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Abstract

Objective: This study aimed to investigate the phytochemical and mineral compositions, antimicrobial activities, and molecular docking of 3 abundant and understudied seaweeds (Cystoseira trinodis, Padina boryana, and Turbinaria triquetra). Methods: Extraction was performed using absolute ethanol, and chemical and mineral compositions were determined using the standard methods of the Association of Official Analytical Chemists. Total phenolics and flavonoids were assessed using the Folin-Ciocalteu and aluminum chloride methods, respectively. Chemical composition was screened using gas chromatography-mass spectrometry (GC-MS), and antimicrobial activity was evaluated using a disc diffusion assay. In addition, molecular docking analysis of the main compounds was performed. Results: The samples had high ash, carbohydrate, and fiber contents. The results showed high amount of Na, K, Mg, Ca, and Fe, with the highest values for Ca. Trace elements such as Cu, Mn, and Zn were found in low amounts in the studied species. The content of total phenolics ranged from 15.54 ± 0.1 to 39.5 ± 0.2 mg gallic acid equivalent/g. For flavonoid, it ranged from 3.4 ± 0.3 to 1.5 ± 0.2 mg rutin/g. GC-MS revealed 9 major compounds, mainly fatty acids, which have been reported to have antimicrobial activity. The antimicrobial results indicated a higher impact of the extracts against bacteria (Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Proteus vulgaris) than against fungi (Candida albicans and Aspergillus fumigatus), and a stronger dose-dependent effect against gram-positive (B. subtilis and S. aureus) bacteria than against gram-negative bacteria (E. coli and P. vulgaris). Molecular docking identified 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester as the highest binding compound for penicillin-binding protein 6, suggesting its potential antimicrobial activity. Conclusion: These findings suggest that these seaweed species could serve as promising sources of functional foods with antimicrobial properties, with potential applications in the nutraceutical and pharmaceutical fields.

Keywords

total phenolics total flavonoids fatty acids antimicrobial activity molecular docking

Introduction

Marine macroalgae, commonly known as seaweed, are a large group of autotrophs that contain diverse bioactive compounds with many biological activities. For decades, seaweeds have been used as food and medicine in many cultures, particularly in East Asia. They are considered healthy foods because of their low lipid content, high mineral and vitamin contents, and richness in polysaccharides and unsaturated fatty acids. In addition, they contain high dietary fibers compared to terrestrial plants, which can reach 60% of their dry weight (d.wt.).¹ Seaweeds acquire minerals and elements from their surrounding marine environments. The mineral content in seaweeds may reach 40% of the d.wt., making them a great source of minerals compared to terrestrial plants.² The most abundant essential elements are sodium, potassium, calcium, magnesium, chloride, sulfur, phosphorus, while iron, copper, zinc, manganese, cobalt, and nickel are the most abundant trace elements.³ In addition to their nutritional composition, seaweeds are rich sources of bioactive compounds with high biological potential.⁴ Primary metabolites, such as pigments, proteins, fibers, and lipids, have been reported to have positive effects on several conditions such as diabetes, obesity, cancer, and cardiovascular diseases.^1,2

Seaweeds produce large quantities of secondary metabolites with multiple functions.⁵ This occurs as a consequence of exposure to many environmental stresses such as changes in light, salinity, temperature, water current, predation, and environmental pollution.⁶ Phenolic compounds, alkaloids, terpenes, fatty acids, and sterols are considered the most important seaweed secondary metabolites that possess various pharmaceutical properties, including antioxidant, anticancer, anti-inflammatory, antimicrobial, and antiviral activities.⁷ The chemical composition of seaweeds can vary according to the species, season, and geographical and physiological variations.⁵

Brown algae are a valuable source of nutraceutical and pharmaceutical compounds. Cystoseira trinodis, Padina boryana, and Turbinaria triquetra are 3 brown seaweeds that are used in food and folk medicine in many cultures.^8–10 The species have been studied for their bioactive compounds and bioactivities. Phlorotannin have been isolated from C. trinodis.¹¹ The structure characteristics of fucoidan have been studied in P. boryana.¹² Several bioactive compounds have been identified in Turbinaria sp, including fatty acids, sterols, and phenolic compounds.¹² The studied species have been detected in number of bioactivities, including antimicrobial, antioxidant, anti-inflammatory, and anticancer.^8–12

Currently, the world is facing the problem of microbial resistance to several antibiotics, which raises the public health priority of exploring and developing new antibiotics from natural compounds. These compounds are thought to be more effective, less toxic, cheaper, have fewer side effects, and higher bioavailability.¹³ Numerous publications from diverse parts of the world have evaluated the antimicrobial property of seaweed extracts against a broad spectrum of microorganisms, and have indicated that seaweed is a promising source of natural compounds with antimicrobial activity.^14,15

The Egyptian Red Sea is composed of diverse seaweed communities, with abundant brown seaweeds.¹⁶ However, few studies have investigated their potential applications and chemical composition. Currently, research in Egypt is focused on exploring seaweed composition and applications, particularly their antimicrobial activity.^15,17 Marsa Allam is an Egyptian town on the western coast of the Red Sea, with many virgin shores filled with scarcely investigated seaweed. C. trinodis, P. boryana, and T. triquetra are considered the most abundant brown seaweed species on the coast of Marsa Allam, with few publications concerning their composition and antimicrobial activity. Thus, this study was conducted to evaluate the proximate chemical and mineral compositions, analyze the important bioactive compounds, highlight the possible antimicrobial activity of the species, and investigate the molecular docking of the main compounds in the extracts of C. trinodis, P. boryana, and T. triquetra collected from Marsa Allam, Red Sea. The aim of the current work is to suggest the studied species for nutraceutical and pharmaceutical applications.

