, 71.37±15.56, 5.70±1.19 and 2.08±0.57 mg/L, ferulic acid was absorbed best in body of normal rats, with C max being 12-35 times of the other three components. Tanshinone IIA exhibited the lowest C max , indicating that its absorption being worse than the other three components. However, the apparent distribution volume (VZ_F_obs) and clearance rate (CL_F_obs) of caffeic acid and ferulic acid were smaller than those of formononetin. With the largest VZ_F_obs and CL_F_obs, tanshinone IIA possessed a widespread in vivo distribution. The average retention time of four components was 4.40 ± 0.46 ~ 5.78 ± 0.29 h.

Discussion
In this experiment, compared with the normal group, the T max of the four components of NXTC were prolonged in acute blood stasis model rats with no statistical difference and the C max of the four components were decreased, with the decrease of ferulic acid and formononetin being statistically signi cant (P < 0.05), which suggested that the blood circulation disorder caused by acute blood stasis hindered the absorption of the four components. The AUC all of caffeic acid and ferulic acid were signi cantly reduced (P < 0.05) in model group. The AUC INF_obs of ferulic acid was also remarkable reduced (P < 0.01). It was observed that compared with the normal group, the clearance rate of tanshinone IIA in the model group was reduced, which allowed more tanshinone IIA to remain in rats body and play its role in the treatment of blood stasis. Signi cant elevations of the plasma concentration of ferulic acid and caffeic acid (P < 0.05) and increased in the mean retention time (MRT last ) of caffeic acid and ferulic acid (P < 0.05) increased as well. The difference of the above results might be due to the characteristics of "viscosity, concentration, coagulation and aggregation" of the blood in acute blood stasis model rats, which makes the blood viscosity increase, the blood ow slow down, the absorption of components slow down, T max and MRT last prolonged.
Blood stasis syndrome is a pathological state of blood circulation, which is described as slow or accumulation of blood due to xinqi disorder in traditional Chinese medicine (TCM) and TCM believes that the "sadness" of seven emotions (qiqing) and the "cold evil" of six evils (liuxie) are the main causes of acute blood stasis [17]. Now blood stasis is usually understood as a blood system disease, pathological studies show that blood stasis is mainly characterized by cardiovascular and cerebrovascular diseases, such as cerebral infarction, myocardial infarction, coronary heart disease, hypertension and so on [17][18][19]. The e cacy of NXTC capsule is replenishing qi and activating blood, removing blood stasis and dredging collaterals. It is often used in the treatment of cardiovascular and cerebrovascular diseases, which is closely related to its main components caffeic acid, ferulic acid, formononetin and tanshinone IIA.
Numerous studies have demonstrated that caffeic acid and its derivatives have pharmacological effects such as antioxidation, immune regulation, anti-cancer, regulation of cardiovascular and cerebrovascular diseases and protection of brain tissue damage [20][21][22]. Ferulic acid is a metabolite of caffeic acid methylation, which can promote bone marrow hematopoiesis, enhance immunosuppression, protect cardiovascular system, reduce blood lipid, resist arteriosclerosis and inhibit platelet aggregation [23][24][25]. Formononetin has pharmacological actions on improving atherosclerosis and inhibiting the proliferation of vascular smooth muscle cells [26,27]. In recent years, tanshinone IIA has attracted the attention of much more researchers in cardiovascular and cerebrovascular aspects. The protective effects of tanshinone IIA on the heart include preventing the formation of atherosclerosis, preventing myocardial injury and hypertrophy, expanding coronary arteries and related mechanisms of action have been reported [28][29][30][31].
In the pathological state, the pharmacokinetic of drug is different from the normal state due to changes in the physiological state and biochemical response of the body. Given that NXTC is usually used for the treatment of patients with blood stasis, we studied the pharmacokinetics of NXTC in rats with acute blood stasis. The acute state of blood stasis may be caused by abnormal hemorheology, such as the increase of blood viscosity and coagulation degree, blood aggregation, vascular obstruction and so on [32,33]. In the model group, it can be observed from the overall point of view that the T max and C max of the four components were prolonged and decreased, respectively, parameters of AUC all , Vz_F_obs and MRT last were changed as well, indicting that the absorption and metabolism of NXTC were affected in acute blood stasis. These results provide important information for guiding the clinical rational use of drugs.

Conclusion
Herein, we have established the analytical method to probe the pharmacokinetic properties of the four main ingredients caffeic acid, ferulic acid, formononetin and tanshinone IIA of NXTC in acute blood stasis rats compared with the corresponding pro le in normal rats. The parameters demonstrated that the C max of ferulic acid and formononetin decreased statistically signi cantly, the AUC all /AUC INF_obs of ferulic acid and the AUC all of caffeic acid were all signi cantly reduced. The plasma concentration and mean retention time (MRT last ) of ferulic acid and caffeic acid all increased. The alteration of the pharmacokinetics was consistent with the change of the rheological characteristics of the blood in acute blood stasis rats. These investigations may provide experimental basis for the plan adjustment of clinical medication of NXTC for patients with blood stasis. Abbreviations NXTC: Naoxintong Capsule; QC: quality control; ESI: electrospray ion source; MRM: multi response monitoring; IS: internal standard; LOD: limit of detection; LLOQ: lower limit of quanti cation; S/N: signal-to-noise; Cmax: peak concentrations; Tmax: peak time; VZ_F_obs: apparent distribution volume; CL_F_obs: clearance rate; MRTlast: the mean retention time.
Declarations Acknowledgements Not applicable.
Authors' contributions WXL and PPC contributed equally to this work. JFT and XLL conceived and designed the study. HZ was mainly responsible for the instrument operation. WXL and SQZ conducted the data analysis and wrote the manuscript. PPC and MML revised the manuscript. XYW and LN conducted the animal experiment. CXL provided the advice for the study. All authors read and approved the nal manuscript.      with the normal group, * P < 0.05; ** P < 0.01. Figure 1 Chemical structures of four main components in Naoxintong Capsule.

Figure 2
Typical MRM chromatograms of various components in rat plasma.

Figure 3
Blood concentration-time curves of caffeic acid, ferulic acid, menthol and tanshinone IIA in normal and acute blood stasis model rats intragastric administration of NXTC.