Chemical Constituents from Diospyros discolor Willd. and their Acetylcholinesterase Inhibitory Activity

Diospyros discolor Willd. (syn. D. blancoi) belongs to the family of Ebenaceae, and it is locally known as ‘buah mentega’1. D. discolor is used traditionally to treat wounds, snakebites, spider bites, stomachache, diabetes, heart problems, hypertension, dysentery, diarrhea and eczema1. D. discolor was reported to have free radical scavenging, anti-diarrheal, antimicrobial, analgesic and anti-inflammatory activities1-5. Diospyros sp. are rich in naphthoquinones, triterpenes, followed by flavonoid, naphthalene and coumarin-based groups6,7. The triterpenes found in Diospyros sp. were mostly of pentacyclic core especially lupane, ursane and oleanane skeleton6. Many triterpenes in Diospyros sp. showed inhibition against acetylcholinesterase (AChE) and butyrylcholinesterase enzymes in vitro and in vivo. D. discolor was reported to contain triterpenes, yet to be investigated for their AChE inhibitory activity. D. discolor leaves extract showed high acetylcholinesterase (AChE) inhibitory activity with 95.80 ± 1.57 % inhibition during preliminary screening of selected medicinal plants from Taman Herba Perlis8. Therefore, this study is warranted to investigate the chemical constituents from the leaves and stem barks extracts of D. discolor and their AChE inhibitory activity. MATERIALS AND METHODS


INTRODUCTION
Diospyros discolor Willd. (syn. D. blancoi) belongs to the family of Ebenaceae, and it is locally known as 'buah mentega' 1 . D. discolor is used traditionally to treat wounds, snakebites, spider bites, stomachache, diabetes, heart problems, hypertension, dysentery, diarrhea and eczema 1 . D. discolor was reported to have free radical scavenging, anti-diarrheal, antimicrobial, analgesic and anti-inflammatory activities [1][2][3][4][5] . Diospyros sp. are rich in naphthoquinones, triterpenes, followed by flavonoid, naphthalene and coumarin-based groups 6,7 . The triterpenes found in Diospyros sp. were mostly of pentacyclic core especially lupane, ursane and oleanane skeleton 6 . Many triterpenes in Diospyros sp. showed inhibition against acetylcholinesterase (AChE) and butyrylcholinesterase enzymes in vitro and in vivo. D. discolor was reported to contain triterpenes, yet to be investigated for their AChE inhibitory activity. D. discolor leaves extract showed high acetylcholinesterase (AChE) inhibitory activity with 95.80 ± 1.57 % inhibition during preliminary screening of selected medicinal plants from Taman Herba Perlis 8 . Therefore, this study is warranted to investigate the chemical constituents from the leaves and stem barks extracts of D. discolor and their AChE inhibitory activity.

MATERIALS AND METHODS
General experimental procedures 1 H-NMR and APT-NMR spectra were recorded at 500 or 600 MHz and 125 or 150 MHz, respectively, using Bruker 500 Ultrashield Plus (Bruker, Switzerland) and Bruker Ascend 600 (Bruker, Switzerland). FTIR-ATR spectra were recorded on FTIR Spectrometer INVENIO (Bruker, Switzerland). The mass spectra were recorded using LCMS/MS QTOF Agilent Technologies 6520 (Agilent, Santa Clara, USA). The absorbance for in-vitro analysis was obtained by Spectrostar Nano spectrometer (BMG Labtech, Germany). The solvents used for extraction and isolation were of analytical grade solvents. The silica gel used were silica gel 60 PF 254 (1.07747), silica gel 60 (0.040-0.063 mm, 1.09385), silica gel 60 PF 254 containing gypsum (1.07749), and TLC silica gel 60 F 254 aluminium sheets (1.05554). The silica gel and TLC were purchased from Merck (Germany). All chemicals and reagents used for acetylcholinesterase inhibitory activity were purchased from Sigma Aldrich unless stated otherwise.

