Chinese Journal of Natural Medicines 2013, 11(5): 0560−0565
Chinese Journal of Natural Medicines
The identification and pharmacokinetic studies of metabolites of salvianolic acid B after intravenous administration in rats QI Qu, HAO Kun, LI Fei-Yan, CAO Li-Juan, WANG Guang-Ji, HAO Hai-Ping* State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China Available online 20 Sept. 2013
[ABSTRACT] AIM: To identify and quantify the major metabolites of salvianolic acid B (SAB) after intravenous injection in rats. METHODS: LC-IT/TOF-MS was used to identify the metabolites in rat bile, plasma, and urine; LC-MS/MS was used to quantify the two major metabolites. RESULTS: In rat bile, plasma, and urine, nine metabolites were identified, including methylated metabolites of SAB, lithospermic acid (LSA), the decarboxylation and methylation metabolites of LSA, salvianolic acid S (SAS), and dehydrated-SAS. The t1/2 of monomethyl-SAB and LSA were both very short, and monomethyl-SAB had a larger AUC than LSA in rats. CONCLUSION: Nine metabolites were found, the metabolic pathway was described, and the pharmacokinetic profiles of LSA and monomethyl-SAB were studied, thereby clarifying that methylation was the dominant metabolic pathway for SAB in rats. [KEY WORDS] Salvianolic acid B; Metabolites; Identification; Pharmacokinetics
[CLC Number] R969.1
1
[Document code] A
[Article ID] 1672-3651(2013)05-0560-06
Introduction
Danshen, the dry roots of Salvia miltiorrhiza Bunge (Lamiaceae), is a well-known, Chinese traditional medicine which is used to promote blood flow and to resolve blood stasis [1-6]. Salvianolic acid B (SAB), a water-soluble compound extracted from danshen, has been clinically used in patients having coronary vascular diseases with successful results in China. The bioavailability and pharmacokinetics of SAB have been investigated in recent years. SAB had extremely low oral bioavailability [7], however, after intravenous injection, SAB reached the maximal plasma concentration (Cmax) around 910 μg·mL−1; and the half life (t1/2) of SAB was around 105 minutes [8]. SAB mainly distributed in organs like kidney, lung, and liver, and was almost com-
pletely cleared from these tissues after 4 h [9]. SAB was excreted rapidly into rat bile, mostly as methylated metabolites, but only four metabolites were found after detection using HPLC-UV [10]. As a novel drug candidate, it is essential to clarify its metabolism in detail for its further development. Although some metabolites of SAB have been found after injection, the metabolic pathway has not been fully clarified. Therefore, LC-IT/TOF-MS was used to thoroughly identify the metabolites in rat bile, plasma, and urine, and to determine the metabolic pathway of SAB. Furthermore, LC-MS/MS was applied to quantify the plasma MMS and LSA, to investigate the main metabolic pathway of SAB in vivo.
2 2.1
[Received on] 28-Dec.-2012 [Research funding] This project was financially supported by the Natural Science Foundation of Jiangsu Province (Nos. BK2011065, BK2012026), Jiangsu Province Key Lab of Drug Metabolism and Pharmacokinetics (No. BM2012012).
[*Corresponding author] HAO Hai-Ping: Prof., E-mail: hhp_ [email protected], Tel: 86-25-83271129, Fax: 86-25-83271060 These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
Materials and Methods
Reagents, chemicals and animals SAB was supplied by Shanghai Institute of Pharmaceutical Industry (China). LSA, chloromycetin (internal standard), HPLC-grade acetonitrile and methanol were obtained from Sigma (St. Louis, MO, USA). Deionized water was purified by Milli-Q system (Millipore, Milford, MA, USA). Other solvents or reagents were of analytical grade. Sprague–Dawley rats (180–220 g) were obtained from the animal center of China Pharmaceutical University (Nan-
QI Qu,, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
jing, China). They were acclimated for at least 1 week before experiments and allowed water and standard chow ad libitum. The studies were approved by the Animal Ethics Committee of China Pharmaceutical University. 2.2 Bile sample collection and preparation Eight male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. All rats were implanted with a PE-10 cannula into the bile duct under anesthesia with ethyl ether. After blank bile was collected, 100 mg·kg-1 SAB (dissolved in saline) was intravenously administrated to the rats. In order to prevent dilution of the metabolites, the bile was collected at 0–2, 2–4, 4–8, and 8–12 h and stored at −20 °C until analyzed. The rats were given water occasionally during the course of bile collection. For the analysis, bile samples (100 μL) were hydrolyzed with hydrochloric acid (1 mol·L-1) and then extracted with ethyl acetate. After the extraction, the sample was evaporated, and the residue was reconstituted in the mobile phase and detected by LC-IT/TOF-MS. 2.3 Plasma sample collection and preparation Four male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. After 100 mg·kg-1 SAB (dissolved in saline) was intravenously given to the rats, plasma was collected from the orbital venous sinus at 5, 30, 60, and 120 min, and stored at -20 °C until analyzed. For the analysis, plasma samples (50 μL) were prepared and detected in the same manner as described above. 