Symbiotic Synergies: A Comprehensive Analysis of Antibacterial Potency, Antioxidant Proficiency, and Phytochemical Composition in Rhizoclonium hieroglyphicum and Spirogyra varians from Distinct Aquatic Habitats

Symbiotic Synergies: A Comprehensive Analysis of Antibacterial Potency, Antioxidant Proficiency, and Phytochemical Composition in Rhizoclonium hieroglyphicum and Spirogyra varians from Distinct Aquatic Habitats | 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 Research Article Symbiotic Synergies: A Comprehensive Analysis of Antibacterial Potency, Antioxidant Proficiency, and Phytochemical Composition in Rhizoclonium hieroglyphicum and Spirogyra varians from Distinct Aquatic Habitats Syed Mansoor Ahmed, Hajra Hameed, Muhammad Tariq, Afsa Hameed, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4919856/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Terrestrial plants have been the main source of many active agents used in the pharmaceutical and cosmetic industries. Many research studies have focused on different marine organisms like algae, fish, sponges, and crustaceans to obtain bioactive products like proteins and peptides for use in the pharmaceutical and cosmeceutical industries. In contrast to terrestrial plants, marine algae are a very abundant and important source of different constituents that have established beneficial effects on human skin and have been used in the cosmeceutical industry. Samples of algae were collected from two different aquatic sites. Identification of samples was made based on microscopic morphological and other key characteristics of algae, and they were found to be R. hieroglyphicum and S. varians . Extraction was carried out in methanol and in a mixture of DCM (dichloromethane) and methanol. Both samples collected from the two different sites showed significant antibacterial effects in various assays performed using the Disc Diffusion Method and the Well Method. The antioxidant activities of the extracts were also detected using a 2.2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging assay. Quantitative and qualitative analyses showed significant quantities of phenolic and flavonoid contents. The stressful climatic factors lead to the synthesis and production of compounds with secondary metabolites, which are anti-stress and important for their antioxidant and therapeutic effects, resulting in various biological activities. Marine algae Rhizoclonium Spirogyra bioactive components seaweed Figures Figure 1 Introduction A large number of active compounds have been isolated from terrestrial plants and used in the pharmaceutical and cosmetic industries throughout history. During the last four decades, many drugs approved by the FDA were prepared from plant species with medicinal value [ 1 ]. But in the last decade, many research studies have also been focused on different marine organisms for the production and isolation of bioactive products like proteins and peptides. Algae are one of the most important and diverse groups of aquatic organisms and have significant physiological and biochemical properties. They have been used as the source of different active compounds, which include carotenoids, steroids, fatty acids, vitamins, proteins, lectins, polysaccharides, dietary minerals, diverse antioxidants, and halogenated compounds. In contrast to traditional plants, algae do not possess true stems, roots, or leaves. Algae exhibit complex taxonomy, and as new molecular information is discovered over time, they are subjected to rapid change [ 2 ]. Carbohydrates are one of the most abundant and important constituents of marine algae [ 3 , 4 ]. The carbohydrates extracted and isolated from marine algae have many applications in pharmaceuticals and cosmetics due to their important physical and chemical characteristics [ 5 , 6 ]. Shanura et al [ 7 ] screened Gracilaria edulis and its methanol extract to study their anti-inflammatory properties in contradiction to lipopolysaccharide-induced inflammatory response. Koneri and Jha conducted a study on G. edulis and its methanol extract in which they determined their anti-diabetic properties for the treatment of male rats with fructose-induced type 2 diabetes mellitus [ 8 ]. Another study was conducted by Patra and Muthuraman [ 9 ] on ethanol extracts of G. edulis , in which they revealed their anticancer properties in mice for the treatment of ascites tumors. Furthermore, G. edulis was screened to study its antioxidant and hypoglycemic properties in different in vitro mechanisms for the first time in Sri Lanka. The results of the study showed that the evident hypoglycemic activity of G. edulis was produced by inhibiting glucose absorption. The antioxidant and hypoglycemic effects were further confirmed by GC-MS analysis [ 10 ]. In several Asian countries, seaweed has traditionally been used as a food. Important biologically active compounds like fatty acids, vitamins, polysaccharides, phycobilins, carotenoids, phycocyanins, and tocopherol is present in seaweeds. Seaweed has extensive applications in the food, pharmaceutical, and cosmetic industries [ 11 ]. The crude polysaccharides and derivatives obtained from them show various useful bioactivities, including anticoagulant, antitumor, immunomodulatory, and cancer-preventive activities. These also have powerful antibiotic, anti-inflammatory, and antioxidant properties. For instance, strong anticancer and immunomodulating characteristics have been shown by the sulfated polysaccharides (SP) from Enteromorpha and Porphyridium [ 12 ]. Algal species also show antibacterial activity against Staphylococcus and Escherichia coli . Sodium alginate and fucoidan obtained from Laminaria japonica show antibacterial activities against E. coli by adhering to bacteria and killing them [ 13 ]. The growth of Staphylococcus aureus was inhibited by an S-galactan obtained from Chaetomorpha aerea , which could not inhibit the growth of Salmonella enteritidis but restricted the propagation of Staphylococcus aureus [ 14 ] While against S. enteritidis , a notable inhibitory activity was shown by the carrageenans obtained from some seaweeds [ 15 ]. Raposo et al [ 16 ] reported the antibacterial activities of some species from microalgae and explained that these activities are based on the solvent used for the extraction of polymer. The study hypothesizes that marine algae, specifically R. hieroglyphicum and S. varians , contain significant bioactive compounds with antibacterial and antioxidant properties, which could be beneficial in these industries. These findings suggest that the stressful climatic conditions experienced by marine algae lead to the synthesis and production of secondary metabolites, which are crucial for their anti-stress, antioxidant, and therapeutic effects. Consequently, these compounds contribute to various beneficial biological activities, highlighting the potential of marine algae as a valuable resource for the development of pharmaceutical and cosmeceutical products. Materials and Methods Collection of Material Algae samples were collected from two locations: Mohli waterfall in Kotli and Lahri Head in Mirpur, Azad Kashmir. The samples were manually gathered and placed in glass bottles which had been thoroughly washed, dried, and preserved with diluted formalin, and was also kept in a cooler with ice. The algae were identified as R. hieroglyphicum and S. varians through microscopic examination and by observing key characteristics like appearance, color, texture, and smell [ 17 ]. Extraction of Algal Sample After washing and fully drying the algae samples, they were ground into powder using a mortar and pestle. Each sample was split into two equal parts. One part was extracted with methanol, and the other with a mixture of DCM and methanol (1:4). The samples were soaked in these solvents and kept in a dark place at room temperature for 7 days. After 7 days, the mixtures were filtered, and the process was repeated twice more. After the final filtration, the filtrate was left to evaporate until the solvents were completely gone [ 18 ]. Total Flavonoid Content Determination Total flavonoid contents were measured by the method used by Ordonez et al. (2006) with minor modifications [ 19 ]. The stock solution of the algal sample was taken in different concentrations of 10 µL, 7.5 µL, 5 µL, and 2.5 µL in a cuvette. Then, 2.5 ml of distilled, deionized water was added to the cuvette. After that, 180 µL of 5% sodium nitrate (NaNO 3 ) was added, followed by 180 µL of 10% aluminum chloride (AlCl 3 ). After one to two minutes, 1200 µL of one molar sodium hydroxide (NaOH) solution was added. The whole solution was mixed and remained at room temperature for about 20 to 30 minutes. The absorbance of the solution was determined by a spectrophotometer at 510 nm wavelength. Total Phenolic Contents Determination The total phenolic content of algal extracts was determined by the Folin-Ciocalteu assay method [ 20 ]. Different concentrations of the algal extract solution were added in a cuvette and labeled accordingly. Then, in each cuvette, 500 µL of Folin-Ciocalteau reagent and 1400 µL of 7% sodium bicarbonate were added and mixed. After mixing, 980 µL of distilled water was added to all cuvettes and left for 30 minutes to complete the reaction at room temperature. The absorbance was checked at 750 nm wavelength by spectrophotometer. The absorbance of the extract was measured three times, and its mean value was taken. The procedure was repeated for different concentrations of gallic acid used as standards, and their