Results and Discussion

Proximate Chemical Composition

The approximate chemical composition of the 3 algal extracts is listed in Table 1. The results showed high values for ash, carbohydrates, and fibers; moderate values for moisture and protein; and low values for lipids. The moisture content ranged from 6.5 ± 0.07 to 10.3 ± 0.4 g/100 g d.wt. T. triquetra had the highest ash content (25.4 ± 0.006 g/100 g d.wt.), while C. trinodis had the lowest content (13.5 ± 0.01 g/100 g d.wt.). For protein, the range was from 6.2 ± 0.01 in T. triquetra to 10.9 ± 0.9 in P. boryana (g/100 g d.wt.). The carbohydrate and fiber content had the highest values compared to the other components. Total carbohydrate ranged from 39.8 ± 0.5 for P. boryana to 49.2 ± 0.001 g/100 g d.wt. for T. triquetra. For total fiber, T. triquetra had the highest value (26.1 ± 0.06 g/100 g d.wt.), while P. boryana had the lowest value (15.1 ± 0.3 g/100 g d.wt.). The 3 species showed low lipid content, ranged from 1.2 ± 0.006 g/100 g d.wt. in C. trinodis to 2.3 ± 0.09 g/100 g d.wt. in T. triquetra. The results indicated that T. triquetra had the highest ash, carbohydrate, lipid, and fiber content, whereas P. boryana had the highest protein content. The current results are consistent with those of previous studies that showed the chemical composition ranges of seaweeds.^1,18,19 Together with their nutritional importance, primary metabolites have bioactive roles.⁷ Carbohydrates and fibers are known to be important components of seaweeds and may reach 60% of their d.wt. Although macroalgae have high carbohydrate and fiber content, they are not considered a rich energy source because of their low digestibility.¹⁹ Macroalgal carbohydrates and dietary fibers are considered to have remarkable effects on chronic diseases such as obesity, diabetes, cardiovascular diseases, and cancer.⁶ Although marine macroalgal proteins are described as low, especially in brown algae (3%-15% d.wt.), they contain all essential amino acids in higher quantities than terrestrial plants. Seaweed proteins are a valuable source of bioactive peptides with several health benefits, including antimicrobial, antioxidant, antihypertensive, immunomodulatory, and antithrombotic properties. Although seaweeds have a low lipid content, they are recognized by their fatty acid profile, which contains more essential fatty acids, especially omega 3 and omega 6 fatty acids, than terrestrial plants. Seaweed lipids have been indicated for their antimicrobial, antioxidant, and anti-inflammatory properties.^1,13,19,20 The chemical composition of seaweed species varies depending on many factors such as the season and location of collection.¹⁶

Table 1.

Moisture, Ash, Protein, Carbohydrates, Total Lipids, and Crude Dietary Fibers Contents, Expressed as g/100 g Dry Weight.

Algal species	Moisture	Ash	Protein	Carbohydrate	Lipid	Fiber
Cystoseira trinodis	9.7 ^a ± 0.3	13.5 ^c ± 0.01	9.2 ^b ± 0.1	41.8 ^b ± 0.007	1.54 ^b ± 0.009	20.2 ^b ± 0.12
Padina boryana	10.3 ^a ± 0.4	21.7 ^b ± 0.9	10.9 ^a ± 0.9	39.8 ^c ± 0.5	1.2 ^c ± 0.006	15.1 ^c ± 0.3
Turbinaria triquetra	6.5 ^b ± 0.07	25.4 ^a ± 0.006	6.2 ^c ± 0.01	49.2 ^a ± 0.001	2.3 ^a ± 0.09	26.1 ^a ± 0.06

The least significant differences were tested using Duncan's test. In the same column, the mean values carrying different superscripts are significantly different.

Minerals and Trace Metals Composition

Seaweeds are rich sources of minerals and trace elements owing to their ability to accumulate in the surrounding environment. The mineral and trace element content of seaweeds is much higher than that of terrestrial plants. Thus, seaweeds have been used as a functional food source to provide the human body with daily requirements for essential minerals and trace elements.¹⁹ In the present work, high amounts of Na, K, Mg, Ca, and Fe were detected (Table 2). The highest value for Na was found in P. boryana (290 ± 0.25 mg/100 g), while C. trinodis was the lowest (125.84 ± 0.52 mg/100 g). For K, C. trinodis had the highest value (1891.7 ± 0.25 mg/100 g), whereas P. boryana had the lowest value (650 ± 0.25 mg/100 g). The content of Mg ranged from 1787.91 ± 0.1 mg/100 g in C. trinodis to 11213.67 ± 0.2 mg/100 g in P. boryana. The content of Ca was the highest among the minerals that ranged from 16125.35 ± 0.8 mg/100 g in C. trinodis to 28635.9 ± 0.1 mg/100 g in P. boryana. For Fe, C. trinodis had the maximum value (341.03 ± 0.1 mg/100 g), while T. triquetra had the minimum value (137.78 ± 0.01 mg/100 g). Trace elements, such as Cu, Mn, and Zn, were found in low amounts in the studied species. The value for Cu ranged from 1.36 ± 0.07 mg/100 g in C. trinodis to 6.08 ± 0.01 mg/100 g in P. boryana. The maximum value for Mn was found in C. trinodis (0.52 ± 0.2 mg/100 g), while the other 2 species did not show significant differences. Finally, Zn values ranged from 0.41 ± 0.02 mg/100 g in C. trinodis to 3.3 ± 0.06 mg/100 g in P. boryana. The present results are consistent with those reported for mineral and trace metal ranges in seaweeds, which identified high values of Na, K, Mg, Ca, and Fe and low values of Cu, Mn, and Zn.^1,21 Our results show that the studied species could be a good dietary supplement for macroelements and microelements to fulfill the recommended daily mineral doses for humans. The results indicate a considerably high value of Ca in the studied species; thus, they could be recommended for consumption by those at risk of Ca deficiency.

