Natural Product Research Formerly Natural Product Letters
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New alkaloids from the leaves of Evodia rutaecarpa Xiao Xia, Jian-Guang Luo, Rui-Huan Liu, Ming-Hua Yang & Ling-Yi Kong To cite this article: Xiao Xia, Jian-Guang Luo, Rui-Huan Liu, Ming-Hua Yang & Ling-Yi Kong (2016): New alkaloids from the leaves of Evodia rutaecarpa, Natural Product Research, DOI: 10.1080/14786419.2016.1146888 To link to this article: http://dx.doi.org/10.1080/14786419.2016.1146888
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Date: 02 March 2016, At: 12:02
Natural Product Research, 2016 http://dx.doi.org/10.1080/14786419.2016.1146888
New alkaloids from the leaves of Evodia rutaecarpa Xiao Xia, Jian-Guang Luo, Rui-Huan Liu, Ming-Hua Yang and Ling-Yi Kong
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State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, People’s Republic of China
ABSTRACT
One new indole alkaloid (1) and one new indole alkaloidal glycoside (2), together with nine known alkaloids (3–11), were isolated from the leaves of Evodia rutaecarpa. Their structures were determined on the basis of spectroscopic and chemical methods. Compound 4 exhibited potent activity against Pseudomonas aeruginosa with an MIC value of 7.13 μg/ml.
ARTICLE HISTORY
Received 13 November 2015 Accepted 31 December 2015 KEYWORDS
Rutaceae; Evodia rutaecarpa; quinolone alkaloids; indole alkaloids; antibacterial activity
1. Introduction The dried and nearly ripe fruits of Evodia rutaecarpa (Rutaceae) are used as a traditional Chinese medicine for the treatment of headache, abdominal pain, migraines, chill limbs, postpartum hemorrhage, dysmenorrhea, diarrhea, nausea and hypertension (Wang & Liang 2004). Recently, a number of quinolone and indole alkaloids (Zhang et al. 2013; Zhao et al. 2015), besides caffeoylgluconic acids (He et al. 2015) and essential oil components (Cai et al. 2012) have been reported from the fruits of E. rutaecarpa, and some of these alkaloids have exhibited anti-inflammatory (Choi et al. 2006), antimalarial (Raman et al. 2012), antibacterial (Wang et al. 2013) and cytotoxic activities (Zhao et al. 2015). However, only one alkaloid (Nakasato et al. 1962) was isolated from the leaves of E. rutaecarpa so far. Attracted by the bioactive alkaloids from the fruits of E. rutaecarpa, we carried out the study of the leaves of E. rutaecarpa, leading to the isolation of three quinolone alkaloids (9–11) and eight indole
CONTACT Ling-Yi Kong [email protected] Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2016.1146888. © 2016 Taylor & Francis
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Figure 1. Structures of compounds 1 and 2.
alkaloids (1–8), including two new compounds (1 and 2). Here, we report the isolation, structural elucidation of the new compounds and antibacterial activities of all isolates.
2. Results and discussion Compound 1 was isolated as an amorphous solid. Its molecular formula was deduced to be C18H15N3O3 by HR–ESI–MS (m/z 344.1007 ([M + Na]+, Calcd for C18H15N3NaO3, 344.1006)). The absorptions at 3452 (OH) and 1637 cm−1(C=O) in IR, and at 207, 255, 312 nm in UV spectrum implied the presence of a dihydroindolone (Li et al. 2014). The 1H- NMR spectrum of 1 displayed signals of two spin systems for 1,2-disubstitued aromatic rings [δH 8.18 (1H, dd, J = 1.6, 8.0 Hz), 7.80 (1H, ddd, J = 1.6, 7.5, 8.5 Hz), 7.63 (1H, d, J = 7.5 Hz), 7.53 (1H, t, J = 7.5 Hz); 7.32 (1H, d, J = 7.5 Hz), 7.17 (1H, td, J = 1.4, 7.5 Hz), 6.93 (1H, t, J = 7.5 Hz), 6.86 (1H, d, J = 7.5 Hz)]. In addition, four coupling methylene protons at δH 4.17 (2H, t, J = 7.0 Hz), 2.45 (1H, dt, J = 7.0, 14.0 Hz) and 2.38 (1H, dt, J = 7.0, 14.0 Hz), and an olefinic proton at δH 8.15 (1H, s) were also observed. The 13C- NMR spectrum, with the aid of HSQC, showed four aromatic quaternary carbons (δC 149.1, 142.6, 132.5 and 123.0), nine aromatic or olefinic methine carbons (δC 149.2, 135.6, 130.8, 128.5, 127.8, 127.4, 125.1, 123.9 and 111.6), two carbonyl carbons (δC 181.2 and 162.7), one oxygenated quaternary carbon at δC 76.3, as well as two methylene carbons (δC 43.8 and 37.1). Full assignments of all hydrogen and carbon signals achieved by HSQC and HMBC spectra, suggested the existence of an indoloquinazoline alkaloid skeleton similar to wuzhuyuamide I (Zuo et al. 2000). However, in contrast to wuzhuyuamide I, the absence of a signal for carbonyl carbon (δC 151.0) and the occurrence of an additional olefinic proton at δH 8.15 (1H, s), attached to the olefinic methine carbon at δC 149.2 from the HSQC experiment, indicated that the amide group at C-3 (N-14) in wuzhuyuamide I was substituted with a C=N group in 1. This deduction was further confirmed by the HMBC correlations from H-3 (δH 8.15 1H, s) to δC 162.7 (C-21), 149.1 (C-15) and 43.8 (C-5) (Figure S14). Therefore, the planar structure of 1 was established as depicted in Figure 1. The absolute configuration of C-7 in 1 was established as S by its CD spectrum (Figure S5), which exhibited a positive Cotton effect at 238 nm, and a negative Cotton effect at 262 nm (Kitajima et al. 2006). Thus, the structure of 1 was elucidated as shown in Figure 1, and named (S)-7-hydroxysecorutaecarpine. Compound 2 was isolated as a brown powder. Its molecular formula, C24H23N3O7, was determined by the HR–ESI–MS (m/z 466.1606 ([M + H]+, Calcd for C24H24N3O7, 466.1609)). The IR spectrum showed the presence of a conjugated carbonyl group (1640 cm−1). The UV
