**1. Introduction**

Lung cancer is one of the most commonly diagnosed cancers and is also one of the most deadly forms of cancer worldwide [1]. It is reported that 80% of lung cancers belong to the non-small-cell lung

cancer (NSCLC) subtype that can be further divided into two classes: (1) lung adenocarcinoma (LUAD; 50%) and (2) lung squamous cell carcinoma (LUSC; 30%) [2,3]. In Taiwan, lung cancer is currently the most prevalent and most frequent cause of cancer-related mortality [4]. As such, it is crucial to develop novel agents and identify novel targets for the therapeutic treatment of lung cancer in order to improve patient outcomes.

Previous investigations reported that many anti-cancer drugs have drawbacks, such as side effects and toxicity [5]. Thus, there is a need for safer anti-cancer agents, particularly ones that can be manufactured using readily available naturally derived ingredients that cause no or minimal side effects. In recent years, researchers increasingly turned their efforts toward natural bioactive compounds due to their possible therapeutic activity in cancer at non-toxic levels [6]. Fucoidan is a water-soluble fucose-containing sulfated polysaccharide that is most commonly isolated from brown algae [7], and its <sup>α</sup>-<sup>l</sup>-fucose-enriched backbone also contains other monosaccharides, including glucose, xylose, galactose, and mannose [8]. Fucoidan possesses remarkable biological functions, including antioxidant, antitumor, anti-inflammatory, immunoregulatory, and antithrombotic activities [9]. These biological activities vary according to differences in the degree of sulfation, sulfation pattern, glycosidic branches, and molecular weight (MW) of fucoidan [9]. Low-molecular-weight (LMW) fucoidan is a highly sulfated fragment derived from fucoidan, and it received considerable attention due to its strong bioactivities with respect to anti-inflammatory, anticoagulant, antiangiogenic, antithrombosis, antioxidant, and anti-obesity effects [10,11]. In addition, LMW fucoidan is reported to be capable of modulating cell adhesion factor [12] and growth factor [13].

The process of extrusion comprises a short-duration, high-temperature bioreaction that involves mixing, heating, shearing, pressurizing, and shaping. During extrusion, the raw materials undergo mechanical shearing at high temperature with a very low moisture content and, thus, the properties of the extruded products, such as texture, microstructure, color, and flavor, are extensively modified [14]. Extrusion cooking provides numerous advantages, such as easy operation, continuous production, low manpower, high production yield, minimal waste, and a diversity of products [15]. Extruders are traditionally used to produce a wide variety of commonly consumed snacks, including corn curls, breadsticks, flatbreads, extruded corn ball, extruded puffed rice cereals, croutons, and breakfast cereals. Extrusion technology is also widely employed in the production of non-snack food products and other applications, such as biomass processing, and in the chemical, polymer, and energy industries [16]. Previous investigations indicated that extrusion can be successfully employed for the pretreatment of rice straw, which involves accelerating the saccharification of rice straw by enzymatic hydrolysis [17]. Fish scale is a good source for extraction of gelatin (a denatured form of collagen). However, it is known that fish scale is composed of collagen and hydroxyapatite, which are tightly linked together and difficult to separate. Extrusion was also adopted to pretreat fish scale to facilitate the separation of collagen and hydroxyapatite [15,18]. Soybean dregs can be pretreated by extrusion to decrease the quantity of insoluble dietary fiber (IDF) and increase soluble dietary fiber (SDF) in soybean residues [19]. Similarly, extrusion can be applied in the pretreatment of orange pomace to redistribute the IDF to SDF and to obtain a greater amount of soluble dietary fiber [20]. Moreover, the saccharification effect of lignocellulose can be improved by subjecting lignocellulosic biomass to bioextrusion pretreatment [21]. Therefore, we were interested in determining whether extrusion could be used to pretreat brown seaweeds in order to disrupt the natural anti-degradation barriers of seaweeds and enhance the release of polysaccharide from seaweed by water extraction alone. In the present study, we extracted fucoidan from *Sargassum crassifolium* pretreated by single-screw extrusion. The extracted fucoidan (native fucoidan, namely, SC) was then treated with different combinations of ascorbic acid (AA), hydrogen peroxide (H2O2), and AA + H2O2 to obtain degraded fucoidans. The composition, structure, and in vitro anti-lung cancer activity of native and degraded fucoidans were evaluated. This paper presents, for the first time to the authors' knowledge, the in vitro anti-lung cancer activity of native and degraded fucoidans prepared from *S. crassifolium* pretreated by single-screw extrusion. In addition, we attempted to elucidate the underlying mechanisms involved in the fucoidan-induced lung cancer cell death. The result of this study may help to inform future research into the possible applications of degraded fucoidans as natural chemopreventive agents for the adjuvant treatment of cancer, especially lung cancer.
