In Silico and In-vitro assessment of β carotene extracted from Westella botryoides for fighting multidrug resistant bacterial strains

Abstract

Algae are a sustainable natural resource because of their beta-carotene content. Therefore, this study demonstrates the extraction of the bioactive compound beta-carotene as a protein pigment from the green alga Westella botryoides and its potential use as an antibacterial agent against pathogenic bacterial cells. This study investigates the interactions between beta-carotene and bacterial receptors, including S. mutans-3IHK, E. coli-7P2M, S. aureus-5M1A, and Salmonella typhi-7CI4, using molecular docking simulations. The study explores the binding affinity, energetic dynamics, and molecular interactions between β-carotene and these receptors, revealing insights into its potential antimicrobial activities. In addition, the study evaluates the toxicity of β-carotene, its potential nephrotoxic and respiratory toxicities, and other biological functions. The results show that the green alga W. botryoides has a number of natural medicinal compounds. This was the first time that beta-carotene was extracted from it. The concentration was 0.78 µg/mg in the fresh weight of the alga and 132 mg in the dry weight of the alga. This shows that the protein pigment beta-carotene was present in the alga. This was especially true when Westella botryoides grew under a light intensity of 45 µmol, which is when the sample was taken in the stationary phase. The study also found that the highest killing rate (22, 18, 27, and 28%) for all cells was at the highest β-Carotene concentration (500 µg/mL). The lowest killing rates for the cells of S. mutans, S. mutans, E. coli, and P. aeruginosa in this study were 18%, 14%, 10%, 9%, and 6%, accordingly, when various concentrations of β-C were used: 500, 250, 125, 62.5, and 0 µg/mL. These compounds exhibit favorable interactions of binding with bacterial receptors, especially those of Staphylococcus aureus. They also have a toxicity profile that is fairly safe, which makes them good candidates for more research on antimicrobial and therapeutic uses.

