Fitoterapia 82 (2011) 1152–1159

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Comparative pharmacokinetics of dehydroevodiamine and coptisine in ratplasma after oral administration of single herbs and Zuojinwan prescription

Rui Yan⁎, Yin Wang, Wenjin Shen, Youping Liu, Xin DiSchool of Pharmacy, Shenyang Pharmaceutical University, Shenyang, PR China

a r t i c l e i n f o

⁎ Corresponding author at: PO box 54, Shenyang Pha103 Wenhua Road, Shenyang 110016, PR China. Tel.fax: +86 24 2390 2539.

E-mail address: yrchn@163.com (R. Yan).

0367-326X/$ – see front matter © 2011 Elsevier B.V.doi:10.1016/j.fitote.2011.07.012

a b s t r a c t

Article history:Received 21 April 2011Accepted in revised form 10 July 2011Available online 23 July 2011

Zuojinwan, which consists of Rhizoma coptidis–Evodia rutaecarpa powder (6:1,g/g), is atraditional Chinese medicine (TCM) prescription, clinically used for the treatment of gastro-intestinal disorders. This study compared the pharmacokinetics of dehydroevodiamine andcoptisine, themain active ingredients in Zuojinwan, in rats administratedwithwhole prescriptionor single herbs. Multiple blood concentration peaks were observed in the mean plasma–concentration curves. The pharmacokinetic parameterswere quite different between single herbsand Zuojinwan prescription. Moreover, the mean plasma concentration of dehydroevodiamineincreased and the one of coptisine decreased after combining, which was in accord with theclinical principle of TCM.

© 2011 Elsevier B.V. All rights reserved.

Keywords:DehydroevodiamineCoptisineRhizoma coptidis–Evodia rutaecarpa coupleLiquid chromatography–tandem massspectrometryPharmacokinetics

1. Introduction

Traditional Chinese medicine (TCM) mainly uses combina-tions to produce a synergistic effect or antagonistic action. Theanalysis of monoingredient or single herb may not representthe pharmacokinetics of the whole composite prescription.Thus, it is better to investigate thepharmacokinetics ofmultiplecomponents and compare the pharmacokinetic differencesbetween single herbs and the whole prescription.

Rhizoma coptidis–Evodia rutaecarpa couple is one of themost widely used combinations as the core in many compositeformulae [1]. Zoujinwan, which consists of R. coptidis–E. rutaecarpapowder (6:1,g/g), is famous in the clinical practiceof TCM for its excellent treatment of gastro-intestinal disorders[2]. The main bioactive components include berberine, palma-tine, jatrorrhizine and coptisine in R. coptidis; evodiamine,rutecarpine and dehydroevodiamine in E. rutaecarpa.

rmaceutical University: +86 24 2398 6269

All rights reserved.

,;

There was no method published for the determination orpharmacokinetic profiles of dehydroevodiamine in biologicalfluids. Pharmacokinetic studies on the other six ingredients inherb extracts have been carried out [3–8]. A previous study hasalso described the pharmacokinetics of berberine, palmatineand jatrorrhizine in Zuojinwan [9]. However, the pharmacoki-netic studies on coptisine, evodiamine and rutecarpine inZuojinwan, which would be helpful to explore the synergisticeffects on the two herbs were never reported.

The pharmacokinetic research of TCM has been seriouslyblocked by the lack of purified standards all along. It isimpracticable to separate the constituents one by one toobtain standard substances for assays. The present paperdemonstrated a ‘relative reference approach’ that herbextracts, instead of purified standards acted as the referencesubstance. This approach is well suited for the analysis of thecompounds without standards. On this basis, a rapid liquidchromatography–tandem mass spectrometry (LC–MS/MS)method for the determination of dehydroevodiamine andcoptisine in rat plasma was developed and applied to thepharmacokinetics after oral administration of R. coptidis,E. rutaecarpa and Zuojinwan powders to rats independent ofauthentic standards.

