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Molecules 2010, 15, 9205-9213; doi:10.

3390/molecules15129205
OPEN ACCESS

molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article

Alkaloids from Stems of Esenbeckia leiocarpa Engl. (Rutaceae)
as Potential Treatment for Alzheimer Disease
Elaine Monteiro Cardoso-Lopes 1, James Andreas Maier 1, Marcelo Rogério da Silva 1, Luis
Octávio Regasini 2, Simone Yasue Simote 2, Norberto Peporine Lopes 3, José Rubens Pirani 4,
Vanderlan da Silva Bolzani 2 and Maria Cláudia Marx Young 1,*
1

2
3
4

Section of Plant Physiology and Biochemistry, Institute of Botany, Box 3005, 01061-970, São
Paulo, SP, Brazil
Institute of Chemistry, São Paulo State University, Box 355, 14801-970, Araraquara, SP, Brazil
Department of Chemistry and Physics, São Paulo University, 14040-903, Ribeirão Preto, SP, Brazil
Department of Botany, Biosciences Institute, São Paulo University, Box 11.461, 05422-970, São,
Paulo, SP, Brazil

* Author to whom corresponding should be addressed; E-Mail: mcmyoung@yahoo.com.br;
Tel.: +55-11-5073-6300; Fax: +55-11-5073-6300.
Received: 15 November 2010; in revised form: 2 December 2010 / Accepted: 6 December 2010 /
Published: 13 December 2010

Abstract: Esenbeckia leiocarpa Engl. (Rutaceae), popularly known as guarantã,
goiabeira, is a native tree from Brazil. Bioactivity-guided fractionation of the ethanol
stems extract afforded the isolation of six alkaloids: leiokinine A, leptomerine,
kokusaginine, skimmianine, maculine and flindersiamine. All isolated compounds were
tested for acetyl cholinesterase inhibition, in vitro and displayed anticholinesterasic
activity. The alkaloid leptomerine showed the highest activity (IC50 = 2.5 μM), similar to
that of the reference compound galanthamine (IC50 = 1.7 μM). The results showed for the
first time the presence of alkaloids leptomerine and skimmianine in E. leiocarpa (Engl.)
with potent anticholinesterasic activity.
Keywords: Esenbeckia leiocarpa; Rutaceae; alkaloids; acetylcholinesterase inhibitors

dictamnine. sesquiterpenes (5). lignans (2). with positive result for alkaloids (Dragendorff´s and iodoplatinate reagents).64% and 3. one coumarin. quinolonic and indolic alkaloids and furocoumarins. triterpenes (17). hexane and alkaloid fractions of Esenbeckia leiocarpa inhibited acetylcholinesterase activity with an IC50 50. 15 9206 1. benzenoids (13). In a recent review.7 μg/mL.Molecules 2010. 1. Chemical studies of Esenbeckia spp. goiabeira (BA). flavonoids (14). (Rutaceae). 4methoxy-2-(3´-pentyl)quinoline. three lignans. native from Brazil. In a screening of 58 extracts from native Brazilian plants. The ethanol extract. Results and Discussion 2. 260 chemically defined natural molecules were evaluated for acetylcholinesterase inhibition.30%. oxygen heterocycles (5). twelve indole derivatives.2% of acetylcholinesterase activity when tested at 200 μg/mL. exhibited highest level of AChE inhibition and was selected for further purification. Esenbeckia leiocarpa Engl. The compounds tested were classified in alkaloids (139). The chloroform fraction. Acid-base partition was used to separate the crude extract (35. quinoid (1). antã-forte and guarataia (ES) [5].4-dihydro-1-methyl-2-(3´-pentyl)quinolin-4-one. benzoxazinone (1). Previous studies on E. coumarins (18).8 g) of Esenbeckia leiocarpa into two fractions. leiokinine A and leiokinine B. 2. two amides and the methyl 4isoprenyloxy-trans-cinnamate [3. showed that they synthesize a variety of secondary metabolites. since these compounds increase the endogenous levels of acetylcholine to boost cholinergic neurotransmission. 6. maculine. is an ornamental tree widely distributed in Brazilian Dense Ombrophilous Atlantic Forest from South of Bahia to São Paulo states and is commonly known as guarantã (SP).1 ± 0. Effect of crude extract and hexane and alkaloid fractions on acetylcholinesterase inhibition The crude ethanol extract of Esenbeckia leiocarpa stems inhibited 91. the ethanolic crude extract of the stems of Esenbeckia leiocarpa showed a strong acetylcholinesterase inhibition and was chosen for bioassay-guided fractionation in order to isolate the biologically active compounds and evaluate the acetylcholinesterase inhibition of these compounds. Alzheimer´s disease (AD) is the most common cause of progressive cognitive dysfunction that results from a deficiency in cholinergic activity in brain [8]. carotenoid (1) and alycyclic (1) [9-11]. and various compounds have been isolated from it. and these compounds were considered chemical markers of this genus [2-4]. Strong efforts to discover new acetylcholinesterase inhibitors from a vast number of plant species were realized in our laboratory.6. respectively (Figure 1). hexane and chloroform (Alkaloid) and the yield of each fraction was 0.6 μg/mL. such as the alkaloids kokusaginine. monoterpenes (27). respectively. polycyclic (1).0 μg/mL and 1.1. diterpenes (8). particularly quinolinic.7]. Introduction The genus Esenbeckia comprises 30 species distributed in Tropical America [1]. stilbenes (3). Several plant-derived drugs (rivastigmine and galanthamine) that inhibit acetylcholinesterase (AChE) can be used to treat early stages of AD. flindersiamine. proteids (2). leiocarpa have been reported in the literature. sulfur compounds (2). .

