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BY 4.0 license Open Access Published by De Gruyter Open Access November 27, 2023

Antibacterial and biofilm prevention metabolites from Acanthophora spicifera

  • Fitri Budiyanto , Nawal A. Albalawi , Mohamed A. Ghandourah , Tariq R. Sobahi , Magda M. Aly , Hanan F. Althagbi , Samah S. Abuzahrah and Walied M. Alarif EMAIL logo
From the journal Open Chemistry

Abstract

Acanthophora spicifera harbors a diverse array of secondary metabolites with therapeutic potential. The aim of this study is to isolate and characterize secondary metabolites from A. spicifera and then evaluate the antiproliferation, antibacterial, and biofilm prevention properties, followed by an analysis of molecular docking experiments. By employing chromatographic analysis and NMR spectroscopy, the isolated compounds were, the known flavonol, 8-hydroxyquercetagetin (1), three recognized steroids cholest-4-ene-3,6-dione (2), cholest-5-en-3β-ol (3), and 5α-cholestane-3,6-dione (4), and 2-bromohexadecanoic acid (5). These compounds exhibited antimicrobial effects against various Gram-negative and Gram-positive bacteria with inhibition zones ranging from 6.5 ± 0.2 to 17.2 ± 0.12 mm and 7.0 ± 0.4 to 15.3 ± 0.60 mm, respectively. Compounds 1 and 2 inhibited biofilm formation in P. aeruginosa and S. aureus. Compounds 14 demonstrated binding affinity values between −7.5 and −9.4 kcal/mol to protein 1A0G. These binding affinity values were akin to that of amoxicillin, implying that one potential antibacterial mechanism of action of these compounds may involve the inhibition of bacterial cell wall synthesis. All compounds showed no toxicity against Artemia salina and weak activity against Lymphoma and Lewis lung carcinoma cell lines with LD50 > 100 μg/mL.

1 Introduction

The increasing incidence of emerging multidrug-resistant (MDR) bacterial infections has become a significant concern. The primary cause of this issue is the widespread and often inappropriate use of antimicrobial drugs [1]. Among these infections, uropathogenic E. coli and pathogenic ESCAPE bacteria (Enterococcus faecium, S. aureus, Clostridium difficile, Acinetobacter, P. aeruginosa, and Enterobacteriaceae) are prevalent MDR infections in the hospital environment [2]. In 2016, pathogen-related mortality reached approximately 700,000 cases annually, resulting in a staggering societal cost of about US$20 billion each year, with treatment expenses averaging US$50,000 per individual [3]. Addressing Gram-positive and Gram-negative MDR infections presents a dual challenge, with current medications proving inadequate and alternative microbial therapies facing limitations. As of now, there is a lack of safe and effective treatments to combat the global epidemic of MDR bacterial infections [4].

Out of the 22 antibiotics available in the USA before 2022, a significant majority – seventeen – originate from natural products, while the remaining four are the result of synthetic approaches [5]. The count of newly discovered and approved antimicrobial drugs has seen fluctuations. It decreased from 16 approved drugs during 1983–1987 to 7 drugs between 1998–2002. Further, in the period 2018–2022, there were five newly approved drugs; however, they belonged to the same class as existing ones, limiting their efficacy. This trend emphasizes the challenges in developing novel antimicrobial agents with distinct mechanisms of action [5]. Conversely, as of May 2019, a total of 407 projects focused on antibacterial preclinical testing were underway, underscoring the sustained efforts in antibacterial drug development [6]. Notably, marine-derived natural products have emerged as a promising avenue within these preclinical stages. Various categories of marine compounds, such as polyketides, flavonoids, alkaloids, terpenoids, steroids, and phenolic compounds, have demonstrated substantial antibacterial activity against a spectrum of harmful microorganisms [7,8].

Marine red macroalgae offer a broad spectrum of advantages to humans. These algae are frequently harvested for consumption and commercial purposes by specific coastal communities in East and Southeast Asia [9]. Rich in secondary metabolites like steroids, flavonoids, and halogenated compounds, these algae possess diverse pharmacological properties, including antibacterial, anti-inflammatory, and anti-cancer effects [10]. Among red algae, the explored genus Acanthophora stands out and A. spicifera is one of the seven recognized species [11]. Its adaptability allows it to thrive across various climates, from tropical to subtropical regions. However, the study of A. spicifera’s secondary metabolites has been limited to a few compounds like sterols, steroids, and flavonoids [12]. As a result, investigating secondary metabolites within this species holds potential for identifying compounds with potent antibacterial properties [13]. Thus, this research aimed to isolate specific secondary metabolites from A. spicifera, assess their antiproliferation, antibacterial, and biofilm inhibition properties, and molecular docking analysis.

2 Materials and methods

2.1 Chemicals

All chemicals used in this study, i.e., n-hexane, diethyl ether (Et2O), dichloromethane (DCM), ethyl acetate (EtOAc), chloroform (CHCl3), methanol (MeOH), and d-chloroform, were of GC-MS grade and purchased from Sigma-Aldrich, Darmstadt, Germany.

2.2 Alga sample collection

In September 2020, A. spicifera were collected at the end of the dry season from Anyer Bay, Banten, Indonesia. The algae were manually collected from the stony coastline and stored in a dark box (icebox) for sampling. Subsequently, the algae were air-dried at room temperature for a minimum of 1 week to achieve complete dryness. The plant material was identified by Tri Handayani, a seaweed specialist from the Research Center for Oceanography, National Research and Innovation Agency in Jakarta, Indonesia. In cases where extraction was delayed, the dried sample was weighed and stored in a refrigerated environment. The total dry weight of A. spicifera amounted to 198.0 g.

