1 第1章 研究開発の概要 1－1 研究開発の背景・研究目的及び目標
Contents
1. ali 2 CITase DGase DGase BL21 DE3 100 g mL LE 37 12 100 g mL 100 mL Overnight Express Instant TB 25C 50 mM pY pH 7 0 0 22 gm 802. C 1 2 7 8 y CD C I C 1 2 8 C I
3. Vr Vs Vr 2 3 R16 CPA E 44
4. C I C I DC 1 C I C I
5. C I 2 8 C 1 1 4 901 0205 671 4 TEL 098 850 0330 FAX 098 850 0330 E mail sd miya ci bio co jp 904 2234 5 1 TEL 098 982 1100 FAX 098 982 1103 E mail toka ttc co jp 2 1 2 1 1 1 CI C I TTC RER
6. ID ID No 71 2 w 10 1 6 DM3 5pzm CE 2 3 1 87 0 H 12
7. CI 10 CI L CD L CI CI 22 23 CITase CI DGase ZEE CI 10 L CI L CI L CI 7 8 9 CI S CD CI plus CI B 24 CI CI CI
8. E 2 7 1 2 4 2 5 BELC 5 Ze s 45 1 2 2 8 2 9 1 2 4 3 L b CRASHER 28 AAE D DNR 3 0 3 1 1 2 4 4 Appendix 4 1 4 2 5 1 5 2 3 2 1 2 33 1 2 3 4 1 2 4 5
9. 1 GRR 3 RE MEE 1 3 1 GRH 1 A No 116 Ch 2
10. 1000 mg kg 10 mL kg OPECD 420 2000 mg kg 2000mg kg 1000mg kg 1mL 16 Bx 2000 mg kg 100g Zac 2 7 0 14 1 1
11. JFM 4 1 C I E 4 30 0 2 Filter 4 CTtase 2 0 1 C I C 1 2 0 2 DGase
12. CD C I C I 5 C I 1993 3 C I 13
13. CI C I CI 50 c Sem fid 1 2 1 2 1 N H 4 PE Ji NY n V vr A B PL
14. C 1 CD CH Buffer 3 45 6 0 40 20 0 0 820 J 40 4 Peo 8 0 10 0 12 0 40 1 a cD B cD
15. C I C I a CD RE dc 2 5 47 1 6 1 sls a CD vcD a CD B cD v CD 26
16. 3 5 1 2 36 1 2 3 7 1 2 4 6 4 1 1 A No 116 638 239 1 2 75 5 6 38 SD y C I 500 1000mg kg 28 1 2 3
17. C 1 Y 6000 4000 3000 Total CI ug ml 2000 1000 0 6 12 18 24 30 36 42 48 hr 10 1 C I 1200 1000 P 800 600 400 200 0 0 6 12 18 24 30 36 42 48 hr 10 2 I 0 6 12 18 24 30 36 42 48 hr 10 3 pH C 150 200L C I p 5 5 o FM 0C478 20 mU ml
18. C I 2 6 C 1 C I 46
19. C I E C I C 1 C I 2 jp 2b C I pH 10 25 50 100 mU ml 1 2 Buffer Ca pH 4 0 4 5 5 0 5 5 6 0 3 6 16 24 15 20 25 30 40 58 99 A 0 001 0 01 0 1 121 C 95 2 C I
20. 1 3 1 C I 2 C I C I C I b K s Qc ZW 1 C I 3 C I Kd bib ei P 4 Ral 2 MF C 1 NF C 1 C I 54 4 50 C I NF C 1
21. C I 500 1000mg kg C 1 ORBE HEEL b 1000mg kg 76 General signs in rats 28days iteration oral administration in subacute toxicity study 2 6 Sex Total animal number Mortality Findings Group No abnormality 6 P 0 6 Male control Vehicle om d ow A e e e e e BB Jg E E E E E i o o Q Q Q Q Q G G G o 2 2 2 2 2 DS e e e e e e o gt o gt o LL LL LL d 2S RS a Dead treated b In parentheses indicate the number of cases General signs in male rats 28days iteration oral administration in subacute 2 7 1 toxicity study 22 23 24 25 26 27 28 29 19 20 21 Day 10 11 12 13 14 15 16 17 18 9 NN NN NN NN N NN NN NN NN NN NN NN NN N N NN NN NN N NN N N N N N N NN NN NN NN AN N N N NN NN NN N NN NN N N N N NN NN N
22. 33 3 1 3 3 2 2010 CI Plus CI 7 C1 16 CI 17 3 2 9 3 1 3 3 3 3 4 51 3 5 ERE TTC 23g HRL TTC 1 TTC 2 CI 7 CI 14 CI 56 4 54 4 50 38 3 39 7 L CI Mutant R CITase CI MR CI CI Plus MR CI CI 26 7 2010 CI Plus 10 CI XE 19 5 2009 CI Plus fg E ENTO WOZIE UL 2 Mutant R CITase DGase L CT 90
23. p H5 0 3 25C 16hr C I CI CI C I pH C1I 30 mU ml 5 0C478 CI x 18 24 Ui 8 E PN us y E HRH pH 5 0
24. C I 5000 4000 T a BiHft Bit G8 3000 3 S 2000 o 1000 0 0 6 12 18 24 30 hr 6 C I CI C I C I C I C I 2 30 mU m1 250C 16hr C 1 24hr C 1 7 1
25. 49 CITase DGase CI DeGase CITase 2 50 mM pH5 5 40C 100 10 TLC Silica gel 60 F 20 alminium sheets 5 x 7 5 cm MERCK HX801501 1 1 70 30 2 5 CI CI 7 CI 8 CI 9 CI 10 CI 11 CI 12 18 L CI 1 EIE M 22 50 mM pH5 5 DGase CITase 37C 70 rpm 207 24 CI
26. 10 80 30 0 80 0 60 0 40 660nm 0 20 0 00 HERAK 10 10 DMSO BD Plate 30 80 4 17 1 00478 0 05 0 04 0 03 Cltase U mD 0 00 HERAK 10 10 DMSO BD Plate 30 80 4 17 1 00478 CItase 31 1 0 0C478 3 1 99890 3 0 5 1 S CItase DGase 3 3 3 D D 3 D
27. 14 16 17 C I GR Beo CI SMGAR H dA uu Hp C 1 C I
28. E DNET 0C478 C I C 3 1 2 2 C 1 1 2 PREVELE 2 BR ANTA 16 24hr 25 50 mU ml TC 3 6hr 3 6hr 25 50 mU ml 2 2 16 24hr 30 mU ml
29. C 1 C 1 7 C 1 8 C 1 9 C 1 10 C I 11 C I 12 1u mol 1unit U 0C478 2 3 0 Fes i 8 9 uu l lt 12218 40m1 100m1 2ml 500L EL 30 0 4ml 1 H 3 1 0 2m1 HI Bir 30 2001 30 3 H 140rpm 500L
30. T3040 DGase 598K Dcase T 3040 CITase DGase CI 4 T 3040 598K CITase 40 T 3040 CI 1 2 1 CI Hee 1 CI 2 Dase CI DGase CI E 40 I AL E BE CIT
31. amp T 2 CDCI CITase CITaserDGase Bacillus circulans T 3040 2 40 Luria Bertani pH8 0 T 3040 30 1 4 80 25 mM pH7 0 10 40 CI CITase CI CITase DGase lt gt 4
32. DL CI REN 30 2j 16 24hr 20 30C C 40C 16hr C E 35C 30C 2 4 1 amp RUTC I 2 C I 16 24hr 20 30C 35C Jes 5000 4000 3000 E 2000 1000 0 0 3 6 9 12 15 18 21 24 hr B 3 1 29 C I1 5000 4000 3 3000 E 2000 1000 0 3 6 9 12 15 18 21 24 hr 3 2 19 C 1 Total CI ug ml Total CI ug ml 6000 500
33. 600 Da NF C 1 C 7 15 54 4 50 50 C I NF HP20 C C I 50 C I 50 C I 5 600 Da NF CI 5 k hr 33L 52L 25 2 2 CI 2 2 1 CI TTC
34. 61 ID TD No 3 1 5 ETO 2 3 10 m 22 4 18 26C 50 30 70 uS 26 1 6 Ed 5 um
35. 64 19 General Group Control Vehicle CI 1000 mg kg CI 2000 mg kg a Dead treated b In parentheses indicate the number of cases 2 0 1 signs in rats Female Male Female Male Female toxicity study Group Control Vehicle CI 1000 mg kg CI 2000 mg kg N Normal Animal No DNo 1 101 102 103 104 105 201 202 203 204 301 302 303 304 305 N N 10 N N N N 11 N N 18 N N 8 N N 6 N N 65 N N 13 N N 17 N N 16 N N 4 N N 9 N N 14 N N 1 N N after single oral administration in acute toxicity study 9 N N Total animal number 3 N N Time after administration 4 N N hour 5 6 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N 65 Mortality 0 5 0 5 0 5 0 5 0 5 0 5 3 5 N NN N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N Findings n No abnormality 5 No abnormality 5 No abnormality 5 No abnormality 5 No abnormality 5 No abnormality 5 General signs in male rats after single oral administration in acute Day after administration 8 9 10 11 13 13 20 2 Group Control Vehicle CI 1000 mg k
