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Phytochemistry Letters 4 (2011) 30–33 Contents lists available at ScienceDirect Phytochemistry Letters journal

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Phytochemistry Letters

journal homepage: www.elsevier.com/locate/phytol

Letters journal homepage: www.elsevier.com/locate/phytol Cytochrome P450 2D6 inhibitory constituents of Lunasia

Cytochrome P450 2D6 inhibitory constituents of Lunasia amara

Subehan, Naoto Takahashi, Shigetoshi Kadota, Yasuhiro Tezuka *

Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

ARTICLE INFO

Article history:

Received 22 June 2010 Received in revised form 21 October 2010 Accepted 23 October 2010 Available online 5 November 2010

Keywords:

Lunasia amara Rutaceae Acridone alkaloids Cytochrome P450 2D6 (CYP2D6) Herb–drug interaction

ABSTRACT

From a MeOH extract of Lunasia amara , which showed the cytochrome P450 2D6 (CYP2D6) inhibition, 14 acridone alkaloids including two new alkaloids [5-hydoroxygraveoline ( 1 ) and 8-methoxyifflaiamine ( 2 )] were identified. Among the 14 acridone alkaloids, 5-hydoroxygraveoline ( 1 ) and lunamarine ( 3 ) showed moderate inhibition selective for CYP2D6, with IC 50 values of 4.7 m M and 1.8 m M, respectively. 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

1. Introduction

Cytochrome P450s (CYPs) are the principal enzymes responsi- ble for the oxidative metabolism of drugs and other xenobiotics, as well as several endogenous substances in mammalian tissues. Drug metabolism plays a critical role in the termination of drug action and influences the resident time of toxic chemicals within the body. Thus, the effects of oxidations by CYPs can be manifested in poor drug bioavailability and various acute and chronic toxicities, including birth defects, cancer susceptibility, and adverse drug interactions ( Guengerich, 2001 ). In human liver microsomes, CYP3A4 is the most abundant enzyme. Recent investigations have revealed that more than 50% of drugs used clinically are oxidized by CYP3A4, and about 30% of drugs are metabolized by CYP 2D6 (CYP2D6) ( Rendic and di Carlo, 1997 ). Inhibition of these enzymes is presumably the major mechanism responsible for several drug–drug interactions. Fur- ther, an inhibitor can also be used as a drug-sparing agent when taken concomitantly with conventional medicines, to decrease the dosage and financial cost of expensive drug regimes ( Clarke and Jones, 2002 ). Most of conventional pharmacokinetic literature deals with drug–drug interactions, but recently interactions between herbal agents and prescription drugs have drawn attention ( Izzo, 2004 ). These interactions occur since herbs are often self-administered along with therapeutic drugs. The popularity of herbal medicinal

* Corresponding author. Tel.: +81 76 434 7627; fax: +81 76 434 5059. E-mail address: tezuka@inm.u-toyama.ac.jp (Y. Tezuka).

products makes it important to understand the potential interac- tions between them and prescribed drugs, because almost all herbal medicines contain a mixture of pharmacologically active constituents. Indeed, several reports have demonstrated that natural compounds and herbal products may cause pharmacoki- netic interactions with Western drugs used clinically when they are administered simultaneously ( Nebel et al., 1999; Taylor and Wilt, 1999 ). We previously reported the CYP3A4 inhibitory constituents of Kampo medicinal herbs, Schizandra Fructus ( Iwata et al., 2004a ) and Evodia Fructus ( Iwata et al., 2005 ); and the Indonesian Jamu herbal medicines, Zingiber aromaticum ( Usia et al., 2004 ), Piper cubeba ( Usia et al., 2005a ), Cinnamomum burmani ( Subehan et al., 2008 ), and Foeniculum vulgare ( Subehan et al., 2007 ). In addition, we also reported the CYP2D6 inhibitory constituents of Cathar- anthus roseus ( Usia et al., 2005b ) and Piper nigrum ( Subehan et al., 2006 ). In our continuing study on the mechanism-based inhibition of Indonesian medicinal plants, we found the MeOH extract of Lunasia amara Blanco (Rutaceae) inhibited CYP2D6 (IC 50 9.1 m g/ mL). L. amara is a popular traditional medicine, used extensively in Indonesia either as a sole extract or in a mixture of several herbs for the treatment of swollen limbs, skin diseases, and inflamed or irritated eyes, while the wood decoction is known as an aphrodisiac in South Sulawesi Province ( PT Eisai Indonesia, 1995 ). Lunacridine from L. amara has been reported to be a DNA-intercalating topoisomerase II inhibitor ( Prescott et al., 2007 ), but there is no report on CYP2D6 inhibitory constituents of this plant. In our continuing research, we examined the active constituents of L. amara , which we report in this study.

