Fitoterapia 92 (2014) 270–273 Contents lists available at ScienceDirect Fitoterapia journal homepage: www.elsevier.com/locate/fitote New furoquinoline alkaloids from the leaves of Evodia lepta Jirapast Sichaem a, Apapond Jirasirichote a, Krittakorn Sapasuntikul a, Suttira Khumkratok b, Pattara Sawasdee a, Thi My Lien Do c, Santi Tip-pyang a,⁎ a b c Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand Department of Organic Chemistry, University of Science, National University—Ho Chi Minh City, 748355 Ho Chi Minh City, Viet Nam a r t i c l e i n f o Article history: Received 8 October 2013 Accepted in revised form 29 November 2013 Available online 9 December 2013 Keywords: Evodia lepta Rutaceae Furoquinoline alkaloids AChE and BChE activities a b s t r a c t Three new furoquinoline alkaloids, leptanoines A–C (1–3) along with three known compounds (4–6) were isolated from the leaves of Evodia lepta. Their structures were identified by interpretation of their spectroscopic data as well as comparison with those reported in the literature. In addition, all isolated compounds were evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities. Compound 4 showed the highest inhibitory activity towards BChE with an IC50 value of 47.9 μM. On the other hand, Compound 5 showed the highest inhibitory activity towards AChE with an IC50 value of 69.1 μM. © 2013 Elsevier B.V. All rights reserved. 1. Introduction 2. Experimental Evodia lepta (Spreng.) Merr., locally known as “Plia Kra Ting” in Thailand, is a shrub and herbaceous plant belonging to the family Rutaceae, and is widely distributed in many areas of Southeast Asia (Vietnam, China and Indonesia) [1]. Notably, Plia Kra Ting is a traditional medicinal plant used to treat arthritis, fever, chickenpox, epidemic influenza, meningitis, infectious hepatitis, and antipruritic, depurative and febrifuge diseases [2,3]. Several phytochemical studies from the extracts of E. lepta have been performed, with extracts revealing the presence of quinoline-type alkaloids, flavonoids, dichromans, dichromenes and a coumarin [4–9]. In the current investigation we describe the isolation, and structural elucidation of three new furoquinoline alkaloids, leptanoines A–C (1–3), together with three known compounds (4–6) from the leaves of E. lepta, as well as an evaluation of their AChE and BChE activities. 2.1. General experimental procedures ⁎ Corresponding author. Tel.: +66 2 218 7625; fax: +66 2 218 7598. E-mail address: Santi.Ti@chula.ac.th (S. Tip-pyang). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.002 1D and 2D NMR spectra were recorded on a Bruker 400 AVANCE spectrometer, and the chemical shifts were reported in parts per million (ppm) using TMS as the internal standard. Adsorbents such as silica gel 60 (Merck) were used for column chromatography and in radial chromatography (chromatotron model 7924T, Harrison Research). Merck silica gel 60F254 plates were used for TLC. HRESIMS spectra were obtained using a Bruker MICROTOF model mass spectrometer. IR data was obtained using a Nicolet 6700 FT-IR spectrometer using KBr discs. UV–visible absorption spectra were taken on a UV-2550 UV–vis spectrometer (Shimadzu, Kyoto, Japan). 2.2. Plant material The leaves of E. lepta were collected from Chom Thong district, Chiang Mai province, Thailand, in July 2013. The plant material was identified by Ms. Suttira Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, and a specimen retained as a reference (Khumkratok no. 1-32). J. Sichaem et al. / Fitoterapia 92 (2014) 270–273 2.3. Extraction and isolation Air-dried and finely powdered leaves (1 kg) of E. lepta were sequentially extracted at room temperature for six days with MeOH (2 × 5 l). The extract was evaporated in vacuo to obtain the MeOH crude extract (112 g). After removal of solvent under reduced pressure, the MeOH crude extract was subjected to preliminary vacuum liquid chromatography (VLC) over silica gel (Merck