Metabolite Profiling, Pharmacokinetics, and In Vitro Glucuronidation of Icaritin in Rats by Ultra-Performance Liquid Chromatography Coupled with Mass Spectrometry

Icaritin is a naturally bioactive flavonoid with several significant effects. This study aimed to clarify the metabolite profiling, pharmacokinetics, and glucuronidation of icaritin in rats. An ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) assay was developed and validated for qualitative and quantitative analysis of icaritin. Glucuronidation rates were determined by incubating icaritin with uridine diphosphate glucuronic acid- (UDPGA-) supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. A total of 30 metabolites were identified or tentatively characterized in rat biosamples based on retention times and characteristic fragmentations, following proposed metabolic pathway which was summarized. Additionally, the pharmacokinetics parameters were investigated after oral administration of icaritin. Moreover, icaritin glucuronidation in rat liver microsomes was efficient with CLint (the intrinsic clearance) values of 1.12 and 1.56 mL/min/mg for icaritin-3-O-glucuronide and icaritin-7-O-glucuronide, respectively. Similarly, the CLint values of icaritin-3-O-glucuronide and icaritin-7-O-glucuronide in rat intestine microsomes (RIM) were 1.45 and 0.86 mL/min/mg, respectively. Taken altogether, dehydrogenation at isopentenyl group and glycosylation and glucuronidation at the aglycone were main biotransformation process in vivo. The general tendency was that icaritin was transformed to glucuronide conjugates to be excreted from rat organism. In conclusion, these results would improve our understanding of metabolic fate of icaritin in vivo.


Introduction
Herba Epimedii, the dried aerial parts of Epimedium L. (Berberidaceae), are a widely used Chinese medicine for impotence, bone loss, and cardiovascular diseases [1][2][3]. Prenylflavonoids are reported to be a group of major active constituents present in Epimedium for the antioxidative stress, anti-inflammatory, antitumor, and antiosteoporosis activities [4][5][6][7][8]. Icaritin is the common aglycone with many biological effects, especially antiosteoporosis activities [5,7]. Besides, icaritin could induce cell death in activated hepatic stellate cells through mitochondrial activated apoptosis and ameliorate the development of liver fibrosis in rats [9]. Meanwhile, icaritin is able to target androgen receptor and androgen receptor COOH-terminal truncated splice variants, to inhibit androgen receptor signaling and tumor growth with no apparent toxicity [10]. Additionally, icaritin has neuroprotective effects against MPP + -induced toxicity in MES23.5 cells. IGF-I receptor mediated activation of PI3K/Akt and MEK/ERK1/2 pathways are involved in the neuroprotective effects of icaritin against MPP + -induced neuronal damage [11]. Recently, icaritin had been shown as a potential agent for the treatment of systemic lupus erythematosus [12]. These biological activities above had stimulated increasing interests in the in vivo metabolism of icaritin or its related prenylflavonoids. Poor bioavailability of prenylated flavonoids results from their poor intrinsic permeation and transporter-mediated efflux by the human intestinal Caco-2 model and the perfused rat intestinal model [13]. Meanwhile, it is shown that Epimedium flavonoids could be hydrolyzed into secondary glycosides or aglycone by intestinal flora or enzymes, thereby enhancing their absorption and antiosteoporosis activity [14]. So far, numerous researches of total prenylflavonoids or individual flavonoid had been conducted in the fields of in vivo metabolites profiling, biliary excretion, and pharmacokinetics [15][16][17][18][19]. Generally, the in vivo 2 Journal of Analytical Methods in Chemistry metabolism of Herba Epimedii extracts or its prenylflavonoids could easily be metabolized in gastrointestinal tract following deglycosylation reaction. Additionally, icaritin was easily metabolized into glucuronidation conjugates to be preferentially eliminated and excreted from rat organism [16,18,20]. Though the data on metabolic researches of icaritin abounds, its metabolic profile is not so clear. It is essential to systematically characterize the in vivo metabolites in order to better understand its mechanism of action. Hence, the present study aimed to conduct the metabolites screening, quantitative determination, and in vitro glucuronidation of icaritin.
Recently, liquid chromatography coupled with mass spectrometry (LC-MS) had been widely introduced to rapidly screen trace components in biological samples [21,22]. In this study, icaritin-related metabolites were analyzed based on characteristic fragmentation by UPLC-MS after oral administration. Meanwhile, possible disposing pathway of icaritin was proposed. Furthermore, a UPLC-MS method was developed and applied to perform the pharmacokinetics of icaritin. Moreover, glucuronidation rates were determined by incubating icaritin with uridine diphosphate glucuronic acid-(UDPGA-) supplemented rat liver microsomes (RLM) and rat intestine microsomes (RIM). Kinetic parameters were derived by appropriate model fitting. Icaritin was subjected to significant hepatic and gastrointestinal glucuronidation.

