Antibacterial and Antioxidant Activities of Triterpenoids and Cyclic 1,7-Diarylheptanoids from the Stem Bark of <i>Myrica salicifolia</i>: A Combined Experimental and Computational Study

Gashaw, Abraham Dilnesa; Bedane, Kibrom Gebreheiwot; Demissie, Taye B.; Ombito, Japheth O.; Yaya, Estifanos Ele; Desta, Mekonnen Abebayehu

doi:https://doi.org/10.1155/2024/2013446

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Research Article | Open Access

Volume 2024 | Article ID 2013446 | https://doi.org/10.1155/2024/2013446

Antibacterial and Antioxidant Activities of Triterpenoids and Cyclic 1,7-Diarylheptanoids from the Stem Bark of Myrica salicifolia: A Combined Experimental and Computational Study

Abraham Dilnesa Gashaw,¹Kibrom Gebreheiwot Bedane,¹Taye B. Demissie,²Japheth O. Ombito,²Estifanos Ele Yaya,¹and Mekonnen Abebayehu Desta¹

Academic Editor: Liviu Mitu

Received25 Jul 2023

Revised09 Jan 2024

Accepted18 Jan 2024

Published12 Feb 2024

Abstract

Myricasalicifolia A Rich (Myricaceae) is a tree growing in Central and East Africa. Traditionally, the plant is used to treat malaria, respiratory disorders, inflammations, and infections. A new compound, 3β-O-trans-caffeoylisomyricadiol (7), was isolated from MeOH : CHCl₃ (2 : 1) extract of the stem bark of Myrica salicifolia along with seven known compounds, namely, myricanone (1), myricanol (2), myricanol-11-O-β-D-xylopyranoside (3), taraxerone (4), taraxerol (5), myricadiol (6), and methyl-β-D-glucopyranoside (8). This is the first report of the isolation of taraxerene-type triterpenes from this plant. The structures were determined by a comprehensive analysis of 1D/2D NMR spectroscopy, HR-MS, and by comparison with literature data. The compounds showed a wide range of DPPH scavenging activities from very weak (IC₅₀ value = 282.61 μM) to very strong (IC₅₀ = 13.48 μM). Antibacterial activities of the compounds were evaluated using the disk diffusion agar method, where some of the compounds showed modest antibacterial activities against S. pyogenes and S. aureus at 250 μg/mL. Compounds 2, 3, and 7 were assessed for their in silico molecular docking analysis. The lowest binding affinity for compound 7 was found to be −7.26 to −10.35 kcal/mol against PqsA protein of P. aeruginosa, pyruvate kinase (PK) enzyme of S. aureus, LuxS protein of S. pyogenes, and DNA gyrase B of E. coli, which showed better binding affinity compared to the standard drug ampicillin (−7.36 to −8.03 kcal/mol) and ciprofloxacin (−6.19 to −6.83 kcal/mol). In silico ADMET predictions revealed that compounds 3 and 8 met all the requirements for pharmacokinetic properties.

1. Introduction

Myrica salicifolia A Rich (Myricaceae) is a deciduous shrub with a trunk diameter of up to 1 m and typically 3–10 meters tall and can grow up to 20 meters in height. It is found in Central and East Africa, including Ethiopia, Zambia, Kenya, Burundi, Malawi, Uganda, Madagascar, Zaire, Rwanda, Tanzania, and also in Saudi Arabia [1, 2]. In Ethiopia, it is known by the local names “Shinet” or “Kalava” in Amharic, “Abay,” “Kataba,” “Radji” or “Tona” in Affan Oromo, and “Nihibi” in Tigrigna [3, 4]. The plant has a long history of use as a traditional medicine in Tanzania, where it is used to treat a variety of illnesses, including pneumonia, chronic constipation, cryptococcal meningitis, herpes zoster, stomach pain, and headaches [5, 6]. M. salicifolia has also been used to treat erectile dysfunction and male sexual impotence in Uganda [7]. In Ethiopia, it is used to treat lung diseases, inflammation, and skin diseases [8].

Compounds isolated from M. salicifolia have shown a range of biological activities. Marealle et al. reported the antimycobacterial activity of compounds isolated from M. salicifolia against three nonpathogenic mycobacterial species. Maslinic acid showed the highest MICs value of 17, 28, and 56 μg/mL against Mycobacterium madagascariense, standard Mycobacterium tuberculosis strain H₃₇RV, and rifampicin-resistant M. tuberculosis clinical isolates, respectively [9]. It has been linked to antimalarial [4], antiplasmodial [10], strong analgesic effects [11], and lowering blood glucose effects [12]. Recently, Rehman and coworkers reported that the root extract of M. salicifolia inhibited the activation of IL-6 and TNF-α in the colonic tissues of the UC model in rats [13]. A preliminary phytochemical analysis of its stem bark [3], root [4], and leaves [14] revealed the presence of polyphenols, saponins, glycosides, unsaturated sterols, triterpenes, alkaloids, tannins, flavonoids, proteins, and carbohydrates. Several cyclic diarylheptanoids were isolated from a methanolic extract of Morella salicifolia bark [15].

The objectives of this study include a comprehensive phytochemical investigation of the stem bark, assessment of antibacterial and antioxidant properties of the isolated compounds, and in silico molecular docking study of the isolated compounds against PqsA of P. aeruginosa, pyruvate kinase (PK) of S. aureus, LuxS of S. pyogenes, and DNA gyrase B of E. coli.

2. Materials and Methods

2.1. General Experimental Procedures

NMR experiments (1D and 2D) were recorded on a 600.0 MHz Bruker Avance III spectrometer using DMSO, MeOD, CDCl₃ solvents, and TMS as an internal reference. Mass spectra were recorded on a high-resolution mass spectrometer (Thermo Scientific, USA) equipped with an electrospray ionization (ESI) ion source. The matrix-assisted laser desorption/ionization high resolution mass spectrometry (MALDI-HR-MS) was conducted using an Applied Biosystems 4800 Proteomics Analyzer equipped with an Nd/YAG laser (λ = 335 nm), operated at a repetition rate of 200 Hz on a Bruker New ultrafleXtremeTM instrument. FT-IR (PerkinElmer) in the range 4000−400 cm⁻¹ (resolution: 4 cm⁻¹, number of scans: 4) was used using KBr discs. For column chromatography, silica gel 60 (70–230 mesh ASTM) and Sephadex LH−20 (18−111 μm, GE Healthcare Bio-Sciences AB, Sweden) were used. The isolation process was monitored by TLC (precoated sheets, ALUGRAM, Xtra SIL G/UV₂₅₄, 20 × 20 cm, coated with silica gel 60 fluorescent indicator, Germany), which was visualized under UV light and also sprayed with vanillin and/or cerium molybdate stain followed by heating gently for a few seconds.

2.2. Plant Material

The stem bark of M. salicifolia was collected from Debre Sina, Northern Shewa, Amhara region, 192 km from Addis Ababa, Ethiopia, in July 2020 by Dr. Mekonnen Abebayehu. The plant specimens were identified by Mr. Melaku Wendafrash, Addis Ababa University, Ethiopia. A voucher specimen of the plant material has been deposited in the National Herbarium, Department of Botany, Addis Ababa University, with a voucher/specimen number of MA/2007/12.

