A bioactive cycloartane triterpene from Garcinia hombroniana

Abstract The dichloromethane bark extract of Garcinia hombroniana yielded one new cycloartane triterpene; (22Z,24E)-3β-hydroxycycloart-14,22,24-trien-26-oic acid (1) together with five known compounds: garcihombronane G (2), garcihombronane J (3), 3β acetoxy-9α-hydroxy-17,14-friedolanostan-14,24-dien-26-oic acid (4), (22Z, 24E)-3β, 9α-dihydroxy-17,14-friedolanostan-14,22,24-trien-26-oic acid (5) and 3β, 23α-dihydroxy-17,14-friedolanostan-8,14,24-trien-26-oic acid (6). Their structures were established by the spectral techniques of NMR and ESI-MS. These compounds together with some previously isolated compounds; garcihombronane B (7), garcihombronane D (8) 2,3’,4,5’-tetrahydroxy-6-methoxybenzophenone (9), volkensiflavone (10), 4’’-O-methyll-volkensiflavone (11), volkensiflavone-7-O-glucopyranoside (12), volkensiflavone-7-O-rhamnopyranoside (13), Morelloflavone (14), 3’’-O-methyl-morelloflavone (15) and morelloflavone-7-O-glucopyranoside (16) were evaluated for cholinesterase enzymes inhibitory activities using acetylcholinesterase and butyrylcholinesterase. In these activities, compounds 1–9 showed good dual inhibition on both the enzymes while compounds 10–16 did not reasonably contribute to both the cholinesterases inhibitory effects. Graphical abstract


In vitro anticholinesterase activities
Compounds isolated in the current studies (1-6) and that isolated previously (7-16) were evaluated for anticholinesterase activities. In this investigation, only compounds 1-9 displayed more than 50% inhibition. Compounds 10-16 which showed less than 50% inhibition were considered inactive. Compound 9 was the significant inhibitor of AChE (IC 50 10.3 μM) compared to the reference drug, galanthamine (IC 50 2.05 μM), followed by 7, 4 and 1 which, respectively, showed an inhibition against AChE with the IC 50 of 16.3, 16.7 and 17.9 μM. Compounds 3 and 8 (IC 50 = 27. 9 and 25.0 μM) were almost two times less active than compounds 9 and 7. In case of inhibitory activity against BChE, compounds 4, 5 and 7 showed good potency with IC 50 values of 16.1, 19.7 and 21.5 μM comparable to galanthamine (IC 50 = 19.24 μM). Compounds 1-3, and 6 and 9 in BChE inhibition showed only moderate activity with IC 50 of 28.4, 25.0, 31.4, 34.6 and 31.5 μM. In comparison with physostigmine, these compounds (1-9) showed less inhibition against AChE and BChE both. It is also worth to mention that compounds 1, 3 and 6-9 showed more selectivity towards AChE with selectivity indices of 1.58, 1.12, 1.47, 1.31, 1.58 and 3.05, while compounds 2 and 4 were more selective for BChE with selectivity indices of 1.40 and 1.03, respectively. Table 1 summarises the detailed % inhibition, IC 50 and the selectivity indices of tested compounds and standard drugs. AChE-specific inhibitors might be useful at the earlier stages of AD whereby AChE enzyme has dominant role in splitting of acetylcholine. However, as the disease progresses, the role of BChE becomes more prominent and therefore a dual inhibitor is expected to be beneficial at both stages.

Plant materials
The plant (G. hombroniana) was collected from Penang Botanical Garden, Malaysia, and has been deposited at the herbarium of this Garden with a voucher specimen (PBGK12).

General experimental procedure
Silica gel 60 (0.040-0.063 mm) was used as an adsorbent for CC. Merck TLC pre-coated aluminium plates, silica gel 60 F 254 , were used for partitioning and detection. Melting points were determined using a Stuart Scientific Melting Point SMP 1 (UK). UV spectra were measured using a Perkin-Elmer, Lambda 25 UV/Vis spectrometer. IR spectra were recorded in KBr and also by direct placement of finely powdered samples on the eye of the Perkin-Elmer (USA) 2000 FT-IR spectrophotometer. One-dimensional and two-dimensional NMR experiments were performed at room temperature using a Bruker Ascend 500 MHz ( 1 H) and 125 MHz ( 13 C) spectrometer (Bruker Biospin, Switzerland). A Tecan Infinite 200 Pro Microplate spectrometer was used for the evaluation of the cholinesterase inhibitory assays.

Cholinesterase inhibitory assay
Cholinesterase inhibitory activities of the compounds 1-14 were determined by slightly modified Ellman's assay (Ellman et al. 1961). In this assay, 140 μL of 0.1 M sodium phosphate buffer (pH 8.0) was added to 96 wells microplate followed by 20 μL of test samples and 20 μL of 0.09 units/mL AChE enzyme. After 15 min of pre-incubation at room temperature, 10 μL of 10 mM DTNB was added into each well following the addition of 10 μL of 14 mM of ATCI. Absorbance of the coloured end product was measured using Tecan Infinite 200 Pro Microplate spectrometer at 412 nm at 30 min after initiation of the enzymatic reaction. Absorbance of the test samples was corrected by subtracting the absorbance of their respective blank. BChE inhibitory assay was carried out with the same procedure using the BChE enzyme and S-butyrylthiocholine chloride as substrate. Galanthamine and Physostigmine were taken as reference standards. The test samples and the standards were prepared in DMSO at the initial concentration of 1 mg/mL. The concentration of DMSO in final reaction mixture was 1%. Initial cholinesterase inhibitory activity of the compounds was evaluated at 50 μg/mL. Compounds with 50% or more inhibition of concentrations (50.0, 25.0, 12.5, 6.25 and 3.125 μM) were further evaluated for the determination of 50% inhibitory concentration (IC 50 ).

Statistical analyses
All data were analysed and expressed as means ± standard deviation of three replicates. The differences between the assayed values were analysed using one-way analysis of variance, followed by Tukey's HSD test at 95% and 99% confidence interval with SPSS software, version 19.0 (SPSS Inc., Chicago, USA). IC 50 were calculated using GraphPad Prism 6.02 (GraphPad Software Inc., La Jolla, USA).

Conclusions
In this study, one new cycloartane triterpene and five known triterpenoids were isolated from dichloromethane extract of G. hombroniana. These compounds and some previously isolated triterpenes, benzophenone and biflavonoids from G. hombroniana were evaluated for cholinesterase enzymes inhibitory activities. In this in vitro enzymatic study, only compounds 1-9 showed moderate cholinesterase inhibitory effects with more than 50% inhibition against both AChE and BChE. Compound 9 was the significant inhibitor of AChE followed by 7, 4 and 1. In BChE inhibition, compounds 4 and 5 showed good potency comparable to galanthamine. In view of the obtained results, it can be concluded that further future phytochemical and pharmacological studies using advanced experimental techniques on the bark of G. hombroniana is needed.