1874-3900/$ – see front matter 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi: 10.1016/j.phytol.2010.10.006

[ ( ) T D $ F I G

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Subehan et al. / Phytochemistry Letters 4 (2011) 30–33 31 Fig. 1. Structures of new acridone

Fig. 1. Structures of new acridone alkaloids.

2. Results and discussion

The bark of L. amara was extracted with MeOH, and the MeOH extract was subjected to column chromatography to give two new acridone alkaloids, 5-hydroxygraveroline ( 1 ) and 8-methoxyif- flaiamine ( 2 ), together with 12 known ones ( Fig. 1 ). The known alkaloids were identified by analyzing their spectral data and comparing them with those found in the literatures. They were lunamarine ( 3 ) ( Goodwin et al., 1959a ), ifflaiamine ( 4 ) ( Chamber- lain and Grundon, 1971 ), orixalone A ( 5 ) ( Ito et al., 2004 ), lunidonine ( 6 ) ( Ru¨ egger and Stauffacher, 1963 ), lunacridine ( 7 ) ( Barr et al., 1994; Brown et al., 1980 ), lunidine ( 8 ) ( Ru¨ egger and Stauffacher, 1963 ), N -methylflindersine ( 9 ) ( Kamperdick et al., 1999 ), lunacrine ( 10 ) ( Goodwin et al., 1959b; Ramesh et al., 1984 ), balfourodine ( 11 ) ( Brown et al., 1980 ), lunine ( 12 ) ( Goodwin et al., 1959b ), lunasine ( 13 ) ( Price, 1959 ), and balfourodinium ( 14 ) ( Rideau et al., 1979 ). Compound 1 was obtained as yellow powder, having a molecular formula of C 17 H 13 NO 4 . The 1 H NMR spectrum of 1 showed signals due to six aromatic protons and an isolated olefinic proton ( d H 6.20, s) together with those of a hydrogen-bonded hydroxyl ( d H 14.70, s), a methylenedioxy ( d H 6.08, s, 2H), and a methyl ( d H 3.60, s) group. The 13 C NMR spectrum of 1 showed 17 carbon signals consisting of seven sp 2 methines, three oxygen- substituted sp 2 quaternary carbons, four sp 2 quaternary carbons, a methylenedioxy carbon ( d C 101.8, t), a N -methyl carbon ( d C 38.0, q), and a carbonyl carbon ( d C 181.9, s). Analysis of the COSY correlations indicated that the six aromatic protons consist of a 1,2,3-trisubstituted benzene ring ( d H 7.56, t, J = 8.1 Hz; d H 6.87, d, J = 8.1 Hz; d H 6.85, d, J = 8.1 Hz) and a 1,2,4-trisubstituted benzene ring ( d H 6.94, d, J = 8.1 Hz; d H 6.88, dd, J = 8.1, 1.8 Hz; d H 6.85, d, J = 1.8 Hz) ( Table 1 ). These spectral data suggested that compound 1 was likely a quinolone alkaloid similar to lunamarine ( 3 ) isolated from the same extract, except for the presence of an oxygen- substituent at C-5 or C-8 instead of that at C-7 in 3 . The presence of the signal due to the hydrogen-bonded hydroxyl proton ( d H 14.70, s) suggested the locations of the hydroxyl and the carbonyl groups at C-5 and C-4, respectively, which was confirmed by the HMBC correlations ( Fig. 2 ). Thus, compound 1 was determined to be 5- hydroxygraveroline.

Table 1 1 H (400 MHz) and 13 C (100 MHz) NMR data for new acridone alkaloids 1 and 2 in CDCl 3 .

