27 6 2010 11 Vol 27 Journal of the Graduate School of the Chinese Academy of Sciences November 1002-1175 2010 06-0768-10 No 6 2010 * 100049 2010 4 20 2010 5 4 Liu X X Tian W X Ma X F Inhibitory effects of strawberry extracts on animal fatty acid synthase J Journal of the Graduate School of the Chinese Academy of Sciences 2010 27 6 768-777 FAS FAS FAS A A HPLC-MS 3T3- L1 R285 5 fatty acid synthase FAS E C 2 3 1 85 FAS NADPH A A FAS FAS FAS 1-3 Loftus FAS C75 C75 FAS A Y FAS FAS 4 5 γ PPARγ CCAAT / C / EBPα FAS 6 FAS RNA FAS C / EBPα PPARγ mrna FAS FAS * 30572252 30670455 O95101PY00 E-mail maxiaofeng@ gucas ac cn
6 769 7 FAS Fragaria ananassa Duch Daniel EM 8 Shiow Y A549 JB6 P + TPA 9 FAS FAS 1 1 1 A Ac-CoA A Mal-CoA Ⅱ NADPH DTT DMSO 5- IAF DTNB 3-isobutyl-1-methylxanthine IBMX Insulin Dexamethasone O catechin quercetin kaempferol HPLC Sigma TOMY GL-20 CPZOBZ Ultrospec 4300 pro UV-Vis spectrophotometer CS501-SP HPLC-MS Agilent 1100 Agilent LC / MSD Trap XCT ESI 4000V 100 ~ 2000m / z 40psi 10L / min 325 DEAE DE-23 Whatman Blue Sephorose 6B Sephadex-LH20 Pharmacia FW100 202-00AB DW-1 Philips HR1861 RE52 SHB-Ⅲ KQ300B Shimazu AY120 FAS Tian 10 FAS SDS- 1 2 SF SL 95% 75% 50% 25% 7 1 20 2h 50% 3-20 1 3 FAS 1 3 1 100mmol / L KH 2 PO 4 -K 2 HPO 4 1mmol / L EDTA 1mmol / L DTT ph7 0 5μmol / L Ac-CoA 10μmol / L Mal-CoA 35 μmol / L NADPH 37 15 ~ 20 μg FAS 2mL - NADPH 340 nm FAS
770 27 1 3 2 100 mmol / L KH 2 PO 4 -K 2 HPO 4 1 mmol / L EDTA ph 7 0 100 mmol / L 35 μmol / L NADPH 37 20 μg FAS 2 ml - NADPH 340 nm 11 1 4 1 4 1 FAS A A 0 A / A 0 Remaining activity R A R A 1 4 2 FAS 50% IC 50 1 4 3 FAS 25 A t A 0 A t / A 0 R A k obs k obs / I 1 5 HPLC HPLC 12 254nm - 1 6 3T3-L1 3T3-L1 3mL 2d 2d 4 ~ 6d 520nm 2 2 1 FAS SF SL 7 FAS 1 a IC 50 50% 23 26mg / L SF 7 46mg / L SL SF SL 50% FAS IC 50 1 b IC 50 1 61mg / L SF 2 55mg / L SL FAS 50% 50% SF-AE SL-AE 2 2 SF-AE SL-AE FAS SF-AE SL-AE FAS KR 2 SF-AE KR IC 50 13 SL-AE KR 20 KR FAS
6 771 1 2 3 4 95% 5 75% 6 50% 7 25% FAS IC 50 a FAS 1 2 3 4 FAS IC 50 b 50% FAS 1 FAS IC 50 SF-AE IC 50 = 1 61mg / L KR IC 50 = 20 18mg / L SL-AE IC 50 = 2 55mg / L KR IC 50 = 48 07mg / L 2 SF-AE SL-AE FAS KR IC 50 2 3 SL-AE FAS SF SL FAS
772 27 SF-AE SL-AE FAS 3 FAS k obs / I = 0 0718min - 1 g / L - 1 SF-AE FAS SL-AE FAS 2 4 SF-AE SL-AE SF-AE SL-AE FAS FAS 3 A Ac-CoA A Mal-CoA FAS 380mg / L SL-AE NADPH 4 SF-AE Ac- 0 0696g / L k obs = 0 005min - 1 CoA NADPH 3 SL-AE FAS Mal-CoA Mal-CoA SF-AE Ac-CoA Mal-CoA NADPH 2 5μmol / L 10μmol / L 32μmol / L a Ac-CoA SF-AE 0mg / L 2 0mg / L 4 7mg / L 6 7mg / L b Mal-CoA SF-AE 0mg / L 1 4mg / L 2 5mg / L 4 5mg / L c NADPH SF-AE 0mg / L 1 4mg / L 2 5mg / L 4 0mg / L SL-AE Ac-CoA Mal-CoA NADPH 2 5μmol / L 10μmol / L 32μmol / L d Ac-CoA SL-AE 0mg / L 1 7mg / L 3 3mg / L 6 0mg / L e Mal-CoA SL-AE 0mg / L 3 0mg / L 5 3mg / L 7 5mg / L f NADPH SL-AE 0mg / L 2mg / L 4 2mg / L 6 7mg / L 4 SF-AE SL-AE FAS
