Callyspongia spp.: Secondary Metabolites, Pharmacological Activities, and Mechanisms

One of the most widespread biotas in the sea is the sponge. Callyspongia is a sponge genus found in the seas, making it easily available. In this review, the pharmacological activity and mechanism of action of the secondary metabolites of Callyspongia spp. are addressed, which may lead to the development of new drugs and targeted therapeutic approaches. Several scientific databases, such as Google Scholar, PubMed, ResearchGate, Science Direct, Springer Link, and Wiley Online Library, were mined to obtain relevant information. In the 41 articles reviewed, Callyspongia spp. was reported to possess pharmacological activities such as cytotoxicity against cancer cell lines (36%), antifungal (10%), anti-inflammatory (10%), immunomodulatory (10%), antidiabetic and antiobesity (6%), antimicrobial (8%), antioxidant (4%), antineurodegenerative (4%), antihypercholesterolemic (2%), antihypertensive (2%), antiparasitic (2%), antiallergic (2%), antiviral (2%), antiosteoporotic (2%), and antituberculosis (2%) activities. Of these, the antioxidant, antituberculosis, and anti-inflammatory activities of Callyspongia extract were weaker compared with that of the control drugs; however, other activities, particularly cytotoxicity, show promise, and the compounds responsible may be developed into new drugs.


Introduction
The ocean, which covers 71% of the earth's surface, regulates our climate and contains abundant resources [1]. The sea encompasses a large area, but it is well connected, and the temperature is less extreme compared with that on land. Although containing more biodiversity, only 16% of all species have been identified [2].
One of the most ubiquitous sea organisms is the sponge. Sponges are often abundant in shallow water habitats, making them a unique biodiversity component [3]. They are one of the most diverse sessile organisms, with approximately 8876 valid species identified worldwide. Each has its unique characteristics, while some features are shared [4].
Callyspongia belongs to the family Callyspongidae. More than 60 species are widely distributed in the tropical sea [5]. It is also found in the Indian, Western Atlantic, and Eastern Pacific oceans, including Indonesia [6], the Red sea [7,8], Cuba [4], Barbados [9], Brazil [10,11], and Ecuador [12]. At a depth of 6-10 m below sea level, Callyspongia spp. can live under coral reefs, ranging from moderate to damaged conditions, or in habitats dominated by hard coral, sand, and coral rubble [13].
Sponges from the Callyspongia genus are formed from primary, secondary, and tertiary spongin fibers [4]. Callyspongia sponges are encrusting, form a single erect branch or a mass of round branches, and many are bifurcated. The longest branch that has been observed is  [15], (d) Callyspongia samarensis [3], (e) Callyspongia aerizusa.
Other pharmacological activities of sponge compounds include antibacterial, antihyperlipidemic, antiproliferative, immunomodulatory, and anti-inflammatory effects have been reported, including Callyspongia spp. [26,27]. Sponges contain multiple primary and secondary metabolites, such as fatty acids, alkaloids, steroids, nucleotides, peptides, polyacetylenes, and terpenoids. A total of 212 compounds have been isolated from Callyspongia spp. and their structures and bioactivities have been presented [28].
This review summarizes the potential pharmacological activities exhibited by Callyspongia spp. compounds that may be developed into new drugs. We also discuss the related mechanisms that may contribute to targeted therapy.

Materials and Methods
The literature review of Callyspongia spp. was based on topics related to pharmacological activity and the mechanism of action of secondary metabolites contained therein. This review was conducted with a qualitative and quantitative approach to obtain information from several scientific databases, including Google Scholar, PubMed, ResearchGate, Science Direct, Springer Link, and the Wiley Online Library. Several keywords, such as "Callyspongia sp.", "metabolites", and "pharmacology activity", were used to procure relevant resources. The inclusion criterion for selecting articles was that they should describe the isolation and functional studies of secondary metabolites from Callyspongia sponges. Articles describing the isolation and activities of fungi or bacteria in Callyspongia species were excluded. The abstracts were carefully read to identify and select relevant articles. From 72 identified articles after screening information sources, 41 published between 1980 and 2021 were selected and reviewed ( Figure 2). been reported, including Callyspongia spp. [26,27]. Sponges contain multiple primary and secondary metabolites, such as fatty acids, alkaloids, steroids, nucleotides, peptides, polyacetylenes, and terpenoids. A total of 212 compounds have been isolated from Callyspongia spp. and their structures and bioactivities have been presented [28]. This review summarizes the potential pharmacological activities exhibited by Callyspongia spp. compounds that may be developed into new drugs. We also discuss the related mechanisms that may contribute to targeted therapy.