1153R. Yan et al. / Fitoterapia 82 (2011) 1152–1159

2. Experimental

2.1. Chemicals and reagents

Methanol, formic acid, acetonitrilewere of chromatographicgrade from the Yuwang Chemical Factory (Shandong, China).Deionized water was purified by use of an Alpha-Q water-purification system (Millipore, Bedford, MA, USA) for thepreparation of samples and buffer solution. All other reagentswere of analytical grade. R. coptidis and E. rutaecarpa werepurchased from the Sifang Pharmacy (Shenyang, China).

2.2. Instrumentation conditions

2.2.1. Qualitative analysisQualitative analysis was operated on a Thermo-Electron

LCQ linear ion-trap mass spectrometer (Thermo-Electron, SanJose, CA, USA)fittedwith anelectrospray ionization source overthe mass range from m/z 50 to 2000 in the positive ionizationmode. Xcalibur1.2 data analysis system was used. The sprayvoltage was set to 4.2 kV. The capillary voltage was fixed at13 V. The heated capillary temperature was fixed at 210 °C.Nitrogen used as the sheath and the auxiliary gas was set to 70and 20 arbitrary units, respectively. The isolationwidth forMSn

was 1.0 Da.The HPLC system consisted of an Agilent 1100 series

equippedwith a Agilent 1100 series photodiode-array detector(PDA) and autosampler Data analysis (Agilent, Palo Alto, CA).Chromatographic separationwas carriedout onaDiamonsil C18(150×4.6 mm I.D., 5 μm, Dikma) with an EasyGuard C18Security guard column (8×4.0 mm I.D., Dikma). The mobilephases consisted of 5 mM ammonium formate (0.025% formicacid) (A) andacetonitrile (B) usinga gradient elution of 25%Bat0 min, 30% B at 5 min, 40% B at 15 min, 55% B at 20 min (v/v), ata flow rate of 0.5 mL/min.

2.2.2. Quantitative analysisTheHPLC systemconsisted of a LC-10ADvpPump (Shimadzu,

Kyoto, Japan) and a SIL-HTA Autosampler (Shimadzu, Kyoto,Japan). Chromatographic separation was carried out on aDiamonsil C18 (150×4.6 mm, 5 μm, Dikma) column with aEasyGuard C18 Security guard column (8×4.0 mm I.D., Dikma)kept at20 °C. Themobilephaseconsistsofwater (containing0.3%formic acid)/acetonitrile (30:70, v/v), at a flow rate of0.45 mL/min.

Mass spectrometric detection was performed on a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer(San Jose, CA, USA) equipped with an ESI source in the positiveionization mode. The MS operating conditions were optimizedas follows: the spray voltage: 4200 V; the heated capillarytemperature: 320 °C; the sheath gas (nitrogen): 30 Arb; theauxiliary gas (nitrogen): 5 Arb; the collision gas (argon)pressure: 1.2 mTorr. Data acquisition was performed byXcalibur 2.0 software. Peak integration and calibration wereperformed using LCquan software. Quantificationwas obtainedby using SRM mode of the transitions at m/z 302→287 fordehydroevodiamine, atm/z 320→292 for coptisine and atm/z172→128 formetronidazole (IS) respectively,with a scan timeof 0.3 s per transition.

2.3. Preparation of the standard and quality control(QC) samples

R. coptidis powder 0.1 g mixed with 95 mL hydrochloricacid:methanol (1:100, v/v) was heated in 60 °Cwater bath for15 min, following with ultrasonic extracting for 30 min. Thesolution was placed overnight at room temperature andfiltered through 50 μm filter paper, then adjusted to 100 mLwith methanol. The filtrate acted as a stock standard solutionwith an apparent concentration of 1.0 mg H/mL, where Hmeant herb [10].

E. rutaecarpapowder0.2 gwas immersedwith 90 mLethanolfor 1 h, following with ultrasonic extracting (250W, 33 kHz) for40 min, cooling. The solution was filtered then adjusted to100mL with ethanol. The filtrate acted as a stock standardsolution with an apparent concentration of 2.0 mg H/mL, whereH meant herb [10].

