Acetylcholine esterase inhibitors and melanin synthesis inhibitors from Salvia officinalis

Accepted Manuscript

Acetylcholine esterase inhibitors and melanin synthesis inhibitors from Salvia officinalis Amal Sallam , Amira Mira , Ahmed Ashour , Kuniyoshi Shimizu PII: DOI: Reference:

S0944-7113(16)30089-7 10.1016/j.phymed.2016.06.014 PHYMED 52043

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

15 March 2016 12 June 2016 18 June 2016

Please cite this article as: Amal Sallam , Amira Mira , Ahmed Ashour , Kuniyoshi Shimizu , Acetylcholine esterase inhibitors and melanin synthesis inhibitors from Salvia officinalis, Phytomedicine (2016), doi: 10.1016/j.phymed.2016.06.014

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Acetylcholine esterase inhibitors and melanin synthesis inhibitors from Salvia officinalis Amal Sallama,*, Amira Miraa, Ahmed Ashoura and Kuniyoshi Shimizub a

Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura,

35516 Egypt. b

Fukuoka, Japan.

*Corresponding author.

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Department of Agro-environmental Sciences, Faculty of Agriculture, Kyushu University,

Amal Sallam, Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura, 35516 Egypt. Tel. +201092017949; fax. +2 050 2247496.

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E-mail address: [email protected] ABSTRACT

Background: Salvia officinalis is a traditionally used herb with a wide range of medicinal applications. Many phytoconstituents have been isolated from S. officinalis, mainly phenolic diterpenes, which possess

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many biological activities.

Purpose: This study aimed to evaluate the ability of the phenolic

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diterpenes of S. officinalis to inhibit acetylcholine esterase (AChE) as well as their ability to inhibit melanin biosynthesis in B16 melanoma cells.

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Methods: The phenolic diterpenes isolated from the aerial parts of S. officinalis were tested for their effect on melanin biosynthesis in B16

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melanoma cell lines. They were also tested for their ability to inhibit AChE using Ellman’s method. Moreover, a molecular docking experiment

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was used to investigate the binding affinity of the isolated phenolic diterpenes to the amino acid residues at the active sites of AChE. Results:

Seven

phenolic

diterpenes―sageone,

12-methylcarnosol,

carnosol, 7b-methoxyrosmanol, 7a-methoxyrosmanol, isorosmanol and epirosmanol―were isolated from the methanolic extract of the aerial parts of S. officinalis. Isorosmanol showed a melanin-inhibiting activity as potent as that of arbutin. Compounds 7a-methoxyrosmanol and

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isorosmanol inhibited AChE activity by 50% and 65%, respectively, at a concentration of 500 µM. Conclusions: The results suggest that isorosmanol is a promising natural compound for further studies on development of new medications which might be useful in ageing disorders such as the declining of cognitive

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functions and hyperpigmentation.

Keywords

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Salvia officinalis; phenolic diterpenes; melanin; acetylcholine esterase.

Abbreviations: S. officinalis, Salvia officinalis; AChE, acetylcholine esterase; AD, Alzheimer's disease; AChEIs, acetylcholine esterase inhibitors; FDA, U.S. Food and Drug Administration; Aβ, amyloid-β peptide;

Rp

HPLC,

reversed

H and

phase

high

performance

liquid

13

C-NMR, proton and carbon-13 nuclear

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chromatography;

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magnetic resonances; ACTI, acetylthiocholine iodide; MTT, thiazolyl blue

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tetrazolium bromide; DTNB, 5,5-dithiobis [2-nitrobenzoic acid]; NaOH, sodium hydroxide; DMSO, dimethyl sulfoxide; EMEM, Eagle’s Minimum Essential Medium; FBS, fetal bovine serum; CO2, carbon dioxide; PBS,

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phosphate-buffered saline.

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Introduction

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Alzheimer’s disease (AD) is an age-related, irreversible neurodegenerative disorder characterized by progressive memory loss and impairment in cognitive function, often accompanied by behavioral disturbances. In elderly people, AD is the most frequently occurring form of dementia, especially if considered alongside concomitant cerebrovascular disease. AD is associated with loss of cholinergic synapses in the hippocampus and neocortex, resulting in deficiencies in the neurotransmitter, acetylcholine (ACh). Inhibition of acetylcholinesterase (AChE), the enzyme responsible 2

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for the hydrolysis of ACh, elevates ACh levels, and thus is considered a promising strategy for temporarily addressing AD symptoms, such as memory loss and confusion, though not for curing Alzheimer’s disease or stopping it from progressing (Mesulam et al. 2004). As two of the U.S. Food and Drug Administration (FDA)-approved acetylcholinesterase (AChEIs)—galantamine

and

rivastigmine—are

naturally

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inhibitors

derived, the potential for plants to yield other therapeutic agents has stimulated extensive research into the discovery of new AChEIs (Howes and Perry 2011).

