Comparative Pharmacokinetics of Five Rhubarb Anthraquinones in Normal and Thrombotic Focal Cerebral Ischemia-Induced Rats
Su-xiang Feng,1,2Jian-sheng Li,3*Ling-bo Qu,1,4*Yan-mei Shi 1and Di Zhao 2
1College of Chemistry and Molecular Engineering,Zhengzhou University,Zhengzhou 450001,China 2
College of Pharmacy,Henan University of Traditional Chine Medicine,Zhengzhou 450008,China 3
Institute of Geriatrics,Henan University of Traditional Chine Medicine,Zhengzhou 450008,China 4
College of Chemistry and Chemical Engineering,Henan University of Technology,Zhengzhou 450001,China
A comparative oral pharmacokinetic study of five anthraquinones (aloe-emodin,emodin,rhein,chrysophanol and physcion)from the extract of Rheum palmatum L.was performed in normal and thrombotic focal cerebral ischemia (TFCI)-induced rats.The plasma samples were clari fied through solid pha extraction prior to simultaneous deter-mination of the anthraquinones with a validated high-performance liquid chromatography -fluorescence system.The results indicated that the C max ,t 1/2and AUC 0-t ,of aloe-emodin,rhein,emodin and chrysophanol in
TFCI-induced rats were nearly double,whereas the CL values were remarkably decread (p <0.05)over tho of the normal rats.The plasma drug concentration –time data of five anthraquinones to rats fitted a two-compartment open model.The five anthraquinones in rat plasma were absorbed quickly and eliminated slowly in both groups.The obtained results could be helpful for evaluating the impact of the ef ficacy and safety of the drug in clinical applications.Copyright ©2012John Wiley &Sons,Ltd.
Keywords:rhubarb extract;anthraquinones;pharmacokinetics comparison;normal rats;thrombotic focal cerebral ischemia-induced rats.
INTRODUCTION
Rhubarb (Rhei rhizoma)is one of the important herbal drugs ud in many traditional Chine medicines for treating obstipation,gastrointestinal indigestion,diarrhea and jaundices in China and other Asian countries for thousands of years (Tang and Einbrand,1992;Newall et al .,1996).The main active components in rhubarb are aloe-emodin,rhein,emodin,chrysophanol,physcion (shown in Fig.1)and their glucosides.A number of studies showed that rhubarb can improve the nervous function of acute ischemic stroke (Liu et al .,2008)and alleviate cerebral edema (Tang et al .,2006).Z
hang et al .found physcion could decrea the expressing of interleukin-1beta and intercellular adhesion molecule-1to protect brain tissue from cerebral ischemia-reperfusion injury (Zhang et al .,2005).Chrysophanol could improve the impairments of memory with cerebral ischemia reper-fusion and increa the brain index (Li et al .,2010a).Lately,a great amount of works has been done on rhubarb anthraquinones (Li et al .,2010b;Li et al .,2011;Liu et al .,2010).They found that rhubarb anthraquinones possd of protective effects against focal cerebral ischemia by decreasing plasminogen and fibrinogen,prolonging blood coagulation or resisting the aggregation and adhesion of platelet.Therefore,the potency of five anthraquinones makes it a promising candidate for devel-opment as a novel anti-cerebral ischemia drug.
Several pharmacokinetic studies on anthraquinones have been performed on healthy volunteers (Lee et al .,2003;Zhu et al .,2005)and normal animals (Gong et al .,2011;Yan et al .,2009;Yan and Ma,2007),but very little on the diad conditions.It has been reported that the elimination rate of puerarin,a principal bio-active component of Pueraria lobata (Willd .)Ohwi ,was signi ficantly slower in the cerebral ischemia reperfu-sion rat than in the normal rat (Yan et al .,2005).C max ,AUC 0–t ,Vd and CL of hydroxysaf flower yellow A,an active marker component of Carthamus tinctorius L.,showed obvious differences between the normal and acute blood stasis rats (Tian et al .,2010).Th
e purpo of this study was to investigate the pharmacokinetics in rats and compare the blood levels of the anthraquinones between normal and thrombotic focal cerebral ischemia (TFCI)-induced rats after oral administration of rhubarb extract.The results may provide uful information for clinical applications.
