Pharmacokinetic study of isoquercitrin in rat plasma after intravenous administration at three different doses

The aim of this study is to develop a simple and specific HPLC method using vitexin as the internal standard to investigate the pharmacokinetics of isoquercitrin (ISOQ) after three different doses administrated intravenously to rats. The pharmacokinetic parameters were calculated by both compartmental and non-compartmental approaches. The results showed that ISOQ fitted a three-compartment open model. The values of AUC increased proportionally within the range of 5-10 mg·kg-1. Moreover, a half-life, b half-life, aCL, MRT0-t and MRT0→∞ of ISOQ in rats showed significant differences between 20 mg·kg-1 and other doses, indicating that ISOQ presented dose-dependent pharmacokinetics in the range of 5-10 mg·kg-1 and non-linear pharmacokinetics at higher doses.


INTRODUCTION
The leaves of Crataegus pinnatifida Bge.var.major recorded in the Chinese Pharmacopoeia are a well-known traditional Chinese medicine and contain many compounds such as chlorogenic acid, vitexin-4''-O-glucoside, vitexin-2''-O-rhamnoside, vitexin, rutin, hyperoside, isoquercitrin (ISOQ), and quercetin (Ying et al., 2009).In recent years, ISOQ, a flavonol glycoside, has attracted a great deal of attention because of its numerous biological and pharmacological activities, including antiinflammatory activity (Rogerio et al., 2007), antioxidant activity in vitro and in vivo (Silva et al., 2009), being effective at attenuating the death of RGC-5 cells in culture caused by exposure to hydrogen peroxide (H 2 O 2 ), and treating glaucoma (Jung et al., 2010).Moreover, many methods including HPLC-UV (Bramati, Aquilano, Pietta, 2003), LC-DAD and LC-MS (Maria et al., 2007), CZE-UV (Jing et al., 2007), SPE-HPLC (Lai et al., 2007) have been reported in the literature for the quantification of ISOQ in various herbal medicines.Also, in vitro and in vivo analyses of ISOQ have been reported (Chang et al., 2005).However, there was little attention paid to the pharmacokinetic study of ISOQ after intravenous administration at three different doses, and therefore the aim of this study is to investigate the pharmacokinetics of ISOQ employing a validated HPLC method with an internal standard (I.S.).To the best of our knowledge, this is the first report of a pharmacokinetic study following the intravenous administration of ISOQ in rats at multiple doses.

Reagents and chemicals
ISOQ and the internal standard, vitexin, were both isolated from leaves of C. pinnatifida Bge.var major in our laboratory; the purities of both were found to be over 98% by HPLC analysis.The leaves were collected in Shenyang, Liaoning Province, China and identified by Prof. Bing Wang.Voucher specimens (20111020) were maintained at the Liaoning University of Tradition Chinese Medicine.The chemical structures of ISOQ and vitexin, confirmed by 1 H and 13 C-nuclear magnetic resonance spectroscopy, are shown in Figure 1.
The water used in all experiments was purified by a Milli-Q® Biocel Ultrapure Water System (Millipore, Bedford, MA, USA).Methanol and acetonitrile were both of HPLC grade and purchased from Damao (Chemical Reagent Plant, Tianjin, China).All other reagents were of analytical grade (Jinfeng Chemical Factory, Tianjin, China).

Isolation and identification
A sample (5 kg) of the leaves of C. pinnatifida Bge.var major was extracted twice using 50 L of 60% aqueous ethanol each time.The crude extract was concentrated and then passed through a porous-polymer resin (AB-8, Tianjin, China).The fraction eluted with 70% ethanol was evaporated under reduced pressure to obtain an extract and subjected to silica-gel column chromatography eluted with ethyl acetate:butanone:formic acid:water (6:2:1:0.2).Fractions of similar composition pooled on the basis of TLC analysis (UV monitoring at 365 nm) were repeatedly subjected to silica gel column chromatography and sublimated on a Sephadex LH-20 column to obtain 0.6 g of ISOQ with a purity of 98%, which was checked by HPLC.The chemical structures of ISOQ confirmed by 1 HNMR, 13 CNMR and MS data were consistent with previous reports (Zhang, Xu, 2001;Wang et al., 2004).