2.3 Extraction and isolation

The dried A. spicifera material was ground to a fine powder and macerated in a mixture of n-hexane, acetone, and methanol (1:2:1 ratio) for 3 days. Filtration using Whatman No. 41 cellulose filter paper was used to separate the organic solution from its solid component. Subsequently, the organic solvent was removed via a vacuum rotary evaporator, resulting in the collection of the oily organic extract (11.7 g) in a flask. For further fractionation, an open chromatographic column containing pre-purified silica oxide was employed. The material was fractionated using organic solvents, with the mobile phase polarity gradually increased by using solvents of ascending polarity such as Et2O, EtOAc, DCM, and MeOH, starting with non-polar n-hexane. Each fraction was obtained using 50 mL of solvent, generating a total of 137 fractions. Identical fractions were pooled for subsequent purification using preparative thin layer chromatography (PTLC) [14]. The process of isolating pure compounds involved combining fractions with similar TLC profiles in PTLC for further purification.

Fractions 28–34, obtained by eluting the chromatographic column with a mixture of n-hexane–Et2O (8:2), exhibited an identical TLC color pattern. Combining these fractions and subsequently repurifying them through PTLC (n-hexane–Et2O, 95:5) resulted in the isolation of compound 4 as a brown band (0.94 mg, Rf = 0.25) from the lower section, and compound 5 as a brown band (0.58 mg, Rf = 0.43) from the upper section. Upon elution using the n-hexane–EtOAc (7:3) mixture, fractions 65–70 were obtained. These fractions were collectively repurified using PTLC (n-hexane–Et2O, 6:4), leading to the isolation of compound 2 as a yellow band (1.79 mg, Rf = 0.49) and compound 3 as a dark red band (1.62 mg, Rf = 0.36). By eluting with a DCM–MeOH (7:3) mixture, fractions 127–131 were obtained, and in the repurification of those fractions using PTLC (DCM–MeOH, 85:15), compound 1 was isolated as a brown band (0.82 mg, Rf = 0.41). The purified samples were completely dried under a vacuum environment using a rotary evaporator. The dried and purified samples underwent characterization using NMR Bruker® (850 Hz) with CDCl3 as the solvent.

2.4 Spectral data

8-Hydroxyquercetagetin (1): Yellow powder; 1H NMR (CDCl3, 850 MHz) δ: 7.37 (1H, bs, 2.5 Hz, H-2′), 7.15 (1H, dd, J = 8.7, 2.5 Hz, H-6′), 7.55 (1H, d, J = 8.7, H-5′). 13C NMR (CDCl3, 850 Hz) δ: 146.4 (C-2), 133.8 (C-3), 176.1 (C-4), 154.1 (C-5), 146.4 (C-6), 147.9 (C-7), 147.8 (C-8), 124.5 (C-9), 104.2 (C-10), 123.9 (C-1′), 116.0 (C-2′), 144.6 (C-3′), 147.1 (C-4′), 116.4 (C-5′), 119.1 (C-6′).

Cholest-4-ene-3,6-dione (2): White needle; 1H NMR (CDCl3, 850 MHz) δ: 1.9 (2H, m, H-1), 2.55 (2H, ddd, J = 2.55, 5.27, and 14.87, H-2), 6.19 (1H, s, H-4), 2.55 (2H, dd, J = 12.41, and 16.06, H-7), 1.94 (1H, m, H-8), 1.39 (1H, m, H-9), 1.90 (2H, m, H-11), 1.68 (1H, ddd, J = 2.6, 5.3, and 7.9, H-12), 1.77 (1H, m, H-14), 1.52 (2H, m, H-15), 1.53 (2H, m, H-16), 1.27 (1H, m, H-17), 0.74 (3H, s, H-18), 1.19 (3H, s, H-19), 1.36 (1H, m, H-20), 0.94 (3H, d, 6.7, H-21), 1.31 (2H, m, H-22), 1.15 (2H, m, H-23), 1.14 (2H, m, H-24), 1.2 (1H, m, H-25), 0.891 (6H, d, 6.6, H-26), 0.895 (3H, d, 6.6, H-27). 13C NMR (CDCl3, 850 Hz) δ: 35.5 (C-1), 34.0 (C-2), 199.6 (C-3), 125.5 (C-4), 161.1 (C-5), 202.4 (C-6), 46.8 (C-7), 34.2 (C-8), 51.0 (C-9), 39.8 (C-10), 20.9 (C-11), 39.1 (C-12), 42.5 (C-13), 56.0 (C-14), 24.0 (C-15), 28.0 (C-16), 56.6 (C-17), 11.9 (C-18), 17.5 (C-19), 35.7 (C-20), 18.6 (C-21), 36.1 (C-22), 23.8 (C-23), 39.5 (C-24), 29.7 (C-25), 22.6 (C-26), and 22.8 (C-27).

