Genotoxicity and cytotoxicity evaluation of brown algae (Cystoseira indica) extract in human gingival fibroblast (HGF) and lung cancer cell lines (A549)

Genotoxicity and cytotoxicity evaluation of brown algae (Cystoseira indica) extract in human gingival fibroblast (HGF) and lung cancer cell lines (A549) | 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 Genotoxicity and cytotoxicity evaluation of brown algae ( Cystoseira indica ) extract in human gingival fibroblast (HGF) and lung cancer cell lines (A549) Emran Habibi, Sahar Sheikhzadeh, Hesamoddin Arabnozari, Mohammad Shokrzadeh, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4545987/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 Cancer, particularly lung cancer, remains a leading cause of mortality worldwide, highlighting the need for new remedies. The brown algae species, C. indica , has gained attention for its rich phytochemical composition and pharmacological potential. This study evaluated the genotoxic and cytotoxic effects of C. indica extract on human gingival fibroblast (HGF) and lung cancer (A549) cell lines. Algae materials were extracted using sequential maceration, and fucoxanthin content was determined via High-Performance Liquid Chromatography (HPLC). Cytotoxic and genotoxic effects were assessed using MTT and comet assays, with statistical analyses performed using GraphPad Prism software. The algal sample contained 3.077 μg of fucoxanthin per 1g in n -hexane-acetone extract and 4.32 μg of fucoxanthin per 1g in ethanolic extract. n -Hexane-acetone and cold water extracts at 5000 µg/mL concentration exhibited the highest antioxidant activities in the DPPH assay with IC 50 values of 306.15 ± 18.46 μg/mL and 8370 ± 2460 μg/mL, respectively. n -Hexane-acetone extract induced 50.66% apoptosis and hot water extract caused 54.97% apoptosis at 100 µg/mL. C. indica offers unique metabolites with potential pharmaceutical applications, especially as cytotoxic agents against cancer. The n -hexane-acetone extract, rich in flavonoids and phenolics, showed significant antioxidant and anticancer effects, inducing notable apoptosis in A549 cancer cells, suggesting further investigation for anticancer use. Algae Antioxidant Cystoseira indica Cytotoxicity Fucoxanthin Genotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cancer is one of the most devastating diseases globally, characterized by an annual incidence of over 10 million new cases worldwide 1 . Mutations in the oncogenes with a dominant gain of function over tumor suppressor genes and malfunction of tumor suppressor genes are considered the main reasons for cancer development 2 . Lung cancer (LC), characterized by uncontrolled cell growth in the lung tissue, often manifests without symptoms in its early stages, leading to delayed diagnosis and advanced disease presentation, associated with severe complications and increased mortality 3 . Survival rates vary significantly depending on cancer type and characteristics. The high mortality rate of LC is attributed to its widespread incidence, low survival rates, and propensity for metastasis, which exacerbates patient outcomes and poses significant challenges to survival 4 . LC stands as the primary contributor to cancer-related mortality globally, with prevalence being highest among men and ranking fourth among women. Notably, in Iran, it holds the second position for cancer-related deaths among males and the third position among females 5 . Various treatment modalities are available for LC, such as surgery, radiation therapy, chemotherapy, and targeted therapy. Treatment recommendations depend on factors like cancer type and stage. Despite advancements in diagnosis and therapy over the last 25 years, the prognosis for LC patients remains unsatisfactory 6 . Recently, there has been an increased interest in searching for plant-derived compounds capable of minimizing chemotherapy-induced toxicity in normal cells while preserving their antineoplastic activity 7 . In recent years, seaweeds and their extracts have emerged as sources of diverse bioactive compounds with potential medicinal properties. These compounds, including polyphenols, polysaccharides, meroterpenoids, and terpenoids, exhibit various biological activities such as anti-inflammatory, antibiotic, antiviral, cytotoxic, and antimitotic effects 8 . The Cystoseira C. Agardh, 1820, a polyphyletic genus of marine macroalgae within the Sargassaceae family, is distributed along the Atlantic-Mediterranean coasts and comprises approximately 40 species. These species produce a diverse array of secondary metabolites, including terpenoids, fatty acids, steroids, and polysaccharides. Thus, the members of this genus offer the potential for the discovery of novel compounds with biomedical applications. Extracts from different Cystoseira species have been investigated for various activities, such as antimicrobial, antiviral, antifungal, antioxidant, cytotoxic, and antitumoral properties, suggesting their promising biomedical relevance 9 . Cystoseira indica (Thivy & Doshi), a tropical brown algae found in southwest Asia, including India, Iran, Oman, Pakistan, Qatar, and Yemen exhibits faster growth and higher biomass when cultivated in the tidal zone of Chabahar Bay, Iran, compared to other algae species in the region. Chabahar Bay, situated in the southeastern part of Iran and the northern region of the Oman Sea, experiences strong ultraviolet radiation due to its tropical location, leading to the production of reactive oxygen species (ROS) and other oxidizing agents. In response to these environmental conditions, marine algae in warm waters alter their metabolism to produce antioxidants as a protective mechanism 10 . C. indica displays significant pharmacological properties ascribed to its rich phytochemical composition, particularly high levels of total phenolics and potent antioxidant activity in which the extracts from C. indica effectively inhibit cell growth in HeLa (cervical carcinoma), and HT-29 (human colon adenocarcinoma), and particularly MCF-7 (human breast adenocarcinoma) 11 . The methanolic and chloroform extracts of C. indica exhibited potent antioxidant and cytotoxic effects against colorectal cancer cells, suggesting their potential as an anticancer agent for preclinical and clinical studies 12 . With lung cancer being widespread globally and considering the documented anticancer properties of brown algae, this research aimed to investigate the genotoxicity and cytotoxicity of hydroalcoholic extract of brown algae C. indica in human gingival fibroblast (HGF) and lung cancer cell lines (A549). 2. Results The results of the preliminary phytochemical analysis for identifying some major groups of compounds in C. indica algae are summarized in Table 1 . Table 1 Phytochemical test results Presence of a group of compounds in the extract Phytochemical test Saponins + Terpenoids + Steroids + Anthraquinone + Alkaloids - Flavonoids and phenols + 2.1 Quantification of phytoconstituents The total phenol compounds were quantified as gallic acid equivalents using a standard curve equation (y = 0.0031x + 0.1114, r 2 = 0.9497). The concentration of phenolics in the extracts, was dependent on the solvent used in the extraction, as shown in Table 2 . Phenolic compounds were not observed in the ethanolic and the aqueous extracts of C. indica and higher concentrations needed to be utilized. Similarly, the total flavonoid contents were determined as mg quercetin equivalent/g of extract powder using a standard curve equation (y = 0.0019x + 0.0848, r 2 = 0.9757). The concentration of flavonoids in the extracts depended on the solvent used in the extraction, as shown in Table 2 . The n -hexane-acetone extract showed a higher flavonoid content compared to the ethanolic extract. In the aqueous extract, calculation was not feasible for cold and hot aqueous extracts, and higher concentrations must be utilized. 2.2 Antioxidant (DPPH radical-scavenging) assay Half-maximal inhibitory concentration (IC 50 ) for DPPH was examined on algal samples extracted using hexane-acetone, ethanol, hot water, and cold water, according to the standard curve equation (y = 1.6214x − 0.8933, r 2 = 0.962). Following the results that are shown in Table 2 different extracts of C. indica algae exhibited the highest percentage of inhibition at a concentration of 5000 µg/mL. Table 2 The total phenolics, flavonoids contents, and scavenging activity of different extracts from C. Indica. Each value is expressed as means ± standard deviation; P value ˂ 0.0001 for all extraction; n. d.= not detected. Samples DPPH radical scavenging assay (IC 50 value, µg/mL) Total phenolics content (mg GAEs/g of dry weight) Total flavonoids content (mg quercetin/g of dry weight) Cold water extract 8370 ± 2460 n. d. n. d. Hot water extract 16450 ± 1790 n. d. n. d. Ethanolic extract 22580 ± 4690 n. d. 3.9 ± 1.06 n -Hexane-acetone extract 306.15 ± 18.46 14.16 ± 0.46 12.43 ± 1.39 Ascorbic acid (Standard) 30.95 ± 0.71 - - 2.3 Determination of fucoxanthin by HPLC method After injecting various extract concentrations, the maximum absorption peak was observed at 450 nm between 2.7 and 3.7 min, indicating fucoxanthin with maximum intensity. The HPLC chromatogram for standard fucoxanthin resulting from a concentration of 10 µg/mL of the standard is shown in Fig. 1 . Figure 2 illustrates that adding diluted concentration from the standard to the ethanolic sample increased the peak height corresponding to the absorption region of fucoxanthin. This suggests that the absorption peak of the sample matched fucoxanthin, and its intensity increased with the standard. As shown in Fig. 3 , after adding a diluted concentration of the standard to the hexane-acetone sample, an increase in the peak height present in the absorption region of fucoxanthin is observed. By calculating the area under the standard curve (y = 53407x + 9778.4, r 2 = 0.9992) of the sample and applying it to the line equation, it was determined that the algal sample contained 3.077 µg of fucoxanthin per 1g in n -hexane-acetone extract and 4.32 µg of fucoxanthin per 1g in ethanolic extract. 2.4 Oxidative Stress Parameters 2.4.1 Measurement of Lipid Peroxidation: The concentration of malondialdehyde (MDA) in micromolar at various concentrations of algae extracts compared to the control group is shown in Fig. 4 . The control group refers to cells that were only incubated with a complete culture medium for 1 hour. One-way analysis of variance (ANOVA) results for concentrations of 10, 50, and 100 µg/mL in cold water, hot water, and ethanolic extracts, as well as concentrations of 5 to 100 µg/mL in n -hexane-acetone extracts, were statistically significant. According to the analysis results, a significant increase in lipid peroxidation was observed at these three concentrations compared to the control group. 2.4.2 Measurement of Glutathione (GSH) Concentration: Figure 5 depicts the mean optical absorption measured for intracellular glutathione (GSH) using a spectrophotometer at a wavelength of 412 nm at various concentrations of algae extracts compared to the control group. One-way ANOVA analysis for each of the three concentrations (10, 50, and 100 µg/mL) in cold and hot water extracts, as well as for concentrations ranging from 5 to 100 µg/mL in n -hexane-acetone and ethanol extracts, showed significant results. According to the analysis, a significant decrease in intracellular glutathione levels was observed at these three concentrations compared to the control group. 2.4.3 Measurement of intracellular production of ROS: According to Fig. 6 , the mean ROS levels measured by fluorometer at 485 nm excitation and 530 nm emission at various concentrations of extracts are compared to the control group. One-way ANOVA analysis for each of the three concentrations (5 to 100 µg/mL) of cold water, ethanol, and hexane-acetone extracts, as well as concentrations ranging from 10 to 100 µg/mL of hot water extract, showed significant results. According to the analysis, a significant increase in ROS production was observed at these three concentrations compared to the control group. 