## Biomedical Applications
### Cytotoxic and Anticancer Activity  
New biomaterials with economic and environmental benefits are required. In this context, toxicity must be determined and considered when determining stability and effectiveness. Given that ulvan may be used as a supplement, nutraceutical, and therapeutic agent, it must be tested to determine potential cytotoxicity and therapeutic dose range that can be used. Cytotoxicity testing is carried out in vitro with cells or in vivo using experimental animals [89; 103]. Recently, the anticancer effect of ulvan has been developed from _U. lactuca, U. australis, U. rigida_, and _U. ohnoi_[60; 107; 120]. The National Institutes of Health (USA) has developed standard methods for evaluating potential cytotoxic effects of compounds or extracts using human cancer cell lines [87]. These cell lines include HepG2 (hepatocellular carcinoma), MCF7 (human breast cancer), and HeLa (cervical cancer) [87]. Other toxicity tests use rat lung cells (L929) [89], mammalian L6 cells [65; 90; 121]. HaCaT keratinocytes, and 3T3 fibroblasts [80]. The most widely used method is the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-((4-sulfophenyl)-2H-tetrazololium (MTT) assay [80; 122; 80]. The results obtained from HepG2, MCF7, and HeLa cells have shown decreased cell viability directly proportional to the increased ulvan concentration [87]. Human L92 cells were still metabolically active and did not show a decrease in viability after 72 h exposure to ulvan [104]. Studies usually examine cellular metabolic activity using the MTT assay to determine cell number and assess proliferation by quantifying double-stranded DNA (dsDNA) and total protein [105]. Ulvan extracts have been found to be safe for mammalian L6 cells as a control because they do not promote cytotoxicity (half maximal inhibitory concentration [IC\({}_{50}\)] less than 90 mg/mL) even at the highest tested concentration. Ulvan did not show toxicity at 10,000 mg/mL in 3T3 cells [80]. Based on the description of the cytotoxic test that has been described, ulvan from _U. lactuca_ decreases viability in cancer cells and does not show a decrease in viability in healthy cells. Cancer occurs due to the abnormal growth of cells and body tissues. Cancer is initiated by various endogenous and exogenous factors that often cause oxidative damage to DNA, a process that produces mutations that disrupt the normal regulation of cell proliferation, differentiation, and apoptosis pathways [124]. Several studies have tested the anticancer activity [68; 76], specifically for anti-breast cancer [13; 60; 87; 88], anti-colon [59; 88], and anti-cervical cancer activity [87], by investigating ulvan in toxicity and cell viability. In vitro, ulvan has been tested on several cancer cell lines: HepG2 [31; 60; 87; 88] Caco-2 (human colon cancer) [88], LS174 (human colon cancer), A549 (human lung carcinoma), Fem-x (malignant melanoma) K562 (chronic myelogenous leukemia) [76], HEp-2 (laryngeal epidermoid carcinoma), NCI-H292 (human lung mucoepidermoid carcinoma) [68], and HeLa (cervical cancer) [87]. It has also been tested in rats injected with carcinogens such as diethylnitrosamine (DENA, 200 mg/kg, intraperitoneally) [83] and 7,12-dimethylbenz2[a]anthracene (DMBA) [13]. Testing the anticancer activity of ulvan has had different outcomes. Ulvan may be a chemopre agent for liver cancer. Sulfated polysaccharides in ulvan can inhibit hepatocellular carcinoma proliferation and induce apoptosis. Sulfated polysaccharides protect against DNEA-induced liver damage by reducing oxidative stress [83]. They also promote improvement in mice subjected to DMBA treatment by augmenting apoptosis, suppressing oxidative stress and inflammation, and enhancing the antioxidant defense system [13]. Ulvan showed weaker cytotoxic activity against cells A459 and LS174 (IC\({}_{50}\) > 200 mg/mL), while it was more effective in preventing moderate cytotoxic effects in Fem-x and K562 cells (IC\({}_{50}\) 74.73 and 82.24 mg/mL, respectively) [76].