1. INTRODUCTION
In continuing our study on chemical and pharmaceutical aspects of Etlingera (Zingiberaceae) growing in the Wallace area (Southeast Sulawesi province, Indonesia), Etlingera rubroloba rhizome is an interesting sample and to date, has not been reported yet. Previous research on this genus conducted by our group consisted of E. calophrys [1–3], Etlingera elatior rhizomes [4–7], E. alba rhizomes [8–11], E. rubroloba fruits [12,13], and E. rubroloba stems [14,15].
Fruits of E. rubroloba have potential immunomodulatory activity on macrophage phagocytosis and interleukin-12 levels in Bacillus Calmette–Guérin Vaccine ( BCG)-stimulated Balb/C mice [12]. Two compounds, namely sinaphyl alcohol diacetate and ergosterol peroxide have been isolated from E. rubroloba fruits, which were potential as an immunomodulator agent [13]. Two compounds from E. rubroloba stems, sinapyl alcohol diacetate and stigmasterol, have potential as antihyperuricemia [14] and antioxidant and anti-inflammatory activities revealed by sinapyl alcohol acetate from this tissue [15]. Steroids and diarylheptanoids isolated from the rhizomes and stems of E. calophrys have been reported to exhibit antimicrobial and radical scavenging activities [2,3]. Another diarylheptanoids from E. alba rhizomes, called 1,7-diphenyl-6-heptene-3-one, can be developed as anti-metatastic for Tripel-Negative Breast Cancer [11]. The most popular of diarylheptanoids is curcumin, which is produced by plants such as rhizomes of Curcuma longa and C. xanthorrhiza (Zingiberaceae). This compound exhibits antidepressant potential through its potent antioxidant properties [16,17].
Based on a literature review of ScienceDirect and SpringerLink databases, no previous studies have reported the chemical or pharmacological aspects of E. rubroloba rhizomes. In particular, information on its chemical profile [using UPLC- High-Resolution Mass Spectrometry (HRMS)], isolation and structure elucidation, antioxidant and anti-inflammatory activities, and antidepressant potential is still lacking.
Therefore, this study aims to comprehensively investigate the chemical constituents and pharmacological properties of E. rubroloba rhizomes through integrated chemical analysis, biological assays, and in silico approaches. The novelty of this work lies in providing the first complete chemical and pharmacological characterization of E. rubroloba rhizomes, which not only expands the understanding of bioactive metabolites in the genus Etlingera but also supports its potential development as a natural source for novel antidepressant and anti-inflammatory agents.
2. MATERIAL AND METHODS
2.1 Plant material and preparation of the extract
Etlingera rubroloba rhizomes were collected from Punggaluku Village, Laeya District, South Konawe Regency, Southeast Sulawesi Province. The sample was identified and deposited at the BRIN Research Center for Biology, Cibinong, Indonesia, under the registration number 956/IPH.1.01/If.07/V/2019. The dried samples (1 kg) were macerated with methanol (Merck) for 72 hours and concentrated using a vacuum rotary evaporator (Stuart RE300, USA) to get 28 g of methanol extract.
2.2 UPLC-HRMS analysis of metabolites in the extract
HRMS is employed to identify chemical compounds, including metabolites and complex organic compounds in plant samples. The secondary metabolite content of the methanol extract was analyzed following previously reported methods [18]. The dried extract was reconstituted in MeOH–H2O (1:1, v/v) to a final concentration of 1 mg/ml, centrifuged at 10,000 rpm for 10 minutes, and filtered through a 0.22 µm PTFE membrane prior to analysis. UPLC separation was performed using an ACQUITY BEH C18 column (2.1 × 100 mm, 1.7 µm) with a mobile phase consisting of water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B), applying a linear gradient from 5% to 95% B within 15 minutes at a flow rate of 0.3 ml/min. Injection volume was 5 µl, and the column temperature was maintained at 40°C. High-resolution Mass spectrometry was carried out on a Q-Exactive Orbitrap equipped with an Electrospray ionization source in positive and negative modes. Full scan spectra were acquired in the range m/z 100–1500 at 70,000 resolutions, followed by data-dependent MS/MS at 17,500 resolution using stepped collision energies of 20, 40, and 60 eV. The instrument was externally calibrated daily, and quantification was performed using six-point calibration curves from reference standards (quercetin) with caffeine (10 µg/ml) as an internal standard. Raw data were processed using Compound Discoverer 3.2 for peak alignment, deconvolution, and metabolite annotation based on accurate mass, isotopic distribution, and MS/MS spectra, further confirmed by comparison with mzCloud and authentic standards where available.
2.3 Isolation of secondary metabolite compounds
The methanol extract (20 g) was fractionated using vacuum liquid chromatography (VLC) with a 10 cm diameter column, silica gel GF254 as stationary phase (250 g), and a mobile phase consisting of an n-hexane:ethyl acetate mixture in varying ratios [8:2, 7:3, 5:5, 4:6, 2:8 (v/v)] and 100% methanol (200 ml each). This process yielded six fractions (1–6) weighing 4.24, 3.35, 0.36, 0.43, 2.56, and 8.22 g, respectively. The purification of fraction 3 by radial chromatography (RC) to get compound ER1, and in fraction 4, identified as compound ER2. Fraction 5 was further separated using RC with a silica gel GF254 stationary phase containing gypsum and a mobile phase of n-hexane:ethyl acetate [7:3 (v/v)] and 100% ethyl acetate, to get compounds ER3 and ER4. Fraction 6 was also separated using RC with a silica gel GF254 adsorbent and eluent of n-hexane:ethyl acetate [5:5 (v/v)] and 100% ethylacetate. This resulted in six subfractions, with TLC analysis identifying a single spot in subfraction 3 as compound ER5 and in subfraction 5 as compound ER6. Purity analysis for each compound was performed using TLC with a mobile phase of n-hexane:ethyl acetate [4:6 (v/v)].
