Pharmacokinetics and Biliary Excretion of Fisetin in Rats - Journal of

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Bioactive Constituents, Metabolites, and Functions

Pharmacokinetics and Biliary Excretion of Fisetin in Rats Miao-Chan Huang, Thomas Y. Hsueh, Yung-Yi Cheng, Lie-Chwen Lin, and Tung-Hu Tsai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00917 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Journal of Agricultural and Food Chemistry

ABSTRACT

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The hypothesis of this study is that fisetin and phase II conjugated forms of fisetin may partly

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undergo biliary excretion. To investigate this hypothesis, the male Sprague-Dawley rats were used

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for the experiment and the bile duct was cannulated with polyethylene tubes for bile sampling. The

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pharmacokinetic results demonstrated that the average area under the curve (AUC) ratios (k % =

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AUCconjugate / AUCfree-form) of fisetin, its glucuronides, and its sulfates were 1:6:21 in plasma and

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1:4:75 in bile, respectively. Particularly, the sulfated metabolites were the main forms that underwent

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biliary excretion. The biliary excretion rate (kBE % = AUCbile / AUCplasma) indicates the amount of

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fisetin eliminated by biliary excretion. The biliary excretion rates of fisetin, its glucuronide

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conjugates and its sulfate conjugates were approximately 144, 109 and 823%, respectively, after

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fisetin administration (30 mg/kg, i.v.). Besides, the biliary excretion of fisetin is mediated by

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p-glycoprotein.

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Keywords: fisetin; biliary excretion; p-glycoprotein; sulfation; phase II conjugation; glucuronidation;

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polyhydroxy flavonoids.

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INTRODUCTION

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Flavonoids are a group of polyphenolic compounds that provide plants with defense and promote the

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attraction of pollinators1,2 and that benefit human health. One series of flavonoids contains several

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hydroxyl groups at the core structure; these polyhydroxy flavonoids include quercetin, fisetin,

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kaempferol, luteolin, apigenin, and chrysin. Polyhydroxy flavonoids have been reported to have

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multiple

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anti-neurodegeneration

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(3,3’,4’,7-tetrahydroxyflavone; Figure 1), is ubiquitous in a wide variety of plants and common

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fruits3,6, such as apple, strawberry, grape, and onion. Kimiria et al. reported that the daily Japanese

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diet includes approximately 0.4 mg/day fisetin7. Due to its multiple bioactive properties, fisetin is

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now considered a health-promoting factor8. Fisetin contributes to improving human health, so

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numerous commercial food supplements containing fisetin are marketed. From the perspective of the

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structure-activity relationship (SAR) contributing to its multiple bioactivities, the 3-OH group of

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fisetin mostly contributes to antioxidative activity, and the 3’-OH and 4’-OH groups and the double

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bond between the 2- and 3-carbon are capable of enhancing antioxidation activity9. Fisetin is

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potentially able to reduce inflammation because of the 4-keto and 7-OH groups at its core structure10.

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Sagara demonstrated that fisetin is neuroprotective and capable of stimulating the differentiation of

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nerve cells via the phosphorylation of ERKs, with greater potency than quercetin, luteolin, and

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isorhamnetin11. Several studies were then launched on the potential therapeutic effects of fisetin

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against neurodegenerative diseases such as Alzheimer's disease12, Parkinson’s disease13 and

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Huntington’s disease14. In addition, fisetin exerts antineoplastic activity against prostate cancer15 and

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lung cancer16, as documented in mouse studies.

bioactivities,

including activity3-5.

antioxidation, Moreover,

one

anti-inflammatory, polyhydroxy

anticancer, flavonoid,

and fisetin

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The resulting in vivo pharmacologic effects of a drug depend on its pharmacokinetics, namely its

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absorption, distribution, metabolism, and excretion (ADME) profile. An important pharmacokinetic 3


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study revealed that fisetin is absorbed rapidly and goes through extensively phase II conjugation via

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sulfation and glucuronidation17. Furthermore, in a tissue distribution study of fisetin in mice, fisetin

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levels were highest in kidney, followed by intestine and liver18. Moreover, the multiple-peak

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phenomenon is also found in other flavonols such as quercetin19 and kaempferol20, and their

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conjugated flavonols are probably eliminated through biliary excretion21,22, which is the

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best-discussed mechanism of reabsorption. Among the factors critical for drug reabsorption, drug

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characteristics and drug efflux transporters are considered to dominate in enterohepatic circulation23.

