Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection

Phytomedicine 22 (2015) 487–497

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Systematic considerations for a multicomponent pharmacokinetic study of Epimedii wushanensis herba: From method establishment to pharmacokinetic marker selection Caihong Wang1, Caisheng Wu1, Jinlan Zhang∗, Ying Jin State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China

a r t i c l e

i n f o

Article history: Received 14 October 2014 Revised 9 February 2015 Accepted 17 February 2015

Keywords: Multicomponent pharmacokinetics Metabolic pathway Pharmacokinetic markers Biotransformation Prenylflavonoids Epimedium

a b s t r a c t Background: Prenylflavonoids are major active components of Epimedii wushanensis herba (EWH). The global pharmacokinetics of prenylflavonoids are unclear, as these compounds yield multiple, often unidentified metabolites. Purpose: This study successfully elucidated the pharmacokinetic profiles of EWH extract and five EWHderived prenylflavonoid monomers in rats. Study design: The study was a comprehensive analysis of metabolic pathways and pharmacokinetic markers. Methods: Major plasma compounds identified after oral administration of EWH-derived prototypes or extract included: (1) prenylflavonoid prototypes, (2) deglycosylated products, and (3) glucuronide conjugates. To select appropriate EWH-derived pharmacokinetic markers, a high performance liquid chromatography– tandem mass spectrometry (HPLC–MS/MS) method was established to simultaneously monitor 14 major compounds in unhydrolyzed plasma and 10 potential pharmacokinetic markers in hydrolyzed plasma. Results: The pharmacokinetic profiles indicated that the glucuronide conjugates of icaritin were the principle circulating metabolites and that total icaritin accounted for 99% of prenylflavonoid exposure after administration of EWH-derived materials to rats. To further investigate icaritin as a prospective pharmacokinetic marker, correlation analysis was performed between total icaritin and its glucuronide conjugates, and a strong correlation (r > 0.5) was found, indicating that total icaritin content accurately reflected changes in the exposure levels of the glucuronide conjugates over time. Therefore, icaritin is a sufficient pharmacokinetic marker for evaluating dynamic prenylflavonoid exposure levels. Next, a mathematical model was developed based on the prenylflavonoid content of EWH and the exposure levels in rats, using icaritin as the pharmacokinetic marker. This model accurately predicted exposure levels in vivo, with similar predicted vs. experimental area under the curve (AUC)0–96 h values for total icaritin (24.1 vs. 32.0 mg/L h). Conclusion: Icaritin in hydrolyzed plasma can be used as a pharmacokinetic marker to reflect prenylflavonoid exposure levels, as well as the changes over time of its glucuronide conjugates. Crown Copyright © 2015 Published by Elsevier GmbH. All rights reserved.

Introduction Herbal medicines have been employed in traditional Chinese medicine for more than a thousand years, and their use is in line with the “multiple components-multiple targets” concept of complicated diseases. They are now gaining popularity in modern medicine (Xin et al. 2011) due to the limitations of the conventional “one component-one target” concept of drug development; however, for

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1

Corresponding author. Tel.: +86 10 83154880; fax: +86 10 63017757. E-mail address: [email protected] (J. Zhang). The authors equally contributed to this research.

http://dx.doi.org/10.1016/j.phymed.2015.02.004 0944-7113/Crown Copyright © 2015 Published by Elsevier GmbH. All rights reserved.

herbal medicines, there are concerns regarding quality control (QC) and standardization of medicine formulations, the presence and proportion of active constituents, and the mechanisms of drug action (Hu et al. 2013). Multicomponent pharmacokinetics is essential for the clarification of the widespread metabolic features and pharmacological components of herbal medicines. However, herbal medicines do not lend themselves easily to the study of pharmacokinetic properties, given their structural diversity, large concentration ranges, and complex degradation dynamics in vivo, as well as the lack of authentic pharmacokinetic standards for herbal medicines (Wu et al. 2012). In light of these shortcomings of herbal medicines, this investigation used a marker compound approach to characterize the pharmacokinetics of

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herbal medicines. A critical concern for this approach is how to select and determine the most appropriate herbal constituent to act as a pharmacokinetic marker. The selected marker compound is typically a principle and pharmacologically active constituent of the herb in question that is representative of most of the herbal constituents. Nonetheless, previous pharmacokinetic studies that used the most abundant compound as a marker have been questioned because this method often does not accurately describe correlations between the levels of marker compounds and circulating metabolites; thus, the results obtained by this method may not reflect the pharmacokinetics of herbal medicines. Therefore, phytochemistry and metabolism should be integrated in the screening of marker compounds. Due to the complicated composition of herbal medicines, an array of phytochemicals could be absorbed and metabolized into more metabolites, and the metabolic pathways of dozens of metabolites are difficult to clarify. Therefore, analyses of the metabolism and pharmacokinetics of herbal medicines should start with pure compounds and then expand to multiple components. Herein, we propose a systematic and logical approach for pharmacokinetic marker selection and use Epimedii wushanensis herba (EWH) as a model herb to clarify what should be considered when determining pharmacokinetic markers of herbal medicines. EWH, the dried leaf of the aerial parts of Epimedium wushanense T.S. Ying (family Berberidaceae), is traditionally used in East Asian countries as a tonic, aphrodisiac, and antirheumatic medicine (Chinese Pharmacopoeia Commission 2010). To date, prenylflavonoids with an isopentenyl group at the 8-position (Fig. 1),