36. pH 5 5 93 95C Lik 30 2 30 mU ml 25C a ER C I 8 oc 5000 4000 3000 em 5 S 2000 o 1000 0 0 6 12 18 24 30 hr 8 gw C I Na Buffer CaCl C I Na Buffer CaCl C I Buffer CaC1
37. C Plus30 50 C I C I C I C 7 C I 4 C 1I CI C 1
38. 0D 660nm 27 iv CItase 1 200 1 2hr 500 1 CI 7 CI 8 CI 9 C I 10 C I 11 C 1 0 45um C I 12 15 000rpm 5 2 0 2gum CItase CItase E T 40 25041 100mM 25mM 50 ul 1 5ml 40 10 2 15 000rpm 5 CITase HPLC C 1 CITase C I
39. 3 2 4 D31 XH18cm 1 p CE 2 DM3 gt 2 1 8 R18 0 5 amp JE mg kg mg mL 1 0 0 5 101 105 5 151 155 2 1000 100 5 201 205 5 251 255 C 3 2000 200 5 301 305 5 351 355 62 2 5 2 6
40. 1 6 0 0 1 3 7 10 14 14 mL Eni nk 63 3 StatLight 3 4 Bartlett 1 way ANOVA 5 4 4 1 1 9 20 1 2 2000 1000 mg kg 14
41. No mg dL Sidi 5 5 250 750ull Std2 10 500 50041 Std4 30 96Well MicroPI 30 Std5 40 5001 1250 se s I ER ate 577 20001 15 15 1 A505nm DC I C I 2 2 CD 0C478 mw B RUBRI E IF 1 CI mos CI N 100 mU ml C I
42. CI 0 1 2 5 3 CI Amide 80 Tosoh L CI 100 L CI 2009 HPLC CI 7 8 9 10 11 12 2 9 CI n CI 7 CI 650 nm 12 30 CI 7 CI 8 CI 9 CI 10 30 3 1 CI 10 CI 18 E CI P1us 7 1 16 CI 9 1
43. T 3040 CITase DGase 598K DGase T 3040 DGase 598K DGase E 598K DGase T 3040 DGase 1 3 1 4 DGase c 1 1 a l 4 a 1 2 gt a 1 3 gt a 1 6 8 1 2 40 T 3040 CI CI mg g 40 C1 7 CI 8 CI 9 CI 10 CI 11 CI 12 1 2 1 39 3 4 0 7 LE X2 2 9 0 19 4 13 1 7 2 8 3 10 6
44. T 40 6 0C478 6 T 40 T 40 1 0g Bacto Peptone 1 0g Yeast ext 0 1g NaCl 0 5g 100ml p H8 0 l 616 2ml 50 1 1 3 30 3 AEIR 140rpm LERZ 504 1 16 2ml 1 30 3 140rpm 26 i 30C lml 30C 10 80 KERAEZ ELE 3 000rpm 5 20 6 1ml 80C 10 DMSO 80 10 6 80 DMSO lml
45. CI Plus 1 7 4 3 650 nm CI 7 L CI 2 5 1 2009 CI Plus 2010 23 C3 C3 C3 56 Chlorophyll a Chlorophyll b 400 700 500 600 Wellenl nge nm 7291716 a ga 3 8 2
46. 1 1 28 g 16 o 74 x 100 3 Bartlett p gt 0 05 3 0 05 Kruskal Wallis p 0 10 Dunnett 5 4 4 1
47. D CI CI 3 ES i ER HERE GEIL A E WHJ 210 DGase o 1 4 e 1 6 eo 1 6 42 DGase SORORE E CTITase Dcase X WE E l2 6 CI E CD CI i4 DE E 2 BRA ERD 2 4
48. QIETRO RE QI RT E IR 52 3 AST UV ALT UV y GTP NU Na K Cl Ca P JS ALP Glucose T Cho TG T Bil Crea A G A G ratio piu H pin AL Biuret P LN 0CPC BCG BCG Biuret 10 O OH OR b TB Urease indophenol EUER
49. 4 7 15H 3 9 5 CI CI Plus 2009 CI Plus 2010 MR CI 2012 CI 57 7B y aCD BCD yCD Cl 8 Cl 9 Cl 10 CI 11 CI 12 0 1 0 1 0 1 01 01 01 01 01 0 1 0 1 L CI TTC 1 0 195 0 5 0 555 05 0 5 05 05 05 05 40 CI 58 650 nm 0 1 2 3 4 5 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 9 7 0 1 amp Cl 10 0 1 CD Cl 11 0 1 gt vCD Cl 120 1 41 CI CD TH8 L C 01 e 05 QCkPlus09 0 596 CI Plis1O 0 596 CI 7 L CI 01 05 1 25 01 05 1 CI Plus09 2 5 0 1 05 1 59 CI Plus10 25 0 1 02 1 2 5 650 nm 650 nm 0 5 10 15 650 nm 60 650 nm 43 CI EZ 2 4
50. EIC 8 0 5 0 0 6 40 qe a 2 Ws 1n4 41 R ao E E E 8 B 1 20 0 2 h 10 z 0 0 0 0 r r r r 0 0 6 8 8 9 2 8 8 8 9 8 8 8 9 lt E a gt o a gt gt E a gt 1 1 1 27 AZER J 4 2 4 0 2 0 T 41 40 4 40 Bf 39 3 8 35 por 2 WEE t6 EEDD t NM 3 6 15 35 M D MB DM D 34 no TA 0 5 32 99 8 CI CI CI 28 C I 48 2 4 3 C I 1 CI CI
51. 7 7 FE 22C 4 18 26C 50 30 70 W26XD31XH18cm REAK 1 2 6 2 4 2 5 25 mg kg mg ml 1 0 0 6 111 116 6 161 166 2 500 50 6 211 216 6 261 266 C I 3 1000 100 6 311 316 6 361 366 2 5 6 2 1
52. L CI CI 7 CI 26 L CI Ed 7 CI 53 8 46 2 CI 27 CI Cl 7 C4zHzoOss 1134 f H2 Cl 8 C4sHsoO4o 1296 Cl 9 Cs4HsoO4s 1458 OH C 10 CeoHiooOso 1620 HO Cl 11 CssHiioOss 1782 OH ng 12 1 12 CzzHizoOso 1944 Cyclodextran Bl29 CI 7 8 9 10 11 12 30 gt 20 1 1 B 10 3 0 0 10 20 30 min 30 1 7 8 9 10 11 12 HPLC Amide 80 Tosoh 52 1000 mV 0 10 20 40 50 30 L CI 2012 CI C1 7 Cl 9 LCl CI10AE ZJLJ AM V XA NATUS 096 100 0 31 L CI 2012 HPLC Amide 80 Tosoh CI rr CI 7 CL16 0 10 20 30 40 50 E t 400 HBDase Glucoamy lase 9 1 33 3 En 1 E E 10 20 30 40 50 Cl Plus 2009 3 2 CI Plus
53. 1 2 3 0C478 0C478 0C478 C I DE 2 0C478 0C478 10 T 40 1 0C478
54. C 200L 17hr 0C478 C I C I C I Na Buffer CaCl C 0 REIN IRURE C CI C I 4000 3000 2000 Total CI ug ml 1000 Buffer Ca Buffer Ca 12 Buffer Ca
55. L CI ec CI Preparative C18 125AA 55 105 3 5 CI 7 CI 8 CI 9 20 TL CI L CI 50 CI CI HBDase 40C 1 E 2 10 mg ml WEGE creo 15 000Xg 10 TSK gel Amido 80 4 6 X 250 mm Tosoh HPLC CI SS 1
56. 7 2 C C 1 au Total CI ug ml Glc ug ml 5000 4000 3000 2000 1000 0 001 0 01 0 1 A 07 1 C 1 500 400 300 200 100 0 001 0 01 0 1 A 07 2 4 SZ ROMEL LEC I EECBKOT eg Buffer C I o
57. 4 2 4 4 1 2 21 22 1 2 TRUCO seen C I 2000 1000 mg kg 14 C I 2000 1000 mg kg d 4 3 2 3 2 4 1 2 C I 2000 1000 mg kg 1 z 5 REMEC I I 2000 1000mg kg jos JEEV KARR T TIRA Holes I 2000 mg kg
58. FX 12001 A amp D 1 1 8 52 3 CD RBC DV t O SLS MCV RBC Ht RBC Hb MCHC Hb Platelet D C WBC DC 73 2 9 2 2 amp amp GG O O OG O OG O 6 Q O O 6 6 GO
59. 40 25 L 2 0 5 M 5 L 20 L 40 C 100 5 HPLC HPLC B circulans T 3040 DGase B circulans T 3040 DNA FA ZIY KV Paenibacillus sp 598K DGase DNA 598K DGase T 3040 DGase 38 DGase DGase gt 1 0 5 mL 0 5 0 1M 0 3 mL 0 02 0 1 m 0 1 mL 0 25 uM 50C
60. 10L MF 100L C 1 30 2hr 1 6 1 2 150L C I 30 MF C I
61. CI 105C 10 20 20C 1 w 25 U mg 4 5 mg 46 4 U mg 1 5 mg 55 16 105 20 CI 1 2 v v 50 mL 200 mL 1 3 1 BIBRO VERE 0 3 C1 7 CI 8 CI L CI Q L CI
62. 6000 5000 4000 Total CI ug ml 0 6 12 18 24 30 36 42 hr 1 1 2000 C 3 C I ug ml 622 180 101 0 3381 39nr 808 1272 C I Na Buffer CaCl C I 00478 C I 9 1 Na Buffer CaCl 150L C I JFM o J F M 0C478 25 mU m1 30 35 C 2 1 2 C I Na Buffer CaC1 1501 18hr
63. WE I 50 C 1 4 E E 2 CI 2 3 0C478 ri HAT 2 1 f hs E 60L ji 0 61 U mL 0 6U ml IT 41 E