2.4 Structure identification
The structures of the isolated compounds were determined using Nuclear Magnetic Resonance (NMR) spectroscopic techniques. The 1H NMR and 13C NMR spectra were recorded on a Jeol JNM-ECZ500R/S1 NMR spectrometer (Japan) and a Bruker NMR spectrometer (USA). Molecular weight of each compounds were based on UPLC-HRMS data.
2.5 Biological activity
Antioxidant properties were evaluated using previously reported methods, focusing on the inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals [19]. For the DPPH assay, a 0.1 mM DPPH solution in methanol was freshly prepared and adjusted to an absorbance of approximately 0.9–1.0 at 517 nm. Samples were prepared as serial dilutions (1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 µg/ml) and mixed with an equal volume of the DPPH solution in a 96-well microplate. The mixtures were incubated for 30 minutes at room temperature in the dark, and absorbance was measured at 517 nm. For the ABTS assay, the ABTS radical cation (ABTS•+) was generated by reacting 7 mM ABTS with 2.45 mM potassium persulfate for 16 hours in the dark at room temperature, and then diluted with ethanol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. Aliquots of the samples at different concentrations were mixed with the ABTS•+ working solution, incubated for 6 min, and absorbance was measured at 734 nm. Trolox was used as the positive control, while reagent blanks and vehicle controls were included for correction. Replicates (n per group): each treatment group (sample, positive control, and vehicle control) was tested at eight concentrations, with n = 3 technical replicates per concentration in one run. Radical scavenging activity was expressed relative to the control, and IC50 values were determined from concentration response curves by linear regression.
The anti-inflammatory activity was assessed using the bovine serum albumin (BSA) protein denaturation inhibition method adapted from previous studies [20]. A stock solution of 5% (w/v) BSA was prepared in 0.05 M Tris-phosphate buffered saline (pH 6.5). Samples were dissolved in methanol (final solvent concentration ≤1% v/v) and prepared as serial dilutions (1.56, 3.13, 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 µg/ml). In a 96-well microplate, BSA solution was mixed with the samples, incubated at 37°C for 15 minutes, then heated at 70°C for 5 minutes to induce protein denaturation, followed by cooling to room temperature. Turbidity was measured at 660 nm using a microplate reader. Methylprednisolone was used as the positive control; vehicle control and sample blanks (sample without BSA) were included for correction. Replicates (n per group): each treatment group (samples, positive control, and vehicle control) was tested at eight concentrations, with n = 3 technical replicates per concentration. Protein stabilization was expressed as the percentage of inhibition relative to the vehicle control. Potency was summarized as IC50 values obtained from concentration-response curves.
2.6 Statistical analysis
The collected data were presented as mean ± SD and statistically analyzed by using IBM SPSS Statistics V26.0 software with one-way ANOVA analysis. The significance was exhibited at p-value < 0.05.
2.7 In silico study
The three-dimensional structures of the target proteins in this study the Monoamine Oxidase (MOA) (PDB ID: 2BXS), dopamine transporter (DAT) (PDB ID: 4M48) [21], and serotonin transporter (SERT) (PDB ID: 5I6X) [22] were obtained from the Protein Data Bank. The molecular structures of the seven compounds identified in E. rubroloba rhizomes were retrieved from the PubChem database. Protein and ligand structures were prepared using AutoDockTools v1.5.6, following standard protocols [23]. Preparation of target proteins involved removing water molecules and bound ligands, protonation, and the addition of Kollman charges to ensure accurate electrostatic properties [24]. To ensure docking reliability, known inhibitors from the co-crystal structures were employed as positive controls: clorgyline for MAO, nortriptyline for DAT, and paroxetine for SERT. Docking simulations were conducted using AutoDock Vina [25] with an exhaustiveness parameter set to 64 to ensure thorough conformational sampling, generating nine binding poses for each ligand. The top-ranked pose based on binding affinity was selected for interaction analysis using Discovery Studio Visualizer.
Docking grids were defined to encompass the active site residues identified from the co-crystalized ligands, with the following parameters: MAO (grid center: x = 10.192, y = 125.199, z = 51.599; grid size: 20 × 20 × 20 Å), DAT (grid center: x = −39.808, y = −1.239, z = 55.308; grid size: 25 × 25 × 25 Å), and SERT (grid center: x = −31.847, y = −20.608, z = 2.120; grid size: 25 × 25 × 25 Å). The docking protocol was validated through redocking of the co-crystallized ligands into their respective binding sites. The Root Mean Square Deviation values between the docked and crystallographic poses were 1.220 Å for clorgyline with MAO, 0.578 Å, or nortriptyline with DAT, and 0.723 Å for paroxetine with SERT, all below the 2 Å threshold, indicating accurate reproduction of experimental binding modes (Fig. 5).
 | Figure 5. Visualization of the co-crystallized ligands (pink) overlapping with their redocked conformations (blue) in the respective binding sites. The RMSD values between the docked and crystallographic poses were (A) 1.220 Å for clorgyline on MAO, (B) 0.578 Å for nortriptyline on DAT, and (C) 0.723 Å for paroxetine on SERT. Green and pink dashed lines represent hydrogen bonds and hydrophobic interactions. [Click here to view] |
3. RESULTS AND DISCUSSION
The extraction results revealed that the yield of E. rubroloba rhizome methanol extract was 2.8%. The chromatogram from the chemical compound analysis of the extract (Fig. 1), based on UPLC-HRMS data within the retention time (RT) range of 0–25 minutes, displayed distinct peaks.
