Metabolites and Bioactivity of the Marine Xestospongia Sponges (Porifera, Demospongiae, Haplosclerida) of Southeast Asian Waters

Sponges are aquatic, spineless organisms that belong to the phylum Porifera. They come in three primary classes: Hexactinellidae, Demospongiae, and Calcarea. The Demospongiae class is the most dominant, making up over 90% of sponge species. One of the most widely studied genera within the Demospongiae class is Xestospongia, which is found across Southeast Asian waters. This genus is of particular interest due to the production of numerous primary and secondary metabolites with a wide range of biological potentials. In the current review, the antioxidant, anticancer, anti-inflammatory, antibacterial, antiviral, antiparasitic, and cytotoxic properties of metabolites from several varieties of Southeast Asian Xestospongia spp. were discussed. A total of 40 metabolites of various natures, including alkaloids, fatty acids, steroids, and quinones, were highlighted in X. bergquistia, X. testudinaria, X. muta, X. exigua, X. ashmorica and X. vansoesti. The review aimed to display the bioactivity of Xestospongia metabolites and their potential for use in the pharmaceutical sector. Further research is needed to fully understand their bioactivities.


Introduction
Sponges, aquatic animals of the phylum Porifera, have existed for millions of years as the simplest multicellular organisms. They are filter feeders and are known for their unique species diversity and morphological complexity [1,2]. Sponge species number over 8000 and are found in temperate, tropical, and polar regions, inhabiting a wide range of freshwater and marine habitats [3]. They are an important source of metabolites. More than 5300 distinct metabolites produced by sponges and the accompanying microbes are known and more than 200 novel sponge metabolites are reported each year [3]. Alkaloids, fatty acids, sterols, terpenoids, polyketones, macrolides, quinines, glucosides, and peptides are a few examples of novel metabolites that have been identified from marine sponges [4][5][6][7][8][9].
Xestospongia spp. can be found in a variety of habitats in the Southeast Asia region. They are typically found in shallow tropical coral reefs but can also be found in deeper waters. They are often found attached to sand, rocks, corals, or other benthic substrates [22][23][24][25][26]. In terms of ecology, Xestospongia spp. play a key role in the coral reef ecosystem. They are filter feeders, which means they filter water through their bodies to obtain food particles. The huge size of these sponges is particularly crucial because body size is mechanistically connected to pumping and nutrition cycling. This process also helps to remove excess nutrients and sediment from the water, which can help maintain the overall health condition of the coral reef ecosystem [27,28]. Xestospongia spp. also provide important habitats for other organisms, such as algae, bacteria, fish, crustaceans, and other invertebrates, which use the sponges as a place to hide, breed, and forage for food ( Figure 2). Some Xestospongia spp. also form symbiotic relationships with other prokaryotes [23,[29][30][31]. Xestospongia spp. are classified as follows within the phylum Porifera. Kingdom: Animalia (animals), Phylum: Porifera (sponges), Class: Demospongiae (demosponges), Order: Haplosclerida, Family: Xestospongiidae, Genus: Xestospongia. There are over 30 Xestospongia spp., including X. bergquistia (Fromont, 1991) [16], X. testudinaria (Lamarck, 1815) [17], X. muta (Schmidt, 1870) [18], X. exigua (Kirkpatrick, 1900) [19], X. ashmorica (Hooper, 1984) [20] and X. vansoesti [21] etc.