Animals.
Male Sprague-Dawley rats (180∼220) g were provided by Guangdong Medical Laboratory Animal Center. The rats were kept in an animal room at constant temperature (24 ± 2) ∘ C and humidity (60 ± 5)% with 12 h of light/dark per day and free access to water and food. The animal protocols were approved and conducted in accordance with the guidelines of Laboratory Animal Ethics Committee of Zhengzhou University.

Samples Collection and Preparation for Qualitative Analysis.
After the rats were fasted for 12 h with free access to water before experiments, icaritin dissolved in 0.3% sodium carboxymethyl cellulose solution was orally administrated to rats at a dose of 100 mg/kg. Blood samples were collected from external jugular vein into heparinized tubes and were separated by centrifuging at 13800 for 10 min at 4 ∘ C, respectively. Bile samples were collected and recorded during 0-24 h period after an abdominal incision anesthetized with 10% aqueous chloral hydrate. The urine and feces samples were collected separately during 0-24 h period after oral administration. Small intestinal samples were obtained after oral administration for 24 h. All blank samples were obtained in the same way.
Before experiments, all biosamples were stored at −20 ∘ C. In this work, solid phase extraction method was applied to pretreat all samples. Before use, C18 columns (3 cm 3 , 60 mg) were first preconditioned and equilibrated with 3 mL of methanol and 3 mL of water, respectively. Urine samples were evaporated and concentrated at 40 ∘ C under reduced pressure. Feces samples and small intestinal samples were dried in air and stirred into powder. And then they were treated with an ultrasonic bath for 30 min. The filtrate was combined and evaporated to dryness at 40 ∘ C in vacuum. The residue was reconstituted with water. Plasma, urine, bile, feces, and small intestinal samples were loaded on pretreated columns. The residue was reconstituted in 200 L of 60% methanol and filtered through a 0.22 m membrane until injection.

Samples Preparation for Quantitative Analysis.
Plasma sample (200 L) was treated with methanol (1.2 mL), after which the mixture was vortex-mixed for 30 s and centrifuged at 13800 for 10 min at 4 ∘ C. The supernatant was then transferred and evaporated to dryness using N 2 at room temperature. The residue was dissolved in 200 L of 60% methanol and was then injected into the UPLC-MS system. 2.6. Glucuronidation Assay. Icaritin was incubated with RLM and RIM to determine the rates of glucuronidation as published references previously [23]. Briefly, the incubation mixture mainly contained 50 mM Tris-hydrochloric acid buffer (pH = 7.4), 0.88 mM MgCl 2 , 22 g/mL alamethicin, 4.4 mM saccharolactone, and 3.5 mM UDPGA. The reaction was terminated by adding ice-cold acetonitrile. The samples were vortexed and centrifuged at 13800 for 10 min. The supernatant was subjected to UPLC-MS analysis. All experiments were performed in triplicate.

UPLC-MS Conditions.
UPLC was performed using an ACQUITY6 UPLC system (Waters, Milford, MA, USA). Separation was achieved on a Waters BEH C18 column (1.7 m, 2.1 × 50 mm) maintained at 35 ∘ C. The mobile phase consisted of water (A) and acetonitrile (B) (both containing 0.1% formic acid), and the flow rate was 0.5 mL/min. The gradient elution program was as follows: 0 min, 15% B; 3 min 35% B; 7 min 60% B; 8 min 100% B. An aliquot of 4 L sample was then injected into the UPLC-MS system.
The UPLC system was coupled to a Waters Xevo TQD (Waters, Milford, MA, USA) with electrospray ionization.
The operating parameters were as follows: capillary voltage, 2.5 kV (ESI+); sample cone voltage, 30.0 V; extraction cone voltage, 4.0 V; source temperature, 100 ∘ C; desolvation temperature, 300 ∘ C; and desolvation gas flow, 800 L/h. The method employed lock spray with leucine enkephalin (m/z 556.2771 in positive ion mode and m/z 554.2615 in negative ion mode) to ensure mass accuracy.

