Structural Pharmacokinetics of Polymethoxylated ... - ACS Publications

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Structural Pharmacokinetics of Polymethoxylated Flavones in Rat Plasma Using HPLC-MS/MS Jing-Ting Huang,†,⊥ Yung-Yi Cheng,†,⊥ Lie-Chwen Lin,‡ and Tung-Hu Tsai*,†,§,∥ †

Institute of Traditional Medicine, National Yang-Ming University, Taipei 112, Taiwan National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan § School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan ∥ Department of Chemical Engineering, National United University, Miaoli 36063, Taiwan ‡

ABSTRACT: The purpose of this study was to investigate the pharmacokinetics of the polymethoxylated flavonoids kumatakenin, pachypodol, and retusin, which contain two, three, or four methoxy substitutions, using a validated ultra-highperformance liquid chromatography−tandem mass spectrometry (UHPLC-MS/MS) method in rats. The pharmacokinetic results demonstrated that the elimination half-lives for kumatakenin, pachypodol, and retusin were 30 ± 11.6, 39.4 ± 19.5, and 106.9 ± 26 min, respectively, for the low dose group and 54.5 ± 16.5, 33.8 ± 10, and 134.6 ± 34.7 min for the high dose group. The results suggested that the area under the curve values (AUC) for the analytes did not correlate with the number of methoxy groups. Pachypodol had the lowest AUC, which may have been correlated with lipophilicity, for both the low and high dose groups. In conclusion, the polymethoxylated flavonoid pachypodol is more hydrophilic than kumatakenin or retusin, which were correlated with the pharmacokinetic results. KEYWORDS: polymethoxylated flavonoids, UHPLC-MS/MS, kumatakenin, pachypodol, retusin, pharmacokinetics, rat plasma

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structure and the pharmacokinetics of compounds.9 The structural pharmacokinetics we used were meant to describe the relationship between the properties (e.g., logP, logS, tPSA, etc.) of a molecule and its pharmacokinetic behavior. The number of methoxy groups in the two polymethoxylated flavones nobiletin and tangertin are five and six, respectively, and the difference between them is a methoxy substitution at the 3′- position. A study by Yuen showed that the clearance and half-life of nobiletin and tangertin were significantly different.10 These results suggested that a methoxy group may be the key aspect that affects the pharmacokinetics of polymethoxylated flavones. Therefore, we hypothesized that the pharmacokinetics of polymethoxylated flavones may be correlated with the number of methoxy groups or with the lipophilicity of an analyte. The reasons that we chose these three particular polymethoxylated flavones for this study were as follows: (1) To investigate the effects of the number of methoxy groups on the pharmacokinetics of the three flavones studied. The number of methoxy groups on kumatakenin, pachypodol, and retusin are two, three, and four, respectively. (2) To investigate whether the position of their methoxy groups affects their pharmacokinetics. The difference between kumatakenin and pachypodol was a methoxy group substituted at the 3′-position. Comparing pachypodol and retusin, the hydroxy group at the 4′-position was replaced by a methoxy group. Furthermore, these three polymethoxylated flavonoids were suitable for an

INTRODUCTION The chemical structure of polymethoxylated flavonoids results from the methylation of polyhydroxylated flavonoids, leading to increased metabolic stability and membrane transport in the intestine and liver, improving oral bioavailability.1 At present, polyhydroxylated flavonoids have been thought to have health promoting properties, including anticancer,2 anti-inflammatory,3 and neuroprotective properties.4 Three polymethoxylated flavonoids, kumatakenin, pachypodol, and retusin, were isolated from a methanolic extract of Melicope semecarpifolia. Kumatakenin is a kaempferol derivative that has two methoxy groups in the 3- and 7-positions and two hydroxy groups in the 5- and 4′positions. Previous reports have shown that kumatakenin was a strong inhibitor of hind paw edema and a potent antinociceptive agent in mice.5 Pachypodol is a quercetin derivative that has three methoxy groups in the 3-, 7-, and 3′- positions and two hydroxy groups in the 5- and 4′-positions. Recent pharmacological research indicated that pachypodol has antipicornavirus properties and can inhibit plus-strand RNA synthesis in poliovirus6 and restrain the growth of the CaCo-2 colon cancer cell line in vitro.7 Retusin is also derived from quercetin and has four methoxy groups in the 3-, 7-, 3′-, and 4′positions and one hydroxyl group in the 5-position. Retusin was shown to be a potential cytotoxic agent for the breast cancer resistance protein (BCRP), overexpressing MCF-7 MX100.8 A structure−activity relationship describes the relationship between the chemical structure of a molecule and its biological activity in general terms. The pharmacokinetic behavior and pharmacodynamic properties reflect the action of a chemical or a drug on a biological system. Quantitative structure− pharmacokinetic relationships (QSPKRs) provide a statistical description and predict the relationship between the molecular © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