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absorptions at 207, 354 nm suggested the existence of a conjugated structure (alkaloid aglycone). The 1H -NMR spectrum of 2 displayed signals of two aromatic proton systems, including three protons of a 1,2,3-trisubstituted aromatic ring at δH 7.81 (1H, dd, J = 1.0, 8.0 Hz), 7.59 (1H, dd, J = 1.0, 8.0 Hz) and 7.39 (1H, t, J = 8.0 Hz), and four protons of a 1,2-disubstituted aromatic ring at δH 7.12–7.68, as well as a N–H at δH 11.54 (1H, s) and four methylene protons at δH 4.49 (1H, dt, J = 6.8, 13.5 Hz), 4.41 (1H, dt, J = 6.8, 13.5 Hz) and 3.20 (2H, t, J = 6.8 Hz). The 13C- NMR spectrum of 2 showed signals of a carbonyl group at δC 160.3, fifteen sp2 carbon signals and two methylene carbons at δC 40.8 and 18.9. The detailed assignments of all hydrogen and carbon signals were achieved by its HSQC and HMBC spectra. In the HMBC spectrum (Figure S14), the key correlations from H-13 (δ 11.54) to C-12a (δ 138.5), C-13a (δ 127.3), C-8b (δ 125.0), C-8a (δ 117.5); from H-7 (δ 4.41, 4.49) to C-5 (δ 160.3), C-13b (δ 144.1), C-8a (δ 117.5), C-8 (δ 18.9); from H-8 (δ 3.20) to C-13a (δ 127.3), C-8b (δ 125.0), C-8a (δ 117.5), C-7 (δ 40.8) were observed. These signal patterns were similar to those of 1-hydroxyrutaecarpine (Danieli et al. 1974), the major difference being the existence of an additional glucosyl unit (δH 5.00 (1H, d, J = 7.8 Hz, H-1′), 3.23–3.77 (6H, m); δC 102.0, 77.3, 76.1, 73.5, 69.9, 60.9). Acid hydrolysis of 2 with 2 M trifluoroacetic acid (TFA) offered a sugar, which was identified as d-glucose by its optical rotation and TLC comparison with an authentic sample. The 3J (1, 2) coupling constant (7.8 Hz) indicated a β-d-glucoside. The sugar unit was connected to C-1 through a C–O linkage as deduced by the HMBC from H-1′ (δH 5.00, d, J = 7.8 Hz) to C-1 (δC 152.2) (Figure S14). Thus, the structure of compound 2 was established as 1-O-β-d-glucopyranosylrutaecarpine. By comparing physical and spectroscopic data with literature values, the nine known compounds were identified as rutaecarpine (3) (Wattanapiromsakul et al. 2003), 7β-hydroxyrutaecarpine (4) (Wu et al. 1995), 7,8-dehydrorutaecarpine (5) (Ikuta et al. 1998), 14-formyldihydrorutaecarpine (6) (Kamikado et al. 1978), dehydroevodiamine (7) (Schramm & Hamburger 2014), hortiamine (8) (Schramm & Hamburger 2014), flindersine (9) (Ahmad 1984), tabouensinium chloride (10) (Wabo et al. 2005), euocarpine E (11) (Wang et al. 2013), respectively. All the compounds (1–11) were evaluated for their antibacterial activities against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 25922. Compound 4 exhibited potent activity against P. aeruginosa with an MIC value of 7.13 μg/ml, and compound 11 exhibited moderate activities against S. aureus, B. subtilis and E. coli with MIC values of 29.29, 14.17 and 28.12 μg/ml, respectively. These results indicated that the hydroxyl in indoloquinazoline alkaloids and the side chain in quinolone alkaloids were important for their antibacterial activities, which was in agreement with previous report (Wang et al. 2013).
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). CD spectra were recorded on a JASCO J-810 spectrometer (Jasco, Tokyo, Japan). UV spectra were obtained using a UV-2450 UV/vis spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra (KBr disks, in cm−1) were recorded on a Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). 1D and 2D NMR spectra were recorded on a Bruker Avance III NMR spectrometer (Bruker, Karlsruhe, Germany) using standard pulse sequences (1H, 500 MHz; 13C, 125 MHz) and
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TMS as an internal standard. HR–ESI–MS spectra were collected using an Agilent 6520B UPLCQ-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was run on a Shimadzu LC-6A equipped with a Shim-pack RP-C18 column (20 × 200 mm2, i.d., 10 μm, Shimadzu, Tokyo, Japan). Column chromatography was performed using ODS RP-C18 (40–63 μm, Fuji, Japan) and Sephadex LH-20 (40−70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden). The fractions were monitored by TLC, and spots were visualised on silica gel plates sprayed with Dragendorff’s reagent.