absorbance was measured. Disk Diffusion Method In the disk diffusion method, five different strains of bacteria were used: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus , and Bacillus pumilus . Nutrient medium was used for bacterial colonization, which was autoclaved along with Petri plates [ 21 ]. In the laminar flow hood, all Petri plates were labeled, and autoclaved nutrient agar media were poured when the temperature of the media reached between 40°C and 45°C in petri plates with a desirable strain of bacterial suspension. Rifampicin (a synthetic antibiotic) was used as the standard by soaking in a disk and placing it at the midpoint of the petri dish. All plates were enclosed with parafilm to avoid impurities. The plates were placed in an incubator set at 37°C for 20 to 24 hours [ 22 ]. After 24 hours, zones of inhibition in millimeters were measured from the circumference and the area free from bacterial growth around disks [ 23 ]. Interpretations were noted three times for each type of bacterial species against each sample, and the mean value was then calculated. The antibiotic effects against selective bacterial strains were plotted in graphs. Antioxidant Activity Antioxidant activity was checked by using different quantities of algal extracts in DMSO, in separated and labeled test tubes. Then, 1440 µL of freshly prepared DPPH solution, prepared by adding 0.012 grams of DPPH to 100 ml of methanol, was added to the plant extract in the test tube. The solution was mixed and left for about 30 minutes to complete the reaction between DPPH and plant extract. In another test tube, 1.5 mL of methanol and 1.4 mL of DPPH were mixed and used as blanks. Absorbance was measured after 30 minutes by a spectrophotometer at 517 nm, and as a positive control, ascorbic acid was used. The IC 50 values were then calculated by the method mentioned previously [ 24 ]. The scavenging activity was measured using the following formula: Data Statistics and Analysis All experiments were performed in triplicate to ensure the reliability and accuracy of the results. Statistical analysis was conducted using SPSS software (Version 27). Data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the means of different groups, and significant differences between means were determined using Tukey's post-hoc test at a significance level of p < 0.05. Graphical representations of the data were created using MS Excel to illustrate the differences in the antioxidant, antibacterial, and total phenolic and flavonoid content between the samples. Additionally, IC 50 values for antioxidant activity were calculated using non-linear regression analysis. Results and Discussion Sample Collection and Extraction The samples of algae R. hieroglyphicum and S. varians were collected from two different sites of Azad Kashmir and extracted in two different solvents, methanol and a mixture of methanol and DCM, for the assessment of phytochemical and biological activities for the first time. Phytochemical Analysis Qualitative phytochemical analysis for different chemical compounds was performed on extracts of S. varians and R. hieroglyphicum . Qualitative analysis of phytochemicals showed that almost similar phytochemicals were detected in all the samples. Among the four different extracts, R. hieroglyphicum extracted in the DCM-methanol mixture showed the most phytochemicals. Saponins were not detected in any of the tested samples. Flavonoids were detected in all four extracts with equal intensity. Steroids were detected in both extracts of R. hieroglyphicum , but they didn’t show their presence in the extracts of S. varian s in methanol. Glycosides, alkaloids, and tannins also showed their presence in all of the tested samples, while the tannins were more intensely detected in R. hieroglyphicum than in S. varians . Results were in concordance with previous reports where S. varians and R. hieroglyphicum extracts showed significant bioactivities in the form of antimicrobial activity and antioxidant properties, and these activities were related to the versatile group of phytochemicals present in these algal extracts. Total Phenolic and Flavonoid Content Following the procedure for the flavonoid and phenolic content of the extracts, plots were obtained against concentration vs. optical density. Flavonoid and phenolic content varied according to the algal species, i.e., S. varians or R. hieroglyphicum , and to the solvents used for extraction. Total phenolic content in the case of R. hieroglyphicum was higher in the DCM methanolic extract (297 mg GAE/g) than in the methanolic extract (191.2 mg GAE/g). Total flavonoid content, on the other hand, was nearly identical in both R. hieroglyphicum extracts: 34.59 mg RE/g in DCM: methanolic extract and 33.7 mg RE/g in methanol extract. Similarly, in the case of S. varians , total phenolic content was higher in DCM: methanolic extract (183.5 mg GAE/g extract) than in methanolic extract (60.4 mg GAE/g extract). Total flavonoid content, on the other hand, was higher in DCM: methanolic extract (35.38 mg RE/g) as compared to the methanolic extract (24.05 mg RE/g extract). Antioxidant Activity The graph of antioxidant activities showed that the dosage of algal extract is correlated with antioxidant activity. In the present study, it was recognized that the methanol extracts of S. varians and R. hieroglyphicum had favorable antioxidant potential and varied bioactive phytochemicals. It can be utilized as a source of a possible natural antioxidant for numerous determinations. Although many past studies are available on the bioactive chemical composition, antioxidant capacity, medicinal properties, cytotoxicity, etc. of the algae present in different parts of Pakistan, no scientific research still documents the algal species showing bioactive phytochemicals and therapeutic effects from Azad Kashmir as far as our information goes. Recent studies by other researchers have documented a strong positive association between the total phenolic content and antioxidant effects in algal species, which supports our results [ 25 ]. The resistance gained by algal cellular and physical compounds against such oxidative contest showed that the intracellular antioxidants are supposed to be responsible for the defensive role as an inherent mechanism for sustenance [ 26 ]. Both S. varians and R. hieroglyphicum contain a number of compounds having antioxidant potential. Antioxidant activities can be measured by different methods but in the present study, DPPH free radical scavenging activity method was used. The antioxidant activity of S. varians and R. hieroglyphicum was observed and considerable variations were observed between these two species [ 27 , 28 ]. From the present study, it was recognized that the methanol extract of S. varians and R. hieroglyphicum had favorable antioxidant prospective and varied bioactive phytochemicals. It can be utilized as a source of a possible natural antioxidant for numerous determinations. Recent studies by other researchers have documented a strong positive association between the total phenolic content and antioxidant effects in algal species that give support to our results [ 29 ]. Antibacterial Activities Regarding the application for bacterial infected skin, all four extracts were tested against five different bacterial strains, two of which are gram-negative strains, i.e., S. aureus and E. coli , and three gram-positive bacterial strains, i.e., B. subtilis , B. pumilus , and P. aeruginosa [ 30 – 32 ]. The methanolic extract of S. varians showed lower antibacterial activity compared to the methanolic extracts of R. hieroglyphicum , having a zone of inhibition of 13 mm against both gram-negative bacterial strains, i.e., E. coli and P. aeruginosa , and gram-positive strains, i.e., B. subtilis, B. pumilus , and S. aureus , with inhibition zones of 11 mm to 14 mm. S. varians extracted in a mixture of DCM and methanol shown the highest antibacterial activity against both gram-negative and gram-positive bacteria, with inhibition zones measured at 22 mm against S. aureus , 21 mm against E. coli , 16 mm against B. subtilis , 20 mm against B. pumilus , and 22 mm against P. aeruginosa . Both extracts of R. hieroglyphicum , i.e., methanolic extract (RM) and the one extracted in DCM and methanol (RDCM), showed similar activity against both gram-negative and gram-positive bacteria. The inhibition zone shown by the methanolic extract was measured at 17 mm against E. coli , 17.3 mm against P. aeruginosa , 16 mm against S. aureus , 17.2 mm against B. pumilus , and 16.9 mm against B. subtilis . Extracts of S. varians exhibited good antibacterial activity, but the results were less promising compared to R. hieroglyphicum extracts. The sterols extracted from Chara wallichii showed activity against various bacterial species [ 33 ], indicating that freshwater green algae may also show antibacterial effects. The results indicate that algae displayed antimicrobial activity depending on the solvents used for extraction, methods of extraction, bacterial strains, and culturing conditions. Importance of Natural Antioxidants Overall, algae exhibit complex taxonomy and are subjected to rapid change as new molecular information is discovered with time [ 34 ]. During the last few years, natural antioxidants have gained importance because oxidative stress production is ultimately a significant factor of disorder [ 35 ]. Antioxidants are important for the neutralization of free radicals by various methods and prevent the body from diseases such as aging, liver