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Pharmacokinetics of phenolic compounds of Danshen extract in rat blood and brain by microdialysis sampling
Journal of Ethnopharmacology 136 (2011) 129–136
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Pharmacokinetics of phenolic compounds of Danshen extract in rat blood and brain by microdialysis sampling Yun-Jing Zhang a,b , Liang Wu a,b , Qun-Lin Zhang a,b,∗ , Jun Li a,b , Fang-Xiong Yin a,b , Ye Yuan a,b a b
School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, PR China Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei, Anhui 230032, PR China
a r t i c l e
i n f o
Article history: Received 28 August 2010 Received in revised form 17 February 2011 Accepted 11 April 2011 Available online 16 April 2011 Keywords: Danshen extract Phenolic compound Microdialysis Pharmacokinetics High-performance liquid chromatography Chemiluminescence
a b s t r a c t Aim of the study: To evaluate the pharmacokinetics of phenolic compounds after oral administration of Danshen extract in rat brain. Materials and methods: Blood and brain microdialysis probes were inserted into jugular vein and cerebral cortex of rat under anesthesia and perfused with ringer’s solution at the rate of 2.0 and 0.8 L/min, respectively. Blank microdialysates were collected after 2 h post-implantation equilibrium time. Danshen extract (danshensu 40 mg/kg BW, protocatechuic aldehyde 149 mg/kg BW, and salvianolic acid B 50 mg/kg BW) was administrated intragastrically, and then blood and brain microdialysates were collected at 15 and 30 min time intervals for 4 h, respectively. The concentrations of phenolic compounds were determined by high-performance liquid chromatography coupled with chemiluminescence detection. Pharmacokinetic parameters were estimated using non-compartmental methods. Results: Danshensu and protocatechuic acid could be detected in both blood and brain microdialysates, while protocatechuic aldehyde and salvianolic acid B were not detected. Brain-to-blood (AUCbrain /AUCblood ) distribution ratio were 0.25 ± 0.04 and 0.09 ± 0.02 for danshensu and protocatechuic acid, respectively. Conclusions: Danshensu can readily permeate the blood brain barrier after oral administration of Danshen extract, and protocatechuic acid is a potential oxidative metabolite of protocatechuic aldehyde. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Danshen, the dry root and rhizome of the labiatae plant Salvia miltiorrhiza Bge., is one of the most widely used traditional Chinese medicine (TCM), for the treatment of cardiovascular and cerebrovascular diseases. The chemical constituents of Salvia miltiorrhiza mainly include water-soluble phenolic compounds and diterpenoid quinines. The widely use of Danshen water decoction in clinical medication indicates that water-soluble phenolic compounds may be the most important constituents responsible for the therapeutic effects of Danshen. Up to today, more than 30 phenolic compounds have been isolated and structurally identified from Danshen, among which danshensu (DSS), protocatechuic aldehyde (PAL), protocatechuic acid (PA) and salvianolic acid B (SalB) (Fig. 1) are the most effective active ingredients. In the pharmacological and clinical studies, these phenolic compounds were found to be strong antioxidants and free radical scavengers (Zhao et al., 2008),
∗ Corresponding author at: School of Pharmacy, Anhui Medical University, Meishan Road 81, Hefei, Anhui 230032, PR China. Tel.: +86 551 5161115; fax: +86 551 5167735. E-mail address: [email protected] (Q.-L. Zhang). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.04.023
and could improve blood circulation (Liu and Lu, 1999), reduce the area of cerebral infarct (Lo et al., 2003), and inhibit the renin angiotensin system (Kang et al., 2002). Along with the deepening of pharmacological investigation, phenolic compounds of Danshen were found to have brain protective effects, such as anti-apoptotic (Li et al., 2008), ameliorating the impairment of cerebral ischemia (Yang et al., 2009), and improving the learning and memory deficit of senile dementia (Zhang et al., 2005b). Numerous pharmacokinetic studies were performed for better understanding the pharmacological effects of water-soluble components in Danshen (Li et al., 2005; Zhou et al., 2005; Hou et al., 2007; Guo et al., 2008; Gao et al., 2009). The concentrationtime data of DSS in rat plasma were found to be best fitted to a two-compartment open model, and its absorption, distribution, and elimination of were relatively moderate following administration of a single oral dose of Danshen extract (Liu et al., 2010). It was reported that methylation was the main pathway of DSS metabolism in rats after oral administration (Zhang et al., 2008). The pharmacokinetic profiles of PAL in rat serum after oral administration of Radix Salviae miltiorrhizae extract showed a double peak pattern (Ye et al., 2003). SalB, another major phenolic compound in Danshen, was absorbed quickly with maximum concentration obtained 0.5 h and eliminated rapidly from plasma after oral admin-
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Fig. 1. Chemical structures of danshensu (DSS), protocatechuic aldehyde (PAL), protocatechuic acid (PA), and salvianolic acid B (SalB).
istration to rats (Zhang et al., 2005a). The bioavailability of SalB was found to be extremely low in rats (Chen et al., 2005; Wu et al., 2006), beagle dogs (Han et al., 2009) and rabbits (Ma and Wang, 2007). Although pharmacokinetics of phenolic compounds in Danshen have been extensively studied, little is known about the pharmacokinetic profiles of phenolic compounds in brain. The relevant pharmacokinetic properties of phenolic compounds or its active metabolites include the ability to be absorbed from the site of administration and to pass through the blood brain barrier (BBB). However, only a few literatures (Xu et al., 2007a; Zheng et al., 2007; Luo et al., 2009) have reported the distribution of water-soluble phenolic compounds of Danshen in brain. Luo et al. (2009) found that DSS, PAL, and SalB were detected in rat serum but could not be detected in brain tissue homogenate after oral administration of water extract of Danshen (DSS 32.4 mg/kg BW, PAL 6.0 mg/kg BW, SalB 36.2 mg/kg BW), due to the limited sensitivity of UV detection in this study. However, it is debatable whether these phenolic compounds can penetrate the BBB. It was reported that DSS could be detected in CSF after oral administration of water extract of Danshen (10 g/kg) to New Zealand rabbits, but the pharmacokinetic parameters of DSS in brain were not mentioned (Zheng et al., 2007). SalB could be detected in rat brain after oral administration of total phenolic acids (500 mg/kg) and SalB (300 mg/kg) extracted from the roots of Salvia miltiorrhiza, but the concentration of SalB was below the quantification limit (Xu et al., 2007a). In the abovementioned studies, the limited sensitivity of UV detection prohibits trace level determination of analytes in the brain, while detection by LC/MS involves relatively tedious sample pretreatment. Besides, the sample collection by tissue homogenizing cannot allow continuous monitoring of analytes, and the drawing of cerebrospinal fluid might disturb the physiological state of animal due to loss of the body fluid. For the pharmacokinetics study of drugs in brain, intracerebral microdialysis is the only technique which offers the possibility to continuously monitor the local BBB transport of unbound drugs in tested animals, under physiological and pathological conditions (de Lange et al., 1997). Biological samples obtained using conventional sampling method contain protein-bound and unbound drugs. However, only the unbound fraction of drug is therapeuti-
cally active. Microdialysis offers protein-free clean samples which do not require purification prior to analysis, because the dialysis membrane of probe is only permeable to protein-unbound small molecules. Microdialysis also presents the advantages of sampling from multiple sites of the same animal, avoiding the effect of biological volume changes, and allowing for analysis without pretreatment. Currently, the Food and Drugs Administration of USA and the European Union’s Committee emphasize the value of human-tissue drug concentration data and support using clinical microdialysis to obtain pharmacokinetic information (Brunner and Derendorf, 2006), demonstrating the potential and importance of microdialysis. However, until now, the application of microdialysis to the pharmacokinetic study of Danshen is extremely limited. Only Tsai group (Chen et al., 2005) developed an in vivo microdialysis sampling method for the monitoring the protein-unbound SalB of Danshen in rat blood and bile after intravenous administration of SalB at about 98% purity. In our previous work, the pharmacokinetic properties of DSS in rat brain after single intravenous administration of Danshen injection with microdialysis sampling were studied (Zhang et al., 2010). The present work is aimed to reveal the BBB penetration and pharmacokinetics of protein-unbound phenolic compounds in rat brain after oral administration of hydrophilic extract of Danshen by simultaneous microdialysis in blood and brain. A highly sensitive HPLC method coupled with chemiluminescence (CL) detection was developed for the determination of DSS, PA, PAL, and SalB in rat microdialysates from blood and brain. As far as we know, it is the first time to report the pharmacokinetic profiles of DSS and metabolite PA in rat brain as well as their blood-to-brain distribution ratio after oral administration of Danshen extract. 2. Materials and methods 2.1. Chemicals and solutions A stock solution of luminol (1.0 × 10−2 mol/L) was prepared by dissolving luminol (Merck, Darmstadt, Germany) in sodium hydroxide solution (0.10 mol/L) and stored at least 7 days before
