In vitro and molecular docking studies on a novel Brevibacillus borstelensis NOB3 bioactive compounds as anticancer, anti-inflammatory, and antimicrobial activity

ABSTRACT Cancerous tumor growth and inflammation stimulate the search for novel anticancer drugs. This pioneering study isolated Brevibacillus borstelensis NOB3 from a solar saltern and tested its bioactive substances for anticancer, antimicrobial, and anti-inflammatory activity. An in-silico test against HeLa cells was followed by an in-vitro MTT test. Agar well diffusion was used to examine the activity of the bioactive compounds of B. borstelensis NOB3 against spectrum human pathogenic bacteria and fungi. The highest clear zone was found with Bacillus subtilis (30 mm), followed by Escherichia coli and Candida albicans (28 mm); Pseudomonas aeruginosa and Staphylococcus aureus had a smaller zone of inhibition (25 mm). The minimum inhibitory concentration (MIC), was 31.25 g/ml against E. coli, followed by 62.5 g/ml against B. subtilis, P. aeruginosa, and C. albicans. Twenty-two bioactive substances were identified by (GC-MS). The binding energy of pyrazine, n-hexadecenoic acid, and oleic acid was measured for the cervical cancer target protein, and the findings were ‒5.1 kcal/mol, ‒4.6 kcal/mol, and ‒4.7 kcal/mol, respectively. In-vitro tests revealed potential action against the HeLa cell line with a half-maximal inhibitory concentration (IC50) of 68.46 g/ml. The novel halophilic B. borstelensis NOB3 has the potential to produce bioactive compounds for drug development.


Introduction
The growth and progression of cancerous tumors, as well as the inflammation associated with the tumors, is a primary cause of morbidity and death worldwide [1].In 2020, it was predicted that there would be 604,000 new cases of cervical cancer in women worldwide and 342,000 deaths due to the disease.It was thought that approximately 90% of these cases and deaths would occur in countries in which people had low or medium incomes [2].In 2023, 1,958,310 new cancer cases and 609,820 cancer deaths were projected to occur in the United States [3].Many cancer therapies, such as surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy, and combinations of these treatments, are time-consuming and expensive.The effectiveness of a cancer therapy depends on how well it destroys cancer cells while having minimal side effects on healthy cells [4].Bacteria can produce a wide variety of bioactive molecules, including alkaloids and antibiotics, which have a variety of biological and therapeutic functions, some of which can be useful in helping humans and animals remain healthy [5].
Brevibacillus sp. are among the species known for producing multiple short-sequence microbial peptides, along with antimicrobial agents such as glycopeptides, brevibacillin, and bacteriocin [6].Brevilaterin B and other peptides extracted from Brevibacillus laterosporus S62-9 were recognized for their anticancer, antibacterial, and antifungal activities [7].Similar effects were generated by Brevibacillus brevis EGS9, as reported previously [8].Bogorol, a peptide isolated from B. laterosporus JX-5, exhibited potent antibacterial and anticancer activities [9].Brevibacillus was described as a facultatively anaerobic, Grampositive, endospore-forming bacterium [10], and thus was further reclassified from Bacillus [11].Brevibacillus can be found in various environments, including oceans, rivers, sediments, waters of hot springs, soils, and composts [12].Microorganisms, especially halophilic or halotolerant bacteria, from severe environments have recently received much attention for their ability to produce enzymes and their use as antimicrobial agents [13][14][15][16][17].Some of these severe saline environments are present in Egypt, such as the lakes of Wadi El-Natrun [18], Solar Lake on the Sinai coast of the Gulf of Aqaba [19], and Mariout Lake near the city of Alexandria [20].Mariout Lake is one of the Nile Delta's most anthropogenically degraded and eutrophic wetlands due to the outflows it receives from the industrial and urban sectors [21].A solar saltern is present in the North Sinai region [22].Halophilic bacterial bioactive metabolites prevent the failure of other available medications [23].B. borstelensis strain BCS-1 produces optically pure D-amino acid amidases [24].These substances are used to make agrochemicals, insecticides, food additives, and medications [25].The final goal of this work was to determine the activity of bioactive compounds extracted from the newly identified B. borstelensis NOB3, which was isolated from the North Sinai solar saltern, characterize these bioactive compounds by using GC-MS analysis, and investigate the druglikeness of these compounds.Finally, the study area would be evaluated to determine whether it was a good source of strains with a variety of good qualities that could be used to make biological antibacterial and anticancer drugs.