such as epimedin (Epi) A, EpiB, EpiC, baohuoside I (BaoI), and icariin, have been considered the main active components of EWH (Chen et al. 2011; Kang et al. 2012b). Recently, we developed a high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS)-based strategy to identify 115 EWH components and metabolites after oral administration of five prenylflavonoid prototypes, or EWH extracts, in rats (Jin et al. 2013). The wealth of identified components indicated that multiple EWH constituents were readily absorbed and metabolized in this animal model, which potentially contributes to the pharmacokinetics of the extract. Therefore, the present study established an HPLC–tandem mass spectrometry (MS/MS) method to explore the global pharmacokinetics of EWH through comprehensive characterization of its metabolites and after selection of potential pharmacokinetic markers. A strategy for potential marker screening is shown in Fig. 2. First, the pharmacokinetic profiles of 14 major metabolites were monitored in unhydrolyzed plasma after oral administration of EWH extract. Then, the pharmacokinetic parameters of 10 potential marker compounds were obtained in hydrolyzed plasma. Finally, a correlation analysis of the pharmacokinetic profiles of the detected compounds in the unhydrolyzed and hydrolyzed plasma was performed. One of our most important findings was that glucuronide conjugates of icaritin were the main circulating metabolites after dosing; furthermore, total icaritin content accurately reflected both systemic exposure levels and dynamic changes of prenylflavonoids in rats. Icaritin, characterized by high abundance and a strong correlation with prenylflavonoid levels (r > 0.5), could be used as a marker compound for the pharmacokinetics of EWH extract. In addition, the pharmacokinetic properties of icaritin were incorporated into an integrated mathematical model to predict the exposure level of EWH in rats. The predicted and experimental AUC (area under the curve) values showed strong agreement, further supporting the hypothesis that icaritin in hydrolyzed plasma can be used as a pharmacokinetic marker for the dynamic exposure levels of prenylflavonoids of EWH. Materials and methods Chemicals, reagents, and materials The compounds used for ex vivo analysis were as follows: EpiA, EpiB, EpiC, sagittatoside B (SagB), and BaoI, which were separated and purified by Professor Baolin Guo (Institute of Medicinal Plants Development, Chinese Academy of Medical Sciences and Peking Union Medical College), and 2 -O-rhamnosyl icariside (2 -rha-icaII), which was purified in our laboratory. The purity of these compounds determined by HPLC was >99% (Fig. S1). Sagittatoside A (SagA) and icaritin were purchased from Baoji Herbest Bio-Tech Co., Ltd. (Baoji, China). Icariside I (IcaI) was obtained from Shanghai Yuanye BioTechnology Co., Ltd. (Shanghai, China). Icariin, carbamazepine (internal standard-1 (IS-1)), and glibenclamide (internal standard-2 (IS-2)) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). In addition, the five prenylflavonoid monomers for the in vivo analysis (EpiA, EpiB, EpiC, icariin, and BaoI) were purchased from Baoji Herbest Bio-Tech Co., Ltd. The structures of the ten compounds and their metabolites are shown in Fig. 1. β -glucuronidase (3,695,000 units/g solid) was obtained from Sigma–Aldrich Co (St. Louis, MO, USA). All solvents and other chemicals used were of the highest grade available. Preparation and evaluation of EWH extracts

Fig. 1. Chemical structures of ten potential pharmacokinetic markers (compounds 1–10), 16 metabolites (M1–16), and two internal standards (IS-1 and IS-2).

Epimedium wushanense T.S. Ying were collected from Chongqing (one autonomous regions of China) and identified by Professor Baolin Guo. The dried leaf of the aerial parts (30 g) was extracted with deionized water (450 ml) by reflux for 1.5 h in a water bath at 100 °C and

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Fig. 2. The main process for pharmacokinetic marker discovery after administration of EWH extract to rats. Compounds in hydrolyzed plasma with an overwhelming proportion and strong correlation (r > 0.5) with compounds in unhydrolyzed plasma can potentially be used as pharmacokinetic markers.

filtered. The extraction was repeated twice. The pooled extract was filtered and evaporated under reduced pressure to a final volume of 60 ml and a concentration of 0.5 g/ml. EWH extract was characterized using the method described by Wu et al. (2008a) , and four peaks were identified as EpiA, EpiB, EpiC, and icariin. Detailed mass data of the four peaks of the EWH extract and the reference compounds are shown in Tables S1 and S2. The concentrations of the four main compounds (EpiA, EpiB, EpiC, and icariin) and the other minor flavonoids (2 -rha-icaII, SagB) were determined through the method described by Wu et al. (2008b) . The concentration of total flavonoids was determined using the ultraviolet–visible spectrophotometry method for Epimedii Folium described in the Chinese Pharmacopoeia Commission (2010) . Data are listed in Table S3. The resulting EWH extract was used as oral liquid, 1 ml of which contained 1 ml of aqueous extract from Epimedium wushanense T.S. Ying (family Berberidaceae), dried leaf of the aerial parts (equivalent to 0.5 g raw EWH herbal material, corresponding to 0.85 mg EpiA, 0.95 mg EpiB, 11.85 mg EpiC, 14.9 mg icariin, 1.25 mg 2 -rha-icaII and SagB, 16.45 mg total flavonoids), with water as extraction solvent. Instrumentation and HPLC–MS/MS analytical conditions The HPLC–MS/MS assay was performed on an Agilent 6410B triple quadrupole LC–MS system. Chromatographic separation was achieved using a Capcell Pak C18 MG II column (3 μm, 2.0 mm × 50 mm). The mobile phase consisted of solvent A (acetonitrile plus water (25:75, v/v) with 0.3% acetic acid) and solvent B (acetonitrile plus methanol (40:50, v/v) with 0.3% acetic acid) delivered at a flow rate of 0.4 ml/min. The gradient elution was performed with

