| 其他摘要 | Sea cucumbers are soft-bodied, slow-moving echinoderms that typically inhabit relatively short distances in a suitable environment. When stressed, sea cucumbers, particularly the species Apostichopus japonicus, rapidly expel their viscera as defense, potentially increasing toxin levels in the surrounding environment and enhancing their chemical defense efficiency. To acquire the toxic secondary metabolites, we conducted a comprehensive analysis to identify and characterize the chemical composition of the viscera of A. japonicus. The secondary metabolites were isolated through a series of techniques, including silica gel chromatography, reversed-phase C18, Sephadex LH-20, preparative thin-layer chromatography, and semi-preparative high-performance liquid chromatography. The aglycone of holostane-type triterpene glycosides was obtained through acid hydrolysis. The type and absolute configuration of carbohydrates were determined by GC-MS. A total of 60 compounds were identified through a combination of various spectrometric and spectroscopic techniques, including 1H NMR, 13C NMR, 1H-1H COSY, NOESY, HSQC, HMBC, HR-ESI-MS, and ESI-MS/MS. Among the identified compounds, there were 19 triterpene glycosides, with compounds 1 and 2 representing non-holostane type triterpene glycosides, and compounds 3 to 19 comprising holostane type triterpene glycosides, one holostane type aglycone, nine steroids, four sulfate esters, one dipeptide, fourteen nucleosides, three monosaccharides, and nine nitrogenous compounds. Notably, eleven new compounds were determined, including ten new triterpene glycosides and one novel sulfated olefinic.
The structure of ten new triterpene glycosides were elucidated as 3ꞵ-O-[ꞵ-D-glucopyranosyl-(1→2)-ꞵ-D-xylopyranosyl]-20-hydroxylanosta-7(8),25(26)-diene-18(16)-lactone (1), 3ꞵ-O-[ꞵ-D-quinovopyranosyl-(1→2)-ꞵ-D-xylopyranosyl]-20-hydroxylanosta-7(8),25(26)-diene-18(16)-lactone (2), 3ꞵ-O-[ꞵ-D-glucopyranosyl-(1→2)-ꞵ-D-xylopyranosyl]-holosta-9(11),25(26)-dien-16-one (3), 3ꞵ-O-[ꞵ-D-quinovopyranosyl-(1→2)-ꞵ-D-xylopyranosyl]-holosta-9(11),25(26)-dien-16-one (4), 3β-O-{2-O-[β-D-quinovopyranosyl]-4-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-β-D-xylopyranosyl}holosta-9(11),25(26)-dien-16-one (5), 3β-O-{2-O-[β-D-glucopyranosyl]-4-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-9(11),25(26)-dien-16-one (6), 3β-O-{2-O-[β-D-glucopyranosyl-(1→4)-β-D-quinovopyranosyl]-4-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-9(11),25(26)-dien-16-one (8), 3β-O-{2-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl]-4-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-9(11),25(26)-dien-16-one (9), 3β-O-{2-O-[β-D-xylopyranosyl-(1→4)-β-D-glucopyranosyl]-4-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-9(11),25(26)-dien-16-one (10), 3β-O-{2-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl]-4-O-[3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-glucopyranosyl]-β-D-xylopyranosyl}-holosta-9(11),25(26)-dien-16-one (12).
The toxicity of triterpene glycosides isolated from the viscera of A. japonicus was evaluated by hemolytic activity and zebrafish embryotoxicity. The results of hemolytic activity revealed that holostane type triterpene glycosides containing six sugar units possessed strong hemolytic activity, with LC50 values ranging from 0.54 to 3.95 μM. The holostane type triterpene glycosides with five sugar units possessed moderately strong hemolytic activity, with LC50 values ranging from 4.48 to 10.49 μM. The holostane type triterpene glycosides with four sugar units possessed relatively weak hemolytic activity, with LC50 values ranging from 13.7 to 21.2 μM. The holostane type triterpene glycosides with two sugar units, the non-holostane type triterpene glycosides with two sugar units, and aglycone showed no hemolytic activity at a concentration of 100 μM. In the zebrafish embryotoxicity test, it showed that the holostane-type triterpene glycosides with four, five, and six sugar units exerted strong toxic effects on zebrafish embryos (96 hpf-LC50 0.091‒0.536 μM). Non-holostane type triterpene glycoside compound 1 exposure showed a moderately strong toxic effect on zebrafish embryos (96 hpf-LC50 41.5 μM). Non-holostane type triterpene glycoside 2 and aglycone exhibited no mortality and moderate teratogenic toxicity to zebrafish embryos at a concentration of 100 μM. The structure-activity relationships between triterpene glycosides and toxicity showed that the number, type, and linkage of sugar units to aglycone in triterpene glycosides were critical for both hemolytic activity and zebrafish embryotoxicity.
Holotoxin A1, the most abundant triterpene glycoside found in the viscera of A. japonicus, exhibits strong hemolytic activity and zebrafish embryotoxicity. In this study, we utilized Holotoxin A1 to investigate the neurotoxic mechanism in zebrafish embryos. Our findings demonstrated a significant inhibition of acetylcholinesterase (AChE) activity in zebrafish larvae. Furthermore, the expression levels of gap43 were markedly upregulated, while the expression levels of elavl3, gfap, syn2a, and α1-tubulin were all significantly down-regulated after being treated with triterpene glycosides in zebrafish larvae. These results strongly confirm that triterpene glycoside exposure treatment induces developmental toxicity and neurotoxicity in zebrafish larvae.
This study aimed to evaluate the hemolytic activity and zebrafish embryotoxicity of triterpene glycosides, elucidate the relationship between the chemical structure and toxic effects of triterpene glycosides, screen the most active triterpene glycoside, Holotoxin A1, and explore the mechanism of neurotoxicity on zebrafish embryos. By combining chemical and toxicological methodologies, we examined the potential toxic effects of triterpene glycosides in chemical defense, providing scientific foundations for understanding the putative chemical defense role of triterpene glycosides for A. japonicus. |
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