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Hyperpigmentation is a common, harmless skin condition in which patches of skin become darker in color than the surrounding area due to excess production of melanin. A common form of hyperpigmentation is age spots, which are dark, uneven patches of skin that appear in response to sunlight exposure. Several factors such as sun exposure, inflammation,

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free radicals and hormonal changes cause reductions in the number of melanocytes in elderly people. As a result, melanocytes are stimulated to

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overproduce melanin, and the excess melanin is unevenly distributed in the epidermis, causing hyperpigmentation (Matt et al. 2007; Videira et al.

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2013).

The medicinal use of plants and their phytoconstituents, especially those

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long used in folk medicine, is becoming very popular worldwide. S. officinalis (common sage) is a common culinary herb native to the

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Mediterranean region and Europe. Known in Arabic as mairamia, S. officinalis has been traditionally used to treat digestive disorders, circulation disturbances, bronchitis, cough, asthma, and angina and to reduce excessive perspiration. It is also used as a mouth wash for the treatment of inflammations of the mouth and throat mucosa such as gingivitis and pharyngitis (Saad and Said 2011; Hamidpour et al. 2013). Scientific researches have reported that hydroalcoholic and methanolic 3

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extracts of S. officinalis and several isolated compounds showed a wide range of biological activities (Devansh 2012; Hamidpour et al. 2013), such as antibacterial, antifungal (Gracia et al. 2012; Stefanovic et al. 2012; Abdelkader et al. 2014), antioxidant (Rasmy et al. 2012; Neagu et al. 2014), anti-inflammatory

(Baricevic et al. 2001), anti-angiogenic

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(Keshavarz et al. 2010) and anti-cancer activities (Janicsak et al. 2011), in addition to having anti-diabetic potential (Christensen et al. 2010).

S. officinalis has been reported to produce many biologically active compounds, mainly phenolic diterpenoids (Masahiro et al. 1994; Masahiro et al. 1997; Fischedick et al. 2013), triterpenoids (Topçu et al. 2006) and

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polyphenolic compounds (Lu et al. 2000).

The alcohol extract of S. officinalis is known to be effective in the management of mild-to-moderate Alzheimer’s disease (Akhondzadeh et al. 2003; Sandra et al., 2014) through improvement of cognition and memory with no adverse effects, even after many years of use (Perry et al.

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1999). Several researches have suggested that S. officinalis extract improves memory and cognition through dose-dependent inhibition of

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AChE (Kennedy 2006; Russo et al. 2013) and by exerting a neuroprotective effect against Aβ-induced toxicity (Iuvone et al. 2006).

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However, there is a lack of research about the effect of the individual phenolic diterpenes comprising the major bioactive phytoconstituents of S.

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officinalis in the inhibition of AChE, and thus in the possible management of AD. In the present study, therefore, we evaluated individual phenolic

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diterpenes as AChEIs.

Although skin-whitening creams containing S. officinalis are used (Toshitsugu and Juko, 1995), there are no detailed data or investigations on the phytochemical constituents of S. officinalis responsible for this antimelanogenesis effect. We thus also evaluated the effect of several isolated phenolic diterpenes from the aerial parts of S. officinalis as melanin synthesis inhibitors, with the aim of developing effective 4

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treatments against hyperpigmentation (age spots). Together, therefore, the present experiments evaluated the ability of several isolated phenolic diterpenes from the aerial parts of S. officinalis as both AChE and melanin

Materials and methods General experimental procedures

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synthesis inhibitors.

Rp HPLC was performed using a Cholester column (4.6 mm × 250 mm) at 210 nm and 1 ml/min 1H and 13C-NMR spectra were recorded on a Bruker

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AV 400 spectrometer (400 and 100 MHz for 1H and 13C, respectively).

