Profiling of the Compounds Absorbed in Human Plasma and Urine After Oral Administration of a Traditional Japanese (Kampo) Medicine Daikenchuto (DKT)

DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 DMD FastThis article Forward. has not beenPublished copyedited on and August formatted. 5,The 2010 final version as doi:10.1124/dmd.110.033589 may differ from this version.

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Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT)

Jun Iwabu, Junko Watanabe, Kazuhiro Hirakura, Yoshinori Ozaki, Kazuhiro Hanazaki

Department of Surgery, Kochi Medical School, Nankoku, Kochi, Japan (J.I., K.Ha); Downloaded from

Tsumura Laboratories (J.W.); Pharmaceutical & Quality Research Dept (K.Hi.); Kampo

Research Planning Dept (Y.O.), Tsumura & Co., Ami, Ibaraki , Japan. dmd.aspetjournals.org

at ASPET Journals on September 23, 2021

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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RUNNING TITLE PAGE

Running title: PK study of a traditional Japanese medicine daikenchuto

Address correspondence to: Kazuhiro Hanazaki, M.D., Ph.D. Department of Surgery,

Kochi Medical School, Kohasu-Okocho, Nankoku, Kochi 783-8505, Japan. E-mail:

[email protected] , Phone: 81-88-880-2370, Fax: 81-88-880-2371 Downloaded from

The number of words in text: 3888 dmd.aspetjournals.org The number of Tables: 5

The number of Figures: 5

The number of References: 32 at ASPET Journals on September 23, 2021

The number of words in ABSTRACT: 252

The number of words in INTRODUCTION: 775

The number of words in DISCUSSION: 1005

Abbreviations: DKT, daikenchuto; ESI, electrospray ionization; HAS, hydroxyl-α-sanshool; HBS, hydroxyl-β-sanshool; HPLC, high-performance liquid chromatography; IR, infrared; LC, liquid chromatography; MRM, multiple reaction monitoring; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NMR, nuclear magnetic resonance; POI, postoperative ileus; RT, retention time; TRP, transient receptor potential; UGT, UDP-glucuronosyl transferase. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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ABSTRACT

Daikenchuto (DKT, TU-100), a pharmaceutical grade traditional Japanese (Kampo) medicine, has been widely used for the treatment of various gastrointestinal disorders including postoperative ileus, and has been integrated into the modern medical care system in Japan as a prescription drug. DKT is a multi-herbal medicine consisting of Japanese

pepper (zanthoxylum fruit), processed ginger and ginseng with maltose as an additive. Downloaded from

Despite substantial research on the pharmacological activities of DKT and its ingredients, the lack of studies on absorption, distribution, metabolism, and excretion (ADME) of DKT dmd.aspetjournals.org has made it difficult to obtain a consistent picture of its mechanism of action. In the present study, we constructed an analysis procedure consisting of 7 conditions of liquid chromatography (LC) and mass spectrometric (MS) analysis, which enabled the at ASPET Journals on September 23, 2021 identification of 44 ingredients of DKT component herbs. We investigated the plasma and urine profiles of these ingredients 0.5 – 8 hours after oral administration of 15.0 g of DKT in 4 healthy volunteers. The results indicated that 1) hydroxyl-α-sanshool (HAS) and

[6]-shogaol, the prominent peaks in plasma derived from Japanese pepper and ginger, respectively, were detected at 0.5 hour, and thereafter decreased throughout the sampling period, 2) ginsenoside Rb1, a prominent peak derived from ginseng, increased gradually during the sampling period, 3) glucuronide conjugates of hydroxylsanshools, shogaols and gingerols were detected in plasma and urine, and 4) no obvious differences between samples from the 2 male and the 2 female individuals were observed.

These results provide a strong basis for future studies on pharmacokinetics and pharmacology of DKT. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Introduction

Kampo medicines, also known as traditional Japanese medicines, have gained a unique status in Japan (Motoo et al., 2009). Kampo medicines are prepared as a hot water extract of a mixture of medicinal herbs whose prescriptions originate in ancient China and have been modified and developed continuously in Japan. Many of the Kampo medicines are

now manufactured on a modern industrial scale, whereby the quality and quantity of Downloaded from ingredients are standardized under strict, scientific quality controls. More than 100 Kampo medicines have been approved as prescription drugs by the Ministry of Health, Welfare and dmd.aspetjournals.org Labor of Japan and are used clinically for the treatment of a wide variety of diseases. The majority of physicians who have been educated in Western medicine use Kampo medicines in daily practice. at ASPET Journals on September 23, 2021