Cholest-5-en-3β-ol (3): White needle; 1H NMR (CDCl3, 850 MHz) δ: 1.89 (2H, m, H-1), 2.51 (2H, ddd, J = 2.55, 5,27, 14.87, H-2), 3.55 (1H, ddd, J = 4.5, 9.69, and 18.81, H-3), 1.68 (2H, m, H-4), 5.37 (1H, t, J = 2.5, H-6), 2.09 (2H, dd, J = 12.41, 16.06, H-7), 1.90 (1H, m, H-8), 1.39 (1H, m, H-9), 1.88 (2H, m, H-11), 1.68 (2H, ddd, J = 2.6, 5.3, 7.9, H-12), 1.70 (1H, m, H-14), 1.52 (2H, m, H-15), 1.58 (2H, m, H-16), 1.30 (1H, m, H-17), 0.7 (3H, s, H-18), 1.03 (3H, s, H-19), 1.41 (1H, m, H-20), 0.93 (3H, d, J = 6.5, H-21), 1.36 (2H, m, H-22), 1.20 (2H, m, H-23), 1.14 (2H, m, H-24), 1.1 (1H, m, H-25), 0.88 (6H, d, J = 6.6, H-26 and H-27). 13C NMR (CDCl3, 850 MHz) δ: 38.1 (C-1), 32.6 (C-2), 71.0 (C-3), 43.4 (C-4), 143.2 (C-5), 120.9 (C-6), 32.3 (C-7), 32.3 (C-8), 51.5 (C-9), 37.6 (C-10), 21.7 (C-11), 40.8 (C-12), 43.7 (C-13), 56.8 (C-14), 24.3 (C-15), 29.1 (C-16), 57.1 (C-17), 11.9 (C-18), 19.4 (C-19), 36.2 (C-20), 18.8 (C-21), 35.8 (C-22), 24.8 (C-23), 40.5 (C-24), 28.2 (C-25), 22.5 (C-26), 22.9 (C-27).

5α-Cholestane-3,6-dione (4): White needle; 1H NMR (CDCl3, 850 MHz) δ: 1.82 (2H, m, H-1), 2.27–2.29 (2H, m, H-2), 1.68 (2H, m, H-4), 2.38 (1H, m, H-5), 2.05 (2H, dd, J = 12.41, 16.06, H-7), 1.94 (1H, m, H-8), 1.39 (1H, m, H-9), 1.90 (2H, m, H-11), 1.68 (2H, ddd, J = 2.6, 5.3, 7.9, H-12), 1.77 (1H, m, H-14), 1.52 (2H, m, H-15), 1.59 (2H, m, H-16), 1.27 (1H, m, H-17), 0.74 (3H, s, H-18), 1.02 (3H, s, H-19), 1.36 (1H, m, H-20), 0.89 (3H, d, J = 6.5, H-21), 1.31 (2H, m, H-22), 1.15 (2H, m, H-23), 1.14 (2H, m, H-24), 1.2 (1H, m, H-25), 0.86 (3H, d, J = 6.0, H-26), 0.87 (3H, d, J = 6.0, H-27). 13C NMR (CDCl3, 850 MHz) δ: 38.2 (C-1), 37.2 (C-2), 209.9 (C-3), 36.4 (C-4), 58.0 (C-5), 208.6 (C-6), 46.3 (C-7), 38.2 (C-8), 53.1 (C-9), 41.1 (C-10), 21.9 (C-11), 39.8 (C-12), 43.1 (C-13), 56.8 (C-14), 25.1 (C-15), 28.2 (C-16), 57.7 (C-17), 12.8 (C-18), 12.9 (C-19), 36.9 (C-20), 18.8 (C-21), 37.2 (C-22), 24.9 (C-23), 40.0 (C-24), 28.1 (C-25), 22.3 (C-26), 22.9 (C-27).

2-Bromohexadecanoic acid (5): Light yellow oil. 1H NMR (CDCl3, 850 MHz) δ: 4.11 (1H, m, H-2), 2.07 (2H, m, H-3), 1.2–1.36 (22H, brs, H-4 to H-14), 1.55 (2H, m, H-15), 0.89 (3H, t, J = 6.6, H-16). 13C NMR (CDCl3, 850 MHz) δ: 171.3 (C-1), 63.1 (C-2), 32.8 (C-3), 27.9 (C-4), 29.4 (C-5), 29.7 (C-6 to C-13), 31.9 (C-14), 22.7 (C-15), and 14.1 (C-16).

2.5 Biological assays

2.5.1 Cell culture

The two culture cell lines, Lymphoma and Lewis carcinoma, were obtained from the American Type Culture Collection and cultured as previously shown [15].

2.5.2 Antibacterial assay

The antimicrobial activities of the isolated compounds 15 from A. spicifera were detected against some Gram-positive and Gram-negative bacteria, obtained from King Fahd General Hospital, Jeddah, Saudi Arabia using the agar well diffusion method of Holder and Boyce [16].

2.5.3 Biofilm assay

All collected bacterial isolates were screened and assayed for biofilm formation using the Crystal Violet Staining method described by Christensen et al. and the ability to form biofilm was measured [17]. Broth cultures were incubated in 96-well microplates under standard conditions and in the presence of the tested compound. The biofilm biomass was assayed as described before and the percentage of inhibition was estimated [18].

2.5.4 Cell viability assay

Cell viability was assayed as previously reported [19].

2.6 Molecular docking experiment

2.6.1 Protein target identification and selection

To identify potential protein targets for each isolated compound, three established pharmacophore matching web servers were employed: PharmMapper (http://www.lilab-ecust.cn/pharmmapper/), SwissTargetPrediction (http://www.swisstargetprediction.ch/index.php), and Super-PRED (https://prediction.charite.de/index.php). Through these platforms, a compilation of more than 300 proteins exhibiting bioactivity with our isolated compounds was gathered. Subsequently, these macromolecules were subjected to evaluation using the STRING web server (https://string-db.org/) provided by the Swiss Institute of Bioinformatics [20]. This analysis aimed to discern the most robust connections among the collected proteins and the isolated compounds. Utilizing the network analysis generated by the STRING web server, a total of six proteins were chosen for the docking model. These selected proteins fulfill specific parameters, making them suitable candidates for molecular docking analysis. Those selected proteins belonged to D-amino acid aminotransferase (PDB ID: 1A0G), tyrosyl-tRNA synthetase (PDB ID: 3G7B, 1JIJ, and 1P7P), tyrosinase (PDB ID: 4D87), and geranyl diphosphate C-methyltransferase (PDB ID: 3VC1) (Table 1).

