Evaluating the Flavonoid-Enriched fraction of Padina tetrastromatica a brown Seaweed as a Marine Chemotherapeutic Prospect Integrated MTT Cytotoxic Profiling in A375 Melanoma Cells.

Evaluating the Flavonoid-Enriched fraction of Padina tetrastromatica a brown Seaweed as a Marine Chemotherapeutic Prospect Integrated MTT Cytotoxic Profiling in A375 Melanoma Cells. | 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 Evaluating the Flavonoid-Enriched fraction of Padina tetrastromatica a brown Seaweed as a Marine Chemotherapeutic Prospect Integrated MTT Cytotoxic Profiling in A375 Melanoma Cells. Dr. Prakash Yoganandam G, Dr. Sittharthan V, Dr. Muthukumaran M, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8452077/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 Background: As marine-derived compounds are considered a new finding in recent days, largely due to too little exploration on their source. Padina tetrastromatica a brown seaweed was picked, in which only the polysaccharide compound that has been analysed so far. Still, their instrumental analysis data disclosed the presence of secondary metabolites, whose activity in various areas is less. In this study, we have explored the anticancer properties of the flavonoid-rich fraction made from Padina tetrastromatica . Methods: Anticancer property of seaweed extract was determined through MTT assay in A375 cell line. whose results are computed based on absorbance read at a wavelength of 570 nm. Result: Significant anticancer activity was observed at 50 µg/mL in seaweed & 4.82 µg/mL in standard Cisplatin from the concentrations used in analysis from 3.12 µg/mL to 100 µg/mL. Conclusion: Based on the attained result, it is clearly indicated that the flavonoid-rich fraction of these seaweeds has promising activity as reported in standard, which can be considered for future studies with these outcomes. Cell Survival and Cell Death Padina tetrastromatica Extraction Flavonoidal fraction MTT assay Cell viability Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Cancer remains grievousand cause mortality worldwide.Melanoma, clinically classified as cutaneous melanoma, is a highly aggressive skinmalignancy that arises from the pigment-producing melanocytes. However, the common sites of melanoma are the neck and face. Global estimates indicate the approximately 1.5 million new cases were reported in 2022. In addition nearly 104,960 new cases are expected to be diagnosed annually with 8,500 deaths attributed to melanoma each year (around 5,000 men and 3,000 women) ( 1 )( 2 ). Mostly defective genes and mutations in tumour suppressor genes cause the cells to undergo rapid cell division over time. Particularly, chronic exposure to solar ultraviolet radiation is recognized as the principle environmental risk factor contributing to the development of cutaneous melanoma( 3 ). Meanwhile, a recent study in 2022 revealed that an important trait of melanoma is neither part of UV exposure, and that creates opportunities for recognising new therapeutic targets for treating this changing factor in the growing population. Lately, data on the most prevalent tumours in North America revealed that advancements in anticancer treatment reduced the incidence of cancer-related deaths by 11–13% between 2010 and 2014( 4 ). Taking certain preventive phytochemicals, minerals, and antioxidants are found to a lower risk of cancer, based on several epidemiological studies, by combining conventional therapy, it can produce synergistic effect in curing( 5 – 10 ). Currently, extensive research has focused on identifying antitumor compounds from terrestrial plants, which has downside in their stability, solubility that create challenges for treating this melanoma( 11 – 14 ). Since marine species has developed a wide variety of unique defence against their surroundings( 15 , 16 ). Among them macroalgae are considered to be the potential source in therapeutic preparations. In recent days seaweeds are utilised in drug development because of its reported bioactive constituents like poly unsaturated fatty acids, proteins, polysaccharides, and pigments which are reported to have antioxidant, anti-inflammatory, antineoplastic, anticoagulant activity( 17 – 19 ). Among them, Padina tetrastromatica a marine brown seaweed has been selected for anti-tumour activity in melanoma cell line. But as per the authors knowledge in selected seaweed, it is clearly denoted that methanolic extract of this marine algae are having anticancer activity that are conducted in various cell lines other than melanoma cell line( 20 ). In order to achieve new breakthrough in melanoma treatment, compound with abundant antioxidant properties is required, as it produce more reactive oxygen species. Hence, current study focused on evaluate the anticancer activity of flavonoid rich fraction of Padina tetrastromatica in melanoma cell line (A375) which is earlierly reported to have high level of total phenolic fraction in it( 21 ). MATERIALS AND METHODS Extraction: Sinceseaweedsdoesnothaveanyrooting systembuttheyhadanattachmentportionbywhich