TSINGHUA SCIENCE AND TECHNOLOGY ISSN 1007-0214 06/20 pp394-399 Volume 12, Number 4, August 2007

Different Kinetics of Puerarin in Plasma of Normal and Depressed Rats After Oral Administration of Chinese Medicine TZ18

SHEN Jia (申 佳), MENG Zhen (孟 甄), SU Hui (苏 慧), XING Dongming (邢东明), DING Yi (丁 怡), DU Lijun (杜力军)**

Laboratory of Pharmaceutical Sciences, Department of Biological Sciences and Biotechnology,

Tsinghua University, Beijing 100084, China

Abstract: The objective of this study is to quantify the puerarin in rat plasma following oral administration of

TZ18 and compare the pharmacokinetics characteristics of puerarin in normal rats with that in depression

model rats. A high performance liquid chromatography method was used to quantify the puerarin due to its

good selectivity and linearity (coefficient correlation, r2 = 0.9991) within the tested range (0.028-0.889 µg·mL−1).

Intra- and inter-day precision coefficients of variation and accuracy bias were acceptable (Maximum coeffi-

cient of variation was 5.74% for intra-day and 3.09% for inter-day) over the entire range. The recoveries

were found to be 98.3%, 101.4%, and 103.4% for concentrations of 0.028, 0.222, and 0.444 µg·mL−1, re-

spectively. The concentration-time curves for both normal rats and depression model rats were fit to a two-

compartment model with the first order absorption. The results show significant differences in the main phar-

macokinetic parameters of peak time, peak concentration, and the area under the concentration-time curve

between the two kinds of rats.

Key words: puerarin; depression rat model; pharmacokinetics

Introduction

Puerariae radix (the root of Pueraria lobata ohwi), the major ingredient of the Chinese medicine TZ18 used to treat depression, has been used for centuries as a Chinese traditional herb. The ethanol extract of puer-ariae radix showed significant anti-depression effects in mice[1]. Puerarin (7-hydroxy-3-(4-hydroxyphenyl)-1-benzopyran-4-one 8-(β-D-glucopyranoside), Fig. 1), is the most abundant and the main active isoflavone glycoside found in puerariae radix and is often used as the puerariae radix marker compound in pharmacoki-netic studies for quality control. Puerarin has exhibited various pharmacological effects such as inhibiting

platelet aggregation[2], hypertension[3], hyperlipide-mia[4] arteriosclerosis[5], and oxygenation[6] and pro-tecting cerebral cortical neurons from damage by glutamine and N-methyl-D-aspartic acid (NMDA)[7]. Puerarin concentrations in plants and plasma can be measured by high performance liquid chromatography (HPLC) with ultraviolet[8,9] or fluorescence[10,11] detec-tion or the liquid chromatography-mass spectrometric (LC-MS) method[12,13]. The pharmacokinetics proper-ties of puerarin have been studied in human beings[14], dogs[15], and rabbits[12] after oral administration and in rats following intravenous administration[16]. However, there is little information about the kinetic characteris-tics of puerarin in animal disease models until now. The present study uses a sensitive and accurate HPLC method to measure puerarin in rat plasma after oral administration of the encapsulated TZ18 to investigate

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Received: 2006-10-18

To whom correspondence should be addressed. E-mail: lijundu@tsinghua.edu.cn; Tel: 86-10-62773636

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the variation of the kinetics of puerarin in normal and depression model rats.

Fig. 1 Structure of puerarin

1 Materials and Methods 1.1 Chemicals and reagents

Puerarin standard (purity 98%) was purchased from the National Chemicals and Biological Products Institute (Beijing, China). Encapsulated TZ18 containing 6.78% puerarin was produced by our laboratory. Phosphoric acid and potassium dihydrogen phosphate were ana-lytical grade materials. Methanol and acetonitrile (HPLC grade) were purchased from Baker (J. T. Baker, USA). Deionized water was used throughout the experiment.

