MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study

Accepted Manuscript Title: Development and validation of a UFLC-MS/MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study Author: Shijun Yue Liang Wu Cheng Qu Yuping Tang Yi Jin Shujiao Li Juan Shen Xuqin Shi Chenxiao Shan Xiaobing Cui Li Zhang Haijun Yang Li Qian Dawei Qian Jin-ao Duan PII: DOI: Reference:

S1570-0232(15)30188-4 http://dx.doi.org/doi:10.1016/j.jchromb.2015.09.013 CHROMB 19622

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

23-3-2015 9-9-2015 11-9-2015

Please cite this article as: Shijun Yue, Liang Wu, Cheng Qu, Yuping Tang, Yi Jin, Shujiao Li, Juan Shen, Xuqin Shi, Chenxiao Shan, Xiaobing Cui, Li Zhang, Haijun Yang, Li Qian, Dawei Qian, Jin-ao Duan, Development and validation of a UFLC-MS/MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.09.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development and validation of a UFLC-MS/MS method for the determination of anhydrosafflor yellow B in rat plasma and its application to pharmacokinetic study Shijun Yuea, Liang Wua, Cheng Qua, Yuping Tanga* [email protected], Yi Jina, Shujiao Lia, Juan Shena, Xuqin Shia, Chenxiao Shanb, Xiaobing Cuib, Li Zhanga, Haijun Yangc, Li Qiana, Dawei Qiana, Jin-ao Duana* [email protected] a

Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and

National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, and Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210023, China b

Analytical Instrumentation Center, School of Pharmacy, Nanjing University of Chinese Medicine,

Nanjing 210023, China c

Jiangsu Jiankang Vocational College, Nanjing, 210029, China

*

Corresponding author. Tel./Fax: +86 25 85811695.

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HIGHLIGHTS 1. A sensitive UFLC-MS/MS method for the quantification of anhydrosafflor yellow B (AHSYB) in rat plasma has been developed and validated. 2. The method was successfully applied to the pharmacokinetic study of AHSYB in normal and acute blood stasis syndrome rats. 3. The oral bioavailability of AHSYB was very low and the AUC0-t, AUC0-∞ and F were all significantly lower (P < 0.05) in acute blood stasis syndrome rats.

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ABSRTRACT A sensitive ultrafast liquid chromatography coupled with triple quadrupole mass spectrometric (UFLC-MS/MS) method for the quantification of anhydrosafflor yellow B (AHSYB), a major active water-soluble pigment from Carthamus tinctorius, in rat plasma has been developed and validated. Sample preparation was achieved by protein precipitation of plasma with four volumes of methanol. Rutin was used as the internal standard (IS). The analytes were separated using a C18 column with an 8 min gradient elution, followed by mass spectrometric detection using negative electrospray ionization (ESI−) in multiple reaction monitoring (MRM) mode. The method was linear in the concentration range of 25-10000 ng/mL for AHSYB. Intra-day and inter-day precision variation was less than 6.5%. The relative error of accuracy was within ± 9.4%. The mean recovery of AHSYB was higher than 70.9%. The established method was successfully applied to the pharmacokinetic study after intravenous (2.5 mg/kg) and oral (30 mg/kg) dosing of AHSYB in normal rats. And the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats were also investigated. The results showed that the compound was poorly absorbed (~ 0.3%) and the AUC0-t, AUC0-∞ and F were all significantly lower (P < 0.05) in acute blood stasis syndrome rats, suggesting that disease condition may alter the body metabolism by enhancing metabolite enzyme activity. Keywords: Carthamus tinctorius; AHSYB; Pharmacokinetic study; UFLC-MS/MS; Acute blood stasis syndrome.

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1. Introduction Carthamus tinctorius L. has been used as a food additive, a natural pigment and an herbal medicine in oriental countries for thousands of years [1]. The dried florets of Carthamus tinctorius (“Honghua” in Chinese), as a common traditional Chinese medicine (TCM), have mainly been taken as decoction to treat stroke, coronary heart disease and angina pectoris in TCM prescriptions [2], therefore the water-soluble components should be responsible for the observed therapeutic effects. The pharmacological studies demonstrated that Honghua water extract had lots of effects, such as anti-coagulant [3], vasodilation [4], anti-oxidant [5], neuroprotection [6-8], immunosuppressant [9] and antagonizing renal fibrosis [10]. It is worth mentioning that Honghua water extract has been developed as an intravenous injection to be extensively applied to treat cardiovascular

diseases

in

Chinese

hospitals

[11].