theyattachedatrocksthus theplants werecollected andextraneousmaterials are removed bywashingrigorously with seawaterandagainwashedwithnormaltapwater.Samplewerethenshade dried and powdered in anelectric mixer ( 22 – 24 ). These powdered sample are than stored in air tight containerfor lateruse. The finepowder of Padina teterastromatica (5g) were macerated with 150ml of methanol (1:3). Extracted fraction is made flavonoid rich with separating funnel bypartionating organic layer with aqueous layer. After extraction filtrate were initially separated with pet.ether and then they allowed to stand for 30min afterward aqueous layer were collected and separated using n-hexane allowed from which aqueous layer arecollected for further processing( 21 ). ANTICANCER ACTIVITY: Previously anti-oxidant activity of this flavonoidal fraction was presented along with total flavonoid concentration and total phenolic concentration( 21 ), which provide a base for this cytotoxic estimation. The cytotoxic activity of flavonoid rich fraction of Padina tetrastromatica was done through MTT assay in Human melanoma cell (A375 cell line). Generally,MTT assay is simplest method in demonstrating the anticancer activity with reproducible results, where cell viability is indicated in terms of purple colour formed due to formation of formazan that are measured spectrophotometrically, reflects the volume of active cells and thereby indicate the extent of cytotoxic induced by the test sample. Preparation of test solution For cytotoxic evaluation, the flavonoidal fraction was prepared as a series of concentrationfrom 3.125-100µg/mLusing appropriate dilution method. Cell culture preparation: For the preparation of cell line, human melanoma cell line (A375) was procured fromNational Centre for Cell Science. Cells were cultured with growth medium supplementing 10% heat-inactivated fatalbovine serum, penicillin (100 IU/mL), streptomycin (100 µg/mL). Cultures wereraised at 37 o Cin a humidified incubatorwith 5% CO 2 . Upon reaching adequate confluency, cells were detached with a trypsin- EDTA solution and evaluated for viability. Subsequently, 50 thousandviable cells per well wereplaced into 96 well plates and incubated for 1dayunder standard culture condition ( 25 – 27 ). Procedure The monolayer cells that were grown were made to detach with a trypsin, and cell numbers were set at 1.0 x 10 5 cells per millilitre by using complete grown medium supplemented with 10%fatal bovine serum. Aliquots of 100 microliters of the cell suspension were dispensed into each well of a 96-well microplate, resulting in approximately 50,000 cells per well. After 24 hours, only a fractional monolayer could be seen. The liquid above cells, were flicked away and the layer wererinsed with fresh medium. After this, into wells of the plate, 100 microliters of test drug solutions of different concentrations were put onto each monolayer. The plates were put in incubation at temperature of 37 degrees Celsius for a day in an environment having five percent CO 2 . Once incubation was done, all test solution was thrown away from wells and 100 microliters of a MTT solution (which consists of 5 mg per 10 ml of MTT added in PBS) went into every well. The plates underwent another four hours incubation at 37 0 C in condition of 5% CO2( 28 – 31 ). After four hours, supernatant fluid was taken out and 100 microliters of the DMSO were poured so plates could be moved slightly to help the formazan become dissolved. The microplate reader was used to get the absorbance at 570 nanometres wavelength. The inhibition percentage was found with a specific calculation and the drug concentration needed to stop cell growth by a factor of 50 (IC50) was figured by dose-response graph generated for each treatment group. IC 50 Value: The half maximal inhibitory concentration (IC 50 ) is quantitative parameter used to describe the potency of a test compoundinsuppressing adefinedbiological or chemical reaction. It represents the concentration at which drug decrease cell viability by 50% when compared with untreated controls. In the present study, IC 50 value were determined by plotting compound concentration against percentage growth inhibition, followed by non-linear regression analysis to generate a sigmoidal dose-response curve. This method enables reliable comparison of cytotoxicity efficacy among test samples( 22 , 23 , 32 ). RESULTS AND DISCUSSION The cytotoxic potential of flavonoidal fraction of Padina tetrastromatica (FRPD)was evaluated against A375 human melanoma cells using the MTT colorimetric assay, with cisplatin as a reference standard. This assay measures cell viability based on mitochondrial dehydrogenase activity, which enzymatically MTT into insoluble formazan crystals in active cells. The intensity of the formazan colour, measured spectrophotometrically at 570 nm, provide a direct measure of the number of viable cells remaining after treatment. Quantitative Evaluation of Cytotoxicity Cells were exposed to increasing concentrations of FRPD (3.12–100 µg/ml) and cisplatin for 1 day, and