2.3. Extraction and Isolation

The collected fresh stem bark of M. salicifolia was air-dried at room temperature. The dried plant materials were ground using an electric grinder to obtain a fine powder. The powder (1.41 kg) was soaked and homogenized exhaustively using MeOH and CHCl₃ mixture at a ratio of 2 : 1. The mixture was filtered using a Buchner funnel with Whatman filter paper. The filtrates were concentrated using a rotary evaporator under reduced pressure at a temperature of 40°C. The concentrated crude extracts were allowed to dry to a constant weight at room temperature and furnished a mass of 98 g. 26.4 g of the crude extract was dissolved in chloroform and adsorbed on 25 g of silica gel, mixed well, and dried by using a rotary evaporator in order to make slurry. The sample was loaded to a glass column (55 cm × 4 cm) packed with silica gel (330 g, 70–230 mesh ASTM) and fractionated using the gradient solvent system of pet ether : CHCl₃ (25 : 75 to 10 : 90, v/v), CHCl₃ : EtOAc (90 : 10 to 0 : 100, v/v), and EtOAc : MeOH (95 : 5 to 20 : 80, v/v) to give 25 fractions each 200 mL. Fractions with similar TLC profiles were combined to give twenty fractions (MS-1 to MS-20). Fraction MS-7 (5.3 g) was subjected to column chromatography over silica gel (120 g) using the gradient solvent system of pet ether : CHCl₃ (0 : 100 to 5 : 95, v/v) and CHCl₃ : EtOAc (0 : 100 to 5 : 95, v/v) to give 14 subfractions, which were combined into four groups (MS-7-1 to MS-7-4) based on their TLC profile. Subfraction MS-7-1 (205.5 mg) was applied to a column chromatography packed with Sephadex LH-20 and eluted using CHCl₃ : MeOH (1 : 1) to give compound 1 (12.4 mg). Fraction MS-9 (1.2 g) was subjected to column chromatography over silica gel (40 g) using gradient of pet ether: CHCl₃ (23 : 77 to 0 : 100, v/v) and CHCl₃ : EtOAct (98 : 2 to 65 : 35, v/v) to give 10 subfractions, which were combined into three groups (MS-9-1 to MS-9-3) based on their TLC profile. Subfraction MS-9-2 (170.7 mg) was subjected to column chromatography over Sephadex LH-20 (90 g) using CHCl₃ : MeOH (2 : 1) as eluent to give compound 2 (8.8 mg). Fraction MS-16 (2.1 g) was rechromatographed over silica gel (100 g) eluted using CHCl₃ : EtOAc (10 : 90 to 5 : 95), followed by EtOAc : MeOH (97 : 3 to 70 : 30, v/v). Seven fractions (MS-16-1 to MS-16-7) were collected. Subfraction MS-16-4 (87 mg) was allowed to pass over Sephadex LH-20 column (90 g) eluted with CHCl₃ : MeOH (2 : 1), and the fractionated eluents were concentrated to give compound 3 (13 mg). The subfractions MS-1 (325.5 mg) and MS-6 (102.5 mg) from pet ether : CHCl₃ (30 : 70, v/v) and pet ether : CHCl₃ (10 : 90, v/v) were dissolved in MeOH and left overnight and afforded compound 4 (10.2 mg) and compound 5 (7.6 mg), respectively. Subfraction MS-7-3 (102.5 mg) was dissolved in MeOH and left overnight; the insoluble part afforded compound 6 (42.12 mg) as a white solid. The major fraction MS-11 was concentrated and dissolved in acetone to give a precipitate named compound 7 (11.2 mg). Fraction MS-20 (1.42g) was rechromatographed over silica gel (100 g) eluted with EtOAc : MeOH mixtures (96 : 4 to 40 : 60, v/v). Five fractions (MS-20-1 to MS-20-5) were collected. Subfraction MS-20-4 (23 mg) was applied to the Sephadex LH-20 column (90 g) and eluted with CHCl₃ : MeOH (2 : 1), and the collected fractions were concentrated to give compound 8 (5.2 mg).

2.4. Spectroscopic Data

Myricanone (1): a white powder; m.p. 193–195°C; IR (KBr) 3409.2, 2934, 2857.2, 1704.2, 1604.2, 1457.4, 1110.2, 1045.2 cm⁻¹ (Figure S1); ¹H-NMR data: see Table 1;¹³C-NMR data: see Table 2; HR-ESI-MS m/z 379.15160 [M + Na]⁺ (calc. for C₂₁H₂₄O₅, 379.15159).

Myricanol (2): a white powder; m.p. 102–110°C; IR (KBr) 3369.8, 2924.2, 2855.6, 1610.8, 1585, 1502.4, 1444, 1405.2, 1352.6, 1235.8, 1059.8, 962 cm⁻¹ (Figure S17); ¹H-NMR data: see Table 1; ¹³C-NMR data: see Table 2; HR-ESI-MS m/z 381.16707 [M + Na]⁺, (calc. for C₂₁H₂₆O₅, 381.16724).

11-O-β-D-xylopyranosylmyricanol (3): a white powder; m.p. 231-232°C; IR (KBr) 3390.8, 2927.8, 2849.6, 1501.8, 1459, 1406.2, 1347, 1229.4, 1069.2, 1039.4 cm⁻¹ (Figure S26); ¹H-NMR data: see Table 1; ¹³C-NMR data: see Table 2; HR-ESI-MS m/z 513.20956 [M + H]⁺ (calc. for C₂₆H₃₄O₉, 513.20950).

Taraxerone (4): a white powder; m.p. 272–274°C; IR (KBr) 2928.4, 2847.8, 1709, 1474.6, 1378.6, 1153.6, 1009.6 cm⁻¹ (Figure S35); ¹H-NMR data: see Table 1; ¹³C-NMR data: see Table 2; MALDI-MS (positive-ion mode) m/z 449.9 [M + Na + H]²⁺ (calc. for [C₃₀H₄₈ONaH]²⁺, 449.7).

Taraxerol (5): a white powder; m.p. 257–260°C; IR (KBr) 3430.8, 3051.4, 2929, 2860.4, 1751.2, 1620, 1449.2, 1385.8, 1081.4 cm⁻¹ (Figure S44); ¹H-NMR data: see Table 1; ¹³C-NMR data: see Table 2; MALDI-MS (positive-ion mode) m/z 449.9 [M + Na]⁺ (calc. for [C₃₀H₅₀ONa]⁺, 449.73).

Myricadiol (6): a white powder; m.p. 266–268°C; IR (KBr) 3392, 2936, 2855, 1644.4, 1461.2, 1382, 1018.2 cm⁻¹ (Figure S53); ¹H-NMR data: see Table 1; ¹³C-NMR data: see Table 2; MALDI-MS (positive-ion mode) m/z 442.1 [M]⁺ (calc. for C₃₀H₅₀O₂, 442.7).