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Various factors contribute to the ability to process a compound by biliary extraction, such as

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chemical structural characteristics, molecular weight, lipophilicity, and polarity. Shapiro and Ling’s

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report demonstrated that P-glycoprotein (P-gp) contains two drug transport binding sites, including

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the Hoechst 33342 affinity site (H-site) and the rhodamine 123 affinity site (R-site)24. Most P-gp

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substrates can be classified into two types based on their preferential interaction site; for example,

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quercetin and colchicine preferentially interact with the H-site24. However, Loo and Clark’s study

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reported that quercetin is an R-site substrate. Moreover, the literature indicates that flavonols with 3-

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and 5-substitution of hydroxy groups and a 2,3-double bond probably have higher binding affinity to

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P-gp25. In the absence of these essential functional groups, a 4’-methoxy group slightly enhances the

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binding affinity, which is slightly decreased by a 4’-hydroxy group25. To sum up, fisetin contains a

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3-hydroxyl group and a 2,3-double bond, implying it certainly has high binding affinity to P-gp. In

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addition to chemical structural characteristics of ingested molecules, other features critically related

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to biliary extraction are molecular weight, lipophilicity, and polarity26,27. The molecular weight of

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fisetin is 286.24 g/mole, which is within the molecular weight cutoff for biliary excretion in rats28.

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The octanol-water partition coefficient (log P, a parameter characterizing lipophilicity) of fisetin is

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1.97, as estimated by the free online software molinspiration that can predict the log P value,

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indicating that fisetin is a lipophilic molecule in a neutral environment. Based on the structural

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characteristics, molecular weight, lipophilicity, and double-peak phenomenon, fisetin could undergo 4



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biliary excretion and be modulated by P-gp because of its high affinity for P-gp.

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After reading the above reports and surveying the literature on the biliary excretion of fisetin in the

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PubMed and Reaxys databases, no related documents were identified. Here, we hypothesized that

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fisetin and its conjugates metabolism might undergo biliary excretion similar to other polyhydroxy

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flavonoids. To clarify this hypothesis, the aims of our study were as follows: (1) to develop a

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high-performance liquid chromatography coupled with photodiode array (HPLC-PDA) method for

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analyzing fisetin levels in plasma and bile; (2) to ascertain the pharmacokinetics of fisetin and its

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phase II metabolites in plasma and bile; and (3) to investigate the role of P-gp on the biliary

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excretion of fisetin through concurrent treatment with the p-glycoprotein inhibitor cyclosporin A

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(CsA).

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MATERIALS AND METHODS

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Reagents and Materials

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Fisetin (3,3’,4’,7-tetrahydroxyflavone), quercetin (internal standard, IS), L-ascorbic acid, heparin,

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urethane, acetic acid, polyethylene glycol (PEG) 400, β-glucuronidase (type B-1 from bovine liver)

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and sulfatase (type H-1 from Helix pomatia) were purchased from Sigma-Aldrich (St. Louis, MO,

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USA). Cyclosporine (CsA; Sandimmune) was purchased from Novartis (Basel, Switzerland).

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Sodium acetate anhydrous, formic acid and methanol were obtained from E. Merck (Darmstadt,

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Germany). Ethyl acetate was purchased from Macron Fine Chemicals (PA, USA). Triple-deionized

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water was prepared by the Milli-Q system (Millipore, Billerica, MA, USA). All solvents used were

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liquid chromatography grade.

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Liquid Chromatography Conditions

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Liquid chromatography was carried out on a Shimadzu (Kyoto, Japan) HPLC system equipped with

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an LC-20AT pump, a SIL-20AC autosampler, and an SPD-M20A PDA detector. Separation was

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achieved on a reversed-phase Diamonsil plus C18 column (150 × 4.6 mm, five µm; Dikma, China)

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using isocratic elution with a mobile phase of 55% solvent A (0.1% formic acid in water) and 45%

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solvent B (0.1% formic acid in methanol). The detection wavelength for fisetin and quercetin (IS)

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was at 360 nm. The flow rate was set at 0.8 mL/min, and the injection volume was 10 µL.

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Calibration Curves and Quality Control (QC) Samples

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A standard stock solution of fisetin was prepared by dissolving 1 mg of fisetin in 1 mL of methanol

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(1 mg/mL). The quercetin stock solution was prepared by dissolving 2 mg of quercetin in 1 mL of

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methanol (2 mg/mL); the stock solutions were stored at -20 °C. For calibration curves, a series of

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working solutions was prepared by spiking aliquots of fisetin stock solution into blank matrices

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(methanol, plasma, and bile) at a final concentration of 0.5, 1, 5, 10, and 50 µg/mL with 20 µg/mL

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quercetin. Working solutions of QC samples at low, medium, and high concentrations (0.5, 5 and 50

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µg/mL) were prepared in the same manner.