Xestospongia spp. can be found in a variety of habitats in the Southeast Asia region. They are typically found in shallow tropical coral reefs but can also be found in deeper waters. They are often found attached to sand, rocks, corals, or other benthic substrates [22][23][24][25][26]. In terms of ecology, Xestospongia spp. play a key role in the coral reef ecosystem. They are filter feeders, which means they filter water through their bodies to obtain food particles. The huge size of these sponges is particularly crucial because body size is mechanistically connected to pumping and nutrition cycling. This process also helps to remove excess nutrients and sediment from the water, which can help maintain the overall health condition of the coral reef ecosystem [27,28]. Xestospongia spp. also provide important habitats for other organisms, such as algae, bacteria, fish, crustaceans, and other invertebrates, which use the sponges as a place to hide, breed, and forage for food ( Figure 2). Some Xestospongia spp. also form symbiotic relationships with other prokaryotes [23,[29][30][31]. However, Xestospongia spp. are facing potential threats to their survival. These include pollution, rise in temperature due to climate changes and associated diseases such as generalised necrosis, cyclic spotted bleaching, sponge orange band, tissue hardening condition and tissue wasting disease [32][33][34][35]. These threats can have negative impacts on the overall health of coral reef ecosystems and therefore on Xestospongia spp. as well [36,37].
Xestospongia species are found in tropical and subtropical waters throughout Southeast Asia ( Figure 3). They are noticed in several places in Indonesia, including Pecaron Bay, Pasir Putih, Situbondo, East Java, Sabang Island, North Sulawesi, and Bandung. They can frequently be seen in deep waters, lagoons, and coral reefs [38][39][40][41]. Xestospongia spp. are observed in the Philippines in several places, including the Manila Channel off Mindoro Island [42]. They are located in Malaysia in several places, including Sepanggar and Gaya Islands, Sabah, Mentigi Island, Johor, Bidong Island, Terengganu, and Langkawi [43][44][45]. In Thailand, they are found in Chon Buri and Rayong Provinces [46] while in Vietnam, they are found in Ha Long Bay and Khanh Hoa [47,48]. However, Xestospongia spp. are facing potential threats to their survival. These include pollution, rise in temperature due to climate changes and associated diseases such as generalised necrosis, cyclic spotted bleaching, sponge orange band, tissue hardening condition and tissue wasting disease [32][33][34][35]. These threats can have negative impacts on the overall health of coral reef ecosystems and therefore on Xestospongia spp. as well [36,37].
Xestospongia species are found in tropical and subtropical waters throughout Southeast Asia ( Figure 3). They are noticed in several places in Indonesia, including Pecaron Bay, Pasir Putih, Situbondo, East Java, Sabang Island, North Sulawesi, and Bandung. They can frequently be seen in deep waters, lagoons, and coral reefs [38][39][40][41]. Xestospongia spp. are observed in the Philippines in several places, including the Manila Channel off Mindoro Island [42]. They are located in Malaysia in several places, including Sepanggar and Gaya Islands, Sabah, Mentigi Island, Johor, Bidong Island, Terengganu, and Langkawi [43][44][45]. In Thailand, they are found in Chon Buri and Rayong Provinces [46] while in Vietnam, they are found in Ha Long Bay and Khanh Hoa [47,48].
It is also worth noting that the diversity of Xestospongia spp. in Southeast Asia is not well understood, and more research is needed to fully understand the diversity, distribution, and abundance of these species in the region.
This review comprehensively addresses the secondary metabolites of marine sponges of the genus Xestospongia in Southeast Asian waters with potential bioactive properties, including antioxidant, anticancer, antiplasmodial, antibiotic, antibacterial, antifungal, cardiotonic, cytotoxic, antimalarial, and antihelminthic properties. The review collects and compares information from peer-reviewed articles from 1974 to 2022 on secondary metabolites isolated from Xestospongia spp. The information was retrieved from several internet databases (PubMed, Web of Science, Scopus, and registries including dissertations and proceedings). The database was searched for marine sponges, Xestospongia, Indo-Pacific, It is also worth noting that the diversity of Xestospongia spp. in Southeast Asia is not well understood, and more research is needed to fully understand the diversity, distribution, and abundance of these species in the region.
