ORYZA CERAMIDE -PTPCDP8TP20CD -WSPWSP8 -LL0.8 -PCPC8PC20 -WSPCWSPC8 -LCLC0.8 Ver.13.0HS
ORYZA CERAMIDE Ver.13.0HS 1. 2. 20 NMR 4 ( 1) 1
Ver.13.0HS HO CH 2 OH O O OH OH OH C CH CH H 2 NH C O R C H C H (CH 2) 2 C H C H (CH 2) 8 CH 3 R: 1. -(CH 2 ) 7 CH:CHCH 2 CH:CH(CH 2 ) 4 CH 3 2. -(CH 2 ) 14 CH 3 3. -(CH 2 ) 7 CH:CH(CH 2 ) 7 CH 3 4. -(CH 2 ) 16 CH 3 1 2 HPLC ( 2 No.7) 3 3 2
3. Ver.13.0HS 1884 Thudichum 120 ( ) ( 4) 4060 % ( 5) 4 26% 10% 5 40% 24% 9 ( 6) 3
Ver.13.0HS 6 CER 9 ( 7) 5) ( 8) (Lati 4) ) () 4
Ver.13.0HS 60 50 40 30 20 10 0 10 30 50 70 7 G. Imokawa, J. Invest. Dermatol., 96, 523, 1991 90 150 100 50 0 8 4. Schmelz 3060 1% Nyberg 5
5. 5-1 5-1-1 (in vivo) Ver.13.0HS GCFr, 99% -PTFGC 9A TEWL 9B FGC256 mg/kg10%sds 1 1 3 TEWL 12 SDS TEWL 9B 9C SDS TEWL GCFrFGC TEWL 9. GCFr -PTFGC TEWL A) SDS B) SDS TEWLC) 12 SDS TEWL n=5-6 6
Ver.13.0HS 16 SDS TLC 10 1, 2Cer 1, 2 GlcCerEOS ( 1 )A/B4 GlcCer GCFr 1 EOS, A/B 1SDS GCFr 11 7
Ver.13.0HS 10. TLC Cho: FFA: PE: Cer: (190:9:1):10% 8%, GlcCer: (40:12:1), 0.1% 10% 1. GCFr SDS Cho FFA Cer 1 Cer 2 Normal 0.711±0.094 0.469±0.097 0.139±0.028 0.112±0.024 Control 0.669±0.057 0.390±0.054 0.158±0.028 0.126±0.022 GCFr (3 mg/kg) 0.732±0.026 0.623±0.096 0.184±0.026 0.137±0.021 GCFr (10 mg/kg) 0.840±0.119 0.724±0.088* 0.211±0.036* 0.135±0.020 GlcCer (EOS) GlcCer A/B PE Normal 0.035±0.005 0.044±0.007 0.071±0.020 Control 0.034±0.007 0.042±0.005 0.064±0.008 GCFr (3 mg/kg) 0.032±0.009 0.033±0.008 0.103±0.018 GCFr (10 mg/kg) 0.016±0.004* 0.017±0.006* 0.069±0.011 Cho: FFA: PE: CerGlcCer n=5-6*: p<0.05. 8
Ver.13.0HS 11. GCFr Cer GlcCer 50 µmcer GlcCer 9
Ver.13.0HS 12 KGKG GCFr Control GCFr 10 mg/kg 123,000 SC:SL:SG:KG: 13 GCFr Control GCFr10 mg/kg Control GCFr10 mg/kg 10
Ver.13.0HS 14 GCFr Control GCFr10 mg/kg 14300,000 15 cornified envelope GCFr Control GCFr10 mg/kg 15. cornified envelope3,000 GCFr 11
Ver.13.0HS 16 Western blotting 7 GCSase-GCaseGCFr GCaseGCSase GCFr 1 11 16. GCFr GCSase- GCase A) 10% SDS-PAGE Western blotting. B) control 1 12
Ver.13.0HS 5-1-2 in vitro [GlcCer (d18:2), 3] 3 17Cer 1, Cer 2, GlcCer (EOS) GlcCer A/B 2 17. TLC Cho: FFA: Cer:GlcCer:PE: 2. GlcCer (d18:2) Cho FFA Cer 1 Cer 2 (µg/ml) Control - 0.238±0.036 0.053±0.006 0.048±0.021 0.109±0.011 GlcCer (d18:2) 1 0.247±0.016 0.066±0.010 0.091±0.003* 0.071±0.015 3 0.263±0.025 0.108±0.018* 0.076±0.001 0.113±0.003 10 0.308±0.016 0.108±0.018* 0.076±0.006 0.132±0.002* GlcCer (EOS) GlcCer A/B PE Control - 0.034±0.004 0.020±0.004 0.052±0.017 GlcCer (d18:2) 1 0.049±0.010 0.020±0.009 0.069±0.007 3 0.081±0.009* 0.067±0.003** 0.117±0.017* 10 0.084±0.005* 0.049±0.010* 0.104±0.010 Cho: FFA: PE:Cer GlcCer n=3 *: p<0.05, **: p<0.01. 13
