Abstract - This study was carried out to investigate the phytochemicals from Polyalthia sumatrana and their acetylcholinesterase inhibitory activity. Fractionation and purification of the leaves of P. sumatrana led to the isolation and identification of five alkaloids; boldine (1), norboldine (2), liriodenine (3), predicentrine (4), laurotetanine (5) together with β-sitosterol (6), β-sitostenone (7), vanillin (8), vanillic acid (9) and cinnamic acid (10). The structures of these compounds were obtained by analysis of their spectroscopic data, as well as the comparison with that of reported data. Acetylcholinesterase inhibitory activity revealed that all isolated alkaloids were found to inhibit AChE with percentage inhibition values ranged from 45.0 to 80.6%.
Keywords - Annonaceae, Polyalthia sumatrana, alkaloid, acetylcholinesterase, Alzheimer
I. INTRODUCTION
Alzheimer's disease (AD) is the most common form of dementia in elderly people, accounting for 60%–80% of the total dementia cases worldwide (Bondi et al., 2017). AD is a multifactorial, neurodegenerative, progressive, and fatal disorder characterized by loss of cholinergic neurons in the hippocampus and cerebral cortex mainly affecting cholinergic neurotransmission. Based on the cholinergic hypothesis, which states that low production of acetylcholine (ACh) initiates AD, the first drugs approved to treat AD were cholinesterase enzyme inhibitors (IChE), which are responsible for the hydrolysis of ACh (Jahn, 2013). Plant secondary metabolites have been a continuous source of new approaches for the treatment of AD, especially alkaloids. For instance, physostigmine (Physostigma venenosum), is an IChE and allosteric modulator of the central nicotinic receptor. Galanthamine (Galanthus woronowii) is a selective inhibitor of AChE, an allosteric modulator of the central nicotinic receptor, inhibits Aβ aggregation, and promotes hippocampal neurogenesis. Berberis (Berberis mahonia) is an IChE, inhibits the formation and aggregation of Aβ, and reduces hyperphosphorylation of tau protein in some in vitro systems (Briggs et al., 2016). However, these drugs only provide asymptomatic and palliative pharmacological effects, becoming moderate and wearing off with continuous use (Nordberg and Svensson, 1998). To date, a paradigm shift towards a multi-targeted modulation approach in the management of neurodegenerative diseases is in line with the prevalent characteristic natural product derivatives with multi-targeting properties. Therefore, the search for new AChE inhibitors is of great interest.
The genus Polyalthia is one of the largest and notable genera in the Annonaceae family and consists of about 120 species of shrubs and trees. The species have been used in many tropical countries as folklore medicine for the treatment of rheumatic fever, malaria, hepatitis, pneumonia, dysmenorrheal, globus hystericus, and syphilis (Li and Gilbert, 2011; Shakri et al., 2020). Polyalthia sumatrana is locally known as buah sasak in Malaysia and distributed mainly in Thailand, Peninsular Malaysia, Sumatra, Java, and Borneo. It is a sub-canopy tree up to 23 m tall and found in undisturbed forests up to 700 m altitude, usually in mixed dipterocarp forests (Burkill, 1966). We have recently reported on the chemical compositions of the essential oil from this species (Shakri et al., 2020). The GC and GC-MS analysis of P. sumatrana leaf essential oil resulted in the identification of thirty components (91.8%) with a high concentration of sesquiterpene hydrocarbons (84.8%). The oil was characterized by the abundance of bicyclogermacrene (18.8%), cis-calamenene (14.6%), β-elemene (11.9%), and α-cubebene (10.7%). As part of our continuing search to explore natural compounds from Malaysian Polyalthia species, we have investigated the phytochemical study of the leaves of P. sumatrana collected from Malaysia.
II. METHODS
A. Plant material
The leaves of P. sumatrana were collected from Behrang, Perak in September 2019 and identified by Shamsul Khamis from Universiti Kebangsaan Malaysia (UKM). The voucher specimen (SK30/19) was deposited at UKM Herbarium.
B. General experimental procedures
Soxhlet extraction technique was applied to extract the phytochemicals from the dried sample using different polarity solvents (n-hexane, ethyl acetate, and methanol). Vacuum liquid chromatography (VLC) was performed on Merck silica gel 60 (230-400 mesh) while column chromatography (CC) on Merck silica gel 60 (70-230 mesh) was the stationary phase. Thin-layer chromatography (TLC) analysis was performed on Merck pre-coated silica (SiO2) gel F254 plates (0.2 mm thickness) to detect and monitor the presence of compounds in the samples. The TLC and PTLC spots were visualized under UV light (254 and 366 nm) followed by spraying with Dragendorff’s reagent for an alkaloid detection. Melting points were measured using melting point apparatus equipped with a microscope, Leica Gallen III and were uncorrected. The 1H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were recorded on a Bruker Avance 400 Spectrometer. Chemical shifts were reported in ppm and CDCl3 as the solvent. The residual solvent was used as an internal standard. The IR spectra were recorded on Perkin Elmer ATR and 1600 spectrophotometer series as KBr disc. The mass spectra were obtained from LCMS-IT-TOF, Shimadzu.