2.4 Urine sample collection and preparation Four male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. After 100 mg·kg-1 SAB (dissolved in saline) was intravenously given to the rats, they were placed in separate metabolic cages and urine was collected at 0–6, 6–12, and 12–24 h. Also, urine samples (100 μL) were prepared and detected in the same manner as described above. 2.5 The pharmacokinetic study of LSA and MMS Eight male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. After 100 mg·kg-1 SAB (dissolved in saline) was intravenously given to the rats, plasma was collected from the orbital venous sinus at 0.08, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, and 8 h, and stored at –20 °C until analyzed. For the analysis, plasma samples (50 μL) with chloromycetin (internal standard) were prepared and detected by LC-MS/MS as described below. MMS was semi-quantified using the standard curves of SAB because of the lack of standards. The pharmacokinetic parameters were estimated by the non-compartmental method using Phoenix WinNonlin v 6.1 software (Pharsight, Sunnyvale, CA, USA). 2.6 Instruments and analytical condition LC-IT/TOF-MS system: The liquid chromatograph-mass spectrometer was equipped with an auto-injector (SIL-20AC), a Shimadzu solvent delivery pump (LC-20AB), a Shimadzu CTO-20AC column oven, a Shimadzu DGU-20A online degasser, and a semi-micro mixer. The separation of SAB and its metabolites was carried out on an Agilent C18 column (3.5
μm, 150 mm × 2.0 mm) at 40 °C. The flow rate was 0.2 mL·min-1. The mobile phase consisted of acetonitrile (B) and purified water containing 0.025% formic acid (A). The timed program was described as follows: 5% B (0–3 min) – 90% B (50 min) – 5% B (55 min) – STOP (60 min). The MS acquisition was performed in the negative ion scan mode. The optimized MS operating conditions were selected as follows: CDL (curved desolvation line) temperature: 200 °C; applied voltage: +4.5 kV/–3.5 kV; CDL voltage: constant mode; analysis mode: MS1 measurement; Measurement range: auto MS/MS mode; MS m/z 100–1 000; MS/MS m/z 50–1 000; ion accumulation time: 30 msec; drying gas pressure：0.1 MPa; each setting of voltage for ionization mode is optimized by auto-tuning. LC-MS/MS system: The analysis was performed using a Shimadzu HPLC (Kyoto, Japan) system coupled with an AB Sciex 4000 QTRAP mass spectrometer with a TurboIonSpray source (Applied Biosystems, Foster City, CA, USA). LC separation was performed using a Hypersil C18 (5 μm, 250 mm × 4.6 mm, Dalian Elite Company, Liaoning Province, China). A linear gradient elution of mobile phase A (methanol) and B (0.5% aqueous formic acid and 1.5 mmol·L-1 ammonium acetate) was used, starting with 15% A and 85% B to reach 67.5% A at 8.5 min, with recovery to the initial conditions in 0.5 min. The detection was stopped at 15 min and the mobile phase was delivered at a flow rate of 0.6 mL·min-1. LC-MS/MS was performed in the negative selected-ion monitoring mode. SAB ， LSA, MMS, and chloromycetin (internal standard) were detected at m/z 717.9 to 519.1, 537.2 to 294.9, 731.2 to 533.4, and 321.1 to 151.9, respectively.
3 3.1
Results and Discussion
Metabolic profiles in rat bile In the previous in vitro impurity analysis of SAB, no impurity was found, so the metabolites could be found through the comparison of blank bile sample and the bile sample collected after SAB was given to the rats. After detecting the bile samples at different times, it was found that the bile sample at 0–2 h had the most metabolites. The resulting total ion chromatograms (TIC) of bile sample at 0–2 h, and the extracted ion chromatograms (XIC) of bile metabolites are shown in Fig. 1. Seven metabolites, M1, M2, M3, M4, M5, M6, and M7, were identified from the bile sample. Metabolites M1, M2, M3, M6, and M7 have 3, 5, 3, 2, and 3 isomers, respectively. 3.2 Metabolic profiles in plasma After detecting plasma samples at different times, the plasma sample at 5 min had the largest number of metabolites. The resulting TIC of the plasma sample at 5 min and the XIC of plasma metabolites are shown in Fig. 2. Five metabolites, M1, M2, M3, M8, and M9, were identified from plasma sample. Metabolites M1, M2, M8, and M9 have 4, 2, 4, and 2 isomers, respectively.
QI Qu, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
3.3
Metabolic profiles in urine After comparing the results of the urine samples at different times after detection, the urine sample at 0–6 h had the most metabolites. The resulting TIC of the urine sample at 0–6 h and the XIC of urine metabolites are shown in Fig. 3. Three metabolites, M1, M2, and M3, were identified from the urine sample, and M2 had two isomers.
