Characterization of Chemical Constituents of Luffa operculata ( Cucurbitaceae )

A mixture of new ceramides (1, 2, 3, 4 and 5) together with a binary mixture of ceramides with long chain alkyl (6 and 7), triterpenoid (10) and steroids (11 and 12) have been isolated from bark of the fruits and of the stems of Luffa operculata (Cucurbitaceae). The structures were elucidated by comprehensive spectroscopic analysis including H and C NMR, DEPT (distortionless enhancement by polarization transfer), COSY (correlated spectroscopy), HMQC (heteronuclear multiple quantum coherence), HMBC (heteronuclear multiple bond connectivity), IR (infrared), HR-ESI-MS (electrospray ionization-high resolution mass spectra) and LR-MS (low resolution electron ionization mass spectra) experiments. All the ceramides are reported for the first time in Cucurbitaceae and this is the first report of the rare triterpene 10 isolated from Luffa operculata. The ceramides 6 and 7 showed a high acetylcholine esterase inhibitory effect.


Introduction
As a part of our continuing chemical studies on plants of Cucurbitaceae family, we have investigated the bark of the fruits and the stems of Luffa operculata specie.L. operculata Cogn.(Cucurbitaceae), locally known as "cabacinha", a perennial shrub widely distributed in Northeastern Brazil where an aqueous solution from its fruits has been used in popular medicine for the treatment of sinusitis [1].In the previous paper [2], we reported the isolation and structure elucidation of triperpenes cucurbitane type from these fruits.In this paper, we report the isolation and structure elucidation of ceramides (1-5, 6 and 7), triterpene oleanane type (10) and steroids (11 and 12) from the bark of the fruits and stems of this plant.In plants, recent studies indicate that ceramides may be involved in signal transduction, membrane stability, host-pathogen interactions, and stress responses [3].The compound 6 and 7, as well as the steroids mixture (12), showed an acetylcholine esterase inhibitory effect.Inhibition of acetylcholinesterase (AchE) is used as a strategy for the treatment of Alzheimer's disease (AD), a neurodegenerative malady characterized by cognitive impairment and personality changes.One of the most promising approaches for treating this disease is to enhance the acetycholine level in rain using acetylcholine esterase (AChE) plant-derived inhibitors [4].In this work we report an evaluation of the cholinesterase inhibition effect of the ceramides 6 and 7 following the methodology of Elmann, adapted by Rhee [5] for the layer chromatography (TLC).

General Procedures
1 H and 13 C NMR spectra were recorded on Bruker DPX 300 and DRX 500 spectrometers in CDCl 3 , with TMS as an internal standard.DEPT and all 2D experiments (COSY, HMQC and HMBC) with standard Bruker pulse sequence; IR spectra were carried out on Perkin-Elmer 2000 series FT-IR; electrospray ionization mass spectra (HR-ESI-MS) obtained in mass spectrometer model LCMS-IT-TOF (225-07100-34, Shimadzu) and on a QP5050 (Shimadzu) instrument at 70 eV for low resolution; melting point were measured on Mettler Toledo FP90 apparatus, uncorrected; the spots were visualized by spraying with a mixture of vanillin-perchloric acid ethanol.

Extraction and Isolation of Constituents
Luffa operculata stems were collected in Acarape County, Brazil and identified in the Departamento de Biologia do Centro de Ciências da Universidade Federal do Ceará (UFC).A voucher specimen (N˚ 43.056) was deposited at Departamento de Biologia (UFC) Prisco Bezerra Herbarium.The air-dried stems (935 g) were powdered and extracted at room temperature with hexane and EtOH.The hexane extract (4.1 g) was subjected to column chromatography (CC) on silica gel (Si gel) 60 (230 -400 mesh) using hexane, CH 2 Cl 2 , EtOAc and MeOH as solvents.The CH 2 Cl 2 fraction (2.56 g) was further subjected to CC on Si gel 60 (230 -400 mesh) to yield a material (17.5 mg) white greasy (1 -5) and 11 (102 mg).The AcOEt fraction (4.95 g) of EtOH extract (22.5 g) was successively chromatographed on Si gel column to afford 10 (7.5 mg) as white powder.The air-dried bark of fruits (195.8 g) were powdered and extracted at room temperature with hexane and EtOH.The EtOH extract (10.5 g) was subjected to CC on silica gel 60 (230 -400 mesh) using CH 2 Cl 2 , EtOAc and EtOH as solvents.The CH 2 Cl 2 fraction (0.29 g) was successively chromatographed on Si gel column to afford 12 (21 mg) as a white powder, while the AcOEt fraction (0.59 g) after successively chromatographed on Si gel column afforded 6 and 7 (24.5 mg) a white solid.

Results and Discussion
The CH 2 Cl 2 fraction of the hexane extract of the stems of L. operculata was chromatographed on silica gel column to yield a white greasy material.Its IR spectrum disclosed bands due to methylene and methyl (γ max 2923/ 2853 cm -1 and δ max 1462/1380 cm -1 ), carbonyl (γ max 1737 cm -1 ) groups, as well as bands of C -O/C -N (γ max 1172 cm -1 ) bounds.The LR-MS displayed a cluster of four 14-amu-apart ion peaks at m/z 311, 297, 283, 269 and 255 indicative of a mixture of homologous compounds (Scheme 1).In agreement, the NMR data (