Standard working solutions at the concentrations of 10, 20,50, 200, 500, 2000, 5000 ng H/mL for coptisine and 100, 200,500, 2000, 5000, 20,000, 50,000 ng H/mL for dehydroevodia-mine were prepared by serially diluting the stock standardsolution with methanol. A 200 ng/mL working solution ofinternal standard (IS) was similarly prepared by diluting ametronidazole stock solution with methanol. Calibrationstandards were prepared by spiking 100 μL of the appropriatestandard working solutions into 50 μL blank plasma. Themixture was disposed according to “Sample preparation” exceptfor adding only 50 μL methanol. Quality control (QC) sampleswere similarly prepared at low, medium and high levels (200,2000, 40,000 ng H/mL for dehydroevodiamine and 20, 200,4000 ng H/mL for coptisine). The samples were prepared priorto use during validation and pharmacokinetic study. All thesolutions were stored at−20 °C.

2.4. Sample preparation

Rat plasma 50 μL was mixed with 50 μL internal standardsolution (200 ng/mL), 150 μL methanol and 100 μL acetoni-trile. After vortex-mixing 2 min, the mixture was centrifugedat 10,000 rpm for 5 min. The supernatant was separated outand blown to dryness with nitrogen at 40 °C. Then the residuewas reconstituted in 100 μL mobile phase and a 10 μL aliquotof the final testing samples was injected onto the LC–MSsystem for analysis.

2.5. Method validation

The method was validated according to the currentlyaccepted USA Food and Drug Administration (FDA) bioanaly-tical method validation guidance.

Method linearity was evaluated by analyzing calibrationstandards in duplicate at each concentration level over threeconsecutive days. The accuracy and precision were assessed byanalyzing QC samples in six replicates at three concentrationlevels on three validation days. The extraction recovery wasevaluated at three concentration levels and for the IS at oneconcentration level by comparing thepeak areas of the analytesobtained from six plasma samples with the analytes spikedbefore and after extraction. Matrix effect was evaluated bycomparing the peak areas of the analytes obtained from sixplasma samples with the analytes spiked after extraction to

1154 R. Yan et al. / Fitoterapia 82 (2011) 1152–1159

those for the neat standard solutions at the same concentra-tions. The stability of dehydroevodiamine and coptisine in ratplasma at low and high concentration levels was evaluatedunder a variety of storage and process conditions.

The effects of the coexisting components in the extractsolutions were investigated using the standard additionmethod at three concentrations. 25 μL blank plasma andappropriate standard extract solution at equivalent concen-trations were added to 25 μL blood samples of six rats afteradministration of Zuojinwan, the concentration of which hadbeen determined. The samples were then extracted asdescribed in “Sample preparation”, taking the volume of theadded standard extract solution into account. The standardaddition recoveries of the analytes were calculated from theoriginal quantity (Wori), the added quantity (Wadd) and thefound quantity (Wfou) as follows:

recovery %ð Þ = Wfou−Wori

Wadd× 100%: ð1Þ

2.6. Pharmacokinetic application

Six male Sprague–Dawley rats (250±20 g) were fastedfor 12 h prior to experiment. The rats were split into threegroups to complete the crossover design for pharmacokineticexperiment with a washout period of 7 days. The powder ofR. coptidis, E. rutaecarpa and Zuojinwan was suspended in0.1% carboxymethyl cellulose sodium (CMC-Na) aqueoussolution and was administered to the rats (1.08 g R. coptidisand 0.18 g E. rutaecarpa powder/kg body weight) by oralgavage. Blood samples (150 μL) were obtained from the oculichorioideae vein before dosing and subsequently at 10, 20, 45,90, 150, 180, 210, 300, 420, 480, 720 and 1440 min followingadministration, transferred to a heparinized Eppendorf tubeand centrifuged at 12,000 rpm for 5 min. The supernatantswas frozen at −20 °C until analysis. Pharmacokinetic param-eters were calculated using Drug and Statistics 2.0 (DAS 2.0)(Mathematical Pharmacology Professional Committee ofChina, Shanghai, China).