Materials and Methods
The literature review of Callyspongia spp. was based on topics related to pharmacological activity and the mechanism of action of secondary metabolites contained therein. This review was conducted with a qualitative and quantitative approach to obtain information from several scientific databases, including Google Scholar, PubMed, Re-searchGate, Science Direct, Springer Link, and the Wiley Online Library. Several keywords, such as "Callyspongia sp.", "metabolites," and "pharmacology activity", were used to procure relevant resources. The inclusion criterion for selecting articles was that they should describe the isolation and functional studies of secondary metabolites from Callyspongia sponges. Articles describing the isolation and activities of fungi or bacteria in Callyspongia species were excluded. The abstracts were carefully read to identify and select relevant articles. From 72 identified articles after screening information sources, 41 published between 1980 and 2021 were selected and reviewed ( Figure 2).

Results
Sixteen pharmacological activities have been reported for Callyspongia spp. These activities along with their descriptions are listed in Table 1.

Results
Sixteen pharmacological activities have been reported for Callyspongia spp. These activities along with their descriptions are listed in Table 1.

Discussion
We have discussed the pharmacological activities of Callyspongia spp. that have been previously reported.
Compounds, such as callyspongiamide A and B as well as disamide A (Figure 3), exert antihypercholesterolemic activity, which can also lead to an antiobesity effect by inhibiting sterol O-acyltransferase (SOAT), the enzyme that catalyzes the formation of cholesteryl ester [76]. In addition, other sterols may be used as activators or substrates of this enzyme [77], which implicates it as a potential drug target [61] in hypercholesterolemia; however, the underlying mechanism remains unknown.
Callyspongia sp. also contains β-Sitosterol. This compound exhibits potent antidiabetic activity related to insulin receptor activation and increased glucose transporter 4 (GLUT-4) translocation to adipose tissue [78,79]. In addition, these compounds can potentially maintain glucose homeostasis through sensitization of insulin resistance by increasing the expression of peroxisome proliferator-activated receptor and GLUT-4 ( Figure 4) [80]. Another study on HFD-fed and sucrose-induced type-2 diabetic rats indicates that β-Sitosterol enhances the glycemic regulation [32,79].

Discussion
We have discussed the pharmacological activities of Callyspongia spp. that have been previously reported.

Antidiabetic, Antihypercholesterolemic, and Antiobesity
The active compound from Callyspongia truncata, callyspongynic acid (Figure 3), shows higher antidiabetic activity by inhibiting α-glucosidase with an IC50 of 0.25 μg/mL [29,30] compared with acarbose (IC50 1.3 μg/mL) [70]. Inhibiting this enzyme reduces caloric intake by attenuating appetite, suppressing hunger, and increasing satiety [71,72], thereby supporting weight loss [73] to a moderate level [74]. It is also one of the targets of diabetes therapy. Compared with α-amylase, inhibiting α-glucosidase can improve hyperglycemia, especially postprandial hyperglycemia, by decreasing glucose production ( Figure 3) [75].  Compounds, such as callyspongiamide A and B as well as disamide A (Figure 3), exert antihypercholesterolemic activity, which can also lead to an antiobesity effect by inhibiting sterol O-acyltransferase (SOAT), the enzyme that catalyzes the formation of cholesteryl ester [76]. In addition, other sterols may be used as activators or substrates of this enzyme [77], which implicates it as a potential drug target [61] in hypercholesterolemia; however, the underlying mechanism remains unknown.
In a cell-based testing assay, the IC50 values of callyspongiamide A against SOAT 1 and SOAT 2 were 0.78 ± 0.19 and 2.8 ± 0.72 μM, those of callyspongiamide B were 1.2 ± Callyspongia sp. also contains β-Sitosterol. This compound exhibits potent antidiabetic activity related to insulin receptor activation and increased glucose transporter 4 (GLUT-4) translocation to adipose tissue [78,79]. In addition, these compounds can potentially maintain glucose homeostasis through sensitization of insulin resistance by increasing the expression of peroxisome proliferator-activated receptor and GLUT-4 ( Figure 4) [80]. Another study on HFD-fed and sucrose-induced type-2 diabetic rats indicates that β-Sitosterol enhances the glycemic regulation [32,79]. The methanolic extract of Callyspongia samarensis also exerts antidiabetic activity by enhancing the activity of AMP-activated protein kinase (AMPK) with an EC50 of 14.47 μg/mL, which is more potent compared with the positive control aspirin (EC50 100 μg/mL). This activity may originate from compounds with phenolic groups in the extract [31]. AMPK is an important target for treating type-2 diabetes because its activation affects various aspects of cellular metabolism. It increases glucose metabolism, uptake in the bone and muscle, fatty acid oxidation in the bone, muscle, and liver, mitochondrial oxidative capacity, and insulin sensitivity, whereas it decreases fatty acid synthesis in the liver through GLUT-4 expression (Figure 4) [81][82][83][84].