Fig. 3 HPLC chromatograms of the blank urine of the rats (a), the urine sample of rats after SAB iv dose (b), and the XIC of the metabolites (c) 3.4
Fig. 1 HPLC chromatograms of the blank bile of the rats (a), the bile sample of rats after SAB iv dose (b), and the XIC of the metabolites (c)
Fig. 2 HPLC chromatograms of the blank plasma of the rats (a), the plasma sample of rats after SAB iv dose (b), and the XIC of the metabolites (c)
Fragmentation mechanism of SAB The information supplied by LC-IT/TOF-MS, including molecular ions ([M − H]−), fragment ions, and assigned identity is summarized and presented in Table 1. SAB was observed as its deprotonated molecule [M − H]− at m/z 717.14, and the product ion was 519.4. As shown in Table 1, the [M − H]− and the product ion of M1 were at m/z 731.15 and 533.10, both 14 amu heavier than that of SAB, suggesting that M1 might be monomethyl-SAB. Similarly, the [M − H]− and the product ions of M2 and M3 were 28 And 42 amu heavier than that of SAB, revealing that they might be dimethyl-SAB and trimethyl- SAB, respectively. On the other hand, the [M − H]− ion of M4 was only m/z 537.10, 180 amu lighter than that of SAB. Considering that SAB had the tendency to lose caffeic acid (180 amu) to produce LSA [11], M4 could be identified as LSA. For M5, the [M − H]− ion was 44 amu lighter than that of M4, indicating that M5 might be obtained through the loss of one carboxyl group of M4, generating salvianolic acid A (SAA). Based on the mechanism described above, M6 and M7 were identified as monomethyl-LSA and dimethyl-LSA, respectively. The metabolite M8 was found in previous studies and named it salvianolic acid S after SAB was orally given to rats [11], and M9 is the dehydrated metabolite of M8 based on the ionic mass information. Finally, the metabolic pathway proposed for SAB is presented in Fig. 4. 3.5 The plasma pharmacokinetic profiles of SAB, LSA, and MMS The plasma concentration-time profiles of SAB, LSA,
QI Qu,, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
and MMS are shown in Fig. 5, and the parameters are presented in Table 2. After intravenous injection of 100 mg·kg−1 Table 1
SAB to rats, the t1/2 of LSA and MMS were both very short, (2.73 ± 0.59) h for LSA and (0.83 ± 0.17) h for MMS,
LC-IT/TOF-MS data and identification of metabolites of SAB in rats
Peak No.
Rt/min
[M − H]− ion
Fragment ions
Diff
M1-1
41.89
731. 143 9
533.102 7
−1.78
M1-2
42.82
731.157 9
533.099 5
7.52
M1-3
43.30
731.156 4
533.105 7
5.47
M1-4
44.17
731.154 5
533.101 1
M2-1
47.29
745.176 1
M2-2
45.50
M2-3
Assigned identify
Bile
Plasma
Urine
+
+
+
+
+
−
+
+
−
2.87
−
+
−
547.117 1
10.87
+
+
+
745.169 4
533.101 9
1.88
+
+
+
47.81
745.1640?
547.117 1
−5.37
+
−
−
M2-4
48.57
745.175 5
#
10.06
+
−
−
M2-5
49.20
745.169 5
547.118 9
2.01
+
−
−
M3-1
51.05
759.182 9
559.117 1
−1.05
+
+
+
M3-2
51.51
759.185 7
#
2.63
+
−
−
M3-3
52.29
759.185 6
#
2.5
+
−
−
M4
35.18
537.098 3
295.055 5
7.08
LSA
+
−
−
M5
35.23
493.110 4
295.057 8
−7.3
SAA
+
−
−
+
−
−
+
−
−
+
−
−
+
−
−
Monomethyl-SAB
Dimethyl-SAB
Trimethyl-SAB
M6-1
40.02
551.110 6
507.124 5
0.91
M6-2
35.72
551.110 8
309.072 4
1.27
M7-1
46.11
565.129 7
521.138 3
6.9
M7-2
40.36
565.127 0
309.072 7
2.12
M7-3
46.68
565.125 2
#
−1.06
+
−
−
M8-1
28.15
539.116 3
297.072 7
−5.94
−
+
−
M8-2
30.11
539.116 5
295.056 5
−4.82
−
+
−
M8-3
27.31
539.115 4
#
−7.61
−
+
−
M8-4
29.50
539.115 4
#
−7.61
−
+
−
M9-1
40.52
521.112 4
#
6.72
−
+
−
M9-2
41.42
521.107 5
289.117 3
−2.69
−
+
−
Monomethyl-LSA
Dimethyl-LSA
SAS
Dehydrated-SAS
#: the information was not obtained; +: the metabolite could be found in the sample; −: the metabolite could not be found in the sample
Table 2 The pharmacokinetic parameters of LSA, Monomethyl-SAB and SAB after 100 mg·kg−1 SAB was intravenously given to rats Parameters Ke