Quinochalcone

C-glycosides,

quinone-containing chalcones that are oxidized at the A ring, are regarded as the main active and characteristic

compounds

in

the

water

extract.

Among

quinochalcone

C-glycosides,

hydroxysafflor yellow A (HSYA) has been demonstrated to restrain the conglomeration of platelets, promote blood circulation, remove blood stasis, promote metabolism, and anti-oxidation [1]. Hence, HSYA is chosen as an active marker component for quality control of Honghua in Chinese Pharmacopoeia [12] and has been developed into powder injection. Except for HSYA, anhydrosafflor yellow B (AHSYB) was reported to be half as much as HSYA, the contents of which varied from 1.29 to 10.43 mg/g [13]. Meanwhile, AHSYB could inhibited ADP-induced platelet aggregation [14], exhibited more significant anti-oxidative effects than HSYA in vitro [15], and possessed certain activity against H2O2-induced cytotoxicity in cultured H9c2 cells [16]. However, to the best of our knowledge, there is no paper to focus on qualitative evaluation and 4

quantitative determination of AHSYB in biological specimen, such as plasma, urine, tissue and bile. In this study, an accurate and sensitive UFLC-MS/MS method for the determination of AHSYB in rat plasma was developed. The developed method was validated and applied to the succeeding pharmacokinetic study after intravenous (2.5 mg/kg) and oral (30 mg/kg) dosing of AHSYB in normal rats. Blood stasis syndrome is widely common in TCM clinic. Honghua has the function of promoting blood circulation by removing blood stasis and alleviating pain in TCM theory and clinic [17]. Many researches also demonstrated that Honghua and its active components, including HSYA and safflor yellow B could improve hemorheological events and exert anticoagulant effects [13,18-20]. Furthermore, HSYA have proved to be with high uptake and eliminated slowly in rats with blood stasis syndrome [21]. Hence, it is of interest to investigate the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats.

2.

Experimental

2.1.

Chemicals and materials

AHSYB and rutin were previously isolated and identified from Honghua in our laboratory, and identified by MS, 1H-NMR,

13

C-NMR and UV spectra. The purities of AHSYB and rutin (IS)

were > 98%, determined by HPLC analysis. Their structures were shown in Fig. 1. Acetonitrile (MeCN) and formic acid were HPLC-grade from Merck (Darmstadt, Germany), and ultrapure water (18 MΩ cm) was obtained from the Milli-Q system (Millipore, Bedford, MA, USA).

2.2. Instrumentation and chromatographic conditions 5

Chromatographic analysis was performed using a Shimadzu UFLC system (Kyoto, Japan) equipped with two LC-20AD pumps, a SIL-30AC autosampler and a CTO-20AC column oven. A Thermo Hypersil GOLD column (50 mm × 2.1 mm, 1.9 μm) was employed for all the analyses. The mobile phase consisted of MeCN (A) and 0.01% formic acid aqueous solution (B) using a gradient elution, which programmed as follows: 10% A at 0−1 min, 10-40% A at 1−4 min, 40−80% A at 4−5 min, 80% A at 5−5.5 min, and then an immediate reduction to 10% A at 5.7 min, 10% A at 5.7−8.0 min for equilibration of the column. The mobile phase was directly delivered into the negative-ion mode (ESI−) source at 0.2 mL/min with 2 μL per injection. The column and the sample manager temperature were kept constant at 35 °C and 4 °C, respectively. Mass spectrometry detection was performed using AB SCIEX QTRAP 5500. The analytes were determined and quantified by multiple-reaction monitoring (MRM) mode in ESI-. Mass spectrometry was operated with an optimized spray voltage at -5000 V and turbo spray temperature at 500 °C. The nebulizer gas (gas 1), auxiliary gas (gas 2) and curtain gas were at 30, 40 and 30 psi, respectively. The precursor-to-product ion pairs, the optimized declustering potential (DP) and collision energy (CE) for each analyte were listed in Table 1.