Antihypertensive
Callypyrone A and callypyrone B (Figure 3) from Callyspongia diffusa exhibit antihypertensive activity by inhibiting angiotensin I-converting enzyme (ACE), which leads to a reduction in angiotensin production. Because angiotensin can constrict blood vessels and increase the heart work rate [85], ACE inhibition results in vasodilation and a decrease in blood pressure ( Figure 5). The IC50 values of these two compounds against ACE were 0.48 mM and 0.57 mM, respectively [33], weaker than the standard drug, captopril (IC50 0.36 mM) [33]. From the results, Callypyrone A and callypyrone B are not considered antihypertensive. The methanolic extract of Callyspongia samarensis also exerts antidiabetic activity by enhancing the activity of AMP-activated protein kinase (AMPK) with an EC 50 of 14.47 µg/mL, which is more potent compared with the positive control aspirin (EC 50 100 µg/mL). This activity may originate from compounds with phenolic groups in the extract [31]. AMPK is an important target for treating type-2 diabetes because its activation affects various aspects of cellular metabolism. It increases glucose metabolism, uptake in the bone and muscle, fatty acid oxidation in the bone, muscle, and liver, mitochondrial oxidative capacity, and insulin sensitivity, whereas it decreases fatty acid synthesis in the liver through GLUT-4 expression (Figure 4) [81][82][83][84].

Antihypertensive
Callypyrone A and callypyrone B (Figure 3) from Callyspongia diffusa exhibit antihypertensive activity by inhibiting angiotensin I-converting enzyme (ACE), which leads to a reduction in angiotensin production. Because angiotensin can constrict blood vessels and increase the heart work rate [85], ACE inhibition results in vasodilation and a decrease in blood pressure ( Figure 5). The IC 50 values of these two compounds against ACE were 0.48 mM and 0.57 mM, respectively [33], weaker than the standard drug, captopril (IC 50 0.36 mM) [33]. From the results, Callypyrone A and callypyrone B are not considered antihypertensive.
Niphatoxin C significantly affects the viability of pre-monocytic THP 1 cells, which express the P2X7 receptor [65]. Activation of this receptor promotes inflammation by releasing inflammatory cytokines, such as IL-18 and IL-1β, and by activating the NLRP3 inflammasome [87,88]. Thus, an antagonist of this receptor may inhibit the secretion of these cytokines (Figure 7). Furthermore, it can inhibit allograft rejection [87], sterile liver inflammation [88], and can potentially treat inflammatory diseases, such as osteoarthritis, rheumatoid arthritis, and chronic obstructive pulmonary disease [28,37].

