Arch Pharm Res Vol 33, No 2, 225-230, 2010DOI 10.1007/s12272-010-0206-5

225

Pharmacokinetics, Tissue Distribution and Excretion of Coumarin Components from Psoralea corylifolia L. in Rats

Liang Feng1*, Ling Wang2, and Xuehua Jiang2

1Department of Pharmacy, ChengDu University of TCM, No. 37, Shier Qiao Street, Chengdu 610041 Sichuan, P.R.China and 2Department of Pharmacy, West China Center of Medical Science, the Sichuan University, Chengdu 610041,China

(Received June 17, 2009/Revised October 1, 2009/Accepted December 16, 2009)

Coumarin components from Psoralea corylifolia L. are novel drugs in which psoralen and isop-soralen are the active components. The pharmacokinetics, tissue distribution and excretion ofthe two compounds were studied by liquid chromatography-tandem mass spectrometry afterintravenous administration to Wistar rats. The elimination half-lives of psoralen and isopsor-alen were 4.88 and 5.35 h. After dosing, the area under the curves of the tissues decreased inthe following order: liver > lung > heart > kidney > spleen > brain for psoralen; and kidney >lung > liver > heart > spleen > brain for isopsoralen. After dosing, 51.27% of psoralen and56.25% of isopsoralen were excreted as prototype, and urine was the major excretion route. Inaddition, the pharmacokinetics of psoralen and isopsoralen after oral administration to Wistarrats were also studied. The elimination half-lives of psoralen and isopsoralen were 4.13 and5.56 h, and their relative bioavailabilities were 61.45% and 70.35%. Overall, the results showthat coumarin components from P. corylifolia L. have high oral bioavailability, they are rap-idly and widely distributed into tissues after intravenous administration, but they are slowlycleared and excreted. Key words: Psoralen, Isopsoralen, Wistar rats, Pharmacokinetics, Distribution, Excretion

INTRODUCTION

Buguzhi, The dried fruit of the medicinal plantPsoralea corylifolia L., is a traditional Chinese medi-cine used for the treatment of coronary artery disease,osteoporosis, bacterial infection, asthma, gynaecologi-cal bleeding, vitiligo and psoriasis (Jia and Zhao,2001). Recent studies have confirmed the pharma-cological activity of the fat-soluble components ofBuguzhi, which are the main active constituents (Ahn,1998). Macropore resin separation has been used toisolate psoralen, isopsoralen, psoralidin, isopsoralidin,and corylidin from Buguzhi (Luo and Guo, 1998).Recently, the coumarin components from P. corylifoliaL., in which psoralen and its isomeride isopsoralen areactive components, are used as novel drugs.

The pharmacological activities of psoralen andisopsoralen have been widely researched. These twocompounds draw attentions recently because of theirabilities to arouse drug interaction through inhibitionof cytochrome P450. In vitro experiments have con-firmed that psoralen is a competitive and mechanism-based inhibitor of CYP3A4 (Guo and Yamazoe, 2004).On the other hand, previous studies have indicatedthat psoralen has low oral bioavailability and thatafter oral administration to rat, it is absorbed slowlyinto blood (Ciaravino, 2001). Isopsoralen is alsoabsorbed slowly. However, the pharmacokinetics,distribution and the excretion fate in blood of the twocompounds are still unclear. Therefore, in the presentstudy, we investigated the pharmacokinetics andtissue distribution of psoralen and isopsoralen as wellas their excretion following intravenous administra-tion of coumarin components from P. corylifolia L. torats.

Correspondence to: Liang Feng, Department of Pharmacy,ChengDu University of TCM, No. 37, Shier Qiao Street,Chengdu 610041 Sichuan, P.R. ChinaTel: 86-028-85501370, Fax: 86-028-85503024E-mail: postgraduate@vip.sina.com

226 L. Feng et al.

MATERIALS AND METHODS

MaterialsPsoralen, isopsoralen (Fig. 1), and the corresponding

internal standard bifonazole (all were > 98% pure)were purchased from the National Institute for theControl of Pharmaceutical and Biological Products,China. Coumarin components were prepared from thefruit of Psoralea corylifolia L., which contains psoralen(31.26%) and isopsoralen (48.13%). Acetonitrile andchloroform (HPLC grade) were purchased from Agilent(Agilent Co.). Physiological saline was purchased fromthe Yongtai pharmaceutical manufactory (ChengDu).All other reagents used in the study were of analyticalgrade at least. Deionized distilled water was used forthe preparation of all solutions.

