vary depending around the combination of the constituent crude drugs. Consequently, it is important to understand the pharmacokinetics 
of individual components when administered as rikkunshito. In this study, we performed a pharmacokinetic study of rikkunshito in humans having a specific focus on ingredients involved in the ghrelin enhancer effect. We initial performed an exploratory pharmacokinetic study of four healthy adult volunteers to recognize the standard 32 ingredients (S1 Table) detected within the plasma or urine. Subsequent, a randomized crossover study was performed to investigate the pharmacokinetics of eight active components derived from rikkunshito, which had been chosen with reference to the exploratory pharmacokinetic study and its pharmacological effect, in the plasma following a single oral administration of a clinical dose of rikkunshito in 21 healthy adult volunteers. We also measured atractylodin carboxylic acid, an atractylodin metabolite pharmacologically as potent as atractylodin (S1 Fig), though it was not measured within the exploratory pharmacokinetic study. Additionally, the pharmacokinetic parameters of every MX69 ingredient have been calculated determined by the outcomes.
Important ingredients and their biological activities related to ghrelin enhancer activity in rikkunshito. Supply Citri unshiu pericarpium Glycyrrhizae radix Atractylodis lanceae rhizoma Poria doi:ten.1371/journal.pone.0133159.t001 Important active ingredients Hesperetin Heptamethoxyflavone Isoliquiritigenin Atractylodin Pachymic acid Ghrelin signal enhancement effect [1] Ghrelin metabolizing enzyme inhibitory impact [17] Identified pharmacology activities Ghrelin secretion promoting activity [14, 15]
Tsumura rikkunshito extract granules for prescription (item code TJ-43, Tsumura & Co. lot numbers E24652 and H05142, Tokyo, Japan) have been used for the investigational product. It was manufactured according to GMP, and adapted to factory release test. The sample of your investigational drug used within this study is retained in Tsumura & Co. 7.5 g of this herbal preparation contains 4.0 g of dried extract obtained by spray drying of a hot water extract of a mixture of eight crude drugs: 4.0 g of Atractylodis lanceae rhizoma (Compositae; atractylodes lancea rhizome), 4.0 g of Ginseng radix (Araliaceae; ginseng), 4.0 g of Pinelliae tuber (Araceae; pinellia tuber), 4.0 g of Poria (Polyporaceae; poria sclerotium), 2.0 g of Zizyphi fructus (Rhamnaceae; jujube), 2.0 g of C. unshiu pericarpium (Rutaceae; citrus unshiu peel), 1.0 g of G. radix (Leguminosae; glycyrrhiza), and 0.5 g of Zingiberis rhizoma (Zingiberaceae; ginger). The standard components contained in rikkunshito and digoxin had been supplied by Tsumura & Co. Atractylodin and atractylenolide III had been supplied by Tsumura & Co. and Wako Pure 17764671 Chemical Industries, Ltd. (Osaka, Japan). Erythromycin was purchased from Wako Pure Chemical Industries, Ltd. (-Warfarin-d5 was purchased from C/D/N ISOTOPES INC. (Pointe-Claire, Quebec, Canada). Other chemicals had been purchased from commercial sources.
The trials had been conducted at the Kochi Medical School in two periods: first trial, between April 2012 and March 2013 and the second trial, between September 2013 and May 2014, and these were approved by the Ethical Committee Kochi Medical School. The trials have been registered at the Japan Pharmaceutical Information Center (JAPIC; #CTI-121801 and -142522). The trials had been conducted in accordance with ethical norms prescribed within the Declaration of Helsinki and good clinica