Antifungal
Callyaerin A, B, and E ( Figure 3) from Callyspongia aerizusa were shown to potently inhibit Candida albicans, with zones of inhibition of 25-30 mm, 15 mm, and 20 mm, respectively using the same concentration. Callyaerin A and E were more potent than callyaerin B [6].  Figure 3) from Callyspongia siphonella exhibits anti-inflammatory activity against rat paw edema that was similar to the control drug, cortisone. The activity was measured by a reduction in edema volume of 19.5 ± 7.3 mL for callysterol and 17.0 ± 7.0 mL for cortisone, whereas the negative control was 61.9 ± 4.7 mL [38]. Callyspongia crassa extracts also showed anti-inflammatory effects with a 61.47% inhibition of protein denaturation [34]. Alkaloids are considered responsible for these anti-inflammatory mechanisms [89], which vary according to the metabolite. The alkaloid that has been identified from ethanolic extract of Callyspongia siphonella was 5-bromo trisindoline and 6-bromo trisindoline [7]. Although the specific mechanism of 5-bromo trisindoline and 6-bromo trisindoline is unknown, some indole alkaloids were known to interfere with the nuclear factor-κB and c-Jun N-terminal kinase signaling pathways [90,91], preventing the synthesis or action of specific pro-inflammatory cytokines, and suppressing histamine release and nitric oxide production ( Figure 7) [92,93]. Alkaloids are effective for treating inflammatory bowel disease [94][95][96][97].

Antifungal
Callyaerin A, B, and E ( Figure 3) from Callyspongia aerizusa were shown to potently inhibit Candida albicans, with zones of inhibition of 25-30 mm, 15 mm, and 20 mm, respectively using the same concentration. Callyaerin A and E were more potent than callyaerin B [6].
Gelliusterol E from Callyspongia aff. implexa also exerts activity against chlamydial fungi in a dose-dependent manner by inhibiting the formation and growth of chlamydial inclusions. At the highest concentration tested (40 µM), no inclusions were observed, similar to the effect of the control, tetracycline. Thus, this compound not only inhibits the formation of Chlamydia, but also affects its development cycle [39]. In addition, the structure of gelliusterol E is similar to that of cholesterol, which is needed for the growth of Chlamydia trachomatis. Furthermore, this compound inhibits lipid acquisition and fungal growth [98].

Antimicrobial and Antiparasitic
Isoakaterpine compounds from Callyspongia sp. exert antiparasitic activity by inhibiting adenosine phosphoribosyltransferase, one of the functional routes in Leishmania adenine metabolism, with an IC 50 of 1.05 µM [11,104], resulting in death of the parasite (Figure 9). Besides antiparasitic activity, the subgenus Callyspongia possesses antituberculosis activity resulting from callyaerins A and B ( Figure 5) isolated from Callyspongia aerizusa. Their MIC90 values (2 μM and 5 μM, respectively) were less effective compared with the controls, rifampicin (<1 μM), ethambutol (1.25 μM), and isoniazid (0.625 μM). Beside the weaker activity compared with the control, there is no in vivo data to support this activity. These compounds inhibited the growth of Mycobacterium tuberculosis as evidenced by reduced cell viability using the resazurin dye reduction method and measuring cell fluorescence [105].
Callyspongia crassa extract potently inhibited Bacillus subtilis and Staphylococcus aureus with zones of inhibition of 16-25 mm (at concentration 500 μg/mL), 9-15 mm and 16-25 mm (at concentration 250 μg/mL) respectively, while exhibiting high activity against marine bacteria. The IC50 of the extract was determined by a microdilution test and ranged from 5 μg/mL to 500 μg/mL. Callyspongia crassa is the most active among the Red sea sponges against Bacillus subtilis, with an LC50 18.2 ± 3.56 μg/mL, but was weak against Staphylococcus aureus with an LC50 215.2 ± 32.9 μg/mL [60]. Callyaerin A also exhibits antimicrobial activity against Escherichia coli and Staphylococcus aureus, with zones of inhibition of 10-15 mm and 9 mm, respectively, whereas callyaerin E has activity against Esch- Besides antiparasitic activity, the subgenus Callyspongia possesses antituberculosis activity resulting from callyaerins A and B ( Figure 5) isolated from Callyspongia aerizusa. Their MIC 90 values (2 µM and 5 µM, respectively) were less effective compared with the controls, rifampicin (<1 µM), ethambutol (1.25 µM), and isoniazid (0.625 µM). Beside the weaker activity compared with the control, there is no in vivo data to support this activity. These compounds inhibited the growth of Mycobacterium tuberculosis as evidenced by reduced cell viability using the resazurin dye reduction method and measuring cell fluorescence [105].
Callyspongia crassa extract potently inhibited Bacillus subtilis and Staphylococcus aureus with zones of inhibition of 16-25 mm (at concentration 500 µg/mL), 9-15 mm and 16-25 mm (at concentration 250 µg/mL) respectively, while exhibiting high activity against marine bacteria. The IC 50 of the extract was determined by a microdilution test and ranged from 5 µg/mL to 500 µg/mL. Callyspongia crassa is the most active among the Red sea sponges against Bacillus subtilis, with an LC 50 18.2 ± 3.56 µg/mL, but was weak against Staphylococcus aureus with an LC 50 215.2 ± 32.9 µg/mL [60]. Callyaerin A also exhibits antimicrobial activity against Escherichia coli and Staphylococcus aureus, with zones of inhibition of 10-15 mm and 9 mm, respectively, whereas callyaerin E has activity against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus, with zones of inhibition of 9-11 mm, 15-17 mm, and 9-10 mm, respectively [6].

