Toxicogenic algae in the ocean can produce various toxins, which accumulate in marine organisms and enter the human body along the food chain. Among them, cyclic imines (CIs) are a novel class of lipophilic marine biotoxins, which are also known as “fast-acting toxins” due to their acute toxicity causing rapid death in mice injected intraperitoneally. As a typical kind of CIs, gymnodimines (GYMs) have been frequently detected in global waters in recent years. However, toxicological studies on GYMs have been limited due to the extreme scarcity of reference materials for GYMs, resulting in the uncertainty of their toxic effects on humans.

In this study, we established a technological process for the large-scale culture of GYM-producing algae using a photobioreactor, which can significantly increase the maximum growth density of the algae and shorten the growth cycle. The efficiency of toxin acquisition can be increased by 4 ~ 6 times compared with that of the traditional culture technique. Then, a multistep chromatographic separation technique was used to isolate and purify GYMs, and a certain amount of the GYM-A reference materials were obtained. The purity of GYM-A was 95.6% by quantitative nuclear magnetic resonance (qNMR), and the samples were homogeneous and stable at -20 ℃ for at least one year.

The neurotoxicity of GYM-A in mice was verified by intraperitoneal injection with an LD₅₀ of 86 μg kg^-1. Subsequently, the distribution of GYM-A in different tissues of mice was investigated, and it was found that GYM-A was predominantly distributed in the intestines and liver of mice, while the concentrations were higher in spleen and kidneys. Additionally, GYM-A was cytotoxic with concentration-dependent pattern to mouse mammary adenocarcinoma cells (4T1) and human colorectal adenocarcinoma cells (Caco-2), with mean IC₅₀ values of 1.39 and 2.79 μmol L^-1, respectively. GYM-A significantly induced apoptosis and oxidative stress in both cell lines, leading to the activation of caspase-3 and the collapse of mitochondrial membrane potential, suggesting that GYM-A can induce apoptosis via the mitochondrial pathway. The present study can effectively alleviate the current situation of the lack of GYM-A reference material in China, and provide technical support for the certification of GYM-A reference material. Meanwhile, the biological toxicity evaluation will provide a theoretical basis for the medicinal value development of GYMs.