0% solvent B from 0 to 3.5 min. The percentage of solvent B was increased starting at 3.8 min to reach 36% B at 7.0 min and 56% at 7.3 min. It was maintained at 56% until 11 min and was then increased again to reach 95% at 12 min. The percentage of solvent B was maintained at 95% until 12.5 min, was quickly returned to 0% at 12.6 min, and was maintained at 0% until 14.5 min. The column temperature was maintained at 35 °C, and the sample injection volume was 3 μl. The optimum operating parameters of the ESI interface in the positive mode were as follows: source = ESI; polarity = positive; nebulizer = 40 psi; dry gas = 9.2 L/min; dry temp = 325 °C; capillary voltage = 4000 V; and multiplier voltage (delta EMV) = 380 V. Quantification was achieved using the multiple reaction monitoring (MRM) mode with the given operating parameters (Table 1). Sample preparation For the detection of 14 major compounds in unhydrolyzed rat plasma, an IS working solution in acetonitrile (500 μl) was directly added to the unhydrolyzed plasma sample (50 μl) for precipitation. For the quantitation of 10 potential pharmacokinetic markers in hydrolyzed plasma, β -glucuronidase (20 μl, 17.5 units/μl) was freshly dissolved in acetic acid buffer (pH 4.4; 0.2 M) and added to the rat plasma sample (50 μl). The mixture was centrifuged for 3 min at room temperature (23 °C), mixed by vortexing for 30 s, and incubated at 37 °C for 3 h. Five hundred microliters of IS working solution was then added to stop the reaction. The unhydrolyzed and hydrolyzed rat plasma mixtures were thoroughly vortex-mixed for 30 s and then centrifuged for 10 min. The supernatant was dried under a stream

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C. Wang et al. / Phytomedicine 22 (2015) 487–497 Table 1 Optimized MS parameters for prenylflavonoids in hydrolyzed and unhydrolyzed rat plasma. Analytesa , b ∗

EpiA EpiC∗ EpiB∗ Icariin∗ 2 -rha-icaII∗ SagA∗ SagB∗ BaoI∗ IcaI∗ Icaritin∗ M5 M6 M7 M8 IS-1∗ IS-2∗ a b

Precursor ion m/z

MS1 Res

Product ion m/z

MS2 Res

Dwell (ms)

Fragmentor (V)

CE (eV)

838.8 822.8 808.8 676.8 660.8 676.8 646.8 514.8 531.8 368.9 691 545 545 721 236.8 494

widest widest widest widest widest widest widest widest widest widest widest widest widest widest widest widest

368.9 368.9 368.9 368.9 368.9 368.9 368.9 368.9 368.9 313 369 369 369 369 194 169

Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit Unit

35 35 35 35 35 35 35 35 35 35 20 20 20 20 35 35

150 150 150 140 120 120 120 100 110 150 120 140 140 150 120 90

40 42 40 32 15 10 10 10 20 28 34 21 21 34 18 15

Compounds monitored in hydrolyzed plasma are marked with an asterisk (∗). All compounds with and without (∗) a reference standard were monitored in unhydrolyzed plasma

of nitrogen gas. The residue was reconstituted in ethanol plus water (50 μl; 70:30, v/v) and centrifuged at 15,493 × g for 10 min at room temperature. An aliquot (3 μl) of the supernatant was injected into the HPLC–MS/MS system for analysis.

Method application and pharmacokinetic study Male Wistar rats weighing 200 ± 20 g were obtained from Vital River Laboratories (Beijing, China). Animals were maintained in an environmentally controlled breeding room for 1 week prior to the initiation of the experiment and fed standard laboratory food and water ad libitum. Prior to the experiment, the rats were fasted overnight but were provided with unrestricted access to water. All animal procedures were in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People’s Republic of China. The rats were either given EpiA, EpiB, EpiC, icariin, and BaoI at 50 mg/kg in saline or EWH extract (5 g/kg, weight of the raw EWH herbal material per unit body weight) via oral administration. The dose of EWH extract given to the rats was based on the high clinical dose recommended for humans (15–30 g/day) from “Shi Yong Zhong Yao Ci Dian” (Tian 2002). According to the dose level of EWH extract for rats (5 g/kg) and the quality control content of EpiC (>1%) by the Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia Commission 2010), the doses of the five monomers were all set as 50 mg/kg. The animals had free access to water during the experiment. Blood samples were collected from the ophthalmic venous plexus at 5 min, 15 min, 45 min, 1.5 h, 3 h, 5 h, 8 h, 12 h, 16 h, 24 h, 36 h, 48 h, 72 h, and 96 h after compound/EWH administration. Alternate eyes were used at every time point for repeated blood sampling. Plasma was obtained by centrifugation of the blood at 1721 × g for 5 min. All plasma samples were stored at −80 °C until use.

Data analysis Pharmacokinetic parameters were estimated by a noncompartmental analysis using the Drug and Statistics (DAS) 2.0 software. Kendall’s tau-b correlation coefficients were calculated using the SPSS Statistics software, version 21 (SPSS Inc., Chicago, IL, USA) and were analyzed for two-tailed significance for the correlation analysis. Correlation results were considered statistically significant at P < 0.01.