3. Results and discussion

3.1. Qualitative analysis

LC–MSn was used for the identification of the constituentsin herbs. Through full-scan ESI mass spectrum in the positiveion mode, dehydroevodiamine was found in E. rutaecarpaextract and plasma samples after administration ofE. rutaecarpa; coptisine was found in R. coptidis extract andplasma samples after administration of R. coptidis; both werefound in plasma samples after administration of Zuojinwan.

Table 1Precursor and product ions of dehydroevodiamine and coptisine in LC–MSn experim

m/z MS2 MS3 MS4 tR (m

302.24 287.35 259.01 8.49320.21 318.19 (292.29) 290.20 262.28 8.40

Neither evodiamine nor rutecarpine were found in plasmasamples. The ion at m/z 302 at 8.49 min was identified asdehydroevodiamine by referring to the MSn spectra with thosereported in literature [11]; The ion at m/z 320 at 8.40 min wasidentified as coptisineby referring to theMSn spectrawith thosereported in literature [12] (Table 1). The chemical structures ofanalytes and IS were shown in Fig. 1. The typical full-scan ESImass spectra of analytes and IS were described in Fig. 2.

3.2. Quantitative analysis

3.2.1. Optimization of LC–MS/MS for quantitative analysisDifferent mobile phases consisting of methanol–water or

acetonitrile–water were attempted. It was found that acetoni-trile provided higher mass spectral signal and lower back-groundnoise thanmethanol. The addition of acidicmodifiers tothe mobile phase had appropriate enhancement on theionization efficiency of the analytes under ESI conditions.Finally, a mobile phase consisting of water (containing 0.3%formic acid)/acetonitrile (30:70, v/v) was chosen to achievesymmetrical peak shapes and short chromatographic run time,and to minimize the matrix effect.

TheMS/MS detection of analytes was optimized by infusinga standard extract solution into the mobile phase using asyringe pump. Someparameters suchas spray voltage, capillarytemperature, source CID, sheath gas (nitrogen) pressure,auxiliary gas (nitrogen) pressure, collision gas (argon) pres-sure, and collision energy were optimized. The other MSparameters were adopted from the recommended values forthe instrument. The product ion mass spectra of analytes wereobtained with maximum sensitivity as shown in Fig. 2.

3.2.2. Method validationThe typical chromatograms of a blank, a spiked plasma

sample with the reference extract (10.0 ng H/mL for coptisineand 100.0 ng H/mL for dehydroevodiamine) and IS (200 ng/mL)and plasma obtained 180 min after oral administration ofZuojinwan were presented in Fig. 3. All samples were found tobe of no interference at the retention times of the analytes or theIS.

Typical equations of the calibration curve using weighted(1/x2) least squares linear regression were as following: Y=−6.496×10−3+8.283×10−5X, r2=0.9804, over the range100.0–50,000.0 ng H/mL (dehydroevodiamine), Y=4.129×10−3+1.641×10−3X, r2=0.9822, over the range 10.0–5000.0 ng H/mL (coptisine). The calibration curves showedexcellent linearity in rat plasma. The precision and accuracydata corresponding to LLOQ are shown in Table 2. The values ofaccuracy and precision were within recommended limits. Theextraction recoveries determined for the analytes were shownin Table 2. Themean extraction recovery of the IS at 200 ng/mLwas 75.7±7.7%. The matrix effect at the concentration of

ents.

in) Formula MW Identification

C19H16N3O+ 302.35 DehydroevodiamineC19H14NO4

+ 320.32 Coptisine

A100

60

80

302.3

287.4

[M]+

NH

N O

unda

nce

1155R. Yan et al. / Fitoterapia 82 (2011) 1152–1159

2000 ng H/mL for dehydroevodiamine, 200 ng H/mL for copti-sine and 200 ng/mL for IS in six different sources of rat plasmawere 110.3±4.5%, 106.2±8.3% and 98.2±9.4%, respectively.The ionization suppression/enhancement was negligible. Thestability study showed that dehydroevodiamine and coptisinewere stable during these tests (Table 3). The standard additionrecoveries in Table4 indicated thegoodaccuracyof themethod.