Coumarin components solution: 50 mg coumarincomponents dissolved in 50 mL mixed solvent whichconsists of 80% physiological saline and 20% pro-pylene glycol.

Animal studiesMale Wistar rats (weighing 230~270 g) were pur-

chased from the Laboratory Animal Center of theChengDu University of TCM. The rats were accli-mated to standard housing and environmental condi-tions (25oC, 60% relative humidity, and 12-h light/dark cycle) for 1 week. The Sichuan Ethical Committeeof Experimental Animal approved all procedures ofthe study. At the end of the experiment, pentobarbitalsodium was used for euthanasia of the animals.

PharmacokineticsFive male rats received a single intravenous admini-

stration of 2.0 mg/kg coumarin components solution.After dosing, blood samples (0.3 mL) were collectedfrom the vein at 0.08, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0,12.0, 18.0 and 24.0 h. Heparin sodium for anticoagula-tion, plasma was isolated from the blood by centri-fugation at 15000 rpm for 3 min. All plasma sampleswere stored at -20oC until analysis.

In additional five rats, blood samples (0.3 mL) werecollected from the vein at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0,3.0, 3.5, 4.0, 6.0, 12.0 and 24.0 h after oral admini-stration of 9.12 mg/kg coumarin components solution.

Plasma was isolated and stored as described before.0.3 mL acetonitrile (containing 1 µg/mL bifonazole)

was added into 0.1 mL plasma, then they were blend-ed and centrifugalized at 15000 rpm for 3 min, thesupernatant fluid was removed into a test tube anddried at 40oC in nitrogen stream. Following, the residuewas redissolved into 0.1 mL acetonitrile and analyzedby liquid chromatography-tandem mass spectrometry(LC-MS/MS).

Data were analyzed using the 3P97 program (ChineseMathematics & Pharmacological society) with a two-compartment model, and a weighting function of 1/C2

for data fitting and parameter estimation.

Tissue distribution studiesFifty-five male rats were given a single intravenous

administration of 2.0 mg/kg coumarin componentssolution through the femoral vein. At 0.08, 0.25, 0.5,1.0, 2.0, 3.0, 4.0, 6.0, 12.0, 18.0 and 24.0 h after dosing,five rats were killed, tissues (heart, liver, spleen, lung,kidney and brain) were promptly removed andwashed with physiological saline. Each tissue samplewas diluted with 4 volumes (v/w) of physiological saline(containing 1 µg/mL bifonazole) and homogenized, andthen 1.5 mL acetonitrile (containing 1 µg/mL bifona-zole) was added into 0.5 mL homogenate, psoralenand isopsoralen were isolated and conserved asdescribed for plasma.

Excretion studiesFive male rats were placed in separate metabolic

cages and received a single intravenous administra-tion of 2.0 mg/kg coumarin components solution.Urine and feces were collected at 0 to 48 h after dosingfor analysis.

The volume of each urine sample was measured,and then each sample was diluted with 2 volumes (v/v) of chloroform (containing 5 µg/mL bifonazole) toextract psoralen and isopsoralen. Following, chloro-form was isolated and dried at 40oC in nitrogenstream, then, the residue was redissolved into 0.5 mLacetonitrile and stored at -20oC until analysis. Eachfeces sample was weighted and homogenized in 5volumes (v/w) of chloroform (containing 5 µg/mLbifonazole) to extract psoralen and isopsoralen. Thenthe organic layer was isolated and processed asdescribed for urine.

