Isolation, Characterization, and Biomedical Potential of Phycoerythrin Phycobiliprotein from Kappaphycus alvarezii (Doty) Doty ex Silva: Antimicrobial, Antioxidant, and Anticancer Activities

Isolation, Characterization, and Biomedical Potential of Phycoerythrin Phycobiliprotein from Kappaphycus alvarezii (Doty) Doty ex Silva: Antimicrobial, Antioxidant, and Anticancer Activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Isolation, Characterization, and Biomedical Potential of Phycoerythrin Phycobiliprotein from Kappaphycus alvarezii (Doty) Doty ex Silva: Antimicrobial, Antioxidant, and Anticancer Activities Harinathan Balasundaram, Parthasarathy Seethapathy, Subbiah Sankaralingam, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5880917/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Kappaphycus alvarezii (Doty) Doty ex Silva, a red seaweed widely cultivated for carrageenan polysaccharide, is also a potential source of the valuable pigment phycoerythrin (PE). Therefore, this study aims to extract phycoerythrin from K. alvarezii , evaluate its antimicrobial, antioxidant, and anticancer activities, and identify its biomedical potential for future therapeutic applications. The protein content of phycoerythrin pigment extracted from K. alvarezii was found to be 69.84% and showed excellent antimicrobial activity against Klebsiella oxytoca and Proteus mirabilis , with a minimum inhibition zone of 11 mm. It showed significant in vitro antioxidant activity, as analyzed using total antioxidant, hydrogen peroxide scavenging, reducing power, DPPH, and ABTS assays. Further, the pigment exhibited potent cytotoxicity against a human lung cancer cell line, with an IC 50 value of 131.7 µg mL -1 . Furthermore, increasing the concentration of phycoerythrin pigment decreased the cell proliferation and induced apoptosis, as confirmed by Annexin V/PI staining. Comprehensive characterization using FT-IR, HPLC, and GC-MS analysis revealed the nature of pigment and functional groups, highlighting its potential for biomedical applications. The molecular docking of K. alvarezii -derived compounds revealed significant binding affinities with 13 antibacterial target proteins. These results highlight the potential of K. alvarezii bioactive compounds as promising antibacterial agents. The phycoerythrin extract from K. alvarezii demonstrated potent antimicrobial, antioxidant, and anticancer properties, with significant cytotoxicity against lung cancer cells and confirmed apoptosis induction. Structural analysis revealed its bioactive composition, emphasizing its potential as a natural therapeutic agent. These findings support its potential application in biomedical and pharmaceutical industry. Biological sciences/Plant sciences Earth and environmental sciences/Ocean sciences Bioactive molecules Lung cancer Molecular docking Phycoerythrin Pigment analysis Red seaweed Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Introduction Marine organisms produce a diversified array of bioactive compounds including carbohydrates, lipids, proteins, polyphenols, minerals, amines, amides, antioxidants, and pigments with unique molecular structures and varied biological functions [ 1 ]. Currently, these bioactive compounds are recognized as valuable sources for the food and health care sectors [ 2 ]. Therefore, there has been a rising attention in the research and development focusing on commercial exploitation of marine organisms derived bioactive primary and secondary metabolites as an ecofriendly and effective alternate for synthetic drugs [ 3 ]. In recent years, the discovery of unique bioactive substances with significant dietary, nutraceutical and medicinal advantages has attracted significant attention to marine algae. Marine algae are essential components of the food chain, constituting over 70% of the global living biomass [ 2 ]. Marine algae are photosynthetic organisms both macroscopic and microscopic, that have garnered attention due to their immense potential as a sustainable and environmentally friendly source for several bioactive compounds [ 4 ]. Marine algae or seaweed are growing habitually through autotrophic, mixotrophic, or heterotrophic ways [ 5 ]. The potential of marine algae to be cultivated in fresh, saline and waste waters, along with their fast multiplication, presents them as an attractive substitute for diverse products, thereby conserving time and resources [ 6 ]. Seaweeds are divided into three categories based on their photosynthetic pigments: green macroalgae, brown macroalgae, and red macroalgae. Among these, red macroalgae account for over 60% of the 30,000 tons of algae produced globally [ 7 ]. A variety of red macroalgae are rich in bioactive compounds, making them a preferred choice for the production of commercially valuable bioproducts such as biofertilizers, biofuels, colorants, cosmetics, food, hydrocolloids, nutraceuticals, pharmaceuticals, plant stimulants and thickeners [ 8 ]. Kappaphycus alvarezii (Doty) Doty ex. Silva (elkhorn sea moss) of family Solieriaceae, is a red seaweed (macroalgae) and is the primary carrageenophyte, commercially known as “cottoni”. It is mostly cultivated to produce commercial kappa -carrageenan hydrocolloids in both habitual and non-indigenous areas and has gained potential economic value in the seaweed industry. This macroalgae is the fifth most cultivated in the world, especially in various tropical countries of Asia and Africa, including India [ 7 ], due to its fast growing rates, simple cultivation process, high biomass yield in a short time with high polysaccharide content, and low investment requirements [ 9 ]. It also serves as a food source for local populations in Southeast Asia and is believed to possess various health benefits. The yellowish, reddish, green, and brown colors of K. alvarezii are based on the levels of phycoerythrin protein pigment [ 10 ]. The highly variable chemical components and quantities in K. alvarezii are influenced by the cultivation conditions, including water temperature, salinity, radiation, environmental variables, light level, profundity, and wave strength. On average, K. alvarezii consists of 50.8% carbohydrates, 15.6% ash, 12.4% sulphated groups, 3.3% proteins, 3.3% lipids, and 3.0% insoluble aromatics [ 8 ]. The literature indicates extensive use of K. alvarezii for carrageenan extraction. Furthermore, reports suggest that extracts of K. alvarezii possesses a multitude of essential compounds and high-value molecules with antioxidant and anti-inflammatory, antitumor, antiviral, antidiabetic, antihyperlipidemic activities, and for neural dysfunction properties [ 7 , 11 , 12 ]. Cancer is a fatal disease that poses a significant risk to individuals worldwide, ranking as the second most common cause of mortality in numerous countries. In 2022, the global community experienced 20 million new cases of cancer and 9.7 million fatalities associated with this disease [ 13 ]. Lung cancer ranks among the most common cancers and is a primary contributor to cancer-related fatalities worldwide [ 14 ]. Patients diagnosed with lung cancer encounter a higher risk of meeting bacterial infections, which can vary from mild cases to those that pose significant threats to life. Both bacteria and viruses have the capability to trigger inflammatory cells and activate inflammatory signaling pathways. In particular, Escherichia coli (Migula 1895) Castellani and Chalmers 1919, Pseudomonas aeruginosa (Schröter 1872) Migula 1900, Staphylococcus aureus Rosenbach 1884, and Klebsiella sp. are gram-negative bacteria that are often found in the lungs of cancer patients [ 15 , 16 ]. These bacteria are often found along with other factors like age-related illnesses, large tumors, and rapid clinical decline [ 17 ]. The currently available cancer medications are come with more physical and psychological side effects like pain, vomiting, diarrhea, exhaustion, and nausea; thus, the cancer therapies cannot be considered safe [ 18 ]. Consequently, it is essential to explore novel anticancer medications sourced from nature that are safe, affordable, and less detrimental. The anti-cancer capability of K. alvarezii could be due to polysaccharides, nutrients, and other metabolites with antioxidant potential [ 19 ]. The phycobiliprotein (PBPs) family includes phycoerythrin (PE), which are macromolecular compounds in most macroalgal species. These proteins assemble to form a super-molecular protein complex called phycobilisome, with linkers joining the sub-units [ 20 ]. PBPs from red macroalgae species are essential as anticancer and anti-bacterial drugs. They have the potential to enhance the efficacy of conventional anticancer medications while simultaneously mitigating their adverse effects. Additionally, they can function as photosensitizers for the treatment of infected cells [ 21 ]. Microorganisms, medicinal plants, and marine algae are the primary sources of most cancer medications. Although red seaweed proteins and pigments hold significant prospects for the advancement of nutraceuticals and pharmaceuticals for cancer treatments, substantial gaps in our understanding remain [ 11 , 22 ]. Multiple factors, including their source, extraction methods, and human physiological reactions, effectiveness of algal compounds, requiring a thorough understanding to fully harness their advantages and commercial exploitation. Furthermore, the uses of these pigments and their derivatives in the context of cancer treatment remain largely unexamined. The present study aims to highlight the anticancer effects of phycoerythrin derived from K. alvarezii through its application in human lung cancer cell lines. This article examines the potential of phycoerythrin (PE), a red protein-pigment complex derived from K. alvarezii , as a promising candidate with potential for anticancer drug with a possible treatment for lung cancer. The purified phycoerythrin was tested in vitro for its antimicrobial, antioxidant and anticancer activities, and K. alvarezii derived metabolites was evaluated for the first time through virtual screening and molecular docking analyses. Materials and methods Source of seaweed The red seaweed K. alvarezii was collected alive and healthy from the Mandapam coast (9.2770°N, 79.1252°E) in Rameshwaram, India, and the formal identification was confirmed. We meticulously cleaned the seaweed samples in seawater to remove any superfluous material, including epiphytes and non-living matter, before transporting them to the laboratory in plastic containers. Then, they were thoroughly cleaned and rinsed with sterile distilled water prior to the extraction process. Extraction of phycoerythrin K. alvarezii was cut into small fragments, ground using a sterile pestle and mortar, and transferred to a 250 mL beaker. Sodium phosphate buffer (pH 7.4) was prepared using the required chemicals. The buffer was added to the ground seaweed, and the mixture was subject to a freeze-thaw cycle at -20°C and 37°C. The sample was subsequently centrifuged at 8000 rpm for 15 min. The pellet was discarded, and the supernatant was analyzed for phycoerythrin pigments utilizing a UV/ Vis spectrophotometer at wavelengths of 562, 615, and 652 nm (Beckman, USA) [ 23 ]. Purification of phycoerythrin The isolated phycoerythrin pigment was saturated with ammonium sulfate 60% concentration by continuous stirring at 4°C. Ammonium sulfate was slowly added until saturation was achieved. The phycoerythrin extract was stirred overnight and then centrifuged at 8000 rpm for 15 min at 4°C. The obtained pellet was collected, dried, and prepared for further analysis. To partially purify the phycoerythrin, the extract was subjected to dialysis. The pellet obtained from ammonium sulfate precipitation was placed in a dialysis bag and dialyzed against sterile distilled water until the bag shrank, and the pigment was purified. Preliminary purification was further conducted using gel permeation chromatography with a Sephadex G-100 column. To achieve this process, a 30 × 2 cm column was prepared and equilibrated with 150 mL of sodium phosphate buffer (pH 7). A 10 mL dialyzed and filtered phycoerythrin sample was loaded onto the column. A linear gradient of sodium phosphate buffer, ranging from pH 5 to 8.2, was used to elute the sample. Fractions of 5 mL were collected at a constant flow rate of 20 mL h -1 . The absorption spectrum of the purified phycoerythrin was measured using a UV/ Vis spectrophotometer in the wavelength range of 300–750 nm. The total protein concentration was determined using Lowry's method. Characterization of phycoerythrin Fourier transform infrared (FT-IR) spectroscopy analysis The Perkin-Elmer FT-IR instrument was used to examine IR spectroscopy of phycoerythrin, facilitating the analysis of various sulfate, carboxyl, and hydroxyl groups contained in the purified phycoerythrin pigment. High-performance liquid chromatography (HPLC) analysis The phycoerythrin was separated using a C18 system column (LC-10VP Shimadzu) and eluted with sterile distilled water at a flow rate of 1.0 mL min -1 at 20°C. A refractive index detector was used to keep track of the separated components. HPLC was used to examine the sugar composition of the seaweed extract after it had been hydrolyzed and treated with methanol. The column was calibrated using molecular mass standards and a standard curve was established. Gas chromatography–mass spectrometry (GC-MS) analysis The GC-2010 (GCM-QP 2010) SHIMADZU gas chromatography apparatus, equipped with a flame-ionization detector (FID) and a split injector, was used to analyze phycoerythrin. High-purity helium served as the carrier gas at a flow rate of 1.40 mL min -1 . The column temperature was maintained at 200°C, while the splitter temperature was set to 240°C. A 1 µL sample of dichloromethane was injected through a glass-lined splitter with a split ratio of 1:90. Absorption was measured within the mass-to-charge (m/z) range of 40 to 800. To facilitate visualization by comparing the mass spectra of the components with those from the NIST 14 mass spectral database. Antibacterial activity of phycoerythrin The antibacterial activity of phycoerythrin was evaluated using the well-diffusion method against seven clinical pathogens such as Bacillus subtilis (Ehrenberg 1835) Cohn 1872, Escherichia coli , Klebsiella oxytoca (Flügge 1886) Lautrop 1956, Proteus mirabilis Hauser 1885, Pseudomonas aeruginosa , Staphylococcus aureus , and Streptococcus pyogenes Rosenbach 1884. These bacterial strains were obtained from the Department of Microbiology, Ayya Nadar Janaki Ammal College, Sivakasi, India. Using a sterile cotton swab, 24-hour-old bacterial cultures were aseptically swabbed on Mueller-Hinton agar plates. Wells were loaded with phycoerythrin at a concentration of 30 µg, while Ampicillin at 50 µg served as positive control. The plates were incubated at 35°C for 24 h. After incubation, the diameter of the inhibitory zones around each well was measured (mm) and recorded. In vitro antioxidant activity of phycoerythrin Determination of total antioxidant capacity The total antioxidant activity of phycoerythrin was assessed using the method described by [ 24 ]. A reaction mixture was prepared by combining 3.0 mL of reagent solution (containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) with 0.3 mL of the phycoerythrin sample. The mixture was incubated in a water bath at 95°C for 90 min. After 15 min of cooling, the absorbance of all samples was measured at 695 nm, along with ascorbic acid as a control. Determination of reducing power The reducing power of phycoerythrin was determined by using the method described by [ 25 ]. A 4 mL reaction mixture, consisting of phycoerythrin samples at various concentrations in phosphate buffer (0.2 M, pH 6.6), was incubated with 1% (w/v) potassium ferricyanide at 50°C for 20 min. The reaction was stopped by adding 10% (w/v) trichloroacetic acid (TCA) solution. The resulting solution was mixed with distilled water and 0.1% (w/v) ferric chloride solution. The absorbance was then measured at 700 nm using a UV/ Vis spectrophotometer. Hydrogen peroxide scavenging assay The free radical scavenging activity of phycoerythrin was determined using the hydrogen peroxide assay [ 26 ]. A 10 mM hydrogen peroxide solution was prepared in phosphate-buffered saline (0.1 M, pH 7.4). To perform the assay, 1 mL of the extract at varying concentrations (100, 250, 500, 750, and 1000 µg) was mixed with 2 mL of the hydrogen peroxide solution. The mixture was incubated at 37°C for 10 min. The absorbance was measured at 230 nm using a UV/ Vis spectrophotometer, with a blank (without hydrogen peroxide) used as the reference. DPPH radical scavenging assay The free radical scavenging activity of phycoerythrin was assessed using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method using spectrophotometry [ 27 ]. DPPH, a simple and reliable reagent, was used to evaluate the oxidizable groups in natural or synthetic antioxidants. A 0.1 mM DPPH solution in methanol was prepared, and 1 mL of this solution was added with 3 mL of varying concentrations of phycoerythrin (100, 250, 500, 750, and 1000 µg). The absorbance was measured at 517 nm after 10 min. ABTS inhibition assay The ability of phycoerythrin to scavenge the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical was determined using the method described in [ 28 ]. ABTS was made by combining 5 mL of 7 mM ABTS with 88 µL of 140 mM potassium persulfate, and the mixture was kept in the dark at room temperature for 16 h. The solution was then diluted with 50% ethanol until its absorbance at 734 nm reached 0.7 ± 0.05. For the assay, 5 mL of the prepared ABTS solution was mixed with 0.1 mL of phycoerythrin at various concentrations (100, 250, 500, 750, and 1000 µg). The final absorbance was measured at 743 nm using a UV/ Vis spectrophotometer. In vitro anticancer activity of phycoerythrin Cell culture The human lung cancer cell line (A549) was purchased from the National Centre for Cell Science (NCCS), Pune, and cultured in liquid Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with Fetal Bovine Serum (FBS) 10% (v/v) (Biochrom, Berlin, Germany), penicillin 100 µg/mL, and streptomycin sulphate 100 