|Budget Amount *help
¥15,940,000 (Direct Cost: ¥15,100,000、Indirect Cost: ¥840,000)
Fiscal Year 2007: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
Fiscal Year 2006: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 2005: ¥7,000,000 (Direct Cost: ¥7,000,000)
Xylem parenchyma cells of trees have an unique adaptation mechanism to subzero temperatures which is called as deep supercooling. In order to clarify mechanism of deep supercooling, supercooling capability of xylem crude extracts from several trees that contained deep supercooling xylem parenchyma cells, was measured by droplet freezing assay. The results showed that crude xylem extracts from these trees had anti-ice nucleation activity, which promotes supercooling of water droplets. Among tree species examined, xylem crude extracts from Katsura-tree showed highest anti-ice nucleation activity. Therefore, the causative substances for the anti-ice nucleation activity were analyzed by using Katsura-tree. As a result of purification by liquid-liquid extraction, silica gelcolumn chromatography and HPLC, existence of diverse kinds of numerous anti-ice nucleation substances in xylem of Katsura-tree was suggested. As a part of them, we could identify 4 kinds of flavonol glycosides with high a
nti-ice nucleation activity. These flavonol glycosides were quercetin-3-0-glucoside, kaempferol-7-0-glucoside, 8-methoxykaempferol-3-0-glucoside and kaempferol-3-0-glucoside with anti-ice nucleation activity of 2.8C, 9.0C, 3.4C and 4.0C, respectively. Among already-known anti-ice nucleation substances, anti-ice nucleation activity of kaempferol-7-0-glucoside was highest. This is first discovery that flavonol glucosides have anti ice nucleation activity. The accumulation of flavonoids in cytoplasm of XPCs was also comfirmed by fluorescence microscopy. These results strongly indicated that anti-ice nucleation substances had important role to supercooling of XPCs.
We confirmed that these anti-ice nucleation flavonoids could facilitate supercooling of 250cc water at-7.5C for 1 week, and applied to a abroad patent as "supercooling facilitating substances". Based upon such supercooling capability, these substances were applied for cold preservation of animal organs with successful I result for long time preservation of the function at supercooling state. We also used these substances to reduce concentration of vitrification solutions (VS2) for successful cryopreservation of biological tissues. Due to lowering concentration of VS2, successful preservation was achieved. We are currently considering application of these substances to wide variety of technologies that use "unfrozen water". Less