Isolation of the novel halophilic strain Brevibacillus borstelensis NOB3
From our previous study Brevibacillus borstelensis NOB3 was isolated from North Sinai solar salt produced from Bardaweil lagoons, identified by molecular methods, and published on GenBank under accession number ON073840 [22].We employed this strain in our current investigation and used the guanine and cytosine (G+C) content of the biological sequence and other physiological tests to evaluate and highlight the novelty of our strain versus other strains of B. borstelensis.

Screening for antagonistic activity
From the results of our previous investigation, 30 halophilic bacterial isolates were collected and tested for enzymatic activity [22].Using the agar cylinder method with little modification, all these isolates that were isolated from the same environment in our previous work were screened for their antagonistic potential toward one another [26].The indicator strains were raised in suitable liquid media, harvested when they were in the late exponential phase of growth, and then evaluated by spreading suspensions on agar plates of suitable medium and the potential strains inoculated as a spot on the surface above indicator strains and after incubation for 3-5 days, the inhibition zone's degree was assessed.

Phylogenetic tree construction
The halophilic Brevibacillus borstelensis NOB3 sequences were aligned by Cluster W for phylogenetic analysis, and MEGA.X software employed neighbor-joining techniques to determine the evolutionary tree [22,27]

Extraction of the bioactive compounds from Brevibacillus borstelensis NOB3
In 250 mL of halophilic broth media, Brevibacillus borstelensis NOB3 was grown to produce the metabolites crude extract.For 3-5 days, the cultures were kept in an incubator at 37°C.The extracellular metabolites were extracted three times with ethyl acetate and then separated using a separating funnel following centrifugation at 3000 rpm for 20 min.A rotary evaporator was used to separate and evaporate the organic phases until they were completely dry.For additional research, the crude extract was concentrated and kept at 4°C [28].

Assay of the antimicrobial activity
The agar-well diffusion method was used to assess the Brevibacillus borstelensis NOB3 extract's antimicrobial properties [29].For bacterial cultures, nutrient broth was used; for fungal cultures, a broth containing 1% peptone and 2% dextrose was utilized.75 mL of the medium was poured into petri plates to create them, and the agar was allowed to set.Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 90,274), Candida albicans (ATCC 10,221), and Mucor reinelloids were the references microorganisms obtained from Ain Shams Hospital, and they served as the test's target pathogens.A sterile cotton swab was used to disperse a freshly made microbial inoculum (1 mL) uniformly across the entire agar surface.The extract was then put into a well that had been created with a sterile cork borer (6 mm).After one hour at room temperature, petri plates were incubated at the proper conditions depending on the organism being examined.Parallel controls were carried out where the well was filled with extract.The plates were examined for zones of inhibition, and the outcomes were compared to those of the positive control using Gentamicin (30 g/mL).

Determination of the minimum inhibitory concentration (MIC)
The smallest concentration required to stop an organism's ability to develop visibly is known as the minimum inhibitory concentration (MIC).By using the microdilution method, the MIC of the Brevibacillus borstelensis NOB3 extract was determined [30].The MIC was calculated via serial dilution with broth media using one ml of the extract (100 g/ml in DMSO).Every test sample and growth control (which contained broth and DMSO but not the antimicrobial extract) were injected with 10 mL of bacterial suspension containing 5 × 10 6 CFU/mL for the examined strains.In the case of bacterial cultures, all samples were incubated at 37°C for 18-24 hours and at 27°C for 72 hours.