 | Figure 1. UPLC-HRMS chromatogram of the methanol extracts of the E. rubroloba rhizome. [Click here to view] |
The analysis revealed 48 major compounds present in the rhizomes of E. rubroloba. The chemical content plays a significant role in contributing to the biological properties of the species. By understanding the chemical composition, the study of metabolite compounds with potential biological activity can be carried out, facilitating the isolation of these compounds. Six isolated compounds from E.rubroloba rhizome consist of ER1, ER2, ER3, ER4, ER5, and ER6, displayed at (Fig. 2), with 1H NMR and 13C NMR spectra could be summarized as follows.
 | Figure 2. Isolation of secondary metabolite compounds from E. rubroloba rhizomes. [Click here to view] |
ER1. White powder; C29H48O. 1H NMR (CDCl3, 500.159 MHz, chemical shift δ in ppm, coupling constant J in Hz) 5.34 (1H, m, H-6), 5.14 (1H, dd, 8.5; 15.5, H-22), 5.00 (1H, dd, 8.5; 15.5, H-23), 3.34 (1H, m, H-3), 1.00 (3H, s, H-18), 0.91(3H, d, 7, H-21), 0.83 (3H, m, H-29), 0.82 (3H, m, H-29), 0.80 (3H, m, H-29), 0.67 (3H, s, H-19). 13C NMR (CDCl3, 125.765 MHz, chemical shift δ in ppm) 140.9 (C-5), 138.4 (C-23), 129.4 (C-22), 121.8 (C-6), 71.9 (C-3), 56.9 (C-14), 56.1 (C-17), 50.2 (C-9), 45.9 (C-24), 42.4 (C-4), 42.4 (C-13), 40.6 (C-20), 39.9 (C-12), 37.4 (C-1), 36.7 (C-10), 32.0 (C-2), 32.0 (C-7), 31.7 (C-8), 29.3 (C-27), 28.4 (C-16), 26.2 (C-28), 24.4 (C-15), 23.2 (C-21), 21.2 (C-11), 20.0 (C-26), 19.5 (C-27), 18.9 (C-19), 12.1 (C-18) and 12.0 (C-29).
ER2. White powder; C29H48O2. 1H NMR ((CD3)2CO, 700.302 MHz, chemical shift δ in ppm, coupling constant J in Hz) 5.69 (1H, s, H-4), 4.30 (1H, d, 8.6 Hz, H-6), 1.41 (3H, s, H-19), 0.98 (3H, d, 6.5 Hz, H-21), 0.88 (3H, d, 7.3 Hz, H-29), 0.87 (3H, d, 6.9 Hz, H-26), 0.85 (3H, d, 6.9 Hz, H-27) and 0.79 (3H, s, H-18); 13C NMR ((CD3)2CO, 176.108 MHz, chemical shift δ in ppm) 200.7 (C-3), 168.6 (C-5), 125.3 (C-4), 72.2 (C-6), 56.0 (C-17), 55.8 (C-14), 53.8 (C-9), 45.8 (C-24), 42.9 (C-13), 39.8 (C-12), 39.6 (C7), 37.9 (C-10), 37.1 (C-1), 36.0 (C-20), 33.9 (C-2), 33.7 (C-22), 29.7 (C-8), 29.1 (C-25), 28.0 (C-16), 25.8 (C-23), 23.9 (C15), 22.8 (C-28), 20.8 (C-11), 19.2 (C-26), 18.7 (C-19), 18.4 (C-27), 18.2 (C-21), 11.4 (C-18) and 11.3 (C-29).
ER3. Yellow oil; C20H24O3. 1H NMR (CDCl3, 500.159 MHz, chemical shift δ in ppm, coupling constant J in Hz) 7.26 (2H, m, H-3”/H-5”), 7.17 (1H, m, H-4”), 7.14 (2H, m, H-2”/H-6”), 6.81 (1H, d, 8.0, H-5’), 6.67 (1H, d, 1.5, H-2’), 6.64 (1H, dd, 2.0 & 8.0, H-6’), 3.84 (3H, s, H-7’), 2.81 (2H, t, 7.5, H-1), 2.67 (2H, t, 7.5, H-2), 2.38 (2H, t, 7.0, H-4), 2.59 (2H, t, 7.0, H-7) and 1.59 (4H, m, H-5/H-6); 13C NMR (CDCl3, 125.765 MHz, chemical shift δ in ppm) 210.3 (C-3), 146.4 (C-3′), 143.9 (C-4′), 142.2 (C-1″), 133.1 (C-1′), 128.4 (C-3”/C-5″), 128.3 (C-2”/C-6″), 125.8 (C-4”), 120.8 (C-6′), 114.4 (C-5′), 111.1 (C-2’), 55.9 (C-7’), 44.7 (C-2), 42.9 (C-4), 35.8 (C-7), 31.0 (C-6), 29.6 (C-1) and 23.4 (C-5).