cirrhosis, cancer, and atherosclerosis, etc. [ 36 ]. Both natural and synthetic antioxidants are available in the market. Synthetic antioxidants like butylhydroxyanisole have shown harmful effects nowadays for the human body [ 37 ]. The natural antioxidants have gained attention for their minimal harmful effects [ 38 ]. Intracellular Antioxidant Mechanisms The resistance gained by algal cellular and physical compounds against such oxidative contest showed that the intracellular antioxidants are supposed to be responsible for the defensive role as an inherent mechanism for sustenance [ 38 ]. Both S. varians and R. hieroglyphicum contain a number of compounds having antioxidant potential. Antioxidant activities can be measured by different methods but in the present study, DPPH free radical scavenging activity method was used. The antioxidant activity of S. varians and R. hieroglyphicum was observed and considerable variations were observed between these two species [ 40 , 41 ]. From the present study, it was recognized that the methanol extract of S. varians and R. hieroglyphicum had favorable antioxidant prospective and varied bioactive phytochemicals. It can be utilized as a source of a possible natural antioxidant for numerous determinations. Recent studies by other researchers have documented a strong positive association between the total phenolic content and antioxidant effects in algal species that give support to our results [ 42 ]. Our study contributes to the growing body of evidence on the bioactive potential of algae, particularly from regions like Azad Kashmir, which remain underexplored in scientific research. By showcasing the phytochemical, antioxidant, and antibacterial properties of S. varians and R. hieroglyphicum , we underscore the potential of these algae as sources of natural bioactive compounds for various applications. Future research should aim to isolate and characterize specific phytochemicals responsible for these activities, paving the way for their potential therapeutic and industrial applications. Conclusion The antioxidant activities of the algal extracts were reported in this study for the first time. The present study showed excellent antioxidant capacities, strong free radical scavenging capacity, and a significant quantity of total phenolic compounds in the samples, which could be utilized as an effective natural source of antioxidants. Moreover, it has gelling ability, so it could be useful in cosmetic, nutritional, and pharmaceutical products. However, the sulfate content and antioxidant activity relationship need to be investigated further. It is obvious from the current study that the S. varians and R. hieroglyphicum present in this region are reservoirs of novel bioactive compounds that could be used sustainably as a huge prosperity for the discovery and development of new drugs against various human diseases, especially oxidative stress-induced disorders. However, there is a need to investigate the cytoprotective properties of the algal extract in the cell culture system for proper authentication of the therapeutic and prophylactic potential and in-vitro antioxidant strength of this bio resource. Declarations Conflicts of Interest: The authors declare no conflict of interest. Author Contribution Syed Mansoor Ahmed: Conceptualization, Data analysis, Writing-Reviewing & Editing. Hajra Hameed: Experimental design, Sample collection and identification, Writing-Original draft, Writing-Reviewing & Editing. Muhammad Tariq: Supervision, Writing-Reviewing & Editing. Afsa Hameed: Antibacterial and antioxidant assays, Data analysis, Writing-Original draft. Muhammad Shakeeb Sharif: Phytochemical analyses, Data visualization, Writing-Original draft. Dunia A Al Farraj: Funding, Writing-Reviewing & Editing. Reem A. Aljeidi: Project supervision, Resources. Afshan Afareen :Literature review, Background research, Methodology, Writing-Original draft. Abdul Waheed: Experimental procedures, Phytochemical characterization, Statistical analysis, Writing-Original draft, Writing-Reviewing & Editing. Acknowledgement The authors extend their appreciation to the Researchers supporting project number (RSP2024R190), King Saud University, Riyadh, Saudi Arabia Data Availability The datasets underpinning the findings presented in this article are accessible upon request. Please feel free to reach out to the corresponding author for further information regarding data availability and access procedures. 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Antioxidants in oak (Quercus sp.): Potential application to reduce oxidative rancidity in foods. Antioxidants, 12(4), 861–883. Deshmukh, R.K., Gaikwad, K.K. (2024). Natural antimicrobial and antioxidant compounds for active food packaging applications. Biomass Conversion and Biorefinery, 14(4), 4419–4431. Bhowmick, S., Mazumdar, A., Moulick, A., Adam, V. (2020). Algal metabolites: An inevitable substitute for antibiotics. Biotechnology Advances, 43, 107571–107592. Jain, A. (2022). Algae-mediated synthesis of biogenic nanoparticles. Advances in Natural Sciences: Nanoscience and Nanotechnology, 13(4), 043001–043026. Cacace, E., Kim, V., Varik, V., Knopp, M., Tietgen, M., Brauer-Nikonow, A., Inecik, K., Mateus, A., Milanese, A., Mårli, M.T. (2023). Systematic analysis of drug combinations against Gram-positive bacteria. Nature Microbiology, 8(11), 2196–2217. Keramane, B., Del Pilar Sánchez-Camargo, A., Montero, L., Laincer, F., Bedjou, F., Ibañez, E. (2023). Pressurized liquid extraction of bioactive extracts with antioxidant and antibacterial activity from green, red and brown Algerian algae. Algal Research, 76, 103293–103312. Tables Table 1 Phytochemicals presence in the extracts of S. varians and R. hieroglyphicum are expressed as + + detected strongly; + detected; - not detected. Whereas; SM: Spirogyra sample extracted in methanol; SDCM: Spirogyra sample extracted in methanol and dichloromethane mixture; RM: Rhizoclonium sample extracted in methanol; RDCM: Rhizoclonium sample extracted in methanol and dichloromethane mixture Samples SM SDCM RM RDCM Total Phenolic Content (TPC) Linear Regression equation Y = 0.0273x + 0.177 Mean Absorbance of Sample Solution (y) 0.342 ± 0.007 0.678 ± 0.011 0.699 ± 0.003 0.990 ± 0.007 Total Phenolic Content (TPC) mgGAE/g 60.4 183.5 191.2 297 Total Flavonoid Content (TFC) Linear Regression equation y = 0.0503x-0.019 Mean Absorbance of Sample Solution (y) 0.102 ± 0.010 0.159 ± 0.006 0.152 ± 0.009 0.155 ± 0.004 Total Flavonoid Content (TFC) mgRE/g 24.05 35.38 33.7 34.59 Phytochemicals Alkaloids ++ + ++ + Glycosides + + + + Flavonoids ++ + ++ ++ Saponins - - - - Tannins + + ++ ++ Steroids + - ++ + Terpenoids + - + + Additional Declarations No competing interests reported. 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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-4919856","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345218067,"identity":"7e85d66d-0ae5-4d2a-9321-63177ad91f86","order_by":0,"name":"Syed Mansoor Ahmed","email":"","orcid":"","institution":"Mirpur University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Syed","middleName":"Mansoor","lastName":"Ahmed","suffix":""},{"id":345218068,"identity":"e4f09997-2541-4232-98e1-f643707cddf0","order_by":1,"name":"Hajra Hameed","email":"data:image/png;base64,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","orcid":"","institution":"Shenzhen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hajra","middleName":"","lastName":"Hameed","suffix":""},{"id":345218069,"identity":"5e4fa44c-363d-47d9-8931-bc62c09a2cfc","order_by":2,"name":"Muhammad Tariq","email":"","orcid":"","institution":"Mirpur University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Tariq","suffix":""},{"id":345218070,"identity":"28047cf9-85d7-4eb5-a10e-ff1af9cd18c7","order_by":3,"name":"Afsa Hameed","email":"","orcid":"","institution":"Mirpur University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Afsa","middleName":"","lastName":"Hameed","suffix":""},{"id":345218071,"identity":"ea7a494c-cbdf-4688-8050-11a29560b6b5","order_by":4,"name":"Muhammad Shakeeb Sharif","email":"","orcid":"","institution":"Scuola Superiore Meridionale","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Shakeeb","lastName":"Sharif","suffix":""},{"id":345218072,"identity":"d64cb0c4-e06a-4699-b95c-94f654c3cd9c","order_by":5,"name":"Dunia A Al Farraj","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dunia","middleName":"A Al","lastName":"Farraj","suffix":""},{"id":345218073,"identity":"a3fb57e3-bd85-47cd-b5b7-408ba196e1d4","order_by":6,"name":"Reem A. Aljeidi","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Reem","middleName":"A.","lastName":"Aljeidi","suffix":""},{"id":345218079,"identity":"3e5d240a-d876-4449-96cd-c340a1f5e994","order_by":7,"name":"Afshan Afareen","email":"","orcid":"","institution":"University of Kotli","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Afshan","middleName":"","lastName":"Afareen","suffix":""},{"id":345218081,"identity":"429b82fc-785a-49e0-af0a-6b99e52ad410","order_by":8,"name":"Abdul Waheed","email":"","orcid":"","institution":"Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abdul","middleName":"","lastName":"Waheed","suffix":""}],"badges":[],"createdAt":"2024-08-15 14:23:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4919856/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4919856/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":64474713,"identity":"49eaef3a-c7c0-4059-afe3-bd449926d4c6","added_by":"auto","created_at":"2024-09-13 15:13:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67767,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Total phenolic contents of the samples determined by the Folin-Ciocalteu assay using Gallic acid as a standard; (B) Total flavonoid contents of the samples in mgRE/g. (C) Antioxidant activity (DPPH radical scavenging activity) of algal extracts and standard ascorbic acid Each data point represents the mean of three experiments with a standard error of the mean (mean ± SEM, n = 3). (D) Half the maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of algal extracts. (E) The antibacterial activity of the positive control, SM, SDCM, RM, and RDCM was evaluated by the disc diffusion method. The results show the zones of inhibition of different samples against model Gram-positive and Gram-negative bacterial strains. All the activities were evaluated three times. All values are given as mean ± SEM.