New antioxidant and cholinesterase inhibitory constituents from Lonicera quinquelocularis

A phytochemical investigation on the ethyl acetate soluble fraction of Lonicera quinquelocularis (whole plant) led to the isolation of three new and two known compounds. The crude extract and the various soluble fractions were screened for cytotoxic activities. The ethyl acetate soluble fraction was found to be more cytotoxic. The chloroform soluble fraction showed moderate activity while the other fractions and crude extract were weakly cytotoxic. The isolated compounds were tested for antioxidant, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Almost all the compounds showed profound antioxidant activities in DPPH free radical scavenging assay. The 50% inhibitory effect (IC50) values of compounds 3 and 5 against AChE were determined to be 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.

Column chromatography (CC) was carried out using silica gel of 230-400 mesh (E.Merck, Darmstadt, Germany).Ceric sulphate and potassium permanganate solutions were used as visualization reagents.The UV spectra (λmax nm) were recorded on Shimadzu UV-2700 spectrophotometer (Shimadzu, Japan) in EtOH.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 CDCl3 as solvents.

Plant
The
Fraction C (4 g) was again subjected to a series of silica gel column chromatography eluted with n-hexane, n-hexane-EtOAc and EtOAc in increasing order of polarity to afford compound 1 and to a preparative TLC using n-hexane:EtOAc (4:1) as solvent system to afford compounds 2 and 3, respectively.

Identification of compounds
Compounds 1 and 2 were isolated as colourless oils and were identified as bis (2-ethylhexyl) phthalate (Nair et al., 2012) and dioctyl phthalate (Sultan et al., 2010).Identification of compounds 3, 4 and 5 were based on spectroscopic data analysis.

Cytotoxic activity
Cytotoxic activities of the extract and fractions were determined using brine-shrimp (Artemia salina) lethality bioassay (Meyer et al., 1982).Artificial sea water was prepared by dissolving 3.7 g of sea salt per liter of double distilled water and was filtered."Sea" water was placed in small tank, and brine shrimp eggs (1 mg) was added and was darkened by covering with aluminium coil.Twenty milligrams of concentrated sample was dissolved in 2 ml of CHCl3 (20 mg/2 ml (10 mg/ml)) and transferred to 500, 50 and 5 μl vials corresponding to 1000, 100 and 10 μg/ml, respectively.Three replicates were prepared for each concentration making a total of nine vials.The vials containing the sample were concentrated, dissolved in DMSO (50 μl) and 5 ml "sea water" was added to each.Then, ten shrimps were added per vial.All the vials were incubated at 37°C for 24 h and the brine shrimps that survived were counted.The activities of these extract and fractions were compared with standard drugs Ampicillin (Mc Laughlin and Anderson, 1988;Rashid et al., 2002).The data was analyzed with a Finney computer program to determine LD50 values with 95% confidence interval.
Acetylthiocholine iodide and butyrylthiocholine chloride were used as substrates to assay acetylcholinesterase and butyrylcholinesterase, respectively.DTNB was used for the measurement of cholinesterase activity.0.2 mM DTNB in 62 mM sodium phosphate buffer (pH 8.0, 880 μl), test compound solution (40 μl) and acetylcholinesterase or butyrylcholinesterase solution (40 μl) were mixed and incubated for 15 min (25°C).The reaction was then initiated by the addition of acetylthiocholine or butyrylthiocholine (40 μl), respectively.The hydrolysis of acetylthiocholine and butyrylthiocholine were monitored by the formation of 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).All the reactions were performed in triplicate in a BMS spectrophotometer (USA).The concentrations of test compounds that inhibited the hydrolysis of substrates (acetylthiocholine and butyrylthiocholine) by 50% (IC50) were determined by monitoring the effect of increasing concentrations of these compounds in the assays on the inhibition values.The final DMSO concentration in the reaction mixture was 6%.

DPPH radical scavenging activity
The DPPH assay was performed according to the standard procedure with little modifications (Gymfi et al., 1999).The fresh stock solution was prepared by dissolving 3 mg DPPH with 100 ml of methanol and then stored at 20°C.The working solution was obtained by diluting DPPH solution with methanol to obtain an absorbance of about 0.980 (± 0.02) at 517 nm using the spectrophotometer.A 900 µl aliquot of this solution was mixed with 100 µl of the compounds (10 µg/ml).The same was repeated with positive reference, that is, ascorbic acid (10 µg/ml).The solution in the test tubes were shaken well and incubated in dark for 30 min at room temperature.Then, the absorbance was taken at 517 nm.The scavenging activity was calculated from the percentage of DPPH radical scavenged as the following equation: Scavenging effect (%) = [(control absorbance-sample absorbance) / (control absorbance)] ×100.

RESULTS AND DISCUSSION
The crude ethanol extract (430 g) was successively extracted with n-hexane (F 1 ), chloroform (F 2 ), ethyl acetate (F 3 ) and n-butanol (F 4 ).All these fractions and the crude extract were tested for their cytotoxic activities (Brine shrimp lethality assay).The F3 was found to be more effective and showed high activities, F2 and showed optimum while F1 and F4 showed low lethality in brine shrimp assay (Table 1).
Compounds 1 to 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 (Zhang, 2004).The percentage of inhibition was first determined at 0.1 mM.Compounds for which enzyme inhibition was greater than 50% were subsequently assayed for IC 50 (50% inhibitory effect) determination.Among the isolated compounds, 3 and 5 showed most effective inhibitory activity against AChE and BChE as compared to 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 μM, while against BChE, were measured as 5.98 and 9.84 μM, respectively.Compounds 2 and 4 showed weak inhibition profile against AChE and BChE (Table 2).
The DPPH radical scavenging activity assay was performed to evaluate the antioxidant property of the isolated compounds with positive reference ascorbic acid (10 µg/ml) according to the standard procedure with some modifications (Gymfi et al., 1999).All isolated compounds in (Figure 2) showed higher antioxidant property than ascorbic acid.Furthermore, the compound 5 was found more active than others.It may be due to different structure of the compounds.

Conclusion
Several antioxidant and anticholinesterase active compounds were isolated from L. quinquelocularis, which which showed good activity.The ethyl acetate soluble fraction was found to be more cytotoxic.In the present work, several new compounds from ethyl acetate soluble fraction which showed good activities in cholinesterase inhibition and DPPH radical scavenging assays were isolated.This species is as one of the ingredient of the traditional medicine in some part of the world.

Table 1 .
Cytotoxic activities of crude extract and fractions.

Table 2 .
AChE and BChE inhibitory activities of compound 1-5 from L. quinquelocularis (IC50, μM).Positive control used in the assays.Data shown are values from triplicate experiments. b