December 1, 2016 March 2, 2017 March 2, 2017 March 2, 2017 DOI: 10.1021/acs.jafc.6b05390 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

plasma samples were cleaned with liquid−liquid extraction (LLE) by adding 500 μL of ethyl acetate containing 0.1% formic acid. After vortexing for 5 min, the samples were centrifuged at 9000g for 10 min at 4 °C, and the supernatant was collected immediately. This step was repeated once more, and the supernatant was evaporated to dryness at 40 °C in a centrifuge evaporator. The dried residue was dissolved in a mobile phase of water and acetonitrile (35:65, v/v) containing 0.1% formic acid. After vortexing for 5 min, the samples were centrifuged at 9000g for 10 min at 4 °C and filtered through a 0.22-μm filter. Finally, 5 μL of the filtrate was analyzed by UHPLC-MS/MS. UHPLC-MS/MS Analysis. The UHPLC-MS/MS analysis system consisted of a Shimadzu LC-20AD HPLC system (Shimadzu, Kyoto, Japan) and an LCMS-8030 triple-quadrupole mass spectrometer (Shimadzu) equipped with an electrospray ionization interface. The chromatographic separation of all analytes was performed using a C18e column (100 × 2.1 mm, 2 μm; Merck Hibar HR Purospher STAR RP-18 end-capped, Darmstadt, Germany). The column oven was maintained at 35 °C. The autosampler temperature was set at 4 °C. The mobile phase included water and acetonitrile (35:65, v/v), both containing 0.1% formic acid. The flow rate was 0.2 mL/min, and the injection volume was 5 μL. The following MS/MS parameters were set: electrospray ionization interface, positive ion mode; desolvation line temperature, 250 °C; heat block temperature, 400 °C; interface voltage, 4.5 kV; detector voltage, 1.86 kV; collision-induced dissociation (CID) gas, 230 kPa; nebulizing gas flow, 3.0 L/min; drying gas flow, 15.0 L/min; nebulizing gas and drying gas, both nitrogen; and the collision gas was argon. Samples were quantified in the selective reaction monitoring (SRM) mode, with monitoring transitions m/z 315.1 → 300.1 for kumatakenin, m/z 345.1 → 330.1 for pachypodol, m/z 359.1 → 344.1 for retusin, and m/z 353.2 → 238.1 for 5-methoxyflavone. Method Validation. The US Food and Drug Administration (FDA) guidelines for the industry provide general recommendations for the validation of a bioanalytical method.20 Fundamental validations were performed for method development and establishment according to these guidelines, including a calibration curve, accuracy, precision, the lower limit of quantification (LLOQ), the matrix effect, recovery, and stability with spiked samples. The calibration standards were individually diluted from stock solutions of kumatakenin, pachypodol, and retusin. After spiking with blank plasma, samples of 2.5, 5, 10, 50, 100, and 500 ng/mL concentrations were treated as described in Sample Preparation. The results were calculated from the peak area ratios and an internal standard, and each calibration curve of the linear correlation coefficient (r2) should be greater than 0.995. Accurate and precise measurements were obtained by replication for each concentration six times per day (intraday) and over six consecutive days (interday), at least three times within the range of expected concentrations. The calculation formula for accuracy is (bias, %) = [(Cobs − Cnom)/Cnom] × 100%, and for precision it is (relative standard deviation, R.S.D., %) = [standard deviation (SD)/Cobs] × 100%. Compared to the nominal concentration, the mean value should be within ±15% except at the LLOQ, at which it should be within ±20%. The matrix effect and recovery were analyzed at three concentrations (low, medium, and high) for kumatakenin, pachypodol, and retusin, and at 10 ng/mL for 5-methoxyflavone (IS) in three different sets as follows. Set 1. The standard solution (10 μL) was mixed with the mobile phase (90 μL) including acetonitrile and water (65:35, v/v) and was acidified with 0.1% formic acid. Set 2. Blank plasma (90 μL) was processed by liquid−liquid extraction as previously described and then mixed with the standard solution (10 μL). After the supernatant was evaporated, dissolved in the mobile phase (100 μL), and filtered through a 0.22 μm filter, the mixture was transferred to an autosampler. Set 3. The standard solution (10 μL) was mixed with blank plasma (90 μL) before sample preparation. After evaporation, dissolution, and filtration, the mixture was transferred to an autosampler.