Results and discussion Comprehensive analysis of prenylflavonoid metabolic pathways and selection of potential pharmacokinetic markers As noted above, EWH is extensively metabolized, and it exhibits multicomponent absorption properties (Jin et al. 2013). Therefore, metabolic pathways were first explored after oral administration of the five prenylflavonoid prototypes in rats. All five compounds showed similar metabolism in vivo, especially with regards to deglycosylation and glucuronidation (Fig. 3). Furthermore, all five compounds were biotransformed into BaoI through deglycosylation. BaoI was then metabolized to icaritin via the loss of the rhamnose residue, and icaritin was biotransformed into M6–M8 via the conjugation of glucuronic acid at the 7-O or 3-O positions or both. In some cases, BaoI was also metabolized to M5 via the conjugation of a glucuronic acid residue at the 7-O position. In addition to deglycosylation and glucuronidation, some of the prototypes underwent demethylation and acetylation. Notably, all glucuronide conjugates were formed from deglycosylated metabolites. Therefore, deglycosylated metabolites (SagA, SagB, 2 -rha-icaII, IcaI, and icaritin) were the key metabolic intermediates bridging the prenylflavonoid prototypes and the glucuronide conjugates. Prenylflavonoid prototypes and their metabolites were detected in plasma samples after the administration of EWH extract by our previously established method (Jin et al. 2013). Among the metabolites detected, four glucuronide conjugates (M5–M8) were the most abundant, while the five prototypes (EpiA, EpiB, EpiC, icariin, and BaoI) and five deglycosylated products (SagA, SagB, 2 -rha-icaII, IcaI, and icaritin) were relatively scarce. In contrast to the above mentioned 14 compounds, the levels of the demethylated and acetylated products (M1, M2, M4, M9, M10, and M13–M15) were so low (Fig. S2) that they were not considered further. Thus, this study focused on the pharmacokinetic profiles of EpiA, EpiB, EpiC, icariin, BaoI, SagA, SagB, 2 -rha-icaII, IcaI, icaritin, and M5–M8 and on the deglycosylation and glucuronidation pathways in the metabolism of components of EWH extract. Because glucuronide-conjugated metabolites are difficult to prepare for quantification, their corresponding deglycosylated metabolites, which are generated by β -glucuronidase, were employed as prospective pharmacokinetic markers to profile the pharmacokinetics of the glucuronide conjugates. Thus, ten prenylflavonoids were selected as potential pharmacokinetic markers in hydrolyzed plasma: the five prototypes and the five deglycosylated metabolites.

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Fig. 3. Metabolic pathways of prenylflavonoids. Deglycosylated metabolites and their respective glucuronide conjugates are shown in pink rectangles, and demethylated and acetylated metabolites are shown in blue rectangles. Metabolites with no obvious correlation are shown in yellow rectangles. The “−glc” label refers to the loss of glycoside, and the “+glA” label refers to the conjugation of glucuronic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Establishment of the HPLC–MS/MS method Optimization of mobile phase and MS parameters Optimization of the MS parameters for the ESI source under the positive ion mode was performed early in the development of this method. Pure compounds were used to select the ion pairs and to optimize the MS conditions. For metabolites without a reference standard (M5–M8), MRM ion pairs were set according to fragmentation patterns (Jin et al. 2013), and detection parameters were optimized by comparing different settings in the MRM mode. The optimized MS parameters are shown in Table 1. HPLC conditions were optimized for efficient separation of the 14 major compounds. According to our previous work (Wu et al. 2011), a rapid and straightforward separation method was established by using acetonitrile plus water with 0.3% acetic acid as the mobile phase. Good separation was achieved for some of the prenylflavonoid glycosides (e.g., EpiA, EpiB, EpiC, and icariin), but the matrix effect of icaritin was >50%. The addition of methanol to the mobile phase markedly reduced the icaritin matrix effect, and the combination of methanol plus acetonitrile at a proportion of 5:4 (v/v) rendered the matrix effect acceptable (98–101%) by attenuating the elution capacity of the mobile phase. However, icariin and EpiC, which were eluted from 0 to 3.5 min, yielded overlapping peaks in the presence of methanol, and under these conditions, EpiC produces the same ion pair as icariin (677/369). The EpiC contribution to the icariin peak was nearly 10% of the injected icariin concentration (Fig. S3). To explore this phenomenon, product ion scans of m/z 838.8 (EpiA), 808.8 (EpiB), 823.8 (EpiC), and 676.8 (icariin) were performed. Fig. S4 shows that all of these four precursor ions produced the same dominant product ions of m/z 677, 531, and 369, corresponding to icariin, IcaI, and icaritin. This result suggested that EpiA-C has the ability to fragment within the source into an icariin precursor ion via the loss of glucose, xylose, and rhamnose. This discovered cross-talk could not be reduced by optimizing the mass parameters; thus, the only way to reduce the