Plasma Sample Preparation
10 μL of I.S. (vitexin, 22.6 μg•mL -1 ), 10 μL of acetic acid, and 500 μL of methanol were successively pipetted into the 100 μL plasma samples, followed by vortex mixing for 1 min.The supernatant was separated and evaporated to dryness under a stream of nitrogen at 40 ºC after being centrifuged at 890 × g for 15 min.The residue was constituted in 100 μL of mobile phase and centrifuged at 15,092 × g for 10 min.A 20 μL aliquot of each supernatant was analyzed by HPLC.

Selectivity
Selectivity was shown by comparing chromatograms of blank plasma obtained from rats prior to dosing with those of corresponding standard plasma samples spiked with ISOQ and I.S., and plasma samples from rats after the intravenous administration of ISOQ.

Preparation of standards and quality control samples
Standard stock solutions of ISOQ and I.S. were prepared in methanol to yield the concentrations of 160 and 226 μg•mL -1 , respectively.The working solutions were prepared with diluted stock solution to concentrations over the range of 0.4-160 μg•mL -1 .All working solutions were stored at 4 ºC.Seven calibrators (0.2, 0.4, 0.8, 2, 5, 20 and 80 μg•mL -1 ) of ISOQ were prepared by adding standard working solutions (50 μL) and the working solution I.S. (22.6 μg•mL -1 , 10 μL) to drug-free rat plasma.The quality control (QC) samples were prepared at low (0.6 μg•mL -1 , 3 times of the lower limit of quantitation), high (60 μg•mL -1 , 75% of the upper limit of quantitation) and medium (6 μg•mL -1 , near the geometric mean of low and high concentration) concentrations in bulk and aliquots were stored at -20 ºC prior to analysis.

Linearity and LOD
The linearity was evaluated over the concentration range of 0.2-80 μg•mL -1 at seven levels of ISOQ.The calibration curves for ISOQ in plasma were generated by plotting the peak area ratio of ISOQ to I.S. versus the nominal concentrations in the standard plasma samples.The regression equation was obtained by weighted (1/c 2 ) least square linear regression.The limit of detection (LOD) was determined by a signal-to-noise ratio of 3. The lower limit of quantification (LLOQ) was defined as the lowest concentration of ISOQ in the calibration curves, giving an acceptable accuracy (R.E.) within ±20% and a precision (R.S.D.) that did not exceed 20%.

Precision and accuracy
The accuracy and precision of the method were evaluated with QC samples at three concentrations and using five replicates on three consecutive days.The intraand inter-assay precisions were assessed by determining the quality control samples at three concentration levels of ISOQ (0.6, 6 and 60 μg•mL -1 ).For the intra-day validation, five replicates of the QC plasma samples were analyzed on the same day.For the inter-day validation, five replicates of the QC plasma samples were analyzed on three different days.The precision was expressed as the R.S.D. which should be less than 15%, except at the LLOQ where it should not exceed 20%; the accuracy of the assay was determined by comparing the means of the determined ISOQ concentrations with the nominal concentrations.The mean percentage deviation from the nominal values was expressed as the R.E. which should be within ±15% of the nominal value, except at the lower limit of quantification where it should not exceed ±20%.

Extraction recovery
The extraction efficiency was determined by comparing the peak areas of ISOQ from a blank plasma with a known concentration added with the peak areas of a blank plasma with the same concentration of ISOQ added after protein precipitation (n = 6).

Stability
Five aliquots of unextracted QC samples at low, medium and high concentrations were subjected to the conditions below.A short-term stability experiment was carried out at ambient temperature (25 °C) for 24 h and long-term stability was tested by storage at -20 °C for one month.QC samples were subjected three freeze (-20 °C)-thaw (room temperature) cycles for determining freeze-thaw stability.Then, the samples were processed and analyzed.The concentrations obtained were compared with the nominal values of QC samples.

Animals and pharmacokinetic study
Male Wistar rats (weight 250 ± 20 g) were obtain from the Laboratory Animal Center of Liaoning University of Traditional Chinese Medicine (Shenyang, China).Before the experiments, all rats were kept in a controlled environment for one week and had free access to standard laboratory food and water.The rats were fasted 12-16 h prior to administration of the ISOQ.All animal studies were performed according to the Guidelines for the Care and Use of Laboratory Animals, which was approved by the Committee of Ethics of Animal Experimentation of Liaoning University of Traditional Chinese Medicine.
Three groups (five rats/group) were randomly assigned to receive ISOQ solution via a tail vein injection at doses of 5, 10 and 20 mg•kg -1 , respectively.ISOQ was dissolved in normal saline containing 20% propylene glycol-water (v/v).Blood samples (0.3 mL) were collected into heparinized tubes from the vena orbitalis at times of 2,5,10,15,20,30,45,60,90,120 and 180 min after intravenous administration and then centrifuged at 890 × g for 15 min.The obtained plasma was stored at -20 °C until analysis.