investigation of the structure−pharmacokinetic relationships of polymethoxylated flavonoids. Many studies have indicated that kumatakenin, pachypodol, and retusin could be extracted from various plants.11−15 However, an analytical method such as LC-MS/MS quantification has only been applied for pachypodol.16 No results were found from a PubMed search with the keywords kumatakenin or retusin and liquid chromatography−tandem mass spectrometry. Furthermore, no study has been done on the development of the qualitative or quantitative detection of kumatakenin, pachypodol, and retusin from biological samples or an investigation of their pharmacokinetics. In the present study, kumatakenin, pachypodol, and retusin were isolated from Melicope semecarpifolia, which is a native Taiwanese species of Rutaceae, a medium-sized evergreen shrub. Melicope semecarpifolia is well distributed in low and moderate altitude forests in Taiwan and the Philippines17 and is a food source for Papilio paris larvae in Taiwan. The bioactive compounds of Melicope semecarpifolia include furoquinoline alkaloids, acetophenones, coumarins, and flavonoids, which have exhibited antiplatelet aggregation, as well as cytotoxic, antifungal, and antiinflammation activities.18,19 The aim of this study was not only to develop a sensitive, convenient, rapid UHPLC-MS/MS method for the determination of kumatakenin, pachypodol, and retusin in rat plasma but also to assess the pharmacokinetics of polymethoxylated flavonoids in rats. Because the differences between pachypodol and kumatakenin or retusin were based on the methoxy groups in the 3′- or 4′-positions, we hypothesized that the pharmacokinetics of these flavonoids might be correlated with the number of methoxy groups or the lipophilicity of the analyte.

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MATERIALS AND METHODS

Chemicals and Reagents. The kumatakenin (5,4′-dihydroxy-3,7dimethoxyflavone), pachypodol (5,4′-dihydroxy-3,7,3′-trimethoxyflavone), and retusin (5,4′-hydroxy-3,7,3′,4′-tetramethoxyflavone) reference standards were isolated from the flowers and stems of Melicope semecarpifolia.19 Urethane, heparin sodium salt, polyethylene glycol 400, and 5-methoxyflavone (an internal standard, IS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Triple deionized water was acquired from Millipore (Bedford, MA, USA) and used in all experiments. Formic acid was obtained from E. Merck (Darmstadt, Germany). Acetonitrile and ethyl acetate were purchased from J.T. Baker (Center Valley, PA, USA). Experimental Animals. Male Sprague−Dawley rats (230 ± 30 g) were acquired from the Laboratory Animal Center of the National Yang-Ming University (Taipei, Taiwan). The rats were housed under standard conditions with a 12-h light/dark cycle. Laboratory rodent diet 5001 (PMI Feeds, Richmond, IN, USA) and water were freely given at all times. The experimental protocol was approved by the Institutional Animal Care and Use Committee of National Yang-Ming University (IACUC number: 1041110). The animals were initially anesthetized via the intraperitoneal administration of urethane (1 g/kg), and polyethylene tubing was fixed in the right jugular vein for blood collection and in the right femoral vein for drug administration. Kumatakenin, pachypodol, and retusin were administered intravenously (i.v.) in polyethylene glycol with ethanol and normal saline (7:2:1) at a dose of 3 or 10 mg/kg. A 300 μL blood sample was collected from the right jugular vein at 5, 15, 30, 60, 90, 120 180, 240, and 360 min after drug administration. The blood samples were stored in Eppendorf vials rinsed with heparin and centrifuged at 9000g for 10 min at 4 °C to obtain the supernatants, which were stored at −20 °C before sample preparation. Sample Preparation. Plasma samples (90 μL) were spiked with 5methoxyflavone (100 ng/mL, 10 μL) as an internal standard. The rat B

