Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near -40°C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree (Cercidiphyllum japonicum), of which XPCs exhibited deep supercooling to -40°C, were prepared by methanol extraction. The crude extracts were purified by liquid-liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8-9.0°C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-

Deep supercooling xylem parenchyma cells (XPCs) of katsura tree (Cercidiphyllum japonicum) contain four kinds of flavonol glycosides with high supercooling-facilitating (anti-ice nucleation) activities. These flavonol glycosides have very similar structures, but their supercooling-facilitating activities are very different. In this study, we analyzed the supercooling-facilitating activities of 12 kinds of flavonol glycosides in order to determine the chemical structures that might affect supercooling-facilitating activity. All of the flavonol glycosides tested showed supercooling-facilitating activity, although the magnitudes of activity differed among the compounds. It was clear that the combination of the position of attachment of the glycosyl moiety, the kind of attached glycosyl moiety and the structure of aglycone determined the magnitude of anti-ice nucleation activity. However, there is still some ambiguity preventing the exact identification of features that affect the magnitude of supercooling-facilitating activity.

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