mean optical density (OD) values were used to calculate cell viability percentages (Table.1). Both compounds showed a decrease in viability that depends on the concentration. Flavonoidal fraction exhibited a gradual reduction in cell survival from 95.96% at 3.12µg/mL to 27.92% at 100 µg/mL, whereas cisplatin produces a much sharper decline- from 66.22% at 3.26% at 100µg/mL.The calculated IC 50 values were 50.15µg/mL for flavonoidal fraction and 4.82µg/mL for cisplatin, respectively. These findings indicate that cisplatin remains significantly more potent thanFRPDthat demonstrated a measurable dose-dependent cytotoxic effects suggesting its potential as a promising anticancer compound. Table 1 Cell viability (Concentration versus % of cell viability) Concentration (µg/mL) Flavonoidal fraction – Mean OD (after blank) Viability % Cisplatin – Mean OD (after blank) Viability % Untreated control 1.2057 100.00 1.2057 100.00 3.12 1.1570 95.96 0.7983 66.22 6.25 1.0130 84.02 0.5490 45.53 12.5 0.9437 78.27 0.2760 22.89 25 0.8173 67.79 0.1690 14.02 50 0.6897 57.20 0.0917 7.60 100 0.3367 27.92 0.0393 3.26 The resulting dose- dependent curve (Fig. 1 ) clearly demonstrates a concentration- dependent decrease in viability for both compounds. Cisplatin displayed sharp inhibition of cell proliferation, reaching below 10% viability at 50µg/mL, while flavonoidal fractions showed a gradual decline to about 28% viability at 100µg/mL. the computed IC 50 values were 50.15µg/mL for FRPD and 4.82 µg/mL for cisplatin, confirming that although Cisplatin is more potent, flavonoidal fraction possesses measurable cytotoxic activity. Morphological Analysis (Based on 24 hours microscopic Observation) Direct microscopic imaging (Fig.2, 3&4) from the experimental data revealed progression morphological damage with increasing drug concentration. Flavonoidal fraction-treated cells displayed visible cytoplasmic shrinkage and rounding at 12.5–50 µg/mL, while at 100 µg/mL most cells detached, lost membrane integrity, and showed apoptotic blebbing in Fig. 3 cisplatin-treated cells showed more severe morphological deterioration even at low concentrations (6.25 µg/mL), with near-complete detachment at 25–100 µg/mL, confirming its strong cytotoxic potency in Fig. 4 These visual observations corroborate the MTT data, confirming that FRPD induces cell death in a dose – dependent manner, most likely through apoptotic pathways rather that necrosis. Interpretation and Literature Correlation The modern cytotoxicity of FRPD could be attributed to its interaction with mitochondrial enzymes, which results in decreased energy metabolism and the initiation of apoptotic signalling. The characteristic cellular shrinkage and membrane blebbing observed in microscopy support this hypothesis. Similar findings were reported and demonstrated that MTT reduction is a valid indicator of mitochondrial function and cell viability( 24 , 33 , 34 ). Although the cytotoxic activity of flavonoidal fraction is less than that of standard cisplatin, the results indicate a clear dose- dependent antiproliferative effects. FRPDas a promising lead molecule for further optimisation, potentially offering reduced toxicity compared with conventional chemotherapeutics. CONCLUSION In conclusion, the present study confirms that flavonoidal fraction of Padina tetrastromatica exerts significant cytotoxic effects on A375 melanoma cells, as evidenced by both the MTT assay and morphological alteration. With an IC50 value of 50.15 µg/ml, FRPD demonstrated measurable anticancer potential. The combination of quantitative and qualitative evidence supports the hypothesis that FRPD induces apoptotic – mediated cell death. Further studies should aim to characterise the underlying molecular mechanism and assess in vivo efficacy. References What Is Melanoma Skin Cancer? | What Is Melanoma? Available from: https://www.cancer.org/cancer/types/melanoma-skin-cancer/about/what-is-melanoma.html Tattoos and risk of cutaneous melanoma and non-melanoma skin cancer in France. Available from: https://www.iarc.who.int/cancer-type/skin-cancer Raimondi S, Suppa M, Gandini S (2020) Melanoma Epidemiology and Sun Exposure. 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06:01:13","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91869,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/d431d9dae59ebb6a1a524125.png"},{"id":99319175,"identity":"b29f215e-caba-436d-a5a1-871d50aa5223","added_by":"auto","created_at":"2025-12-31 16:36:33","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66747,"visible":true,"origin":"","legend":"","description":"","filename":"rs84520770structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/459490303d43af770cb509e5.xml"},{"id":99318693,"identity":"49fdf123-88b6-4b11-a46b-0583594eb08e","added_by":"auto","created_at":"2025-12-31 16:33:58","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74810,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/697dec6a56bacff515e82244.html"},{"id":99271108,"identity":"ec27449f-2334-433d-b500-92293314686a","added_by":"auto","created_at":"2025-12-31 06:01:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":36820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMTT assay of A375 cell line treated with Flavonoidal fraction of Padina tetrastromatica and Cisplatin for 24 hours, showing percentage cell viability versus concentration (µg/mL).