Introduction

Environmental sustainability, comprising 17 targets, focuses on the efficient utilization of natural resources and health of ecosystems. Algae are known, as a renewable source for these natural resources by means of bioactive substances like beta-carotene (β-C) (Neekhra et al. 2022; Gaidarenko 2018). Their intrinsic properties render them an attractive source of food, which is authentically natural and yet both numerous and integrated in its solutions. They can also contribute to achieving environmental sustainability goals, improving environmental quality, and promoting sustainable economies, significantly increasing the market share of these products. Algal compounds, including green algae, including β Carotene (β-C), are considered natural alternatives in the pharmaceutical industry (Freitas et al. 2021; Manowattana et al. 2020; Herrero et al. 2013). Carotenoids (β-carotene, Zeaxanthin, lutein) are natural organic pigments that contribute to photosynthesis and are secondary metabolites, Algae represent the primary natural source of β-carotene in aquatic ecosystems (Patel et al. 2022; Pereira et al. 2021). In algae such as green algae and higher plants, these compounds are C40 isoprenoids, containing 40 carbon atoms and lacking an O2 (Zulfiqar et al. 2021; Wang et al. 2022). They are unsaturated hydrocarbon compounds belonging to the carotenoid group (alpha, beta, lycopene, xanthophyll) found naturally in green algae such as Westella botryoides (West) De Wildeman. Their chemical formula is C40H56, with a molecular mass of 536.88 g/mol. They contain an extended chain of consolidate paired bonds across the molecule, which reduces the energy gap between electron levels, enabling them to absorb visible light, giving them antioxidant properties (as an antioxidant for bacteria and cancer). They have two beta-ionone rings on each end, allowing them to be converted to vitamin A (retinol) upon absorption [10 Recent research have proven the efficacy of β-Carotene as a natural antioxidant to determine the inhibition zones using the disk diffusion assay against several types of bacteria, including pathogenic and antibiotic-resistant bacterial strains (Bereksi-Reguig et al. 2024; Coyago-Cruz et al. 2024; Rafeeq et al. 2020) β-C is a fat-solvable provitamin, the lively form of which is vitamin A. It has antibacterial activity due to its antimicrobial properties. It disrupts cell membranes and inhibits bacterial enzymes, reducing the cells’ ability to reproduce and grow, and promoting the production of ROXs that lead to bacterial cell death (Taie and Al-Katib 2020). This also applies to the beta-carotene F1 pigment extracted from Westella botryoides and Chlorella, and to its effectiveness against bacteria as well as fungi. In contrast, the F2 substrate showed a lower effect against them. The highest concentration of the pigment was found in the acetone extract of Chlorella algae, while the lowest concentration was found in the substrate after isolation of the cyanobacterium Cloeocapsa (F2G) (Cakmak et al. 2014). The bioactivity and biochemical composition of different extracts (aqueous, methanolic, and ethanolic) was determined from Dunaliella salina and their antimicrobial and antioxidant properties evaluated against pathogenic microorganisms using diffusion and dilution assay methods. The antioxidant power was evaluated by using the method of beta-carotene/linoleic acid. The findings indicated that D. salina extracts may serve as potential natural antimicrobials and antioxidants for the food, feed, and pharmaceutical sectors, as well as in the biodiesel industry due to their high unsaturated fatty acid concentration (Kim et al. 2019) In their study of a Gram-negative bacterium; Heliobacter pylori using different concentration of β-C, the lowest inhibition rate was recorded at 5–10 μM CM-LC whereas highest death-rate appeared at 50–100 μM CM. This is due to elevated ROX as β-C blocked fluorescent cell growth. These findings suggest that the physiological impacts of β-C might be dependent on concentration ranges and dosage is a key determinant factor in regulating mortality. Chlorella vulgaris is also abundant in natural antioxidants, including the β-C, however its stiff cell wall makes its application as a food additive limited (Sarkar et al. 2021). Its bactericidal activity towards Staphylococcus aureus is believed to be a natural antibiotic. (Garima Pandey et al. 2018) have performed a study, whereby the quality of herbal medicines depended on their natural secondary metabolites concentration that may be altered relative to growth of microbial susceptibility to the extracts in disk diffusion. Meanwhile, bacterial and antioxidant functions of β-C were characterized according to the MIC value with the broth dilution method against Escherichia coli O157:H7 and its reducing power, as well as DPPH free radical scavenging capacity. The findings indicated that the concentration of compounds was different by altitude; they had a better antibacterial effect toward S. aureus, E. coli and Salmonella typhi. Recent studies have shown the ability to assess the environmental impact of β-C algal products as a beneficial element in recognizing potential adverse impacts of bacteria and reducing their impact on the ecosystem (Souza Silente et al. 2024). Products such as W. botryoides could be marketed as natural remedies for killing or inhibiting certain bacteria, given their accessibility and low cost, thereby reducing environmental pressure by eliminating their effects. β-carotene is also economically viable because it can be obtained naturally without adverse effects on the ecosystem. It also acts as a negative factor, limiting the growth of some bacteria, especially pathogenic ones, by disrupting bacterial metabolism (Souza Silente et al. 2024; Nazir et al. 2024; Mawed et al. 2022; Rymbai et al. 2011). The current study aimed to evaluate the effects of environmental factors, such as light, on beta-carotene production by a green algal species and to test its biological effectiveness as an antibacterial agent against pathogenic bacteria. This was done through using a species of green algae, Westella botryoides, isolated from local environments, for experimental purposes. A study of the effect of light intensity (65) µmol/m²/s using white LEDs on beta-carotene production by the alga under study. Detecting beta-carotene produced by the algae using high-performance liquid chromatography (HPLC). This study aimed to find a molecular pathway to address the problem of pathogenic bacteria’s resistance to chemical treatments using the natural compound beta-carotene. This was achieved by simultaneously addressing two key gaps: Firstly, by sustainably extracting beta-carotene from the alga Westella botryoides and using it as an antibacterial agent through molecular activity; and second, by computationally verifying the effectiveness of beta-carotene as a bacterial inhibitor against various pathogenic bacteria using our methods, The time to identification of the extracted natural compound relative to the imported standard was measured, along with the natural compound concentration in the algae and the development of a calibration and identification curve. Evaluating the antibacterial efficacy of β Carotene against bacteria that are pathogenic and four species of S. aureus and S. mutans. E. coli, Salmonella typhi. Moreover, finally, evaluating the binding interactions between the natural beta-carotene compound from the studied algae and several bacterial receptors using molecular docking simulations, assessing its antimicrobial potential by examining its toxicological properties and its other biological activities, and demonstrating its effectiveness in potential therapeutic applications by examining its antibacterial efficacy.

2-Materials and methods

2–1—Collection and cultivation of the free-range algae Westella botryoides and biomass production

In this study, pure cultures of the green alga Westella botryoides were obtained from the Advanced Environmental Laboratory inside the Department of Life Sciences at the College of Education, the University of Al-Qadisiyah. For verification of the purification of the isolates, a sample of the algal isolate was cultivated on a solid culture medium for bacterial analysis and incubated at 37 °C for 72 h to ascertain its purity. The cultures were thereafter propagated on CH-10 culture medium for biomass production using a batch culture method. 10 mL of the purified algal isolate was incorporated into 400 mL of the culture medium, and then incubated in an algal growth chamber at 25 ± 2 °C, 65 µmol/m²/s, and a light intensity of 8:16 light: dark cycle. The cultures were stirred at least twice daily to prevent algal growth on the walls. The incubation of algal samples continued until their growth decreased and they died (AL-Obaidy and Leelo 2025; AL-Obaidy et al. 2022).

Extraction of natural β Carotene by HPLC

Method described by Barba et al. (2006) was used to measure the natural beta-carotene content in the algae Westella botryoides, using the standard beta-carotene pigment, produced by SIGMA-ALDRICH (USA). After culturing the algae and allowing them to reach the numerical plateau, beta-carotene was extracted, detected, and quantified as described in Table 1. Samples were obtained and centrifuging at 10,000 rpm for 30 min. The sediment was weighed and its fresh weight calculated. 5 mL of a solution comprising hexane, acetone, and ethanol at a ratio of 50–25-25 was added. The sediment was then crushed using a mechanical homogenizer and cooled. The specimen was thereafter subjected to centrifugation at 8,000 rpm for 30 min. The filter was removed before being injected into the centrifuge. The filter was removed, and the process was repeated on the sediment until it turned white. The filtrate was then collected and allowed to settle. The upper hexane layer was removed. The hexane layer was then evaporated at 40 °C (24). The resultant extract was weighed and subsequently dissolved in a solution of THF, ACN, and methanol, ACN, and methanol (15–30-55). The resulting excerpt was then injected into the apparatus.