3β-O-trans-caffeoylisomyricadiol (7): a white powder; m.p. 292–294°C; IR (KBr) 3434.2, 2937.8, 2868.4, 1695.6, 1616.6, 1526.4, 1471, 1389.8, 1278.6, 1221.6, 1159.2, 1001.8, 808 cm⁻¹ (Figure S62); ¹H-NMR data: see Table 3; ¹³C-NMR data: see Table 3; MALDI-MS m/z 897.8 as [M + (DHB-H₂O)₂ + Na-2H]⁺ (calc. for C₃₉H₅₆O₅, 897.0).

Methyl-β-D-glucopyranose (8): a colorless crystal; m.p. 104–106°C; IR (KBr) 3420, 2928.2, 2858.6, 1643.8, 1454.8, 1071, 1027 cm⁻¹ (Figure S70); ¹H-NMR data: see Table 1; ¹³C-NMR data: see Table 2; HR-ESI-MS m/z 217.06848 [M + H]⁺ (calc. for C₇H₁₄O₆, 217.06826).

2.5. Antioxidant Activity Assays by 2, 2-Diphenyl-1-Picrylhydrazyl (DPPH)

The antioxidant activities of the isolated compounds were assessed using DPPH radical scavenging activity with ascorbic acid as the positive control as per the previously described method [16]. All extracted compounds were separately dissolved in DMSO (1 mg/mL) and serially diluted with methanol, followed by addition of 0.004% (4 mg/100 mL) methanolic solution of DPPH to get 3.12, 6.25, 12.5, 25, and 50 μg/mL, and the absorbance of each dilution, after 30 minutes, was measured at 517 nm. An ascorbic acid solution of the same concentration (3.12 µg/mL to 50 μg/mL) was prepared in similar fashion and measured.

The DPPH radical scavenging activity of each of the tested compounds was reported as percentage inhibition using the following formula:where A control is the absorbance of DPPH solution and B sample is the absorbance of the test sample (DPPH solution plus compound).

The DPPH solution was used as a negative control. The relative half-maximal inhibitory concentration (IC₅₀) values were calculated using the plotted % RSA and double-verified using trusted online computational tools [17]. The final antioxidant activity of each compound was expressed as the IC₅₀ value.

2.6. Antibacterial Activity

The four common standard ATCC strains, Staphylococcus aureus (S. aureus, ATCC 25923), Escherichia coli (E. coli, ATCC 25922), Streptococcus pyogenes (S. pyogenes, ATCC 19615), and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) were obtained from Adama Science and Technology University (ASTU). Experiments were conducted in close consultation with the Microbiology Laboratory of the Applied Biology Department of ASTU.

The in-vitro antibacterial susceptibility test was determined by the disc diffusion method [18]. The medium was prepared by dissolving 38 g of Mueller–Hinton agar in 1000 mL of distilled water in 2.5 L flask. The flask was placed on a hot plate with a magnetic stirrer, and the mixture was heated slowly until the powder was completely dissolved and then autoclaved at 121°C for 15 min. The autoclaved medium was poured into sterile Petri dishes (20 mL/plate), and the plates were allowed to solidify under sterile conditions at room temperature. Then, the plates were seeded with an overnight grown culture approximately 1.5 × 10⁸ CFU/mL by swabbing evenly on the surface of the medium with a sterile cotton swab.

The isolated compounds at concentrations of 250 μg/mL and 500 μg/mL were prepared by dissolving in 10% 10% aqueous DMSO [19, 20]. 6.0 mm in diameter discs made of Whatman filter paper No. 1 were infused with the solutions of the isolated compounds and placed on the surface of the medium by gently pressing down to ensure contact with the MHA. Plates were inverted and incubated at 37°C for 24 hours. Ampicillin was used as a standard antibiotic positive control [21], while 10% aqueous DMSO was used as a negative control. After incubation, the inhibition zones were evaluated by measuring the diameter (mm) of the clear zone around the discs. All the experiments were performed in triplicate and interpreted using the CLSI zone diameter interpretative standards [18] and compared with 10 μg/mL controls.

2.7. Computational Methods

2.7.1. Molecular Docking

The interaction and binding affinity of the isolated compounds was investigated against PqsA protein of P. aeruginosa (PDB: 50E4), pyruvate kinase (PK) enzyme of S. aureus (PDB: 3T07), LuxS protein of S. pyogenes (PDB: 1INN), and DNA gyrase of E. coli (PDB: 6F86). The crystal structures of the proteins were downloaded from the protein data bank and processed by removing the cocrystallized ligands, deleting water molecules, and adding polar hydrogen and cofactors according to the AutoDock 4.2.6 (MGL tools 1.5.7) procedure [22]. After cleaning the proteins, only polar hydrogens and the Kollman charges were introduced. Nonpolar hydrogen atoms were merged, and Gasteiger partial atomic charges were assigned. In line with the experiment, we used ampicillin as a standard control drug. The grid center coordinates were 70, 70, and 70 pointing in the x, y, and z directions, respectively, with a grid point spacing of 0.375 Å. The center grid box was 9.393, −0.025, and 13.018 Å. 50 different conformations were generated for each targeted isolated compound. Furthermore, to gain deeper insights into the biological activities of the isolated compounds, we conducted a comparative analysis with the binding interactions of ciprofloxacin against the aforementioned proteins. This comparative assessment aims to provide a comprehensive understanding of the potential activities exhibited by the isolated compounds in comparison to the well-known antibacterial agent ciprofloxacin. The conformation of the compounds with the lowest binding free energy was selected to analyze the interactions with the receptors using the Discovery Studio Visualizer.

2.7.2. In-Silico Prediction of the Pharmacokinetics and Physicochemical Properties

Swiss ADME [23], an online prediction tool, was used to predict the pharmacokinetics of the isolated compounds. Parameters such as lipophilicity, molecular weight, water solubility, hydrogen bond donors and acceptors, bioavailability score, gastrointestinal absorption, and blood-brain barrier permeability were computed [23]. The OSIRIS property explorer and ProTox online server [24] were used to determine the oral toxicity and LD₅₀ values of the investigated compounds.

3. Results and Discussion

3.1. Structure Elucidation of Isolated Compounds

The chloroform-methanol (2 : 1) extract of the stem bark of M. salicifolia was subjected to column chromatography to afford eight compounds 1–8 (Figure 1). Three diarylheptanoids were isolated and characterized as myricanone (1) [25], myricanol (2) [25], and myricanol-11-O-β-D-xylopyranoside (3) [26]. Four triterpenoids, taraxerone (4) [27], taraxerol (5) [28], myricadiol (6) [29], and 3β-O-trans-caffeoylisomyricadiol (7), were isolated. One pyranoside, methyl-β-D-glucopyranoside (8), was also isolated and characterized [30, 31]. This is the first report of compounds 4, 5, 6, 7, and 8 from M. salicifolia.