C. Extraction and isolation
The dried powder leaves (350 g) of P. sumatrana were ground and extracted exhaustively for 12 hours by Soxhlet extraction with hexane, followed by dichloromethane (DCM). The extraction of alkaloids was carried out in the usual manner, which has been described in detail and gave 14 g of crude alkaloid. The crude alkaloid was submitted to exhaustive column chromatography over silica gel using DCM gradually enriched with methanol (MeOH) to yield 18 fractions. Fractions were combined on the basis of TLC behavior. Fractions 4-7 (2.0 g), afforded two alkaloids identified as (1) (10 mg) (PTLC; DCM:MeOH 92:8; Rf 0.25) and (2) (15 mg) (PTLC; DCM:MeOH 95:5; Rf 0.40). Fraction 11-13 (1.9 g) produced (3) (12 mg) (PTLC; DCM:MeOH 95:5; Rf 0.50) and (4) (15 mg) (PTLC; DCM:MeOH 94:6; Rf 0.52). Fraction 12-15 (2.0 g) produced (5) (16 mg) (PTLC; EtOAc:MeOH 96:4; Rf 0.45). The hexane extract was separated on a silica gel column using a gradient Hex:DCM to give β-sitosterol (6) (30 mg), β-sitostenone (7) (22 mg), vanillin (8) (25 mg), vanillic acid (9) (22 mg) and cinnamic acid (10) (21 mg).
D. Acetylcholinesterase (AChE) inhibitory activity
AChE inhibitory activities were measured by slightly modifying the
spectrophotometric method (Salleh et al. 2016a). Electric eel AChE was
used, while acetylthiocholine iodides were employed as substrates of the
reaction. 5,5’-Dithio-bis(2-nitrobenzoic) acid (DTNB) was used for the
measurement of the AChE activity. Briefly, 140 µL of sodium phosphate buffer
(pH 8.0), 20 µL of DTNB, 20 µL of the compound (concentration of 1 mg/mL) and
20 µL of AChE solution were added by multichannel automatic pipette in a
96-well microplate and incubated for 15 min at 25°C. The reaction was then
initiated with the addition of 10 µL of acetylthiocholine iodide. Hydrolysis of
acetylthiocholine iodide was monitored by the formation of the yellow
5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with
thiocholines, catalyzed by enzymes at 412 nm utilizing a 96-well microplate
reader (Epoch Micro-Volume Spectrophotometer). Percentage inhibition (I%) of
AChE was determined by comparison of reaction rates of samples relative to
blank sample (ethanol in phosphate buffer pH = 8) using the formula: I% = [ E –
S / E ] × 100; where E is the activity of enzyme without test sample and S is the
Figure 1- Chemical structures of isolated compounds
activity of the enzyme with the test sample. Galantamine (1 mg/mL) was used as a positive control. Analyses were performed in triplicate and the result was expressed as means ± SD of triplicate. Data obtained from the acetylcholinesterase activity are expressed as mean values. Statistical analyses were carried out by employing one-way ANOVA (p>0.05).
III. RESULTS
Herbal plants act as a significant source for discovering new compounds with potential therapeutic activities. The Polyalthia genus is considered to be of medicinal importance because of the presence of clerodane diterpenoids and alkaloids in various parts of the plant. Besides, chemical constituents from Polyalthia species have also resulted in the isolation and identification of flavonoids, acetogenin, and terpenoids. In view of the attributed medicinal properties, studies were undertaken on the leaves parts of P. sumatrana which resulted in the isolation and structure elucidation of alkaloids, steroids, and benzoic acid derivatives. Five alkaloids have been successfully isolated which are boldine (1), norboldine (2), liriodenine (3), predicentrine (4), laurotetanine (5), together with β-sitosterol (6), β-sitostenone (7), vanillin (8), vanillic acid (9) and cinnamic acid (10). The chemical structures of isolated compounds as shown in Fig. 1.
All secondary metabolites were identified by analyzing their spectroscopic data and comparing them with the literature data. To the best of our knowledge, all compounds were isolated for the first time from this species. These isolated alkaloids have been previously reported from several Polyalthia species. Compounds (1) and (4) have been isolated previously from P. cauliflora (Jossang et al., 1984), while compound (3) was isolated from P. laterifolia (Azziz et al., 2020), P. insignis (Lee et al., 1997), and P. nemoralis (Lu et al., 2009). In addition, compounds (2) and (5) were reported previously from P. longifolia (Chen et al., 2000). Furthermore, alkaloids compounds were also reported from other Annonaceae genera such as Annona (Dahiya and Dahiya, 2021), Fissistigma (Pham et al., 2020), Miliusa (Son, 2019), Alphonsea (Bakri et al., 2017), and Xylopia (Silva et al., 2015). The presence of those valuable alkaloids in various species enriches their chemical diversity and provides evidence for chemotaxonomic studies of Polyalthia species and the family Annonaceae as well.
