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Metabolic and pharmacokinetic studies of scutellarin in rat plasma, urine, and feces.

AIM
To study the metabolic and pharmacokinetic profile of scutellarin, an active component from the medical plant Erigeron breviscapus (Vant) Hand-Mazz, and to investigate the mechanisms underlying the low bioavailability of scutellarin though oral or intravenous administration in rats.


METHODS
HPLC method was developed for simultaneous detection of scutellarin and scutellarein (the aglycone of scutellarin) in rat plasma, urine and feces. The in vitro metabolic stability study was carried out in rat liver microsomes from different genders.


RESULTS
After a single oral dose of scutellarin (400 mg/kg), the plasma concentrations of scutellarin and scutellarein in female rats were significantly higher than in male ones. Between the female and male rats, significant differences in AUC, t(max2) and C(max2) for scutellarin were found. The pharmacokinetic parameters of scutellarin in the urine also showed significant gender differences. After a single oral dose of scutellarin (400 mg/kg), the total percentage excretion of scutellarein in male and female rats was 16.5% and 8.61%, respectively. The total percentage excretion of scutellarin and scutellarein in the feces was higher with oral administration than with intravenous administration. The in vitro t(1/2) and CL(int) value for scutellarin in male rats was significantly higher than that in female rats.


CONCLUSION
The results suggest that a large amount of ingested scutellarin was metabolized into scutellarein in the gastrointestinal tract and then excreted with the feces, leading to the extremely low oral bioavailability of scutellarin. The gender differences of pharmacokinetic parameters of scutellarin and scutellarein are due to the higher CL(int) and lower absorption in male rats.


Introduction
Breviscapines are flavones extracted from the medical plant Erigeron breviscapus (Vant) Hand-Mazz. One of the active components of breviscapine is scutellarin (C 21 H 18 O 12 ; 4',5,6-hydroxyl-flavone-7-glucuronide). Scutellarin is hydrolyzed in vivo to form scutellarein (4',5,6,7-hydroxyl-flavone), an aglycone of scutellarin, and it is further metabolized to form conjugated metabolites. Scutellarin has been extensively used for the clinical treatment of cardiovascular and cerebrovascular diseases in China for many years. Pharmacological research indicates that scutellarin is associated with antihypertrophic effects [1] , fibrinolysis and anticoagulant effects [2] , protective effects in the brain and heart [3] , hypercholesterolemia suppression [4] , and endothelium-independent relaxation induction [5] . Scutellarin produces an anti-inflammatory effect via actions on arachidonic acid metabolism [6] . The compound has also been shown to have some anti-HIV effects [7] and to attenuate hepatocellular damage [8] . It is interesting that scutellarin exhibits weak estrogenic properties [9,10] , similar to genistein and apigenin. Many phytoestrogen compounds, such as genistein, apigenin, daidzein, glycitein, and their metabolites, have been demonstrated pharmacokinetic differences between male and female in humans and rats.
Gender differences affect the metabolism, excretion, and bioavailability of soy isoflavones in humans and rats [11] . To date, the pharmacokinetic profiles of scutellarin and scutellarein in different genders are unknown. Therefore, this study represents to investigate preliminary pharmacokinetics in male and female rats following administration of single oral and intravenous doses of scutellarin.
There are increasing benefits for scutellarin use in patients; however, problems with its analysis in biological samples have been reported in many studies. Pharmacological profiles of the parent drug and its metabolites in plasma [12,13] , tissues [14] , www.nature.com/aps Xing JF et al Acta Pharmacologica Sinica npg urine [15] , and bile [16] in humans and rats have been evaluated using high-performance liquid chromatography (HPLC) with ultraviolet detector or HPLC with mass spectrometer. After oral administration of scutellarin, only a minute amount of the compound was detected in human blood [13] , and its bioavailability was estimated to be only 10.67% [17] . However, substantial amounts of scutellarein were present in blood and urine in humans [18] . Scutellarein has similar pharmacological effects to scutellarin, but it is more potent than scutellarin. Previous studies have shown that scutellarin consistently displays double peaks in its plasma concentration-time curves [14] . Although the pharmacokinetics and metabolism of scutellarin have been extensively studied in humans and animals, the reasons for its extremely low oral bioavailability have not been elucidated. Prior to this study, there was very limited information available on the excretion dynamics of scutellarin. Most studies focused on excretion through the urine [19] with less emphasis on excretion through the feces. Scutellarin and scutellarein were found to be negligible in the urine in this present study.
Hao et al [20] asserted that the gastrointestinal tract played an important role in scutellarin's bioavailability. Accordingly, fecal excretion of scutellarin and scutellarein was investigated in this study, and this is the first report on the fecal excretion process and the pharmacokinetics in different genders of rats for these compounds. This study provides evidence that improving the formulation design of scutellarin in a drug delivery system would result in better bioavailability and efficacy. were used to process the samples.