Pharmacokinetic Application.
After fasting with free access to water for 12 h, icaritin was given to rats as a dosage of 100 mg/kg. Plasma samples were then obtained at 0.083, 0.25, 0  (1) and (2), respectively. Appropriate models were selected by visual inspection of the Eadie-Hofstee plot [24]. Model fitting and parameter estimation were performed by Graphpad Prism V5 software (San Diego, CA).
The parameters were as follows. is the formation rate of product. max is the maximal velocity. m is the Michaelis constant and [ ] is the substrate concentration. si is the substrate inhibition constant. The intrinsic clearance (CL int ) was derived by max / m for Michaelis-Menten and substrate inhibition models. (2)

Fragment Pattern of Icaritin.
As had already been reported in the previous study [16],

Screening of Metabolites.
On the basis of MS/MS fragmentation pattern, the metabolites were deduced, clarifying the general metabolism in vivo. The extracted ion chromatograms (EICs) of prototype (M0) and metabolites (M1∼ M30) were shown in Figure 1, while the individual EICs of M1∼M30 were exhibited in Figure S2. The UV, MS, and MS/MS data of M0∼M30 were all exhibited in Table 1.  Figure  S1b), which indicated that M15 and M23 were the hydration products at isopentene group of icaritin and agreed with previous study of icariin [18]. .0721 were attributed to obvious loss of CH 3 (15.0235 Da) and C 4 H 6 (54.0470 Da) group, respectively, which indicated that the dehydrogenation position was at isopentene group [18].     Figure S1g) were tentatively characterized as the glucose glycosylation conjugate of M20. These glycosylation reactions were the same as the metabolism of epimedin C in rats reported in reference (Liu et al., 2011). By comparing with references, M14, M17, and M21 were identified as epimedin C, icariside I, and icariside II, respectively. And the MS/MS spectra of M17, M19, and M20 were shown in Figures S1h-S1j, respectively.

Method Validation.
The method was validated for specificity, linearity, extraction recovery, matrix effects, precision, accuracy, and stability according to the US Food Drug Administration guidelines for bioanalytical method validation [25].
Specificity was determined by comparing the chromatograms obtained for six blank plasma samples, blank plasma samples spiked with standard solutions at LLOQ concentrations, and drug plasma samples obtained 4 h after oral administration. As shown in Figure S3, no interference peaks were detected at the retention times of icaritin.
The LOD and LOQ were calculated as 3-fold and 10-fold of the ratio of signal-to-noise, respectively. The LLOQ was defined as the lowest concentration in the calibration curve with accuracy of 80∼120% and precision of 20%. Calibration curves were acquired by plotting peak area ( ) versus respective plasma concentrations ( ) using a 1/ 2 weighting factor and linear least-squares regression analysis. A series of standard solutions were used to generate calibration curve. The correlation coefficients ( 2 ) of calibration curves were greater than 0.9926 within 2.0∼512.0 ng/mL and LLOQ was 2.0 ng/mL. The regression equations, correlation coefficients, and LLOQ were shown in Table S1.
The experiments to evaluate matrix effect and recovery were conducted by the protocol [26]. According to the protocol, the peak areas from QC samples at three concentrations were defined as A1; those from extracted control plasma reconstituted with standard solutions at 4.0, 64.0, and 256.0 ng/mL were A2. The responses of icaritin found by direct injection of the corresponding pure reference standards at three QC levels were A3. The matrix effect and recovery were calculated as follows: matrix effect (%) = A2/A3 × 100%. Recovery (%) = A1/A2 × 100%. The results (as shown in Table S2) illustrated that matrix effect was between 89.1% and 113.5%, and the recovery was from 96.3% to 102.7%.
The accuracy and inter/intraday precision of the method were evaluated by determining six replicates of QC samples on three consecutive days. The measured concentrations of QC samples were determined with a calibration curve obtained on the same day. Relative error and relative standard deviation were used to describe accuracy and inter/intraday precision, respectively. They both should not exceed 15%. As exhibited in Table S3, the intraday and interday precision were less than 13.2% and 10.2%, respectively, while the intraday and interday precision of LLOQ were no more than 17.4% and 15.6%, respectively.
Stability of icaritin in rat plasma was assessed under different conditions at three concentration levels, including extracted samples for 12 h at room temperature, kept at −20 ∘ C for 60 h, three cycles of freezing at −20 ∘ C and thawing at 25 ∘ C, and plasma sample at room temperature for 8 h. Each was compared by three QC replicates of the same concentration with a calibration curve in the same day. The RE was within 13.8% and RSD was less than 11.3%. Stability results (Table  S4) indicated that icaritin were stable under different storage conditions.