µg/mL. The cells were maintained in an atmosphere of 5% CO 2 and 100% relative humidity at 37°C. MTT assay for cell cytotoxicity The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphentyltetrazoliumbromide] assay was performed to evaluate the in vitro cytotoxicity of phycoerythrin on A549 cells, as described in [ 29 ]. Cultured A549 cells were harvested by trypsinization and collected into a 15 mL tube. The cells were then plated into a 96-well tissue culture plate at a density of 1 × 10⁵ cells mL -1 at the rate of 200 µL well -1 in DMEM media with 10% FBS and 1% antibiotic solution for 24–48 h at 37°C. After incubation, the medium was replaced with serum-free DMEM, and wells were rinsed with sterile Phosphate Buffer Saline (PBS) and treated with various doses of phycoerythrin. Each sample was performed in triplicate, and the cells were cultured for 24 h at 37°C in a humidified 5% CO 2 incubator. Following, MTT (20 µL at 5 mg mL -1 ) was added to each well after the incubation period, and the cells were incubated for another 2–4 h until purple precipitates were visible under an inverted microscope observation. Finally, the medium was aspirated within the wells together with MTT (220 µL) and rinsed with 200 µL of 1x PBS. The formazan crystals were dissolved by adding 100 µL of Dimethylsulfoxide (DMSO) to each well, and the plate was gently agitated for 5 min. Absorbance at 570 nm was measured using a microplate reader (Thermo Fisher Scientific, USA), and the percentage cell viability and IC 50 values were calculated using GraphPad Prism 6.0 software (USA). Apoptosis assay A549 cells were planted at a density of 5×10 5 cells mL -1 in a 96-well tissue culture plate in DMEM media with 10% FBS and 1% antibiotic solution for 24–48 h at 7°C. After incubation, the medium was replaced with serum-free DMEM, and wells were rinsed with PBS before being treated with 44.33 µg mL -1 of phycoerythrin. Then, the plate was incubated at 37°C in a 5% CO 2 incubator for 24 h. Following the incubation, 10 µL Alexa Fluor and 10 µL propidium iodide were added to the wells, gently mixed, and incubated for 15 min. Subsequently, 400 µL of 1x Annexin binding buffer was added and gently mixed. The plate was centrifuged at 800 rpm for 2 min, and the cells were inspected using a fluorescence microscope with a fluorescent filter within 1 h [ 30 ]. Computational analysis Target preprocessing In the present study, the multitargeted proteins were chosen for the antimicrobial activity as Spore coat polysaccharide biosynthesis protein [PDB Id 1H7L], Staphylococcus aureus tyrosyl-tRNA synthetase [PDB id 1JIJ], Isoleucyl-tRNA synthetase [PDB ID QU3], Transcriptional regulator qacR [PDB ID 1RKW], HTH-Type Transcriptional Regulator MgrA [PDB ID 2BV6], YcgJ protein from Bacillus subtilis [PDB ID 2GLU], Dihydropteroate synthase [PDB ID 2VEG], Processed Glycerol Phosphate Lipoteichoic Acid Synthase 2 [PDB ID 2W8D], DNA Gyrase Subunit B, DNA Gyrase Subunit A [PDB ID 2XCT], DNA topoisomerase 4 subunit A [PDB ID 3RAE], Dihydrofolate reductase [PDB ID 3SRW], Penicillin-binding protein 3 [PDB ID 3VSL], Transcriptional regulator MvfR [PDB ID 4JVC] subjected for the molecular docking studies. These multitargeted proteins were retrieved from the Protein Data Bank (PDB). The preprocessing steps involved assigning bond orders, adding hydrogens, creating zero-order bonds to metals and disulfide bonds, converting seleno-methionines to methionine, and filling missing side chains using the Protein Preparation Wizard of the Maestro platform. The structures were then refined by optimizing hydrogen bonds and minimizing the structures using the OPLS4 force field (Maestro, Schrödinger 2021-2, NY, USA). Molecular docking The flexible ligand docking parameter was enabled via Glide's XP (extra precision) function, and the target proteins and ligand molecules were docked using Maestro's Glide docking module. We determined the optimistic pose of the ligand-protein complex molecule by examining the interaction between the ligand and protein during docking using the XP pose viewer. We used the ligand interaction module to acquire the 2D interaction diagram. We subsequently analyzed the acquired XP pose to investigate the binding interactions of ligand molecules with the target protein [ 31 , 32 ]. Ligand preparation The compound extracted from K. alvarezii , including (1,2-Benzisothiazol-3-amine, 2-Ethylacridine, 2,4-Dimethylbenzo[h]quinoline, 2-bromobutyloxychalcone), has been analyzed and profiled using GC-MS. We subsequently examined the 3D structures of the resultant compounds, retrieving them in the 3D structure data file format from the PubChem databases. We employed the LigPrep module to preprocess the structures, using the OPLS4 force field to minimize energy and generate 32 different stereoisomeric and tautomeric states (Schrödinger Release 2021-2: LigPrep, Schrödinger, LLC, NY, 2021). Statistical analysis The differences in the measured mean values between the triplicate observations were assessed for statistical significance using the student’s two-tailed t test. A p value less than 0.05 was deemed significant. Results and discussion K. alvarezii holds significant importance as a red macroalga due to its bioactive compounds, which hold significant therapeutic potential. Different fields have utilized their diverse, unique, biologically active substances. The evaluation focused on the antioxidant, antimicrobial, and anticancer properties of the extract from K. alvarezii . We conducted a chromatographic examination of its phycoerythrin pigment to gain a deeper understanding of the metabolic constituents involved. Extraction and purification of phycoerythrin pigment Phycoerythrin was successfully extracted from microalgae using cell disruption methods such as sonication, mechanical maceration, and lysozyme treatments [ 21 ]. The centrifuged phycoerythrin pigment extracted from a K. alvarezii exhibited a red color. The purification and recovery involved four steps. The purity ratio of phycoerythrin, successively with ammonium sulfate (151.24 µg mL -1 , purity 2.24, recovery 74%) dialysis (338.71 µg mL -1 , purity 3.39, recovery 35%), and column chromatography yielded highest concentration and purity (554.12 µg mL -1 , purity 5.63, recovery 11%) when compared to crude pigment were reported in Table 1 . K. alvarezii phycoerythrin was found to have a protein content of 69.84%. Similar findings were seen by previous researchers [ 33 , 34 ]. The purification of R-phycoerythrin from the crude extract of Gracilaria gracilis was achieved using a one-step chromatographic method with a phosphate buffer of 20 mM at pH 7.1 [ 35 ], resulting in a high purity index, with an A565/A280 ratio of 3.25. This was accomplished through DEAE Sepharose fast flow column on anion-exchange chromatography, utilizing the fraction obtained at 200 mM NaCl. Compared to this study, our sequential purification process improves both phycoerythrin purity and concentration. Still, there is a trade-off between purity and recovery, with significant losses arising at each stage. Further, process optimization may be necessary to balance recovery efficacy with required purity levels. The selection of the appropriate cell disruption strategy significantly influences the recuperation of the entire process. Gel filtration expanded bed absorption chromatography, hydroxyapatite chromatography, and ion exchange chromatography are among the chromatographic technologies employed for purification. Table 1 Phycoerythrin pigment purification and recovery steps Step Volume (mL) PE (µg mL -1 ) Purity (A620/A280) Recovery (%) Crude pigment 100 78.43 0.95 100 Ammonium sulfate precipitation of pigment 15 151.24 2.24 74 Dialysis of pigment 10 338.71 3.39 35 Sephadex G-100 Column chromatography of pigment 5 554.12 5.63 11 Characterization of phycoerythrin FT-IR analysis The phycoerythrin bond and functional group analysis of FT-IR is shown in Fig. 1 . The peak 3446.56 cm -1 shows in O–H stretch and H–bonded stretching vibration presence of alcohols and phenols. The peak 3130.25 cm -1 was present in the O–H stretching vibration functional group of carboxylic acids. The peak was 2920.99 cm -1 C–H stretching vibration presence of alkanes. The peak 1719.42 cm -1 C = O stretching vibration presence of alpha, beta–unsaturated esters. The peak 1610.45 cm -1 , 1528.48 cm -1 and 1443.62 cm -1 C–C stretch (in–ring) presence of aromatics. The peak 1383.83 cm -1 , 1264.25 cm -1 , 1139.85 cm -1 and 1056.92 cm -1 C–O stretch presence of alcohols, carboxylic acids, esters, ethers. The peak 994.24 cm -1 , 951.81 cm -1 , 865.01 cm -1 , 816.80 cm -1 , 761.83 cm -1 and 619.11 cm -1 =C–H bend alkenes. The peak 535.21 cm -1 C–Br stretching vibration presence of alkyl halides. The FT-IR spectra is used to examine functional groups or chemical bonds within a molecule of an interaction system; it is used to analyze the functional groups or chemical bonds present in that molecule. Prior to extraction, the spectra display the absorption bands of DES at 1472 cm⁻¹ and dialyzed R-PE at 1074 cm⁻¹. FT-IR analysis of C-phycoerythrin (CPE) showed significant changes in the amide III, indicating interactions within the side chains. The protein backbone, on the other hand, seemed to have stayed mostly the same [ 36 ]. HPLC analysis HPLC analysis of phycoerythrin showed in Fig. 2 . The obtained peaks were 2.110 (kaempferitrin), 2.823 (γ- tocopherol), 3.060 (β-sitosterol) and 4.353 (corilagin). Similar types of compounds were detected in a rapid two-step chromatographic technique for the purification of B-phycoerythrin from Porphyridium cruentum (S.F.Gray) Nägeli is presented [ 37 ]. The separation of its α-, β-, and γ-subunits was successfully accomplished using a reversed-phase HPLC gradient semipreparative approach. This involved a C4 large-pore column, and a solvent system made up of 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile. GC-MS analysis GC-MS analysis of the phycoerythrin revealed the following compounds such as 16.832 (-2-bromobutyloxychalcone), 17.024 (2-Ethylacridine), 17.072 (1,2-Benzisothiazol-3-amine) and 17.148 (Benzo[h]quinoline, 2,4-dimethyl-) within the peaks shown in Fig. 3 . According [ 38 ], researchers collected Gracilaria corticata from Mandapam and subsequently identified and characterized it using GC-MS. Researchers prepared and analyzed the methanol extract using GC-MS to identify the various bioactive compounds present in the seaweed. The analysis identified a variety of bioactive compounds, including undecane, 2-decyloxirane, methyl n-tridecanoate, n-hexadecanoic acid, eicosanoic acid, nonanoic acid, oleic acid, pentadecanoic acid, bicycle [3.2.1] oct-3-en-2-one, 3,8-dihydroxy-1-1methoxy-7-(7-methoxy-1, 3 benzodioxol-5-yl)-6-methyl-5, N-(5-chloro-2-hydroxyphenyl) dodecanamide, and cholesta-8,24-dien-3-ol, 4-methyl. This research work examined the bioactive compounds derived from the methanol extract of seaweed through GC-MS analysis, highlighting their significant antimicrobial and free radical scavenging activities. Antibacterial activity of phycoerythrin The antibacterial activity of phycoerythrin (30 µg) was evaluated against seven clinical pathogens. The maximum zone of suppression was found on Klebsiella oxytoca (20 mm), followed by Escherichia coli (17 mm), and Pseudomonas aeruginosa (18 mm), though generally less effective than Ampicillin (50 µg), which exhibited inhibition zones of 17–23 mm shown in Table 2 . Similarly [ 39 ], the phycoerythrin of Microchaetes has antibacterial activity against human pathogens. Increasing the concentrations of phycoerythrin to 0.1 mg mL⁻¹ resulted in a higher percentage of inhibition across all bacterial strains tested. The antibacterial action of macroalgae involves changing the permeability of pathogen cells. This alteration leads to the loss of macromolecules, disrupts membrane functions, and ultimately results in the destruction of the bacterial cells [ 40 , 41 ]. Research has demonstrated the effectiveness of seaweeds such as K. alvarezii, Kappaphycus striatum , and Ulva lactuca L. with antagonistic effects against human pathogenic bacteria [ 42 ]. Table 2 Antibacterial activity of phycoerythrin S. No Bacterial pathogens Ampicillin 50 µg (mm) Phycoerythrin 30 µg (mm) 1 Bacillus subtilis 17 12 2 Escherichia coli 20 17 3 Klebsiella oxytoca 23 20 4 Proteus mirabilis 19 11 5 Pseudomonas aeruginosa 21 18 6 Staphylococcus aureus 21 13 7 Streptococcus pyogenes 18 15 Antioxidant activity of phycoerythrin pigment The phycoerythrin pigment showed a total antioxidant capacity of 73.27 ± 0.43%, reducing power of 71.09 ± 0.23%, hydrogen peroxide scavenging activity of 67.54 ± 0.41%. Also, it showed 58.13 ± 0.99% activity in the DPPH, and 61.92 ± 0.34% in the ABTS assay, portraying its antioxidant potential (Table 3 ). Previous studies also proved the antioxidant potential of phycoerythrin from multiple seaweed sources. According to [ 43 ], hydrophilic PBPs from red macroalgae, have antioxidant activity against free radicals and selenium in vitro and in vivo . According to [ 44 ], phycoerythrin has antioxidant, anti-inflammatory, and hepatoprotective effects that are advantageous to human health. Algal antioxidants are divided into two types [ 45 ]. The water-soluble antioxidants include vitamins, PBPs, and polyphenols, while the fat-soluble antioxidants consist of carotenoids and tocopherol. The scavenging amount of DPPH radical by purified phycoerythrin varied with concentration, showing a concentration-dependent relationship, with an IC 50 value of 0.043 mg mL -1 , in contrast to 0.031 mg mL -1 for ascorbic acid [ 39 ]. This indicates that phycoerythrin serves as an effective free radical scavenger, exhibiting DPPH scavenging properties comparable to those of ascorbic acid. The scavenging percentage of the ABTS radical by natural phycoerythrin varies with concentration. Phycoerythrin exhibited an IC 50 of 0.023 mg mL -1 , in contrast to Standard Butylated Hydroxyl Toluene (BHT), which had an IC 50 of 0.031 mg mL -1 . Phycoerythrin exhibits comparable ABTS scavenging activities to BHT and serves as a potent free radical scavenger [ 39 ]. The radical scavenging capacity of extracts of Nostoc linckia at 5 mg l -1 improved with increasing PBPs content [ 46 ]. PBPs inhibits the generation of reactive oxygen species and hence the risk of diseases including cancer, diabetes, inflammation, and neurological diseases [ 47 ]. Table 3 Percentage of in vitro antioxidant activity of phycoerythrin S. No Antioxidant activity Phycoerythrin 1 Total antioxidant capacity 73. 27 ± 0.43% 2 Reducing power 71.09 ± 0.23% 3 Hydrogen peroxide scavenging assay 67.54 ± 0.41% 4 DPPH 58.13 ± 0.99% 5 ABTS 61.92 ± 0.34% Anticancer action of phycoerythrin pigment Cytotoxicity of cells by MTT assay This study utilized the MTT assay to investigate the ways phycoerythrin affects the growth of A549 cancer cells. The cells endured culture for a duration of 24 h across a range of phycoerythrin concentrations. The viability of the cells was assessed through the MTT assay following a 24-hour incubation period. The alterations in the morphology of human lung cancer cells were observed following treatment with varying concentrations of phycoerythrin, as detailed in Table 4 . The test showed a clear relationship between dosage and the level of toxicity in A549 human lung cancer cells. The cell viability showed a decline from 91.24 ± 0.09% at a concentration of 50 µg mL -1 to 46.36 ± 0.16% at 500 µg mL -1 . Significant reductions were also observed at 200 µg mL -1 (74.29 ± 0.07%) and 400 µg mL -1 (54.85 ± 0.11%), emphasizing its promise as a potential anticancer agent. The concentrations of phycoerythrin at 50 µg mL -1 and 100 µg mL -1 indicated the presence of viable human lung cancer cells. The concentration of 100 µg mL -1 of phycoerythrin showed the existence of both viable and non-viable cells. The levels of phycoerythrin between 200 and 400 µg mL -1 indicated a minimal count of visible cells (Fig. 4 ). Table 4 Viability of human lung cancer cells on cytotoxicity of phycoerythrin S. No Concentration (µg mL -1 ) Cell viability (%) 1 Control 100 ± 0.02 2 50 91.24 ± 0.09 3 100 82.59 ± 0.12 4 200 74.29 ± 0.07 5 300 69.04 ± 0.14 6 400 54.85 ± 0.11 7 500 46.36 ± 0.16 Human lung cancer cells treated with phycoerythrin experience growth inhibition. The tested sample exhibited an IC 50 value of 131.7 µg mL⁻¹. Ultimately, as the concentration of phycoerythrin increased, the presence of the cell steadily reduced. It is important to note that phycoerythrin demonstrates a more potent effect against human lung cancer cells shown in Table 5 . Similarly, the various solvent extracts of Gracilaria edulis were evaluated against cancer cell lines for their anti-proliferative capabilities [ 48 ]. The G. edulis ethyl acetate extract (GEEA) at a concentration of 100 µg mL -1 demonstrated a notable and highly significant reduction in growth within the A549 lung cancer cell line model. Additionally, when assessing a control group against a GEEA extract-treated group at different dosages (40, 60, 80, and 100 µg mL -1 ) after 48 h, a minimal level of LDH release was observed. The cell cytotoxicity assay [ 39 ], revealed that phycoerythrin extracted from Michrochaete acts as an anticancer agent in the Hep G2 cell line at specific doses (20–160 g mL -1 ), demonstrating an IC 50 value of 105.77 g mL -1 . In a similar way, anticancer efficacy of Gracilaria cortica phycoerythrin was reported against the HepG2 cell line [ 49 ]. Table 5 Tested phycoerythrin shows IC 50 values Log (inhibitor) Phycoerythrin (µg mL -1 ) Best-fit values LogIC 50 2.119 HillSlope -1.017 IC 50 131.7 Std. Error LogIC 50 0.05416 HillSlope 0.1334 95% Confidence Intervals LogIC 50 2.008 to 2.230 HillSlope -1.290 to -0.7434 IC 50 102.0 to 170.0 Goodness of Fit Degrees of Freedom 28 R square 0.8809 Absolute sum of squares 3584 Sy.x 11.31 Number of points Analyzed 30 Phycoerythrin induces apoptosis in cancer cells The evaluation of apoptotic cells involved staining phycoerythrin with human lung cancer cells, as illustrated in Fig. 5 . The control cells exhibited the highest number of viable cells, as indicated by green-coloured fluorescence. The percentage of apoptotic