MATERIALS AND METHODS
Materials and reagents.The reference standards of aloe-emodin,rhein,emodin,chrysophanol,physcion and 1,8-dihydroxyanthraquinone (internal standard (IS))were purchad from the National Institutes for Food and Drug Control (Beijing,China).The purity of all the compounds was more than 98%on high-performance liquid chromatography (HPLC).Solid pha extraction (SPE)cartridges (C 18,3mL,packed with 200mg of 50m m particle sizes)were purchad from Bonna-Agela Technologies (Tianjin,China).HPLC grade methanol
*Correspondence to:Jian-sheng Li,Institute of Geriatrics,Henan Univer-sity of Traditional Chine Medicine,Zhengzhou,Henan Province 450008,China;Ling-bo Qu,College of Chemistry and Chemical Engineering,Henan University of Technology,Zhengzhou,Henan Province 450001,China.E-mail:li_;qulingbo@zzu.edu
PHYTOTHERAPY RESEARCH Phytother.Res.27:1489–1494(2013)
Published online 26November 2012in Wiley Online Library ()DOI :10.1002/ptr.4890
was purchad from Dikma Technology Inc.(Beijing,China).Puri fied water was obtained from a Millipore Milli-Q system (Bedford,MA,USA).The other chemi-cals were all of analytical grade.Rheum palmatum L .,collected from Sichuan,was purchad from Henan Medical Limited Company (Zhengzhou,China)and authenticated by Dr.Sui-qing Chen (College of Pharmacy,Henan University of Traditional Chine Medicine).Voucher specimens (No.Rhu.20110301)were deposited in the desiccators of our laboratory at Henan University of Traditional Chine Medicine (Zhengzhou,China).
Chromatographic condition.HPLC system (Waters,USA)was equipped with a 2695-paration module compod of an auto-sampler,a 2475-fluorescence detector and an Empower-2chromatography analytical workstation.The chromatographic paration was per-formed on a Venusil XBP C 18(L)column (4.6Â250mm,5m m,Bonna-Agela Technologies,Tianjin,China)protected by a Waters curity guard C 18column (4.6Â20mm,5m m).The mobile pha consisted of a mixture
of methanol-0.1%phosphoric acid water (75:25,v/v)at a flow rate of 1.0mL/min.The analysis was performed at 30 C.The column eluent was monitored with a fluorescence detector at wavelengths of 435nm and 515nm for excitation and emission,respectively.
Preparation of rhubarb extract.Rhubarb extract was prepared in our laboratory.Brie fly,the powder of Rheum palmatum L .was extracted twice using 75%ethanol,and then filtered.The combined filtrate was evaporated and re fluxed with 8%hydrochloric acid for 0.75h,then concentrated to dryness.The extraction was loaded to column chromatography on polyamide and eluted with 40%and 95%ethanol in turn.After-ward,the fraction eluted with 95%ethanol was collected and evaporated by rotary evaporation below 45 C.Finally,the residue was evaporated to dryness at 40 C.The quantitative analysis of anthraquinones was determined by internal standardization under the same chromatography conditions above.The con-tent of aloe-emodin,rhein,emodin,chrysophanol and physcion in the rhubarb extract was 87,115,231,263and 176mg/g,respectively.
Animals.Healthy male Sprague –Dawley (SD)rats weighing 250–280g were purchad from Henan Laboratory Animal Centra (Zhengzhou,China)with the licen number SCXK2010-0002,which were houd in an air-conditioned room at a temperature of 22Æ2 C,a relative humidity of 50Æ10%.They were fed standard laboratory chow and had access to water ad libitum for at least one week.Prior to
admin-istration,all the rats were fasted with free access to water for 12h.Animal experiments were approved by the Ethics Committee for the U of Experimental Animals in Zhengzhou University做梦梦到怀孕了
黄鳝的做法Preparation of plasma sample.SPE cartridges were equilibrated with 2mL methanol and 2mL puri fied water.A 200m L plasma sample was piped into a test tube and 50m L 1,8-dihydroxyanthraquinone (1.6m g/mL)as IS was added.Then,2mL methanol was introduced to the mixture.The tube was shaken quickly for 5min and then centrifuged at 12,000rpm for 15min at 4 C (Refrigerated Centrifuge 3-18k,Sigma,German).The supernatant was collected carefully and diluted with 5mL 0.1%phosphoric acid.Then,the mixture was loaded into the cartridge.After washing the SPE col-umn with 2mL puri fied water,the analytes were eluted with 2mL methanol.Afterward,the eluate was evapo-rated to dryness under a stream of nitrogen at room temperature.Finally,the residue was dissolved in 100m L methanol,and 20m L of the mixture was injected into HPLC system for analysis.
