Anti-Fouling Effects of Saponin-Containing Crude Extracts from Tropical Indo-Pacific Sea Cucumbers

Abstract

Sea cucumbers are bottom dwelling invertebrates, which are mostly found on subtropical and tropical sea grass beds, sandy reef flats, or reef slopes. Although constantly exposed to fouling communities in these habitats, many species are surprisingly free of invertebrate epibionts and microfouling algae such as diatoms. In our study, we investigated the anti-fouling (AF) activities of different crude extracts of tropical Indo-Pacific sea cucumber species against the fouling diatom Cylindrotheca closterium. Nine sea cucumber species from three genera (i.e., Holothuria, Bohadschia, Actinopyga) were selected and extracted to assess their AF activities. To verify whether the sea cucumber characteristic triterpene glycosides were responsible for the observed potent AF activities, we tested purified fractions enriched in saponins isolated from Bohadschia argus, representing one of the most active anti-fouling extracts. Saponins were quantified by vanillin-sulfuric acid colorimetric assays and identified by LC-MS and LC-MS/MS analyses. We were able to demonstrate that AF activities in sea cucumber extracts were species-specific, and growth inhibition as well as attachment of the diatom to surfaces is dependent on the saponin concentration (i.e., Actinopyga contained the highest quantities), as well as on the molecular composition and structure of the present saponins (i.e., Bivittoside D derivative was the most bioactive compound). In conclusion, the here performed AF assay represents a promising and fast method for selecting the most promising bioactive organism as well as for identifying novel compounds with potent AF activities for the discovery of potentially novel pharmacologically active natural products.

Chemical Defense Mechanisms and Ecological Implications of Indo-Pacific Holothurians

“…Furthermore, we observed that among the two relatively heavy saponin molecules isolated from B. argus , bivittoside D ( m / z 1426.698) showed stronger cytotoxic effects and deterred fish feeding significantly, whereas bivittoside C ( m / z 1410.703) did not reveal antifeeding activities ( Figure 10 ). Similarly, in a previous study, we observed that the same saponin molecule (i.e., bivittoside D ) showed a strong antifouling activity [ 40 ], indicating that the presence/absence of a particular functional group within the saponin molecule considerably affects its bioactivity. To assess the differences in chemical defenses between the different sea cucumber families in more detail, we looked at the bioactivities of each family separately.…”

“…Preliminary antimicrobial screening of each SPE fraction from the three most active sea cucumbers showed that the SPE Fraction B and Fraction C (see above) of B. argus had pronounced activities and were further purified using semi-preparative HPLC (Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA) equipped with a PDA detector (Agilent, G4212-60008, Santa Clara, CA, USA). Thus, as described in Kamyab et al, 2020 [ 40 ], we used a C 18 column (Pursuit XRs 5 µm, 250 mm × 10 mm, Agilent, Santa Clara, CA, USA) with a pre-column (2.7 µm, 2.1 mm × 5 mm, Agilent, Santa Clara, CA, USA). The two eluents of “A”, consisting of 95% H 2 O and 0.1% of formic acid 98% (Carl Roth GmbH), and “B” consisting of ACN and 0.1% formic acid, were used to provide a linear gradient of initial A:B (50%:50%).…”

“…Following methods described in a previous study [ 40 ], we analyzed the major saponin compounds in the butanol fractions of the three most active sea cucumber crude extracts, namely B. argus, H. fuscopunctata and S. chloronotus . In brief, an aliquot of each butanol fraction and purified compound was analyzed by ultra-performance liquid chromatography–high resolution mass spectrometry (UPLC-HRMS) using a Waters Acquity UPLC BEH C 18 column (1.7 µm, 2.1 mm × 50 mm) for chromatographic separation.…”

“…The above described morphological and behavioral defense mechanisms are often combined with chemical defenses via production of bioactive molecules [ 33 ]. These molecules have been shown to act not only as antipredatory defense but also as potent antimicrobial and antifungal compounds [ 34 , 35 , 36 , 37 , 38 , 39 , 40 ] that can control associated microbial communities or prevent pathogenic attacks [ 37 , 41 ]. Among the wide spectrum of bioactive compounds present in several orders of sea cucumbers (i.e., Holothuroidea, Echinodermata; [ 34 , 42 , 43 ]), triterpene glycosides (or saponins) are a potent and prominent example for such bioactive defense molecules.…”

Triterpene Glycosides from the Far Eastern Sea Cucumber Thyonidium (=Duasmodactyla) kurilensis (Levin): The Structures, Cytotoxicities, and Biogenesis of Kurilosides A3, D1, G, H, I, I1, J, K, and K1

“…The investigations of the triterpene glycosides from different species of sea cucumbers have a range of goals. Among them are the drug discoveries based on the promising candidates, demonstrating the target bioactivity [ 1 , 2 , 3 , 4 , 5 , 6 ], the solving of some taxonomic problems of the class Holothuroidea based on the specificity of the glycosides having characteristic structural peculiarities for the certain systematic groups [ 7 , 8 , 9 , 10 ], the ascertaining of biologic and ecologic functions of these metabolites [ 11 , 12 , 13 , 14 , 15 ], and the discovery of novel compounds, especially minor ones, that can be the “hot metabolites” clarifying the biosynthetic pathways of triterpene glycosides [ 16 , 17 , 18 ].…”