h
−1
LSA
Monomethyl-SAB
SAB
0.26 ± 0.06
0.86 ± 0.15
0.28 ± 0.07
t1/2
h
2.73 ± 0.59
0.83 ± 0.17
2.59 ± 0.62
AUC(0-t)
μg·h·mL−1
4.35 ± 1.05
36.34 ± 8.43
140.10 ± 30.06
AUC(0-Inf)
μg·h·mL−1
4.84 ± 2.20
36.54 ± 11.18
149.47 ± 32.65
MRT
h
3.04 ± 0.32
1.26 ± 0.35
1.87 ± 0.17
33.10 ± 10.43
16.96 ± 7.87
1.04 ± 0.64
132.69 ± 40.58
17.66 ± 3.86
4.31 ± 1.80
−1
CL
L·(kg·h)
Vd
−1
L·kg
QI Qu, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
Fig. 4
Proposed metabolic pathways of SAB in normal rats
Fig. 5 The plasma concentration-time profiles of LSA, MMS, and SAB after intravenous injection of 100 mg·kg−1 SAB to rats (mean ± SD, n = 8)
QI Qu,, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
indicating that the metabolites were cleared quickly in rats. The AUC(0-Inf) of MMS [(36.54 ± 11.18) μg·h·mL−1] was higher than LSA [(4.84 ± 2.20) μg·h·mL−1], accounting for almost 30% of that of SAB [(149.47 ± 32.65) μg·h·mL−1], revealing that the methylated metabolites were dominant in rats. Moreover, the concentration-time profile of MMS showed two peaks, which may be caused by enterohepatic circulation, or the different metabolic rates of MMS by different rats.
4
Conclusions
In the present study, more metabolites of SAB were found than in previous studies, and allowed the depiction of the metabolic pathway after intravenous administration to rats. Also, the quantification of the major metabolites showed that methylation was the dominant metabolic pathway for SAB in rats. The illustration of the metabolic pathway and quantification of main metabolites provides a better understanding of the pharmacokinetics of SAB, which it is hoped will promote its further clinical development.
References [1]
[2]
[3]
Du GH, Qiu Y, Zhang JT. Salvianolic acid B protects the memory functions against transient cerebral ischemia in mice [J]. J Asian Nat Prod Res, 2000, 2(2): 145-152. Ho JHC, Hong CY. Salvianolic acids: small compounds with multiple mechanisms for cardiovascular protection [J]. J Biomed Sci, 2011, 18(1): 30-34. Chen YL, Hu CS, Lin FY, et al. Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipopro-
tein-E-deficient mouse aorta [J]. J Cell Biochem, 2006, 98(3): 618-631. [4] Han JY, Fan JY, Horie Y, et al. Ameliorating effects of compounds derived from Salvia miltiorrhiza root extract on microcirculatory disturbance and target organ injury by ischemia and reperfusion [J]. Pharmacol Ther, 2008, 117(2): 280-295. [5] Li M, Zhao C, Wong RN, et al. Inhibition of shear-induced platelet aggregation in rat by tetramethylpyrazine and salvianolic acid B [J]. Clin Hemorheol Microcirc, 2004, 31(2): 97-103. [6] Shi CS, Huang HC, Wu HL, et al. Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells [J]. Thromb Res, 2007, 119(6): 769-775. [7] Zhang Y, Akao T, Nakamura N, et al. Extremely low bioavailability of magnesium lithospermate B, an active component from Salvia miltiorrhiza, in rat [J]. Planta Med, 2004, 70(2): 138-142. [8] Wu YT, Chen YF, Hsieh YJ, et al. Bioavailability of salvianolic acid B in conscious and freely moving rats [J]. Int J Pharm, 2006, 326(1): 25-31. [9] Li X, Yu C, Lu Y, et al. Pharmacokinetics, tissue distribution, metabolism, and excretion of depside salts from Salvia miltiorrhiza in rats [J]. Drug Metab Dispos, 2007, 35(2): 234239. [10] Zhang Y, Akao T, Nakamura N, et al. Magnesium lithospermate B is excreted rapidly into rat bile mostly as methylated metabolites, which are potent antioxidants [J]. Drug Metab Dispos, 2004, 32(7): 752-757. [11] Xu M, Guo H, Han J, et al. Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography-mass spectrometry [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 858 (1-2): 184-198.