2.3. Samples Preparation

The stock solutions of AHSYB and rutin were both prepared in methanol at concentrations of 0.5 mg/mL. Working solutions of AHSYB with concentrations in the range of 250-100000 ng/mL were obtained by diluting the stock solution with methanol. An IS working solution of 2000 ng/mL was obtained by diluting corresponding stock solution with methanol. All the solutions were stored at 4 °C and brought to room temperature before use. The calibration standards were prepared by spiking drug-free rat plasma (90 μL) with a working 6

solution (10 μL) to yield AHSYB concentrations of 25, 50, 100, 250, 500, 1000, 2500, 5000 and 10000 ng/mL. Quality control (QC) samples were prepared in the same way as calibration standards at nominal AHSYB concentrations of 50, 500 and 5000 ng/mL. 2.4.

Sample pretreatment

One step protein precipitation with methanol was used to extract the analytes from plasma. After thawed to room temperature, rat plasma sample (100 μL) was pipetted into 1.5 mL EP tubes followed by IS (10 μL) and methanol (390 μL). The mixture was vortex-mixed for 3 min and then centrifuged at 15000 rpm for 10 min to separate the precipitated protein. The supernatant was transferred to fresh 1.5 mL EP tube and centrifuged at 15000rpm for another 10 min. At last, 2 μL of the resulting supernatant was injected for UFLC-MS/MS analysis. Once AHSYB concentrations in some samples exceed the upper limit of the standard curve, the resulting supernatants will be diluted proportionately.

2.5. Method validation

2.5.1. Selectivity and specificity Blank plasma samples from six different rats were processed without AHSYB and IS to ensure the absence of interfering peaks. Each blank sample was analyzed and compared with those obtained by spiking AHSYB (25 ng/mL) and IS (50 ng/mL) into the corresponding blank plasma sample.

2.5.2.

Linearity and lower limit of quantification (LLOQ)

The calibration curve was constructed by plotting the ratio of peak area of AHSYB to that of IS versus the nominal concentration of calibration standards and fitted to linear regression (y = ax + b)

7

using 1/x as the weighting factor. The concentrations of analytes in test samples were calculated by the regression parameters from the calibration curves. The LLOQ was assessed as the lowest concentration on the calibration curve that could be quantitatively determined with an acceptable precision variation less than 20% and an accuracy error within ± 20%, which was established based on six replicate calibration curves.

2.5.3.

Precision and accuracy

The precision and accuracy of the assay were evaluated by analyzing QC samples at three concentrations. To determine the intra- and inter-day precision and accuracy, six replicates were analyzed at each concentration level on three consecutive validation days. The calibration curve constructed on the same testing day was used for the quantification of each sample. The criteria for acceptability of the data included the relative standard deviation (RSD) less than 15% and relative error (RE) within ± 15%.

2.5.4.

Recovery and matrix effect

The extraction recoveries of AHSYB from rat plasma at three QC levels following protein precipitation were measured individually by comparing the mean peak areas obtained from six plasma samples spiked with the analytes prior to extraction with those added after extraction. The ratio gives the percentage recovery. The matrix effect of AHSYB was measured by comparing the response of the solution at three different QC levels spiked in plasma matrix v.s. ultrapure water after the same extraction process using 4 volumes of methanol (n=6); and for IS, matrix effect was also investigated at two concentration levels (20 and 100 ng/mL).

2.5.5. Stability

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QC samples at three concentrations were subjected to analysis following the conditions given below: at room temperature for 6 h, in autosampler at 4°C for 24 h, undergo three freeze-thaw cycles (-20°C to room temperature as one cycle) and at -20°C for 6 days. All stability testing QC samples were determined using freshly prepared calibration curve.

2.5.6.

Quality control

A series of QC samples at three concentrations were evenly distributed across the sample test sequence to ensure results’ reliability.

2.6.