cells that tested positive for Annexin V/PI in human lung cancer cells treated with varying dosages of phycoerythrin (203.4 µg mL -1 ) increased in accordance with the dosage levels. Cells treated with fluorouracil demonstrated the presence of both viable and non-viable cells. In addition to identifying the morphological changes associated with apoptosis, we also assessed the apoptosis rate using the annexin V test. Annexin V has a strong and specific attraction for damaged and compromised plasma membranes, making apoptotic and necrotic cells stand out. The recent study demonstrated that phycoerythrin-induced apoptotic cell death in A549 lung cancer cells. Cell apoptosis [ 50 ], refers to a form of programmed cell death that relies on energy. Caspase belongs to the family of enzymes known for their specificity to aspartic acid and homocysteine proteases. These enzymes play a crucial role in degrading antiapoptotic proteins, leading to the release of mitochondrial cytochrome C. Molecular docking analysis Computational biology combines computational techniques with biological systems to tackle disease-related challenges. Virtual screening, a key tool, reduces drug development costs and time by predicting receptor-ligand binding and affinities through docking and scoring. Molecular docking enhances drug discovery by accurately identifying ligand orientations in protein binding sites and predicting interaction strengths, significantly advancing the field [ 32 ]. By utilizing molecular docking, targeting the presence of antibacterial proteins in these compounds for the first time with the K. alvarezii extracted bioactive compounds. The 13 antibacterial multitarget proteins were docked with 4 compounds detected in GC-MS analysis of the phycoerythrin, revealing that a lower docking score corresponds to higher binding affinity (Table 6 ; Fig. 7 – 19 ). Molecular docking of K. alvarezii-derived compounds revealed significant binding affinities with 13 antibacterial target proteins. Among the results, the protein DNA gyrase subunits A (GyrA) and B (GyrB) showed a binding score of -1.13 kcal/mol with 1,2-Benzisothiazol-3-amine. Staphylococcus aureus tyrosyl-tRNA synthetase protein scored − 2.86 kcal/mol with 1,2-Benzisothiazol-3-amine, while dihydropteroate synthase and transcriptional regulator qacR scored − 1.17 kcal/mol and − 1.21 kcal/mol, respectively, with 2-bromobutyloxychalcone. Penicillin-binding protein 3 (-1.41 kcal/mol), DNA topoisomerase 4 subunit A (-1.66 kcal/mol), and isoleucyl-tRNA synthetase (-3.10 kcal/mol) also showed strong interactions with 1,2-Benzisothiazol-3-amine. Other substantial interactions included HTH-type transcriptional regulator MgrA (-2.39 kcal/mol) and processed glycerol phosphate lipoteichoic acid synthase 2 (-3.51 kcal/mol) with 2,4-dimethylbenzo[h]quinoline, and YcgJ protein from Bacillus subtilis (-2.52 kcal/mol) with 2-bromobutyloxychalcone. Particularly, transcriptional regulator MvfR (-4.88 kcal/mol) interacted strongly with 1,2-Benzisothiazol-3-amine, and dihydrofolate reductase exhibited the highest binding affinity (-5.24 kcal/mol) with 2-ethylacridine. In general, 1,2-Benzisothiazol-3-amine and 2-bromobutyloxychalcone demonstrated strong interactions with multiple targets, while 2-ethylacridine showed the highest affinity with dihydrofolate reductase (-5.24 kcal/mol), emerged as the most potent compound. Table 6 The molecular docking of the antimicrobial target proteins with the compounds Compound ID Compound Name Amino acid Interaction Bond Length Glide Score Glide Energy 1H7L - Spore coat polysaccharide biosynthesis protein SpsA 89966 1,2-Benzisothiazol-3-amine HIS 159, TYR 77. [2.03]; [4.21, 3.70]. -3.44 -20.03 610161 2-Ethylacridine PHE 233, TYR 11, TYR 77, ARG 76. [5.03]; [5.49]; [5.21, 5.84, 5.45]; [5.31]. -3.90 -24.14 610182 2,4-Dimethylbenzo[h]quinoline ARG 76, LEU 80. [5.17]; [4.81]. -4.95 -27.35 91733949 2-bromobutyloxychalcone THR 9, ASP 99, TYR 11, ARG 76, TYR 77. [2.21]; [2.50, 2.59]; [5.55]; [5.18, 5.12]; [5.24]. -4.63 -37.07 1JIJ - Staphylococcus aureus tyrosyl-tRNA synthetase 89966 1,2-Benzisothiazol-3-amine ALA 39, ASP 40. [4.38,4.23]; [2.74,1.94]. -2.86 -22.97 610161 2-Ethylacridine ASP 195, TYR 36, CYS37, GLY 192. [4.51]; [5.31]; [2.94,5.17,5.04]; [2.80,2.92]. -3.82 -27.65 610182 2,4-Dimethylbenzo[h]quinoline ASP 80, TYR 36, LEU 70, CYS 37. [4.45]; [5.26]; [3.81]; [4.05]. -3.94 -32.71 91733949 2-bromobutyloxychalcone LEU 70, GLN 190, CYS 37, GLY 193, GLN196. [4.80]; [2.08]; [4.93]; [3.08]; [2.77]. -4.79 -46.30 1QU3 - Isoleucyl-tRNA synthetase (IleRS) 89966 1,2-Benzisothiazol-3-amine GLU 554. [1.73]. -3.10 -22.76 610161 2-Ethylacridine GLY 554, TRP 562, TRP 528, [1.64]; [5.41, 4.94]; [4.78]. -7.92 -35.62 610182 2,4-Dimethylbenzo[h]quinoline PRO 56, GLU 554, TRP 562, ASP 557, HIS 64. [5.26]; [2.41]; [5.97, 5.13]; [4.46, 5.22]; [5.47]. -6.25 -30.45 91733949 2-bromobutyloxychalcone TRP 528, GLY 555, GLU 554, ASN 70, HIS 67, PRO 57. [5.97]; [2.14]; [2.84]; [2.68]; [4.24]; [5.37]. -4.71 -40.99 1RKW - Transcriptional regulator qacR 89966 1,2-Benzisothiazol-3-amine HIS 42, GLU 52. [1.93]; [3.67, 3.49]. -1.98 -16.57 610161 2-Ethylacridine GLU 52, LYS 115, LYS 118, LEU 119. [4.52]; [4.21, 4.58, 5.17, 4.00, 4.21]; [4.31]; [5.16]. -2.95 -31.49 610182 2,4-Dimethylbenzo[h]quinoline LYS 115, GLU 52. [4.24]; [4.40, 3.62, 3.70]. -1.80 -19.45 91733949 2-bromobutyloxychalcone ASN 55, LYS 118, LYS 115. [2.32]; [4.36]; [3.08,4.92]. -1.21 -26.36 2BV6 - HTH-type transcriptional regulator MgrA 89966 1,2-Benzisothiazol-3-amine ALA 63, LEU 62, LEU 42, PRO 39, TYR 38. [2.19, 4.96]; [2.81, 4.48]; [5.42]; [4.90]; [5.50]. -3.06 -17.13 610161 2-Ethylacridine LEU 42, PRO 39, LEU 62, ALA 63. [4.91]; [4.84]; [5.01]; [2.26, 5.12, 5.03]. -2.75 -20.26 610182 2,4-Dimethylbenzo[h]quinoline ALA 63, LEU 62, LEU 42, PRO 39, TYR 38. [5.10, 4.93, 3.95, 2.25]; [5.37]; [5.13]; [5.28, 4.88]; [5.30] -2.39 -19.83 91733949 2-bromobutyloxychalcone VAL 116, TYR 38, GLN 19, LEU 134, PHE 41, LEU 42. [4.94]; [3.00]; [2.05, 2.98]; [4.23]; [4.32]; [4.99]. -3.89 -30.05 2GLU - YcgJ protein from Bacillus subtilis 89966 1,2-Benzisothiazol-3-amine PRO 119, TYR 117. [5.12]; [2.08]. -2.85 -15.11 610161 2-Ethylacridine LEU 145, LEU 167, PRO 168, GLN 149. [5.27]; [4.96]; [5.12, 4.86]; [2.14, 2.82]. -2.61 -18.78 610182 2,4-Dimethylbenzo[h]quinoline TYR 159, GLN 149, LEU 145, GLU 120. [5.35]; [1.94]; [4.48, 5.09]; [4.21, 4.48]. -3.35 -20.95 91733949 2-bromobutyloxychalcone PRO 168, GLU 120, PRO 119. [4.42]; [2.90]; [2.57, 4.82]. -2.52 -30.91 2VEG - Dihydropteroate synthase 89966 1,2-Benzisothiazol-3-amine ASP 221, GLN 276, ARG 275, LLE 272. [1.92]; [3.06]; [4.15, 4.13]; [5.42]. -3.22 -16.19 610161 2-Ethylacridine ARG 275, LLE 272, ASP 221. [3.88, 5.36, 3.95]; [4.85]; [2.07]. -4.24 -2312 610182 2,4-Dimethylbenzo[h]quinoline ARG 275, ASP 221. [4.39, 4.59]; [3.87, 3.68]. -2.71 -23.28 91733949 2-bromobutyloxychalcone ARG 275. [5.45, 4.01]. -1.17 -26.44 2W8D - Processed glycerol phosphate lipoteichoic acid synthase 2 89966 1,2-Benzisothiazol-3-amine TRP 350, ASN 478, HIS 479, PHE 413. [5.23, 4.85, 3.53, 4.21]; [3.02]; [2.45]; [5.04]. -4.05 -21.07 610161 2-Ethylacridine TRP 350, ASN 478, HIS 479. [4.97, 4.03, 5.01, 3.78, 4.46]; [3.10]; [4.79, 4.07]. -4.58 -24.35 610182 2,4-Dimethylbenzo[h]quinoline HIS 479, PHE 349, ASN 478, TYR 380, PHE 413, TRP 350. [4.31]; [4.74]; [2.68]; [5.09]; [504]; [4.19, 5.08, 5.15, 4.87, 4.48, 3.44]. -3.51 -18.95 91733949 2-bromobutyloxychalcone TPO 297, TRP 350, TYR 380, PHE 349, ASN 478, PHE 413. [4.02]; [2.27]; [5.64]; [4.32]; [2.07]; [4.76]. -7.16 -36.62 2XCT - DNA gyrase subunit B, DNA gyrase subunit A 89966 1,2-Benzisothiazol-3-amine LLE 1452, LYS 1375, ALA 1378, GLU 1382, LEU 1449. [5.16]; [3.55, 4.20]; [5.32]; [1.92]; [5.07]. -0.97 -11.96 610161 2-Ethylacridine GLU 1382, LEU 1449, LYS 135, LLE 1452. [1.52]; [5.13, 4.76]; [3.97]; [4.17]. -2.69 -27.77 610182 2,4-Dimethylbenzo[h]quinoline LLE 1452, LYS 1375, ALA 1378, LEU 1449, GLU 1382. [5.16]; [5.14]; [3.65]; [4.97, 4.57]; [5.51]. -2.86 -22.49 91733949 2-bromobutyloxychalcone LLE 1452, LEU 1449, ASN 474. [4.97]; [5.28, 5.38]; [2.11]. -1.13 -26.97 3RAE - DNA topoisomerase 4 subunit A 89966 1,2-Benzisothiazol-3-amine PRO 113, ARG 117, MET 116, ALA 115. [2.95]; [4.72]; [5.45]; [4.13, 3.91]. -1.66 -24.21 610161 2-Ethylacridine PRO 112, TYR 82, ALA 115, MET 116. [5.02]; [4.42]; [4.68]; [4.39]. -2.95 -31.49 610182 2,4-Dimethylbenzo[h]quinoline ASP 83, MET 116, ALA 115. [4.01]; [5.52]; [4.71, 4.65]. -3.64 -29.51 91733949 2-bromobutyloxychalcone ALLA 115, MET 116, ARG 117. [4.76]; [5.30]; [2.40]. -2.79 -34.49 3SRW - Dihydrofolate reductase 89966 1,2-Benzisothiazol-3-amine LLE 15, LEU 21, ALA 8, PHE 93. [2.07]; [5.15, 5.43]; [2.14, 2.27,5.13]; [5.18, 5.63]. -6.08 -22.26 610161 2-Ethylacridine LEU 21, LEU 29, PHE 93, LLE 15. [4.46, 4.35]; [5.14]; [5.28]; [4.70]. -5.24 -278994 610182 2,4-Dimethylbenzo[h]quinoline PHE 93, LLE 15, LEU 21. [4.87]; [5.36]; [4.51, 5.10]. -5.63 -29.9 91733949 2-bromobutyloxychalcone VAL 132, VAL 17, LLE 15, ALA 8, LEU 21, ASN 19. [5.43]; [2.61]; [5.28]; [1.99, 5.32]; [4.84]; [2.59]. -7.13 -41.79 3VSL - Penicillin-binding protein 3 89966 1,2-Benzisothiazol-3-amine ASN 501, VAL 493, LYS 494. [1.94]; [5.34, 3.25]; [4.87, 3.98, 4.27]. -1.41 -19.70 610161 2-Ethylacridine LYS 494, ASN 501, PRO 500, TYR 278. [4.91, 3.07, 2.81]; [3.01, 2.59]; [5.20] [4.97]. -3.22 -24.34 610182 2,4-Dimethylbenzo[h]quinoline LYS 494, ASN 501, PRO 500. [4.76, 5.38]; [2.76, 2.15]; [5.30, 5.45]. -2.75 -22.93 91733949 2-bromobutyloxychalcone TYR 278, LYS 494, HIS 259, LEU 576. [4.61]; [1.98]; [5.56]; [5.06]. -4.66 -35.39 4JVC - Transcriptional regulator MvfR 89966 1,2-Benzisothiazol-3-amine LEU 197, GLN 194, ARG 209, LEU 207, LLE 236, LEU 208. [2.63]; [2.90]; [2.07]; [2.58]; [2.81, 3.98]; [4.77, 5.14]. -4.88 -27.06 610161 2-Ethylacridine LLE 236, ALA 102, ALA 168, LLE 149, LEU 208. [4.92, 3.87, 4.17]; [4.27]; [4.16, 3.25]; [4.95, 4.05]; [4.99, 4.94]. -6.71 -19.70 610182 2,4-Dimethylbenzo[h]quinoline LLE 149, ALA 168, LLE 236, VAL 211, LEU 208. [5.32]; [4.52]; [4.54, 3.98, 4.12, 5.45]; [5.48]; [4.80,4.87]. -6.71 -28.29 91733949 2-bromobutyloxychalcone ALA 102, PRO 238, ALA 168, LLE 236, LLE 149, LEU 208, ARG 209. [4.37]; [4.96] [3.22]; [5.29, 2.87]; [4.31]; [5.14]; [4.02]; -7.26 -34.37 Conclusions The scope of the present paper was to assess the potential of seaweed as a novel source for marine pharmaceuticals. This study successfully isolated and purified phycoerythrin from K. alvarezii using a process that included ammonium sulfate precipitation, dialysis, and column chromatography. In the FT-IR test, many different functional groups were found. In the HPLC test, bioactive compounds such as kaempferitrin, β-sitosterol, and 2-bromobutyloxychalcone were found. The antioxidant activity of phycoerythrin was very high, with a total antioxidant capacity of 73.27% and strong scavenging abilities in the DPPH and ABTS tests. Its antibacterial activity was most effective against K. oxytoca (20 mm) and moderately active against other pathogens. MTT assays verified that cytotoxicity against A549 human lung cancer cells was dose-dependent, and Annexin V/PI staining revealed increased apoptotic activity. Molecular docking further emphasized the potential of phycoerythrin as a natural source of bioactive compounds for therapeutic applications, highlighting its potential in antimicrobial target protein interactions. The outcome of this study demonstrates the potential of the red seaweed K. alvarezii against cancer cell lines, supporting the way forward for the development of phycoerythrin as anti-cancer drug with pharmaceutical prospective. Clinical trial number Not applicable Statement We implemented all methods in accordance with pertinent regulations and guidelines. The authors have not conducted any experiments on humans or utilized human tissue samples. The ethics committee of the institute approved all experimental protocols and panelists involved in the study. Declarations The authors declare that data supporting the findings of this study will be available from the corresponding author upon reasonable request. Clinical trial number Not applicable Statement We implemented all methods in accordance with pertinent regulations and guidelines. The authors have not conducted any experiments on humans or utilized human tissue samples. The ethics committee of the institute approved all experimental protocols and panelists involved in the study. Acknowledgement Authors would like to express their gratitude to Ayya Nadar Janaki Ammal College, Tamil Nadu, for providing the necessary facilities to conduct this research, as well as the International Centre for Genetic Engineering and Biotechnology, New Delhi for their assistance with molecular docking. The authors extend their deep appreciation to Researchers Supporting Project number (RSPD2025R741), King Saud University, Riyadh, Saudi Arabia. Authors contributions H.B., G.R.P. and D.P. conceived and designed the experiments. P.S. and S.S. drafted the main manuscript text. H.B., S.M., M.M., J.P. and P.M. conducted the extraction, GC-MS analysis, and antibacterial, antioxidant, and anticancer experiments. M.A., S.V. and P.S. carried out the molecular docking analyses. D.P. and A.P. supervised the research project. F.U., H.O.E., M.N., A.A.F., M.A.R. and I.M.M. contributed to funding acquisition, manuscript review and editing, and project administration. Funding Researchers Supporting Project number (RSPD2025R741), King Saud University. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests Additional information Correspondenceand requests for materials should be addressed to D.P. References Menaa F, Wijesinghe U, Thiripuranathar G, Althobaiti NA, Albalawi AE, Khan BA, et al. Marine algae-derived bioactive compounds: A new wave of nanodrugs? Mar Drugs. 2021;19:1–36. Ahmed N, Sheikh MA, Ubaid M, Chauhan P, Kumar K, Choudhary S. 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Zandi K, Tajbakhsh S, Nabipour I, Rastian Z, Yousefi F, Sharafian S, et al. In vitro antitumor activity of Gracilaria corticata (a red alga) against jurkat and molt-4 human cancer cell lines. African J Biotechnol. 2010;9:6787–90. Logue SE, Martin SJ. Caspase activation cascades in apoptosis. Biochem Soc Trans. 2008;36:1–9. Additional Declarations No competing interests reported. Supplementary Files Annexure.docx Cite Share Download PDF Status: Published Journal Publication published 31 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 Mar, 2025 Reviews received at journal 25 Feb, 2025 Reviews received at journal 18 Feb, 2025 Reviewers agreed at journal 04 Feb, 2025 Reviews received at journal 02 Feb, 2025 Reviewers agreed at journal 02 Feb, 2025 Reviewers agreed at journal 31 Jan, 2025 Reviewers invited by journal 31 Jan, 2025 Editor assigned by journal 30 Jan, 2025 Editor invited by journal 30 Jan, 2025 Submission checks completed at journal 28 Jan, 2025 First submitted to journal 22 Jan, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5880917","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":408210589,"identity":"74d7c7d2-cfba-499e-925f-d250b607856d","order_by":0,"name":"Harinathan Balasundaram","email":"","orcid":"","institution":"Krishna Institute of Allied Sciences","correspondingAuthor":false,"prefix":"","firstName":"Harinathan","middleName":"","lastName":"Balasundaram","suffix":""},{"id":408210590,"identity":"226cb66a-0070-4086-ab36-fd9a742dd7b2","order_by":1,"name":"Parthasarathy Seethapathy","email":"","orcid":"","institution":"Amrita School of Agricultural Sciences, Amrita Vishwa Vidyapeetham","correspondingAuthor":false,"prefix":"","firstName":"Parthasarathy","middleName":"","lastName":"Seethapathy","suffix":""},{"id":408210591,"identity":"58720ef6-5e7f-42a9-a2f4-8aeb29fe28f6","order_by":2,"name":"Subbiah Sankaralingam","email":"","orcid":"","institution":"Virudhunagar Hindu Nadars and Senthikumara Nadar College","correspondingAuthor":false,"prefix":"","firstName":"Subbiah","middleName":"","lastName":"Sankaralingam","suffix":""},{"id":408210592,"identity":"a087cc01-49f1-4dcd-b235-8b07f3c57c13","order_by":3,"name":"Shunmugiah Mahendran","email":"","orcid":"","institution":"Ayya Nadar Janaki Ammal College (Autonomous)","correspondingAuthor":false,"prefix":"","firstName":"Shunmugiah","middleName":"","lastName":"Mahendran","suffix":""},{"id":408210593,"identity":"edc0ddac-55ba-4df7-8492-bc1fae44381f","order_by":4,"name":"Murugan Mareeswaran","email":"","orcid":"","institution":"Ayya Nadar Janaki Ammal College (Autonomous)","correspondingAuthor":false,"prefix":"","firstName":"Murugan","middleName":"","lastName":"Mareeswaran","suffix":""},{"id":408210594,"identity":"dcf5134b-87e7-4b9b-b196-a564da6c00e5","order_by":5,"name":"Jeyaraj Pandiarajan","email":"","orcid":"","institution":"Ayya Nadar Janaki Ammal College (Autonomous)","correspondingAuthor":false,"prefix":"","firstName":"Jeyaraj","middleName":"","lastName":"Pandiarajan","suffix":""},{"id":408210595,"identity":"bc4d1a23-9f83-4b34-89af-4d82afeff86c","order_by":6,"name":"Pandiaraj Maheswari","email":"","orcid":"","institution":"Ayya Nadar Janaki Ammal College (Autonomous)","correspondingAuthor":false,"prefix":"","firstName":"Pandiaraj","middleName":"","lastName":"Maheswari","suffix":""},{"id":408210596,"identity":"cc3d8ed8-c103-4e96-baa7-6f55f91d4822","order_by":7,"name":"Sakthivel Venkatesh","email":"","orcid":"","institution":"Virudhunagar Hindu Nadars and Senthikumara Nadar College","correspondingAuthor":false,"prefix":"","firstName":"Sakthivel","middleName":"","lastName":"Venkatesh","suffix":""},{"id":408210597,"identity":"7d4525c6-6063-458b-8219-5974ae7756e3","order_by":8,"name":"Malaisamy Arunkumar","email":"","orcid":"","institution":"International Centre for Genetic Engineering and Biotechnology (ICGEB)","correspondingAuthor":false,"prefix":"","firstName":"Malaisamy","middleName":"","lastName":"Arunkumar","suffix":""},{"id":408210598,"identity":"583ffa49-e7da-4c25-97fc-0132eb6f2c59","order_by":9,"name":"Girish R. 