Research Article |
Corresponding author: Suciati Suciati ( suciati@ff.unair.ac.id ) Academic editor: Magdalena Kondeva-Burdina
© 2024 Suciati Suciati, Erlinda Rhohmatul Laili, Hamizah Haula, Lidya Tumewu, Nitra Nuengchamnong, Nungruthai Suphrom, Aty Widyawaruyanti.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Suciati S, Laili ER, Haula H, Tumewu L, Nuengchamnong N, Suphrom N, Widyawaruyanti A (2024) Phytoconstituents, Antioxidant, and cholinesterase inhibitory activities of the leaves and stem extracts of Artocarpus sericicarpus. Pharmacia 71: 1-8. https://doi.org/10.3897/pharmacia.71.e112499
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The study aimed to investigate the antioxidant and cholinesterase inhibitory activities of the leaves and stems of Artocarpus sericicarpus and to analyse the phenolic compounds in the extracts. The modified Ellman’s method was used to determine the cholinesterase inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The antioxidant properties were evaluated using DPPH and ABTS methods. The total phenolic content (TPC) was measured by spectrometric assay, and compound identification was carried out by LC-MS/MS analysis. The results showed that the leaf and stem extracts of A. sericicarpus exerted significant inhibitory effects against AChE and BChE, as well as antioxidant activities. The stem ethanolic extract exhibited the highest potency against AChE and BChE with IC50 values of 5.81 and 11.46 µg/mL, respectively. The leaf and stem ethanolic extracts gave higher antioxidant activities and TPC compared to the water-based extracts. The LC-MS/MS analysis indicated the presence of phenolic compounds, such as flavones, flavonols, flavanones, prenylated chalcones, and xanthones in the extracts.
Artocarpus sericicarpus, cholinesterase inhibitor, antioxidant, phenolic compounds
Among several neurological disorders, Alzheimer’s disease (AD) is a progressive neurodegenerative disease that commonly affects elderly people. It is reported that more than 50 million people are suffering from AD worldwide, and the number is expected to increase every year (
Medicinal plants have been used widely as a source of therapeutic substances. Plant secondary metabolites have played a significant role in the development of medicine for various therapeutic targets, including neurological disorders (
The leaves and stem of A. sericicarpus were collected from Balikpapan Botanical Garden, East Kalimantan, Indonesia in 2015. The voucher specimens (BPN-03) were kept at the Institute of Tropical Diseases, Universitas Airlangga. The plant was identified by Purwodadi Botanical Garden, East Java, Indonesia, with identification letter number: 0074/IPH.06/HM/XII/2015.
The reagents used for cholinesterase assays were acetylcholinesterase from electric eel (AChE type VI-S), acetylthiocholine iodide (ATCI), horse-serum butyrylcholinesterase (BChE), butyrylthiocholine iodide (BTCI), 5,5´-dithiobis[2-nitrobenzoic acid] (DTNB), bovine serum albumin (BSA), Tris buffer, and galantamine. The chemicals used for antioxidant assays were 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2´-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and potassium persulfate. Folin-Ciocalteu’s phenol reagent, sodium carbonate, and standard gallic acid were used for the determination of total phenolics. All reagents were obtained from Sigma-Aldrich.
Freshly collected leaves and stems of A. sericicarpus were air-dried at room temperature for approximately seven days and then pulverized. Ten grams of the powdered leaves and stems were each extracted with ethanol using maceration protocol. The samples were soaked in the solvent (100 mL) for 24 hours, followed by vacuum filtration. The extraction process was repeated twice. Another extraction was carried out in water. Ten grams of the powdered leaves and stems were each soaked in water (100 mL) in an ultrasonic bath for 3 × 10 mins, frequency 50 Hz. The extract and residue were then separated by filtration. The residue was re-extracted using the same protocol twice. All collected filtrates from the ethanolic and water extracts were evaporated in a rotary evaporator to obtain crude ethanolic and water extracts.
The assay was performed using a modified Ellman’s protocol as has been reported in our previous publications (
The 50% inhibitory concentration (IC50) was calculated using GraphPad Prism 8.0 software (Dotmatics, USA) using log concentrations as axis and % inhibition as ordinate.