cross-talk was efficient chromatographic separation of EpiA-C from icariin. As described in this section, acetonitrile plus water with acetic acid, which effectively separated EpiA-C from icariin, was used as the mobile phase. Collectively, to overcome EpiA-C/icariin cross-talk, a ternary mobile phase (acetic acid, water, and acetonitrile) was used to improve the resolution from 0 to 3.5 min. At the same time, a quaternary mobile phase consisting of acetic acid, water, acetonitrile, and methanol was used to decrease the matrix effects of icaritin from 3.5 to 12.5 min (Fig. 4A). As a result, a quaternary gradient separation effect was achieved using the binary liquid pumps through the optimization of the composition and mixing time of the mobile phases A and B. Based on a previous report (Liang et al. 2010), segmented MRM was used to shorten the duty cycle and increase the sensitivity of this method (Fig. S5). Different responses were observed between the two MRM segments, and two internal standards were chosen for two different segments (Fig. 4B). The individual MRM chromatograms are shown in Fig. S6. Evaluation of phospholipid contribution to icaritin matrix effects As noted in the previous section, the matrix effect of icaritin was markedly reduced with the optimization of the mobile phase. Therefore, we explored the cause of the matrix effects of icaritin. Matrix effects are generally described as the impact of co-eluting constituents from the matrix on the ionization of a specific analyte (Trufelli et al. 2011). Phospholipids are the main group of endogenous components known to precipitate matrix effects (Little et al. 2006). Little et al. (2006) monitored glycerophosphocholine-containing phospholipids (GPChos) using high-energy, in-source collision-induced dissociation to yield m/z 184 ions. Additional phospholipid classes (phosphatidic acid-containing phospholipids (PAs) and phosphatidyl glycerolcontaining phospholipids (PGs)) were monitored using the MRM mode. The late retention time of icaritin in the present experiment suggests that co-elution of phospholipids most likely instigated the

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Fig. 4. Representative MRM chromatograms. Chromatograms are shown for (A) ten prenylflavonoids (40 ng/ml) in plasma, and (B) blank rat plasma spiked with IS-1 (2 ng/ml) and IS-2 (80 ng/ml). Compounds 1–10 represent EpiA, EpiB, EpiC, icariin, SagA, IcaI, SagB, 2 -rha-icaII, BaoI, and icaritin.

icaritin matrix effect, which was >50%. Therefore, a resolution analysis was performed for the icaritin and phospholipid peaks to validate our supposition. Accordingly, the precursor ion scan mode was used to monitor GPChos, and the MRM mode was used to monitor representative PA and PG. Detailed MS parameters are described in Section 4 of the supplementary material. The addition of methanol to acetonitrile (5:4, v/v) for the mobile phase resulted in good resolution of the analytes from the phospholipids (Fig. 5A), while the mobile phase of pure acetonitrile eluted the analytes together with the phospholipids (Fig. 5B). In combination with the method validation data, these results indicated that the observed icaritin matrix effects were indeed largely due to phospholipids. Quantitation of total prenylflavonoids in plasma by enzyme hydrolysis As described above, glucuronide-conjugated metabolites were profiled using β -glucuronidase-mediated hydrolysis of their corresponding deglycosylated metabolites. The optimum condition for the stability of the free prenylflavonoid glycosides was an enzyme concentration of 5 U/μl in acetate buffer solution (pH 4.4; 0.2 M), and no glucuronide conjugates were detected after enzyme digestion (see supplementary material). Validation of the quantitation method The established methods were validated using the US Food and Drug Administration (FDA) guidelines (U.S. Department of Health and Human Services 2013). The calibration curves (Table S4) covered wide ranges (80-fold) for all analytes and showed good correlation coefficients (r > 0.997). The matrix effects were determined by comparing the peak responses of the post-extraction spiked samples with those of the pure standards prepared in ethanol plus water (70:30, v/v) and were found to be negligible under the conditions used in these experiments. The ratios of the peak responses were within acceptable

ranges (95.5–105%) (Table S5), and the matrix effects of the two ISs were 92.6% and 95.5%. The extraction efficiency was determined by comparing the peak responses of the spiked samples extracted as QC samples with those of the post-extraction spiked samples and ranged from 75.2% to 100% (Table S3), and the extraction efficiencies of the two ISs were 86.3% and 90.7%. Intra- and inter-batch precision and accuracy values for each analyte are listed in Table S6. The relative standard deviation (RSD) values of the intra-batch and inter-batch values were less than 12.6% and 14.7%, respectively. The stability of the analytes was evaluated by the analysis of QC samples at three concentrations (Table S7). Analytes maintained under the conditions of this study were stable. Pharmacokinetic profiles of 14 major compounds in unhydrolyzed plasma after oral administration of five prenylflavonoid prototypes or EWH extract Plasma prenylflavonoids and glucuronide conjugates of deglycosylated metabolites (M5–M8) were detected after the oral administration of the five prenylflavonoid prototypes or EWH extract in rats. The concentration–time curves are shown in Fig. S7, and the pharmacokinetic parameters of the detected compounds are summarized in Tables S8 and S9. Two distinct pharmacokinetic patterns for the detected compounds emerged an early phase, wherein the Tmax (time after drug administration to reach maximum plasma concentration) values of five compounds (EpiA, EpiB, EpiC, BaoI, and M5, the glucuronide conjugate of BaoI) were all <1 h, and a late phase, wherein the icaritin-derived glucuronide conjugates (M6–M8) reached their highest concentrations at 8–16 h. After oral administration of the five prenylflavonoid prototypes, M6–M8 were the most abundant plasma compounds and exhibited the highest AUC0–96 h values, followed by M5, EpiA, EpiB, EpiC, and BaoI (Fig. S8). The other 6/14 compounds