Ver.13.0HS GlcCer (d:18:2) 18. GlcCer (d:18:2) Cer GlcCer 50 µmcer GlcCer 14
Ver.13.0HS GlcCer (d:18:2) GCase GCSase 19 19. GlcCer (d:18:2) GCSase-GCaseTGase A) 10% SDS-PAGE Western blotting. B) control 1 15
5-2 (in vitro) 5-2-1 B16 Ver.13.0HS B16 () 20 (%) 40 30 20 10 100g/ (%) 50 40 30 20 10 0 0 1 2 3 4 5 1. 2. 3. C 4. 5. -1 0 1 10 µg/ml 100 20. B16 (2 10 3 cells/ml) (60 mm dish) 10% D-MEM 24 () 4 2 2N NaOH (371 ) 450 nm C : 100 % : 95 % 16
Ver.13.0HS 5-2-2 (melan-a) * 21, 22 * 1 N HCl (70/18 ) n- % of control % of control 0 5 10 20 (µg/ml) 0 5 10 20 (µg/ml) 21. % of control % of control 0 5 10 20 (µg/ml) 0 5 10 20 (µg/ml) 22. 17
Ver.13.0HS 1) (melan-a cells, 1 10 4 cells/well) 96well plate 200 nm TPA 10% RPMI1640 24 ( 95%) 4 1% tritonx-100 PBS (90µlwell) 1 10M -DOPA PBS (10µlwell) (137) 475nm 2) melan-a cells (3 10 5 cellswell) 90 mm plate 6 well plate 1) 1N NaOH (500µl) (10030 ) 405nm 5-3 (in vitro) Merrill Futerman 1-3) in vitro 1. 2. 3. 4. ) 3% 23. ( 3%) 35 40% 8 18
Ver.13.0HS 5-4 3 300 µg/ml (163%) : 1. 95% 2. 95% 3. 95% 4. 95% 1. 2. 3. 4. 5. 24. (300 µg/ml) HS-K 10 %1000 unit/ml 100 µg/ml RITC80-7 (5%CO 2, 37 ) 96 1 10 5 cells (100 µl/well) 1% (FBS) 1000 unit/ml 100 µg/ml RITC80-7 24 11 µl 3 mg/ml (EtOH 0.5 %) 72 Cell Counting kit-8 450 nm 5-5 4) 19
Ver.13.0HS 5-6 5-6-1 RBL-2H3 RBL-2H3 3 RBL-2H3 (%) 87.3 2.2 82.2 5.9 70.8 5.9 64.2 5.4 : 1 µg/ml, S.E., n = 6 IgE () RBL-23 ( 1 µg/ml) 10 30 5-6-2 compound48/80 (ddy, ) compound 48/80 ( 25)compound 48/80 25. compound 48/80 (ddy, ) 4 (00.150.3 0.5%) 3 3%compound 48/80 30 20
Ver.13.0HS 5-7 () 3 5-8 α P. Acnes QOL QOL 5-8-1 1834 VISIA Skindex-16 QOL 5-8-2 26 21
µ Ver.13.0HS 26. *: p<0.05 P. Acnes 27 P. Acnes 27. 28 28. *: p<0.05 22
Ver.13.0HS 29 29. Skindex-16 QOL Skindex-16 16 QOL -100 QOL 30 QOL 30. QOL *: p<0.05 23
Ver.13.0HS 5-9 5-9-1 7) F344 (5 ) (AOM) 1 2 aberant crypt foci (ACF) mucin depleted foci (MDF) (G 1 CM) 4 23 (G 1 CM initiation ) ACF MDF 1 () 8 ACF MDF 1 45 (G 1 CM post-initiation ) ( 4) PCNA Caspase-3 ( 31) 2 (ACF MDF) 4 AOM PCNA Group Conc. N Number of ACF Number of MDF (ppm) 4 w 8w 4w 8w 1 0 12 7517 11133 9.25.2 17.16.6 2 100 12 46 9 p<0.01 9028 0.88 p<0.05 10.85.9 p<0.05 3 250 12 4514 p<0.01 7520 p<0.05 0.80.4 p<0.05 6.82.7 p<0.05 4 100 12-8928 - 10.05.1 5 250 12-7815 p<0.05-9.63.1 p<0.01 6 250 3, 6 0 0 0 0 7 0 3, 6 0 0 0 0 24 Caspase-3