Chromatographic conditions
An ODS-2 HYPERSIL C 18 analytical column (250 mm×4.6 mm, 5 μm) (Thermo Scientific Corporation, USA) with a C 18 guard column (4 mm×3 mm, 5 μm) (Phenomenex Corporation, Torrance, CA, USA) was used in chromotography. The mobile phase consisted of methanol and water at a ratio of 1:1 (v/v), and the pH was adjusted to 2.5 with phosphoric acid (1 mol/L). The mobile phase was delivered at a flow rate of 1 mL/min. The diode array detector was operated at 335 nm. npg samples to cover the respective calibration standard ranges. All samples were stored at 4 ºC and brought to room temperature before use.

Preparation of plasma samples
Quercetin stock solution (10 µL; 2.5 μg/mL) was transferred to a 10 mL tapered glass tube and evaporated to dryness under a stream of nitrogen. Plasma (100 μL) and phosphoric acid (50 μL; 1 mol/L) solutions were then added to the glass tube and mixed for 1 min before being extracted with acetic ether (1 mL) by vortex mixing for another 3 min. The glass tubes were then centrifuged at 3000 revolutions per minute for 10 min. The upper organic phase was transferred to a clean tube and evaporated to dryness at 35 ºC under a stream of nitrogen. The residue was immediately reconstituted in the mobile phase (100 μL) and centrifuged at 10 000 revolutions per minute at 4 ºC for 10 min. The supernatant (80 μL) was transferred into an autosampler vial, and 50 μL of it was injected into the HPLC system.

Preparation of the urine and feces samples
The blank urine and feces samples were collected from rats housed in metabolic cages. Quercetin (10 μL; 2.5 μg/mL) was transferred to a 10 mL tapered glass tube and evaporated to dryness under a stream of nitrogen. The collected urine samples were centrifuged at 10 000 revolutions per minute for 10 min, and the urine sample (200 μL) and phosphoric acid solution (100 μL; 1 mol/L) were added to the glass tube. This mixture was sonicated for 1 min, vortexed for 5 min, and centrifuged at 10 000 revolutions per minute for 10 min. Then the mixture (20 μL) was injected into the HPLC system for analysis.
The rat feces were first dried at room temperature and ground to powder with a mortar. The dry feces powder (0.2 g) was then sonicated in methanol (2 mL) for 10 min. Distilled water (1 mL) was then added to the sample, and another sonication step was performed. The samples were centrifuged at 10 000 revolutions per minute for 10 min, and the supernatant was filtered through a 0.45 μm membrane and transferred into an autosampler vial. Samples (20 μL) were injected into the HPLC system for analysis.
Mechanistic study on gender differences Liver microsomal preparation Pooled rat liver microsomes from both genders were prepared according to the method reported previously [21] . Three male and three female rats (weighing 200 to 220 g; 3-4 months old) were euthanized with CO 2 after a 24-h fasting period. The pooled, extracted liver microsomes were suspended in Tris buffer (0.1 mol/L; pH 7.4) and stored at -80 ºC before use. Rat microsomal protein contents were determined by a Nanodrop 1000.

Liver microsomal incubations
The scutellarin stock solution in dimethyl sulfoxide was added to Tris buffer (0.1 mol/L; pH 7.4) with the rat liver microsomes. The mixture was first shaken for 5 min to equilibrate in a shaking water bath at 37 ºC. Incubation was then initiated by adding a β-NADPH solution. The final concentrations of scutellarin, NADPH and the microsomal protein in the incubation mixture (1.5 mL) were 30 µmol/L, 1 mmol/L and 2 mg/mL, respectively. The percentage of dimethyl sulfoxide in the incubation mixture was kept less than 0.5% (v/v). Incubation sample mixtures (50 µL) were collected at 0, 10, 20, 30, 40, 50, 60, and 90 min. The reactions were terminated with ice-cold methanol (100 µL) to precipitate the proteins, and the samples were subsequently centrifuged at 16 000×g for 15 min. Negative controls were prepared by adding NADPH, which was followed by immediate termination using ice-cold methanol and considered the 0 min aliquots. Supernatants (50 µL) were collected and analyzed by HPLC.

Pharmacokinetic and statistical analyses
The pharmacokinetic parameters in plasma and urine were estimated using the DAS2.0 software (Drug and Statistics, version 2.0, Mathematical Pharmacology Professional Committee of China, Shanghai) [22] . Differences in the plasma scutellarin concentrations at each time point and differences in the pharmacokinetic parameters (AUC, t 1/2 , t max , C max ) in the plasma and urine between male and female rats were analyzed using ANOVA and a Student's t-test with the SSPS 11.5 software pack. P<0.05 was considered statistically significant. Data are presented as the mean±SD.