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dilution. The working H2 O2 solution was prepared fresh daily from 30% (w/w) H2 O2 (Suzhou Chemical Reagent Company, China). A stock solution (1%, w/w) of HAuCl4 was prepared by dissolving HAuCl4 ·4H2 O (Shanghai Chemical Reagent Company, China) in water. The reference compounds of DSS and SalB were purchased from Shanghai Winherb Medical Science Company (Shanghai, China), and PA and PAL were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Stock solutions of danshensu (0.1790 mg/mL), protocatechuic acid (0.2160 mg/mL), protocatechuic aldehyde (0.2000 mg/mL), and SalB (0.2064 mg/mL) were prepared in methanol. These stock solutions were kept in the dark at 4 ◦ C. Danshen crude drug was purchased from a local pharmacy. Methanol was of HPLC grade, and all other chemicals were of analytical-reagent grade. The ultrapure water (18.3 M cm, Millipore, USA) was used to prepare the mobile phases of HPLC and all the solutions. The mobile phases of HPLC were prepared fresh daily and filtered through a 0.22 m membrane filter (Bandao, Shanghai, China). The perfusate for blood and brain microdialysis was Ringer’s solution (122 mmol/L NaCl, 3 mmol/L KCl, 0.4 mmol/L KH2 PO4 , 1.2 mmol/L MgSO4 , 25 mmol/L NaHCO3 , and 1.2 mmol/L CaCl2 ). 2.2. Animals Adult, male Spague-Dawley rats (300 ± 20 g) were supplied by the Laboratory Animal Center of Anhui Medical University, housed in an air-conditioned room (temperature, 22–25 ◦ C; relative humidity, 55 ± 5%), and kept on a light/dark cycle of 12/12 h. Free access to food and mains drinking water was allowed throughout the study except for fasting 18 h before the experiment. Animal studies were performed according to the Guidelines for the Care and Use of Laboratory Animals approved by the Committee of Ethics of Animal Experimentation of Anhui Medical University (Hefei, China). 2.3. Preparation of Danshen extract In view of the thermolability of phenolic compounds, the acid percolation process (Qu, 2004) was chosen to prepare Danshen extract. The coarse powder of Danshen crude drug was soaked in 8 volumes of 0.01% HCl overnight and then the coarse powder together with the leachate was percolated with 14 volumes of water (pH 2.0). The percolate, which is a hydrophilic extract, was collected and loaded onto an AB-8 macroporous resin packed column. The column was washed with 5 times of column volume of water (pH 2.0) to remove the unabsorbed impurities, and followed by 6 times of column volume of 25% ethanol (pH 2.0) and 4 times of column volume of 40% ethanol (pH 2.0) to elute phenolic compounds. The eluent of 25% and 40% ethanol (pH 2.0) was combined and condensed under vacuum to obtain Danshen extract. The components and contents of Danshen extract were analyzed by a validated HPLC–CL method against the reference standards of DSS, PAL, and SalB, and were determined as 8.0, 29.8, and 10.0 mg/mL of DSS, PAL, and SalB, respectively (n = 5). 2.4. Instruments and chromatographic and CL conditions The HPLC–CL detection system consists of an HPLC system and a CL detection system, as shown in Fig. 2. The HPLC system is Shimadzu LC-20A series (Shimadzu Corporation, Japan), including a quaternary pump, a vacuum degasser, a thermostated column compartment, a prominence diode array detector (DAD), a manual sample valve injector with a 20 L loop, and an analytical column (Shim-pack ODS, 250 mm × 4.6 mm I.D. 5 m; Shimadzu). The CL detection is performed with a flow injection CL system (Remax,
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Xi’an, China), which is composed of a model IFFM-E peristaltic pump, a mixing tee, and a model IFFS-A CL detector equipped with a flat glass coil (used as reaction coil and detection cell) and a photomultiplier operated at −400 V. The analysis was performed on a ODS column at 30 ◦ C with gradient elution at a flow rate of 1.0 mL/min. The mobile phase was composed of aqueous phosphoric acid (0.1%, v/v)–methanol (92:8, v/v). Detection by DAD was set at 280 nm. The column effluent from DAD was first mixed on-line with HAuCl4 solution via a PEEK tube, and then combined with H2 O2 and luminol solution in a mixing tee, respectively (Fig. 2). All the CL solutions were delivered by a peristaltic pump at a flow rate of 2.0 mL/min. The microdialysis system (CMA, Stockholm, Sweden) consists of a CMA/400 microinjection pump, a CMA/150 temperature controller, and a CMA/470 refrigerated fraction collector. 2.5. Microdialysis experiments Rats were anesthetized with chloral hydrate (300 mg/kg, i.p.) and remained anesthetized throughout the experimental period. The rats’ body temperature was maintained at 37 ◦ C with a heating blanket. Surgical sites were shaved and cleaned with povidone–iodine. The probe for blood sampling (MAB 7.8.10, membrane length, 10 mm, molecular weight cut-off 15 kDa) was implanted within the jugular vein/right atrium and perfused with Ringer’s solution at a flow rate of 2.0 L/min. Rat was then mounted on a stereotaxic frame and the brain microdialysis probe (CMA/12, membrane length, 4 mm, molecular weight cut-off 20 kDa) was implanted in the cerebral cortex (coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to midline, 3.5 mm from dura) and perfused with Ringer’s solution at 0.8 L/min. The proper placement of probe in the cerebral cortex was confirmed by making an incision at the site of probe implantation after completion of studies. Danshen extract was administered orally (5 mL/kg, DSS 40 mg/kg, PAL 149 mg/kg, and SalB 50 mg/kg) after a 2 h post-surgical stabilization period. The microdialysis samples of blood and brain were collected every 15 and 30 min, respectively, for 4 h. The collected samples were kept at 4 ◦ C and analyzed within 48 h. The blood dialysate samples of 15, 30, 45 and 60 min were diluted 4 times with Ringer’s solution prior to analysis. 2.6. In vivo recovery assessment of microdialysis probes To estimate the in vivo recoveries, a retrodialysis calibration technique was utilized. The microdialysis probe was inserted into the rat’s jugular vein and cerebral cortex under anesthesia, and then the Ringer’s solution containing the phenolics (DSS, PA, and PAL) was perfused through the probe at the flow rate of 2.0 L/min and 0.8 L/min, respectively by the microinjection pump after a stabilization period of 2 h post probe implantation. The perfusate (Cperf ) and dialysate (Cdial ) concentrations of six phenolics were determined by the HPLC–CL method. The in vivo relative recovery (Rdial ) of each phenolic compound across the microdialysis probe was calculated by the following equation: Rdial = (Cperf − Cdial )/Cperf. The in vivo recovery assessment was done after the feeding experiment when phenolic compounds in the microdialysates were undetectable. The concentrations of phenolic compounds (Cm ) were converted to free-form concentrations (Cf ) as follows: Cf = Cm /Rdial . 2.7. Pharmacokinetic data analysis The drug concentration data (after being converted to free form concentrations) were processed by Drug and Statistics (DAS 2.0) (Mathematical Pharmacology Professional Committee of China, Shanghai, China).
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Fig. 2. Schematic diagram of the HPLC–CL system used for the determination of phenolic compounds.
2.8. Method validation 2.8.1. Selectivity For the pharmacokinetic study, blank blood and brain microdialysates from rats and blank Ringer’s solution spiked with DSS, PA and PAL were evaluated for the peak interference. 2.8.2. Linearity and sensitivity The linearity of the HPLC–CL method was evaluated by the correlation coefficients (r) of the calibration curves generated with more than 10 standard mixtures, using Ringer’s solution spiked with DSS, PA, and PAL. For each phenolic compound, an external standard method was used to establish the calibration curve. The quantitative determination was based on the relative CL intensity I = IS − I0 , where IS is the CL intensity of phenolic compound and I0 is the intensity of blank signal. Calibration curves were constructed by plotting logarithm of the phenolic concentration (lg C) against the logarithm of the relative CL intensity (lg I). All calibration curves were required to have a correlation value of at least 0.990. The signal-to-noise ratio (S/N) of 3 was set as the threshold for calculating the detection limit. 2.8.3. Precision and accuracy The validation was performed in Ringer’s solution. Precision was expressed as the relative standard deviation (RSD) of each calculated concentration while accuracy was calculated as the percentage of the found concentration to the added concentration. The intra-day precision was tested with six spiked samples at three concentration levels (DSS, 4.58, 22.91, and 114.56 ng/mL, PA, 6.91, 27.65, and 331.52 ng/mL, PAL, 9.60, 25.60, and 480.00 ng/mL). The inter-day precision and accuracy were evaluated by injecting five times every day on three consecutive days. 3. Results 3.1. Validation of HPLC–CL method 3.1.1. Selectivity DSS, PA, and PAL were completely separated with the retention times of 19.8, 22.0, and 31.6 min, respectively. Representative chromatograms of blank blood and brain dialysate are shown in Fig. 3a and b. Ringer’s solution spiked with DSS, PA, and PAL are shown in Fig. 3c, and microdialysis samples from rat blood and
brain collected 1.5 h after post administration of Danshen extract are presented in Fig. 3d and e, respectively. The biological matrix from rat blood or brain dialysate did not interfere with the analytes. 3.1.2. Linearity and sensitivity Calibration curves showed good linear relationship over the ranges of 2.29–179.0, 3.46–345.6, and 4.80–800.0 ng/mL with the detection limits of 0.29, 0.52, and 0.80 ng/mL for DSS, PA, and PAL, respectively. The calibration equations for DSS, PA, and PAL are: lg I = 0.6142 lg C + 2.0338, lg I = 0.7902 lg C + 1.9416, and lg I = 0.9704 lg C + 1.7908 with the correlation coefficients (r) of 0.9993, 0.9999, and 0.9984, respectively. The proposed HPLC–CL method has much lower detection limits than those of HPLC–UV method, which are at 43.0–65.0 ng/mL level (Wang et al., 2007; Yuan et al., 2009). The method is also more sensitive than LC–MS/MS, whose detection limits for DSS and PAL were reported to be 2.5 and 2.0 ng/mL, respectively (Li et al., 2005). 3.1.3. Precision and accuracy The precision and accuracy data in Table 1 indicate that the present HPLC–CL method has good accuracies and reproducibilities for DSS, PA, and PAL at three levels of concentrations. The accuracies ranged from 90.1 to 103.2% with the relative standard deviations (RSD) not more than 5.5%, which were well within the acceptance criteria of bioanalysis. 3.2. In vitro recovery and delivery The microdialysis probes recoveries by gain and delivery by loss in vitro in 4 h were determined. The perfusing rates for blood and brain microdialysis probes 2.0 L/min and 0.8 L/min, respectively. The microdialysis samples of blood and brain probe were collected every 15 and 30 min, respectively. The RSDs of recoveries by gain in 4 h of blood probe for DSS, PA, and PAL were 3.1%, 3.4%, and 3.9%, respectively, and for brain probe, the recoveries by gain were 3.3%, 4.3% and 3.0% for DSS, PA, and PAL, respectively. The RSDs of deliveries by loss in 4 h of blood probe for DSS, PA, and PAL were 3.2%, 2.4%, and 2.9%, respectively, and for brain probe, the recoveries by gain were 2.3%, 3.2% and 3.1% for DSS, PA, and PAL, respectively. The above results indicate that the recoveries and deliveries are stable during 4 h sampling time. The RSD between recoveries and deliveries of DSS, PA, and PAL were 5.5%, 4.8%, and 6.5%, respectively, which indicates that
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Fig. 3. HPLC–CL chromatograms of (a) blank microdialysate from rat’s blood; (b) blank microdialysate from rat’s brain; (c) blank Ringer’s solution spiked with DSS (71.56 ng/mL), PA (43.20 ng/mL), and PAL (32.00 ng/mL); (d) blood and (e) brain microdialysate collected 1.5 h after administration of Danshen extract detected under the conditions of Section 2.4. UNK = unknown compounds.
the recoveries and deliveries are equivalent for the analytes, thus the recoveries determined by loss can be used for the calibrations of the probes.
3.3. In vivo recoveries of the probes The in vivo recoveries of blood and brain probes for DSS, PA, and PAL were examined and summarized in Table 2. In the further study, the in vivo recoveries were used for the calibration of probes.
3.4. Analytes’ stabilities in the dialysate It was found phenolic compounds are subject to degradation catalyzed by the high concentrations of metal ions in the perfusate. To avoid degradation, 2.0 L H3 PO4 was added in the collecting vials in advance, and then no significant degradation of analytes in Ringer’s solution with H3 PO4 was observed after 48 h at 20 ◦ C. Thus the determination of DSS, PA, and PAL in microdialysis samples could be accomplished in 48 h.