Table 1

Target proteins from some selected bacteria with suitable properties for docking experiments of the isolated compounds

PDB ID Protein structure Resolution (Å) R-value free R-value work Structure weight (kDa)
1A0G Bacillus sp. D-amino acid aminotransferase 2.00 0.263 0.216 65.04
3G7B S. aureus Gyrase B co-complex with its inhibitor 2.30 0.275 0.209 43.08
1JIJ S. aureus TyrRS in complex with SB-239629 3.20 0.305 0.257 48.07
1P7P Methionyl-tRNA synthetase from Escherichia coli complexed with methionine phosphonate 1.80 0.225 0.195 63.21
4D87 Tyrosinase from Bacillus megaterium in complex with SDS 3.50 0.293 0.259 70.96
3VC1 Geranyl diphosphate C-methyltransferase from Streptomyces coelicolor A3 (2) 1.82 0.194 0.167 430.83

2.6.2 Docking experiment

To conduct docking experiments, the 3D structures of the proteins were sourced from the RCSB Protein Data Bank (PDB) (https://www.rcsb.org), while the 3D conformers of the isolated compounds were acquired from the PubChem Data Bank (https://pubchem.ncbi.nlm.nih.gov). These proteins underwent preparation and optimization before the docking experiments using AutoDock4 and AutoDock1.5.7 [21]. This preparation encompassed the removal of water molecules, the addition of polar hydrogen, and the incorporation of Kollman charges. The isolated compounds were also optimized as ligands using the same software. Subsequently, the docking experiments were conducted using the aforementioned software. To validate the results of the docking experiments, Chimera® version 1.17.1 was employed [22]. The docking outcome showcasing the highest binding affinity between each isolated compound and the respective proteins was then visualized using the Discovery Studio Visualizer client, BIOVIA.

2.6.3 Docking validation

To ensure the reliability of the docking procedure, a re-docking experiment involving the native ligand was executed. The native ligand was extracted from the protein through the utilization of Chimera® version 1.17.1 [22]. Prior to conducting the re-docking experiment, the proteins underwent optimization. This involved the elimination of water molecules, the addition of polar hydrogen, and the incorporation of Kollman charges, all performed using AutoDock4 and AutoDock1.5.7 [21]. The extracted native ligands were also optimized using the software. Subsequently, the re-docking experiment was conducted for both the proteins and their respective native ligands. The outcomes of the re-docking experiments were superimposed onto the docked native ligands. The validity of the docking result was established when the root mean square deviation (RMSD) value was less than 2 Å. The RMSD values of the re-docking experiments were as follows: 1A0G (0.749 Å), 1JIJ (1.969 Å), 3G7B (1.607 Å), 3VC1 (1.73 Å), 4D87 (1.82 Å), and 1P7P (1.792 Å).

3 Results and discussions

3.1 Metabolites identification

Fractionation of the organic extract of the red alga A. spicifera using different planar chromatographic techniques successfully resulted in isolation of a flavonol compound, 8-hydroxyquercetagetin (1), three cholestane derivatives, cholest-4-ene-3,6-dione (2), cholest-5-en-3β-ol (3), and 5α-cholestane-3,6-dione (4), along with the halogenated fatty acid, 2-bromohexadecanoic acid (5). This is the first report of isolation of 8-hydroxyquercetagetin (1) from marine organisms. Compound 1 was first reported from the terrestrial plant, petals of Tagetes erecta [24] (Figure 1).

Figure 1 
                  The molecular structures of compounds 1–5 isolated from A. spicifera.
Figure 1

The molecular structures of compounds 1–5 isolated from A. spicifera.

Compound 1 was isolated as a yellow powder substance. Compound 1 showed to be active under short UV lamp in TLC which referred to the presence of a conjugated system-containing structure. The 1H NMR spectrum indicated the presence of three aromatic proton signals resonating at δ H 7.55 (d, 8.7 Hz), 7.37 (brs, 2.5 Hz), and 7.15 (dd, 8.7, 2.5 Hz) ppm. In addition, several other signals were observed as broad ones in the range of 7–13 ppm, these signals were assigned mainly to hydroxyl functions. The 13C NMR spectrum of compound 1 indicated the presence of 15 carbon signals, all of which fall in the range from δ C 104.2 to 176.1 ppm. DEPT spectrum indicated the presence of 3 methine carbons resonating at δ C 116.0, 116.4, and 119.1 and 12 quaternary carbons including a conjugated carbonyl carbon resonating at 176.1, 9 oxygenated carbons resonating at δ C 124.5, 133.8, 144.6, 146.4, 146.4, 147.1, 147.8, 147.9, and 154.1 ppm. The obtained spectral data strongly suggested that compound 1 belongs to flavonoids and more specifically to flavonols owing to the absence of absorption due to H-3 in the 1H NMR spectrum. Literature survey showed that the obtained data are identical to that of 8-hydroxyquercetagetin described by Alvarado-Sansininea [23]. Based on these findings, compound 1 was identified as 8-hydroxyquercetagetin (Figure 1).

