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(1)hon p.1 [100%]. YAKUGAKU ZASSHI 131(10) 1519―1525 (2011)  2011 The Pharmaceutical Society of Japan. 1519. Article. 学生実習における卵黄を用いたリポソーム調製の試み  . Preparation of Liposome Using Egg Yolk Ikumi SUGIYAMAand Yasuyuki SADZUKA School of Pharmacy, Iwate Medical University, 211 Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate 0283694, Japan （Received January 24, 2011; Accepted July 20, 2011; Published online July 26, 2011） In this study, 162 students in the 6 year Pharmacy Program at the School of Pharmacy, Iwate Medical University were asked to prepare liposomal preparations using chicken egg yolk and to evaluate their properties with the aim of developing novel liposomes. High-purity lecithins are generally used for preparing liposomes but they are expensive. On the other hand, egg yolk has various components, including lecithin and cholesterol, which are important components for the formation of liposomes, so it was hypothesized that liposomes prepared from egg yolk may participate in the formation of cell membrane in chicks. Both liposomes from egg yolk (YL) and from lecithin (LL) exhibited Malthesian crosses using a polarizing microscope and multilamellar vesicles were observed, conˆrming that liposomal preparations from egg yolk were useful. The particle size of YL was about 100 nm with one peak. Furthermore, the YL are believed to be viable under diŠerent conditions because the particle size did not change when they were prepared in buŠers having diŠerent pH values. The results of these experiments indicate that liposomal preparations from egg yolk can serve as natural materials, although some obstacles remain. This is a unique approach for carrying out practical training in our 6 year pharmaceutical science program. Key wordsliposome; egg yolk; polarized light; Malthesian cross. 緒. . 言. _;"uv35"k. 3" /WM

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(13) 3 ßSü _s". 法. w5 W]^4/Lecithin-lipo-. W / Lecithin from. some: LL 0 )6 LL 35pQ[3 9.0. )6S¯3!. V/ 10 mM W(XY4/ pH 4.0 0Ze. )6Òò dSü. þ)" # W$ S "ü%" )p"#Sü$ &)6. 10 mM (XY4/ pH 7.0 045. !(`. ( LL4 îï LL7 a&)6 3.. ビタミン含有リポソームの調製. {b. *+"+<3"#$P¡c<|"d Table 1. yolk9*. Nutritional Value per 100 g of Raw Chicken EggComposition. Volume g. Water Protein Fat saturated fatty acid unsaturated fatty acid cholesterol Carbohydrate Others. 48.2 16.5 33.5 9.2 9.2 1.4 0.1 1.7. e¢). »¼*+ B1f B2. d]'¾BîïB1f C ".

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(26) hon p.4 [100%]. 1522. Vol. 131 (2011). Fig. 2.. Malthesian Crosses of Various Types. a Double halo. b Conspicuous cross in strong halo. c Four leaves shape. d Single halo. e Elimination center of sphere.. 56)35B1fê56 _s3#" 3qr8c)p1"q roð 8d. 5ú©d. Table 2. Lipid. kd5 8úYZ()6%8& klmndoð5´. Contained drug model. BuŠer pH. Sample name. Particle size mm. . 4.0 7.0. YL4 YL7. 88.017.7 66.713.4. Ribo‰avin. 4.0 7.0. YL4-B2 YL7-B2. 90.115.6 94.129.8. Ascorbic acid. 4.0 7.0. YL4-C YL7-C. 85.330.1 65.820.7. . 4.0 7.0. LL4 LL7. 120.332.4 79.124.1. Ribo‰avin. 4.0 7.0. LL4-B2 LL7-B2. 106.226.6 152.744.8. Ascorbic acid. 4.0 7.0. LL4-C LL7-C. 244.9176.0 95.114.4. B1f56. ) YL p1"qr8L#µ¶8] . Particle Size of Each Liposome. Egg Yolk. "¡c<8·5 8®&. ()6 粒度分布測定. 3.. É"~ "c. èæ Table 2 3)65("pk lmnoðH3 20 F";<=GâwÑ)) . ~ O 100 nm d]Ñ)6 W45. Lecithin. ) LL "~ YL 3¨ ¬5¸8Y &()6XY4" pH 3~ 3¬¹ Z(%Ñ)8. .

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(39) $Ar¼. Ê. $½'7 tC. tÒÇ ÉË6. ÍCÁ&. S{@9. ñ. Âaª. tÂÁ¬. tÒÆ . Õ. . }4T. ÒZ¨89. . É. ÎÏ. Sk Ò ". Ö×Z£. ÔØ. REFERENCES. ÒòþüxÃ¬°*°±" 1 q. 謝辞. . [ w¥. A % . Zq. é . Â. ÉÂ. Ð{²±. SJ\. /üýd] ®&()6. . Z7%. tÒ[. *°±8 6 H² . 8³Z( p3. À. {. {$0. [. =7. $»é 8. H. / . «n . oA²³. tó. {$. §@n. ®ñ"¯. !. { . . £¤¥. +¥cª. ¬õs. µ¶åæ8]Ñ) ô®6"åæcq. . ¦i\¯. oA°±. . e. {t%/. WÅJô. e . i=¯. ¦ëk. qrs. {$ Ù. . . YZ^. l7. 8/g. 4%. Y. ×$cd. {$}. 7. ñ. z¯[. {$0E. k34 3£5õe"ïð8t;. Y]. ×$oê. 7% 20 F";<=. a. WñX. . QR. 4 5. Hashida M., Takakura Y., ``Seitainai Yakubutsu Soutatsu Gaku,'' ed. by Ikada Y., Sangyo Tosho, Tokyo, 1994, pp. 3493. Inoue K., Nojima S., ``The Liposomes,'' eds. by Nojima S., Sunamoto J., Inoue K., Nankodo Co., Ltd., Tokyo, 1988, pp. 119. Yasuda T., ``The Liposomes,'' eds. by Nojima S., Sunamoto J., Inoue K., Nankodo Co., Ltd., Tokyo, 1988, pp. 246276. Fang J. Y., Sung C. K., Lin H. H., Fang C. L., J. Control. Release, 60, 110 1999 . Cevc G., Vierla U., J. Control. Release, 141, 277299 2010 ..

(40) hon p.7 [100%]. No. 10. 6 7. 8 9. 10. Betz G., Aeppli A., Menshutine N., Leuenberger H., Int. J. Pharm., 296, 4454 2005 . Sunamoto J., Sato T., Hirota M., Fukushima K., Hiratani K., Hara K., Biochim. Biophys. Acta, 898, 323330 1987 . Massing U., Cicko S., Ziroli V., J. Control. Release, 125, 1624 2008 . Clinical Nutrition Research Institute, ``Jouyoryo Meyasu Shokuhin Seibun Hayamihyo,'' 3rd ed., Ishiyaku Publishers, Inc., Tokyo, 2006, pp. 384385. Shinoda K., Friberg S., ``Emulsions and Solubilization,'' A Wiley-Interscience, New. 1525. 11 12. 13. 14 15. York, 1986, pp. 159169. Hato M., Minamikawa H., Lungmuir, 12, 16581665 1996 . Hato M., Minamikawa H., Tamada K., Baba T., Tanabe Y., Adv. Colloid Interface Sci., 80, 233270 1999 . Niraula B. B., Seng N. T., Misran M., Colloids Surf. A Physicochem. Eng. Asp., 236, 7 22 2004 . Li D. S., Chono S., Huang L., J. Control. Release, 126, 7784 2008 . Takeuchi H., Yamamoto H., Kawashima Y., Adv. Drug Deliv. Rev., 47, 3954 2001 ..

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