Acetyl and butyryl cholinesterase inhibitory sesquiterpene lactones from Amberboa ramosa

Background Alzheimer’s disease (AD) is characterized by a progressive memory loss that leads to a profound emotional disturbance in later stages. As no safe and effective drug is yet available for the treatment of AD, secondary metabolites from plants may be instrumental in meeting this challenge. Keeping in view this point we evaluated sesquiterpenes of medicinal plant Amberboa ramosa for their cholinesterase inhibitory activity. Results Four sesquiterpene lactones have been isolated from the ethyl acetate soluble fraction of Amberboa ramosa. In which one compound Amberbin C (1) was found to be new while other three Amberin (2), Amberbin A (3), and Amberbin B (4) were previously reported ones. The structures of the isolated compounds were elucidated using different spectroscopic techniques. Isolated compounds were tested for their inhibitory potential against acetyl cholinesterase and butyryl cholinesterase enzymes. All compounds showed excellent inhibitory activities against acetyl cholinesterase and butyryl cholinesterase. Conclusions A new sesquiterpene lactone has been isolated and fully characterized, the sesquiterpene lactones from Amberboa ramosa showed good inhibitory activities against acetyl cholinesterase and butyryl cholinesterase enzymes, this study indicated that sesquiterpene lactone can become interesting lead molecules in drug development against Alzheimer’s disease (AD).


Background
Cholinesterases (ChE) form an important class of enzymes intimately connected with the nervous system. Acetylcholine (ACh) was first synthesized in 1867. In 1906 it was detected in the adrenal gland of human tissue as a neurotransmitter, which transmits signals from one nerve cell to another. Cholinesterases ChE inactivate acetylcholine by hydrolyzing it into choline and acetic acid. Cholinesterase inhibitor increases the availability of ACh for nerve cell communications [1]. Alzheimer's disease (AD) is characterized by a progressive memory loss that leads to a profound emotional disturbance in later stages. The disease is accompanied by dysfunctions in cholinergic neurotransmission of the central nervous system [2]. Hence, cholinesterase inhibitors may act as potential leads in the discovery of therapeutics for such nervous system disorders.
As the anti cholinesterase activity of chloroform soluble fraction from Amberboa ramosa has previously been reported [3], keeping in view this point we evaluated the guaianolides (sesquiterpenes) isolated from chloroform soluble fraction of Amberboa ramosa for their cholinesterase inhibitory activity. The genus Amberboa belongs to the family Compositeae and comprises of six species. Amberboa ramosa is an annual herb which belongs to genus Amberboa and family Compositeae. It is mainly found in Pakistan and India. Amberboa ramosa has tonic, aperient, febrifuge, deobstruent, cytotoxic and antibacterial activities [4]. Literature survey revealed that triterpenoids, flavonoids, steroids and sesquiterpene lactones have previously been reported from this species [4,5]. In this study we have isolated four guaianolides (sesquiterpenes) out of which Amberbin C (1) was identified as a new compound while other three Amberin (2) [6], Amberbin A (3) and Amberbin B (4) [7] were previously been reported from the same source. All structures were elucidated by using different spectroscopic techniques like UV, IR, EI-MS, 1D and 2D NMR techniques.

Biological evaluation
The Compound (1) and (2) showed good inhibitory potential towards acetyl cholinesterase. Compound (2) also showed potent activity against butyryl cholinesterase. Compound (3), and (4) showed significant activity against acetyl cholinesterase and potent activity against butyryl cholinesterase (see Table 2). Compound (1) also showed moderate butyryl cholinesterase inhibitory activity. All four compounds share similar basic skeleton, they differ only due to the substituents present at C-8 and C-10. Compound 3 which have O-acetyl group at C-8 and OH group at C-10 showed moderate inhibitory potential towards both cholinestrease enzymes. Compound 4 only differs from Compound 3 due to the presence of an α-D-glucose group at C-10, the introduction of glucose moeity significantly increases the cholinestrease inhibitory activity. While in case of compound 2 which have glucose moeity at C-8 and O-acetyl at C-10 a significant decrease in acetylcholinestrease inhibitory activity occures, while butrylcholinestrease inhibitory activity was almost the same. Simillarly in case of compound 1 which have glycerol moeity at C-10 significant anti-acetylcholinestrease activity was observed. On the basis of these results we can conclude that the effect of substituents at C-10 is more important towards acetylcholinestrease enzyme, mostly activity enhances with the presence of substituents.

Conclusions
A new sesquiterpene lactone has been isolated and fully characterized, the sesquiterpene lactones from Amberboa ramosa showed good inhibitory activities against acetyl cholinesterase and butyryl cholinesterase enzymes, this study indicated that sesquiterpene lactone can become interesting lead molecules in drug development against Alzheimer's disease (AD).

