Journal of Multidisciplinary Applied Natural Science ACCEPTED MANUSCRIPT • OPEN ACCESS Cytotoxic Activity of Green Seaweed Halimeda tuna Methanolic Extract Against Lung Cancer Cells To cite this article before publication: A. Husni, M. Gazali, N. Nurjanah, R. Syafitri, A. Matin, and Z. Zuriat. (2023). J. Multidiscip. Appl. Nat. Sci. in press. https://doi.org/10.47352/jmans.2774-3047.172. Manuscript version: Accepted Manuscript Accepted Manuscript is “the version of the article accepted for publication including all changes made as a result of the peer review process, and which may also include the addition to the article by Pandawa Institute of a header, an article ID, a cover sheet and/or an ‘Accepted Manuscript’ watermark, but excluding any other editing, typesetting or other changes made by Pandawa Institute and/or its licensors” This Accepted Manuscript is © 2023 The Author(s). 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View the article online for updates and enhancements. https://doi.org/10.47352/jmans.2774-3047.172 https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.47352/jmans.2774-3047.172 Cytotoxic Activity of Green Seaweed Halimeda tuna 1 Methanolic Extract Against Lung Cancer Cells 2 3 Amir Husni1,a); Mohamad Gazali2,6b); Nurjanah Nurjanah3,c); Rina Syafitri4,d), 4 Abdul Matin5,6e); Zuriat Zuriat7,f) 5 6 1Department of Fisheries, Universitas Gadjah Mada, Yogyakarta-55281 (Indonesia) 7 2Department of Marine Science, Teuku Umar University, West Aceh-23681 (Indonesia) 8 3Department of Aquatic Product Technology, IPB University, Bogor-16680 (Indonesia) 9 4Department of Agribusiness, Teuku Umar University, West Aceh-23681 (Indonesia) 10 5Department of Food Processing and Engineering, Chattogram Veterinary and Animal Sciences University, 11 Chattogram-4225 (Bangladesh) 12 6Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Kuala Terengganu-21030 (Malaysia) 13 7Department of Fisheries, Teuku Umar University, West Aceh-23681 (Indonesia) 14 15 a)a-husni@ugm.ac.id 16 b)Correspondence: mohamadgazali@utu.ac.id 17 c)nurjanah@apps.ipb.ac.id 18 d)rinasyafitri@utu.ac.id 19 e)abmatin@cvasu.ac.bd 20 f)zuriat@utu.ac.id 21 22 ORCIDs: 23 First AUTHOR : http://orcid.org/0000-0003-3327-9848 24 Second AUTHOR : https://orcid.org/0000-0001-7575-3582 25 Third AUTHOR : https://orcid.org/0000-0001-7925-1782 26 Fourth AUTHOR : https://orcid.org/0000-0002-2334-4397 27 Fifth AUTHOR : https://orcid.org/0000-0003-3029-4989 28 Sixth AUTHOR : https://orcid.org/0009-0000-6296-1788 29 30 ACKNOWLEDGEMENT 31 32 This research supported by the Ministry of Education and Culture Republic Indonesia 33 through DRTPM Dikti Grant for the Inter-University Cooperation Research Scheme (PKPT) 34 with Contract Number 037/UN59.7/PG.02.00.PT/2022. 35 36 AUTHOR CONTIBUTIONS 37 38 Conceptualization, A.M. and M.G.; Methodology, A.M and M.G; Software, A.M.; 39 Validation, A.H., M.G. and A.M.; Formal Analysis, A.M. and M.G.; Investigation, A.H.; 40 Resources, M.G.; Data Curation, M.G.; Writing–Original Draft Preparation, M.G.; Writing – 41 Review & Editing, A.M. M.G. A.M. and N.N., R.S., and Z.Z.; Visualization, .X.; Supervision, 42 A.M. and N.; Project Administration, M.G.; Funding Acquisition, M.G. 43 A CC EP TE D M A N U SC RI PT mailto:a-husni@ugm.ac.id mailto:mohamadgazali@utu.ac.id mailto:nurjanah@apps.ipb.ac.id mailto:rinasyafitri@utu.ac.id/ mailto:abmatin@cvasu.ac.bd mailto:zuriat@utu.ac.id http://orcid.org/0000-0003-3327-9848 https://orcid.org/0000-0001-7575-3582 https://orcid.org/0000-0001-7925-1782 https://orcid.org/0000-0002-2334-4397 https://orcid.org/0000-0003-3029-4989 https://orcid.org/0009-0000-6296-1788 1 CONFLICT OF INTEREST 2 3 The authors declare no conflict of interest. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 A CC EP TE D M A N U SC RI PT Cytotoxic Activity of Green Seaweed Halimeda tuna 1 Methanolic Extract Against Lung Cancer Cells 2 3 Abstract. Lung cancer is a malignant tumor that attacks the lungs generated by carcinogenic 4 free radicals such as cigarette smoke. Seaweed contains bioactive compounds that have the 5 potential to reduce cancer-causing free radicals. This study aimed to determine the 6 phytochemical content and cytotoxic activity of Halimeda tuna seaweed extract against lung 7 cancer cells (A549). The H. tuna sample was macerated using methanol for 24 h. Cytotoxic 8 test of H. tuna crude extract used the MTT test against A549. The crude extract was 9 phytochemically tested and analyzed using gas chromatography–mass spectrometry (GC-MS). 10 The results showed that the H. tuna crude extract had cytotoxic activity against A549 with an 11 IC50 value of 2771 µg/mL. The phytochemical test showed that H. tuna crude extract contained 12 flavonoids and steroids. GC-MS spectra showed the presence of fatty acid compounds 13 including palmitic acid, oleic acid, myristic acid, palmitoleic acid and stearic acid. Based on 14 the results can be concluded that H. tuna extract had cytotoxic activity against A549 with low 15 cytotoxicity to be used as a chemo-preventive agent. 16 17 Keywords: anticancer; cytomorphology; flavonoid; green seaweed; steroid 18 19 1. INTRODUCTION 20 21 Lung cancer is one of the most dangerous deadly diseases. The death rate from lung 22 cancer worldwide can reach one million people annually; even in Indonesia, this disease is 23 ranked 4th in the world [1]. Cancer is a metabolic syndrome that is one of the leading causes 24 of death and morbidity worldwide. Primary cancer-triggering factors include genetic, 25 epigenetic, environmental, and hormonal that cause mutations [2]. The leading cause of lung 26 cancer is caused by long-term exposure to carcinogenic substances, especially substances that 27 enter through the respiratory process, such as air pollution and cigarette smoke. Many have 28 reported that lung cancer is associated with smoking habits. As many as 65% of the risk of lung 29 cancer is suffered by males, especially those aged over 40 years [1]. The most effective cancer 30 treatment, namely chemotherapy, still has various side effects, such as nausea, hair loss, pain, 31 fatigue, and diarrhea. In the long term, these symptoms can harm the patient's quality of life 32 and are at risk of death [3]. 33 A CC EP TE D M A N U SC RI PT Most Asian people use complementary medicine such as dietary supplements, herbal 1 products, and other traditional treatments [4]. One of the herbal medicines or natural 2 ingredients from the fisheries sector is seaweed. Seaweed contains various secondary 3 metabolites, such as flavonoids, phenolics, and tannins [5]. Seaweed also contains phenolic 4 compounds, polysaccharides, polyunsaturated fatty acids (PUFAs), proteins, vitamins, and 5 minerals. These compounds show biological activity and have the potential to be used as drugs 6 to ward off cancer, tumors, thrombosis, diabetes, inflammation, and other degenerative 7 diseases [5-8]. These bioactive compounds can be used as antioxidant, anticancer, antibacterial, 8 anti-inflammatory, and antiviral agents [9]. 9 Several studies have shown that seaweed from the Halimeda genus consists of bioactive 10 compounds, including polyphenols, diterpenes, fatty acids, and sterols, that show anticancer 11 activities [10,11]. One potential seaweed species as an anticancer is the green seaweed 12 Halimeda tuna from Aceh waters. Previous research has been carried out related to the 13 bioactivity of seaweed originating from Aceh waters, such as H. macroloba [12], H. opuntia 14 [13], and H. tuna [14]. Green seaweed is abundant in Indonesia and mainly used in the food 15 sector, however, green seaweed is rarely used in the pharmaceutical and health fields. Research 16 shows that green seaweed contains bioactive compounds such as alkaloids, flavonoids, tannins, 17 saponins, and steroids [15]. Some of these bioactive compounds can potentially reduce free 18 radicals that cause cancer. Several studies have been conducted on the cytotoxic activity of 19 green seaweed, namely Boergesenia forbesii, which has high cytotoxic activity so it has the 20 potential to become an anticancer [16]. Puc et al. [17] reported that H. tuna has cytotoxic 21 activity against cervical cancer cells (HeLa), laryngeal cancer cells (Hep-2), and 22 nasopharyngeal cancer cells (KB). Several species of Halimeda sp. contain halimedatrial 23 compounds (diterpenetrialdehyde), which have cytotoxic activity [18], so they have the 24 potential as anticancer. However, the content of seaweed bioactive compounds can vary 25 depending on the type of species, age of harvest, and environmental conditions of the habitat 26 or place of growth [19]. Therefore, this study aimed to determine the anticancer activity of 27 green seaweed H. tuna methanol extract against lung cancer cells (A549). 