Compound 2 was isolated as white needles material. It gave a positive Liebermann–Burchard test of steroids. 1H NMR spectrum showed the presence of five methyl resonances, a characteristic feature unique to the cholestane scaffold. This methyl in compound 2 appeared as singlets at H-18 (δ H: 0.74 ppm) and H-19 (δ H: 1.19 ppm); and doublet at H-21 (δ H: 0.94 ppm), H-26 (δ H: 0.891 ppm), and H-27 (δ H: 0.895 ppm). An olefinic proton occurred as a singlet at δ H 6.19 ppm, which might be a part of α,β-unsaturated carbonyl moiety. 13C NMR indicated the presence of 27 carbon signals which were categorized by DEPT experiment into five methyl, ten methylene, seven methine, and five quaternary carbons. The quaternary carbons were allocated to one olefinic (δ C 161.1 ppm), two carbonyls (δ C 202.4, 199.6 ppm), and two carbon signals at the up-field region (δ C 42.5 and 39.8 ppm). The methine carbons were allocated to one olefinic (δ C 125.5 ppm) and six in the upfield region. Meanwhile, two carbonyl groups appeared at δ C: 199.6 ppm (C-3) and δ C: 202.4 ppm (C-6). Thus, by comparing the spectra of the previous report, compound 2 was identified as cholest-4-ene-3,6-dione [25].

Compound 3 was isolated as white needle material. It gave a positive Liebermann–Burchard test of steroids. The 1H NMR spectra of compound 3 were similar to those of compound 2. 1H NMR spectra showed the presence of methyl signal patterns resembling cholestane structure. Five methyls appeared as singlets at H-18 (δ H: 0.70 ppm) and H-19 (δ H: 1.03 ppm); and doublet at H-21 (δ H: 0.93 ppm), H-26 (δ H: 0.88 ppm), and H-27 (δ H: 0.88 ppm). An olefinic proton appeared at δ H 5.37 ppm and an oxygenated methine proton appeared at δ H 3.55 ppm. 13C NMR indicated the presence of 5 methyl, 11 methylene, 8 methine, and 3 quaternary carbons. The quaternary carbons were allocated to one olefinic (δ C 143.2 ppm), and two at the upfield region (δ C 43.7 and 37.6 ppm). One methine carbon at δ C 71.0 ppm was attached to a hydroxyl group, and one olefinic methine proton appeared at δ C 120.9 ppm, while the other five proton signals appeared at the upfield region. The spectra of compound 3 were identical to the report [25]; thus, compound 3 was determined as cholest-5en-3β-ol.

Compound 4 was isolated as white needles material. It gave a positive Liebermann–Burchard test of steroids. Compound 4 possessed a cholestane structure based on the observation of five distinct methyl resonances, a distinctive characteristic specific to the cholestane scaffold. This methyl in compound 4 appeared as singlet at H-18 (δ H: 0.74 ppm) and H-19 (δ H: 1.02 ppm); and doublet at H-21 (δ H: 0.89 ppm), H-26 (δ H: 0.86 ppm), and H-27 (δ H: 0.87 ppm). 13C NMR indicated the presence of 5 methyl, 11 methylene, 7 methine, and 4 quaternary carbons. Two oxygenated carbonyls appeared at δ C 209.9, and 208.6 ppm, and two quaternary carbon signals occurred at the up-field region (δ C 41.1 and 43.1 ppm). Thus, by comparing the spectra of the previous report, compound 4 was determined as 5α-cholestane-3,6-dione [25].

Compound 5 was isolated as a light-yellow oily substance. The 1H-NMR spectrum indicated the presence of a triplet methyl signal at δ H 0.89 ppm (t, 6.6 Hz), a methine proton resonating at 4.11 ppm, and a huge number of proton signals appeared at δ H 1.2–1.36 ppm, at δ H 1.55, and at 2.07 ppm; 13C NMR spectrum of compound 5 indicated the presence of 16 carbon signals, classified by DEPT experiment into one quaternary carbon at δ C 171.3 ppm, a halogenated methine at δ C 63.12, a methyl carbon at δ C 14.1, and another 14 carbons for methylene at 29.7 ppm. The in-hand spectral data referred undoubtedly to the presence of a halogenated long chain fatty acid. The identity of compound 5 was obtained by comparing with the literature data, compound 5 was identified as 2-bromohexadecanoic acid [26].

3.2 Biological activity

Table 2 represents the antibacterial activity (diameter of inhibition zone, mm) of the isolated compounds (1–5) against various Gram-negative and Gram-positive bacteria to assess their antibacterial properties (Table 2). All compounds demonstrated the ability to hinder the growth of both types of bacteria, leading to inhibition zone diameters ranging from 6.5 ± 0.2 to 17.2 ± 0.12 mm for Gram-negative bacteria and 7.0 ± 0.4 to 15.3 ± 0.60 mm for Gram-positive bacteria (Table 2). Notably, compound 1 displayed the broadest inhibition zone, followed by compound 2, across both categories of bacteria. Compounds 3 and 4 exhibited similar inhibition zones, whereas compound 5 displayed the narrowest zones. Considering that compounds 1 and 2 displayed the broadest inhibition zones, their bioactivities were subjected to further testing to evaluate their potential for inhibiting biofilm formation against P. aeruginosa and S. aureus. Among the two compounds, compound 1 emerged as the superior candidate in terms of inhibition activity. This was evident from the decreased viability of both P. aeruginosa and S. aureus after exposure to compound 1, resulting in biofilm formation reductions of 65% and 64%, respectively (Table 3). Regrettably, neither compounds 1 nor 2 exhibited cytotoxic properties against Lymphoma and Lewis lung cancer cell lines and showed to be nontoxic to Artemia salina (Table 4).