Pharmacokinetics Application.
The mean concentrationtime profiles of these bioactive components were shown in Figure 2. The main pharmacokinetic parameters were illustrated in Table 2. In this study, max was (294.5 ± 22.7) ng/mL Journal of Analytical Methods in Chemistry    Table 3.

Discussion
Normally, only the prototypes or metabolites in blood with a high enough exposure in target organs for a finite period of time are considered as potential effective components for therapeutic benefits [30]. In this study, M0, M1, and M13 were the main xenobiotics in plasma (Figure 1(a)), which may be the potential in vivo effective components directly. After circulation, M2, M5, M13, M23, and M28 were passed out with the urine (Figure 1(b)).
Due to poor oral bioavailability, several components were limited to be absorbed in blood. But they could influence intestinal dysfunction to exert efficacy by their prototypes, secondary metabolites, or finally the aglycone in intestinal  tract [31]. Massive metabolites containing M6, M8, M13, M17, M25, M28, and M30 were detected in rat feces and small intestinal samples (Figure 1(d)). Moreover, icaritin underwent phase II metabolism by main conjugating enzymes including UDP-glucuronosyltransferases (UGTs) to produce extensive mono-or diglucuronic acid conjugates. In rat bile, M3, M6, M13, M18, and M24 mainly were biotransformed in rat liver and excreted into bile (Figure 1(c)). Characterization of icaritin glucuronidation assumed a great role in the understanding of its pharmacokinetics and bioavailability. Oral bioavailability is a major factor in determining the biological actions of icaritin in vivo following oral administration of the compound [32]. This study suggested that the oral bioavailability of icaritin would be influenced by first-pass glucuronidation in the liver. The glucuronidation activity was obtained by kinetic profiling and modeling. Kinetic profiling required the determination of the rates of icaritin glucuronidation at a series of icaritin concentrations. The relative activities of RLM and RIM toward icaritin glucuronidation were evaluated by the derived CL int values ( Table 3). Use of CL int (= max / m ) as an indicator of enzymes activity was advantageous, because (1) CL int represents the catalytic efficiency of the enzyme and is independent of the substrate concentration; (2) compared with other kinetic parameters such as m and max , CL int is more relevant in an attempt to predict hepatic clearance in vivo [33]. Therefore, CL int values were used to determine icaritin glucuronidation activity in this study.
Based on the metabolite profiles, the metabolic pathways of icaritin were proposed and shown in Figure 4(a), and the metabolic sites were shown in Figure 4(b). In summary, icaritin was hard to be absorbed into the rat blood. In small intestine, icaritin could form flavonoid glycoside by the sequential glycosylation metabolism. Meanwhile, icaritin could easily conjugate with a glucuronic acid to form phase II metabolites in liver, which indicated that the biliary clearance was one of the major routes of excretion. Phase I metabolism of icaritin mainly included demethylation, dehydrogenation, and hydration. The general tendency was that the saponins were metabolized and transformed into the high polar metabolites to be eliminated and excreted from the rat organism.

Conclusion
As a result, a total of 30 metabolites were identified or tentatively characterized based on the retention time behaviors and fragmentation patterns. Dehydrogenation at isopentenyl group and glycosylation and glucuronidation at the flavonoid aglycone were the main biotransformation process of icaritin in vivo. Meanwhile, a validated method was successfully applied to a pharmacokinetic study. Moreover, icaritin glucuronidation in RLM was efficient with CL int values of 1. 12   study could provide an experimental basis to understand the metabolic fate of icaritin in rat.