# Water-Insoluble Black Pigment Released from the Octocoral _Sinularia flexibilis_
## 4 Discussion  
Marine organisms are potential sources of natural pigments that are useful substitutes for existing synthetic or land-organism pigments in various fields, such as food, cosmetic, and pharmaceutical industries, because of their excellent biocompatibility, safety, and stability [13]. Algae, microorganisms, and animals are major pigment sources in marine environments. Furthermore, these metabolites can be classified into eight major  
Figure 8: Observation of the collected black precipitate with the naked eye and with a microscope. (**a**) Black pigment released from _S. flexibilis_ was collected from the bottom of a 1.5 mL centrifuge tube; (**b**) the black pigment entangled with Symbiodiniaceae (brown spheres) as observed under microscopy. Figure 7: The amount of black pigment released by different kinds of octocorals. (**a–e**) Tentacles about 1 cm long from five different species of octocorals were collected in 6-well plates; (**f–j**) these octocorals were cut into small pieces and incubated in FSW (36%); (**k–o**) after 20 h, the amounts of black pigment were observed. (**a,f,k**) _S. flexibilis;_ (**b,g,l**) _S. brassica;_ (**c,h,m**) _S. sandensis;_ (**d,i,n**) _S. compressa;_ (**e,j,o**) _Klyzum simplex._classes: chlorophylls, carotenoids, phycobiliproteins, tetrapyrroles, quinones, azulenes, indigoids, and melanin [10; 13]. Melanin is a group of complex, natural, and dark-colored pigments [11; 14; 15; 16]. It plays multiple roles [17; 18] in the field of biomedicine and biological functions [19; 20]. It is also a very promising drug due to its good biocompatibility and biodegradability [21]. Thus, finding new sources of black pigment in cultured marine animals may be a promising alternative for producing sustainable melanin. However, the extraction and purification of melanin are highly dependent on the source. Since melanin is always bound to other cellular components, it can be a challenging task to isolate pure melanin [22]. Previous studies found that corals not only have chlorophyll-a and carotenoid pigments from symbiotic dinoflagellates [23] but also can perform melanin synthesis to put up a cytotoxic defense, form a protective barrier, and provide structural support [5]. The coagulation pathway also induces clot formation to seal wounds and prevent infections [24]. Although corals can produce melanin in the tissues, no report has mentioned that they can release melanin into a solution. In this study, we found aquaculture corals can produce black pigments in the tissues and they are insoluble in five common organic solvents (methanol, acetone, toluene, ethyl acetate, and dimethyl sulfoxide) and water. This article is the first to report that corals can release a black pigment that dyes the human skin black. Moreover, this black pigment can be continuously collected through coral aquaculture. Although the chemical structure of _S. flexibilis'_ black pigment is still not clear, the chemical characterization of the pigment is similar to that of melanin. Melanin is a mixture of different tyrosine derivatives [25]. Marine invertebrates, such as sea cucumbers and cephalopods, also produce melanin [11]. A previous study used the alkali-soluble acid precipitation method to purify melanin and was able to solubilize sepia melanin with 10 M NaOH and precipitate the melanin with 5 M HCl [26]. To purify sea cucumber melanin, researchers have also used NaOH to dissolve the melanin and then adjusted the pH of the supernatant to about 2.0 to precipitate the melanin [27]. These approaches inspired us (as we encountered obstacles in our attempt to dissolve the octocoral's black pigment) to conduct a follow-up study in the future to mix different solvents or adjust the pH values of the solutions to dissolve it. Doing so will enable us to obtain liquid samples needed for spectroscopic experiments that will give us insight into the structure of the black pigment released from _S.