Table 2.

Major Minerals and Trace Elements Measured as mg/100 g Dry Weight.

Elements	Cystoseira trinodis	Padina boryana	Turbinaria triquetra
Na	125.84 ^c ± 0.52	290 ^a ± 0.25	141.47 ^b ± 0.75
K	1891.7 ^a ± 0.25	650 ^c ± 0.25	980.3 ^b ± 0.15
Mg	1787.91 ^c ± 0.1	11213.67 ^a ± 0.2	3171.40 ^b ± 0.3
Ca	16125.35 ^c ± 0.8	28635.9 ^a ± 0.1	27355.62 ^b ± 0.2
Fe	341.03 ^a ± 0.1	165.49 ^b ± 0.01	137.78 ^c ± 0.01
Cu	1.36 ^c ± 0.07	6.08 ^a ± 0.01	3.9 ^b ± 0.06
Mn	0.52 ^a ± 0.2	0.17 ^b ± 0.02	0.24 ^b ± 0.01
Zn	0.41 ^c ± 0.02	3.3 ^a ± 0.06	2.4 ^b ± 0.06

The least significant differences were tested using Duncan's test. In the same row, the mean values carrying different superscripts are significantly different.

Total Phenolic and Flavonoid Content

Marine macroalgae have developed several secondary metabolites as defense mechanisms to survive in competitive environments. Phenolics and flavonoids are among the most important bioactive compounds produced by seaweeds.¹⁴ The average values for total phenolic and flavonoid contents of the macroalgal extracts in the present study are presented in Table 3. Total phenolic contents were from 15.54 ± 0.1 mg gallic acid equivalent (GAE)/g in P. boryana to 39.5 ± 0.2 mg GAE/g in T. triquetra. For flavonoids, the maximum value was for T. triquetra (3.4 ± 0.3 mg rutin/g) and the minimum was for P. boryana (1.5 ± 0.2 mg rutin/g). The phenolic and flavonoid content in the current study were found higher than some studies, on the same species, from different parts of the world.^22,23 Meanwhile, the content were found to be lower than other studies.²⁴ The limitation that lead to the observed variation in phenolic and flavonoid contents could be attributed to several factors, including light intensity, water temperature, nutrient availability, and geographical location. Furthermore, the choice of extraction solvent, in this case, absolute ethanol, could also influence the extraction efficiency and subsequently the quantification of these compounds. Different solvents may selectively extract different classes of phytochemicals, leading to variations in the measured phenolic and flavonoid contents.²⁵ Several bioactivities of seaweeds, including antioxidant and antimicrobial, are related to phenolics and flavonoids.^13,26

Table 3.

Measurements of Total Phenolic Content in mg Gallic Acid Equivalent (GAE)/g and Total Flavonoids in mg Rutin/g.

Algal species	Total phenolic content (mg GAE/g)	Flavonoids (mg rutin/g)
Cystoseira trinodis	17.3 ^b ± 0.1	2.3 ^b ± 0.02
Padina boryana	15.54 ^c ± 0.1	1.5 ^c ± 0.2
Turbinaria triquetra	39.5 ^a ± 0.2	3.4 ^a ± 0.3

The least significant differences were tested using Duncan's test. In the same column, the mean values carrying different superscripts are significantly different.

GC-MS Analysis

The GC-MS analysis for the extracts indicated nine major components in each extract (Figure 1 and Table 4). The 3 extracts are sharing 5 compounds (ethyl pentadecanoate; heptadecanoic acid, ethyl ester; hexadecanoic acid, ethyl ester; 1,2-benzenedicarboxylic acid; and 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester). The maximum area % for those 5 compounds are found in T. triquetra, while the minimum area % for them were found in C. trinodis. The compound 1,2-benzenedicarboxylic acid, 3-nitro-, was found in both T. triquetra and C. trinodis, with a higher area % in the former. Three compounds, stigmasta-5,24(28)-dien-3-ol, (3á)-; methyl sulfinyl methane; and 9-octadecenoic acid (z)-, ethyl ester, were only observed in C. trinodis. Compounds 7-methyl-z-tetradecen-1-ol acetate; cyclopropanedecanoic acid, à(acetyloxy)-2-hexyl-, methyl ester; 1-heptatriacotanol; and 9,12,15-octadecatrienoic acid, 2,3-dihydroxypropyl ester, (z,z,z)- were present only in P. boryana. Compounds 3,3-dimethyl-4-(3,3,4,4 tetramethyloxetan-2 ylidene) butan-2-one; 6-hexadecenoic acid, 7methyl, methyl ester (z); and 7-isopropyl-10-methyl-1, 5-dithiaspiro [5.5] undecane-2-carboxylic acid 1-oxide were found only in T. triquetra. As shown in Table 5, the compounds belonged mostly to the fatty acid group, together with other compounds including phthalates, fucosterols, and terpenoids. Table 5 summarizes the previously reported bioactivity of these compounds, including their antioxidant, antimicrobial, anti-inflammatory, and anticancer activities.

Figure 1.

Gas chromatography-mass spectrometry chromatogram representing the chemical profile for the 3 seaweed extracts: Cystoseira trinodis (A), Padina boryana (B), and Turbinaria triquetra (C).