Table 1 Precisions and accuracies of DSS, PA and PAL by HPLC–CL method (¯x ± s). Phenolic compounds
Added (ng/mL)
Intra-day found (ng/mL) (n = 5)
DSS
4.58 22.91 114.56 6.91 27.65 331.52 9.60 25.60 480.00
4.53 21.76 115.60 6.72 25.64 332.42 8.65 26.170 482.01
PA
PAL
± ± ± ± ± ± ± ± ±
0.24 1.05 2.41 0.12 0.95 14.01 0.45 1.32 3.97
Recovery (%)
RSD (%)
Inter-day found (ng/mL) (n = 3)
98.87 94.97 100.91 97.28 92.74 100.27 90.10 102.23 100.42
5.30 4.82 2.08 1.86 3.69 4.21 5.20 5.04 0.82
4.52 22.00 118.19 6.86 25.10 339.87 8.99 25.10 483.19
± ± ± ± ± ± ± ± ±
0.24 0.67 1.96 0.24 0.92 4.12 0.49 0.59 9.53
Recovery (%)
RSD (%)
98.65 96.01 103.17 97.80 91.07 102.52 93.66 98.05 100.67
5.31 3.04 1.67 3.55 3.63 1.21 5.47 2.35 1.98
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Table 2 In vivo recoveries of blood and brain microdialysis probes for DSS, PA, and PAL. Phenolic compounds
In vivo recovery (%) (¯x ± s, n = 5)
Concentration (ng/mL)
Blood probe DSS
PA
PAL
27.54 344.32 Average 55.30 331.52 Average 48.00 480.00 Average
34.85 34.93 34.89 36.08 36.74 36.41 34.40 35.26 34.83
± ± ± ± ± ± ± ± ±
1.39 1.28 0.06 0.68 1.19 0.46 3.06 2.17 0.61
Brain probe 26.63 28.33 27.48 34.29 37.16 35.73 21.25 23.55 22.40
± ± ± ± ± ± ± ± ±
2.04 1.33 1.20 1.46 0.34 2.03 2.12 1.53 1.63
Fig. 5. Unbound PA concentration versus time curves of blood and brain after administration of Danshen extract to SD rats (¯x ± s, n = 6) [ blood, brain].
DSS could be detected up to 4 h after oral administration. The brain distribution ratio (AUCbrain /AUCblood ) of DSS was calculated to be 0.25 ± 0.04. PA appeared in blood rapidly following oral administration, reached a peak at 0.5 h, eliminated from blood with the mean residence time less than 1 h [(0.60 ± 0.01) h], and was undetectable in blood or brain 2.0 h after administration. PA penetrated the BBB with a brain distribution ratio (AUCbrain /AUCblood ) of 0.09 ± 0.02. 4. Discussion 4.1. Optimization of HPLC–CL system Fig. 4. Unbound DSS concentration versus time curves of blood and brain after administration of Danshen extract to SD rats (¯x ± s, n = 6) [ blood, brain].
3.5. Pharmacokinetic study After oral administration of Danshen extract (DSS 40 mg/kg, PAL 149 mg/kg, and SalB 50 mg/kg), DSS and PA were detected in the rats’ blood and brain microdialysates while no PAL or SalB was observed. The mean blood and brain concentration-time profiles of DSS and PA are illustrated in Figs. 4 and 5, respectively. The main blood and brain pharmacokinetic parameters of DSS and PA estimated by statistical moment method are given in Table 3. The maximum blood concentration of DSS was observed after 0.5 of administration. DSS was distributed rapidly to brain with the maximum brain concentration being achieved 1.0 h after dosing. Table 3 Pharmacokinetic parameters of DSS and PA in rat blood and brain after administration of Danshen extract (5 mL/kg, i.g., x¯ ± s, n = 6). Parameters Blood AUC0–∞ (g h/mL) MRT (h) tmax (h) Cmax (g/mL) t1/2 (h) Brain AUC0–∞ (g h/mL) MRT (h) tmax (h) Cmax (g/mL) t1/2 (h) Brain-to-blood distribution AUCbrian /AUCblood
DSS
PA
4.46 4.31 0.5 1.14 3.00
± ± ± ± ±
0.99 1.11 0.0 0.17 1.16
1.18 0.60 0.5 1.62 0.42
± ± ± ± ±
0.45 0.01 0.0 1.20 0.01
1.10 2.28 1.0 0.28 1.60
± ± ± ± ±
0.20 1.14 0.0 0.14 0.70
0.13 1.34 1.0 0.09 0.90
± ± ± ± ±
0.03 0.01 0.0 0.07 0.10
0.25 ± 0.04
0.09 ± 0.02
Several compositions of mobile phases have been tested for HPLC separation, such as methanol–acetic acid–water, methanol–formic acid, and acetonitrile–phosphoric acid. It was found that the mobile phase consisting of a mixture of methanol and aqueous 0.1% phosphoric acid (8:92, v/v) was not only suitable for the good separation of DSS, PA, and PAL but also compatible with the HAuCl4 –luminol–H2 O2 CL reaction. The good resolution of HPLC separation was achieved within 33 min. To obtain the maximal efficiency of CL system and stability of CL signals, the effects of configurations, type and pH of the buffer solution, concentrations of luminol, H2 O2 , and HAuCl4 , and flow rate on the CL intensities were investigated. The stable and maximal relative CL signals were obtained under the conditions of NaHCO3 –Na2 CO3 buffer solution of pH 10.83, 2.0 × 10−5 mol/L luminol, 5.0 × 10−5 mol/L H2 O2 , 4.0 × 10−5 g/mL HAuCl4 , and the flow rate of 2.0 mL/min using the configuration in Fig. 2. 4.2. Optimization of microdialysis system Effects of perfusing rate on the recoveries of probes were evaluated. Blood and brain microdialysis probes were immersed in Ringer’s solution containing known concentrations of the analytes (DSS, 275.46 ng/mL, PA, 331.520 ng/mL, and PAL, 480.000 ng/mL), and perfused with blank Ringer’s solution at various flow rates (2.5, 2.0, and 1.5 L/min for blood probe; 1.0, 0.8, and 0.6 L/min for brain probe). For each perfusing rate, three microdialysis samples were collected with 30 min time interval and the recoveries under each perfusing rate were determined. The results in Table 4 indicate that the recoveries of the probes increased with the decreased perfusing rate. The effects of analytes’ concentration on the recoveries of probes were also investigated. Blood and brain microdialysis probes were
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Table 4 Effects of perfusing rate on in vitro recovery of the probes (¯x ± s, n = 3). Phenolic compounds
DSS
PA
PAL
Blood probe
Brain probe
Perfusing rate (L/min)
Recovery (%)
2.5 2.0 1.5 2.5 2.0 1.5 2.5 2.0 1.5
28.86 31.23 35.40 20.08 23.83 27.08 29.72 32.43 35.45
immersed in Ringer’s solution containing different concentrations of DSS, PA, and PAL, and perfused with blank Ringer’s solution at the flow rate of 2.0 L/min for blood probe and 0.8 L/min for brain probe. For each concentration, three microdialysis samples were collected with 30 min time interval and the recoveries under each perfusing rate were determined. As shown in Table 5, the recoveries for DSS, PA, and PAL showed a non concentration-related pattern within the examined range. At low perfusing rate, the diffusions of the solution inside and outside the probe membranes are almost balanced, producing relatively high recovery. However, it takes longer sampling time to collect sufficient sample volume for HPLC analysis, resulting in reduced time resolution and concentration-time information. Conversely, excessively high perfusing rate will impair the normal physiological state of rats. Considering the above factors, the perfusing rate of 2.0 and 0.8 L/min was chosen for blood and brain probe with sampling interval of 15 and 30 min, respectively. 4.3. Pharmacokinetic study In the preliminary experiment of this study, SalB was not observed in rat blood and brain microdialysates after oral administration of Danshen extract. Thus in the further experiments, we cut out the detection of SalB by HPLC to shorten the analysis time. The oral bioavailability of SalB was calculated about 2.3%, and the protein binding rate of SalB in vivo was determined to be 83.78 ± 10.5% (Wu et al., 2006). As protein-bound compounds cannot penetrate the membrane of the microdialysis probes, only very small amounts of free SalB could occur in the microdialysates. The relatively low oral bioavailability together with high protein binding rate might result in undetectable SalB levels in the microdialysates after oral administration of Danshen extract. The pharmacokinetic data show that DSS is capable of permeating the BBB, which is in conformity with our results of previous study after administration of Danshen injection (Zhang et al., 2010), implying BBB permeability to DSS independent of dosage or administration route. The good BBB permeability of DSS is probably due Table 5 Effects of analytes’ concentration on in vitro recovery of the probes. Phenolic compounds
DSS
PA
PAL
Blood probe
Brain probe
Concentration (ng/mL)
Recovery (%)
27.54 344.32 Average 55.30 331.52 Average 48.00 480.00 Average
29.63 31.23 30.43 23.34 23.83 23.59 31.22 32.43 31.83
± ± ± ± ± ± ± ± ±
2.25 2.68 1.13 0.87 0.89 0.35 1.87 3.02 0.86
Recovery (%) 22.08 24.06 23.07 17.54 17.67 17.60 19.26 18.83 19.05
± ± ± ± ± ± ± ± ±
2.14 1.12 1.40 2.12 4.83 0.09 2.09 1.31 0.30
± ± ± ± ± ± ± ± ±
0.83 1.68 1.01 1.12 0.89 1.57 0.98 2.02 2.73
Perfusing rate (L/min)
Recovery (%)
1.0 0.8 0.6 1.0 0.8 0.6 1.0 0.8 0.6
11.26 23.83 28.78 15.48 17.67 23.05 13.13 18.83 24.70
± ± ± ± ± ± ± ± ±
1.08 0.89 0.59 2.15 2.83 3.49 0.97 1.31 1.48
to its small molecular weight and low protein binding rate of 5% (Yang et al., 2007). This finding supports that DSS can protect the brain injury in rats. PAL was not detected in rat blood and brain, despite the high concentration measured in the Danshen extract. Also, PAL was not detected in human serum after an i.v. drip infusion of 250 mL Danshen injection with PAL ≥ 3 mg (Li et al., 2005). These results suggest that PAL might be extensively metabolized and degraded rapidly in vivo. It is reported that orally administered PAL was quickly oxidized to PA in the phase I metabolism and PAL occurred in the plasma mainly in the form of its conjugates (Xu et al., 2007b). Thus in this study, PA, which is not detected in Danshen extract but observed in rat blood and brain, is assumed to be the oxidation metabolite from PAL. In view of that the conjugated PAL will elute earlier than PAL due to its higher polarity, we speculate that the unknown peaks appeared at around the retention time of 10–15 min may be conjugated PAL, as can be seen in Fig. 3d and e. However, such speculation needs further confirmation by LC–MS. Besides, TCM is a complicated system, in which components may affect absorption, distribution, metabolism, and excretion of each other. The concurrent components in Danshen extract may depress the absorption and accelerate the elimination of PAL (Song et al., 2007). All the above factors and the relatively high protein binding rate of 54.5–74.0% of PAL (Yang et al., 2007) made it almost impossible to detect PAL in the microdialysates. These findings might help to identify that the active component responsible for brain effects in Danshen extract is PA instead of PAL.
