Impact of Co-Culture on the Metabolism of Marine Microorganisms.

Natural products from plants have been listed for hundreds of years as a source of biologically active molecules. In recent years, the marine environment has demonstrated its ability to provide new structural entities. More than 70% of our planet's surface is covered by oceans, and with the technical advances in diving and remotely operated vehicles, it is becoming easier to collect samples. Although the risk of rediscovery is significant, the discovery of silent gene clusters and innovative analytical techniques has renewed interest in natural product research. Different strategies have been proposed to activate these silent genes, including co-culture, or mixed fermentation, a cultivation-based approach. This review highlights the potential of co-culture of marine microorganisms to induce the production of new metabolites as well as to increase the yields of respective target metabolites with pharmacological potential, and moreover to indirectly improve the biological activity of a crude extract.

Purification and characterization of a novel thermostable anticoagulant protein from medicinal leech Whitmania pigra Whitman.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of cardiovascular disease (CVD) is increasing worldwide. Despite significant improvements in novel targeted treatment agents, natural products purified from medicinal animals with minimal side effects have attracted much attention. Several native proteins explored from suck-blood leeches, such as non-thermostable hirudin and its variants, revealed potent anticoagulant activity. Traditional Chinese medicine clinics have proved that non-suck-blood leech Whitmania pigra Whitman (W. pigra) also played notable roles in CVD treatments even after decoction. However, only a few natural proteins and peptides have been identified from the fresh material of this medicinal species. AIM OF THE STUDY: We aimed to purify and characterize thermostable anticoagulant proteins from W. pigra for further development of a therapeutic agent for thrombosis. MATERIALS AND METHODS: W. pigra crude extract was prepared by decoction in water. Anticoagulant proteins were purified by DEAE cellulose DE-52, Sephadex G-75, and reversed-phase liquid chromatography sequentially and analyzed by SDS-PAGE and LC-MS/MS for structural information. In addition, we conducted in vitro anticoagulant experiments, including plasma recalcification time (PRT) assay, fibrinolytic assay, activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (Fib) assay, and cell viability assays. Furthermore, a carrageenan-induced chronic thromboembolism model was employed in ICR mice, and four coagulation factors (APTT, PT, TT, and Fib) activities were determined after intragastric administration. RESULTS: The anticoagulant protein WP-77 has a relative molecular weight of ca. 20.8 kDa. It was effective over a broad temperature range from 20 °C to 100 °C and a pH 2-8 condition. The anticoagulant activity of WP-77 was retained after incubation with pepsin but was greatly inhibited by trypsin (P < 0.01). It significantly prolonged APTT and TT (P < 0.05) but had little effect on PT and Fib in vitro. Furthermore, WP-77 of a low concentration resulted in the recovery of injured EA.hy926 by thrombin. The protein also significantly prolonged APTT and TT (P < 0.01) and inhibited thrombus formation in carrageenan-induced thrombosis mice, demonstrating its antithrombotic effect in vivo. CONCLUSION: Our results suggest that WP-77 from W. pigra plays a distinct role in treating thrombotic diseases, and it is an essential substance of anticoagulant activity of non-suck-blood medicinal leeches. This thermostable anticoagulant protein could be a promising candidate for the development of clinical antithrombosis medicines.

Indonesia-Based Study of the Clinical and Cost-Saving Benefits of Subcutaneous Allergen Immunotherapy for Children with Allergic Rhinitis in Private Practice.