64. 1 0 5 p 2 0 3 4 CI MF BAI E D 4 35 H 0 2 Filter CItase U ml 0 5 10 2 0 1 00478 CItase 5000 RAE m 4000 E 0 2 Filter d 3 3000 O S 2000 1000 0 1 5 10 20 2 0C478 CI 36 0 12 2 0 09 g 6 yr m 0
65. C I 20 2 1 2 CI TTC 1 RUE Ze FH TE C T 200L C I C I C I MF C 50 600Da NF C 2 CI 1 3 10 C 1 8 000rpm 15C Xf C I
66. DO p CItase 0 10 U ml UF 6 10 0 61 U ml 1 1 D S 1 S JFM 60 L 0 6 U ml CTtase 0 6 1 0 U ml DO 96 E D S 1 S 1 1 JFM CItase 40 60 80 hr 19 JFM 00478 DO pH 100 1 78 7 6 174 1 7 2 1 6 8 6 6 U ml 0 10 0 61 34 pH 0C478
67. 100 5 HPLC 60 v v 60 DGase CITase CI Dase E E HPLC 40 Paenibacillus sp 598K DGase DGase T 3040 DNA TE FEL B circulans T 3040 DGase 233 598K DGase 598K DGase 62 6
68. CItase DGase 4 1 8 1 2 4C CTtase DGase D S 1 S CItase 35 DGase 3 D S 1 S 0C478 BD Plate 10 9 cfu ml 18 3 D S 1 S 0 15 0 12 ON iB R 0 09 2 0 06 Wr 1 Ag 0 00 0 7 14 21 28 35 4 1 8 1 D S 1 S5 CItase 32 0 40 TN 2 E R 030
69. DGase 0 6 WM 22 250 1 100mM 25mM 200 1 50 ul 1 5m 40 3hr 10 15 000rpm 5 0 45um 2 CI Wako DGase 1 1nmol 1unit U 75 8 9 T 40 30 0C478 40ml T 40 200ml 0 4ml 30 140rpm 3 150ml OIRO 500ml 8 3ml
70. Hoe M BE C I 28 2 2 1 I 54 4 C I 2 2 21 18 j 22 PE 18 5 6 Wy gt b Jel SD SPF 3 4 pE 162 57 176 8g 125 231 7 244 3g 170 27 187 6g 47 140 6g 1
71. 15 000rpm 5 BZ 0 45um CI 7 C I 8 CI 9 C I 10 C 1 11 C 1 12 C 1 CAD RD HPLC CITase 0 CI v 15 000rpm 5 0 45 wm CT Wako 2 vi 15 000rpm 5 0 45um pH pH AS 212 Tes 2
72. 30 DED AMEC I 1 5 BERI e OO 16 1 C 1 16 2 C I 2hr QukER 600Da NF C IL 600D a NF CI 5 C 1 7 12 14 0 35 49 600D a NF
73. 30 35 2 2001 1 1 200L C 1 17hr CI 10 10L C I C I 4 C 1 15 13 C I C I C I riam alli
74. 1 7 E17 2 3 6 1 8 ICPA 30 CPA Change of membrane Potential caused by Adsorption 3 2 4 e CD 8 CD CD C 1
75. CI kn 2 4 1 TTC 1 TUBE C C I CI BE 50 2 i 3 000rpm 15 10 8 5 2g
76. C 1 C I Na Buffer CaCl OC I 2 30 m m1 25 TORR WKE oe C Water Na Buffer 15 CaCl 9 1 9 2 pH Buffer pH 24hr pH 0C478 Ze Hv THEE CO C 1 Dp H Buffer CaCl C I 6000 5000 Total CI ug
77. 2 4 1 C I 1 CI 2 2 1 WRiBUMSEUONUEGSRUE C I 51 4 WA CT 2 2 Jcl SD SPF ME 15 pt 15 pE 5 6 212 9 226 4 g 153 0 168 8 g 7
78. 55 y v 55 45 CI HBDase CI HBDase 1 ml min 30C0 H 23 ELULT 7 8 CI 9 10 11 1 12 eR CI CI 12 CI 70 CI 56 CI 2 4 gs 2 1 C1I 12 300 1 700 1 CI
79. SL REN 1 1 ER BR A577 A E T A597 EE T ABRE ECT ACE
80. 108 7 111 5 801 48 9 15 7 61 19 6 32 1 72 7 6 4 112 19 632 42 9 12 8 68 20 3 29 8 77 3 8 2 113 10 811 50 1 16 6 62 20 5 33 1 70 3 92 114 2 716 410 15 6 61 20 1 332 872 11 5 Vehicle 115 8 807 49 1 16 7 61 20 7 34 0 87 3 11 3 116 9 809 413 16 2 59 20 0 340 729 97 772 67 47 62 15 60 62 00 20 20 32 70 77 95 9 38 50 70 10 2 56 1 44 3 10 0 39 1 58 7 55 1 93 211 4 832 57 5 16 8 69 20 2 292 78 0 3 6 212014 790 48 9 15 8 62 200 32 3 88 1 5 7 213013 771 49 0 15 5 64 20 1 316 944 8 8 CI 214 17 770 47 3 15 5 61 20 1 32 8 76 2 7 6 500mg ke 215 20 742 45 6 14 9 61 20 1 32 7 74 2 8 5 216 16 845 49 3 16 1 58 19 1 327 90 7 6 2 Mean 791 67 49 60 15 77 62 50 19 93 31 88 83 60 6 73 SD 39 59 4 11 0 64 3 73 0 41 1 39 8 51 1 97 311 6 793 53 6 16 2 68 204 30 2 78 6 78 3812 1 797 48 1 16 2 60 20 3 33 7 75 2 11 0 313 3 793 48 4 15 8 61 19 9 326 88 8 92 CI 314 18 803 49 5 16 6 62 20 7 33 5 83 6 11 0 1000mg k 315 12 764 46 9 154 61 20 2 32 8 80 7 8 1 316 21 840 487 15 9 58 18 9 326 815 6 3 Mean 798 33 49 20 16 02 61 67 20 07 32 57 81 40 8 90 50 448 2 32 0 41 3 39 0 63 125 4 61 1 87 3 3 2 Measuring data of blood and serum Measurement item Group ps K Ht Hb MCV MCH MCHG Platelet 10 p D 8 91 1 pg 10 D oS D 161 6 768 50 1 154 65 20 1 30 7 84 1 40 162 1 741 492 154 66 20 6 313 66 5 10 1 163 10 775 53 8 15 8 6
81. 200L C 1 C I C I 50 2 CI B C I 50 CI E 3 AMEC I C I C 1
82. 30C 140rpm 3 60L JFM 30C 150rpm Air 1 5L min 3 3 0C478 10 BD 10 BD Leda M 0C478 1 7 1 10 80 CItase 1 7 2 0C4788 C I 30 10 80 0C4788
83. 14 0 0 6 0 5 2 4 C I C I 2 5 C I
84. SD 0 1 0 1 0 9 0 0 0 2 0 0 0 0 Mean 1 2 1 5 13 0 0 7 3 3 0 1 3 4 Male 6 CI SD 0 1 0 1 1 2 0 1 0 2 0 0 0 3 500mg kg Mean 0 9 12 8 1 0 5 2 0 0 1 0 2 Female 6 80 0 1 0 1 0 6 0 1 0 1 0 0 0 0 Mean 1 3 1 6 12 8 0 8 3 3 0 1 3 6 Male 6 CI SD 0 1 0 1 1 2 0 1 0 3 0 0 0 3 1000mg kg Mean 0 9 14 8 5 0 5 2 0 0 1 0 2 Female 6 80 0 1 0 3 1 6 0 1 0 2 0 0 0 0 Unit g P 0 05 Significant difference from Vehicle male by Dunnett test 35 2 Relative weight Group Sex iotalianimal Heart Lung Liver Spleen Kidney Adrenal Testis or number gland Ovary Mal 6 Mean 0 31 0 36 3 02 0 18 0 77 0 02 0 74 ale control 80 0 01 0 02 0 15 0 01 0 09 0 00 0 07 Vehicle Mean 0 37 0 49 3 15 0 20 0 81 0 04 0 06 Female 6 80 0 02 0 03 0 12 0 01 0 05 0 00 0 01 ii 6 Mean 0 29 0 36 3 07 0 18 0 79 0 02 0 81 ale CI 80 0 01 0 01 0 17 0 02 0 06 0 00 0 07 500mg kg Mean 0 35 0 50 3 31 0 22 0 80 0 04 0 06 Female 6 8 0 02 0 03 0 18 0 02 0 04 0 01 0 00 6 0 31 0 39 3 10 0 18 0 80 0 02 0 87 ale CI 80 0 01 0 02 0 11 0 03 0 04 0 00 0 06 1000mg kg Mean 0 37 0 56 3 38 0 21 0 81 0 03 0 06 Female 6 SD 0 02 0 15 0 22 0 02 0 04 0 01 0 01 Unit P lt 0 05 Significant difference from Vehicle male by Dunnett test 87 36 1 Organ weight Group prs Heart Lung Liver Spleen Kidney Adrenal gland
85. 1 1 1 C I CD CD o 1 4 C 1I oc 1 6 7717 CH gt CHsOH CHz X HOH oTa 0 lt OH RO o 097 0 3 Y CH 0H OH HOL N i N j OH o AW K OH A HOO N of V OH A OH V _ v 3 i b oH Ha HO 0 o 0 cd d OH T O oH NOH A 7 N p DE f f CH2 O A cmo CHzo 0 CH 8 C 1 CD
86. 30 ey CD 727 34M B CD y CD C I Plus30 CD C I C I Plus30 ii 1 5 1 5 2 24g 0 045 g 1L 43 i 2 3 16
87. 80C BD Plate 4 C 7 BD Plate BD Plate 30C 5 1 HE Plate 4C 20 7 BD Plate 0 3g Bacto Tryptone 1 0g Yeast ext 0 1g NaCl 0 5g Bacto Agar 1 5g 100ml pH ii 10 6 10 DMSO ff 6 BD Plate T 40 4 6 0C478