Selectivity
Six individual, blank rat plasma, urine, and feces samples were analyzed to determine if anything in the matrix interfered with the analytes. Under the optimized HPLC conditions, scutellarin, an unknown metabolite, scutellarein, and quercetin were separated chromatographically with retention times of 5.1, 6.4, 8.9, and 11 min, respectively. No interference was observed at these retention times (Figure 1).
Linearity of the calibration curves and lower limit of quantifica tion Calibration curves were constructed based on the peak area ratios of the analytes to the internal standard versus the concentrations. These were made using a weighted (1/c) liner regression analysis. Good linear relationships were established for both scutellarin and scutellarein in the plasma (0.1 mL), urine (0.2 mL), and fecal (0.2 mL) homogenates. The lower limits of quantification (LLOQ) were defined as signalto-noise ratios (S/N) greater than 10, and they were evaluated by analyzing six replicates of the biological samples spiked with scutellarin and scutellarein. The results of the calibration curves and LLOQ are summarized in Table 1.

Precision and accuracy
Intra-day accuracy and precision were evaluated by analyzing the quality control (QC) samples (n=5) at different time points in the same day. They were determined by repeated   npg scutellarein, respectively) and in the urine and feces (20.5, 5.13, and 1.28 μg/mL for scutellarin and 5.2, 1.3, and 0.32 μg/mL for scutellarein, respectively). Absolute recovery was determined by measuring the peak-area ratio of a post-extracted sample against the non-extracted samples. The results of these analyses are shown in Table 2.

Stability
Scutellarin has a structure almost identical to baicalin and is stable in acidified biological samples [23] ; however, baicalin lacks the 4'-hydroxyl group that scutellarin has. Oxidationreduction reactions, which are mediated by phenol radicals, are the major cause for the degradation of these compounds in biological samples. Previous studies have shown that acidic conditions can stabilize these flavonoids in solutions and biological samples [24] . The pH is responsible for degrading baicalin [24] but not the matrix. Therefore, in this study, scutellarin and scutellarein were stabilized by adjusting the pH to 2.5 in all samples with phosphoric acid (1 mol/L). The stability of scutellarin and scutellarein during sample storage and processing was evaluated by analyzing the QC samples. At room temperature for 8 h, the scutellarin and scutellarein concentrations in the biological samples varied by less than ±9.90%, and the responses did not vary by more than 7.74% after 24 h of storage at 4 ºC. The concentration variations were within ±11.13% of the nominal concentrations after three cycles of freezing at -20 ºC, and thawing at 20 ºC showed no significant loss of scutellarin or scutellarein. The processed samples were reconstituted in the mobile phase and placed at room temperature (25 ºC) in the autosampler for 8 h, and scutellarin and scutellarein showed very good postpreparation stabilities with RSDs less than 10.43%. When the processed samples were stored at -20 ºC for two weeks, scutellarin and scutellarein showed good stabilities at the concentrations studied with RSDs of ±11.44%.

Pharmacokinetic and excretion studies in rats Pharmacokinetics
The mean plasma concentration-time curves exhibited double peaks in both male and female rats after oral administration of scutellarin (400 mg/kg) (Figures 2 and 3). Significantly higher plasma concentrations of scutellarin were measured in females   (Figure 2). Similar results were also noted for scutellarein in female rats ( Figure 3). Significant differences for scutellarin in AUC, t max2 , and C max2 were noted between the male and female rats (P<0.05 or P<0.01) ( Table 3).

Excretion
The urinary pharmacokinetic parameters of scutellarin and suctellarein after the oral administration of 400 mg/kg scutellarin and intravenous administration of 40 mg/kg scutellarin are shown in Table 4 and Table 5, and the urinary excretion rates of scutellarin and scutellarein are shown in Figure 4 and Figure 5. The percentages of the accumulated excretion of scutellarin and scutellarein in urine and feces are shown in Figure 6-9. Table 4 and Table 5 show that the excretion percentage of scutellarein was lower than that of scutellarin. Scutellarin was excreted quickly, but scutellarein was excreted slowly from 0     (Figures 4 and 5). The elimination half-life of scutellarein was much longer than scutellarin with oral administration. The cumulative amounts of scutellarin recovered in the urine were 0.53% in male rats and 0.96% in female rats (P<0.05), and they were 0.11% and 0.02% in male and female rats, respectively, for scutellarein (P<0.05, Figure 6). After intravenous administration of scutellarin (40 mg/kg), the cumulative amounts of scutellarin recovered in the urine were 3.72% and 6.42% in male and female rats, respectively (P<0.05, Figure 7). The amounts of scutellarin and scutellarein recovered in the feces were much higher than those recovered from the urine after oral scutellarin administration ( Figure 8); however, less amounts were recovered by the intravenous route than the oral route (Figure 9). These results indicate that most of the scutellarein that was excreted in the feces was unabsorbed after oral administration of scutellarin. Although the plasma level of scutellarein was very low, a substantial amount of scutellarein was recovered in the feces after oral administration. Thus, we conclude that the bulk of scutellarin is hydrolyzed by the intestinal microflora or transformed by enzymes in the gastrointestinal tract into scutellarein, which leads to the low bioavailability. Hao et al [20] hypothesized that the site of    npg the first-pass effect was not the liver but in the gastrointestinal tract. Therefore, scutellarein could be partially excreted in the feces without being absorbed. The percentages of scutellarin and scutellarein that were cumulatively excreted were different according to gender for both oral and intravenous administrations (Figures 8 and 9).