Table 2

Antibacterial activity (diameter of inhibition zone, mm) of the tested fractions compared to Amoxicillin (control antibiotic) on some MDR bacteria using agar well diffusion method

Bacterium Antimicrobial activity
1 2 3 4 5 #control
Gram-negative bacteria
E. coli 17.2 ± 0.12* 15.2 ± 0.13 11.5 ± 0.3 12.0 ± 0.15 7.2 ± 0.4 21 + 0.49
K. pneumoniae 16.2 ± 0.22* 14.0 ± 0.29 11.3 ± 1.1 11.5 ± 0.25 6.5 ± 0.2 21 + 0.29
P. mirabilis 16.3 ± 0.12* 15.3 ± 1.33 11.4 ± 1.1 11.3 ± 0.20 7.7 ± 0.5 19 + 0.98
P. aeruginosa 15.0 ± 1.10* 13.5 ± 2.30 11.3 ± 0.9 11.3 ± 0.01 7.3 ± 0.5 17 + 0.89
Gram-positive bacteria
E. faecalis 14.3 ± 0.45* 10.3 ± 0.49 11.9 ± 1.1 10.2 ± 0.19 9.5 ± 1.0 27 + 0.99
MRSA 14.1 ± 0.49* 10.1 ± 0.66 11.2 ± 1.0 10.1 ± 0.10 9.7 ± 0.0 17 + 0.77
S. aureus 15.3 ± 0.60* 10.3 ± 0.29 11.0 ± 1. 0 10.1 ± 0.12 9.5 ± 0.1 29 + 0.33
Streptococcus pyogenes 12.3 ± 0.12* 10.3 ± 0.11 11.0 ± 1.2 10.2 ± 0.22 7.0 ± 0.4 25 + 0.39

MRSA: methicillin-resistant Staphylococcus aureus, #: Amoxicillin (10 µg/mL), *: significant results compared to control.

Table 3

Effect of compounds 1 and 2 on the biofilm formation by P. aeruginosa and S. aureus detected by percentage of cell viability compared to control (untreated, 100 % viability)

Tested bacterium Compound no.
1 2
P. aeruginosa 65 80
S. aureus 64 70

Control: distilled water.

Table 4

Toxicity and antitumor activity of compounds 1 and 2 compared to bleomycin

Compound Toxicity against Artemia salina (LD50, μg/mL) Antitumor activity (LD50, μg/mL)
Lymphoma Lewis lung carcinoma
1 nd nd nd
2 nd nd nd
Bleomycin (positive control) ≥002 0.02 ± 0.001 nd

nd = not detected (LD50 > 100 μg/mL).

Compound 1 was categorized as a flavonoid with hydroxyl substitutions at positions C3, C5, C6, C7, C8, C3′, and C4′. This specific pattern of substitution is likely instrumental in contributing to the compound’s antibacterial activity. The arrangement of substitutions profoundly impacts the antibacterial potential of flavonoids. The complete hydroxylation of ring A in compound 1 renders the compound highly polar, facilitating intermolecular interactions such as hydrogen bonding. This elevated polarity was substantiated through the isolation of compound 1 from a polar solvent (DCM–MeOH, 7:3). The heightened polarity of compound 1 amplifies its attraction to polar or hydrophilic segments within bacterial cell membranes and proteins. Consequently, this heightened affinity has the potential to augment compound 1’s capacity to perturb bacterial structures. Meanwhile, substitutions like prenylation and geranylation at C6, along with hydroxylation at C5, C7, C3', and C4', tend to enhance the antibacterial efficacy. Conversely, methoxylation at C5 and C3' is known to reduce this activity [26].

Flavonoids emerge as a prominent class for their antibacterial properties. Historically, folk medicine has long recognized the efficacy of Tagetes minuta in Argentina and Tripleurospermum disciforme in Iran for their anti-infectious attributes. T. minuta, for instance, harbors the flavonol quercetagetin-7-arabinzylgalactoside, contributing to its antibacterial potency. Flavonols, present in various plants, are synthesized as protective agents against both abiotic and biotic stressors, including extreme environmental conditions and predatory activities. Flavonoids operate through diverse antibacterial mechanisms, including membrane disruption, inhibition of cell envelope synthesis, inhibition of nucleic acid synthesis, and restraint of bacterial toxins [27]. An illustrative example is the flavonoid glycoside sourced from Graptophyllum grandulosum, which exerts antibacterial properties by triggering cytoplasmic membrane disruption and inducing cell lysis mechanisms [28]. However, flavonols featuring hydroxyl substitutions only exhibit comparably weaker antibacterial activities when measured against conventional antibiotics. For instance, the conventional antibiotic gentamicin demonstrates a minimum inhibitory concentration (MIC) value of 0.2–0.8 μg/mL against Escherichia coli and Staphylococcus aureus, while kaempferol exhibits MIC values of 16–63 μg/mL [29].