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3.2. Plant material The leaves of E. rutaecarpa were collected from the botanical garden of China Pharmaceutical University, Jiangsu province, China, and identified by Prof Mian Zhang, China Pharmaceutical University, China. A voucher specimen (NO.20141009) is deposited in the Department of Natural Products Chemistry, China Pharmaceutical University, Nanjing, China.
3.3. Extraction and isolation Air-dried leaves of E. rutaecarpa (1.5 kg) were extracted with 95% EtOH three times (3 h each) under conditions of reflux to give a crude extract (140 g). The extract, suspended in 2% aq. HCl, was partitioned with petroleum ether. The acidic water soluble layer was adjusted to pH 9–10 with ammonia liquor, and then partitioned with CH2Cl2. The CH2Cl2 extract (430 mg) was fractionated into fourteen fractions (Fr. 1–Fr. 14) by ODS column chromatography with CH3OH–H2O (30:100 to 100:0). Compound 8 (5.6 mg, tR = 17.8 min) was obtained from preparative HPLC [45% CH3OH/H2O; 10 ml/min (containing 0.05% TFA v/v)] of Fr. 3. Fr. 5 was subjected to preparative HPLC [75% CH3OH/H2O; 10 ml/min (containing 0.05% TFA v/v) ] to give 11 (7.6 mg, tR = 21.4 min). Fr. 7 was purified by Sephadex LH-20 (CH3OH) to obtain 9 (1.7 mg) and 6 (2.3 mg). Fr. 8 was subjected to preparative HPLC [25% CH3CN/H2O; 10 ml/min (containing 0.05% TFA v/v)] to furnish 7 (5.2 mg, tR = 11.9 min) and 1 (3 mg, tR = 21.6 min). Fr. 12 was exposed to Sephadex LH-20 (CH3OH) to give 3 (3.6 mg) and 4 (3.0 mg). Fr. 13 was subjected to Sephadex LH-20 (CH3OH) to obtain 5 (2.3 mg) and a mixture, which was subjected to preparative HPLC [35% CH3CN/H2O; 10 ml/min (containing 0.05% TFA v/v)] to give 10 (3.5 mg, tR = 12.8 min) and 2 (2.7 mg, tR = 18.9 min).
3.3.1. (S)-7-hydroxysecorutaecarpine (1) + C18H15N3O3, white powder, [𝛼]25 D = −5.8 (c 0.09, CH3OH); HR–ESI–MS m/z: 344.1007 [M + Na] , −1 (Calcd for C18H15N3NaO3, 344.1006); IR (KBr) νmax: 3452, 2924, 1637, 1473, 1384, 1205, 699 cm ; UV (CH3OH), λmax nm (log ε): 207 (4.53), 255 (3.97), 264 (3.96), 299 (3.73), 312 (3.68); CD λmax nm (Δε): 207 (−12.6), 238 (+8.6), 262 (−2.4); 1H- NMR (500 MHz, CD3OD): δ 8.18 (1H, dd, J = 1.6, 8.0 Hz, H-19), 8.15 (1H, s, H-3), 7.80 (1H, ddd, J = 1.6, 7.5, 8.5 Hz, H-17), 7.63 (1H, d, J = 7.5 Hz, H-16), 7.53 (1H, t, J = 7.5 Hz, H-18), 7.32 (1H, d, J = 7.5 Hz, H-9), 7.17 (1H, td, J = 1.4, 7.5 Hz, H-11), 6.93 (1H, t, J = 7.5 Hz, H-10), 6.86 (1H, d, J = 7.5 Hz, H-12), 4.17 (2H, t, J = 7.0 Hz, H-5), 2.45 (1H, dt, J = 7.0, 14.0 Hz, Hb-6), 2.38 (1H, dt, J = 7.0, 14.0 Hz, Ha-6); 13C- NMR (125 MHz, CD3OD): δ 181.2 (C-2), 162.7 (C-21), 149.2 (C-3), 149.1 (C-15), 142.6 (C-13), 135.6 (C-17), 132.5 (C-8), 130.8 (C-11), 128.5 (C-18), 127.8 (C-16), 127.4 (C-19), 125.1 (C-9), 123.9 (C-10), 123.0 (C-20), 111.6 (C-12), 76.3 (C-7), 43.8 (C-5), 37.1 (C-6).