Art 7730) eluting with increasing polarity using CH2Cl2, EtOAc and MeOH to afford four major fractions (A–D). Fraction B was subjected to silica gel column chromatography eluting with 100% CH2Cl2 to give five sub-fractions (B1–B5). Sub-fraction B2 was purified by radial chromatography (chromatotron) using 100% CH2Cl2 to obtain 1 (20 mg), 2 (12 mg) and 4 (9 mg). Sub-fraction B3 was purified by chromatotron using CH2Cl2–EtOAc (1:0–0:1) to afford 3 (20 mg) and 5 (9 mg). Finally, sub-fraction B4 was subjected to silica gel column chromatography eluting with CH2Cl2 and EtOAc gradient systems to afford 6 (13 mg). The chemical structures of these compounds were determined using various spectroscopic methods (1D and 2D NMR, IR, UV and MS). 2.4. AChE and BChE activity assays Stock solutions of the test compounds (1–6), and the one reference standard anti-ChE compound (galantamine) were each prepared in 50 mM Tris–HCl (pH 8.0) containing ≤10% (v/v) methanol. The AChE and BChE inhibitory activity assays were performed using a modified Ellman's colorimetric method, as previously reported [10]. Acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), AChE from electric eels (type VI-S, EC 3.1.1.7), BChE from horse serum (EC 3.1.1.8), and galantamine hydrobromide was obtained from Sigma-Aldrich. In a 96-well microplate, 25 μl of 1.5 mM ATCI or BTCI, 125 μl of 3 mM DTNB, 50 μl of 50 mM Tris–HCl buffer (pH 8) and 25 μl of the test compound were added to each of triplicate wells followed by 25 μl of either AChE (0.3 U/ml) or BChE (1 U/ml). The absorbance was then measured at 415 nm every 5 s over a 2 min period using a Sunrise microplate reader (P-Intertrade Equipments, Australia). A negative control reaction was carried out using the same volume of solvent (25 μl of 10% (v/v) methanol in 50 mM Tris–HCl pH 8.0), instead of the test compound. The velocities of the reactions were measured. Enzyme activity was calculated as a percentage of the velocity observed in the presence of the test compound compared to that of the control assay. 3. Results and discussion The MeOH crude extract was initially subjected to silica gel column chromatography, with several sub-fractions being further purified to afford six pure furoquinoline alkaloids (1–6) (Fig. 1). The structures of compounds 1–3 are new while the remaining compounds (4–6) are known compounds including melineurine (4) [11], skimmianine (5) [12] and 7-hydroxydictamnine (6) [13], which were identified by comparison of their spectroscopic data with those reported in the literature. 271 Leptanoine A (1) was obtained as a pale yellow gum and the molecular formula C17H15O3N was deduced from the HRESIMS (m/z 282.1104 [M + H]+) spectrum. The 13C NMR spectrum exhibited a total of 17 carbon resonances, attributed to one methyl, one methoxyl, one methylene, seven methine and seven quaternary carbon centers. The 1H NMR spectrum revealed the presence of a downfield methoxy substituent at δ 4.44 at C-4, two coupled doublet resonances at δ 7.59 and δ 7.06 (each 1H, J = 2.6 Hz), characteristic of H-2 and H-3 of a disubstituted furan ring, and three aromatic protons [δH 8.21 (d, J = 9.2 Hz, H-5), 7.17 (dd, J = 9.2, 2.4 Hz, H-6) and 7.50 (d, J = 2.4 Hz, H-8)] indicating the presence of a 4,7-dioxyfuroquinoline nucleus [14]. This nucleus was also confirmed by the HMBC correlations of H-2 to C-3a (δC 102.7) and C-3 (δC 104.9), H-3 to C-2 (δC 142.5) and C-9a (δC 164.5), H-5 to C-7 (δC 158.5) and C-8a (δC 147.4), and H-6 to C-4a (δC 115.0), C-7 (δC 158.5) and C-8 (δC 110.5) (Fig. 2). The remaining signals in the 1H NMR spectrum indicated the existence of a prenyl-derived (C5) side chain. The presence of a \OCH_CHC(CH3)_CH2 chain was shown from the correlations observed between the methyl protons at δH 1.92 and carbon centers δC 138.9 (C-3′) and 115.1 (C-4′), and the methine proton at δH 6.24 (1H, d, J = 12.2 Hz, H-2′) with carbon centers δC 143.1 (C-1′), 138.9 (C-3′) and 115.1 (C-4′). Moreover, H-1′ and H-2′ were assigned to be in a cis-configuration from their coupling constants (J = 12.2 Hz) and was also found this chain as natural in umbelliferone[3′-hydroxymethyl- 1t.