Compound 1 was isolated as a white powder with the melting point of 193–195°C. The IR spectrum (Figure S8) revealed a broad absorption band centered at 3369.8 cm⁻¹ due to O-H stretching vibration, 2924.2 cm⁻¹ and 2855.6 cm⁻¹ due to C-H stretching vibration, 1704.2 cm⁻¹ due to C=O stretching of aliphatic ketone, 1614.8 cm⁻¹ and 1585.0 cm⁻¹ due to C=C stretching vibrations, 1110.2 cm⁻¹–1045 cm⁻¹ due to C-O, and 750 cm⁻¹–1000 cm⁻¹ due to the aromatic CH group. The molecular formula was established as C₂₁H₂₄O₅ by the sodiated-molecular-ion peak in the HR-ESI-MS at m/z 379.1516 [M + Na]⁺ (calcd for C₂₁H₂₄O₅Na, 379.1516) (Figure S7). The ¹H-NMR spectrum (Figure S1) of the aromatic region confirmed the presence of four aromatic protons, with a singlet at 6.31 (H-19) and three mutually coupled protons at 6.95 (H-15), 6.72 (H-16), and 6.51 (H-18), indicating the presence of one proton in one aromatic ring and three in 1,2,4-relative positions in the other aromatic ring. In addition, the observed four oxygenated aromatic carbons at 140.4 (C-4), 148.5 (C-3), and 149.1 (C-5) supported the hypothesis that one of the aromatic rings has the pyrogallol oxidation pattern. Three aromatic proton signals were observed as an ABX system at 6.95 (dd, J = 8.2, 2.4 Hz, H-15), 6.72 (d, J = 8.2 Hz, H-16), and 6.51 (d, J = 2.5 Hz, H-18) in the ¹H-NMR spectra (Table 1). The presence of two methoxy groups was deduced from the ¹H-NMR spectra at 3.76 (3H-21) and 3.74 (3H-20) and the ¹³C-NMR spectra at 60.9 (C-21) and 60.5 (C-20). The HMBC showed a correlation between the deshielded methoxyl groups ( 3.74 (H-20) and 3.76 (H-21)) and the aromatic carbons at C-148.5 (C-3) and C-140.3 (C-4), respectively, indicative of a di-ortho-substitution. The ¹³C-NMR, DEPT, and HSQC spectra (Figures S2, S3, and S5) revealed 21 carbon atoms corresponding to two methyls, six methylenes, four methines, and eight quaternary carbon signals, as well as the typical peaks of a carbonyl carbon and twelve aromatic carbons (Table 2). In the HMBC spectrum (Figure S6), OH-protons at 8.87 and 8.62 are attached to C-5 ( 149.1) and C-17 ( 152.7), respectively, thus supporting the attachment of OH-to the phenol ring. In the ¹H-NMR, the two hydroxy groups were resonating highly down field at δ 8.85 (s, OH-5) and 8.70 (s, OH-17) than expected because when a five, six, and seven-membered conjugated ring and the π-delocalization of the hydrogen bonded heteroconjugated fragment result in a resonance-assisted with an intramolecular hydrogen bond in the compound structure, δΟΗ resonance in DMSO-d6 is more deshielded, i.e., the hydrogen bond is stronger than the expected aromatic systems [32]. The COSY spectra (Figure S4) showed correlations of protons H-7/H-8/H-9/H-10 and H-12/H-13. Six methylene carbons at 45.9 (C-10), 42.4 (C-12), 28.8 (C-13), 27.2 (C-7), 24.5 (C-8), and 21.8 (C-9) and one carbonyl carbon at 213.8 (C-11) were also identified and linked together in a chain, as demonstrated by the absence of a carbon resonance associated with a point of branching. The HMBC correlations of the methylene protons H-9, H-10, H-12, and H-13 with C-11 revealed that C-10 and C-12 are connected through a C=O group at the 11-position, while HMBC correlations between 2.56 (H-7) and 149.1 (C-5), 122.3 (C-6), 129.1 (C-19), and between 2.82 (H-13), 128.3 (C-15), and 131.4 (C-14) were used to link the aliphatic chain with the diphenyl moiety, and consequently compound 1 was determined as myricanone (Figure 1) by comparison of these data (Tables 1 and 2) and the literature data [25].

Compound 2 was isolated as an amorphous white powder with a melting point of 102–110°C. The IR spectra (Figure S17) of compound 2 revealed a broad absorption band at 3369.8 cm⁻¹ due to O-H stretching vibration, 2924.2 cm⁻¹ and 2855.6 cm⁻¹ due to C-H stretching vibration of the aliphatic chain, and 1610.8 cm⁻¹–1444.0 cm⁻¹ due to C=C stretching vibrations of aromatic. Its molecular formula, C₂₁H₂₆O₅, was determined from HR-ESI-MS at m/z 381.1670 [M + Na]⁺ (calcd for C₂₁H₂₆O₅Na, 381.1672) (Figure S16). The ¹H-and DEPT-NMR spectrum (Figures S9 and S11) of compound 2 was very similar to that of 1, except the aliphatic side chain contains OH at C-11. This observation was supported by a methine proton at 3.98 bonded to C-11 ( 67.1). Furthermore, methane proton signal at 3.98 showed HMBC correlations with C-10 and C-12. Its ¹³C-NMR spectra (Figure S10 and Table 2) were similar to those of compound 1, except for position 11. In contrast, the carbonyl group at C-11 ( 213.8) in 1 was replaced by a hydroxyl group ( 67.1). The ¹H and ¹³C-NMR values for all the protons and carbons were assigned on the basis of 2D-NMR (Figure S12–S15) correlations and were given in Tables 1 and 2. As a result, compound 2 was identified as myricanol by comparison of these data found in the literature [25].

Compound 3 was isolated as an amorphous white powder with a melting point of 231-232°C. The IR (Figure S26) of compound 3 revealed a broad absorption band at 3390.8 cm⁻¹ due to O-H stretching vibration, absorptions at 2927.8 cm⁻¹ and 2849.6 cm⁻¹ due to C-H stretching, 1501.8 cm⁻¹–1406.2 cm⁻¹ due to C=C stretching vibration of aromatics, and 1352.6 cm⁻¹ and 1235.8 cm⁻¹ due to C-O-C stretching vibrations of aromatic methoxy. The molecular formula was established to be C₂₆H₃₄O₉ from HR-ESI-MS at m/z 513.2095 [M + Na]⁺ (calcd for C₂₆H₃₄O₉Na, 513.2095) (Figure S25). The ¹H and ¹³C-NMR data of compound 3 (Tables 1 and 2) were similar to those of compound 2, except for an additional sugar moiety at C-11. The anomeric proton and carbon of the sugar moiety resonated at 3.98 (d, J = 7.4 Hz, H-1′) and 101.4 (C-1′) in the ¹H, ¹³C-NMR, and DEPT spectra (Figures S18–S20), respectively. The remaining sugar carbons characteristic of D-xylose appeared at 66.1 (5′), 70.1 (C-4′), 73.6 (C-2′), and 77.0 (C-3′) [33]. The relative configuration of the xylose sugar moiety was also assigned by ¹H-¹H coupling constants, HSQC, and NOESY correlations (Figures S21, S22 and S24). H-1′ ( 3.98) and H-2′ ( 2.89) were located at axial positions which were confirmed by the large coupling constant value of ³JH1′-H2′ (7.4 Hz). The coupling constant of 8.9 Hz between H-2′ ( 2.89) and H-3′ ( 3.05) also indicated their axial-axial relationship. The large coupling value (8.9 Hz) between H-3′ and H-4′ revealed that H-3′ ( 3.05) and H-4′ ( 3.21) are located on the axial positions of the sugar. Furthermore, the NOESY correlations of H-1′ ( 3.98) and H-3′ ( 3.05) assigned that these two protons are found in the same plane, while H-2′ ( 2.89) and H-4′ ( 3.21) protons are found on the other side, so that the OH at H-3′ is trans to H-2′ and H-4′. If two adjacent hydrogen atoms in a six-membered ring are trans (i.e., diaxial), a value of 7–10 Hz should be observed for JH-H, while only 2–4 Hz should be observed if the hydrogen atoms are gauche (i.e., axial-equatorial) [34]. The attachment of the sugar moiety at C-11 was supported by the HMBC (Figure S23) correlation between the anomeric proton at 3.98 (H-1′) and C-11 ( 75.4). Hence, based on the above data (Tables 1 and 2) and comparison with the literature data, compound 3 was proposed to be 11-O-β-D-xylopyranosylmyricanol [26].