BACKGROUND: Until now, the cost of allergy treatment in the insured public health care system and the non-insured self-financing private health care system in Indonesia has not been well documented and published, as well as the cost of allergy treatment with subcutaneous immunotherapy. OBJECTIVE: To evaluate the clinical and cost benefits of allergic rhinitis treatment in children with subcutaneous immunotherapy in a non-insured self-financing private health care system. METHODS: A retrospective cohort study conducted from 2015 until 2020 that compared the clinical improvement and health care costs over 18 months in newly diagnosed AR children who received SCIT versus matched AR control subjects who did not receive SCIT, with each group consisting of 1098 subjects. RESULTS: A decrease in sp-HDM-IgE level (kU/mL) from 20.5 + 8.75 kU/mL to 12.1 + 3.07 kU/mL was observed in the SCIT group. To reduce the symptom score of allergic rhinitis by 1.0 with SCIT, it costs IDR 21,753,062.7 per child, and for non-SCIT, it costs IDR 104,147,878.0 per child. Meanwhile, to reduce the medication score (MS) by 1.0 with SCIT, it costs IDR 17,024,138.8, while with non-SCIT, it costs IDR 104,147,878.0. Meanwhile, to lower combination symptoms and medication score (CSMS) by 1.0, with SCIT, it costs IDR 9,550,126.6, while with non-SCIT, it costs IDR 52,073,938.9. CONCLUSIONS: In conclusion, this first Indonesia-based study demonstrates substantial health care cost savings associated with SCIT for children with AR in an uninsured private health care system and provides strong evidence for the clinical benefits and cost-savings benefits of AR treatment in children.

Anti-tumor effect of Huaier extract against neuroblastoma cells in vitro.

Huaier extract, the main active constituent proteoglycan, has anti-tumor activity in various experimental and clinical settings. However, the potential anti-neuroblastoma and associated mechanisms have not been investigated. Therefore, in this study, we aimed to elucidate the potential role of Huaier extract in 3 human neuroblastoma cell lines. Our study demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability in a dose-dependent manner. Huaier extract induced cell cycle arrest at G0/G1 phase in neuroblastoma and decreased the cell cycle related protein expression of cyclin D3. Western blotting analysis also showed that Huaier extract induced neuroblastoma cell apoptosis and autophagy. Signaling analysis indicated that Huaier extract suppressed the MEK/ERK and mTOR signaling pathways simultaneously. In conclusion, we verify that Huaier extract causes cell proliferation inhibition, apoptosis, autophagy, and cell cycle arrest in G0/G1 phase via MEK/ERK and mTOR signaling. Huaier extract may act as a complementary agent for treating neuroblastoma.

Polysaccharides from Pleurotus eryngii: Selective extraction methodologies and their modulatory effects on THP-1 macrophages.

Polysaccharides from P. eryngii mushroom were selectively extracted using low-cost technologies (water at different conditions of temperature and pressure). Mannogalactan was the main polysaccharide in cold-water extracted fraction (CWEF), while a linear (1→6)-ß-d-glucan was the main polymer in hot-water extracted fraction (HWEF). Autoclave-extracted fraction (AEF) contained a mixture of at least four different α- and ß-glucans. The report of linear (1→6)-ß-glucan and linear (1→3)-ß-glucan is a new finding for P. eryngii fruiting bodies. The immunostimulatory properties of the fractions on THP-1 macrophages were studied. All fractions at 50, 250 and 500 µg/mL were not cytotoxic and produced different stimulus on NO, IL-1ß and IL-10 secretion by the cells. Thus, our results showed that it is possible to concentrate different P. eryngii polysaccharides in selected fractions using a simple and low-cost procedure. Since biological effects depends on the polysaccharide structure, this technique allows the obtainment of fractions with distinct immunomodulatory activities.

Agaricus bisporus Crude Extract: Characterization and Analytical Application.