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Sample Preparation

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Enzymatic hydrolysis and Quantification of glucuronide/sulfate conjugates of fisetin in plasma and

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bile/liquid-liquid extraction for the biological samples

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To quantify the glucuronide/sulfate conjugates of fisetin, both plasma and bile samples underwent

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enzymatic hydrolysis prior to further sample preparation with a modified reported method17. Briefly, 6



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a 50-µL aliquot of plasma or sample was mixed with the same volume of β-glucuronidase (1000

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units/mL in acetate buffer, pH 5.0) or sulfatase (1000 units/mL in acetate buffer, pH 5.0) and 25 µL

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of ascorbic acid (100 mg/mL). Then, the samples were incubated in a 37 °C water bath for one hour

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under anaerobic conditions, immediately placed on ice to terminate the hydrolysis reaction, and

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acidified with 25 µL of 0.1% formic acid in water. After the samples were spiked with 250 µL of

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ethyl acetate and vortexed for 5 minutes, they were centrifuged at 9,000 × g for 10 minutes at 4 °C.

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Next, the ethyl acetate layer was transferred to another propylene tube, and this extraction procedure

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was done twice before the ethyl acetate layer of the samples was evaporated under N2. Before

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analysis, the samples were reconstituted with 45 µL of methanol and 5 µL of quercetin (200 µg/mL)

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and filtered through a 0.22 µm filter (Merck, Darmstadt, Germany). The bile samples were prepared

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in the same manner. To obtain the concentration of fisetin conjugates, the concentration of free-form

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fisetin before hydrolysis is used as baseline for correction, and the baseline-corrected concentration

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were calculated using the following formula: Cconjugate = (Cfree-form·post-hydrolysis + Cfree-form). The duration

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of enzymetic hydrolysis was determined in a pilot experiment. The collected 5-min biosamples

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including plasma and bile were incubated with or without glucuronidases or sulfatases at 37 °C for 0,

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0.5, 1 or 2 hours. Briefly, the treated plasma samples were completely hydrolyzed after incubating

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with two enzymes for half an hour. For the bile samples, the sulfatase hydrolysis reached maximum

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concentration of total free-form fisetin after treating half hour, and hydrolyzed with glucuronidase

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took one hour to reach the reaction balance. Based on our primary experiment, the incubated duration

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of all biosamples were one hour.

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Extraction of fisetin in plasma and bile

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All samples were prepared in a similar manner; approximately 50 µL of plasma or bile samples was

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mixed with 50 µL of acetate buffer to simulate the enzyme solution. Then, this sample solution was

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combined with 25 µL of ascorbic acid solution, 25 µL of water containing 0.1% formic acid and 250 7


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µL of ethyl acetate; the resulting mixture was vortexed for 5 minutes and then centrifuged at 9,000 ×

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g for 10 minutes at 4 °C. The evaporation and reconstitution procedures were the same as those for

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the samples of fisetin conjugates. The plasma samples were diluted 10-fold prior to glucuronidase

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and sulfatase hydrolysis, while the sample incubated without enzyme was diluted 5-fold. The bile

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samples were diluted 50-fold prior to glucuronidase and sulfatase hydrolysis, and the sample

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incubated without enzyme was diluted 10-fold.

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Method Validation

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The advanced analysis method in our experiment was fully validated regarding linearity, precision,

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accuracy, extraction recovery and stability according to US Food and Drug Administration

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guidance29.

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Linearity

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The plasma and bile calibration curves were generated by plotting the peak area ratio of analyte to IS

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against the nominal concentrations. The linearity was assessed using weighted (1/x2) linear

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least-squares regression analysis, and the correlation coefficient (r2) should be greater than 0.995.

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The signal to noise ratio (S/N) of the limit of detection (LOD) was defined as greater than 3, and that

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of the lowest limit of quantification (LLOQ) was defined as greater than 10. The accuracy and

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precision of each calibration sample should be within ± 15%, except the LLOQ, which should be

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within ± 20%.

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Accuracy and precision

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The accuracy and precision were evaluated by analyzing six replicates of QC samples on the same 8



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day (intra-day) and on six different days (inter-day). The accuracy is presented as the percent bias to

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show the degree to which observed concentration deviated from the nominal concentration [Bias% =

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(Cobs – Cnom) / Cnom]. The precision was calculated by dividing the standard deviation (S.D.) by the

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observed concentration, and this value is expressed as the percent relative standard deviation (R.S.D.)

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[R.S.D.% = S.D./Cobs]. The acceptable criteria for both accuracy and precision are ±15% (±20% for

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LLOQ).

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Extraction recovery

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The extraction recovery was assessed as the ratio of the peak area of fisetin spiked into matrices to

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that of fisetin spiked into methanol using three replicates of QC samples.

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Stability

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To evaluate analyte stability in matrices during the experiment, the observed concentrations were

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compared using QC samples before and after handling under different conditions: autosampler (12

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hours at 10 °C), short-term (4 hours at room temperature), long-term (14 days at -20 °C), and three

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free-thaw cycles (freeze at -20 °C for 24 hours and then thaw at room temperature).