2.5 The cytotoxicity of different algae extracts on HGF and A549 cancer cell lines: Figure 7 shows the cell toxicity levels of various concentrations of algae extracts on two cell lines, A549 and HGF compared to the control group. The cell viability for cold and hot water and ethanol extracts showed significant differences from the control group at concentrations ranging from 10 to 100 µg/mL, while for the hexane-acetone extract, significant differences were observed at concentrations ranging from 5 to 100 µg/mL compared to the control group. 2.6 The genotoxicity of different algae extracts on HGF and A549 cancer cell lines : To determine the optimal genotoxic concentrations of algae extracts, HGF and A549 cancer cell lines were treated with varying concentrations of C. indica (5, 10, 50, and 100 µg/mL) for 1 hour before conducting the comet assay. The results obtained from the comet assay were measured based on the Tail moment criterion and compared with the results of the control group. Results of the visual scoring and percentage of total DNA damage induced by different concentrations of algae extracts are shown in Fig. 8 . The data indicate that cold and hot water and ethanol extracts showed significant differences from the control group at concentrations ranging from 10 to 100 µg/mL, while for the n -hexane-acetone extract, significant differences were observed at concentrations ranging from 5 to 100 µg/mL compared to the control group. 2.7 Apoptosis level The results obtained from flow cytometry for measuring the induced apoptosis by different extracts of Cystoseira indica algae are presented in Fig. 9 . According to the figure, the cold water extract results in 28.1% apoptosis in A549 lung cancer cells, while the hot water extract induces 54.97% apoptosis, the ethanolic extract leads to 8.83% apoptosis, and the hexane-acetone extract causes 50.66% apoptosis at the highest concentration (100 µg/mL) respectively. 3. Discussion The global cancer report highlights a significant and abrupt surge in cancer incidence, with cancer ranking as the second leading cause of death globally 1 . Lung cancer is a major health concern due to its high mortality rate, making it the leading cause of cancer-related deaths worldwide. Despite advances in treatment, its impact remains significant, highlighting the urgent need for effective prevention strategies, early detection methods, and innovative therapies to improve patient outcomes and reduce the burden of this disease 13 . Various compounds with diverse applications, such as antibiotic, antiviral, antifungal, and anticancer effects, have been identified and extracted from macroalgae. Regarding seaweeds, they are primitive plant-like organisms lacking roots, stems, and leaves, yet they boast a rich composition of vitamins, minerals, trace elements, proteins, and bioactive compounds. 12 , 14 In the present study, phytochemical screening of the seaweeds showed the presence of saponins, terpenoids, steroids, anthraquinone, flavonoids, and phenols. These compounds demonstrate diverse mechanisms of action, such as inhibiting cancer cell growth and metastasis, as well as inducing cancer cell death 15 . This research aimed to investigate the genotoxicity and cytotoxicity of hydroalcoholic extract of brown algae C. indica in HGF and A549 lung cancer cell lines. The strong positive correlation between the polyphenolic content of algae and its antioxidant activity has been extensively documented 16 , 17 . Phenolic compounds, commonly found in plants including seaweeds, are known for their diverse biological activities, particularly their antioxidant properties 14 . Consequently, the high antioxidant activities of the extracts can likely be attributed to the content of total phenolic compounds. Furthermore, in this study, C. indica exhibited antioxidant activity, possibly attributable to its elevated polyphenolic content (14.16 ± 0.46 mg GAE/g). Total flavonoids in the seaweeds ranged from 3.9 to 12.43 mg/g. Flavonoids, known for their antioxidant and free radical scavenging properties, are key natural phenols with broad chemical and biological activities 18 . They serve as antioxidants against various reactive oxygen species and inhibit lipid peroxidation, suggesting potential therapeutic use against diverse diseases 19 . These compounds, including flavonoids, and polyphenols, found in significant amounts in C. indica , play a preventive role against diseases by scavenging free radicals. Based on the results obtained from the study and the analysis of phytochemical, cytotoxic, and genotoxic tests, the hexane-acetone extract, containing a higher concentration of compounds such as phenols and flavonoids, exhibits greater potential for antioxidant and anticancer properties. In this study, the antioxidant effect of algae extract was also investigated using the DPPH radical scavenging assay. The results exhibited 29.84%, 11.5%, and 43.17% radical scavenging activity for the cold water, ethanolic, and hexane-acetone extract, respectively at a concentration of 5000 µg/mL. Therefore, algae extract can serve as a potent antioxidant and protector against free radicals in cells, given its natural and safe composition with high scavenging capacity against free radicals. Additionally, it increases antioxidant enzyme levels in the body's immune system, making it a potentially useful supplement to aid the body's defense system against oxidative stress. The Akt/mTOR pathway is considered as a potential therapeutic target for treating malignant tumors, particularly in chemotherapy and also fucoxanthin could be considered a promising antitumor agent, as it induces autophagy by inhibiting the Akt/mTOR signaling pathway 20 . Also, the main active compounds involved in the DPPH free-radical-scavenging method in C. indica and N. zanardinii were identified as polyphenols and fucoxanthin 10 . The recent surge in seaweed bioactives research has primarily focused on their antioxidant properties, driven by their potential applications as preservatives, protectors against oxidation in food and cosmetics, and functional ingredients for health purposes 21 , 22 . Brown algae, in particular, exhibit significantly higher antioxidant potential compared to red and green algae. Additionally, they contain unique compounds absent in terrestrial sources. In vitro, antioxidant chemical assays, utilized to assess their efficacy in preventing lipid oxidation in foods, have demonstrated that crude extracts, fractions, and pure components derived from brown algae are either comparable or superior to synthetic antioxidants 23 . The findings of these studies are consistent with the present research. Multiple mechanisms are involved in genotoxicity, including increased production of reactive oxygen species (ROS), reduction in antioxidant capacity by decreasing GSH levels, and increased MDA production 24 . In this study, the genotoxic effects of aqueous, hexane-acetone, and ethanolic extracts of C. indica algae at various concentrations were investigated using the comet assay method. With increasing concentrations of these algae extracts, the Tail Moment value increased in both cell lines compared to the control group. Additionally, ROS levels increased with increasing concentrations of the extracts. Moreover, GSH production decreased with increasing extract concentrations. Lipid peroxidation assessment also showed an increase in MDA levels with increasing extract concentrations. These results indicate that C. indica algae exhibit significant genotoxic effects on both cancerous and normal cells. Results of cytotoxic activity of certain brown algae on cancer cell lines HT-29, Caco-2, T47D, MDA-MB468, and NIH 3T3 demonstrated that extracts from various species of brown algae ( Colpomenia sinuosa, Cystoseira myrica , Sargassum swartzii ) collected from the Persian Gulf exhibit apoptotic properties and cytotoxicity 25 . Additionally, in vitro antitumor activity of a brown algae species called Padina pavonia on uterine and breast cancer cell lines investigation revealed that the methanolic extract of P. pavonia displayed cytotoxic activity with an IC 50 of 45.86 µg/mL for uterine cancer cells and 59.74 µg/mL for breast cancer cells, suggesting apoptotic and cytotoxic properties of the algae extract 26 . Furthermore, examination of the apoptotic effects of fucoxanthin derived from brown algae on the HL-60 cell line indicated that the compound induced apoptosis through cell cycle disruption in HL-60 cells 27 . In this study, the cytotoxic effect of C. indica on the cancer cell line (A549) and the normal gum cells (HGF) was examined using the MTT assay. It was observed that with increasing concentrations of various extracts, the survival rates of both cancerous and normal cells significantly decreased compared to the control group, indicating the cytotoxicity of this alga on these cell lines. Based on the results obtained from flow cytometry, the concentrations of 100 µg/mL of hot water and hexane-acetone extracts induced 54.97% and 50.66% apoptosis in A549 cancer cells, respectively, making them preferable options for further anticancer studies, especially at higher concentrations. These findings are consistent with the results of cell viability measurements using the MTT assay. 4. Conclusion C. indica is identified as a substantial source of biologically active metabolites with promising applications in pharmaceuticals and medicinal compounds. The study emphasizes its potential for developing cytotoxic agents targeting cancer cell lines. Notably, the n-hexane-acetone extract, which is rich in phenols and flavonoids, exhibited superior antioxidant and anticancer properties. The results showed significant induction of apoptosis in A549 cancer cells at concentrations of 100 µg/mL for both hot water and n-hexane-acetone extracts, indicating their potential for further anticancer research, particularly at elevated doses. This study provides valuable insights into the antioxidant, cytotoxic, and genotoxic characteristics of C. indica extracts, reinforcing their potential as candidates for anticancer studies and applications. 5. Experimental Section 5.1 General Experimental Procedures HGF and lung cancer cell lines (A549) were prepared by the Pasteur Institute of Iran (Iran) according to the ATCC number and cultured in the cell culture laboratory of Sari Pharmacy Faculty. Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma Aldrich (USA). Methanol 80%, ethanol 96%, and sodium sulfate (Na 2 SO 4 ) were purchased from Merck (Germany). Dulbecco’s Modified Eagle’s medium (DMEM) was purchased from GIBCO (USA). Penicillin G Procaine (400000 IU), streptomycin, cisplatin, and all other chemicals and solvents were of the highest grade commercially available. 5.2 Sampling and Identification The brown algae, C. indica was collected along the shores of the Chabahar Bay (25° 17′ 28″ N, 60° 38′ 15″E). Voucher specimens (MAZ-MA-214) were deposited in the herbarium of the School of Pharmacy and Pharmaceutical Sciences of Mazandaran University of Medical Sciences. They were identified by the Agricultural and Natural Resources Research Center. Following collection, epiphytes were removed, and any necrotic parts were removed. The algal samples underwent surface cleaning followed by rinsing with seawater and distilled water. Subsequently, they were dried in the shade and ground into a fine powder. Clean algae pieces were frozen and stored at -20°C for chemical analysis. 5.3 Extraction Algae materials were extracted by sequential maceration. A total of 300 g of dried seaweed C.indica was put in the Erlenmeyer flask, at first the solvent hexane-acetone (1:3 v/v) was added to the Erlenmeyer flask. After that, the sample was macerated for 24 h at room temperature. The steps were repeated at least three times. Then the liquid extract was evaporated with a rotary evaporator at 30–35 ℃ to remove the solvent contained in the extract to obtain a thick extract of C. indica . After that, the reconstituted residue was macerated in ethanol and also in cold water separately for the continued extraction step. These samples were macerated for 3x24h at room temperature. Then liquid ethanol and cold water extract were evaporated with a rotary evaporator to remove the solvent contained in the extract to obtain a thick extract. Finally, the residue of the seaweed sample was extracted by hot water in the reflux method (3x 6h). All of these concentrated extracts were dried and powdered by freeze dryer and were stored in glass containers, sealed, and kept in the refrigerator away from heat and light 28 . 