Mechanism study on gender differences
Gender-specific rat liver microsomes were used in an in vitro metabolic study of scutellarin. These microsome experiments were performed in triplicate at eight time points between 0 and 90 min. Scutellarin was rapidly metabolized in both genders, and the starting concentrations of scutellarin that remained in female and male rat liver microsomes were 16.16%±1.02% and 8.08%±1.90%, respectively. The calculated in vitro scutellarin t 1/2 in female and male rats were 75.5±1.78 min and 75.6±0.33 min, respectively. The CL int values for scutellarin in male rats (5.56±0.11 mL·min -1 ·kg -1 ) was much higher than that in female rats (4.51±0.02 mL·min -1 ·kg -1 , P<0.01).

Discussion
Scutellarin, an unknown metabolite and scutellarein were detected simultaneously in rat plasma 5 min after oral administration of scutellarin. This indicates that scutellarin is immediately absorbed and rapidly transformed into other metabolites in vivo. Scutellarin and the unknown metabolite can be hydrolyzed completely by β-glucuronidase from bovine liver (essentially sulfatase-free) but not by sulfatase from Aerobacter aerogenes (no β-glucuronidase activity at pH 7). Huang et al [25] reported that scutellarin was hydrolyzed and transformed into scutellarein. Therefore, after hydrolysis by β-glucuronidase, the scutellarin peak disappeared, but the scutellarein peak increased. Additionally, when the unknown metabolite disappeared, another compound formed after glucuronidase hydro-lysis, and the retention time of the second unknown metabolite was 5.72 min [ Figure 1, panel B (a)]. After oral administration of scutellarin, its plasma concentrations at 20 and 40 min and at 6, 8, and 16 h were significantly higher in female than in male rats (Figure 2), and the AUC (0-t) and C max2 for scutellarin were much higher in female than in male rats. Similar results were also noted for scutellarein in female rats (Figure 3). Due to the absence of calibration standards for the unknown metabolite, its plasma concentrations could not be quantified. It is interesting to note that the plasma levels, which were estimated by the peak ratios, appeared to be higher in females than in males. Identifying the structural and pharmacological effects of the unknown metabolite warrants further investigation. The plasma concentrations of scutellarein were very sporadic and low, and they were quantified at a few time points. However, the scutellarein concentrations still appeared to be higher in female than in male rats, which was similar to the findings for scutellarin and the unknown metabolite.
After oral administration, the plasma concentration-time profile for scutellarin in rats was present as double peaks ( Figure 2). The first peak was at 0.18 and 0.43 h in male and female rats, respectively. This suggests that scutellarin is absorbed in the stomach or upper intestinal lumen and mucosa to produce the first concentration peak because the lower pH conditions favor the liposolubility of scutellarin and its absorption. The second peak emerged at 5.6 and 8.8 h in male and female rats, respectively, and its peak concentration was higher than the first peak. These results indicate that scutellarin is transformed in the inferior part of the intestines and the colon by bacterial enzymes into the corresponding aglycone, which is absorbed. The aglycone is then followed by a regioselective glucuronidation to transform scutellarin to enter the bloodstream [13] .
The observed gender effect on the pharmacokinetics of scutellarin is similar to another flavonoid, genistein [26,27] . It is suggested that these effects on genistein are due to higher CYP1A2 activity in male rats [27,28] . Similar to other flavonoids, scutellarin could also be metabolized by CYP1A2 enzymes. The involvement of CYP1A2 in the metabolism of scutellarin will be studied in the future.

Conclusion
This study demonstrates that a substantial amount of scutellarin is converted to scutellarein before absorption. The presystemic transformation of scutellarin could be partially responsible for its low bioavailability. The gender effect was identified in the pharmacokinetics of scutellarin, and the plasma levels of scutellarin were much higher in female than in male rats. The observed differences in plasma, urine, and feces levels could be due to gender-related differences in enzyme activity.