DOI: 10.1021/acs.jafc.6b05390 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry The results were calculated from the peak area where the matrix effect was Set2/Set1 × 100%, the recovery was Set3/Set2 × 100%, and the process efficiency was Set3/Set1 × 100%, which should be consistent, precise, and reproducible. Stability was evaluated at three concentrations (low, medium, and high) for kumatakenin, pachypodol, and retusin and was analyzed under the following conditions: a processed sample was stored in an autosampler for 6 h at 4 °C and on a benchtop for 4 h at room temperature, subjected to a freeze−thaw cycle three times for 24 h at −20 °C and then thawed at room temperature, and stored for 20 days (long-term storage) at −20 °C. Compared to a freshly prepared sample, the relative error of each condition should be within ±15%. The results indicated that the analytes remained stable under all of these conditions. Pharmacokinetic Parameters and Statistical Analysis. WinNonlin Standard Edition (Version 1.1, Scientific Consulting Inc., Apex, NC, USA) was used to calculate the experimental results using a noncompartment model for the pharmacokinetic parameters. The parameters included the maximum concentration of drug (Cmax), the elimination half-life (t1/2), the area under the concentration−time curve (AUC), the clearance (Cl), and the mean residence time (MRT). The drug concentration versus time profiles were calculated using SigmaPlot (version 13.0). The results were further analyzed using one-way ANOVA using SPSS (version 20.0). A P value 10. The limit of detection (LOD) for kumatakenin, pachypodol, and retusin was 0.5 ng/mL with an S/N > 3. The intraday and interday accuracy (% bias) and precision (% RSD) were determined at concentrations of 2.5, 5, 10, 50, 100, and 500 ng/mL for kumatakenin and pachypodol and at 5, 10, 50, 100, and 500 ng/mL for retusin. The results of these analyses are shown in Table 1. The mean values of all the bias and RSD values were within ±15% except for the LLOQ, which was within ±20%, although it was still within an acceptable range. C


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Figure 2. Representative SRM chromatograms of (A) a blank plasma sample; (B) a blank plasma sample spiked with kumatakenin (100 ng/mL); (C) a plasma sample at 30 min after administration of kumatakenin (3 mg/kg, i.v.); (D) a blank plasma sample; (E) a blank plasma sample spiked with pachypodol (100 ng/mL); (F) a plasma sample at 30 min after administration of pachypodol (3 mg/kg, i.v.); (G) a blank plasma sample; (H) a blank plasma sample spiked with retusin (100 ng/mL); and (I) a plasma sample at 30 min after administration of retusin (3 mg/kg, i.v.). Retention times of the analytes: kumatakenin, 2.4 min; pachypodol, 3.1 min; and retusin, 5.2 min.

and the 5-methoxyflavone concentration was 10 ng/mL. The results are shown in Table 2. The matrix effects of kumatakenin were 80.45 to 83.39%, similar to pachypodol, which ranged from 86.58 to 88.45%, and retusin, which ranged from 95.74 to

Three concentrations (low, medium, and high) were evaluated for matrix effects and recovery. The kumatakenin and pachypodol concentrations tested were 2.5, 50, and 500 ng/mL, the retusin concentrations were 5, 50, and 500 ng/mL, D


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Table 1. Intra- and Inter-day Precision and Accuracy for the Determination of Kumatakenin, Pachypodol, and Retusin Intraday

a

Interday

Nominal conc

Obsd conca

(ng/mL)

(ng/mL)

2.5 5 10 50 100 500

2.45 ± 0.46 5.13 ± 0.50 10.55 ± 0.57 51.37 ± 2.35 97.68 ± 2.92 500.4 ± 0.22

18.77 9.75 5.40 4.57 2.99 0.04

2.5 5 10 50 100 500

2.24 ± 0.39 4.85 ± 0.29 10.18 ± 0.31 51.36 ± 2.19 97.74 ± 3.45 500.1 ± 0.17

17.41 5.98 3.05 4.26 3.53 0.03

5 10 50 100 500

4.63 ± 0.74 9.36 ± 0.42 49.35 ± 1.59 102.8 ± 2.36 499.7 ± 0.31

15.98 4.49 3.22 2.30 0.06

Precision, RSD (%)

Accuracy, Bias (%)

Precision, RSD (%)

Accuracy, Bias (%)

2.25 ± 0.16 4.64 ± 0.37 10.41 ± 1.22 48.54 ± 2.28 99.25 ± 3.85 499.9 ± 0.22

7.11 7.97 11.72 4.70 3.88 0.04

−10.00 −7.20 4.10 −2.92 −0.75 −0.02

2.69 ± 0.39 5.23 ± 0.29 10.52 ± 0.36 50.04 ± 1.62 102.0 ± 3.13 500.5 ± 0.66

14.50 5.54 3.42 3.24 3.07 0.13

7.60 4.60 5.20 0.08 2.00 0.01

4.73 ± 0.28 10.40 ± 0.69 49.06 ± 2.95 101.3 ± 2.41 500.2 ± 0.94

5.92 6.63 6.01 2.38 0.19

−5.40 4.00 −1.89 1.30 0.04

(ng/mL) Kumatakenin −2.0 2.60 5.50 2.74 −2.32 0.08 Pachypodol −10.40 −3.00 1.80 2.72 −2.26 0.02 Retusin −7.40 −6.40 −1.30 2.80 −0.06

Data are expressed as the mean ± SD.