This review comprehensively addresses the secondary metabolites of marine sponges of the genus Xestospongia in Southeast Asian waters with potential bioactive properties, including antioxidant, anticancer, antiplasmodial, antibiotic, antibacterial, antifungal, cardiotonic, cytotoxic, antimalarial, and antihelminthic properties. The review collects and compares information from peer-reviewed articles from 1974 to 2022 on secondary metabolites isolated from Xestospongia spp. The information was retrieved from several internet databases (PubMed, Web of Science, Scopus, and registries including dissertations and proceedings). The database was searched for marine sponges, Xestospongia, Indo-Pacific, Southeast Asia, the South China Sea, bioactivity, morphotypes, haplotypes, secondary metabolites and marine natural products.

Secondary Metabolites in Xestospongia spp. and Their Bioactivity
In Southeast Asian waters, Xestospongia spp. has been found to produce a variety of secondary metabolites of various nature including alkaloids, steroids, fatty acids, quinone etc. These compounds have been shown to have antioxidant, anti-inflammatory, antiparasitic, antitumor, and antimicrobial properties [4][5][6][49][50][51][52][53][54][55][56]. However, more research is needed to fully understand these metabolites' bioactivity and potential uses. Some of the important Xestospongia spp. have been discussed below. (Fromont, 1991) X. bergquistia is an abundant member of the coral reef community found in the Philippines, Indian Ocean, Indonesia, and Malaysia [15,57]. Recent studies have isolated three unique pentacyclic polyhydroxylated steroids, known as xestobergsterol A (1), B (2), and C (3), from the methanol/toluene extract of this species. These compounds are notable for being the first steroids to have five carbocyclic rings. Compounds 1 and 2 have been found to have anti-inflammatory activity, with both displaying potent inhibition of histamine release from rats' mast cells induced by anti-IgE (Immunoglobulin E) in a dose-dependent manner [4][5][6]. The anti-inflammatory activity of the compound 1 was found to be approximately 5200 times stronger than that of disodium cromoglycate, a commonly used antiallergy medication. In addition to their anti-inflammatory properties, compounds 1 and 3 were also found to exhibit cytotoxic activity against L-1210 murine leukaemia cells. Specifically, they displayed IC 50 values of 4.0 µg/mL and 4.1 µg/mL, respectively. However, compound 2 was found to have negligible cytotoxic effects [49,50]. The chemical structures of compounds 1-3 are shown in Figure 4. unique pentacyclic polyhydroxylated steroids, known as xestobergsterol A (1), B (2), and C (3), from the methanol/toluene extract of this species. These compounds are notable for being the first steroids to have five carbocyclic rings. Compounds 1 and 2 have been found to have anti-inflammatory activity, with both displaying potent inhibition of histamine release from rats' mast cells induced by anti-IgE (Immunoglobulin E) in a dose-dependent manner [4][5][6]. The anti-inflammatory activity of the compound 1 was found to be approximately 5200 times stronger than that of disodium cromoglycate, a commonly used antiallergy medication. In addition to their anti-inflammatory properties, compounds 1 and 3 were also found to exhibit cytotoxic activity against L-1210 murine leukaemia cells. Specifically, they displayed IC50 values of 4.0 μg/mL and 4.1 μg/mL, respectively. However, compound 2 was found to have negligible cytotoxic effects [49,50]. The chemical structures of compounds 1-3 are shown in Figure 4. Overall, these findings suggest that compounds 1-3 isolated from X. bergquistia have potential therapeutic applications in the fields of anti-inflammatory and cancer treatment. However, further research is needed to fully understand the properties and potential uses of these compounds. (Schmidt, 1870) X. muta is a species of sponge found on Sabang Island, Indonesia, and other parts of the world. Studies have shown that the tissues of this sponge contain symbionts of the Synechococcus group, which are a type of microorganism that lives in symbiotic relationships with other organisms [37,38].