5.4 Preliminary phytochemical screening The hydro-alcoholic extract from C. indica was screened for a range of phytochemical compounds such as alkaloids, sterols, triterpenoids, saponins, anthraquinone glycosides, and flavonoids 29 . 5.5 Measurement of total phenolic content Determining total phenolic content involved the utilization of the Folin-Ciocalteu reagent coupled with standard spectrophotometry and calibration curve construction with some modifications. A 0.5 mL sample solution was mixed with 2.5 mL of 0.2N Folin–Ciocalteu reagent, followed by adding 2 mL of 20% sodium carbonate solution after 5 minutes, and the mixture was vigorously shaken. Subsequently, the absorbance was measured at 760 nm post a 2-hour incubation at room temperature using a double beam Perkin Elmer UV/Visible spectrophotometer. The quantification was based on a calibration curve created using standard gallic acid concentrations, expressed as milligram gallic acid equivalents per gram of dried algae extract 30 . 5.6 Measurement of total flavonoid content The total flavonoid content was determined using the aluminum chloride colorimetric method with minor adjustments. In this method, 0.5 mL of the methanolic sample extract solution was mixed with 1.5 mL of methanol, 0.1 mL of 10% anhydrous aluminum chloride in methanol, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. Following a 30-minute incubation at room temperature, the absorbance of the samples was measured at 415 nm. A calibration curve was prepared using standard concentrations of a methanolic solution of quercetin. The total flavonoid content was then quantified as milligram equivalents of quercetin per gram of dried algae extract 30 . 5.7 Determination of antioxidant capacity 5.7.1 DPPH assay 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is a widely used method for assessing the radical-scavenging capacity of compounds. DPPH, in its stable form, exhibits a dark purple color, which diminishes upon reduction by antioxidants. In this method, 1 mL of 10 µM DPPH in methanol was added to 4 mL of different concentrations of the extract and ascorbic acid as the standard compound. Then the mixtures were incubated at room temperature in the dark for 30 min. Finally, the absorbance was measured at 517 nm. The percentage of inhibition of DPPH radical was calculated according to the following formula: Scavenging rate= (( \(\:{A}_{0}\) - \(\:{A}_{S}\) )/ \(\:\:{A}_{0}\) ) ×100 A 0 and A S are the absorbance of the DPPH solution without the sample and the absorbance of the sample with DPPH, respectively. Finally, the half-maximal inhibitory concentration (IC 50 ) of DPPH free radical was calculated according to the standard curve 31 . 5.8 Analysis of Fucoxanthin content by HPLC The fucoxanthin concentration in the sample was assessed using a Waters 1525 binary pump, featuring a Waters 2487 dual λ absorbance detector. Reversed-phase high-performance liquid chromatography (RP-HPLC) utilized Isocratic condition (82% methanol: 6.5% CH 2 Cl 2 : 7.5% acetonitrile: 4% H 2 O) as the mobile phase at a flow rate of 1.0 mL/min. Analyses were conducted at 28°C with a C 18 column (5 µm, 4.6 mm x 150 mm). Initially, a sample aliquot was dissolved in the mobile phase and filtered through a 0.22-µm membrane. Subsequently, a fraction of the filtered sample underwent HPLC analysis with a detection wavelength set at 450 nm. Quantification of fucoxanthin content was achieved through a calibration curve prepared using a standard 32 . 5.9 Measurement of Oxidative Stress Parameters 5.9.1 Measurement of Lipid Peroxidation: Lipid peroxidation was assessed by quantifying the formation of thiobarbituric acid reactive substances (TBARs), with malondialdehyde (MDA) concentration in micromolar (µM) units serving as the marker. Tissue homogenates (0.2 mL) were treated with 0.1 mL of Thiobarbituric acid (TBA) reagent, which consisted of 15% w/v trichloroacetic acid (TCA) and 0.3% w/v TBA in 0.5N HCl, followed by thorough vortexing. Subsequently, the samples underwent incubation in boiling water for 30 minutes. After incubation, the samples were cooled in an ice bath, and 0.2 mL of n-butanol was added. The mixture was then centrifuged at 3500 × g for 10 minutes to separate the n-butanol layer. The absorbance of the n-butanol layer was measured at a wavelength of 532 nm, and the amount of TBARs was determined from a standard curve 33 . 5.9.2 Measurement of Glutathione (GSH) Concentration: The reduced level of glutathione (GSH) was evaluated using 5,5ʹ-dithiobis-(2-nitrobenzoic acid) (DTNB) as an indicator. Samples from each group were treated with 20% trichloroacetic acid (TCA) and Ethylenediaminetetraacetic acid (EDTA) to precipitate proteins, followed by centrifugation at 3,500 × g for 15 minutes. 1 ml of resulting supernatants was mixed with 2.5 ml Tris buffer (PH = 8.9) and 0.5 ml of 40% DTNB. The reaction between DTNB and the sulfhydryl groups within the glutathione molecules produced a yellow thiolate anion, which was detectable. Absorbance at 412nm was then measured using a spectrophotometer for each group, enabling the quantification of GSH concentration in micromolar (µM) units 34 . 5.9.3 Measurement of intracellular production of ROS: The measurement of intracellular reactive oxygen species (ROS) production utilized the oxidation-sensitive fluoroprobe 2ˈ,7ˈ-dichlorofluorescin diacetate (DCFH-DA). Upon cellular uptake, DCFH-DA is hydrolyzed by esterases, forming DCFH, which is then oxidized to fluorescent dichlorofluorescein (DCF) by intracellular oxidants, indicating ROS levels. DCFH-DA was diluted in anhydrous DMSO to 10 mM and stored as a stock solution at 4ºC. A working solution of 100 µM was prepared in HBSS and was added to the cells for 30 min at 37 ◦C after the drug exposure. The DCFH-DA was then removed, and each well was washed with HBSS. Cold lysis buffer (1% Triton X-100, 2.5 M NaCl, 10 mM Tris, 0.1 M EDTA, pH 10) was added to the wells, and after 1 min, the solution was collected and centrifuged at 2800 ×g, for 5 minutes; 2 × 100 µl of the supernatant was added to two wells in a 96-well plate (white, clear bottom) and fluorescence was measured in a computerized microplate fluorometer (Biotek, USA) at 485 nm excitation and 530 nm emission. Background fluorescence was subtracted, and the values were expressed as times increased compared to unexposed cells 35 . 5.10 Cell culture Cell lines derived from the A549 cell line were used to study DNA damage and cultured as a monolayer in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin (100 IU/ml) and streptomycin (100µg/ml) mixture. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 and 95% air 35 . 5.11 Cell viability test by MTT assay Cell viability was assessed using the MTT assay, a widely utilized method for measuring cell viability and proliferation. For each concentration of the extract and Cisplatin, 20 µl of DMEM/F12 containing 1×10 5 cells were added to 3 wells. The cells were cultured for 72 hours until they reached the logarithmic growth phase. Throughout the incubation period, cell growth and contamination were monitored every two days. Following incubation, 50µl of each concentration of the fractions of hexane-acetone, ethanol, and algal water extract were added to each well and incubated for an additional 72 hours to ensure adequate exposure to the extracts and Cisplatin. Cells treated with 0.5% DMSO served as the solvent control and were washed with 0.5 ml of sterile normal saline (0.09%). After this, the contents of the wells were discarded, and the cells were incubated with 50 µl of MTT for 4 hours. The MTT solution was then removed, and 50 µl of diluted DMSO was added to each well to dissolve the purple formazan crystals. The plates were shaken for 15 minutes, and the absorbance of the colored colonies was measured using an ELISA reader at wavelengths of 490 nm and 630 nm 1 . 5.12 Genotoxicity assessment by comet assay The comet assay, also known as single-cell gel electrophoresis (SCGE), is a microelectrophoretic method used to directly observe DNA damage in individual cells. Different concentrations of C.indica were added to the cell culture medium and incubated at various time points. Degreased slides were first coated with a layer of 1% normal melting agarose. Subsequently, cells were trypsinized, centrifuged, counted, and mixed with 1% (w/v) low-melting-point agarose in PBS, then placed on top of the first layer. After allowing the agarose to solidify at 4°C for 5–10 minutes, the slide was immersed in an alkaline lysis buffer (pH = 10.0) for 40 minutes to remove cellular proteins and membranes. DNA unwinding occurred for 40 minutes in an electrophoretic alkaline buffer (pH > 13) before horizontal electrophoresis in the same buffer at 25V and 300 mA. Following electrophoresis, the slides were neutralized with a Tris buffer solution (pH = 7.5) for 15 minutes and then stained with ethidium bromide (20µg/ml) before being washed in phosphate-buffered saline. Comet analysis was performed under dark conditions at 400 × magnification using a fluorescent microscope. Image analysis software (comet score) was utilized to score 100 randomly selected comets on each slide, with % DNA in tail, tail length, and tail moment typically assessed to determine DNA damage 35 . 5.13 Detection of apoptosis The annexin V assay relies on annexin V's ability to bind to phosphatidylserine (PS) exposed on the outer membrane leaflet in apoptotic cells. In viable cells, PS is typically located in the inner membrane leaflet, but during apoptosis, it translocates to the outer membrane leaflet, where it becomes accessible for annexin V binding. The annexin V assay was conducted according to the manufacturer's instructions using the Annexin V-FITC kit (eBioscience). Initially, 1 × 10 6 cells were washed with PBS and suspended in a binding buffer. Subsequently, 100 µl of cell suspension was mixed with 5 µl Annexin V-FITC and incubated for 10 minutes in the dark at room temperature. Afterward, the cells were washed with binding buffer, resuspended in 200 µl binding buffer, and stained with 5 µl propidium iodide (PI) solution. Finally, the samples were analyzed using flow cytometry (Partec, Germany) equipped with Flomax software (version 2.4) within a 4-hour timeframe 36 . 5.14 Statistical analysis The findings were reported as mean ± standard deviation (SD). All statistical analyses were done using the GraphPad Prism software, version 3. All experiments were performed in triplicate, and the mean values were used for statistical evaluation. A one-way ANOVA test was utilized, followed by the post hoc Tukey-Krame multiple comprehension test to assess statistical significance. A significance level of P < 0.05 was considered statistically significant. Declarations Acknowledgements and Fundings The authors are grateful to Mazandaran University of Medical Sciences for their financial support. Lutfun Nahar gratefully acknowledges the financial support of the European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and the Czech Agency Grants - Project 23-05474S and Project 23-05389S. Data availability All the data are available in the main text. All the data generated in this study can be obtained from the corresponding authors upon reasonable request. 8. Author contributions Emran Habibi design of study, analysis of data and fund acquisition, Sahar Sheikhzadeh Acquisition of data, Mohammad Shokrzadeh compound isolation and structure validation, Fariborz Sharifianjazi concept of study and data analysis, Satyajit D. Sarker article writing and data analysis, Hesamoddin Arabnozari interpretation of data, Final approval of the version to be published and Lutfun Nahar supervision and fund acquisition. All authors reviewed the manuscript. 