including the storage of processed samples in an autosampler for 6 h at 4 °C and benchtop storage for 4 h at room temperature, for which the values were within ±15% compared to a freshly prepared sample. However, in long-term experiments, including triplicate freeze−thaw cycles for 24 h at −20 °C and thawing at room temperature and long-term storage for 20 days at −20 °C, some of the analytes lacked stability, especially at medium concentrations in freeze−thaw cycles and at a low concentrations in long-term storage. Therefore, the analytical experiments should be completed in a short time. Pharmacokinetic Evaluation. To investigate the effect of flavone methoxy groups on the pharmacokinetics in rats, three flavones with a similar chemical structure and analytical method were chosen. Kumatakenin is 5,4′-dihydroxy-3,7-dimethoxyflavone, and pachypodol has more 3′-methoxy groups than kumatakenin. The structural difference between pachypodol and retusin is only at the 4′- position, where there is a 4′hydroxy in pachypodol and a 4′-methoxy in retusin. Kumatakenin, pachypodol, and retusin were administered at two different dosages of 3 mg/kg and 10 mg/kg to each group (n = 6). Plasma samples were collected and analyzed with a validated UHPLC-MS/MS method. The mean plasma concentration−time profiles for kumatakenin, pachypodol, and retusin in rats after administration at doses of 3 mg/kg and 10 mg/kg are shown in Figure 3. In addition, a noncompartmental model was used to describe the pharmacokinetics of kumatakenin, pachypodol, and retusin. As the results in Table 4 show, there were no significant differences between pachypodol and kumatakenin when administered at 3 mg/kg, but the AUC and Cl differed significantly at the high dosages. Furthermore, the pharmacokinetic parameters of pachypodol and retusin were significantly different, including the AUC, Cl, t1/2, and MRT both at the 3 mg/kg and the 10 mg/kg dosages. The results suggested the rapid elimination of three methylated flavones with a short halflife.

Table 2. Matrix Effect, Recovery, and Process Efficiency of Kumatakenin, Pachypodol, and Retusin in Rat Plasma Nominal conc (ng/mL) 2.5 50 500 2.5 50 500 5 50 500 10 a

Obsd conca

Matrix effect (%)a

Recovery (%)a

Kumatakenin 83.39 ± 3.55 92.17 ± 8.19 81.69 ± 2.05 91.56 ± 3.80 80.45 ± 1.68 94.1 ± 5.57 Pachypodol 86.58 ± 0.77 94.37 ± 9.15 88.45 ± 1.95 88.29 ± 2.10 87.31 ± 4.04 95.22 ± 5.15 Retusin 102.8 ± 7.89 106 ± 8.53 95.74 ± 15.51 99.94 ± 17.65 97.87 ± 6.19 95.94 ± 9.80 5-MF 100.99 ± 0.89 99.39 ± 1.87

Process efficiency (%)a 76.63 ± 4.84 74.78 ± 3.29 75.61 ± 2.86 81.63 ± 7.21 78.12 ± 3.28 82.94 ± 0.66 108.53 ± 1.48 93.86 ± 1.10 93.55 ± 5.07 100.36 ± 1.89


102.8%, and they were slightly higher than those for the other two compounds. There were no significant differences in the analytical range, which was acceptable according to the FDA’s biological method validation guidelines. The recovery of kumatakenin was 91.56 to 94.1, that for pachypodol was 88.29 to 95.22, and that for retusin was 95.94 to 106, which indicated that there were no significant differences in the analytical range. Stability. The stability of kumatakenin, pachypodol, and retusin is summarized in Table 3. Three concentrations (low, medium, and high) were evaluated for stability. The kumatakenin and pachypodol concentrations tested were 2.5, 50, and 500 ng/mL, and the retusin concentrations were 5, 50, and 500 ng/mL. The results indicated that kumatakenin, pachypodol, and retusin were stable in short-term experiments, E


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Journal of Agricultural and Food Chemistry Table 3. Stability Evaluation Data for Kumatakenin, Pachypodol, and Retusin in Rat Plasma

a

Nominal conc (ng/mL)

Freeze−thaw cycle stabilitya

2.5 50 500

−9.62 ± 5.70 −15.89 ± 1.06 −8.71 ± 5.15

2.5 50 500

−7.9 ± 8.87 −12.57 ± 1.52 −8.42 ± 0.14

5 50 500

−15.75 ± 0.50 −19.69 ± 1.39 13.11 ± 4.50

Bench-top Stabilitya Kumatakenin −3.09 ± 0.99 8.14 ± 1.07 7.55 ± 3.28 Pachypodol −3.32 ± 2.66 4.75 ± 1.20 3.62 ± 1.83 Retusin −8.22 ± 2.96 3.59 ± 2.65 −9.79 ± 3.55