Compound 4 was isolated as a white powder with a melting point of 272–274°C. The infrared spectrum (Figure S35) of compound 4 revealed absorptions at 3051.4 cm⁻¹ due to = C-H stretching vibration and absorptions at 2929.0 cm⁻¹ and 2860.4 cm⁻¹ due to C-H stretching vibration of alkanes. The sharp peaks appeared at 1709 cm⁻¹ due to the C=O stretching vibration of carbonyl groups and at 1474 cm⁻¹ and 1378.6 cm⁻¹ due to C=C stretching vibrations and umbrella mode of –CH₃ symmetric bending, respectively. Its molecular formula was deduced to be C₃₀H₄₈O by the MALDI-MS (Figure S34) from the molecular ion peak at m/z 449.9 for [M + Na + H]²⁺ (calc. for [C₃₀H₄₈ONaH]²⁺, 449.7). ¹H-NMR, ¹³C, and DEPT spectra (Figures S27–S29) showed eight singlet methyl signals at chemical shifts of 1.15 (H-27), 1.12 (H-25), 1.11 (H-23), 1.01 (H-24), 0.98 (H-29), 0.94 (H-30), 0.93 (H-26), and 0.85 (H-28), which were associated with the relevant carbon resonances at 33.4 (C-29), 29.9 (C-26), 29.8 (C-28), 26.1 (C-23), 25.6 (C-27), 21.5 (C-24), 21.4 (C-30), and 14.8 (C-25), suggesting the presence of a pentacyclic triterpenoid structure [27]. There were four methine protons, the double (J = 8.2, 3.2 Hz) centered at 5.58 (H-15) is attributable to the olefinic proton at C-15. A multiplet at 1.34 (m), multiplet at 1.53 (m), and a doublet of doublet at 1.01 (14.8, 8.1) are attributable to the methine protons at C-5, C-9, and C-18, respectively. ¹H-NMR and ¹³C spectrum showed ten methylene signals at chemical shifts of 1.40 (m, H₁-1), 1.90 (m, H₂-1), 2.35 (m, H₁-2), 2.60 (m, H₂-2), 1.57 (m, H₁-6), 1.62 (m, H₂-6), 1.04 (m, H₁-7), 1.40 (m, H₂-7), 1.57 (m, H₁-11), 1.69 (m, H₂-11), 1.00 (d, J = 3.7 Hz, H₁-12), 1.34 (m, H₂-12), 1.69 (m, H₁-16), 1.95 (m, H₂-16), 2.10 (dt, J = 13.0, 3.4, H₁-19), 1.39 (m, H₂-19), 1.57 (m, H₁-21), 1.62 (m, H₂-21), 1.34 (m, H₁-22), and 1.39 (m, H₂-22). ¹³C-NMR and DEPT spectra showed 30 carbon signals: eight methyls, ten methylenes, four methines, and four quaternary carbons, of which one of the quaternary carbons at 217.5 (C-3) was a carbonyl carbon. The COSY spectrum (Figure S30) of compound 4 showed correlations between H-1 and H-2, H-6 and H-7, H-15 and H-16, H-21 and H₂-22, H-9 and H-11, H-5 and H₂-6, and H-18 and H-19, which are characteristics of the connectivity sequence in the pentacyclic ring. In HSQC spectra, protons correlate with their directly attached carbon which is shown in Figure S31. The olefin was attributed to C-14 based on long-range HMBC (Figure S32) correlations of the methyl singlets H-26 and H-27 with C-14 and H-16 with C-14. NOESY (Figure S33) correlations showed that H-24/H-25, H-26/H-18, and H-30/H-26/H-28 were found on the same side of the molecule, whereas H-23/H-5, H-5/H-9, and H9/H-27 were found on the opposite side of the molecule. Therefore, the structure of compound 4 was determined to be taraxerone [27].

Compound 5 was isolated as a white powder with a melting point of 257–260°C. Its IR spectrum (Figure S44) at 3430.8 cm⁻¹ was due to O-H stretching vibration, 3051.4 cm⁻¹ due to = C-H stretching vibration, and 1620 cm⁻¹ due to C=C stretching vibrations. Its molecular formula was deduced to be C₃₀H₅₀O using the MALDI-MS at m/z 449.9 [M + Na]⁺ (calc. for C₃₀H₅₀ONa, 449.7) (Figure S43). Its NMR (Figures S36–S38) spectroscopic data were identical to those of 4, except for the absence of carbonyl carbon signals in 5, which was replaced by an OH group at C-3. This was confirmed by the shielded chemical shift of C-3 at 79.1 and the COSY (Figure S39) correlation 3.22 (H-3) with H-2. In HSQC spectrum, protons correlate with their directly attached carbon which is showed in Figure S40. The HMBC (Figure S41) correlations of methyl protons H-23 and H-24 with C-3 also supported the assignment. The relative configuration of OH was determined to be 3β from the NOESY (Figure S42) correlations of H-3 and H-5. Therefore, the structure of compound 5 was elucidated as taraxerol [28].

Compound 6 was isolated as a white amorphous powder with a melting point of 266–268°C. Its molecular formula was deduced to be C₃₀H₅₀O₂ from the MALDI-MS at m/z 442.1 [M]⁺ (calc. for C₃₀H₅₀O₂, 442.7) (Figure S52). Its spectroscopic data (Figures S45–S49) were identical to those of compound 5, except for the presence of a hydroxylated carbon signal at 65.5 (C-28) in the ¹³C-NMR spectrum of compound 6. The presence of this additional hydroxyl group was confirmed by the ¹H-NMR signal for diastereotopic protons at 3.16 and 3.31 (H-28), as shown by the HMBC (Figure S50) correlations with C-17, C-22, and C-16. NOESY (Figure S51) correlations showed that H-24/H-25/H-26 and H-18/H-28/H-30 were all classified as β-oriented due to their appearances on the same side of the molecule. Whereas, the H-3/H-23/H-5, H-5/H-9, and H9/H-27 NOESY correlations showed that they were α-configured. Based on the spectroscopic data (Tables 1 and 2) and comparison with the literature data, compound 6 was determined to be myricadiol [29].