Table 1.

HPLC apparatus components, sample volume, extraction devices and tools, and analysis program

N	Component	Model	Company
1	Colum	C18	Knuaer
2	Detector	Diode array 2.1L	Knuaer
3	Injector and Sample volume	D1357, 20 µl	Knuaer
4	Pump	Gradient, P6,1L	Knuaer

Analysis and separation of β-carotene concentration using liquid chromatography with high performance (HPLC)

After abstraction and quantification of β-carotene, the carotenoid content of the studied algae was determined by liquid chromatography with high performance (HPLC) (AL-Obaidy and Leelo 2022) using β-carotene produced by SIGMA-ALDRICH (USA). Separation was carried out using a C18 column (manufactured by Knauer, Germany) (inner diameter 250 × 4.6 mm, particle size 5 μm, pore size 80 Å). Mobile phase components were obtained from Chem-lab, Belgium. The mobile phase was a solution of methanol (solvent A)/ACN (solvent B) (90/10 v/v) + TEA 9 LM, at a flow rate of 1 mL/min with a wavelength of 475 nm. Each molecule was identified by correlating the 5.2-min retention time of the isolated natural β-carotene with the absorption spectrum of standard β-carotene (Fig. 4). The sample quantity was determined by measuring the integrated peak area, and its purity was confirmed using a calibration curve. The equation, according to Fig. 2, was used: The sample appeared pure and free of impurities (Figs. 3 and 5).

Fig. 4.

UV/Vis absorbance spectrum matching between β-Carotene standard (blue trace) and resolved peak in sample Westella botryoides (red trace)

Fig. 2.

Calibration curve for β-Carotene. The x-axis represents the five concentrations of standard β-Carotene used for matching, and the y-axis represents the peak area of ​​the β-Carotene concentrations in the algae samples of the current study, Westella botryoides

Fig. 3.

HPLC chromatogram of analyzed sample Westella botryoides (red trace) matched with β-Carotene standard (blue trace)

Fig. 5.

Shows a three-dimensional schematic diagram of the UV–VIS absorption spectrum of the standard β-Carotene blue dye in an HPLC device

Antibacterial activity

The antibacterial activity of the synthesized β-Carotene was evaluated versus Gram-negative and Gram-positive strains of bacteria using the technique of diffusion on agar wells (Shindia et al. 2024; Al-Monofy et al. 2025). About 20 mL of Muller– Hinton (MH) agar was carefully allocated onto sterile Petri dishes. The bacterial species were retrieved from the existing cultures using a sterile wire loop. After cultivating the microbes, 6 mm-diameter wells were formed on agar plate using a sterile tip. Multiple concentrations of β-Carotene were employed in the bored wells. The cultured plates containing beta-carotene and the tested organism have been incubated overnight at 37°C before measuring and recording the average widths of the inhibitory zones (Azizi et al. 2021).

Assessment of MIC

The CLSI (2022) microdilution technique was employed to determine the minimal inhibitory concentration (MIC). Stock solutions of β-Carotene were prepared in Mueller–Hinton broth at values ranging from 2 to 1024 µg/mL. The solutions that had been prepared were distributed into the wells of a microtiter plate with 96 wells. All wells, except for the negative control wells, received 10 µL of a suspension of bacteria standardized to a McFarland 0.5 standard. Wells containing bacterial inoculum served as positive controls. E. coli ATCC 25922 functioned as an example standard for quality assurance purposes. The microtiter plate then underwent incubation overnight at 37 °C.

Flow cytometry assay

Flow cytometry was employed to measure reactive oxygen species (ROS) in bacteria following therapy with the substance -carotene at a concentration of 125 μg/mL. Bacterial strains were exposed to β-Carotene for 24 h at 37 °C. The bacterial cells acquired post-centrifugation were subsequently washed and resuspended in PBS to form a uniform culture (volume, 1 mL). The cells were subsequently treated with 50μM 2-dichlorodihydrofluorescein diacetate (DCFH2-DA) in a 1.5 mL volume at 37°C for 45 min, followed by ROS detection. The reactive oxygen species (ROS) level was quantified by flow cytometry; (FACSVerse, BD Biosciences).