6 773 K is = 1 SF-AE SL-AE FAS 4 50mg / L K is mg / L K ii mg / L SF-AE SL-AE SF-AE SL-AE SF-AE SL-AE K ii = 4 14mg / L 1 2 Ac-CoA 4 29 2 49 8 32 / Mal-CoA 4 50 6 42 4 14 12 47 SL-AE Ac- CoA Mal-CoA NADPH 1 84 4 23 3 90 6 58 NADPH 2 5 SF-AE SL-AE HPLC ESI-MS SF-AE SL-AE 5 HPLC 5 a SF-AE HPLC 5 b SF-AE Rt = 35 927 Rt =63 319 SF-AE ESI-MS 2 a 5 1 2 3 4 5 HPLC b SF-AE HPLC c SL-AE HPLC HPLC Agilent 1100 Inertsil ODS3 250mm 4 6mm 5μm A -0 1% B 0 ~ 60min A 3% ~ 30% B 97% ~ 70% 60 ~ 80min A 30% ~ 55% B 70% ~ 45% 80 ~ 85min A 55% B 45% 1mL / min 25 254nm 5 SF-AE SL-AE HPLC
774 27 2 HPLC-MS SF-AE / min / ESI + % ESI - 10 736 1 027 365 341 Caffeoyl-glucose 342 13 16 231 1 546 147 349 675 316 633 - - Galloyl-HHDP glucose 633 14 17 042 1 341 nd 325 p-coumaroyl-glucoside 326 14 19 745 nd 175 C Vitamin C 176 15 28 586 8 009 nd 467 507 935 - Peonidin -araboside 468 29 855 1 123 nd nd unknown 31 374 15 101 nd 447 - Ellagic acid deoxyhexose 448 14 33 36 1 105 619 811 393 787 Tetragalloyl glucose 788 14 34 63 9 049 333 309 345 369 457 Coumaroyl-glucoside 310 13 35 927 28 023 515 301 634 Ellagic acid 302 16 42 308 1 315 nd 447 Kaempferol hexose 448 17 44 981 1 565 340 679 701 nd - Kaempferol glucuronide 462 12 53 729 3 172 469 445 unknown 446 61 570 9 249 373 395 349 431 unknown 372 63 319 11 010 nd nd quercetine 302 SF-AE SL-AE 5 c 3 SF-AE SL-AE HPLC-MS 5 HPLC SF-AE SL-AE 5 SF-AE SL-AE 4 5 SF-AE SL-AE 63 25% 68 87% / / ESI + min % 3 ESI - HPLC-MS SL-AE 8 786 2 524 nd 391 783 - Bis-HHDP glucose 784 14 10 369 0 805 nd 633 - - Galloyl-HHDP glucose 634 14 12 448 3 290 579 601 391 577 B1 Procyanidin B1 578 18 14 627 0 578 579 577 B2 Procyanidin B2 578 18 15 983 1 456 291 289 catechin 290 17 17 759 1 447 nd 325 445 - p-coumaroyl-glucoside 326 14 18 255 3 526 379 355 p-coumaroyl ester 356 14 22 028 0 835 nd 635 - Tris-galloyl-glucose 636 14 28 557 18 300 nd 467 935 - Peonidin -araboside 468 31 416 5 678 nd 447 787 -ellagic acid deoxyhexose 448 14 33 442 4 507 811 393 787 - tetragalloylglucose 788 14 35 833 32 456 303 519 301 Ellagic acid 302 14 37 799 2 603 377 399 nd unknown 376 39 916 2 031 nd 447 469 - Kaempferol hexose 448 17 44 782 0 835 485 701 461 713 - Kaempferol -glucuronide 462 16 63 279 7 926 nd nd quercetin 302 4 HPLC SF-AE SL-AE / min / % / % 28 586 - Peonidin -araboside 8 009 18 300 468 31 374 - Ellagic acid deoxyhexose 15 101 5 678 448 33 360 tetragalloylglucose 1 105 4 507 788 35 927 Ellagic acid 28 023 32 456 302 63 319 quercetin 11 010 7 926 302
6 775 HPLC SF-AE SL-AE 2 6 SF-AE SL-AE SF-AE SL-AE 3T3-L1 6 SF-AE SL-AE 50mg / L 520nm 10mg / L 25mg / L 50mg / L 100mg / L p < 0 001 6 SF-AE SL-AE 3T3-L1 3 3 1 FAS FAS 50% FAS SF- AE IC 50 50% 20 1 61mg / L SL-AE IC 50 2 55mg / L FAS IC 50 = 20mg / L 19 EGCG IC 50 = 24mg / L 20 SF-AE SL-AE FAS EGCG 2 FAS C75 FAS SF-AE Ac-CoA NADPH Mal-CoA Mal-CoA FAS SL-AE Ac-CoA Mal-CoA NADPH FAS Ac-CoA SF-AE SL-AE FAS 3 2 21-3- -3- -3-22-23 24