Boldine (1). Light brown powder; m.p. 162-164ºC; MS m/z 328 [M+, C19H21NO4]; 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): δ 2.54 (3H, s, N-CH3), 2.40-2.74 (3H, m, H-7b, H-4), 2.95-3.12 (4H, m, H-6a, H-5, H-7a), 3.60 (3H, s, 2-OCH3), 3.90 (3H, s, 10-OCH3), 6.65 (1H, s, H-3), 6.80 (1 H, s, H-8), 7.90 (1H, s, H-11); 13C NMR (100 MHz, CDCl3, δ, ppm): δ 28.5 (C-4), 35.0 (C-7), 43.2 (N-CH3), 53.0 (C-5), 56.0 (10-OCH3), 60.5 (1-OCH3), 62.5 (C-6a), 110.0 (C-11), 113.0 (C-3), 114.0 (C-8), 123.5 (C-11c), 126.0 (C-11b), 126.5 (C-11a), 130.0 (C-3a), 130.0 (C-7a), 142.0 (C-1), 145.0 (C-9), 145.2 (C-10), 148.0 (C-2).
Norboldine (2). Brown amorphous solid; m.p. 138-140ºC; MS m/z 314 [M+, C18H19NO4]; 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): δ 2.77-3.80 (6H, m, H-4a/4b/5a/5b/7a/7b), 3.61 (3H, s, 1-OCH3), 3.88 (3H, s, 10-OCH3), 4.14 (1H, dd, J = 14.0, 4.4, H-6a), 6.65 (1H, s, H-3), 6.76 (1H, s, H-8), 8.02 (1H, s, H-11); 13C NMR (100 MHz, CDCl3, δ, ppm): δ 29.2 (C-4), 36.5 (C-7), 43.0 (C-5), 53.8 (C-6a), 56.0 (10-OCH3), 60.2 (1-OCH3), 110.2 (C-11), 113.5 (C-3), 114.0 (C-8), 123.5 (C-11a), 125.0 (C-1a), 128.0 (C-1b), 130.0 (C-7a), 130.5 (C-3a), 141.8 (C-1), 145.0 (C-9), 145.5 (C-10), 148.2 (C-2).
Liriodenine (3). Yellow needles; m.p. 280-282ºC; MS m/z 275 [M+, C17H9NO3]; 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): δ 6.40 (2H, s, OCH2O), 7.16 (1H, s, H-3), 7.59 (1H, td, J = 7.8, 7.2, 1.5, H-9), 7.76 (1H, td, J = 7.2, 1.2, H-10), 7.79 (1H, d, J = 5.1, H-4), 8.59 (1H, dd, J = 7.8, 1.2, H-8), 8.66 (1H, d, J = 7.2, H-11), 8.90 (1H, d, J = 5.1, H-5); 13C NMR (100 MHz, CDCl3, δ, ppm): δ 102.8 (OCH2O), 103.6 (C-3), 108.5 (C-1a), 123.6 (C-3b), 124.7 (C-4), 127.7 (C-11a), 128.9 (C-9), 129.2 (C-8), 131.6 (C-7a), 133.2 (C-11a), 134.3 (C-10), 136.1 (C-3a), 145.3 (C-5), 145.7 (C-6a), 148.5 (C-2), 152.1 (C-2), 182.8 (C-7).
Predicentrine (4). Yellow powder; m.p. 168-170ºC; MS m/z 342 [M+, C20H23NO4]; 1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): δ 2.50 (1H, dd, J = 12.0, 4.0, H-5b) 2.52 (3H, s, N-CH3), 2.54-2.58 (1H, m, H-7b), 2.65 (2H, m, H-4a, 4b), 3.03-3.06 (1H, m, H-7a), 3.12-3.18 (2H, m, H-5a, 6a), 3.65 (3H, s, 1-OCH3), 3.88 (3H, s, 9-OCH3), 3.90 (3H, s, 10-OCH3), 6.58 (1H, s, H-3), 6.80 (1H, s, H-8), 8.05 (1H, s, H-11); 13C NMR (100 MHz, CDCl3, δ, ppm): δ 26.9 (C-4), 32.7 (C-7), 41.6 (N-CH3), 52.0 (C-5), 54.2 (9-OCH3), 54.4 (10-OCH3), 58.3 (1-OCH3), 148.7 (C-2), 61.8 (C-6a), 113.4 (C-3), 110.5 (C8), 110.9 (C-11), 123.6 (C-11a), 124.0 (C-6b), 125.5 (C-11b), 127.7 (C-3a), 128.3 (C-7a), 142.5 (C-1), 147.2 (C10), 147.5 (C-9).
Laurotetanine (5). Brownish amorphous
solid; m.p 124-125°C; MS m/z 328 [M+, C19H21NO4];
1H NMR (400 MHz, CDCl3, δ, ppm, J/Hz): δ 2.71 (2H, m,
H-7), 3.02 (2H, m, H-4), 3.38 (2H, m, H-5), 3.65 (3H, m, 1-OCH3),
3.82 (1H, m, H-6a), 3.88 (3H, m, 2-OCH3), 3.87 (3H, m, 10-OCH3),
6.59 (1H, s, H-3), 6.82 (1H, s, H-8), 8.05 (1H,
Table 1 - Acetylcholinesterase inhibitory activity of isolated alkaloids from P. sumatranaa
aData represent mean ± standard deviation of three replicate experiments; (p < 0.05); bpositive control
s, H-11); 13C NMR (100 MHz, CDCl3, δ, ppm): δ 29.0 (C-4), 36.5 (C-7), 43.0 (C-5), 53.5 (C-6a), 55.8 (10-OCH3), 56.0 (2-OCH3), 60.2 (1-OCH3), 110.5 (C-3), 111.0 (C-11), 113.8 (C-8), 124.0 (C-11a), 126.5 (C-1a), 127.5 (C-1b), 128.5 (C-3a), 129.2 (C-9), 129.5 (C-7a), 144.2 (C-1), 145.5 (C-10), 152.0 (C-2).
β-Sitosterol (6). White needles; m.p 133-134°C; MS m/z 414 [M+, C29H50O]. Based on the NMR spectral data and their comparison with those reported in the literature (Salleh et al. 2016a).
β-Sitostenone (7). White solids; m.p 77-79°C; MS m/z 412 [M+, C29H48O]. Based on the NMR spectral data and their comparison with those reported in the literature (Salleh et al. 2016a).
Vanillin (8). Colourless solid; m.p 80-81°C; MS m/z 152 [M+, C8H8O3]. Based on the NMR spectral data and their comparison with those reported in the literature (Salleh et al. 2016b).
Vanillic acid (9). Colourless solid; m.p 209-212°C; MS m/z 168 [M+, C8H8O4]. Based on the NMR spectral data and their comparison with those reported in the literature (Salleh et al. 2016b).
Cinnamic acid (10). White crystalline; m.p 132-134°C; MS m/z 148 [M+, C9H8O2]. Based on the NMR spectral data and their comparison with those reported in the literature (Wang et al. 2012).
Plant alkaloids have become important sources of nutraceuticals owing to their pharmacological importance especially in the management of neurodegenerative diseases such as Alzheimer's disease. In assessing the therapeutic potentials of plant phytochemicals, in the current study, the isolated alkaloids of P. sumatrana were subjected to the acetylcholinesterase inhibitory activity and the results are shown in Table 1.
The aporphine type alkaloid is an important class of natural AChE inhibitors and there are several sub-type aporphine alkaloids that have been obtained as AChE inhibitors (Loizzo et al., 2008). However, of the five aporphine alkaloids from P. sumatrana, compound (3) showed the highest AChE inhibitory activity. The structure-activity relationship of compound (3) for AChE inhibitory effects revealed that the presence of methylenedioxy might play important roles in the activity of aporphine alkaloids, which was in accordance with a previous study (Dong et al., 2015). In addition, we found that the hydroxyl at C-2 position may also be important to activity. The relationship was also confirmed by another two aporphine alkaloids reported before (Markmee et al., 2006), which had similar structures to compounds (1), (2) and (4) but no hydroxyl at C-2 position and showed weak activity against AChE.
V. CONCLUSIONS
This study revealed that the alkaloids showed promising
AChE inhibitory activity. The isolated compounds should be further evaluated to develop safe agents to be introduced in modern therapy. In addition, a more comprehensive understanding should be made to reveal the mode action of alkaloids which might help understand the possible roles in human physiology.
ACKNOWLEDGEMENT
The authors would like to thank the Department of Chemistry, Faculty of Science and Mathematics, UPSI for research facilities. This research was funded by the Ministry of Education (MOE) through the Fundamental Research Grant Scheme for Research Acculturation of Early Career Researcher (FRGS-RACER) with no. FRGS-RACER/1/2019/STG01/UPSI/1).
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Received: August 18, 2021
Sent to Subject Editor: September 16, 2021
Accepted: October 25, 2021
Recommended by Subject Editor: Laura Briand