Modulation of P-glycoprotein activity by acridones and coumarins from Citrus sinensis

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386 C. BAYET ET AL.

Phytother. Res. 21, 386–390 (2007)

Published online 18 January 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ptr.2081

Modulation of P-glycoprotein Activity by

Acridones and Coumarins from Citrus sinensis

C. Bayet¹, C. Fazio¹, N. Darbour¹, O. Berger¹, I. Raad¹, A. Chaboud¹, C. Dumontet² and D. Guilet¹*

1Université Claude Bernard Lyon 1, Laboratoire de Pharmacognosie, Faculté de Pharmacie, 8 avenue Rockefeller, 69373 Lyon Cedex 8, France

2Université Claude Bernard Lyon-1, Laboratoire de Cytologie Analytique, INSERM U590, Faculté de Médecine de Lyon, 8 avenue Rockefeller, 69880 Lyon Cedex 8, France

Bioguided fractionation of the roots of Citrus sinensis (Rutaceae) led to the isolation and identification of five coumarins, namely, clausarin, suberosin, poncitrin, xanthyletin and thamnosmonin, seven acridones, namely, acrimarine B, 2-methoxycitpressine I, citpressine I, buntanine, acrimarine E, honyumine and acrimarine C, and one terpenoid, namely, limonin. Among these compounds, clausarin, 2-methoxycitpressine I and acrimarine E inhibited P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells over-expressing P- glycoprotein. Copyright © 2007 John Wiley & Sons, Ltd.

Keywords: Citrus sinensis; acridones; coumarins; P-glycoprotein inhibitors.

Received 18 October 2006 Accepted 8 November 2006

* Correspondence to: David Guilet, Université Claude Bernard Lyon 1, Laboratoire de Pharmacognosie, Faculté de Pharmacie, 8 avenue Rockefeller, 69373 Lyon Cedex 8, France.

E-mail: [email protected]

INTRODUCTION

It is established that constituents of some Citrus juices, especially Citrus paradisi (grapefruit) and Citrus sinensis (sweet orange), could affect the pharmacokinetics of diverse chemotherapeutic drugs (Honda et al., 2004;

Romiti et al., 2004; Tian et al., 2002; Ikegawa et al., 2000).

One of the mechanisms implicated in this phenomenon is the ability of several Citrus compounds to inhibit the P-glycoprotein (P-gp) activity. In addition to this effect on the bioavailability of drugs, modulation of P-gp presents another major interest in the anticancer research ﬁeld. In fact, P-gp recognizes a wide range of apolar compounds including anticancer agents and, functioning as efﬂux pump, extrudes them outside the cell (Asschert et al., 1997; Goldstein, 1995). The over- expression of P-gp in tumor cell lines is often associ- ated with the multidrug resistance (MDR) to anticancer agents, a major cause of treatment failure in cancer chemotherapy.

In the research on P-gp inhibitors to reduce the MDR effect, some natural substances belonging to various chemical classes have been already described, such as ﬂavonoids (Di Pietro et al., 2002), coumarins (Barthomeuf et al., 2005), terpenoids (Ramachandran et al., 2003) or alkaloids (Kam et al., 2004). For Citrus species, studies of potential modulators of the P-gp were focused on compounds from aerial parts, in particular from fruit juices. Two chemical groups of Citrus compounds, polymethoxylated ﬂavones (Ikegawa et al., 2000) and furanocoumarins such as bergamottin and derivatives (Wang et al., 2001), have been reported as inhibitors of Pgp-mediated drug function.

As part of our ongoing search for natural P-gp inhibitors (Raad et al., 2006), an investigation of the roots of Citrus sinensis was undertaken. Using bioactivity- guided fractionation procedures, 13 compounds (1–13) have been isolated and their activities on P-gp- mediated drug efﬂux in K562/R7 human leukemic cells overexpressing P-gp were evaluated.