Preparation of blood samples
In vitro anti-inflammatory action has been investigated using the human red blood cell (HRBC) membrane stabilization method [25].Blood was drawn from a healthy human volunteer who had abstained from NSAIDS for two weeks before the experiment, and the blood was then transferred to heparinized tubes.Prior to usage, all blood samples were kept for 24 hours at 4°C.The supernatant was collected after 5 minutes of centrifugation at 3000 rpm.The cell suspension was centrifuged at 3000 rpm for 5 minutes after being cleaned with sterile saline solution (0.9% w/v NaCl).The packed cell volume was measured after this process was carried out three times until the supernatant was clear and colorless.The cellular component was utilized in the experiments after being reconstituted to a 40% (v/v) solution with isotonic phosphate buffered saline (10 mM, pH 7.4).

Hypotonicity-induced hemolysis
The Brevibacillus borstelensis NOB3 extracts used in this test were diluted in distilled water in accordance with [25] with a few minor adjustments (hypotonic solution).Graded dosages of the extract (100, 200, 400, 600, 800, and 1000 g/ ml) were applied to duplicate pairs (per dose) of the centrifuge tubes together with the hypotonic solution (5 ml).Additionally, isotonic solution (5 ml) containing graded doses of the extract (100-1000 g/ml) was added to duplicate pairs (per dose) of centrifuge tubes.5 ml of the vehicle (distilled water) and 5 ml of indomethacin 200 g/ml, respectively, were employed in the control tubes.0.1 ml of erythrocyte suspension was added to each tube and then gently blended.After being incubated for one hour at room temperature (37°C), the solutions were then centrifuged for three minutes at a speed of 1300 rpm g.The absorbance (OD) of the supernatant at 540 nm was computed to determine its hemoglobin concentration using a Spectronic (Milton Roy) spectrophotometer.The percentage hemolysis was calculated by assuming that all hemolysis created in the presence of distilled water is 100%.The percentage of hemolysis inhibition by the extract was calculated as follows: Where OD1 = absorbance of test sample in isotonic solution OD2 = absorbance of test sample in hypotonic solution OD3 = absorbance of control sample in hypotonic Solution

GC-MS analysis of the bioactive compounds from Brevibacillus borstelensis NOB3
The chemical composition of Brevibacillus borstelensis metabolites were performed using Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5 MS (30 m x 0.25 mm x 0.25 µm film thickness).The column oven temperature was initially held at 50 C and then increased by 5°C/min to 230°C hold for 2 min increased to the final temperature 290°C by 30°C/min and hold for 2 min.The injector and MS transfer line temperatures were kept at 250, 260°C respectively; Helium was used as a carrier gas at a constant flow rate of 1 ml/min.The solvent delay was 3 min and diluted samples of 1 µl were injected automatically using Autosampler AS1300 coupled with GC in the split mode.EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40-1000 in full scan mode.The ion source temperature was set at 200°C.The components were identified by comparison of their retention times and mass spectra with those of WILEY 09 and NIST 11 mass spectral database.

Preparation of protein
RCSB Protein Data Bank (http://www), which provides protein structures, was used to retrieve Hela cell line protein structures.On the last stage, hydrogen atoms were supplied to the target protein molecule after all water molecules had been removed.

Docking analysis
The best Cellular Tumor Antigen p53 Ligand Complexes were visualized in 2D structure by BIOVIA Discovery Studio 2021, and Molecular Docking was performed using AutoDock Vina of PyRx virtual screening program [27].

Drug-likeness analysis
Based on Lipinski's rule of five, the compounds' drug-likeness was evaluated.According to the 'Rule of 5', the compounds that are like drugs have a maximum of 10 hydrogen bond acceptors, a maximum of 5 hydrogen bond donors, a minimum molecular mass of 500 Daltons, and a maximum of 2 infractions [28].