General experimental procedures
Melting points were determined on a Gallenkemp apparatus and are uncorrected. IR spectra were measured on a JASCO 302-A spectrophotometer with KBr cells. UV spectra were obtained on a Hitachi UV-3200 spectrophotometer. Optical rotations were measured on a JASCO DIP-360 polarimeter and the 1D and 2D NMR spectra were recorded on a Bruker AMX-400 Spectrometer operating at 400 MHz for 1 H and 100 MHz for 13 C. Electron impact (EI) mass spectra were recorded on JEOL JMS-HX-110 and Varian MAT-311 -A mass spectrometers. The HR-ESI-MS was recorded on a Jeol JMS 600H instrument. Silica gel (230-400 mesh, E. Merck, Darmstadt, Germany) was used for column chromatography. All reagents were obtained from Sigma Aldrich Chemical sand used without further purification. The redistilled and de-ion-ized water was used in all experiments. The spectrophotometer, Spectra max 384 from molecular devices USA is used for inhibition protocol.

Plant material
The whole plant of Amberboa ramosa Jafri (Compositae) was collected in June 2002, from Malir district of Karachi (Pakistan) and identified by Dr. Surraiya Khatoon, Plant Taxonomist, Department of Botany, University of Karachi, where a voucher specimen (no. KU 312 b) has been deposited.

Extraction and isolation
The shade dried plant material (8 kg) was extracted three times with methanol at room temperature. Solvents were evaporated through vacuum distillation. The condensed and crude methanolic extract (217 g) was partitioned between n-hexane and water. The water soluble fraction was further fractionated into chloroform, ethyl acetate and n-butanol soluble fraction. The column chromatography of the EtOAc soluble sub-fraction (90 g) over silica gel, using a mixture of n-hexane/EtOAc (collecting 200 ml for each), afforded six major fractions A (n-hexane: EtOAc 8:2), B (n-hexane: EtOAc 6.5:3.5), C (n-hexane: EtOAc 5.5:4.5) D (n-hexane: EtOAc 4:6), E (n-hexane: EtOAc 3:7) and Fraction F (100% EtOAc). Sub-fraction A: It was subjected to vacuum liquid chromatography (VLC) using the solvent gradient from n-hexane-EtOAc to afford two sub-fractions AA and AB. The sub-fraction AA obtained from n-hexane-EtOAc (8:2) was further purified by column chromatography over silica gel eluting with n-hexane-EtOAc (8.5:1.5) to furnish compound 3 (13.5 mg).
Sub-fraction E: It was subjected to column chromatography over silica gel using mixtures of n-hexane-EtOAc (3:7) as eluent to obtain two major sub-fractions EA -EB. The sub-fraction EA was subjected to further column chromatography over silica gel eluting with n-hexane-EtOAc (3.5:6.5) as eluent to give compound 1 (11 mg), and n-hexane-EtOAc (4:6) to give compound 2 (7.8 mg). The sub-fraction EB was subjected to successive column chromatography over silica gel eluting with n-hexane-EtOAc (3.2:6.8) solvent system to yield compound 4 (9.4 mg).

Enzyme inhibitory assays
In vitro cholinesterase inhibition assay and determination of IC 50 Acetylcholinesterase (electric eel EC 3.1.1.7), butyrylcholinesterase (horse serum E.C. 3.1.1.8), acetylthiocholine iodide, butyrylthiocholine chloride, 5, 5′dithiobis [2-nitrobenzoic-acid] (DTNB) and galanthamine were purchased from Sigma (St. Louis, MO, USA). Buffer and other chemicals were of analytical grade. Acetylcholinesterase and butyrylcholinesterase inhibiting activities were measured by a slightly modified spectrophotometric method [17]. Acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates to assay acetylcholinesterase and butyrylcholinesterase, respectively. 5, 5′-Dithiobis [2-nitrobenzoic-acid] (DTNB) was used for the measurement of cholinesterase activity. 140 μL of (100 mM) sodium phosphate buffer (pH = 8.0), 10 μL of DTNB, 20 μL of test compound solution and 20 μL of acetylcholinesterase or butyrylcholinesterase solution were mixed and incubated for 15 min (25°C). The reaction was then initiated by the addition of 10 μL acetylthiocholine or butyrylthiocholine, respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine was monitored by the formation of the yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine and butyrylthiocholine, respectively, at a wavelength of 412 nm (15 min). Test compounds and control were dissolved in EtOH. All the reactions were performed in triplicate in 96-well micro plates and monitored in a Spectra Max 340 (Molecular Devices, USA) spectrometer. Estimation of IC 50 values: The concentrations of test compounds which inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (IC 50 ) were determined by monitoring the effect of increasing concentrations of these compounds in the assays on the inhibition values. The IC50 values were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA).