Isolation, Identification, and Quantitative Determination of Saponin in Apostichopus japonicus by HPLC-DAD

Apostichopus japonicus is an important invertebrate that is widely used as a tonic food in Asian countries. The purpose of this study is to purify and identify a class of compound, the saponins, from the body wall of A. japonicus , and to establish a new method to determine the quantity of saponins in the sea cucumber. In this study, the saponins of A. japonicus , cladoloside A (CA), were obtained from 80% ethanol extract by column chromatography for the first time and were characterized using the spectral method. The resulting purified saponins were then profiled using 1 HNMR, 13 CNMR, and ESI-MS, which revealed the CA molecular formula of C53H82O2 and contained a triterpenoid backbone, a methylglucopyranosyl moiety, a quinovopyranosyl, and two xylopyranosyls. A method for the quantitative determination of CA, comprising microwave-assisted extraction, high-performance liquid chromatography, and diode array detector method, was established. Extraction efficiency was optimized by changing microwave power, extraction solvent, volume, time, and temperature. Results showed that under the optimum conditions (extraction time of 10 min, temperature of 45°C, and solvent of 25 mL 70% ethanol under 400 W), the detection limit of CA was 0.0015 mg mL −1 , and the recoveries of CA from samples at spiking levels of 10, 20, and 50 µg g −1 ranged from 90.1%–104%. The proposed method was successfully applied to analyze the saponins in different tissues of A. japonicus collected in different seasons. The method developed in this study can provide quantitative technical support for the quality control of A. japonicus .