9. Compliance with Ethical Standards Conflict of interest: The authors declare no conflict of interest. Ethical approval: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. References Shokrzadeh, M., Rajabali, F., Habibi, E. & Modanloo, M. Survey cytotoxicity and genotoxicity of hydroalcoholic extract of Stevia rebaudiana in breast cancer cell line (MCF7) and human fetal lung fibroblasts (MRC-5). J. Cancer Res. Metastasis 1 , 12-17 (2018). Sanjeewa, K. A., Lee, J.-S., Kim, W.-S. & Jeon, Y.-J. The potential of brown-algae polysaccharides for the development of anticancer agents: An update on anticancer effects reported for fucoidan and laminaran. Carbohydrate polymers 177 , 451-459 (2017). Huang, J. et al. Distribution, risk factors, and temporal trends for lung cancer incidence and mortality: a global analysis. Chest 161 , 1101-1111 (2022). Bastani, E. & Shokri, F. Incidence Trend of Lung Cancer in Iran: A Systematic Review and Meta-analysis. Int J Cancer Manag 16 , e135020 (2023). https://doi.org:10.5812/ijcm-135020 Khazaei, S. et al. Epidemiology of lung cancer in Iran: sex difference and geographical distribution. Middle East Journal of Cancer 8 , 223-228 (2017). Lemjabbar-Alaoui, H., Hassan, O. U., Yang, Y.-W. & Buchanan, P. Lung cancer: Biology and treatment options. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1856 , 189-210 (2015). Pratheeshkumar, P. & Kuttan, G. Ameliorative action of Vernonia cinerea L. on cyclophosphamide-induced immunosuppression and oxidative stress in mice. Inflammopharmacology 18 , 197-207 (2010). Mhadhebi, L., Mhadhebi, A., Robert, J. & Bouraoui, A. Antioxidant, anti-inflammatory and antiproliferative effects of aqueous extracts of three mediterranean brown seaweeds of the genus cystoseira. Iranian journal of pharmaceutical research: IJPR 13 , 207 (2014). de Sousa, C. B. et al. Cystoseira algae (Fucaceae): Update on their chemical entities and biological activities. Tetrahedron: Asymmetry 28 , 1486-1505 (2017). Fariman, G. A., Shastan, S. J. & Zahedi, M. M. Seasonal variation of total lipid, fatty acids, fucoxanthin content, and antioxidant properties of two tropical brown algae (Nizamuddinia zanardinii and Cystoseira indica) from Iran. Journal of Applied Phycology 28 , 1323-1331 (2016). Yegdaneh, A., Ghannadi, A. & Dayani, L. Chemical constituents and biological activities of two Iranian Cystoseira species. Research in pharmaceutical sciences 11 , 311 (2016). Taheri, A., Ghaffari, M., Houshmandi, S. & Namavari, M. M. Investigation of the anticancer and antioxidant activity of the brown algae (Cystoseira indica) extract against the colorectal cancer cells. KAUMS Journal (FEYZ) 21 , 317-325 (2017). Cruz, C. S. D., Tanoue, L. T. & Matthay, R. A. Lung cancer: epidemiology, etiology, and prevention. Clinics in chest medicine 32 , 605-644 (2011). El-Din, S. M. M. & El-Ahwany, A. M. Bioactivity and phytochemical constituents of marine red seaweeds (Jania rubens, Corallina mediterranea and Pterocladia capillacea). Journal of Taibah University for Science 10 , 471-484 (2016). Moussavou, G. et al. Anticancer effects of different seaweeds on human colon and breast cancers. Marine drugs 12 , 4898-4911 (2014). Yen, G. C., Duh, P. D. & Tsai, C. L. Relationship between antioxidant activity and maturity of peanut hulls. Journal of Agricultural and Food Chemistry 41 , 67-70 (1993). Karawita, R. et al. Reactive oxygen species scavenging, metal chelation, reducing power and lipid peroxidation inhibition properties of different solvent fractions from Hizikia fusiformis. European Food Research and Technology 220 , 363-371 (2005). Kähkönen, M. P. et al. Antioxidant activity of plant extracts containing phenolic compounds. Journal of agricultural and food chemistry 47 , 3954-3962 (1999). Duan, X.-J., Zhang, W.-W., Li, X.-M. & Wang, B.-G. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food chemistry 95 , 37-43 (2006). Hou, L.-l., Gao, C., Chen, L., Hu, G.-q. & Xie, S.-q. Essential role of autophagy in fucoxanthin-induced cytotoxicity to human epithelial cervical cancer HeLa cells. Acta Pharmacologica Sinica 34 , 1403-1410 (2013). López-Hortas, L. et al. Applying seaweed compounds in cosmetics, cosmeceuticals and nutricosmetics. Marine drugs 19 , 552 (2021). Kumar, Y., Tarafdar, A. & Badgujar, P. C. Seaweed as a source of natural antioxidants: Therapeutic activity and food applications. Journal of Food Quality 2021 , 5753391 (2021). Balboa, E. M., Conde, E., Moure, A., Falqué, E. & Domínguez, H. In vitro antioxidant properties of crude extracts and compounds from brown algae. Food chemistry 138 , 1764-1785 (2013). M. Green, R., Graham, M., RO'Donovan, M., Chipman, J. K. & J. Hodges, N. Subcellular compartmentalization of glutathione: correlations with parameters of oxidative stress related to genotoxicity. Mutagenesis 21 , 383-390 (2006). Khanavi, M. et al. Cytotoxic activity of some marine brown algae against cancer cell lines. Biological Research 43 , 31-37 (2010). Stanojković, T. P. et al. In vitro antitumoral activities of Padina pavonia on human cervix and breast cancer cell lines. J. Med. Plant Res 7 , 419-424 (2013). Hosokawa, M. et al. Apoptosis-inducing effect of fucoxanthin on human leukemia cell line HL-60. Food Science and Technology Research 5 , 243-246 (1999). Paradiso, V. M., Castellino, M., Renna, M., Santamaria, P. & Caponio, F. Setup of an Extraction Method for the Analysis of Carotenoids in Microgreens. Foods 9 , 459 (2020). Talla, E. et al. Antioxidant activity and a new ursane-type triterpene from Vitellaria paradoxa (Sapotaceae) stem barks. European Journal of Medicinal Plants 16 , 1-20 (2016). Vinatoru, M. et al. The use of ultrasound for the extraction of bioactive principles from plant materials. Ultrasonics sonochemistry 4 , 135-139 (1997). Rodríguez-Meizoso, I. et al. Subcritical water extraction of nutraceuticals with antioxidant activity from oregano. Chemical and functional characterization. Journal of pharmaceutical and biomedical analysis 41 , 1560-1565 (2006). Yan, X., Chuda, Y., Suzuki, M. & Nagata, T. Fucoxanthin as the major antioxidant in Hijikia fusiformis, a common edible seaweed. Bioscience, biotechnology, and biochemistry 63 , 605-607 (1999). Chakraborty, S., Singh, O. P., Dasgupta, A., Mandal, N. & Das, H. N. Correlation between lipid peroxidation-induced TBARS level and disease severity in obsessive–compulsive disorder. Progress in Neuro-Psychopharmacology and Biological Psychiatry 33 , 363-366 (2009). Azari, A., Shokrzadeh, M., Zamani, E., Amani, N. & Shaki, F. Cerium oxide nanoparticles protects against acrylamide induced toxicity in HepG2 cells through modulation of oxidative stress. Drug and Chemical Toxicology 42 , 54-59 (2019). Ghassemi-Barghi, N., Varshosaz, J., Etebari, M. & Dehkordi, A. J. Role of recombinant human erythropoietin loading chitosan-tripolyphosphate nanoparticles in busulfan-induced genotoxicity: Analysis of DNA fragmentation via comet assay in cultured HepG2 cells. Toxicology in Vitro 36 , 46-52 (2016). Zamani, E., Shaki, F., AbedianKenari, S. & Shokrzadeh, M. Acrylamide induces immunotoxicity through reactive oxygen species production and caspase-dependent apoptosis in mice splenocytes via the mitochondria-dependent signaling pathways. Biomedicine & Pharmacotherapy 94 , 523-530 (2017). Additional Declarations The authors declare no competing interests. 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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-4545987","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":336581369,"identity":"8f8fc492-7be3-4c25-952c-a4698a5f8283","order_by":0,"name":"Emran Habibi","email":"","orcid":"","institution":"Medicinal Plants Research Center, Mazandaran University of Medical Sciences, Sari, Iran","correspondingAuthor":false,"prefix":"","firstName":"Emran","middleName":"","lastName":"Habibi","suffix":""},{"id":336581370,"identity":"b51192e7-1a0a-497e-9547-52427235c130","order_by":1,"name":"Sahar Sheikhzadeh","email":"","orcid":"","institution":"Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.","correspondingAuthor":false,"prefix":"","firstName":"Sahar","middleName":"","lastName":"Sheikhzadeh","suffix":""},{"id":336581371,"identity":"93fb67c7-45fc-454c-a2cd-182c1232ab6e","order_by":2,"name":"Hesamoddin Arabnozari","email":"","orcid":"","institution":"Student Research Committee, School of Medicine, Babol University of Medical Sciences, Babol, Iran","correspondingAuthor":false,"prefix":"","firstName":"Hesamoddin","middleName":"","lastName":"Arabnozari","suffix":""},{"id":336581372,"identity":"c6339693-f3ef-491e-98a5-5af0860d3505","order_by":3,"name":"Mohammad Shokrzadeh","email":"","orcid":"","institution":"Pharmaceutical Sciences Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences, Sari, Iran.","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"","lastName":"Shokrzadeh","suffix":""},{"id":336581373,"identity":"9e1ffb57-100a-442c-a473-d952969c9e9e","order_by":4,"name":"Fariborz Sharifianjazi","email":"","orcid":"","institution":"School of Science and Technology, The University of Georgia, Tbilisi, Georgia","correspondingAuthor":false,"prefix":"","firstName":"Fariborz","middleName":"","lastName":"Sharifianjazi","suffix":""},{"id":336581374,"identity":"83ca3402-b7fe-4279-8835-6db772a44fb3","order_by":5,"name":"Satyajit D. 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0.0001).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/c8c6abe8db0044742214d913.png"},{"id":61963588,"identity":"4b33b2e5-8308-4a8a-b4fa-801138b4820c","added_by":"auto","created_at":"2024-08-07 15:03:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":119935,"visible":true,"origin":"","legend":"\u003cp\u003eThe level of GSH in HGF and A549 cancer cell lines in exposure to cold water, hot water, ethanolic, and hexane-acetone extract The results are presented as Mean ± SEM and repeated three times independently with similar results. ψψψψ indicates a significant difference from the control group (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/0502f9587bb37aae2d922fdd.png"},{"id":61963593,"identity":"3abfe47f-4716-459f-8ddd-21e52de5e4f2","added_by":"auto","created_at":"2024-08-07 15:03:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":124346,"visible":true,"origin":"","legend":"\u003cp\u003eThe level of ROS in HGF and A549 cancer cell lines in exposure to cold water, hot water, ethanolic, and hexane-acetone extract The results are presented as Mean ± SEM and repeated three times independently with similar results. ψψψψ indicates a significant difference from the control group (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/1d129e7939cad78e8b326063.png"},{"id":61964302,"identity":"b4ae25f3-b52c-49b0-b367-9b61e147221a","added_by":"auto","created_at":"2024-08-07 15:11:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114016,"visible":true,"origin":"","legend":"\u003cp\u003eCell survival rate (percentage) of HGF and A549 cancer cell lines in exposure to cold water, hot water, ethanolic, and hexane-acetone extract The results are presented as Mean ± SEM and repeated three times independently with similar results.\u003c/p\u003e\n\u003cp\u003eψψψψ indicates a significant difference from the control group (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/52f25a0e9b6deddeb539fdc5.png"},{"id":61963591,"identity":"74d32cbf-7736-46e5-a663-5513a549723a","added_by":"auto","created_at":"2024-08-07 15:03:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":54073,"visible":true,"origin":"","legend":"\u003cp\u003eTail moments of HGF and A549 cancer cell lines in exposure to cold water, hot water, ethanolic, and hexane-acetone extract The results are presented as Mean ± SEM and repeated three times independently with similar results.