73 min. [12]. 10. 15 9207 Figure 1.Molecules 2010.9) [M+H]+ = m/z 202 1 (Fr. The fractions 7. 14.0 mg) from E. respectively (Figure 2). Acetylcholinesterase inhibition by ethanol extract. 12) 4 (Fr. 2 (Fr. 11 and 12 presented strong anticholinesterasic activity on TLC bioassay (results not showed) and retention times (tR) of 9.12 min. 9. alkaloid and hexane fractions (0.69 min. Figure 2. Analytical chromatographic profile of alkaloid fraction from E.2 to 100. Chromatographic profile of alkaloid fraction The analytical chromatographic (HPLC) analyses of alkaloid fraction (105. 11) [M+H]+ = m/z 244 + [M+H] = m/z 260 [M+H]+ = m/z 274 .0 μg/mL).7) [M+H]+ = m/z 232 3 (Fr.26 min. 15. They were fractionated by chromatographic procedure and the anticholinesterasic activity of each fraction was tested by TLC bioassay as described by Marston et al. Acetylcholinesterase inhibition (%) 100 ethanol extract alkaloid fraction 75 hexane fraction 50 25 0 0 10 20 30 40 50 60 70 80 90 100 [sample] μg mL-1 2. leiocarpa afforded 14 fractions from which six major compounds were identified. leiocarpa by HPLC and their respectives molecular ion peaks [M+H]+ data in the ESI-MS. 11.2.10) [M+H]+ = m/z 260 5 and 6 (Fr.60 min and 20.