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3.3.2. 1-O-β-d-glucopyranosylrutaecarpine (2) + C24H23N3O7, brown powder, [𝛼]25 D = −58.3 (c 0.035, CH3OH). HR–ESI–MS m/z: 466.1606 [M + H] , (Calcd for C24H24N3O7, 466.1609). UV (CH3OH), λmax nm (log ε): 204 (4.37), 282 (3.82), 292 (3.96), 339 (3.84), 354 (4.10), 371 (4.07), 396 (3.33); IR (KBr) vmax: 3450, 2923, 1640, 1473, 1384, 1255, 1204, 1073, 599 cm−1; 1H- NMR (500 MHz, DMSO-d6): δ 11.54 (1H, s, H-13), 7.81 (1H, dd, J = 1.0, 8.0 Hz, H-4), 7.68 (1H, d, J = 8.0 Hz, H-9), 7.59 (1H, dd, J = 1.0, 8.0 Hz, H-2), 7.56 (1H, d, J = 8.0 Hz, H-12), 7.39 (1H, t, J = 8.0 Hz, H-3), 7.31 (1H, t, J = 7.5 Hz, H-11), 7.12 (1H, t, J = 7.5 Hz, H-10), 4.49 (1H, dt, J = 6.8, 13.5 Hz, Hb-7), 4.41 (1H, dt, J = 6.8, 13.5 Hz, Ha-7), 3.20 (2H, t, J = 6.8 Hz, H-8), 5.00 (1H, d, J = 7.8 Hz, H-1′), 3.77 (1H, m, Hb-6′), 3.53 (1H, m, H-2′), 3.53 (1H, m, Ha-6′), 3.41 (1H, m, H-5′), 3.31 (1H, m, H-3′), 3.23 (1H, m, H-4′). 13C- NMR (125 MHz, DMSO-d6): δ 160.3 (C-5), 152.2 (C-1), 144.1 (C-13b), 138.5 (C-12a), 138.4 (C-14a), 127.3 (C-13a), 126.1 (C-3), 125.0 (C-8b), 124.7 (C-11), 121.8 (C-4a), 120.4 (C-2), 119.9 (C-9), 119.7 (C-10), 119.6 (C-4), 117.5 (C-8a), 112.4 (C-12), 40.8 (C-7), 18.9 (C-8), 102.0 (C-1′), 77.3 (C-5′), 76.1 (C-3′), 73.5 (C-2′), 69.9 (C-4′), 60.9 (C-6′). 3.4. Acid hydrolysis Compound 1 (1 mg) was dissolved in 2 M TFA (3 ml) and then heated in a water bath at 90 °C for 3 h. After extraction with EtOAc (3 × 3 ml), the aq. layer was repeatedly evaporated to dryness with CH3OH until neutral. Co-TLC analysis (EtOAc:CH3OH:H2O:HOAc = 13:2:1:3, Rf = 0.32) in comparison to authentic glucose, together with its optical rotation ([𝛼]25 D = +50.3 (c 0.04, H2O)), indicated the presence of d-glucose.
3.5. Antibacterial assays Antibacterial activity was determined against S. aureus, B. subtilis, P. Aeruginosa and E. coli by the microplate assay method (Pierce et al. 2008). The resulting values were compared with the values of positive controls, penicillin (against S. aureus and B. subtilis, range 0.25–0.50 μg/ ml) and streptomycin (against P. Aeruginosa and E. coli, range 0.12–0.25 μg/ml), under the same conditions.
4. Conclusion In this study, a new indole alkaloid (S)-7-hydroxysecorutaecarpine (1) and a new indole alkaloidal glycoside 1-O-β-d-glucopyranosylrutaecarpine (2), together with nine known compounds (3–11), were isolated from the leaves of E. rutaecarpa. Compound 4 exhibited potent activity against P. aeruginosa with an MIC value of 7.13 μg/ml.
Supplementary material Supplementary material relating to this article is available online, alongside Figures S1–S14.
Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was financially supported by the Program for Changjiang Scholars and Innovative Research Team in University [grant number IRT_15R63]; the Priority Academic Program Development of Jiangsu Higher Education Institutions [grant number PAPD].
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References Ahmad S. 1984. Flindersine from Fagara heitzii. J Nat Prod. 47:391–392. Cai GX, Huang D, Li SX, Xu F, Wang L, Lu YH, Cao L, Wei BY, Li Y, Cai P. 2012. Comparative analysis of essential oil components of Evodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang and Evodia rutaecarpa (Juss.) Benth. Nat Prod Res. 26:1796–1798. Choi YH, Shin EM, Kim YS, Cai XF, Lee JJ, Kim HP. 2006. Anti-inflammatory principles from the fruits of Evodia rutaecarpa and their cellular action mechanisms. Arch Pharm Res. 29:293–297. Danieli B, Palmisano G, Rainoldi G, Russo G. 1974. 1-Hydroxyrutaecarpine from Euxylophora paraënsis. Phytochemistry. 13:1603–1606. He Y, Li J, Wu HH, Chai X, Yang J, Wang YF, Zhang P, Zhu Y, Gao XM. 2015. A new caffeoylgluconic acid derivative from the nearly ripe fruits of Evodia rutaecarpa. Nat Prod Res. 29:1243–1248. Ikuta A, Nakamura T, Urabe H. 1998. Indolopyridoquinazoline, furoquinoline and canthinone type alkaloids from Phellodendron amurense callus tissues. Phytochemistry. 48:285–291. Kamikado T, Murakoshi S, Tamura S. 1978. Structure elucidation and synthesis of alkaloids isolated from fruits of Evodia rutaecarpa. Agric Biol Chem. 42:1515–1519. Kitajima M, Mori I, Arai K, Kogure N, Takayama H. 2006. Two new tryptamine-derived alkaloids from Chimonanthus praecox f. concolor. Tetrahedron Lett. 47:3199–3202. Li YH, He J, Li Y, Wu XD, Peng LY, Du RN, Cheng X, Zhao QS, Li RT. 2014. Evollionines A–C, three new alkaloids isolated from the fruits of Evodia rutaecarpa. Helv Chim Acta. 97:1481–1486. Nakasato T, Asada S, Marui K. 1962. Dehydroevodiamine, main alkaloid from the leaves of Evodia rutaecarpa. Yakugaku Zasshi. 82:619–626. Pierce CG, Uppuluri P, Tristan AR, Wormley FLJ, Mowat E, Ramage G, Lopez-Ribot JL. 2008. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 3:1494–1500. Raman S, Alexandre SL, Nicholas EF. 2012. Identification of novel antimalarial-chemotypes via chemoinformatic compound selection methods for a high-throughput screening program against the novel malarial target, PfNDH2: increasing hit rate via virtual screening methods. J Med Chem. 55:3144–3154. Schramm A, Hamburger M. 2014. Gram-scale purification of dehydroevodiamine from Evodia rutaecarpa fruits, and a procedure for selective removal of quaternary indoloquinazoline alkaloids from Evodia extracts. Fitoterapia. 94:127–133. Wabo HK, Tane P, Connolly JD, Okunji CC, Schuster BM, Iwu MM. 2005. Tabouensinium chloride, a novel quaternary pyranoquinoline alkaloid from Araliopsis tabouensis. Nat Prod Res. 19:591–595. Wang QZ, Liang JY. 2004. Studies on the chemical constituents of Evodia rutaecarpa (Juss.) Benth. Acta Pharm Sin. 39:605–608. Wang XX, Zan K, Shi SP, Zeng KW, Jiang Y, Guan Y, Xiao CL, Gao HY, Wu LJ, Tu PF. 2013. Quinolone alkaloids with antibacterial and cytotoxic activities from the fruits of Evodia rutaecarpa. Fitoterapia. 89:1–7. Wattanapiromsakul C, Forster PI, Waterman PG. 2003. Alkaloids and limonoids from Bouchardatia neurococca: systematic significance. Phytochemistry. 64:609–615. Wu TS, Yeh JH, Wu PL, Chen KT, Lin LC, Chen CF. 1995. 7-Hydroxyrutaecarpine from Tetradium glabrifolium and Tetradium ruticarpum. Heterocycles. 41:1071–1076. Zhang XL, Sun J, Wu HH, Jing YK, Chai X, Wang YF. 2013. A new indoloquinazoline alkaloidal glucoside from the nearly ripe fruits of Evodia rutaecarpa. Nat Prod Res. 27:1917–1921. Zhao N, Li ZL, Li DH, Sun YT, Shan DT, Bai J, Pei YH, Jing YK, Hua HM. 2015. Quinolone and indole alkaloids from the fruits of Euodia rutaecarpa and their cytotoxicity against two human cancer cell lines. Phytochemistry. 109:133–139. Zuo GY, Yang XS, Hao XJ. 2000. Two new indole alkaloids from Evodia rutaecarpa. Chin Chem Lett. 11:127–128.
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New alkaloids from the leaves of Evodia rutaecarpa Xiao Xia, Jian-Guang Luo, Rui-Huan Liu, Ming-Hua Yang & Ling-Yi Kong To cite this article: Xiao Xia, Jian-Guang Luo, Rui-Huan Liu, Ming-Hua Yang & Ling-Yi Kong (2016): New alkaloids from the leaves of Evodia rutaecarpa, Natural Product Research, DOI: 10.1080/14786419.2016.1146888 To link to this article: http://dx.doi.org/10.1080/14786419.2016.1146888
View supplementary material
Published online: 29 Feb 2016.
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Article views: 3
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Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Download by: [Orta Dogu Teknik Universitesi]
Date: 02 March 2016, At: 12:02
Natural Product Research, 2016 http://dx.doi.org/10.1080/14786419.2016.1146888
New alkaloids from the leaves of Evodia rutaecarpa Xiao Xia, Jian-Guang Luo, Rui-Huan Liu, Ming-Hua Yang and Ling-Yi Kong
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State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing, People’s Republic of China
ABSTRACT
One new indole alkaloid (1) and one new indole alkaloidal glycoside (2), together with nine known alkaloids (3–11), were isolated from the leaves of Evodia rutaecarpa. Their structures were determined on the basis of spectroscopic and chemical methods. Compound 4 exhibited potent activity against Pseudomonas aeruginosa with an MIC value of 7.13 μg/ml.
ARTICLE HISTORY
Received 13 November 2015 Accepted 31 December 2015 KEYWORDS
Rutaceae; Evodia rutaecarpa; quinolone alkaloids; indole alkaloids; antibacterial activity
1. Introduction The dried and nearly ripe fruits of Evodia rutaecarpa (Rutaceae) are used as a traditional Chinese medicine for the treatment of headache, abdominal pain, migraines, chill limbs, postpartum hemorrhage, dysmenorrhea, diarrhea, nausea and hypertension (Wang & Liang 2004). Recently, a number of quinolone and indole alkaloids (Zhang et al. 2013; Zhao et al. 2015), besides caffeoylgluconic acids (He et al. 2015) and essential oil components (Cai et al. 2012) have been reported from the fruits of E. rutaecarpa, and some of these alkaloids have exhibited anti-inflammatory (Choi et al. 2006), antimalarial (Raman et al. 2012), antibacterial (Wang et al. 2013) and cytotoxic activities (Zhao et al. 2015). However, only one alkaloid (Nakasato et al. 1962) was isolated from the leaves of E. rutaecarpa so far. Attracted by the bioactive alkaloids from the fruits of E. rutaecarpa, we carried out the study of the leaves of E. rutaecarpa, leading to the isolation of three quinolone alkaloids (9–11) and eight indole
CONTACT Ling-Yi Kong [email protected] Supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2016.1146888. © 2016 Taylor & Francis
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Figure 1. Structures of compounds 1 and 2.
alkaloids (1–8), including two new compounds (1 and 2). Here, we report the isolation, structural elucidation of the new compounds and antibacterial activities of all isolates.