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4919856/v1/e633c6aa76cc219ea3964529.png"},{"id":72824044,"identity":"29ed4a36-545d-4da1-8d76-cd7af7ade97a","added_by":"auto","created_at":"2025-01-02 14:17:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":550616,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4919856/v1/b21e96de-370c-4841-be71-c8181e7fc412.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Symbiotic Synergies: A Comprehensive Analysis of Antibacterial Potency, Antioxidant Proficiency, and Phytochemical Composition in Rhizoclonium hieroglyphicum and Spirogyra varians from Distinct Aquatic Habitats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA large number of active compounds have been isolated from terrestrial plants and used in the pharmaceutical and cosmetic industries throughout history. During the last four decades, many drugs approved by the FDA were prepared from plant species with medicinal value [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. But in the last decade, many research studies have also been focused on different marine organisms for the production and isolation of bioactive products like proteins and peptides. Algae are one of the most important and diverse groups of aquatic organisms and have significant physiological and biochemical properties. They have been used as the source of different active compounds, which include carotenoids, steroids, fatty acids, vitamins, proteins, lectins, polysaccharides, dietary minerals, diverse antioxidants, and halogenated compounds. In contrast to traditional plants, algae do not possess true stems, roots, or leaves. Algae exhibit complex taxonomy, and as new molecular information is discovered over time, they are subjected to rapid change [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Carbohydrates are one of the most abundant and important constituents of marine algae [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The carbohydrates extracted and isolated from marine algae have many applications in pharmaceuticals and cosmetics due to their important physical and chemical characteristics [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Shanura et al [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] screened \u003cem\u003eGracilaria edulis\u003c/em\u003e and its methanol extract to study their anti-inflammatory properties in contradiction to lipopolysaccharide-induced inflammatory response. Koneri and Jha conducted a study on \u003cem\u003eG. edulis\u003c/em\u003e and its methanol extract in which they determined their anti-diabetic properties for the treatment of male rats with fructose-induced type 2 diabetes mellitus [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Another study was conducted by Patra and Muthuraman [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] on ethanol extracts of \u003cem\u003eG. edulis\u003c/em\u003e, in which they revealed their anticancer properties in mice for the treatment of ascites tumors. Furthermore, \u003cem\u003eG. edulis\u003c/em\u003e was screened to study its antioxidant and hypoglycemic properties in different in vitro mechanisms for the first time in Sri Lanka. The results of the study showed that the evident hypoglycemic activity of \u003cem\u003eG. edulis\u003c/em\u003e was produced by inhibiting glucose absorption. The antioxidant and hypoglycemic effects were further confirmed by GC-MS analysis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In several Asian countries, seaweed has traditionally been used as a food. Important biologically active compounds like fatty acids, vitamins, polysaccharides, phycobilins, carotenoids, phycocyanins, and tocopherol is present in seaweeds. Seaweed has extensive applications in the food, pharmaceutical, and cosmetic industries [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The crude polysaccharides and derivatives obtained from them show various useful bioactivities, including anticoagulant, antitumor, immunomodulatory, and cancer-preventive activities. These also have powerful antibiotic, anti-inflammatory, and antioxidant properties. For instance, strong anticancer and immunomodulating characteristics have been shown by the sulfated polysaccharides (SP) from \u003cem\u003eEnteromorpha\u003c/em\u003e and \u003cem\u003ePorphyridium\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Algal species also show antibacterial activity against \u003cem\u003eStaphylococcus\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e. Sodium alginate and fucoidan obtained from \u003cem\u003eLaminaria japonica\u003c/em\u003e show antibacterial activities against \u003cem\u003eE. coli\u003c/em\u003e by adhering to bacteria and killing them [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The growth of \u003cem\u003eStaphylococcus\u003c/em\u003e aureus was inhibited by an S-galactan obtained from \u003cem\u003eChaetomorpha aerea\u003c/em\u003e, which could not inhibit the growth of \u003cem\u003eSalmonella enteritidis\u003c/em\u003e but restricted the propagation of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] While against \u003cem\u003eS. enteritidis\u003c/em\u003e, a notable inhibitory activity was shown by the carrageenans obtained from some seaweeds [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Raposo \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] reported the antibacterial activities of some species from microalgae and explained that these activities are based on the solvent used for the extraction of polymer. The study hypothesizes that marine algae, specifically \u003cem\u003eR. hieroglyphicum\u003c/em\u003e and \u003cem\u003eS. varians\u003c/em\u003e, contain significant bioactive compounds with antibacterial and antioxidant properties, which could be beneficial in these industries. These findings suggest that the stressful climatic conditions experienced by marine algae lead to the synthesis and production of secondary metabolites, which are crucial for their anti-stress, antioxidant, and therapeutic effects. Consequently, these compounds contribute to various beneficial biological activities, highlighting the potential of marine algae as a valuable resource for the development of pharmaceutical and cosmeceutical products.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eCollection of Material\u003c/h2\u003e\n \u003cp\u003eAlgae samples were collected from two locations: Mohli waterfall in Kotli and Lahri Head in Mirpur, Azad Kashmir. The samples were manually gathered and placed in glass bottles which had been thoroughly washed, dried, and preserved with diluted formalin, and was also kept in a cooler with ice. The algae were identified as \u003cem\u003eR. hieroglyphicum\u003c/em\u003e and \u003cem\u003eS. varians\u003c/em\u003e through microscopic examination and by observing key characteristics like appearance, color, texture, and smell [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003eExtraction of Algal Sample\u003c/h2\u003e\n \u003cp\u003eAfter washing and fully drying the algae samples, they were ground into powder using a mortar and pestle. Each sample was split into two equal parts. One part was extracted with methanol, and the other with a mixture of DCM and methanol (1:4). The samples were soaked in these solvents and kept in a dark place at room temperature for 7 days. After 7 days, the mixtures were filtered, and the process was repeated twice more. After the final filtration, the filtrate was left to evaporate until the solvents were completely gone [\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003eTotal Flavonoid Content Determination\u003c/h2\u003e\n \u003cp\u003eTotal flavonoid contents were measured by the method used by Ordonez et al. (2006) with minor modifications [\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e]. The stock solution of the algal sample was taken in different concentrations of 10 \u0026micro;L, 7.5 \u0026micro;L, 5 \u0026micro;L, and 2.5 \u0026micro;L in a cuvette. Then, 2.5 ml of distilled, deionized water was added to the cuvette. After that, 180 \u0026micro;L of 5% sodium nitrate (NaNO\u003csub\u003e3\u003c/sub\u003e) was added, followed by 180 \u0026micro;L of 10% aluminum chloride (AlCl\u003csub\u003e3\u003c/sub\u003e). After one to two minutes, 1200 \u0026micro;L of one molar sodium hydroxide (NaOH) solution was added. The whole solution was mixed and remained at room temperature for about 20 to 30 minutes. The absorbance of the solution was determined by a spectrophotometer at 510 nm wavelength.