MATERIAL AND METHODS

Instrumentation. NMR spectra were recorded on a DRX 500 spectrometer (500 MHz for ¹H and 125 MHz for ¹³C). Solvents were used as internal references (CDCl₃, DMSO-d₆ and acetone-d₆). Mass spectra (EI) were recorded with a Thermo-Finnigan Mat 95XL spectrometer. Optical rotation was measured on a Perkin-Elmer photometer. TLC was carried out using Merck silica gel Si 60 F₂₅₄ 20 × 20 cm plastic sheets, RP- 18 F_254S 20 × 20 cm aluminum sheets and TLC plates on DIOL-F₂₅₄S 10 × 10 cm plates. Preparative TLC was performed on Kieselgel 60 F₂₅₄ (Merck) plates (0.5 mm thickness). Analytical HPLC was carried out on a Thermo Separation Products system equipped with a P-4000 quaternary gradient pump, a UV-6000LP photodiode array detector using analytical 125-4 mm columns packed with Merck Lichrospher 100 RP-18 (5µm). Medium pressure liquid chromatography (MPLC) using a Buchi B688 pump equipped with a Buchi B687 gradient maker was carried out using Merck silica gel 60 (40–63µm) or Lichroprep 60 RP-18 (40–

63µm) with UV detection at 254 and 366 nm.

Plant material. Root bark of Citrus sinensis Obsbeck (Rutaceae), synonym Citrus aurantium var. dulcis (Y.

Tanaka) Hiroe, was collected from Valencia, Spain and identiﬁed by Dr Jose Tormo, Department of Pharmacology, University of Valencia, Spain. A voucher

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specimen is deposited at the herbarium of the Depart- ment of Botany and Pharmacognosie, Claude Bernard University, Lyon, France. Root barks were dried and crude extracts were obtained from the dried powder by successive macerations (24 h) at room temperature with hexane, dichloromethane and methanol which were removed by evaporation under reduced pressure.

Activity-guided compound isolation. The dichlorometh- ane extract (25 g) was fractionated by column chromatography on silica gel 60 (63–200µm), eluting with a hexane–EtOAc gradient (95.5 – 0.100), giving 18 fractions (F1–F18). Fractions F5 and F17 were selected after a drug accumulation assay showing their ability to reduce the P-gp-mediated daunorubicin efﬂux out of cells (33% and 47% respectively, Fig. 1). Fraction F5 (430 mg) was applied to a MPLC column using Lichroprep 60 RP-18 (40–63µm) eluted with MeOH–

H₂O gradient to afford eight fractions. Sub-fractions F5-4, F5-6, F5-7 and F5-8 were puriﬁed by preparative TLC (toluene–AcOEt/80:20), to afford respectively suberosin (1, 7 mg), clausarin (2, 18 mg), poncitrin (3, 3 mg) and xanthyletin (4, 16 mg). Fraction F17 (3.53 g) was separated on a silica gel MPLC column using Lichroprep 60 RP-18 (40 – 63µm) eluted with MeOH-H₂O gradient to afford 12 fractions (F17-1 to F17-12). Modulation of P-gp-mediated daunorubicin efﬂux observed in drug accumulation assay pointed out

the activity in two sub-fractions, F17-2 and F17-3. From fraction F17-2 (1.25 g), thamnosmonin (5, 6.2 mg), limonin (6, 9.2 mg), 2-methoxycitpressine I (7, 10.7 mg), buntanine (8, 2.8 mg), acrimarine C (9, 4.3 mg) and acrimarine E (10, 16.7 mg) were purified by sequential MPLC using Merck silica gel 60 (40–63µm) eluted with a hexane–AcOEt gradient, by preparative TLC (CH₂Cl₂–AcOEt/7:3) and by gel filtration on Sephadex LH-20 with MeOH as mobile phase. From fraction F17-3 (0.617 g), honuymine (11, 1.1 mg), citpressine I (12, 2.2 mg) and acrimarine B (13, 2.2 mg) were also purified by sequential MPLC using Merck silica gel 60 (40–63µm) eluted with hexane–AcOEt gradient, by preparative TLC (CH₂Cl₂:AcOEt – 7:3) and by gel filtration on Sephadex LH-20 with MeOH as the mobile phase.

Suberosin (1). Pale yellow amorphous powder. ESIMS m/z: [M+H]⁺ 245.2. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Mali et al., 2002).

Clausarin (2). White amorphous powder. ESIMS m/z: [M+H]⁺ 381.2. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Anwer et al., 1977; Huang et al., 1997).

Poncitrin (3). White amorphous powder. CIMS m/z:

[M+H]⁺ 327.2. This compound exhibited similar spectroscopic data (¹H) to published values (Tomimatsu et al., 1972; Kumar et al., 1995).

Figure 1. Chemical structures of the natural compounds isolated from Citrus sinenis.

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Xanthyletin (4). White amorphous powder. ESIMS m/z: [M+H]⁺ 229.1. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Mali et al., 2002).

(-)-Thamnosmonin (5). White amorphous powder.