II
In the 1 H -1 H COSY spectrum, the mutual correlations between the signals at δ H 4.38 and 3.66, as well as between the signals at δ H 1.12 and 3.57, supported the fragment II.The linkage of theses partial structures (I and II) to each other was based on additional long-range connectivities observed between the hydrogens at δ H 4.38 (-NCH 2 -) and the carbon atom in δ C 174.20 (C = O) in the 1 H- 13 C HMBC spectrum and resulted in the general structure III, corresponding to amides mixture.Others correlations in the HMBC spectrum were assigned in the Table 1.The AcOEt fraction of the EtOH extract from barc fruit of L. operculata was chromatographed on silica gel column to afford a white solid whose high-resolution high-resolution ESI mass spectrometry in the negative mode displayed two 14-amu-apart quasimolecular ion peaks [M-H] -at m/z 736.5277 and 722.3396, indicative of a binary mixture of homologous compounds.The IR spectrum of this solid disclosed bands at 3336/3218, 2918/2849 and 1621 cm -1 suggestive of OH and/or NH, CH 3 /CH 2 and C = O groups, respectively, as well as bands at 1070/1025 cm -1 of C-O/C-N bound; further bands at 1544, 1466 and 750 cm -1 were attributed to NH, CH 3 /CH 2 and CH 2 groups, respectively.The 13 C and DEPT NMR spectra (Table 2) showed several aliphatic methylenes (δ C 23.28 -36.17) and methyl terminal signal (δ C 14.55) which constructed a long alkane chain.These spectra also revealed the presence of six methine [δ C 53.42; three oxygenated (δ C 72.75, 73.26 and 77.32) and two olefinic (δ C 131.16 and 131.04)] carbons.In addition, signals at δ C 62.36 and 175.64 indicated an oxymethylene carbon and an ester or amide carbonyl, respectively.The 1 H NMR spectrum also revealed characteristic signals for long alkyl chains (δ H 1.27 -1.33) as well as a signal at δ H 8.61 compatible with hydrogen of secondary amide (RCONHR') which, was further substantiated by its 13 C NMR (δ C 175.64) and IR (1621 and 1544 cm -1 ) spectra.In the 1 H-1 H COSY spectrum, the amide hydrogen with resonance at δ H 8.61 coupled to a methine hydrogen at δ H 5.13 (δ C 53.42) which in turn revealed coupling to a methyne carbinolic hydrogen at δ H 4.38 (δ C 77.32) and to a diastereotopic methylene group observed at δ H 4.45 and 4.53 (δ C 62.36).On the other hand, in the HMBC spectrum, the hydrogen resonance at δ H 4.38 showed correlation to the δ C 53.42 (CH), 62.38 (CH 2 ), 73.26 (CH) and 34.55 (CH 2 ).The HMQC spectrum established the association of the methyne carbon at δ C 73.26 with the carbinolic hydrogen at δ H 4.32.This analysis, based on amide function (RCONHR'), allowed to establish the partial structure IV.
The third oxygenate methine carbon at δ C 72.75 was associated to hydrogen in δ H 4.64 by HMQC experiment.In addition, the HMBC spectrum showed that this hydrogen was correlated with carbonyl carbon and with the methylene carbons at δ C 36.17 and 26.19, beta and gama carbons, respectively, to carbonyl function.Thus, a partial structure IV was expanded to V. Based on the above spectral analysis and by comparison with spectral data [IR, NMR ( 1 H and 13 C) and MS] of the literature [3,[6][7][8] the sample was identified as a ceramides mixture with general structure VI. .The E stereochemistry of double bond was determined on the basis of 13 C NMR chemical shift of the methylene carbons adjacent to the olefinic carbons, which is observed at δ C ≈ 27.00 in Z isomers and at δ C ≈ 32.00 in E isomers [3,6].
These data support the structures 6 and 7 proposed for ceramides: The structures of acyl chains were confirmed by analysis of the mixture of products (8 and 9) resulting from methanolysis of 6 and 7.The CG-MS of 8 and 9 was in agreement with structures of 6 and 7, showing the presence of two constituents, which were identified as methyl-2-hydroxydocosanoato (m/z 370 [M + ]) and methyl-2-hydroxyhenicosanoato (m/z 356 [M + ]).The steroids were identified as 24α-etil-5α-colest-7,trans-22-dien-3β-ol [11 (spinasterol)] and a mixture of 24α-ethyl-5α-colest-7,trans-22-dien-3β-ol (11) and 24βethyl-5α-colest-7,trans-22,25-trien-3β-ol (12) from their spectral analysis and by comparison of their physical and spectral data with literature [14,15] values.In the anticholine esterase activity test, fisostgmine was used as positive control (with an inhibition zone of 9 mm) since it is a drug that binds and activates the acetylcholine receptor.Acetylcholine esterase (AChE) hydrolyzes the neurotransmitter acetylcholine at one of the highest known wnzymatic rates.Therefore the anticholine esterase activity of the ceramides (6 and 7) (with aninhibition zone of 12 mm) is relevant as the results below (Table 4).

Conclusions
Many previous studies showed that Luffa operculata is rich in triterpenes cucurbitano type, as expected for a Cucurbitaceae.Although almost all of these metabolites were found only in their fruits, this study showed that the stems and bark of the fruits of this plant are bioproductors of ceramides as well as steroids and triterpenes of another type (oleanane).According to the analysis of spectral data, the mixture of long chain ceramides seems to involve more than two components, requiring a further thorough study about the subject.

The
AcOEt fraction of the EtOH extract of the stems of L. operculata was successively chromatographed on silica gel column to afford 10 as white powder, mp 262˚C -263˚C.The 13 C NMR spectrum of 10 exhibited thirty signals divided by DEPT spectra in nine quarternary carbons, three CH, eleven CH 2 and seven CH 3