Compound 7 was obtained as a white amorphous powder with a melting point of 292–294°C. The molecular formula was found to be C₃₉H₅₆O₅, via the MALDI-MS (Figure S61) with m/z 897.8 for [M + (DHB-H₂O)₂ + Na-2H]⁺ (calc. for C₃₉H₅₆O₅, 897.0). The ¹H, ¹³C-NMR, and DEPT spectra (Figures S54–S56) of compounds 7 (Table 3) and 6 were nearly superimposable for the pentacyclic moiety. Differences were observed due to the signals of a trans-caffeoyl moiety in 7. This group was identified in the ¹H-NMR from two olefinic trans coupled protons at 7.45 (d, J = 15.8 Hz, H-7′) and 6.24 (d, J = 15.9 Hz, H-8′) and three aromatic protons at 6.76 (d, J = 8.1 Hz, H-4′), 7.00 (dd, J = 8.2, 2.1 Hz, H-5′), and 7.04 (d, J = 2.1 Hz, H-1′); the coupling relationship established a 1,3,4-substituted benzene ring [35]. The attachment of the caffeoyl group at C-3 via an ester linkage was deduced from the HMBC (Figure S59) correlation of H-3 ( 4.49) with caffeoyl group carbonyl carbon C-9′ ( 166.8). The relative configuration of the stereogenic center of 7 was deduced from the ¹H-¹H coupling constant (Figure S57) and the NOESY (Figure S60) experiment. The typical coupling constant for H-3 (J = 11.7, 4.5 Hz) in the ¹H-NMR spectrum revealed that H-3 was in the α-orientation. The difference in the multiplicity with a larger coupling constant of H-3 in 7 was in agreement with the respective coupling patterns (axial-equatorial and axial-axial) of H-3 and H₂-2, indicating that H-3 is situated in an axial position [36, 37]. In the HSQC spectrum, protons correlate with their directly attached carbon that is shown in Figure S58. In addition, α-orientation of H-3, H-5, H-9, H-23, and H-27 was suggested by the NOESY interactions of H-3 to H-23/H-5 and H-9 to H-5/H-23/H-27. Thus, the structure of compound 7 was determined to be 3β-(3′, 4′-dihydroxy-trans-cinnamoyloxy)-D-friedoolean-14-en-3α, 28-diol and named with a trivial name 3β-O-trans-caffeoylisomyricadiol.

Compound 8 was isolated as a colorless crystal with a melting point of 104–106°C. A strong broad IR (Figure S70) absorption band characteristic of the bonded-OH group was observed at 3420 cm⁻¹, absorptions at 2928.2 cm⁻¹ and 2858.6 cm⁻¹ due to C-H stretching vibration of CH₃, CH₂, and CH. The molecular formula was established as C₇H₁₄O₆ by an ESI-MS (Figure S69) at m/z 217.06848 [M + Na]⁺ (calcd for C₇H₁₄O₆, 217.06826). Seven carbon atoms were identified by the ¹³C-NMR and DEPT spectra, indicating a sugar moiety. A detailed analysis of 1D and 2D NMR spectra (Figures S63–S68 and Tables 1 and 2) and comparison with the literature indicated that the results were in line with those for methyl-β-D-glucopyranoside. The pyranose moiety′s ¹³C-NMR values were consistent with those seen in the literature [31].

3.2. Antioxidant Activity

The antioxidant capacities of the isolated compounds, expressed as the DPPH free radical inhibition and the IC₅₀ values, are presented in Table 4.

According to antioxidant activity parameters, the IC50 value category is very strong if the IC50 value is <10 μg/mL, strong if the IC50 value is between 10 and 50 μg/mL, mild if the IC50 value is between 50 and 100 μg/mL, weak if the IC50 value is between 100 and 250 μg/mL, and not active if IC50 is above 250 μg/m [38]. The relative IC₅₀ values for the isolated compounds were in the range of 13.48 μM–740.83 μM (Table 4). Compound 2 (IC₅₀ = 13.48 μM) showed the highest antioxidant activity compared to other isolated compounds, while compound 8 (IC₅₀ = 740.83 μM) showed the lowest antioxidant activity. The IC₅₀ values recorded for diarylheptanoid compounds 1, 2, and 3 were 19.55 μM, 13.48 μM, and 97.99 μM, respectively, whereas 282.61 μM, 306.32 μM, 59.31 μM, and 23.95 μM were reported for compounds 4, 5, 6, and 7, respectively. In diarylheptanoid compounds, the most susceptible OH group is the phenolic hydroxyl group, which confirms the importance of the enolic-OH moiety for the highest antioxidant activity of compound 2. The presence of the ketone functional group in compound 1 and the sugar moiety in compound 3 might be responsible for their reduced antioxidant activity in comparison to compound 2. The attached sugar moiety creates steric hindrance in compound 3, which may reduce its antioxidant activity compared to compound 6 [39, 40]. Of the triterpenoid compounds, compound 7 has a phenylpropanoid moiety, which increases its antioxidant activity compared to other terpenes. This suggests that the phenylpropanoid moiety is an essential functional group for the observed antioxidant activity.

3.3. Antibacterial Activity

The in-vitro antibacterial activities of compounds 1–7 against Gram-positive (S. aureus and S. pyogenes) and Gram-negative (E. coli and P. aeruginosa) bacterial strains were examined at doses of 250 μg/mL and 500 μg/mL compared with 10 μg/mL of positive controls. The measured inhibition zones of the compounds are presented in Table 5.

The inhibition zone for the seven isolated compounds against E. coli ranges from 6.5 to 9.5 mm at a concentration of 250 μg/mL. The compound having better activity against E. coli was compound 3 (inhibition zone of 9.0 mm) compared to other isolated compounds. Compounds 3 (10.0 mm) and 7 (10.0 mm) displayed the highest activity against S. pyogenes, while compound 4 showed the lowest inhibition zone (7.0 mm) against S. aureus, followed by compounds 6 (8.5 mm) and 7 (9.0 mm). Against the Gram-negative bacteria P. aeruginosa, the highest activity was recorded for compound 3 (8.5 mm), followed by compound 7 (8.0 mm) at a concentration of 250 μg/mL. The results of the present study generally indicate that compounds 3 and 7 displayed better activities to inhibit the growth of both Gram-positive and Gram-negative bacterial strains compared to other compounds, and the lowest activity was exhibited by compound 4. All the isolated compounds showed relatively weaker activities compared to the standard drugs ampicillin and ciprofloxacin. However, compared to the growth of Gram-negative bacteria, it was more effective against the growth of Gram-positive bacteria. Several studies suggest that the antibacterial activity of polyphenols is generally more effective against Gram-positive than Gram-negative bacteria due to cell walls linked to a molecularly complex outer membrane that slows down the passage of chemicals [42].

The results from this study suggest that the higher antibacterial and antioxidant activity of compounds 3 and 7 could be attributed to the complex phenolic compounds. In the case of compound 3 with the skeletal structure of two aromatic rings linked together with seven carbon chains and possesses strong nucleophilic properties, which may allow it to donate an electron pair to electrophilic functional groups of plasma membrane proteins and/or lipids, probably leading to membrane dysfunction [43]. For the activity displayed by compound 7, the presence of caffeoyl group might be responsible. This seems to be in line with our findings that showed better antibacterial performance of compounds 3 and 7 than the other compounds.