[α]_D−16.3° (acetone, c 0.0675), Lit. [α]D+187° (Chang et al., 1976). ESIMS m/z: [M+H]⁺ 277.0. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Chang et al., 1976).

Limonin (6). White amorphous powder. [α]_D−102.4°

(acetone, c 0.0825), Lit. [α]_D −106° (Ng et al., 1987).

ESIMS m/z: [M+H]⁺ 471.1. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Ng et al., 1987; Zukas et al., 2004).

2-methoxycitpressine I (7). Yellow amorphous powder. ESIMS m/z: [M+H]⁺ 332.1. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Bowen and Patel, 1986).

Buntanine (8). Yellow amorphous powder. ESIMS m/z: [M+H]⁺ 356.0. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Wu, 1988).

Acrimarine C (9). Yellow amorphous powder. [α]_D

−11.6° (acetone, c 0.060), Lit. [α]_D −6.17° (Furukawa et al., 1990). ESIMS m/z: [M+H]⁺ 530.2. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Furukawa et al., 1990).

(-)-Acrimarine E (10). Yellow amorphous powder.

[α]_D−8.6° (acetone, c 0.035), Lit. [α]_D+20.1° (Furukawa et al., 1990). ESIMS m/z: [M+H]⁺ 530.0. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Furukawa et al., 1990).

Honyumine (11). Yellow powder. CIMS m/z: [M+H]⁺ 354.2. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Wu et al., 1986).

Citpressine I (12). Yellow powder. ESIMS m/z: [M+H]⁺ 302.1. This compound exhibited similar spectroscopic data (¹H) to published values (Wu et al., 1982).

Acrimarine B (13). Yellow amorphous powder. [α]_D

−30.0° (acetone, c 0.010), Lit. [α]_D +20.1° (Furukawa et al., 1990). CIMS m/z: [M+H]⁺ 544.1. This compound exhibited similar spectroscopic data (¹H and ¹³C NMR) to published values (Furukawa et al., 1990).

Evaluation of P-glycoprotein drug efﬂux modulation.

Extract, chromatographic fractions and pure compounds were tested in the K562/R7 MDR cell line overexpressing P-glycoprotein according to established protocols (Comte et al., 2001).

Cell culture. The human erythroleukemic cell line K562 was purchased from the American Type Culture Collection. The K562/R7 MDR cell line was obtained by prolonged exposure of K562 cells to doxorubicin.

Cell lines were cultured in RPMI 1640 supplemented with 10% newborn-calf serum, 2 mm glutamine, 200 U/

mL penicillin and 100µg/mL streptomycin. Cells were maintained at 37 °C in a 5% CO₂ atmosphere.

Drug accumulation assay. One million K562/R7 human leukemic cells expressing high levels of P-glycoprotein were incubated for 1 h at 37 °C in 1 mL RPMI 1640 medium containing a ﬁnal concentration of 10µ^M daunorubicin, in the presence or absence of inhibitor.

The cells were then washed twice with ice-cold

phosphate-buffered saline (PBSt), and kept on ice until analysis by flow cytometry on a FACS-II (Centre Leon Berard, Lyon, France). Assays were performed in du- plicate, with at least three separate experiments. CsA, an inhibitor of P-glycoprotein, was used as a positive control at 2µ^M final concentration. Compounds were tested at 10µ^M concentration and mixtures of 2, 7 and 10 were tested with 10µ^M concentration of the pure compounds. Their ability to inhibit P-gp-mediated drug efflux was quantified by comparing the intracellular fluorescence of daunorubicin (DNR) in the presence or absence of tested compounds, and was expressed in comparison with values recorded with the positive control cyclosporin A (CsA)

Activity

( )

= −

− ×

+

F F

product DNR DNR CsA DNR

100

where F_product stands for induced shift in fluorescence of DNR with the presence of coumarin, F_DNR for induced shift in fluorescence of DNR alone in the absence of inhibitor (negative control) and F_(DNR+CsA) for induced shift in fluorescence of DNR with the presence of CsA (positive control). Considering the stability of the cell population (size and granulosity), as shown by the analysis of the biparametric histogram of FACS (FSC-SSC), no cytotoxic effect of natural compounds was observed on the K562/R7 cell line at this concentration.