5. Conclusion An ultrasensitive HPLC–CL method coupled with microdialysis sampling was developed for the simultaneous determination of there phenolic compounds (DSS, PA, and PAL) of Danshen extract in rat blood and brain microdialysates. The proposed method has the advantages of producing less tissue damage, no biological fluid loss, and less physical interference to animals, providing samples with unbound analytes, and allowing for analysis without pretreatment. DSS and metabolite PA were found both in blood and brain after oral administration of Danshen extract (DSS 40 mg/kg, PAL 149 mg/kg, SalB 50 mg/kg), indicating PAL was metabolized into PA. For the first time, the pharmacokinetic profiles of DSS and PA in rat brain after oral administration of Danshen extract were illustrated. This model could be used for the studies of brain pharmacokinetics and BBB permeability for other TCMs.
Acknowledgement This work was supported by Science and Technological Fund of Anhui Province for Outstanding Youth (10040606Y12) and Anhui Provincial Natural Science Foundation (070413113).
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References Brunner, M., Derendorf, H., 2006. Clinical microdialysis: current applications and potential use in drug development. Trends in Analytical Chemistry 25, 674–680. Chen, Y.F., Jaw, I., Shiao, M.S., Tsai, T.H., 2005. Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography. Journal of Chromatography A 1088, 140–145. de Lange, E.C.M., Danhof, M., de Boer, A.G., Breimer, D.D., 1997. Methodological considerations of intracerebral microdialysis in pharmacokinetic studies on drug transport across the blood–brain barrier. Brain Research Reviews 25, 27–49. Gao, D.Y., Han, L.M., Zhang, L.H., Fang, X.L., Wang, J.X., 2009. Bioavailability of salvianolic acid B and effect on blood viscosities after oral administration of salvianolic acids in beagle dogs. Archives of Pharmacal Research 32, 773–779. Guo, X.R., Chen, X.H., Li, L., Shen, Z.D., Wang, X.L., Zheng, P., Duan, F.X., Ma, Y.F., Bi, K.S., 2008. LC–MS determination and pharmacokinetic study of six phenolic components in rat plasma after taking traditional Chinese medicinal-preparation: guanxinning lyophilized powder for injection. Journal of Chromatography B 873, 51–58. Han, D.E., Gao, Z.D., Zhao, D., Wang, L., Li, N., Li, T.T., Wu, L., Chen, X.J., 2009. Liquid chromatography mass spectrometry for the determination of salvianolic acid B, a natural compound from the herb Danshen in rat plasma and application to pharmacokinetic study. Biomedical Chromatography 23, 1073–1078. Hou, Y.Y., Peng, J.M., Chao, R.B., 2007. Pharmacokinetic study of salvianolic acid A in rat after intravenous administration of Danshen injection. Biomedical Chromatography 21, 598–601. Kang, D.G., Yun, Y.G., Ryoo, J.H., Lee, H.S., 2002. Anti-hypertensive effect of water extract of danshen on renovascular hypertension through inhibition of the renin angiotensin system. The American Journal of Chinese Medcine 30, 87–93. Li, X.C., Yu, C., Cai, Y.B., Liu, G.Y., Jia, J.Y., Wang, Y.P., 2005. Simultaneous determination of six phenolic constituents of danshen in human serum using liquid chromatography/tandem mass spectrometry. Journal of Chromatography B 820, 41–47. Li, M., Zhao, M.Q., Kumar Durairajan, S.S., Xie, L.X., Zhang, H.X., Kum, W.F., Goto, S., Liao, F.L., 2008. Protective effect of tetramethylpyrazine and salvianolic acid B on apoptosis of rat cerebral microvascular endothelial cell under high shear stress. Clinical Hemorheology and Microcirculation 38, 177–187. Liu, Q.G., Lu, Z.Y., 1999. Effect of Salvia miltiorrhiza on coronary collateral circulation in dogs with experimental acute myocardial infarction. Journal of Huazhong University of Science and Technology (Medical Sciences) 19, 40–41. Liu, Y., Li, X.R., Li, Y.H., Wang, L.J., Xue, M., 2010. Simultaneous determination of danshensu, rosmarinic acid, cryptotanshinone, tanshinone IIA, tanshinone I and dihydrotanshinone I by liquid chromatographic–mass spectrometry and the application to pharmacokinetics in rats. Journal of Pharmaceutical and Biomedical Analysis 53, 698–704. Lo, C.J., Lin, J.G., Kuo, J.S., Chiang, S.Y., Chen, S.C., Liao, E.T., Hsieh, C.L., 2003. Effect of Salvia miltiorrhiza bunge on cerebral infarct in ischemia–reperfusion injured rats. The American Journal of Chinese Medcine 31, 191–200. Luo, S.Y., Zhong, Z.G., Lin, J.T., Wu, T., 2009. Determination and pharmacokinetic study of danshen in rat plasma and brain after oral administration of danshen extract. Pharmacology and Clinics of Chinese Materia Medica 25, 44–47. Ma, Y.M., Wang, T.M., 2007. A solid-phase extraction method for highperformance liquid chromatographic determination of salvianolic acid B in rabbit plasma: application to pharmacokinetic study. Biomedical Chromatography 21, 217–220. Qu, G.W., 2004. Studies on the Preparation and Quality Control of Total Salvianolic Acid, Pharmaceutical Chemistry. Ocean University of China, Qingdao. Song, M., Hang, T.J., Zhang, Z.X., 2007. Pharmacokinetic interactions between the main components in the extracts of Salvia miltiorrhiza Bge in rat. Acta Pharmaceutica Sinica 17, 493–498. Wang, X.L., Zhang, Z.R., Fu, H.L., Liu, J., Chen, Q., Nie, Y., Deng, L., Gong, T., 2007. Simultaneous determination and pharmacokinetic study of water-soluble and lipid-soluble components of danshen in rat plasma using HPLC–UV method. Biomedical Chromatography 21, 1180–1185.
Wu, Y.T., Chen, Y.F., Hsieh, Y.J., Jaw, I., Shiao, M.S., Tsai, T.H., 2006. Bioavailability of salvianolic acid B in conscious and freely moving rats. International Journal of Pharmaceutics 326, 25–31. Xu, M., Fu, G., Qiao, X., Wu, W.Y., Guo, H., Liu, A.H., Sun, J.H., Guo, D.A., 2007a. HPLC method for comparative study on tissue distribution in rat after oral administration of salvianolic acid B and phenolic acids from Salvia miltiorrhiza. Biomedical Chromatography 21, 1052–1063. Xu, M., Zhang, Z.C., Fu, G., Sun, S.F., Sun, J.H., Yang, M., Liu, A.H., Han, J., Guo, D.A., 2007b. Liquid chromatography–tandem mass spectrometry analysis of protocatechuic aldehyde and its phase I and II metabolites in rat. Journal of Chromatography B 856, 100–107. Yang, X.N., Wang, Y.J., Liu, Y.S., Tang, X., 2007. Effects of Panax quinquefolium protopanaxadiol saponins on the animal and human plasma protein binding of salvianolic acids in vitro. Asian Journal of Pharmaceutical Sciences 2, 170–179. Yang, Y., Xue, X., Liu, Z.P., 2009. Protective Effect of danshensu on focal cerebral ischemia–reperfusion injury in rats. Food and Drug 11, 24–26. Ye, G., Wang, C.S., Li, Y.Y., Ren, H., Guo, D.A., 2003. Simultaneous determination and pharmacokinetic studies on (3,4-dihydroxyphenyl)-lactic acid and protocatechuic aldehyde in rat serum after oral administration of Radix Salviae miltiorrhizae extract. Journal of Chromatographic Science 41, 327–330. Yuan, J.C., Yao, J.H., Han, G.Z., Hu, X.W., 2009. Simultaneous determination of five water-soluble components of danshen in rabbit plasma and its application to pharmacokinetics. In: ICBBE 2009. 3rd International Conference on Bioinformatics and Biomedical Engineering, 2009 , Beijing, pp. 1–4. Zhang, J.L., Yu, H.L., Sheng, Y.X., Li, L., Ye, M., Guo, D.A., 2005a. HPLC determination and pharmacokinetic studies of salvianolic acid B in rat plasma after oral administration of Radix Salviae miltiorrhizae extract. Biomedical Chromatography 19, 15–18. Zhang, T., Wang, C.Y., Tian, J.W., Fu, F.H., Liu, K., 2005b. Protective effect of danshensu on brain injury. Chinese Traditional and Herbal Drugs 36, 1041–1043. Zhang, Z.C., Xu, M., Sun, S.F., Qiao, X., Wang, B.R., Han, J., Guo, D.A., 2008. Metabolic analysis of four phenolic acids in rat by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B 871, 7–14. Zhang, Q.L., Zhang, Y.J., Wu, L., Li, J., Yuan, Y., 2010. Determination of phenolic compounds of Salvia miltiorrhiza in rat brain by liquid chromatography with chemiluminescence detection using microdialysis sampling and application to pharmacokinetic study. Chinese Pharmacological Bulletin 26, 1230–1237. Zhao, G.R., Zhang, H.M., Ye, T.X., Xiang, Z.J., Yuan, Y.J., Guo, Z.X., Zhao, L.B., 2008. Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B. Food and Chemical Toxicology 46, 73–81. Zheng, X.H., Zhao, X., Fang, M.F., Wang, S.X., Wei, Y.M., Zheng, J.B., 2007. Pharmacokinetic effects of Shi herb-Borneol on Jun herb-Salvia Miltiorrhiza. Journal of Xi’an Jiaotong University (Medical Sciences) 28, 170–173. Zhou, L., Zuo, Z., Chow, M.S.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. The Journal of Clinical Pharmacology 45, 1345–1359.