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Fig. 5. Representative total ion current chromatograms for blank rat plasma spiked with a standard mixture of ten prenylflavonoids. (A) Mobile phase B = acetonitrile plus methanol (acetonitrile-to-methanol ratio = 40:50, v/v). (B) Mobile phase B = acetonitrile alone. The red lines in (A) and (B) represent each type of lipid (PA, PG) detected using MRM mode; the green lines represent total GPChos detected using precursor ion scan mode; and the black lines represent the standard mixture of ten prenylflavonoids. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(icariin, SagA, SagB, 2 -rha-icaII, IcaI, and icaritin) were detected at only one or two time points due to their low concentrations. In addition, peak areas were also utilized to calculate the AUC percentage and the relative distribution of each detected compound in the plasma after oral administration of EWH extract. The relative distributions of M6 and M7 were 78.5% and 21.2%, respectively, and those for EpiA-C, BaoI, and M5 were all <0.3% (Fig. 2). In contrast to the results obtained after administration of the five prenylflavonoid prototypes, the diglucuronide conjugate of icaritin (M8) was not detected after the administration of EWH extract. Collectively, the current findings indicate that M6 and M7, the icaritin glucuronide conjugates, were the major coexisting metabolites of the prenylflavonoid prototypes and EWH extract in unhydrolyzed plasma. Pharmacokinetic profiles of ten potential pharmacokinetic markers in hydrolyzed plasma The HPLC–MS/MS method was used to determine the pharmacokinetic profiles of the ten potential pharmacokinetic markers in hydrolyzed plasma after administration of five prenylflavonoid monomers or EWH extract. The concentration–time curves after administration of the five prenylflavonoid prototypes are shown in Fig. 6A. Among the five administered prototypes, icariin alone was not observed in the hydrolyzed plasma, but its deglycosylated metabolite, BaoI, was detected. BaoI exhibited two peaks after icariin administration to rats, one at 0.25 h and the other at 5 h (Fig. 6A). These peaks were similar to those observed after administration of BaoI itself and are consistent with previous reports (Chen et al. 2011; Liu et al. 2000; Zhao et al. 2010), suggesting that icariin is rapidly metabolized to BaoI through the loss of a glucose moiety at the 7-O position and, in turn, BaoI is rapidly reabsorbed by the intestine. The concentration of

BaoI in hydrolyzed plasma at 0.25 h after icariin administration was twice that after BaoI administration, which might explain the poor solubility of BaoI in saline. The time–concentration curves of EpiA, EpiB, and EpiC exhibited peaks at 0.75, 0.08, and 0.08 h, respectively, with Cmax (maximum concentration after dosing) values of 19.4, 8.1, and 4.2 ng/ml, respectively (Fig. 6A). The divergent circulating concentrations of the five prototypes might be due to the presence of different efflux transporters, such as breast cancer resistance protein or multidrug resistanceassociated protein, or to different permeability properties of the prototypes (Chen et al. 2008, 2014). Deglycosylated metabolites (SagA, SagB, and 2 -rha-icaII) produced by direct hydrolysis of prenylflavonoid triglycosides (i.e., EpiA, EpiB, and EpiC) at the 7-O position were only detected at certain time points. This suggests that prenylflavonoid diglycosides (i.e., icariin) are degraded more readily at the 7-O position than triglycosides, and this in agreement with earlier findings (Chen et al. 2008, 2014). Small amounts of the deglycosylated metabolite (IcaI) derived from hydrolysis of prenylflavonoid monomers at the 3-O position were also detected at only one or two time points. Among the five active prenylflavonoid prototypes (EpiA, EpiB, EpiC, icariin, and BaoI) and the five deglycosylated metabolites (SagA, SagB, 2 -rha-icaII, IcaI, and icaritin), icaritin was the only compound to exist in hydrolyzed plasma after the administration of the five prototypes. Icaritin exhibited a significantly higher exposure level than the other nine compounds in hydrolyzed plasma. The concentration– time profiles of icaritin in hydrolyzed plasma after oral dosing of the five prototypes are depicted in Fig. 6B, and the pharmacokinetic parameters of total icaritin are summarized in Table 2. The total icaritin concentration exhibited appropriate pharmacokinetic properties, with Cmax values of 120–840 ng/ml and Tmax values of 8–18 h. The

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Fig. 6. Mean plasma concentration–time profiles. Profiles are shown after administration of (A) and (B) prenylflavonoids or (C) and (D) EWH extract in hydrolyzed plasma (n = 6 male rats). (A) and (C) Concentration–time profiles of prototype prenylflavonoids. (B) and (D) Concentration–time profiles of icaritin.

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Table 2 Pharmacokinetic parameters for prenylflavonoids in hydrolyzed plasma after oral administration of various compounds. Administrated prenylflavonoids

Pharmacokinetic parameter

Measuring unit

Mean ± SDa , P (prototype )

Mean ± SD, I (icaritin)

EpiA

AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax

mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L

0.05 ± 0.09 ± 0.39 ± 0.030 ± 0.03 ± 0.05 ± 0.08 0.005 0.02 ± 0.05 ± 0.08 0.004 0.12 ± 0.17 ± 1.80 ± 0.028 ± 0.11 ± 0.22 ± 5.08 ± 0.024 164 ± 165 ± 4.35 ± 13.5 ±

19.7 22.5 9.46 0.840 2.28 2.96 18.0 0.121 6.22 10.0 16.0 0.420 8.64 8.86 10.7 0.821 5.88 8.24 8.19 0.538 164 165 4.35 13.5

