Corresponding author: Nanang Fakhrudin ( nanangf@ugm.ac.id ) Academic editor: Plamen Peikov
© 2020 Nanang Fakhrudin, Krisna Kharisma Pertiwi, Marce Inggrita Takubessi, Eka Fitri Susiani, Arief Nurrochmad, Sitarina Widyarini, Ari Sudarmanto, Arief Adi Nugroho, Subagus Wahyuono.
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Citation:
Fakhrudin N, Pertiwi KK, Takubessi MI, Susiani EF, Nurrochmad A, Widyarini S, Sudarmanto A, Nugroho AA, Wahyuono S (2020) A geranylated chalcone with antiplatelet activity from the leaves of breadfruit (Artocarpus altilis). Pharmacia 67(4): 173-180. https://doi.org/10.3897/pharmacia.67.e56788
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Platelet plays a crucial role in cardiovascular diseases (CVDs) development. Abnormalities in platelet aggregation provokes thromboembolism, eventually leading to death. In Indonesia, breadfruit (Artocarpus altilis) leaf is traditionally used to treat CVDs. This study aimed to evaluate the antiplatelet activity of A. altilis leaf extract (AAE) and to identify its active compound. A. altilis leaves were extracted with ethanol, and the antiplatelet activity was assessed using ADP-induced platelet aggregation. The major compound was isolated with column chromatography followed by preparative TLC, and the structure was determined on the basis of UV, MS, IR, and NMR spectra. The binding mode of the active compound to platelet receptors was characterized in in silico study. AAE exhibited an antiplatelet activity (IC50 of 252.23 µg/mL). A geranylated chalcone, 2-geranyl-2ʹ,3,3,4ʹ-tetrahydroxydihydrochalcone (GTDC) was identified as the antiplatelet compound (IC50 of 9.09 µM). GTDC actions with P2Y12 platelet receptor involving three amino acid residues.
Artocarpus communis, 2-geranyl-2ʹ, 3, 3, 4ʹ-tetrahydroxydihydrochalcone, thrombosis, P2Y12, P2Y1
The cardiovascular diseases remain the main cause of death globally (
The discovery of antiplatelet agents is a promising approach to treat cardiovascular diseases (
This work aimed to investigate the antiplatelet activity of A. altilis leaf extract and identify the active compound responsible for its antiplatelet activity. This study provided a scientific basis for promoting the traditional usage of A. altilis to treat cardiovascular diseases.
A. altilis leaves were collected from Mlati, Sleman District, Yogyakarta Province of Indonesia. The plant was identified by Dr. Djoko Santosa, a botanist at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia (number BF/192/Ident/Det/VI/2013).
Platelet-rich plasma (PRP) was obtained from 10 healthy participants who met the criteria described previously (
The dried leaf powders of A. altilis (2 kg) were extracted with 20 L of ethanol. The extract was concentrated using a vacuum rotary evaporator, left at room temperature until it was dry (262 g), and partitioned with a mixture of n-hexane : ethyl acetate : methanol : water (3:1:3:1) to yield the upper (94 g) and lower (169 g) fractions. The upper fraction containing chlorophylls and inert nonpolar components was separated, while the lower fraction was collected and dried. The lower fraction was subjected to silica vacuum chromatography (diameter of 7 cm and height of 5 cm) by using the gradient polarity of n-hexane, ethyl acetate, and methanol to yield 16 fractions. The fractions (F9 [12.9 g], F10 [27.5 g], F11 [30.6 g], F12 [25.1 g], and F13 [30.5 g]) were combined, subjected to Sephadex LH-20 column chromatography (height of 40 cm and diameter of 1.5 cm), and eluted with methanol : dichloromethane (9:1) to give 12 fractions. The fractions (F5’ [1.9 g], F6’ [7.0 g], F7’ [9.6 g], F8 [8.0 g], F9 [4.3 g], and F10 [0.8 g]) were combined, further separated through silica gel column chromatography (diameter of 0.5 cm and height of 30 cm), and eluted with n-hexane : ethyl acetate in gradient polarity (10:0 to 0:10) to give 10 fractions. The fractions (F5"–7" [5.97 g]) were separated via preparative TLC and eluted with n-hexane : ethyl acetate (2:1) to yield 2.84 g of pure active compound.
The structure of 2-geranyl-2',3,4,4'-tetrahydroxydihydrochalcone (GTDC) was elucidated on the basis of its spectral data. Spectral UV and IR were recorded on a spectrophotometer (Hitachi U-2800) and an FTIR spectrometer (Perkin Elmer Spectrum 100). LC-MS data were obtained from a Mariner Biospectrometry (Cambridge Scientific, USA) connected to an HPLC instrument (Hitachi L 6200) by using a Q-TOF mass spectrometer (ESI). NMR spectra were obtained using a DELTA 2500M Hz spectrometer (Jeol) running at 500 M Hz (13C-NMR) and 125.76 M Hz (1H-NMR) by using CDCl3 as a solvent and TMS as an internal standard.