Long-term Stabilitya

Processed sample Stabilitya

−37.43 ± 1.06 −11.42 ± 1.53 0.81 ± 3.55

−2.47 ± 4.70 1.32 ± 2.48 0.04 ± 0.01

−56.49 ± 1.65 −9.15 ± 2.46 −5.43 ± 0.29

−2.07 ± 3.95 2.43 ± 2.45 0.003 ± 0.03

−14.53 ± 0.15 −3.05 ± 2.36 11.7 ± 0.88

−1.67 ± 11.48 3.39 ± 0.48 0.04 ± 0.03


Smith’s report, the hydrophilicity of a drug is a key physicochemical property that affects the availability of a drug.25 Modification of a flavone by O-methylation changes its hydrophilicity, polarity, and the electron distribution accordingly. The calculated partition coefficient factor (cLogP) value is a useful established measurement of the hydrophilicity of a compound. The OSIRIS DataWarrior and Molinspiration software were used to calculate the cLogP values, which are summarized in Table 5. Kumatakenin has a higher mean cLogP Table 5. CLogP Values of Kumatakenin, Pachypodol, and Retusin Compound

cLogPa

cLogPb

Kumatakenin Pachypodol Retusin

2.54 2.47 2.75

2.98 2.80 3.11

a

Figure 3. Drug concentration versus time profiles of kumatakenin, pachypodol, and retusin in rat plasma after drug administration (3 mg/ kg, i.v. and 10 mg/kg). Data are presented as the means ± standard deviation (n = 6).

These were experimental values taken from OSIRIS DataWarrior software. bThese were experimental values taken from Molinspiration software.

than pachypodol, and the higher hydrophobicity of kumatakenin may prolong its residence time in rats. The difference between the chemical structures of pachypodol and retusin is a methoxy group substituted at the 4′-position. Retusin has a higher cLogP value than pachypodol, indicating greater hydrophobicity. The results also indicated that the clearance of retusin was significantly lower than that of pachypodol, and retusin had a longer MRT than pachypodol. These results suggested that the retusin tended to be eliminated more slowly than pachypodol. Based on flavanoid metabolism, the conjugation reaction is favored to occur at the polar hydroxyl groups, and reactions with glucuronic acid, sulfate, or glycine have been reported for flavonoids.26 Retusin has a hydroxyl

Flavone metabolism includes phase I and phase II hepatic metabolic pathways. Based on an investigation by Manthey et al., although nobiletin and tangeretin do not contain a hydroxy group, the glucuronidated metabolites of nobiletin and tangeretin were determined in plasma.22 Therefore, even polymethoxylated flavones without hydroxyl groups may be potentially involved in the conjugation reaction. Thus, to enhance the polarity of the hydrophobic flavones for kidney and bile excretion, Wu et al. reported that sulfation and glucuronidation were the most important phase II metabolic pathways.23 The glucuronidation of flavones frequently occurred at the 7-, 3-, 3′-, and 4′-positions.24 According to

Table 4. Pharmacokinetic Parameters of Kumatakenin, Pachypodol, and Retusin in Rat Plasma (3 mg/kg and 10 mg/kg i.v.)b Kumatakenina

a

Parameters

3 mg/kg, i.v.

10 mg/kg, i.v.

C0 (ng/mL) t1/2 (min) AUC (min ng/mL) Cl (mL/min/kg) MRT (min)

553.4 ± 150.9 30 ± 11.6 12720 ± 3207 251.3 ± 63.26* 27.6 ± 4.0

1921 ± 751.8 54.5 ± 16.5 57120 ± 14180** 184.8 ± 39.94** 55.7 ± 15.6

Pachypodola

Retusina

3 mg/kg, i.v.

10 mg/kg, i.v.

3 mg/kg, i.v.

10 mg/kg, i.v.

± ± ± ± ±

1865 ± 402.1 33.7 ± 1.0 33270 ± 5545 309 ± 51.33 21.3 ± 2.3

1065.6 ± 328.5 106.9 ± 26.0 25390 ± 3267*** 120.1 ± 15.11*** 80.3 ± 15.3

1860 ± 537.5 134.6 ± 34.7 52830 ± 13690** 203.9 ± 57.66** 96.3 ± 33.5

775.4 39.4 9530 330.2 24.9

194.9 19.5 1932 76.24 11.5

Data are expressed as the mean ± SD. bCompared with pachypodol, ″*″ P value < 0.05, ″**″ P value < 0.01, and ″***″ P value < 0.001. F