28 29 2. MATERIALS AND METHODS 30 31 2.1. Materials. The materials used in this study were green seaweed H. tuna, methanol 32 (Sigma Aldrich,), ethanol, NaOH, chloroform, anhydrous acetic acid, HCl, FeCl3, NH3, CHCl3, 33 H2SO4, Dragendorfs reagent, Meyer's reagent, Wagner's reagent, lung cancer cells (A549) 34 A CC EP TE D M A N U SC RI PT (BPPT, Tangerang), RPMI medium, Fetal Bovine Serum (FBS), streptomycin penicillin, 1 doxorubicin, fugizone, formazan, MTT, SDS. The tools used in this study included laboratory 2 glasswares, Whatman filter paper no.42, rotary evaporator (DLab RE100-Pro, Germany), 3 nitrogen gas evaporator, hot plate stirrer (F20500011 Velp AREC Heating stirrer, Italy), ELISA 4 microplate reader (Heales MB-580), 96-well microplate, and CO2 incubator (Memmert 5 ICO150Med, Germany). 6 7 2.2. Methods 8 9 2.2.1. Preparation and Identification of Samples. Samples of green seaweed H. tuna were 10 collected from the coast of Lhok Bubon, Samatiga Subdistrict, West Aceh District, Aceh 11 Province. The samples were washed with fresh water to remove the adhering sand and dirt. The 12 wet samples were then dried at room temperature. The wet and dry samples were sent to 13 Universitas Gadjah Mada, Yogyakarta. Fresh seaweed samples were identified at the Plant 14 Systematics Laboratory, Faculty of Biology, Universitas Gadjah Mada, to determine the 15 specific type, whereas, the dry samples were cut into 1 cm pieces using scissors. The seaweed 16 was weighed and stored at -20 °C. 17 18 2.2.2. Extraction of Seaweed. The extraction of H. tuna was carried out according to Yang 19 et al. [24] with modifications. Samples of dried H. tuna were weighed as much as 250 g. The 20 sample was macerated with 2 L of methanol for 24 h at room temperature and then the filtrate 21 was filtered to remove the remaining residue carried. The filtrate was evaporated using a rotary 22 evaporator at a temperature of 40 °C at 60 rpm. The sample was further treated using nitrogen 23 gas to produce an extract in the form of a more concentrated paste and then extracted in the 24 freeze dryer. 25 26 2.2.3. Anticancer Activity Test. An anticancer activity test was conducted to determine 27 whether the extracted sample had the potential as an anticancer of the lungs. The anticancer 28 activity test was carried out based on the method according to Husni et al. [20]. Anticancer 29 activity tests included an A549 culture, cytotoxicity, and cytomorphological testing. A549 30 cancer cells were cultured in RPMI medium, then added 10% FBS, streptomycin, penicillin, 31 and fungizone. Then the mixture was incubated with 5% CO2 at 37°C to obtain an A549 cell 32 culture. Furthermore, the cytotoxicity test was carried out using the MTT method. A549 cells 33 were placed on a 96-well culture microplate that included cancer cell treatment with samples, 34 A CC EP TE D M A N U SC RI PT positive controls with doxorubicin, and negative controls without sample treatment. Then the 1 mixture was incubated with 5% CO2 at 37 °C for 24 h. After that, the media was discarded and 2 then it was mixed with 100 µL MTT and incubated again for 4 h. After that, the purple format 3 was dissolved in 100 µL 10% SDS and allowed to stand for 12 h at room temperature. Cell 4 growth was read using an ELISA microplate reader at a wavelength of 570 nm. The percentage 5 of live cells after exposure to fucoidan was calculated using the following equation 1. 