The platelet aggregation assay was done using turbidimetry-based method (
A docking study was conducted to examine the affinity of GTDC to the ADP receptor (P2Y12) by using MOE-Dock 2015.10 (Chemical Computing Group Inc.) on a computer with Intel Core i5-4460 3.20 GHz, 4.00 GB memory, and a Windows operating system. The crystal structures of the protein complex were taken from the Protein Data Bank (PDB id:4NTJ). Ticagrelor (a selective P2Y12 receptor agonist), ethyl 6-[4-(benzylsulfonylbamoyl) piperidin-1-yl]-5-cyano-2-methylpyridine-3-carboxylate or AZD 1283 (a native ligand), and ADP (a purinergic P2Y12 agonist) were used in this study. In addition, the Triangle Matcher function and London DG were used as the placement method and scoring function, respectively.
The isolated active compound was identified as a geranylated chalcone, 2-geranyl-2',3,4,4'-tetrahydroxydihydrochalcone (GTDC). GTDC appeared as yellowish oil with ESI-MS 433 m/z [M+Na+] (calc. for C25H30O5); UV:342 nm; IR vmax in cm−1: 1450 (CH), 1496 (C-C), 1628 (C=O), 2360 (C=C). 1H-NMR (400 M Hz, CDCL3): δ 6.37 (1H, dd, 2.4 Hz, H-3'), 6.36 (1H, dd, 2.4 Hz, H-5ʹ), 7.57 (1H, d, 8.4 Hz, H-6ʹ), 3.10 (2H, t, H-α), 2.97 (2H, t, H-β), 6.73 (1H, dd, 8 Hz, H-5), 6.71 (1H, dd, 8 Hz, H-6), 3.41 (2H, d, H-1"), 5.17 (1H, t, H-2ʹʹ), 2.04 (2H, m, H-4ʹʹ), 2.06 (2H, m, H-5ʹʹ), 5.00 (1H, t, H-6ʹʹ), 1.65 (3H, s, H-8ʹʹ), 1.79 (3H, s, H-9ʹʹ), 1.57 (3H, s, H-10ʹʹ), 12.75 (1H, s, 2'-OH), 5.88 (1H, s, 4ʹ-OH), 5.44 (1H, s, 3-OH), 5.33 (1H, s, 4-OH). 13C-NMR (400 M Hz, CDCL3): δ 113.86 (C-1'), 165.30 (C-2'), 103.66 (C-3ʹ), 162.63 (C-4'), 107.81 (C-5ʹ), 132.27 (C-6ʹ), 203.87 (C=O), 39.81 (Cα), 27.78 (Cβ), 131.17 (C-1), 126.03 (C-2), 142.51 (C-3), 142.98 (C-4), 112.97 (C-5), 121.55 (C-6), 25.99 (C-1ʹʹ), 121.80 (C-2ʹʹ), 139.03 (C-3ʹʹ), 39.69 (C-4ʹʹ), 26.38 (C-5ʹʹ), 123.76 (C-6ʹʹ), 132.32 (C-7ʹʹ), 25.81 (C-8ʹʹ), 16.35 (C-9ʹʹ), 17.81 (C-10ʹʹ). The NMR spectra were compared with those of a previous study (Table
H No. | GTDC Data |
GTDC ( |
||
1H–NMR | 13C–NMR | 1H–NMR | 13C–NMR | |
δ ppm (∑H; m; J) | Δ ppm | δ ppm (∑H; m; J) | δ ppm | |
1' | – | 113.86 | – | 113.61 |
2' | – | 165.30 | – | 165.18 |
3' | 6.37 (1H; dd; 2.4Hz) | 103.66 | 6.38 | 103.57 |
4' | – | 162.63 | – | 163.17 |
5' | 6.36 (1H; dd; 2.4Hz) | 107.81 | 6.36 | 108.01 |
6' | 7.57 (1H; d; 8.4Hz) | 132.27 | 7.57 | 132.25 |
C=O | – | 203.87 | – | 204.02 |
Α | 3.10 (2H; t) | 39.81 | 3.10 | 39.68 |
Β | 2.97 (2H; t) | 27.78 | 2.97 | 27.81 |
1 | – | 131.17 | – | 132.09 |
2 | – | 126.03 | – | 126.12 |
3 | – | 142.51 | – | 142.52 |
4 | – | 142.98 | – | 142.74 |
5 | 6.73 (1H; dd; 8Hz) | 112.97 | 6.71 | 112.92 |
6 | 6.71 (1H; dd; 8Hz) | 121.55 | 6.65 | 121.34 |
1" | 3.41 (2H; d) | 25.99 | 3.40 | 25.84 |
2" | 5.17 (1H; t) | 121.80 | 5.17 | 121.86 |
3" | – | 139.03 | – | 138.50 |
4" | 2.04 (2H; m) | 39.69 | 2.04 | 39.63 |
5" | 2.06 (2H; m) | 26.38 | 2.06 | 26.40 |
6" | 5.00 (1H; t) | 123.76 | 5.02 | 123.79 |
7" | – | 132.32 | – | 131.25 |
8" | 1.65 (3H; s) | 25.81 | 1.65 | 25.69 |
9" | 1.79 (3H; s) | 16.35 | 1.78 | 16.30 |
10" | 1.57 (3H; s) | 17.81 | 1.57 | 17.71 |
2'–OH | 12.75 (1H; s) | – | 12.84 | – |
4'–OH | 5.88 (1H; s) | – | 5.81 | – |
3–OH | 5.44 (1H; s) | – | 5.81 | – |
4–OH | 5.33 (1H; s) | – | 5.57 | – |
The antiplatelet activities of the extract and the compound were assessed using the ADP-induced platelet aggregation assay. Fig.