88. C I i 0 2M Na Buffer 50mM CaCl pH5 5 1M 1M 0 5M 50mM CaCl 0 2M 0 2M 2 pH 5 5 0 2 Na Buffer 50mM CaCl ii 4 TUER Ag 100ml 121C 15 o Ru AZ iii 00478 0C478 FUERTES Ze 8 000rpm 20 4C EHZ O0 2um 5k Da iv CI
89. 140rpm 140rpm BD Plate 7 0 Iml Plate em T Z WR L 30C 1 28 0 5 100m1 30 X8 T 40 Dextran T 40 1 0g Wako Polypeptone 1 0g Yeast ext 0 1g 0 5g 100ml pH8 0 Xo BH Starch T 40 1 0g Wako Polypeptone 1 0g Yeast ext 0 1g 0 5g 100ml pH8 0 R10 e 4 _ z SUR D Dextran S Starch H y SUBE Ub Dy U L 29 iii CItase DGase
90. 28 72 jv Cf 0 5 2 1 2 2 500mg kg 1000mg kg 10mL kg 10 155 1000mg kg day S OECD 407 500mg kg 1000mg kg 7 1 28 1 1 1 7 14 21 28
91. C I 25 4 0 C 30C 8 000rpm 5 MS n NO TOMY 1 3 BARALAR E ORDRER OVT 21 QUE 600Da NF C 600Da NF 414 4 C I 4 600DaNF E J 1 OMPa 40 2001 79L 20 f 14L 2651 22 2 QO 101 L SR CI AWPEB AED SN 1 5 C 1 200 L MF LAPX404 6 000rpm 5L 20 200L
92. min MR CI CI Bio 2012 3 6 MR CI L CI CITase 2012 HPLC Amide 80 Tosoh CI 0 10 20 30 40 50 70 80 90 100 110 120 60 HBDase Glucoamy lase T M T 0 10 20 30 40 50 60 70 80 90 100 110 120 min L CI 2012 CI 01 7 01 26 ZJL3 A 73 53 8 37 L CI 2012 HPLC Amide 80 Tosoh CI 55 CD CI 3 8 b DR CI Plus 2009 CI Plus 2
93. 2009 HPLC Amide 80 Tosoh E 8 i CI o 25 E 50 mir Tu HBDase Glucoamylase 17 396 9 396 0 10 40 50 20 30 min CI Plus CI Bio 2010 3 3 CI Plus 2010 HPLC Amide 80 Tosoh 53 CI 387r f GI 7 C 14 0 69717798 30 40 50 0 10 20 min me 56 4 38 3 HBDase Glucoamy lase mV min CI TTC 1 2012 3 4 CI TTC 1 TTC 2012 HPLC Amide 80 Tosoh 00 xu AA E CI ALM E GI 7 CI 14 Te F mir cO _ j 54 4 39 7 400 z HBDase Glucoamy lase t io T E 200 H E 1 0 9 0 10 20 30 40 50 min CI TTC 2 2012 13 5 CI TTC 2 TTC 2012 HPLC Amide 80 Tosoh 54 d I azr oe qu 2 O uc 19 5 750 d 500 4 s 250 d Ds 0 10 20 30 40 50
94. o lt G 020 1 R 0 10 0 00 14 21 4C 1 8 2 D S 1 S DGase N 3 1 3 200ml 500ml 500ml 0 4 0 8 ml 2ml 2ml 30C 140rpm 30C 140mpm 30 140 8 7 6 X 10 cfu ml 1 7 X 10 cfu ml 1 3 x 10 cfu ml 18 3 D S 1 S 0478 33 0C478 4 JFM DO p 1 9 0C478 40hr AR 40 60hr
95. 0 35 2 85 0 20 0 74 0 01 0 80 500mg kg 215 20 0 30 0 37 3 28 0 18 0 74 0 02 0 83 216 16 0 26 0 37 2 96 0 15 0 81 0 02 0 75 Mean 0 29 0 36 3 07 0 18 0 79 0 02 0 81 SD 0 01 0 01 0 17 0 02 0 06 0 00 0 07 311 6 0 29 0 39 342 0 20 0 83 0 02 0 83 312 1 0 32 0 39 3 15 0 16 0 81 0 02 0 88 313 3 0 32 0 39 3 01 017 0 75 0 02 0 89 CI 314 18 0 32 0 36 3 13 0 18 0 85 0 02 0 97 1000mg k 315 12 0 32 0 37 3 26 0 15 0 77 0 02 0 78 316 21 0 31 0 41 2 95 0 23 0 78 0 02 0 90 Mean 0 31 0 39 3 10 0 18 0 80 0 02 0 87 SD 0 01 002 0 11 0 03 0 04 0 00 0 06 Unit 37 2 Relative weight Group NN Heart Lung Liver Spleen Kidney Adrenal gland Testis or Ovary 161 6 0 40 0 47 3 24 0 18 0 74 0 03 0 07 162 1 0 37 0 46 3 04 0 19 0 82 0 03 0 06 163 10 0 37 0 51 3 33 0 19 0 86 0 03 0 06 ntr l 164 C11 0 34 0 47 3 20 0 20 0 81 0 04 0 05 Vehicle 165 8 0 36 0 53 3 01 0 21 0 88 0 03 0 07 166 4 0 40 0 51 3 11 0 22 0 78 0 04 0 06 0 37 0 49 3 15 0 20 0 81 0 04 0 06 50 0 02 0 03 0 12 0 01 0 05 0 00 0 01 261 17 0 34 0 46 321 0 24 0 79 0 03 0 06 262 18 0 38 0 54 3 56 0 20 0 79 0 03 0 06 263 13 0 35 0 52 3 43 0 22 0 80 0 04 0 06 CI 264 21 0 34 0 51 3 40 0 24 0 88 0 04 0 07 500mg kg 265 12 0 32 0 48 3 07 0 20 0 78 0 04 0 06 266 2 0 35 0 52 321 0 19 0 75 0 05 0 06 Mean 0 35 0 50 331 0 22 0 80 0 04 0 06 SD 0 02 0 03 0 18 0 02 0 04 0 01 0 00 361 20 0 39 0 45 3 41 0 24 0 78 0 02 0 07 362
96. 14 6 242 24 6 16 7 262 18 25 2 18 4 21 3 20 4 21 2 263 13 20 8 18 9 24 3 24 2 23 1 CI 264 21 17 5 18 8 21 9 20 7 20 9 500mg k 265 12 19 9 20 5 214 17 5 20 9 266 2 27 3 17 6 19 8 19 4 20 9 22 3 18 1 22 2 21 1 20 6 5 3 6 2 0 1 8 2 8 2 1 361 20 11 8 22 1 26 5 25 6 26 8 362 19 214 23 7 19 5 28 2 22 5 363 15 21 1 17 7 19 5 19 3 22 0 CI 364 3 15 3 18 4 20 5 20 5 20 2 1000 365 16 19 1 19 9 18 2 23 2 21 7 366 5 20 9 19 9 18 2 24 3 21 7 Mean 19 3 20 3 20 4 23 5 22 5 5 5 4 2 3 3 1 3 3 2 2 Unit g 32 1 M Measuring data of blood and serum S x Total animal Measurement item number o u D Ht Hb g dl MCV f MCH pg MCHC aou D Mea 7727 476 5 6 62 0 20 2 327 78 0 94 control SD 70 1 2 6 14 3 1 04 1 6 75 19 Vehicle Mean 765 7 50 4 5 6 65 7 20 4 31 0 713 6 6 Female 6 SD 17 8 24 0 4 23 04 0 9 13 9 20 Mean 791 7 496 5 8 62 5 19 9 31 9 83 6 6 7 Cl SD 39 6 41 0 6 3 7 04 14 8 5 20 500mg kg Mean 7448 484 5 1 64 7 20 3 312 714 70 Female 6 SD 36 0 2 0 0 8 10 0 6 0 8 12 3 20 Mitis Mean 798 3 492 6 0 617 20 1 326 814 8 9 Cl SD 24 5 23 0 4 3 4 0 6 1 3 46 1 9 1000mg kg Mean 766 2 50 9 5 5 66 3 20 2 304 81 8 70 Female 6 SD 231 28 0 7 20 04 0 9 14 1 24 83 33euung Ad Wo e2ueJejyip 3ueoyrusIS G0 0 d 20 20 lt 0 oz
97. 67 2 1 Body weight changes in rats after single oral administration in acute toxicity study 7 Total animal Day after administration Group Sex number 0 1 3 7 10 14 218 9 244 8 268 6 310 5 345 6 388 6 Male 5 8 49 51 59 9 8 95 14 8 Control Vehicle Mean 160 1 183 8 192 9 210 8 231 5 242 7 Female 5 SD 46 45 8 2 94 140 10 6 Mean 218 8 245 6 269 7 310 0 346 3 390 9 Male 5 8 43 48 70 13 1 14 8 16 8 CI 000 mg Mean 160 0 180 1 191 9 204 2 217 233 0 Female 5 SD 5 0 42 8 7 9 7 13 2 141 219 0 244 8 266 0 308 2 349 4 392 2 Male 5 SD 5 2 5 7 6 0 66 8 3 174 CI 2000 mg kg Mean 160 2 183 0 191 1 207 8 246 3 Female 5 SD 5 9 7 6 80 75 94 10 6 Unit g Significant differences were not observerd X 22 1 Body weight changes in male rats after single oral administration in acute toxicity study Animal No Day after administration Mud ID No o 0 1 3 1 10 14 101 7 2264 250 7 276 0 324 8 358 7 409 5 102 10 219 6 249 5 2740 3151 351 9 395 7 103 3 219 2 2440 264 6 302 0 338 4 373 8 Control rw Hex Nm vehic 104 11 215 7 240 8 263 7 309 2 342 8 388 8 105 18 218 7 239 5 264 8 301 8 336 3 376 8 Mean 218 9 244 8 268 6 310 5 345 6 388 6 8 49 51 5 9 9 8 95 148 201 8 224 6 251 9 281 7 331 3 369 6 416 9 202 6 220 8 241 7 269 7 310 3 348 5 394 1 208 5 2188 248 5 266 5 3