2. Results and discussion Compound 1 was isolated as an amorphous solid. Its molecular formula was deduced to be C18H15N3O3 by HR–ESI–MS (m/z 344.1007 ([M + Na]+, Calcd for C18H15N3NaO3, 344.1006)). The absorptions at 3452 (OH) and 1637 cm−1(C=O) in IR, and at 207, 255, 312 nm in UV spectrum implied the presence of a dihydroindolone (Li et al. 2014). The 1H- NMR spectrum of 1 displayed signals of two spin systems for 1,2-disubstitued aromatic rings [δH 8.18 (1H, dd, J = 1.6, 8.0 Hz), 7.80 (1H, ddd, J = 1.6, 7.5, 8.5 Hz), 7.63 (1H, d, J = 7.5 Hz), 7.53 (1H, t, J = 7.5 Hz); 7.32 (1H, d, J = 7.5 Hz), 7.17 (1H, td, J = 1.4, 7.5 Hz), 6.93 (1H, t, J = 7.5 Hz), 6.86 (1H, d, J = 7.5 Hz)]. In addition, four coupling methylene protons at δH 4.17 (2H, t, J = 7.0 Hz), 2.45 (1H, dt, J = 7.0, 14.0 Hz) and 2.38 (1H, dt, J = 7.0, 14.0 Hz), and an olefinic proton at δH 8.15 (1H, s) were also observed. The 13C- NMR spectrum, with the aid of HSQC, showed four aromatic quaternary carbons (δC 149.1, 142.6, 132.5 and 123.0), nine aromatic or olefinic methine carbons (δC 149.2, 135.6, 130.8, 128.5, 127.8, 127.4, 125.1, 123.9 and 111.6), two carbonyl carbons (δC 181.2 and 162.7), one oxygenated quaternary carbon at δC 76.3, as well as two methylene carbons (δC 43.8 and 37.1). Full assignments of all hydrogen and carbon signals achieved by HSQC and HMBC spectra, suggested the existence of an indoloquinazoline alkaloid skeleton similar to wuzhuyuamide I (Zuo et al. 2000). However, in contrast to wuzhuyuamide I, the absence of a signal for carbonyl carbon (δC 151.0) and the occurrence of an additional olefinic proton at δH 8.15 (1H, s), attached to the olefinic methine carbon at δC 149.2 from the HSQC experiment, indicated that the amide group at C-3 (N-14) in wuzhuyuamide I was substituted with a C=N group in 1. This deduction was further confirmed by the HMBC correlations from H-3 (δH 8.15 1H, s) to δC 162.7 (C-21), 149.1 (C-15) and 43.8 (C-5) (Figure S14). Therefore, the planar structure of 1 was established as depicted in Figure 1. The absolute configuration of C-7 in 1 was established as S by its CD spectrum (Figure S5), which exhibited a positive Cotton effect at 238 nm, and a negative Cotton effect at 262 nm (Kitajima et al. 2006). Thus, the structure of 1 was elucidated as shown in Figure 1, and named (S)-7-hydroxysecorutaecarpine. Compound 2 was isolated as a brown powder. Its molecular formula, C24H23N3O7, was determined by the HR–ESI–MS (m/z 466.1606 ([M + H]+, Calcd for C24H24N3O7, 466.1609)). The IR spectrum showed the presence of a conjugated carbonyl group (1640 cm−1). The UV
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absorptions at 207, 354 nm suggested the existence of a conjugated structure (alkaloid aglycone). The 1H -NMR spectrum of 2 displayed signals of two aromatic proton systems, including three protons of a 1,2,3-trisubstituted aromatic ring at δH 7.81 (1H, dd, J = 1.0, 8.0 Hz), 7.59 (1H, dd, J = 1.0, 8.0 Hz) and 7.39 (1H, t, J = 8.0 Hz), and four protons of a 1,2-disubstituted aromatic ring at δH 7.12–7.68, as well as a N–H at δH 11.54 (1H, s) and four methylene protons at δH 4.49 (1H, dt, J = 6.8, 13.5 Hz), 4.41 (1H, dt, J = 6.8, 13.5 Hz) and 3.20 (2H, t, J = 6.8 Hz). The 13C- NMR spectrum of 2 showed signals of a carbonyl group at δC 160.3, fifteen sp2 carbon signals and two methylene carbons at δC 40.8 and 18.9. The detailed assignments of all hydrogen and carbon signals were achieved by its HSQC and HMBC spectra. In the HMBC spectrum (Figure S14), the key correlations from H-13 (δ 11.54) to C-12a (δ 138.5), C-13a (δ 127.3), C-8b (δ 125.0), C-8a (δ 117.5); from H-7 (δ 4.41, 4.49) to C-5 (δ 160.3), C-13b (δ 144.1), C-8a (δ 117.5), C-8 (δ 18.9); from H-8 (δ 3.20) to C-13a (δ 127.3), C-8b (δ 125.0), C-8a (δ 117.5), C-7 (δ 40.8) were observed. These signal patterns were similar to those of 1-hydroxyrutaecarpine (Danieli et al. 1974), the major difference being the existence of an additional glucosyl unit (δH 5.00 (1H, d, J = 7.8 Hz, H-1′), 3.23–3.77 (6H, m); δC 102.0, 77.3, 76.1, 73.5, 69.9, 60.9). Acid hydrolysis of 2 with 2 M trifluoroacetic acid (TFA) offered a sugar, which was identified as d-glucose by its optical rotation and TLC comparison with an authentic sample. The 3J (1, 2) coupling constant (7.8 Hz) indicated a β-d-glucoside. The sugar unit was connected to C-1 through a C–O linkage as deduced by the HMBC from H-1′ (δH 5.00, d, J = 7.8 Hz) to C-1 (δC 152.2) (Figure S14). Thus, the structure of compound 2 was established as 1-O-β-d-glucopyranosylrutaecarpine. By comparing physical and spectroscopic data with literature values, the nine known compounds were identified as rutaecarpine (3) (Wattanapiromsakul et al. 2003), 7β-hydroxyrutaecarpine (4) (Wu et al. 1995), 7,8-dehydrorutaecarpine (5) (Ikuta et al. 1998), 14-formyldihydrorutaecarpine (6) (Kamikado et al. 1978), dehydroevodiamine (7) (Schramm & Hamburger 2014), hortiamine (8) (Schramm & Hamburger 2014), flindersine (9) (Ahmad 1984), tabouensinium chloride (10) (Wabo et al. 2005), euocarpine E (11) (Wang et al. 2013), respectively. All the compounds (1–11) were evaluated for their antibacterial activities against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 25922. Compound 4 exhibited potent activity against P. aeruginosa with an MIC value of 7.13 μg/ml, and compound 11 exhibited moderate activities against S. aureus, B. subtilis and E. coli with MIC values of 29.29, 14.17 and 28.12 μg/ml, respectively. These results indicated that the hydroxyl in indoloquinazoline alkaloids and the side chain in quinolone alkaloids were important for their antibacterial activities, which was in agreement with previous report (Wang et al. 2013).