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/bb1122512ccb86470ff9be95.png"},{"id":99271111,"identity":"6aaf589e-5b74-40ab-a341-0314accd6381","added_by":"auto","created_at":"2025-12-31 06:01:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170919,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl cells\u003c/strong\u003e (untreated) appeared healthy, polygonal, and tightly attached to the culture surface.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/605a25d1503552579fdbfce8.png"},{"id":99319750,"identity":"1f1c79e8-5fe0-49ed-9912-407fac7770c2","added_by":"auto","created_at":"2025-12-31 16:37:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":491417,"visible":true,"origin":"","legend":"\u003cp\u003eFlavonoid fraction treated cells.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/0672243943f2264bcfeb12bd.png"},{"id":99788276,"identity":"2de15339-d7fa-440f-901d-1ade9d416195","added_by":"auto","created_at":"2026-01-08 12:45:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1290647,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/d620448a-a0eb-40db-83f8-0b9577d6da16.pdf"},{"id":99319004,"identity":"c50f06e7-fa0c-4b0d-a6a0-a30556b0522a","added_by":"auto","created_at":"2025-12-31 16:35:58","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":689680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8452077/v1/ac79ee1c6271001d7be5182a.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluating the Flavonoid-Enriched fraction of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePadina tetrastromatica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e a brown Seaweed as a Marine Chemotherapeutic Prospect Integrated MTT Cytotoxic Profiling in A375 Melanoma Cells.\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eCancer remains grievousand cause mortality worldwide.Melanoma, clinically classified as cutaneous melanoma, is a highly aggressive skinmalignancy that arises from the pigment-producing melanocytes. However, the common sites of melanoma are the neck and face. Global estimates indicate the approximately 1.5\u0026nbsp;million new cases were reported in 2022. In addition nearly 104,960 new cases are expected to be diagnosed annually with 8,500 deaths attributed to melanoma each year (around 5,000 men and 3,000 women) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Mostly defective genes and mutations in tumour suppressor genes cause the cells to undergo rapid cell division over time. Particularly, chronic exposure to solar ultraviolet radiation is recognized as the principle environmental risk factor contributing to the development of cutaneous melanoma(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Meanwhile, a recent study in 2022 revealed that an important trait of melanoma is neither part of UV exposure, and that creates opportunities for recognising new therapeutic targets for treating this changing factor in the growing population. Lately, data on the most prevalent tumours in North America revealed that advancements in anticancer treatment reduced the incidence of cancer-related deaths by 11\u0026ndash;13% between 2010 and 2014(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Taking certain preventive phytochemicals, minerals, and antioxidants are found to a lower risk of cancer, based on several epidemiological studies, by combining conventional therapy, it can produce synergistic effect in curing(\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Currently, extensive research has focused on identifying antitumor compounds from terrestrial plants, which has downside in their stability, solubility that create challenges for treating this melanoma(\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Since marine species has developed a wide variety of unique defence against their surroundings(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Among them macroalgae are considered to be the potential source in therapeutic preparations. In recent days seaweeds are utilised in drug development because of its reported bioactive constituents like poly unsaturated fatty acids, proteins, polysaccharides, and pigments which are reported to have antioxidant, anti-inflammatory, antineoplastic, anticoagulant activity(\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Among them, \u003cem\u003ePadina tetrastromatica\u003c/em\u003e a marine brown seaweed has been selected for anti-tumour activity in melanoma cell line. But as per the authors knowledge in selected seaweed, it is clearly denoted that methanolic extract of this marine algae are having anticancer activity that are conducted in various cell lines other than melanoma cell line(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In order to achieve new breakthrough in melanoma treatment, compound