ER4. Yellow oil; C20H24O4. 1H NMR (CDCl3, 500.159 MHz, chemical shift δ in ppm, coupling constant J in Hz) 7.00 (2H, m, H-2”, H-6”), 6.82 (1H, d, 8.0, H-5’), 6.74 (2H, m, H-3”, H-5”), 6.67 (1H, d, 2.0, H-2’), 6.65 (1H, dd, 2.0 & 8.0, H-6’), 3.85 (3H, s, H-7’), 2.81 (2H, t, 7.5, H-1), 2.67 (2H, t, 8, H-2), 2.52 (2H, t, 7.5, H-7), 2.38 (2H, t, 7.0, H-4), 1.59 (2H, m, H-5) and 1.54 (2H, m, H-6); 13C NMR (CDCl3, 125.765 MHz, chemical shift δ in ppm) 210.6 (C-3), 153.8 (C-4”), 146.5 (C-3’), 144.0 (C-4’), 134.4 (C-1”), 133.2 (C-1’), 129.5 (C-2”/C-6”), 120.9 (C-6’), 115.3 (C-3”/C-5”), 114.5 (C-5’), 111.2 (C-2’), 56.0 (C-7’), 44.8 (C-2), 43.1 (C-4), 34.9 (C-7), 31.3 (C-6), 29.7 (C-1) and 23.5 (C-5).
ER5. White oil; C13H16O5. 1H NMR (CDCl3, 500.159 MHz, chemical shift δ in ppm, coupling constant J in Hz) 6.61 (2H, s, H-2/H-6), 6.54 (1H, d, 15.5, H-7), 6.30 (1H, dt, 15.5 & 6.0, H-8), 4.31 (2H, d, 6.0, H-9), 3.81 (6H, s, H-10, H-13), 2.32 (3H, s, H-12); 13C NMR (CDCl3, 125.765 MHz, chemical shift δ in ppm) 168.9 (C-11), 152.2 (C-3/C-5), 135.2 (C-1), 130.9 (C-7), 129.0 (C-8), 128.5 (C-4), 103.1 (C-2, C-6), 63.5 (C-9), 56.1 (C-10/C-13) and 20.5 (C-12).
ER6. White powder; C9H8O3. 1H NMR ((CD3)2CO, 400.172 MHz, chemical shift δ in ppm, coupling constant J in Hz) 8,97 (1H, s), 7.63 (1H, d, 15.6, H-7), 7.57 (2H, d, 8.8 Hz, H-2/H-6), 6.91 (2H, d, 8.8 Hz, H-3/H-5) and 6.34 (1H, d, 15.6, H-8). 13C NMR ((CD3)2CO, 400.172 MHz, chemical shift δ in ppm) 167.2 (C-9), 159.6 (C-4), 144.6 (C-7), 130.0 (C-2/C-6), 126.1 (C-1), 115.7 (C3/C-5), and 114.8 (C-8).
Forty-eight major compounds were identified by UPLC-HRMS from rhizomes of E. rubroloba, six of 48 compounds were successfully isolated using chromatographic techniques, and their structures were determined through NMR data interpretation (Table 2) and molecular weight of UPLC-HRMS data (Table 1).
Table 1. Secondary metabolites in the methanol extracts of E. rubroloba rhizome.
| No | RT (min) | Calc. MW | Formula | Compound ame | Area [BPI] | Delta mass (ppm) |
|---|---|---|---|---|---|---|
| 1 | 1.147 | 226.0839 | C??H??O? | 3,4,5-Trimethoxyphenyl acetate | 62,125,033.5 | –0.94 |
| 2 | 5.165 | 164.0473 | C?H?O? | p-Coumaric acid | 212,270,699.9 | –0.46 |
| 3 | 6.042 | 330.1461 | C??H??O? | 1,7-bis(3,4-dihydroxyphenyln) heptan-3-one | 82,656,462.9 | –1.99 |
| 4 | 6.844 | 185.2141 | C??H??N | Tributylamine | 613,921,790.6 | –1.61 |
| 5 | 7.581 | 252.0995 | C??H??O? | 3,5-dimethoxy-4- acetoxycinnamyl alcohol | 841,400,105.1 | –1.25 |
| 6 | 8.700 | 328.1669 | C??H??O? | Crocetin | 143,115,946.1 | –1.65 |
| 7 | 9.044 | 148.0522 | C?H?O? | Cinnamic acid | 72,186,893.4 | –1.54 |
| 8 | 9.046 | 372.1564 | C??H??O? | 1-(2,4-Dihydroxy-5-methoxyphenyl)-7- (3-methoxyphenyl)-3,5-heptanedione | 473,628,904.9 | –2.42 |
| 9 | 9.138 | 316.0577 | C??H??O? | 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-6-methyl-4H-chromen-4-one | 117,59,9157.5 | –1.92 |
| 10 | 9.754 | 317.2921 | C??H??NO? | 2-Amino-1,3,4-octadecanetriol | 593,185,760.9 | –2.72 |
| 11 | 9.780 | 296.1406 | C??H??O? | (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one | 82,488,185.6 | –2.14 |
| 12 | 9.783 | 433.2093 | C??H??NO? | Mycophenolate mofetil | 255,008,161.3 | –1.72 |
| 13 | 9.785 | 356.1616 | C??H??O? | Odoratisol A | 399,075,683.8 | –2.32 |
| 14 | 10.017 | 144.0573 | C??H?O | 1-Naphthol | 305,196,215.1 | –1.69 |
| 15 | 10.245 | 328.1667 | C??H??O? | 1-(3’-methoxy-4’- hydroxyphenyl)-7- (4”-hydroxyphenyl)-3-heptanone | 568,317,610.2 | –2.26 |
| 16 | 10.277 | 234.0889 | C??H??O? | 7-Hydroxy-2-(2-hydroxypropyl)-5-methyl-4H-chromen-4-one | 60,917,917.5 | –1.39 |