leiocarpa The alkaloid fraction of E. leptomerine. In the present paper. The alkaloids leptomerine and kokusaginine had a potent effect . Fractions Fr. In addition.7 mg) [14] and skimmianine (4. ALK 1 to 6. 3 – kokusaginine. 9 (tR = 19.8 mg) [3] and leptomerine (2. with UV detection at 242 nm and flow rate of 8 mL/min. maculine (5) [16] and flindersiamine (6) [6] (Figure 3). kokusaginine (3.). 11 (tR = 26.0 mg) [13]. Isolation and identification of alkaloids from stems of E. 2. 2 – leptomerine. 12 (tR = 34.5 min) and Fr.4. leiocarpa (m = 105. fraction Fr. respectively. 12.5 μM and 46 μM. Anticholinesterasic activity of alkaloids The alkaloids maculine. Their identification was based on analysis of 1D and 2D NMR experiments. O O 5 O 4 CH3 7 N CH3 N 1’ CH3 H3C H3C 3’ O 4a 4 CH3 3’ 2’ 8a N 2 O O 7 3 2 1 O CH3 O CH3 O CH3 O O H3C O 5 2’’ O H3C N O O 4 O 8 N 5 O O H3C N O O 6 2.3. While leptomerine and kokusaginine showed IC50 values of 2. no study on evaluation of acetylcholinesterase activity was performed with species of the genus Esenbeckia.3 mg) [15]. respectively. 13C-NMR data of compounds 1. as well as by comparison with literature data. fractions Fr.4 mM. respectively (Figure 4).17-19].0 mg) was dissolved into MeOH (10 mg/mL) and submitted to RP-HPLC using acetonitrile-water-methanol (10:45:45) as mobile phase.2 min) yielded two furoquinoline alkaloids. 15 9208 2. respectively.4 min) and Fr. leiokinine A and skimmianine presented acetylcholinesterase inhibition with IC50 values of 0. 3 and 4 and 1H-NMR data of compounds 5 and 6 (see Experimental section). 16. 4 – skimmianine.3 min) afforded a mixture of two others furoquinoline alkaloids (3. affording 14 fractions (Fractions 1–14). flindersiamine. this is the first occurrence of leptomerine and skimmianine in Esenbeckia leiocarpa (Engl. 6 – flindersiamine. 10 (tR = 24. leiokinine A (1.3 min) were identified as two 4quinolinone alkaloids. 7 (tR = 15. The alkaloids leptomerine and skimmianine have already been isolated from Haplophyllum heptomerum (Rutaceae) in 1986 [13]. 5. The alkaloids isolated are: 1 – leiokinine A. leiocarpa. 5. 5 – maculine. kokusaginine. the alkaloids isolated from stems of E. Figure 3.2 mg).21 mM and 1. leiokinine A and skimmianine identified in this work were previously isolated from other Esenbeckia species [1. To date.Molecules 2010. From preparative HPLC. Chemical structures of alkaloids with anticholinesterasic activities. However.

Molecules 2010. On the other hand. Raulinoa echinata Cowan. Experimental 3.22]. The ethanol extract (35. The acid aqueous fraction was basified with NH4OH (pH 10) and partitioned with CHCl3 (7 × 60 mL) yielding an active alkaloid .230 g). Acetylcholinesterase inhibition of physostigmine. In another species of Rutaceae. were collected in January 2007 from a specimen cultivated at the “Cidade Universitária . skimmianine and flindersiamine demonstrated weak mutagenic activity [21. A voucher specimen (SPF 1169) was deposited at the Herbarium of the Departamento de Botânica. Figure 4. kokusaginine. SP.1 M. Brazil. José Rubens Pirani. leiokinine A and skimmianine (10-10 to 10-3 M). kokusaginine. Plant material The stems of Esenbeckia leiocarpa Engl.1. The alkaloids maculine. a symbiotic fungus of leaf-cutting ants [23]. this activity was attributed to alkaloids leiokinine A and leiokinine B [3]. the alkaloids isolated from leaves of E. these same alkaloids showed antifungal activity against Leucoagaricus gongylophorus. 2 × 120 mL) and the insoluble portion was filtered off. galanthamine. USP. 3. Instituto de Biociências. Extraction and isolation The dry stems of Esenbeckia leiocarpa (1000 g) were powdered and extracted with ethanol (5 × 1. Brazil. 15 9209 anticholinesterasic similar to reference compounds galanthamine and physostigmine that have 1.5 L) using a maceration method.CUASO”. São Paulo State. São Paulo. respectively. Pectinophora gossypiella.Armando Salles de Oliveira. and identified by Dr. USP. 3.7 μM and 0. Acetylcholinesterase inhibition (%) 100 physostigmine galanthamine leptomerine 80 60 kokusaginine leiokinine A 40 skimmianine 20 0 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 log [M] Previous biological studies with chloroform extract from leaves of E. leptomerine.2.000 ppm) [20].4 μM values of IC50. leiocarpa demonstrated weak antifeedant activity against the pink bollworm.8 g) was re-dissolved in aqueous solution (HCl 0. belizencis Lundell were tested in the brine shrimp toxicity assay and revealed that kokusaginine have mildly toxicity (LC50 = 367 ppm) and flindersiamine did not showed toxicity (LC50 > 1. The acid solution was partitioned with hexane (7 × 60 mL) yielding a hexane fraction (0.

