13 4 2015 7 Chinese Journal of Bioprocess Engineering Vol. 13 No. 4 Jul. 2015 doi 10. 3969 /j. issn. 1672-3678. 2015. 04. 012 1 2 2 2 2 1. 830054 2. 361005 L- 50% IC 50 0. 27 mg /ml K I - K IS 0. 218 0. 313 mg /ml 1 1- -2- DPPH DPPH 50% IC 50 0. 18 mg /ml Q946. 8 A 1672-3678 2015 04-0063 - 05 Inhibition of mushroom tyrosinase by flavonoid from Sorbus tianschanica Ruper in Xinjiang FU Jianhong 1 QI Rui 2 ZHENG Jing 2 ZHOU Chenjie 2 CHEN Qingxi 2 1. Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology College of Life Science Xinjiang Normal University Urumqi 830054 China 2. School of Life Sciences Xiamen University Xiamen 361005 China Abstract Flavonoid was extracted from powder of Sorbus tianschanica Ruper stems under assistance of ultrasonic wave. Adopting enzymatic kinetic method inhibitory action of flavonoid extract on the tyrosinase activity was studied using L-DOPA as substrate. Results showed that flavonoid extract from Sorbus tianschanica Ruper inhibited tyrosinase. Its IC 50 was estimated as 0. 27 mg /ml. The inhibition mechanism was studied and it was found that the reaction was reversible and type of the inhibition belonged to the mixed-type. The inhibition equilibrium constants for inhibitor binding with free enzyme K I and with enzyme-substrate complex K IS were determined as 0. 218 and 0. 313 mg /ml respectively. The DPPH 1 1-diphenyl-2-picrylhydrazyl radical test results showed that flavonoid extract from Sorbus tianschanica Ruper had the antioxidant activity to a certain extent and the inhibitor concentration leading to 50% DPPH free radical activity loss IC 50 was determined to be 0. 18 mg /ml. The above study provides a basis for further development of the fruits and vegetables preservatives and whitening ingredient in the Sorbus tianschanica Ruper. 2015-05 - 09 31260213 XJEDU2012I30 XJSD2013-06 1970 E-mail fjh_719@ 163. com
64 13 Keywords Sorbus tianschanica Ruper flavonoid tyrosinase inhibitory mechanism EC 1. 14. 18. 1 tyrosinase 1 1 1. 1 2 6 680 U /mg L- L- 3 4 DOPA DMSO Sigma 1. 2 KQ5200E SHB-Ⅲ 1 EYELA DU800 DKB-501A 5 1. 3 6 g 300 ml 60% 6 Kubo 7 200 W 30 120 min Xie 8 5% DMSO 3 7 4-15 mg /ml 0 25 50 75 100 μl Sorbus tianshanica Ruprer 3. 0 ml 0 0. 125 0. 25 0. 375 0. 5 mg /ml A C 9 1. 4 11 10 13. 2 mg 60% 0. 528 mg /ml 0 0. 4 0. 8 1. 2 1. 6 2. 0 ml 10 ml 2. 0 1. 6 1. 2 0. 8 0. 4 0 ml 60% 5% NaNO 2 0. 5 ml 6 min 10% Al NO 3 3 0. 5 ml 6 min
4 65 4% NaOH 4. 0 ml 60% 14 3. 0 ml 1. 0 ml 15 min510 nm 0. 1 mol /L ph5. 5 1. 87 ml A 510 60% 0. 1 ml 3 mmol /L DPPH 0. 03 ml 0 ~ 0. 525 mg /ml 1. 5 20 min 517 nm 12 V c DPPH 0. 5 mmol /L L-DOPA 0. 05 mol /L 14 ph = 6. 8 3. 0 ml 2 0. 1 ml 2. 8 ml 0. 1 ml 2. 1 30 A 475 A ρ A = ε ε = 3 700 L / mol cm 11. 425ρ - 0. 0179 R = 0. 999 9 13 U L-DOPA 0. 02 ~ 0. 10 mg /ml 1 μmol 1 = V s /V c 100% 1 V c OD 475 min - 1 V s 6 g OD 475 min - 1 25 60% 300 ml 1. 6 200 W L-DOPA 30 120 min 0. 5 mmol /L 60% 510 nm 2. 75% 2. 2 L-DOPA L-DOPA 1 1 = 1 000A 475 /3. 7 Lineweaver- IC 50 0. 27 mg /ml Burk K m v max 2. 3 L-DOPA K I - K IS 1. 7 1 1- -2- DPPH 2 2
66 13 Fig. 1 2 Fig. 2 2. 4 1 L-DOPA Lineweaver-Burk Effect of flavonoid extract of Sorbus tianschanica 3 3 Ruper stems on activity of diphenolase of tyrosinase K m v max E - ES 3 b 3 c K I 0. 218 mg /ml - K IS 0. 313 mg /ml K I < K IS Determination of the inhibitory mechanism of - flavonoid extract from Sorbus tianschanica Ruper on diphenolase of tyrosinase Fig. 3 1 ~ 5 0 0. 083 0. 167 0. 25 0. 333 mg /ml 3 Detemination of inhibition type and inhibition constant of flavonoid extract from Sorbus tianschanica Ruper on diphenolase of tyrosinase 2. 5 DPPH DPPH
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