Senkyunolide 1

The reference standards of chlorogenic acid, caffeic acid, ethyl gallate, 3-butyl-phthalide, benzoyloxypaeoniflorin, senkyunolide I, gallic acid, oxypaeoniflorin, vanillic acid, albiflorin, paeoniflorin, ferulic acid, 1,2,3,4,6-pentagalloylglucose, benzoylpaeoniflorin, paeonol, and ligustilide (the purities of all standards were above 98.0%) were supplied by Shanghai Yuanye Bio-Technology Co., Ltd. Appropriate amounts of the reference standards were mixed and dissolved in methanol in a 10-mL measuring flask, then filtered and set aside.

Starch, sodium phosphate monobasic (anhydrous, ≧99%) and sodium phosphate dibasic (anhydrous, ≧99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2,3-Bisphosphoglycerate (2,3-BPG) in the form of pentasodium salt was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Dried roots of Angelica Sinensis (Oliv.) Diels were purchased from Yong-Fong Herbal Medicines Inc. (Kaohsiung, Taiwan), while the four phthalide derivatives experimentally investigated in this work, including Z-butylidenephthalide (HPLC grade, >98%), z-ligustilide (HPLC grade, >98%), senkyunolide A (HPLC grade, >98%), senkyunolide I (HPLC grade, >98%) were purchased from Shanghai Yuanye Bio-Technology (Shanghai, China). The AS plant extract was prepared from the dried AS root by immersing 1 g of the dried AS root into 10 mL of DI water after initial cleaning and incubated for 20 minutes at 90 °C to obtain the concentrated AS plant extract of 2.5 mL. This plant extract preparation method was chosen as this is the most common way from which the roots of AS was prepared as the traditional tonic^{1 (link), 65 (link), 66 (link)}. The resulting plant extract was separated from the plant material and further centrifuged at 12,000 × g for 10 minutes to remove any residual fine particulates.