x 200 x 200 PCNA Caspase-3 Ver.13.0HS 31. 20 PCNA Caspase-3 F344 (5 ) 7 32 (AOM 20 mg/kg) 1 2 ACF MDF (ph 2.5) ACF MDF proliferating nuclear antigen (PCNA) cleaved caspase-3 1 : AOM 1 2,3 : 90% (G 1 CM) 100, 250 ppm AOM 1 4 4,5 : AOM 1 2 G 1 CM (100, 250 ppm) 3 6,7 : AOM 250 ppm G 1 CM 0 1 2 4 8 weeks 1 (24rats) CE-2 2 (24rats) CE-2+100ppm G 1CM CE-2 3 (24rats) CE-2+250ppm G 1CM CE-2 4 (12rats) CE-2 CE-2+100ppm G 1CM 5 (12rats) 6 (9rats) CE-2 CE-2+250ppm G 1CM CE-2+250ppm G 1CM 7 (9rats) CE-2 ; AOM s.c. injection (20mg/kg body weight) ; Sacrifice ( diet 2 ) 32 AOM 25
Ver.13.0HS 5-9-2 8) NOD/SCID 5 mm (300 mg/kg/day 24 mg/kg/day) 14 ( 33) TUNEL ( 34) NOD/SCID ( 35) 33. 26
Ver.13.0HS p < 0.001 control glucosylceramide 34. 35. 1) Eva-Maria S. et al., J. Nutr., 124 (5), 702 (1994). 2) Merrill A. H. Jr. et al., FASEB, 3A, 469 (1989). 3) Futerman A. H., CHAPTER 4, Current Topics in Membranes, Vol.40, 93 (1994). 4) Elian L. Fragrance Journal, 23 (1), 81 (1995). 5),, 44 (10), 751 (1995). 6) Lena N. et al., J. Nutr. Biochem., 8 (3), 122 (1997). 7) Inamine M. et al., Cancer Sci., 96, 876-881 (2005). 8) Fujiwara K. et al., Int. J. Clin. Oncol., 16, 133-140 (2011). 27
6. Ver.13.0HS 100 80 60 40 20 100 100 140 140 0 0 30 60 17 36 7. ph ph 100 80 60 40 20 0 1 3 5 7 9 11 13 37 ph 90% (ph 6.8) 100% 8. 28
Ver.13.0HS -PT, PCD () 3050 mg/day 2040 mg/day -P8T () 1119 mg/day 7.515 mg/day -P20CD () 4.57.5 mg/day 36 mg/day -WSP () 3050 mg/day 2040 mg/day -WSP8 () 1119 mg/day 7.515 mg/day -L () 300500 mg/day 200400 mg/day L0.8 () 113188 mg/day 75150 mg/day 9. PT PCD P8T P20CD WSP WSP8 L L0.8 (/100 g) 1.2 g 1.8 g 1.1 g 2.2 g 1.8 g 40.2 g (/100 g) 5.3 g 3.8 g 7.3 g 4.7 g 3.8 g 0.4 g (/100 g) 19.6 g 33.8 g 21.8 g 42.2 g 33.8 g 5.9 g (/100 g) 66.3 g 2.5 g 67.2 g 3.1 g 2.5 g 0.2 g (/100 g) 5.6 g 58.3 g 0.4 g 47.8 g 58.3 g 53.3 g (/100 g) 2.0 g 0.0 g 2.2 g 0.0 g 0.0 g 0.0 g (/100 g) 337 mg 524 mg 328 mg 655 mg 524 mg 52 mg (/100 g) 224kcal 552kcal 232kcal 590kcal 552kcal 268kcal -PT : 19 5 17 307040849-001 : : 11 9 7 : 399080329-002 29
Ver.13.0HS 10. 1 (447 (456 )) 18 9 5 2 (LD 50 ) 30 g 5 ICR 5000 mg/kg 23±2 50±10% 14 LD 50 5000 mg/kg 3 28 ) 4 Slc:ddy 60 mg/kg ( 60 kg 3.6 g/) 223 5020 % 28 60 mg/kg 4 ( ) 30
11. 12. Ver.13.0HS -PTPCDP8TP20CD () -WSPWSP8 () -PCPC8PC20 () -WSPCWSPC8 () 1kg -LL0.8 () -LCLC0.8 () 5kg 13. 31
Ver.13.0HS 14. PTP8T PCDP20CD WSPWSP8 LL0.8 () () INCI PCPC8PC20 Cyclodextrin Oryza Sativa (Rice) Bran Oil Glycosphingolipids WSPCWSPC8 Maltosyl Cyclodextrin Cyclodextrin Maltose Oryza Sativa (Rice) Bran Oil Glyco sphingolipids LC 10 Glycerin Water Polyglyceryl 10 Oleate Lecithin Oryza Sativa (Rice) Bran Oil Glycosphingolipids LC0.8 10 Glycerin Water Polyglyceryl 10 Oleate Oryza Sativa (Rice) Bran Oil Glycosphingolipids 32