Daikenchuto (DKT) is widely prescribed for patients with gastrointestinal obstruction such as postoperative ileus (POI), postoperative intestinal paralysis and adhesive bowel obstructions by a large number of surgeons at medical institutions including all of the university-affiliated hospitals in Japan (Itoh et al., 2002; Ohya et al., 2003; Kono et al.,

2009). Several double-blind placebo-controlled studies on DKT are now in progress in

Japan and the U.S. and very recently, the results of a clinical pharmacological study have clearly indicated that DKT accelerates intestinal transit in healthy humans (Manabe et al.,

2010). Potential beneficial effects of DKT for internal diseases including irritable bowel syndrome and functional constipation in children (Iwai et al., 2007) and for Parkinsonian patients (Sakakibara et al., 2005) have also been investigated.

DKT consists of processed ginger rhizome, ginseng, Japanese pepper (zanthoxylum fruit), DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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and, as an additive, maltose powder. Several experimental studies have focused on the effects of DKT on gastrointestinal motility. DKT has been shown to accelerate delayed intestinal transit induced by intestinal manipulation and/or morphine administration

(Nakamura et al., 2002; Fukuda et al., 2006; Tokita et al., 2007b) presumably via the stimulation of acetylcholine release and serotonin receptors, 5-HT3R and 5-HT4R (Shibata

et al., 1999; Satoh et al., 2001b; Tokita et al., 2007b). On the other hand, DKT has been Downloaded from shown to suppress the overactivity of intestines induced by several stimuli, which may relate to its relaxing effects on the intestinal smooth muscle (Satoh et al., 2001c). Recent dmd.aspetjournals.org studies have addressed the possibility that DKT increases intestinal blood flow and ameliorates colitis via calcitonin gene-related peptide and/or adrenomedullin (Murata et al.,

2002; Kono et al., 2008). Further, several pharmacological effects of DKT ingredients have at ASPET Journals on September 23, 2021 been reported to be antagonized by inhibitors of transient receptor potential (TRP) channels

(Satoh et al., 2001a; Iwasaki et al., 2006; Koo et al., 2007). Recently, HAS has been reported to be an inhibitory ligand for two pore domain potassium channels (KCNK3, etc.) which may regulate the excitability of enteric neurons (Bautista et al., 2008). In spite of substantial research on the pharmacological activities of DKT and its ingredients (Table 1), the lack of information on absorption, distribution, metabolism, and excretion (ADME) of

DKT has made it difficult to obtain a consistent picture of DKT's efficacy and pharmacology.

ADME studies of Kampo medicines are extraordinarily challenging. Generally, like DKT, multiple constituents are contained in a single Kampo formulation while the amount of each constituent is minute. Further, most Kampo medicines are administered orally and DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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exposed to gastric acid, intestinal fluid, bile and intestinal microflora. Consequently, some ingredients are extensively converted to other forms. In addition, it has been well recognized that many Kampo medicines can exert their pharmacological activity only after a metabolic conversion of their ingredients to bioactive forms by these gastrointestinal contents (Ohta et al, 2009). For these reasons, there have been only a few reports on ADME

of Kampo medicines. Downloaded from

However, recent advancements in mass spectrometry (MS) technology have opened the way to profiling and elucidating trace amounts of ingredients contained in these complex dmd.aspetjournals.org medicines. There are reports on analyses of plasma profiles of metabolites after oral dosing of ginger and ginseng (Yan et al., 2007; Jiang et al., 2008; Lee et al., 2009). However, no study on ADME of zanthoxylum fruit, as a single herb or as a part of a combination drug, at ASPET Journals on September 23, 2021 has been reported to our knowledge. Administration of polyherbal compounds may result in a different ADME profile than the combination of several ADME profiles obtained from each individual herb.