Molecular docking technique

The selected proteins were identified for their antibacterial characteristics, and molecular docking was utilised to analyse the relationship between the ligands and the protein, as well as their corresponding binding scores. 3IHK is a PDB code associated with the protein structure of the S. mutans receptor; 7P2M corresponds to the protein structure of the E. coli receptor; 5M1A pertains to the protein structure of the S. aureus receptor; and 7CI4 is linked to the protein structure of the Salmonella typhi receptor. This study investigates the sites of activity of these kinds of proteins to clarify their biological activities. The crystal structures were sourced from the Protein Data Bank at (https://www.rcsb.org/structure/3IHK), (https://www.rcsb.org/structure/7P2M), (https://www.rcsb.org/structure/5M1A), and (https://www.rcsb.org/structure/7CI4), with resolutions of 3.00 Å, 1.16 Å, 2.00 Å, and 2.00 Å, respectively, and are considered appropriate for docking studies. An RMSD (Root Means Square Deviation) value of 2 Å and an energy score of -7 kcal/mol or below are deemed optimal parameters for validating molecular docking studies. The optimised compounds, Bromelain and methyl-penicillanate, were docked onto the receptor’s active region using the Molecular Operating Environment (MOE) software. Following the preparation of the receptors protein, the docking and scoring computations were executed in MOE. To facilitate binding through hydrogen bonding, the ligand and the target sequester water molecules within the active site. Moreover, missing bonds in the protein structure, which were compromised during X-ray diffraction, were corrected, and the protein was protonated. The optimisation of protein structure was improved using assisted model building with energy refinement (AMBER 10) with the extended Hückel theory (EHT) force field.

Toxicological studies

To verify the anticipated toxicological endpoints, such as nephrotoxicity and respiratory toxicity, LD50 experiments should be conducted in animal models. Assays for Biological Activity: In vitro biochemical or cellular assays are used to test specific actions, such as HIF1A inhibition, peroxidase inhibition, or NADPH oxidase inhibition. For comparative analysis, use controlled substances known to have these activities. Pharmacokinetics: Assess the pharmacokinetic characteristics of the substances, such as metabolic stability or BBB penetration (if CNS effect is anticipated). Using the open-source ProTox 3.0 server (https://tox.charite.de/protox3/).

Molecular dynamics simulations

The single-isothermal-isobaric molecular dynamics (NPT) technique employs a real-time Hamiltonian framework that integrates an extra Poincaré transformation into the original Nose-Andersen (NA) Hamiltonian model. This methodology was employed to investigate the molecular motion (MD) of biologically active ligands. The most effective system layout used the FACTS technique and utilized the MMFF94x electromagnetic field with a cylindrical border form, water. to be the solvent of choice, and an offset setting of 6. The molecules of solvent have been enhanced by taking out water molecules that were in excess of four Å away. The experiment lasted for 1500 of ps with an interval step of 0.002 ps. Before that, there was a 100 ps equilibrium phase at T = 300 K degrees Celsius. To make sure the NPA calculations were correct, there were also limits on light bonds.

Statistical analysis

We used GraphPad Prism software (version 7) to do the statistical analyses. An independent samples t-test or a one-way analysis of variance (ANOVA) was utilized for the comparison. ANOVA was followed by Tukey post hoc testing for various group comparisons. All investigations were performed in triplicate, and data are presented as the mean ± standard deviation (SD). A p-value below 0.05 was considered statistically significant.

Results & discussion

Detection of β-carotene using HPLC gradient from Westella botryoides

After selecting the green algae Westella botryoides as shown in Figure 1, ensuring its classification and purity, and culturing it on CH-10 culture medium, Figures 2, 3, 4, and 5 show the concentrations used (1200, 1000, 800, 600, 400, and 200 ng/mL) to build the calibration curve and their corresponding peak area, according to the equation in Figure 2, between the natural β-carotene and the standard of the algae W. botryoides. The results showed a high retention time match of 5.2, and the peak areas were identical between the extracted pure natural β-carotene compound and the standard compound. The results indicate that W. botryoides algae contains a high percentage of sustainable compounds for medical and natural therapeutic applications, including beta-carotene and that its purity after extraction using an HPLC gradient device is high. The results of Tables 2 and 3 and Figure 3 show high values ​​for the concentration of beta-carotene. Carotene in W. botryoides algae, where the concentration reached 0.78 ug/mg in fresh weight and 132 mg in soft moss weight, indicates the presence of the protein pigment beta-carotene, as well as its high values ​​in W. botryoides algae when when the algae were cultivated under a specific light intensity of 45 μmol of light after taking the sample in the numerical stationary phase as in Barba et al. (2006). This indicates that the physiological state of the algae responded to various sustainable environmental factors, including the culture medium and its nutrients (nitrate, phosphate, and salts), as well as in addition to sufficient lighting for growth, which gradually increased with controlled increases in these factors (AL-Obaidy and Leelo 2025; AL-Obaidy and Leelo 2022).

Fig. 1.

The moss Westella botryoides is represented under an optical microscope with 40X magnification

Table 2.

Shows the values ​​of the bioactive compound β-Carotene extracted from Westella botryoides, shown in Peak area, Media size, Soft moss weight, Fresh weight, and Final sample size (ml)

S	Peak area	β-C ug/ml	Media size ml	Soft moss weight mg	β-Cug/mg fresh weight	Final sample size ml
1	1292.736	69.5076568	25	132	0.789859736	1.5

Table 3.