1.2 Animals

Male Wistar rats weighing 180-220 g were purchased from the Laboratory Animal Institute of the Chinese Academy of Medical Sciences (Beijing, China). The animals were housed in an environmentally controlled breeding room (temperature: (25±2)℃ , humidity: 60%±5%, 12 h dark/light cycle ) for 1 week before use and had ad libitum access to water and a standard laboratory diet. All rats were fasted overnight before the experiments. The experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Tsinghua University, China.

1.3 Chromatographic system and conditions

The puerarin in the rat plasma was assayed using an HPLC system consisting of a pump-515E, a UV-/Vis-2487detector, a 20-µL injection loop, and a Millenium-32 workstation for data collection (Waters, USA). Sample analysis was carried out by applying a 10-µL volume to an ODS-3 C18 reversed-phase column (5 µm, 150 mm× 4.6 mm, Elite, China) detected at 252 nm. A mobile

phase of water-acetonitrile (89:11, v/v) was filtered through a 0.45-mm millipore filter and degassed prior to use. The flow rate was 1 mL·min−1 and the temperature was kept at (25±1)℃.

1.4 Preparation of standard solutions

A standard stock solution of the reference puerarin was dissolved in methanol at a concentration of 100 µg·mL−1 and stored at 4℃. The standard working so-lutions were made by serial dilution with methanol. Then, the drug-free rat plasma was spiked with differ-ent working solutions, which were further diluted to give final concentrations of 0.028, 0.111, 0.222, 0.333, 0.444, and 0.889 µg·mL−1 for the calibration curve.

1.5 Experimental model of depression

The depression model rats were produced using the forced swimming test (FST) described by Porsolt et al.[17] with modifications. Briefly, the rats were indi-vidually placed in 30 cm of ambient temperature water ((30±2)℃) in 20-L plastic beakers and allowed to swim for 6 min, with the durations of immobility re-corded during the last 240 s of the test. The duration of immobility is defined as the absence of active, escape-oriented behavior, such as swimming, jumping, rearing, sniffing, or diving. After swimming, the rats were re-moved from the tank, dried with a towel, and returned to their home cages. The forced swimming test was continued for one week and the control animals were tested on the first and last days only. Rats that had ex-tended immobility times were chosen and randomly grouped for individual time tests.

1.6 Drug administration and blood sampling

After fasting overnight, the rats received oral adminis-trated of a single 0.8 g·kg−1 dose of aqueous solution of TZ18 (containing 54.24 mg·kg−1 of puerarin). At each time point (0.25, 0.5, 0.75, 1.0, 1.5, 2, 4, 6, 8, 12 h after gavage), the rats were anesthetized with ether and 5-6 mL blood samples were taken from the ab-dominal aorta. Each blood sample was centrifuged at 1500g for 15 min at 8-10℃. The pH was adjusted to 2.5 by the addition of 0.3 mL 1 mol/L dihydrogen phosphate solution and 30 µL phosphoric acid. Then each tube was vortex-mixed with 5 mL of acetonitrile for 1 min and centrifuged at 3000g for 15 min at 8-10℃.

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The supernatant was evaporated to dryness in a water bath at 37℃. For the analysis, the sample was dis-solved in 0.1 mL of methanol with 10 µL injected into the HPLC system. The same sample handling process was used for recovery and precision determinations.

1.7 Method validation

1.7.1 Calibration curve A calibration curve was derived from the peak height ratio of the puerarin to its concentration using un-weighted linear regression. The puerarin concentra-tions in the samples were determined using the regres-sion parameters obtained from the calibration curve. Calibration standards were included in every analytical batch of samples. 1.7.2 Limit of detection and quantification The limit of detection (LOD) was determined as the lowest concentration that could be detected with ac-ceptable accuracy and precision. This was identified on the plots as three times the noise level. The limit of quantification (LOQ) in the plasma was defined as the lowest concentration on the calibration curve for which the assay precision (coefficient of variation, CV) was lower than 10%. 1.7.3 Recovery The puerarin recovery from the rat plasma was evalu-ated using three different concentrations (0.028, 0.111, and 0.889 µg·mL−1) covering the linear range of the standard curve. After the samples were processed ac-cording to the method described above, the resulting peak areas were compared with those of the puerarin standard carried in methanol to provide the recovery values.