| 17 | 10.479 | 314.0787 | C??H??O? | Myricetin | 68,662,223.3 | –1.2 |
| 18 | 10.706 | 304.2034 | C??H??O? | 16-hydroxytestosterone | 77,014,298.3 | –1.39 |
| 19 | 10.778 | 266.1879 | C??H??O? | Tetranor 12-HETE | 80,630,083.81 | –1.12 |
| 20 | 11.201 | 250.1931 | C??H??O? | 4-tert-Octylphenol monoethoxylate | 119,562,048.3 | –0.73 |
| 21 | 11.204 | 146.0367 | C?H?O? | Coumarin | 96,909,389.2 | –0.65 |
| 22 | 11.413 | 296.1772 | C??H??O? | Ethinylestradiol | 200,143,430.3 | –1.44 |
| 23 | 11.442 | 134.1094 | C??H?? | p-Cymene | 204,277,353.9 | –0.99 |
| 24 | 12.092 | 312.172 | C??H??O? | Yakuchinone A | 701,945,554.6 | –1.85 |
| 25 | 12.232 | 148.1251 | C??H?? | n-Amylbenzene | 76,486,075.4 | –1.03 |
| 26 | 12.237 | 190.1717 | C??H?? | 1,3-Di-tert-butylbenzene | 1,948,646,557.0 | –2.24 |
| 27 | 12.431 | 278.2243 | C??H??O? | α-Eleostearic acid | 71,276,217.3 | –1.15 |
| 28 | 12.512 | 288.2084 | C??H??O? | Testosterone | 64,4105,044.4 | –1.83 |
| 29 | 12.514 | 306.2188 | C??H??O? | 11-Hydroxyetiocholanolone | 170,356,852.4 | –2.18 |
| 30 | 12.557 | 288.2449 | C??H??O | Ethylestrenol | 209,789,603.3 | –1.48 |
| 31 | 12.667 | 314.224 | C??H??O? | Progesterone | 110,511,558.1 | –1.99 |
| 32 | 12.668 | 350.2449 | C??H??O? | Tetrahydro-11-deoxycortisol | 257,115,037.3 | –2.2 |
| 33 | 12.822 | 294.219 | C??H??O? | (10E,12Z)-9-oxooctadeca-10,12-dienoic acid | 121,046,943.7 | –1.61 |
| 34 | 13.335 | 162.1406 | C??H?? | Hexylbenzene | 57,544,975.2 | –1.29 |
| 35 | 13.645 | 348.2294 | C??H??O? | 3,17,21-Trihydroxypregn-2-en-1-one | 69,4250,398.5 | –2.04 |
| 36 | 13.739 | 282.1983 | C??H??O | Dehydroretinaldehyde | 109,425,595.2 | –0.38 |
| 37 | 14.000 | 354.2763 | C??H??O? | 1-Linoleoyl glycerol | 58,923,475.5 | –1.97 |
| 38 | 14.615 | 402.3126 | C??H??O? | 4-Allyl-2-methoxyphenyl palmitate | 61,037,734.5 | –1.88 |
| 39 | 14.623 | 264.2452 | C??H??O | 2-[(5Z)-5-tetradecenyl] cyclobutanone | 373,833,551.1 | –0.57 |
| 40 | 14.897 | 162.1406 | C??H?? | Hexylbenzene | 153,492,893.6 | –1.29 |
| 41 | 15.341 | 284.2134 | C??H??O | (9cis)-Retinal | 5,873,347,815.0 | –2.12 |
| 42 | 15.443 | 272.2499 | C??H?? | syn-labda-8(17),12E,14-triene | 89,658,451.06 | –1.97 |
| 43 | 15.964 | 418.3075 | C??H??O? | Maxacalcitol | 369,254,566.5 | –1.85 |
| 44 | 16.243 | 511.4957 | C??H??NO? | C14-Dihydroceramide | 191,350,557.3 | –1.43 |
| 45 | 16.929 | 428.3647 | C??H??O? | Stigmast-4-en-6β-ol-3-one | 178,739,474.9 | –1.76 |
| 46 | 17.032 | 539.5272 | C??H??NO? | C16-Dihydroceramide | 145,546,559.9 | –0.99 |
| 47 | 18.371 | 424.3337 | C??H??O? | 4,4'-Methylenebis | 74,563,926.84 | –1.1 |
| 48 | 18.904 | 412.3697 | C??H??O | Stigmasterol | 382,572,989.4 | –1.89 |
Table 2. 1H and 13C NMR data of compounds ER1 and ER2 compared with data from literature (ER1* and ER2*)
| No | ER1 | ER1* | ER2 | ER2* | ||||
|---|---|---|---|---|---|---|---|---|
| δC | δH(ΣH, m, J = Hz) | δC | δH(ΣH, m, J = Hz) | δC | δH(ΣH, m, J = Hz) | δC | δH(ΣH, m, J = Hz) | |
| 1 | 37.4 | 37.6 | 37.1 | 37.2 | ||||
| 2 | 32.0 | 32.1 | 33.9 | 33.9 | ||||
| 3 | 71.9 | 3.51 (1H, m) | 72.1 | 3.51 (1H, tdd, 4.5; 4.4; 3.8) | 200.7 | 200.7 | ||
| 4 | 42.4 | 42.4 | 125.3 | 5.69 (1H, s) | 125.4 | 5.82 (1H, s) | ||
| 5 | 140.9 | 141.1 | 168.6 | 168.6 | ||||
| 6 | 121.8 | 5.34 (1H, m) | 121.8 | 5.31 (1H, t, 6.1) | 72.2 | 4.30 (1H, d, 8.6) | 72.3 | 4.34 (1H, m) |
| 7 | 32.0 | - | 31.8 | - | 39.6 | - | 39.6 | - |
| 8 | 31.7 | - | 31.8 | - | 29.7 | - | 29.7 | - |
| 9 | 50.2 | - | 50.2 | - | 53.8 | - | 53.9 | - |
| 10 | 36.7 | - | 36.6 | - | 37.9 | - | 37.9 | - |
| 11 | 21.2 | - | 21.5 | - | 20.8 | - | 20.8 | - |