Glossary BBB: blood brain barrier BW: body weight CL: chemiluminescence CSF: cerebrospinal fluid DSS: danshensu HPLC: high performance liquid chromatography PA: protocatechuic acid PAL: protocatechuic aldehyde SalB: salvianolic acid B TCM: traditional Chinese medicine UNK: unknown
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Pharmacokinetics of phenolic compounds of Danshen extract in rat blood and brain by microdialysis sampling Yun-Jing Zhang a,b , Liang Wu a,b , Qun-Lin Zhang a,b,∗ , Jun Li a,b , Fang-Xiong Yin a,b , Ye Yuan a,b a b
School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, PR China Anhui Key Laboratory of Bioactivity of Natural Products, Anhui Medical University, Hefei, Anhui 230032, PR China
a r t i c l e
i n f o
Article history: Received 28 August 2010 Received in revised form 17 February 2011 Accepted 11 April 2011 Available online 16 April 2011 Keywords: Danshen extract Phenolic compound Microdialysis Pharmacokinetics High-performance liquid chromatography Chemiluminescence
a b s t r a c t Aim of the study: To evaluate the pharmacokinetics of phenolic compounds after oral administration of Danshen extract in rat brain. Materials and methods: Blood and brain microdialysis probes were inserted into jugular vein and cerebral cortex of rat under anesthesia and perfused with ringer’s solution at the rate of 2.0 and 0.8 L/min, respectively. Blank microdialysates were collected after 2 h post-implantation equilibrium time. Danshen extract (danshensu 40 mg/kg BW, protocatechuic aldehyde 149 mg/kg BW, and salvianolic acid B 50 mg/kg BW) was administrated intragastrically, and then blood and brain microdialysates were collected at 15 and 30 min time intervals for 4 h, respectively. The concentrations of phenolic compounds were determined by high-performance liquid chromatography coupled with chemiluminescence detection. Pharmacokinetic parameters were estimated using non-compartmental methods. Results: Danshensu and protocatechuic acid could be detected in both blood and brain microdialysates, while protocatechuic aldehyde and salvianolic acid B were not detected. Brain-to-blood (AUCbrain /AUCblood ) distribution ratio were 0.25 ± 0.04 and 0.09 ± 0.02 for danshensu and protocatechuic acid, respectively. Conclusions: Danshensu can readily permeate the blood brain barrier after oral administration of Danshen extract, and protocatechuic acid is a potential oxidative metabolite of protocatechuic aldehyde. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Danshen, the dry root and rhizome of the labiatae plant Salvia miltiorrhiza Bge., is one of the most widely used traditional Chinese medicine (TCM), for the treatment of cardiovascular and cerebrovascular diseases. The chemical constituents of Salvia miltiorrhiza mainly include water-soluble phenolic compounds and diterpenoid quinines. The widely use of Danshen water decoction in clinical medication indicates that water-soluble phenolic compounds may be the most important constituents responsible for the therapeutic effects of Danshen. Up to today, more than 30 phenolic compounds have been isolated and structurally identified from Danshen, among which danshensu (DSS), protocatechuic aldehyde (PAL), protocatechuic acid (PA) and salvianolic acid B (SalB) (Fig. 1) are the most effective active ingredients. In the pharmacological and clinical studies, these phenolic compounds were found to be strong antioxidants and free radical scavengers (Zhao et al., 2008),
∗ Corresponding author at: School of Pharmacy, Anhui Medical University, Meishan Road 81, Hefei, Anhui 230032, PR China. Tel.: +86 551 5161115; fax: +86 551 5167735. E-mail address: [email protected] (Q.-L. Zhang). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.04.023
and could improve blood circulation (Liu and Lu, 1999), reduce the area of cerebral infarct (Lo et al., 2003), and inhibit the renin angiotensin system (Kang et al., 2002). Along with the deepening of pharmacological investigation, phenolic compounds of Danshen were found to have brain protective effects, such as anti-apoptotic (Li et al., 2008), ameliorating the impairment of cerebral ischemia (Yang et al., 2009), and improving the learning and memory deficit of senile dementia (Zhang et al., 2005b). Numerous pharmacokinetic studies were performed for better understanding the pharmacological effects of water-soluble components in Danshen (Li et al., 2005; Zhou et al., 2005; Hou et al., 2007; Guo et al., 2008; Gao et al., 2009). The concentrationtime data of DSS in rat plasma were found to be best fitted to a two-compartment open model, and its absorption, distribution, and elimination of were relatively moderate following administration of a single oral dose of Danshen extract (Liu et al., 2010). It was reported that methylation was the main pathway of DSS metabolism in rats after oral administration (Zhang et al., 2008). The pharmacokinetic profiles of PAL in rat serum after oral administration of Radix Salviae miltiorrhizae extract showed a double peak pattern (Ye et al., 2003). SalB, another major phenolic compound in Danshen, was absorbed quickly with maximum concentration obtained 0.5 h and eliminated rapidly from plasma after oral admin-
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Fig. 1. Chemical structures of danshensu (DSS), protocatechuic aldehyde (PAL), protocatechuic acid (PA), and salvianolic acid B (SalB).
istration to rats (Zhang et al., 2005a). The bioavailability of SalB was found to be extremely low in rats (Chen et al., 2005; Wu et al., 2006), beagle dogs (Han et al., 2009) and rabbits (Ma and Wang, 2007). Although pharmacokinetics of phenolic compounds in Danshen have been extensively studied, little is known about the pharmacokinetic profiles of phenolic compounds in brain. The relevant pharmacokinetic properties of phenolic compounds or its active metabolites include the ability to be absorbed from the site of administration and to pass through the blood brain barrier (BBB). However, only a few literatures (Xu et al., 2007a; Zheng et al., 2007; Luo et al., 2009) have reported the distribution of water-soluble phenolic compounds of Danshen in brain. Luo et al. (2009) found that DSS, PAL, and SalB were detected in rat serum but could not be detected in brain tissue homogenate after oral administration of water extract of Danshen (DSS 32.4 mg/kg BW, PAL 6.0 mg/kg BW, SalB 36.2 mg/kg BW), due to the limited sensitivity of UV detection in this study. However, it is debatable whether these phenolic compounds can penetrate the BBB. It was reported that DSS could be detected in CSF after oral administration of water extract of Danshen (10 g/kg) to New Zealand rabbits, but the pharmacokinetic parameters of DSS in brain were not mentioned (Zheng et al., 2007). SalB could be detected in rat brain after oral administration of total phenolic acids (500 mg/kg) and SalB (300 mg/kg) extracted from the roots of Salvia miltiorrhiza, but the concentration of SalB was below the quantification limit (Xu et al., 2007a). In the abovementioned studies, the limited sensitivity of UV detection prohibits trace level determination of analytes in the brain, while detection by LC/MS involves relatively tedious sample pretreatment. Besides, the sample collection by tissue homogenizing cannot allow continuous monitoring of analytes, and the drawing of cerebrospinal fluid might disturb the physiological state of animal due to loss of the body fluid. For the pharmacokinetics study of drugs in brain, intracerebral microdialysis is the only technique which offers the possibility to continuously monitor the local BBB transport of unbound drugs in tested animals, under physiological and pathological conditions (de Lange et al., 1997). Biological samples obtained using conventional sampling method contain protein-bound and unbound drugs. However, only the unbound fraction of drug is therapeuti-
cally active. Microdialysis offers protein-free clean samples which do not require purification prior to analysis, because the dialysis membrane of probe is only permeable to protein-unbound small molecules. Microdialysis also presents the advantages of sampling from multiple sites of the same animal, avoiding the effect of biological volume changes, and allowing for analysis without pretreatment. Currently, the Food and Drugs Administration of USA and the European Union’s Committee emphasize the value of human-tissue drug concentration data and support using clinical microdialysis to obtain pharmacokinetic information (Brunner and Derendorf, 2006), demonstrating the potential and importance of microdialysis. However, until now, the application of microdialysis to the pharmacokinetic study of Danshen is extremely limited. Only Tsai group (Chen et al., 2005) developed an in vivo microdialysis sampling method for the monitoring the protein-unbound SalB of Danshen in rat blood and bile after intravenous administration of SalB at about 98% purity. In our previous work, the pharmacokinetic properties of DSS in rat brain after single intravenous administration of Danshen injection with microdialysis sampling were studied (Zhang et al., 2010). The present work is aimed to reveal the BBB penetration and pharmacokinetics of protein-unbound phenolic compounds in rat brain after oral administration of hydrophilic extract of Danshen by simultaneous microdialysis in blood and brain. A highly sensitive HPLC method coupled with chemiluminescence (CL) detection was developed for the determination of DSS, PA, PAL, and SalB in rat microdialysates from blood and brain. As far as we know, it is the first time to report the pharmacokinetic profiles of DSS and metabolite PA in rat brain as well as their blood-to-brain distribution ratio after oral administration of Danshen extract. 2. Materials and methods 2.1. Chemicals and solutions A stock solution of luminol (1.0 × 10−2 mol/L) was prepared by dissolving luminol (Merck, Darmstadt, Germany) in sodium hydroxide solution (0.10 mol/L) and stored at least 7 days before