Ver.13.0HS (Oryza sativa Linne) 3.0 % 3.0 % () (HPLC) 5.0 % (1 g1052 ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 25.00 % 1.13 % 65.60 % 5.00 % 2.00 % 0.57 % 0.50 % 0.20 % 100.00 % 1.0% (3.8%) 33
Ver.13.0HS (Oryza sativa Linne) 3.0 % 3.0 % () (HPLC ) 8.0 % (1 g1052 ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 40 % 60 % 100 % 34
Ver.13.0HS (Oryza sativa Linne) 8.0 % 8.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 25.00 % 1.13 % 65.60 % 5.00 % 2.00 % 0.57 % 0.50 % 0.20 % 100.00 % 35
Ver.13.0HS (Oryza sativa Linne) 20.0 % 20.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 50 % 50 % 100 % 36
Ver.13.0HS (Oryza sativa Linne) 3.0 % 3.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 40 % 60 % 100 % 37
Ver.13.0HS (Oryza sativa Linne) 8.0 % 8.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 40 % 60 % 100 % 38
Ver.13.0HS (Oryza sativa Linne) 0.30 % 0. 30 % () (HPLC ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 40 % 4 % 46 % 10 % 100 % 39
Ver.13.0HS (Oryza sativa Linne) 0.80 % 0. 80 % () (HPLC ) (1) 10 ppm () Pb (2) 1 ppm ( ) As 2 O 3 1 10 3 g () 1 10 2 g ( ) (BGLB ) 40 % 4 % 46 % 10 % 100 % 40
Ver.13.0HS (Oryza sativa Linne) () 3.0 % 0.2 g 2 ml 3.0 % () (HPLC ) 8.0 % (1 g1052 ) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 60 % 37 % 3 % 100 % 41
Ver.13.0HS (Oryza sativa Linne) () 8.0 % 0.2 g 2 ml 8.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 60 % 32 % 8 % 100 % 42
Ver.13.0HS (Oryza sativa Linne) () 20.0 % 0.2 g 2 ml 20.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 50 % 30 % 20 % 100 % 43
Ver.13.0HS (Oryza sativa Linne) () 3.0 % 0.2 g 2 ml 3.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 60 % 37 % 3 % 100 % 44
Ver.13.0HS (Oryza sativa Linne) () 8.0 % 0.2 g 2 ml 8.0 % () (HPLC ) 5.0 % (1 g1052 ) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 60 % 32 % 8 % 100 % 45
Ver.13.0HS (Oryza sativa Linne) () 0.30 % (1) 10 g 50 ml 0.5 g (2) 1 g 5 g 0.5 g 20 ml 2 20 ml 5 ml 10 ml 0.30 % () (HPLC ) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 40.0 % 38.0 % 10 13.0 % 5.0 % 3.7 % 0.3 % 100.0 % 46
Ver.13.0HS (Oryza sativa Linne) () 0.80 % 10 g 50 ml 0.5 g 0.80 % () (HPLC ) (1) 10 ppm ( 2 ) (Pb) (2) 1 ppm ( 3 ) As 2 O 3 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) 46.0 % 40.0 % 10 10.0 % 3.2 % 0.8 % 100.0 % 47
Ver.13.0HS 493-8001 TEL(0586)86-5141() FAX(0586)86-6191 URL/http://www.oryza.co.jp/ E-mail: info@oryza.co.jp OEM 101-0041 1-24-10 5F TEL(03)5209-9150 FAX(03)5209-9151 E-mail: tokyo@oryza.co.jp * * * 48
Ver.13.0HS 1