6 7 % 𝐿𝑖𝑓𝑒 𝑐𝑒𝑙𝑙 = 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑡𝑟𝑒𝑎𝑡𝑚𝑒𝑛𝑡−𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑚𝑒𝑑𝑖𝑢𝑚 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑚𝑒𝑑𝑖𝑢𝑚 × 100% (1) 8 9 2.2.4. Phytochemical assay 10 11 2.2.4.1. Flavonoid. The flavonoid test was carried out to determine the content of flavonoid 12 compounds in the sample. Five mL of 70% ethanol was added to 0.05 g of the extracted sample, 13 then heated and filtered. Then the filtrate was taken, and two drops of 10% NaOH were added. 14 If the color changes to yellow or orange, the sample contains flavonoids. 15 16 2.2.4.2 Saponin. The saponin test was carried out based on the method as described by Lubis 17 et al. [21]. The saponin test was carried out to determine the content of saponin compounds in 18 the sample. A total of 0.05 g of the extracted sample was dissolved into 10 mL of hot distilled 19 water and then shaken vigorously until foamy and cooled. Then 1 drop of 2 M HCl was added. 20 If the foam does not disappear, then the sample contains saponins. 21 22 2.2.4.3 Steroid and Triterpenoid. Steroid and triterpenoid tests were carried out as follows: 23 chloroform was added to 0.05 g of the extracted sample to the drip plate and then allowed to 24 dry. Then ten drops of anhydrous acetic acid were added and stirred until homogeneous. Then 25 three drops of 96% sulfuric acid were added. If it is blue or green, then the sample contains 26 steroids. If it is red or purple, the sample contains triterpenoids [21]. 27 28 2.2.4.4 Tannin. The tannin test was carried out based on the method as described by 29 Widowati et al. [22]. A total of 0.1 g of sample was dissolved in 10 mL of hot distilled water 30 and filtered. Then 5 mL of the sample filtrate was added with 3 drops of 1% FeCl3. If the results 31 show a blue-black color, the sample contains tannins. 32 33 A CC EP TE D M A N U SC RI PT 2.2.5 Gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS analysis was 1 performed to identify the profile of bioactive compounds in H. tuna methanolic extract. The 2 GC-MS analysis was carried out based on the method as described by Hidayah [23]. The 3 sample to be analyzed by GC-MS was first dissolved in 5 mL methanol. Then the GC-MS 4 analysis was carried out by injecting the sample into the injection port at a temperature of 290 5 °C. The volatilized sample was carried by Helium gas at a flow rate of 1 mL/min through the 6 GC column. The initial injection temperature was 80 °C and increased by 10 °C/min with a 7 final temperature of 300 °C. Compounds are detected in the MS system by colliding 8 compounds with electrons to form ionized molecules and record fragmentation patterns [24]. 9 10 2.3 Statistical Analysis. The percentage data of inhibition was then converted to a linear 11 regression equation calculating the IC50 value. The IC50 values of the linear regression results 12 of each sample were statistically tested using SOVS (one-way ANOVA) and Tukey HSD test 13 with a 95% confidence level. 14 15 3. RESULTS AND DISCUSSIONS 16 17 3.1. Yield of Extract. The yield is the result of a comparison between the total mass of H. 18 tuna extract in the form of paste with the initial mass of H. tuna in the form of dried seaweed 19 [30]. The yield of H. tuna methanolic extract obtained was 0.17±0.04%. The methanol extract 20 of H. tuna had a lower yield when compared to the methanol extract of H. macroloba (0.34%) 21 and the ethyl acetate extract of H. macroloba (0.28%), but higher than the n-hexane extract of 22 H. macroloba (0.04%) [25]. Gazali et al. [12] also reported that the yield of ethanol extract of 23 H. macroloba (2.32%) was higher than the yield of ethyl acetate extract (1.26%), and n-hexane 24 extract (1.03%). The difference in yield can be caused by the type of solvent and different 25 species. Different solvents can affect the yield due to the level of polarity. According to Muzaki 26 et al. [30], the yield value decreases along with the decrease in the polarity of the solvent. In 27 addition, the solvent will attract bioactive compounds that have the same polarity. The type of 28 seaweed species also affects the yield because it depends on its compounds. According to 29 Purwaningsih and Deskawati [26], the content of bioactive compounds in seaweed is 30 influenced by the type of species, harvest season, harvest age, and geographical location. 