Antiplatelet activities of AAE and GDTC in ADP-induced platelet aggregation. Sigmoidal dose–response curves showing the antiplatelet activity of AAE (A), GTDC, and ticagrelor (B). The percentage of platelet aggregation was calculated on the basis of the decrease in the aggregation peak. Data were mean ± SD (n = 3); *p < 0.05 relative to the solvent-treated group (set as 100% aggregation).
To obtain further details regarding the antiplatelet activity of GTDC, we assessed the platelet aggregation profile of GTDC in the late phase of aggregation (10 min). Fig.
Curve showing the antiplatelet activity profiles of GTDC in ADP-induced platelet aggregation. A Baseline or peak of platelet aggregation (solvent treatment); B Peak of platelet aggregation in GTDC or ticagrelor treatments; C Platelet disaggregation in GTDC or ticagrelor treatments. Blue line, solvent; black line, 0.1 µM GTDC; red line, 1 µM GTDC; and green line, 0.1 µM tigacrelor.
A molecular docking study was performed to obtain insights into the interaction between GTDC and the ADP receptors responsible for platelet aggregation. The binding of GTDC to the platelet receptors (P2Y1 and P2Y12) was compared with that of ticagrelor. The table in Fig.
Putative interactions of ligands and the binding pockets of P2Y12 receptor. The binding modes of GTDC (A), and ticagrelor (B) at P2Y12 receptor. (C) The overlay-complexes of GTDC (blue) and AZD 1283 (red); and (D) Ticagrelor (blue) and AZD 1283 (red) at the binding sites of P2Y12 receptor. The table shows the docking score of the compounds to P2Y12 receptor.
Preventing cardiovascular diseases by providing promising therapeutic agents is a challenge in biomedical sciences. A. altilis leaves have been traditionally used in Central Java, Indonesia, to treat cardiovascular-related diseases, which are correlated with platelet aggregation. In this study, an antiplatelet in vitro assay was conducted to assess the effectiveness of AAE, and the active compound was characterized. AAE inhibited platelet aggregation with IC50 of 9.09 µM. The active compound was identified as GTDC, which represented a major compound in the extract. Its chromatography profile is provided in Suppl. material 1: Fig. S2. Although the presence of GTDC in A. altilis leaves was described previously (
In this study, we found that GTDC inhibited platelet aggregation and induced the disaggregation of aggregated platelets. This finding was interesting because the efficacy assessment of the antiplatelet agent in ADP-induced platelet aggregation might rely not only on the inhibition of platelet aggregation in the early phase (measured by a decrease in the aggregation peak) but also on the disaggregation activity after the aggregation peak (late phase) (
In addition to the inhibitory effect on platelet aggregation, GTDC demonstrated a strong platelet disaggregation activity similar to that observed in ticagrelor in the late phase of platelet aggregation. Considering the antiplatelet curve profile of GTDC in the late phase of platelet aggregation as illustrated in Fig.
The in silico approach revealed that the interaction between GTDC and P2Y12 receptor involved more amino acid residues compared with that of P2Y1. Thus, the binding of GTDC was more favorable to P2Y12 than to the P2Y1 receptor. The molecular docking study showed that the docking score of GTDC was lower (−11.9185) than that of ADP (−11.3436), suggesting that GTDC could prevent the binding of ADP to P2Y12. This finding explained the stronger binding affinity of GTDC to P2Y12 rather than to the P2Y1 receptor. The structure–activity relationship of chalcone-derived compounds demonstrated that the 4-OH phenolic group and the adjacent geranyl group are essential for the antiplatelet activity in ADP-induced platelet aggregation (
Recent studies and clinical evidence indicated that antagonizing the P2Y12 receptor remains the main target in coronary artery thrombosis as it shows a prolific efficacy (
We found that GTDC, a geranylated chalcone isolated from A. altilis leaves demonstrated a promising antiplatelet activity. It exerts an antiplatelet activity by inhibiting platelet aggregation and inducing platelet disaggregation in the initial and late phases of aggregation, respectively. The docking study indicates that GTDC interacts with the P2Y12 receptor via three amino acid residues.
We thank Setiono and Miranda Pratiwi for excellent technical assistances. This work was used by KKP, MIT, EFS, and AAD for their thesis to obtain their academic degrees.
This work was financially supported by Deputi Bidang Penguatan Riset dan Pengembangan, Kementerian Riset dan Teknologi / Badan Riset dan Inovasi Nasional Republic of Indonesia (Hibah PDUPT: 2733/UN1.DITLIT/DIT-LIT/PT/2020).