The DPPH assay was carried out according to the modified method developed by
The ABTS assay was performed based on
The TPC of the extracts was measured using a slightly modified method developed by
The LC-MS/MS analysis was performed using Agilent 1260 Infinity Series HPLC connected to a QTOF 6540 UHD accurate mass spectrometer. The chromatographic separation was carried out with an analytical C-18 column (Phenomenex Luna C18(2), 150 × 4.6 mm, 5 µm, USA). A 10 µL sample solution (10 mg/mL in methanol) was introduced into the LC system and eluted with a solvent combination of water containing 0.1% formic acid (A), and acetonitrile with 0.1% formic acid (B). A linear gradient from 5 to 95% B in 30 mins, and hold on at this ratio for 10 mins at a flow rate of 0.5 mL/min. The ion source parameter was performed in positive mode with a mass range of m/z 100–1,000 amu. The ESI-MS condition parameters were as follows: drying gas (N2) 7 L/min; dry gas temperature at 350 °C; capillary voltage +3,500 V; and nebulizer pressure at 30 psig. Fragmentations were performed using auto MS/MS with collision energies at 10, 20, and 40 eV. Compound identification was carried out by comparing the MS data, MS/MS fragmentation profiles, and molecular formula with the literature data and databases such as Human Metabolome and MetFrag (https://msbi.ipb-halle.de/MetFrag/) with a maximum error of 5 ppm was accepted (
To perform a GNPS analysis the LC-QTOF-MS/MS data were converted to mmol file format using MSConvert software. The data were then transferred to the GNPS server (gnps.ucsd.edu) to generate the chemical networking map (ID = 79727b25f7a4487d904ba114b79a4319). The networks were created where edges were filtered to have a cosine score above 0.7 and more than 6 matched peaks. Precursor ion mass tolerance was set to 0.2 Da, while MS/MS fragment ion tolerance was set at 0.5 Da. The molecular networking data were visualized with Cytoscape software version 3.9.1. A ball-and-stick layout where nodes represent parent mass and cosine score was reflected by edge thickness (Nothia et al. 2020;
The leaves and stem extracts were screened against AChE and BChE enzymes based on the modified Ellman’s method. The cholinesterase inhibitory activity of the extracts expressed as IC50 values, calculated from the regression equations obtained from the activity of samples at different concentrations, was found to increase dose-dependently (Fig.
ChE inhibitory activity, antioxidant, and total phenolic contents (TPC) of A. sericicarpus extracts.
Samplea | IC50 (µg/mL)b | TPC b (mg GAE/g extract) | |||
---|---|---|---|---|---|
AChE | BChE | DPPH | ABTS | ||
LE | 12.31 ± 0.88 | 22.20 ± 0.60 | 10.54 ± 0.23 | 8.74 ± 0.03 | 217.23 ± 0.80 |
LW | 32.41 ± 1.36 | 35.06 ± 0.23 | 17.61 ± 0.56 | 11.32 ± 0.03 | 157.61 ± 0.95 |
SE | 5.81 ± 0.1 | 11.46 ± 0.10 | 14.42 ± 0.47 | 8.62 ± 0.32 | 215.60 ± 1.08 |
SW | 8.10 ± 0.41 | 16.84 ± 0.26 | 17.43 ± 0.79 | 11.74 ± 0.13 | 140.80 ± 1.87 |
Gal | 0.20 ± 0.01 | 1.33 ± 0.02 | ND | ND | ND |
GA | ND | ND | 2.76 ± 0.02 | 0.97 ± 0.03 | ND |
The antioxidant activity of the extracts was evaluated by DPPH and ABTS tests. Table
The total phenolic contents (TPC) in the extracts were evaluated using a Folin-Ciocalteu reagent. The gallic acid standard curve equation (y = 0.0056× + 0.0492, R2 = 0.9994) was used for the calculation of the TPC content in the extracts. The results as can be seen from Table
To analyze the difference between the leaves and the stem extracts in terms of chemical composition, a molecular networking study was carried out. The GNPS molecular networking (Fig.
LC-MS/MS analysis of phenolic compounds identified in the leaf ethanolic extract of A. sericicarpus.