1.8 Reproducibility

The reproducibility of the method was defined by ex-amining both the intra- and inter-day variances using three different puerarin concentrations (0.028, 0.111, and 0.889 µg·mL−1). The intra-day variance was de-termined by assaying the spiked samples 3 times dur-ing one day with the inter-day variance assayed over three consecutive days. Coefficients of variation were calculated from these values.

1.9 Pharmacokinetic analysis

The puerarin serum concentrations in the rat plasma

were determined after oral administration of the TZ18 (containing 54.24 mg·kg−1 of puerarin). Data from these samples were used to construct pharmcokinetic profiles by plotting the drug concentration versus time. All data were processed by the computer program 3P87 (Chinese Pharmaceutical Association, 1987) with the compartmental pharmacokinetic parameter calcula-tions based on the moment theory. Data were ex-pressed as means ± SD. The student’s t-test assesses the statistical significance which was set at p < 0.05.

2 Results and Discussion 2.1 Selectivity, linearity, detection, and

quantification limits

The HPLC chromatograms of puerarin, blank plasma, plasma spiked with puerarin (1.7 µg·mL−1), and the plasma obtained 30 min after oral administration of TZ18 from normal animals are shown in Fig. 2. The re-tention times of the puerarin standard and the puerarin in the TZ18 were the same. No interference peaks were observed within the time frame in which puerarin was detected (Rs>1.5), which indicated that the procedure selectivity was satisfactory. The calibration curve for the rat plasma was linear over the concentration range 0.028-0.889 µg·mL−1. The mean coefficient correla-tion (r2) was 0.9991, indicating a good linear relation-ship between the peak areas and concentrations. The regression equation for the calibration curve was y=72 652x+923.43 (where y is the puerarin concentra-tion in the plasma and x the area under the curve). LOD was calculated as 0.007 µg·mL−1 with LOQ of 0.028 µg·mL−1 using LOD=yB+3SB and LOQ=yB+ 10SB (where yB is a blank signal, and SB the standard deviation of three peak areas for the minimum puerarin concentration), which is sufficient for routine pharma-cokinetic monitoring.

2.2 Recovery and reproducibility

The puerarin recoveries from the rat plasma were 98.3%, 101.4%, and 103.4% for puararin concentrations of 0.028, 0.222, and 0.444 µg·mL−1 (Table 1). The intra-day and inter-day variances are summarized in Tables 2 and 3. The maximum CV was 5.74% for the intra-day variance and 3.09% for the inter-day variance indicating that the method was quite precise. Moreover, the small difference (≤10%) between the administered

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concentration and the measured concentrations indi-cates that the method’s accuracy is sufficient.

Fig. 2 Typical HPLC chromatograms

Table 1 Puerarin recovery

Spiked conc. (µg/mL)

Measured conc.* (µg/mL)

Recovery (%)

0.028 0.028±0.001 98.3 0.222 0.225±0.004 101.4 0.444 0.459±0.024 103.4

* Each value represents the mean ± SD, n=3.

Table 2 Intra-day assay validation

Spiked conc.(µg / mL)

Measured conc.* (µg / mL)

Accuracy (%)

CV (%)

0.028 0.028±0.002 100.5 5.74 0.222 0.224±0.002 100.8 1.08 0.444 0.436±0.016 98.3 3.68

* Each value represents the mean ± SD, n=3.

Table 3 Inter-day assay validation

Spiked conc.(µg / mL)

Measured conc.* (µg / mL)

Accuracy (%)

CV (%)

0.028 0.028±0.001 99.4 3.09 0.222 0.222±0.004 99.9 1.74 0.444 0.445±0.012 100.2 2.59

* Each value represents the mean ± SD, n=3.