In the present work crude Agaricus bisporus extract (ABE) has been prepared and characterized by its tyrosinase activity, protein composition and substrate specificity. The presence of mushroom tyrosinase (PPO3) in ABE has been confirmed using two-dimensional electrophoresis, followed by MALDI TOF/TOF MS-based analysis. GH27 alpha-glucosidases, GH47 alpha-mannosidases, GH20 hexosaminidases, and alkaline phosphatases have been also detected in ABE. ABE substrate specificity has been studied using 19 phenolic compounds: polyphenols (catechol, gallic, caffeic, chlorogenic, and ferulic acids, quercetin, rutin, dihydroquercetin, l-dihydroxyphenylalanine, resorcinol, propyl gallate) and monophenols (l-tyrosine, phenol, p-nitrophenol, o-nitrophenol, guaiacol, o-cresol, m-cresol, p-cresol). The comparison of ABE substrate specificity and affinity to the corresponding parameters of purified A. bisporus tyrosinase has revealed no major differences. The conditions for spectrophotometric determination have been chosen and the analytical procedures for determination of 1.4 × 10-4-1.0 × 10-3 M l-tyrosine, 3.1 × 10-6-1.0 × 10-4 M phenol, 5.4 × 10-5-1.0 × 10-3 M catechol, 8.5 × 10-5-1.0 × 10-3 M caffeic acid, 1.5 × 10-4-7.5 × 10-4 M chlorogenic acid, 6.8 × 10-5-1.0 × 10-3 M l-DOPA have been proposed. The procedures have been applied for the determination of l-tyrosine in food supplements, l-DOPA in synthetic serum, and phenol in waste water from the food manufacturing plant. Thus, we have demonstrated the possibility of using ABE as a substitute for tyrosinase in such analytical applications, as food supplements, medical and environmental analysis.

Development of an innovative methodology combining chemical fractionation and in vivo analysis to investigate the biological properties of cheese.

With the ever-increasing human lifespan, age-related affections have become a public health issue. The health sector is looking for new bioactive compounds to respond to this demand. The unexplored microbial biodiversity and its metabolites represent a major source of innovative bioactive molecules with health potential. Fermented foods, such as raw-milk cheese, have already been investigated for their rich microbial environment, especially for their organoleptic qualities. But studies remain limited regarding their effects on health and few metabolites of microbial origin have been identified. An efficient methodology was developed in this study to investigate the biological effect of raw-milk cheese, combining a chemical fractionation, to isolate the most metabolites from the cheese matrix, and an in vivo biological test using Caenorhabditis elegans. C. elegans was brought into contact with cheese extracts, obtained by means of chemical fractionation, and with freeze-dried whole cheese by supplementing the nematode growth medium. A longevity assay was performed to evaluate the effects of the extracts on the worms. Our results demonstrate the feasibility of the method developed to bring the worms into contact of the cheese extracts. The evaluation of the effects of the extracts on the longevity was possible. Some extracts showed a beneficial effect as extract W70 for example, obtained with water, which increases the mean lifespan by 16% and extends the longevity by 73% (p < 0.0001).

Colletotrichalactones A-Ca, unusual 5/6/10-fused tricyclic polyketides produced by an endophytic fungus, Colletotrichum sp. JS-0361.

Three unusual polyketides with a 5/6/10-fused ring system, named colletotrichalactones A-Ca (1-3a), were isolated from cultures of the endophytic fungus, Colletotrichum sp. JS-0361, which was isolated from a leaf of Morus alba. Their structures, including their absolute stereochemistries, were completely established using extensive spectroscopic methods together with a chemical reaction utilizing competing enantioselective acylation coupled with LC/MS. Compounds possessing this ring skeleton were previously reported in three studies. Our rigorous chemical investigation revealed the complete configuration of this skeleton, which agreed with the results for glabramycin B with this ring skeleton established by computational chemistry and enantioselective synthesis in previous reports. 1 and 2 had unstable aldehyde groups that were easily converted to acetal groups in the presence of solvents. Meanwhile, compound 3a, with terminal acetal functionality, was deduced to be an artefact originating from compound 3 with a terminal aldehyde group. Compounds 1 and 3a displayed moderate-to-potent cytotoxic activities against MCF7 cells with IC50s of 35.06 and 25.20 µM, respectively.

Identification of a Novel Pyrrole Alkaloid from the Edible Mushroom Basidiomycetes-X (Echigoshirayukidake).

Three pyrrole alkaloid derivatives were isolated from the edible mushroom Basidiomycetes-X (Echigoshirayukidake) by water extraction followed by ethyl acetate fractionation. The chemical structures determined by MS and NMR were 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid (compound I), 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanamide (compound II), and 5-(hydroxymethyl)-1H-pyrrole-2-carboxaldehyde (compound III). Compound I was found to be the major component, followed by compound II, and compound III was the minor component. The dry powder of Basidiomycetes-X contained approximately 825 µg g-1 compound I and 484 µg g-1 compound II. Compound II was found to be a novel pyrrole aldehyde homologue not previously reported and thus is a specific component of this mushroom.

