Factor, Level and drug compatibility ratios Conditions serial number A Factor: RhizomaCoptidis(HL) Herb concentration/μg (crude drug)•mL-1 B Factor: FructusEvodiaeRutaecarpae(WZY) Herb concentration/μg (crude drug)•mL-1 C Factor: Radix Paeoniae Alba(BS) Herb concentration/μg (crude drug)•mL-1 D Factor: 0 A:B:C Ratios Herb concentration/μg (crude drug)•mL-1 1#formula (1#F) 3/168 1/56 3/168 - 3: 1: 3/392 2# formula (2#F) 3/168 2/112 6/336 - 3: 2: 6/616 3# formula (3#F) 3/168 6/336 12/672 - 3: 6: 12/1176 4# formula (4#F) (adopted by China Pharmacopoeia) 6/336 1/56 6/336 - 6: 1: 6/728 5# formula (5#F) 6/336 2/112 12/672 - 6: 2: 12/1120 6# formula (6#F) 6/336 6/336 3/168 - 6: 6: 3/840 7# formula (7#F) 12/672 1/56 12/672 - 12: 1: 12/1400 8# formula (8#F) 12/672 2/112 3/168 - 12: 2: 3/952 9# formula (9#F) 12/672 6/336 6/336 - 12: 6: 6/1344 single HL 672.24       672.24 single WZY   4259.32     4259.32 single BS     35870   35870

Note: 1 All Wuji Wan Formulae and single HL, WZY, BS was prepared in extract, but the dose/concentration was expressed as crude drug. 2 The gradient concentration for detecting IC50 of Wuji Wan on six major CYPs begins at concentrations of Table 1 and dilutes to half concentrations continually until getting 6 gradient concentrations (take 7#F as example, the gradient concentration were 1400, 700, 350, 175, 87.5 and 43.75).

Table 1: Investigational herb conditions design of Wuji Wan, L9(34) orthogonal table and Initial concentration.