Standardized Herbal Extract Production Protocol

The formula to produce 5 g of dried extract of KKT comprised the following 14 components: Astragali Radix (3 g, root of Astragalus propinquus Schischkin or Astragalus mongholicus Bunge), Bupleuri Radix (3 g, root of Bupleurum falcatum L.), Ziziphi Semen (3 g, seed of Ziziphus jujuba var. spinosa [Bunge] Hu ex H.F.Chow), Atractylodis Lanceae Rhizoma (3 g, rhizome of Atractylodes lancea [Thunb.] DC. or Atractylodes chinensis [Bunge] Koidz.), Ginseng Radix (3 g, root of Panax ginseng C.A.Mey.), Poria (3 g, sclerotium of Wolfiporia cocos Ryvarden et Gilbertson), Longan Arillus (3 g, arillus of Dimocarpus longan Lour.), Polygalae Radix (2 g, root of Polygala tenuifolia Willd.), Gardeniae Fructus (2 g, fruit of Gardenia jasminoides J.Ellis), Ziziphi Fructus (2 g, fruit of Ziziphus jujuba var. inermis [Bunge] Rehder), Angelicae Radix (2 g, root of Angelica acutiloba [Siebold & Zucc.] Kitag. or Angelica acutiloba var. sugiyamae Hikino), Glycyrrhizae Radix (1 g, root of Glycyrrhiza uralensis Fisch. ex DC. or Glycyrrhiza glabra L.), Zingiberis Rhizoma (1 g, rhizome of Zingiber officinale Roscoe), and Saussureae Radix (1 g, root of Aucklandia costus Falc.). All raw materials were supplied by Tsumura & Co. (Tokyo, Japan). The dry-powdered extract of KKT (lot number: 351152900 and 361095300) was produced by Tsumura & Co. Briefly, the mixture of the 14 raw materials was extracted in boiling water for 1 h, and the extract was then separated from insoluble waste. The separated extract was concentrated under reduced pressure and then spray-dried to produce the extract powder of KKT (hereafter termed “KKT”). The quality of KKT was confirmed to meet the Japanese Pharmacopoeia and our company’s standards; specifically, the following ingredients were included (with their corresponding ranges): saikosaponin b2 (0.8–3.2 mg); geniposide (27–81 mg); glycyrrhizinic acid (6–18 mg), in 5 g of KKT. All voucher specimens of the raw materials used for each lot of KKT were deposited in the herbarium of Tsumura & Co., with batch numbers (Table S1). For use in the elevated plus maze (EPM) test, KKT was suspended in 10 mL of distilled water immediately prior to use and was orally administered. To administer KKT without manually handling the mice in the IntelliCage experiment, KKT was suspended in tap water in the water bottles at a concentration of 1% (w/v) and placed in the cage. The bottles of KKT suspension were refreshed every 2 to 3 days.