Shows the standard curve for β-Carotene, showing the values ​​for the six diluted concentrations of standard β-carotene, as well as the peak area for each concentration

Standard	peak area	concentration μg/ml
β-C	10880.656	1000
β-C	5770.25	500
β-C	1888.093	100
β-C	132.81	10

Antibacterial efficacy of β-Carotene

The results of the current study Table 4 showed that the natural compound extracted from algae Green Westella botryoides, β-Carotene (β-C), affects the growth of pathogenic bacteria S. mutans, E. coli, Salmonella typhi using different concentrations of β-C 500, 250, 125, 62.5, and 0 µg/mL, if this effect is considered the target of the problem adopted in this study, if the study recorded when using the highest concentration of 500 µg/mL the highest killing rate, reaching 22, 18, 27, 28% for all the above cells, respectively, through the effect on the bacterial cell walls, as β-C can cause damage to the cell wall, which increases its susceptibility to killing, as the study showed that β-C can disrupt some vital pathways within the bacterial cell, as well as the sensitivity of bacteria to the oxidative effects of β-C (Shindia et al. 2024; Al-Monofy et al. 2025; Azizi et al. 2021). β-C concentrations also enhance the killing rates against cells directly or indirectly by creating oppressive environmental conditions around or in the environment of bacteria, with the possibility of killing them. The higher the concentration, the greater the toxic effect and the higher the killing rate, the current study’s results indicated the diameter of the inhibitory zone measured after 24 hours or more, as its effect appears through the stability of bacterial DNA and its effect on it, which causes an increase in the rates of genetic mutations and limits the ability of bacteria to repair their DNA, thus causing damage to the genetic material and killing them. (Astley et al. 2004; Van Helden et al. 2009). β-C also enhances the immune response, since it enhances the activity of immunological cells, including T cells and B cells by stimulating metabolic activity, which stimulates a faster response (Medina-García et al. 2025; El-Shora et al. 2025). β-C also affects the cell membrane, as studies indicate that β-C has the potential to inhibit the bacterial cell membrane (Hagaggi and Abdul-Raouf 2023). The study also showed that β-C can affect bacterial metabolism by disrupting energy production within bacterial cells, preventing bacteria from using certain nutrients, and reducing the ability of bacteria to produce biofilms by disrupting the function of bacterial mitochondria (or their bacterial equivalents). This reduces the ability of bacteria to divide and reproduce, making it easier for the immune system to eliminate them (Chew and Park 2004). β-C furthermore promotes the release of cytokines, They are essential in regulating the immune system’s reaction and improving the effectiveness of immune cells, such as macrophage and T cells, by increasing the body’s ability to recognize and eradicate germs. β-C promotes the synthesis of cytokines such as IL-1β, TNF-α, and IFN-γ, which activate the immune system to eliminate bacterial cells (Al-Monofy et al. 2025; Kondo et al. 2022). It also increases activity by inhibiting antibacterial enzymes. (Martins et al. 2023), and disrupting biofilms and chemical signals used by bacteria to communicate causing a defect in energy production, Some studies also suggests that β-C beta-carotene as a natural protein pigment (Jiang et al. 2024), may change the lipid composition of the bacterial membrane, making it more permeable and unstable, reducing its flexibility and integrity, and causing leakage of its vital components (Bazaid et al. 2022). The study also indicated that β-carotene disrupts the bacterial defense system because it possesses anti-oxidant defense systems, such as the enzyme catalase. The dye inhibited the function of these systems, making the cell unable to protect itself from damage (Miazek et al. 2022). Some research has also indicated that β-C inhibits the production of toxins, which may inhibit genes responsible for producing toxins, such as a-toxin, which bacteria rely on to cause infection (Tufail et al. 2024). While the results of the current study, Table 4 Figures 6, 7, 8, and 9 recorded the lowest percentage of killing bacterial cells S.mutans, S.mutans, E.coli, P.aeruginosa, as it reached 18, 14, 10, 9, 6%, respectively, when using different concentrations of β-C, respectively 500, 250, 125, 62.5, 0µg/ml, as the study showed that the antibacterial effect of the compound depends on the dose and time factor, as the higher the concentration, the higher the killing percentage and vice versa (Stephen et al. 2023), and this is what was shown by the study of Wu et al. (2023). on some bacterial cells, as some conditions affect bacteria, including the time and concentration factor, as the concentration decreases, the β-C decomposes quickly, which reduces its effect or reduces absorption inside the bacterial cell because it has thick cell walls that prevent entry β-C is found in low concentrations, resulting in a lower killing rate. Studies have shown that some compounds have a combined inhibitory effect, such as lipids and proteins, which may interfere with the effect of β-C, resulting in a lower killing rate of bacterial cells (Rocha 2023). Also, a low dose of β-C may not be sufficient to produce a lethal effect (Shastak and Pelletier 2024). Some antibacterial effects depend on the photoactivation of β-C, and a deficiency of some substances may reduce its antibacterial activity (Riley et al. 2023). This can also result in an imbalance in the alkaline or acidic medium, which leads to a decrease in the killing rate (El-Marasy et al. 2024). Bactericidal activity of β-C against S. aureus is believed to be a natural antibiotic, whereby the quality of herbal medicines depended on their natural secondary metabolites concentration that may be altered relative to growth of microbial susceptibility to the extracts in disk diffusion. Meanwhile, bacterial and antioxidant functions of β-C were characterized according to the MIC value with the broth dilution method against E. coli O157:H7 and its reducing power, alongside the DPPH its radical scavenging ability. The antibacterial activities of β-Carotene against tested bacteria are evaluated in liquid culture media using turbidity assays. For Salmonella typhi, and E. coli the MIC was 32 µg/mL. While, for S. aureus, and S. mutans was 16 µg/mL.