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eTotal Phenolic Contents Determination\u003c/h2\u003e\n \u003cp\u003eThe total phenolic content of algal extracts was determined by the Folin-Ciocalteu assay method [\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Different concentrations of the algal extract solution were added in a cuvette and labeled accordingly. Then, in each cuvette, 500 \u0026micro;L of Folin-Ciocalteau reagent and 1400 \u0026micro;L of 7% sodium bicarbonate were added and mixed. After mixing, 980 \u0026micro;L of distilled water was added to all cuvettes and left for 30 minutes to complete the reaction at room temperature. The absorbance was checked at 750 nm wavelength by spectrophotometer. The absorbance of the extract was measured three times, and its mean value was taken. The procedure was repeated for different concentrations of gallic acid used as standards, and their absorbance was measured.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003eDisk Diffusion Method\u003c/h2\u003e\n \u003cp\u003eIn the disk diffusion method, five different strains of bacteria were used: \u003cem\u003eEscherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus\u003c/em\u003e, and \u003cem\u003eBacillus pumilus\u003c/em\u003e. Nutrient medium was used for bacterial colonization, which was autoclaved along with Petri plates [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. In the laminar flow hood, all Petri plates were labeled, and autoclaved nutrient agar media were poured when the temperature of the media reached between 40\u0026deg;C and 45\u0026deg;C in petri plates with a desirable strain of bacterial suspension. Rifampicin (a synthetic antibiotic) was used as the standard by soaking in a disk and placing it at the midpoint of the petri dish. All plates were enclosed with parafilm to avoid impurities. The plates were placed in an incubator set at 37\u0026deg;C for 20 to 24 hours [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e]. After 24 hours, zones of inhibition in millimeters were measured from the circumference and the area free from bacterial growth around disks [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. Interpretations were noted three times for each type of bacterial species against each sample, and the mean value was then calculated. The antibiotic effects against selective bacterial strains were plotted in graphs.\u003c/p\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003eAntioxidant Activity\u003c/h2\u003e\n \u003cp\u003eAntioxidant activity was checked by using different quantities of algal extracts in DMSO, in separated and labeled test tubes. Then, 1440 \u0026micro;L of freshly prepared DPPH solution, prepared by adding 0.012 grams of DPPH to 100 ml of methanol, was added to the plant extract in the test tube. The solution was mixed and left for about 30 minutes to complete the reaction between DPPH and plant extract. In another test tube, 1.5 mL of methanol and 1.4 mL of DPPH were mixed and used as blanks. Absorbance was measured after 30 minutes by a spectrophotometer at 517 nm, and as a positive control, ascorbic acid was used. The IC\u003csub\u003e50\u003c/sub\u003e values were then calculated by the method mentioned previously [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. The scavenging activity was measured using the following formula:\u003c/p\u003e\n \u003cp\u003e\u003cimg 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op/gp89x014MinDR4gkMbkGUTb46mTkdnCMmBPMkiAEkT3PIl3MVwnHwlIGnGDfccEPF4IgnSpTLIIZBJsevsA/tyqCKQQSinbYiH77zBIb+yGA3235V0sQnyzAMwzCMAhELlSgWwFEszKKlS5emUrMjFkipb5VJlw75ris0hS5rR9a9uqSr61NPPRX17ds3Gj58eCplx9CmTZsoFqnRmDFjUimZeeaZZ2SfeAASjR8/PpX6KY8++mjUoUOHKB68ZXUc8YApeuKJJ6LRo0dH3bp1i+KBG6bwqF27dtHKlStlm6T2WrRoURQPBqJYlEexcJe0eOAn7RcPQqJ44BjFAxdJV9auXRt17do1qlu3bjRjxgxJo3zqyT7t27eP4kGGpJc75hJjGIZhGAUCCysRQLBSYmH23UayIZ0lMJOFMN91habQZe3IuleXdHXFj5xJp1h+C0Ws3VLfPoX/wzTcaEjDyp4NPHHh6QEWaXUvUnDlwWp9/vnni3uKEpap0P//+Mc/ilUcFx8m7/L0iDaKhbpsE7YXTyKYX4G/P24tGgkJv39cvJiwy1wPLPa46QB5YUlnP7YhTj3wRIXIPUxGZi4AYTB5QlHumGA3DMMwjALBI3oEC2IGV49CCjWj/MAtBF93fOpx7ygEodjl/zBN3YwQqqEATwcuOuSDCFZhzXwAXGJwbeJlTz5hmQouL+PGjRPfdwYAzCPQ/NLB3AzaCJcgxLo/GMAFCTccXJ1wF9Pjwf0LFx7ScQ9TtytQVyXAjUdFfjljgt0wDMMwCgTiAYsqAgUfaBPsOzdM0iQm+hVXXLFdVJ1iooNF5hJka2XHIo3QRThTZ6zTxLgnCs+ZZ56ZdV/Gjx7hjf8/39NZ4n2YVIrPOROnfT91YO4G/usIciZGq2Bn8vNjjz0mEX54oqDzKQA/fsJjUmdEPoOGcscEu2EYhmEUCFxhNOwiosG3+inZCBijdoAAbt68uUx8TOoLhSCpP2nf44kPk1XT4e+L2wrRfBD5uLIQ3Yewk0S2UYu9kq4PJ6Wns8T7MIEYUc3kZ4S+D6KfY2EQzNMrJnMDn1xrbB+2LWUyKZZ9scSTf7lTJ25c++UwDMMwjAJAFBCsktdcc434+/bp06fixTs+xDUnEgYCxTByBenG0qNHj+3CYgJ9EMGNX/qoUaMqwmr6qPzzBTUx3q+++moJ9di5c2c3cuTISi98Utg3SYgnpeOXjvDnOiA/Iu6E8CIrXGgY4BCRhgg/mg/uLESaIcwogx9i2BNClLrqG4GJckMUHo1cw5Mu3kJLpCTccgjrmVRuWRE3rmEYhmEYBeC1116Lzj//fJRQdOWVV8r/ScRiSLaxxZbqLOvWrUv1qMpMmDAhql+/ftSpU6dozZo1qdTP+OSTTxIjtcQiWKLFxIOAaPbs2dHHH3+cWpM/8cA02n///aNYMEePPPJIKrUy1He//faL4gFGFAv87eo2Z86cqF69elHbtm2jF154QdLuuOOOqFmzZlE8oIhWrVoVffTRR5KuEJGmYcOGsk26cssJc4kxDMMwjAIR31crJtjxnSUJXk4EvBzHlp1rIb54UnouCy4v6ieehPY7vz/6YL0OLeHEMyd+O5NkefIzd+5ciQ5TDNJdF9Q1rC/b4rfOJy4uYb2TjtHfB9/2cJ9yxFxiDMMwDKNA8MZS3BFwQyCqBqHwmPwWwmN8QtnVBiFh1AzIN8KG+hM7tT9dddVV4pZFdBU+/RdoJUE0G17yhVsJL0XChx3fbyLEpHvxV7Zk6xJDnRmM8OKkDh06yHEBfupTpkxxgwYNkpdRETKSCd28yXbgwIEyqXby5MkSylFdzPCHv+eee1yvXr3cEUccIfnzMivaqVyvObOwGznDG/i4YPAhY0Tuww8DFwPriw31wG+NelCuUTz4kaxXr95253tngSgD9DMW+l11II4wbcmr5Y3aByKDyBT8DmKdxJc2CbYphKXVlp13of+oqKW/+UJU+x4W+KR5Er6tFnFLFBgmxuLzzVts27Vr51555RW3bt26HRJhhegwRNFh0nZoEedYiLzEm2r9ybv8jrIf++C77u/DgJg3CTMxlQEzb+EFf5tyYzvBroIr3cLIi4k03MCMnQ8E2znnnCOvVSZuak2BaGLE3bdv3xqth2EYhg/CASHF/RLLYDrBXs7Yg/nSIul8aN/DbUbFqg/9E4gNz8RN8mACKxb2Bg0aiFUasY8Ve/PmzbJtvqgbC586wAjBio8FnGguuORo/QDRzWRuBrnEitcBCPHeW7RoIUKeF1T5+5CGyKc8dCvXZLmzXctdfPHF8niBE6fwPycTYcRsYWYQE6+TRq3tYCneWa2KSfB4Dau2voUshP5DXyH2bDEh7iplXXrppakUo5jcd999buvWrXL+aztJlm/CnRH/l4W+Vx14bEtbch0lYZb38gaRg+BBPGSysBtGofCFqoJgx68boZruN+utt96SiCy33XabO+WUU1z79u0lHQs3QpjIMsuWLZPfveqALznuX5ks9USFadu2rVjLZ8+eXckoTIz2tWvXymCCqDcq2LGc8ySAY8Tf3tekxGh/6KGHZCCACw5vTC13Eoc6nFweiyh6srlpqVjj7Vk8Nqnu4+FSBz9EwzB2Dvh9IwZxTVHT5Ru5E1o3ERO8xAURhbWwNgr2JIEISZZeo2bQuOO4mSS5xCDWCY+IARYxjY871ngFlxLcbrBsE4L0+eefT635lPBch/8zl+PZZ58Vg+fChQvF8Iuf/NKlS8UazkuSyFv3w80FTYnvOj7vEydOdA8++KD4od99991iKcfHnpcqaf8jDYs7bznlKQAGVsq6//77JeQj+vS0006rFCKynEl+NhGT6fEBk2gA0U7D1FY4+eZuYRg7B/y4M6mppqjp8o38CIUAwoPJdVgpOacIn9oA1lpewsPxpBPmhRRFNSH+sQTjfsGLhvRtmuWKGlPx8w5hEDlz5kw3YcIEEes8FcI6jVUemGw6f/58EdxEo8HizeRTXZ9EeO7nzZsnEz5POOEEd9lll4mrGP2HN7526tTJ9e/fX8S4H90F1xWMpD179pTBBHHgjzvuOKnj+PHj3emnn57a8jPU6wNdih7t3r27zLmi7kz+ZuJ3ukg6ZUd8USSyfPlyrhZZQvx1I0eOTKXWLp588skoHpnKMXK8RmW6dOlSEm1D/6vN/dDYcfTp00f6En27Jqjp8o3C8fbbb0cHHHBAtMsuu0j86NoA98S+fftG3bp1i7Zu3ZpKrT28//77USwgJW53ixYtolj8pdaUH2+++Wa0zz77RI0aNYpmzJiRSq3Mhx9+GL333ntRLMLlMxbxFbHP+YzFdaX1bO+vrwq2jwc/FXmESzwokjJDiPvul83CuYkHUKkttiesr+YfC/3UFrWDtBb2bAn9gnj8gQ8moy0WRjqhrztREpgsqCMyJrHqtj48HmbEpXnxnX1DGEkyWZYIDmzHiDKMGkIdKEcjXeAfpfVkP9+flDJ4NMMTBMCvK6wfefC/1i1TpBLK4ngpW7cPF59we/JOOu4kwuPkuPhOHuoTpseu+fNJeToiD6Fs/zyk25Y0jR6T1BbhcaUrN9f65QLtE/ZP/qetQrLpf9SJ9uaYdTvy8/t80kRuv33C9T5VXU+UTz1J1/6p54Dt2Tep3UijXL/e4aJtwvnw+5RCun++tS3YjoXvSYT9IGmpimza3SdsR62zQtsRxgx4O55up8fLJ3UmTQnPG4ufZ7heCc8XpCt/0qRJlfJg8fejXv46//wYNQdWdvxtYyHhNm7cWMmKGN93U98+Iymt1MAnePny5WknDeZKVcectJ52xN967NixYkGtilzalQmNhx12mFh08TAoV6ss1nP8vXlSwCRO3LOS4PgICYnbC5/0WX5DgE/caPz1bO+vrwq2xzqveYQLE2EpMzxH9C+/bBbODU+s0hHWV/PHql8O11bWxAeTSCYLe48ePSrWvfjii6nUKJo5c6ZYpdXqyif/szA6h3vvvbfS/lhGBw4cWGHN1vywNvG2Lc3Lt3iTh7JlyxbZju35zqKWKj6BfSlDy7zhhhtkH8rWbVn8Y4F0VmRtG82fMvWYQksveVJvyvOPLV2ZepzUEVjPvmxL+2YiPE6+s/AWMN2fump9+A5qpU6y7LEP6/z6+OdP24Z08tGywnbQ4yJdy9V2oC5KrvXTdWF5SWjeui3/6/7hOaZu1KGq/kedSGc9aN8gTesPtB/p4f4KabSd3x+yuZ60L5MvdaHeLByXngv+D2EfP289zyx+P2O9thGLbq9tRx6k088oX/ucpvt5AcfHOsrXY9V+wOIffyaybXfQdtS6sF7bjLoqmkfYz9jPr6NPrueVNiP/pHLSle+XodehD8fDurCtQzSPbBfqauTHBx98IO0Xi4ioV69eUTxITa0pTx566KFo0KBBUSxmo6OPPjrt21t3BD179oyGDh1alDpgoe3Xr190+eWXRxs2bEillhdYtYcPHy5Pd/r37x9t3rw5taa4ZGN1N6pHVoJdbzbcGLjBabp/g2Ab0vSGrujNPrwJaR56k2N/vfmSb1JeWrZfLjfSMG/qqYLBz0Nv0qGA0ZtxeDPUG2tYDxWsfjrfSQvronmHN/N0wpb0ME3zThIjSWi9VYywj9ZVb/5hGaSx+Gi54bbp2hf0fCcdV9g2nHPyYZ2SS/0gXXlJ6PGEbUi9/OPItv/RX/k/PC5t/3B/7Qt+/1XI20/P5Xqib5FGW+o1BJoH6T56fNo/FK1feDyQ7pi0Tbi2/HbVMrhWfHK9HpLIpd21DcJ8tc2oj8J+SfkqrGMJyeW8QrpyMpWv7RyeM+BYaH+jdODxPa9Kx72C17EvWbIktab8wK0AAXjJJZdEY8aMEXeRl156qZJA4zvX2k033RRde+210bhx40TkM3ABtr/55pujUaNGRZMmTYpWrFgh6crixYujW265Rdrs3XffjRYtWhSNHz8+mj9/vrh3AK+3HzFihLzi/phjjhFRvWDBAhHZlDNr1iy51l5++eXo1VdfldfWcz/hNfeAkH3ggQekftddd51si+uSzxtvvCEuP7feemvZuv288847Uffu3aPddttNzseOwgR78cnq2ZY+CubNUjj3xzdhmYDAI2aFmNjxTXe7sG+E3AEe8yY9mifEGRCBpmXLlvKdiVfxDWi7vHgLVlzninLJj8fIzBz2IaoNMUSBGcOKRrshfJEPsTxBZ1VXBccZiyDXqFGjVEp6ePwN4SRe2jAEd4v4R08mafhoO+Ci89hjj8n3bODNYsBxax777ruvfB566KHymQneDAZhffz2zQbagOPSycoK55zwdn7IqFzqly+DBw9OffuUsP9k2/947EY/aN26tfxfFdrvwomF9GMm9eR7PWnf4pzoNQS0L6hrl3LnnXfKZ+jO1rVr19S37NE8TjzxxIrrC5jJD2HEk1yuh3Tk0u6jR4+WzzPOOEM+FX53OJe4nFSXXM5rvjBximPm9y78HWUy1gUXXJD6zygFeHzPbwjXYCxWM4bFox+WMoTUw02hefPm8pIa4H+fWExL36Qvct/o16+fmzZtmtxTeXkN65hMGAt5N2TIEHFp0agj7MsEQyYjMgmSexwTHocNGybxwYkoAkQOiQW427Rpk4sH7bIP90xibT/++OOyLh7QissMrmHxAMGdd955Un9+hwhNy+9BPOhwsWCXiZBMUsR1BDgP3I+YGElYTtwwfEr9PCnxAEZcYg466CBxiSkWYXtk4yZjVI+sBDsdnZOjCzddFdoKFxWijJPmLxrXE7gwq4KbEfn4N/90aH4nn3zyduUiaEAv9kKCcOPC5scYf1x8VUPRp+Cnm0RSTFB8BAGhFh6PotvkiwoVPmlrziW+2UkgNkCFX77wpjTIFHlIyaV+uYL45SbKzQMfZvKmDASVCuNc+p8OOOgPwM2DvLTvhVBGly5dJH8VrsBN46yzzkr99ymFup6SSNcnszk/1SWX6yEdubT7qlWr5DOb85kvuZzXfKH+vHOAwReDOYXfH8RKNoOCsC9Vtfj++EZ+8DvGC1wQmOmgrfnNK1WWLFkiv5scCxFiqC++5Fpn+j1iecaMGRLTm/9HjBgh8bLxpyad9YhlxDaCnusX8Q5cM8cff7wM+vlNe/jhh92AAQNkPwYI7AMYFGbOnCnhBxHrhAhE4BO3G4F/5ZVXisGCEIBbtmwRsc9L/ojDvXLlSqkTv3Hcj55++mn5LWUOjs57ITKM/uZixPNDHALppQ7RVPDtp804X2oAyxa/H5Zyn9xZKczskRRc1JzkdEtoLUwiHxGCtT+pPBYGG8WAGyU3ScIOITaGDx+eWlMZ1gM/GD782AA3+hB+bJKOhYWXBVUXRCkT9ohfyo/X5MmTU2s+o5CT13IdNGVTv3xBwHHz4IbBQK9Jkybyo63k0/904iUhqnh/QdI5VfQpw9SpU+UTsMyGFmAoxPWUxFFHHSWfxMP10bfZMemqWORzPaQjm3ZfvXp16ltxyeW85gt5YWW/5JJL5PcHEEF9+vSR71WR1IcyLYX4rdmZ0HbzoV8SA5pB1YoVK1Kp21OKYhBRjoGIyXw8MaPvEa4SgYzVnE/g5TRr1qyRawArPPDEh5B+GzZskCd69FFeigNYr3nhjYKwbtWqlYhMBD4BH3j9PG3JQHWvvfaS7bB8sw0D9r333lvSgPYdOnSoGAN4lT4vyTn22GPF0PP73/9e6oTQp0zOB8fBZEcs0EyKVCibey8TF1nKEcIxzpkzR+rPAEifdGaL3w+r6pPlMICpbRRUsHNzRGxVB3UzSedCk0SmH8JigEUAKzgLjzp5q2e6C4PHeggvfjhUBPOJlZfHd74Lg5KL20uuYE1ApCKiKQcrZVIdCola6H33pHTsiPpxvriRjBw5Uv7v3bt3hTUxl/7Hem4KPOrl9c24V2DVyATr6TfkTz/AOoyITbIAF+J6SoKBJjdQ6qCDFQSgugIV8y21+VwPIbm0O20NtHMxyeW85ota2QGhTjvwW1TIQYGRPwiYUMRg4SSqz7Zt29z06dNTqaVDOMDwoc5Ypflt5GU2/C7zJBmXC8Q6bjG4vPBbikDEAEJUDkAQY43HAMILejp27FghgnFPwY1FBT/uQ6TxSWQd3P5oR64lBgu44gL14akVUU98Qc22RAThFftsjxDX33H+R8Tzkh7uQ7y9U8FAwZs3tQ3Ynxf1YF0v1wgxWNfvuusu98tf/lIGPUbtomCCXUVZ6B+sIAzUKpQJ8mEEDLfffrt8hoTiipt9Ujg3bmjFeKyLTz83Z30knwlusliIGf3zY8ePC5+IxXB/Db+EL1+SUOMY/Ufu+cAPMI/VETmZxIRvva1umSrCcLFJOi4Ejoq3bOuXDyqkgLyxIPIDTX9DQEIu/Y/1iGqeruQyqNCnMXzi83nhhRfK/z6Fup7SocKZvkmf5DsCs1hPpJRcrod05NLuHBfgT5tEIX8fsjmv1UWt7Pzm8VIQzpn2FaM04SUwWJd5W+Ty5ctTqaUB118SuPHwdBOLM9ZyrOAYNnijJCIYcc2C8EZsh1ZzwCKOaEdMI/ZVzK9fv15EvD8HBfGMgCcPXFHYl3CY+GDroFsHBwjq0ALOS46YK4BY56mbQp64uiDKGTw1bNhQ0hl08NSD7ak7cN/heLHQ6z2gHNABB4Mj7rHM8cEgU07HYGRHWsHOaDYXdOIVNxIseCqgEWiIC956le2NRa1ITApRIQfkhVhWYUt+PAbnQuMxGsJSBSHlY9nQSXqFgLqQL+XhUuGLT3UnUPx68GgQazHWeC4uPpMeN/P2Ly4yxAh1962CfMfPj22qw+LFi+WTR5pKKPy07vqoHcurf6w+2fQTJq1yXPgHIkD9vGhT/BV1gJBL/fIBIeWD4Asn0Gbb/xD7gAVHYRu/fkl11fOMBQmSrotCXk8h5EH/QjjjIkSfpD8XY5AUksv1kI5c2v3cc8+VT25k+jRB4X91xUmCPLTdsyGb85oLSeVzfrR/EgBAj88oXegHXM88hcUw4N8rVGyVGvQ9fou5v9Lf8AVHuDNgxLKOEKfuCH5cZ/CdxqUEEPqIZ53MiWhG5LMtvuP8pnKd+Pcy+jkDgKZNm8r/tBHiGcGOFZz8+I1iwSedshGoCHhA4PNUFjGP244P2yLoyZ9BA3XHCs2r83lyoL/nDDwQ8uTBnAPKLwdoV+rNZFtckzhXPEngeI1aRtyZtyPuqJVibYch2NKhocfCJb44K4Wb05BqLEl5U36bVAhGFr7HPxySTywiU1t9SiwCJV239Rd/W7bT9BuC8I16rJRB2YqmUz7HpqHhtDzSSWM/ytL82VaPS/PQ/XUhhBuLXx74bRMuYXi4JPz2oMwQrQ/bUA/qTZruw/+kg38eGjduLOVTZ7YJ20ChLUgnTx/2JZ2FfdmOffnu941c65euvCQ0bB7b0k5+mn8M2fY/+pFuQ1uz6HGRxnd/ex/KY5tM1xb5af7+ErYZZZDOOfL7E9voPn45Wm/agXrowja0h7aNQp7a/v6147dT2P5aBvv5+bEd6dleD0nk2u7aPiy0Eev5ZPt07aV56nrtJyx+24dwLGyT6bzqNpnOV1i+D2m0K/sb5QFvfZw1a1Z08MEHR7Fol2si05sbaxLeEDlnzpyodevWEo4yFsOSTr8bO3Zs1KBBAwmLSPjFWChGQ4YMkdjssWCU64QY6YRjJLzi1KlTo1atWkVTpkyJYqEexQPMKBbqErJRiYV+dNJJJ0W9e/eW2OeEB1y5cmUUC3Mpj3ZbuHCh5E1e8UA7mjt3roSOjAfHkkcs8KPDDz9c6hULcUmDeLAgYR07duwYxUJWwkdSz86dO0ennnpqFA/6ZTvKJAxks2bNohtvvDG6/vrro+eee07WlTrxQCOaOHGiHCNtSHsatZPtBLveTJIWLpiq4EalN04WXxwBNyE/TxbSQvhxoC4qFLg58X8SbMtN2d/WFxbpjsm/CYfpwA2UvEjjBqr46Ryrbs936uALa1+oJi1sH97cyV+FDYtfRibSHacPbaXnwG8njo80/zghbFv2pX58suixpmtLH7bRslk4RvLyyaV+mo+/ZGon1tGWeu5Y/DJ8su1/Wi+24zv7cT5J4zj4PwnSybMqqrqeNN1fqKffzrqQBrS5Hle6RY81qU+RT7rznS5d88vnekgi13anXD3v7EOfTjo3mi/bat9MasukvgBVndcwHxa/zyaVnwT9IKnfGjUHoo/Fx09D+PLq+/r160eDBw+ONm3aJOmlBiKY2PG8eGfYsGEV8cgR34j1OnXqRB06dJC46LBs2bLoyCOPlH676667yj6vv/66rEMQI9K1r7ds2TKaN2+erFMQ/sQ+5yU/KrY3btwYNW3aNNpjjz0qxD0x0hHZ5EM9EPLK+vXrRWwTy33btm2p1M+YPn161KRJE9l39913j84++2wZmCgI+wULFkTNmzePDjzwwGj16tWpNaUPg5pDDjkkGjBgQMXgyqid1OFP3ImNIsGjPh69q4sDk0LwxVOI1IFLAIth7Ah43M1jU3Wn4PGz716CmwiuOPHAoNpuHSF2PVQfzh8+wbgDFNuFyTAMwygNTLAXkWxvrEySMYFi7CjwXycEW6aINmyDT3shBbtdD4WBibIMqjg/hmEYxs5BQcM6GpXBiskE1XBCqg8TZQv1chXDqAqs20yK1JeRJIEVfM899yy4dd2uh/xg0jXnjU/ODRMXixGBxjAMwyhdzMJeRHj5D5EcoEePHvJyCJ+5c+dKWLZsw9kZRnUh2hCh2aBNmzbycg0fogwQLYG3DBba3cKuh/zgaYdGnoGRI0faS40MwzB2MkywFxlCWPEqZkJkYV2Exo0bizDBjzfft1UaRr5gqeXFO/RJ/NShbt260id5QyBhJIuFXQ+5w1MHXkpDO1100UVFfbGVYRiGUZqYYDcMwzAMwzCMEsZ82A3DMAzDMAyjhDHBbhiGYRiGYRgljAl2wzAMwzAMwyhhTLAbhmEYhmEYRgljgt0wDMMwDMMwShgT7IZhGIZhGIZRwphgNwzDMAzDMIySxbn/B1eXeT+gaFmJAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003eData Statistics and Analysis\u003c/h2\u003e\n \u003cp\u003eAll experiments were performed in triplicate to ensure the reliability and accuracy of the results. Statistical analysis was conducted using SPSS software (Version 27). Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the means of different groups, and significant differences between means were determined using Tukey\u0026apos;s post-hoc test at a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Graphical representations of the data were created using MS Excel to illustrate the differences in the antioxidant, antibacterial, and total phenolic and flavonoid content between the samples. Additionally, IC\u003csub\u003e50\u003c/sub\u003e values for antioxidant activity were calculated using non-linear regression analysis.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eSample Collection and Extraction\u003c/p\u003e \u003cp\u003eThe samples of algae \u003cem\u003eR. hieroglyphicum\u003c/em\u003e and \u003cem\u003eS. varians\u003c/em\u003e were collected from two different sites of Azad Kashmir and extracted in two different solvents, methanol and a mixture of methanol and DCM, for the assessment of phytochemical and biological activities for the first time.\u003c/p\u003e \u003cp\u003ePhytochemical Analysis\u003c/p\u003e \u003cp\u003eQualitative phytochemical analysis for different chemical compounds was performed on extracts of \u003cem\u003eS. varians\u003c/em\u003e and R. \u003cem\u003ehieroglyphicum\u003c/em\u003e. Qualitative analysis of phytochemicals showed that almost similar phytochemicals were detected in all the samples. Among the four different extracts, \u003cem\u003eR. hieroglyphicum\u003c/em\u003e extracted in the DCM-methanol mixture showed the most phytochemicals. Saponins were not detected in any of the tested samples. Flavonoids were detected in all four extracts with equal intensity. Steroids were detected in both extracts of \u003cem\u003eR. hieroglyphicum\u003c/em\u003e, but they didn\u0026rsquo;t show their presence in the extracts of \u003cem\u003eS. varian\u003c/em\u003es in methanol. Glycosides, alkaloids, and tannins also showed their presence in all of the tested samples, while the tannins were more intensely detected in \u003cem\u003eR. hieroglyphicum\u003c/em\u003e than in \u003cem\u003eS. varians\u003c/em\u003e. Results were in concordance with previous reports where \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e extracts showed significant bioactivities in the form of antimicrobial activity and antioxidant properties, and these activities were related to the versatile group of phytochemicals present in these algal extracts.\u003c/p\u003e \u003cp\u003eTotal Phenolic and Flavonoid Content\u003c/p\u003e \u003cp\u003eFollowing the procedure for the flavonoid and phenolic content of the extracts, plots were obtained against concentration vs. optical density. Flavonoid and phenolic content varied according to the algal species, i.e., \u003cem\u003eS. varians\u003c/em\u003e or \u003cem\u003eR. hieroglyphicum\u003c/em\u003e, and to the solvents used for extraction. Total phenolic content in the case of \u003cem\u003eR. hieroglyphicum\u003c/em\u003e was higher in the DCM methanolic extract (297 mg GAE/g) than in the methanolic extract (191.2 mg GAE/g). Total flavonoid content, on the other hand, was nearly identical in both \u003cem\u003eR. hieroglyphicum\u003c/em\u003e extracts: 34.59 mg RE/g in DCM: methanolic extract and 33.7 mg RE/g in methanol extract. Similarly, in the case of \u003cem\u003eS. varians\u003c/em\u003e, total phenolic content was higher in DCM: methanolic extract (183.5 mg GAE/g extract) than in methanolic extract (60.4 mg GAE/g extract). Total flavonoid content, on the other hand, was higher in DCM: methanolic extract (35.38 mg RE/g) as compared to the methanolic extract (24.05 mg RE/g extract).\u003c/p\u003e \u003cp\u003eAntioxidant Activity\u003c/p\u003e \u003cp\u003eThe graph of antioxidant activities showed that the dosage of algal extract is correlated with antioxidant activity. In the present study, it was recognized that the methanol extracts of \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e had favorable antioxidant potential and varied bioactive phytochemicals. It can be utilized as a source of a possible natural antioxidant for numerous determinations. Although many past studies are available on the bioactive chemical composition, antioxidant capacity, medicinal properties, cytotoxicity, etc. of the algae present in different parts of Pakistan, no scientific research still documents the algal species showing bioactive phytochemicals and therapeutic effects from Azad Kashmir as far as our information goes. Recent studies by other researchers have documented a strong positive association between the total phenolic content and antioxidant effects in algal species, which supports our results [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The resistance gained by algal cellular and physical compounds against such oxidative contest showed that the intracellular antioxidants are supposed to be responsible for the defensive role as an inherent mechanism for sustenance [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Both \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e contain a number of compounds having antioxidant potential. Antioxidant activities can be measured by different methods but in the present study, DPPH free radical scavenging activity method was used. The antioxidant activity of \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e was observed and considerable variations were observed between these two species [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. From the present study, it was recognized that the methanol extract of \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e