Table 4.

Major Compounds Identified Using Gas Chromatography-Mass Spectrometry (GC-MS) Analysis.

Algal species	Compound name	Area, %	Molecular formula	Molecular weight (Da)
Cystoseira trinodis	Stigmasta-5,24(28)-dien-3-ol, (3á)-	20	C₂₉H4₈O	412
	Ethyl pentadecanoate	18	C₁₇H₃₄O₂	270
	Heptadecanoic acid, ethyl ester	18	C₁₉H₃₈O₂	298
	Hexadecanoic acid, ethyl ester	18	C₁₈H₃₆O₂	284
	Methyl sulfinyl methane	11	C₂H₆OS	78
	9-Octadecenoic acid (z)-, ethyl ester	7	C₂₀H₃₈O₂	310
	1,2-Benzenedicarboxylic acid	7	C₂₄H₃₈O₄	390
	1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	7	C₁₆H₂₂O₄	278
	1,2-Benzenedicarboxylic acid, 3-nitro-	7	C₈H₅NO₆	211
Padina boryana	Ethyl pentadecanoate	29	C₁₇H₃₄O₂	270
	Heptadecanoic acid, ethyl ester	29	C₁₉H₃₈O₂	298
	Hexadecanoic acid, ethyl ester	29	C₁₈H₃₆O₂	284
	1,2-Benzenedicarboxylic acid	13	C₂₄H₃₈O₄	390
	1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	13	C₁₆H₂₂O₄	278
	7-Methyl-Z-tetradecen-1-ol acetate	5.3	C₁₇H₃₂O₂	268
	Cyclopropanedecanoic acid, à(acetyloxy)-2-hexyl-, methyl ester	5	C₂₂H₄₀O₄	368
	1-Heptatriacotanol	5	C₃₇H₇₆O	536
	9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (z,z,z)-	5	C₂₁H₃₆O₄	352
Turbinaria triquetra	Ethyl pentadecanoate	43	C₁₇H₃₄O₂	270
	Heptadecanoic acid, ethyl ester	43	C₁₉H₃₈O₂	298
	Hexadecanoic acid, ethyl ester	43	C₁₈H₃₆O₂	284
	1,2-Benzenedicarboxylic acid	20	C₂₄H₃₈O₄	390
	1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	20	C₁₆H₂₂O₄	278
	1,2-Benzenedicarboxylic acid, 3-nitro-	20	C₈H₅NO₆	211
	3,3-Dimethyl-4-(3,3,4,4 tetramethyloxetan-2 ylidene) butan-2-one	6	C₁₃H₂₂O₂	210
	6-Hexadecenoic acid, 7methyl, methyl ester (z)	6	C₁₈H₃₄O₂	282
	7-Isopropyl-10-methyl-1, 5-dithiaspiro [5.5] undecane-2-carboxylic acid 1-oxide	6	C₁₄H₂₄O₃S₂	304

Shaded rows are for common compounds in the 3 extracts.

Table 5.

GC-MS Compounds Nature and Bioactivity from Previous Reports.

Compound name	Compound nature	Biological activity	References
1,2-Benzenedicarboxylic acid	Phthalate (Plasticizer)	Antimicrobial	^27–29
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	Mono-ethyl hexyl phthalic acid (Plasticizer)	Antimicrobial	²⁹
1,2-Benzenedicarboxylic acid, 3-nitro-	3-Nitrophthalic acid (Plasticizer)	Antimicrobial Anticancer,	²⁹
Cyclopropanedecanoic acid, à-(acetyloxy)-2-hexyl-, methyl ester	Fatty acid ester	Antimicrobial	³⁰
3,3-Dimethyl-4-(3,3,4,4 tetramethyloxetan-2 ylidene) butan-2-one	Monoterpenoid	Flavor, antimicrobial	³¹
Ethyl pentadecanoate	Fatty acid ester	Antimicrobial, antioxidants, hypocholesterolemic, nematicide	³²
1-Heptatriacotanol	Fatty alcohol	Antimicrobial, antioxidant, anti-inflammatory, hypocholesterolemic and anticancer	³³
6-Hexadecenoic acid, 7methyl, methyl ester (z)	Fatty acid ester	Anti-inflammatory, anticancer	³²
Heptadecanoic acid, ethyl ester	Fatty acid ester	Antimicrobial, antioxidant	³⁰
Hexadecanoic acid, ethyl ester	Fatty acid ester	Antimicrobial, antioxidant	³⁰
7-Isopropyl-10-methyl-1, 5 dithiaspiro [5.5] undecane-2-carboxylic acid 1-oxide	Terpenoid	Antimicrobial	³²
Methyl sulfinyl methane	Organic sulfur compound	Antimicrobial, anti-inflammatory, antioxidant and antiviral	³⁴
7-Methyl-Z-tetradecen-1-ol acetate	Fatty acid methyl ester	Antimicrobial, anticancer, anti-inflammatory, hepatoprotective	²⁷
9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (z,z,z)-	Fatty acid	Antimicrobial, anti-inflammatory, anticancer, antidiabetic,	³²
9-Octadecenoic acid (z)-, ethyl ester	Fatty acid ester	Antimicrobial, anti-inflammatory	³²
Stigmasta-5,24(28)-dien-3-ol, (3á)-	Isofucosterol	Antimicrobial, antioxidant, antiasthma, anti-inflammatory, diuretic, anticancer, antiarthritic	³⁵