98. 80 00 6 00 091 8t 90 SZL L S 0Z asc mm 80 06 00 120 6 o st 0 62 0 9Z 76 Lvl 9 L ESE ozz 801 ueaW 4 3w000 1 00 20 61 oz 0 Lz 00 61 00 EY zu 8 0 9911 SOE asc 7 an yz LG 001 0 810 LG ZSh 0 LZZ 0 L6y 916 981 L Y6G 82 99 00 0 vo 21 91 0 vi 89 00 rm 91 gui 90 68 FE Loz asc 5 nm 80 oz LG 06 00 6 g Sb 0 08Z 0 892 SL S ILE g 877Z g0Z 8E ay au009 00 0 62 0 z 0 62 00 LY 00 092 29 v 90 2801 VL 9 asc du 80 Sz L S LOL 700 0G 0 0 8vG L 9G 921 9 6696 872 20 vo I vo 21 0 01 00 00 gv 891 0 60v 89 80Z asc 5 80 9G 96 8720 6v S S 0 92 0 8 9 10 9 82 gzz 6 ueeW elouleA 00 0 91 0 01 0 61 00 t 00 SEE FEL 6 60 69 29 VEE asc sin 199999 yz 96 86 870 LG Stt vic 0 019 g09 SI 066 ovy ueeW oy CIp 3 1 9 7 30 1 e3u 1 5 mw 1 b3u 1 3u 1p 3u TP 8u 3u p 320 17 Qm Qm QO m gts lv 1 d go IO 4 eN e919 NN 8 1 91 1 dl1D A div IW 1SV ej S pue poo q jo Sulansee z zeX 84 3 3 1 Measuring data of blood serum Measurement item Group MM RBC Ht Hb MCV MCH MCHC Platelet 10 u D g d D n pg C
99. NN NN NN NN NN N N NN NN N N N N NN NN N N N N NN NN N N N NN NN N N NN N N NN NN NN N N NN NN N NN NN NN NN NN NN NN NN N N NN N NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN NN N NN NN N N N N N N N N N N N NN N N N NN NNN NN NN NN NN NN NN NN NN NN NN NN NN N NN NN N N N N N N NN NN N N N N NN NN NN N N N N N NN NN NN NN N N NN NN NN NN NN NN NN NN NN NN NN NN NN N 0 Animal No ID No Group control Vehicle 17 18 13 21 12 261 262 263 264 265 266 500mg kg NN NN NN NN N NN NN N N NN NN NN NN N N N NN NN N NN N N NN N Normal 78 500 450 r 350 300 r e Control Vehicle 250 f Cl 500mg kg Mean body weight g 200 r 2 Cl 1000mg kg 1 7 14 21 28 29 Day after administration 4 5 1 Body weight changes in male rats 28days iteration oral administration in subacute toxicity study 400 r 350 r 269 Control Vehicle Mean body weight g 150 r x cl 500mg kg CI 100
100. Testis or Ovary 111 5 42 171 13 52 0 88 410 0 08 3 64 112 19 38 1 59 15 08 0 90 3 21 0 08 3 32 113 C10 42 1 69 12 93 0 89 3 76 0 08 3 70 114 2 28 1 55 13 20 071 287 0 07 3 18 Vehicle 115 8 21 144 1245 0 65 3 12 0 07 3 14 116 9 38 1 59 1244 0 72 3 14 0 08 2 65 35 1 60 13 27 079 3 37 0 08 3 27 SD 0 08 0 10 0 98 0 11 0 46 0 01 0 38 211 4 27 1 51 13 62 0 65 3 66 0 07 3 19 212 14 43 142 11 68 0 75 3 46 0 06 3 68 213 13 31 170 14 33 0 80 3 35 0 07 3 67 CI 214 17 23 149 11 99 0 85 342 0 05 3 38 500mg kg 215 20 29 1 58 14 18 0 79 3 19 0 08 3 57 216 16 05 149 11 98 0 60 327 0 07 3 02 Mean 21 53 12 96 0 74 234 0 07 342 SD 0 10 0 10 1 21 0 10 0 20 0 01 0 27 311 6 29 174 13 97 0 91 3 72 0 08 3 70 312 1 31 59 12 79 0 66 3 28 0 09 3 58 313 3 21 48 11 30 0 64 2 86 0 09 3 36 CI 314 18 38 58 13 59 0 77 3 68 0 09 420 000mg kg 315 12 33 57 1374 0 62 3 26 0 07 3 31 316 21 21 59 11 39 0 90 3 02 0 07 3 46 29 59 12 81 075 3 30 0 08 3 60 SD 0 07 0 08 1 17 0 13 0 34 0 01 0 33 Unitg 36 2 Organ weight Group Heart Lung Liver Spleen Kidney Adrenal gland Testis or Ovary 161 6 1 03 1 22 8 43 0 47 1 92 0 09 0 17 162 1 0 95 1 20 787 0 49 241 0 09 0 15 163 10 1 02 140 9 14 0 53 2 36 0 09 0 17 control 164 C11 0 86 1 18 7 98 0 50 2 02 0 09 0 13 Vehicle 165 8 0 84 1 23 7 01 0 50 2 04 0 08 0 16 166 4 0 87 111 6 83 0 48 1 72 0 09 0 14 Mean 0 93 1 22 7 88
101. ml 6 A N Water E Ca only 1000 f Buffer Ca 0 6 12 18 24 30 36 42 hr 9 1 Na Buffer CaCl C I 8 0 7 0 6 0 pH 5 0 Water 40 only Buffer Ca 3 0 0 6 12 18 24 30 36 42 hr 2 Na Buffer CaCl pH 16 C 1 10L 0C478 CI 2 C I 1 0 c 2 30 mU ml 25C bml C 1 p H
102. 0 4000 3000 2000 1000 0 0 6 12 18 24 30 36 42 48 hr 4 1 CI 5000 4000 3000 2000 1000 0 0 3 6 9 12 15 18 21 24 hr 4 2 C 1 11 n f i 25 4 5 6000 5000 Total CI ug ml 1000 2 6 B o o o w N WA ET 5 30 mU ml DLE ZR 0C478 CI 12 hr pH C 1 C s BERI 2
103. 0 50 2 03 0 09 0 15 SD 0 08 0 10 0 87 0 02 0 21 0 00 0 02 261 17 0 92 1 23 8 68 0 66 213 0 09 017 262 18 0 94 1 31 8 72 0 49 1 94 0 08 0 15 263 13 0 89 1 32 8 70 0 55 204 0 09 014 CI 264 21 0 78 1 18 7 80 0 56 2 02 0 10 0 15 500mg kg 265 12 0 76 142 721 0 47 1 84 0 09 0 14 266 2 0 84 125 177 0 46 1 82 0 11 0 15 Mean 0 86 1 24 8 15 0 53 1 97 0 09 0 15 50 0 07 0 08 0 64 0 08 0 12 0 01 0 01 361 20 1 08 1 22 9 34 0 65 244 0 06 0 20 362 19 141 144 1151 0 58 228 0 10 0 18 363 15 0 82 1 30 749 0 53 201 0 09 0 13 CI 364 3 0 84 1 19 7 30 0 46 1 85 0 07 0 14 1000mg k 365 16 0 79 147 7 61 0 40 1 88 0 10 0 14 366 5 0 92 201 733 0 50 1 91 0 08 0 12 0 93 1 39 8 50 0 52 201 0 08 015 SD 0 14 0 32 1 65 0 09 0 17 0 02 0 03 Unit g 88 37 1 Relative weight Group prs Heart Lung Liver Spleen Kidney Adrenal gland Testis or Ovary 111 5 0 32 0 38 3 01 0 20 0 91 0 02 0 81 112 19 0 29 0 34 3 18 0 0 68 0 02 0 70 113 C10 0 30 0 34 277 0 19 0 80 0 02 0 79 control 114 2 0 30 0 36 3 10 0 17 0 67 0 02 0 75 Vehicle 115 8 0 30 0 36 3 10 0 16 0 78 0 02 0 78 116 9 0 33 0 38 2 97 0 17 0 75 0 02 0 63 Mean 0 31 0 36 3 02 0 18 0 77 0 02 0 74 SD 0 01 002 0 15 0 01 0 09 0 00 0 07 211 4 0 30 0 35 3 19 0 15 0 86 0 02 0 75 212 14 0 29 0 36 2 97 0 19 0 88 0 02 0 94 213 13 0 29 0 38 3 19 0 18 0 75 0 02 0 82 CI 214 17 0 29
104. 010 MR CI L CI CI Plus 2009 CI Plus 2010 L CI 7 MR CI 2 1 30 CI 4 0 L CI 1 2009 CI Plus 2010 L CI 16 I Plus 2009 CI Plus 2010 34 ZDZ CI 7 CI 10 CI 11 CI 12 CI 10 L CI CI TTC 1 TTC 2 MR CI CI Plus 2009 I Plus 2010 16 CD 40 CI 7 CI 12 L CI SH L CI 650