EpiB

EpiC

Icariinb

BaoI

Icaritin

0.02 0.04 0.29 0.018 0.02 0.05

0.01 0.05

0.05 0.12 2.47 0.008 0.02 0.10 4.54 29.5 29.2 2.69 4.47

± ± ± ± ± ± ± ± ± ±

2.80 13.3 10.5 0.321 1.12 1.33 4.90 0.072 5.54 7.70

± ± ± ± ± ± ± ± ± ± ± ± ±

0.401 2.96 2.90 2.07 0.359 5.46 6.00 5.87 0.393 29.5 29.2 2.69 4.47

I/Pc 394

76.0

311

72.0

53.4

1

Values are given as the means ± the SD (n = 6 male rats). Detection of the icariin prototype was conducted by using BaoI. P represents the AUC0–96 h of parental prenylflavonoids; I represents the AUC0–96 h of icaritin; and I/P represents the ratio between icaritin and the parental prenylflavonoids. a

b c

Table 3 Pharmacokinetic parameters for prenylflavonoids in hydrolyzed plasma after oral administration of EWH extract. Administrated prenylflavonoids

Pharmacokinetic parameter

Measuring unit

Mean ± SDa

EpiA

AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax AUC(0−t) AUC(0−) Tmax Cmax

mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L mg/L h mg/L h h mg/L

0.06 ± 0.01 0.29 ± 0.17 1.62 ± 2.02 0.003 ± 0.004 0.01 ± 0.01 0.02 ± 0.01 1.43 ± 1.31 0.004 ± 0.005 0.09 ± 0.03 0.12 ± 0.05 0.33 ± 0.32 0.008 ± 0.009 0.08 ± 0.02 0.20 ± 0.12 0.93 ± 1.99 0.011 ± 0.008 32.0 ± 17.2 32.4 ± 17.1 24.7 ± 12.8 1.54 ± 1.07

EpiB

EpiC

BaoI

Icaritin

a

Values represent the means ± the SD (n = 6 male rats).

AUC0–96 h ratio between icaritin (I) and parental prenylflavonoids (P), with BaoI used to represent the parental form of icariin, was 53– 394 (Table 2), indicating that the exposure level of total icaritin was nearly 50-fold higher than that of the five prototypes and the other four deglycosylated metabolites. Notably, icaritin was not detected in unhydrolyzed plasma. Enzyme hydrolysis was next applied to study the pharmacokinetics of EWH extract. Fig. 6C and Table 3 show the concentration–time profiles and pharmacokinetic parameters of EWH extract. EpiA, EpiB, EpiC, and BaoI concentrations in hydrolyzed plasma reached 3–10 ng/ml within 0.3–1.6 h after EWH extract administration. Total icaritin exhibited two peaks at 16 and 36 h and reached concentrations

of 1165 and 1168 ng/ml, respectively (Fig. 6D). Icaritin still accounted for the overwhelming majority of plasma prenylflavonoid compounds after hydrolysis, and its AUC percentage was 99.2% (Fig. 2). Pharmacokinetic profiles of the five prototypes and EWH extract in both hydrolyzed and unhydrolyzed plasma again indicated that the glucuronide conjugates of icaritin were the main circulating metabolites. Previous studies using parental prenylflavonoid forms as pharmacokinetic markers (Wu at al. 2011) were able to determine the pharmacokinetic properties of the prototypes, but not the systemic exposure levels of the circulating metabolites. Our work demonstrates that icaritin in hydrolyzed plasma and icaritin-derived glucuronide conjugates in unhydrolyzed plasma are all suitable marker compounds that can accurately reflect changes in systemic exposure levels over time after the oral dosing of prenylflavonoids. Compared with icaritinderived glucuronide conjugates, icaritin in hydrolyzed plasma possesses certain merits as a pharmacokinetic marker because it exists in the hydrolyzed plasma of rats after the administration of five different prototypes and because its abundance and availability allow it to be easily quantified. Therefore, total icaritin is proposed as a pharmacokinetic marker to substantiate rat systemic exposure levels to five prenylflavonoids and EWH extract. Collectively, our analyses reduced the number of potential pharmacokinetic markers in hydrolyzed plasma to five: four prototypes with rapid-elimination (EpiA, EpiB, EpiC, and BaoI), representative of intestinal absorption, and one icaritin, whose elimination profile was representative of systemic prenylflavonoid exposure. Correlation analysis of exposure level changes over time between major compounds in unhydrolyzed plasma and potential pharmacokinetic markers in hydrolyzed plasma As described in the previous section, ten potential pharmacokinetic markers were condensed to four prototypes (EpiA, EpiB, EpiC, and BaoI) and one deglycosylated metabolite (icaritin) in hydrolyzed plasma. Next, we explored whether these five potential pharmacokinetic markers could accurately reflect the real concentration changes