Cell line
HeLa cervical cancer cell line was purchased from the Egyptian Vacsera Research Foundation.Fetal bovine serum (FBS) 10% (v/v) was added to Dulbecco's Modified Eagle's Medium (DMEM), which was used to sustain cells.This medium was incubated at 37°C with 5% CO2.To prevent contamination, streptomycin, and penicillin (100 g/ml) were employed.

Sample preparation
Brevibacillus borstelensis NOB3 crude extracts were dissolved in 1 mL of dimethylsulphoxide (DMSO) and then put into sterile containers.These samples were protected from light and kept at ambient temperature.The extract volume was fixed for cytotoxicity tests so that the maximum permissible DMSO content was 0.1% [29].

Anticancer activity by MTT assay
Brevibacillus borstelensis NOB3 crude extract was tested for its anticancer properties using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay [30].The 96 well tissue culture plate was inoculated with 1 X 10 5 cells/ml (100 ul/well) and incubated at 37°C for 24 hours to develop a complete monolayer sheet.And Growth medium was decanted from 96 micro titer plates after confluent sheet of cells were formed, cell monolayer was washed twice with wash media.The two-fold dilutions of tested sample were made in RPMI medium with 2% serum (maintenance medium).And 0.1 ml of each dilution was tested in different wells leaving three wells as control, receiving only maintenance medium.Then the Plate was incubated at 37°C and examined.Cells were checked for any physical signs of toxicity, e.g.partial, or complete loss of the monolayer, rounding, shrinkage, or cell granulation.The MTT solution was prepared (5 mg/ml in PBS) (BIO BASIC CANADA INC), and 20ul MTT solution were added to each well.Place on a shaking table, 150 rpm for 5 minutes, to thoroughly mix the MTT into the media.Incubate (37C, 5% CO2) for 1-5 hours to allow the MTT to be metabolized.And Dump off the media.(Dry plate on paper towels to remove residue if necessary.Resuspend formazan (MTT metabolic product) in 200ul DMSO.Place on a shaking table, 150 rpm for 5 minutes, to thoroughly mix the formazan into the solvent.Finally Read optical density at 560 nm and subtract background at 620 nm.Optical density should be directly correlated with cell quantity.For the treated cell line, the test compound's 50% inhibitory concentration value (IC50) was found.The percentage of cell viability as determined by the formula below: % Of cell viability = 100 (treatment OD ⁄ control OD

Statistical analysis
To identify significant differences, the data were subjected to one-way analysis of variance (ANOVA; Minitab) after being represented as mean SD.The least significant difference multiple-range test was used to further examine these differences (p ≤ 0.05).

Isolation and characterization of halophilic Brevibacillus borstelensis NOB3
Species of the Gram-positive, spore-forming bacteria B. borstelensis NOB3 were identified (Figure 1).It was raised at 37°C with 15% NaCl, and the accession number for it in the GenBank database is ON073840.According to a phylogenetic analysis of the 16S rRNA gene sequences, this strain clustered with members of the Brevibacillus genus with 99.86% similarity (Figure 2).Furthermore, based on physiological tests that made comparisons with the same species as shown in

Antagonistic activity of the novel halophilic Brevibacillus borstelensis NOB3
The antagonistic interactions between each of the isolates collected as described in our previous report [19] were examined and different levels of  activity were noted, but B. borstelensis NOB3 had the most potential antagonistic activities against all other bacterial strains isolated from the same habitats (Table 2).

Antimicrobial activity
Figures 3 and S1 show that the bioactive compounds of B. borstelensis NOB3 were more effective at killing human pathogenic microbial strains of bacteria than the antibiotic gentamicin.
The presence or absence of a zone of inhibition in millimeters was used to evaluate the antibacterial potency.The findings demonstrated that the crude extract had excellent antibacterial efficacy against the examined pathogens.B. subtilis ATCC 6633 had the largest zone of inhibition (30 mm), followed by E. coli and C. albicans (28 mm); P. aeruginosa and S. aureus had a smaller zone of inhibition (25 mm) than the control (see Figure S1).B. borstelensis NOB3 had promising antimicrobial activities against all pathogens tested except Mucor reinelloids.