\u003c/p\u003e\n\u003cp\u003eψψψψ indicates a significant difference from the control group (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/fe6abcd70df6698da9e6058d.png"},{"id":61963594,"identity":"e031afb9-dda8-40e1-97fc-176e2cdfff1f","added_by":"auto","created_at":"2024-08-07 15:03:34","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":207072,"visible":true,"origin":"","legend":"\u003cp\u003eThe apoptosis rate of A549 cancer cell lines in exposure to cold water, hot water, ethanolic, and hexane-acetone extract using flow cytometry\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/55e5bb492ceae3e0d3dea571.png"},{"id":61965547,"identity":"f7d295ce-4aad-43c0-add6-c5581d021598","added_by":"auto","created_at":"2024-08-07 15:27:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1745526,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/4f4efcec-0fe9-4d1c-9123-e66affd33719.pdf"},{"id":61963589,"identity":"3750cee1-fe88-4a3c-8cb3-b2de4b52ae8f","added_by":"auto","created_at":"2024-08-07 15:03:34","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":290855,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4545987/v1/ec5b4a302d1d03c5842b91eb.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eGenotoxicity and cytotoxicity evaluation of brown algae (\u003cem\u003eCystoseira indica\u003c/em\u003e) extract in human gingival fibroblast (HGF) and lung cancer cell lines (A549)\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCancer is one of the most devastating diseases globally, characterized by an annual incidence of over 10\u0026nbsp;million new cases worldwide\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Mutations in the oncogenes with a dominant gain of function over tumor suppressor genes and malfunction of tumor suppressor genes are considered the main reasons for cancer development\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLung cancer (LC), characterized by uncontrolled cell growth in the lung tissue, often manifests without symptoms in its early stages, leading to delayed diagnosis and advanced disease presentation, associated with severe complications and increased mortality\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Survival rates vary significantly depending on cancer type and characteristics. The high mortality rate of LC is attributed to its widespread incidence, low survival rates, and propensity for metastasis, which exacerbates patient outcomes and poses significant challenges to survival\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. LC stands as the primary contributor to cancer-related mortality globally, with prevalence being highest among men and ranking fourth among women. Notably, in Iran, it holds the second position for cancer-related deaths among males and the third position among females\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVarious treatment modalities are available for LC, such as surgery, radiation therapy, chemotherapy, and targeted therapy. Treatment recommendations depend on factors like cancer type and stage. Despite advancements in diagnosis and therapy over the last 25 years, the prognosis for LC patients remains unsatisfactory\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecently, there has been an increased interest in searching for plant-derived compounds capable of minimizing chemotherapy-induced toxicity in normal cells while preserving their antineoplastic activity\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In recent years, seaweeds and their extracts have emerged as sources of diverse bioactive compounds with potential medicinal properties. These compounds, including polyphenols, polysaccharides, meroterpenoids, and terpenoids, exhibit various biological activities such as anti-inflammatory, antibiotic, antiviral, cytotoxic, and antimitotic effects\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eCystoseira\u003c/em\u003e C. Agardh, 1820, a polyphyletic genus of marine macroalgae within the Sargassaceae family, is distributed along the Atlantic-Mediterranean coasts and comprises approximately 40 species. These species produce a diverse array of secondary metabolites, including terpenoids, fatty acids, steroids, and polysaccharides. Thus, the members of this genus offer the potential for the discovery of novel compounds with biomedical applications. Extracts from different \u003cem\u003eCystoseira\u003c/em\u003e species have been investigated for various activities, such as antimicrobial, antiviral, antifungal, antioxidant, cytotoxic, and antitumoral properties, suggesting their promising biomedical relevance\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCystoseira indica\u003c/em\u003e (Thivy \u0026amp; Doshi), a tropical brown algae found in southwest Asia, including India, Iran, Oman, Pakistan, Qatar, and Yemen exhibits faster growth and higher biomass when cultivated in the tidal zone of Chabahar Bay, Iran, compared to other algae species in the region. Chabahar Bay, situated in the southeastern part of Iran and the northern region of the Oman Sea, experiences strong ultraviolet radiation due to its tropical location, leading to the production of reactive oxygen species (ROS) and other oxidizing agents. In response to these environmental conditions, marine algae in warm waters alter their metabolism to produce antioxidants as a protective mechanism\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. indica\u003c/em\u003e displays significant pharmacological properties ascribed to its rich phytochemical composition, particularly high levels of total phenolics and potent antioxidant activity in which the extracts from \u003cem\u003eC. indica\u003c/em\u003e effectively inhibit cell growth in HeLa (cervical carcinoma), and HT-29 (human colon adenocarcinoma), and particularly MCF-7 (human breast adenocarcinoma)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The methanolic and chloroform extracts of \u003cem\u003eC. indica\u003c/em\u003e exhibited potent antioxidant and cytotoxic effects against colorectal cancer cells, suggesting their potential as an anticancer agent for preclinical and clinical studies\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWith lung cancer being widespread globally and considering the documented anticancer properties of brown algae, this research aimed to investigate the genotoxicity and cytotoxicity of hydroalcoholic extract of brown algae \u003cem\u003eC. indica\u003c/em\u003e in human gingival fibroblast (HGF) and lung cancer cell lines (A549).\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003eThe results of the preliminary phytochemical analysis for identifying some major groups of compounds in \u003cem\u003eC. indica\u003c/em\u003e algae are summarized in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhytochemical test results\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePresence of a group of compounds in\u003c/p\u003e\n \u003cp\u003ethe extract\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePhytochemical test\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSaponins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTerpenoids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSteroids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnthraquinone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlkaloids\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFlavonoids and\u003c/p\u003e\n \u003cp\u003ephenols\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Quantification of phytoconstituents\u003c/h2\u003e\n \u003cp\u003eThe total phenol compounds were quantified as gallic acid equivalents using a standard curve equation (y\u0026thinsp;=\u0026thinsp;0.0031x\u0026thinsp;+\u0026thinsp;0.1114, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9497). The concentration of phenolics in the extracts, was dependent on the solvent used in the extraction, as shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Phenolic compounds were not observed in the ethanolic and the aqueous extracts of \u003cem\u003eC. indica\u003c/em\u003e and higher concentrations needed to be utilized.\u003c/p\u003e\n \u003cp\u003eSimilarly, the total flavonoid contents were determined as mg quercetin equivalent/g of extract powder using a standard curve equation (y\u0026thinsp;=\u0026thinsp;0.0019x\u0026thinsp;+\u0026thinsp;0.0848, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9757). The concentration of flavonoids in the extracts depended on the solvent used in the extraction, as shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The \u003cem\u003en\u003c/em\u003e-hexane-acetone extract showed a higher flavonoid content compared to the ethanolic extract. In the aqueous extract, calculation was not feasible for cold and hot aqueous extracts, and higher concentrations must be utilized.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Antioxidant (DPPH radical-scavenging) assay\u003c/h2\u003e\n \u003cp\u003eHalf-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) for DPPH was examined on algal samples extracted using hexane-acetone, ethanol, hot water, and cold water, according to the standard curve equation (y\u0026thinsp;=\u0026thinsp;1.6214x \u0026minus;\u0026thinsp;0.8933, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.962). Following the results that are shown in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e different extracts of\u0026nbsp;\u003cem\u003eC. indica\u003c/em\u003e algae exhibited the highest percentage of inhibition at a concentration of 5000 \u0026micro;g/mL.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThe total phenolics, flavonoids contents, and scavenging activity of different extracts from C. Indica. Each value is expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation; P value ˂ 0.0001 for all extraction; n. d.= not detected.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSamples\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDPPH radical scavenging assay (IC\u003csub\u003e50\u003c/sub\u003e value, \u0026micro;g/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal phenolics content\u003c/p\u003e\n \u003cp\u003e(mg GAEs/g of dry weight)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal flavonoids content\u003c/p\u003e\n \u003cp\u003e(mg quercetin/g of dry weight)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCold water extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8370\u0026thinsp;\u0026plusmn;\u0026thinsp;2460\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en. d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en. d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHot water extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16450\u0026thinsp;\u0026plusmn;\u0026thinsp;1790\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en. d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en. d.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEthanolic extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22580\u0026thinsp;\u0026plusmn;\u0026thinsp;4690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003en. d.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003en\u003c/em\u003e-Hexane-acetone extract\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e306.15\u0026thinsp;\u0026plusmn;\u0026thinsp;18.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAscorbic acid\u003c/p\u003e\n \u003cp\u003e(Standard)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e30.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Determination of fucoxanthin by HPLC method\u003c/h2\u003e\n \u003cp\u003eAfter injecting various extract concentrations, the maximum absorption peak was observed at 450 nm between 2.7 and 3.7 min, indicating fucoxanthin with maximum intensity. The HPLC chromatogram for standard fucoxanthin resulting from a concentration of 10 \u0026micro;g/mL of the standard is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates that adding diluted concentration from the standard to the ethanolic sample increased the peak height corresponding to the absorption region of fucoxanthin. This suggests that the absorption peak of the sample matched fucoxanthin, and its intensity increased with the standard. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, after adding a diluted concentration of the standard to the hexane-acetone sample, an increase in the peak height present in the absorption region of fucoxanthin is observed. By calculating the area under the standard curve (y\u0026thinsp;=\u0026thinsp;53407x\u0026thinsp;+\u0026thinsp;9778.4, r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9992) of the sample and applying it to the line equation, it was determined that the algal sample contained 3.077 \u0026micro;g of fucoxanthin per 1g in \u003cem\u003en\u003c/em\u003e-hexane-acetone extract and 4.32 \u0026micro;g of fucoxanthin per 1g in ethanolic extract.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Oxidative Stress Parameters\u003c/h2\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.1 Measurement of Lipid Peroxidation:\u003c/h2\u003e\n \u003cp\u003eThe concentration of malondialdehyde (MDA) in micromolar at various concentrations of algae extracts compared to the control group is shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The control group refers to cells that were only incubated with a complete culture medium for 1 hour.\u003c/p\u003e\n \u003cp\u003eOne-way analysis of variance (ANOVA) results for concentrations of 10, 50, and 100 \u0026micro;g/mL in cold water, hot water, and ethanolic extracts, as well as concentrations of 5 to 100 \u0026micro;g/mL in \u003cem\u003en\u003c/em\u003e-hexane-acetone extracts, were statistically significant. According to the analysis results, a significant increase in lipid peroxidation was observed at these three concentrations compared to the control group.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.2 Measurement of Glutathione (GSH) Concentration:\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e depicts the mean optical absorption measured for intracellular glutathione (GSH) using a spectrophotometer at a wavelength of 412 nm at various concentrations of algae extracts compared to the control group. One-way ANOVA analysis for each of the three concentrations (10, 50, and 100 \u0026micro;g/mL) in cold and hot water extracts, as well as for concentrations ranging from 5 to 100 \u0026micro;g/mL in \u003cem\u003en\u003c/em\u003e-hexane-acetone and ethanol extracts, showed significant results. According to the analysis, a significant decrease in intracellular glutathione levels was observed at these three concentrations compared to the control group.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\n \u003ch2\u003e2.4.3 Measurement of intracellular production of ROS:\u003c/h2\u003e\n \u003cp\u003eAccording to Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, the mean ROS levels measured by fluorometer at 485 nm excitation and 530 nm emission at various concentrations of extracts are compared to the control group.\u003c/p\u003e\n \u003cp\u003eOne-way ANOVA analysis for each of the three concentrations (5 to 100 \u0026micro;g/mL) of cold water, ethanol, and hexane-acetone extracts, as well as concentrations ranging from 10 to 100 \u0026micro;g/mL of hot water extract, showed significant results. According to the analysis, a significant increase in ROS production was observed at these three concentrations compared to the control group.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 The cytotoxicity of different algae extracts on HGF and A549 cancer cell lines:\u003c/h2\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e shows the cell toxicity levels of various concentrations of algae extracts on two cell lines, A549 and HGF compared to the control group.\u003c/p\u003e\n \u003cp\u003eThe cell viability for cold and hot water and ethanol extracts showed significant differences from the control group at concentrations ranging from 10 to 100 \u0026micro;g/mL, while for the hexane-acetone extract, significant differences were observed at concentrations ranging from 5 to 100 \u0026micro;g/mL compared to the control group.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.6 The genotoxicity of different algae extracts on HGF and A549 cancer cell lines\u003c/strong\u003e:\u003c/p\u003e\n \u003cp\u003eTo determine the optimal genotoxic concentrations of algae extracts, HGF and A549 cancer cell lines were treated with varying concentrations of \u003cem\u003eC. indica\u003c/em\u003e (5, 10, 50, and 100 \u0026micro;g/mL) for 1 hour before conducting the comet assay. The results obtained from the comet assay were measured based on the Tail moment criterion and compared with the results of the control group.\u003c/p\u003e\n \u003cp\u003eResults of the visual scoring and percentage of total DNA damage induced by different concentrations of algae extracts are shown in Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e. The data indicate that cold and hot water and ethanol extracts showed significant differences from the control group at concentrations ranging from 10 to 100 \u0026micro;g/mL, while for the \u003cem\u003en\u003c/em\u003e-hexane-acetone extract, significant differences were observed at concentrations ranging from 5 to 100 \u0026micro;g/mL compared to the control group.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2.7 Apoptosis level\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe results obtained from flow cytometry for measuring the induced apoptosis by different extracts of \u003cem\u003eCystoseira indica\u003c/em\u003e algae are presented in Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e. According to the figure, the cold water extract results in 28.1% apoptosis in A549 lung cancer cells, while the hot water extract induces 54.97% apoptosis, the ethanolic extract leads to 8.83% apoptosis, and the hexane-acetone extract causes 50.66% apoptosis at the highest concentration (100 \u0026micro;g/mL) respectively.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe global cancer report highlights a significant and abrupt surge in cancer incidence, with cancer ranking as the second leading cause of death globally\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Lung cancer is a major health concern due to its high mortality rate, making it the leading cause of cancer-related deaths worldwide. Despite advances in treatment, its impact remains significant, highlighting the urgent need for effective prevention strategies, early detection methods, and innovative therapies to improve patient outcomes and reduce the burden of this disease\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVarious compounds with diverse applications, such as antibiotic, antiviral, antifungal, and anticancer effects, have been identified and extracted from macroalgae. Regarding seaweeds, they are primitive plant-like organisms lacking roots, stems, and leaves, yet they boast a rich composition of vitamins, minerals, trace elements, proteins, and bioactive compounds.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the present study, phytochemical screening of the seaweeds showed the presence of saponins, terpenoids, steroids, anthraquinone, flavonoids, and phenols. These compounds demonstrate diverse mechanisms of action, such as inhibiting cancer cell growth and metastasis, as well as inducing cancer cell death\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This research aimed to investigate the genotoxicity and cytotoxicity of hydroalcoholic extract of brown algae \u003cem\u003eC. indica\u003c/em\u003e in HGF and A549 lung cancer cell lines.\u003c/p\u003e \u003cp\u003eThe strong positive correlation between the polyphenolic content of algae and its antioxidant activity has been extensively documented\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Phenolic compounds, commonly found in plants including seaweeds, are known for their diverse biological activities, particularly their antioxidant properties\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Consequently, the high antioxidant activities of the extracts can likely be attributed to the content of total phenolic compounds. Furthermore, in this study, \u003cem\u003eC. indica\u003c/em\u003e exhibited antioxidant activity, possibly attributable to its elevated polyphenolic content (14.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 mg GAE/g).\u003c/p\u003e \u003cp\u003eTotal flavonoids in the seaweeds ranged from 3.9 to 12.43 mg/g. Flavonoids, known for their antioxidant and free radical scavenging properties, are key natural phenols with broad chemical and biological activities\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. They serve as antioxidants against various reactive oxygen species and inhibit lipid peroxidation, suggesting potential therapeutic use against diverse diseases\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. These compounds, including flavonoids, and polyphenols, found in significant amounts in \u003cem\u003eC. indica\u003c/em\u003e, play a preventive role against diseases by scavenging free radicals.\u003c/p\u003e \u003cp\u003eBased on the results obtained from the study and the analysis of phytochemical, cytotoxic, and genotoxic tests, the hexane-acetone extract, containing a higher concentration of compounds such as phenols and flavonoids, exhibits greater potential for antioxidant and anticancer properties.\u003c/p\u003e \u003cp\u003eIn this study, the antioxidant effect of algae extract was also investigated using the DPPH radical scavenging assay. The results exhibited 29.84%, 11.5%, and 43.17% radical scavenging activity for the cold water, ethanolic, and hexane-acetone extract, respectively at a concentration of 5000 \u0026micro;g/mL. Therefore, algae extract can serve as a potent antioxidant and protector against free radicals in cells, given its natural and safe composition with high scavenging capacity against free radicals. Additionally, it increases antioxidant enzyme levels in the body's immune system, making it a potentially useful supplement to aid the body's defense system against oxidative stress.\u003c/p\u003e \u003cp\u003eThe Akt/mTOR pathway is considered as a potential therapeutic target for treating malignant tumors, particularly in chemotherapy and also fucoxanthin could be considered a promising antitumor agent, as it induces autophagy by inhibiting the Akt/mTOR signaling pathway\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Also, the main active compounds involved in the DPPH free-radical-scavenging method in \u003cem\u003eC. indica\u003c/em\u003e and \u003cem\u003eN. zanardinii\u003c/em\u003e were identified as polyphenols and fucoxanthin\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The recent surge in seaweed bioactives research has primarily focused on their antioxidant properties, driven by their potential applications as preservatives, protectors against oxidation in food and cosmetics, and functional ingredients for health purposes\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Brown algae, in particular, exhibit significantly higher antioxidant potential compared to red and green algae. Additionally, they contain unique compounds absent in terrestrial sources. In vitro, antioxidant chemical assays, utilized to assess their efficacy in preventing lipid oxidation in foods, have demonstrated that crude extracts, fractions, and pure components derived from brown algae are either comparable or superior to synthetic antioxidants\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The findings of these studies are consistent with the present research.\u003c/p\u003e \u003cp\u003eMultiple mechanisms are involved in genotoxicity, including increased production of reactive oxygen species (ROS), reduction in antioxidant capacity by decreasing GSH levels, and increased MDA production\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this study, the genotoxic effects of aqueous, hexane-acetone, and ethanolic extracts of \u003cem\u003eC. indica\u003c/em\u003e algae at various concentrations were investigated using the comet assay method. With increasing concentrations of these algae extracts, the Tail Moment value increased in both cell lines compared to the control group. Additionally, ROS levels increased with increasing concentrations of the extracts. Moreover, GSH production decreased with increasing extract concentrations. Lipid peroxidation assessment also showed an increase in MDA levels with increasing extract concentrations. These results indicate that \u003cem\u003eC. indica\u003c/em\u003e algae exhibit significant genotoxic effects on both cancerous and normal cells.