Amaranthus tricolor crude extract inhibits Cronobacter sakazakii isolated from powdered infant formula.

The purpose of this study was to elucidate the antibacterial activity and possible mechanism of action of Amaranthus tricolor crude extract (ATCE) against Cronobacter sakazakii isolated from powdered infant formula (PIF). The antibacterial activity of ATCE was assessed by measuring the diameter of inhibition zone (DIZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The possible mechanism of action of ATCE was revealed by analyzing the effects of ATCE on growth curves and changes in cell membrane potential, intracellular pH, content of bacterial protein and genomic DNA, and cell morphology. Finally, ATCE was applied to the disinfection of C. sakazakii in biofilm on stainless steel tube. The results showed that the DIZ, MIC, and MBC of ATCE against C. sakazakii strains were from 14.35 ± 0.67 to 14.84 ± 0.67 mm, 20 mg/mL, and 40 mg/mL, respectively. Treatment with ATCE ended the logarithmic growth phase of C. sakazakii, and led to depolarization of the cell membranes, reducing intracellular pH and bacterial protein and genomic DNA contents, and resulting in cytoplasmic leakage and deformation. In addition, ATCE effectively inactivated C. sakazakii in biofilm, reducing viable bacteria by approximately 6.5 log cfu/mL bacterial count after treatment with 1 MIC (1 MIC = 20 mg/mL) of ATCE for 20 min at 25°C. Our findings showed that ATCE inactivated C. sakazakii strains isolated from PIF and has potential as a natural disinfectant to reduce the contamination of PIF by C. sakazakii.

Inhibitory effects of Lentinus edodes mycelia polysaccharide on α-glucosidase, glycation activity and high glucose-induced cell damage.

At present, diabetes and diabetic complications have become one of the serious diseases affecting human health. In this study, the inhibitory effects of Lentinus edodes mycelia polysaccharide (LMP) on α-glucosidase activity, the formation of advanced glycation end products (AGEs) and high glucose-induced human umbilical vein endothelial cells (HUVECs) damage were explored. The interaction between LMP and α-glucosidase and the inhibition against AGEs formation were investigated with spectroscopic techniques. The results revealed that LMP had a reversible inhibition on α-glucosidase activity in a mixed-type manner. When the concentration of LMP was 2.7 mM, the inhibition rate was 34.38 %. LMP quenched the fluorescence of α-glucosidase through the static quenching and formed the LMP-α-glucosidase complex. At 310 K, the number of binding sites (n) and binding constant (Kb) were 1.01 and 3.71 × 104 L mol-1, respectively. In addition, LMP could inhibit the formation of AGEs. Compared with 40 mM glucose treatment group, treatment with 0.05 mM LMP for 48 h increased the cell viability from 70.17% to 91.14% and decreased ROS production from 3.33-fold to 1.21-fold. LMP inhibited high glucose-induced activation of MAPK signaling pathways. These findings may promote the application of LMP in the functional food industry.

Trametes robiniophila Murr in the treatment of breast cancer.

Breast cancer is the leading cause of cancer death among women across the world. Trametes robiniophila Murr (Huaier), a traditional herbal medicine, has been used in China to protect human health for about 1600 years. Recent years, Huaier had been proven to be effective for multiple types of malignancies. This systematic review focused on breast cancer treatment, summarizing the curative function of Huaier aqueous extract and polysaccharides in preclinical researches. Huaier could markedly inhibit breast cancer progression with low toxicity, enhance immune response and increase the sensitivity to radiation and chemotherapy. The therapeutic effect of Huaier granule in clinical studies was also included. This review amalgamated the current studies and highlighted the promising role of Huaier and its polysaccharides as complementary alternative medicine in breast cancer treatment.

Antibacterial Activity of Fruiting Body Extracts from Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes) against Oral Pathogen Streptococcus mutans.