31 32 A CC EP TE D M A N U SC RI PT 3.2 Anticancer Activity. H. tuna methanolic extract was assayed for its cytotoxic activity 1 against A549. The inhibition of the growth of A549 by H. tuna methanolic extract and 2 doxorubicin is presented in Figure 1 while their IC50 is shown in Table 1. The morphological 3 attributes of the cells were monitored under an inverted microscope after the cells were 4 incubated. The morphological attributes of A549 that were exposed and not exposed to H. tuna 5 extract are illustrated in Figure 2. A cytotoxicity test was carried out on H. tuna methanolic 6 extract against A549 to determine whether the sample had potential as an anticancer and 7 directly affected cell death [17]. The MTT test is a method that can be used to determine the 8 toxic properties of a compound. The MTT test results of H. tuna extract, and doxorubicin on 9 A549 (Figure 1) showed that the dose given to cancer cells was directly proportional to the 10 inhibition of cancer cell growth. H. tuna extract with a dose of 500 μg/mL could hinder the 11 growth of cancer cells by 28.72% while doxorubicin (as a standard drug) at a dose of 14 μg/mL 12 could hinder the growth of cancer cells by 39.68% (Figure 1). This is because doxorubicin is a 13 widely used drug for anticancer chemotherapy. However, doxorubicin works non-selectively 14 and is toxic to cancer cells and normal cells [27]. 15 16 17 Figure 1. Effect of concentration of H. tuna and doxorubicin on inhibition of proliferation of 18 lung cancer cell A549. 19 20 Prasetyaningrum et al. [28] indicated that the cytotoxicity of a substance based on its IC50 21 is divided into three levels: potential cytotoxic (IC50 <100 μg/mL), moderate cytotoxic (100 22 μg/mL < IC50 <1000 μg/mL), and low cytotoxic (IC50 >1000 μg/mL). Furthermore, according 23 to the National Cancer Institute [29], a compound can be classified as a strong anticancer agent 24 if its IC50 is <20 μg/mL. The cytotoxicity test on the crude extract of H. tuna showed low 25 cytotoxic values (IC50 value of 2771 µg/mL). A substance with low cytotoxicity can be used 26 0 10 20 30 40 50 0 100 200 300 400 500 In h ib it io n a c ti v it y ( % ) Concentration (μg/mL) H. tuna Doxorubicin A CC EP TE D M A N U SC RI PT as a chemo-preventive agent. The chemo-preventive ability indicates that the crude extracts of 1 H. tuna can be used to prevent and hinder the growth of cancer cells and also trigger apoptosis. 2 Previous research reported the cytotoxicity of brown seaweed fucoidan extracted from 3 Turbinaria conoides species against A549 with IC50 of 396.46 μg/mL [30]. Polysaccharide 4 from Caulerpa taxifolia showed anticancer acitivity against A549 with a relative IC50 of 45.44 5 μg/mL [31]. Methanol extract of brown algae Hormophysa cuneiformis has anticancer activity 6 against A549 with IC50 of 40.97 μg/mL [32]. Factors that can affect the content and activity of 7 bioactive metabolites include sampling location or habitat, genetic variation, sampling time, 8 evolution, and environmental conditions [33]. 9 Doxorubicin is an anticancer medicine and an important agent for the therapy of malignant 10 breast cancer [34]. The anticancer action of doxorubicin has been described with various 11 molecular pathways, covering the interaction mechanism of doxorubicin with DNA, DNA-12 related enzymes, and cell membranes [35]. Another study has shown that Cladosiphon 13 okamuranus fucoidan has strong antiproliferative and apoptotic reactions on MCF-7 cells in 14 certain doses and does not affect normal cell proliferation in human mammalian epithelial cells 15 [36]. The cell pattern is a process that requires high energy and involves four sequential stages 16 that change from the stationary stage (G0 stage) to the proliferation stage (G1, S, G2, and M 17 stage) and return to rest [37]. Fucoidan increases the population of hepatocarcinoma (Huh7) 18 cells at the G0/G1 stage and decreases their population at the S stage; this result indicates that 19 fucoidan can induce the cell pattern to persist at the G0/G1 stage [38]. 