| 12 | 39.9 | - | 39.9 | - | 39.8 | - | 39.7 | - |
| 13 | 42.4 | - | 42.4 | - | 42.9 | - | 42.9 | - |
| 14 | 56.9 | - | 56.8 | - | 55.8 | - | 55.9 | - |
| 15 | 24.4 | - | 24.4 | - | 23.9 | - | 24.0 | - |
| 16 | 28.4 | - | 29.3 | - | 28.0 | - | 28.1 | - |
| 17 | 56.1 | - | 56.2 | - | 56.0 | - | 56.1 | - |
| 18 | 12.1 | 1.00 (3H, s) | 12.2 | 1.03 (3H, s) | 11.4 | 0.79 (3H, s) | 11.5 | 0.75 (3H, s) |
| 19 | 18.9 | 0.67 (3H, s) | 18.9 | 0.71 (3H, s) | 18.7 | 1.41 (3H, s) | 18.8 | 1.38 (3H, s) |
| 20 | 40.6 | - | 40.6 | - | 36.0 | - | 36.0 | - |
| 21 | 23.2 | 0.91 (3H, d, 7) | 21.7 | 0.91 (3H, d, 6.2) | 18.2 | 0.98 (3H, d, 6.5) | 18.3 | 0.93 (3H, d, 6.5) |
| 22 | 129.4 | 5.14 (1H, dd, 8.5; 15.5) | 129.6 | 5.14 (1H, m) | 33.7 | - | 33.8 | - |
| 23 | 138.4 | 5.00 (1H, dd, 8.5; 15.5) | 138.7 | 4.98 (1H, m) | 25.8 | - | 25.9 | - |
| 24 | 45.9 | - | 46.1 | - | 45.8 | - | 45.8 | - |
| 25 | 29.3 | - | 29.6 | - | 29.1 | - | 29.1 | - |
| 26 | 20.0 | 0.82 (3H, m) | 20.2 | 0.82 (3H, d, 6.6) | 19.2 | 0.87 (3H, d, 6.9) | 19.2 | 0.84 (3H, d, 6.1) |
| 27 | 19.5 | 0.80 (3H, m) | 19.8 | 0.80 (3H, d, 6.6) | 18.4 | 0.85 (3H, d, 6.9) | 18.5 | 0.82 (3H, d, 6.1) |
| 28 | 26.2 | - | 25.4 | - | 22.8 | - | 22.9 | - |
| 29 | 12.0 | 0.83 (3H, m) | 12.1 | 0.83 (3H, t, 7.1) | 11.3 | 0.88 (3H, d, 7.3) | 11.4 | 0.85 (3H, t, 6.7) |
ER1*3 ER2*26,2727.
ER1 and ER2 are identified as steroids. It is supported by the carbon NMR spectra (Table 2), specifically identifiable due to the presence of 29 carbon atoms. The carbonyl carbon (C=O) signal appears in a more downfield region (around 200 ppm), while the methyl carbon at positions C18, C19, C26, C27, and C29 appears in the δ ≈10–20 ppm region. The carbon at δ ≈70 ppm, namely C3 (ER1) and C6 (ER2), experiences deshielding due to the presence of oxygen atom substituents, which significantly shifts the carbon chemical shift value to a higher ppm. This shift correlates directly with the chemical shift observed in proton NMR. Through a literature search comparing NMR spectroscopic data, it can be concluded that compound ER1 is Stigmasterol [3], while compound ER2 is Stigmast-4-en-6-ol-3-one [26,27] (Table 2), and with molecular weight is relevant to compounds 25 and 48, respectively, in Table 1.
Diarylheptanoids are compounds consisting of two aromatic rings connected by a seven-carbon chain (C6-C7-C6). The same way as the structure elucidation of steroids, ER3 is Yakuchinone A [3] and ER4 is 1-(3’-methoxy-4’-hydroxyphenyl)-7-(4”-hydroxyphenyl)-3-heptanone [26,27] with a molecular weight of relevant to compounds 24 and 15 in Table 1, respectively. Through a literature search comparing NMR spectroscopic data, it can be observed that compound ER5 is 3,5-dimethoxy-4-acetoxycinnamyl alcohol [11] and compound ER6 is p-Coumaric acid with molecular weight is relevant to compounds 5 and 2 in Table 1, respectively. The molecular structure of the isolated compounds is presented in Figure 3.
 | Figure 3. Structure of isolated compounds from E. rubroloba rhizome. [Click here to view] |
Biological activities of methanol extract and six compounds towards DPPH, ABTS, and BSA displayed in (Fig. 4), and in-silico study as an antidepressant potential of six compounds were shown in Table 3.
 | Figure 4. Biological activity of E. rubroloba methanol extract and isolated secondary metabolite compounds. Data is presented as mean±SD (n=3). * indicates the significancy compared to positif control. [Click here to view] |
Table 3. Binding energies from the docking results of six compounds from the E. rubroloba against the target proteins MAO, DAT, and SERT.