57 (C-6). H-C3’). 1. H-C4-OCH3).2 (6-OCH3).52 (d. Leptomerine (2).3.29 (C-9). 56. 21].0 Hz.0 (7-OCH3) [21].85 (t. 126. H-C2’). J = 2. 126. 1H-NMR [CDCl3.6 (C-3’). J = 8. 1H-NMR [CDCl3.0 and 1.41 (dd. 104.22 (3-OCH3) [3].40 (s. Leiokinine A (1). H-C8-OCH3) [21].7 (C-2’). 109. 59. Preparative HPLC was carried out using Varian Prep-Star 400 system using a Phenomenex C-18 (250 mm × 20. 141. J = 2. 3. H-C2’). 126.5 Hz.02 (s. General methods for compounds identification The alkaloid fraction was analyzed in HPLC Varian Pro Star 310 system equipped with a 20 μL injection loop. 1H-NMR [CDCl3.53 (dd. H-C7).77 (C-10). Maculine (5). 7. H-C2’). 100. 4.4 Hz. H-C7). 142.6 (C-1’). 6. 115. 13C-NMR [CDCl3]: δ 29.41 (s. J = 2. 3.56 (d.93 (N-CH3). using the CDCl3 as an internal standard. 7.11 (C-7). δ (ppm). 6.18 (C-8).39 (s. J (Hz)]: δ 2.17 (d. H-C5). 8.7 (8-OCH3) [15.07 (s. . 56.03 (d. 56.07 (C-5). H-C3’). 123.Molecules 2010. 172. 7.4 Hz.5 Hz. H-C3’). 132. 4.02 (d.4 Hz. H-C3-OCH3) [3]. 3. 4.99 (d. 6. H-C6).97 (H-C1`). H-C2’). H-C6-OCH3). 11. 6.5 Hz.34 (t.97 (s.00 (s. 7. 4.8 (C-3’). 7.7 (C-3’). 8.48 (C-1`).52 (d.2 (C-5). 118. All the fractions were tested in biological test for acetylcholinesterase inhibitor activity. 21]. HPLC analysis of alkaloid fraction afforded 14 fractions.5 Hz. the mobile phase (1 mL/min) was ACN: MeOH: H2O (10:45:45).68 (H-C2`). H-C6). 1. J = 9. H-C2”). 22.36 (tl.19 (s. 112. 126. 115.5 Hz.41 (s. 148. 4. H-C7-OCH3). 140. J = 2.5 Hz. 4. 131. H-C5).6 mm) maintained at 20 °C. HC3’). H-C3’).52 (dl. 7. 7.0 (C-3). 11 and 12 were identified by NMR analyses.182 g). H-C5). J (Hz)]: δ 1. H-C4-OCH3). 9. 7.1 (4-OCH3).47 (s.45 (C-2). 7.60 (m. δ (ppm). 7. H-C5). H-C8). H-C8). 1H-NMR [CDCl3.60 (m. 7. 7. Skimmianine (4). H-C8-OCH3) [14. H-C2”).2 (C-5).95 (d.01 (C-3). 123. δ (ppm).9 (C-3). J = 2.2 (C-2’).4 (N-CH3) [13].53 (d. 15 9210 fraction (1. 13C-NMR [CDCl3]: δ 142. δ (ppm).54 (d. 4. J = 2. J = 8. J = 2. H-C2’). H-C6). H-C7-O CH3) [21]. J = 9.46 (s.8 (C-8).6 (C-2’).21 (s. J (Hz)]: δ 7. 7. 13C-NMR [CDCl3]: δ 27. 59. 1. gHMQC and gHMBC) NMR experiments were recorded on a Varian INOVA 500 spectrometer (11.06 (s.70 (C-7).8 (7-OCH3). 7. 14.0 Hz.15 (C-3`). Kokusaginine (3). J (Hz)]: δ 7.77 (s.00 (d.94 (s.5 Hz. 1H-NMR [CDCl3. 1H-NMR [CDCl3. 102.82 (s. Twenty μL of the sample (1 mg/mL) was injected. H-C1’). 13 C and DEPT) and 2D (1H-1H gCOSY. J = 2. H-N-CH3). Fractions 7.70 (C-8). 61.9 (C-9).36 (s.4 (C-6). H-C8). 10. 0.10 (H-C3`).52 (C6). 142. 3.5 Hz. The column used was a Phenomenex C-18 (250 × 4.23 (C-2`). 27. 34. Flindersiamine (6).03 (s. 112. 13C-NMR [CDCl3]: δ 143.0 mm) preparative column. 105.23 (C-4).7 (C-8a).19 (s.7 (C-10). J = 8. 4. 7. 60.4 (C-4a). J = 8. H-C5). H-C4-OCH3) [16. δ (ppm). 21]. H-NCH3).65 (dt.8 (4-OCH3). H-C3). The 1D – (1H. 104. 34. 7. J (Hz)]: δ 7. 177.39 (s.01 (C-4). δ (ppm). 3.0 and 1. H-C8).7 T) at 500 MHz (1H) and 125 MHz (13C). J (Hz)]: δ 7.4 Hz. H-C5). H-C4-OCH3).63 (d.14 (C-5).