Bm/z

80 120 160 200 240 280 3200

20

40

303.3

288.4

274.4

N

Rel

ativ

e A

bve

Abu

ndan

ce

40

60

80

100 292.3

320.2

[M]+

NH+

O

O

O

3.2.3. Results of pharmacokinetic studyThe method described above was applied to the pharma-

cokinetic study in which plasma concentrations of dehydroe-vodiamine and coptisine were determined for 24 h after oraladministration of single herbs and Zuojinwan (1.08 g R. coptidisand 0.18 g E. rutaecarpa powder/kg body weight). The meanplasma concentration–time profiles (n=6) were representedin Fig. 4. On the whole, the concentration–time curves of eachconstituent were obviously different after administration ofeach corresponding herb alone and Zuojinwan, which impliedthe interaction between R. coptidis and E. rutaecarpa; multiplepeakswere characteristic of the pharmacokinetic profiles of theanalytes, which often happens in the researches on herbs.Multiple peaksmaybe causedbydiverse factors, differentherbswith different reasons [14,15].

NH

N

N

O

(1) dehydroevodiamine

N+

+

O

O

O

O

(2) coptisine

N

N N

OH

O

O

(3) metronidazole

Fig. 1. Chemical structures of (1) dehydroevodiamine (2) coptisine, and(3) metronidazole (IS).

Rel

ati

Rel

ativ

e A

bund

ance

100 140 180 220 260 300

m/z

0

20293.3

321.2256.3 305.2

C

50 70 90 110 130 150 170 190m/z

0

20

40

60

80

100128.2

127.2

172.2

147.2 174.4171.5

[M+H]+

N

NH2

NO

O

Fig. 2. Product ion mass spectra of [M]+ ions of (A) dehydroevodiamine(B) coptisine (C) metronidazole (IS).

Chinese formula tends to act on the overall functional stateof the patient as different fromwesternmedicine. Comparativestudies of pharmacological compounds in Chinese herbalpreparation are very important for elucidating its mechanismof action in different compositions of herbs. There are severalmethods extensively used to investigate drug interactions. Invivo pharmacokinetics studies are more reliable than in vitrochemical studies, and more accurate than pharmacologicalstudies. Many research have demonstrated the alteration inpharmacokinetics of the combined use of multiple herbs or

A

B

C

Fig. 3. Representative SRM chromatograms for analytes in (A) a blank plasma sample (B) a blank plasma spikedwith standard extract at the LLOQ and IS at 200 ng/mL(C) a plasma sample 3 h after administration of Zuojinwan.

1156 R. Yan et al. / Fitoterapia 82 (2011) 1152–1159

compounds. Hence, comparative pharmacokinetic studywouldprovide more useful information for further understanding ofthe combination of herbs.

There were few reports about the pharmacokinetics ofE. rutaecarpa because of the low contents of the constituents,evodiamine and rutaecarpine as the most important pharma-cologically active constituents [6–8]. Zuojinwan is composed ofherb powders other than herb extracts, 1/7 E. rutaecarpa, 6/7

Table 2Intra- and inter-day precision and accuracy data for dehydroevodiamine and coptis

Compound Added(ng H/mL)

Found(ng H/mL)

RSD%

Intra-day

Dehydroevodiamine 100 82.46 17.9200 180.81 14.22000 1826.54 8.140,000 34,920.42 8.8

Coptisine 10 9.57 14.620 19.63 2.9200 195.89 12.64000 4323.74 6.8

Table 3Stability data of dehydroevodiamine and coptisine in rat plasma under various stor

Compound Concentration(ng H/mL)

Short-term (4 h)