MIC of Brevibacillus borstelensis NOB3 bioactive compounds
The microdilution method assessed the microbial toxicity of promising bioactive compounds from B. borstelensis NOB3 against both bacterial and fungal species.The lowest MIC preventing the visible growth of a microorganism in the broth dilution susceptibility test was 31.25 g/ml against E. coli, followed by 62.5 g/ml against B. subtilis, P. aeruginosa, and C. albicans, as shown in Figure 4 Anti-inflammatory assay Figure 5 shows the stability of the membrane of human red blood cells in terms of antiinflammatory activity.B. borstelensis NOB3 bioactive compounds showed concentrationdependent anti-inflammatory activity, and the percentage of protection improved as the concentration of the extract increased.At dosages of 800, 600, 400, and 200 μl the hemolytic inhibition was 96.8%, 84%, 70.8%, and 61.9%, respectively.Distilled water, a hypotonic solution, had 0% hemolytic inhibition as a positive control.

GC-MS analysis of the bioactive compounds extracted from promising Brevibacillus borstelensis NOB3
The GC-MS evaluation of B. borstelensis NOB3 bioactive compounds (Figure 6) revealed that 22 aliphatic and aromatic secondary metabolite chemicals were present; this was evidenced by several peaks in the GC-MS chromatogram (Table 3).The compound 1,2-benzenedicarboxylic acid was the main compound, with the highest peak, found in   the crude extract of B. borstelensis NOB3; it made up 34.19% of the total components.It was followed by oleic acid at 10.7%, benzylhexahydropyrrolo at 9.9%, isobutylhexahydropyrrolo at 7.5%, and n-hexadecenoic acid at 5.71%.

Molecular docking investigation of bioactive substances against a specific protein involved in cervical cancer
An assessment of drug-like properties, based on Lipinski's rule of five, was performed on chemicals identified in B. borstelensis NOB3.The outcome showed that all 29 compounds could adhere to Lipinski's rule, although the four compounds chosen for molecular docking research against the cervical cancer protein were the most effective (Figure 7).According to our investigation and molecular docking tests, the four drugs' binding affinity scores to the cervical cancer protein ranged from 3.1 to 6.4 kcal/mol (Table 4).The best complexes were p53-5, low-bond energy (-4, -4, 7 kcal/mol), followed by p53-7,9-di-tert-butyl -1-oxaspiro (4,5) deca-6,9-diene-2,8-dione (Figure 8).

Anticancer activity (in vitro)
The effect of bioactive compounds extracted from B. borstelensis NOB3 on cancer cell viability was determined by an MTT assay against cervical HeLa cells.The mitochondrial succinate dehydrogenase enzyme converts MTT into insoluble formazan crystals (purple), which are then solubilized by DMSO and evaluated spectrophotometrically.This assay is based on colorimetry.This assay is frequently utilized to determine cell viability and to evaluate the cytotoxic effects of medicines on cell lines in vitro since a decrease shown by the MTT can only occur in metabolically active cells.The outcomes demonstrated that B. borstelensis NOB3 ethyl acetate extracts had promising antitumor efficacy against cervical HeLa cells.HeLa cells were cultivated in a 96-well plate at different extract dosages, and viability rates were measured following exposure to the extract to ascertain the degree of inhibition of HeLa cell proliferation.The ethyl acetate extract of B. borstelensis contained 62.5 g/ml of cell-free NOB3, which decreased the viability of HeLa cells by 37.5%, as shown in Figure 9 In  addition, as the extract concentration increased, the cell viability dropped sharply, as demonstrated in Figure 10 Based on these findings, the IC 50 value for inhibiting the availability of the HeLa cell line was 68.46 g/ml.