大鼠静脉注射丹酚酸 B 后代谢产物的鉴定及血浆药动学研究 亓
蕖，郝
琨，李飞燕，曹丽娟，王广基，郝海平*
中国药科大学天然药物活性组分与药效国家重点实验室, 药物代谢动力学重点实验室, 南京 210009 【摘 要】 目的：鉴定丹酚酸 B 静注后在大鼠体内的代谢产物并对主要代谢物进行定量。方法：应用 LC-IT/TOF-MS 对大 鼠胆汁、血浆和尿液中的代谢产物进行分析鉴定；应用 LC-MS/MS 对两个主要代谢产物进行定量，得到血浆药时曲线。结果： 在大鼠胆汁、血浆和尿液中，共发现了九种代谢产物，包括丹酚酸 B 的甲基化产物，紫草酸及紫草酸的脱羧和甲基化产物，丹 酚酸 S 及其脱水产物；药动学研究表明紫草酸和单甲基-丹酚酸 B 的 t1/2 都较小，单甲基-丹酚酸 B 与紫草酸相比有较大的 AUC。 结论: 丹酚酸 B 静注后，在大鼠体内共发现了 9 种代谢产物, 绘制了代谢通路图并对紫草酸和单甲基-丹酚酸 B 的药物代谢动力 学进行了研究，阐明了丹酚酸 B 在大鼠体内主要的代谢方式是甲基化。 【关键词】 丹酚酸 B; 代谢产物; 鉴定; 药物代谢动力学 【基金项目】 江苏省自然科学基金面上项目及江苏省杰出青年科学基金项目(Nos. BK2011065, BK2012026), 江苏省重点实验室 平台提升项目(No. BM2012012)
Chinese Journal of Natural Medicines
The identification and pharmacokinetic studies of metabolites of salvianolic acid B after intravenous administration in rats QI Qu, HAO Kun, LI Fei-Yan, CAO Li-Juan, WANG Guang-Ji, HAO Hai-Ping* State Key Laboratory of Natural Medicines, Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China Available online 20 Sept. 2013
[ABSTRACT] AIM: To identify and quantify the major metabolites of salvianolic acid B (SAB) after intravenous injection in rats. METHODS: LC-IT/TOF-MS was used to identify the metabolites in rat bile, plasma, and urine; LC-MS/MS was used to quantify the two major metabolites. RESULTS: In rat bile, plasma, and urine, nine metabolites were identified, including methylated metabolites of SAB, lithospermic acid (LSA), the decarboxylation and methylation metabolites of LSA, salvianolic acid S (SAS), and dehydrated-SAS. The t1/2 of monomethyl-SAB and LSA were both very short, and monomethyl-SAB had a larger AUC than LSA in rats. CONCLUSION: Nine metabolites were found, the metabolic pathway was described, and the pharmacokinetic profiles of LSA and monomethyl-SAB were studied, thereby clarifying that methylation was the dominant metabolic pathway for SAB in rats. [KEY WORDS] Salvianolic acid B; Metabolites; Identification; Pharmacokinetics
[CLC Number] R969.1
1
[Document code] A
[Article ID] 1672-3651(2013)05-0560-06
Introduction
Danshen, the dry roots of Salvia miltiorrhiza Bunge (Lamiaceae), is a well-known, Chinese traditional medicine which is used to promote blood flow and to resolve blood stasis [1-6]. Salvianolic acid B (SAB), a water-soluble compound extracted from danshen, has been clinically used in patients having coronary vascular diseases with successful results in China. The bioavailability and pharmacokinetics of SAB have been investigated in recent years. SAB had extremely low oral bioavailability [7], however, after intravenous injection, SAB reached the maximal plasma concentration (Cmax) around 910 μg·mL−1; and the half life (t1/2) of SAB was around 105 minutes [8]. SAB mainly distributed in organs like kidney, lung, and liver, and was almost com-
pletely cleared from these tissues after 4 h [9]. SAB was excreted rapidly into rat bile, mostly as methylated metabolites, but only four metabolites were found after detection using HPLC-UV [10]. As a novel drug candidate, it is essential to clarify its metabolism in detail for its further development. Although some metabolites of SAB have been found after injection, the metabolic pathway has not been fully clarified. Therefore, LC-IT/TOF-MS was used to thoroughly identify the metabolites in rat bile, plasma, and urine, and to determine the metabolic pathway of SAB. Furthermore, LC-MS/MS was applied to quantify the plasma MMS and LSA, to investigate the main metabolic pathway of SAB in vivo.
2 2.1
[Received on] 28-Dec.-2012 [Research funding] This project was financially supported by the Natural Science Foundation of Jiangsu Province (Nos. BK2011065, BK2012026), Jiangsu Province Key Lab of Drug Metabolism and Pharmacokinetics (No. BM2012012).