Antiviral
Callyspongia crassa and Callyspongia siphonella extracts exhibited cytotoxic effects on Vero cells, which were cultured for the isolation and multiplication of enterovirus and hepatitis A virus, with MICs of 9.765 µg/mL and 0.625 µg/mL, respectively. The maximum non-toxic concentrations of these extracts were 4.9 and 0.3 µg/mL, respectively. Callyspongia crassa crude extract had an antiviral activity of 85.3%, whereas the antiviral activity of Callyspongia siphonella extract was 16.4% [34].

Immunomodulatory
Callyspongia extract at doses of 300 mg/kg and 400 mg/kg body weight, increased S. aureus-induced production of interferon-γ (IFN-γ) (455.265 pg/mL and 384.319 pg/mL) and tumor necrosis factor-α (TNF-α)(954 pg/mL and 1042 pg/mL) in male Wistar rats. It was more effective compared with 0.5% carboxymethyl cellulose sodium as negative control (160.314 pg/mL for INF-γ and 785.5 pg/mL for TNF-α) and bay leaf extract as positive control (353.486 pg/mL for INF-γ and 976 pg/mL for TNF-α) [68]. β-Sitosterol compounds from Callyspongia spp. modulate the activity of dendritic cells and increase the viability of peripheral blood mononuclear cells [67]. Siphonodiol, callyspongidiol, and 14,15-dihydrosphonodiol modulate the function of dendritic cells for T1 cell proliferation as well as IL-2 and IFN-γ production [66]. IL-2, along with other ILs, regulates innate and adaptive immunity by promoting an increase in the population of various immune cells [109,110]. Meanwhile, IFN-γ activates macrophages and enhances their immune response [111]. Callyspongia extract can stimulate the branch of the immune system involved in forming a receptor complex with gp130 to eventually inhibit the bioactivity of IL-6 ( Figure 10) [112,113]. Metabolites 2023, 13, x FOR PEER REVIEW 23 of 31 Figure 10. Immunomodulatory mechanisms of compounds from the subgenus Callyspongia.

Antineurodegenerative
• β-secretase 1 Selectively inhibiting β-secretase 1 in specific subcellular compartments is an effective strategy to reduce the accumulation of neurotoxic amyloid plaques [114]. The methanol extract of Callyspongia samarensis significantly and non-competitively inhibited βsecretase 1 (IC50 99.82 μg/mL). An acute oral toxicity test revealed that the extract was nontoxic, with an LD50 value of less than 2000 mg/kg. Moreover, an unknown compound in the extract, with a mass/charge ratio of 337.9 [M + H]+, was able to permeate the bloodbrain barrier, making it a suitable candidate for developing central nervous system drugs [31].

Antineurodegenerative
• β-secretase 1 Selectively inhibiting β-secretase 1 in specific subcellular compartments is an effective strategy to reduce the accumulation of neurotoxic amyloid plaques [114]. The methanol extract of Callyspongia samarensis significantly and non-competitively inhibited β-secretase 1 (IC 50 99.82 µg/mL). An acute oral toxicity test revealed that the extract was non-toxic, with an LD 50 value of less than 2000 mg/kg. Moreover, an unknown compound in the extract, with a mass/charge ratio of 337.9 [M + H]+, was able to permeate the blood-brain barrier, making it a suitable candidate for developing central nervous system drugs [31].