Preparation of calibration standards and quality control samples.Stock solutions of aloe-emodin,rhein,emodin,chrysophanol and physcion were prepared by dissolving the accurately weighed reference substance in metha-nol,respectively,and an appropriate amount of 1,8-dihydroxyanthraquinone as IS was dissolved in methanol to a final concentration of 1.6m g/mL.
The working solutions of calibration curve were prepared by mixing and diluting the above stock solu-tions with methanol.Seven calibration samples were prepared by spiking the standard mixture into 200m L blank plasma in tubes containing 50m L IS.The calibra-tion curves of aloe-emodin,rhein,emodin,chrysophanol and physcion were linear over the range of 22.8–2224,40.0–19520,30.2–7360,21.0–2048and 24.2–2368ng/mL,
respectively.
Figure 1.The chemical structure of five anthraquinones.
1490S.-X.FENG ET AL .
The quality control(QC)samples were prepared from blank plasma at high,medium and low concentra-tions forfive anthraquinones.All plasma samples were procesd according to the procedures described above. Method validation.The linearity of calibration curve
was determined by plotting the peak ratio of each anthraquinone over the IS against the respective con-centration of aloe-emodin,rhein,emodin,chrysophanol and physcion with the lower limit of quantification (LLOQ)established as the lowest concentration point.The limit of detection(LLOD)was determined at a signal-to-noi ratio of3.
The accuracy of the established method was assd by exterminating QC samples at low,medium and high concentrations and the intra-day accuracy,by assaying the six replicates at each concentration level on the same day while inter-day accuracy was assayed by analyzing the six duplicates duringfive concutive days.
The extraction recoveries of anthraquinones in rat plasma were determined by comparing the peak area ratios obtained with the ratios of standard samples at the same three concentrations as QC samples.The recovery of IS was determined in the same way. The stability of anthraquinones in rat p
lasma was evaluated under a variety of storage and handling conditions using the low,medium and high QC sam-ples in six replicates.Freeze–thaw stability was tested on three freeze(À20 C)/thaw(room temperature) cycles concutively.Storage stability was assd by analyzing samples that were kept atÀ20 C for 30days.
Pharmacokinetic study.Rats were randomly divided into two groups,normal and TFCI-induced model group.TFCI-induced rats were duplicated through the occlusion of middle cerebral artery(MCA)by using the embolus of rat autologous blood blot inrted with nylon thread(Zhang et al.,1997;Li et al.,2006). Unilateral stroke was induced in male rats by embolic occlusion of the right MCA.Briefly,animals were anesthetized with10%chloral hydrate.Under sterile conditions,the right carotid artery comprising the external and internal carotid arteries was expod and isolated.A silicon-coated nylon thread(30mm long, 0.26–0.30mm in diameter)was carefully inrted into the right middle carotid artery via external carotid artery,and the nylon thread was pulled out to an appropriate length to allow blood to coagulate.Rectal temperature was monitored and maintained at37 C to38 C throughout the experiment.After occlusion of the bloodflow for60min,the nylon thread was withdrawn from the external carotid artery to allow reperfusion for24h.During the24h period,the rats were put in the cages parately and fed with water and food.
Each rat was orally administered at a single intra-gastric gavage do of300mg/kg rhubarb extract. The do of300mg/kg is ten times to the effective do of rats(the do recommended for human is6 mg/kg,amounting to300–360mg/d to a60kg person). The blood samples(0.5mL)were collected into heparinized tubes from the tail vein before and after oral administration at time points of0,0.083,0.17, 0.333,0.5,0.75,1.0,2.0,4.0,6.0,8.0,12.0and24.0h,then centrifuged them immediately for20min at 3000rpm to parate plasma.Each plasma sample was stored in0.5mL polypropylene tubes atÀ70 C prior to analysis.
The maximum plasma concentration(C max)and the time to reach C max(T max)were noted directly from the obrved concentrations versus time data.The phar-macokinetic parameters such as t1/2(half-life),CL(total body clearance)and AUC0–t(area under curve)were calculated by drug and statistics software,version2.0 (Shanghai,China).
Statistical analysis.The statistical analysis was per-formed with SPSS for Windows Version16.0(SPSS Inc.,Chicago,IL,USA),and statistical analys were made using the two-tailed Student’s t-test.