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(5) Simpson, B. S.; Claudie, D. J.; Smith, N. M.; Gerber, J. P.; McKinnon, R. A.; Semple, S. J. Flavonoids from the leaves and stems of Dodonaea polyandra: A Northern Kaanju medicinal plant. Phytochemistry 2011, 72, 1883−1888. (6) Arita, M.; Philipov, S.; Galabov, A. S. Phosphatidylinositol 4kinase III beta is the target of oxoglaucine and pachypodol (Ro 09− 0179) for their anti-poliovirus activities, and is located at upstream of the target step of brefeldin A. Microbiol. Immunol. 2015, 59, 338−347. (7) Ali, H. A.; Chowdhury, A.; Rahman, A. K.; Borkowski, T.; Nahar, L.; Sarker, S. D. Pachypodol, a flavonol from the leaves of Calycopteris floribunda, inhibits the growth of CaCo 2 colon cancer cell line in vitro. Phytother. Res. 2008, 22, 1684−1687. (8) Pick, A.; Müller, H.; Mayer, R.; Haenisch, B.; Pajeva, I. K.; Weigt, M.; Bö nisch, H.; Müller, C. E.; Wiese, M. Structure−activity relationships of flavonoids as inhibitors of breast cancer resistance protein (BCRP). Bioorg. Med. Chem. 2011, 19, 2090−2102. (9) Mayer, J. M.; Waterbeemd, H. Development of Quantitative Structure-Pharmacokinetic Relationships. Environ. Health Persp 1985, 61, 295−306. (10) Yuen, H. Q.; Hwang, Q. H.; Zhang, X. Y.; Zhou, Z. X. Cellular Antioxidant Activity and Pharmacokinetic Study of Polymethoxylated Flavonoids in Extract of Citrus reticulata Chachi Peel. Food Sci. Technol. Res. 2014, 20, 629−637. (11) Yang, Y.; Kinoshita, K.; Koyama, K.; Takahashi, K.; Tai, T.; Nunoura, Y.; Watanabe, K. Anti-emetic principles of Pogostemon cablin (Blanco) Benth. Phytomedicine 1999, 6, 89−93. (12) Afifi, F.; Al-Khalil, S.; Abdul-Haq, B.; Mahasneh, A.; Al-Eisawi, D.; Sharaf, M.; Wong, L.; Schiff, P. Antifungal flavonoids from Varthemia iphionoides. Phytother. Res. 1991, 5, 173−175. (13) Dungerdorzh, D.; Petrenko, V. Kumatakenin from Astragalus membranaceus. Chem. Nat. Compd. 1972, 8, 382−382. (14) González-Vázquez, R.; King Díaz, B.; Aguilar, M. I.; Diego, N.; Lotina-Hennsen, B. Pachypodol from Croton ciliatoglanduliferus Ort. as water-splitting enzyme inhibitor on thylakoids. J. Agric. Food Chem. 2006, 54, 1217−1221. (15) Puripattanavong, J.; Tewtrakul, S. Antiallergic and antiinflammatory compounds from Aglaia andamanica leaves. Songklanakarin. J. Sci. Technol. 2015, 37, 37−41. (16) Zhao, Y.; Yu, Z.; Fan, R.; Gao, X.; Yu, M.; Li, H.; Wei, H.; Bi, K. Simultaneous determination of ten flavonoids from Viscum coloratum grown on different host species and different sources by LC-MS. Chem. Pharm. Bull. 2011, 59, 1322−1328. (17) Chen, J.-J.; Duh, C.-Y.; Huang, H.-Y.; Chen, I.-S. Furoquinoline alkaloids and cytotoxic constituents from the leaves of Melicope semecarpifolia. Planta Med. 2003, 69, 542−546. (18) Chen, I.-S.; Chen, H.-F.; Cheng, M.-J.; Chang, Y.-L.; Teng, C.M.; Tsutomu, I.; Chen, J.-J.; Tsai, I.-L. Quinoline alkaloids and other constituents of melicopes emecarpifolia with antiplatelet aggregation activity. J. Nat. Prod. 2001, 64, 1143−1147. (19) Lin, L.; Chou, C.; Chen, K.; Chen, C. Studies on the constituents of the flowers and stems of Evodia merrillii. Chin. Pharm. J. 1992, 44, 125−31. (20) U.S. FDA. Guidance for Industry, Bioanalytical Method Validations; U.S. Department of Health and Human Services, Food and Drug Administration Center for Drug Evaluation and Research (CDER): Washington, DC, USA, 2013. (21) McDowall, R. Sample preparation for biomedical analysis. J. Chromatogr., Biomed. Appl. 1989, 492, 3−58. (22) Manthey, J. A.; Cesar, T. B.; Jackson, E.; Mertens-Talcott, S. Pharmacokinetic Study of nobiletin and tangeretin in rat serum by high-performance liquid chromatography-electrospray ionization-mass spectrometry. J. Agric. Food Chem. 2011, 59, 145−151. (23) Wu, B.; Basu, S.; Meng, S.; Wang, X.; Zhang, S.; Hu, M. Regioselective sulfation and glucuronidation of phenolics: Insights into the structural basis of conjugation. Curr. Drug Metab. 2011, 12, 900− 16. (24) Boersma, M. G.; van der Woude, H.; Bogaards, J.; Boeren, S.; Vervoort, J.; Cnubben, N. H.; van Iersel, M. L.; van Bladeren, P. J.; Rietjens, I. M. Regioselectivity of phase II metabolism of luteolin and