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Elansary","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Hosam","middleName":"O.","lastName":"Elansary","suffix":""},{"id":408210603,"identity":"7d566710-ef5a-4bbf-a76a-2984019a6ae9","order_by":14,"name":"Muhammad Nazim","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Nazim","suffix":""},{"id":408210604,"identity":"b533638d-f031-452d-bf9a-1866781b6749","order_by":15,"name":"Adel A. Fickak","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Adel","middleName":"A.","lastName":"Fickak","suffix":""},{"id":408210605,"identity":"57302955-1dca-43b7-b89d-70af274d63ea","order_by":16,"name":"Mohamed A. Rashwan","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Mohamed","middleName":"A.","lastName":"Rashwan","suffix":""},{"id":408210606,"identity":"9e140892-81b2-420f-b510-f59e7c204dca","order_by":17,"name":"Ihab Mohamed Moussa","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Ihab","middleName":"Mohamed","lastName":"Moussa","suffix":""}],"badges":[],"createdAt":"2025-01-22 12:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5880917/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5880917/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-11899-7","type":"published","date":"2025-07-31T16:12:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75118556,"identity":"9fb22eed-3c99-47a9-ba98-f5ff3be4318d","added_by":"auto","created_at":"2025-01-30 16:47:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1040075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eKappaphycus alvarezii\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/d932bfa52ce8399adeed347f.png"},{"id":75118553,"identity":"b426e2c4-1e25-4ec3-9b4c-487cb942e779","added_by":"auto","created_at":"2025-01-30 16:47:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107898,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR analysis of phycoerythrin\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/ce846c013c5c4d88459ee915.png"},{"id":75119256,"identity":"3f82f0e8-820d-4c6a-a201-83560fd7a643","added_by":"auto","created_at":"2025-01-30 16:55:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90209,"visible":true,"origin":"","legend":"\u003cp\u003eHPLC analysis of phycoerythrin\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/bae865d2c75739fa53ee7aa9.png"},{"id":75118573,"identity":"99f8c6e5-5136-4cdb-a8dc-cf19189a96db","added_by":"auto","created_at":"2025-01-30 16:47:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38666,"visible":true,"origin":"","legend":"\u003cp\u003eGC-MS analysis of phycoerythrin\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/d8ac5145fc074afbc494c609.png"},{"id":75118548,"identity":"fdb4e735-5a37-4ab3-b809-df2b5b1bdf84","added_by":"auto","created_at":"2025-01-30 16:47:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165367,"visible":true,"origin":"","legend":"\u003cp\u003eThe morphological view of human lung cancer cells assesses the proliferation of cell inhibitory concentrations (50, 100, 200, 300, 400, and 500 µg mL\u003csup\u003e-1\u003c/sup\u003e) of phycoerythrin pigment.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/cefe5f665f66e37b1b7e2fb4.png"},{"id":75118550,"identity":"6bb3bfa5-ed27-4b0f-802d-eff93ac9bc75","added_by":"auto","created_at":"2025-01-30 16:47:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":51460,"visible":true,"origin":"","legend":"\u003cp\u003ePhycoerythrin produces programmed cell death in human lung cancer cells. (a) Control cells exhibited a green color, signifying that the cells were alive; (b) Cells treated with phycoerythrin at a concentration of 203.4 μg mL⁻¹ displayed live cells (green color), dead cells (red color), and apoptotic cells (yellow color).\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/db54dcfc45ae2bd56bd31744.png"},{"id":75118576,"identity":"b269e27e-40ce-4309-b759-0da72791567d","added_by":"auto","created_at":"2025-01-30 16:47:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":315000,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Spore coat polysaccharide biosynthesis protein SpsA docked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinolone (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/0ce2f7e68359ba65ab0971fe.png"},{"id":75118616,"identity":"b2f64af5-4dc5-4bb6-8191-98fe9e9a0aa7","added_by":"auto","created_at":"2025-01-30 16:47:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":320619,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein \u003cem\u003eS. aureus \u003c/em\u003etyrosyl-tRNA synthetase docked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture8.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/5c90a350efe879fb6174d3fe.png"},{"id":75118583,"identity":"5a2b485f-da26-4b10-b1c4-228c3e7a833d","added_by":"auto","created_at":"2025-01-30 16:47:18","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":312704,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Isoleucyl-tRNA synthetase docked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture9.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/14fd90974b301cbb5c9cb00a.png"},{"id":75118587,"identity":"698c77f0-0c0d-4580-bef8-dd9ad2ab13eb","added_by":"auto","created_at":"2025-01-30 16:47:18","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":276454,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Transcriptional regulator qacR\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture10.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/feb0e42ccd409e50ae98757d.png"},{"id":75119290,"identity":"5a4f359d-00b4-4935-929b-103a911e82ff","added_by":"auto","created_at":"2025-01-30 16:55:21","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":297724,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein HTH-Type Transcriptional Regulator MgrA docked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture11.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/cee9316b6aea4d072154bfa3.png"},{"id":75118555,"identity":"917de8f6-4510-4e09-8bec-c366f1ff47b6","added_by":"auto","created_at":"2025-01-30 16:47:16","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":292848,"visible":true,"origin":"","legend":"\u003cp\u003eYcgJ protein from \u003cem\u003eBacillus subtilis\u003c/em\u003e [PDB ID 2GLU] with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture12.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/dff0419a180493d0e258de17.png"},{"id":75119261,"identity":"a3223ae8-7305-45a3-9d4a-2b319dee37c8","added_by":"auto","created_at":"2025-01-30 16:55:17","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":279765,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Dihydropteroate synthase\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture13.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/f81d60cbad2aa831bc49b9d0.png"},{"id":75118551,"identity":"0dfbcaf0-d788-43bd-b684-61e96f6985d1","added_by":"auto","created_at":"2025-01-30 16:47:15","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":323908,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Processed Glycerol Phosphate Lipoteichoic Acid Synthase 2\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture14.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/b2c88b0db6e87a086bb0ff27.png"},{"id":75119279,"identity":"1b71186a-7d1a-44ca-9667-4b7b20eaed4d","added_by":"auto","created_at":"2025-01-30 16:55:19","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":260432,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein DNA Gyrase Subunit B, DNA Gyrase Subunit A\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture15.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/662b97cf0b4bbc89ffe7d59e.png"},{"id":75119297,"identity":"e91d979b-c602-4d41-9f8a-b8a5a0079702","added_by":"auto","created_at":"2025-01-30 16:55:21","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":310978,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein DNA topoisomerase 4 subunit A\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture16.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/f3499835c8526a7b59537daf.png"},{"id":75118605,"identity":"95190027-9fa9-4392-9c8f-0dd382a6bc7c","added_by":"auto","created_at":"2025-01-30 16:47:19","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":359365,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Dihydrofolate reductase\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture17.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/f2058884d8eb4089620d218f.png"},{"id":75118589,"identity":"cb0edaf3-1aa9-4b1a-b304-69bb5e6ceeba","added_by":"auto","created_at":"2025-01-30 16:47:18","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":972244,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein \u003cem\u003ePenicillin-\u003c/em\u003ebinding protein 3 docked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture18.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/baf184951ccb92352a3faea2.png"},{"id":75118590,"identity":"857a3382-568b-449b-be37-b3ebeaab8194","added_by":"auto","created_at":"2025-01-30 16:47:18","extension":"png","order_by":19,"title":"Figure 19","display":"","copyAsset":false,"role":"figure","size":319748,"visible":true,"origin":"","legend":"\u003cp\u003eThe target protein Transcriptional regulator MvfR\u003cem\u003e \u003c/em\u003edocked with the compound of three- and two-dimensional structure such as 1,2-Benzisothiazol-3-amine (a, b), 2-Ethylacridine (c, d), 2,4-Dimethylbenzo[h]quinoline (e, f), 2-bromobutyloxychalcone (g, h) respectively.\u003c/p\u003e","description":"","filename":"Picture19.png","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/d286ff8b71481f9806f879d3.png"},{"id":88268154,"identity":"41276d34-a812-46d7-8d22-caea98c71ac0","added_by":"auto","created_at":"2025-08-04 16:49:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8622299,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/c9af0f36-87bc-458a-bafc-7031204962b7.pdf"},{"id":75119259,"identity":"46a871b5-60fd-4908-b8f4-932770cb1c6c","added_by":"auto","created_at":"2025-01-30 16:55:17","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8454530,"visible":true,"origin":"","legend":"","description":"","filename":"Annexure.docx","url":"https://assets-eu.researchsquare.com/files/rs-5880917/v1/911d53aa64d09789dad99f48.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Isolation, Characterization, and Biomedical Potential of Phycoerythrin Phycobiliprotein from Kappaphycus alvarezii (Doty) Doty ex Silva: Antimicrobial, Antioxidant, and Anticancer Activities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMarine organisms produce a diversified array of bioactive compounds including carbohydrates, lipids, proteins, polyphenols, minerals, amines, amides, antioxidants, and pigments with unique molecular structures and varied biological functions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Currently, these bioactive compounds are recognized as valuable sources for the food and health care sectors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, there has been a rising attention in the research and development focusing on commercial exploitation of marine organisms derived bioactive primary and secondary metabolites as an ecofriendly and effective alternate for synthetic drugs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In recent years, the discovery of unique bioactive substances with significant dietary, nutraceutical and medicinal advantages has attracted significant attention to marine algae.\u003c/p\u003e \u003cp\u003eMarine algae are essential components of the food chain, constituting over 70% of the global living biomass [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Marine algae are photosynthetic organisms both macroscopic and microscopic, that have garnered attention due to their immense potential as a sustainable and environmentally friendly source for several bioactive compounds [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Marine algae or seaweed are growing habitually through autotrophic, mixotrophic, or heterotrophic ways [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The potential of marine algae to be cultivated in fresh, saline and waste waters, along with their fast multiplication, presents them as an attractive substitute for diverse products, thereby conserving time and resources [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Seaweeds are divided into three categories based on their photosynthetic pigments: green macroalgae, brown macroalgae, and red macroalgae. Among these, red macroalgae account for over 60% of the 30,000 tons of algae produced globally [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A variety of red macroalgae are rich in bioactive compounds, making them a preferred choice for the production of commercially valuable bioproducts such as biofertilizers, biofuels, colorants, cosmetics, food, hydrocolloids, nutraceuticals, pharmaceuticals, plant stimulants and thickeners [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e (Doty) Doty ex. Silva (elkhorn sea moss) of family Solieriaceae, is a red seaweed (macroalgae) and is the primary carrageenophyte, commercially known as \u0026ldquo;cottoni\u0026rdquo;. It is mostly cultivated to produce commercial \u003cem\u003ekappa\u003c/em\u003e-carrageenan hydrocolloids in both habitual and non-indigenous areas and has gained potential economic value in the seaweed industry. This macroalgae is the fifth most cultivated in the world, especially in various tropical countries of Asia and Africa, including India [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], due to its fast growing rates, simple cultivation process, high biomass yield in a short time with high polysaccharide content, and low investment requirements [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It also serves as a food source for local populations in Southeast Asia and is believed to possess various health benefits. The yellowish, reddish, green, and brown colors of \u003cem\u003eK. alvarezii\u003c/em\u003e are based on the levels of phycoerythrin protein pigment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The highly variable chemical components and quantities in \u003cem\u003eK. alvarezii\u003c/em\u003e are influenced by the cultivation conditions, including water temperature, salinity, radiation, environmental variables, light level, profundity, and wave strength. On average, \u003cem\u003eK. alvarezii\u003c/em\u003e consists of 50.8% carbohydrates, 15.6% ash, 12.4% sulphated groups, 3.3% proteins, 3.3% lipids, and 3.0% insoluble aromatics [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The literature indicates extensive use of \u003cem\u003eK. alvarezii\u003c/em\u003e for carrageenan extraction. Furthermore, reports suggest that extracts of \u003cem\u003eK. alvarezii\u003c/em\u003e possesses a multitude of essential compounds and high-value molecules with antioxidant and anti-inflammatory, antitumor, antiviral, antidiabetic, antihyperlipidemic activities, and for neural dysfunction properties [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCancer is a fatal disease that poses a significant risk to individuals worldwide, ranking as the second most common cause of mortality in numerous countries. In 2022, the global community experienced 20\u0026nbsp;million new cases of cancer and 9.7\u0026nbsp;million fatalities associated with this disease [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Lung cancer ranks among the most common cancers and is a primary contributor to cancer-related fatalities worldwide [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Patients diagnosed with lung cancer encounter a higher risk of meeting bacterial infections, which can vary from mild cases to those that pose significant threats to life. Both bacteria and viruses have the capability to trigger inflammatory cells and activate inflammatory signaling pathways. In particular, \u003cem\u003eEscherichia coli\u003c/em\u003e (Migula 1895) Castellani and Chalmers 1919, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (Schr\u0026ouml;ter 1872) Migula 1900, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e Rosenbach 1884, and \u003cem\u003eKlebsiella\u003c/em\u003e sp. are gram-negative bacteria that are often found in the lungs of cancer patients [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These bacteria are often found along with other factors like age-related illnesses, large tumors, and rapid clinical decline [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The currently available cancer medications are come with more physical and psychological side effects like pain, vomiting, diarrhea, exhaustion, and nausea; thus, the cancer therapies cannot be considered safe [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Consequently, it is essential to explore novel anticancer medications sourced from nature that are safe, affordable, and less detrimental. The anti-cancer capability of \u003cem\u003eK. alvarezii\u003c/em\u003e could be due to polysaccharides, nutrients, and other metabolites with antioxidant potential [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The phycobiliprotein (PBPs) family includes phycoerythrin (PE), which are macromolecular compounds in most macroalgal species. These proteins assemble to form a super-molecular protein complex called phycobilisome, with linkers joining the sub-units [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. PBPs from red macroalgae species are essential as anticancer and anti-bacterial drugs. They have the potential to enhance the efficacy of conventional anticancer medications while simultaneously mitigating their adverse effects. Additionally, they can function as photosensitizers for the treatment of infected cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroorganisms, medicinal plants, and marine algae are the primary sources of most cancer medications. Although red seaweed proteins and pigments hold significant prospects for the advancement of nutraceuticals and pharmaceuticals for cancer treatments, substantial gaps in our understanding remain [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Multiple factors, including their source, extraction methods, and human physiological reactions, effectiveness of algal compounds, requiring a thorough understanding to fully harness their advantages and commercial exploitation. Furthermore, the uses of these pigments and their derivatives in the context of cancer treatment remain largely unexamined. The present study aims to highlight the anticancer effects of phycoerythrin derived from \u003cem\u003eK. alvarezii\u003c/em\u003e through its application in human lung cancer cell lines. This article examines the potential of phycoerythrin (PE), a red protein-pigment complex derived from \u003cem\u003eK. alvarezii\u003c/em\u003e, as a promising candidate with potential for anticancer drug with a possible treatment for lung cancer. The purified phycoerythrin was tested \u003cem\u003ein vitro\u003c/em\u003e for its antimicrobial, antioxidant and anticancer activities, and \u003cem\u003eK. alvarezii\u003c/em\u003e derived metabolites was evaluated for the first time through virtual screening and molecular docking analyses.