Plant material

The dry aerial parts of S. officinalis (3 kg) were purchased from the local market in Cairo, Egypt in 2012. The aerial parts were compared with a reference sample kept at the Pharmacognosy Department, Faculty of

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Pharmacy, Mansoura University, Egypt. A voucher specimen (2012SO/01) was placed at the Herbarium of the Pharmacognosy Department,

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Faculty of Pharmacy, Mansoura University.

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Extraction and isolation of compounds The powdered plant material (3 kg) was extracted with methanol (10 l ×

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3), and the collected methanolic extracts were evaporated under reduced pressure, yielding a dark green viscous residue (500 g). The methanolic

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extract residue was fractionated using n-hexane, chloroform and ethyl acetate, respectively. The n-hexane fraction (130 g) was chromatographed on a silica gel column using a petroleum ether : ethyl acetate gradient elution, then purified on a Sephadex LH20 column using methylene chloride for elution, yielding compound 1 (3.4 mg). The chloroform fraction (15 g) was chromatographed on a silica gel column using methylene chloride : methanol (100:0 to 0:100), yielding 5 groups, I-V.

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Group I, eluted with 100% methylene chloride, yielded on evaporation compound 2 (10 mg). Group II, eluted with methylene chloride : methanol (99:1), was further purified on a silica gel column using methylene chloride : methanol (100:0 and 99:1), yielding compound 3 (10 mg). Group III, eluted with methylene chloride : methanol (97:3), was purified

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on the Sephadex LH20 column, yielding Group III A and Group III B. Group III A (20 mg) was further purified by Rp HPLC using acetonitrile : water yielding compound 4 (Rt 33 min) and compound 5 (Rt 35 min). Group IV, eluted with methylene chloride : methanol (95:5), was purified on a Sephadex LH20 column, yielding compound 6. Group V, eluted with

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methylene chloride : methanol (95:5), was purified on a silica gel column using petroleum ether : ethyl acetate (100:0 to 0:100), yielding compound 7.

Reagents for biological assays

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Acetylthiocholine iodide (ACTI) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Thiazolyl blue tetrazolium bromide (MTT),

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galantamine hydrobromide and AChE from Electrophorus electricus (electric eel), 500 U/mg, were purchased from Sigma (St. Louis, MO, USA). 5,5-Dithiobis [2-nitrobenzoic acid] (DTNB), NaOH and DMSO

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were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

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Media for cell lines Eagle’s Minimum Essential Medium (EMEM) was purchased from Nissui

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Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Gibco BRL (Tokyo, Japan). Buffer A (50 mM Tris-HCl, PH 8, 0.1% BSA) and Buffer B (50 mM TrisHCl, pH = 8, 0.1 M NaCl, 0.02 M MgCl2.6 H2O) were used. Cell line A mouse B16 melanoma cell line was obtained from RIKEN Cell Bank. The cells were maintained in EMEM supplemented with 10% (v/v) fetal 6

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bovine serum (FBS) and 0.09 mg/ml theophylline. The cells were incubated at 37 ºC in a humidified atmosphere of 5% CO2.

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Melanin biosynthesis inhibitory activity assay The assay was carried out according to Ashour et al. (2013). The mouse B16 melanoma cells were cultured in a pair of 24-well plates (one plate for melanin content determination and the other for cell viability testing), at a density of 1 × 105 cells/well. After 24 h, the medium was replaced with

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998 µl of fresh medium and 2 µl of the test compound at maximum solubility (n = 3). The final concentrations of the tested compounds are shown in Table 1. Arbutin was used as a positive control at a concentration of 50 mg/ml, while DMSO was used as a negative control. Cells were incubated for an additional 48 h, then a fresh medium-

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containing sample was added. After further incubation for 24 h, the remaining adherent cells were assayed. For melanin content determination,

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the cell pellet obtained after media removal and washing with phosphatebuffered saline (PBS) was dissolved in 1 ml of 1N NaOH and the color produced after overnight incubation in the dark was measured by a

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microplate reader at 405 nm to determine the melanin content in the cells. The results obtained from the cells treated with the test samples were

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analyzed as a percentage of the results obtained from the control culture. On the other hand, the effects of the tested compounds on cell viability

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were determined by testing their cytotoxicity on B16 melanoma cells using an MTT assay. So, for the other 24-well culture plate, 50 µl of MTT reagent in PBS (5 mg/ml) was added to each well. The plate was incubated in a humidified atmosphere of 5% CO2 at 37°C for 4 h. After the medium was discarded, 1.0 ml isopropyl alcohol (containing 0.04 N HCl) was added, and the absorbance was measured at 570 nm after overnight incubation in the dark. 7