Mean RSD% RE%

Dehydroevodiamine 200 221.40 3.4 10.740,000 41,130.05 10.5 2.8

Coptisine 20 19.27 5.1 −3.74000 4473.30 1.5 11.8

R. coptidis. The plasma concentrations of most constituents inE. rutaecarpa, including evodiamine and rutaecarpine, werebelow the limits of detection. Dehydroevodiamine, one of theimportant active constituents in E. rutaecarpa, shows strongMSresponse, resulting from one positive charge in the molecule(Fig. 1). No pharmacokinetic study has been published fordehydroevodiamine in biological fluids yet. Table 5 lists thepharmacokinetic parameters of dehydroevodiamine and

ine in rat plasma (3 days, six replicates per day).

Relative error%

Recovery %

Inter-day Mean SD

/ −17.5 / /13.1 −9.6 71.1 9.712.8 −8.7 70.8 7.713.8 −12.7 62.5 2.4/ −4.3 / /6.0 −1.9 74.0 10.815.9 −2.1 73.7 119.3 8.1 69.4 5.1

age conditions (n=3).

Three freeze–thaw Post-preparative (24 h)

Mean RSD% RE% Mean RSD% RE%

163.84 15.1 −8.1 198.94 12.9 −0.542,962.88 7.8 7.4 39,920.73 14.4 −0.221.05 6.0 5.2 19.25 7.6 −3.83976.33 13.5 −0.6 4377.96 3.6 9.4

A

B

0 5 10 15 20 250

10000

20000

30000

40000

50000

60000

70000

Con

cent

ratio

n (n

gH/m

L)

Time (h)

Evodia rutaecarpa Zuojinwan

0 5 10 15 20 250

1000

2000

3000

4000

5000

6000

7000

Con

cent

ratio

n (n

gH/m

L)

Time (h)

Rhizoma coptidis Zuojinwan

Fig. 4. Mean plasma concentration–time curves of (A) dehydroevodiamine(B) coptisine after oral administration of single herbs and Zuojinwanpowders.

1157R. Yan et al. / Fitoterapia 82 (2011) 1152–1159

coptisine. According to definitions, pharmacokinetic parame-ters except AUC and Cmax are independent of plasmaconcentrations so that independent of standards. In the relativeapproach, AUC and Cmax are not comparable between differentanalytes but comparable between the same analyte in differentsamples. Theplasmaconcentrationof dehydroevodiamineafteroral administration of Zuojinwan was much higher than thatafter oral administration of E. rutaecarpa alone. Cmax increasedfrom 15,383±7166 to 40,992±21,052 (μg H/L); AUC in-creased from 68,134±19,162 to 186,715±39,211 (μg H/L h).It seemed that R. coptidis could enhance the absorption andbioavailability of dehydroevodiamine. The enhancement ofabsorption and AUC was often observed in the researches onTCM [16–18]. However, the mechanism is still unknown andsystematic research shouldbedone to elucidate it. Probably, thealkaloids in R. coptidis aremore apt to be degraded by intestinalbacterial than dehydroevodiamine. Thus the competitiveinhibition between dehydroevodiamine and other alkaloidsmight reduce the degradation and increase the concentration ofdehydroevodiamine in the intestine, which finally enhance thebioavailability of dehydroevodiamine. The proof is that quitelow plasma concentration and low bioavailability of orallyadministered a single constituent resulting from extensivelymetabolism [13]. The bioavailability upgrade of dehydroevo-diamine is favorable for the warm nature of E. rutaecarpa toweaken the cold nature of R. coptidis.