Discussion
More and more natural therapeutics are being discovered as researchers look for alternative therapeutics that employ microbial species and have significant benefits for the biotechnological and biological disciplines [4].
Extreme ecosystems, such as soda lakes and hypersaline environments, provide a lot of potential for bioprospecting, which aims to find new enzymes or genes for novel biotechnological applications [31].New species are frequently reported, indicating that microbial diversity and activity increase in hypersaline settings [32][33][34].Rocks, dust, aquatic environments, and the viscera of numerous insects and mammals are just a few places where Brevibacillus has been found [35][36][37][38].B. borstelensis NOB3 had never been identified in the North Sinai solar saline environment created by the Bardawil lagoons.We selected this area for our research because it has a unique physicochemical profile and contains multiple novel species, and because research  on the area was lacking [22].Numerous researchers have chosen new locations for the isolation of novel antibiotic-producing bacteria, as well as those containing advantageous microbial populations, which are strongly present in these unusual and alluring settings [39,40].
We investigated the bioactive components extracted from the novel strain B. borstelensis NOB3 for antagonistic and antibacterial activity.This was in line with other research that had shown that numerous microorganisms have promising antibacterial potential [41].When bacteria are competing for survival in  EGYPTIAN JOURNAL OF BASIC AND APPLIED SCIENCES a biological system, those with antimicrobial properties are thought to have a competitive advantage.These findings agree with those of [42].Moreover, numerous studies have shown the existence of Gram-negative bacteria, particularly marine Bacillus species, which are frequently isolated from sediments and invertebrates [43].Members of the genus Bacillus, which are known to produce metabolites with antibacterial and antifungal effects or with other effects that are generally cytotoxic, appeared to have significant potential as novel antimicrobial compounds in the marine environment [44,45].Numerous investigations  had focused on testing halophilic microorganisms for antibacterial efficacy against human pathogenic microorganisms [46].One study [47] reported that the major public health concern of our day was the development of resistance to antibacterial medications by both Gram-positive and Gram-negative bacteria.All these concerns have led researchers to try to discover new bioactive compounds made by communities of microbes in marine, endophytic, and terrestrial environments.The main goal of our research was to find ways to identify active ingredients that were antibacterial, anti-inflammatory, and anti-cancerous.Fatty acids and their derivatives, as well as benzoquinones, pyrroles, and others, were among the bioactive chemicals discovered by GC-MS analysis, in comparison with the findings of [48][49][50].Because of their low toxicity, high biodegradability, and great stability at high temperatures, these chemicals not only had strong antibacterial activity but were also ecologically friendly [50].Like the researchers of [51], we reported that bio-extracts contained pharmaceutically relevant bioactive compounds that were efficient against isolates that were multidrug-resistant.In our study, we showed that hexadecenoic acid had a variety of antifungal and antibacterial properties against urinary tract infections.This result was in line with two other studies [47,52].Chronic disorders, including cancer, diabetes, and other inflammatory conditions such as chronic inflammatory diseases, are responsible for 50% of all fatalities worldwide [53].Our results were in line with those of [54] in that we showed the concentrationdependent anti-inflammatory activity of the bioactive components of the novel strain B. borstelensis NOB3, with an increase in the level of protection as the concentration of the extract increased.Adult enterocytes and immunocytes treated with saturated fatty acids generated by Bifidobacterium infant is showed anti-inflammatory properties [55,56].Additionally, plants may have anti-inflammatory properties because of compounds released by bacterial endophytes [57].The most prevalent viral infection of the reproductive system, human papillomavirus infection, causes a significant majority (>95%) of cervical malignancies [58].Therefore, we investigated B. borstelensis NOB3's bioactive compounds by in-silico and in-vitro studies.
We showed an antiproliferative effect on HeLa cells and suggested the presence of potent cytotoxic and anticancer components in this extract.These outcomes supported those of [59], who found that the inhibition of the HeLa cell line consistently increased with an increasing concentration of an extract.According to our findings, the IC50 was 68.46 g/ml which agrees with [60].The crude extract was toxic (active) if the LC50 value was less than 1000 g/ml and nontoxic (inactive) if the LC50 value was larger than 1000 g/ml.
Techniques such as de novo drug development and virtual screening are potent ways of finding leading compounds with specific biological activity.An anticancer examination of the crude extract of B. borstelensis NOB3 against the cervical cancer (HeLa) cell line may thus be due to these compounds and the derivatives of lipids and other compounds from B. borstelensis extracts, which were discovered to have anticancer and antibacterial components in earlier investigations [59,61] and in our findings.According to the study by [62], pyrrole,1,2 pyrazine 1,4, dione,hexahydro 3-(2-methylpropyl) (PPDHMP) showed dose-dependent anticancer activity in vitro against lung (A549) and cervical (HeLa) cancer cells.Therefore, therapy with the monounsaturated fatty acid oleic acid increased lipid buildup in healthy cells, as would be predicted.Given that cancer cells have higher levels of lipid buildup, which causes cell death, and lower levels of proliferation, migration, and invasion, this supported a positive role for oleic acid in cancer cells [63].However, prior studies had also revealed that the Bacillus species exhibited good bioactivity as cytotoxic agents.Four bacterial cultures were isolated from the sea sponge Amphimedon ochracea.The human cancer cell lines HepG2 (hepatocellular carcinoma), HCT (colon carcinoma), and MCF-7 (breast carcinoma) were all sensitive to the cytotoxic action of the bacteria B. subtilis, Bacillus safensis, and Lactobacillus murinus [64].Another probable source for the isolation of a cytotoxic substance was Bacillus sp.Three new cyclic acyl peptides were successfully recovered from the fermentation of the ethyl acetate extract from Bacillus sp.This chemical affects human intestinal tumor cells (HCT-116) [65].Moreover, previous studies [66] showed that some Halomonas species produce substances that may be able to kill cancer cells.Antibacterial compounds derived from B. borstelensis were the subjects of the first article [67], while the bioactivity of B. laterosporus toward human cancer cells was the subject of the second [68].Additionally, our findings showed that the effectiveness of cytotoxic drugs in preventing colony formation in HeLa cells depended on the cells' capacity to survive by establishing a colony following treatment [69].In addition to earlier studies demonstrating a clear association between inflammation and cancer growth and progression, several clinical trials are currently examining the use of antiinflammatory medicines in combination with chemotherapeutic treatments in the treatment of cancer [70].Notably, in our research, bioactive compounds from B. borstelensis NOB3 were able to prevent blood hemolysis and act as anti-inflammatory agents; these results were comparable with those of [71][72][73].