Statistical analysis
The AChE inhibitory activity data were expressed as mean ± standard deviation. All the data were subjected to one-way analysis of variance (ANOVA) completed with Tukey's post hoc test and p<0.05 was considered as statistically significant using IBM SPSS Statistic version 20. The IC 50 was obtained by plotting nonlinear-regression curve of percentage AChE inhibitory activity against logarithm of compound concentration using GraphPad Prism statistical software version 6.01.
A new flavonol (1) was obtained along with compound (2) as a mixture in the form of brown powder. A molecular formula of C 18 O 8 ). The FTIR-ATR spectrum showed a broad peak of hydroxyl (O-H) at 3384 cm -1 , a strong peak of carbonyl (C=O) at 1719 cm -1 , medium peak of aromatic (C=C) at 1609 cm -1 and a strong peak of C-O stretch at 1214 cm -1 .
The 1 H-NMR spectrum revealed three aromatic proton signals. A pair of meta-coupled signals resonated at δ H 6.99 (1H, d, J=1.8 Hz) and 7.05 (1H, d, J=1.8 Hz) assignable as H-6 and H-8 of ring A. A singlet aromatic proton signal at δ H 7.45 integrated for two protons was assigned as H-2' and H-6' of ring B. A singlet appeared at δ H 3.90 integrated for six protons is assigned to two methoxy protons at C-3' and C-5' of ring B while another singlet signal at δ H 3.81 is attributed to methoxy protons at C-5 of ring A. The integration of methoxy protons and aromatic protons of ring B suggesting these protons are arranged in symmetrical manner. The APT-NMR spectrum displayed 14 carbon signals including two signals of methoxy carbon at δ C 56.7 (5-OCH 3 ) and 56.9 (3'/5'-OCH 3 ), three signals of methine carbon at δ C 106.4 (C-6), 112.0 (C-8) and 108.8 (C-2'/6') and nine signals of quaternary carbons. Carbonyl carbon was observed at δ C 172.4 (C-4) while C-9 was observed at δ C 152.1. The chemical shift for C-3 was not detected. The absence of typical singlet aromatic proton signal assignable at C-3 as well as chemical shift value for C-2 suggesting this compound is of flavonol moiety 18 .  The assignment of H-8 of ring A was confirmed based on its HMBC correlations to C-7 and C-10 while the placement of H-6 was determined based of its HMBC correlations to C-5, C-7 and C-10. The hydroxyl group is located at C-7 based on HMBC cross peaks between H-6 and H-8 with C-7. Meanwhile the methoxy group was assigned to C-5 based on correlations observed between H-6 and C-5. The singlet proton signal of H-2'/6' showed HMBC cross peaks with C-1' , C-2, C-3'/5' and C-4' which confirmed their placement at ring B. The connection of ring B to ring C was deduced based on HMBC correlations of H-2'/6' to C-1' and C-2. Two methoxy groups were assigned at C-3' and C-5' by observing HMBC cross peaks of H-2' and H-6' with C-3' and C-5' . The location of hydroxyl group at C-4' was confirmed based on 3 J correlations of H-2' and H-6' with C-4' . Even though no HMBC correlation was observed to confirm the assignment of C-4 and C-9 at ring C, their chemical shift values are quite typical of flavonol moiety 19 . Close inspection of all spectroscopic data confirmed that compound 1 is 7,4'-dihdroxy-5, 3' ,5'-trimethoxyflavonol.
A flavonoid (4) and six triterpenes (7-12) from the leaves and stem barks of D. discolor were examined for AChE inhibitory activity. All the compounds exhibited positive AChE inhibitory activity at 10 µM concentration, but only stigmast-4-en-3-one (7) showed inhibition of more than 50% (Table 1). When evaluated for AChE inhibitory activity in dose-dependent manner, it gave an IC 50 value of 11.77 ± 2.11 µM. Some of the compounds isolated in the present study showed moderate inhibition concentration against AChE. 20,21 while kaempferol (3) and ß-sitosterol-3-O-glucopyranoside (10) were previously reported to have low inhibition concentration against AChE 20,22,23 .

CONCLUSIONS
D. discolor (syn. D. blancoi) was found to inhibit AChE during random screening. Phytochemical study on the leaves and stem barks of D. discolor yielded a new flavonol, 7,4'-dihydroxy-5,3' ,5'trimethoxyflavonol (1) along with five known flavonoids and six known triterpenes. The compounds examined for AChE inhibitory activity showed moderate inhibiton concentration except for stigmast-4-en-3-one (7). It is postulated that the AChE inhibitory activity of the extract of D. discolor is due to synergistic effect of the phytochemicals collectively.

CONFLICTS OF INTEREST
None.