LC-MS/MS analysisPlasma samples, tissue samples and excretion sam-

ples were analyzed by LC-MS/MS method. The systemwas composed of an HPLC apparatus (Shimadzu)with a 5-µm Diamonsil C18 column (150 mm × 2.1 mmFig. 1. The chemical structures of psoralen and isopsoralen

The ADME of Coumarin Components from Psoralea corylifolia L. 227

i.d., Dikma) coupled to an API-2000 triple-quadruplemass spectrometer equipped with an electrosprayionization source. The mobile phase for the HPLCcolumn was 30% acetonitrile/70% water. For thedetermination of psoralen and isopsoralen, thepositive ionization mode was selected, along with thefollowing conditions: nebulizer gas, 8 L/min; curtaingas, 7 L/min; collision gas, 8 L/min; ionspray voltage,3.5 kV; temperature of ion source, 100oC; temperatureof heated gas, 300oC. The mass spectrometer wasoperated in multiple reactions monitoring (MRM)mode, with monitoring of the precursor-to-product iontransitions of m/z 186→102 for psoralen, m/z 186→158 for isopsoralen (Fig. 2), and m/z 310→243 forbifonazole. Blank plasma, tissue homogenate, urineand feces were used for the preparation of matrix-

matched calibration curves so as to avoid differentmatrix effects. Linear ranges of calibration curveswere 0.017 to 54.3 µg/mL for psoralen with a lowestdetectable limit of 6 ng/mL, and 0.011 to 67.7 µg/mLfor isopsoralen with a lowest detectable limit of 4 ng/mL.

RESULTS

PharmacokineticsFig. 3 shows the concentration-time curves of psoralen

and isopsoralen after intravenous administration. Theconcentration-time curves were appropriately describ-ed by a two-compartment model, and the results ofpharmacokinetic parameters are summarized in TableI. The AUC0-t values of psoralen and isopsoralen were99.20 and 132.39 µg·h/mL, respectively. The t1/2βvalues of psoralen and isopsoralen were 3.88 and 3.25h, respectively. The result for V of psoralen was muchhigher than that of isopsoralen.

Fig. 4 shows the concentration-time curves of psora-

Fig. 2. The product ion scan of psoralen and isopsoralen. (A)psoralen, (B) isopsoralen.

Fig. 3. The mean blood concentrations of psoralen andisopsoralen in rats after intravenous administration (n = 5,mean ± S.D.)

Table I. Blood pharmacokinetic parameters of psoralenand isopsoralen in rats after intravenous administration(n = 5, mean ± S.D.)

Parameters Psoralen IsopsoralenAUC0-t (µgh/mL) 99.20 ± 27.44 132.39 ± 26.69MRT (h) 5.95 ± 21.06 4.91 ± 20.86V (1/kg) 8.97 ± 23.32 5.68 ± 22.71t1/2α (h) 1.13 ± 20.33 1.51 ± 20.46t1/2β (h) 4.88 ± 21.96 5.35 ± 22.27Cl (1/h/kg) 1.04 ± 20.34 0.86 ± 20.43

228 L. Feng et al.

len and isopsoralen after oral administration. Thecurves were also adequately described by a two-com-partment model, and obvious enterohepatic circula-tion could be observed which result in double-peakphenomenon in the curves. The results of pharma-cokinetic parameters are summarized in table II. Thet1/2α values of psoralen and isopsoralen were 0.68 and0.74 h, respectively. The AUC0-t values of the two com-pounds were 277.98 and 424.71 µg·h/mL, respectively,and their relative bioavailabilities were 61.45% and70.35%, respectively.

Tissue distributionThe tissue concentrations of psoralen and isopsora-

len determined at 11 time points after intravenousadministration are shown in Fig. 5 and 6. The highestconcentration of psoralen was found in liver, andlowest was found in brain. The high concentration ofisopsoralen was found in heart, liver, lung and kidney.The two compounds could be determined in tissues

even 24 h after dosing.The pharmacokinetic parameters for psoralen and

isopsoralen in individual tissues are summarized inTable III. The mean Cmax values of the two compoundsin all tissues analyzed were obtained at 0.08 h afterdosing. Their t1/2β values in all tissues were more than3.5 h. The AUC0-t values for the individual tissuesdecreased in the following order: liver > lung > heart> kidney > spleen > brain for psoralen; and kidney >lung > liver > heart > spleen > brain for isopsoralen.

Excretion studyThe recovery of psoralen and isopsoralen in urine

Fig. 4. The mean blood concentrations of psoralen and iso-psoralen in rats after oral administration (n = 5, mean ±S.D.)