 

1

2

1 H

13 C

1 H

13 C

1

8.86 br s

2

155.2 s

160.9 s

3

6.20 s

111.0 d

116.9 s

4

181.9 s

162.7 s

5

162.9 s

7.29 d (8.0)

110.0 d

6

6.85 d (8.1)

108.9 d

7.10 t (8.0)

121.6 d

7

7.56 t (8.1)

134.3 d

6.94 d (8.0)

114.6 d

8

6.87 d (8.1)

104.6 d

145.7 s

4a

114.0 s

129.6 s

 

8a

142.9 s

112.5 s

1

0

129.0 s

2

0

6.85 d (1.8)

122.7 d

4.61 q (6.6)

91.5 d

3

0

148.1 s

44.2 s

4

0

149.0 s

1.49 s (3H) 1.27 s (3H) 1.45 d (6.6) (3H)

20.3 q

5

0

6.94 d (8.1) 6.88 dd (8.1, 1.8) 3.60 s (3H)

108.8 d

25.6 q

6 0 N–CH O–CH O–CH 2 –O OH

3

3

108.9 d

14.5 q

38.0 q

 

3.86 s (3H)

56.0 q

6.08 s (2H) 14.70 s

101.8 t

Assignments are based on COSY, HMQC, and HMBC experiments.

Compound 2 was isolated as a colorless amorphous solid having a molecular formula of C 15 H 16 NO 3 . Its IR spectrum showed absorptions corresponding to NH (3400 cm 1 ) and carbonyl groups (1650 cm 1 ). The 1 H NMR spectrum of 2 revealed signals due to three aromatic protons ( d H 7.29, d, J = 8.0 Hz; d H 7.10, t, J = 8.0 Hz; d H 6.94, d, J = 8.0 Hz), two quaternary methyls ( d H 1.49, 1.27, both 3H, s), and an (O)–CH–CH 3 group ( d H 4.61, q, J = 6.6 Hz; d H 1.45, d, J = 6.6 Hz), together with those of a methoxyl ( d H 3.86, 3H, s) and an amino proton ( d H 8.86, br s) ( Table 1 ). The 13 C NMR spectrum showed 15 carbon signals including three sp 2 methines, two oxygen-substituted sp 2 quaternary carbons, three sp 2 quater- nary carbons, an oxygen-substituted methine, a quaternary sp 3 carbon, and three methyls, together with those of a methoxyl ( d C 56.0, q) and a carbonyl carbon ( d C 162.7, s) ( Table 1 ). These signals suggested that 2 was a quinolone alkaloid similar to ifflaiamine ( 4 ) ( Chamberlain and Grundon, 1971 ), but 2 differed from 4 due to the presence of a methoxyl group at C-5 or C-8 and the lack of the N - methyl group of 4 . Then, the HMBC correlations H-6/C-4a, H-6/C-8, H-5/C-8a, and OCH 3 /C-8 confirmed the location of the methoxyl group at C-8. These and other HMBC correlations confirmed the structure of 2 ( Fig. 3 ). We could not determine the absolute stereochemistry at C-2 0 due to the small amount obtained. However, 2 should have the same absolute stereochemistry as 4 because 2 showed the same levorotatory optical rotation ([ a ] D 30 8 ). Thus, compound 2 was concluded to be N -demethyl-8- methoxyifflaiamine. All compounds isolated were tested for their inhibitory activities on CYP3A4 and CYP2D6. Among the 14 compounds

[ ( ) T D $ F I G ]

and CYP2D6. Among the 14 compounds [ ( ) T D $ F I G ]

[ ( ) T D $ F I G ]

the 14 compounds [ ( ) T D $ F I G ] [ ( )

32

Subehan et al. / Phytochemistry Letters 4 (2011) 30–33

Table 2 Inhibitory activities (IC 50 m M) of isolated alkaloids in the metabolism mediated by CYP3A4 and CYP2D6.