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSource of seaweed\u003c/h2\u003e \u003cp\u003eThe red seaweed \u003cem\u003eK. alvarezii\u003c/em\u003e was collected alive and healthy from the Mandapam coast (9.2770\u0026deg;N, 79.1252\u0026deg;E) in Rameshwaram, India, and the formal identification was confirmed. We meticulously cleaned the seaweed samples in seawater to remove any superfluous material, including epiphytes and non-living matter, before transporting them to the laboratory in plastic containers. Then, they were thoroughly cleaned and rinsed with sterile distilled water prior to the extraction process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtraction of phycoerythrin\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eK. alvarezii\u003c/em\u003e was cut into small fragments, ground using a sterile pestle and mortar, and transferred to a 250 mL beaker. Sodium phosphate buffer (pH 7.4) was prepared using the required chemicals. The buffer was added to the ground seaweed, and the mixture was subject to a freeze-thaw cycle at -20\u0026deg;C and 37\u0026deg;C. The sample was subsequently centrifuged at 8000 rpm for 15 min. The pellet was discarded, and the supernatant was analyzed for phycoerythrin pigments utilizing a UV/ Vis spectrophotometer at wavelengths of 562, 615, and 652 nm (Beckman, USA) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003ePurification of phycoerythrin\u003c/h3\u003e\n\u003cp\u003eThe isolated phycoerythrin pigment was saturated with ammonium sulfate 60% concentration by continuous stirring at 4\u0026deg;C. Ammonium sulfate was slowly added until saturation was achieved. The phycoerythrin extract was stirred overnight and then centrifuged at 8000 rpm for 15 min at 4\u0026deg;C. The obtained pellet was collected, dried, and prepared for further analysis. To partially purify the phycoerythrin, the extract was subjected to dialysis. The pellet obtained from ammonium sulfate precipitation was placed in a dialysis bag and dialyzed against sterile distilled water until the bag shrank, and the pigment was purified. Preliminary purification was further conducted using gel permeation chromatography with a Sephadex G-100 column. To achieve this process, a 30 \u0026times; 2 cm column was prepared and equilibrated with 150 mL of sodium phosphate buffer (pH 7). A 10 mL dialyzed and filtered phycoerythrin sample was loaded onto the column. A linear gradient of sodium phosphate buffer, ranging from pH 5 to 8.2, was used to elute the sample. Fractions of 5 mL were collected at a constant flow rate of 20 mL h\u003csup\u003e-1\u003c/sup\u003e. The absorption spectrum of the purified phycoerythrin was measured using a UV/ Vis spectrophotometer in the wavelength range of 300\u0026ndash;750 nm. The total protein concentration was determined using Lowry's method.\u003c/p\u003e\n\u003ch3\u003eCharacterization of phycoerythrin\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFourier transform infrared (FT-IR) spectroscopy analysis\u003c/h2\u003e \u003cp\u003eThe Perkin-Elmer FT-IR instrument was used to examine IR spectroscopy of phycoerythrin, facilitating the analysis of various sulfate, carboxyl, and hydroxyl groups contained in the purified phycoerythrin pigment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHigh-performance liquid chromatography (HPLC) analysis\u003c/h2\u003e \u003cp\u003eThe phycoerythrin was separated using a C18 system column (LC-10VP Shimadzu) and eluted with sterile distilled water at a flow rate of 1.0 mL min\u003csup\u003e-1\u003c/sup\u003e at 20\u0026deg;C. A refractive index detector was used to keep track of the separated components. HPLC was used to examine the sugar composition of the seaweed extract after it had been hydrolyzed and treated with methanol. The column was calibrated using molecular mass standards and a standard curve was established.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGas chromatography–mass spectrometry (GC-MS) analysis\u003c/h3\u003e\n\u003cp\u003eThe GC-2010 (GCM-QP 2010) SHIMADZU gas chromatography apparatus, equipped with a flame-ionization detector (FID) and a split injector, was used to analyze phycoerythrin. High-purity helium served as the carrier gas at a flow rate of 1.40 mL min\u003csup\u003e-1\u003c/sup\u003e. The column temperature was maintained at 200\u0026deg;C, while the splitter temperature was set to 240\u0026deg;C. A 1 \u0026micro;L sample of dichloromethane was injected through a glass-lined splitter with a split ratio of 1:90. Absorption was measured within the mass-to-charge (m/z) range of 40 to 800. To facilitate visualization by comparing the mass spectra of the components with those from the NIST 14 mass spectral database.\u003c/p\u003e\n\u003ch3\u003eAntibacterial activity of phycoerythrin\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of phycoerythrin was evaluated using the well-diffusion method against seven clinical pathogens such as \u003cem\u003eBacillus subtilis\u003c/em\u003e (Ehrenberg 1835) Cohn 1872, \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e (Fl\u0026uuml;gge 1886) Lautrop 1956, \u003cem\u003eProteus mirabilis\u003c/em\u003e Hauser 1885, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, and \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e Rosenbach 1884. These bacterial strains were obtained from the Department of Microbiology, Ayya Nadar Janaki Ammal College, Sivakasi, India. Using a sterile cotton swab, 24-hour-old bacterial cultures were aseptically swabbed on Mueller-Hinton agar plates. Wells were loaded with phycoerythrin at a concentration of 30 \u0026micro;g, while Ampicillin at 50 \u0026micro;g served as positive control. The plates were incubated at 35\u0026deg;C for 24 h. After incubation, the diameter of the inhibitory zones around each well was measured (mm) and recorded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eantioxidant activity of phycoerythrin\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of total antioxidant capacity\u003c/h2\u003e \u003cp\u003eThe total antioxidant activity of phycoerythrin was assessed using the method described by [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. A reaction mixture was prepared by combining 3.0 mL of reagent solution (containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) with 0.3 mL of the phycoerythrin sample. The mixture was incubated in a water bath at 95\u0026deg;C for 90 min. After 15 min of cooling, the absorbance of all samples was measured at 695 nm, along with ascorbic acid as a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of reducing power\u003c/h2\u003e \u003cp\u003eThe reducing power of phycoerythrin was determined by using the method described by [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A 4 mL reaction mixture, consisting of phycoerythrin samples at various concentrations in phosphate buffer (0.2 M, pH 6.6), was incubated with 1% (w/v) potassium ferricyanide at 50\u0026deg;C for 20 min. The reaction was stopped by adding 10% (w/v) trichloroacetic acid (TCA) solution. The resulting solution was mixed with distilled water and 0.1% (w/v) ferric chloride solution. The absorbance was then measured at 700 nm using a UV/ Vis spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHydrogen peroxide scavenging assay\u003c/h2\u003e \u003cp\u003eThe free radical scavenging activity of phycoerythrin was determined using the hydrogen peroxide assay [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. A 10 mM hydrogen peroxide solution was prepared in phosphate-buffered saline (0.1 M, pH 7.4). To perform the assay, 1 mL of the extract at varying concentrations (100, 250, 500, 750, and 1000 \u0026micro;g) was mixed with 2 mL of the hydrogen peroxide solution. The mixture was incubated at 37\u0026deg;C for 10 min. The absorbance was measured at 230 nm using a UV/ Vis spectrophotometer, with a blank (without hydrogen peroxide) used as the reference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDPPH radical scavenging assay\u003c/h2\u003e \u003cp\u003eThe free radical scavenging activity of phycoerythrin was assessed using the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) method using spectrophotometry [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. DPPH, a simple and reliable reagent, was used to evaluate the oxidizable groups in natural or synthetic antioxidants. A 0.1 mM DPPH solution in methanol was prepared, and 1 mL of this solution was added with 3 mL of varying concentrations of phycoerythrin (100, 250, 500, 750, and 1000 \u0026micro;g). The absorbance was measured at 517 nm after 10 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eABTS inhibition assay\u003c/h2\u003e \u003cp\u003eThe ability of phycoerythrin to scavenge the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical was determined using the method described in [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. ABTS was made by combining 5 mL of 7 mM ABTS with 88 \u0026micro;L of 140 mM potassium persulfate, and the mixture was kept in the dark at room temperature for 16 h. The solution was then diluted with 50% ethanol until its absorbance at 734 nm reached 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. For the assay, 5 mL of the prepared ABTS solution was mixed with 0.1 mL of phycoerythrin at various concentrations (100, 250, 500, 750, and 1000 \u0026micro;g). The final absorbance was measured at 743 nm using a UV/ Vis spectrophotometer.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eanticancer activity of phycoerythrin\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe human lung cancer cell line (A549) was purchased from the National Centre for Cell Science (NCCS), Pune, and cultured in liquid Dulbecco's Modified Eagle Medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with Fetal Bovine Serum (FBS) 10% (v/v) (Biochrom, Berlin, Germany), penicillin 100 \u0026micro;g/mL, and streptomycin sulphate 100 \u0026micro;g/mL. The cells were maintained in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and 100% relative humidity at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMTT assay for cell cytotoxicity\u003c/h2\u003e \u003cp\u003eThe MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphentyltetrazoliumbromide] assay was performed to evaluate the \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity of phycoerythrin on A549 cells, as described in [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Cultured A549 cells were harvested by trypsinization and collected into a 15 mL tube. The cells were then plated into a 96-well tissue culture plate at a density of 1 \u0026times; 10⁵ cells mL\u003csup\u003e-1\u003c/sup\u003e at the rate of 200 \u0026micro;L well\u003csup\u003e-1\u003c/sup\u003e in DMEM media with 10% FBS and 1% antibiotic solution for 24\u0026ndash;48 h at 37\u0026deg;C. After incubation, the medium was replaced with serum-free DMEM, and wells were rinsed with sterile Phosphate Buffer Saline (PBS) and treated with various doses of phycoerythrin. Each sample was performed in triplicate, and the cells were cultured for 24 h at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. Following, MTT (20 \u0026micro;L at 5 mg mL\u003csup\u003e-1\u003c/sup\u003e) was added to each well after the incubation period, and the cells were incubated for another 2\u0026ndash;4 h until purple precipitates were visible under an inverted microscope observation. Finally, the medium was aspirated within the wells together with MTT (220 \u0026micro;L) and rinsed with 200 \u0026micro;L of 1x PBS. The formazan crystals were dissolved by adding 100 \u0026micro;L of Dimethylsulfoxide (DMSO) to each well, and the plate was gently agitated for 5 min. Absorbance at 570 nm was measured using a microplate reader (Thermo Fisher Scientific, USA), and the percentage cell viability and IC\u003csub\u003e50\u003c/sub\u003e values were calculated using GraphPad Prism 6.0 software (USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eA549 cells were planted at a density of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells mL\u003csup\u003e-1\u003c/sup\u003e in a 96-well tissue culture plate in DMEM media with 10% FBS and 1% antibiotic solution for 24\u0026ndash;48 h at 7\u0026deg;C. After incubation, the medium was replaced with serum-free DMEM, and wells were rinsed with PBS before being treated with 44.33 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e of phycoerythrin. Then, the plate was incubated at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 24 h. Following the incubation, 10 \u0026micro;L Alexa Fluor and 10 \u0026micro;L propidium iodide were added to the wells, gently mixed, and incubated for 15 min. Subsequently, 400 \u0026micro;L of 1x Annexin binding buffer was added and gently mixed. The plate was centrifuged at 800 rpm for 2 min, and the cells were inspected using a fluorescence microscope with a fluorescent filter within 1 h [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eComputational analysis\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eTarget preprocessing\u003c/h2\u003e \u003cp\u003eIn the present study, the multitargeted proteins were chosen for the antimicrobial activity as Spore coat polysaccharide biosynthesis protein [PDB Id 1H7L], \u003cem\u003eStaphylococcus aureus\u003c/em\u003e tyrosyl-tRNA synthetase [PDB id 1JIJ], Isoleucyl-tRNA synthetase [PDB ID QU3], Transcriptional regulator qacR [PDB ID 1RKW], HTH-Type Transcriptional Regulator MgrA [PDB ID 2BV6], YcgJ protein from \u003cem\u003eBacillus subtilis\u003c/em\u003e [PDB ID 2GLU], Dihydropteroate synthase [PDB ID 2VEG], Processed Glycerol Phosphate Lipoteichoic Acid Synthase 2 [PDB ID 2W8D], DNA Gyrase Subunit B, DNA Gyrase Subunit A [PDB ID 2XCT], DNA topoisomerase 4 subunit A [PDB ID 3RAE], Dihydrofolate reductase [PDB ID 3SRW], Penicillin-binding protein 3 [PDB ID 3VSL], Transcriptional regulator MvfR [PDB ID 4JVC] subjected for the molecular docking studies. These multitargeted proteins were retrieved from the Protein Data Bank (PDB). The preprocessing steps involved assigning bond orders, adding hydrogens, creating zero-order bonds to metals and disulfide bonds, converting seleno-methionines to methionine, and filling missing side chains using the Protein Preparation Wizard of the Maestro platform. The structures were then refined by optimizing hydrogen bonds and minimizing the structures using the OPLS4 force field (Maestro, Schr\u0026ouml;dinger 2021-2, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking\u003c/h2\u003e \u003cp\u003eThe flexible ligand docking parameter was enabled via Glide's XP (extra precision) function, and the target proteins and ligand molecules were docked using Maestro's Glide docking module. We determined the optimistic pose of the ligand-protein complex molecule by examining the interaction between the ligand and protein during docking using the XP pose viewer. We used the ligand interaction module to acquire the 2D interaction diagram. We subsequently analyzed the acquired XP pose to investigate the binding interactions of ligand molecules with the target protein [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLigand preparation\u003c/h2\u003e \u003cp\u003eThe compound extracted from \u003cem\u003eK. alvarezii\u003c/em\u003e, including (1,2-Benzisothiazol-3-amine, 2-Ethylacridine, 2,4-Dimethylbenzo[h]quinoline, 2-bromobutyloxychalcone), has been analyzed and profiled using GC-MS. We subsequently examined the 3D structures of the resultant compounds, retrieving them in the 3D structure data file format from the PubChem databases. We employed the LigPrep module to preprocess the structures, using the OPLS4 force field to minimize energy and generate 32 different stereoisomeric and tautomeric states (Schr\u0026ouml;dinger Release 2021-2: LigPrep, Schr\u0026ouml;dinger, LLC, NY, 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe differences in the measured mean values between the triplicate observations were assessed for statistical significance using the student\u0026rsquo;s two-tailed t test. A \u003cem\u003ep\u003c/em\u003e value less than 0.05 was deemed significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cp\u003e \u003cem\u003eK. alvarezii\u003c/em\u003e holds significant importance as a red macroalga due to its bioactive compounds, which hold significant therapeutic potential. Different fields have utilized their diverse, unique, biologically active substances. The evaluation focused on the antioxidant, antimicrobial, and anticancer properties of the extract from \u003cem\u003eK. alvarezii\u003c/em\u003e. We conducted a chromatographic examination of its phycoerythrin pigment to gain a deeper understanding of the metabolic constituents involved.