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Acetylcholine esterase inhibitory assay Acetylcholine esterase inhibitory (AChEI) activity was measured using Ellman’s method (Ellman et al. 1961; Mukherjee et al. 2007; Mira et al. 2015). The substrate acetylthiocholine iodide (ACTI) is hydrolyzed by

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AChE into acetate and thiocholine. In neutral and alkaline media; thiocholine reacts with 5,5-Dithiobis [2-nitrobenzoic acid] (DTNB) to give yellow-colored

2-nitro-5-thiobenzoate,

which

can

be

detected

spectrophotometrically at 405 nm. Briefly, in a 96-well plate, 25 µl of 15 mM ACTI, 125 µl of 3 mM DTNB in buffer B, 50 µl of buffer A and 25

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µl of tested compound (dissolved in 25% DMSO) were mixed, and the absorbance was measured ten times at 16-second intervals using a microplate reader (Biotek, Winooski, VT, USA) at 405 nm. Then, 25 µl of AChE (0.25 U/ml in buffer A) was added and the absorbance was measured ten times every 16 seconds. A solution of 25% DMSO was used

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as a negative control. Absorbance was plotted against time and the enzyme activity was calculated from the slope of the obtained line and expressed

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as a percentage compared to an assay using a buffer without any inhibitor. To avoid any increase in absorbance due to the color of the compounds or spontaneous hydrolysis of the substrate, the absorbance before addition of

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the enzyme was subtracted from the absorbance after adding the enzyme.

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Molecular docking

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A docking experiment was used to investigate the binding affinity of the tested compounds to the binding residues of the active site of AChE. The crystal structure of aged phosphorylated AChE was downloaded from the RCSB Protein Data Bank (PDB, code 1CFJ) and imported into the workspace of Molegro Virtual Docker (MVD 6.0; Molegro, Aarhus, Denmark) software. All cofactors, ligands and water molecules were removed from the protein structure before docking. The docking experiment was adjusted as previously described (Hai-Bang and Shimizu, 8

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2014). The three-dimensional structures of ligands were drawn using Chemsketch 12.0 software (Cambridge Soft Corporation, Cambridge, MA, USA) and saved in the mol2 format. Compounds were docked inside a sphere with a 15 Å radius centered at the largest cavity (volume 228.352, surface 546.56 Å) detected by the program. The steps of the docking

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experiment were performed as previously described (Ratnavali et al. 2011). The binding residues were determined using the LigPlot+ V.1.4 program. Statistical analysis

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The IC50 values of the isolated compounds in the AChEI assay were calculated using Probit Analysis (SPSS Version 16.0 for Windows; SPSS Inc., Chicago, IL, USA) for five different concentrations in three independent experiments. Cell viability was expressed as a percentage of MTT reduction, assuming that the viability of cells treated with 1%

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DMSO as a negative control was 100%. Figures were built in Microsoft Excel 2010. All values are the means ± SDs of three independent

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experiments. Data were analyzed for statistical significance using one-way ANOVA, followed by Dunnet’s test as a post-hoc test with GraphPad

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Prism 5.0 software for Windows (San Diego, CA, USA).

Results and Discussion

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This study evaluated both the acetylcholine esterase inhibitory activity and

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the melanin synthesis inhibitory activity of phenolic diterpenoids isolated from the aerial parts of S. officinalis.

By using different chromatographic techniques, we isolated seven phenolic diterpenes (1-7) from the methanolic extract of the aerial parts of S. officinalis (Fig. 1). Identification of the isolated phenolic diterpenes was achieved by comparison of their 1H and

13

C NMR spectroscopic data

(Figs. S1-S12) with those reported in the literature. The compounds were 9

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identified as sageone (1) (Tada et al. 1994), 12-methylcarnosol (2), carnosol (3) (Inatani et al., 2015), 7b-methoxyrosmanol (4), 7amethoxyrosmanol (5) (Ahmed et al. 2006), isorosmanol (6) and epirosmanol (7) (Nakatani and Inatani, 1984). This is the first report to describe the isolation of both 7b-methoxyrosmanol (4) and 7a-

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methoxyrosmanol (5) from the methanolic extract of the aerial parts of S. officinalis.