2.3 Pharmacokinetics of puerarin in normal and depression model rat plasma

The depression rats were induced as above. The swimming stress significantly extended the immobility time by 20%-30% and depression in the rats as de-scribed by Sakakibaba et al.[18] Administration of amitriptyline hydrochloride could alleviate the symp-toms (Data not shown). Thus, these depressed rats were used as the model animals for the pharmacokinet-ics studies of puerarin. The validated method was suc-cessfully used to analyze the puerarin concentration variation over time profile in rat plasma shown in Fig. 3. After oral administration of the TZ18, the puerarin plasma level could be detected after 15 min in both the normal and the depression model rats, with α and β half-lives (T1/2) of 0.226 h and 5.93 h in normal ani-mals and 1.22 h and 1.914 h in the model animals. The concentrations were below the quantitative limit (0.028 µg·mL−1) in both sets of animals 8 h after gavage. The pharmacokinetic parameters derived from the data by the 3P87 program are listed in Table 4. The data shows significant differences in the pharmacokinetic parameters between the depression model rats and normal rats, although both puerarin plasma concentra-tion-time courses were best fit by a two-compartment open model. Compared with the normal rats, the de-pressed rats showed a little slower but better absorp-tion of the puerarin. The peak height in the depression model animals was 40% higher than that in the normal animals. In addition, the depression model rats had a lower clearance and a longer mean puerarin retention time.

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Fig. 3 Mean puerarin serum concentrations after oral administration of TZ18 (containing 54.24 mg/kg of puerarin). Each point and bar represents the mean ±SD. Compared with normal rats: *, p<0.05; **,

p<0.01 (n=5).

Table 4 Pharmacokinetic parameters of puerarin in normal and depression model rat plasma after oral administration of TZ18 at a dose containing 54.24 mg/kg of puerarin (n=5)

Estimate (mean±SD) Parameter

Normal Model AUC0-α (µg / (min·mL )) 25.62±12.66 69.3±17.34** CL(s)(mL / (min·kg)) 36.17±13.00 16.67±1.63** Cmax (µg / mL) 0.232±0.078 0.326±0.057*

Vd (mL / kg) 1.780±0.390 1.533±0.105 T1/2α (min) 13.56±3.84 73.20±45.54*

T1/2β (min) 355.80±262.80 114.84±18.54*

Tmax (min) 28.84±1.32 52.14±11.34*

MRT (min) 125.40±11.40 166.26±9.24*

Note: Vd, volume of distribution; AUC0-α, the area under the concentra-

tion-time curve; T1/2α, distribution half-life; T1/2β, elimination half-life; MRT,

mean residence time; CL is the elimination clearance. * p<0.05, ** p<0.01,

n=5.

3 Conclusions

A sensitive, economical, and accurate HPLC method was developed to determine puerarin plasma concen-trations in rats after oral administration. The method was used to compare the puerarin pharmacokinetics in normal rats with that in depression model rats. As the major component of puerariae radix, these puerarin kinetic studies help clarify the characteristics of the Chinese medicine TZ18. The puerarin kinetics in depression model rats differed from those in normal rats perhaps due to the physiological change induced by the stress. The higher adsorption of puerarin in the depressed animals may explain the better anti-depression effects of TZ18.

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Department of Industrial Engineering Won Award of IIE The Department of Industrial Engineering of Tsinghua University won the Innovations in Curriculum Award of In-stitute of Industrial Engineering (IIE) at the annual meeting of IIE held in Nashville, Tennessee on May 20, 2007. The subject of the award-winning project is Logistics Laboratory. Competing with American universities, the pro-ject won the first place and became the only winner of this award. It is the first time for IIE to set up such an award. The Department of Industrial Engineering was established in 2001 with Dr. Gavriel Salvendy as the founding chair professor and department head. The Logistics Laboratory was set up in 2004.

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