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Experimental animals

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All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC)

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of National Yang-Ming University, Taipei, Taiwan (IACUC Approval No: 1050201). Male specific

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pathogen-free Sprague-Dawley rats (220-260 g) were obtained from the Laboratory Animal Center

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of the National Yang-Ming University, Taipei, Taiwan. The animals were housed in an

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environmentally controlled room with 12:12-hour light-dark cycle and had free access to food 9


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(Laboratory Rodent Diet 5001, PMI Feeds, Richmond, IN, USA) and water.

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Pharmacokinetic Study

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The male Sprague-Dawley rats were anesthetized with urethane (1 g/kg; 1 mL/kg; i.p.) and remained

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anesthetized throughout the experimental period. Polyethylene tubes (PE-50) were implanted in the

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jugular vein for blood sampling and in the femoral vein for dosing. The bile duct was impacted with

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two polyethylene tubes (PE-10) for bile sampling, with one to the liver and the other to the

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duodenum, and the two tubes could be connected with an adapter to maintain bile flow while not

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sampling.

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Fisetin (30 mg/kg) dissolved in PEG 400 was administered via the femoral vein by i.v. bolus

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injection for the control group (n = 6). The CsA-treated group (n = 6) received an i.v. injection of

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CsA (20 mg/kg) via the femoral vein 10 minutes before fisetin administration.

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In the control group, serial blood samples (200 µL) were collected into heparin-rinsed polypropylene

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tubes at 5, 15, 30, 45, 60, 75, 90, 120 and 180 minutes after dosing, and bile was sampled at 0-10,

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10-20, 25-35, 40-50, 55-65, 70-80, 85-95, 115-125 and 175-185 minutes after fisetin dosing; blood

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and bile were collected in the CsA-treated group until 240 minutes. After sampling, the bile samples

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were tightly sealed with parafilm and frozen at -20 °C. The blood samples were centrifuged at 9,000

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× g for 10 minutes at 4 °C, and the separated plasma samples were tightly sealed with parafilm and

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frozen at -20 °C until analysis.

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Data Analysis

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The pharmacokinetic parameters were determined using WinNonlin (version 1.1; Scientific 10



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Consulting Inc., Apex, NC, USA), and the compartment model of fisetin in plasma was selected

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based on Akaike’s Information Criterion (AIC). Statistics were processed on SigmaPlot (Systat

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Software, London, UK), and comparisons were made using one-way ANOVA. Significance was

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defined as p < 0.05. Data are presented as the mean ± standard deviation (S.D.).

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RESULTS AND DISCUSSION

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Method validation

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The optimized HPLC-PDA method was validated for analysis of free-form fisetin and resulted in

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good linearity (r2 > 0.995) ranging from 0.5 – 50 µg/mL, and the LLOQ for fisetin was 0.5 µg/mL in

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both plasma and bile. The LOD was 0.05 µg/mL in plasma and 0.1 µg/mL in bile. The extraction

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recoveries (%) for the low, mid and high concentration QC samples were 85.04 ± 3.34, 86.49 ± 2.14

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and 85.41 ± 0.40 in rat plasma and 77.82 ± 3.45, 76.03 ± 3.20 and 77.53 ± 1.95 in rat bile,

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respectively. The method validation results are summarized in Supplementary Tables S1 and S2. The

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selectivity of the developed method toward fisetin in rat plasma and bile was evaluated by comparing

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the chromatograms of blank matrices with those of blank matrices spiked with standard samples or

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with biosamples collected after fisetin dosing. Endogenous components in rat plasma and bile did not

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interfere with the fisetin and quercetin signals, indicating good selectivity, and the chromatographs

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are shown in Supplementary Figures S1 and S2.

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The intra-day accuracy ranged from -8.71% to 18.37% in plasma and from -4.21% to 8.46% in bile,

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and the intra-day precision was within 5% in plasma and bile. The inter-day accuracy was within 8%

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in plasma and bile, and the inter-day precision ranged from 0.56% to 15.82% in plasma and from

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0.26% to 12.34% in bile. The results are summarized in Supplementary Table S3. The developed

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method and processes satisfy the consistency criteria for quantification on the same day and on 11


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different days. The inter- and intra-day accuracy and precision of fisetin in rat plasma and bile are in

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the acceptable range, that is, the developed method has adequate repeatability for detecting fisetin in

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the matrices.

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Stability tests were conducted to exam the stability of fisetin in the matrix before and after sample

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preparation under various circumstances. The results are summarized in Supplementary Table S4.

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The stability of both plasma and bile QC samples in the autosampler was within 11% and met the

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criteria of the US-FDA. After short-term storage (room temperature for 4 hours), the change in fisetin

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concentration ranged from -79.19% to -47.53% in plasma and from -96.59% to 19.13% in bile. The

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stability after short-term storage at 0 °C (on ice) for 1 hour was within 12% in plasma and bile.

