ver5.1 HS 1. ALPHA LIPOIC ACID () S S COOH HS 1. α - SH COOH - ( 1) CoA -( 2)- - --P80 (--WSP8-L1-WSPC8 -LC1) - 1

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ORYZA OIL & FAT CHEMICAL CO., LTD. ALPHA LIPOIC ACID --P () --P80 () --WSP8 () --L1 () --PC () --PC80 () --WSPC8 () --LC1 () ver. 5.1HS

ver5.1 HS 1. ALPHA LIPOIC ACID 16 3 31 0331009 - () 19 5 24 0524001 S S COOH HS 1. α - SH COOH - ( 1) CoA -( 2)- - --P80 (--WSP8-L1-WSPC8 -LC1) - 1

ver5.1 HS [] [] CO 2 [] [] - CoA [] [-] - CO 2 H 2 O e - [] ATP e - Q10 [] H 2 O e - ATP* * 3-2. - 2

ver5.1 HS 2. () - 1) () - 2) 2004 Nature Medicine- ( ) 3) - 4) 1) Saengsirisuwan V., Perez F. R., Sloniger J. A. Maier T., Henriksen E. J. Interaction of exercise training and α-lipoic acid on insulin signaling in skeletal muscle of obese Zucker rats. Am. J. Physiol. Endocrinol. Metab., 287, E529-536 (2004). 2) Burke D. G., Chilibeck P. D., Parise G., Tarnopolsky M. A., Candow D. G. Effect of α-lipoic acid combined with creatine monohydrate on human skeletal muscle creatine and phosphagen concentration. Int. J. Spot. Nutr. Exerc. Metab., 13, 294-302 (2003). 3) Kim M. S., Park J. Y., Namkoong C., Jang P. G., Ryu J. W., Song H. S., Yun J. Y., Namgoong I. S., Ha J., Park I. S., Lee I. K., Viollet B., Youn J. H., Lee H. K., Lee K. U. Anti-obesity effects of α-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase. Nat. Med., 10, 727-733 (2004). 4) Dicter N., Madar Z., Tirosh O. α-lipoic acid inhibits glycogen synthesis in rat soleus muscle via its oxidative activity and the uncoupling of mitochondria. J. Nutr., 132, 3001-3006 (2002). 3

ver5.1 HS (1) (in vitro) L6-24 - ( 3) 3. α- (L6) (±S.E., n=6) L6 96-24 MTT (2) (in vivo) - 24 in vivo ( 4) 4. - (±S.E., n=6) (ddy5 ) - (0.05 0.1%) (MF, ) 24 4

ver5.1 HS (3) (in vitro) - (3T3-L1) ( ) 5) ( 5) - 3- (GPDH) 3T3-L1 GPDH - ( 6) - 5) Cho K. J., Moon H. E., Moini H., Packer L., Yoon D. Y., Chung A. S. α-lipoic acid inhibits adipocyte differentiation by regulating pro-adipogenic transcription factors via mitogen-activated protein kinase pathway. J. Biol. Chem., 278, 34823-34833 (2003). Control 1 µg/ml 3 µg/ml 10 µg/ml 5. α- 6. - GPDH (n=2-3) 5

ver5.1 HS 3T3-L1 2 (1 µg/ml) (0.25 µm) (0.5 mm) - 2 - (1 µg/ml) 1 7 3T3-L1 GPDH [ () ] (4) (in vivo) - 13 7 - (0.1%) 7. - (n=5) (ddy5 ) - (0.1%) (MF, ) 13 (MK-770M) 10 (5 rpm) 1 1 (5) (in vitro) (HepG2) (L6) 1 -CPTACOX AMPK PPAR ( 9) 6

ver5.1 HS 8. CPT CoA ACOX camp AMPK PPAR - CPT - CPT - -ACOX ACOX - AMPK PPARα PPARγ β-actin - CPTACOX AMPK PPAR ( 9) 7