105. 04 8 332 9 380 2 CI 204 13 216 2 240 1 264 8 296 0 334 0 372 6 1000 mg kg 205 17 213 4 245 7 265 6 307 5 346 5 390 7 Mean 218 8 245 6 269 7 310 0 346 3 390 9 SD 4 3 4 8 13 1 148 16 8 301 16 224 6 2494 272 9 308 2 353 1 393 4 302 4 223 9 248 3 269 5 313 4 355 2 407 8 308 9 218 5 2484 267 9 315 6 357 6 410 6 CI 304 14 2149 236 5 258 0 299 4 339 4 3741 2000 mg kg 305 1 2129 241 2 261 7 304 2 341 6 375 0 Mean 219 0 244 8 266 0 308 2 349 4 392 2 8 52 5 7 6 0 6 6 8 3 174 Unit g Significant differences were not observerd 68 2 2 2 Body weight changes in female rats after single oral administration in acute toxicity study Animal No Day after administration diii ID 0 1 3 1 10 14 151 2 165 3 1831 192 5 152 14 164 5 1894 199 8 153 4 1586 178 8 185 9 ere 154 9 1574 187 2 202 5 155 11 154 9 180 8 184 0 226 5 Mean 160 1 183 8 192 9 231 5 8 46 45 8 2 94 140 251 15 167 0 185 9 204 1 215 1 231 0 252 19 162 4 174 8 185 2 198 1 207 0 253 13 159 1 179 7 182 8 1911 200 8 212 6 CI 254 1 157 9 182 2 196 7 210 8 228 0 243 1 tid 255 10 153 8 1781 190 9 205 7 221 240 3 Mean 160 0 180 1 191 9 204 2 233 0 8 5 0 42 8 7 97 141 351 6 168 8 195 0 202 1 244 1 352 17 162 0 185 3 193 1 254 2 353 7 160 4 181 1 193 6 212 2 258 8 CI 354 3 156 7 176 0 1821 197 0 231 9 2000 mg kg 355 8 153 0 177 5 184 4 2
106. 05 3 242 4 160 2 183 0 191 1 207 8 246 3 8 5 9 16 8 0 7 7 9 4 10 6 Unit g Significant differences were not observerd 3 23 Autopsy in rats after single oral administration in acute toxicity study Group Sex Total animal number Findings Male 5 No abnormality 5 Control Vehicle Female 5 No abnormality 5 Male 5 No abnormality 5 CI 1000 mg kg Female 5 No abnormality 5 Male 5 No abnormality 5 CI 2000 mg kg Female 5 No abnormality 5 a In parentheses indicate the number of cases 69 2 4 1 Autopsy in male rats after single oral administration in acute toxicity study Animal No Group ID No Findings 101 7 102 10 Control Vehicle 108 3 104 11 105 18 201 8 202 6 CI 203 5 1000 mg kg 204 13 205 17 301 16 302 4 CI 303 9 2000 mg kg 304 14 2 a Z uu uz zziz zzz zz N Normal 2 4 2 Autopsy in female rats after single oral administration in acute toxicity study Animal No TEN Group ID No Findings 151 2 N 152 14 N Control F Vehicle 153 4 N 154 9 N 155 11 N 251 15 N 252 19 N CI 253 13 N 1000 mg kg 254 1 N 255 10 N 351 6 N 352 17 N CI 353 7 N 2000 mg kg 354 3 N 355 8 N N Normal 70 2 4 2 REB BKI BECIOZYMeRU 28 1
107. 06 js e 2 Filter 0 00 0 5 10 20 3 00478 CItase 9 0 1 0E 08 8 0 10E 06 7 0 E 1 0 04 20 im E 10E 02 5 0 1 0E 00 4 0 Egg m pum 0 2 Filter pH_ pH pH _0 2Filter 20 4 00478 pH 37 2 2 1 CI im 1 C I Dase C 1 CITase CI CITase DGase CI DGase
108. 0mg kg 1 7 14 21 28 29 Day after administration 45 2 Body weight changes in female rats 28days iteration oral administration in subacute toxicity study 79 3 28 Body weight changes in rats 28days iteration administration in subacute toxicity study Total animal Day after administration Group Sex number 1 7 14 21 28 29 us Mean 241 5 3058 3755 4297 4706 4393 ale conti SD 4 3 6 8 11 7 20 9 27 0 28 9 Vehicle Mean 1809 2090 231 6 2554 2669 2492 Female 6 SD 8 7 15 5 20 1 20 7 19 7 20 0 T 2409 3068 3657 4083 4476 4211 ale Cl SD 5 0 8 4 11 6 16 4 19 8 20 1 500mg kg Mean 1783 2063 2306 2486 26411 245 8 Female 6 SD 7 3 13 8 17 6 17 7 13 4 14 5 T Mean 2424 2902 3631 4089 4422 4124 ale Cl SD 7 6 40 4 21 2 25 6 28 5 27 2 1000mg kg Mean 1763 2040 2321 2524 2695 2496 Female 6 SD 7 8 12 0 14 8 29 0 34 9 31 5 Unit g Significant differences were not observerd 29 1 Body weight changes in male rats 28days iteration oral administration in subacute toxicity study Animal No Day after administration Group ID 1 7 14 21 28 29 111 5 249 1 312 9 387 0 447 8 488 8 449 1 112 19 243 9 307 7 382 8 445 3 502 1 474 1 113 10 2406 313 0 385 0 448 1 486 5 467 5 114 2 237 8 304 9 375 9 427 1 465 3 425 3 Vehicle 115 8 238 8 297 3 360 0 400 4 430 0 401 5 116 9 238 5 298 7 3624 409 4 450 6 4
109. 1 va Spi 89 z 89 ge 911 1 291 880 62 29 ZOL 201 9v 91 zeo 812 61 68 see 12 tl 9 191 6 CP 0P Jp 8u pwu Mu 1 09 1 09 7 TP 8u pu Pu Qp 3u 1 1 Gm Qm tei UD lv eo Io eN e919 Nn L 91 1 esoop dlD dv 11 isv ey pue DO01d 40 B1BD Sul Jnseey 200 220 681 290 702 SVO 00 681 000 9Z EF 978 ZSO 9911 YSS 6y0E 05 940 erz 899 166 ZEOL 86101 EL 920 922 196v 0845 09811 19069 C8Z2 099b ueow ZLO EZ SS 06 901 201 ZG vvl 820 6 21 SLL 289 021 12 91 eLo vz US 06 gol 01 6v vvl 620 voz 19 621 z 98 1 92 ESL 21 918 4 3w0001 8L0 Sz US 088 ror 66 Fe 120 z 68 oL LZ i 18 zz ORNS vz ss gol 66 101 8v gpl 820 esz zs v 021 9 9 62 061 glg sr0 68 L6 soL 8 ShL vzo voz 62 os z le LE ZLI 1 218 LO 92 re sel 101 09 07 Szo 8zz zz 8 be ell 9 700 080 262 EE0 SZZ LTO 887 00 21 000 86 9Z 2279 82 990 02801 S01 egee 05 LYZ 2176 S901 0201 19001 209 19691 120 8 vZ 8 vG 1998 09211 081 EE699 9161 Yeaw 8L0 Sz US 98 gol 101 9v 9r 920 602 18 6 nm Lev Lt 921 9 91Z LO z v 06 201 6v vl vzo v6l 06 6 SLL i tz ZSL 02 S12 yasuooe 080 vz vs v6 01 6v ve 080 82 z
110. 18 3 Mean 2415 305 8 375 5 429 7 470 6 439 3 50 43 6 8 117 20 9 270 28 9 211 4 249 8 321 1 378 8 421 9 459 9 426 9 212 14 2412 306 0 356 5 397 0 428 6 392 7 213 13 242 5 309 4 376 9 422 6 470 2 449 3 CI 214 17 237 9 303 4 361 3 399 8 443 0 420 6 500mg kg 215 20 236 9 305 3 370 9 423 1 462 7 432 0 216 16 2369 295 5 350 0 385 3 421 3 405 0 Mean 2409 306 8 365 7 408 3 441 6 421 1 SD 50 84 11 6 16 4 19 8 20 1 311 6 257 6 319 9 389 0 434 8 480 5 448 4 312 1 241 7 303 2 374 8 417 8 433 3 406 0 313 3 237 2 302 9 339 8 374 3 409 1 39 0 Cl 314 18 240 9 307 8 375 7 433 0 465 7 433 6 000mg kg 315 12 238 5 298 2 363 2 411 8 450 6 421 8 316 21 2384 209 2 336 1 381 5 413 8 385 6 Mean 2424 290 2 363 1 408 9 442 2 412 4 SD 76 40 4 212 25 6 28 5 272 Unit g Significant differences were not observerd 80 29 1 weight changes in female rats 28days iteration oral administration in subacute toxicity study Animal No Day after administration Group ID 1 7 14 21 28 29 161 6 192 6 228 3 256 2 275 0 278 0 260 3 162 1 186 0 207 3 231 7 264 1 277 0 258 5 163 10 1841 226 4 253 1 275 9 288 1 274 5 Santol 164 11 1811 205 4 222 0 253 9 271 7 249 7 Vehicle 165 8 171 3 193 5 222 6 2403 0 249 7 2327 166 4 170 1 1927 203 7 2233 236 3 219 5 Mean 180 9 209 0 231 6 2554 266 9 249 2 50 87 15 5 20 1 20 7 197 20 0 261 17 1915 231 5 261 1 282 0 285 5 270 1 2