RESULTS AND DISCUSSION
Assay validation
The typical chromatography offive anthraquinones and IS are prented in Fig.2,which indicates thatfive ana-lytes and IS are not detected in plasma blank(A)and could be well parated and detected in plasma spiked (B),plasma in normal rats(C)and TFCI-induced rats (D)after oral administration.In addition,there also appeared veral unknown peaks,and they do not interfere with the paration and analysis of thefive analytes and IS,which may be caud by the unknown compounds existing in the plasma sample matrix.The regression equations,correlation coefficient,linearity range were listed in Table1.The calibration curves showed good linearity over the concentration range with a correlation coefficient(r)greater than0.9972. The LLOD were estimated to be7.6,13.3,10.1,7.0 and8.1ng/mL for aloe-emodin,rhein,emodin,chryso-phanol and physcion,respectively.The LLOQ of aloe-emodin,rhein,emodin,chrysophanol and physcion were established as22.8,40.0,30.2,21.0and24.2ng/mL in rat plasma,respectively.
The mean extraction recovery rates of aloe-emodin, rhein,emodin,chrysophanol and physcion in rat plasma were94.4–97.9%,96.4–110.4%,94.2–98.8%, 94.6–97.3%and96.0–98.6%with RSD less than 7.4%.The average recovery of the IS was96.2Æ3.7%. The mean RSD values in the intra-and inter-day preci-sions ranged3.3–9.1%and1.8–9.1%,respectively. The results indicated that an acceptable
precision and accuracy of the prent method was obtained to deter-mination offive anthraquinones in rat plasma.
Stability of the anthraquinones in rat plasma was evaluated by analyzing the low,medium and high QC samples.The RSD values were3.4–8.6%forfive ana-lytes after three freeze/thaw cycles and3.9–9.5%for 30days stored atÀ20 C.The results indicated that the analytes were all stable under the above storage and process conditions.
Pharmacokinetics study
The validated method was applied to a pharmacokinetic study in normal and TFCI-induced rats.The mean
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COMPARATIVE PHARMACOKINETICS OF FIVE RHUBARB ANTHRAQUINONES
Figure 2.HPLC chromatograms of rat plasma samples:(A)blank plasma;(B)plasma spiked with aloe-emodin (1),rhein (2),internal standards (I.S.),emodin (3),chrysophanol (4)and physcion (5);(C)plasma
sample at collected 0.5h in normal rats after oral administration of rhubarb extract;(D)plasma sample at collected 0.75h in TFCI-induced rats after oral administration of rhubarb extract.
Table 1.Regression equation,correlation coef ficient (r )and linearity range of anthraquinones in rat plasma samples历史是什么
Analytes Regression equation Correlation coefficient (r)
Linearity range (ng/mL)
Aloe-emodin y =0.2186+0.0039x 0.998722.8–2224Rhein y =0.1406+0.0015x 0.999740.0–19,520Emodin
y =0.4618+0.0039x 0.997230.2–7360Chrysophanol y =0.2275+0.0019x 0.999121.0–2048Physcion
y =0.2913+0.0057x
四时田园杂兴教案0.9995
24.2–
2368
点滴关怀Figure 3.Mean concentration –time curves of five anthraquinones in normal rats and TFCI-induced rats (A)aloe-emodin;(B)rhein;(C)emodin;(D)chrysophanol;(E)physcion.1492S.-X.FENG ET AL .
plasma concentration –time curves (n =6)of five anthra-quinones are illustrated in Fig.3,and the main
pharma-cokinetic parameters of the five compounds in rats are shown in Table 2.The plasma drug concentration –time data of aloe-emodin,rhein,emodin,chrysophanol and physcion after a single oral administration of rhubarb extract to rats fitted a two-compartment open model.Comparison of the pharmacokinetic data of normal and TFCI-induced rats showed that there were signi fi-cant differences (P <0.05)in the main pharmacokinetic parameters such as t 1/2,AUC 0-t ,C max and CL.
In summary,the results of prent study indicated dis-ea condition in fluenced the potential pharmacokinetic of rhubarb anthraquinones.Under the pathologic condi-tion,rhubarb anthraquinones had a better absorption effect in TFCI-induced rats with higher concentrations and larger AUC 0Àt than normal rats,and it proved the rationality of using it in cerebrovascular dia,which would improve the therapeutic ef ficacy.The C max and AUC 0Àt of aloe-emodin,rhein,emodin and chrysopha-nol in TFCI-induced rats were nearly double than normal rats after a single oral do of rhubarb extract,while the CL remarkably decread (P <0.05)compared with normal rats.The time cours showed that five anthraqui-nones were absorbed rapidly in rat plasma in 5min after oral administration.However,the elimination of anthra-quinones was slow both in normal and TFCI-induced rats.The t 1/2of the five anthraquinones was long and still detectable at 48h for aloe-emodin and rhein,32h for emodin,60h for chrysophanol and physcion.Similar rearch was wide spread in other drugs or other dia models (Deng et al .,2008;Lu et al .,2002).