The edible medicinal mushroom Flammulina velutipes (enokitake) has many applications as food and medicine, but its application in dentistry is unknown. This study aims to investigate the inhibitory effect of fruiting body extracts from F. velutipes on the growth and adhesion of Streptococcus mutans, the main cause of human caries, in vitro. Of the four extracts (named TG01 from water, TG02 from 95% ethanol, TG03 from 50% ethanol, and TG04 from ethyl acetate), TG03 had significant antibacterial activity (MIC = 10 mg/mL; MBC = 20 mg/mL). Planktonic growth and biofilm formation in S. mutans was repressed by TG03 at 5 mg/mL and above. Meanwhile, cytotoxicity analysis showed that TG03 was not toxic to human oral keratinocyte cells. HPLC-QQQ-MS analysis showed that the TG03 extract contained a large amount of arabitol, a sucrose substitute that reduces the development of caries. Thus, F. velutipes extracts can effectively inhibit the growth of the oral pathogen S. mutans without cytotoxicity against human oral keratinocytes. Therefore, F. velutipes is a good candidate for the development of oral hygiene agents to control dental caries.

Antioxidant and Genotoxic Effects of Aqueous and Methanol Extracts from Two Edible Mushrooms from Turkey in Human Peripheral Lymphocytes.

This study reported the genetic and oxidative effects of aqueous and methanol extracts from two edible mushrooms, Lepista nuda (Bull.) Cooke and Pleurotus ostreatus (Jacq.) P. Kummer, in cultured human lymphocytes. Chromosome aberration (CA) and micronucleus (MN) assays were used for genotoxic influences estimation. In addition, the changes of total antioxidant capacity (TAC) and total oxidative stress (TOS) in the cells were monitored. The fungal extracts at all applied concentrations did not indicate significant differences (p > 0.05) in CA and MN analyses. Furthermore, while the treatments with maximum concentration of aqueous extract of L. nuda statistically (p < 0.05) increased TAC especially, TOS levels in the cells were reduced by them in comparison with negative control. Based on TAC analysis, low IC50 values belonging to aqueous (5.43 mg/L) and methanol (10.88 mg/L) extracts of L. nuda were remarkable. Our data demonstrated that the extracts obtained from P. ostreatus and especially L. nuda can be a new resource for therapeutics with their nonmutagenic and antioxidant features.

Antioxidant and Neuroprotector Influence of Endo-Polyphenol Extract from Magnesium Acetate Multi-Stage Addition in the Oak Bracket Medicinal Mushroom, Phellinus baumii (Agaricomycetes).

The objective of this study was to explore the effect of magnesium acetate (MA) addition on the endo-polyphenol yield by Phellinus baumii and establish a feasible additive strategy. The optimal three-point MA addition strategy (0.05 g/L concentration of MA added at 0 h and 6 h, 0.9 g/L concentration of MA added at 12 h) was employed to obtain maximum endo-polyphenol yield. The maximum endo-polyphenol production was reached at 1.22 g/L, which was 1.39-fold higher than that of the control. Additionally, the endo-polyphenol showed stronger antioxidant activity in vitro compared with the control, including DPPH· scavenging capacity (78.76%) and Trolox equivalent antioxidant capacity (TEAC) (32.28 µmol Trolox/g sample). HPLC analysis showed that the endo-polyphenol production of the crude ethanol extracts was significantly higher than that of the control. Hispidin was isolated and identified from the ethanol extract of the culture mycelia from Ph. baumii with the three-point MA addition strategy. Hispidin showed a strong ability to scavenge DPPH free radicals and TEAC, equivalent to positive (vitamin C) value of 89.41% and 75.98%, respectively. Furthermore, hispidin protected H2O2-induced PC12 cells injured by decreased oxidative stress level. These results indicated that the MA multi-stage addition strategy was dependable, and could be used to develop new natural antioxidants for foods or medicines.

Antitumor Effects of Extract of the Oak Bracket Medicinal Mushroom, Phellinus baumii (Agaricomycetes), on Human Melanoma Cells A375 In Vitro and In Vivo.