Melanin biosynthesis inhibitory activity assay

Compounds (1-7) were assayed using B16 melanoma cells to evaluate the

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inhibition of melanin biosynthesis and cell viability at their maximum solubility using arbutin as a positive control. For compounds sageone (1), carnosol (3), 7b-methoxyrosmanol (4), 7a-methoxyrosmanol (5) and epirosmanol (7), the maximum solubility was 80 µg/ml, while compounds 12-methylcarnosol (2) and isorosmanol (3) showed a maximum solubility

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of 40 µg/ml. The ability of these compounds to inhibit melanin formation in B16 melanoma cells was evaluated at various concentrations (Table 1).

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The effect of the tested compounds on cell viability reflects the cytotoxic effect of these compounds on B16 melanoma cells. A compound that can lower melanin content in the tested cells (inhibit melanin biosynthesis)

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without decreasing the cell viability (i.e., without a cytotoxic effect) of the tested cell line would be a good candidate for use as an antimelanogenesis

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agent. By contrast, a compound that lowers melanin content but causes cytotoxicity is certainly not preferable as an antimelanogenesis agent. The

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results of the assay are shown in (Table 1). Taking into account the cytotoxicity, the most active compound exhibiting melanin synthesis inhibition with no cytotoxicity was compound 6 (the melanin content in melanoma cells was decreased to be 50%) at a concentration of 10 µg/ml. Compound 6 is a promising candidate for use as a melanin synthesis inhibitor with results better than that of the positive control arbutin, which showed 49.3% melanin content at a concentration of 100 µg/ml keeping 10

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cell viability at 86.5%, while compound 6 showed 50.4% melanin content at a concentration of 10 µg/ml keeping cell viability at 105.3% (Table 1). Although compounds 3, 5 and 7 decreased melanin content, they possessed high cytotoxic activity. Compound 5 at 20 µg/ml concentration lowered the melanin content in cells moderately and showed low cytotoxic Compound 1 showed moderate melanin synthesis inhibitory

activity but was the most cytotoxic.

Acetylcholine esterase inhibitory assay

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activity.

Acetylcholine esterase inhibitory activity was measured using Ellman’s

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method, and the results are shown in Table 2. At a concentration of 500 µM, compounds 1, 2, 3, 4 and 7 did not inhibit acetylcholine esterase, while compounds 5 and 6 inhibited acetylcholine esterase by 50% and 65%, respectively. The IC50 values were measured to be 482.36 and 354.21 µM for compounds 5 and 6, respectively. A molecular docking

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experiment (Fig. 2) was used to investigate the binding affinity of the isolated compounds and the amino acid residues at the active sites, the

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peripheral anionic and esteratic sites, and the oxyanionic holes of AChE (Harel et al. 1993; Shafferman et al. 1992). Compounds 5 and 6 showed the highest binding scores (Table 2 and Fig. 2). Compound 6 had a

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hydrophobic interaction with important amino acid residues at the catalytic residues (His440, Ser200, Phe288 and Phe290) and oxyanionic hole

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(Gly119 and Gly118) in addition to hydrogen bonding with the anionic site (Tyr121 and Trp84). Compound 5 showed less interaction through

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hydrophobic binding with one of the amino acid residues at the catalytic site (His440) and two of the anionic site residues (Asp72 and Phe330) and hydrogen bonding with another anionic site residue (Trp84). Compounds 1, 2, 3 and 7 showed less interaction with the important amino acid residues at the active sites. Beta binding of the methoxy group in compound 4 resulted in complete loss of activity. This could be explained by loss of the

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interaction with His440, Asp72 and Phe330 when compared with the alpha binding of the methoxy group in compound 5 (Fig. 2).

Due to the limited number of acetylcholine esterase inhibitors and melanin synthesis inhibitors currently available, the present study is significant for

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its isolation of natural products that could potentially be optimized as one or both types of inhibitors. Our results suggest that S. officinalis should be evaluated for its potential control of hyperpigmentation through melanin synthesis inhibition. The present study suggests the broad scope of the potential uses of this culinary herb (S. officinalis), including the of

Alzheimer’s

hyperpigmentation.

Conclusion

disease

and

the

treatment

of

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management

In this research, seven known phenolic diterpenes―sageone, 12-

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methylcarnosol, carnosol, 7b-methoxyrosmanol, 7a-methoxyrosmanol, isorosmanol and epirosmanol―were isolated from the aerial parts of S.