RESULTS AND DISCUSSION

Bioguided fractionation of the roots of Citrus sinensis Osbeck led to the isolation and identification of five coumarins (1–5), limonin (6) and seven acridones (7–13). Their structures were determined by means of spectrometric methods, including 1D and 2D NMR experiments and MS analysis, and, except for 5 and 10, were confirmed by comparison with values in the literature. The [α]_D values recorded for thamnosmonin (5) and acrimarine E (10) differed from those already reported (5, [α]_D −16.3° vs +187° in Chang et al., 1976 and 10, [α]_D −8.6° vs +20.1° in Furukawa et al., 1990) suggesting that the compounds isolated from the roots of Citrus sinensis were respectively the diastereoisomer and enantiomer derivatives of the metabolites previ- ously described. To our knowledge, except for the suberosin (1), xanthyletin (4) and limonin (6) previ- ously mentioned in this species (Wu and Furukawa, 1983), all the other compounds in this study are reported here for the first time in the roots of Citrus sinensis. It should be noted that acridones are charac- teristic alkaloids of Citrus and are found exclusively in the Rutaceae family and particularly in the roots (Michael, 2005). The literature survey indicated that only four acridinones, citrusinine I, citrusinine II, citracridone I and citbrasine, have been isolated in the Citrus sinensis species, specifically from the roots of C. sinensis var. brasiliensis (Wu and Furukawa, 1983). These four compounds were not found in our phytochemical investigation, suggesting some chemical disparity depending on the variety studied.

To evaluate their ability to inhibit P-gp-mediated drug efﬂux, compounds were tested in a daunorubicin accumulation assay in K562/R7 human leukemic cells

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expressing high levels of P-glycoprotein. Among these compounds, clausarin (2), 2-methoxycitpressine I (7) and acrimarine E (10) showed significant activities comprising between 45% and 65% compared with cyclosporin A (Fig. 2). Preliminary observation of structure-activity relationships indicated that activity was largely influenced by slight modifications of the substitution pattern. For the coumarinic group (1–5), as already observed (Raad et al., 2006), substitution of lactonic ring by hydrophobic moiety, in this case an α,α-dimethylallyl group, increased the activity (2 vs 3).

Considering the acridone group, signiﬁcant variations in activity were recorded by comparing the P-gp mediated drug efﬂux of derivatives with different substitutions

in 2-position (7 vs 11 and 12) and also in 7-position (10 vs 13). The methoxy group is then a favorable factor for activity when located at the 2-position whereas it has an unfavorable effect at the 7-position compared with a hydroxyl group (10 vs 13). Finally, the prenyl group, known in xanthones and flavonoids as a favorable substituent for the inhibition of Pgp-mediated drug efflux because of its lipophilicity (Comte et al., 2001), failed to appear in our experiments as an active moiety when it is located at the 3-position (8). No significant variation in activity was recorded by comparing the activity of pure compounds (2, 7 and 10) and their different mixtures suggesting a lack of synergetic effect for these compounds on P-pg activity.

REFERENCES

Anwer F, Shoeb A, Kapil RS, Popli SP. 1977. Clausarin – a novel coumarin from Clausena pentaphylla (Roxb.) DC. Experientia 33: 412–413.

Asschert J, De Vries E, Van der Kolk D, Muller M, Vellenga E.

1997. The combined effects of IL 3 and PSC 833 on daunorubicin and mitoxantrone cytotoxicity in two growth factor-dependent leukemia cell lines. Leukemia 11: 680–686.

Barthomeuf C, Grassi C, Demeule M, Fournier C, Boivin D, Beliveau R. 2005. Inhibition of P-glycoprotein transport function and reversion of MDR1 multidrug resistance by cnidiadin. Cancer Chemother Pharmacol 56: 173–181.

Bowen H, Patel YN. 1986. Acridone alkaloids from Pleiosper- mium alatum (Rutaceae). Phytochemistry 25: 429–431.

Chang PTO, Cordell GA, Aynilian GH, Fong HHS, Farnsworth NR. 1976. Alkaloids and coumarins of Thamnosmoma montana. Lloydia 39: 134 –140.

Comte G, Daskiewicz JB, Bayet C et al. 2001. C-isoprenylation of flavonoids enhances binding affinity toward P- glycoprotein and modulation of cancer cell chemoresistance.

J Med Chem 44: 763–768.

Di Pietro A, Conseil G, Perez-Victoria JM et al. 2002. Modula- tion by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters. Cell Mol Life Sci 59: 307–322.

Furukawa H, Ito C, Mizuno T et al. 1990. Spectrometric elucidation of acrimarines, the first naturally occurring acridone-coumarin dimers. J Chem Soc Perkin Trans I: 1593–

1599.