Xestospongia muta
A manzamine alkaloid, named manzamine C (4) ( Figure 5), was isolated from X. muta, and displayed inhibition activity against human pancreatic cell carcinoma (PANC-1) under glucose starvation conditions with IC50 values of 10 μM, whereas no growth inhibition was observed up to 100 μM under the general culture conditions. Additionally, the compound 4 also exhibited strong antileishmanial and antimalarial activity against drug-sensitive and drug-resistant strains of Plasmodium [58,59]. These findings suggest that compound 4 has the potential as a treatment for these diseases, and further research is needed to explore this possibility. Overall, these findings suggest that compounds 1-3 isolated from X. bergquistia have potential therapeutic applications in the fields of anti-inflammatory and cancer treatment. However, further research is needed to fully understand the properties and potential uses of these compounds.

Xestospongia muta (Schmidt, 1870)
X. muta is a species of sponge found on Sabang Island, Indonesia, and other parts of the world. Studies have shown that the tissues of this sponge contain symbionts of the Synechococcus group, which are a type of microorganism that lives in symbiotic relationships with other organisms [37,38].
A manzamine alkaloid, named manzamine C (4) ( Figure 5), was isolated from X. muta, and displayed inhibition activity against human pancreatic cell carcinoma (PANC-1) under glucose starvation conditions with IC 50 values of 10 µM, whereas no growth inhibition was observed up to 100 µM under the general culture conditions. Additionally, the compound 4 also exhibited strong antileishmanial and antimalarial activity against drug-sensitive and drug-resistant strains of Plasmodium [58,59]. These findings suggest that compound 4 has the potential as a treatment for these diseases, and further research is needed to explore this possibility. In addition to the pure secondary metabolites the fractions of the solvent extract of X. muta also displayed protective properties. The X. muta collected in coastal Terengganu (Malaysia) was found to have cardiovascular protective properties. The study showed that various fractions of this species contain various fatty acids with cardiovascular protective activity, with Fraction-7 being the most notable. Fraction 7 was obtained from the methanol extract of X. muta, and gas chromatography and mass spectrometry (GCMS) analysis of this fraction indicated the presence of 58 compounds. In vitro research in HepG2 cells displayed that fraction-7 of X. muta boosted the expression of Scavenger receptor class B type I (SR-BI) mRNA by 129%. SR-BI is the primary receptor for high-density lipoprotein In addition to the pure secondary metabolites the fractions of the solvent extract of X. muta also displayed protective properties. The X. muta collected in coastal Terengganu (Malaysia) was found to have cardiovascular protective properties. The study showed that various fractions of this species contain various fatty acids with cardiovascular protective activity, with Fraction-7 being the most notable. Fraction 7 was obtained from the methanol extract of X. muta, and gas chromatography and mass spectrometry (GCMS) analysis of this fraction indicated the presence of 58 compounds. In vitro research in HepG2 cells displayed that fraction-7 of X. muta boosted the expression of Scavenger receptor class B type I (SR-BI) mRNA by 129%. SR-BI is the primary receptor for high-density lipoprotein (HDL) cholesterol, which is crucial for preventing atherosclerosis [44]. (Kirkpatrick, 1900) X. exigua is abundant in tropical Southeast Asia, the Western South Pacific, Papua New Guinea, and Australia [53,60,61]. In natural product chemistry, X. exigua has attracted a lot of attention due to the diversity of secondary metabolites that have been isolated from it. Since the 1980s, over 24 different bioactive metabolites have been identified from this species, and these metabolites have been found to display a wide range of bioactivities, including vasodilation, cytotoxicity, and antibacterial effects [53,[61][62][63][64][65].