Table 4.

Antibacterial activity of β-Carotene

Mean of Inhibition Zone of inhibition (mm)
sample		0 µg/ml	62.5 µg/ml	125 µg/ml	250 µg/ml	500 µg/ml
S.aureus	β-Carotene	0	10.56	13.53	18.3	22.8
S.mutans		0	8.66	11.2	18.0	21.57
E.coli	β-Carotene	0	4.0	5.3	16	22
Salmonella typhi		0	4.76	5.66	10.2	15.43

Fig. 6.

Antibacterial activity of (β-Carotene) against Salmonella typhi. A, Control. B, 62.5 µg/ml. C, 125 µg/ml. D, 250 µg/ml. E, 500 µg/ml

Fig. 7.

Antibacterial activity of (β-Carotene) against E. coli. A, Control. B, 62.5 µg/ml. C, 125 µg/ml. D, 250 µg/ml. E, 500 µg/ml

Fig. 8.

Antibacterial activity of (β-Carotene) against S. aureus. A, Control. B, 62.5 µg/ml. C, 125 µg/ml. D, 250 µg/ml. E, 500 µg/ml

Fig. 9.

Antibacterial activity of (β-Carotene) against S. mutans. A, Control. B, 62.5 µg/ml. C, 125 µg/ml. D, 250 µg/ml. E, 500 µg/ml

Scanning electron microscopy (SEM) was utilized to investigate morphological alterations in bacterial cell membranes after treatment with beta-carotene. with Beta-carotene. Clear differences were observed between untreated control bacteria and Beta-carotene -treated bacterial strains. The membranes of treated bacteria appeared surface damage and leakage. The results indicate that beta-carotene interacts with the cell membrane of bacterial species that are both Gram-positive and Gram-negative, inducing pore formation that ultimately leads to bacterial cell death, as illustrated in Figure 10. Bacterial ROS generations were measured using the DCFDA assay following exposure to β-Carotene, as presented in Figure 11. Flow cytometry results indicated that the untreated bacterial control group exhibited minimal ROS production. In contrast, treatment with β-Carotene at concentration 125 μg/mL^-1 resulted in a marked increase in ROS levels.

Fig. 10.

SEM images of bacterial strains treated with beta-carotene

Fig. 11.

β-Carotene induce ROS generation in bacterial strains. A, control bacteria. B, treated bacteria

Performance against antimicrobial

The molecular docking outcomes presented in Table 5 and Figures 12, 13, 14, and 15 for beta-carotene complexes with different bacterial receptors (S. mutans-3IHK, E. coli-7P2M, S. aureus-5M1A, Salmonella typhi-7CI4) provide valuable insights into the interaction between beta-carotene and the bacterial receptors. Below is a discussion based on the provided data: The binding affinity score (negative values) reflects the strength of interaction between beta-carotene and the receptor. Lower (more negative) scores indicate stronger binding. The strongest binding is observed with S. aureus-5M1A (-8.0156 kcal/mol), followed by S. mutans-3IHK (-5.6381 kcal/mol), E. coli-7P2M (-6.1465 kcal/mol), and Salmonella typhi-7CI4 (-6.8665 kcal/mol). This suggests that beta-carotene interacts most strongly with S. aureus, possibly because the receptor-ligand interactions are better. RMSD values show how much the ligand’s shape changes during docking simulations. A stable and accurate docking conformation has a smaller RMSD value. The RMSD values, which range from 2.2510 Å (S. aureus-5M1A) to 2.4997 Å (E. coli-7P2M), show that all of the complexes have fairly stable docking poses. Atoms that are involved: In each case, beta-carotene interacts with different atoms in the receptors, such as carbon (C), oxygen (O), nitrogen (N), and others. Alkyl, hydrogen-bond donor (H-donor), Pi-Alkyl, Pi-sigma, and H-Pi interactions are all different. The reality that β-carotene can bind to different receptor types in different ways indicates that it is elastic and may function in an array of environments. Types of Interactions: Interactions with S. mutans-3IHK are mostly between alkyl and hydrogen-bond donors (ILE 181, ILE 102). The binding distance (3.29 Å and 2.66 Å) shows that the interactions are close, which adds to the overall binding affinity of -25.7743 kcal/mol. E. coli-7P2M: PHE 196, ARG 192, and ALA 188 all have different kinds of interactions with each other, like alkyl, Pi-Alkyl, and Pi-sigma. The binding distances (1.97, 3.18, and 2.33 Å) show that beta-carotene binds strongly. S. aureus-5M1A: complicated interactions including the alkyl, Pi-alkyl, and Pi- sigma interactions with the residues such as LEU 224, LYS 219, and others. The binding distances (1.02 to 2.15 Å) indicate that the relationship between them is stable and strong. Salmonella typhi-7CI4: β-carotene communicates with LYS 142, PHE 160, and other proteins through Pi-Alkyl and H-Pi reactions. The binding dimensions, ranging from 2.00 to 2.95 Å, indicate the bond’s attraction is low. Interaction Energy, also known as The interaction’s energies (E, kcal per mol) indicate how much energy every interaction adds to the system. The total energy (or total E, kcal per mol) is the sum of all the combined energies. S. aureus-5M1A shows the most negative total energy (-28.4026 kcal/mol), reflecting the most substantial binding, followed by S. mutans-3IHK (-25.7743 kcal/mol), E. coli-7P2M (-26.5486 kcal/mol), and Salmonella typhi-7CI4 (-21.567 kcal/mol). What the Data Means: The overall binding affinity and interaction energies show that beta-carotene binds best to S. aureus, then to E. coli and S. mutans, and finally to Salmonella typhi, which has the weakest interaction. This arrangement might . The interaction distances, particularly those between 1.0 and 3.0 Å, suggest close and specific binding, which is typical of powerful receptor-drug interactions. The docking results show that beta-carotene powerful and firm bonds with all four bacteria’s receptor types. The greatest binding occurred with S. aureus-5M1A. The data show that beta-carotene might have antibacterial effects, particularly against S. aureus, E. coli, and S. mutans. This makes it a good candidate for more research as an antimicrobial agent. Nevertheless, further experimental validation is necessary to substantiate these in silico predictions and evaluate the practical utility of beta-carotene in medication development (Manochkumar et al. 2022).