-buten-1′-yl]-ether [15]. This prenylderived residue was shown to be attached to C-7 of the furoquinoline framework due to the correlation of H-1′ (δH 6.92, 1H, d, J = 12.2 Hz) with C-7 at δC 158.5. The proposed structure for (Z)-4-methoxy-7-[(3-methylbuta1,3-dienyloxy)]furo[2,3-b]quinoline has not been previously reported, and this compound is named herein as leptanoine A (Fig. 1). Leptanoine B (2) was isolated as a pale yellow gum. It was assigned the molecular formula C18H17O4N from its quasimolecular ion peak at m/z 334.1016 [M + Na]+ in the HRESIMS, corresponding to eleven degrees of unsaturation. The 1H NMR spectrum indicated sharp singlets attributable to one methyl group at δH 1.92, two methoxy groups at δH 4.03 and 4.46, and two aromatic protons at δH 7.54 and 7.54. The 1 H–1H COSY NMR spectrum showed cross peaks between the two protons at δH 7.60 ppm and 7.06 ppm (J = 2.1 Hz) which were assigned to be H-2 and H-3 of a disubstituted furan ring, and these were attached to the carbon centers at δC 143.1 and 104.7, respectively. A comparison of the 1H and 13C NMR spectra of compound 2 with those of leptanoine A (1) showed that compound 2 also possessed a furoquinoline nucleus with the same side chain, except for the presence of one methoxy group at C-6 which was proved through HMBC correlations between the methoxy protons at δH 4.46 and δC 148.3 (C-6). The presence of the 3-methylbuta-1,3-dienyloxyl chain was confirmed from the HMBC correlations of H-5′ (δH 1.92, s) to C-2′ (δC 119.2), C-3′ (δC 138.7) and C-4′ (δC 115.4), and H-2′ [δH 6.33 (1H, d, J = 12.2 Hz)] to carbons C-1′ (δC 142.5), C-3′ (δC 138.7) and C-4′ (δC 115.4). This chain was also linked to C-7 of the furoquinoline nucleus, as confirmed by the correlation of H-1′ (δH 6.87) to C-7 at δC 150.3. Thus, the chemical structure of 2 was assigned as (Z)-4,6-dimethoxy- 272 J. Sichaem et al. / Fitoterapia 92 (2014) 270–273 R1 OCH3 5 4a 3 3a 6 2 1' 2' O 7 3' N O R2 5' 4' 8a 9a 1 R1 = R 2 = H 2 R1 = OCH 3 ; R 2 = H OCH 3 OCH 3 R1 O R1 O N O N R2 R2 5 R1 = R 2 = OCH 3 6 R1 = OH; R 2 = H 3 R1 = OCH 3; R 2 = H 4 R1 = R 2 = H Fig 1. Structures of 1–6 isolated from the leaves of E. lepta. which is attached at C-7, as in 2, as shown by the HMBC correlation between the downfield allylic 2H-1′ protons at δH 4.76 (d, J = 4.0 Hz) and the oxygenated aromatic carbon center C-7 (δC 152.8). This new compound was therefore assigned the name of 4,6-dimethoxy-7-(3-methylbut-2-enyloxy) furo[2,3-b]quinolone and given the trivial name of leptanoine C (Fig. 1). The isolated compounds were evaluated for their AChE and BChE activities. The AChE and BChE data showed that compound 4 showed highest inhibitory activity towards BChE with an IC50 value of 47.9 μM (Table 2). On the other hand, compound 5 showed the highest inhibitory activity towards AChE with an IC50 value of 69.1 μM. 7-[(3-methylbuta-1,3-dienyloxy)]furo[2,3-b]quinoline, herein named as leptanoine B (Fig. 1). Leptanoine C (3) was isolated as a pale yellow gum. Its molecular formula was determined as C18H20NO4 through its pseudomolecular ion peak at m/z 314.1384 [M + H]+ in the HRESIMS spectrum, and this was supported by 13C NMR data which indicated the presence of eighteen carbon centers (Table 1). The spectral pattern of 3 closely resembles that of 2 except that in 3 the methylene carbon resonated at δC 66.2 (C-1′) and methyl carbon at δC 26.0 (C-4′), whereas in 2 they occur at δC 142.5 (C-1′) and δC 115.4 (C-4′). The O-prenyl-derived group at C-7 in 2 was replaced by a 3′-methyl-2′-butenyl ether side chain in 3, OCH 3 O N O 1 OCH 3 H 3CO O N 2 O OCH 3 H 3CO O N 3 Fig. 2. Selected HMBC (arrow curves) and COSY (bold lines) correlations in compounds 1–3. O 273 J. Sichaem et al. / Fitoterapia 92 (2014) 270–273 Table 1 1 H and 13C NMR data for 1–3 in CDCl3. Position 1 2 Table 2 Inhibitory activity of the isolated compounds (1–6) from E. lepta against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). 