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Therefore, in the short-term storage, fisetin is more stable in plasma than in bile, and its stability at 0

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°C (on ice) for an hour is in the acceptable range. After three freeze-thaw cycles, the change in fisetin

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concentration was -17.34% to 8.41% in plasma and -37.95% to -1.19% in bile. Long-term storage

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changed the fisetin concentration from -39.20% to -21.07% in plasma and from -49.44% to 18.51%

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in bile.

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The significant changes in QC samples might be attributed to the strong antioxidative activity of

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fisetin. Wang et al.30 revealed that fisetin is unstable in aqueous basic solutions at temperatures

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higher than 37 °C, but the proteins present in matrices might inhibit fisetin degradation and thus

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stabilize it in aqueous solution. In our pilot study (data not shown), biological samples were placed at

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room temperature for 0.5, 1 and 2 hours before sample preparation or processed immediately; there

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was no significant change in fisetin concentration in the samples, indicating that fisetin is relatively

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stable in biological samples for 2 hours at ambient temperature. The variations in fisetin stability in

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QC samples and biological samples might be related to the use of methanol as the solvent in the

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working solution; methanol, a common solvent used in protein precipitation, can denature proteins in

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matrices31 and may have reduced the stability of fisetin. After recognizing that fisetin was unstable 12



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under the operating conditions, the collected biological samples were processed within an hour, and

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the entire preparation process was performed on ice to minimize the decomposition of fisetin.

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Preliminary Identification of Unidentified Fisetin Metabolites

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An unidentified signal peak was observed in the HPLC chromatograph while analyzing the processed

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samples with or without enzymatic hydrolysis by fisetin glucuronidases or sulfatases. As shown in

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Supplementary Figures S3 and S4, peak 3 was present behind the internal standard, quercetin,

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indicating that it was probably more lipophilic than quercetin or fisetin. For preliminary

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identification, this peak was collected and roughly scanned by UPLC-MS/MS (Waters, Milford, MA,

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USA) in a range from 100 to 500 Daltons in positive mode. Three significant ion peaks were

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observed in the full mass spectrum scan, but only the peak at m/z 299 was stable enough for further

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analysis. The selected signal was fragmentized with a collision energy of 20 eV, resulting in three

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major mass fragments at m/z 136.99, 286.07 and 301.10 (Supplementary Figure S5a). The molecular

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weight of the unidentified compound was 300 Daltons and could be cleaved into two different

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product ions, 286.07 and 136.99 (Supplementary Figure S5b). In accordance with the characteristic

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fragmentation pattern of flavonoids32, the systematic CO2 loss at the C-ring C-O bond and 3-4 single

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bond32 produces a crucial product ion, m/z 136.99, as shown in Supplementary Figure S6. Neutral

305

loss fragments are important clues for identifying the probable structure of the selected target. For

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example, neutral loss fragments are widely used to clarify phase II conjugation metabolites; the loss

307

of 176 Daltons is considered to indicate a glucuronidated metabolite, the loss of 80 Daltons implies a

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sulfated metabolite, and the loss of 14 Daltons commonly suggests methylation33,34. The mass change

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of the parent compound (m/z 300) and the fragment ion (m/z 286.07) was 14 Daltons, suggesting that

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fisetin has two methylated metabolites and is methylated at 3’-OH or 4’-OH. Compared with the

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results of Touil’s study18, we demonstrated that fisetin probably produces two methylated metabolites

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and a series of conjugated metabolites. 13


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Pharmacokinetic Study

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Samples were analyzed as concentration-time profiles, as illustrated in Figures 2 and 3. After the

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administration of fisetin (30 mg/kg, i.v.), the concentration of fisetin in both plasma and bile

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decreased rapidly within 30 minutes, after which fisetin continuously diminished within 60 minutes

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until the levels below the LLOQ level. Fisetin glucuronides and sulfates appeared within 15 minutes

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after dosing, but their levels declined relatively slowly and continuously above the LLOQ level until

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the last sampling time point in the control group. This phenomenon of the blood concentration of

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fisetin rapidly decreasing and being relatively lower than that of its phase II conjugated metabolites

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is similar to the metabolism of luteolin35, probably caused by extensive first-pass metabolism in the

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liver and intestine35.

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WinNonlin software was employed to calculate the pharmacokinetic parameters summarized in Table

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1 based on the concentrations at serial sampling time points. When comparing the AIC of the one-

328

and two-compartment models, the two-compartment model was adequate for describing the

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pharmacokinetics of fisetin in plasma because of the lower AIC. The pharmacokinetic parameters of

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fisetin in bile and of its conjugated products in plasma and bile were calculated with the

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non-compartment model. Besides, to avoid the effect of gastrointestinal oral first pass effect, the

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fisetin (30 mg/kg) was given intravenously. The average area under the curve (AUC) values of fisetin,

333

its glucuronides, and its sulfates in plasma were 275.9, 1719, and 6429 µg min/mL, respectively, and

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the ratio was 1:6:21. Moreover, the average AUC values of fisetin, its glucuronides, and its sulfates

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in bile were 402.5, 1810 and 29170 µg min/mL, respectively, and the ratio was 1:4:75. Interestingly,

336

the AUC of fisetin sulfates in bile was five-fold that in plasma.