The above information displayed that the metabolites detected in X. exigua have a diverse range of chemical structures, including alkaloids, quinones, and sterols, with protective activity such as indoleamine-2,3-dioxygenase inhibition, vasodilation, and cytotoxicity against human cancer cell lines. Some metabolites demonstrated anti-inflammatory and antibacterial properties. Crude extract of X. exigua also showed cytotoxic and antioxidant properties. These results demonstrated the potential of X. exigua as a source of bioactive metabolites for further research.  Similarly, the crude extract of X. exigua also displayed protective activity. The cytotoxic and antioxidant activities of the crude extract of X. exigua collected from Pecaron Bay, Pasir Putih, Situbondo, East Java, Indonesia, were determined. The cytotoxic assay was carried out using MTT methods for cancerous cells, including HT-29, T47D, and Casky, while the antioxidant assay was conducted using the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The ethanol extract of X. exigua (1 mg/mL) showed cytotoxic activity

Xestospongia testudinaria (Lamarck, 1815)
X. testudinaria is a well-known sponge species that is common across the Indo-Pacific and dominates coral reef sponge ecosystems in Indonesia. The function of the so-called "giant barrel sponge" in the reef ecology as well as its different bioactive substances have been extensively researched. Important metabolites include nonanedioic acid (azelaic acid) (26), which displayed bactericidal activity in a variety of Gram-positive and Gram-negative bacteria such as Propionibacterium acnes, Staphylococcus epidermidis, S. aureus, Pseudomonas aeruginosa, Escherichia coli, Corynebacterium diphtheriae and Proteus mirabilis [67]. It also displayed potent anti-inflammatory and antioxidant properties. Compound 26 treatment dramatically decreased the number of inflammatory papules and pustules in individuals. It promoted the transcription of genes involved in the production of proinflammatory cytokines, including IL-1β, IL-6 or TNFα [67]. Tetradecanoic acid (27), at concentrations of 1.0, 2.5, and 5.0 ppm, displayed larvicidal activity against Aedes aegypti and Culex quinquefasciatus mosquitoes' larvae with LC 50 values of 14.08 and 25.10 ppm, respectively [68]. Trans phytol (28), at a concentration of 10 µg/mL showed aromatase inhibition activity with an IC 50 value of 1 µM. The compound 28 reduced aromatase mRNA and protein expression levels in human ovarian granulosa-like KGN cells [69]. In vertebrates, the sole enzyme that catalyzes the biosynthesis of estrogens is aromatase. Overexposure to estrogens causes endometrial, ovarian and breast cancer, so lowering estrogen levels by inhibiting aromatase becomes a possibility in the prevention and treatment of estrogen-mediated cancer [69,70]. In another study, 28 displayed antioxidant properties, at a concentration of 7.2 µg/mL displayed 59.89 and 62.79% scavenging capacity of DPPH• and ABTS•+ (2,2 -azino-bis(3ethylbenzthiazoline-6-sulphonic acid), respectively. The compound 28 (administered via the intraperitoneal (i.p.) route at doses of 25, 50 and 75 mg/kg), reduced lipid peroxidation (LP) and nitrite (NO 2 − ) levels and elevated reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities in the Swiss mouse hippocampus [71]. Pentadecanoic acid (29) displayed anticancer effects against human breast carcinoma MCF-7/stem-like cells (SC). Furthermore,29 reduced interleukin-6 (IL-6)-induced JAK2/STAT3 signalling. In populations of CD44+/CD24 stem-like cells derived from human breast cancer cells, JAK2/STAT3 signalling is essential for maintenance [72,73]. Thus, targeting JAK2/STAT3 signalling is seen as a viable therapeutic approach. Additionally, 29 caused a cell cycle arrest at the sub-G1 phase and aided caspase-dependent death in MCF-7/SC cells [72,73]. Palmitic acid (30) displayed anticancer properties against the human prostate cancer cell lines PC3 and DU145 as well as the subcutaneous xenograft model. Both in vitro and in vivo, 30 reduce the development of prostate cancer cells. The exposure of 30 induced G1 phase arrest, linked with the downregulation of cyclin D1 and p-Rb and upregulation of p27 [54]. The compound 30 displayed anticancer properties against murine colorectal carcinoma (CT-26) and (MC-38) cell lines [55]. It also demonstrated antiviral activity against viremia of carp virus (SVCV) infection using Zebrafish (Danio rerio) model. The findings showed