776 27 16 HPLC-MS SF-AE SL-AE 5 SF-AE 11 010% SL-AE 7 926% FAS IC 50 1 30mg / L 25 SF-AE 28 023% SL-AE 32 456% FAS IC 50 1 31mg / L SF-AE SL-AE FAS IC 50 2 SF-AE SF-AE IC 50 1 61mg / L SF-AE 40% 2 FAS SF-AE FAS SF- AE SL-AE FAS 3 3 FAS 3T3-L1 SF-AE SL-AE 50mg / L 100mg / L FAS SF-AE SL-AE FAS FAS 2 FAS FAS 1 Tian W X The control of animal fat level and fatty acid synthase activity J Chemistry of Life 1994 14 1-5 in Chinese J 1994 14 1 1-5 2 Li M Shi Y Tian W X Factors influencing the levels of fatty acid synthase complex activity in fowl J Biochem Mol Biol Int 1999 47 1 63-69 3 Tian W X Dong Y Quan H et al The dependence of fat level of hen on activity of fatty acid synthase in lever on different ages J Chinese Biochemical Journal 1996 12 234-236 in Chinese J 1996 12 2 234-236 4 Loftus T M Jaworsky D E Frehywot G L et al Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors J Science 2000 288 5475 2379-2381 5 Flier J S The adipocyte storage depot or node on the energy information superhighway J Cell 1995 80 1 15-18 6 Rosen E D Molecular mechanisms of adipocyte differentiation J Ann Endocrinol Paris 2002 63 1 79-82 7 Schmid B Rippmann J F Tadayyon M et al Inhibition of fatty acid synthase prevents preadipocyte differentiation J Biochem Biophys Res Commun 2005 328 4 1073-1082 8 Daniel E M Stoner G D The effects of ellagic acid and 13-cis-retinoic acid on N-nitrosobenzylmethylamine-induced esophageal tumorigenesis in rats J Cancer Lett 1991 56 2 117-124 9 Shiow Y W Inhibitory effect on activator protein-1 nuclear factor-kappa B and cell transformation by extracts of strawberries Fragaria ananassa Duch J Agra Food Chem 2005 53 10 4187-4193 10 Tian W X Wang Y S Hsu R Y Affinity labeling of chicken liver fatty acid synthase with chloroacetyl-coa and bromopyruvate J Biochim Biophys Acta 1989 998 3 310-316 11 Tian W X Jiang R F Wu H B et al The substrate inhibition by NADPH and kinetics of fatty acid synthase from duck liver J Chinese Biochemical Journa1 1994 10 4 413-419 in Chinese NADPH J 1994 10 4 413-419
6 777 12 Seeram N P Adams L S Blackberry black raspberry blue berry red raspberry and strawberry extracts inhubit growth and stimulate apoptosis of human cancer cells in vitro J J Agric Food Chem 2006 54 25 9329-9339 13 Wulf J S Rühmann S Rego I et al Nondestructive application of laser-induced fluorescence spectroscopy for quantitative analyses of phenolic compounds in strawberry fruits Fragaria ananassa J Agric Food Chem 2008 56 9 2875-2882 14 Kati H Ilana R Harri K et al Non-targeted analysis of spatial metabolite composition in strawberry Fragaria ananassa flowers J Phytochemistry 2008 69 13 