Antimicrobial Activity

As microbial resistance to antibiotics grows rapidly, there is an urgent need to develop new antibiotics from natural sources. Seaweeds have been widely investigated worldwide for their ability to produce a wide range of bioactive compounds with antimicrobial activity.¹³ In the present study, the antimicrobial activities of the 3 extracts were tested against several microbial pathogens, and the results are presented in Table 6. The extracts effectively suppressed the growth of the tested microorganisms, especially at higher concentrations (200 µg/disc). In general, the effect against bacteria was greater than that against fungi, and the effect against gram-positive bacteria was greater than that against gram-negative bacteria in correlation with the dose. The lower concentrations of the 3 extracts did not show any activity against the tested fungi. C. trinodis extract showed the highest activity (10 ± 0.12) against Aspergillus fumigatus. For Candida albicans, the effect for the 3 extracts was very close (10 ± 0.17, 9.5 ± 0.12, 10 ± 0.1 for C. trinodis, P. boryana, and T. triquetra, respectively) with the same effect for C. trinodis and T. triquetra. T. triquetra extract was the most effective extract against the gram-positive bacterium Staphylococcus aureus, whereas the extract of C. trinodis was the most effective extract against Bacillus subtilis. For gram-negative bacteria, the C. trinodis extract had the lowest effect against Escherichia coli that did not show any effect at the low concentration (100 µg/disc). The greatest effect against E. coli was observed for T. triquetra extract. The maximum inhibition for Proteus vulgaris was due to the extracts of both C. trinodis and P. boryana at the low concentration (100 µg/disc), while at the high concentration (200 µg/disc) P. boryana extract had the highest effect. In the present study, algal samples were found to contain considerable amounts of phenols and flavonoids, which have been shown to possess antimicrobial activity.^5,36 In addition, GC-MS analysis of the crude extracts of the 3 species revealed several major compounds with known antimicrobial activities (Table 5). There are some limitations to the current results; the antimicrobial activity of the extracts could be due to the effect of certain compounds or as a synergetic effect of several compounds. Moreover, the study identifies potential antimicrobial compounds, which require more study for their efficacy and safety require. In addition, in vitro and in vivo studies are required to evaluate the pharmaceutical properties and potential toxicity of these compounds before considering their application in pharmaceutical or nutraceutical products. Finally, the efficacy of the natural crude extracts can differ significantly depending on the used solvent, the method of extraction, and the presence of synergistic or antagonistic compounds within the extract. In contrast, ketoconazole and gentamycin (positive controls) are highly purified, single compound with well-established antimicrobial properties. Therefore, optimization of the extraction technique, fractionation, and isolation of certain compounds of the crude extract will enhance the yield and efficacy of the active compounds allowing for more direct comparison to the established antibiotics.

Table 6.

Antimicrobial Activity of the 3 Seaweed Ethanol Extracts.

Algal samples		Cystoseira trinodis	Padina boryana	Turbinaria triquetra	Positive control	Negative control (ethanol)
Microorganisms					Positive control
Fungi					Ketoconazole
Aspergillus fumigatus	1	-ve	-ve	-ve	17 ± 0.2	-ve
Aspergillus fumigatus	2	10^a± 0.12	8^c± 0.16	9^b± 0.07	17 ± 0.2	-ve
Candida albicans	1	-ve	-ve	-ve	20 ± 0.12	-ve
Candida albicans	2	10^a± 0.17	9.5^b± 0.12	10^a± 0.1	20 ± 0.12	-ve
Gram-positive bacteria					Gentamycin
Staphylococcus aureus	1	8^c± 0.13	12^b± 0.07	14^a± 0.1	24 ± 0.06	-ve
Staphylococcus aureus	2	12.5^c± 0.6	13.5^b± 0.12	17^a± 0.12	24 ± 0.06	-ve
Bacillus subtilis	1	12^a± 0.07	8^c± 0.12	9^b± 0.06	26 ± 0.06	-ve
Bacillus subtilis	2	15^a± 0.12	10^c± 0.12	11^b± 0.1	26 ± 0.06	-ve
Gram-negative bacteria					Gentamycin
Escherichia coli	1	-ve	10^b± 0.1	13^a± 0.06	30 ± 0.12	-ve
Escherichia coli	2	8^c± 0.1	12^b± 0.12	16^a± 0.3	30 ± 0.12	-ve
Proteus vulgaris	1	10^a± 0.12	10^a± 0.06	8^b± 0.12	25 ± 0.17	-ve
Proteus vulgaris	2	12^b± 0.17	12.5^a± 0.12	10^c± 0.2	25 ± 0.17	-ve

For treatment: 1 refers to 100 µg/disc algal extract concentration and 2 refers to 200 µg/disc. Negative control: ethanol. -ve: negative. Positive control: ketoconazole (100 µg/disc) was used for fungi and gentamycin (4 µg/disc) for bacteria. The least significant differences were tested using Duncan's test. In the same row, the mean values carrying different superscripts are significantly different.

Molecular Docking

Molecular docking is a powerful tool in drug discovery and design, which can predict the binding affinity and interactions between compounds and target proteins.^37,38 A molecular docking study was performed to investigate the binding mode of the GC-MS identified compounds to the crystal structures of penicillin-binding protein 6 (PBP6) from E. coli (PDB ID: 3ITA), UDP-N-acetylenolpyruvoylglucosamine reductase (Murb) from S. aureus (PDBID: 1HSK), and secreted aspartyl proteinase-5 (SAP5) from C. albicans. The 3 microorganisms have chosen as a representative for the 3 studied groups, gram-negative bacteria, gram-positive bacteria, and fungi. The 3 microorganisms are well studied in the basis of antibiotic interaction and molecular docking. The 3 chosen proteins are considered potential targets for antimicrobial agents and drug discovery.