that a low concentration of 30 modulates the infection and reduced Zebrafish mortality [56]. At concentrations ranging from 0.1 to 1.0 mg/mL, 9,12-octadecadienoic acid (linoleic acid) (31) demonstrated antibacterial activity against five Gram-positive bacteria, including Bacillus cereus, B. pumilus, B. subtilis, Micrococcus kristinae, and S. aureus, but was inactive against Gram-negative species (Enterobacter cloacae, E. coli, Klebsiella pneumoniae, P. aeruginosa and Serratia marcescens) [74]. Heneicosane (32) at a concentration of 10 µg/mL displayed excellent antimicrobial potential against S. pneumoniae (zone of inhibition = ZOI = 31 ± 0.64 mm) and Aspergillus fumigatus (ZOI = 29 ± 0.86 mm) respectively [75]. 1-iodohexadecane (hexadecyl iodide or cetyl iodide) (33) reduced 2,4-Dinitrochlorobenzene-Induced Atopic Dermatitis (AD) (a chronic inflammatory dermal) in mice. The treatment with bioactive alkane at a concentration of 100µg/mL for 21 days improved AD-like skin lesions, suppressed epidermal thickness and elevated filaggrin [76,77]. The above-mentioned long-chain fatty acids from X. testudinaria are displayed in Figure 7.
In another study, the organic (methanol-dichloromethane) extracts of X. testudinaria from Langkawi, Malaysia, displayed antimicrobial activity against S. aureus, B. cereus, and E. coli with ZOI of 11.5, 12 and 9 mm, while no antimicrobial activity was noticed in the aqueous extract of the same species against the same bacteria [45]. Similarly, the symbiotic bacteria extract isolated from X. testudinaria was found to possess antibacterial activity against different types of bacteria, including S. aureus, P. aeruginosa, E. coli, and Salmonella typhi, as conducted by the disc diffusion dilution method. The n-hexane, ethyl acetate, and n-butanol fractions of the extract demonstrated antibacterial activities. Additionally, phytochemical screening of the extract revealed the existence of important metabolites, such as alkaloids, steroids, and triterpenoids [78]. The chloroform fraction of methanol extract of X. tesdudinaria displayed anticancer properties against the HeLa cell line using the MTT In another study, the organic (methanol-dichloromethane) extracts of X. testudinaria from Langkawi, Malaysia, displayed antimicrobial activity against S. aureus, B. cereus, and E. coli with ZOI of 11.5, 12 and 9 mm, while no antimicrobial activity was noticed in the aqueous extract of the same species against the same bacteria [45]. Similarly, the symbiotic bacteria extract isolated from X. testudinaria was found to possess antibacterial activity against different types of bacteria, including S. aureus, P. aeruginosa, E. coli, and Salmonella typhi, as conducted by the disc diffusion dilution method. The n-hexane, ethyl acetate, and n-butanol fractions of the extract demonstrated antibacterial activities. Additionally, phytochemical screening of the extract revealed the existence of important metabolites, such as alkaloids, steroids, and triterpenoids [78]. The chloroform fraction of methanol extract of X. tesdudinaria displayed anticancer properties against the HeLa cell line using the MTT method. The fraction displayed anticancer activity with IC 50 values of 2.273 ppm. The GC-MS analysis indicated the presence of 21 metabolites [77]. Another study reported the antioxidant, anti-inflammatory, and immunomodulatory properties of the methanolic extract of X. testudinaria. against carrageenan-induced rat hind paw edema. The methanolic extract of X. testudinaria at a concentration of 100 mg/kg significantly decreased percentage increase in paw weight after carrageenan injection. The histopathological observation indicated that the extract administration reduced inflammatory cell infiltrate and capillary congestion. The extract boosted reduced glutathione, glutathione peroxidase, and catalase activities while decreasing malondialdehyde (MDA) and nitric oxide (NO) levels. Inflammatory cytokines such as tumour necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 were also lowered [79]. The extract of X. testudinaria also possessed poisonous effects on brine shrimp (Artemia salina), with LC 50 values ranging from 0.56 to 6.99 µM [80,81]. In addition to this, the extract of X. testudinaria from Bandung, Indonesia, mixed with the marine sponge Melophlus sarasinorum to form a scaffold for bone tissue, promoting the growth and division of bone cells. The study displayed promising results for bone tissue engineering [41].