ver5.1 HS CPT ACOX PPAR AMPK β-actin α-µg/ml 0 1 3 10 100 α-µg/ml 0 1 3 10 100 9α-β-actin 10- S.E., n=6 ( 10)- - -- Triglyceride (% of control) 100 100 Triglyceride (% of control) 80 80 60 60 40 40 20 20 0 0 0 1 3 10 30 100 0 1 3 10 α-lipoic acid (µg/ml) α-lipoic acid (µg/ml) 30 100 1. (HepG2) 12 - DMSO 24 (L6) 12 8 - DMSO 24 RNA (RNeasy micro QIAGEN ) RNA (SuperScript IIIInvitrogen ) DNA (PCR) 2. (HepG2) (L6) 96 1 () 8

ver5.1 HS (6) - 8-100 mg 10-200 mg 4 - (100 mg/) 4 (p<0.01) (p<0.05) ( 2 ) - (200 mg/) BMI (p<0.05) ( 3 ) - (100 200 mg/) 4-2. - (100 mg) / (kg) 65.512.2 65.512.1 4 / 7 (%) 19.15.8 18.65.9 6 / 7 BMI (kg/m 2 ) 22.24.0 22.24.0 3 / 7 Ω 49066 48055 5 / 7 (%) 12.72.6 12.53.2 6 / 7 (%) 0.918.3 0.918.2 4 / 7 (cm) 77.210.9 77.612.1 2 / 7 (cm) 95.58.9 92.17.3 7 / 7 / 0.810.06 0.840.08 2 / 7 (mm) 13.34.0 14.64.3 1 / 7 (mg/dl) 87.3100.7 69.318.0 p<0.01 6 / 7 (mg/dl) 199.428.1 204.036.1 2 / 7 HDL- (mg/dl) 54.616.3 54.1144.8 1 / 7 (mg/dl) 155.0127.5 198.3206.3 1 / 7 (mg/dl) 219.933.6 228.750.4 4 / 7 (mg/dl) 0.820.11 0.870.131 p<0.05 6 / 7 (g/dl) 7.110.34 7.160.29 4 / 7 7 (1 ) 9

ver5.1 HS 3. - (200 mg) (kg) 71.015.3 70.414.0 6 / 10 (%) 22.36.6 21.96.4 6 / 10 BMI (kg/m 2 ) 24.45.4 24.34.9 4 / 10 Ω 48977 48168 5 / 10 (%) 14.65.8 14.35.2 6 / 7 (%) 4.413.4 4.412.4 4 / 10 (cm) 82.111.4 82.29..5 3 / 10 (cm) 96.08.11 95.48.8 7 / 10 / 0.850.05 0.860.03 1 / 10 (mm) 16.77.3 16.37.6 6 / 10 (mg/dl) 97.028.4 94.029.9 5 / 10 (mg/dl) 200.226.6 203.126.5 4 / 10 HDL- (mg/dl) 58.422.7 57.320.3 3 / 10 (mg/dl) 164.4117.7 118.165.5 4 / 10 (mg/dl) 238.341.4 230.633.5 5 / 10 (mg/dl) 0.840.15 0.860.16 p<0.05 8 / 10 (g/dl) 7.210.2 7.320.1 5 / 10 10 10

ver5.1 HS 11- (100 mg) 11

ver5.1 HS 3. (1) (in vitro) - B16 ( 12) 12. - (B16) (±S.E., n=6) B16 2 mmmem (10%100 units/ml100 µg/ml) (5 10 4 /ml) 24 500 µl - (55 µl) 3 PBS (300 µl) 96 (415 nm 700 nm) (in vivo) - 13 8 10 (- 0 mg/kg) (0 ) - (25 50 mg/kg) ( 14 ) - in vitro in vivo 12