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eExtraction and purification of phycoerythrin pigment\u003c/h2\u003e \u003cp\u003ePhycoerythrin was successfully extracted from microalgae using cell disruption methods such as sonication, mechanical maceration, and lysozyme treatments [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The centrifuged phycoerythrin pigment extracted from a \u003cem\u003eK. alvarezii\u003c/em\u003e exhibited a red color. The purification and recovery involved four steps. The purity ratio of phycoerythrin, successively with ammonium sulfate (151.24 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e, purity 2.24, recovery 74%) dialysis (338.71 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e, purity 3.39, recovery 35%), and column chromatography yielded highest concentration and purity (554.12 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e, purity 5.63, recovery 11%) when compared to crude pigment were reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eK. alvarezii\u003c/em\u003e phycoerythrin was found to have a protein content of 69.84%. Similar findings were seen by previous researchers [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The purification of R-phycoerythrin from the crude extract of \u003cem\u003eGracilaria gracilis\u003c/em\u003e was achieved using a one-step chromatographic method with a phosphate buffer of 20 mM at pH 7.1 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], resulting in a high purity index, with an A565/A280 ratio of 3.25. This was accomplished through DEAE Sepharose fast flow column on anion-exchange chromatography, utilizing the fraction obtained at 200 mM NaCl. Compared to this study, our sequential purification process improves both phycoerythrin purity and concentration. Still, there is a trade-off between purity and recovery, with significant losses arising at each stage. Further, process optimization may be necessary to balance recovery efficacy with required purity levels. The selection of the appropriate cell disruption strategy significantly influences the recuperation of the entire process. Gel filtration expanded bed absorption chromatography, hydroxyapatite chromatography, and ion exchange chromatography are among the chromatographic technologies employed for purification.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhycoerythrin pigment purification and recovery steps\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStep\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVolume (mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePE (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePurity (A620/A280)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrude pigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmmonium sulfate precipitation of pigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e151.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDialysis of pigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e338.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSephadex G-100 Column chromatography of pigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e554.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of phycoerythrin\u003c/h2\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eFT-IR analysis\u003c/h2\u003e \u003cp\u003eThe phycoerythrin bond and functional group analysis of FT-IR is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The peak 3446.56 cm\u003csup\u003e-1\u003c/sup\u003e shows in O\u0026ndash;H stretch and H\u0026ndash;bonded stretching vibration presence of alcohols and phenols. The peak 3130.25 cm\u003csup\u003e-1\u003c/sup\u003e was present in the O\u0026ndash;H stretching vibration functional group of carboxylic acids. The peak was 2920.99 cm\u003csup\u003e-1\u003c/sup\u003e C\u0026ndash;H stretching vibration presence of alkanes. The peak 1719.42 cm\u003csup\u003e-1\u003c/sup\u003e C\u0026thinsp;=\u0026thinsp;O stretching vibration presence of alpha, beta\u0026ndash;unsaturated esters. The peak 1610.45 cm\u003csup\u003e-1\u003c/sup\u003e, 1528.48 cm\u003csup\u003e-1\u003c/sup\u003e and 1443.62 cm\u003csup\u003e-1\u003c/sup\u003e C\u0026ndash;C stretch (in\u0026ndash;ring) presence of aromatics. The peak 1383.83 cm\u003csup\u003e-1\u003c/sup\u003e, 1264.25 cm\u003csup\u003e-1\u003c/sup\u003e, 1139.85 cm\u003csup\u003e-1\u003c/sup\u003e and 1056.92 cm\u003csup\u003e-1\u003c/sup\u003e C\u0026ndash;O stretch presence of alcohols, carboxylic acids, esters, ethers. The peak 994.24 cm\u003csup\u003e-1\u003c/sup\u003e, 951.81 cm\u003csup\u003e-1\u003c/sup\u003e, 865.01 cm\u003csup\u003e-1\u003c/sup\u003e, 816.80 cm\u003csup\u003e-1\u003c/sup\u003e, 761.83 cm\u003csup\u003e-1\u003c/sup\u003e and 619.11 cm\u003csup\u003e-1\u003c/sup\u003e =C\u0026ndash;H bend alkenes. The peak 535.21 cm\u003csup\u003e-1\u003c/sup\u003e C\u0026ndash;Br stretching vibration presence of alkyl halides. The FT-IR spectra is used to examine functional groups or chemical bonds within a molecule of an interaction system; it is used to analyze the functional groups or chemical bonds present in that molecule. Prior to extraction, the spectra display the absorption bands of DES at 1472 cm⁻\u0026sup1; and dialyzed R-PE at 1074 cm⁻\u0026sup1;. FT-IR analysis of C-phycoerythrin (CPE) showed significant changes in the amide III, indicating interactions within the side chains. The protein backbone, on the other hand, seemed to have stayed mostly the same [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eHPLC analysis\u003c/h2\u003e \u003cp\u003eHPLC analysis of phycoerythrin showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The obtained peaks were 2.110 (kaempferitrin), 2.823 (γ- tocopherol), 3.060 (β-sitosterol) and 4.353 (corilagin). Similar types of compounds were detected in a rapid two-step chromatographic technique for the purification of B-phycoerythrin from \u003cem\u003ePorphyridium cruentum\u003c/em\u003e (S.F.Gray) N\u0026auml;geli is presented [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The separation of its α-, β-, and γ-subunits was successfully accomplished using a reversed-phase HPLC gradient semipreparative approach. This involved a C4 large-pore column, and a solvent system made up of 0.05% trifluoroacetic acid (TFA) in water and 0.05% TFA in acetonitrile.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eGC-MS analysis\u003c/h2\u003e \u003cp\u003eGC-MS analysis of the phycoerythrin revealed the following compounds such as 16.832 (-2-bromobutyloxychalcone), 17.024 (2-Ethylacridine), 17.072 (1,2-Benzisothiazol-3-amine) and 17.148 (Benzo[h]quinoline, 2,4-dimethyl-) within the peaks shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. According [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], researchers collected \u003cem\u003eGracilaria corticata\u003c/em\u003e from Mandapam and subsequently identified and characterized it using GC-MS. Researchers prepared and analyzed the methanol extract using GC-MS to identify the various bioactive compounds present in the seaweed. The analysis identified a variety of bioactive compounds, including undecane, 2-decyloxirane, methyl n-tridecanoate, n-hexadecanoic acid, eicosanoic acid, nonanoic acid, oleic acid, pentadecanoic acid, bicycle [3.2.1] oct-3-en-2-one, 3,8-dihydroxy-1-1methoxy-7-(7-methoxy-1, 3 benzodioxol-5-yl)-6-methyl-5, N-(5-chloro-2-hydroxyphenyl) dodecanamide, and cholesta-8,24-dien-3-ol, 4-methyl. This research work examined the bioactive compounds derived from the methanol extract of seaweed through GC-MS analysis, highlighting their significant antimicrobial and free radical scavenging activities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntibacterial activity of phycoerythrin\u003c/h3\u003e\n\u003cp\u003eThe antibacterial activity of phycoerythrin (30 \u0026micro;g) was evaluated against seven clinical pathogens. The maximum zone of suppression was found on \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e (20 mm), followed by \u003cem\u003eEscherichia coli\u003c/em\u003e (17 mm), and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (18 mm), though generally less effective than Ampicillin (50 \u0026micro;g), which exhibited inhibition zones of 17\u0026ndash;23 mm shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Similarly [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], the phycoerythrin of Microchaetes has antibacterial activity against human pathogens. Increasing the concentrations of phycoerythrin to 0.1 mg mL⁻\u0026sup1; resulted in a higher percentage of inhibition across all bacterial strains tested. The antibacterial action of macroalgae involves changing the permeability of pathogen cells. This alteration leads to the loss of macromolecules, disrupts membrane functions, and ultimately results in the destruction of the bacterial cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Research has demonstrated the effectiveness of seaweeds such as \u003cem\u003eK. alvarezii, Kappaphycus striatum\u003c/em\u003e, and \u003cem\u003eUlva lactuca\u003c/em\u003e L. with antagonistic effects against human pathogenic bacteria [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibacterial activity of phycoerythrin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBacterial pathogens\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmpicillin 50 \u0026micro;g (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePhycoerythrin 30 \u0026micro;g (mm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eKlebsiella oxytoca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eProteus mirabilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus pyogenes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant activity of phycoerythrin pigment\u003c/h2\u003e \u003cp\u003eThe phycoerythrin pigment showed a total antioxidant capacity of 73.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%, reducing power of 71.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23%, hydrogen peroxide scavenging activity of 67.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41%. Also, it showed 58.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99% activity in the DPPH, and 61.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34% in the ABTS assay, portraying its antioxidant potential (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Previous studies also proved the antioxidant potential of phycoerythrin from multiple seaweed sources. According to [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], hydrophilic PBPs from red macroalgae, have antioxidant activity against free radicals and selenium \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. According to [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], phycoerythrin has antioxidant, anti-inflammatory, and hepatoprotective effects that are advantageous to human health. Algal antioxidants are divided into two types [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The water-soluble antioxidants include vitamins, PBPs, and polyphenols, while the fat-soluble antioxidants consist of carotenoids and tocopherol. The scavenging amount of DPPH radical by purified phycoerythrin varied with concentration, showing a concentration-dependent relationship, with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.043 mg mL\u003csup\u003e-1\u003c/sup\u003e, in contrast to 0.031 mg mL\u003csup\u003e-1\u003c/sup\u003e for ascorbic acid [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. This indicates that phycoerythrin serves as an effective free radical scavenger, exhibiting DPPH scavenging properties comparable to those of ascorbic acid. The scavenging percentage of the ABTS radical by natural phycoerythrin varies with concentration. Phycoerythrin exhibited an IC\u003csub\u003e50\u003c/sub\u003e of 0.023 mg mL\u003csup\u003e-1\u003c/sup\u003e, in contrast to Standard Butylated Hydroxyl Toluene (BHT), which had an IC\u003csub\u003e50\u003c/sub\u003e of 0.031 mg mL\u003csup\u003e-1\u003c/sup\u003e. Phycoerythrin exhibits comparable ABTS scavenging activities to BHT and serves as a potent free radical scavenger [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The radical scavenging capacity of extracts of \u003cem\u003eNostoc linckia\u003c/em\u003e at 5 mg l\u003csup\u003e-1\u003c/sup\u003e improved with increasing PBPs content [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. PBPs inhibits the generation of reactive oxygen species and hence the risk of diseases including cancer, diabetes, inflammation, and neurological diseases [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage of \u003cem\u003ein vitro\u003c/em\u003e antioxidant activity of phycoerythrin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAntioxidant activity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePhycoerythrin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal antioxidant capacity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e73. 27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReducing power\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e71.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHydrogen peroxide scavenging assay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e67.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDPPH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e58.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eABTS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e61.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eAnticancer action of phycoerythrin pigment\u003c/h2\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eCytotoxicity of cells by MTT assay\u003c/h2\u003e \u003cp\u003eThis study utilized the MTT assay to investigate the ways phycoerythrin affects the growth of A549 cancer cells. The cells endured culture for a duration of 24 h across a range of phycoerythrin concentrations. The viability of the cells was assessed through the MTT assay following a 24-hour incubation period. The alterations in the morphology of human lung cancer cells were observed following treatment with varying concentrations of phycoerythrin, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The test showed a clear relationship between dosage and the level of toxicity in A549 human lung cancer cells. The cell viability showed a decline from 91.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09% at a concentration of 50 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e to 46.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16% at 500 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e. Significant reductions were also observed at 200 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e (74.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07%) and 400 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e (54.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11%), emphasizing its promise as a potential anticancer agent. The concentrations of phycoerythrin at 50 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e and 100 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e indicated the presence of viable human lung cancer cells. The concentration of 100 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e of phycoerythrin showed the existence of both viable and non-viable cells. The levels of phycoerythrin between 200 and 400 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e indicated a minimal count of visible cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eViability of human lung cancer cells on cytotoxicity of phycoerythrin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS. No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eConcentration (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCell viability (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e91.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e82.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e74.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e69.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e54.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e46.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHuman lung cancer cells treated with phycoerythrin experience growth inhibition. The tested sample exhibited an IC\u003csub\u003e50\u003c/sub\u003e value of 131.7 \u0026micro;g mL⁻\u0026sup1;. Ultimately, as the concentration of phycoerythrin increased, the presence of the cell steadily reduced. It is important to note that phycoerythrin demonstrates a more potent effect against human lung cancer cells shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Similarly, the various solvent extracts of \u003cem\u003eGracilaria edulis\u003c/em\u003e were evaluated against cancer cell lines for their anti-proliferative capabilities [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The \u003cem\u003eG. edulis\u003c/em\u003e ethyl acetate extract (GEEA) at a concentration of 100 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e demonstrated a notable and highly significant reduction in growth within the A549 lung cancer cell line model. Additionally, when assessing a control group against a GEEA extract-treated group at different dosages (40, 60, 80, and 100 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e) after 48 h, a minimal level of LDH release was observed. The cell cytotoxicity assay [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], revealed that phycoerythrin extracted from Michrochaete acts as an anticancer agent in the Hep G2 cell line at specific doses (20\u0026ndash;160 g mL\u003csup\u003e-1\u003c/sup\u003e), demonstrating an IC\u003csub\u003e50\u003c/sub\u003e value of 105.77 g mL\u003csup\u003e-1\u003c/sup\u003e. In a similar way, anticancer efficacy of \u003cem\u003eGracilaria cortica\u003c/em\u003e phycoerythrin was reported against the HepG2 cell line [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTested phycoerythrin shows IC\u003csub\u003e50\u003c/sub\u003e values\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLog (inhibitor)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePhycoerythrin (\u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eBest-fit values\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLogIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.119\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHillSlope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-1.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e131.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eStd. Error\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLogIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05416\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHillSlope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1334\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003e95% Confidence Intervals\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLogIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.008 to 2.230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHillSlope\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-1.290 to -0.7434\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102.0 to 170.