Method development
To obtain suitable retention time and good separation for the analysis, many mixed solutions were used as the mobile phase, such as methanol-water (40:60, 45:55) and methanol-acetonitrile-water (30:5:65, 35:5:60, 25:10:65); 0.1% formic acid was added in the solvent system after numerous trials to improve the peak shape and resolution.A mixture of methanol-acetonitrile-0.1% aqueous formic acid (35:5:60, v/v/v) was finally selected for use in this study.
The UV absorption spectra of ISOQ have two maximum absorptions at 256 nm and 358 nm and that of I.S. at 269 and 331 nm.The interferences from endogenous substances in the plasma were observed when the wavelength was set at 256 nm; therefore, the detection wavelength was set at 360 nm because of no interference appeared and it was found to be suitable for the analysis of ISOQ and I.S.
A suitable internal standard should be similar to the analyte, ISOQ, either structurally or chemically.Hence, vitexin-4''-O-glucoside, vitexin-2''-O-rhamnoside, hyperoside and vitexin were considered as the internal standards, with vitexin finally being chosen as the internal standard because the optimum resolution and retention time could be obtained when compared with analyte.
To simultaneously acquire high extraction recovery and precision of ISOQ and I.S., several solvents such as acetonitrile and methanol were applied to precipitate protein in different ratios during the preliminary investigation; the highest recovery occurred using 500 μL of methanol.10, 20 and 30 μL of acetic acid were added to the plasma to avoid dissociation of the analyte, and a good peak shape was finally obtained when 10 μL of acetic acid was added to the plasma.

Selectivity
To determine the selectivity of this method, blank rat plasma, plasma spiked with ISOQ and I.S. and plasma samples from rats after intravenous doses of ISOQ were analyzed; results are shown in Figure 2. The chromatograms showed that there were no interfering peaks in the region of the peaks of the analyte and I.S.The retention times of ISOQ and I.S. were approximately 6.8 min and 10.6 min, respectively.The total run time was 15.0 min.

Calibration curve and quality control samples
The evaluation of the linearity was performed with a seven-point calibration curve over the concentration range of 0.2-80 μg•mL -1 .The slope and intercept of the calibration graphs were calculated by weighted (1/c 2 ) least squares linear regression.The regression equation of the calibration curves was typically: y = 0.2426x -0.0242, and r was 0.9961, where y is the peak area ratio of ISOQ to I.S., and x is the plasma concentration of ISOQ.The limit of detection (LOD) was 0.06 μg•mL -1 , which was determined by a signal-to-noise ratio (S/N) of 3. The lower limit of quantification defined as the lowest concentration on the calibration curve, was 0.2 μg•mL -1 with the precision and accuracy within 20%, as verified by repeated analysis.doses, mainly because the metabolic enzyme of the drug or carrier of drug membrane-permeable process is saturated at high concentrations; i.e. the catalytic capability of metabolic enzymes or the transportation capability of a carrier is saturated when the dosage and the concentration in vivo exceed a certain limit (Leon, Andrew, 1993).

CONCLUSIONS
A simple and specific HPLC method was developed for the determination of ISOQ in rat plasma, which was successfully applied to an in vivo kinetic study in rats.ISOQ presented dose-dependent pharmacokinetics in the range of 5-10 mg•kg -1 and non-linear pharmacokinetics at higher doses.The validated method contributes not only to the determination of ISOQ in rat plasma but also to our understanding of the pharmacokinetic characteristics of ISOQ over the multiple doses in rats after intravenous administration.The compartmental and non-compartmental approach, respectively.* P < 0.05 by one-way ANOVA compared with 5 and 10 mg•kg -1 dose of isoquercitrin.

TABLE II -
Pharmacokinetic parameters of isoquercitrin in rats (mean ± S.D., n = 5) after the intravenous administration of doses of 5, 10 and 20 mg•kg -1