\u003c/p\u003e \u003cp\u003eResults of cytotoxic activity of certain brown algae on cancer cell lines HT-29, Caco-2, T47D, MDA-MB468, and NIH 3T3 demonstrated that extracts from various species of brown algae (\u003cem\u003eColpomenia sinuosa, Cystoseira myrica\u003c/em\u003e, \u003cem\u003eSargassum swartzii\u003c/em\u003e) collected from the Persian Gulf exhibit apoptotic properties and cytotoxicity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Additionally, in vitro antitumor activity of a brown algae species called \u003cem\u003ePadina pavonia\u003c/em\u003e on uterine and breast cancer cell lines investigation revealed that the methanolic extract of \u003cem\u003eP. pavonia\u003c/em\u003e displayed cytotoxic activity with an IC\u003csub\u003e50\u003c/sub\u003e of 45.86 \u0026micro;g/mL for uterine cancer cells and 59.74 \u0026micro;g/mL for breast cancer cells, suggesting apoptotic and cytotoxic properties of the algae extract\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, examination of the apoptotic effects of fucoxanthin derived from brown algae on the HL-60 cell line indicated that the compound induced apoptosis through cell cycle disruption in HL-60 cells\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, the cytotoxic effect of \u003cem\u003eC. indica\u003c/em\u003e on the cancer cell line (A549) and the normal gum cells (HGF) was examined using the MTT assay. It was observed that with increasing concentrations of various extracts, the survival rates of both cancerous and normal cells significantly decreased compared to the control group, indicating the cytotoxicity of this alga on these cell lines. Based on the results obtained from flow cytometry, the concentrations of 100 \u0026micro;g/mL of hot water and hexane-acetone extracts induced 54.97% and 50.66% apoptosis in A549 cancer cells, respectively, making them preferable options for further anticancer studies, especially at higher concentrations. These findings are consistent with the results of cell viability measurements using the MTT assay.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003e \u003cem\u003eC. indica\u003c/em\u003e is identified as a substantial source of biologically active metabolites with promising applications in pharmaceuticals and medicinal compounds. The study emphasizes its potential for developing cytotoxic agents targeting cancer cell lines. Notably, the n-hexane-acetone extract, which is rich in phenols and flavonoids, exhibited superior antioxidant and anticancer properties. The results showed significant induction of apoptosis in A549 cancer cells at concentrations of 100 \u0026micro;g/mL for both hot water and n-hexane-acetone extracts, indicating their potential for further anticancer research, particularly at elevated doses. This study provides valuable insights into the antioxidant, cytotoxic, and genotoxic characteristics of \u003cem\u003eC. indica\u003c/em\u003e extracts, reinforcing their potential as candidates for anticancer studies and applications.\u003c/p\u003e"},{"header":"5. Experimental Section","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e5.1 General Experimental Procedures\u003c/h2\u003e\n \u003cp\u003eHGF and lung cancer cell lines (A549) were prepared by the Pasteur Institute of Iran (Iran) according to the ATCC number and cultured in the cell culture laboratory of Sari Pharmacy Faculty. Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), and 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma Aldrich (USA). Methanol 80%, ethanol 96%, and sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) were purchased from Merck (Germany). Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM) was purchased from GIBCO (USA). Penicillin G Procaine (400000 IU), streptomycin, cisplatin, and all other chemicals and solvents were of the highest grade commercially available.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e5.2 Sampling and Identification\u003c/h2\u003e\n \u003cp\u003eThe brown algae, \u003cem\u003eC. indica\u003c/em\u003e was collected along the shores of the Chabahar Bay (25\u0026deg; 17\u0026prime; 28\u0026Prime; N, 60\u0026deg; 38\u0026prime; 15\u0026Prime;E). Voucher specimens (MAZ-MA-214) were deposited in the herbarium of the School of Pharmacy and Pharmaceutical Sciences of Mazandaran University of Medical Sciences. They were identified by the Agricultural and Natural Resources Research Center. Following collection, epiphytes were removed, and any necrotic parts were removed. The algal samples underwent surface cleaning followed by rinsing with seawater and distilled water. Subsequently, they were dried in the shade and ground into a fine powder. Clean algae pieces were frozen and stored at -20\u0026deg;C for chemical analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e5.3 Extraction\u003c/h2\u003e\n \u003cp\u003eAlgae materials were extracted by sequential maceration. A total of 300 g of dried seaweed \u003cem\u003eC.indica\u003c/em\u003e was put in the Erlenmeyer flask, at first the solvent hexane-acetone (1:3 v/v) was added to the Erlenmeyer flask. After that, the sample was macerated for 24 h at room temperature. The steps were repeated at least three times. Then the liquid extract was evaporated with a rotary evaporator at 30\u0026ndash;35 ℃ to remove the solvent contained in the extract to obtain a thick extract of \u003cem\u003eC. indica\u003c/em\u003e. After that, the reconstituted residue was macerated in ethanol and also in cold water separately for the continued extraction step.\u003c/p\u003e\n \u003cp\u003eThese samples were macerated for 3x24h at room temperature. Then liquid ethanol and cold water extract were evaporated with a rotary evaporator to remove the solvent contained in the extract to obtain a thick extract. Finally, the residue of the seaweed sample was extracted by hot water in the reflux method (3x 6h). All of these concentrated extracts were dried and powdered by freeze dryer and were stored in glass containers, sealed, and kept in the refrigerator away from heat and light\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e5.4 Preliminary phytochemical screening\u003c/h2\u003e\n \u003cp\u003eThe hydro-alcoholic extract from \u003cem\u003eC. indica\u003c/em\u003e was screened for a range of phytochemical compounds such as alkaloids, sterols, triterpenoids, saponins, anthraquinone glycosides, and flavonoids\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e5.5 Measurement of total phenolic content\u003c/h2\u003e\n \u003cp\u003eDetermining total phenolic content involved the utilization of the Folin-Ciocalteu reagent coupled with standard spectrophotometry and calibration curve construction with some modifications. A 0.5 mL sample solution was mixed with 2.5 mL of 0.2N Folin\u0026ndash;Ciocalteu reagent, followed by adding 2 mL of 20% sodium carbonate solution after 5 minutes, and the mixture was vigorously shaken. Subsequently, the absorbance was measured at 760 nm post a 2-hour incubation at room temperature using a double beam Perkin Elmer UV/Visible spectrophotometer. The quantification was based on a calibration curve created using standard gallic acid concentrations, expressed as milligram gallic acid equivalents per gram of dried algae extract\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e5.6 Measurement of total flavonoid content\u003c/h2\u003e\n \u003cp\u003eThe total flavonoid content was determined using the aluminum chloride colorimetric method with minor adjustments. In this method, 0.5 mL of the methanolic sample extract solution was mixed with 1.5 mL of methanol, 0.1 mL of 10% anhydrous aluminum chloride in methanol, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water. Following a 30-minute incubation at room temperature, the absorbance of the samples was measured at 415 nm. A calibration curve was prepared using standard concentrations of a methanolic solution of quercetin. The total flavonoid content was then quantified as milligram equivalents of quercetin per gram of dried algae extract\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e5.7 Determination of antioxidant capacity\u003c/h2\u003e\n \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\n \u003ch2\u003e5.7.1 DPPH assay\u003c/h2\u003e\n \u003cp\u003e2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay is a widely used method for assessing the radical-scavenging capacity of compounds. DPPH, in its stable form, exhibits a dark purple color, which diminishes upon reduction by antioxidants. In this method, 1 mL of 10 \u0026micro;M DPPH in methanol was added to 4 mL of different concentrations of the extract and ascorbic acid as the standard compound. Then the mixtures were incubated at room temperature in the dark for 30 min. Finally, the absorbance was measured at 517 nm. The percentage of inhibition of DPPH radical was calculated according to the following formula:\u003c/p\u003e\n \u003cp\u003eScavenging rate= ((\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{0}\\)\u003c/span\u003e\u003c/span\u003e-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{A}_{S}\\)\u003c/span\u003e\u003c/span\u003e)/\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:{A}_{0}\\)\u003c/span\u003e\u003c/span\u003e) \u0026times;100\u003c/p\u003e\n \u003cp\u003eA\u003csub\u003e0\u003c/sub\u003e and A\u003csub\u003eS\u003c/sub\u003e are the absorbance of the DPPH solution without the sample and the absorbance of the sample with DPPH, respectively. Finally, the half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of DPPH free radical was calculated according to the standard curve\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e5.8 Analysis of Fucoxanthin content by HPLC\u003c/h2\u003e\n \u003cp\u003eThe fucoxanthin concentration in the sample was assessed using a Waters 1525 binary pump, featuring a Waters 2487 dual \u0026lambda; absorbance detector. Reversed-phase high-performance liquid chromatography (RP-HPLC) utilized Isocratic condition (82% methanol: 6.5% CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e: 7.5% acetonitrile: 4% H\u003csub\u003e2\u003c/sub\u003eO) as the mobile phase at a flow rate of 1.0 mL/min. Analyses were conducted at 28\u0026deg;C with a C\u003csub\u003e18\u003c/sub\u003e column (5 \u0026micro;m, 4.6 mm x 150 mm). Initially, a sample aliquot was dissolved in the mobile phase and filtered through a 0.22-\u0026micro;m membrane. Subsequently, a fraction of the filtered sample underwent HPLC analysis with a detection wavelength set at 450 nm. Quantification of fucoxanthin content was achieved through a calibration curve prepared using a standard\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n \u003ch2\u003e5.9 Measurement of Oxidative Stress Parameters\u003c/h2\u003e\n \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n \u003ch2\u003e5.9.1 Measurement of Lipid Peroxidation:\u003c/h2\u003e\n \u003cp\u003eLipid peroxidation was assessed by quantifying the formation of thiobarbituric acid reactive substances (TBARs), with malondialdehyde (MDA) concentration in micromolar (\u0026micro;M) units serving as the marker. Tissue homogenates (0.2 mL) were treated with 0.1 mL of Thiobarbituric acid (TBA) reagent, which consisted of 15% w/v trichloroacetic acid (TCA) and 0.3% w/v TBA in 0.5N HCl, followed by thorough vortexing. Subsequently, the samples underwent incubation in boiling water for 30 minutes. After incubation, the samples were cooled in an ice bath, and 0.2 mL of n-butanol was added. The mixture was then centrifuged at 3500 \u0026times; g for 10 minutes to separate the n-butanol layer. The absorbance of the n-butanol layer was measured at a wavelength of 532 nm, and the amount of TBARs was determined from a standard curve\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e5.9.2 Measurement of Glutathione (GSH) Concentration:\u003c/h2\u003e\n \u003cp\u003eThe reduced level of glutathione (GSH) was evaluated using 5,5ʹ-dithiobis-(2-nitrobenzoic acid) (DTNB) as an indicator. Samples from each group were treated with 20% trichloroacetic acid (TCA) and Ethylenediaminetetraacetic acid (EDTA) to precipitate proteins, followed by centrifugation at 3,500 \u0026times; g for 15 minutes. 