with abundant antioxidant properties is required, as it produce more reactive oxygen species. Hence, current study focused on evaluate the anticancer activity of flavonoid rich fraction of \u003cem\u003ePadina tetrastromatica\u003c/em\u003e in melanoma cell line (A375) which is earlierly reported to have high level of total phenolic fraction in it(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExtraction:\u003c/h2\u003e \u003cp\u003eSinceseaweedsdoesnothaveanyrooting systembuttheyhadanattachmentportionbywhich theyattachedatrocksthus theplants werecollected andextraneousmaterials are removed bywashingrigorously with seawaterandagainwashedwithnormaltapwater.Samplewerethenshade dried and powdered in anelectric mixer (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These powdered sample are than stored in air tight containerfor lateruse. The finepowder of \u003cem\u003ePadina teterastromatica\u003c/em\u003e(5g) were macerated with 150ml of methanol (1:3). Extracted fraction is made flavonoid rich with separating funnel bypartionating organic layer with aqueous layer. After extraction filtrate were initially separated with pet.ether and then they allowed to stand for 30min afterward aqueous layer were collected and separated using n-hexane allowed from which aqueous layer arecollected for further processing(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eANTICANCER ACTIVITY:\u003c/h3\u003e\n\u003cp\u003ePreviously anti-oxidant activity of this flavonoidal fraction was presented along with total flavonoid concentration and total phenolic concentration(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), which provide a base for this cytotoxic estimation. The cytotoxic activity of flavonoid rich fraction of Padina tetrastromatica was done through MTT assay in Human melanoma cell (A375 cell line). Generally,MTT assay is simplest method in demonstrating the anticancer activity with reproducible results, where cell viability is indicated in terms of purple colour formed due to formation of formazan that are measured spectrophotometrically, reflects the volume of active cells and thereby indicate the extent of cytotoxic induced by the test sample.\u003c/p\u003e\n\u003ch3\u003ePreparation of test solution\u003c/h3\u003e\n\u003cp\u003eFor cytotoxic evaluation, the flavonoidal fraction was prepared as a series of concentrationfrom 3.125-100\u0026micro;g/mLusing appropriate dilution method.\u003c/p\u003e\n\u003ch3\u003eCell culture preparation:\u003c/h3\u003e\n\u003cp\u003eFor the preparation of cell line, human melanoma cell line (A375) was procured fromNational Centre for Cell Science. Cells were cultured with growth medium supplementing 10% heat-inactivated fatalbovine serum, penicillin (100 IU/mL), streptomycin (100 \u0026micro;g/mL). Cultures wereraised at 37\u003csup\u003eo\u003c/sup\u003eCin a humidified incubatorwith 5% CO\u003csub\u003e2\u003c/sub\u003e. Upon reaching adequate confluency, cells were detached with a trypsin- EDTA solution and evaluated for viability. Subsequently, 50 thousandviable cells per well wereplaced into 96 well plates and incubated for 1dayunder standard culture condition (\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eProcedure\u003c/h3\u003e\n\u003cp\u003eThe monolayer cells that were grown were made to detach with a trypsin, and cell numbers were set at 1.0 x 10\u003csup\u003e5\u003c/sup\u003e cells per millilitre by using complete grown medium supplemented with 10%fatal bovine serum. Aliquots of 100 microliters of the cell suspension were dispensed into each well of a 96-well microplate, resulting in approximately 50,000 cells per well. After 24 hours, only a fractional monolayer could be seen. The liquid above cells, were flicked away and the layer wererinsed with fresh medium. After this, into wells of the plate, 100 microliters of test drug solutions of different concentrations were put onto each monolayer. The plates were put in incubation at temperature of 37 degrees Celsius for a day in an environment having five percent CO\u003csub\u003e2\u003c/sub\u003e. Once incubation was done, all test solution was thrown away from wells and 100 microliters of a MTT solution (which consists of 5 mg per 10 ml of MTT added in PBS) went into every well. The plates underwent another four hours incubation at 37\u003csup\u003e0\u003c/sup\u003eC in condition of 5% CO2(\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). After four hours, supernatant fluid was taken out and 100 microliters of the DMSO were poured so plates could be moved slightly to help the formazan become dissolved. The microplate reader was used to get the absorbance at 570 nanometres wavelength. The inhibition percentage was found with a specific calculation and the drug concentration needed to stop cell growth by a factor of 50 (IC50) was figured by dose-response graph generated for each treatment group.