**3. Purification Technique of Collagens from Marine Invertebrates**

The molecules in invertebrates are complex, and therefore the purification of any specific molecule from this group is tricky. An individual species is required to apply different techniques, as the characterization of their components is multifaceted. For instance, soft corals have sclerites and soft tissue (unlike the stony corals) comprising complex organic matrices [14]. For these complexities, it was difficult to purify molecules; however, our group successfully purified the molecules [11,14], including the functional extracellular matrix proteins (e.g., ECMP-67), enzymes, calcium-binding proteins, and glycoproteins (see Figure 3 for examples). Glycoprotein in the extracellular matrix protein is a key component of collagen (Figure 2) that plays the main role in the biological process of collagen in invertebrates. Applying similar techniques, we recently investigated coralline algae, which have a high concentration of both chitin and collagen biopolymers and are functional in both soluble and insoluble organic matrix fractions (Figures 4 and 5) [5,9].

Coralline algal concentrations of the soluble organic matrix (0.9%) and insoluble organic matrix (4.5%) fractions are significantly higher than those of other marine invertebrates such as soft corals, with a soluble organic matrix and insoluble organic matrix of 0.03% and 0.05%, respectively [56,72]. The evidence of purified collagen in the coralline skeletons was also shown by X-ray diffraction (XRD) analysis (Figure 4). Jiang et al. [73] identified mineral crystals in collagen fibrils in a different marine invertebrate. The findings by Jiang et al. support our XRD results, and this technique has been revealed as a promising tool in analyzing collagens in the mineralization process. The results obtained by XRD demonstrated that XRD will become an important tool to study biological materials like collagen from the ECM of invertebrates. Such a high concentration of collagen present in the organic matrices of marine calcifiers presents the opportunity for future drug development in bone-related disease, and,

moreover, both chitin and collagen present in the same species can take a significant role in drug design of other related diseases, because these two polymers are commonly used in drug design [74–83].

At present, the methods for the isolation and purification of collagens from the octocorals have been significantly improved. A patented protocol on the collagen purification from the soft coral [54] is now on the market. Since this method is patented, it is not open to the public. However, there are several publications by the same research group that currently exist in the literature, in which they established the methods in purifying collagens (including collagen types I and II) from the soft coral [2,3]. The development of these new technologies, along with the technologies established by our group as mentioned above, will be extremely beneficial for purifying functional collagens from these marine organisms.

Despite the importance of collagenous marine sponge skeletons being documented, the techniques for the purification of collagens from this group are not well-established yet because of their insolubility and mineralization, which might cause difficulties in its separation and characterization [84,85]. However, researchers are trying to resolve these issues, and numerous investigations have so far been reported in this group [47,50,53,86,87]. Recently, Pozzolini et al. [47] established several new methods to purify collagenous fibrillar suspensions from the *Chondrosia reniformis* demosponge. The authors demonstrated that the obtained fibrillar collagens are extensively useful for tissue engineering and regenerative medicine, as well as in antioxidant activity.

There are some techniques that have been established in purifying collagens from the invertebrates; however, a proteomic approach might be a useful tool to learn more about the collagen and its functions in detail. Proteomics have already been established as an important tool for the detection, characterization, and analysis of pharmaceutically useful proteins from marine organisms, and this approach provides the most precise evaluation of protein identities, abundance, composition, and protein expression profiling [5,26,88–90]. Therefore, in regard to collagen, the proteomics approach could be a promising toolkit in the near future. The overview regarding marine collagen of invertebrates stated above allows us to understand some newly developed techniques and suitable methods for extracting and purifying collagen, as well as for applying proteomics approach for medical applications.

#### **4. Future Applications of Invertebrate Collagens in Medical Field**

The marine ecosystem provides suitable and numerous diversified resources for human health in comparison to the terrestrial ecosystem. In the last few decades, marine resources, especially invertebrates, have been recognized to be a promising source for many drugs (e.g., Cytarabine, Vidarabine, and Halichondrin B) [91]. According to the discussion above (Sections 1–3), marine invertebrates and related calcifying organisms such as soft and hard corals, sponge, mollusk, sea urchin, and coralline algae could be a major source of medicines over the next decades. However, extraction and purification of collagen for the purpose of medical application of these resources is still under investigation developing. Despite some impressive work having been performed on collagenous sponges and corals [1–4,6,7,15–17,47,54,91–93], an intensive study is necessary with these two groups and other invertebrates to use these huge apposite resources in future years. The potential of marine invertebrates for collagen could be realized by developing new technologies; indeed, there are many methods such as proteomics, computer-aided design, bioinformatics, and combinatorial synthesis that are now being applied.

The biological diversity of marine invertebrates and complex protein and peptide components direct us toward discovery of many new drugs for various therapeutic areas, including bone-related disease (e.g., osteoporosis) [94]. Besides cancer, microbial infections, and inflammation, drug discovery for bone-related disease is the biggest challenge of the current century, and collagen extraction from marine invertebrates shows new promise in fighting against this and other related diseases.