3.4. Computational Study

3.4.1. Molecular Docking Investigation

The in silico molecular docking study of the isolated compounds (2, 3, and 7) against PqsA protein of P. aeruginosa, pyruvate kinase (PK) enzyme of S. aureus, LuxS protein of S. pyogenes, and DNA gyrase of E. coli are discussed. The three isolated compounds 2, 3, and 7 interacted (Figures 2 and 3) with the main amino acids of pyruvate kinase of S. aureus (PDB: 3T07 and have minimum binding energy ranging from −6.95 to −10.05 kcal/mol, with the best result achieved for compound 7 (−10.05 kcal/mol)) (Table 6) compared to the standard drugs ampicillin and ciprofloxacin, and it has good interaction affinities with the residual amino acids (Figure 4 and 5). Compound 7 has a stronger binding energy with the lowest inhibition constant () 0.04 μM than the standard drugs since it interacts through four hydrogen bonds between residues Met1, Asn29, Asp58, and Arg443 with the oxygen of hydroxyl and oxygen of ketone of the isolated compounds (Figure 6). On the other hand, the hydrogen bond interaction of ampicillin and ciprofloxacin was with Asn29, Lys59, Ile60, Pro418 and Met1, Asp58, and Lys59 (Figures 4 and 5), respectively. Moreover, hydrophobic (π-Sigma/π-Alkyl) and van der Waals interactions between compound 7 and residues Glu60, Pro118, Gly65, Pro79, Gly119, and Glu124 were observed, which are different from the standard drug.

The three isolated compounds 2, 3, and 7 interacted (Figures 7–9) with the main amino acids of pqsA of p. aeruginosa (PDB: 50E4) and have minimum binding energy ranging from −7.35 to −10.35 kcal/mol, with the best result achieved for compound 7 (−10.35 kcal/mol) (Table 6), with the lowest inhibition constant () 0.03 μM than ampicillin and ciprofloxacin since it interacts through different hydrophobic and van der Waals interactions. But hydrogen bond with Tyr163 for compound 7 showed less binding scores compared to ampicillin and ciprofloxacin which interacts through four hydrogen bonds: Thr164, Asp382, Arg372, Arg397 and Thr164, Thr304, Arg372, Arg397 (Figures 10 and 11), respectively. The three isolated compounds 2, 3, and 7 interacted (Figures 12–14) with the main amino acids of LuxS of S. pyogenes (PDB: 1INN) and have minimum binding energy ranging from −5.51 to −7.45 kcal/mol, with the best result achieved for compound 7 (−7.45 kcal/mol) (Table 7). It also showed hydrophobic/π-cation interactions with Glu60 and Pro118 of a phenyl and methyl groups of the compound, respectively. On the other hand, Gly65, Pro79, Gly119, and Glu124 showed van der Waals residual interactions (Figure 14), while ampicillin and ciprofloxacin form H-bonds with Asp76, Ser78, and Gly81 and Asp76, respectively, and hydrophobic and van der Waals interactions with various amino acid residues at the active sites are presented (Figures 15 and 16).

The isolated compounds 2, 3, and 7 interacted with the main amino acid residues of the DNA gyrase of E. coli (PDB: 6F86) through H-bonds, van der Waals, and π-sigma/π-alkyl (hydrophobic) interactions (Figures 17–19). Compound 7 forms H-bonds with Val71, and hydrophobic and van der Waals interactions with various amino acid residues at the active sites are presented in Table 7 and Figure 19, while ampicillin and ciprofloxacin form H-bonds with Asn46 and Asp73, and Asp49, Gly77, and Arg136, respectively, and hydrophobic and van der Waals interactions with various amino acid residues at the active sites are presented in Figures 20 and 21. The binding scores range from −6.27 to −9.72 kcal/mol with the best binding energy achieved for compound 7 with −6.72 kcal/mol and inhibition constant () 0.07 μM, while for ampicillin and ciprofloxacin, the binding energies were −7.52 kcal/mol and −6.19 kcal/mol and inhibition constants () were 3.07 µM and 28.85 µM, respectively. The significant binding interaction of compound 7 with the target proteins could suggest possible activity of these isolated compounds against S. aureus, p. aeruginosa S. pyogenes, and E. coli. Interestingly, the standard exhibits higher antibacterial activity despite having a lower binding energy than compound 7. This divergence in antibacterial activity could be attributed to specific interactions crucial for the antibacterial efficacy, potentially linked to the complete solubility of the isolated compounds. To understand the precise nature of interactions between the compounds and the target proteins, further investigations are imperative. A more in-depth analysis, especially in-vivo bioassay study, is required to discern the factors contributing to the observed variations in antibacterial activity, ensuring a comprehensive understanding of the underlying mechanisms at play. As preliminary evidence, the two-dimensional interactions projections make it clear that, in comparison to the two compounds, the standard exhibits a significantly higher number of specific hydrogen bond interactions with the target protein residues which might be linked to its activity. A similar situation was encountered in the work of [44, 45] where good antibacterial and antitubercular activities of isolated compounds and ligands are observed as a result of specific interactions with important protein residues despite smaller binding energies.

3.4.2. In Silico Pharmacokinetics and Toxicity Analysis

The drug-likeness of the isolated compounds was characterized according to “Lipinski’s rule of five.” As per Lipinski’s rule, the potential compounds should have the following physicochemical properties [46], such as (i) total polar surface area (TPSA), which should not be >140 Å; (ii) a number of hydrogen bond donors ≤5, number of hydrogen bond acceptors ≤10; (iv) a molecular mass ≤500 Da; and (v) log P not ≥5. The Swiss ADME-computed results (Table 8) showed that compounds 1–3 and 8 in the present study satisfy Lipinski’s rule of five with zero violations [47]. Except for compound 7, all the studied compounds recorded a lipophilicity (iLogP) value of less than five (range from 1.25 to 4.77), indicating their optimal lipophilicities for good oral and intestinal absorption. Compounds 1–3, 4–7, and 8 presented log S values of −4.47 to −4.84, −7.50 to −9.57, and 0.76, respectively. The drug-likeness of the isolated compounds was characterized according to “Lipinski’s rule of five.” As per Lipinski’s rule, the potential compounds should have the following physicochemical properties [46], such as (i) total polar surface area (TPSA) which should not be >140 Å, (ii) a number of hydrogen bond donors ≤5, number of hydrogen bond acceptors ≤10, (iv) a molecular mass ≤500 Da, and (v) log P not ≥5. The Swiss ADME-computed results (Table 8) showed that compounds 1–3 and 8 in the present study are satisfying Lipinski’s rule of five with zero violations [47]. Except for compound 7, all the studied compounds recorded a lipophilicity (iLogP) value of less than five (range from 1.25 to 4.77), indicating their optimal lipophilicities for good oral and intestinal absorption. Compound 1–3, 4–7, and 8 presented log S values of −4.47 to −4.84, −7.50 to −9.57, and 0.76, respectively. According to Sepay et al. [48], log S values between 0 and −2 indicate very good solubility, −2 to −4 indicate good solubility, −4 and −6 indicate moderate solubility, and less than −6 indicate low solubility. Considering this model, we can conclude that compounds 1, 2, and 3 are moderately water-soluble, and compound 8 is likely to be very good water-soluble and promising for oral administration. The total polar surface area (TPSA) influences the permeability and bioavailability of a molecule. All the TPSA values of compounds (17.07–138.07) are found to be less than the cut-off value (140 Å2), which indicates their excellent absorption in the intestine. Compounds 1–3 and 8 met all the requirements of the physicochemical and ADMET data.