Antiosteoporotic
Neviotine A and D are isolated triterpene-type compounds from Callyspongia siphonella. These compounds possess antiosteoporotic activity by inhibiting receptor activator of nuclear factor-kB ligand (Rankl) with IC50 values of 32.8 μM and 12.8 μM (quercetin as positive control: 25 μg/mL) [69]. The interaction between Rankl and Rank receptor translocate the tumor necrosis factor receptor-associated factors (TRAF6) to the RANK cytoplasmic domain, results in the activation of ERK, p38, and JNK via activation of signaling cascades and downstream targets. Thus, AP-1 and NF-kB transcription factors were activated and stimulated the formation and activity of osteoclasts, which affect resorption activity [118]. Neviotine A and D inhibit cell differentiation into multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, which was upregulated via RANKL-induced osteoclastogenesis ( Figure 12) [69,119].

Antiosteoporotic
Neviotine A and D are isolated triterpene-type compounds from Callyspongia siphonella. These compounds possess antiosteoporotic activity by inhibiting receptor activator of nuclear factor-kB ligand (Rankl) with IC 50 values of 32.8 µM and 12.8 µM (quercetin as positive control: 25 µg/mL) [69]. The interaction between Rankl and Rank receptor translocate the tumor necrosis factor receptor-associated factors (TRAF6) to the RANK cytoplasmic domain, results in the activation of ERK, p38, and JNK via activation of signaling cascades and downstream targets. Thus, AP-1 and NF-kB transcription factors were activated and stimulated the formation and activity of osteoclasts, which affect resorption activity [118]. Neviotine A and D inhibit cell differentiation into multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, which was upregulated via RANKL-induced osteoclastogenesis ( Figure 12) [69,119].

Conclusions
In the 41 articles we reviewed, the pharmacological activities that Callyspongia spp. is reported to possess include cytotoxic against cancer cell line (36%), antifungal (10%), antiinflammatory (10%), immunomodulatory (10%), antidiabetic and antiobesity (6%), antimicrobial (8%), antioxidant (4%), antineurodegenerative (4%), antihypercholesterolemic (2%), antihypertensive (2%), antiparasitic (2%), antiallergic (2%), antiviral (2%), antiosteoporotic (2%), and antituberculosis (2%) activities ( Figure 13). The most studied pharmacological activity is cytotoxicity against cancer cell lines. Most of the research was limited to in vitro testing and there is insufficient in vivo data to support such activity. In addition, not all secondary metabolites responsible for certain activities have been identified. Several activities require modification and further study because of a lack of testing or low activity. For example, the antiallergic activity of Callyspongia sp. predicted from in silico results or the antioxidant, antituberculosis, and anti-inflammatory activities of Callyspongia extract were weaker compared with those of the control drugs. Although many promising compounds with a high potential to become drugs remain to be comprehensively evaluated in vivo, Callyspongia with its known mechanisms of action, such as antidiabetic and cytotoxic effects, may be further developed for targeted therapy.

Conclusions
In the 41 articles we reviewed, the pharmacological activities that Callyspongia spp. is reported to possess include cytotoxic against cancer cell line (36%), antifungal (10%), antiinflammatory (10%), immunomodulatory (10%), antidiabetic and antiobesity (6%), antimicrobial (8%), antioxidant (4%), antineurodegenerative (4%), antihypercholesterolemic (2%), antihypertensive (2%), antiparasitic (2%), antiallergic (2%), antiviral (2%), antiosteoporotic (2%), and antituberculosis (2%) activities ( Figure 13). The most studied pharmacological activity is cytotoxicity against cancer cell lines. Most of the research was limited to in vitro testing and there is insufficient in vivo data to support such activity. In addition, not all secondary metabolites responsible for certain activities have been identified. Several activities require modification and further study because of a lack of testing or low activity. For example, the antiallergic activity of Callyspongia sp. predicted from in silico results or the antioxidant, antituberculosis, and anti-inflammatory activities of Callyspongia extract were weaker compared with those of the control drugs. Although many promising compounds with a high potential to become drugs remain to be comprehensively evaluated in vivo, Callyspongia with its known mechanisms of action, such as antidiabetic and cytotoxic effects, may be further developed for targeted therapy.