Chemical constituents from Diospyros discolor Willd. and their acetylcholinesterase inhibitory activity / Norhafizoh Abdul Somat

Diospyros discolor Willd. (Ebenaceae), also known as ‘buah mentega’ in Malay, is widely distributed in South East Asia. Traditionally, the plant was used for various health complaints such as diarrhea, dysentery, stomatitis, snake bites, spider bites, diabetes, and eczema in South East Asia. Many compounds, especially triterpenes in Diospyros sp. were reported to inhibit acetylcholinesterase and butrylcholinesterase enzyme 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 (95.80 ± 1.57 %) AChE inhibitory activity at the concentration of 100 μg/mL. Therefore, D. discolor was further evaluated for its chemical constituents and bioactivities. The leaves and stem barks of D. discolor were air-dried, powdered, and successively extracted using n-hexane, dichloromethane, and methanol. The solvents were evaporated off to obtain dried crude extracts. Exhaustive purification employing various chromatographic techniques including vacuum liquid chromatography (VLC), column chromatography (CC), radial chromatography (RC), and preparative thin layer chromatography (PTLC) afforded a new flavonol, 7,4'-dihydroxy-5,3',5'-trimethoxyflavonol (149), along with seven known triterpenes; betulin (2), betulinic acid (3), ursolic acid (27), β-sitosterol (91), β-sitosterol-3-O-glucopyranoside (92), stigmasterol (94), stigmast-4-en-3-one (95), four flavonols; kaempferol (109), hyperin (116), astragalin (122), isoquercitrin (123) and a flavan-3-ol; (+)-epicatechin (140) from the leaves and stem bark of D. discolor. Their structures were determined by comprehensive NMR, FTIR and MS analysis. The methanol extracts of leaves and stem barks of D. discolor did not show significant antiproliferation against SH-SY5Y cell when incubated at different sample concentration ranging from 1000 to 3.90625 ppm at 24-, 48- and 72-hours incubation time. However, the methanol extract of stem barks exhibit significant antiproliferative activity at 125, 500 and 1000 ppm concentration at 72 hours incubation. The compounds (2, 3, 91, 92, 94, 95, 122) were tested for AChE inhibitory activity, but only stigmast-4-en-3-one (95) showed the highest AChE inhibitory activity among the compounds tested with an IC50 value of 11.77 ± 2.11 μM. Compound (95) was previously isolated from D. maritima stem and D. eriantha heartwood, but this is the first time reported from D. discolor and studied against AChE. In conclusion, the phytochemical study on D. discolor resulted in a new flavonol (149) and twelve known compounds of triterpenes and flavonoids previously reported in Diospyros sp. Even though D. discolor extracts showed high percent inhibition against AChE enzyme, the compounds isolated showed moderate inhibition.