Pharmacokinetic study

Fifteen Sprague-Dawley rats (220-250 g) were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai), and kept in an environmentally controlled breeding room (temperature: 20 ± 2 °C, humidity: 60 ± 5%, 12 h dark-light cycle) for 1 week before the experiment operated. In present study, animal welfare and experimental procedures were strictly in accordance with the guide for the Care and Use of Laboratory Animals (US National Research Council, 1996) and the related ethics regulations of Nanjing University of Chinese Medicine. The rats were divided into three groups at random (normal oral group, normal intravenous group and acute blood stasis group, respectively) and fasted for 12 h with free access to water prior to the experiment. The rats in acute blood stasis group were hypodermically injected with adrenaline hydrochloride twice at dose of 0.8 mg/kg before next-day pharmacokinetic experiment, time interval for 4h. After 2 h receiving the first hypodermic injection, the rats were put in 0-2 °C ice water to swim 4 min, causing acute blood stasis model [22]. The dosage preparation was made by dissolving appropriate amount of AHSYB in normal saline solution and then given to rats in

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normal oral group and acute blood stasis group at a single oral dose (30 mg/kg) by gastric gavage and in intravenous group at a single intravenous dose (2.5 mg/kg) by rapid injection via the catheter. Aliquots of 300 μL blood samples were immediately collected in heparinized polyethylene tubes before dosing and at 0.083, 0.33, 0.67, 1, 2, 3, 4, 6, 8, 10 and 12 h after dosing from fossa orbitalis vein of the rats in normal oral group and acute blood stasis group. In intravenous group, aliquots of 300 μL blood samples were immediately collected in heparinized polyethylene tubes before dosing and at 0.017, 0.083, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 12 h after dosing from fossa orbitalis vein. After centrifugation at 3000 rpm for 10 min, 100 μL plasma was finally obtained and stored at -20 °C until analysis.

2.7.

Statistical analysis

To

determine

the

pharmacokinetic

parameters

of

AHSYB

in

different

groups,

concentration-time data were analyzed with a non-compartmental method using DAS 2.0 software (Mathematical Pharmacology Professional Committee of China, Shanghai, China). Absolute bioavailability (F) was calculated based on the AUC0→∞ obtained after oral and intravenous administration at the equivalent dose. All data were presented as mean ± SD. The statistically significant differences between different groups were carried out by Student’s t-test.

3.

Results and discussion

3.1.

UFLC-MS/MS optimization

In our study, different mobile phase compositions (methanol-water, MeCN-water, MeCN-0.1% formic acid aqueous solution, MeCN-0.05% formic acid aqueous solution, MeCN-0.01% formic

10

acid aqueous solution) were assayed. The data showed that the optimized mobile phase consisted of MeCN and 0.01% formic acid aqueous solution, which significantly improve the shape of peak and the responses of analytes. In addition, MeCN could not only improve resolution of analytes from polar matrix components, but also shorten the analysis time. The negative ionization mode was chosen since the responses of analytes were higher than those in the positive ionization mode. Moreover, parameters such as spray voltage, turbo spray temperature, gas1 and gas 2, curtain gas, DP and CE were optimized to obtain the highest intensity of deprotonated molecule of analytes. The precursor-to-product ion pairs for MRM detection were generated and optimized by product ion scan procedure, which was embedded in the Analyst® software.

3.2. Method validation

3.2.1. Selectivity and specificity Representative chromatograms obtained from the blank plasma sample, blank plasma sample added with AHSYB (at LLOQ) or IS and treated plasma sample were shown in Fig. 2. The retention time of AHSYB and IS were 4.37 and 4.29 min, respectively. The results indicated that there is no significant interference from endogenous ingredients with AHSYB and IS in the plasma samples.

3.2.2.

Linearity and LLOQ

The linearity was investigated with duplicate injections of calibration standards (viz., 25-10000 ng/mL) on six consecutive validation days. Satisfactory linearity were observed for AHSYB, and the typical regression equation of the calibration curves using 1/x as weighting factor was y =

11

0.26140 x – 0.00116 (r2 = 0.99959). Consequently, the LLOQ for AHSYB was regarded as 25 ng/mL.

3.2.3.

Precision and accuracy

Table 2 summarized the intra- and inter-day precision and accuracy of the analytical method. In this assay, the intra- and inter-day precision variation of AHSYB at each QC level was within 1.4-6.5%. The RE of accuracy was within ± 9.4%. These results indicated that the developed method has satisfactory accuracy, precision and reproducibility.