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dilution. The working H2 O2 solution was prepared fresh daily from 30% (w/w) H2 O2 (Suzhou Chemical Reagent Company, China). A stock solution (1%, w/w) of HAuCl4 was prepared by dissolving HAuCl4 ·4H2 O (Shanghai Chemical Reagent Company, China) in water. The reference compounds of DSS and SalB were purchased from Shanghai Winherb Medical Science Company (Shanghai, China), and PA and PAL were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Stock solutions of danshensu (0.1790 mg/mL), protocatechuic acid (0.2160 mg/mL), protocatechuic aldehyde (0.2000 mg/mL), and SalB (0.2064 mg/mL) were prepared in methanol. These stock solutions were kept in the dark at 4 ◦ C. Danshen crude drug was purchased from a local pharmacy. Methanol was of HPLC grade, and all other chemicals were of analytical-reagent grade. The ultrapure water (18.3 M cm, Millipore, USA) was used to prepare the mobile phases of HPLC and all the solutions. The mobile phases of HPLC were prepared fresh daily and filtered through a 0.22 m membrane filter (Bandao, Shanghai, China). The perfusate for blood and brain microdialysis was Ringer’s solution (122 mmol/L NaCl, 3 mmol/L KCl, 0.4 mmol/L KH2 PO4 , 1.2 mmol/L MgSO4 , 25 mmol/L NaHCO3 , and 1.2 mmol/L CaCl2 ). 2.2. Animals Adult, male Spague-Dawley rats (300 ± 20 g) were supplied by the Laboratory Animal Center of Anhui Medical University, housed in an air-conditioned room (temperature, 22–25 ◦ C; relative humidity, 55 ± 5%), and kept on a light/dark cycle of 12/12 h. Free access to food and mains drinking water was allowed throughout the study except for fasting 18 h before the experiment. Animal studies were performed according to the Guidelines for the Care and Use of Laboratory Animals approved by the Committee of Ethics of Animal Experimentation of Anhui Medical University (Hefei, China). 2.3. Preparation of Danshen extract In view of the thermolability of phenolic compounds, the acid percolation process (Qu, 2004) was chosen to prepare Danshen extract. The coarse powder of Danshen crude drug was soaked in 8 volumes of 0.01% HCl overnight and then the coarse powder together with the leachate was percolated with 14 volumes of water (pH 2.0). The percolate, which is a hydrophilic extract, was collected and loaded onto an AB-8 macroporous resin packed column. The column was washed with 5 times of column volume of water (pH 2.0) to remove the unabsorbed impurities, and followed by 6 times of column volume of 25% ethanol (pH 2.0) and 4 times of column volume of 40% ethanol (pH 2.0) to elute phenolic compounds. The eluent of 25% and 40% ethanol (pH 2.0) was combined and condensed under vacuum to obtain Danshen extract. The components and contents of Danshen extract were analyzed by a validated HPLC–CL method against the reference standards of DSS, PAL, and SalB, and were determined as 8.0, 29.8, and 10.0 mg/mL of DSS, PAL, and SalB, respectively (n = 5). 2.4. Instruments and chromatographic and CL conditions The HPLC–CL detection system consists of an HPLC system and a CL detection system, as shown in Fig. 2. The HPLC system is Shimadzu LC-20A series (Shimadzu Corporation, Japan), including a quaternary pump, a vacuum degasser, a thermostated column compartment, a prominence diode array detector (DAD), a manual sample valve injector with a 20 L loop, and an analytical column (Shim-pack ODS, 250 mm × 4.6 mm I.D. 5 m; Shimadzu). The CL detection is performed with a flow injection CL system (Remax,
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Xi’an, China), which is composed of a model IFFM-E peristaltic pump, a mixing tee, and a model IFFS-A CL detector equipped with a flat glass coil (used as reaction coil and detection cell) and a photomultiplier operated at −400 V. The analysis was performed on a ODS column at 30 ◦ C with gradient elution at a flow rate of 1.0 mL/min. The mobile phase was composed of aqueous phosphoric acid (0.1%, v/v)–methanol (92:8, v/v). Detection by DAD was set at 280 nm. The column effluent from DAD was first mixed on-line with HAuCl4 solution via a PEEK tube, and then combined with H2 O2 and luminol solution in a mixing tee, respectively (Fig. 2). All the CL solutions were delivered by a peristaltic pump at a flow rate of 2.0 mL/min. The microdialysis system (CMA, Stockholm, Sweden) consists of a CMA/400 microinjection pump, a CMA/150 temperature controller, and a CMA/470 refrigerated fraction collector. 2.5. Microdialysis experiments Rats were anesthetized with chloral hydrate (300 mg/kg, i.p.) and remained anesthetized throughout the experimental period. The rats’ body temperature was maintained at 37 ◦ C with a heating blanket. Surgical sites were shaved and cleaned with povidone–iodine. The probe for blood sampling (MAB 7.8.10, membrane length, 10 mm, molecular weight cut-off 15 kDa) was implanted within the jugular vein/right atrium and perfused with Ringer’s solution at a flow rate of 2.0 L/min. Rat was then mounted on a stereotaxic frame and the brain microdialysis probe (CMA/12, membrane length, 4 mm, molecular weight cut-off 20 kDa) was implanted in the cerebral cortex (coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to midline, 3.5 mm from dura) and perfused with Ringer’s solution at 0.8 L/min. The proper placement of probe in the cerebral cortex was confirmed by making an incision at the site of probe implantation after completion of studies. Danshen extract was administered orally (5 mL/kg, DSS 40 mg/kg, PAL 149 mg/kg, and SalB 50 mg/kg) after a 2 h post-surgical stabilization period. The microdialysis samples of blood and brain were collected every 15 and 30 min, respectively, for 4 h. The collected samples were kept at 4 ◦ C and analyzed within 48 h. The blood dialysate samples of 15, 30, 45 and 60 min were diluted 4 times with Ringer’s solution prior to analysis. 2.6. In vivo recovery assessment of microdialysis probes To estimate the in vivo recoveries, a retrodialysis calibration technique was utilized. The microdialysis probe was inserted into the rat’s jugular vein and cerebral cortex under anesthesia, and then the Ringer’s solution containing the phenolics (DSS, PA, and PAL) was perfused through the probe at the flow rate of 2.0 L/min and 0.8 L/min, respectively by the microinjection pump after a stabilization period of 2 h post probe implantation. The perfusate (Cperf ) and dialysate (Cdial ) concentrations of six phenolics were determined by the HPLC–CL method. The in vivo relative recovery (Rdial ) of each phenolic compound across the microdialysis probe was calculated by the following equation: Rdial = (Cperf − Cdial )/Cperf. The in vivo recovery assessment was done after the feeding experiment when phenolic compounds in the microdialysates were undetectable. The concentrations of phenolic compounds (Cm ) were converted to free-form concentrations (Cf ) as follows: Cf = Cm /Rdial . 2.7. Pharmacokinetic data analysis The drug concentration data (after being converted to free form concentrations) were processed by Drug and Statistics (DAS 2.0) (Mathematical Pharmacology Professional Committee of China, Shanghai, China).
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Fig. 2. Schematic diagram of the HPLC–CL system used for the determination of phenolic compounds.
2.8. Method validation 2.8.1. Selectivity For the pharmacokinetic study, blank blood and brain microdialysates from rats and blank Ringer’s solution spiked with DSS, PA and PAL were evaluated for the peak interference. 2.8.2. Linearity and sensitivity The linearity of the HPLC–CL method was evaluated by the correlation coefficients (r) of the calibration curves generated with more than 10 standard mixtures, using Ringer’s solution spiked with DSS, PA, and PAL. For each phenolic compound, an external standard method was used to establish the calibration curve. The quantitative determination was based on the relative CL intensity I = IS − I0 , where IS is the CL intensity of phenolic compound and I0 is the intensity of blank signal. Calibration curves were constructed by plotting logarithm of the phenolic concentration (lg C) against the logarithm of the relative CL intensity (lg I). All calibration curves were required to have a correlation value of at least 0.990. The signal-to-noise ratio (S/N) of 3 was set as the threshold for calculating the detection limit. 2.8.3. Precision and accuracy The validation was performed in Ringer’s solution. Precision was expressed as the relative standard deviation (RSD) of each calculated concentration while accuracy was calculated as the percentage of the found concentration to the added concentration. The intra-day precision was tested with six spiked samples at three concentration levels (DSS, 4.58, 22.91, and 114.56 ng/mL, PA, 6.91, 27.65, and 331.52 ng/mL, PAL, 9.60, 25.60, and 480.00 ng/mL). The inter-day precision and accuracy were evaluated by injecting five times every day on three consecutive days. 3. Results 3.1. Validation of HPLC–CL method 3.1.1. Selectivity DSS, PA, and PAL were completely separated with the retention times of 19.8, 22.0, and 31.6 min, respectively. Representative chromatograms of blank blood and brain dialysate are shown in Fig. 3a and b. Ringer’s solution spiked with DSS, PA, and PAL are shown in Fig. 3c, and microdialysis samples from rat blood and
brain collected 1.5 h after post administration of Danshen extract are presented in Fig. 3d and e, respectively. The biological matrix from rat blood or brain dialysate did not interfere with the analytes. 3.1.2. Linearity and sensitivity Calibration curves showed good linear relationship over the ranges of 2.29–179.0, 3.46–345.6, and 4.80–800.0 ng/mL with the detection limits of 0.29, 0.52, and 0.80 ng/mL for DSS, PA, and PAL, respectively. The calibration equations for DSS, PA, and PAL are: lg I = 0.6142 lg C + 2.0338, lg I = 0.7902 lg C + 1.9416, and lg I = 0.9704 lg C + 1.7908 with the correlation coefficients (r) of 0.9993, 0.9999, and 0.9984, respectively. The proposed HPLC–CL method has much lower detection limits than those of HPLC–UV method, which are at 43.0–65.0 ng/mL level (Wang et al., 2007; Yuan et al., 2009). The method is also more sensitive than LC–MS/MS, whose detection limits for DSS and PAL were reported to be 2.5 and 2.0 ng/mL, respectively (Li et al., 2005). 3.1.3. Precision and accuracy The precision and accuracy data in Table 1 indicate that the present HPLC–CL method has good accuracies and reproducibilities for DSS, PA, and PAL at three levels of concentrations. The accuracies ranged from 90.1 to 103.2% with the relative standard deviations (RSD) not more than 5.5%, which were well within the acceptance criteria of bioanalysis. 3.2. In vitro recovery and delivery The microdialysis probes recoveries by gain and delivery by loss in vitro in 4 h were determined. The perfusing rates for blood and brain microdialysis probes 2.0 L/min and 0.8 L/min, respectively. The microdialysis samples of blood and brain probe were collected every 15 and 30 min, respectively. The RSDs of recoveries by gain in 4 h of blood probe for DSS, PA, and PAL were 3.1%, 3.4%, and 3.9%, respectively, and for brain probe, the recoveries by gain were 3.3%, 4.3% and 3.0% for DSS, PA, and PAL, respectively. The RSDs of deliveries by loss in 4 h of blood probe for DSS, PA, and PAL were 3.2%, 2.4%, and 2.9%, respectively, and for brain probe, the recoveries by gain were 2.3%, 3.2% and 3.1% for DSS, PA, and PAL, respectively. The above results indicate that the recoveries and deliveries are stable during 4 h sampling time. The RSD between recoveries and deliveries of DSS, PA, and PAL were 5.5%, 4.8%, and 6.5%, respectively, which indicates that
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Fig. 3. HPLC–CL chromatograms of (a) blank microdialysate from rat’s blood; (b) blank microdialysate from rat’s brain; (c) blank Ringer’s solution spiked with DSS (71.56 ng/mL), PA (43.20 ng/mL), and PAL (32.00 ng/mL); (d) blood and (e) brain microdialysate collected 1.5 h after administration of Danshen extract detected under the conditions of Section 2.4. UNK = unknown compounds.
the recoveries and deliveries are equivalent for the analytes, thus the recoveries determined by loss can be used for the calibrations of the probes.
3.3. In vivo recoveries of the probes The in vivo recoveries of blood and brain probes for DSS, PA, and PAL were examined and summarized in Table 2. In the further study, the in vivo recoveries were used for the calibration of probes.
3.4. Analytes’ stabilities in the dialysate It was found phenolic compounds are subject to degradation catalyzed by the high concentrations of metal ions in the perfusate. To avoid degradation, 2.0 L H3 PO4 was added in the collecting vials in advance, and then no significant degradation of analytes in Ringer’s solution with H3 PO4 was observed after 48 h at 20 ◦ C. Thus the determination of DSS, PA, and PAL in microdialysis samples could be accomplished in 48 h.