Table II. Blood pharmacokinetic parameters of psoralenand isopsoralen in rats after oral administration (n=5,mean ± S.D.)

Parameters Psoralen IsopsoralenAUC0-t (µgh/mL) 277.98 ± 67.96 424.71 ± 102.70Cmax (µg/mL) 25.19 ± 24.53 37.49 ± 229.72V/F (1/kg) 7.20 ± 22.66 6.43 ± 221.15t1/2α (h) 0.68 ± 20.17 0.74 ± 220.27t1/2β (h) 4.13 ± 21.28 5.56 ± 221.07Cl/F (mL/h) 1.26 ± 20.37 1.09 ± 220.17Relative bioavailability 61.45% 70.35%

Fig. 5. Tissue distribution of psoralen in rats after in-travenous administration (n = 5, mean ± S.D.)

Fig. 6. Tissue distribution of isopsoralen in rats afterintravenous administration (n = 5, mean ± S.D.)

The ADME of Coumarin Components from Psoralea corylifolia L. 229

and feces after intravenous administration are shownin Table IV. In the urine, the mean recovery of psora-len and isopsoralen was 47.03% and 53.93%, respec-tively. In the feces, over a 48-h period, the meanrecovery of the two compounds was only 4.24% and2.32%, respectively. On the whole, 51.27% of psoralenand 56.25% of isopsoralen was excreted as prototype.Most of the two compounds were excreted within 12 hafter dosing, with the urine being the major route ofexcretion.

DISCUSSION

In this study, we found that the pharmacokineticsand distribution of psoralen and isopsoralen in wistarrats fit best to a two-compartment model. The t1/2βvalues of the two compounds in blood after intra-venous administration were more than 4.5 h, andtheir t1/2β values in blood after oral administrationwere more than 4.0 h, indicating that psoralen andisopsoralen are slowly eliminated.

Psoralen was reported to have obvious enterohepa-tic circulation (Tsai and Hsin, 2006). In the liver,about 60% to 70% of psoralen was combined withglucuronic acid and sulfuric acid. The combinationwas excreted into the small intestine through bileduct and hydrolyzed, and then the prototype wasregenerated and reabsorbed into blood (Stolk andSiddiqui, 1988). In this study, we found that after oral

administration, the enterohepatic circulation of psoralenwas quite obvious, the tmax of its concentration-timecurve was 2.4 and 4.1 h after dosing, but the entero-hepatic circulation of isopsoralen was unobvious.

Psoralen was reported to have low bioavailability inrats (Huang et al., 1991; Huang and Yang, 1991).After oral administration of 50 mg/kg psoralen to rats,the peak concentration of psoralen was only 100 µg/mLin blood, which was much less than that after in-travenous administration. But in this study, the oralbioavailability of psoralen was about 61.45%, whichwas extremely high. This discrepancy may be attri-buted to the dose used, indicating that the absorptionof psoralen may be a non-linear process, and activetransport may be involved in. But further studies arenecessary to investigate the relationship between doseand bioavailability.

Coumarin components from P. corylifolia L. wereused in clinics for the treatment of coronary arterydisease and asthma, and the study also found highAUC0-t values of psoralen and isopsoralen in heart andlung, the pharmacodynamic effects may be related tothe high distribution level. But a low level of AUC0-twas found in brain, suggesting that these two com-pounds can not efficiently cross blood-brain barrier.The maximal concentrations of psoralen and isopsora-len in the tissues were found at 5 min after dosing,indicating their distribution rate from blood intotissues was extremely rapid. The two compounds could

Table III. Tissue pharmacokinetic parameters of psoralen and isopsoralen in rats after intravenous administration (n= 5, mean ± S.D.)