had favorable antioxidant prospective and varied bioactive phytochemicals. It can be utilized as a source of a possible natural antioxidant for numerous determinations. Recent studies by other researchers have documented a strong positive association between the total phenolic content and antioxidant effects in algal species that give support to our results [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAntibacterial Activities\u003c/p\u003e \u003cp\u003eRegarding the application for bacterial infected skin, all four extracts were tested against five different bacterial strains, two of which are gram-negative strains, i.e., \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, and three gram-positive bacterial strains, i.e., \u003cem\u003eB. subtilis\u003c/em\u003e, \u003cem\u003eB. pumilus\u003c/em\u003e, and \u003cem\u003eP. aeruginosa\u003c/em\u003e [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The methanolic extract of \u003cem\u003eS. varians\u003c/em\u003e showed lower antibacterial activity compared to the methanolic extracts of \u003cem\u003eR. hieroglyphicum\u003c/em\u003e, having a zone of inhibition of 13 mm against both gram-negative bacterial strains, i.e., \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eP. aeruginosa\u003c/em\u003e, and gram-positive strains, i.e., \u003cem\u003eB. subtilis, B. pumilus\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e, with inhibition zones of 11 mm to 14 mm. \u003cem\u003eS. varians\u003c/em\u003e extracted in a mixture of DCM and methanol shown the highest antibacterial activity against both gram-negative and gram-positive bacteria, with inhibition zones measured at 22 mm against \u003cem\u003eS. aureus\u003c/em\u003e, 21 mm against \u003cem\u003eE. coli\u003c/em\u003e, 16 mm against \u003cem\u003eB. subtilis\u003c/em\u003e, 20 mm against \u003cem\u003eB. pumilus\u003c/em\u003e, and 22 mm against \u003cem\u003eP. aeruginosa\u003c/em\u003e. Both extracts of \u003cem\u003eR. hieroglyphicum\u003c/em\u003e, i.e., methanolic extract (RM) and the one extracted in DCM and methanol (RDCM), showed similar activity against both gram-negative and gram-positive bacteria. The inhibition zone shown by the methanolic extract was measured at 17 mm against \u003cem\u003eE. coli\u003c/em\u003e, 17.3 mm against \u003cem\u003eP. aeruginosa\u003c/em\u003e, 16 mm against \u003cem\u003eS. aureus\u003c/em\u003e, 17.2 mm against \u003cem\u003eB. pumilus\u003c/em\u003e, and 16.9 mm against \u003cem\u003eB. subtilis\u003c/em\u003e. Extracts of \u003cem\u003eS. varians\u003c/em\u003e exhibited good antibacterial activity, but the results were less promising compared to \u003cem\u003eR. hieroglyphicum\u003c/em\u003e extracts. The sterols extracted from \u003cem\u003eChara wallichii\u003c/em\u003e showed activity against various bacterial species [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], indicating that freshwater green algae may also show antibacterial effects. The results indicate that algae displayed antimicrobial activity depending on the solvents used for extraction, methods of extraction, bacterial strains, and culturing conditions.\u003c/p\u003e \u003cp\u003eImportance of Natural Antioxidants\u003c/p\u003e \u003cp\u003eOverall, algae exhibit complex taxonomy and are subjected to rapid change as new molecular information is discovered with time [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. During the last few years, natural antioxidants have gained importance because oxidative stress production is ultimately a significant factor of disorder [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Antioxidants are important for the neutralization of free radicals by various methods and prevent the body from diseases such as aging, liver cirrhosis, cancer, and atherosclerosis, etc. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Both natural and synthetic antioxidants are available in the market. Synthetic antioxidants like butylhydroxyanisole have shown harmful effects nowadays for the human body [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The natural antioxidants have gained attention for their minimal harmful effects [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIntracellular Antioxidant Mechanisms\u003c/p\u003e \u003cp\u003eThe resistance gained by algal cellular and physical compounds against such oxidative contest showed that the intracellular antioxidants are supposed to be responsible for the defensive role as an inherent mechanism for sustenance [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Both \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e contain a number of compounds having antioxidant potential. Antioxidant activities can be measured by different methods but in the present study, DPPH free radical scavenging activity method was used. The antioxidant activity of \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e was observed and considerable variations were observed between these two species [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. From the present study, it was recognized that the methanol extract of \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e had favorable antioxidant prospective and varied bioactive phytochemicals. It can be utilized as a source of a possible natural antioxidant for numerous determinations. Recent studies by other researchers have documented a strong positive association between the total phenolic content and antioxidant effects in algal species that give support to our results [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study contributes to the growing body of evidence on the bioactive potential of algae, particularly from regions like Azad Kashmir, which remain underexplored in scientific research. By showcasing the phytochemical, antioxidant, and antibacterial properties of \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e, we underscore the potential of these algae as sources of natural bioactive compounds for various applications. Future research should aim to isolate and characterize specific phytochemicals responsible for these activities, paving the way for their potential therapeutic and industrial applications.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe antioxidant activities of the algal extracts were reported in this study for the first time. The present study showed excellent antioxidant capacities, strong free radical scavenging capacity, and a significant quantity of total phenolic compounds in the samples, which could be utilized as an effective natural source of antioxidants. Moreover, it has gelling ability, so it could be useful in cosmetic, nutritional, and pharmaceutical products. However, the sulfate content and antioxidant activity relationship need to be investigated further. It is obvious from the current study that the \u003cem\u003eS. varians\u003c/em\u003e and \u003cem\u003eR. hieroglyphicum\u003c/em\u003e present in this region are reservoirs of novel bioactive compounds that could be used sustainably as a huge prosperity for the discovery and development of new drugs against various human diseases, especially oxidative stress-induced disorders. However, there is a need to investigate the cytoprotective properties of the algal extract in the cell culture system for proper authentication of the therapeutic and prophylactic potential and in-vitro antioxidant strength of this bio resource.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSyed Mansoor Ahmed: Conceptualization, Data analysis, Writing-Reviewing \u0026amp; Editing. Hajra Hameed: Experimental design, Sample collection and identification, Writing-Original draft, Writing-Reviewing \u0026amp; Editing. Muhammad Tariq: Supervision, Writing-Reviewing \u0026amp; Editing. Afsa Hameed: Antibacterial and antioxidant assays, Data analysis, Writing-Original draft. Muhammad Shakeeb Sharif: Phytochemical analyses, Data visualization, Writing-Original draft. Dunia A Al Farraj: Funding, Writing-Reviewing \u0026amp; Editing. Reem A. Aljeidi: Project supervision, Resources. Afshan Afareen :Literature review, Background research, Methodology, Writing-Original draft. Abdul Waheed: Experimental procedures, Phytochemical characterization, Statistical analysis, Writing-Original draft, Writing-Reviewing \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors extend their appreciation to the Researchers supporting project number (RSP2024R190), King Saud University, Riyadh, Saudi Arabia\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe datasets underpinning the findings presented in this article are accessible upon request. Please feel free to reach out to the corresponding author for further information regarding data availability and access procedures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChavda, V.P., Patel, A.B., Mistry, K.J., Suthar, S.F., Wu, Z.-X., Chen, Z.-S., Hou, K. (2022). Nano-drug delivery systems entrapping natural bioactive compounds for cancer: recent progress and future challenges. 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Journal of Pharmaceutical Chemistry, 2, 169\u0026ndash;182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMi\u0026ntilde;o, K., P\u0026eacute;rez, A., Iza-Garc\u0026iacute;a, T.X., Cabascango, D.G., Gordillo, C., Gordillo, A., Vispo, N., Machado, A., Zamora-Ledezma, C., Alexis, F. (2024). Antimicrobial cellulose hydrogels against gram-positive and gram-negative bacteria. Frontiers in Soft Matter, 4, 1362677\u0026ndash;1362682.