111. 19 0 37 0 48 3 80 0 19 0 75 0 03 0 06 363 15 0 35 0 56 3 20 0 23 0 86 0 04 0 06 CI 364 3 0 37 0 52 3 18 0 20 0 10 0 03 0 06 1000mg k 365 16 0 35 0 52 3 39 0 18 0 84 0 04 0 06 366 5 0 39 0 86 3 32 0 21 0 82 0 03 0 05 Mean 0 37 0 56 3 38 0 21 0 81 0 03 0 06 SD 0 02 0 15 0 22 0 02 0 04 0 01 0 01 Unit 89 3 38 Autopsy in rats Group control Vehicle Ci 500mg kg CI 1000mg kg 28days iteration oral administration in subacute toxicity study Sex Male Female Male Female Male Female Total animal number a In parentheses indicate the number of cases 90 Findings No abnormality 6 No abnormality 6 No abnormality 6 No abnormality 6 No abnormality 6 No abnormality 6 39 1 Autopsy in male rats 28days iteration oral administration in subacute toxicity study Animal No ID No 1115 112 19 control 113 10 Vehicle 114 2 115 8 N 116 9 Withering of a spermary Right 211 4 N 212 14 CI 213 13 500mg kg 214 17 215 20 216 16 311 6 312 1 CI 313 3 1000mg kg 314 18 315 12 316 21 Group Findings 2 2 2 2 2 222221222 gt 2 39 1 Autopsy in male rats 28days iteration oral administration in subacute toxicity study Animal No ID No 161 6 162 1 control 163 10 Vehicle 164 11 165 8 166
112. 3 29 2 44 7 32 9 19 4 18 6 20 7 4 40 4 52 0 45 9 27 2 24 7 25 9 40 mg g TRIR CI 7 CI 8 CI 9 CI 10 CI 11 CI 12 0 8 15 1 8 0 0 0 1 8 60 9 1 2 9 2 0 1 5 13 2 9 0 15 1 4 8 2 1 1 6 14 7 12 3 18 1 3 6 3 0 2 0 E 40 CI U mg Dex40 Dex Glc SS SS Glc 16 IG Glc Glc Suc Fujioligo Dextrin 21 T 3040 40 CI CI U mg 22 40 Dex40 RATEN SS 1G Dex40 IG Glc T 3040 40 CI Dex40 dex amp 55 IG dex _Dex40 ss SS 55 G Glc dex Dex40 Glc dex 41 1 3 eo DGase mo1 min mg 0 IE 0
113. 4 261 17 262 18 CI 263 13 500mg kg 264 21 265 12 266 2 361 20 362 19 CI 363 15 1000mg kg 364 3 365 16 366 5 Group Findings 222222122222 2 2 222 2 2 91
114. 62 18 1805 210 3 237 8 251 6 266 4 244 7 263 13 1768 203 9 232 6 246 5 272 6 253 7 CI 264 21 173 6 202 6 220 9 2317 250 1 229 5 500mg k amp 265 12 170 3 192 5 213 5 241 8 253 8 234 6 266 2 1712 197 0 2174 238 0 256 2 2422 Mean 1783 206 3 230 6 248 6 264 1 245 8 50 73 13 8 17 6 173 134 14 5 361 20 185 8 219 3 241 5 260 9 293 5 213 6 362 19 1859 2172 253 2 305 8 329 4 302 9 363 15 1719 204 3 228 8 2327 256 9 233 7 CI 364 3 1674 197 8 219 1 229 5 241 3 2297 1000mg k 365 16 174 4 190 2 218 5 233 0 242 9 2247 366 5 1724 194 9 225 5 252 5 253 1 233 0 Mean 1763 204 0 232 1 2524 269 5 249 6 50 78 12 0 148 29 0 34 9 31 5 Unit g Significant differences were not observerd 30 Food consumption in rats 28days iteration oral administration in subacute toxicity study Total animal Day after administration Group Sex number 0 1 7 8 14 15 21 22 27 28 Mean 272 28 3 31 5 31 7 31 8 Male 6 control 8 1 6 2 2 1 4 3 2 2 5 Vehicle Mean 23 7 20 2 21 8 22 0 20 6 Female 6 SD 7 7 1 6 3 4 3 2 2 3 Mean 25 3 28 8 31 0 28 2 31 3 Male 6 CI 8 27 1 8 2 9 1 6 27 500mg kg Mean 223 18 1 222 21 1 20 6 Female 6 8 3 6 20 1 8 2 8 21 26 2 28 8 28 7 30 4 30 8 Male 6 CI 8 27 1 5 40 20 2 3 000mg kg Mean 19 3 20 3 20 4 23 5 22 5 Female 6 8 54 2 3 3 1 3 3 2 2 Unit g 81 3 1 1 Food consumption in male rats 28days iteration oral administration in
115. 64 100 0 11 17 0 08 13 X14 DGase ST mM 0 61 1 4 Glc 1108 7 100 01 1 2 G1c 436 3 39 Glc 1 3 Glc 369 44 33 Gle a 1 6 Glc 22 4 2 Glc Glc N D Glc f 1 4 Glc N D Glc 8 1 2 61 N D 61 B 1 3 G1c N D 61 8 1 6 G1c N D E EE CI 50 CI CI H CI CI 7 CI 10 CI 11 CI 12 4 AE CI 1 WE DGase CI E
116. 9 204 294 99 9 6 5 164 11 785 52 9 16 4 67 20 9 310 794 67 Vehicle 165 8 741 48 0 152 65 20 5 317 60 6 5 3 166 4 778 48 6 154 62 19 8 317 734 6 8 Mean 765 67 5043 15 60 65 67 20 38 30 97 77 32 6 57 50 17 75 2 38 0 44 2 34 0 39 0 86 13 94 2 04 261017 683 45 3 142 66 20 8 313 59 7 34 262 18 764 49 1 15 1 64 19 8 30 8 88 8 77 263 13 734 412 142 64 19 3 30 1 784 79 CI 264 21 756 50 0 15 3 66 20 2 30 6 62 6 9 3 500mg k amp 265 12 742 477 15 4 64 20 8 32 3 59 9 6 0 266 2 790 50 9 16 3 64 20 6 32 0 78 7 75 Mean 744 83 4837 15 08 64 67 2025 31 18 71 35 6 97 50 36 00 2 04 0 80 1 03 0 61 0 85 12 26 2 04 361 20 726 413 144 65 194 29 8 55 9 3 6 362 19 789 55 0 15 9 70 20 2 28 9 87 5 10 0 363 15 778 524 15 9 67 20 4 30 3 719 8 9 CI 364 3 765 50 1 15 6 65 204 311 844 77 1000mg k 365 16 783 51 8 15 9 66 20 3 30 7 88 8 6 3 366 5 756 48 8 15 3 65 20 2 314 96 4 52 Mean 766 17 50 90 1545 66 33 20 15 30 37 81 82 6 95 50 23 08 2 75 0 70 1 97 0 38 0 92 14 05 2 38 85 100 120 120 621 161 080 ort 700 265 000 1911 9681 848Z se0 101 176 oroz 50 LY 86 006 200 19201 26 00991 0g0 18 EE 92 LIEL L9vII 061 LIESE 0022 80 ueeW 290 E 58 901 66 201 69 Sp 820 892 ro ve 16 9L z LOE oz 0 9 99 LLO vz ss 96 001 901 89 Syl LTO z 88 66 z 89 z L6 91 89 34 3w0001 Sz 85 61 66 01 8
117. EAD 0 EMO 4 ERED TTR 0C478 Bh E 2 1 CI 0C478 C pH OC478 CItase 2 Na CaCl Buffer pH5 5 C 1 DGase CItase 2