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIC\u003csub\u003e50\u003c/sub\u003e Value:\u003c/h2\u003e \u003cp\u003eThe half maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) is quantitative parameter used to describe the potency of a test compoundinsuppressing adefinedbiological or chemical reaction. It represents the concentration at which drug decrease cell viability by 50% when compared with untreated controls. In the present study, IC\u003csub\u003e50\u003c/sub\u003evalue were determined by plotting compound concentration against percentage growth inhibition, followed by non-linear regression analysis to generate a sigmoidal dose-response curve. This method enables reliable comparison of cytotoxicity efficacy among test samples(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eThe cytotoxic potential of flavonoidal fraction of \u003cem\u003ePadina tetrastromatica\u003c/em\u003e(FRPD)was evaluated against A375 human melanoma cells using the MTT colorimetric assay, with cisplatin as a reference standard. This assay measures cell viability based on mitochondrial dehydrogenase activity, which enzymatically MTT into insoluble formazan crystals in active cells. The intensity of the formazan colour, measured spectrophotometrically at 570 nm, provide a direct measure of the number of viable cells remaining after treatment.\u003c/p\u003e\n\u003ch3\u003eQuantitative Evaluation of Cytotoxicity\u003c/h3\u003e\n\u003cp\u003eCells were exposed to increasing concentrations of FRPD (3.12\u0026ndash;100 \u0026micro;g/ml) and cisplatin for 1 day, and mean optical density (OD) values were used to calculate cell viability percentages (Table.1). Both compounds showed a decrease in viability that depends on the concentration. Flavonoidal fraction exhibited a gradual reduction in cell survival from 95.96% at 3.12\u0026micro;g/mL to 27.92% at 100 \u0026micro;g/mL, whereas cisplatin produces a much sharper decline- from 66.22% at 3.26% at 100\u0026micro;g/mL.The calculated IC\u003csub\u003e50\u003c/sub\u003e values were 50.15\u0026micro;g/mL for flavonoidal fraction and 4.82\u0026micro;g/mL for cisplatin, respectively.\u003c/p\u003e \u003cp\u003eThese findings indicate that cisplatin remains significantly more potent thanFRPDthat demonstrated a measurable dose-dependent cytotoxic effects suggesting its potential as a promising anticancer compound.\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\u003eCell viability (Concentration versus % of cell viability)\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\u003eConcentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlavonoidal fraction \u0026ndash; Mean OD (after blank)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eViability %\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCisplatin \u0026ndash; Mean OD (after blank)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eViability %\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUntreated control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.2057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.2057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.1570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e95.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.7983\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e66.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.0130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e84.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.9437\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e78.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.2760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.8173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e67.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1690\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.6897\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0917\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.3367\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0393\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.26\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\u003eThe resulting dose- dependent curve (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) clearly demonstrates a concentration- dependent decrease in viability for both compounds. Cisplatin displayed sharp inhibition of cell proliferation, reaching below 10% viability at 50\u0026micro;g/mL, while flavonoidal fractions showed a gradual decline to about 28% viability at 100\u0026micro;g/mL. the computed IC\u003csub\u003e50\u003c/sub\u003e values were 50.15\u0026micro;g/mL for FRPD and 4.82 \u0026micro;g/mL for cisplatin, confirming that although Cisplatin is more potent, flavonoidal fraction possesses measurable cytotoxic activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMorphological Analysis (Based on 24 hours microscopic Observation)\u003c/h2\u003e \u003cp\u003eDirect microscopic imaging (Fig.2, 3\u0026amp;4) from the experimental data revealed progression morphological damage with increasing drug concentration.