| No | Compounds | Binding Energies (kcal/mol) | ||
|---|---|---|---|---|
| MOA | DAT | SERT | ||
| 1 | Stigmast-4-en-6β-ol-3-one | –11.9 | –10.7 | –10.5 |
| 2 | Stigmasterol | –11.5 | –10.7 | –10.1 |
| 3 | 1-(3’-methoxy-4’- hydroxyphenyl)-7- (4”-hydroxyphenyl)-3-heptanone | –9.1 | –8.3 | –8.7 |
| 4 | Yakuchinone A | –8.5 | –8.2 | –8.5 |
| 5 | 3,5-dimethoxy-4- acetoxycinnamyl alcohol | –6.9 | –6.8 | –6.6 |
| 6 | p-Coumaric acid | –6.7 | –6.8 | –6.7 |
| 7 | Clorgyline | –6.6 | N.A | N.A |
| 8 | Nortriptyline | N.A | –9.9 | N.A |
| 9 | Paroxetine | N.A | N.A | –10.2 |
Methanol extract of E. rubroloba (ER) rhizome and some isolated compounds demonstrated strong antioxidant and anti-inflammatory activities. The statistical analysis showed that there were significant differences between several treatment groups and the positive control (p < 0.05). This indicates that the activity exhibited by the test samples was significantly lower than that of the positive control. Conversely, in certain treatment groups, the difference with the positive control was not significant (p > 0.05), suggesting that the activity produced was comparable to the effectiveness of the positive control.
In the DPPH and ABTS radical scavenging assays (Fig. 4), most of the test samples exhibited p < 0.05 when compared with the positive control, indicating significant differences. This confirms that although the samples possessed activity, the potential they demonstrated was still lower than that of the standard control. Meanwhile, in the BSA denaturation inhibition assay, some test groups showed p > 0.05, indicating that their biological activity was comparable to the positive control.
Two steroid compounds, stigmasterol (ER1) and Stigmast-4-en-6β-ol-3-one (ER2), showed no significant difference compared to methylprednisolone as the control. Overall, the statistical results reinforced the validity of the finding that the positive control consistently provided higher effects. However, samples that showed p > 0.05 compared to the positive control deserve particular attention, as they potentially possess effectiveness equivalent to the standard comparator.
This synergistic action of antioxidant and anti-inflammatory properties presents an opportunity to explore their potential as antidepressant agents. The link between antioxidants, anti-inflammation, and antidepressants lies in the capacity of these compounds to address two key components of depressionoxidative stress and inflammation. By reducing oxidative damage and inflammation, compounds with antioxidant and anti-inflammatory properties can improve neurotransmitter balance and alleviate depressive symptoms [28].
Docking results of six compounds against target proteins MAO, DAT, and SERT to predict their antidepressant activity revealed that stigmast-4-en-6β-ol-3-one and stigmasterol were the most notable compared to other compounds (Table 3). Stigmast-4-en-6β-ol-3-one and stigmasterol demonstrated the highest potential as antidepressants, based on their binding affinities, particularly against MAO, with binding energies of −11.9 and −11.5 kcal/mol, respectively. In general, all compounds from the E. rubroloba rhizome exhibited promising potential against the MAO target compared to Clorgyline. Notably, stigmast-4-en-6β-ol-3-one and stigmasterol showed stronger affinities than Nortriptyline and Paroxetine against the DAT and SERT targets.
In targeting MAO, stigmast-4-en-6β-ol-3-one exhibited a binding energy of −11.9 kcal/mol, which is lower than that of stigmasterol (−11.5 kcal/mol) and the positive control clorgyline (−6.6 kcal/mol). Stigmast-4-en-6β-ol-3-one formed a hydrogen bond with Cys406 and demonstrated hydrophobic interactions with key residues such as Phe352, Val210, Cys323, Leu337, Ile335, Phe208, Tyr69, Tyr407, and Tyr444 (Fig. 6A), indicating strong binding at the MAO active site [22]. This suggests that stigmast-4-en-6β-ol-3-one may serve as a more efficient inhibitor, potentially contributing significantly to the inhibition of this enzyme. Although stigmasterol did not form hydrogen bonds, it still displayed stable hydrophobic interactions with residues similar to those of stigmast-4-en-6β-ol-3-one, indicating sufficient binding stability (Fig. 6B).
 | Figure 6. Molecular interactions of (A) stigmast-4-en-6β-ol-3-one and (B) stigmasterol from E. rubroloba rhizome with MAO. [Click here to view] |
MAO is an enzyme responsible for the oxidative deamination of neurotransmitters such as serotonin, dopamine, and norepinephrine. There are two major isoforms: MAO-A and MAO-B. Inhibition of MAO, particularly MAO-A, increases neurotransmitter concentrations at synapses by reducing their degradation, which is important in mood regulation [29]. In the context of molecular docking, both stigmast-4-en-6β-ol-3-one and stigmasterol exhibited lower binding energies than the standard inhibitor clorgyline, indicating their potential to inhibit MAO activity. Inhibition of this enzyme would elevate serotonin, dopamine, and norepinephrine levels in the brain, which are directly linked to improved mood and reduced depression symptoms.