[*Corresponding author] HAO Hai-Ping: Prof., E-mail: hhp_ [email protected], Tel: 86-25-83271129, Fax: 86-25-83271060 These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
Materials and Methods
Reagents, chemicals and animals SAB was supplied by Shanghai Institute of Pharmaceutical Industry (China). LSA, chloromycetin (internal standard), HPLC-grade acetonitrile and methanol were obtained from Sigma (St. Louis, MO, USA). Deionized water was purified by Milli-Q system (Millipore, Milford, MA, USA). Other solvents or reagents were of analytical grade. Sprague–Dawley rats (180–220 g) were obtained from the animal center of China Pharmaceutical University (Nan-
QI Qu,, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
jing, China). They were acclimated for at least 1 week before experiments and allowed water and standard chow ad libitum. The studies were approved by the Animal Ethics Committee of China Pharmaceutical University. 2.2 Bile sample collection and preparation Eight male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. All rats were implanted with a PE-10 cannula into the bile duct under anesthesia with ethyl ether. After blank bile was collected, 100 mg·kg-1 SAB (dissolved in saline) was intravenously administrated to the rats. In order to prevent dilution of the metabolites, the bile was collected at 0–2, 2–4, 4–8, and 8–12 h and stored at −20 °C until analyzed. The rats were given water occasionally during the course of bile collection. For the analysis, bile samples (100 μL) were hydrolyzed with hydrochloric acid (1 mol·L-1) and then extracted with ethyl acetate. After the extraction, the sample was evaporated, and the residue was reconstituted in the mobile phase and detected by LC-IT/TOF-MS. 2.3 Plasma sample collection and preparation Four male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. After 100 mg·kg-1 SAB (dissolved in saline) was intravenously given to the rats, plasma was collected from the orbital venous sinus at 5, 30, 60, and 120 min, and stored at -20 °C until analyzed. For the analysis, plasma samples (50 μL) were prepared and detected in the same manner as described above. 2.4 Urine sample collection and preparation Four male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. After 100 mg·kg-1 SAB (dissolved in saline) was intravenously given to the rats, they were placed in separate metabolic cages and urine was collected at 0–6, 6–12, and 12–24 h. Also, urine samples (100 μL) were prepared and detected in the same manner as described above. 2.5 The pharmacokinetic study of LSA and MMS Eight male SD rats, weighing 180–220 g, were fasted for 12 h before dosing. After 100 mg·kg-1 SAB (dissolved in saline) was intravenously given to the rats, plasma was collected from the orbital venous sinus at 0.08, 0.17, 0.33, 0.5, 0.75, 1, 2, 4, 6, and 8 h, and stored at –20 °C until analyzed. For the analysis, plasma samples (50 μL) with chloromycetin (internal standard) were prepared and detected by LC-MS/MS as described below. MMS was semi-quantified using the standard curves of SAB because of the lack of standards. The pharmacokinetic parameters were estimated by the non-compartmental method using Phoenix WinNonlin v 6.1 software (Pharsight, Sunnyvale, CA, USA). 2.6 Instruments and analytical condition LC-IT/TOF-MS system: The liquid chromatograph-mass spectrometer was equipped with an auto-injector (SIL-20AC), a Shimadzu solvent delivery pump (LC-20AB), a Shimadzu CTO-20AC column oven, a Shimadzu DGU-20A online degasser, and a semi-micro mixer. The separation of SAB and its metabolites was carried out on an Agilent C18 column (3.5
μm, 150 mm × 2.0 mm) at 40 °C. The flow rate was 0.2 mL·min-1. The mobile phase consisted of acetonitrile (B) and purified water containing 0.025% formic acid (A). The timed program was described as follows: 5% B (0–3 min) – 90% B (50 min) – 5% B (55 min) – STOP (60 min). The MS acquisition was performed in the negative ion scan mode. The optimized MS operating conditions were selected as follows: CDL (curved desolvation line) temperature: 200 °C; applied voltage: +4.5 kV/–3.5 kV; CDL voltage: constant mode; analysis mode: MS1 measurement; Measurement range: auto MS/MS mode; MS m/z 100–1 000; MS/MS m/z 50–1 000; ion accumulation time: 30 msec; drying gas pressure：0.1 MPa; each setting of voltage for ionization mode is optimized by auto-tuning. LC-MS/MS system: The analysis was performed using a Shimadzu HPLC (Kyoto, Japan) system coupled with an AB Sciex 4000 QTRAP mass spectrometer with a TurboIonSpray source (Applied Biosystems, Foster City, CA, USA). LC separation was performed using a Hypersil C18 (5 μm, 250 mm × 4.6 mm, Dalian Elite Company, Liaoning Province, China). A linear gradient elution of mobile phase A (methanol) and B (0.5% aqueous formic acid and 1.5 mmol·L-1 ammonium acetate) was used, starting with 15% A and 85% B to reach 67.5% A at 8.5 min, with recovery to the initial conditions in 0.5 min. The detection was stopped at 15 min and the mobile phase was delivered at a flow rate of 0.6 mL·min-1. LC-MS/MS was performed in the negative selected-ion monitoring mode. SAB ， LSA, MMS, and chloromycetin (internal standard) were detected at m/z 717.9 to 519.1, 537.2 to 294.9, 731.2 to 533.4, and 321.1 to 151.9, respectively.