337 338

The biotransformation rate (k %) was estimated by the ratio of the AUC of the conjugated compound 14



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to that of the free-form compound using the following formula: k % = AUCconjugate / AUCfree-form.

340

Therefore, the biotransformation rates (shown in Table 2) for fisetin glucuronides in plasma and bile

341

were 641.7 ± 215.4% and 599 ± 467.8%, implying that the difference in fisetin plasma and biliary

342

biotransformation is not significant. On the other hand, the biotransformation rates for fisetin sulfates

343

were 2601 ± 2539% and 8795 ± 4304% in plasma and bile; the biliary transformation rate of fisetin

344

sulfate was significantly higher than that in plasma. In addition to the biotransformation rate, the

345

biliary excretion rate is a vital factor that should be considered.

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The biliary excretion rate (kBE %) indicates the amount of a drug excreted from circulation into bile,

348

and the formula is kBE % = AUCbile / AUCplasma. The biliary excretion rates of fisetin, its glucuronide

349

and its sulfate were approximately 144%, 109%, and 823%, respectively. Taken together, the half-life

350

of the free-form of fisetin was shorter than that of its glucuronides and sulfates whether in plasma or

351

bile, which reveals that free fisetin is rapidly transformed into its major conjugated form through

352

phase II metabolism in the liver, in agreement with the findings of Shia17. The fact that fisetin, its

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glucuronides and its sulfates immediately enter the bile explains the double-peak phenomenon.

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Briefly, fisetin sulfate was the most predominant metabolic product in both plasma and bile because

355

it was abundantly produced and extensively excreted into bile. This observation matched those of

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Mullen19, who reported that major metabolites of quercetin were sulfated and glucuronidated

357

conjugates, although methylated quercetin was also observed. Also, the rapid and extensive

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metabolism and biliary excretion contribute to the low bioavailability of phenolics previously

359

discussed in a recent study36. In conclusion, the main metabolic pathway of fisetin is phase II

360

conjugation, which is consistent with the metabolism of other flavonoids37,38.

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Some ATP-binding cassette transporters on apical membranes dominate biliary excretion39,40. To

363

better understand the mechanism of biliary excretion, the P-gp inhibitor cyclosporin A (CsA) was

364

used to block P-gp function and thus clarify the impact of P-gp on the biliary excretion of fisetin. 15


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365

Prior exposure to CsA (20 mg/kg, i.v.) inhibited P-gp and increased the levels of fisetin, its

366

glucuronides and its sulfates. Fisetin was detectable for 75 minutes in plasma (Figure 2) because of

367

the prolonged half-life. Based on the results shown in Table 1, the AUC of the free-form of fisetin

368

increased in plasma but decreased in bile, suggesting that the biliary excretion of the free-form of

369

fisetin was inhibited by P-gp. Additionally, the same phenomenon was observed regarding the biliary

370

excretion of fisetin glucuronides.

371 372

As aforementioned, the lower biliary excretion rate of fisetin and fisetin glucuronides in the

373

CsA-treated group compared with the control group indicates that fisetin and its glucuronides might

374

be substrates of P-gp. Indeed, the inhibition of P-gp might result in the obstruction of biliary

375

excretion of fisetin and its glucuronides, thus reducing the AUC of fisetin and its glucuronides.

376

Fisetin and its glucuronides might be retained in plasma and partly metabolized into glucuronides

377

and sulfates. The higher AUC of fisetin sulfates in bile than in plasma suggests that sulfated fisetin

378

might enter bile by means other than P-gp-mediated transport. In addition, a higher distribution

379

volume and lower clearance of fisetin in plasma were found in the CsA-treated group compared with

380

the control group. P-gp is expressed in some organs, such as the liver, kidney, brain and intestine41.

381

The organ distribution of fisetin in mice mainly involves the liver, intestine, and kidney18; therefore,

382

P-gp suppression might block not only the excretion of fisetin into bile but also its renal excretion

383

and distribution among other pathways, thus reducing the clearance of fisetin in plasma.

