New Cholinesterase Inhibitory Constituents from Lonicera quinquelocularis

A phytochemical investigation on the ethyl acetate soluble fraction of Lonicera quinquelocularis (whole plant) led to the first time isolation of one new phthalate; bis(7-acetoxy-2-ethyl-5-methylheptyl) phthalate (3) and two new benzoates; neopentyl-4-ethoxy-3, 5-bis (3-methyl-2-butenyl benzoate (4) and neopentyl-4-hydroxy-3, 5-bis (3-methyl-2-butenyl benzoate (5) along with two known compounds bis (2-ethylhexyl phthalate (1) and dioctyl phthalate (2). Their structures were established on the basis of spectroscopic analysis and by comparison with available data in the literature. All the compounds (1–5) were tested for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities in dose dependent manner. The IC50 (50% inhibitory effect) values of compounds 3 and 5 against AChE were 1.65 and 3.43 µM while the values obtained against BChE were 5.98 and 9.84 µM respectively. Compounds 2 and 4 showed weak inhibition profile.

The compound 3 was obtained as colourless oil and the IR spectrum of this compound showed strong absorption bands at 1725 cm 21 (ester carbonyl), 1610 cm 21 (aromatic) and 1130-1200 cm 21 (ether C-O). The UV bands were observed at 232 and 296 nm corresponding to the unsaturated carbonyl and aromatic ring respectively. The HR-EIMS ( Fig. S3e in File S1) analysis showed molecular ion peak at m/z 562.7642 which was corresponded in establishing the formula C 32 H 50 O 8 S3c in File S1) were seen to show pronounce vicinal correlation between H-1a' and H-1b' which confirmed the branched nature of the phthalate. The triplet for H-7' showed that there was no such branching near to correlate with it. In addition the HMBC clearly correlated the H-1' to the ester moiety on one side and H-7' with the ester moiety on the other side.
The 13 C NMR spectrum ( Fig. S3b in File S1) of compound 3 showed presence of 18 carbon atoms while their multiplicity was determined by DEPT experiment which indicated three methyl, six methylene, four methine and three quaternary carbons. The two carbonyl carbons resonated at d 171.22 (C-1) and 167.75 (C-9') respectively. The aromatic carbons displayed signals at d  (Table 1) The HMBC correlations ( Fig. 2 and S3d in File S1) were in conformation with the assigned structure of compound 3. The H-1' protons showed 3 J correlation with C-1 (171.22) and 2 J correlation with C-2' (38.76) and C-11' (23.76). H-7' was observed to have 3 J correlation with C-9' (167.75) and 2 J correlation with C-6' (30.39). Similarly 2 J correlation was observed between H-10' and C-9' of carbonyl carbon. The carbons and protons ( 1 H/ 13 C) connectivity along with some important HMBC correlations of compound 3 are shown in Table 1. On the basis of these evidences the structure of compound 3 was assigned as bis (7-acetoxy-2-ethyl-5methylheptyl phthalate.
The compound 4 was isolated as amorphous powder and its molecular formula was established as C 24 H 36 O 3 by HR-EIMS analysis ( Fig. S4c in File S1) corresponding to the molecular weight (C-1), 129.3 (C-2, C-6), 124.8 (C-3, C-5) and two ethoxy carbons at d 68.3 and 15.5 respectively. Five isoprene carbons displayed signals at d 133.5 (C-3'), 121.5 (C-2'), 27.6 (C-1'), 24.7 (C-5') and 19.4 (C-4') and three signals for neopentyl group of ester moiety were observed at d 83.2 (C-3''), 29.4 (C-4'') and 23.1 (C-5''). The ester carbonyl carbon was observed at d 163.0. The assignments of the 1 H and 13 C NMR data were supported by 2D experiments (Fig. 3) and by comparing the data with the known derivatives of related benzoates [25]. In HMBC spectrum of compound 4 (  Table 2 which ultimately established the structure of compound 4 as neopentyl-4ethoxy-3, 5-bis (3-methyl-2-butenyl benzoate. The compound 5 was isolated as amorphous white solid and from its HR-EIMS analysis (Fig. S5c in File S1), a parent ion peak at m/z 344.6403 (calcd. 344.2549) was observed which suggested that the molecular formula would be C 22     Through the analysis and comparison of the data with the similar known benzoate ester [26] the structure of compound 5 was assigned as neopentyl-4-hydroxy-3, 5-bis [3-methyl-2-butenyl] benzoate. In a bioassay-guided search for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors from medicinal plants, it is interested in this study to identify AChE and BChE inhibiting small molecules from herbal medicinal plants. Compounds 1-5 from L. quinquelocularis were tested against AChE and BChE, which represent the most attractive target for drug design and discovery of mechanism-based inhibitors for the treatment of neurone degenerative disorders such as Alzheimer's disease [27]. The percentage of inhibition was first determined at 0.1 mM. The compounds which had enzyme inhibition greater than 50% were subsequently assayed for IC 50 (50% inhibitory effect) determination. Among the isolated compounds, 3 and 5 showed most effective inhibition activity against AChE and BChE as compared to the standard drugs; allanzanthane and galanthamine in a dose dependent manner. The IC 50 values of compounds 3 and 5 against AChE were determined to be 1.65 and 3.43 mM, while against BChE, were measured as 5.98 and 9.84 mM respectively. The compounds 2 and 4 showed weak inhibition profile against AChE and BChE (Table 3).