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGoodness of Fit\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDegrees of Freedom\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR square\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.8809\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAbsolute sum of squares\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3584\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSy.x\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNumber of points\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalyzed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003ePhycoerythrin induces apoptosis in cancer cells\u003c/h2\u003e \u003cp\u003eThe evaluation of apoptotic cells involved staining phycoerythrin with human lung cancer cells, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The control cells exhibited the highest number of viable cells, as indicated by green-coloured fluorescence. The percentage of apoptotic cells that tested positive for Annexin V/PI in human lung cancer cells treated with varying dosages of phycoerythrin (203.4 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e) increased in accordance with the dosage levels. Cells treated with fluorouracil demonstrated the presence of both viable and non-viable cells. In addition to identifying the morphological changes associated with apoptosis, we also assessed the apoptosis rate using the annexin V test. Annexin V has a strong and specific attraction for damaged and compromised plasma membranes, making apoptotic and necrotic cells stand out. The recent study demonstrated that phycoerythrin-induced apoptotic cell death in A549 lung cancer cells. Cell apoptosis [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], refers to a form of programmed cell death that relies on energy. Caspase belongs to the family of enzymes known for their specificity to aspartic acid and homocysteine proteases. These enzymes play a crucial role in degrading antiapoptotic proteins, leading to the release of mitochondrial cytochrome C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular docking analysis\u003c/h3\u003e\n\u003cp\u003eComputational biology combines computational techniques with biological systems to tackle disease-related challenges. Virtual screening, a key tool, reduces drug development costs and time by predicting receptor-ligand binding and affinities through docking and scoring. Molecular docking enhances drug discovery by accurately identifying ligand orientations in protein binding sites and predicting interaction strengths, significantly advancing the field [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. By utilizing molecular docking, targeting the presence of antibacterial proteins in these compounds for the first time with the \u003cem\u003eK. alvarezii\u003c/em\u003e extracted bioactive compounds. The 13 antibacterial multitarget proteins were docked with 4 compounds detected in GC-MS analysis of the phycoerythrin, revealing that a lower docking score corresponds to higher binding affinity (Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e19\u003c/span\u003e). Molecular docking of K. alvarezii-derived compounds revealed significant binding affinities with 13 antibacterial target proteins. Among the results, the protein DNA gyrase subunits A (GyrA) and B (GyrB) showed a binding score of -1.13 kcal/mol with 1,2-Benzisothiazol-3-amine. \u003cem\u003eStaphylococcus aureus\u003c/em\u003e tyrosyl-tRNA synthetase protein scored \u0026minus;\u0026thinsp;2.86 kcal/mol with 1,2-Benzisothiazol-3-amine, while dihydropteroate synthase and transcriptional regulator qacR scored \u0026minus;\u0026thinsp;1.17 kcal/mol and \u0026minus;\u0026thinsp;1.21 kcal/mol, respectively, with 2-bromobutyloxychalcone. Penicillin-binding protein 3 (-1.41 kcal/mol), DNA topoisomerase 4 subunit A (-1.66 kcal/mol), and isoleucyl-tRNA synthetase (-3.10 kcal/mol) also showed strong interactions with 1,2-Benzisothiazol-3-amine. Other substantial interactions included HTH-type transcriptional regulator MgrA (-2.39 kcal/mol) and processed glycerol phosphate lipoteichoic acid synthase 2 (-3.51 kcal/mol) with 2,4-dimethylbenzo[h]quinoline, and YcgJ protein from \u003cem\u003eBacillus subtilis\u003c/em\u003e (-2.52 kcal/mol) with 2-bromobutyloxychalcone. Particularly, transcriptional regulator MvfR (-4.88 kcal/mol) interacted strongly with 1,2-Benzisothiazol-3-amine, and dihydrofolate reductase exhibited the highest binding affinity (-5.24 kcal/mol) with 2-ethylacridine. In general, 1,2-Benzisothiazol-3-amine and 2-bromobutyloxychalcone demonstrated strong interactions with multiple targets, while 2-ethylacridine showed the highest affinity with dihydrofolate reductase (-5.24 kcal/mol), emerged as the most potent compound.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe molecular docking of the antimicrobial target proteins with the compounds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmino acid Interaction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBond Length\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlide Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGlide Energy\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e1H7L - Spore coat polysaccharide biosynthesis protein SpsA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHIS 159,\u003c/p\u003e \u003cp\u003eTYR 77.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.03]; [4.21, 3.70].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-20.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePHE 233,\u003c/p\u003e \u003cp\u003eTYR 11,\u003c/p\u003e \u003cp\u003eTYR 77,\u003c/p\u003e \u003cp\u003eARG 76.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.03]; [5.49];\u003c/p\u003e \u003cp\u003e[5.21, 5.84, 5.45]; [5.31].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-24.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eARG 76,\u003c/p\u003e \u003cp\u003eLEU 80.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.17]; [4.81].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-27.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTHR 9,\u003c/p\u003e \u003cp\u003eASP 99,\u003c/p\u003e \u003cp\u003eTYR 11,\u003c/p\u003e \u003cp\u003eARG 76,\u003c/p\u003e \u003cp\u003eTYR 77.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.21];\u003c/p\u003e \u003cp\u003e[2.50, 2.59];\u003c/p\u003e \u003cp\u003e[5.55]; [5.18, 5.12]; [5.24].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-37.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e1JIJ - \u003cem\u003eStaphylococcus aureus\u003c/em\u003e tyrosyl-tRNA synthetase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALA 39,\u003c/p\u003e \u003cp\u003eASP 40.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.38,4.23]; [2.74,1.94].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASP 195,\u003c/p\u003e \u003cp\u003eTYR 36,\u003c/p\u003e \u003cp\u003eCYS37,\u003c/p\u003e \u003cp\u003eGLY 192.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.51]; [5.31];\u003c/p\u003e \u003cp\u003e[2.94,5.17,5.04];\u003c/p\u003e \u003cp\u003e[2.80,2.92].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-27.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASP 80,\u003c/p\u003e \u003cp\u003eTYR 36,\u003c/p\u003e \u003cp\u003eLEU 70,\u003c/p\u003e \u003cp\u003eCYS 37.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.45]; [5.26];\u003c/p\u003e \u003cp\u003e[3.81]; [4.05].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-32.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLEU 70,\u003c/p\u003e \u003cp\u003eGLN 190,\u003c/p\u003e \u003cp\u003eCYS 37,\u003c/p\u003e \u003cp\u003eGLY 193,\u003c/p\u003e \u003cp\u003eGLN196.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.80]; [2.08];\u003c/p\u003e \u003cp\u003e[4.93]; [3.08];\u003c/p\u003e \u003cp\u003e[2.77].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-46.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e1QU3 - Isoleucyl-tRNA synthetase (IleRS)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGLU 554.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[1.73].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGLY 554,\u003c/p\u003e \u003cp\u003eTRP 562,\u003c/p\u003e \u003cp\u003eTRP 528,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[1.64]; [5.41, 4.94]; [4.78].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-35.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRO 56,\u003c/p\u003e \u003cp\u003eGLU 554,\u003c/p\u003e \u003cp\u003eTRP 562,\u003c/p\u003e \u003cp\u003eASP 557,\u003c/p\u003e \u003cp\u003eHIS 64.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.26]; [2.41];\u003c/p\u003e \u003cp\u003e[5.97, 5.13];\u003c/p\u003e \u003cp\u003e[4.46, 5.22];\u003c/p\u003e \u003cp\u003e[5.47].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-30.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTRP 528,\u003c/p\u003e \u003cp\u003eGLY 555,\u003c/p\u003e \u003cp\u003eGLU 554,\u003c/p\u003e \u003cp\u003eASN 70,\u003c/p\u003e \u003cp\u003eHIS 67,\u003c/p\u003e \u003cp\u003ePRO 57.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.97]; [2.14];\u003c/p\u003e \u003cp\u003e[2.84]; [2.68];\u003c/p\u003e \u003cp\u003e[4.24]; [5.37].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-40.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e1RKW - Transcriptional regulator qacR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHIS 42,\u003c/p\u003e \u003cp\u003eGLU 52.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[1.93];\u003c/p\u003e \u003cp\u003e[3.67, 3.49].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-16.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGLU 52,\u003c/p\u003e \u003cp\u003eLYS 115,\u003c/p\u003e \u003cp\u003eLYS 118,\u003c/p\u003e \u003cp\u003eLEU 119.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.52]; [4.21, 4.58, 5.17, 4.00, 4.21]; [4.31];\u003c/p\u003e \u003cp\u003e[5.16].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-31.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLYS 115,\u003c/p\u003e \u003cp\u003eGLU 52.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.24]; [4.40, 3.62, 3.70].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-19.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASN 55,\u003c/p\u003e \u003cp\u003eLYS 118,\u003c/p\u003e \u003cp\u003eLYS 115.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.32]; [4.36];\u003c/p\u003e \u003cp\u003e[3.08,4.92].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-26.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e2BV6 - HTH-type transcriptional regulator MgrA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALA 63,\u003c/p\u003e \u003cp\u003eLEU 62,\u003c/p\u003e \u003cp\u003eLEU 42,\u003c/p\u003e \u003cp\u003ePRO 39,\u003c/p\u003e \u003cp\u003eTYR 38.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.19, 4.96];\u003c/p\u003e \u003cp\u003e[2.81, 4.48];\u003c/p\u003e \u003cp\u003e[5.42]; [4.90];\u003c/p\u003e \u003cp\u003e[5.50].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-17.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLEU 42,\u003c/p\u003e \u003cp\u003ePRO 39,\u003c/p\u003e \u003cp\u003eLEU 62,\u003c/p\u003e \u003cp\u003eALA 63.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.91]; [4.84];\u003c/p\u003e \u003cp\u003e[5.01]; [2.26, 5.12, 5.03].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-20.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALA 63,\u003c/p\u003e \u003cp\u003eLEU 62,\u003c/p\u003e \u003cp\u003eLEU 42,\u003c/p\u003e \u003cp\u003ePRO 39,\u003c/p\u003e \u003cp\u003eTYR 38.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.10, 4.93, 3.95, 2.25]; [5.37];\u003c/p\u003e \u003cp\u003e[5.13]; [5.28, 4.88]; [5.30]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-19.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVAL 116,\u003c/p\u003e \u003cp\u003eTYR 38,\u003c/p\u003e \u003cp\u003eGLN 19,\u003c/p\u003e \u003cp\u003eLEU 134,\u003c/p\u003e \u003cp\u003ePHE 41,\u003c/p\u003e \u003cp\u003eLEU 42.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.94]; [3.00];\u003c/p\u003e \u003cp\u003e[2.05, 2.98];\u003c/p\u003e \u003cp\u003e[4.23]; [4.32];\u003c/p\u003e \u003cp\u003e[4.99].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-30.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e2GLU - YcgJ protein from \u003cem\u003eBacillus subtilis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRO 119,\u003c/p\u003e \u003cp\u003eTYR 117.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.12]; [2.08].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-15.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLEU 145,\u003c/p\u003e \u003cp\u003eLEU 167,\u003c/p\u003e \u003cp\u003ePRO 168,\u003c/p\u003e \u003cp\u003eGLN 149.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.27]; [4.96];\u003c/p\u003e \u003cp\u003e[5.12, 4.86];\u003c/p\u003e \u003cp\u003e[2.14, 2.82].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-18.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTYR 159,\u003c/p\u003e \u003cp\u003eGLN 149,\u003c/p\u003e \u003cp\u003eLEU 145,\u003c/p\u003e \u003cp\u003eGLU 120.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.35]; [1.94];\u003c/p\u003e \u003cp\u003e[4.48, 5.09];\u003c/p\u003e \u003cp\u003e[4.21, 4.48].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-20.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRO 168,\u003c/p\u003e \u003cp\u003eGLU 120,\u003c/p\u003e \u003cp\u003ePRO 119.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.42]; [2.90];\u003c/p\u003e \u003cp\u003e[2.57, 4.82].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-30.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e2VEG - Dihydropteroate synthase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASP 221,\u003c/p\u003e \u003cp\u003eGLN 276,\u003c/p\u003e \u003cp\u003eARG 275,\u003c/p\u003e \u003cp\u003eLLE 272.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[1.92]; [3.06];\u003c/p\u003e \u003cp\u003e[4.15, 4.13];\u003c/p\u003e \u003cp\u003e[5.42].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-16.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eARG 275,\u003c/p\u003e \u003cp\u003eLLE 272,\u003c/p\u003e \u003cp\u003eASP 221.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[3.88, 5.36, 3.95]; [4.85]; [2.07].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2312\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eARG 275,\u003c/p\u003e \u003cp\u003eASP 221.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.39, 4.59];\u003c/p\u003e \u003cp\u003e[3.87, 3.68].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-23.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eARG 275.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.45, 4.01].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-26.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e2W8D - Processed glycerol phosphate lipoteichoic acid synthase 2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTRP 350,\u003c/p\u003e \u003cp\u003eASN 478,\u003c/p\u003e \u003cp\u003eHIS 479,\u003c/p\u003e \u003cp\u003ePHE 413.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.23, 4.85, 3.53, 4.21]; [3.02];\u003c/p\u003e \u003cp\u003e[2.45]; [5.04].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-21.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTRP 350,\u003c/p\u003e \u003cp\u003eASN 478,\u003c/p\u003e \u003cp\u003eHIS 479.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.97, 4.03, 5.01, 3.78, 4.46]; [3.10]; [4.79, 4.07].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-24.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHIS 479,\u003c/p\u003e \u003cp\u003ePHE 349,\u003c/p\u003e \u003cp\u003eASN 478,\u003c/p\u003e \u003cp\u003eTYR 380,\u003c/p\u003e \u003cp\u003ePHE 413,\u003c/p\u003e \u003cp\u003eTRP 350.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.31]; [4.74];\u003c/p\u003e \u003cp\u003e[2.68]; [5.09];\u003c/p\u003e \u003cp\u003e[504]; [4.19, 5.08, 5.15, 4.87, 4.48, 3.44].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-18.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPO 297,\u003c/p\u003e \u003cp\u003eTRP 350,\u003c/p\u003e \u003cp\u003eTYR 380,\u003c/p\u003e \u003cp\u003ePHE 349,\u003c/p\u003e \u003cp\u003eASN 478,\u003c/p\u003e \u003cp\u003ePHE 413.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.02]; [2.27];\u003c/p\u003e \u003cp\u003e[5.64]; [4.32];\u003c/p\u003e \u003cp\u003e[2.07]; [4.76].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-36.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e2XCT - DNA gyrase subunit B, DNA gyrase subunit A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLLE 1452,\u003c/p\u003e \u003cp\u003eLYS 1375,\u003c/p\u003e \u003cp\u003eALA 1378,\u003c/p\u003e \u003cp\u003eGLU 1382,\u003c/p\u003e \u003cp\u003eLEU 1449.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.16]; [3.55, 4.20]; [5.32];\u003c/p\u003e \u003cp\u003e[1.92]; [5.07].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-11.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGLU 1382,\u003c/p\u003e \u003cp\u003eLEU 1449,\u003c/p\u003e \u003cp\u003eLYS 135,\u003c/p\u003e \u003cp\u003eLLE 1452.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[1.52]; [5.13, 4.76]; [3.97];\u003c/p\u003e \u003cp\u003e[4.17].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-27.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLLE 1452,\u003c/p\u003e \u003cp\u003eLYS 1375,\u003c/p\u003e \u003cp\u003eALA 1378,\u003c/p\u003e \u003cp\u003eLEU 1449,\u003c/p\u003e \u003cp\u003eGLU 1382.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.16]; [5.14];\u003c/p\u003e \u003cp\u003e[3.65]; [4.97, 4.57]; [5.51].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLLE 1452,\u003c/p\u003e \u003cp\u003eLEU 1449,\u003c/p\u003e \u003cp\u003eASN 474.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.97]; [5.28, 5.38]; [2.11].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-26.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e3RAE - DNA topoisomerase 4 subunit A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRO 113,\u003c/p\u003e \u003cp\u003eARG 117,\u003c/p\u003e \u003cp\u003eMET 116,\u003c/p\u003e \u003cp\u003eALA 115.