Compounds 2–4 are classified within the steroid class, differing primarily in their carbonyl and double bond patterns. Compound 2 features two carbonyl groups (at C3 and C6) and a single double bond (C4–C5). Compound 3, on the other hand, possesses one hydroxyl group (at C3) and a double bond (C5–C6), while compound 4 exhibits two carbonyl groups (at C3 and C6) exclusively. Notably, compound 2 displayed superior antibacterial activity when compared to compounds 3 and 4. This suggests that the combination of carbonyl and double bond functionalities contributes to heightened antibacterial properties. Moreover, an increase in the number of double bonds elevates the lipophilic nature of steroids, influencing their capacity to interact with bacterial cell membranes. Simultaneously, the carbonyl group may participate in diverse chemical reactions, including covalent binding to bacterial proteins. In terms of existing synthetic steroids used as antibacterial agents, cationic steroids can elicit both lethal and non-lethal effects [30]. The antibacterial action of cationic steroids is achieved by triggering alterations in bacterial morphology, inducing genetic stress, or causing depolarization of bacterial membranes [31]. Notably, the steroid compounds and derivatives derived from natural sources demonstrate favorable compatibility with normal cells. Natural steroids possess antibacterial potential, often in a dose-dependent manner [32], against various pathogenic bacteria such as Enterobacter spp., E. coli, K. pneumoniae, P. aeruginosa, Acinetobacter baumannii, Enterococcus spp., and S. aureus. These steroids have the capacity to diminish the expression of virulence factors in S. aureus, including capsule biosynthesis protein C (capC), cell surface factors (fnbB), staphylococcal enterotoxin A (sea), leucotoxins E–D (LukE–D), alpha-hemolysin (hla), and beta-hemolysin (hlb) [33].

Compound 5 is categorized as a halogenated fatty acid. The inclusion of bromine (Br) within the compound has the potential to elevate its lipophilicity, thereby impacting both its solubility and its interactions with lipid-based structures within bacteria. Nevertheless, despite these factors, compound 5 exhibited lower antibacterial activity in comparison to the other compounds examined in this study. This disparity may be attributed to its comparatively simpler molecular structure in contrast to the others, even though these halogenated compounds obtained from red algae have exhibited antibacterial properties. Notably, iso-obtusol and elatol, extracted from Laurencia majuscula in Malaysia, demonstrated potent antibacterial activity against Salmonella species, K. pneumoniae, and S. epidermidis [34]. The hexane extract of red alga from Malaysia displayed potent antibacterial effects against P. aeruginosa. Notably, the extract is rich in n-hexadecanoic acid and oleic acid, both contributing to its antibacterial activity [35].

3.3 Molecular docking

Amoxicillin, the standard drug in this study, displayed binding affinity ranging from −6 kcal/mol (3CV1) to −8.6 kcal/mol (1P7P and 1JIJ). Compound 1 exhibited the highest binding affinity towards 1JIJ, recording −10.5 kcal/mol. Compounds 2–4 showcased their strongest binding affinity towards 1P7P, with values between −8.9 and −9.5 kcal/mol. In contrast, compound 5 demonstrated the most substantial binding affinity towards 3VC1, with a value of −6.7 kcal/mol (Figure 2).

Figure 2 
                  The binding affinity of isolated compounds 1–5 from A. spicifera to the selected proteins.
Figure 2

The binding affinity of isolated compounds 1–5 from A. spicifera to the selected proteins.

As per Aranda and Rivas [36], the mechanism of action for amoxicillin involves inhibiting cell wall synthesis. In the molecular docking experiment, amoxicillin displayed a robust binding affinity to protein 1A0G. This observation substantiates the notion that amoxicillin interferes with cell wall synthesis. Protein 1A0G is involved in the metabolism of D-amino acids, particularly D-glutamate and D-alanine, crucial for constructing the peptidoglycan layer of the bacterial cell wall [37].

Compound 1 demonstrated the highest binding affinity towards 1JIJ, establishing five hydrogen bonds with the protein linked to ASP40 (2.26 Å), ASP177 (2.53 Å), THR75 (2.02 Å, 2.40 Å), and LYS84 (2.29 Å). Additionally, one electrostatic interaction, specifically a Pi-anion interaction, was noted with ASP40 (4.77 Å). Furthermore, a Pi-Alkyl interaction (hydrophobic interaction) occurred with LEU70 (5.37 Å). For compound 2, the most robust binding affinity was observed with 1P7P, featuring three hydrogen bonds and seven hydrophobic interactions. The hydrogen bonds were established between oxygen atoms of compound 2 and HIS21 (2.79 Å), VAL326 (2.48 Å), and GLY23 (3.69 Å). Hydrophobic interactions arose from the carbon atoms of compound 2, manifesting as alkyl interactions with ALA12 (3.83 Å) and ILE297 (4.43 Å, 5.06 Å), as well as Pi-alkyl interactions with TYR15 (3.57 Å), TRP253 (4.87 Å, 5.04 Å), and TYR325 (5.30 Å).

Similarly, compound 3 displayed a strong binding affinity to 1P7P, forming two hydrogen bonds and ten hydrophobic interactions. The hydrogen bonds were between oxygen atoms of compound 3 and GLY324 and TYR325. Hydrophobic interactions were categorized as Pi-Sigma (C-29 to ILE297), alkyl (ring center to ILE297 and ALA12), and Pi-alkyl interactions (C-25 to TYR15 and TRP253; C-17 to HIS21, C-22 to TRP253). Compound 4 also exhibited a potent binding affinity to 1P7P, featuring three hydrogen bonds and seven hydrophobic interactions. The hydrogen bonds occurred between oxygen atoms of compound 4 and HIS21 (2.93 Å), VAL326 (2.51 Å), and GLY23 (3.75 Å). Simultaneously, hydrophobic interactions were observed with ILE297, ALA12, TYR15, TRP253, and TYR325, maintaining distances between 3.55 and 5.33 Å.

In the case of compound 5, the most substantial binding affinity was towards 3VC1, characterized by three hydrogen bonds and ten hydrophobic interactions. Two hydrogen bonds originated from oxygen atoms of compound 5 to LEU128 (2.63 Å) and TRP29 (3.54 Å), and another hydrogen bond was established from Br to SER174 (3.79 Å). Ten hydrophobic interactions emerged from carbon atoms of compound 5 to MET176, ILE218, CYS224, TRP29, TYR177, and PHE222, with distances ranging from 4.37 to 5.47 Å. Additionally, an unfavorable acceptor–acceptor interaction occurred from oxygen atoms of compound 5 to GLN132 (Table 5).