3 3.1
Results and Discussion
Metabolic profiles in rat bile In the previous in vitro impurity analysis of SAB, no impurity was found, so the metabolites could be found through the comparison of blank bile sample and the bile sample collected after SAB was given to the rats. After detecting the bile samples at different times, it was found that the bile sample at 0–2 h had the most metabolites. The resulting total ion chromatograms (TIC) of bile sample at 0–2 h, and the extracted ion chromatograms (XIC) of bile metabolites are shown in Fig. 1. Seven metabolites, M1, M2, M3, M4, M5, M6, and M7, were identified from the bile sample. Metabolites M1, M2, M3, M6, and M7 have 3, 5, 3, 2, and 3 isomers, respectively. 3.2 Metabolic profiles in plasma After detecting plasma samples at different times, the plasma sample at 5 min had the largest number of metabolites. The resulting TIC of the plasma sample at 5 min and the XIC of plasma metabolites are shown in Fig. 2. Five metabolites, M1, M2, M3, M8, and M9, were identified from plasma sample. Metabolites M1, M2, M8, and M9 have 4, 2, 4, and 2 isomers, respectively.
QI Qu, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
3.3
Metabolic profiles in urine After comparing the results of the urine samples at different times after detection, the urine sample at 0–6 h had the most metabolites. The resulting TIC of the urine sample at 0–6 h and the XIC of urine metabolites are shown in Fig. 3. Three metabolites, M1, M2, and M3, were identified from the urine sample, and M2 had two isomers.
Fig. 3 HPLC chromatograms of the blank urine of the rats (a), the urine sample of rats after SAB iv dose (b), and the XIC of the metabolites (c) 3.4
Fig. 1 HPLC chromatograms of the blank bile of the rats (a), the bile sample of rats after SAB iv dose (b), and the XIC of the metabolites (c)
Fig. 2 HPLC chromatograms of the blank plasma of the rats (a), the plasma sample of rats after SAB iv dose (b), and the XIC of the metabolites (c)
Fragmentation mechanism of SAB The information supplied by LC-IT/TOF-MS, including molecular ions ([M − H]−), fragment ions, and assigned identity is summarized and presented in Table 1. SAB was observed as its deprotonated molecule [M − H]− at m/z 717.14, and the product ion was 519.4. As shown in Table 1, the [M − H]− and the product ion of M1 were at m/z 731.15 and 533.10, both 14 amu heavier than that of SAB, suggesting that M1 might be monomethyl-SAB. Similarly, the [M − H]− and the product ions of M2 and M3 were 28 And 42 amu heavier than that of SAB, revealing that they might be dimethyl-SAB and trimethyl- SAB, respectively. On the other hand, the [M − H]− ion of M4 was only m/z 537.10, 180 amu lighter than that of SAB. Considering that SAB had the tendency to lose caffeic acid (180 amu) to produce LSA [11], M4 could be identified as LSA. For M5, the [M − H]− ion was 44 amu lighter than that of M4, indicating that M5 might be obtained through the loss of one carboxyl group of M4, generating salvianolic acid A (SAA). Based on the mechanism described above, M6 and M7 were identified as monomethyl-LSA and dimethyl-LSA, respectively. The metabolite M8 was found in previous studies and named it salvianolic acid S after SAB was orally given to rats [11], and M9 is the dehydrated metabolite of M8 based on the ionic mass information. Finally, the metabolic pathway proposed for SAB is presented in Fig. 4. 3.5 The plasma pharmacokinetic profiles of SAB, LSA, and MMS The plasma concentration-time profiles of SAB, LSA,
QI Qu,, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
and MMS are shown in Fig. 5, and the parameters are presented in Table 2. After intravenous injection of 100 mg·kg−1 Table 1
SAB to rats, the t1/2 of LSA and MMS were both very short, (2.73 ± 0.59) h for LSA and (0.83 ± 0.17) h for MMS,
LC-IT/TOF-MS data and identification of metabolites of SAB in rats
Peak No.
Rt/min
[M − H]− ion
Fragment ions
Diff
M1-1
41.89
731. 143 9
533.102 7
−1.78
M1-2
42.82
731.157 9
533.099 5
7.52
M1-3
43.30
731.156 4
533.105 7
5.47
M1-4
44.17
731.154 5
533.101 1
M2-1
47.29
745.176 1
M2-2
45.50
M2-3
Assigned identify
Bile
Plasma
Urine
+
+
+
+
+
−
+
+
−
2.87
−
+
−
547.117 1
10.87
+
+
+
745.169 4
533.101 9
1.88
+
+
+
47.81
745.1640?