3.2.4.

Recovery and matrix effect

As shown in Table 2, the extraction recovery of AHSYB in plasma at three QC levels ranged from 70.9-84.9%. With regard to matrix effect, all the calculated values were between 81.4% and 108.3% (Table 3). No significant matrix interference for AHSYB and IS was observed, indicating that no co-eluting substance influenced the ionization of the analytes.

3.2.5. Stability The stability results of AHSYB at three concentrations under a variety of storage and process conditions were summarized in Table 4. The results demonstrated that the stabilities of AHSYB were acceptable at room temperature for 6 h, in autosampler at 4°C for 24 h, three freeze-thaw cycles and at -20°C for 6 days. All RSD values were less than 15% and RE values were within ± 11.3%.

3.3.

Pharmacokinetic study

The sensitive and reproducible method was successfully applied to the pharmacokinetic study after intravenous (2.5 mg/kg) dosing of AHSYB in normal rats and oral (30 mg/kg) dosing in 12

normal and acute blood stasis syndrome rats. The concentration-time curves of AHSYB are shown in Fig. 3. Meanwhile, the major pharmacokinetic parameters are listed in Table 5. As shown in the results, the uptake of AHSYB was quickly and the half-lives in both normal oral group and acute blood stasis group were very short, and 90% AHSYB were eliminated within 6 h. Besides, the very low oral bioavailability (F: 0.17% ~ 0.3%) of AHSYB in both normal group and acute blood stasis group could be due to the potential hydrolysis in the gastrointestinal tract, poor permeability through the intestinal epithelial membrane and first-pass effect in the liver, which is corresponding to the bioavailability (F: 1.2%) of HSYA as a class III drug according to the biopharmaceutics classification system [23]. In previous study, AHSYB was demonstrated to degrade into HSYA at high temperature (> 60 °C) or extreme pHs (pH ≤ 3.0 or > 7.0) [14]. So we tried to monitor the phenomenon during the pre-experiment and the formal experiment. Unfortunately, the degradation reaction was not occurred as no HSYA was detected in rat plasma. Many studies have demonstrated that disease condition could cause the alteration of pharmacokinetic parameters [24-26]. In the present study, there were no statistically significant differences (P > 0.05) in the T1/2 and Tmax between normal and acute blood stasis syndrome rats, both receiving 30 mg/kg AHSYB, which indicated that the absorption and elimination rate of AHSYB were not significantly altered in acute blood stasis syndrome. Meanwhile, the Cmax values of AHSYB in acute blood stasis group were lower than that in normal oral group, although no significant difference was observed due to high standard deviation. On the other side, the AUC0-t, AUC0-∞ and F were all significantly lower (P < 0.05) in acute blood stasis syndrome rats. We hypothesized that acute blood stasis syndrome may enhance metabolic enzyme activity and AHSYB maybe not only in prototype form but also converted into more active metabolites. 13

However, this hypothesis needs to be further investigated by both metabolic enzyme activity and metabolite profiling studies.

4. Conclusions In this study, an accurate and sensitive UFLC-MS/MS method for the determination of AHSYB in rat plasma was developed and validated for the first time. The preliminary pharmacokinetic behavior of AHSYB in rats after intravenous and oral administration was firstly elucidated. The oral bioavailability (F) of AHSYB was demonstrated to be very low, which is most likely due to the hydroxyl groups in its molecular structure. Meanwhile, the pharmacokinetic properties of AHSYB in rats with acute blood stasis and the differences between normal and acute blood stasis syndrome rats were also investigated. It was found that the AUC0-t, AUC0-∞ and F were all significantly lower (P < 0.05) in acute blood stasis syndrome rats, which indicated that acute blood stasis syndrome may enhance metabolic enzyme activity and AHSYB maybe not only in prototype form but also converted into more active metabolites. The achieved comparative pharmacokinetic results provide primary data and scientific basis for the further ADME work and identification of AHSYB metabolites.

