Separation and Characterization of Chlorophyll Degradation Products in Silkworm Using HPLC‐UV‐APCI‐MS

Abstract

By use of HPLC‐UV‐APCI‐MS, the chemical nature of chlorophyll degradation products (CpD) in the excreta of the silkworm (Bombyx mori) was clarified in relation to its photosensitizing efficacy in photodynamic therapy (PDT) of tumors in vitro. Besides CpD from silkworm excreta aged in nature for more than a year, mulberry leaves, substances in the digestive system of silkworms fed on the mulberry leaves, and fresh excreta less than 24 hrs old were analyzed and compared. The CpDs isolated were separated and characterized, and their photosensitizing efficacy in experimental PDT was evaluated. Among the agents characterized by HPLC‐UV‐APCI‐MS, pheophorbide‐a and pyropheophorbide‐a exerted the most potent photosensitizing effect in vitro. Although these pigments were detected in silkworm excreta, the amounts extracted were significantly higher in materials aged for more than one year. Yields from fresh excreta or digestive tract contents were lower than from stored excreta. None of those CpDs were detected in mulberry leaves. Natural environments presumably provide favorable conditions for the degradation of chlorophyll to pheophorbide‐a and/or pyropheophobide‐a.

Keywords:

• Chlorophyll degradation
• Silkworm excreta
• Pheophorbide‐a
• Pyropheophorbide‐a
• Photosensitizing effect
• Photodynamic therapy (PDT)
• HPLC‐MS

Acknowledgments

This work was supported by grant No. 20000068 from the National Institute of Agricultural Science and Technology. The authors thank Dr. Jong‐Shin Yoo and Dr. Jin‐Young Kim for the use of a mass spectrometer from the Korea Basic Science Institute, by the Ministry of Science and Technology of the Republic of Korea.