Compound

IC 50 (m M)

CYP3A4

CYP2D6

1

> 100

4.7

2

32.5

>

100

3

46.8

1.8

4

69.7

>

100

5

> 100

>

100

6

23.9

>

100

7

> 100

95.5

8

71.1

>

100

9

47.8

>

100

10

57.6

>

100

11

> 100

>

100

12

46.9

21.8

13

> 10

7.6

14

61.7

14.5

Ketoconazole a

0.2

Quinidine a

0.05

a Positive controls.

tested, 1 and 3 showed moderate inhibition selective for CYP2D6,

with IC 50 values of 4.7 and 1.8 m M, respectively ( Table 2 ). It is known that substrates for CYP2D6 contain a basic nitrogen atom at

˚

5–7 A from the oxidation site and that the aromatic rings are coplanar ( Meyer et al., 1986; Wolff et al., 1985 ). Moreover, many potent CYP inhibitors are nitrogen-containing drugs, including imidazole, pyridines, and quinolines, which can inhibit CYP through binding to the prosthetic heme iron or to the lipophylic region of the protein ( Lin and Lu, 1998 ). On the other hand, the methylenedioxyphenyl ring could be metabolized by CYP2D6 to generate carbene intermediates, which can then form a strong covalent complex with iron center of the heme in cytochromes ( Bertelsen et al., 2003; Murray, 2000 ). Thus, the presence of the quinolone group and/or the methylenedioxyphenyl ring in 1 and 3 might be correlated with CYP inhibition.

3. Experimental

3.3. Extraction and isolation

Fresh wood and bark of L. amara (4 kg) were crushed, extracted with MeOH, evaporated under reduced pressure followed by lyophilization to give a MeOH extract (128 g). Part of the MeOH extract (20 g) was subjected to silica gel column chromatography with a CHCl 3 –MeOH solvent system, yielding seven fractions [fr. 1:

CHCl 3 eluate, 836 mg; fr. 2: CHCl 3 –MeOH (90:10) eluate, 1.6 g; fr. 3:

CHCl 3 –MeOH (80:20) eluate, 343 mg; fr. 4: CHCl 3 –MeOH (70:30) eluate, 5.4 g; fr. 5: CHCl 3 –MeOH (60:40) eluate, 3.3 g; fr. 6: CHCl 3 – MeOH (60:40) eluate, 2.5 g; fr. 7: MeOH eluate, 5.5 g]. Fraction 2 was separated by repetitive normal-phase prep. TLC with hexane–EtOAc (7:3) to afford 5-hydroxygraveroline (1 , 3.0 mg), orixalone A ( 5, 15.2 mg), lunidonine ( 6, 15.0 mg), and N -methylflindersine (9 , 21.9 mg). Fraction 3 was purified repeatedly by normal-phase prep. TLC with EtOAc–toluene (1:5) to give ifflaiamine (4, 32.8 mg), lunacridine (7, 3.5 mg), lunidine (8 , 17.0 mg), and 9 (34.2 mg), while fraction 4 was repeatedly subjected to normal-phase prep. TLC with EtOAc–toluene (2:8) to give 8-methoxyifflaiamine ( 2, 4.0 mg), 4 (6.9 mg), 9 (13.9 mg), lunacrine (10, 31.4 mg), and lunine (12, 3.5 mg). Fraction 5 was separated by MPLC on silica gel with CH 2 Cl 2 – MeOH (0–60% of MeOH), followed by normal-phase prep. TLC with CH 2 Cl 2 –MeOH (88:12), reversed-phase prep. TLC with acetone–H 2 O (2:1), and/or normal-phase prep. TLC with CH 2 Cl 2 –MeOH–NH 3 (85:15:2), to give lunamarine (3 , 3.2 mg) and balfourodine (11, 14.7 mg). Fraction 6 was purified by a combination of normal-phase prep. TLC with CH 2 Cl 2 –MeOH (85:15) and reversed-phase prep. TLC with CH 3 CN–H 2 O (2:3) to give lunasine (13, 25.9 mg) and balfourodinium ( 14 , 12.3 mg).

3.3.1. 5-Hydroxygraveroline ( 1 )

Yellow powder; HR-FAB-MS m / z 296.0890 [M+H] + (calc. for C 17 H 14 O 4 N, 296.0923); 1 H and 13 C NMR (400 MHz and 100 MHz,

CDCl 3 ): Table 1 .

