Central composite design-based response surface methodology for optimization of tetrodotoxin extraction from the horseshoe crab Carcinoscorpius rotundicauda

Tetrodotoxin analogues (TTXs) used in research and development were originated from biological sources for the last decades. Among TTXs-bearing aquatic animals, horseshoe crab Carcinoscorpius rotundicauda from Vietnam's Coast was reported to contain a high level of TTXs, indicating a promising source for extraction and purification of TTXs. In this work, horseshoe crab C. rotundicauda collected from Can Gio Coast, Vietnam was used for TTX extraction using homogenization-assisted method with acetic acid solvent. The extraction process was analysed and optimized by single-factor investigation and response surface methodology (RSM). Single-factor investigation pointed out that the optimal conditions for TTX recovery yield from C. rotundicauda's soft tissues were homogenization time of 20 min, acetic acid concentration of 1.0%, solvent/matrix ratio of 4 mL/g, extraction temperature of 70 & DEG;C, extraction time of 30 min and number of extraction of two times. The most significant factors including acetic acid concentration, solvent/matrix ratio and extraction temperature were utilized for constructing the RSM model to predict TTX recovery yield. The maximal TTX recovery yield predicted from RSM model was 97.3% under solvent/matrix ratio of 4 mL/g, extraction temperature of 70 & DEG;C and acetic acid concentration of 1% (v/v). The RSM model displayed a high stability and precision for prediction of TTX recovery yield from experiments of 1-10 kg C. rotundicauda's soft tissues/batch extractions producing low errors (-1.95% to 1.85%). TTXs in C. rotundicauda's soft tissues were elucidated by HPLC-MS/MS, revealing the presence of three analogues TTX, 4epi-TTX and Anh-TTX with their contents of 43.02, 22.54 and 45.07 & mu;g/g, respectively.