group at the 5-position, but this hydroxyl group forms an intramolecular hydrogen bond with a carbonyl group, which suggests that this hydroxyl group will not react in a conjugation reaction. In conclusion, the results indicate that a methoxy group replaced the hydroxyl group in the 4′- position, enhancing hydrophobicity, which extended the elimination time from the body. In our opinion, the pharmacokinetics of flavonoids may be affected by the methoxy group if a substitution occurs at a site at which a conjugation reaction is favored, which will retard the metabolizing rate of a flavonoid. This may explain why the metabolizing rate of retusin was lower than that of pachypodol. A rapid and sensitive UHPLC-MS/MS analytical method for the quantification of kumatakenin, pachypodol, and retusin in biological samples was developed. According to the FDA’s biological method validation guidelines (2001), all validation parameters, including the matrix effect and recovery in rat plasma, were acceptable. The structure−pharmacokinetics relationship of the three methoxylated flavones was characterized by the methoxy-substituted position that would change the physicochemical properties of the compounds, possibly affecting the metabolism and elimination, especially when substituted at the 4′-position. Our investigation successfully determined the pharmacokinetics of kumatakenin, pachypodol, and retusin and provided a constructive contribution to the understanding of the structure−pharmacokinetics relationship of methoxylated flavones.

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AUTHOR INFORMATION

Corresponding Author

*Fax: (886-2) 2822 5044; Tel.: (886-2) 2826 7115; E-mail: [email protected]. ORCID

Yung-Yi Cheng: 0000-0001-7473-2835 Lie-Chwen Lin: 0000-0001-9518-9018 Tung-Hu Tsai: 0000-0002-9007-2547 Author Contributions ⊥

J.-T.H. and Y.-Y.C. contributed equally.

Funding

Funding for this study was provided in part by research grants from the Ministry of Science and Technology of Taiwan (MOST 105-2113-M-010-004). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the PK Lab members who assisted with and supported this study. REFERENCES

(1) Walle, T. Methoxylated flavones, a superior cancer chemopreventive flavonoid subclass? Semin. Cancer Biol. 2007, 17, 354−62. (2) Silalahi, J. Anticancer and health protective properties of citrus fruit components. Asia Pac. J. Clin. Nutr. 2002, 11, 79−84. (3) Lin, N.; Sato, T.; Takayam, Y.; Mimaki, Y.; Sashida, Y.; Yano, M. Novel antiinflammatory actions of nobiletin, a citrus polymethoxy flavonoid, on human synovial fibroblasts and mouse macrophages. Biochem. Pharmacol. 2003, 65, 2065−2071. (4) Datla, K. P.; Christidou, M.; Widmer, W. W.; Rooprai, H. K.; Dexter, D. T. Tissue distribution and neuroprotective effects of citrus flavonoid tangeretin in a rat model of Parkinson’s disease. NeuroReport 2001, 12, 3871−3875. G


Article

Journal of Agricultural and Food Chemistry quercetin by UDP-glucuronosyl transferases. Chem. Res. Toxicol. 2002, 15, 662−70. (25) Alex, A. A.; Storer, R. I. Drugs and their Structural Motifs. Metabolism, pharmacokinetics, and toxicity of functional groups: impact of the building blocks of medicinal chemistry: impact of chemical building blocks on ADMET, 1st ed..; Smith, D. A.; Royal Society of Chemistry: Cambridge, UK, 2010; Chapter 1, pp 1−60. (26) Hollman, P. C. H.; Katan, M. B., Absorption, metabolism, and bioavailability of flavonoids. In Flavonoids in Health & Disease; RiceEvans, C, Packer, L, Eds.; Marcel Dekker: New York, 1998; pp 483− 522.

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