3.3.2. N-Demethyl-8-methoxyifflaiamine ( 2 )

Colorless amorphous solid; ½ a 24 30 ( c 0.25, CHCl 3 ); HR-FAB- MS m / z 260.1322 [M+H] + (calc. for C 15 H 18 NO 3 , 260.1287); 1 H and 13 C NMR (400 MHz and 100 MHz, CDCl 3 ): Table 1 .

D

3.1. General

Optical rotation was measured with a JASCO DIP-140 digital polarimeter. HR-FAB-MS was recorded on a JEOL JMS-700T mass spectrometer using glycerol as matrix. The 1 H and 13 C NMR spectra were recorded at 400 MHz and 100 MHz, respectively, on a JEOL JNM-LA400 spectrometer. Chemical shifts are reported in d (ppm) using TMS as internal standard and coupling constants ( J ) were measured in Hz. Column chromatography was carried out on silica gel (70–230 mesh, Kanto Chemical Co. Ltd., Tokyo, Japan). Medium pressure liquid chromatography (MPLC) was conducted with a Bu¨ chi MPLC apparatus on silica gel. Normal or reversed- phase TLC was performed on Merck precoated silica gel 60F 254 plates or on Merck RP-18F 254 plates, and spots were visualized by using ceric sulfate spray reagent. All reagents used were analytical grade.

3.2. Plant material

Wood and bark of L. amara Blanco was obtained at Gadjah Mada University, Yogyakarta, Indonesia, in September 2007, and was identified by Mr. Sri Suhadiyah, Sulawesi Biodiversity Foundation, Makassar, Indonesia. A voucher sample (TMPW25679) is held at the Museum for Materia Medica, Analytical Research Center for Ethnomedicines, Institute of Natural Medicine, University of Toyama, Toyama, Japan.

3.4. CYP inhibition assay

[ O -Methyl- 14 C]dextromethorphan (55 mCi/mmol, radiochemi- cal purity > 99%) and [ N -methyl- 14 C]erythromycin (55 mCi/mmol, radiochemical purity > 99%) were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). b -Nicotinamide adenine dinucleotide phosphate (NADP + , oxidized form), glucose- 6-phosphate (G-6-P), and G-6-P dehydrogenase were purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Human liver microsomes (HLM) were obtained from XenoTech, LCC (Lenexa, KS, USA) and stored at 80 8 C prior to use. Inhibitory activity on the metabolism mediated by CYP2D6 and CYP3A4 was determined using a radiometric measurement of [ 14 C]formaldehyde formed by the reaction with [ O -methyl- 14 C]- dextromethorphan or [ N -methyl- 14 C]erythromycin as a substrate ( Iwata et al., 2004b ). Briefly, in disposable culture tubes (13 100 mm; Iwaki, Tokyo, Japan) containing phosphate buffer (0.1 M, pH 7.4), 50 m L of HLM (4 mg protein/mL), sample, and purified water were added in 500 m L of total incubation volume. After a preincubation for 5 min in shaking water bath at 37 8 C, the reaction is initiated by addition of 50 m L of the NADPH-generating system (4.2 mg/mL of NADP + in a solution of 100 mM G-6-P, 100 mM MgCl 2 , and 10 U/mL G-6-P dehydrogenase). Substrate solution (50 m L) was added and incubated for 20 min (for CYP2D6) or for 10 min (for CYP3A4) in a shaking water bath at 37 8 C. The reaction was stopped by addition of 125 m L of 10% trichloroacetic

Subehan et al. / Phytochemistry Letters 4 (2011) 30–33

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acid, and the solution was centrifuged at 3000 rpm for 10 min at room temperature. The supernatant was applied to Envi-carb solid phase extraction columns (Supelco, PA, UK) and was eluted with two volumes of ultrapure water (2 500 m L). After adding 10 mL of Clear-sol I (Nacalai Tesque, Inc., Kyoto, Japan), the eluted radioactivity was quantified by liquid scintillation counting LS 6500 (Beckman, CA, USA). Correction was made for radioactivity eluted from control incubation in which HLM and NADPH- generating systems had been omitted. The assays were performed in triplicate for all test specimens, and remaining activity was analyzed using the WinNonlin software (ver.3.1; Pharsight Corporation; Mountain View, CA, USA).

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