On the DAT, stigmast-4-en-6β-ol-3-one and stigmasterol exhibited identical binding energies of −10.7 kcal/mol, which is lower than the control nortriptyline (−9.9 kcal/mol). Stigmast-4-en-6β-ol-3-one interacts through strong hydrophobic interactions with key residues such as Val120, Ala117, Ala479, Tyr124, Phe325, and Phe319 (Fig. 7A) at the DAT binding site [30], indicating the compound’s ability to effectively inhibit dopamine reuptake. Stigmasterol, on the other hand, forms hydrogen bonds with Phe325 and Gly425, potentially enhancing the stability of transporter inhibition. Additionally, it also forms hydrophobic interactions with residues Ala479, Phe319, Arg52, Tyr124, Ala48, and Val120 (Fig. 7B). In comparison, nortriptyline forms more hydrogen bonds (Phe43, Asp46, and Phe319) and hydrophobic interactions (Tyr124, Val120, and Ala479), but with a higher binding energy.
 | Figure 7. Molecular interactions of (A) stigmast-4-en-6β-ol-3-one and (B) stigmasterol from E. rubroloba rhizome with DAT [Click here to view] |
The DAT is responsible for the reuptake of dopamine from the synapse back into the presynaptic neuron after its release. Dopamine plays a key role in mood regulation, reward, and motivation [31]. Inhibition of DAT can elevate dopamine levels in the synapse, potentially enhancing motivation and happiness—both aspects often disrupted in depressed patients [32]. Docking simulations revealed that stigmast-4-en-6β-ol-3-one and stigmasterol have high affinities for DAT, with lower binding energies compared to nortriptyline (a tricyclic antidepressant). DAT inhibition by these compounds would increase dopamine levels in the brain, contributing to antidepressant effects. This aligns with the dopaminergic dysfunction theory of depression, which suggests that dopamine deficits contribute to symptoms of anhedonia and reduced motivation in depression.
On the SERT, stigmast-4-en-6β-ol-3-one displayed a binding energy of −10.5 kcal/mol, which is lower than that of stigmasterol (−10.1 kcal/mol) and the control paroxetine (−10.2 kcal/mol). Stigmast-4-en-6β-ol-3-one demonstrated strong hydrophobic interactions with residues Tyr176, Ala169, Phe341, Ile172, Val343, Tyr95, and Phe335 at the SERT binding site (Fig. 8A) [22], similar to the inhibitory mechanism observed with DAT, indicating its potential to inhibit serotonin reuptake. Stigmasterol also showed good binding stability through interactions with similar residues as stigmast-4-en-6β-ol-3-one (Fig. 8B). Although paroxetine exhibited slightly lower affinity, it formed hydrogen bonds with Ala96 and Ser336, providing additional stability to SERT inhibition. Overall, stigmast-4-en-6β-ol-3-one appears to be superior to paroxetine in terms of binding energy, suggesting its potential as a stronger serotonin reuptake inhibitor with relevant clinical applications for increasing serotonin levels and alleviating depressive symptoms.
 | Figure 8. Molecular interactions of (A) stigmast-4-en-6β-ol-3-one and (B) stigmasterol from E. rubroloba rhizome with SERT. [Click here to view] |
The SERT is a protein responsible for the reuptake of serotonin from the synaptic cleft back into the presynaptic neuron. Serotonin is a key neurotransmitter involved in mood regulation, anxiety, and well-being [33]. Selective serotonin reuptake inhibitors (SSRIs) function by blocking SERT, thereby increasing serotonin levels in the synapse and improving depression symptoms [34]. Molecular docking showed that stigmast-4-en-6β-ol-3-one and stigmasterol have very low binding energies toward SERT, comparable to or even lower than paroxetine, an SSRI. Inhibition of SERT by these compounds may elevate serotonin concentrations in the synapse, contributing to mood improvement and the reduction of depression symptoms.
4. CONCLUSION
Firstly, six of the 48 major compounds of E. rubroloba rhizome have been isolated and identified: Stigmasterol (ER1), Stigmast-4-en-6β-ol-3-one (ER2), Yakuchinone A (ER3), 1-(3’-methoxy-4’-hydroxyphenyl)-7-(4”-hydroxyphenyl)-3-heptanone (ER4), 3,5-dimethoxy-4-acetoxy cinnamyl alcohol (ER5), and p-Coumaric acid (ER6). ER3 and ER4 demonstrate strong antioxidant potential, while ER1 and ER2 exhibit significant anti-inflammatory properties. Computational analysis suggested that ER1 and ER2 have potential interactions with MAO, DAT, and SERT, which may be relevant to neurotransmission pathways involved in depression. These findings indicate a possible role of ER1 and ER2 in influencing dopamine and serotonin systems, warranting further in vitro validation.
5. ACKNOWLEDGMENT
We would like to thank to The Ministry of Education, Culture, Research, and Technology of the Republic of Indonesia for a research grant under scheme “KATALIS” Research 2024, with contract No. 151/UN29.20/PG/2024.
6. AUTHOR CONTRIBUTIONS
All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; agreed to submit to the current journal; gave final approval of the version to be published; and agreed to be accountable for all aspects of the work. All the authors are eligible to be an author as per the International Committee of Medical Journal Editors (ICMJE) requirements/guidelines.
7. CONFLICTS OF INTEREST
The authors report no financial or any other conflicts of interest in this work.
8. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
9. DATA AVAILABILITY
All data generated and analyzed are included in this research article.
10. PUBLISHER’S NOTE
All claims expressed in this article are solely those of the authors and do not necessarily represent those of the publisher, the editors and the reviewers. This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
11. USE OF ARTIFICIAL INTELLIGENCE (AI)-ASSISTED TECHNOLOGY
The authors declare that they have not used artificial intelligence (AI)-tools for writing and editing of the manuscript, and no images were manipulated using AI.
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