General Experimental Procedure
The melting point was determined by using Kofler hot-stage apparatus (Reichert, Vienna, Austria). Aluminium TLC plates (20620, 0.5 mm thick) pre-coated with silica gel 60 F 254 (0.2 mm layer thickness; E. Merck, Darmstadt, Germany) were used for TLC to check the purity of the compounds. Column chromatography (CC) was carried out using silica gel of 230-400 mesh (E. Merck, Darmstadt, Germany). Preparative TLC Glass plates (20620, 2 mm thick) pre-coated with silica gel 60 F 254 (0.5 mm layer thickness; E. Merck, Germany) were used for the purification of semi pure compounds. Ceric sulphate and potassium permanganate solutions were used as visualization reagents. The UV spectra (l max nm) were recorded on Shimadzu UV-2700 spectrophotometer (Shimadzu, Japan) in ethanol. Mass Spectra was recorded on Bruker TOF Mass spectrometers (Billerica, USA) using electrospray ionisation (ESI). The 1 H NMR and 13 C NMR spectra were recorded on a Bruker DPX-400 NMR spectrometer (Billerica, USA) (400 MHz for 1 H and 100 MHz for 13 C-NMR), using CDCl 3 as solvents. Further assignments were made by DEPT, COSY, HMQC and HMBC experiments.

Plant Material
The whole plant of Lonicera quinquelocularis was collected from Bara Galli, Hazara division, District Mansehra, in June 2009. It was identified by Professor Dr. Manzoor Ahmad, Plant Taxonomist, Department of Botany, Government Degree College Abbotabad, Pakistan. As the field from where the plant has been collected was open, no authority was responsible to issue permission and the study did not involve endangered and   protected species. Likewise there was no restriction from land owner on usability of this specie and the permission was given from the land owner for the collection of this specie. After collection, the voucher specimen has been deposited in the herbarium of the Department of Botany, Government Degree College Abbottabad, Pakistan where a voucher specimen has been deposited in herbarium (Accession No. C-0013).

Extraction and Isolation
The shade dried whole plant of L. quinquelocularis (13 kg) was ground and extracted with ethanol at room temperature (3X25 L). The combined ethanolic extract was evaporated under reduced pressure to obtain a thick greenish gummy material (crude). It was suspended in water and was successively partitioned into various soluble fractions of n-hexane (151 g), chloroform (147 g), ethyl acetate (109 g), and n-butanol (53 g) with suitable solvents respectively.
The Acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates for investigation of acetylcholinesterase and butyrylcholinesterase assays, respectively. The 5,59-Dithiobis[2-nitrobenzoic-acid] (DTNB) was used for the measurement of cholinesterase activity. The 0.2 mM DTNB in 62 mM sodium phosphate buffer (pH 8.0, 880 mL), test compound solution (40 mL) and acetylcholinesterase or butyrylcholinesterase solution (40 mL) were mixed and incubated for 15 minutes (25uC). The reaction was then initiated by the addition of acetylthiocholine or butyrylthiocholine (40 mL), respectively. The hydrolysis of acetylthiocholine and butyrylthiocholine were monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as a 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). All the reactions were performed in triplicate using a BMS spectrophotometer (USA). The concentrations of test compounds that inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) up to 50% (IC 50 ) were determined by monitoring the effect of increasing concentrations of these compounds in the assays on the inhibition values. The final concentration of DMSO in the reaction mixture was maintained at 6%.

Conclusion
The phytochemical studies of Lonicera quinquelocularis; medicinal plant were carried out using latest chromatographic and spectroscopic techniques. Three new and two known compounds were obtained from the ethyl acetate soluble fraction in which compound 3 and 5 showed pronounced cholinesterase inhibition activities with IC 50 1.65 and 3.43 mM against AChE and 5.98 and 9.84 mM against BChE respectively in dose dependent manner using reference drugs. The compounds 2 and 4 showed weak inhibition profile while the compound 1 was found inactive for inhibition activity. The studies also showed the structure activity relationship between the new structural features and the corresponding enzymes activities. Lonicera quinquelocularis is one of the ingredient of the traditional medicine in some part of the world. Therefore, furthure investigation on this medicnal plant is recommended to exploit its hidden medicinal values.