RTa (mins) | [M+H]+ | Product ions m/z | Formula | Exact mass | Diff (ppm) | Proposed Compounds |
---|---|---|---|---|---|---|
9.984 | 355.1029 | 163.0385, 145.0285, 135.0438, 117.0326, 89.0381 | C16H18O9 | 355.1024 | -1.52 | Chlorogenic acid |
9.986 | 163.0391 | 145.0278, 135.0438, 117.0336, 89.0379, 77.0382 | C9H6O3 | 163.0390 | -0.79 | Umbelliferone |
11.815 | 611.1624 | 465.1022, 345.0609, 303.0502, 255.0862, 129.0539, 85.0280 | C27H30O16 | 611.1607 | -2.84 | Rutin |
12.455 | 449.1086 | 349.0291, 287.0553, 139.0654, 95.0836 | C21H20O11 | 449.1087 | -1.7 | Kaempferol glucoside |
12.473 | 465.1031 | 303.0500, 145.0491, 127.0388, 85.0274, 69.0320 | C21H20O12 | 465.1028 | -0.75 | Isoquercetin |
15.313 | 517.1713 | 499.1622, 337.1083, 283.0613, 127.0407, 85.0280 | C26H28O11 | 517.1704 | -1.67 | Luteone glucoside |
19.157 | 427.2115 | 409.2032, 337.1449, 299.1657, 257.1549, 227.1067, 185.0963, 71.0495 | C25H30O6 | 427.2115 | 0.04 | Broussoflavan A |
19.436 | 409.2012 | 353.1403, 257.1551, 231.1031, 201.0920, 173.0964, 123.0442, 69.0699 | C25H28O5 | 409.2010 | -0.61 | Paratocarpin J |
21.349 | 423.1808 | 271.1329, 253.1232, 201.0927, 137.0234 | C25H26O6 | 423.1802 | -1.38 | Kuwanon F |
22.526 | 409.2015 | 391.1921, 337.1450, 257.1546, 239.1441, 185.0965, 157.1019, 123.0433 | C25H28O5 | 409.2010 | -1.34 | Paratocarpin D |
22.669 | 409.2017 | 337.1448, 257.1550, 239.1441, 185.0966, 185.0969, 123.0433 | C25H28O5 | 409.2010 | -1.83 | Paratocarpin E |
25.548 | 339.1231 | 283.0618, 189.0919, 165.0186, 123.0089 | C20H18O5 | 339.1227 | -1.18 | Paratocarpin K |
25.58 | 409.2015 | 353.1403, 257.1548, 201.0920, 69.0697 | C25H28O5 | 409.2010 | -1.34 | Paratocarpin L |
27.581 | 391.1910 | 373.1820, 281.1551, 263.1440, 239.1446, 221.1338, 197.0974, 169.1015, 119.0489 | C25H26O4 | 391.1904 | -1.57 | Paratocarpin B |
28.298 | 391.1912 | 337.1450, 241.1599, 209.0969, 185.0964, 155.0865, 137.0238, 69.0698 | C25H26O4 | 391.1904 | -2.08 | Paratocarpin C |
LC-MS/MS analysis of phenolic compounds identified in the stem ethanolic extract of A. sericicarpus.
RTa (mins) | [M+H]+ | Product ions m/z | Formula | Exact mass | Diff (ppm) | Proposed Compounds |
---|---|---|---|---|---|---|
13.537 | 583.1804 | 437.1241, 301.0711, 191.0334, 129.0545, 85.0280, 71.0487 | C30H30O12 | 583.1810 | 1.03 | Epicatechin 3-O-(2-trans-cinnamoyl-beta-D-allopyranoside) |
22.173 | 437.1609 | 395.1127, 381.0959, 363.0812, 339.0500, 113.0561, 79.0168 | C25H24O7 | 437.1595 | -3.25 | Artonin J |
22.402 | 383.1137 | 365.1026, 341.0668, 323.0563, 295.0603, 83.0856 | C21H18O7 | 383.1125 | -3.06 | Artonin K |
26.193 | 435.1432 | 393.0982, 321.0393, 219.0291, 163.0379, 121.0282 | C25H22O7 | 435.1438 | 1.45 | Artobiloxanthone |
26.667 | 437.1587 | 381.0978, 363.0873, 335.0914, 283.0949, 189.0178 | C25H24O7 | 437.1595 | 1.78 | Artonin E |
31.612 | 503.2081 | 461.1608, 447.1446, 405.0976, 231.0827, 139.1107 | C30H30O7 | 503.2064 | -3.32 | Artonin A |
32.083 | 449.1246 | 407.0736, 393.0974, 379.0817, 337.0723, 67.0358 | C25H20O8 | 449.1231 | -3.35 | Artonin M |
32.702 | 503.2070 | 447.1450, 405.0976, 311.2965, 261.0760, 213.0554, 55.0161 | C30H30O7 | 503.2064 | -1.13 | Artonin B |
36.124 | 503.2072 | 447.1452, 373.1676, 191,0297, 79.0522 | C30H30O7 | 503.2064 | -1.53 | Artonin P |
Phenolic compounds have been well-documented to play a significant role in the antioxidant activities of medicinal plants. The redox capacity of the phenolic compound is the primary contributor to its antioxidant activity, enabling it to effectively scavenge and counteract free radicals, break down peroxide, and extinguish singlet or triplet oxygen. Research findings indicate that the antioxidative potential of phenolic compounds is contingent upon the number and configuration of hydroxyl groups present in the compound (
The findings of our study are in accordance with those reported in the previous study of Artocarpus spp. The antioxidant potential of several Artocarpus has been reported (
Artocarpus sericicarpus leaf and stem extracts exhibited significant cholinesterase inhibitory activity against AChE and BChE, as well as antioxidant activity. The ethanolic extracts of the leaves and stems were more effective as antioxidants and cholinesterase inhibitors than their water-based counterparts. The phenolic compounds, such as flavones, flavonols, flavanones, chalcones, and xanthones present in the extracts, may contribute to the antioxidant and cholinesterase inhibitory activities of the extracts.
Authors acknowledge Balikpapan Botanic Garden Indonesian Institute of Sciences for sample collection and Universitas Airlangga for research grant PUF 2022 contract number 545/UN3.15/PT/2022.