(L ) -2-4 -6-8 -10-12 0 mg/kg 1 mg/kg 25 mg/kg 50 mg/kg -2 0 2 4 6 8 10 12 0-14 ver5.1 HS 13. (±SD, n=3) 1 mg/kg 25 mg/kg 50 mg/kg 14. 8 - (4 ) (0 ) 2 (2 ) 0 3 4 [ () ] (UVB2000 mj/cm 2 ) - 2 10 (0 ) 8 10 (L*) () 13

ver5.1 HS (2) (in vitro) - NB1RGB 15-120 110 100 90 80 Control 1 5 25 15. - NB1RGB (±S.E., n=6) NB1RGB 96-2 MTT (in vitro) Control - (1 µg/ml) ( 16) - () [TESTSKIN () ] - 3 6 14

ver5.1 HS Control - (1g/mL) 16. - ( 400 ) 4. SOD 17. - SOD 18. - DPPH 15

ver5.1 HS 5. - (--WSP8 WSPC8) - (WSP8 WSPC8) 19. --WSP8 WSPC8 6. α --WSP8 (- 8)- -P (- 100) - 30 mg/kg (SD 7 ) - (n=3)- -WSP8 --P 3 (AUC) --WSP8 --P 3 --WSP8 --WSP8 - - 3 3 --WSP8-20. - ( ) 16

ver5.1 HS 7. (1) --P 601 15 80100120 1 25-25 21. - (2) ph - 0.2 60pH 1 1 -- ph 22. - ph 17

ver5.1 HS 8. α 0.2 g/100g 0.0 g/100g 1 99.8 g/100g 0.0 g/100g 0.0 g/100g 2 898 kcal/100g 3 0.0 g/100 1 mg/100g 1) 6.25 2) ( 15 176 ) 100 (+++) 3) ( 15 176 ) 4; 9; 4; 2 16 9 2 200408200016 9. α (1) (LD 50 ) ddy-ld 50 405 mg/kg 277 mg/kg60 kg 16.624.3 g (2) / OECD No.404 (2002 4 24 ) Commission Directive 2004/73/EC 3 () /- 0.5 g 4 1244872 Draize p.i.i. (primary irritation index) 3 1 () 24 p.i.i. 0.0 - / Safepharm Laboratories Limited 2005 8 11 1600/007 (3) 3 () 14 0.1 1 10-0.05L Draize 3 14-2005 8 23 C-I168 18

ver5.1 HS (4) OECD No.405 2002 4 24 Commission Directive 2004/73/EC 3 () - 70mg 1244872 71421 3 7 1 21 2 Kay 48 26.0 - ( 18 5) Safepharm Laboratories Limited 2005 8 11 1600/008 (5) OECD No.429 2002 4 24 Commission Directive 2004/73/EC 1 4 (CBA/Ca ) 1025 50- (LLNA Assay) 1025 50 - Safepharm Laboratories Limited 2005 8 11 1600/009 (6) (Ames ) OECD No.471 Commission Directive 2000/32/EC (Ames ) Salminella typhimurium (TA1535TA1537TA98TA100) Escherichia coli (WP2uvrA) 10 S9-Mix 505000g/- Safepharm Laboratories Limited 2005 8 12 1600/010 (7) 1 5 ppm - 2 5 ppm - 3 -tert- 5 ppm - 4 5 ppm - 5 5 ppm - 6-5 ppm - 7 1 ppm - 16 12 1 304110371-001 19

ver5.1 HS 10. α (1) - -- () 2 - A () - B ( ) 23 - A B R 1 S S : A R 1 = R 2 = H or R 2 n COOH B : R 1 = R 2 = H or COOR 3 R 3 = H or Et 23. - (2) - () - B () 500 mg/kg - B 24 2005 7 15 IB05039 in press. 11.α -50100 mg 1 200 mg 20

ver5.1 HS 12.α --P --P80 --WSP8 --L1 --PC --PC80 --WSPC8 - - - - () --LC1-13. --P, P80, WSP8 () --PC, PC80, WSPC8 () 5 kg --L1 () --LC1 () 5 kg 14. (15) (- -L1-LC1) 5 21

ver5.1 HS 15. --P --P80 --WSP8 --L1 --PC --WSPC8 --LC1 INCI Thioctic acid INCIMaltosilcyclodextrin (and) Thioctic acid INCI Polyglyceryl-10 Myristate (and) Propylene Glycol Caprylate (and) Glycerin (and) Thioctic acid (and) Alcohol -10 PG (2007 11 ) 100g (g) 0.01 0.01 22