Thus, in addition to the protective nature of the pure secondary metabolites of X. testudinaria, the crude extract and fractions of the sponge also displayed a wide range of protective properties, including antimicrobial, anticancer, antioxidant, anti-inflammatory, and immunomodulatory properties. Phytochemical screening of these extracts and fractions further revealed the presence of important metabolites. These results demonstrated the potential of X. testudinaria as a source of biologically active compounds with a range of therapeutic applications.
2.5. Xestospongia ashmorica (Hooper, 1984) X. ashmorica is a marine sponge found in the Manila Channel off Mindoro Island in the Philippines. In 1996, researchers isolated three alkaloids from this sponge, named manzamine A (34), manzamine E (35), and manzamine F (36). These alkaloids were found to have cytotoxic properties against L5178 mouse lymphoma cells at a concentration range of 0.3 to 20 µg/mL. The median effective dose (ED50) for compounds 34-36 were 1.8, 6.6 and 2.3 g/mL respectively, showing that all the compounds were active against the selected cell line [42]. The structures of the compounds 34-36 from X. ashmorica are illustrated in Figure 8.
indicated that the extract administration reduced inflammatory cell infiltrate and capillary congestion. The extract boosted reduced glutathione, glutathione peroxidase, and catalase activities while decreasing malondialdehyde (MDA) and nitric oxide (NO) levels. Inflammatory cytokines such as tumour necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 were also lowered [79]. The extract of X. testudinaria also possessed poisonous effects on brine shrimp (Artemia salina), with LC50 values ranging from 0.56 to 6.99 μM [80,81]. In addition to this, the extract of X. testudinaria from Bandung, Indonesia, mixed with the marine sponge Melophlus sarasinorum to form a scaffold for bone tissue, promoting the growth and division of bone cells. The study displayed promising results for bone tissue engineering [41].
Thus, in addition to the protective nature of the pure secondary metabolites of X. testudinaria, the crude extract and fractions of the sponge also displayed a wide range of protective properties, including antimicrobial, anticancer, antioxidant, anti-inflammatory, and immunomodulatory properties. Phytochemical screening of these extracts and fractions further revealed the presence of important metabolites. These results demonstrated the potential of X. testudinaria as a source of biologically active compounds with a range of therapeutic applications. (Hooper, 1984) X. ashmorica is a marine sponge found in the Manila Channel off Mindoro Island in the Philippines. In 1996, researchers isolated three alkaloids from this sponge, named manzamine A (34), manzamine E (35), and manzamine F (36). These alkaloids were found to have cytotoxic properties against L5178 mouse lymphoma cells at a concentration range of 0.3 to 20 μg/mL. The median effective dose (ED50) for compounds 34-36 were 1.8, 6.6 and 2.3 g/mL respectively, showing that all the compounds were active against the selected cell line [42]. The structures of the compounds 34-36 from X. ashmorica are illustrated in Figure 8.