Table 5

Visualization of the docking experiment of the isolated compounds 1–5 with the selected protein having the highest binding affinity

3D model 2D interaction Amino acid residue
Compound 1 docking on 1JIJ
Active residue:
HB = ASP40 (2.26 Å), ASP177 (2.53 Å), THR75 (2.02; 2.40 Å), LYS84 (2.29 Å)
HI = LEU70 (5.37 Å)
EI = ASP40 (4.77 Å)
Compound 2 docking on 1P7P
Active residue:
HB = HIS21 (2.79 Å), VAL326 (2.48 Å), GLY23 (3.69 Å)
HI = ALA12 (3.82 Å), ILE297 (4.43; 5.06 Å), TYR 15 (3.57 Å), TRP253 (4.87; 5.04 Å), TYR325 (5.30 Å)
EI = –
Compound 3 docking on 1P7P
Active residue:
HB = GLY3243 (2.52 Å), TYR325
HI = TYR15 (3.98; 4.01 Å), ILE297 (5.08; 4.34 Å), ALA12 (4.99; 5.41 Å), HIS21 (5.04 Å), TRP 253 (4.87; 5.14; 5.00 Å)
EI = –
Compound 4 docking on 1P7P
Active residue:
HB = HIS21 (2.93 Å), VAL326 (2.52 Å), GLY23 (3.75 Å)
HI = ILE297 (5.13; 4.41 Å), ALA12 (3.91 Å)
TYR15 (3.55 Å), TRP253 (5.10; 4.85 Å), TYR325 (5.33 Å)
EI = –
Compound 5 docking on 3VC1
Active residue:
HB = LEU128 (2.63), TRP29 (3.54), SER174 (3.79)
HI = MET176 (4.95; 4.36; 5.16 Å), ILE218 (5.46 Å), CYS224 (4.81 Å), TRP29 (5.20; 4.37 Å), TYR177 (5.47 Å), PHE222 (4.71; 4.95 Å)
EI = –

HB: hydrogen bond.

EI: Electrostatic interaction.

HI: hydrophobic interaction.

Interestingly, the binding affinities of flavonol compound 1 and steroid compounds 2–4 to protein 1A0G closely paralleled the binding affinity of amoxicillin to the same protein. This alignment strongly suggested that flavonol compound 1 and steroid compounds 2–4 may share a common antibacterial mechanism, achieved by inhibiting bacterial cell wall production. To further elucidate the potential antibacterial mechanisms of action of the compounds, additional protein targets were included in the docking experiment. Compounds 1–4 exhibited significant binding activity towards 1JIJ and 1P7P, providing valuable insights into their efficacy against different protein targets. Proteins 1JIJ and 1P7P belong to the aminoacyl-tRNA synthetase group, responsible for overseeing the synthesis of charged tRNAs [38,39]. These proteins are integral to the translation process by activating amino acids [39]. Hence, it is plausible that these compounds may possess inhibitory properties affecting protein synthesis. Protein 3G7B is DNA gyrase, which holds significant roles in DNA transcription, recombination, and replication processes [40]. Protein 4D87 serves as a tyrosinase, contributing to melanin production [41]. Finally, protein 3VC1 functions as a methyltransferase, modifying acyclic isoprenoid diphosphate [42]. Indeed, the investigation into the mechanism of action concerning the antibacterial activity of compounds 1–5 necessitates in-depth examination through laboratory experiments.

4 Conclusion

The red alga, A. spicifera, is a warehouse of different metabolites originating from a variety of biosynthetic pathways. Five secondary metabolites were isolated, identified as 8-hydroxyquecertagetin (1), cholest-4-ene-3,6-dione (2), cholest-5-en-3β-ol (3), and 5α-cholestane-3,6-dione (4), and 2-bromohexadecanoic acid (5). Amongst the isolated compounds, the flavonol (1) displayed remarkable antibacterial activities against a set of Gram-negative bacteria, E. coli, K. pneumonia, P. mirabilis, and P. aeruginosa (inhibition zone: 15.0 ± 1.10–17.2 ± 0.12 mm), and also against a number of Gram-positive bacteria including E. faecalis, MRSA, S. aureus, and S. pyogenes (inhibition zone: 12.3 ± 0.4–15.3 ± 0.60 mm). Whereas, the other coisolated compounds showed weak to moderate antibacterial activities. Moreover, compound 1 showed to inhibit biofilm formation of both P. aeruginosa and S. aureus, with cell viability 65 and 64%, respectively.

Acknowledgements

The authors extend their sincere gratitude to the Department of Marine Chemistry and the Department of Chemistry, King Abdulaziz University, Saudi Arabia for generously providing the necessary facilities for this study. Special appreciation is expressed to Ms. Tri Handayani (Research Center for Oceanography, National and Innovation Agency, Indonesia) for her invaluable assistance in specimen identification.

  1. Funding information: This research received no funding.

  2. Author contributions: All authors contributed equally as main contributors to this manuscript. W.M.A. and F.B.: conceptualization. F.B., N.A.A., M.A.A., H.F.A., and S.S.A.: investigation, data curation, visualization, and writing – original draft. F.B., M.A.A., M.A.G., T.R.S., and W.M.A.: supervision, visualization, and writing – review and editing.

  3. Conflict of interest: The authors declare no competing financial interest.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2023-09-01
Revised: 2023-11-07
Accepted: 2023-11-09
Published Online: 2023-11-27

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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