547.117 1
−5.37
+
−
−
M2-4
48.57
745.175 5
#
10.06
+
−
−
M2-5
49.20
745.169 5
547.118 9
2.01
+
−
−
M3-1
51.05
759.182 9
559.117 1
−1.05
+
+
+
M3-2
51.51
759.185 7
#
2.63
+
−
−
M3-3
52.29
759.185 6
#
2.5
+
−
−
M4
35.18
537.098 3
295.055 5
7.08
LSA
+
−
−
M5
35.23
493.110 4
295.057 8
−7.3
SAA
+
−
−
+
−
−
+
−
−
+
−
−
+
−
−
Monomethyl-SAB
Dimethyl-SAB
Trimethyl-SAB
M6-1
40.02
551.110 6
507.124 5
0.91
M6-2
35.72
551.110 8
309.072 4
1.27
M7-1
46.11
565.129 7
521.138 3
6.9
M7-2
40.36
565.127 0
309.072 7
2.12
M7-3
46.68
565.125 2
#
−1.06
+
−
−
M8-1
28.15
539.116 3
297.072 7
−5.94
−
+
−
M8-2
30.11
539.116 5
295.056 5
−4.82
−
+
−
M8-3
27.31
539.115 4
#
−7.61
−
+
−
M8-4
29.50
539.115 4
#
−7.61
−
+
−
M9-1
40.52
521.112 4
#
6.72
−
+
−
M9-2
41.42
521.107 5
289.117 3
−2.69
−
+
−
Monomethyl-LSA
Dimethyl-LSA
SAS
Dehydrated-SAS
#: the information was not obtained; +: the metabolite could be found in the sample; −: the metabolite could not be found in the sample
Table 2 The pharmacokinetic parameters of LSA, Monomethyl-SAB and SAB after 100 mg·kg−1 SAB was intravenously given to rats Parameters Ke
h
−1
LSA
Monomethyl-SAB
SAB
0.26 ± 0.06
0.86 ± 0.15
0.28 ± 0.07
t1/2
h
2.73 ± 0.59
0.83 ± 0.17
2.59 ± 0.62
AUC(0-t)
μg·h·mL−1
4.35 ± 1.05
36.34 ± 8.43
140.10 ± 30.06
AUC(0-Inf)
μg·h·mL−1
4.84 ± 2.20
36.54 ± 11.18
149.47 ± 32.65
MRT
h
3.04 ± 0.32
1.26 ± 0.35
1.87 ± 0.17
33.10 ± 10.43
16.96 ± 7.87
1.04 ± 0.64
132.69 ± 40.58
17.66 ± 3.86
4.31 ± 1.80
−1
CL
L·(kg·h)
Vd
−1
L·kg
QI Qu, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
Fig. 4
Proposed metabolic pathways of SAB in normal rats
Fig. 5 The plasma concentration-time profiles of LSA, MMS, and SAB after intravenous injection of 100 mg·kg−1 SAB to rats (mean ± SD, n = 8)
QI Qu,, et al. /Chinese Journal of Natural Medicines 2013, 11(5): 560−565
indicating that the metabolites were cleared quickly in rats. The AUC(0-Inf) of MMS [(36.54 ± 11.18) μg·h·mL−1] was higher than LSA [(4.84 ± 2.20) μg·h·mL−1], accounting for almost 30% of that of SAB [(149.47 ± 32.65) μg·h·mL−1], revealing that the methylated metabolites were dominant in rats. Moreover, the concentration-time profile of MMS showed two peaks, which may be caused by enterohepatic circulation, or the different metabolic rates of MMS by different rats.
4
Conclusions
In the present study, more metabolites of SAB were found than in previous studies, and allowed the depiction of the metabolic pathway after intravenous administration to rats. Also, the quantification of the major metabolites showed that methylation was the dominant metabolic pathway for SAB in rats. The illustration of the metabolic pathway and quantification of main metabolites provides a better understanding of the pharmacokinetics of SAB, which it is hoped will promote its further clinical development.
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大鼠静脉注射丹酚酸 B 后代谢产物的鉴定及血浆药动学研究 亓
蕖，郝
琨，李飞燕，曹丽娟，王广基，郝海平*
中国药科大学天然药物活性组分与药效国家重点实验室, 药物代谢动力学重点实验室, 南京 210009 【摘 要】 目的：鉴定丹酚酸 B 静注后在大鼠体内的代谢产物并对主要代谢物进行定量。方法：应用 LC-IT/TOF-MS 对大 鼠胆汁、血浆和尿液中的代谢产物进行分析鉴定；应用 LC-MS/MS 对两个主要代谢产物进行定量，得到血浆药时曲线。结果： 在大鼠胆汁、血浆和尿液中，共发现了九种代谢产物，包括丹酚酸 B 的甲基化产物，紫草酸及紫草酸的脱羧和甲基化产物，丹 酚酸 S 及其脱水产物；药动学研究表明紫草酸和单甲基-丹酚酸 B 的 t1/2 都较小，单甲基-丹酚酸 B 与紫草酸相比有较大的 AUC。 结论: 丹酚酸 B 静注后，在大鼠体内共发现了 9 种代谢产物, 绘制了代谢通路图并对紫草酸和单甲基-丹酚酸 B 的药物代谢动力 学进行了研究，阐明了丹酚酸 B 在大鼠体内主要的代谢方式是甲基化。 【关键词】 丹酚酸 B; 代谢产物; 鉴定; 药物代谢动力学 【基金项目】 江苏省自然科学基金面上项目及江苏省杰出青年科学基金项目(Nos. BK2011065, BK2012026), 江苏省重点实验室 平台提升项目(No. BM2012012)