Table 5.

Findings of beta-carotene molecular docking complexes with the Salmonella typhi-7CI4, E. coli-7P2M, S. aureus-5M1A, and S. mutans-3IHK Receptors

Bonds between (Beta-Carotene) with active site residues
Compd NO	Score (kcal/mol)	RMSD (Å)	Compd Atoms	Receptor atoms	Receptor residues	Interaction	d (Å)	E (kcal/mol)	Total E (kcal/mol)
S. mutans-3IHK	-5.6381	2.3976	C 17 O 18	OD2 NH1	ILE 181 (A) ILE 102 (A)	Alkyl H-donor	3.29 2.66	-1.4 -2.3	-25.7743
E. coli-7P2M	-6.1465	2.4997	C 13 C 30 C 21	OG NE2 NE	PHE 196 (A) ARG 192 (A) ALA 188 (A)	Alkyl Pi-Alkyl Pi-sigma	1.97 3.18 2.33	-1.0 -0.9 -2.1	-26.5486
S. aureus-5M1A	-8.0156	2.2510	C C C C	OD2 OD2 NE NE	LEU 224 (A) LYS 219 (A) PRO 350 (A) VAL 217 (A)	Alkyl Pi-Alkyl Alkyl Pi-Alkyl	2.03 1.02 2.15 1.01	-3.1 -2.6 -1.6 -2.9	-28.4026
Salmonella typhi-7CI4	-6.8665	2.2713	C C C C C	NE OE1 OE1 OE2 OE1	LYS 142 (A) PHE 160 (A) ASP 196 (A) LYS 261 (A) MET 62 (A)	Pi-Alkyl H-Pi Alkyl Alkyl Pi-Alkyl	2.95 2.11 2.02 2.00 2.17	-1.0 -3.7 -5.5 -4.8 -4.5	-21.567

Fig. 12.

Visualisations for Molecular Docking. A depiction of the highest-ranked conformation from the docking simulation of β-Carotene with the S. mutans-3IHK receptor. A three-dimensional representation of the amino acids around the most stable conformer and engaging with it

Fig. 13.

Depictions of Molecular Docking (a) The top-ranked conformer’s binding location from the docking simulator with the E. coli-7P2M receptors and β-carotene. A three-dimensional depiction of amino acids encircling and engaging with their most stable conformer

Fig. 14.

Representations of Molecular Docking The top-ranked conformer’s binding site from the docking simulation utilizing the S. aureus-5M1A receptors and β-carotene. b A three-dimensional representation of the amino acids’ configuration and relationship with a highly stable conformer

Fig. 15.

Depictions of Molecular Docking a The binding site of the top-ranked conformer from the docking simulation using the Salmonella typhi-7CI4 Receptor and β-Carotene. b A three-dimensional depiction of the manner in which the amino acids encircle and interact with the most stable conformer