ver5.1 HS P α- (1,2-dithiolane-6-pentanoic acid) 98.0 % 331 335 nm 278 283 nm α- 98.0 % (HPLC) 60 63 () 0.5 % (1 g40, 4 ) 0.1 % (1 g) (1) 6,8-Epitrithiooctanoic acid 0.1 % (HPLC) (2) 2.0 % () (3) Pb 10 ppm () (4) As 2 O 3 1 ppm () 1 10 3 /g () 1 10 2 /g ( ) (BGLB ) (1) 0.1 % (GC) (2) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) 100 % 23

ver5.1 HS P80 α- (1,2-dithiolane-6-pentanoic acid) 80.0 % 331 335 nm 278 283 nm α- 80.0 % (HPLC) 0.5 % (1 g40, 4 ) 0.1 % ( 1 g) (1) 6,8-Epitrithiooctanoic acid 0.1 % (HPLC) (2) 10 ppm () (3) 1 ppm () 1 10 3 /g () 1 10 2 /g ( ) (BGLB ) (1) 0.1 % (GC) (2) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) 80 % 20 % 100 % 24

ver5.1 HS WSP8 α- (1,2-dithiolane-6-pentanoic acid) 8.0 % 331 335 nm 278 283 nm α- 8.0 % (HPLC) 9.0 % (1 g40, 4 ) 0.10 % ( 1 g) (1) 10 ppm () (2) 1 ppm () 1 10 3 /g () 1 10 2 /g ( ) (BGLB ) (1) 0.1 % (GC) (2) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) 8 % 92 % 100 % 25

ver5.1 HS L1 α- (1,2-dithiolane-6-pentanoic acid) α- 10.0 % α- 10.0 % (HPLC) (1) 10 ppm () (2) 1 ppm () 1 10 3 /g () 1 10 2 /g ( ) (BGLB ) (0.1 ppm ) (GCMS) 10 % 2 % 50 % 23 % 15 % 100 % 26

ver5.1 HS PC α- (1,2-dithiolane-6-pentanoic acid) 98.0 % 331 335 nm 278 283 nm α- 98.0 % (HPLC) 60 63 () 0.5 % (1 g40, 4 ) 0.1 % (1 g) (1) 6,8-Epitrithiooctanoic acid 0.1 % (HPLC) (2) 2.0 % () (3) Pb 10 ppm ( 2 ) (4) As 2 O 3 1 ppm ( 3 ) 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) (1) 0.1 % (GC) (2) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) 100 %, 27

ver5.1 HS PC80 α- (1,2-dithiolane-6-pentanoic acid) 80.0 % 331 335 nm 278 283 nm α- 80.0 % (HPLC) 0.5 % (1 g40, 4 ) 0.1 % (1 g) (1) 6,8-Epitrithiooctanoic acid 0.1 % (HPLC) (2) Pb 10 ppm ( 2 ) (3) As 2 O 3 1 ppm ( 3 ) 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) (1) 0.1 % (GC) (2) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) 80 % 20 % 100 %, 28

ver5.1 HS WSPC8 α- (1,2-dithiolane-6-pentanoic acid) 8.0 % 331 335 nm 278 283 nm α- 8.0 % (HPLC) 9.0 % (1 g40, 4 ) 0.10 % (1 g) (1) Pb 10 ppm ( 2 ) (2) As 2 O 3 1 ppm ( 3 ) 1 10 2 /g () 1 10 2 /g ( ) (BGLB ) (1) 0.1 % (GC) (2) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) (1 ppm ) (GCMS) 92 % 8 % 100 %, 29

ver5.1 HS LC1 - (1,2-dithiolane-6-pentanoic acid) - 10.0 % - 10.0 % (HPLC) (1) 10 ppm ( 2 ) Pb (2) 1 ppm ( 3 ) As 2 O 3 110 2 /g () 110 2 /g ( ) (BGLB ) (0.1 ppm ) (GCMS) -10 50 % 23 % 15 % 10 % 2 % 100 %, 30

493-8001 TEL(0586)86-5141() FAX(0586)86-6191 URL/http://www.oryza.co.jp/ E-mail: info@oryza.co.jp 101-0041 1-24-10 5F TEL(03)5209-9150 FAX(03)5209-9151 * * * * Ver. 5.1 (pp.25-32) 2004 10 5 2011 9 15