Conclusion
This study provided the first identification of the novel strain B. borstelensis NOB3, which was isolated from the North Sinai saltern.It could be the source of bioactive compounds with broadspectrum activity against different types of human pathogenic fungal and bacterial species that have become resistant to multiple drugs.Additionally, GC-MS analysis showed that B. borstelensis NOB3 bioactive components demonstrated a variety of metabolic abilities and adaptable biochemical effects through the formation of various bioactive chemical structures.These structures may be important in several pharmaceutical industries and biotechnological processes and have anti-cancer activity against HeLa cell line proliferation in vitro and in silico.
Overall, our research suggested that the Sinai solar saltern should be further investigated using a culture-dependent methodology and a cultureindependent strategy to discover more extremophilic species that might be essential for producing pharmaceutically useful microbes, including potentially effective anti-microbial, antiinflammatory, and anticancer agents.

Figure 8 .
Figure 8. Interaction of the four compounds to the active sites of cervical cancer protein.

Table 1 .
Major features of strains NOB3 and species of the genus Brevibacillus.

Table 3 .
GC-MS analysis revealed bioactive compound in an ethyl acetate extract of Brevibacillus borstelensis NOB3.

Table 4 .
In silico docking analysis of bioactive compounds of Brevibacillus borstelensis NOB3against cervical cancer protein.