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Fig. 7. Correlation analysis between potential pharmacokinetic markers in the plasma with enzyme hydrolysis and major compounds in plasma without enzyme hydrolysis at 14 different time points. Points in different colors and shapes represent different rats. (A)–(E) n = 2; (F) n = 4; t = total. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

over time of prenylflavonoid prototypes and metabolites in unhydrolyzed plasma. Thus, a correlation analysis was performed between the concentration of potential pharmacokinetic markers in hydrolyzed plasma and the intensity of major metabolites in unhydrolyzed plasma at each investigated time point (Fig. 7). EpiA served as a representative triglycoside in the correlation analysis. EpiA levels in hydrolyzed plasma exhibited a strong positive correlation with EpiA levels in unhydrolyzed plasma (Kendall’s tau-b coefficient r = 0.771, p < 0.01, n = 28) and a poor correlation with M5–M8 levels in unhydrolyzed plasma (r < 0.270, p > 0.05, n = 28). However, good correlation existed between total icaritin and its glucuronide-conjugated metabolites, M6–M8, in unhydrolyzed plasma (r > 0.524, p < 0.01, n = 28) (Fig. 7A). Similar results were obtained after oral administration of EpiB (Fig. 7B) and EpiC (Fig. 7C). Icariin served as a representative of diglycosides, but because icariin is rapidly biotransformed into BaoI, the latter was used for correlation analysis. Total BaoI levels showed good correlation with free BaoI levels (r = 0.519, p < 0.01, n = 28) and M5 levels in unhydrolyzed plasma (r = 0.725, p < 0.01, n = 28) but poor correlations with the icaritin glucuronide conjugates, M6–M8 (r < 0.249, p > 0.05, n = 28). The level of icaritin in hydrolyzed plasma changed in parallel with the levels of M6–M8 in unhydrolyzed plasma (r > 0.707, p < 0.01, n = 28) (Fig. 7D). Similar results were obtained after oral administration of BaoI (Fig. 7E). Thus, we conclude that the pharmacokinetics of the main metabolites in the prenylflavonoid metabolic pathway (Fig. 3) can be profiled by potential pharmacokinetic markers in hydrolyzed plasma.

The exposure levels of five prototype prenylflavonoids, with BaoI level representing icariin level, in hydrolyzed rat plasma only indicated their corresponding exposure levels in unhydrolyzed rat plasma. In contrast, the exposure level of icaritin in hydrolyzed rat plasma could indicate the exposure levels of multiple glucuronide conjugates in unhydrolyzed rat plasma after the administration of the five prenylflavonoid monomers in rats. Similar correlation results were obtained after the oral administration of EWH extract (Fig. 7F). Given the overwhelming abundance, the coexistence in hydrolyzed plasma after the administration of EWH-derived materials, and the correlation of multiple icaritin metabolites, icaritin attracted more attention than EpiA, EpiB, EpiC, and BaoI as a potential pharmacokinetic marker. The epimedin compounds were more suitable as prodrugs, as they undergo intestinal hydrolysis by intestinal enzymes and microflora, have low intrinsic permeability, and undergo active efflux transport (Chen et al. 2008 2014). Hence, icaritin is most likely the major prenylflavonoid form absorbed into the body and then undergoes extensive glucuronide conjugation. Icaritin glucuronide conjugates in the plasma show weak estrogen-like activity (Wong et al. 2009), but their activity is enhanced if they are deconjugated to icaritin in target tissues. Icaritin exhibits many pharmacological effects both in vitro and in vivo, including enhancement of osteoblastic differentiation, suppression of osteoclastic differentiation, and stimulation of pro-estrogenic/anti-estrogenic activities (Huang et al. 2007a,b; Kang et al. 2012a,b; Wong et al. 2009). Icaritin also interacts more strongly with the estrogen receptor

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Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.02.004. References

Fig. 8. Mean plasma concentration–time profiles of icaritin in hydrolyzed plasma after administration of icaritin (n = 6 male rats).

in vitro than EpiA, EpiB, EpiC, or icariin (Kang et al. 2012a), although in vivo studies have revealed that icariin and icaritin can both significantly increase the duration of the estrus cycle (Kang et al. 2012b). Nonetheless, our findings support the selection of icaritin rather than the parental prenylflavonoid forms as a pharmacokinetic marker. To further investigate the pharmacokinetics of icaritin, the compound was administered to rats at a concentration of 50 mg/kg, and its pharmacokinetic profile in hydrolyzed plasma was compared with that of five prenylflavonoid glycosides. The pharmacokinetic parameters of icaritin are summarized in Table 2, and its concentration–time curves are presented in Fig. 8. The AUC0–96 h of total icaritin after its administration was more than eight-fold higher than the corresponding values after administration of the prenylflavonoid glycosides. Meanwhile, the time–concentration curve after administration of icaritin exhibited only one peak at 4.35 h, indicating a lower degree of hydrolysis by intestinal enzymes/microflora and higher permeability than the other glycosides. Construction of a mathematical prediction model for exposure levels of EWH extract To construct the prediction model for EWH extract exposure levels, an integrated pharmacokinetic model was established based on the exposure levels of prenylflavonoid constituents in vivo (calculated by icaritin, the pharmacokinetic marker) and prenylflavonoid content in EWH extract. This model used the AUC0–96 h of total icaritin after the administration of the five prenylflavonoid glycosides (50 mg/kg) as constants and their respective contents in EWH extract as variances. The equation was as follows:

Predicted AUCtotal icaritin =



AUCj0−96 h × Cj

In this equation, j represents total icaritin after administration of EpiA, EpiB, EpiC, icariin, or BaoI, and Cj represents the parental drug content for each prototype in EWH extract (g/100 g). The predicted AUC0–96 h value for total icaritin exposure level (24.1 mg/L h) was close to the experimental value (32.0 mg/L h), indicating that the prediction model can comprehensively anticipate the exposure level of EWH extract and other natural medicines. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgment We thank the National Mega-Project for Innovative Drugs (2012ZX09301002-001) for their financial support of this work.

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