20 21 Table 1. IC50 values of H. tuna extract and doxorubicin against cancer cells A549 22 Sample IC50 (µg/mL) H. tuna extract 2771a Doxorubicin 24.13b a/bDifferent letters show a significant difference (p<0.05) 23 24 The differences in the morphological attributes of A549 to H. tuna extract and not exposed 25 to H. tuna extract are illustrated in Figure 2. The morphological characteristics of A549 26 exposed to H. tuna extract and the control cells not exposed to H. tuna extract differed. The 27 morphological attributes of MCF-7 cells in the control cells not exposed to H. tuna extract were 28 observed as an irregular polygonal and attached to the substrate. The morphological 29 characteristics of the cells that were exposed to H. tuna extract varied, that is, the cells shrank, 30 were round, and had limited distribution patterns compared with those of the control cells. This 31 A CC EP TE D M A N U SC RI PT change in shape was consistent with that observed by Kim et al. [39] who stated that MC3T3 1 osteoblast cells exposed to fucoidan for 4 h have altered morphological characteristics, i.e., 2 from an irregular shape to a round form with smaller sizes. 3 4 A B C Figure 2. Morphology of A549 lung cancer cells without sample treatment (A), given a 5 sample of H. tuna extract 250 µg/mL (B), and given a standard doxorubicin 14 µg/mL (C). 6 7 3.3 Phytochemical Content. The phytochemical test aims to identify chemical compounds 8 in samples such as flavonoids, steroids, saponins, tannins, and alkaloids. Many of these 9 chemical compounds are found in seaweed. The results of the phytochemical test were shown 10 in Table 2. 11 12 Table 2. Phytochemical analysis of H. tuna crude extract 13 Phytochemicals Result Indicator Flavonoid ++ Yellow/orange color Steroid +++ Blue-green color Triterpenoid - Red – purple color Saponin - Foam Alkaloid + Orange precipitate Tannin - Blue-black color + : low, ++ : moderate, +++ : high 14 15 According to Nome et al. [15], flavonoids were found in almost all types of green 16 macroalgae but with different levels, such as Codium sp., Caulerpa sp., and Ulva sp. Similarly, 17 the steroids found in the green macroalgae Caulerpa sp., Halimeda sp., Enteromorpha sp., and 18 Codium sp. Alkaloids are also found in green macroalgae such as Ulva sp. and Caulerpa sp., 19 but little was found in Halimeda sp., Enteromorpha sp., and Codium sp. Gazali et al. [40] 20 reported that alkaloids, flavonoids, saponins, and tannins were found in the macroalga 21 Chaetomorpha crassa. Based on the research of Widowati et al. [22], Gracilaria salicornia 22 contains flavonoids, saponins, and steroids, Halimeda gracilis contains steroids and saponins, 23 A CC EP TE D M A N U SC RI PT and H. macroloba contains flavonoids and steroids. Gazali et al. [12] reported that H. opuntia 1 seaweed contains alkaloids, steroids, saponins, flavonoids, phenols, and tannins. Gazali et al. 2 [13] reported that the phytochemical test results showed that the H. tuna fractions were positive 3 for alkaloids, flavonoids, steroids, and phenol hydroquinone compounds. Flavonoids are 4 secondary metabolites with anticancer activity [41] because these compounds contain 5 quercetin, genistein, or flavopiridol which can be used as cancer drugs [42]. Flavonoids as 6 anticancer have a mechanism of inhibition of DNA topoisomerase I/II activity, decreased 7 expression of Bcl-2 and Bcl-xl genes, and activation of endonucleases [43]. Flavonoids also 8 have the biological ability to chelate metals, inhibiting cancer cell growth [44]. Flavonoids are 9 polar and are mostly produced from green seaweed, so these compounds are generally easily 10 soluble in polar solvents such as methanol [45]. 