C. C. pH 8.2 to 100. Mafezoli. Quím. buffer B: 50 mM Tris-HCl. Then 25 μL of 0. F. pH 8. flindersiamine. Bolzani are grateful to CNPq for research fellowships.R. 125 μL of 3 mM DTNB in buffer C. . containing 0. Barbosa. 03/02176-7)..Molecules 2010.. J. 3. Pirani. Nunes. containing 0. On the other hand. Oliveira. B. [26] and Galanthus nivalis Lumikello. Nova 2007.biotasp. Brazil. The IC50 values were calculated by means of regression analysis. Acknowledgements This work was funded by grants of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) within the BIOTA/FAPESP – The Biodiversity Virtual Institute Program (www.0 μg mL-1) were added and the absorbance was measured at 405 nm every 30 s for three times. Young and V. 30. Mattos.G. 25 μL of 15 mM acetylthiocholine iodide (ATCI) in water.5. buffer C: 50 mM Tris-HCl. respectively. This work demonstrated that these alkaloids presented potent anticholinesterasic activity and must be tested in pharmacological models in vivo to determine if they have activity on memory of mice and pre clinical studies before human application. Statistical analysis of data Data were presented as means ± SEM of experiments realized at least in triplicate. M.org. alkaloid or hexane fractions (0..) Trev. M. In the 96-well plates..br). leiocarpa. galanthamine isolated from Huperzia serrata (Thunb. maculine and leiokinine A were previously isolated from E. The following buffers were used. Barros-Filho.02 M MgCl2·6H2O. Buffer A: 50 mM Tris-HCl.1 M NaCl and 0. grant no. There are many compounds identified from higher plants with acetylcholinesterase inhibition activity as huperzine A. and used for dementia treatment. J. M.. References and Notes 1. 25 μL of plant extract. Anticholinesterasic assay Acetylcholinesterase activity was measured using a 96-well microplate reader [24] based on Ellman´s method [25].4. Elaine Monteiro Cardoso-Lopes thanks PRODOC/CAPES for providing a post-doctoral fellowship through the postgraduate programme on “Biodiversidade Vegetal e Meio Ambiente” of Instituto de Botânica of São Paulo. 4. 15 9211 3. 1589-1591.22 U/mL of the enzyme were added and the absorbance was again read every 10 min for two times. but none have been found to be related to acetycholinesterase inhibition.. this is the first occurrence of leptomerine and skimmianine in Esenbeckia leiocarpa. Secondary Metabolites of Esenbeckia almawillia Kaastra (Rutaceae). The percentage of inhibition was calculated by comparison with the rates for the sample to a blank (10% MeOH in Buffer A).C. Andrade-Neto. F.. 50 μL of buffer B.F. Some biological activities have been reported for the alkaloids isolated in this work.A. pH 8.1% bovine serum albumin V fraction (BSA). M.M. Conclusions The alkaloids kokusaginine. S. M.

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