118. N N N N N NN N NN NN N NN NN N N N N NN NN NN NN N NN N NN NN NN NN N NN NN NN NN NN NN NN N N N NN NN NN N NN NN NN N N NN NN N N NN NN N N N N N NN N N N N NN NN NN N N N NN NN NN N N N NN N N N N N N NN NN NN N N NN N NN NN N NN NN N N N N NN NN NN NN N N NN N NN NN N N N N N N N NN N N NN NN NN N NN NN NN NN N N N N NN NN NN N NN NN NN NN NN N N NN NN NN N NN NN N N N N N N NN N N N N NN NN NN NN NN N NN NN NN NN NN NN NN NN N N N NN NN N N N N N NN N N N N NN NN NN N N N NN NN NN N NN NN NN NN NN NN NN N N NN N NN NN N NN N N N N N N NN NN NN NN AN N NN NN NN NN N NN NN NN NN NN NN NN NN NN AN N N NN NNN N N NN N N NN NN NN N NN 1 Animal No ID Group control Vehicle N Normal 77 General signs in female rats 28days iteration oral administration in subacute 2 7 2 toxicity study 12 13 14 11 10 12 13 6 7 8 Day 8 9 10 11 NN NN NN NN N N NN NN NN N N NN NN NN N NN NN N N N N N N NN NN N N N N NN NN N NN NN NN NN NN NN NN NN N N NN NN NN NN N N N N N N NN NN N N N N NN NN NN NN N N NN NN NN NN NN NN NN NN N NN NN NN N N N N NN NN NN
119. ase CITase CITase 2 1 40 39 CI CITase DGase 40 CI 40 T CI 40 TRAER CEE 7 CO DHRIB amp 48 40 CI CI CI dE Sint 2 2 E 2 CI 1
120. g CI 2000 mg kg N Normal General signs in female rats after single oral toxicity study Animal No ID 151 352 353 354 355 2 14 4 15 19 13 1 10 0 N N N N N N 1 N N N N N N 2 N 3 N 4 N 2 N N N N N N N N N N N N Time after administration hour 6 N N N N N N N N N N N N N N N 66 zZ N zZ N z z zZ N zZ zZ administration in acute a N 7 N 8 N N N N zZ N Day after administration 9 N N N N 10 11 13 N N N N N N N N N N 14 N N N N N N N N Mean body weight g 300 100 300 to 200 g 100 Mean body weight e Control Vehicle N acute toxicity study 3 CI 1000 mg kg CI 2000 mg kg 6 7 8 9 10 11 19 Day after administration 4 4 1 Body weight changes in male rats after single oral administration in Control Vehicle CI 1000 mg kg CI 2000 mg kg 1 1 1 1 1 acute toxicity study 6 7 8 9 10 11 Day after administration 4 4 2 Body weight changes in female rats after single oral administration in
121. nm 4 1 CD CD CD L CI 3 20 CD CD CI T CI 10 CI 11 CI 102 TH L CI CI PTus 2009 CI Plus 2010 5 CI CT Plus 2009 CT P1us 2010 20 5 CI CI 7 L CI CI Plus 2009 CI Plus 2010 4 2 C1 7 L CI 2 5 23 CI Plus 2009 0 5 CI Plus 2010 1 2 5
122. s 6 SLL z 62 00 FAO 85 601 OL 66 ge 9 1 LTO LZ ze 89 z SLS Lu 00 amp 1 amp 180 92 85 96 96 201 rs tz0 TEZ Sz es zu z 968 Lz vel 1 92 29 vol 901 16 161 81 6v 09 soL 68 SE 191 S00 ZrO 810 91 0g0 860 620 181 00 67 000 16t 6011 048Z se0 59 69 6088 05 r70 BEZ 299 56 SLOL 870l S0G 06 vzo oriz oro 0019 09 08111 061 0066 1942 0071 ueow LO EZ rg 201 6v zvl vzo roz 8L 6 z 705 Sz 661 6 911 ELO vz US SL 001 01 8v 020 22 56 v9 Zbl 656 z 86 8 911 alolyaN LO Ez vs zol 66 OL vs vl oeo viz 6 v 18 12 Sel 2 11 Iouoo 940 901 zol 201 Lb 9 1 20 voz 89 99 s6 z LIS 82 991 01 990 ez gs am Lol 201 Lvl Szo 02 iz SL z 68v le LEL 61 211 180 92 gs OL 66 OL 2 ShL el 19 06 te 8 S61 DA 198 8 JP TP 8u Qp7 3u 1 P um p w Pu pu PAu TP 8u 1 1 am 171 171 gv di d eo Io z eN e919 18 1 91 euo ssosnID dlp A dW 11 15 ES We Wn4ss pue jo 86 35 1 Organ weight Group Sex Total animal Heart Lung Liver Spleen Kidney Adrenal Testis 9f number gland Ovary Mean 1 3 1 6 13 3 0 8 3 4 0 1 3 3 Male 6 control 8 0 1 0 1 1 0 0 1 0 5 0 0 0 4 Vehicle Mean 0 9 12 79 0 5 20 0 1 0 2 Female 6
123. subacute toxicity study Group Animal Intakelday o o SSS ID No 0 1 7 8 14 15 21 22 27 28 111 5 29 9 32 1 33 6 29 9 33 0 112 19 284 29 2 31 2 36 9 32 2 113 10 259 27 5 31 9 33 9 33 1 114 2 25 7 28 3 324 30 3 32 7 Vehicle 115 8 26 6 26 7 30 0 279 26 8 116 9 26 7 25 9 29 9 31 3 33 1 Mean 272 28 3 31 5 81 7 31 8 50 16 22 14 3 2 2 5 211 4 27 6 28 0 30 9 26 9 340 212 14 20 6 27 3 27 9 27 5 30 8 213013 26 6 31 6 29 9 30 3 34 2 Cl 214 17 280 30 3 31 6 28 8 29 4 500mg k 215 20 24 8 27 6 36 3 297 32 3 216016 243 27 7 29 3 26 2 27 3 Mean 25 3 28 8 31 0 28 2 31 3 SD 27 1 8 2 9 1 6 2 7 311 6 29 8 28 5 27 9 31 1 33 3 312 1 25 5 29 5 33 6 32 9 32 6 313 3 21 9 30 0 22 8 28 0 28 1 Cl 314 18 24 9 29 4 32 4 28 9 32 3 000mgkg 315 12 28 0 29 6 29 2 32 1 30 6 316 21 26 9 25 9 26 3 29 1 27 9 Mean 262 28 8 28 7 30 4 30 8 50 27 1 5 40 20 23 Unit g 82 3 1 2 toxicity study Food consumption in female rats 28days iteration oral administration in subacute Animal Intake day Group ID 0 1 7 8 14 15 21 22 27 28 161 6 24 8 20 5 22 5 24 9 172 162 1 14 4 19 4 21 9 22 8 21 3 163 10 20 3 17 7 21 6 25 3 18 7 control 164 11 33 0 22 5 26 5 21 9 23 3 Vehicle 165 8 17 5 20 0 15 9 20 7 20 6 166 4 32 1 21 3 22 4 16 6 22 6 Mean 23 7 20 2 21 8 22 0 20 6 5 77 1 6 3 4 3 2 2 3 261 17 23 1
124. v vl 180 92 zz 6L 861 952 61 101 Sz 85 06 66 01 v Lvl LeO 802 vL 09 544 oz Sll 91 9 6L0 92 65 86 OL 001 8 Spi LTO 61 6L 26 921 6 61 29 280 Lz 09 vL 201 6 Vl 620 82 9v zL sei z 9vv 61 02 19 00 SrO TZ 10 91 LEO LE I 900 289 000 1911 91 9Lvl 290 98 8 ersz qs g80 097 19 568 0F0L 49201 18 BA ze 0 S612 8890 0910 091 1 1 88LLZ 00 098EI uesW 180 12 89 rg 66 1 vvl 0g0 ro 08 ZL 961 82 81 611 2 992 980 zg 56 S01 Fe vl ezo Le ol 6 LLL oLz LL LEL 2 992 34 3w008 180 Sz 95 78 zol 01 vv Syl veo vie 62 LL LI voz oz zLl DNS 80 Sz ge L8 66 OL 2 Spi veo 998 6L LL 081 z 0 92 gll 92 780 65 v6 ZOL 201 1 Lvl zy0 ove ez 18 WE A 808 oz 811 81 292 8L0 82 vo ru 601 101 rs Lvl 820 ZZ vy soL veL 962 12 L9L 01 192 100 120 560 921 LUL 0 S01 500 90 000 08 2891 bel 560 88 07 LL 9 7800 ASF 8 0 ESZ 096 566 8001 Ez820I 099bL 820 orsz oro 8881 099L EEIOL 061 800 EE6EI UPN 980 29 08 v6 voL 9v 5 820 vec I 9L z eze LI LEL 7 991 060 92 ge v8 01 8v vel ezo vez Lz 86 LL 292 61 8 991 LO Ez vs 06 001 OL rs Lvl 020 602 ro SL z8 s6 LSE gz 9ll II v9l jenuop Sz 85 ru OL 101 rs 9 1 00 8L 96 201 z zee 1Z 921 01 891 80 Sz 901 001 0
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