\u003c/p\u003e\u003cp\u003e \u003cb\u003eFlavonoidal fraction-treated cells\u003c/b\u003e displayed visible cytoplasmic shrinkage and rounding at 12.5\u0026ndash;50 \u0026micro;g/mL, while at 100 \u0026micro;g/mL most cells detached, lost membrane integrity, and showed apoptotic blebbing in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ecisplatin-treated cells\u003c/b\u003e showed more severe morphological deterioration even at low concentrations (6.25 \u0026micro;g/mL), with near-complete detachment at 25\u0026ndash;100 \u0026micro;g/mL, confirming its strong cytotoxic potency in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese visual observations corroborate the MTT data, confirming that FRPD induces cell death in a dose \u0026ndash; dependent manner, most likely through apoptotic pathways rather that necrosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInterpretation and Literature Correlation\u003c/h2\u003e \u003cp\u003eThe modern cytotoxicity of FRPD could be attributed to its interaction with mitochondrial enzymes, which results in decreased energy metabolism and the initiation of apoptotic signalling. The characteristic cellular shrinkage and membrane blebbing observed in microscopy support this hypothesis. Similar findings were reported and demonstrated that MTT reduction is a valid indicator of mitochondrial function and cell viability(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the cytotoxic activity of flavonoidal fraction is less than that of standard cisplatin, the results indicate a clear dose- dependent antiproliferative effects. FRPDas a promising lead molecule for further optimisation, potentially offering reduced toxicity compared with conventional chemotherapeutics.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn conclusion, the present study confirms that flavonoidal fraction of \u003cem\u003ePadina tetrastromatica\u003c/em\u003e exerts significant cytotoxic effects on A375 melanoma cells, as evidenced by both the MTT assay and morphological alteration. With an IC50 value of 50.15 \u0026micro;g/ml, FRPD demonstrated measurable anticancer potential. The combination of quantitative and qualitative evidence supports the hypothesis that FRPD induces apoptotic \u0026ndash; mediated cell death. Further studies should aim to characterise the underlying molecular mechanism and assess in vivo efficacy.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWhat Is Melanoma Skin Cancer? | What Is Melanoma? 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Cells 13(22):1838\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao L, Gonz\u0026aacute;lez-Cardenete MA, Prieto-Garcia JM (2023) In Vitro Cytotoxic Effects of Ferruginol Analogues in Sk-MEL28 Human Melanoma Cells. Int J Mol Sci 24(22):16322\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"School of Pharmacy, Sri Balaji Vidyapeeth (Deemed to be University) Karaikal Campus","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":"Padina tetrastromatica, Extraction, Flavonoidal fraction, MTT assay, Cell viability","lastPublishedDoi":"10.21203/rs.3.rs-8452077/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8452077/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAs marine-derived compounds are considered a new finding in recent days, largely due to too little exploration on their source. \u003cem\u003ePadina tetrastromatica\u003c/em\u003ea brown seaweed was picked, in which only the polysaccharide compound that has been analysed so far. Still, their instrumental analysis data disclosed the presence of secondary metabolites, whose activity in various areas is less. In this study, we have explored the anticancer properties of the flavonoid-rich fraction made from \u003cem\u003ePadina tetrastromatica\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Anticancer property of seaweed extract was determined through MTT assay in A375 cell line. whose results are computed based on absorbance read at a wavelength of 570 nm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResult: \u003c/strong\u003eSignificant anticancer activity was observed at 50 µg/mL in seaweed \u0026amp; 4.82 µg/mL in standard Cisplatin from the concentrations used in analysis from 3.12 µg/mL to 100 µg/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Based on the attained result, it is clearly indicated that the flavonoid-rich fraction of these seaweeds has promising activity as reported in standard, which can be considered for future studies with these outcomes.\u003c/p\u003e","manuscriptTitle":"Evaluating the Flavonoid-Enriched fraction of Padina tetrastromatica a brown Seaweed as a Marine Chemotherapeutic Prospect Integrated MTT Cytotoxic Profiling in A375 Melanoma Cells.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 06:01:08","doi":"10.21203/rs.3.rs-8452077/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":"8f6ddab7-e48d-4ab3-8849-3165d4d9121a","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60224055,"name":"Cell Survival and Cell Death"}],"tags":[],"updatedAt":"2025-12-31T06:01:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-31 06:01:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8452077","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8452077","identity":"rs-8452077","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}