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a JASCO P-1020 polarimeter (Jasco, Tokyo, Japan). CD spectra were recorded on a JASCO J-810 spectrometer (Jasco, Tokyo, Japan). UV spectra were obtained using a UV-2450 UV/vis spectrophotometer (Shimadzu, Tokyo, Japan). IR spectra (KBr disks, in cm−1) were recorded on a Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany). 1D and 2D NMR spectra were recorded on a Bruker Avance III NMR spectrometer (Bruker, Karlsruhe, Germany) using standard pulse sequences (1H, 500 MHz; 13C, 125 MHz) and
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TMS as an internal standard. HR–ESI–MS spectra were collected using an Agilent 6520B UPLCQ-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Preparative HPLC was run on a Shimadzu LC-6A equipped with a Shim-pack RP-C18 column (20 × 200 mm2, i.d., 10 μm, Shimadzu, Tokyo, Japan). Column chromatography was performed using ODS RP-C18 (40–63 μm, Fuji, Japan) and Sephadex LH-20 (40−70 μm; Amersham Pharmacia Biotech AB, Uppsala, Sweden). The fractions were monitored by TLC, and spots were visualised on silica gel plates sprayed with Dragendorff’s reagent.
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3.2. Plant material The leaves of E. rutaecarpa were collected from the botanical garden of China Pharmaceutical University, Jiangsu province, China, and identified by Prof Mian Zhang, China Pharmaceutical University, China. A voucher specimen (NO.20141009) is deposited in the Department of Natural Products Chemistry, China Pharmaceutical University, Nanjing, China.
3.3. Extraction and isolation Air-dried leaves of E. rutaecarpa (1.5 kg) were extracted with 95% EtOH three times (3 h each) under conditions of reflux to give a crude extract (140 g). The extract, suspended in 2% aq. HCl, was partitioned with petroleum ether. The acidic water soluble layer was adjusted to pH 9–10 with ammonia liquor, and then partitioned with CH2Cl2. The CH2Cl2 extract (430 mg) was fractionated into fourteen fractions (Fr. 1–Fr. 14) by ODS column chromatography with CH3OH–H2O (30:100 to 100:0). Compound 8 (5.6 mg, tR = 17.8 min) was obtained from preparative HPLC [45% CH3OH/H2O; 10 ml/min (containing 0.05% TFA v/v)] of Fr. 3. Fr. 5 was subjected to preparative HPLC [75% CH3OH/H2O; 10 ml/min (containing 0.05% TFA v/v) ] to give 11 (7.6 mg, tR = 21.4 min). Fr. 7 was purified by Sephadex LH-20 (CH3OH) to obtain 9 (1.7 mg) and 6 (2.3 mg). Fr. 8 was subjected to preparative HPLC [25% CH3CN/H2O; 10 ml/min (containing 0.05% TFA v/v)] to furnish 7 (5.2 mg, tR = 11.9 min) and 1 (3 mg, tR = 21.6 min). Fr. 12 was exposed to Sephadex LH-20 (CH3OH) to give 3 (3.6 mg) and 4 (3.0 mg). Fr. 13 was subjected to Sephadex LH-20 (CH3OH) to obtain 5 (2.3 mg) and a mixture, which was subjected to preparative HPLC [35% CH3CN/H2O; 10 ml/min (containing 0.05% TFA v/v)] to give 10 (3.5 mg, tR = 12.8 min) and 2 (2.7 mg, tR = 18.9 min).