11 Steroids are non-polar secondary metabolites, so they are easily extracted by polar solvents 12 such as methanol [15]. Steroids have anticancer activity as these compounds have aromatase 13 enzymes and sulfatase inhibitors that can inhibit the growth of cancer cells [46]. Steroids, as 14 anticancer agents, damage mitochondrial membrane permeability in cancer cells and cause cell 15 death or necrosis [47]. In addition, steroids can also capture reactive species such as superoxide 16 and chelate metals [48]. The content of chemical compounds in seaweed can be influenced by 17 environmental factors where it grows because the bioactive compounds formed are a natural 18 response to environmental conditions where they grow, resulting in various types of chemical 19 compounds. The ability of seaweed to produce secondary metabolites that are bioactive 20 compounds can occur due to extreme environmental conditions [15]. 21 22 3.4 GC-MS Analysis. GC-MS analysis showed a GC spectra chromatogram with seven 23 peaks (Figure 3) representing the bioactive compounds interacting with the GC column. The 24 peak obtained was only a little and not too high, with the results of comparison with the 25 database having a slight similarity. The bioactive activity and utilization of compounds were 26 obtained from the NCBI web and previous studies. Compounds belonging to the flavonoid 27 group were flemichapparin A [49]. The steroids identified in the extract consisted of stigmasta, 28 androst-4-ene-3,17-dione, estra-1,3,5(10)-trien-17-one, 5-alpha-androstan-17-one, and 1-29 docosanol [50]. Some compounds that include fatty acids include palmitic acid, hexadecanoic 30 acid, octadecanoic acid, lauric acid, 4-hexenoic acid, and dodecanoic acid [46]. The list of 31 information on the identified compounds and the activity of the metabolite compounds from 32 the H. tuna extract is explained further in Table 3. 33 34 A CC EP TE D M A N U SC RI PT Table 3. Results of identification of compound components of H. tuna methanol extract 1 Peak RT Area (%) Component Group Activity SI 1 12.308 32.34 stigmasta- 5,22-dien-3- ol Steroid Antioxidant, antimycobacterial (tuberculosis), Anticancer, inhibition of chemocarcinogen [51] 19 2 13.572 7.73 androst-4- ene-3,17- dione Steroid Osteoporosis, antiinfectives, hyperglycemia (antidiabetic) [52] 21 3 18.010 5.03 1-docosanol Steroid Antiviral [53] 57 4 19.683 0.01 1- hexadecanol Fatty Alcohol Antioxidant, antimicrobial [54] 61 5 20.441 27.41 14-beta-h- pregna Steroid Cancer Prevention [55] 63 6 20.883 6.78 dodecanoic acid Fatty Acid Antimicrobial, relieve neuro-inflammatory [56] 34 7 21.065 20.73 hexadecanoic acid Fatty Acid Anti-inflammatory, antiviral, antioxidant [57] 70 RT: Retention Time, SI: Similarity Index 2 3 The activity of volatile compounds, as listed in Table 2, many compounds have anticancer-4 related bioactivity. The main compound with a large percentage of area is found at peaks 1, 5, 5 and 7, with an area of 32.34%, 27.41%, and 20.73%, respectively. According to Singla and 6 Dubey [58], in predicting compounds using GC-MS, if the similarity value is low (SI<90%) 7 then the component should not be considered because it is less accurate. In this study, the 8 compound with the greatest similarity index was 70, so the compound with the largest 9 percentage area and the greatest similarity was used. 10 A CC EP TE D M A N U SC RI PT The active compound in peak 1 is stigmasta-5,22-dien-3-ol, with activities including 1 antioxidant, antimycobacterial (anti-tuberculosis bacteria), anti-inflammatory, anticancer, and 2 inhibition of chemocarcinogens. The compound stigmasta-5,22-dien-3-ol belongs to the 3 stigmasteroid group [59]. Stigmasta-5,22-dien-3-ol has been found in the genus Halimeda 4 seaweed, precisely in H. opuntia, with a percentage of 54.74% as the most dominant compound 5 [60]. In this study, the stigmasta compound only had an SI of 19 so it might not be accurate 6 and have little effect on anticancer activity. 