Spray-dried, water-extracted GJG powder was obtained from Tsumura & Co. (Tokyo, Japan). GJG was approved in 1986 as a drug for clinical use by the Japanese

Ministry of Health, Labour and Welfare. It is produced at the Shizuoka plant which meets Japanese pharmaceutical GMP (good manufacturing practice). The local pharmaceutical administration of Shizuoka Prefecture assesses the GMP status of the plant every 5 years. The plant has had permission for pharmaceutical production for more than 30 years, and the production process has been well validated. Since active substances are still ambiguous, quality control is conducted by quantitation of major components. In the case of GJG, paeoniflorin (moutan bark), loganin (Rehmannia root), and total alkaloids (processed aconite root) are chosen as marker compounds for quality control. Paeoniflorin, loganin, and total alkaloids in 1 g of GJG extract powder used in our experiments were 2.11, c-Met inhibitor Selleckchem GSK3 inhibitor 1.58, and 0.11 mg, respectively. In 10 lots (a total of 20 lots) produced before and behind this lot, paeoniflorin, loganin, and total alkaloids were within ± 10% of the range of this content, and quality was managed satisfactorily. Other physicochemical properties, e.g. loss on drying, water content, ash, heavy metals, etc., were also examined in all lots.

GJG extract is listed in the Japanese Pharmacopeia, and the material used in this study met that description. The general manufacturing procedure of GJG extract powder is as follows. Ten kinds of botanical raw materials are crushed and then weighed in accordance with the mixing ratio as shown in Table S1. The mixture of botanical raw materials is extracted 12 times with ion-exchanged water for 60 min at 100 °C. The extract is centrifuged to obtain a supernatant, which is then concentrated in vacuo. The

concentrated extract solution is dried by a spray dryer. The standard yield of extract powder is around 16% of the total weight of botanical raw materials. A three-dimensional high-performance liquid chromatography (HPLC) profile of a methanol solution of GJG was performed according to our aminophylline previous procedure ( Hattori et al. 2010) and is shown in Fig. 1. 3D-HPLC analysis and LC/MS analysis of the crude drugs involved in GJG are shown in Figs. S1–S3. Seven-week-old male SAMP8 mice were purchased from SLC, Inc. (Shizuoka, Japan) and divided into 2 groups: those fed a normal diet (powdered mouse food; Oriental Yeast Co. Ltd. (Tokyo, Japan; P8 + N group; n = 10)); and those fed a normal diet supplemented with 4% (w/w) GJG (P8 + GJG group; n = 10). As controls, 7-week-old male SAMR1 mice were purchased from SLC and also divided into 2 groups: those fed a normal diet (R + N group; n = 10) and those fed a normal diet supplemented with 4% (w/w) GJG (R + GJG group; n = 11). General conditions and body weight were recorded for all mice.