The skin absorption of molecules is indicated by the skin permeability value () in cm/s. The more negative the value of log , the less skin absorption [23]. The skin permeability, , values of all isolated compounds (−1.97 to −9.37 cm/s) infer low skin permeability. In addition to that, absorption and distribution of drug molecules are measured by gastrointestinal (GI) and blood brain barrier (BBB) permeation. The Swiss ADME prediction parameters (Table 9) have shown that compounds 1, 2, and 3 have high gastrointestinal (GI) absorption, and none of the compounds have blood brain barrier (BBB) permeation except for compound 1. Many different drugs’ absorption, distribution, and clearance are affected by the P-GP. Therefore, permeability glycoprotein substrate identification is crucial for the identification and optimization of potential medicines. The findings demonstrate that all isolated compounds were not substrates of permeability glycoprotein (P-gp) except for compounds 1, 3, and 8. When CYP enzymes are inhibited, inhibitory drug metabolism is compromised. Understanding the interaction of compounds with cytochromes P450 enzymes is crucial for the liver’s drug metabolism. The prediction result exhibits that all the compounds are found to be noninhibitors for CYP2C19 and CYP2C9. Compound 1 is a potential inhibitor for CYP1A2, and compounds 2–8 are noninhibitors. For CYP2D6, the compounds 3–8 are noninhibitors, and the other compounds 1-2 are potential inhibitors. For CYP3A4, compounds 1–6 and 8 are noninhibitors, and compound 7 is a potential inhibitor. According to ADME prediction parameters, compounds 3 and 8 met all the requirements for pharmacokinetic properties. The toxicity assessment was evaluated using the OSIRIS property explorer and ProTox-II online server to obtain the values of LD₅₀ and molecular target toxicity probabilities (Table 10).

The result of the predictions represents the probability of each molecule to causing toxicity over specific targets. Acute toxicity prediction results such as LD₅₀ values and toxicity class classification (1 (toxic) to 6 (nontoxic)) reveal that the isolated compounds 1–4 and 6 have shown toxicity class classification 4 and 5, respectively, indicating harmful if swallowed. The results found for the compounds 5 (LD₅₀ = 7000 mg/kg), 7 (LD₅₀ = 9960 mg/kg), and 8 (LD₅₀ = 23000 mg/kg) indicate that the dose administered in this study would not be toxic. The toxicological prediction gives results for endpoints such as hepatotoxicity, carcinogenicity, mutagenicity, immunogenicity, and cytotoxicity. All compounds were predicted to be nonhepatotoxic, noncytotoxic, noncarcinotoxic, and nonirritant. Compounds 2, 4, and 8 were predicted to be nonimmunotoxic, and compounds 3–8 were to be nonmutagenic. However, the compounds 1, 3, 5, 6, and 7 have shown immunotoxicity, and compounds 1 and 2 were found to have mutagenicity.

3.5. Chemotaxonomic Significance

Previous phytochemical studies revealed that species of the Myrica genus are rich in diarylheptanoids, dihydrochalcones, triterpenoids, and flavonoids [49]. In our present study, eight compounds, including three diarylheptanoids derivatives (1, 2, 3), pentacyclic triterpenoid derivatives (4, 5, 6, 7), and one pyranoside derivative (8), were isolated from the stem bark of M. salicifolia. Compound 7 (3β-O-trans-caffeoylisomyricadiol) was identified for the first time, and this is the first report of compounds 4–7 from M. salicifolia. Furthermore, compound 8 is reported here in the Myrica genera. The diarylheptanoids and triterpenoids have been previously isolated from the Myrica genera, including myricanone (1) [50], myricanol (2) [50], and myricanol-11-O-β-D xylopyranoside (3) [51] from M. rubra, taraxerone (4), taraxerol (5), and myricadiol (6) from M. cerifera [52, 53]. Thus, compounds 4–8 from M. salicifolia, suggesting that their occurrence could be used to verify the chemotaxonomic relationship of M. salicifolia and other species of Myrica, might serve as valuable chemotaxonomic makers for the identification of M. salicifolia. Comprehensive phytochemical investigations involving an expanded series of compounds could help define the chemotaxonomic significance of species belonging to the genus Myrica.

4. Conclusions

In this study, a new compound, 3β-O-trans-caffeoylisomyricadiol (7), was isolated from the chloroform-methanol extract of M. salicifolia stem bark for the first time. According to the results of the antioxidant test, compounds 2, 3, and 7 demonstrated high antioxidant activities compared to the others. It is noteworthy that compounds 7 and 8 have not been reported from the Myrica genus and therefore suggest new findings on the chemotaxonomic information of the genus and additional constituents for the chemical diversity of M. salicifolia. The present study also demonstrated that compounds 3 and 7 from the stem bark of M. salicifolia possessed modest antibacterial activity against four strains, and compound 2 showed very strong antioxidant activity based on antioxidant activity parameters. Of all the compounds docked, compound 7 showed better binding affinity, with the active site of PqsA protein of P. aeruginosa and pyruvate kinase (PK) enzyme of S. aureus, with inhibition constant () 0.04 μM and 0.03 μM, respectively. In silico pharmacokinetics studies showed that the isolated compounds 1–3 and 8 satisfy Lipinski’s rule of five with zero violations. As a result, we deduced that the current study would bring a new scientific report on the new isolated compound and other compounds.

Abbreviations

ADMET:	Absorption, distribution, metabolism, excretion, and toxicity
ATCC:	American type culture collection
CLSI:	Clinical and Laboratory Standards Institute
DHB:	2,5-dihydroxybenzoic acid
1D-NMR:	One-dimensional nuclear magnetic resonance spectroscopy
2D-NMR:	Two-dimensional nuclear magnetic resonance spectroscopy
% [DPPH]:	Percent of DPPH concentration; DPPH: 2, 2-diphenyl-1-picrylhydrazyl
ESI-MS:	Electrospray ionization mass spectroscopy
HR-ESI-MS:	High resolution electrospray ionization mass spectroscopy
IC50:	Half maximal inhibitory concentration
TLC:	Thin layer chromatography
TMS:	Tetramethylsilane
UV-Vis:	Ultraviolet-visible.

Data Availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

ADG performed lab work, data analysis, and writing of the draft manuscript and reviewing; KGB conducted NMR measurements, data analysis, and reviewing; TBD conducted docking studies, editing, and reviewing; JOO performed editing and reviewing; EEY and MAD conducted conceptualization and supervision and collected resources. All authors have read and approved the final manuscript.

Acknowledgments

The Departments of Chemistry of Addis Ababa University and the University of Botswana are greatly acknowledged for providing all the necessary research facilities and support. Abraham Dilnesa is grateful to the Ethiopian Forest Development Institute for sponsoring his study.

Supplementary Materials

Mass spectra, ¹H, ¹³C and 2D NMR spectra of compounds 1–8 are available as supplementary materials. (Supplementary Materials)

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Copyright © 2024 Abraham Dilnesa Gashaw et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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