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMumtaz, S., Mumtaz, S., Ali, S., Tahir, H., Kazmi, S., Mughal, T., Younas, M. (2021). Evaluation of antibacterial activity of vitamin C against human bacterial pathogens. Brazilian Journal of Biology, 83, e247165.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, S.S., Shastri, S.L., Venkatesh, R., Pradeepa, K., Krishna, V. (2019). Antibacterial potential of Caesalpinia bonducella extracts and their isolated phytoconstituents: in vitro and in silico analysis. Journal of Applied Biology and Biotechnology, 7(3), 41\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZia, I., Zafar, H., Shahzad, M.I., Meraj, M., Kazmi, J.H. (2017). Assessment of sea water inundation along Daboo creek area in Indus Delta Region, Pakistan. Journal of Ocean University of China, 16, 1055\u0026ndash;1071.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinkel, T., Holbrook, N.J. (2000). Oxidants, oxidative stress and the biology of ageing. Nature, 408(6809), 239\u0026ndash;252.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuleman, M., Khan, A., Baqi, A., Kakar, M.S., Ayub, M. (2019). Antioxidants, its role in preventing free radicals and infectious diseases in human body. Pure and Applied Biology (PAB), 8(1), 380\u0026ndash;399.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOth\u0026oacute;n-D\u0026iacute;az, E.D., Fimbres-Garc\u0026iacute;a, J.O., Flores-Sauceda, M., Silva-Espinoza, B.A., L\u0026oacute;pez-Mart\u0026iacute;nez, L.X., Bernal-Mercado, A.T., Ayala-Zavala, J.F. (2023). Antioxidants in oak (Quercus sp.): Potential application to reduce oxidative rancidity in foods. Antioxidants, 12(4), 861\u0026ndash;883.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeshmukh, R.K., Gaikwad, K.K. (2024). Natural antimicrobial and antioxidant compounds for active food packaging applications. Biomass Conversion and Biorefinery, 14(4), 4419\u0026ndash;4431.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhowmick, S., Mazumdar, A., Moulick, A., Adam, V. (2020). Algal metabolites: An inevitable substitute for antibiotics. Biotechnology Advances, 43, 107571\u0026ndash;107592.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain, A. (2022). Algae-mediated synthesis of biogenic nanoparticles. Advances in Natural Sciences: Nanoscience and Nanotechnology, 13(4), 043001\u0026ndash;043026.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCacace, E., Kim, V., Varik, V., Knopp, M., Tietgen, M., Brauer-Nikonow, A., Inecik, K., Mateus, A., Milanese, A., M\u0026aring;rli, M.T. (2023). Systematic analysis of drug combinations against Gram-positive bacteria. Nature Microbiology, 8(11), 2196\u0026ndash;2217.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeramane, B., Del Pilar S\u0026aacute;nchez-Camargo, A., Montero, L., Laincer, F., Bedjou, F., Iba\u0026ntilde;ez, E. (2023). Pressurized liquid extraction of bioactive extracts with antioxidant and antibacterial activity from green, red and brown Algerian algae. Algal Research, 76, 103293\u0026ndash;103312.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \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 \u003cdiv class=\"SimplePara\"\u003ePhytochemicals presence in the extracts of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eS. varians\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eR. hieroglyphicum\u003c/span\u003e are expressed as +\u0026thinsp;+\u0026thinsp;detected strongly; + detected; - not detected. Whereas; SM: \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSpirogyra\u003c/span\u003e sample extracted in methanol; SDCM: \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eSpirogyra\u003c/span\u003e sample extracted in methanol and dichloromethane mixture; RM: \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eRhizoclonium\u003c/span\u003e sample extracted in methanol; RDCM: \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eRhizoclonium\u003c/span\u003e sample extracted in methanol and dichloromethane mixture\u003c/div\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\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSamples\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eSM\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003eSDCM\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003eRM\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003eRDCM\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal Phenolic Content (TPC)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eLinear Regression equation\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eY\u0026thinsp;=\u0026thinsp;0.0273x\u0026thinsp;+\u0026thinsp;0.177\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMean Absorbance of Sample Solution (y)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.342\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.678\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.699\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.990\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal Phenolic Content (TPC) mgGAE/g\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e60.4\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e183.5\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e191.2\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e297\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal Flavonoid Content (TFC)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eLinear Regression equation\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003ey\u0026thinsp;=\u0026thinsp;0.0503x-0.019\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMean Absorbance of Sample Solution (y)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.102\u0026thinsp;\u0026plusmn;\u0026thinsp;0.010\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.159\u0026thinsp;\u0026plusmn;\u0026thinsp;0.006\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.152\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTotal Flavonoid Content (TFC) mgRE/g\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e24.05\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e35.38\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e33.7\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e34.59\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cdiv class=\"SimplePara\"\u003ePhytochemicals\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eAlkaloids\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eGlycosides\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eFlavonoids\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSaponins\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTannins\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eSteroids\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e++\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eTerpenoids\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e-\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cdiv class=\"SimplePara\"\u003e+\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Marine algae, Rhizoclonium, Spirogyra, bioactive components, seaweed","lastPublishedDoi":"10.21203/rs.3.rs-4919856/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4919856/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTerrestrial plants have been the main source of many active agents used in the pharmaceutical and cosmetic industries. Many research studies have focused on different marine organisms like algae, fish, sponges, and crustaceans to obtain bioactive products like proteins and peptides for use in the pharmaceutical and cosmeceutical industries. In contrast to terrestrial plants, marine algae are a very abundant and important source of different constituents that have established beneficial effects on human skin and have been used in the cosmeceutical industry. Samples of algae were collected from two different aquatic sites. Identification of samples was made based on microscopic morphological and other key characteristics of algae, and they were found to be \u003cem\u003eR. hieroglyphicum\u003c/em\u003e and \u003cem\u003eS. varians\u003c/em\u003e. Extraction was carried out in methanol and in a mixture of DCM (dichloromethane) and methanol. Both samples collected from the two different sites showed significant antibacterial effects in various assays performed using the Disc Diffusion Method and the Well Method. The antioxidant activities of the extracts were also detected using a 2.2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging assay. Quantitative and qualitative analyses showed significant quantities of phenolic and flavonoid contents. The stressful climatic factors lead to the synthesis and production of compounds with secondary metabolites, which are anti-stress and important for their antioxidant and therapeutic effects, resulting in various biological activities.\u003c/p\u003e","manuscriptTitle":"Symbiotic Synergies: A Comprehensive Analysis of Antibacterial Potency, Antioxidant Proficiency, and Phytochemical Composition in Rhizoclonium hieroglyphicum and Spirogyra varians from Distinct Aquatic Habitats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-13 15:13:41","doi":"10.21203/rs.3.rs-4919856/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"263ce1e4-a2eb-4208-9b98-cdd9ba2341bc","owner":[],"postedDate":"September 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-02T14:09:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-13 15:13:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4919856","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4919856","identity":"rs-4919856","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}