环境描写句子CONCLUSIONS
A study was conducted to evaluate and compare the pharmacokinetics differences of rhubarb anthraquinones in normal and TFCI-induced rats.The results showed that the main pharmacokinetic parameters of five anthraqui-nones such as the C max ,t 1/2,AUC 0-t ,and CL were signi fi-cantly different between normal rats and TFCI-induced rats.It indicated dia in fluenced the potential pharma-cokinetic of rhubarb anthraquinones.In the pathologic condition,a better absorption of the anthraquinones was found in TFCI-induced rats than normal rats,and it proves the rationality of using it in cerebrovascular dis-ea,which would improve the therapeutic ef ficacy.The five anthraquinones in rat plasma were absorbed quickly and eliminated slowly in both groups after oral adminis-tration of rhubarb extract.The obtained results could be helpful for evaluating the impact of the ef ficacy and safety of the drug in clinical applications.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.81274179)and the Foundation for University Young Teachers by Henan Province (No.2009GGJS-065).
Con flict of Interest
The authors have declared that there is no con flict of interest.
T a b l e 2.P h a r m a c o k i n e t i c p a r a m e t e r s o f fiv e a n t h r a q u i n o n e s i n n o r m a l r a t s a n d T F C I -i n d u c e d r a t s a f t e r o r a l a d m i n i s t r a t i o n o f r h u b a r b e x t r a c t (m e a n ÆS D ,n =6).
P a r a m e t e r s
A l o e -e m o d i n
R h e i n
E m o d i n
C h r y s o p h a n o l P h y s c i o n
n o r m a l r a t s
T F C I -i n d u c e d r a t s n o r m a l r a t s
T F C I -i n d u c e d r a t s
n o r m a l r a t s
T F C I -i n d u c e d r a t s n o r m a l r a t s
T F C I -i n d u c e d r a t s n o r m a l r a t s T F C I -i n d u c e d r a t s
A U C (0-t )(m g h /m L )1.24Æ0.093.10Æ0.06*17.79Æ0.2132.72Æ0.12*2.46Æ0.269.18Æ0.31*1.49Æ0.234.83Æ0.19*1.57Æ0.351.24Æ0.09A U C (0-1)(m g h /m L )1.59Æ0.164.67Æ0.39*20.30Æ0.3045.44Æ0.33*2.95Æ0.3211.16Æ0.38*1.92Æ0.348.06Æ0.31*2.19Æ0.413.29Æ0.60C m a x (m g /m L )0.18Æ0.110.46Æ0.18*5.01Æ0.168.91Æ0.19*0.48Æ0.251.80Æ0.21*0.22Æ0.180.65Æ0.30*0.26Æ0.220.21Æ0.18T m a x (h )0.47Æ0.150.36Æ0.19*0.50Æ0.000.54Æ0.180.47Æ0.150.54Æ0.190.75Æ0.000.83Æ0.150.70Æ0.140.50Æ0.00*t 1/2(h )10.54Æ0.2114.63Æ0.706.80Æ0.4114.23Æ0.77*9.05Æ0.269.61Æ0.4610.86Æ0.4820.99Æ0.74*12.91Æ0.1939.12Æ0.86*M R T (0-t )(h )8.03Æ0.058.64Æ0.127.12Æ0.117.03Æ0.117.65Æ0.087.66Æ0.138.14Æ0.128.97Æ0.068.10Æ0.099.41Æ0.07*M
R T (0-1)(h )14.76Æ0.2021.01Æ0.6810.14Æ0.3318.15Æ0.70*12.33Æ0.2512.83Æ0.4115.08Æ0.4528.55Æ0.75*17.51Æ0.1955.13Æ0.87*C L (L /h /k g )16.78Æ0.176.15Æ0.29*1.83Æ0.300.82Æ0.26*25.27Æ0.267.24Æ0.47*44.74Æ0.3010.57Æ0.28*27.35Æ0.3622.14Æ0.58V d (L /k g )
248.5Æ0.10108.1Æ0.36*16.38Æ0.16
14.10Æ0.48
不买社保319.0Æ0.27
93.28Æ0.55*649.2Æ0.28275.3Æ0.44*489.9Æ0.30
762.1Æ0.40*
*P <0.05,v e r s u s n o r m a l r a t s .
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荆门高铁站
COMPARATIVE PHARMACOKINETICS OF FIVE RHUBARB ANTHRAQUINONES