As to coptisine, three peaks were observed in the plasmaconcentration–time curves after administration of Zuojinwan,while a single peak after administration of R. coptidis alone(Fig. 4). The similar result had happened in Deng's study [9].They reported the pharmacokinetic profiles of berberine,palmatine and jateorrhizine after administration of Zuojinwanwith similar three peaks, which probably due to the similarityof their molecular structures. Deng discussed that distributionre-absorption and enterohepatic circulation might contributeto multiple blood concentration peaks of protoberberinealkaloids after oral administration of Zuojinwan. If theconcentration in tissues ismuch higher than that of the plasma,it is possible for the drug to transfer from tissues to plasma,which causes another peak in plasma. The alkaloids which hadbeen already metabolized would excrete and re-absorbed. Wealso noticed that Zuojinwan was composed of powders ofherbs. The absorption rate of alkaloids was limited by theirdissolution rate fromherb powders. E. rutaecarpamay decreasethe dissolution rate of the alkaloids in R. coptidis. It would takemore time for the constituents todissolve outof deep layer thansurface layer of herb powders. The second and third peakswereprobably produced by the absorption of the deep layerconstituents. Double peaks in the pharmacokinetic profiles ofdehydroevodiamine may be caused in the same way. Thecombination may decrease the dissolution rate of the constit-

Table 4Standard addition recovery data of dehydroevodiamine and coptisine (n=6).

Plasma sample Dehydroevodiamine

Added (ng H) Re

45 min after administration 5 9390 min after administration 75 91150 min after administration 2 91

uents in herbs. In addition, another probable reason wasmetabolism. Zuo [13] reported that oneprotoberberine alkaloidin R. coptidis could be one of the metabolites of anotherprotoberberine alkaloid in rat, which could contribute to theoccurrence of the second and third peaks. According to Table 5,the tmax after administration of Zuojinwan is longer than that ofR. coptidis; the Cmax after administration of Zuojinwan is lowerthan that of R. coptidis; AUCdecreases a little after combination.It was surmised that E. rutaecarpa could moderate the potencyand prolong the action time of R. coptidis. “Essentials of MateaMedica”, an ancient TCM book, recorded that the warm nature

Coptisine

covery (%) Added (ng H) Recovery (%)

.3±4.9 250 92.9±3.9

.3±5.6 1000 94.2±3.8

.2±3.1 750 91.8±2.7

Table 5Mean pharmacokinetic parameters of dehydroevodiamine and coptisine in rat plasma (n=6).

Compound p.o. AUC0–t (μg H/L h) AUC0–∞ (μg H/L h) MRT0–t (h) MRT0–∞ (h) t1/2z (h) tmax (h) Cmax (μg H/L)

Dehydroevodiamine Evodia rutaecarpa 68,130±17,451 68,134±19,162 4.6±0.7 4.6±5.6 1.6±6.4 3.5±3.0 15,383±7166Zuojinwan 186,698±46,442 186,715±39,211 4.9±0.7 4.9±3.6 1.8±5.4 1.5±1.1 40,992±21,052

Coptisine Rhizoma coptidis 5446±1312 5454±1408 3.8±1.1 3.8±1.1 3.4±1.2 0.8±0.1 4585±1001Zuojinwan 4654±1948 4669±4470 4.3±1.1 4.3±9.0 2.8±5.6 1.5±0.9 2848±1056

1158 R. Yan et al. / Fitoterapia 82 (2011) 1152–1159

of E. rutaecarpa could attenuate the bitter cold nature ofR. coptidis thus protect the stomach fromthe repulsion betweenillness and medicines.

In summary, the interactions between R. coptidis andE. rutaecarpa caused obvious alteration of the pharmacoki-netic profiles of dehydroevodiamine and coptisine; R. coptidisstrengthened the effects of E. rutaecarpa while E. rutaecarpaweakened the effects of R. coptidis; the enhancement ofbioavailability is an important, but not the only effect ofcombination. The connotation of formula is so complex thatcould hardly be elucidated only through qualitative analysis.Appropriate mathematical models are required to character-ize its inner structure.