PBP5 and PBP6 is the main dd-carboxypeptidases enzymes in E. coli, which involved in cell wall maturation and cell shape formation. The distinguished feature of PBP6 from PBP5 is its role in the peptidoglycan stabilization in the bacterial stationary phase.^39,40 MurB enzyme is responsible for catalyzing the formation of UDP-N-acetylmuramic acid (UDP-MurNAc) through reducing the enolpyruvyl to a lactyl ether. UDP-MurNAc serves as the building block for peptidoglycan synthesis and consequently the formation of the bacterial cell wall.⁴¹ PBP6 and MurB is the target proteins for the antibiotics that work through the inhibition of bacterial cell wall formation.^40,41 The SAP5 is one of the secreted aspartyl proteinase produced by C. albicans. The enzyme involved in the cell colonization, penetration, and mucosal infection of C. albicans. The enzyme is expressed during the development of germ tubes and the fungus hyphae. Thus, SAP5 is contributing to the virulence of the fungus. Inhibiting SAP5 activity has been explored as a potential strategy for antifungal drugs.⁴²

Table 7 summarizes the ligand-receptor interactions of nine compounds with PBP6 protein. The binding affinity of the compounds ranged from −12.9 to −26.3 kcal/mol. The compound 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester, was docked with the highest binding affinity and formed 2 H-bond interactions with the leucine residue at position 304 (Leu304) and the alanine residue at position 306 (Ala306) as key interactive amino acid residues, like the co-crystallized ligand. Table 8 summarizes the ligand-receptor interactions of 8 compounds with MurB. The binding affinity of the compounds ranged from −11.3 to −14.4 kcal/mol. The compound 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester, was docked with the highest binding affinity and formed 2 H-bond interactions with aspartic residue at position 86 (Asp86) and one H-bond with threonine residue at position 221 (Thr221). Table 9 summarizes the ligand-receptor interactions of 8 compounds with SAP5. The binding affinity ranged from −9.8 to −14.8 kcal/mol. The compound 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester, was docked with the highest binding affinity and formed 2 H-bond interactions with serine residue at position 82 and 143 (Ser82, Ser143) and one H-bond with asparagine residue at position 80 (Asn80). Figures 2, 3, and 4 shows the binding of 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester to the PBP6, MurB, and SAP5 protein active site, respectively. The results validation has been checked with the root-mean-square deviation (RMSD) calculation that was lower than 2.5 Å. The presence of the hydrogen bounds between the target proteins and the compound influencing the specificity and stability of the compound and suggest the success of drug design and development.⁴³

Figure 2.

Binding disposition of the docked 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (yellow-colored) inside the binding site of PBP6 protein of Escherichia coli (represented with ribbon). Hydrogen bond distances (black dashed line) were measured in Å with the key amino acid residues.

Figure 3.

Binding disposition of the docked 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (yellow-colored) inside the binding site of MurB of Staphylococcus aureus (represented with ribbon). Hydrogen bond distances (black dashed line) were measured in Å with the key amino acid residues.

Figure 4.

Binding disposition of the docked 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (yellow-colored) inside the binding site of SAP5 from Candida albicans (represented with ribbon). Hydrogen bond distances (black dashed line) were measured in Å with the key amino acid residues.

Table 7.

Summary of Ligand-Receptor Interactions of the Docked Compounds towards Penicillin-Binding Protein 6 (PBP6) from Escherichia coli (PDBID: 3ITA).

Compound	Binding affinity score (kcal/mol)	Ligand-receptor interactions
Ampicillin ^a	−14.23	2 H-bond with Leu304 and Ala306
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	−26.3	2 H-bond with Leu304 and Ala306
1,2-Benzenedicarboxylic acid	−21.4	2 H-bond with Leu304 and Ala306
1,2-Benzenedicarboxylic acid, 3-nitro-	−18.6	2 H-bond with Ala306
7-Isopropyl-10-methyl-1	−18.6	2 H-bond with Leu304 and Ala306
3,3-Dimethyl-4-(3,3,4,4 tetramethyloxetan-2 ylidene) butan-2-one	−16.5	1 H-bond with Ala306
7-Methyl-z-tetradecen-1-ol acetate	−16.3	1 H-bond with Ala306
6-Hexadecenoic acid, 7methyl, methyl ester (z)	−15.3	1 H-bond with Ala306
9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (z,z,z)-	−14.3	1 H-bond with Ala306
Methyl sulfinyl methane	−12.9	1 H-bond with Ala306

Docking was carried out using Autodock Vina. Results were validated by root-mean-square deviation calculation that was lower than 2.5 Å.

^a Co-crystallized ligand with PBP6 protein.

Table 8.

Summary of Ligand-Receptor Interactions of the Docked Compounds towards MurB (PDBID: 1HSK) from Staphylococcus aureus.

Compound	Binding affinity score (kcal/mol)	Ligand-receptor interactions
Flavin-adenine dinucleotide ^a	−10.4	6 H-bond with Ser82, Ser143, Gly81, Gly79, Asn83 and Asn80
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	−14.4	1 H-bond with Thr221
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	−14.4	2 H-bond with Asp86
7-Isopropyl-10-methyl-1	−14.1	1 H-bond with Ser82
7-Methyl-z-tetradecen-1-ol acetate	−13.4	1 H-bond with Gly81
6-Hexadecenoic acid, 7methyl, methyl ester (z)	−12.5	1 H-bond with Gly81
3,3-Dimethyl-4-(3,3,4,4 tetramethyloxetan-2 ylidene) butan-2-one	−12.4	None
1,2-Benzenedicarboxylic acid, 3-nitro-	−12.3	1 H-bond with Ser82
1,2-Benzenedicarboxylic acid, 3-nitro-	−12.3	1 H-bond with Gly81
9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (z,z,z)-	−11.9	1 H-bond with Gly81
1,2-Benzenedicarboxylic acid	−11.3	1 H-bond with Ser82
1,2-Benzenedicarboxylic acid	−11.3	1 H-bond with Asn80

Docking was carried out using Autodock Vina. Results were validated by root-mean-square deviation calculation that was lower than 2.5 Å.