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officinalis. The acetylcholine esterase inhibitory activity and the melanin synthesis inhibitory activity of the compounds were evaluated. Isorosmanol showed a high anti-melanogenesis activity compared to that

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of arbutin without causing cytotoxicity to the cells. Isorosmanol and 7amethoxyrosmanol showed moderate acetylcholine esterase inhibitory

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activity. The results suggest that isorosmanol is a promising natural compound for further studies on development of new medications which

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might be useful in ageing disorders such as the declining of cognitive functions and hyperpigmentation.

Supplementary material Supplementary data relating to this article includes Figs. S1-S12.

Acknowledgments 12

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Authors would like to thank Prof. Dr. Hiroshi Morita, Professor of Pharmacognosy, Hoshi University, Japan and Dr. Alfarius Eko Nugroho, Hoshi University, Japan for their kind help in performing spectroscopic analyses of the isolated compounds. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-

Conflict of interest There is no conflict of interest.

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Table legends

Table 1 Effects of isolated compounds on melanin biosynthesis and cell proliferation of B16 melanoma cells. Data presented as means ±SD (n = 3), MC, melanin content (%); CV, cell viability (%). Arbutin was used as a

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positive control at 100 µg/ml, CV = 86.5 ± 5.5, MC = 49.3 ± 4.3.

Table 2 Inhibitory activity of compounds (1-7) in the AChEI assay

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Fig. 1. Structures of compounds 1-7

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Fig. 2. Binding residues of the isolated compounds in AChE assay using

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LigPlot+ v.1.4 program.

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Graphical abstract

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Table 1 Effects of isolated compounds on melanin biosynthesis and cell proliferation of B16 melanoma cells. Data presented as means ± SD (n = 3), MC, melanin content (%); CV, cell

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viability (%). Arbutin was used as a positive control at 100 µg/ml, CV = 86.5 ± 5.5, MC = 49.3 ± 4.3.

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compound 1 compound 1 compound 1 compound 2 compound 2 compound 2 compound 3 compound 3 compound 3 compound 4 compound 4 compound 4 compound 5 compound 5 compound 5 compound 6

Concentration (µg/ml) 80 µg/ml 40 µg/ml 20 µg/ml 40 µg/ml 20 µg/ml 10 µg/ml 80 µg/ml 40 µg/ml 20 µg/ml 80 µg/ml 40 µg/ml 20 µg/ml 80 µg/ml 40 µg/ml 20 µg/ml 40 µg/ml

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Compound

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Melanin content (MC) (%) 43.8 ± 6.8 30.5 ± 2.5 28.06 ± 0.8 27.7 ± 1.7 27.9 ± 0.6 37.2 ± 2.7 28.7 ± 1.1 27.6 ± 1.6 32.3 ± 4.0 28.06 ± 1.09 36.03 ± 1.6 41.2 ± 1.8 46.2 ± 12.5 77.4 ± 6.3 65.3 ± 9.5 29.9 ± 2.7

Cell viability (CV) (%) 4.8 ± 0.04 6.06 ± 1.4 23.6 ± 5.4 15.3 ± 0.4 14.7 ± 5.5 45.4 ± 12.7 13.8 ± 2.2 31.8 ± 3.0 26.4 ± 8.1 13.8 ± 7.1 43.4 ± 4.6 75.4 ± 4.5 37.3 ± 0.4 72.3 ± 4.7 93.5 ± 13.6 31.8 ± 2.8

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37.1 ± 3.2 50.4 ± 4.5 32.5 ± 2.1 31.4 ± 0.8 27.5 ± 1.7

20 µg/ml 10 µg/ml 80 µg/ml 40 µg/ml 20 µg/ml

61.1 ± 3.8 105.3 ± 3.0 20.4 ± 4.8 16.4 ± 3.7 24.1 ± 11.9

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compound 6 compound 6 compound 7 compound 7 compound 7

Table 2 Inhibitory activity of compounds (1-7) in the AChEI assay: Compound % inhibition at 500 µM

inactive

-59.68 ± 0.41

inactive

-25.63 ± 0.42

compound 4

inactive

-22.06 ± 0.12

compound 5

50 ± 1.21 (IC50 = 482.36 ± 2.81) 65 ± 1.74 (IC50 = 354.21 ± 3.12) inactive

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inactive

Binding Energy (kcal/mol) -55.92 ± 0.82

compound 2

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compound 3

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compound 1

compound 6

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compound 7

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-113.57 ± 0.54 -72.49 ± 0.34