β-carotene and toxicological properties

Table 6 suggests a complex interaction between β-carotene and various receptors associated with toxicity pathways. The radar chart in Figures 16 and 17 shows the chances of different toxicity-related receptors. The chart shows that β-carotene has strong interactions with important receptors like CYP2E1 (which is strongly linked to hepatotoxicity), CYP3A4, and CYP2D6, among others. This means that beta-carotene may interact with metabolic pathways that are involved in drug metabolism and toxicity. Beta-carotene exhibits diverse interactions within multiple organ systems. For instance, nephrotoxicity: β-carotene’s impact on kidney function or toxicity is regarded as inactive in this context, indicating that its correlation with nephrotoxic effects is negligible according to this prediction. Toxicity and cancer-causing effects on the lungs: It also seems to have little effect and possible dangers related to respiratory toxicity and cancer, which means it is relatively safe in these areas. Nutritional Toxicity: β-carotene is a type of Vitamin A, so its effect on nutritional toxicity can be complicated (Koklesova et al. 2020). Too much beta-carotene can be toxic, but this is not common at normal levels of consumption. The second and third figures show how β-carotene binds to important receptors like the Aryl Hydrocarbon Receptor (AR) and Estrogen Receptor Alpha (ER). This is important for understanding how β-carotene changes gene expression and cellular pathways. Based on what we know, enzymes like CYP2E1 and CYP3A4 are involved. It’s essential to think ab 450 enzymes. The toxicology data provided suggests that beta-carotene is relatively safe concerning significant organ toxicity; however, it may still present minimal toxicity risks that necessitate further investigation. Beta-carotene’s interactions with different receptors also suggest that it could be used as a medicine, but its bioactivity may depend on how it interacts with certain receptors.

Table 6.

In Silico virtual screening results of β-Carotene, Including LD50, predicted biological activities, targets, classifications, and toxicity predictions

Fig. 16.

The toxicity radar chart is designed to visually represent the confidence in beta-carotene’s favorable toxicity results compared to the average of its class

Fig. 17.

The network chart quickly illustrates the connections between the selected Beta-Carotene and their predicted activities

β-carotene doesn’t kill bacteria directly, but it can help the body fight off bacterial infections by boosting the immune system, working with cellular enzymes, and acting as an antioxidant. If you take beta-carotene as a dietary supplement or as part of a treatment plan with other antibacterial agents, it may help improve clinical outcomes against bacterial infections (Maurya et al. 2020; Heikal et al. 2023; Jing et al. 2022).

Simulations of molecular dynamics

Figure 18 shows how the energy of different proteins changes over time when they interact with β-carotene in molecular dynamics simulations. The energy (in kcal/mol) of Beta-Carotene interacting with one of the proteins—Salmonella typhi-7CI4, E. coli-7P2M, S. aureus-5M1A, and S. mutans-3IHK—is shown in each image over time (in picoseconds). The changes in energy shown in each graph are due to the interactions between β-carotene and the proteins. The energy changes between -4400 and -4200 kcal/mol in the interaction between Beta-Carotene and S. mutans and S. aureus show that the interaction is fairly stable. This means that Beta-Carotene binds to these proteins in a fairly steady way. In contrast, the larger changes in energy between -5300 and -5100 kcal/mol when interacting with E. coli suggest a more flexible or dynamic interaction. This could mean that this protein reacts more flexibly to Beta-Carotene (Heikal et al. 2023; Jing et al. 2022; Pérez-Gálvez et al. 2020; Lavelli and Sereikaitė 2022). The interaction between Beta-Carotene and Salmonella typhi changed in a way that was similar to the changes seen with S. mutans and S. aureus, going from -4400 to -4200 kcal/mol. This suggests that the interaction between Beta-Carotene and Salmonella typhi is fairly stable. In general, these changes show that the way Beta-Carotene interacts with proteins depends a lot on the type of protein and how its molecules behave. More significant changes, like those seen with E. coli, may mean that the interactions are more flexible or change quickly. On the other hand, smaller changes mean that the interactions are more stable. These simulations show us how Beta-Carotene interacts with different proteins in a useful way. They can also help us come up with new ways to treat infections or design drugs [54-65].

Fig. 18.

(β-carotene) shows the Energy dynamics with proteins, the S. mutans-3IHK, E. coli-7P2M, S. aureus-5M1A, Salmonella typhi-7CI4 Receptors over time

Conclusion

The findings provide evidence of the antibacterial activity of the natural product beta-carotene produced sustainably from algae Westella botryoides against pathogenic bacteria. Results of molecular docking the molecular docking study showed that Beta-Carotene has a strong interaction with bacterial receptors and particularly in the case of S. aureus, which suggest Beta-Carotene’s possible role as an antimicrobial agent. Toxicity The toxicity of Beta-Carotene is very low with the exception of a small increase in resistance to lung cancer; even at high doses (as provided by supplements), but there may be an increase in risk for stomach or prostate cancers. This result could provide the theoretical basis for treating liver fibrosis with Beta-Carotene to construction copy DNA.

Author contribution

H. Al., A. H. Al, Z. A. O., H. A. Al., M. S. J., B. B., and A. A. S.: Composition of the initial draft, methodology, investigation, and formal evaluation. H. Al., A. And.M. S. J.: Principal Concept, Data Analysis, and Oversight. H. Al., A. H. Al, Z. A. O., H. A. Al., M. S. J., and S. Gh.: Writing, review and editing, visualization, and data curation. All writers evaluated the text.

Funding

This study received no funding.

Data availability

All data produced in this investigation are incorporated inside this article.

Declarations

Conflict of interest

The authors declare no competing interests.