Acknowledgments. This work was supported by National Natural Science Foundation of China (81274058, 81573714), National Key Technology R&D Program (2008BAI51B01), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20113237110010), Key Research Project in Basic Science of Jiangsu College and University (12KJA360002). This research was also financially supported by A Project Funded by the Priority Academic Program Development of 14

Jiangsu Higher Education Institutions (PAPD).

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Figure Captions Fig. 1. Chemical structures of AHSYB and rutin (IS). Fig. 2. Representative chromatograms for AHSYB (1) and IS (2) in (A) blank plasma; (B) blank plasma sample spiked with AHSYB (25 ng/mL) and IS (50 ng/mL); (C) plasma sample collected at 15 min after intravenous administration of 2.5 mg/kg AHSYB. Fig. 3. Mean plasma concentration-time curves of AHSYB in rats (n = 5).

19

HO HO HO

OH O

O HO O

OH OH O

OH OH HO

HO O

H

O

HO

OH

O

H

H

OH

H

OH

OH HO

OH

CH2OH

AHSYB

rutin (IS)

Fig. 1

Fig. 2

Fig. 3

Tables Table 1. The molecular weight (MW), MRM transitions, DP, CE and retention times (R.T.) of AHSYB and IS. Compds.

MW

MRM transitions

DP (V)

CE (eV)

R.T. (min)

IS

610

609.0 → 300.0

-180

-40

4.29

AHSYB

1044

1043.2 → 449.1

-100

-40

4.37

20

Table 2. Precision, accuracy and recovery for the analysis of AHSYB in rat plasma (n = 6). Nominal concentration (ng/mL) 50 500 5000

Intra-day

Inter-day

Measured concentration (ng/mL)

Precision (RSD, %)

Accuracy (RE, %)

Measured concentration (ng/mL)

Precision (RSD, %)

Accuracy (RE, %)

49.8 ± 3.1 466.9 ± 6.8 5244.7 ± 322.3

6.3 1.4 6.5

-0.4 -6.6 4.9

54.7 ± 1.9 480.0 ± 15.4 5282.2 ± 313.8

3.8 3.1 6.3

9.4 -4.0 5.6

21

Recovery (%) 84.9 ± 11.4 70.9 ± 2.1 71.0 ± 4.7

Table 3. Matrix effect of AHSYB and IS in rat plasma (n = 6). Compds. AHSYB

IS

Concentration (ng/mL) 50 500 5000 20 100

Matrix effect (%) 93.6 ± 12.2 89.5 ± 6.1 93.3 ± 1.6 98.0 ± 10.3 91.1 ± 2.6

22

Table 4. Stability of AHSYB under different storage conditions (n = 6). Storge conditions

6 h at room temperature

24 h in autosampler

3 freeze-thaw cycles

6 days storage at -20 °C

Concentration (ng/mL) Nominal 50 500 5000 50 500 5000 50 500 5000 50 500 5000

Measured 48.9 ± 7.6 460.0 ± 35.9 4739.8 ± 252.2 52.9 ± 1.0 443.5 ± 13.7 4697.0 ± 434.1 51.0 ± 4.5 469.9 ± 18.8 4946.8 ± 258.2 54.6 ± 3.5 507.7 ± 47.5 5119.2 ± 369.8

RSD (%)

RE (%)

15.0 7.2 5.0 2.0 2.8 8.7 9.0 3.8 5.2 7.1 9.5 7.4

-2.2 -8.0 -5.2 5.9 -11.3 -6.1 2.0 -6.0 -1.1 9.2 1.5 2.4

23

Table 5. Main pharmacokinetic parameters of AHSYB in rats (n = 5). Parameter

Unit

Normal intravenous

Normal oral

Acute blood stasis

AUC(0-t)

ng h/mL ng h/mL h ng/mL h %

10175.20 ± 1505.10 10179.60 ± 1509.76 1.05 ± 0.16 -

349.67 ± 35.57 370.80 ± 64.21 1.49 ± 1.04 145.30 ± 24.37 0.44 ± 0.19 0.30 ± 0.05

169.00 ± 77.00∗ 207.75 ± 79.30∗ 1.38 ± 0.86 100.44 ± 56.35 0.35 ± 0.24 0.17 ± 0.06∗

AUC(0-∞) T1/2 Cmax Tmax F ∗

P < 0.05, versus normal oral rats.

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