1 ml of resulting supernatants was mixed with 2.5 ml Tris buffer (PH\u0026thinsp;=\u0026thinsp;8.9) and 0.5 ml of 40% DTNB. The reaction between DTNB and the sulfhydryl groups within the glutathione molecules produced a yellow thiolate anion, which was detectable. Absorbance at 412nm was then measured using a spectrophotometer for each group, enabling the quantification of GSH concentration in micromolar (\u0026micro;M) units\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e5.9.3 Measurement of intracellular production of ROS:\u003c/h2\u003e\n \u003cp\u003eThe measurement of intracellular reactive oxygen species (ROS) production utilized the oxidation-sensitive fluoroprobe 2ˈ,7ˈ-dichlorofluorescin diacetate (DCFH-DA). Upon cellular uptake, DCFH-DA is hydrolyzed by esterases, forming DCFH, which is then oxidized to fluorescent dichlorofluorescein (DCF) by intracellular oxidants, indicating ROS levels. DCFH-DA was diluted in anhydrous DMSO to 10 mM and stored as a stock solution at 4\u0026ordm;C. A working solution of 100 \u0026micro;M was prepared in HBSS and was added to the cells for 30 min at 37 ◦C after the drug exposure. The DCFH-DA was then removed, and each well was washed with HBSS. Cold lysis buffer (1% Triton X-100, 2.5 M NaCl, 10 mM Tris, 0.1 M EDTA, pH 10) was added to the wells, and after 1 min, the solution was collected and centrifuged at 2800 \u0026times;g, for 5 minutes; 2 \u0026times; 100 \u0026micro;l of the supernatant was added to two wells in a 96-well plate (white, clear bottom) and fluorescence was measured in a computerized microplate fluorometer (Biotek, USA) at 485 nm excitation and 530 nm emission. Background fluorescence was subtracted, and the values were expressed as times increased compared to unexposed cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e5.10 Cell culture\u003c/h2\u003e\n \u003cp\u003eCell lines derived from the A549 cell line were used to study DNA damage and cultured as a monolayer in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin (100 IU/ml) and streptomycin (100\u0026micro;g/ml) mixture. The cells were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% air\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e5.11 Cell viability test by MTT assay\u003c/h2\u003e\n \u003cp\u003eCell viability was assessed using the MTT assay, a widely utilized method for measuring cell viability and proliferation. For each concentration of the extract and Cisplatin, 20 \u0026micro;l of DMEM/F12 containing 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were added to 3 wells. The cells were cultured for 72 hours until they reached the logarithmic growth phase. Throughout the incubation period, cell growth and contamination were monitored every two days. Following incubation, 50\u0026micro;l of each concentration of the fractions of hexane-acetone, ethanol, and algal water extract were added to each well and incubated for an additional 72 hours to ensure adequate exposure to the extracts and Cisplatin. Cells treated with 0.5% DMSO served as the solvent control and were washed with 0.5 ml of sterile normal saline (0.09%). After this, the contents of the wells were discarded, and the cells were incubated with 50 \u0026micro;l of MTT for 4 hours. The MTT solution was then removed, and 50 \u0026micro;l of diluted DMSO was added to each well to dissolve the purple formazan crystals. The plates were shaken for 15 minutes, and the absorbance of the colored colonies was measured using an ELISA reader at wavelengths of 490 nm and 630 nm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\n \u003ch2\u003e5.12 Genotoxicity assessment by comet assay\u003c/h2\u003e\n \u003cp\u003eThe comet assay, also known as single-cell gel electrophoresis (SCGE), is a microelectrophoretic method used to directly observe DNA damage in individual cells. Different concentrations of \u003cem\u003eC.indica\u003c/em\u003e were added to the cell culture medium and incubated at various time points. Degreased slides were first coated with a layer of 1% normal melting agarose. Subsequently, cells were trypsinized, centrifuged, counted, and mixed with 1% (w/v) low-melting-point agarose in PBS, then placed on top of the first layer. After allowing the agarose to solidify at 4\u0026deg;C for 5\u0026ndash;10 minutes, the slide was immersed in an alkaline lysis buffer (pH\u0026thinsp;=\u0026thinsp;10.0) for 40 minutes to remove cellular proteins and membranes. DNA unwinding occurred for 40 minutes in an electrophoretic alkaline buffer (pH\u0026thinsp;\u0026gt;\u0026thinsp;13) before horizontal electrophoresis in the same buffer at 25V and 300 mA. Following electrophoresis, the slides were neutralized with a Tris buffer solution (pH\u0026thinsp;=\u0026thinsp;7.5) for 15 minutes and then stained with ethidium bromide (20\u0026micro;g/ml) before being washed in phosphate-buffered saline. Comet analysis was performed under dark conditions at 400 \u0026times; magnification using a fluorescent microscope. Image analysis software (comet score) was utilized to score 100 randomly selected comets on each slide, with % DNA in tail, tail length, and tail moment typically assessed to determine DNA damage\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec30\" class=\"Section2\"\u003e\n \u003ch2\u003e5.13 Detection of apoptosis\u003c/h2\u003e\n \u003cp\u003eThe annexin V assay relies on annexin V\u0026apos;s ability to bind to phosphatidylserine (PS) exposed on the outer membrane leaflet in apoptotic cells. In viable cells, PS is typically located in the inner membrane leaflet, but during apoptosis, it translocates to the outer membrane leaflet, where it becomes accessible for annexin V binding. The annexin V assay was conducted according to the manufacturer\u0026apos;s instructions using the Annexin V-FITC kit (eBioscience). Initially, 1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were washed with PBS and suspended in a binding buffer. Subsequently, 100 \u0026micro;l of cell suspension was mixed with 5 \u0026micro;l Annexin V-FITC and incubated for 10 minutes in the dark at room temperature. Afterward, the cells were washed with binding buffer, resuspended in 200 \u0026micro;l binding buffer, and stained with 5 \u0026micro;l propidium iodide (PI) solution. Finally, the samples were analyzed using flow cytometry (Partec, Germany) equipped with Flomax software (version 2.4) within a 4-hour timeframe\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\n \u003ch2\u003e5.14 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe findings were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). All statistical analyses were done using the GraphPad Prism software, version 3. All experiments were performed in triplicate, and the mean values were used for statistical evaluation. A one-way ANOVA test was utilized, followed by the post hoc Tukey-Krame multiple comprehension test to assess statistical significance. A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements and Fundings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Mazandaran University of Medical Sciences for their financial support.\u0026nbsp;Lutfun Nahar gratefully acknowledges the financial support of the\u0026nbsp;European Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and the Czech Agency Grants - Project 23-05474S and Project 23-05389S.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data are available in the main text. All the data generated in this study can be obtained from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e8. Author contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEmran Habibi\u003c/strong\u003e design of study, analysis of data and fund acquisition, \u003cstrong\u003eSahar Sheikhzadeh\u003c/strong\u003e Acquisition of data, \u003cstrong\u003eMohammad Shokrzadeh\u003c/strong\u003e compound isolation and structure validation, \u003cstrong\u003eFariborz Sharifianjazi\u003c/strong\u003e concept of study and data analysis, \u003cstrong\u003eSatyajit D. Sarker\u003c/strong\u003e article writing and data analysis, \u003cstrong\u003eHesamoddin Arabnozari\u003c/strong\u003e interpretation of data, Final approval of the version to be published and \u003cstrong\u003eLutfun Nahar\u0026nbsp;\u003c/strong\u003esupervision and fund acquisition. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e9. Compliance with Ethical Standards\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShokrzadeh, M., Rajabali, F., Habibi, E. \u0026amp; Modanloo, M. Survey cytotoxicity and genotoxicity of hydroalcoholic extract of Stevia rebaudiana in breast cancer cell line (MCF7) and human fetal lung fibroblasts (MRC-5). \u003cem\u003eJ. Cancer Res. Metastasis\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 12-17 (2018).\u003c/li\u003e\n\u003cli\u003eSanjeewa, K. A., Lee, J.-S., Kim, W.-S. \u0026amp; Jeon, Y.-J. 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Acrylamide induces immunotoxicity through reactive oxygen species production and caspase-dependent apoptosis in mice splenocytes via the mitochondria-dependent signaling pathways. \u003cem\u003eBiomedicine \u0026amp; Pharmacotherapy\u003c/em\u003e \u003cstrong\u003e94\u003c/strong\u003e, 523-530 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Mazandaran University of Medical Sciences","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":"Algae, Antioxidant, Cystoseira indica, Cytotoxicity, Fucoxanthin, Genotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-4545987/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4545987/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer, particularly lung cancer, remains a leading cause of mortality worldwide, highlighting the need for new remedies. The brown algae species, \u003cem\u003eC. indica\u003c/em\u003e, has gained attention for its rich phytochemical composition and pharmacological potential. This study evaluated the genotoxic and cytotoxic effects of \u003cem\u003eC. indica\u003c/em\u003e extract on human gingival fibroblast (HGF) and lung cancer (A549) cell lines. Algae materials were extracted using sequential maceration, and fucoxanthin content was determined via High-Performance Liquid Chromatography (HPLC). Cytotoxic and genotoxic effects were assessed using MTT and comet assays, with statistical analyses performed using GraphPad Prism software. The algal sample contained 3.077 μg of fucoxanthin per 1g in \u003cem\u003en\u003c/em\u003e-hexane-acetone extract and 4.32 μg of fucoxanthin per 1g in ethanolic extract. \u003cem\u003en\u003c/em\u003e-Hexane-acetone and cold water extracts at 5000 µg/mL concentration exhibited the highest antioxidant activities in the DPPH assay with IC\u003csub\u003e50\u003c/sub\u003e values of 306.15 ± 18.46 μg/mL and 8370 ± 2460 μg/mL, respectively. \u003cem\u003en\u003c/em\u003e-Hexane-acetone extract induced 50.66% apoptosis and hot water extract caused 54.97% apoptosis at 100 µg/mL. \u003cem\u003eC. indica\u003c/em\u003e offers unique metabolites with potential pharmaceutical applications, especially as cytotoxic agents against cancer. The \u003cem\u003en\u003c/em\u003e-hexane-acetone extract, rich in flavonoids and phenolics, showed significant antioxidant and anticancer effects, inducing notable apoptosis in A549 cancer cells, suggesting further investigation for anticancer use.\u003c/p\u003e","manuscriptTitle":"Genotoxicity and cytotoxicity evaluation of brown algae (Cystoseira indica) extract in human gingival fibroblast (HGF) and lung cancer cell lines (A549)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-07 15:03:29","doi":"10.21203/rs.3.rs-4545987/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":"106c2ca8-686a-446b-85d5-4214bf87d012","owner":[],"postedDate":"August 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-07T15:03:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-07 15:03:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4545987","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4545987","identity":"rs-4545987","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}