3.2.4. A tentative pharmacokinetic model of TCMThe pharmacokinetic process is usually thought to be

continuous and depicted with a smooth and continuousconcentration–time curve, initial dose D0 decreasing graduallyuntil undetectable. Actually, the process can also be treated asdiscrete one, which consists of numerous micro processes. Theinteraction between medicine and body is too complex so thatcan only be denoted with a general function f(⋅). In a microprocess, D0 interacts with body, yielding D1. In the next microprocess, D1 interacts with body, yielding D2,…… In this way, apharmacokinetic can be regarded as a series of iterationprocesses, Dn= f(Dn−1). Quantum mechanism has pointedout that energy cannot be divided infinitely but has a minimalunit. All continuous processes are not really continuous, butcomposed of numerous micro processes. It is deduced thatelimination of drugs cannot be divided infinitely but has aminimal unit, that one unit elimination equals to iterating once.It is concluded that a discrete process is closer to the truth thana continuousprocess. In thisway, amedicine's pharmacokineticproperties depend on the mathematical properties of itscorresponding iteration function.

As to somenonlinear iteration functions, numberswith veryminusculedifferenceswouldbecompletedifferentafter severaltimes iteration, which is called chaos in mathematics. Chaosleads to nonlinear pharmacokinetic of drugs. In this case, minorfluctuation of dose will cause large scale fluctuation on thestable bloodconcentration, andoverdose ormissing-dose couldlead abnormal blood concentration exceed or below clinicrange.

TCM has created a valid method to avoid chaos twothousand years ago. The pharmacokinetic process of TCMcould also be regarded as a series of iteration, but morecomplex. If a TCM prescription contains n+1 activeconstituents, its dosage vector could be expressed as D=(D0, D1, D2,…, Dn). TCM is a system where constituents reactwith each other. After iterating once, the dosage vectorbecomes D'= f (D0, D1, D2, …, Dn)=(D0′, D1′, D2′,…, Dn′).Fundamentally, the changes are not caused by the absolute

dosage of a constituent but by the ratio between the dosagesof different constituents. Hence dosage vector can besubstituted with a percentage vector through normalization,X=(X0, X1,X2,…, Xn),where0≤Xi =

Di∑Di

≤1,∑Xi=X0+X1+X2+⋯+Xn=1. The results after iterating also become thepercentage vector X'= f (X0, X1, X2,…, Xn)=(X0′, X1′, X2′,…,Xn′). The domain of percentage vector is an n-dimensionalvector space Sn with radium 1. There is no evidencesuggesting that any ratio could not exist, so the iteration ofdisposition is a continuous mapping from Sn to Sn. Accordingto the Brouwer fixed points theorem (If f :Sn→Sn is acontinuous correspondence from Sn to Sn, f has fixed points.),the iteration has fixed points, namely f (X0, X1, X2,…, Xn)=(X0, X1, X2,…, Xn). As for TCM pharmacokinetic, that meansthat the active constituents will come to a stable ratiogradually.

When the stable ratio reached, m1m2

= m01

m02⇒m

01−m1

m02−m2

= m1m2,

becausedm1=dtdm2=dt

=

m01−m1

Δtm0

2−m2

Δt

=m

01−m1

m02−m2

, sodm1=dtdm2=dt

=m1

m2

and dm1dt : dm2

dt : ··· : dmndt =m1 : m2 : ··· : mn = km1 : km2 : ··· :

kmn (wherem is the concentration of constituents in plasma;t is time; suffix is the serial number of constituents, k is aconstant), then the concentration of constituent i would bemi

t=mi0ekt. The equation means that when a stable ratio

reached, each active constituent would follow first orderelimination kinetics with the same rate constant k, thus theterminal of each concentration–time curve can be describedas linear elimination approximately.

In conclusion, the composition of active constituents in aTCM preparation tends to reach a stable ratio in vivo. When anequilibrium point reached, the ratio between different activeconstituents will keep constant. Before equilibrium, theelimination rate of constituents will keep varying, whichcould cause multiple peaks in the concentration–time curves.In ancient China, there was no precise apparatus to controlthe dosage. It was the equilibrium point that ensured theactivity of TCM. As long as the equilibrium point wasdetermined, the real composition of TCM preparations couldbe found out. However, pharmacokinetic data are easy to beaffected so that stricter and delicate experiments are requiredto minimize the errors.

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