^a Co-crystallized ligand with MurB protein.

Table 9.

Summary of Ligand-Receptor Interactions of the Docked Compounds towards Secreted Aspartyl Proteinase-5 (SAP5) (PDBID: 2QZX) from Candida albicans.

Compound	Binding affinity score (kcal/mol)	Ligand-receptor interactions
Pepstatin ^a (Oligopeptides)	−11.2	4 H-bond with Gly85, Gly34, Asp32 and Thr221
1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester	−14.8	2 H-bond with Ser82, and Ser143 1 H-bond with Asn80
1,2-Benzenedicarboxylic acid, 3-nitro-	−13.7	1 H-bond with Asn83
1,2-Benzenedicarboxylic acid	−13.6	1 H-bond with Gly153
7-Isopropyl-10-methyl-1	−11.3	2 H-bond with Gly81 and Gly79
6-Hexadecenoic acid, 7methyl, methyl ester (z)	−11.2	2 H-bond with Gly153 and Gly79
9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (z,z,z)-	−11.1	1 H-bond with Gly153
7-Methyl-z-tetradecen-1-ol acetate	−10.2	1 H-bond with Asn80
3,3-Dimethyl-4-(3,3,4,4 tetramethyloxetan-2 ylidene) butan-2-one	−9.8	None

Docking was carried out using AutoDock Vina. Results were validated by root-mean-square deviation calculation that was lower than 2.5 Å.

^a Co-crystallized ligand with SAP5 protein.

Although molecular docking is important for predicting the binding affinity between ligands and target proteins, it is important to note that docking results are theoretical and may not always accurately reflect the actual binding affinity in vivo. Moreover, molecular docking gives information on the antimicrobial ability of single compounds without paying attention to the synergetic interactions between different compounds within the extracts.

It is important to point that additional molecular dynamics studies are required to confirm the ligand binding interactions of the compounds. In order to go deeply into the mode of action, future work of compound isolation must be performed and further molecular docking tools are required before suggesting the compounds in pharmaceutical applications.

Conclusion

The present study was conducted on 3 rarely investigated brown seaweeds collected from Marsa Allam, Red Sea. Their chemical composition, mineral and trace element contents, antimicrobial action of the extracts, and molecular docking of the main detected compounds were evaluated. We concluded that the 3 seaweed extracts may be valuable sources of nutrients, especially carbohydrates and fiber. The richness of minerals and trace elements in the 3 extracts, particularly calcium, suggests that they are excellent natural supplements for minerals. The results indicated that the 3 extracts possessed high phenol and flavonoid content. GC-MS analysis revealed valuable compounds, mostly fatty acids, which were noted for their bioactivity, particularly as antimicrobial compounds. The 3 extracts showed dose-dependent antimicrobial activity against the tested microorganisms (B. subtilis, S. aureus, E. coli, P. vulgaris, C. albicans, and A. fumigatus), with a relatively high effect against gram-positive bacteria (B. subtilis and S. aureus). For molecular docking, 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester was found to have the highest binding energy and formed 2 H-bond interactions with Leu304 and Ala306. These results suggest that the 3 extracts are sources of nutritional and bioactive compounds with antimicrobial activity. Further research should be conducted on the separation and purification of bioactive compounds to elucidate their mode of action.

Materials and Methods

Algae Collection and Preparation

The collection site of the 3 seaweed species (C. trinodis, P. boryana, and T. triquetra) was at the tidal and intertidal zones of Marsa Alaam, Red Sea, Egypt, in late March 2021. Species were selected based on their abundance during collection. The samples were washed in seawater, thoroughly washed with fresh water, air-dried in the shadow, pulverized, and stored until further usage. One hundred grams of each sample was mixed with 500 mL absolute ethanol, placed in a shaking incubator overnight at 37°C, and the extract was collected. This process was repeated 3 times or until the extract became clear and then combined. The extract was filtered and evaporated until dry under reduced-pressure vacuum. A 50 mg/mL stock solution of each crude extract was prepared using absolute ethanol.

Proximate Chemical Composition

The moisture, ash, fiber, and lipid contents of the 3 samples were determined using the official methods of the Association of Official Analytical Chemists (AOAC).⁴⁴ The method described by Lowry et al was used for the protein extraction.⁴⁵ The carbohydrate assay described by Hedge and Hofreiter was used.⁴⁶ All results are expressed as g/100 g d.wt.

Minerals and Trace Metals Composition

Minerals and trace elements (Na, K, Mg, Ca, Fe, Cu, Mn, and Zn) were measured by flame atomic absorption spectrometry. One gram of each sample was dried to ash, digested with 1:1 nitric and perchloric acid, diluted with distilled water, and filtered prior to the analysis.⁴⁴ Flame atomic absorption spectrometry (Perkin Elmer 301) was used for this analysis, and the concentrations were expressed as mg/100 g d.wt. Synthetic standard metals were used for calibration.