Xestospongia vansoesti (Bakus & Nishiyama, 2000)
The compounds salsolinol (37), norsalsolinol (38), cis-4-hydroxysalsolinol (39), and trans-4-hydroxysalsolinol (40) were isolated from the marine sponge X. vansoesti found in North Sulawesi, Indonesia. These compounds are all tetrahydroisoquinoline alkaloids. Compound (37) showed cytotoxicity against various cancer cell lines such as murine leukaemia (L1210) with IC 50 values of 8 mg/mL, human amnion (FL) with IC 50 values of 13 mg/mL, human oral epidermoid carcinoma (KB) with IC 50 values of 20 mg/mL and human lung adenocarcinoma (A549) with IC 50 values of 27 mg/mL, respectively. Additionally, both compounds 37 and 38 inhibited the activity of the proteasome with IC 50 values of 50 and 32 mg/mL, respectively, and were also cytotoxic to human cancer cell lines (HeLa) with IC 50 values of 17 and 7 mg/mL However, compounds 39 and 40 were found to have no protosome inhibitory effect and no cytotoxicity against HeLa [39]. The chemical structures of compounds 37-40 are exhibited in Figure 9. lung adenocarcinoma (A549) with IC50 values of 27 mg/mL, respectively. Add both compounds 37 and 38 inhibited the activity of the proteasome with IC50 val and 32 mg/mL, respectively, and were also cytotoxic to human cancer cell line with IC50 values of 17 and 7 mg/mL However, compounds 39 and 40 were found no protosome inhibitory effect and no cytotoxicity against HeLa [39]. The chemi tures of compounds 37-40 are exhibited in Figure 9.  Table 1 below summarizes the metabolites detected in the extracts of variou of Xestospongia, including their bioactivity, class, molecular formula (MF), and m weight (MW).   Table 1 below summarizes the metabolites detected in the extracts of various species of Xestospongia, including their bioactivity, class, molecular formula (MF), and molecular weight (MW).

Summary of Xestospongia Metabolites
Xestospongia spp., found in Southeast Asian waters, are a rich source of secondary metabolites with potential for application in various industries, such as pharmaceuticals, biotechnology, and agriculture. Among the species, X. exigua displayed a high number of metabolites with bioactivity, followed by X. testudinaria, X. bergquistia, X. ashmorica, X. vansoesti, and X. muta. Despite the potential application, there is still limited knowledge about Xestospongia spp. and the metabolites they produce. To address this knowledge gap, Southeast Asian nations must collaborate on research. The primary objectives of the research should be the identification, classification, and biosynthetic and biotechnological potential of natural compounds from Xestospongia spp.
The efficiency of the bioactive secondary metabolites discovered in Xestospongia spp. must also be determined by clinical study. Additionally, the generation of metabolites by the host sponge and its microbial population must also be investigated individually to confirm the source of these metabolites, which will not only assist in establishing the origin of the metabolites but also shed light on the sponge's host microbial community's biochemistry.
Further, to aid future identification and metabolomics studies, a mass spectrometry database for Xestospongia spp. related metabolites should be established. This will provide a valuable resource for researchers studying these species, allowing for rapid identification and characterization of new natural products.
Overall, further research on Xestospongia spp. is necessary to fully understand the potential of these species and their secondary metabolites.

Conclusions
The genus Xestospongia within the Demospongiae class of sponges from Southeast Asian waters is a rich source of biologically active compounds. This review highlights the various structural diversity of the metabolites, including alkaloids (xestospongin, araguspongine, exiguamine, motuporamine, manzamine, salsolinol and norsalsolinol), fatty acids (nonanedioic acid, phytol, pentadecanoic acid, palmitic acid, and 9,12-octadecadienoic acid), steroids (xestobergsterol, clionasterol and 5α,8α-epidioxy-24αethylcholest-6-en-βbol), alkane (heneicosane and iodohexadecane), and quinones (halenaquinone and exiguaquinol), that have been isolated from Xestospongia spp. and found to exhibit a wide range of bioactivity. These compounds displayed antioxidant properties, which means they can protect cells from damage caused by free radicals. They also demonstrated anticancer properties, which could make them useful in the development of new cancer therapies. Additionally, the compounds displayed anti-inflammatory properties, which could be beneficial in the treatment of chronic diseases such as rheumatoid arthritis and asthma. They also indicated anti-bacterial, antiviral, antiparasitic, and cytotoxic properties.