Table 1 Precisions and accuracies of DSS, PA and PAL by HPLC–CL method (¯x ± s). Phenolic compounds
Added (ng/mL)
Intra-day found (ng/mL) (n = 5)
DSS
4.58 22.91 114.56 6.91 27.65 331.52 9.60 25.60 480.00
4.53 21.76 115.60 6.72 25.64 332.42 8.65 26.170 482.01
PA
PAL
± ± ± ± ± ± ± ± ±
0.24 1.05 2.41 0.12 0.95 14.01 0.45 1.32 3.97
Recovery (%)
RSD (%)
Inter-day found (ng/mL) (n = 3)
98.87 94.97 100.91 97.28 92.74 100.27 90.10 102.23 100.42
5.30 4.82 2.08 1.86 3.69 4.21 5.20 5.04 0.82
4.52 22.00 118.19 6.86 25.10 339.87 8.99 25.10 483.19
± ± ± ± ± ± ± ± ±
0.24 0.67 1.96 0.24 0.92 4.12 0.49 0.59 9.53
Recovery (%)
RSD (%)
98.65 96.01 103.17 97.80 91.07 102.52 93.66 98.05 100.67
5.31 3.04 1.67 3.55 3.63 1.21 5.47 2.35 1.98
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Table 2 In vivo recoveries of blood and brain microdialysis probes for DSS, PA, and PAL. Phenolic compounds
In vivo recovery (%) (¯x ± s, n = 5)
Concentration (ng/mL)
Blood probe DSS
PA
PAL
27.54 344.32 Average 55.30 331.52 Average 48.00 480.00 Average
34.85 34.93 34.89 36.08 36.74 36.41 34.40 35.26 34.83
± ± ± ± ± ± ± ± ±
1.39 1.28 0.06 0.68 1.19 0.46 3.06 2.17 0.61
Brain probe 26.63 28.33 27.48 34.29 37.16 35.73 21.25 23.55 22.40
± ± ± ± ± ± ± ± ±
2.04 1.33 1.20 1.46 0.34 2.03 2.12 1.53 1.63
Fig. 5. Unbound PA concentration versus time curves of blood and brain after administration of Danshen extract to SD rats (¯x ± s, n = 6) [ blood, brain].
DSS could be detected up to 4 h after oral administration. The brain distribution ratio (AUCbrain /AUCblood ) of DSS was calculated to be 0.25 ± 0.04. PA appeared in blood rapidly following oral administration, reached a peak at 0.5 h, eliminated from blood with the mean residence time less than 1 h [(0.60 ± 0.01) h], and was undetectable in blood or brain 2.0 h after administration. PA penetrated the BBB with a brain distribution ratio (AUCbrain /AUCblood ) of 0.09 ± 0.02. 4. Discussion 4.1. Optimization of HPLC–CL system Fig. 4. Unbound DSS concentration versus time curves of blood and brain after administration of Danshen extract to SD rats (¯x ± s, n = 6) [ blood, brain].
3.5. Pharmacokinetic study After oral administration of Danshen extract (DSS 40 mg/kg, PAL 149 mg/kg, and SalB 50 mg/kg), DSS and PA were detected in the rats’ blood and brain microdialysates while no PAL or SalB was observed. The mean blood and brain concentration-time profiles of DSS and PA are illustrated in Figs. 4 and 5, respectively. The main blood and brain pharmacokinetic parameters of DSS and PA estimated by statistical moment method are given in Table 3. The maximum blood concentration of DSS was observed after 0.5 of administration. DSS was distributed rapidly to brain with the maximum brain concentration being achieved 1.0 h after dosing. Table 3 Pharmacokinetic parameters of DSS and PA in rat blood and brain after administration of Danshen extract (5 mL/kg, i.g., x¯ ± s, n = 6). Parameters Blood AUC0–∞ (g h/mL) MRT (h) tmax (h) Cmax (g/mL) t1/2 (h) Brain AUC0–∞ (g h/mL) MRT (h) tmax (h) Cmax (g/mL) t1/2 (h) Brain-to-blood distribution AUCbrian /AUCblood
DSS
PA
4.46 4.31 0.5 1.14 3.00
± ± ± ± ±
0.99 1.11 0.0 0.17 1.16
1.18 0.60 0.5 1.62 0.42
± ± ± ± ±
0.45 0.01 0.0 1.20 0.01
1.10 2.28 1.0 0.28 1.60
± ± ± ± ±
0.20 1.14 0.0 0.14 0.70
0.13 1.34 1.0 0.09 0.90
± ± ± ± ±
0.03 0.01 0.0 0.07 0.10
0.25 ± 0.04
0.09 ± 0.02
Several compositions of mobile phases have been tested for HPLC separation, such as methanol–acetic acid–water, methanol–formic acid, and acetonitrile–phosphoric acid. It was found that the mobile phase consisting of a mixture of methanol and aqueous 0.1% phosphoric acid (8:92, v/v) was not only suitable for the good separation of DSS, PA, and PAL but also compatible with the HAuCl4 –luminol–H2 O2 CL reaction. The good resolution of HPLC separation was achieved within 33 min. To obtain the maximal efficiency of CL system and stability of CL signals, the effects of configurations, type and pH of the buffer solution, concentrations of luminol, H2 O2 , and HAuCl4 , and flow rate on the CL intensities were investigated. The stable and maximal relative CL signals were obtained under the conditions of NaHCO3 –Na2 CO3 buffer solution of pH 10.83, 2.0 × 10−5 mol/L luminol, 5.0 × 10−5 mol/L H2 O2 , 4.0 × 10−5 g/mL HAuCl4 , and the flow rate of 2.0 mL/min using the configuration in Fig. 2. 4.2. Optimization of microdialysis system Effects of perfusing rate on the recoveries of probes were evaluated. Blood and brain microdialysis probes were immersed in Ringer’s solution containing known concentrations of the analytes (DSS, 275.46 ng/mL, PA, 331.520 ng/mL, and PAL, 480.000 ng/mL), and perfused with blank Ringer’s solution at various flow rates (2.5, 2.0, and 1.5 L/min for blood probe; 1.0, 0.8, and 0.6 L/min for brain probe). For each perfusing rate, three microdialysis samples were collected with 30 min time interval and the recoveries under each perfusing rate were determined. The results in Table 4 indicate that the recoveries of the probes increased with the decreased perfusing rate. The effects of analytes’ concentration on the recoveries of probes were also investigated. Blood and brain microdialysis probes were
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Table 4 Effects of perfusing rate on in vitro recovery of the probes (¯x ± s, n = 3). Phenolic compounds
DSS
PA
PAL
Blood probe
Brain probe
Perfusing rate (L/min)
Recovery (%)
2.5 2.0 1.5 2.5 2.0 1.5 2.5 2.0 1.5
28.86 31.23 35.40 20.08 23.83 27.08 29.72 32.43 35.45
immersed in Ringer’s solution containing different concentrations of DSS, PA, and PAL, and perfused with blank Ringer’s solution at the flow rate of 2.0 L/min for blood probe and 0.8 L/min for brain probe. For each concentration, three microdialysis samples were collected with 30 min time interval and the recoveries under each perfusing rate were determined. As shown in Table 5, the recoveries for DSS, PA, and PAL showed a non concentration-related pattern within the examined range. At low perfusing rate, the diffusions of the solution inside and outside the probe membranes are almost balanced, producing relatively high recovery. However, it takes longer sampling time to collect sufficient sample volume for HPLC analysis, resulting in reduced time resolution and concentration-time information. Conversely, excessively high perfusing rate will impair the normal physiological state of rats. Considering the above factors, the perfusing rate of 2.0 and 0.8 L/min was chosen for blood and brain probe with sampling interval of 15 and 30 min, respectively. 4.3. Pharmacokinetic study In the preliminary experiment of this study, SalB was not observed in rat blood and brain microdialysates after oral administration of Danshen extract. Thus in the further experiments, we cut out the detection of SalB by HPLC to shorten the analysis time. The oral bioavailability of SalB was calculated about 2.3%, and the protein binding rate of SalB in vivo was determined to be 83.78 ± 10.5% (Wu et al., 2006). As protein-bound compounds cannot penetrate the membrane of the microdialysis probes, only very small amounts of free SalB could occur in the microdialysates. The relatively low oral bioavailability together with high protein binding rate might result in undetectable SalB levels in the microdialysates after oral administration of Danshen extract. The pharmacokinetic data show that DSS is capable of permeating the BBB, which is in conformity with our results of previous study after administration of Danshen injection (Zhang et al., 2010), implying BBB permeability to DSS independent of dosage or administration route. The good BBB permeability of DSS is probably due Table 5 Effects of analytes’ concentration on in vitro recovery of the probes. Phenolic compounds
DSS
PA
PAL
Blood probe
Brain probe
Concentration (ng/mL)
Recovery (%)
27.54 344.32 Average 55.30 331.52 Average 48.00 480.00 Average
29.63 31.23 30.43 23.34 23.83 23.59 31.22 32.43 31.83
± ± ± ± ± ± ± ± ±
2.25 2.68 1.13 0.87 0.89 0.35 1.87 3.02 0.86
Recovery (%) 22.08 24.06 23.07 17.54 17.67 17.60 19.26 18.83 19.05
± ± ± ± ± ± ± ± ±
2.14 1.12 1.40 2.12 4.83 0.09 2.09 1.31 0.30
± ± ± ± ± ± ± ± ±
0.83 1.68 1.01 1.12 0.89 1.57 0.98 2.02 2.73
Perfusing rate (L/min)
Recovery (%)
1.0 0.8 0.6 1.0 0.8 0.6 1.0 0.8 0.6
11.26 23.83 28.78 15.48 17.67 23.05 13.13 18.83 24.70
± ± ± ± ± ± ± ± ±
1.08 0.89 0.59 2.15 2.83 3.49 0.97 1.31 1.48
to its small molecular weight and low protein binding rate of 5% (Yang et al., 2007). This finding supports that DSS can protect the brain injury in rats. PAL was not detected in rat blood and brain, despite the high concentration measured in the Danshen extract. Also, PAL was not detected in human serum after an i.v. drip infusion of 250 mL Danshen injection with PAL ≥ 3 mg (Li et al., 2005). These results suggest that PAL might be extensively metabolized and degraded rapidly in vivo. It is reported that orally administered PAL was quickly oxidized to PA in the phase I metabolism and PAL occurred in the plasma mainly in the form of its conjugates (Xu et al., 2007b). Thus in this study, PA, which is not detected in Danshen extract but observed in rat blood and brain, is assumed to be the oxidation metabolite from PAL. In view of that the conjugated PAL will elute earlier than PAL due to its higher polarity, we speculate that the unknown peaks appeared at around the retention time of 10–15 min may be conjugated PAL, as can be seen in Fig. 3d and e. However, such speculation needs further confirmation by LC–MS. Besides, TCM is a complicated system, in which components may affect absorption, distribution, metabolism, and excretion of each other. The concurrent components in Danshen extract may depress the absorption and accelerate the elimination of PAL (Song et al., 2007). All the above factors and the relatively high protein binding rate of 54.5–74.0% of PAL (Yang et al., 2007) made it almost impossible to detect PAL in the microdialysates. These findings might help to identify that the active component responsible for brain effects in Danshen extract is PA instead of PAL.
