Alkaloids from Stems of Esenbeckia leiocarpa Engl. (Rutaceae) as Potential Treatment for Alzheimer Disease

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.


Introduction
The genus Esenbeckia comprises 30 species distributed in Tropical America [1]. Chemical studies of Esenbeckia spp. showed that they synthesize a variety of secondary metabolites, particularly quinolinic, quinolonic and indolic alkaloids and furocoumarins, and these compounds were considered chemical markers of this genus [2][3][4].
Strong efforts to discover new acetylcholinesterase inhibitors from a vast number of plant species were realized in our laboratory. In a screening of 58 extracts from native Brazilian plants, 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.

Effect of crude extract and hexane and alkaloid fractions on acetylcholinesterase inhibition
The crude ethanol extract of Esenbeckia leiocarpa stems inhibited 91.1 ± 0.2% of acetylcholinesterase activity when tested at 200 μg/mL. Acid-base partition was used to separate the crude extract (35.8 g) of Esenbeckia leiocarpa into two fractions, hexane and chloroform (Alkaloid) and the yield of each fraction was 0.64% and 3.30%, respectively. The ethanol extract, hexane and alkaloid fractions of Esenbeckia leiocarpa inhibited acetylcholinesterase activity with an IC 50 50.7 μg/mL, 6.0 μg/mL and 1.6 μg/mL, respectively ( Figure 1). The chloroform fraction, with positive result for alkaloids (Dragendorff´s and iodoplatinate reagents), exhibited highest level of AChE inhibition and was selected for further purification.

Chromatographic profile of alkaloid fraction
The analytical chromatographic (HPLC) analyses of alkaloid fraction (105.0 mg) from E. leiocarpa afforded 14 fractions from which six major compounds were identified. They were fractionated by chromatographic procedure and the anticholinesterasic activity of each fraction was tested by TLC bioassay as described by Marston et al. [12]. The fractions 7, 9, 10, 11 and 12 presented strong anticholinesterasic activity on TLC bioassay (results not showed) and retention times (t R ) of 9.
To date, no study on evaluation of acetylcholinesterase activity was performed with species of the genus Esenbeckia. In the present paper, the alkaloids isolated from stems of E. leiocarpa, leiokinine A and skimmianine presented acetylcholinesterase inhibition with IC 50 values of 0.  Previous biological studies with chloroform extract from leaves of E. leiocarpa demonstrated weak antifeedant activity against the pink bollworm, Pectinophora gossypiella, this activity was attributed to alkaloids leiokinine A and leiokinine B [3]. On the other hand, the alkaloids isolated from leaves of E. belizencis Lundell were tested in the brine shrimp toxicity assay and revealed that kokusaginine have mildly toxicity (LC 50 = 367 ppm) and flindersiamine did not showed toxicity (LC 50 > 1,000 ppm) [20]. The alkaloids maculine, kokusaginine, skimmianine and flindersiamine demonstrated weak mutagenic activity [21,22]. In another species of Rutaceae, Raulinoa echinata Cowan, these same alkaloids showed antifungal activity against Leucoagaricus gongylophorus, a symbiotic fungus of leaf-cutting ants [23].

Plant material
The stems of Esenbeckia leiocarpa Engl. were collected in January 2007 from a specimen cultivated at the "Cidade Universitária -Armando Salles de Oliveira-CUASO", USP, São Paulo State, Brazil, and identified by Dr. José Rubens Pirani. A voucher specimen (SPF 1169) was deposited at the Herbarium of the Departamento de Botânica, Instituto de Biociências, USP, São Paulo, SP, Brazil.

Extraction and isolation
The dry stems of Esenbeckia leiocarpa (1000 g) were powdered and extracted with ethanol (5 × 1.5 L) using a maceration method. The ethanol extract (35.8 g) was re-dissolved in aqueous solution (HCl 0.1 M, 2 × 120 mL) and the insoluble portion was filtered off. The acid solution was partitioned with hexane (7 × 60 mL) yielding a hexane fraction (0.230 g). The acid aqueous fraction was basified with NH 4 OH (pH 10) and partitioned with CHCl 3 (7 × 60 mL) yielding an active alkaloid fraction (1.182 g). All the fractions were tested in biological test for acetylcholinesterase inhibitor activity.

General methods for compounds identification
The alkaloid fraction was analyzed in HPLC Varian Pro Star 310 system equipped with a 20 μL injection loop. The column used was a Phenomenex C-18 (250 × 4.6 mm) maintained at 20 °C. Twenty μL of the sample (1 mg/mL) was injected, the mobile phase (1 mL/min) was ACN: MeOH: H 2 O (10:45:45). Preparative HPLC was carried out using Varian Prep-Star 400 system using a

Anticholinesterasic assay
Acetylcholinesterase activity was measured using a 96-well microplate reader [24] based on Ellman´s method [25]. In the 96-well plates, 25 μL of 15 mM acetylthiocholine iodide (ATCI) in water, 125 μL of 3 mM DTNB in buffer C, 50 μL of buffer B, 25 μL of plant extract, alkaloid or hexane fractions (0.2 to 100.0 μg mL -1 ) were added and the absorbance was measured at 405 nm every 30 s for three times. Then 25 μL of 0.22 U/mL of the enzyme were added and the absorbance was again read every 10 min for two times. The percentage of inhibition was calculated by comparison with the rates for the sample to a blank (10% MeOH in Buffer A). The following buffers were used. Buffer A: 50 mM Tris-HCl, pH 8; buffer B: 50 mM Tris-HCl, pH 8, containing 0.1% bovine serum albumin V fraction (BSA); buffer C: 50 mM Tris-HCl, pH 8, containing 0.1 M NaCl and 0.02 M MgCl 2 ·6H 2 O.

Statistical analysis of data
Data were presented as means ± SEM of experiments realized at least in triplicate. The IC 50 values were calculated by means of regression analysis.

Conclusions
The alkaloids kokusaginine, flindersiamine, maculine and leiokinine A were previously isolated from E. leiocarpa. On the other hand, this is the first occurrence of leptomerine and skimmianine in Esenbeckia leiocarpa. There are many compounds identified from higher plants with acetylcholinesterase inhibition activity as huperzine A, galanthamine isolated from Huperzia serrata (Thunb.) Trev. [26] and Galanthus nivalis Lumikello, respectively, and used for dementia treatment.
Some biological activities have been reported for the alkaloids isolated in this work, but none have been found to be related to acetycholinesterase inhibition. 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.