3 δH (ppm), J δC δH (ppm), δC δH (ppm), δC (Hz) (ppm) J (Hz) (ppm) J (Hz) (ppm) 2 3 3a 4 4a 5 6 7 8 8a 9a 1′ 2′ 3′ 4′-cis 4′-trans 5′ 4-OCH3 6-OCH3 7.59, d (2.6) 7.06, d (2.6) – – – 8.21, d (9.2) 7.17, dd (9.2, 2.4) – 7.50, d (2.4) – – 6.92, d (12.2) 6.24, d (12.2) – 4.95, s 4.89, s 1.92, s 4.44, s 142.5 143.1 102.7 157.2 115.0 124.3 7.60, d (2.1) 7.07, d (2.1) – – – 7.54, s 142.8 103.4 156.2 114.9 101.5 7.59, d (2.6) 7.06, d (2.6) – – – 7.46, s 116.6 – 148.3 – 148.7 158.5 110.5 – 7.54, s 150.3 111.6 – 7.48, s 152.8 106.8 147.4 164.5 143.1 – – 6.87, d (12.2) 6.33, d (12.2) – 4.95, s 4.90, s 1.92, s 4.46, s 4.03, s 142.4 163.8 142.5 – – 4.76, d (4.0) 5.59, t (6.6) – 1.80, s – 1.80, s 4.46, s 4.00, s 139.0 162.0 66.2 104.9 118.3 138.9 115.1 19.1 59.2 104.7 119.2 138.7 115.4 19.2 59.1 56.3 104.9 103.4 156.0 112.9 100.5 119.2 138.8 26.0 18.5 59.3 56.2 Compound 1 2 3 4 5 6 Galantamine IC50 (μM) AChE BChE N200 N200 N200 95.3 69.1 N200 5.6 56.8 99.4 N200 47.9 130.2 151.7 26.3 Acknowledgments The authors are grateful to the Graduate School of Chulalongkorn University for a Postdoctoral Fellowship (Ratchadaphiseksomphot Endowment Fund) to JS, the Higher Education Research Promotion and National Research University Project of Thailand, the Office of the Higher Education Commission and the Ratchadaphiseksomphot Endowment Fund (FW645A) for partially supporting this project. We also thank Dr Christopher Smith of the Department of Chemistry, Faculty of Science, Chulalongkorn University, for his editorial comments. Appendix A. Supplementary data 4. Spectroscopic data of compounds Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2013.12.002. 4.1. Leptanoine A (1) References Pale yellow gum; UV (MeOH) λmax (log ε): 245 (4.7), 324 (3.9) nm; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) are shown in Table 1; positive ion HRESIMS m/z: [M + H]+ 282.1104 for C17H15O3N + H (calcd. 282.1130). [1] Yoon JY, Jeong HY, Kim SH, Kim HG, Nam G, Kim JP, et al. Methanol extract of Evodia lepta displays Syk/Src-targeted anti-inflammatory activity. J Ethnopharmacol 2013;48:999–1007. [2] Juan JS, Lee NH. Chinese medicinal herbs of Hong Kong; 1981 58. [3] Gunawardana YAGP, Cordell GA, Ruangrungsi N, Chomya S, Tantivatana P. Traditional medicinal plants of Thailand VII. Alkaloids of Evodia lepta and Evodia gracilis. 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[12] Cardoso-Lopes EM, Maier JA, Da Silva MR, Regasini LO, Simote SY, Lopes NP, et al. Alkaloids from stems of Esenbeckia leiocarpa Engl. (Rutaceae) as potential treatment for Alzheimer disease. Molecules 2010;15:9205–13. [13] Tillequin F, Baudouin G, Ternoir M, Koch M, Pusset J, Sevenet T. Plantes de nouvelles-calédonie. LXX VIII. Alcaloïdes des tiges feuillées de Melicope lasioneura. J Nat Prod 1982;45:486–8. [14] Tarus PK, Coombes PH, Crouch NR, Mulholland DA, Moodley B. Furoquinoline alkaloids from the southern African Rutaceae Teclea natalensis. Phytochemistry 2005;66:703–6. [15] Hamerski D, Beier RC, Kneusel RE, Matern U, Himmelspacht K. Accumulation of coumarins in elicitor-treated cell suspension cultures of Ammi majus. Phytochemistry 1990;29:1137–42. 4.2. Leptanoine B (2) Pale yellow gum; UV (MeOH) λmax (log ε): 243 (4.7), 323 (4.1), 335 (4.0) nm; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) are shown in Table 1; positive ion HRESIMS m/z: [M + Na]+ 334.1016 for C18H17O4N + Na (calcd. 334.1055). 4.3. Leptanoine C (3) Pale yellow gum; UV (MeOH) λmax (log ε): 243 (4.7), 323 (4.2), 334 (4.1) nm; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) are shown in Table 1; positive ion HRESIMS m/z: [M + Na]+ 314.1385 for C18H19O4N + H (calcd. 314.1392). Conflict of Interest The authors have declared that there is no conflict of interest.