Total Phenolics Content Measurement

The Folin-Ciocalteu method was used to estimate the total phenolic content.⁴⁷ A mixture of 1 mL of 0.1 mg/mL algal extract, 1 mL 95% ethanol, 5 mL distilled water, and 50% Folin-Ciocalteu reagent (0.5 mL) was prepared for each extract. The mixture was incubated at 21°C to 23°C for 5 min, followed by adding 1 mL of 5% Na₂CO₃ solution. The solutions were mixed and incubated for 1 h in the dark. Subsequently, absorbance was measured at 700 nm. Different concentrations of gallic acid were used to generate a standard curve, and the results were expressed as gallic acid equivalents (mg GAE/g).

Flavonoid Content Measurement

An aluminum chloride colorimetric assay was used with some modifications for flavonoid estimation.⁴⁸ Briefly, 0.5 mL of each sample extract at 1 mg/mL was added to 1.5 mL of distilled water and mixed with 0.5 mL of 5% sodium nitrite. After 6 min, 0.15 mL of 10% aluminum-chloride was added, and 6 min later, 2 mL of 4% sodium hydroxide was added and the total volume was adjusted to 5 mL with distilled water. After 15 min, the solution was vortexed and the absorbance was measured against a newly prepared reagent blank at 510 nm. Different concentrations of rutin were used to generate standard curves. The results were expressed as rutin equivalents (mg rutin/g).

GC-MS Analysis

Chemical analyses of the 3 crude ethanolic extracts were performed using GC-MS (ISQ LT GC-MS Model, Thermo Fisher Scientific, Waltham, MA, USA). The Turbomass software was used to analyze the mass spectra and chromatograms. The extract content was identified using linked-library comparisons.

Antimicrobial Activity Measurement

The antimicrobial activities of the 3 extracts against selected microorganisms were assessed using a disc diffusion assay.⁴⁹ Standard isolates of B. subtilis (ATCC813106) and S. aureus (ATCC25923) were gram-positive bacteria, E. coli (ATCC25955) and P. vulgaris (ATCC13315) were gram-negative bacteria, and C. albicans (ATCC10231) and A. fumigatus were fungi. A suspension (100 μL) containing 10⁸ CFU/mL of the bacteria and 10⁶ CFU/mL of the fungus was spread on Mueller-Hinton agar and Sabouraud dextrose agar medium, respectively. The discs were loaded with 100 and 200 μg/disc of each algal extract and placed on the inoculated agar. Absolute ethanol was used as a negative control. Ketoconazole (100 µg/disc) and gentamycin (4 µg/disc) were used as positive controls for fungi and bacteria, respectively. The cultured plates were incubated at 37°C (24 h for bacteria and 48 h for the fungi). The antimicrobial activity was evaluated by measuring the inhibition zone (mm) of the extracts against the tested organisms.

Molecular Docking

To elucidate the virtual binding mechanism of the identified compounds, a molecular docking study of penicillin-binding protein 6 (PBP6) from the E. coli (PDB ID: 3ITA), UDP-N-acetylenolpyruvoylglucosamine reductase (Murb) from S. aureus (PDBID: 1HSK), and secreted aspartyl proteinase-5 (SAP5) from C. albicans (PDBID: 2QZX). Active sites were performed. The protein structures were optimized by adjusting the amino acids with missing atoms or alternative positions, and the ligand structures were built, optimized, and energetically favored using the Maestro software. Molecular docking studies were performed following routine work of preparing the appropriate formats of receptors and ligands, determination of grid box dimensions box of 10 Å in the x, y, and z axes centered on the ligand, and docking with binding activities in terms of binding energies and ligand-receptor interactions following the routine work as discussed. Autodock Vina software was used to validate molecular docking calculations. Finally, Chimera was utilized as the visualization software for the assessment of drug-target interactions. The results validation was checked using the RMSD.

Statistical Analysis

The statistical analysis was performed using SPSS (SPSS Inc., Version 11.5) and Microsoft Excel (2010). The significance difference was tested using analysis of variance. The least significant differences were tested using Duncan's test. All values are presented as the mean ± SD of triplicate. Statistical significance was set at P < .05.

Footnotes

Authors’ Contributions

NO contributed to conceptualization, methodology, validation, formal analysis, investigation, writing—original draft, writing—review and editing, and visualization. MSN was involved in methodology, formal analysis, and investigation. H-KK and JWH were involved in formal analysis, resources, and funding acquisition. Y-SK was involved in conceptualization, methodology, validation, formal analysis, resources, writing—original draft, writing—review and editing, visualization, supervision, project administration, and funding acquisition. All authors have critically read and approved the final manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This research was supported by Brain Pool program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (RS-2023-00221435) and by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2023-00270936).

Ethical Approval

This article does not contain any studies with human or animal subjects.

Informed Consent

This article does not contain any studies with human or animal subjects.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

ORCID iDs

Mohamed S. Nafie

Young-Sang Koh

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Chemical Composition,Antimicrobial Activity,and Molecular Docking of the Main Phytoconstituents from Three Rarely Investigated Marine Macroalgae

Abstract

Keywords

Introduction

Results and Discussion

Proximate Chemical Composition

Minerals and Trace Metals Composition

Total Phenolic and Flavonoid Content

GC-MS Analysis

Antimicrobial Activity

Molecular Docking

Conclusion

Materials and Methods

Algae Collection and Preparation

Proximate Chemical Composition

Minerals and Trace Metals Composition

Total Phenolics Content Measurement

Flavonoid Content Measurement

GC-MS Analysis

Antimicrobial Activity Measurement

Molecular Docking

Statistical Analysis

Footnotes

Authors’ Contributions

Declaration of Conflicting Interests

Funding

Ethical Approval

Informed Consent

Statement of Human and Animal Rights

ORCID iDs

References