5. Conclusion An ultrasensitive HPLC–CL method coupled with microdialysis sampling was developed for the simultaneous determination of there phenolic compounds (DSS, PA, and PAL) of Danshen extract in rat blood and brain microdialysates. The proposed method has the advantages of producing less tissue damage, no biological fluid loss, and less physical interference to animals, providing samples with unbound analytes, and allowing for analysis without pretreatment. DSS and metabolite PA were found both in blood and brain after oral administration of Danshen extract (DSS 40 mg/kg, PAL 149 mg/kg, SalB 50 mg/kg), indicating PAL was metabolized into PA. For the first time, the pharmacokinetic profiles of DSS and PA in rat brain after oral administration of Danshen extract were illustrated. This model could be used for the studies of brain pharmacokinetics and BBB permeability for other TCMs.
Acknowledgement This work was supported by Science and Technological Fund of Anhui Province for Outstanding Youth (10040606Y12) and Anhui Provincial Natural Science Foundation (070413113).
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References Brunner, M., Derendorf, H., 2006. Clinical microdialysis: current applications and potential use in drug development. Trends in Analytical Chemistry 25, 674–680. Chen, Y.F., Jaw, I., Shiao, M.S., Tsai, T.H., 2005. Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography. Journal of Chromatography A 1088, 140–145. de Lange, E.C.M., Danhof, M., de Boer, A.G., Breimer, D.D., 1997. Methodological considerations of intracerebral microdialysis in pharmacokinetic studies on drug transport across the blood–brain barrier. Brain Research Reviews 25, 27–49. Gao, D.Y., Han, L.M., Zhang, L.H., Fang, X.L., Wang, J.X., 2009. Bioavailability of salvianolic acid B and effect on blood viscosities after oral administration of salvianolic acids in beagle dogs. Archives of Pharmacal Research 32, 773–779. Guo, X.R., Chen, X.H., Li, L., Shen, Z.D., Wang, X.L., Zheng, P., Duan, F.X., Ma, Y.F., Bi, K.S., 2008. LC–MS determination and pharmacokinetic study of six phenolic components in rat plasma after taking traditional Chinese medicinal-preparation: guanxinning lyophilized powder for injection. Journal of Chromatography B 873, 51–58. Han, D.E., Gao, Z.D., Zhao, D., Wang, L., Li, N., Li, T.T., Wu, L., Chen, X.J., 2009. Liquid chromatography mass spectrometry for the determination of salvianolic acid B, a natural compound from the herb Danshen in rat plasma and application to pharmacokinetic study. Biomedical Chromatography 23, 1073–1078. Hou, Y.Y., Peng, J.M., Chao, R.B., 2007. Pharmacokinetic study of salvianolic acid A in rat after intravenous administration of Danshen injection. Biomedical Chromatography 21, 598–601. Kang, D.G., Yun, Y.G., Ryoo, J.H., Lee, H.S., 2002. Anti-hypertensive effect of water extract of danshen on renovascular hypertension through inhibition of the renin angiotensin system. The American Journal of Chinese Medcine 30, 87–93. Li, X.C., Yu, C., Cai, Y.B., Liu, G.Y., Jia, J.Y., Wang, Y.P., 2005. Simultaneous determination of six phenolic constituents of danshen in human serum using liquid chromatography/tandem mass spectrometry. Journal of Chromatography B 820, 41–47. Li, M., Zhao, M.Q., Kumar Durairajan, S.S., Xie, L.X., Zhang, H.X., Kum, W.F., Goto, S., Liao, F.L., 2008. Protective effect of tetramethylpyrazine and salvianolic acid B on apoptosis of rat cerebral microvascular endothelial cell under high shear stress. Clinical Hemorheology and Microcirculation 38, 177–187. Liu, Q.G., Lu, Z.Y., 1999. Effect of Salvia miltiorrhiza on coronary collateral circulation in dogs with experimental acute myocardial infarction. Journal of Huazhong University of Science and Technology (Medical Sciences) 19, 40–41. Liu, Y., Li, X.R., Li, Y.H., Wang, L.J., Xue, M., 2010. Simultaneous determination of danshensu, rosmarinic acid, cryptotanshinone, tanshinone IIA, tanshinone I and dihydrotanshinone I by liquid chromatographic–mass spectrometry and the application to pharmacokinetics in rats. Journal of Pharmaceutical and Biomedical Analysis 53, 698–704. Lo, C.J., Lin, J.G., Kuo, J.S., Chiang, S.Y., Chen, S.C., Liao, E.T., Hsieh, C.L., 2003. Effect of Salvia miltiorrhiza bunge on cerebral infarct in ischemia–reperfusion injured rats. The American Journal of Chinese Medcine 31, 191–200. Luo, S.Y., Zhong, Z.G., Lin, J.T., Wu, T., 2009. Determination and pharmacokinetic study of danshen in rat plasma and brain after oral administration of danshen extract. Pharmacology and Clinics of Chinese Materia Medica 25, 44–47. Ma, Y.M., Wang, T.M., 2007. A solid-phase extraction method for highperformance liquid chromatographic determination of salvianolic acid B in rabbit plasma: application to pharmacokinetic study. Biomedical Chromatography 21, 217–220. Qu, G.W., 2004. Studies on the Preparation and Quality Control of Total Salvianolic Acid, Pharmaceutical Chemistry. Ocean University of China, Qingdao. Song, M., Hang, T.J., Zhang, Z.X., 2007. Pharmacokinetic interactions between the main components in the extracts of Salvia miltiorrhiza Bge in rat. Acta Pharmaceutica Sinica 17, 493–498. Wang, X.L., Zhang, Z.R., Fu, H.L., Liu, J., Chen, Q., Nie, Y., Deng, L., Gong, T., 2007. Simultaneous determination and pharmacokinetic study of water-soluble and lipid-soluble components of danshen in rat plasma using HPLC–UV method. Biomedical Chromatography 21, 1180–1185.
Wu, Y.T., Chen, Y.F., Hsieh, Y.J., Jaw, I., Shiao, M.S., Tsai, T.H., 2006. Bioavailability of salvianolic acid B in conscious and freely moving rats. International Journal of Pharmaceutics 326, 25–31. Xu, M., Fu, G., Qiao, X., Wu, W.Y., Guo, H., Liu, A.H., Sun, J.H., Guo, D.A., 2007a. HPLC method for comparative study on tissue distribution in rat after oral administration of salvianolic acid B and phenolic acids from Salvia miltiorrhiza. Biomedical Chromatography 21, 1052–1063. Xu, M., Zhang, Z.C., Fu, G., Sun, S.F., Sun, J.H., Yang, M., Liu, A.H., Han, J., Guo, D.A., 2007b. Liquid chromatography–tandem mass spectrometry analysis of protocatechuic aldehyde and its phase I and II metabolites in rat. Journal of Chromatography B 856, 100–107. Yang, X.N., Wang, Y.J., Liu, Y.S., Tang, X., 2007. Effects of Panax quinquefolium protopanaxadiol saponins on the animal and human plasma protein binding of salvianolic acids in vitro. Asian Journal of Pharmaceutical Sciences 2, 170–179. Yang, Y., Xue, X., Liu, Z.P., 2009. Protective Effect of danshensu on focal cerebral ischemia–reperfusion injury in rats. Food and Drug 11, 24–26. Ye, G., Wang, C.S., Li, Y.Y., Ren, H., Guo, D.A., 2003. Simultaneous determination and pharmacokinetic studies on (3,4-dihydroxyphenyl)-lactic acid and protocatechuic aldehyde in rat serum after oral administration of Radix Salviae miltiorrhizae extract. Journal of Chromatographic Science 41, 327–330. Yuan, J.C., Yao, J.H., Han, G.Z., Hu, X.W., 2009. Simultaneous determination of five water-soluble components of danshen in rabbit plasma and its application to pharmacokinetics. In: ICBBE 2009. 3rd International Conference on Bioinformatics and Biomedical Engineering, 2009 , Beijing, pp. 1–4. Zhang, J.L., Yu, H.L., Sheng, Y.X., Li, L., Ye, M., Guo, D.A., 2005a. HPLC determination and pharmacokinetic studies of salvianolic acid B in rat plasma after oral administration of Radix Salviae miltiorrhizae extract. Biomedical Chromatography 19, 15–18. Zhang, T., Wang, C.Y., Tian, J.W., Fu, F.H., Liu, K., 2005b. Protective effect of danshensu on brain injury. Chinese Traditional and Herbal Drugs 36, 1041–1043. Zhang, Z.C., Xu, M., Sun, S.F., Qiao, X., Wang, B.R., Han, J., Guo, D.A., 2008. Metabolic analysis of four phenolic acids in rat by liquid chromatography–tandem mass spectrometry. Journal of Chromatography B 871, 7–14. Zhang, Q.L., Zhang, Y.J., Wu, L., Li, J., Yuan, Y., 2010. Determination of phenolic compounds of Salvia miltiorrhiza in rat brain by liquid chromatography with chemiluminescence detection using microdialysis sampling and application to pharmacokinetic study. Chinese Pharmacological Bulletin 26, 1230–1237. Zhao, G.R., Zhang, H.M., Ye, T.X., Xiang, Z.J., Yuan, Y.J., Guo, Z.X., Zhao, L.B., 2008. Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B. Food and Chemical Toxicology 46, 73–81. Zheng, X.H., Zhao, X., Fang, M.F., Wang, S.X., Wei, Y.M., Zheng, J.B., 2007. Pharmacokinetic effects of Shi herb-Borneol on Jun herb-Salvia Miltiorrhiza. Journal of Xi’an Jiaotong University (Medical Sciences) 28, 170–173. Zhou, L., Zuo, Z., Chow, M.S.S., 2005. Danshen: an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use. The Journal of Clinical Pharmacology 45, 1345–1359.
Glossary BBB: blood brain barrier BW: body weight CL: chemiluminescence CSF: cerebrospinal fluid DSS: danshensu HPLC: high performance liquid chromatography PA: protocatechuic acid PAL: protocatechuic aldehyde SalB: salvianolic acid B TCM: traditional Chinese medicine UNK: unknown