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.95]; [4.72];\u003c/p\u003e \u003cp\u003e[5.45]; [4.13, 3.91].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-24.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePRO 112,\u003c/p\u003e \u003cp\u003eTYR 82,\u003c/p\u003e \u003cp\u003eALA 115,\u003c/p\u003e \u003cp\u003eMET 116.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.02]; [4.42];\u003c/p\u003e \u003cp\u003e[4.68]; [4.39].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-31.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASP 83,\u003c/p\u003e \u003cp\u003eMET 116,\u003c/p\u003e \u003cp\u003eALA 115.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.01]; [5.52];\u003c/p\u003e \u003cp\u003e[4.71, 4.65].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-29.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALLA 115,\u003c/p\u003e \u003cp\u003eMET 116,\u003c/p\u003e \u003cp\u003eARG 117.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.76]; [5.30];\u003c/p\u003e \u003cp\u003e[2.40].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-34.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e3SRW - Dihydrofolate reductase\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLLE 15,\u003c/p\u003e \u003cp\u003eLEU 21,\u003c/p\u003e \u003cp\u003eALA 8,\u003c/p\u003e \u003cp\u003ePHE 93.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.07]; [5.15, 5.43]; [2.14, 2.27,5.13];\u003c/p\u003e \u003cp\u003e[5.18, 5.63].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLEU 21,\u003c/p\u003e \u003cp\u003eLEU 29,\u003c/p\u003e \u003cp\u003ePHE 93,\u003c/p\u003e \u003cp\u003eLLE 15.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.46, 4.35]; [5.14]; [5.28];\u003c/p\u003e \u003cp\u003e[4.70].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-278994\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePHE 93,\u003c/p\u003e \u003cp\u003eLLE 15,\u003c/p\u003e \u003cp\u003eLEU 21.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.87]; [5.36];\u003c/p\u003e \u003cp\u003e[4.51, 5.10].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-29.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVAL 132,\u003c/p\u003e \u003cp\u003eVAL 17,\u003c/p\u003e \u003cp\u003eLLE 15,\u003c/p\u003e \u003cp\u003eALA 8,\u003c/p\u003e \u003cp\u003eLEU 21,\u003c/p\u003e \u003cp\u003eASN 19.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.43]; [2.61];\u003c/p\u003e \u003cp\u003e[5.28]; [1.99, 5.32]; [4.84];\u003c/p\u003e \u003cp\u003e[2.59].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-41.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e3VSL - Penicillin-binding protein 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eASN 501,\u003c/p\u003e \u003cp\u003eVAL 493,\u003c/p\u003e \u003cp\u003eLYS 494.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[1.94]; [5.34, 3.25]; [4.87, 3.98, 4.27].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-19.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLYS 494,\u003c/p\u003e \u003cp\u003eASN 501,\u003c/p\u003e \u003cp\u003ePRO 500,\u003c/p\u003e \u003cp\u003eTYR 278.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.91, 3.07, 2.81]; [3.01, 2.59]; [5.20]\u003c/p\u003e \u003cp\u003e[4.97].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-24.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLYS 494,\u003c/p\u003e \u003cp\u003eASN 501,\u003c/p\u003e \u003cp\u003ePRO 500.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.76, 5.38];\u003c/p\u003e \u003cp\u003e[2.76, 2.15];\u003c/p\u003e \u003cp\u003e[5.30, 5.45].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-22.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTYR 278,\u003c/p\u003e \u003cp\u003eLYS 494,\u003c/p\u003e \u003cp\u003eHIS 259,\u003c/p\u003e \u003cp\u003eLEU 576.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.61]; [1.98];\u003c/p\u003e \u003cp\u003e[5.56]; [5.06].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-35.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e4JVC - Transcriptional regulator MvfR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e89966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,2-Benzisothiazol-3-amine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLEU 197,\u003c/p\u003e \u003cp\u003eGLN 194,\u003c/p\u003e \u003cp\u003eARG 209,\u003c/p\u003e \u003cp\u003eLEU 207,\u003c/p\u003e \u003cp\u003eLLE 236,\u003c/p\u003e \u003cp\u003eLEU 208.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[2.63]; [2.90];\u003c/p\u003e \u003cp\u003e[2.07]; [2.58];\u003c/p\u003e \u003cp\u003e[2.81, 3.98];\u003c/p\u003e \u003cp\u003e[4.77, 5.14].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-27.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Ethylacridine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLLE 236,\u003c/p\u003e \u003cp\u003eALA 102,\u003c/p\u003e \u003cp\u003eALA 168,\u003c/p\u003e \u003cp\u003eLLE 149,\u003c/p\u003e \u003cp\u003eLEU 208.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.92, 3.87, 4.17]; [4.27];\u003c/p\u003e \u003cp\u003e[4.16, 3.25];\u003c/p\u003e \u003cp\u003e[4.95, 4.05];\u003c/p\u003e \u003cp\u003e[4.99, 4.94].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-19.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e610182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,4-Dimethylbenzo[h]quinoline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLLE 149,\u003c/p\u003e \u003cp\u003eALA 168,\u003c/p\u003e \u003cp\u003eLLE 236,\u003c/p\u003e \u003cp\u003eVAL 211,\u003c/p\u003e \u003cp\u003eLEU 208.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[5.32]; [4.52];\u003c/p\u003e \u003cp\u003e[4.54, 3.98, 4.12, 5.45]; [5.48];\u003c/p\u003e \u003cp\u003e[4.80,4.87].\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-28.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e91733949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-bromobutyloxychalcone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eALA 102,\u003c/p\u003e \u003cp\u003ePRO 238,\u003c/p\u003e \u003cp\u003eALA 168,\u003c/p\u003e \u003cp\u003eLLE 236,\u003c/p\u003e \u003cp\u003eLLE 149,\u003c/p\u003e \u003cp\u003eLEU 208,\u003c/p\u003e \u003cp\u003eARG 209.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e[4.37]; [4.96]\u003c/p\u003e \u003cp\u003e[3.22]; [5.29, 2.87]; [4.31];\u003c/p\u003e \u003cp\u003e[5.14]; [4.02];\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-34.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe scope of the present paper was to assess the potential of seaweed as a novel source for marine pharmaceuticals. This study successfully isolated and purified phycoerythrin from \u003cem\u003eK. alvarezii\u003c/em\u003e using a process that included ammonium sulfate precipitation, dialysis, and column chromatography. In the FT-IR test, many different functional groups were found. In the HPLC test, bioactive compounds such as kaempferitrin, β-sitosterol, and 2-bromobutyloxychalcone were found. The antioxidant activity of phycoerythrin was very high, with a total antioxidant capacity of 73.27% and strong scavenging abilities in the DPPH and ABTS tests. Its antibacterial activity was most effective against \u003cem\u003eK. oxytoca\u003c/em\u003e (20 mm) and moderately active against other pathogens. MTT assays verified that cytotoxicity against A549 human lung cancer cells was dose-dependent, and Annexin V/PI staining revealed increased apoptotic activity. Molecular docking further emphasized the potential of phycoerythrin as a natural source of bioactive compounds for therapeutic applications, highlighting its potential in antimicrobial target protein interactions. The outcome of this study demonstrates the potential of the red seaweed \u003cem\u003eK. alvarezii\u003c/em\u003e against cancer cell lines, supporting the way forward for the development of phycoerythrin as anti-cancer drug with pharmaceutical prospective.\u003c/p\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eStatement\u003c/h2\u003e \u003cp\u003eWe implemented all methods in accordance with pertinent regulations and guidelines. The authors have not conducted any experiments on humans or utilized human tissue samples. The ethics committee of the institute approved all experimental protocols and panelists involved in the study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare that data supporting the findings of this study will be available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe implemented all methods in accordance with pertinent regulations and guidelines. The authors have not conducted any experiments on humans or utilized human tissue samples. The ethics committee of the institute approved all experimental protocols and panelists involved in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors would like to express their gratitude to Ayya Nadar Janaki Ammal College, Tamil Nadu, for providing the necessary facilities to conduct this research, as well as the International Centre for Genetic Engineering and Biotechnology, New Delhi for their assistance with molecular docking. The authors extend their deep appreciation to Researchers Supporting Project number (RSPD2025R741), King Saud University, Riyadh, Saudi Arabia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.B., G.R.P. and D.P. conceived and designed the experiments. P.S. and S.S. drafted the main manuscript text. H.B., S.M., M.M., J.P. and P.M. conducted the extraction, GC-MS analysis, and antibacterial, antioxidant, and anticancer experiments. M.A., S.V. and P.S. carried out the molecular docking analyses. D.P. and A.P. supervised the research project. F.U., H.O.E., M.N., A.A.F., M.A.R. and I.M.M. contributed to funding acquisition, manuscript review and editing, and project administration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearchers Supporting Project number (RSPD2025R741), King Saud University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondenceand requests for materials should be addressed to D.P.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMenaa F, Wijesinghe U, Thiripuranathar G, Althobaiti NA, Albalawi AE, Khan BA, et al. 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RSC Adv. 2016;6:16615\u0026ndash;23.\u003c/li\u003e\n\u003cli\u003eRe R, Nicoletta P, Anna P, Ananth P, Min Y, Catherine R-E. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med. 1999;26 (9-10):1231\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eMohan H, Ramalingam V, Lim JM, Lee SW, Kim J, Lee JH, et al. E-waste based graphene oxide/V2O5/Pt ternary composite: Enhanced visible light driven photocatalyst for anti-microbial and anti-cancer activity. Colloids Surfaces A Physicochem Eng Asp. 2020; 125469.\u003c/li\u003e\n\u003cli\u003eRamalingam V, Raja S, Harshavardhan M. In situ one-step synthesis of polymer-functionalized palladium nanoparticles: An efficient anticancer agent against breast cancer. Dalt Trans. 2020;49:3510\u0026ndash;8.\u003c/li\u003e\n\u003cli\u003eLeslie VA, Mohammed Alarjani K, Malaisamy A, Balasubramanian B. Bacteriocin producing microbes with bactericidal activity against multidrug resistant pathogens. J Infect Public Health. 2021;14:1802\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eMalaisamy AK, Balasubramanian B, Tamilselvan PY, Sakthivel V, Venkatachalapathi S, Bhotla HK. Probing marine macroalgal phlorotannins as an antibacterial candidate against \u003cem\u003eSalmonella typhi\u003c/em\u003e: Molecular docking and dynamics simulation approach. Curr Plant Biol. 2024;100418.\u003c/li\u003e\n\u003cli\u003eSharmila G, Nikitha VS, Ilaiyarasi S, Dhivya K, Rajasekar V, Kumar NM, et al. Ultrasound assisted extraction of total phenolics from \u003cem\u003eCassia auriculata\u003c/em\u003e leaves and evaluation of its antioxidant activities. Ind Crops Prod. 2016;84:13\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eBenavides J, Rito-Palomares M. Bioprocess intensification: A potential aqueous two-phase process for the primary recovery of B-phycoerythrin from \u003cem\u003ePorphyridium cruentum\u003c/em\u003e. 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Vitamin B12, a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells. Cytotechnology. 2006;52:181\u0026ndash;7.\u003c/li\u003e\n\u003cli\u003eValuta A, Cepoi L, Rudi L, Bulhac I, Bourosh P, Bologa O. Phycobiliprotein accumulation in Cyanobacterium \u003cem\u003eNostoc linckia\u003c/em\u003e and modification of antioxidant activity. Ann Oradea Univ Biol Fascicle. 2015;21:13\u0026ndash;9.\u003c/li\u003e\n\u003cli\u003eLi S, Ji L, Shi Q, Wu H, Fan J. Advances in the production of bioactive substances from marine unicellular microalgae \u003cem\u003ePorphyridium\u003c/em\u003e spp. Bioresour Technol. 2019;292 June:122048.\u003c/li\u003e\n\u003cli\u003eSakthivel R, Muniasamy S, Archunan G, Devi KP. \u003cem\u003eGracilaria edulis\u003c/em\u003e exhibit antiproliferative activity against human lung adenocarcinoma cell line A549 without causing adverse toxic effect \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Food Funct. 2016;7:1155\u0026ndash;65.\u003c/li\u003e\n\u003cli\u003eZandi K, Tajbakhsh S, Nabipour I, Rastian Z, Yousefi F, Sharafian S, et al. In vitro antitumor activity of \u003cem\u003eGracilaria corticata\u003c/em\u003e (a red alga) against jurkat and molt-4 human cancer cell lines. African J Biotechnol. 2010;9:6787\u0026ndash;90.\u003c/li\u003e\n\u003cli\u003eLogue SE, Martin SJ. Caspase activation cascades in apoptosis. Biochem Soc Trans. 2008;36:1\u0026ndash;9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bioactive molecules, Lung cancer, Molecular docking, Phycoerythrin, Pigment analysis, Red seaweed","lastPublishedDoi":"10.21203/rs.3.rs-5880917/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5880917/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eKappaphycus alvarezii\u003c/em\u003e (Doty) Doty ex Silva, a red seaweed widely cultivated for carrageenan polysaccharide, is also a potential source of the valuable pigment phycoerythrin (PE). Therefore, this study aims to extract phycoerythrin from \u003cem\u003eK. alvarezii\u003c/em\u003e, evaluate its antimicrobial, antioxidant, and anticancer activities, and identify its biomedical potential for future therapeutic applications. The protein content of phycoerythrin pigment extracted from \u003cem\u003eK. alvarezii\u003c/em\u003e was found to be 69.84% and showed excellent antimicrobial activity against \u003cem\u003eKlebsiella oxytoca\u003c/em\u003e and \u003cem\u003eProteus mirabilis\u003c/em\u003e, with a minimum inhibition zone of 11 mm. It showed significant \u003cem\u003ein vitro\u003c/em\u003e antioxidant activity, as analyzed using total antioxidant, hydrogen peroxide scavenging, reducing power, DPPH, and ABTS assays. Further, the pigment exhibited potent cytotoxicity against a human lung cancer cell line, with an IC\u003csub\u003e50\u003c/sub\u003e value of 131.7 \u0026micro;g mL\u003csup\u003e-1\u003c/sup\u003e. Furthermore, increasing the concentration of phycoerythrin pigment decreased the cell proliferation and induced apoptosis, as confirmed by Annexin V/PI staining. Comprehensive characterization using FT-IR, HPLC, and GC-MS analysis revealed the nature of pigment and functional groups, highlighting its potential for biomedical applications. The molecular docking of \u003cem\u003eK. alvarezii\u003c/em\u003e-derived compounds revealed significant binding affinities with 13 antibacterial target proteins. These results highlight the potential of \u003cem\u003eK. alvarezii\u003c/em\u003e bioactive compounds as promising antibacterial agents. The phycoerythrin extract from \u003cem\u003eK. alvarezii\u003c/em\u003e demonstrated potent antimicrobial, antioxidant, and anticancer properties, with significant cytotoxicity against lung cancer cells and confirmed apoptosis induction. Structural analysis revealed its bioactive composition, emphasizing its potential as a natural therapeutic agent. These findings support its potential application in biomedical and pharmaceutical industry.\u003c/p\u003e","manuscriptTitle":"Isolation, Characterization, and Biomedical Potential of Phycoerythrin Phycobiliprotein from Kappaphycus alvarezii (Doty) Doty ex Silva: Antimicrobial, Antioxidant, and Anticancer Activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-30 16:47:08","doi":"10.21203/rs.3.rs-5880917/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-27T04:26:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-25T21:28:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-18T10:49:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328992215871761431452920727169315051859","date":"2025-02-05T01:22:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-02T14:32:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"116556966510070609123478168707249644476","date":"2025-02-02T13:08:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25046310604441531027085478724154699464","date":"2025-01-31T12:19:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-01-31T10:58:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-30T07:33:10+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-01-30T06:49:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-28T13:24:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-22T12:32:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd092706-262b-46cb-88fa-bc5c8c4f647c","owner":[],"postedDate":"January 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":43523193,"name":"Biological sciences/Plant sciences"},{"id":43523194,"name":"Earth and environmental sciences/Ocean sciences"}],"tags":[],"updatedAt":"2025-08-04T16:39:34+00:00","versionOfRecord":{"articleIdentity":"rs-5880917","link":"https://doi.org/10.1038/s41598-025-11899-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-31 16:12:55","publishedOnDateReadable":"July 31st, 2025"},"versionCreatedAt":"2025-01-30 16:47:08","video":"","vorDoi":"10.1038/s41598-025-11899-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-11899-7","workflowStages":[]},"version":"v1","identity":"rs-5880917","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5880917","identity":"rs-5880917","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}