Melanoma is among the most aggressive and treatment-resistant human cancers. Phellinus baumii, a famous medicinal mushroom, has been used to treat different diseases, including cancer, in China and other east Asian countries. The purpose of this research was to explore its anticancer effects against melanoma, and the mechanisms that might be involved. CCK-8 assay exhibited that extracts of Ph. baumii (EPB) strongly inhibited cell viability of A375 melanoma cancer cell. Typical morphological changes of cell apoptosis were observed in EPB-treated A375 cells in Hoechst staining assay. Flow cytometry analysis indicated that EPB significantly induced A375 cells apoptosis and the cell cycle was disrupted in S phase. EPB increased the expression of Bax, and decreased Bcl-2 in A375 cells. EPB remarkably caused mitochondrial membrane potential collapse and induced a mitochondrial-dependent apoptosis in A375 cells evidenced by caspase-3 activation, followed by PARP cleavage. More importantly, EPB has shown a strong inhibitory effect on the migration and aggression of the A375 cells through the healing of the wound and transwell assay. In vivo, EPB was also found to strongly inhibit the growth of tumors in BALB/c nude mice. Our results indicated that Ph. baumii might be a natural therapeutic product for aggressive melanoma because it could induce apoptosis and inhibit metastasis in A375 cells.

Advantageous use of SFC for separation of crude therapeutic peptides and peptide libraries.

A simple SFC/MS (Supercritical Fluid Chromatography/Mass Spectrometry) method set was developed to effectively screen separations of various crude synthetic peptide products of pharmaceutical interest. Additives to the modifier methanol which were successful for these separations were found to include TFA (trifluoroacetic acid) and ammonia mixed with TFA, each at 0.1 % (v/v) composition in methanol. A final screening column set consisted of 2-ethylpyride (2-EP), 4-ethylpyridine (4-EP) and cross-linked diol (Luna™ HILIC) stationary phases. Small, linear and macrocyclic peptides with fewer than ten residues could all be eluted with good performance under at least one of the method conditions comprising the final screening protocol. For larger peptides either the 4-EP and HILIC columns with the above additives provided a good initial screening method set without 2-EP. The gradient was slightly longer and shifted to higher polarity relative to the gradients for smaller peptides. This method was often successful to elute large, hydrophilic peptides up to a 41-mer with acceptable peak profiles although the largest peptides with most ionizable residues were most challenging. For these peptides generally HILIC column performance was better with TFA + ammonia in the modifier than with TFA, while 4-EP performance was usually improved with TFA relative to TFA + ammonia.

Effects of a pyroglutamyl pentapeptide isolated from fermented barley extract on atopic dermatitis-like skin lesions in hairless mouse.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic and eczematous skin lesions. The skin of AD patients is generally in a dried condition. Therefore, it is important for AD patients to manage skin moisturization. In this study, we examined the effects of orally administered fermented barley extract P (FBEP), which is prepared from a supernatant of barley shochu distillery by-product, on stratum corneum (SC) hydration and transepidermal water loss (TEWL) in AD-like lesions induced in hairless mice using 2,4,6-trinitrochlorobenzene. Oral administration of FBEP increased SC hydration and decreased TEWL in the dorsal skin of this mouse model. Further fractionation of FBEP showed that a pyroglutamyl pentapeptide, pEQPFP comprising all -L-form amino acids, is responsible for these activities. These results suggested that this pyroglutamyl pentapeptide may serve as a modality for the treatment of AD.

Activity of Antioxidant Defense Enzymes in Rats with Experimental Allergic Encephalomyelitis.

We studied activities of antioxidant system enzymes in tissues of rats with experimental allergic encephalomyelitis. It was shown that the development of pathology is accompanied by deformation of the neurons and axonal degeneration, intensification of free radical oxidation, exhaustion of the reduced glutathione pool, and multidirectional changes in activities of antioxidant enzymes in rat tissues. The observed imbalance in the antioxidant defense system can be associated with excessive glutathione utilization in the glutathione transferase reaction and different severity of the pathological process in the brain and spinal cord. The received data necessitate the search for compounds that can prevent inhibition of antioxidant system components in order to analyze the possibility of their use in the treatment of multiple sclerosis.

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

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.