7 The active compounds in peak 5 include 14-beta-H-pregna with a similarity of 63, which 8 has antidiabetic and cancer-preventive properties. Compound 14-beta-H-pregna belongs to 9 steroids [61]. Compound 14-beta-H-pregna was found with an area of 55% in green seaweed 10 extract Chlorella vulgaris [62]. Compound 14-beta-H-pregna is a component of the medicinal 11 plant Verbascum pseudoholotricum or mullein with a similarity of 98. Mullein has antioxidant, 12 anti-inflammatory and anti-bacterial activity [63]. 13 Compounds in peak 7 include hexadecanoic acid, octadecanoic acid, dodecanoic acid, and 14 octadecane, a group of fatty acids. Fatty acid bioactivity includes anti-inflammatory, antiviral, 15 antioxidant, antimicrobial, and antibiotic [64]. Fatty acids function as antioxidants so that they 16 can reduce reactive oxygen species and act as preventive agents for diseases caused by reactive 17 oxygen species, such as cancer [37]. 18 Research by Nazaruddin et al. [60] on the GC-MS test proved the presence of Hexadecanoic 19 acid in H. opuntia. The hexadecanoic acid in Halimeda has antioxidant effects and is cytotoxic 20 against the colorectal cancer cell line HCT-116. The retention time of Hexadecanoic acid in 21 this study was 50.91 min. Nazaruddin et al. [11] researched H. macroloba using the GC-MS 22 test with the Shimadzu QP2010 Plus GC-MS system. One of the compounds found is 23 hexadecanoic acid. Hexadecanoic acid retention time at two different peaks had values of 24 21.039 and 20.548 min, respectively. The RT value of the GC-MS test on H. tuna in this study 25 for hexadecanoic acid had a retention time of 21.065 min with a similarity index of 70 so it 26 was more similar to the results of the GC-MS test on H. macroloba. 27 28 A CC EP TE D M A N U SC RI PT 1 Figure 3. Chromatogram of H. tuna methanolic extract 2 3 The H. tuna extract contains several fatty acid compounds. This is because, in addition to 4 secondary metabolites, seaweed also contains primary metabolites such as protein, 5 carbohydrates, fat, crude fiber, macro minerals, and several vitamins. Differences in the content 6 of chemical compounds in seaweed can be influenced by the type of species and their habitat 7 [15]. Secondary metabolites have been shown to have high bioactivity. However, fatty acids 8 are also known to have antioxidant activity [65], so they are thought to have the potential to 9 have cytotoxic activity. According to Asbanu et al. [66], several fatty acids have antioxidant 10 bioactivity such as octadecanoic acid (stearic acid), hexadecanoic acid (palmitic acid), 11 tetradecanoic acid (myristic acid), and 9-octadecenoic acid (oleic acid). In general, the 12 Halimeda genus shifts the production of protein and fat primary metabolites to increase the 13 production of halimedatrial and halimedatetraacetate secondary metabolites, so that the 14 bioactive compounds of these secondary metabolites are higher than their primary metabolites 15 [67]. However, this is also influenced by environmental conditions where it grows, resulting in 16 a variety of compound content [68]. In addition, the solvent used is methanol, which is a 17 universal polar solvent so that it can attract all compounds, both polar and non-polar 18 compounds, such as fats [69]. Methanol is also one of the most widely used solvents in the 19 extraction process of organic compounds such as oils or fats [70], so fatty acids can be carried 20 away in the extraction process. 21 22 23 24 25 A CC EP TE D M A N U SC RI PT 4. CONCLUSIONS 1 2 H. tuna methanolic extract was obtained in 0.17±0.04% yield. H. tuna extract had cytotoxic 3 activity against lung cancer cells (A549) with IC50 2771 µg/mL and potentially can be used as 4 a chemo-preventive agent. Based on cytomorphological observations, changes in the 5 morphology of cancer cells were seen before and after being treated with H. tuna extract 6 samples. Metanolic extract of H. tuna have contents palmitic acid, oleic acid, palmitoleic acid, 7 myristic acid, and stearic acid. 8 9 REFERENCES 10 11 [1] J. Joseph and L. W. A. Rotty. (2020). "Kanker paru: laporan kasus". Medical Scope 12 Journal. 2 (1): 17–25. 10.35790/msj.v2i1.31108. 13 [2] R. L. Siegel, K. D. Miller, and A. Jemal. (2016). "Cancer statistics, 2016". 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