2463-2481 15 Wang Y Y Jiang G B Hu W Z et al Determination of vitamin C in strawberry by high performance liquid chromatography J Journal of Dalian University 2006 27 1 21-22 in Chinese C J 2006 27 1 21-22 16 Seeram N P Lee R Scheuller H S et al Identification of phenolic compounds in strawberries by liquid chromatography electrospray ionization mass spectroscopy J Food Chem 2006 97 1 1-11 17 Vasco C Riihinen K Ruales J et al Phenolic compounds in rosaceae fruits from ecuador J J Agric Food Chem 2009 57 4 1204-1212 18 Mudnic I Modun D Brizic I et al Cardiovascular effects in vitro of aqueous extract of wild strawberry Fragaria vesca L leaves J Phytomedicine 2009 16 5 462-469 19 Vance D Goldberg I Mitsuhashi O et al Inhibition of fatty acid synthetases by the antibiotic cerulenin J Biochem Bhys Res Commun 1972 48 3 649-656 20 Wang X Tian W X Green tea epigallocatechin gallate a natural inhibitor of fatty acid synthase J Biochem Biophys Res Commun 2001 288 5 1200-1206 21 Hannum S M Potential impact of strawberries on human health a review of the science J Crit Rev Food Sci Nutr 2004 44 1 1-17 22 Gil M I Holcroft D M Kader A A Changes in strawberry anthocyanins and other polyphenols in response to carbon dioxide treatments J J Agric Food Chem 1997 45 5 1662-1667 23 Cordenunsi B R Genovese M I Nascimento J R O et al Effects of temperature on the chemical composition and antioxidant activity of three strawberry cultivars J Food Chem 2005 91 1 113-121 24 Maas J L Galletta G J Ellagic acid an anticarcinogen in fruits especially in strawberry J Hortscience 1991 26 1 10 25 Li B H Tian W X Inhibitory effects of flavonoids on animal fatty acid synthase J J Biochem 2004 135 1 85-91 Inhibitory effects of strawberry extracts on animal fatty acid synthase LIU Xiao-Xin TIAN Wei-Xi MA Xiao-Feng College of Life Sciences Graduate University Chinese Academy of Sciences Beijing 100049 China Abstract Fatty acid synthase FAS has been considered as a potential new therapeutic target for obesity In the present study we show that the components of both strawberry Fragaria ananassa Duch fruit dreg and strawberry leaves that extracted with 50% ethanol and partitioned by ethyl acetate are potent FAS inhibitors The ethyl acetate fractions of strawberry fruit dregs SF-AE show noncompetitive inhibition with the substrate Mal- CoA while the ethyl acetate fractions of strawberry leaves SL-AE show competitive inhibition with Ac-CoA Based on the HPLC-MS analysis we conclude that ellagic acid and quercetin are the efficient components SF- AE and SL-AE could reduce lipids accumulation in 3T3-L1 preadipocyte with dose-dependence These results may be of great value for application of strawberry extracts to obesity prevention and cure Key words fatty acid synthase strawberry inhibitor kinetic assay anti-obesity