384 385

In conclusion, an HPLC-PDA method capable of determining fisetin in rat plasma and bile was

386

developed and successfully applied to investigate the pharmacokinetics and biliary excretion of

387

fisetin, its glucuronides and its sulfates. Accordingly, fisetin was metabolized into sulfated and

388

glucuronidated products, and fisetin sulfates were the predominant metabolites in both plasma and

389

bile. Although the glucuronidation and sulfation of fisetin represent the primary metabolic pathways,

390

fisetin can be methylated at 3'-OH or 4'-OH and subsequently undergo glucuronidation or sulfation. 16



391

Moreover, the role of P-gp in the biliary excretion of fisetin was ascertained by pre-treatment with

392

CsA. Our finding suggests that P-gp modulates the biliary excretion of fisetin and its glucuronides,

393

while the biliary excretion of fisetin sulfates might occur via alternative mechanisms. Consequently,

394

fisetin metabolites in bile and plasma would be metabolized into sulfates or glucuronides or remain

395

in the mono-methylated form before undergoing biliary excretion or distribution in the blood; these

396

metabolic patterns are presented in Figure 4.

397 398

ABBREVIATIONS USED

399

AIC, Akaike’s information criterion; ATP, adenosine triphosphate; AUC, area under the

400

concentration−time curve; Cmax, maximum plasma concentration; Cl, clearance; CsA, cyclosporine A;

401

HPLC-PDA, high-performance liquid chromatography-photodiode array detection; IS, internal

402

standard; LLOQ, lower limit of quantification; OH, hydroxyl; P-gp, p-glycoprotein; R.S.D., relative

403

standard deviation.

404 405

ACKNOWLEDGMENTS

406

We appreciate the PK Lab members who assisted and supported this study.

407 408

SUPPORTING INFORMATION

409

The method validation results for fisetin in plasma and bile and the MS2 spectra of unidentified

410

fisetin metabolites are provided. These materials are available free of charge at http://pubs.acs.org.

411 412

REFERENCES

413

1. Bennett, R.N.; Wallsgrove, R.M. Secondary metabolites in plant defence mechanisms. New

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Phytol. 1994, 127, 617-633.

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2. Harborne, J. B.; Grayer, R. N. The anthocyanins. The flavonoids: advances in research since

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39. Kalliokoski, A, Niemi, M. Impact of OATP transporters on pharmacokinetics. Br J Pharmacol 2009, 158, 693-705. 40. Hennessy, M.; Spiers, J. A primer on the mechanics of P-glycoprotein the multidrug transporter. Pharmacol Res 2007, 55, 1-15. 41. Thiebaut, F.; Tsuruo, T.; Hamada, H.; Gottesman, M. M.; Pastan, I.; Willingham, M. C. Cellular

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localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues.

520

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521 522

FUNDING

523

Funding for this study was provided in part by research grants from the Ministry of Science and

524

Technology of Taiwan (MOST 106-2113-M-010-002).

525 526

22



527

Figure Captions

528 529

Figure 1. Chemical structure of fisetin (3,3',4',7-tetrahydroxyflavone).

530 531

Figure 2. Rat plasma concentration-time profiles of fisetin (30 mg/kg, i.v., ), fisetin (30 mg/kg, i.v.)

532

pretreated with cyclosporin A (CsA, 10 mg/kg, i.v., ), fisetin (30 mg/kg, i.v.) incubated with

533

β-glucuronidase (), fisetin (30 mg/kg, i.v.) pretreated with CsA (10 mg/kg, i.v.) and incubated with

534

β-glucuronidase (), fisetin (30 mg/kg, i.v.) incubated with sulfatase (), and fisetin (30 mg/kg, i.v.)

535

pretreated with CsA (10 mg/kg, i.v.) and incubated with sulfatase ().

536 537

Figure 3. Rat bile concentration-time profiles of fisetin (30 mg/kg, i.v., ), fisetin (30 mg/kg, i.v.)

538

pretreated with cyclosporin A (CsA, 10 mg/kg, i.v., ), fisetin (30 mg/kg, i.v.) incubated with

539

β-glucuronidase (), fisetin (30 mg/kg, i.v.) pretreated with CsA (10 mg/kg, i.v.) and incubated with

540

β-glucuronidase (), fisetin (30 mg/kg, i.v.) incubated with sulfatase (), and fisetin (30 mg/kg, i.v.)

541

pretreated with CsA (10 mg/kg, i.v.) and incubated with sulfatase ().

542 543

Figure 4. Proposed metabolic pathway of fisetin and its glucuronides, sulfates, and O-methylated

544

metabolites and their distribution in plasma and bile.

545 546 547

23


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Page 23 of 31


Table 1. Pharmacokinetic parameters of fisetin (30 mg/kg, i.v.) in rat plasma and bile. Fisetin (30 mg/kg, i.v.)

Fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.)

free form

glucuronide

sulfate

free form

glucuronide

sulfate

AUC (min·µg/mL)

275.9 ± 44.56

1719 ± 535.7

6429 ± 5690

894.5 ± 324.6*

5806 ± 2397*

7858 ± 3681

Cmax (µg/mL)

73.94 ± 23.90

27.06 ± 9.09

29.10 ± 18.59

92.45 ± 31.78

54.84 ± 21.55

42.59 ± 14.68

Tmax (min)

-

6.67 ± 4.08

16.67 ± 14.72

-

10.33 ± 7.23

32.50 ± 19.94

T1/2 α (min)

1.72 ± 0.69

-

-

5.94 ± 1.94

-

-

T1/2 β (min)

11.29 ± 2.43

-

-

436.7 ± 321.6*

-

-

T1/2 (min)

-

134.4 ± 61.06

208.8 ± 200.5

-

164.0 ± 77.96

130.6 ± 37.46

Vd (mL)

953.5 ± 191.6

3615 ± 2185

1595 ± 743.0

3145 ± 5626

1200 ± 335.5

797.0 ± 221.1

Cl (mL/min)

111.2 ± 18.19

18.82 ± 5.52

9.57 ± 8.64

37.51 ± 13.64

6.07 ± 2.73*

4.71 ± 2.34

AUC (min·µg/mL)

402.5 ± 228.2

1810 ± 1065

29170 ± 6235

278.2 ± 46.35

1460 ± 933.2

50120 ± 24990

Cmax (µg/mL)

38.30 ± 25.28

114.4 ± 94.66

1052 ± 480.3

18.47 ± 7.59

60.42 ± 45.25

1787 ± 944

Tmax (min)

5.00 ± 0.00

6.67 ± 4.08

8.33 ± 5.16

5.00 ± 0.00

17.50 ± 6.12

10.00 ± 5.48

Plasma

Bile

24



Page 24 of 31

T1/2 (min)

24.83 ± 24.68

24.59 ± 9.90

50.97 ± 21.03

12.81 ± 1.81*

59.48 ± 17.82*

80.04 ± 45.10

Vd (mL)

3389 ± 3842

784.1 ± 453.2

75.55 ± 25.70

2015 ± 259.4

2318 ± 1393

73.83 ± 38.85

Cl (mL/min)

88.80 ± 32.59

21.41 ± 10.60

1.08 ± 0.31

110.3 ± 17.86

30.47 ± 21.75

0.72 ± 0.31

kBE (%)

143.7 ± 71.17

109.4 ± 55.96

822.6 ± 585.5

34.94 ± 13.64*

35.16 ± 34.15*

754.9 ± 506.7

Data are presented as the mean ± S.D. (n = 6). Control group: fisetin (30 mg/kg, i.v.) only; CsA-treated group: pretreatment with cyclosporine (CsA, 20 mg/kg, i.v.) before fisetin (30 mg/kg, i.v.); AUC: area under the concentration-time curve; T1/2: half-life; Vd: volume of distribution; CL: clearance; Ratio of biliary excretion: kBE (%) = AUCbile /AUCplasma; *P < 0.05, significant difference compared with the control group.

25


Page 25 of 31


Table 2. Biotransformation of fisetin (30 mg/kg, i.v.) in rats. Fisetin (30 mg/kg, i.v.) Fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) Plasma

Bile

Plasma

Bile

kGlu (%)

640.7 ± 215.4

599.2 ± 467.8

779.0 ± 498.0

544.9 ± 349.2

kSul (%)

2601 ± 2539

8795 ± 4304*

974.4 ± 668.8

17990 ± 9012*

Data are presented as the mean ± S.D. (n = 6). Control group: fisetin (30 mg/kg, i.v.) only. CsA-treated group: pretreatment with cyclosporine (CsA, 20 mg/kg, i.v.) before fisetin (30 mg/kg, i.v.). Ratio of biotransformation into glucuronides: kGlu (%) = AUCGlucuronides / AUCparent. Ratio of biotransformation into sulfates: kSul (%) = AUCSulfates /AUCparent. *P < 0.05, significant difference compared with plasma.

26



Figure 1.

27


Page 26 of 31

Page 27 of 31


Plasma fisetin (µg/mL)

fisetin (30 mg/kg, i.v.) fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) fisetin (30 mg/kg, i.v.) + β-glucuronidase fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) + β-glucuronidase fisetin (30 mg/kg, i.v.) + sulfatase fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) + sulfatase

100

10

1

0.1 0

60

120

180

Time (min) Figure 2.

28


240


Page 28 of 31

fisetin (30 mg/kg, i.v.) fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) fisetin (30 mg/kg, i.v.) + β-glucuronidase fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) + β-glucuronidase fisetin (30 mg/kg, i.v.) + sulfatase fisetin (30 mg/kg, i.v.) + CsA (20 mg/kg, i.v.) + sulfatase

Bile fisetin (µg/mL)

1000 100 10 1 0.1 0.01 0

60

120

180

Time (min) Figure 3.

29


240

Page 29 of 31


Figure 4.

30



Graphic for Table of Contents

31


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Pharmacokinetics and Biliary Excretion of Fisetin in Rats - Journal of

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