Goldstein LJ. 1995. Clinical reversal of drug resistance. Curr Probl Cancer 19: 65 –124.

Honda Y, Ushigome F, Koyabu N et al. 2004. Effects of grapefruit juice and orange juice components on P-glycoprotein- and MRP2-mediated drug efflux. Br J Pharmacol 143: 856–864.

Huang SC, Wu PL, Wu TS. 1997. Two coumarins from the root bark of Clausena excavata. Phytochemistry 44: 179–

181

Ikegawa T, Ushigome F, Koyabu N et al. 2000. Inhibition of P-glycoprotein by orange juice components, polymethoxy- flavones in adriamycin-resistant human myelogenous leukemia (K562/ADM) cells. Cancer Lett 160: 21–28.

Kam TS, Sim KM, Pang HS, Koyano T, Hayashi M, Komiyama K. 2004. Cytotoxic effects and reversal of multidrug resistance by ibogan and related indole alkaloids. Bioorg Med Chem Lett 14: 4487–4489.

Kumar V, Niyaz NMM, Wickramatatne DBM. 1995. Coumarins from stem bark of Paramignya monophylla. Phytochemistry 38: 805–806.

Mali RS, Joshi PP, Sandhu PK, Manekar-Tilve A. 2002.

Efficient syntheses of 6-prenyl-coumarins and linear pyranocoumarins: total synthesis of suberosin, toddaculin, O-methylapigravin, (O-methylbrosiperin), O-methylbalsami- ferone, dihydroxanthyletin, xanthyletin and luvangetin. J Chem Soc Perkin Trans 1: 371–376.

Michael JP. 2005. Quinoline, quinazoline and acridone alkaloids. Nat Prod Rep 22: 627–646.

Figure 2. Effect of fractions (F5 and F17), pure compounds 1–13 and mixtures of compounds 2, 7 and 10 from Citrus sinensis on the daunorubicin (DNR) accumulation in K562/R7 human leukemic cells. Each column represents mean ± SE of three experiments. CsA, cyclosporine A.

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a coumarin from the root and stem barks of Tetradium glabrifolium. J Nat Prod 50: 1160 –1163.

Raad I, Terreux R, Richomme P et al. 2006. Structure-activity relationship of natural and synthetic coumarins inhibiting the multidrug transporter P-glycoprotein. Bioorg Med Chem 14: 6979–6987.

Ramachandran C, Rabi T, Fonseca HB, Melnick SJ, Escalon EA.

2003. Novel plant triterpenoid drug amooranin overcomes multidrug resistance in human leukemia and colon carci- noma cell lines. Int J Cancer 105: 784 –789.

Romiti N, Tramonti G, Donati A, Chieli E. 2004. Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2. Life Sci 76:

293–302.

Tian R, Koyabu N, Takanaga H, Matsuo H, Ohtani H, Sawada Y.

2002. Effects of grapefruit juice and orange juice on the intestinal efflux of P-glycoprotein substrates. Pharm Res 19: 802–809.

Tomimatsu T, Hashimoto M, Shingu T, Tori K. 1972. Studies

on the chemical components of Rutaceae plants – VI: Com- ponents of the root of Poncirus trifoliata Raf. Tetrahedron 28: 2003–2010.

Wang EJ, Casciano CN, Clement RP, Johnson WW. 2001.

Inhibition of P-glycoprotein transport function by grapefruit juice psoralen. Pharm Res 18: 432–438.

Wu TS. 1988. Alkaloids and coumarins of Citrus grandis.

Phytochemistry 27: 3717–3718.

Wu TS, Furukawa H. 1983. Acridone alkaloids. VII. Constituents of Citrus sinensis Osbeck var. brasiliensis Tanaka. Chem Pharm Bull 31: 901–906.

Wu TS, Huang SC, Jong, TT, Lai JS, Furukawa H. 1986.

Honyumine, a new linear pyrano-acridone alkaloid from Citrus grandis Osbeck. Heterocycles 24: 41–43.

Wu TS, Kuoch CS, Furukawa H. 1982. Constituents of Citrus depressa (Rutaceae). Characterizations of five new acridone alkaloids. Heterocycles 19: 273–277.

Zukas AA, Breksa III AP, Manners GD. 2004. Isolation and characterization of limonoate and nomilinoate A-ring lactones. Phytochemistry 65: 2705–2709.