3.3.1. (S)-7-hydroxysecorutaecarpine (1) + C18H15N3O3, white powder, [𝛼]25 D = −5.8 (c 0.09, CH3OH); HR–ESI–MS m/z: 344.1007 [M + Na] , −1 (Calcd for C18H15N3NaO3, 344.1006); IR (KBr) νmax: 3452, 2924, 1637, 1473, 1384, 1205, 699 cm ; UV (CH3OH), λmax nm (log ε): 207 (4.53), 255 (3.97), 264 (3.96), 299 (3.73), 312 (3.68); CD λmax nm (Δε): 207 (−12.6), 238 (+8.6), 262 (−2.4); 1H- NMR (500 MHz, CD3OD): δ 8.18 (1H, dd, J = 1.6, 8.0 Hz, H-19), 8.15 (1H, s, H-3), 7.80 (1H, ddd, J = 1.6, 7.5, 8.5 Hz, H-17), 7.63 (1H, d, J = 7.5 Hz, H-16), 7.53 (1H, t, J = 7.5 Hz, H-18), 7.32 (1H, d, J = 7.5 Hz, H-9), 7.17 (1H, td, J = 1.4, 7.5 Hz, H-11), 6.93 (1H, t, J = 7.5 Hz, H-10), 6.86 (1H, d, J = 7.5 Hz, H-12), 4.17 (2H, t, J = 7.0 Hz, H-5), 2.45 (1H, dt, J = 7.0, 14.0 Hz, Hb-6), 2.38 (1H, dt, J = 7.0, 14.0 Hz, Ha-6); 13C- NMR (125 MHz, CD3OD): δ 181.2 (C-2), 162.7 (C-21), 149.2 (C-3), 149.1 (C-15), 142.6 (C-13), 135.6 (C-17), 132.5 (C-8), 130.8 (C-11), 128.5 (C-18), 127.8 (C-16), 127.4 (C-19), 125.1 (C-9), 123.9 (C-10), 123.0 (C-20), 111.6 (C-12), 76.3 (C-7), 43.8 (C-5), 37.1 (C-6).
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3.3.2. 1-O-β-d-glucopyranosylrutaecarpine (2) + C24H23N3O7, brown powder, [𝛼]25 D = −58.3 (c 0.035, CH3OH). HR–ESI–MS m/z: 466.1606 [M + H] , (Calcd for C24H24N3O7, 466.1609). UV (CH3OH), λmax nm (log ε): 204 (4.37), 282 (3.82), 292 (3.96), 339 (3.84), 354 (4.10), 371 (4.07), 396 (3.33); IR (KBr) vmax: 3450, 2923, 1640, 1473, 1384, 1255, 1204, 1073, 599 cm−1; 1H- NMR (500 MHz, DMSO-d6): δ 11.54 (1H, s, H-13), 7.81 (1H, dd, J = 1.0, 8.0 Hz, H-4), 7.68 (1H, d, J = 8.0 Hz, H-9), 7.59 (1H, dd, J = 1.0, 8.0 Hz, H-2), 7.56 (1H, d, J = 8.0 Hz, H-12), 7.39 (1H, t, J = 8.0 Hz, H-3), 7.31 (1H, t, J = 7.5 Hz, H-11), 7.12 (1H, t, J = 7.5 Hz, H-10), 4.49 (1H, dt, J = 6.8, 13.5 Hz, Hb-7), 4.41 (1H, dt, J = 6.8, 13.5 Hz, Ha-7), 3.20 (2H, t, J = 6.8 Hz, H-8), 5.00 (1H, d, J = 7.8 Hz, H-1′), 3.77 (1H, m, Hb-6′), 3.53 (1H, m, H-2′), 3.53 (1H, m, Ha-6′), 3.41 (1H, m, H-5′), 3.31 (1H, m, H-3′), 3.23 (1H, m, H-4′). 13C- NMR (125 MHz, DMSO-d6): δ 160.3 (C-5), 152.2 (C-1), 144.1 (C-13b), 138.5 (C-12a), 138.4 (C-14a), 127.3 (C-13a), 126.1 (C-3), 125.0 (C-8b), 124.7 (C-11), 121.8 (C-4a), 120.4 (C-2), 119.9 (C-9), 119.7 (C-10), 119.6 (C-4), 117.5 (C-8a), 112.4 (C-12), 40.8 (C-7), 18.9 (C-8), 102.0 (C-1′), 77.3 (C-5′), 76.1 (C-3′), 73.5 (C-2′), 69.9 (C-4′), 60.9 (C-6′). 3.4. Acid hydrolysis Compound 1 (1 mg) was dissolved in 2 M TFA (3 ml) and then heated in a water bath at 90 °C for 3 h. After extraction with EtOAc (3 × 3 ml), the aq. layer was repeatedly evaporated to dryness with CH3OH until neutral. Co-TLC analysis (EtOAc:CH3OH:H2O:HOAc = 13:2:1:3, Rf = 0.32) in comparison to authentic glucose, together with its optical rotation ([𝛼]25 D = +50.3 (c 0.04, H2O)), indicated the presence of d-glucose.
3.5. Antibacterial assays Antibacterial activity was determined against S. aureus, B. subtilis, P. Aeruginosa and E. coli by the microplate assay method (Pierce et al. 2008). The resulting values were compared with the values of positive controls, penicillin (against S. aureus and B. subtilis, range 0.25–0.50 μg/ ml) and streptomycin (against P. Aeruginosa and E. coli, range 0.12–0.25 μg/ml), under the same conditions.
4. Conclusion In this study, a new indole alkaloid (S)-7-hydroxysecorutaecarpine (1) and a new indole alkaloidal glycoside 1-O-β-d-glucopyranosylrutaecarpine (2), together with nine known compounds (3–11), were isolated from the leaves of E. rutaecarpa. Compound 4 exhibited potent activity against P. aeruginosa with an MIC value of 7.13 μg/ml.
Supplementary material Supplementary material relating to this article is available online, alongside Figures S1–S14.
Disclosure statement No potential conflict of interest was reported by the authors.
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Funding This work was financially supported by the Program for Changjiang Scholars and Innovative Research Team in University [grant number IRT_15R63]; the Priority Academic Program Development of Jiangsu Higher Education Institutions [grant number PAPD].
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