Psoralen IsopsoralenAUC0-t Cmax t1/2β AUC0-t Cmax t1/2β

µgh/mL µg/g h µgh/mL µg/g hHeart 30.72 ± 10.83 5.07 ± 0.88 3.92 ± 1.79 24.22 ± 18.55 3.10 ± 0.44 4.84 ± 1.03Liver 52.30 ± 15.23 4.95 ± 1.05 5.89 ± 2.24 27.28 ± 13.14 3.01 ± 1.30 5.51 ± 1.76Spleen 9.56 ± 14.17 1.36 ± 0.42 6.38 ± 1.47 11.17 ± 14.73 1.33 ± 0.27 3.73 ± 0.69Lung 33.22 ± 16.76 3.15 ± 0.52 4.33 ± 1.15 28.20 ± 11.16 3.43 ± 0.75 3.62 ± 1.01Kidney 29.96 ± 13.44 4.25 ± 0.77 4.59 ± 1.48 33.25 ± 12.49 3.72 ± 0.56 4.04 ± 0.79Brain 4.68 ± 11.65 0.77 ± 0.20 5.51 ± 0.87 6.14 ± 12.66 0.80 ± 0.16 5.12 ± 2.08

Table IV. Recovery of psoralen and isopsoralen in urine and feces of rats after intravenous administration (n=5, mean± S.D.)

Time after administration

Psoralen Isopsoralenurine feces urine feces

0 to 4 h 14.24 ± 14.01 1.38 ± 0.47 19.14 ± 4.12 0.56 ± 0.194 to 12 h 27.59 ± 16.58 2.17 ± 1.21 30.81 ± 8.63 1.52 ± 0.6412 to 24 h 14.26 ± 11.21 0.69 ± 0.33 13.85 ± 1.25 0.24 ± 0.2024 to 48 h 10.94 ± 10.46 - 10.14 ± 0.09 -0 to 48 h 47.03 ± 10.64 4.24 ± 2.21 53.93 ± 8.12 2.32 ± 1.44

230 L. Feng et al.

be detected in tissues even 24 h after dosing with thet1/2β of more than 3.5 h, indicating their eliminationfrom tissues was relatively slow.

After intravenous administration, urine was themajor route of excretion, but about 4.24% of psoralenand 2.32% of isopsoralen could be detected in thefeces, indicating that a small quantity of the twocompounds was excreted into the intestine throughbile duct. Most of the psoralen and isopsoralen wasexcreted within 12 h, but 0.94% of psoralen and 0.14%of isopsoralen could be detected in excretions (urineand feces) during 24 to 48 h after dosing, indicatingthe excretion of the two compounds from blood wasslow.

In conclusion, the coumarin components from P.corylifolia L. have high oral bioavailability. They arereadily and rapidly distributed to most tissues but cannot efficiently cross the blood-brain barrier. Theelimination of the components in blood and tissues arerelatively slow, and the major route of elimination isexcretion in urine.

REFERENCES

Jia, L. and Zhao, M. R., Applied studies on trace element in

looking for new active substance. Acta. Bot. Boreal.-Occident. Sin., 21, 579-583 (2001).

Ahn, B. Z., Inhibitory effect of radix saponions on adhesionof some Solid tumor cells and relation to hemolytic actionscreening of 232 herbal drugs from anti-cell adhesion.Planta Med., 64, 220-224 (1998).

Luo, Z. D. and Guo, Y. H., Optimal extraction of Psoraleacorylifolia L. Acta. Pharmacol. Sin., 16, 705-708 (1998).

Guo, L. Q. and Yamazoe, Y., Inhibition of cytochrome P450by furanocoumarins in grapefruit juice and herb almedicines. Acta. Pharmacol. Sin., 2, 129-136 (2004).

Ciaravino, V., Pharmacokinetic and toxicology assessmentof intercept (S-59 and UVA treated) platelets. Hum. Exp.Toxicol., 20, 533-550 (2001).

Tsai, W. J. and Hsin, W. C., Antiplatelet flavorioids fromseeds of Psoralea corylifolia L. J. Nat. Prod., 59, 671-672(2006).

Stolk, L. M. and Siddiqui, A. H., Biopharmaceutics, pharma-cokinetics and pharmacology of psoralen. Gen. Pharma-col., 19, 649-653 (1988).

Huang, X., Jiang, Y. P., and Wang, Y. P., Fit of compartmentmodel of psoralen and bioavailability. J. Fourth. Mil. Med.Univ., 5, 349-351 (1991).

Huang, X. and Yang Y. C., Detection of psoralcn by reversed-phase HPLC in whole blood. J. Fourth. Mil. Med. Univ., 3,210-213 (1991).