In this study, we determined the analytical conditions for 44 possible constituents of DKT, including HAS and hydroxyl-β-sanshool (HBS), by using liquid chromatography-tandem

MS (LC-MS/MS). These 44 constituents were subsequently investigated in human plasma and urine from 4 healthy volunteers after oral administration of 15.0 g of DKT. Further, the conjugated forms of DKT metabolites were analyzed by digesting plasma samples with enzymatic hydrolysis. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Materials and Methods

Chemicals and Reagents. TSUMURA Daikenchuto Extract Granules (TU-100) was manufactured by TSUMURA & CO. Fifteen grams of TSUMURA Daikenchuto extract granules contains 1.25 g of a dried extract prepared from a mixture of three herbs (5.0 g of processed ginger, 3.0 g of ginseng and 2.0 g of Japanese pepper), and 10.0 g of maltose. Downloaded from Acetonitrile and acetic acid of HPLC grade, and ginsenoside Rh1 were purchased from

Wako Pure Chemical Industries, Ltd., (Osaka, Japan). Ginsenoside F1 and

(20S)-protopanaxadiol were obtained from LKT Laboratories Inc. (St. Paul, dmd.aspetjournals.org

MN). Water was purified using a pure water supply system (MILLI-Q, Nihon

Millipore Ltd.). Oasis HLB μElution Plate (Nihon Waters K.K) was used as the

solid phase extraction (SPE) plate. β-Glucuronidase (EC 3.2.1.31) was obtained from at ASPET Journals on September 23, 2021

Sigma-Aldrich (St. Louis, MO).

Authentic Standards. The following authentic standards were isolated or synthesized by

Tsumura & Co.: hydroxy-α-sanshool, hydroxy-β-sanshool, γ-sanshool, [6]-gingerol,

[6]-shogaol, [10]-gingerol, [10]-gingerdione, [10]-shogaol, [8]-gingerol, [6]-paradol,

[8]-shogaol, [10]-dehydrogingerdione, [6]-gingerol 4’-O-glucronide, [6]-shogaol

4’-O-glucronide, [8]-gingerol 4’-O-glucronide, [8]-shogaol 4’-O-glucronide, [10]-gingerol

4’-O-glucronide, [10]-shogaol 4’-O-glucronide, ginsenoside Rg1, ginsenoside Rb1, ginsenoside F2, ginsenoside Rh2, ginsenoside Re, ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Rg3, ginsenoside Rf, ginsenoside Rg2, (20R)-ginsenoside

Rg3, [6]-gingerdiol, (20R)-ginsenoside Rh1,

5-hydroxy-9-(4-hydroxy-3-methoxyphenyl)-7-oxonanoic acid, DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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4-(4-hydroxy-3-methoxyphenyl)butanoic acid,

(E)-9-(4-hydroxy-3-methoxyphenyl)-7-oxonon-4-enoic acid, [6]-dehydroparadol,

(20R)-ginsenoside Rh2 , (20S)-protopanaxatriol, (20R)-protopanaxatriol, compound K,

(20R)-protopanaxadiol.

All of these authentic standards were reliably identified with spectral methods such as

NMR, mass and IR spectrometries. The structural formulas of the authentic standards are Downloaded from provided in Supplementary Figure S1.

Clinical Trial Design. dmd.aspetjournals.org This study was conducted in accordance with the ethical principles of the Declaration of

Helsinki, and consistent with Good Clinical Practice guidelines. The study obtained approval from an independent ethics committee at the Kochi Medical School before at ASPET Journals on September 23, 2021 recruitment commenced. Prior to initiation of study procedures, all volunteers gave their written informed consent for participation in the study.

Four healthy volunteers (2 males and 2 females) aged 21-32 (25.0±4.2), with a body mass index of 18.5-24.1 (20.8±2.3) kg/m2 participated in this open label study. The demographics of the participants are summarized in Table 2. Participants were orally administered 15.0 g of TSUMURA Daikenchuto Extract Granule and were fasted from 12 hours before and 4 hours after administration. Each participant then consumed a standardized meal. In addition, the participants refrained from Japanese pepper, ginseng and ginger-containing foods from

3 days before the study until completion of the study. Blood samples (20 mL each) were collected from the medial cubital vein into evacuated tubes containing heparin just before and at 0.5, 1, 2, 4, and 8 hours after administration and were immediately centrifuged (3000 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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rpm, 15 min). Urine samples (50 mL each) were collected the day before the administration and at 0-4 hours, and 4-8 hours after administration. Plasma and urine fractions were stored at -80°C until analysis.

Preparation of the standard solution. Each reference substance was dissolved separately with acetonitrile/water (1:1, v/v) or acetonitrile to prepare standard stock solutions (1000

µg/mL). After mixing 0.1 mL of the respective standard stock solutions, the resulting Downloaded from solution was diluted with 0.2 v/v% acetic acid aqueous solution/acetonitrile (1:1, v/v) to make 10 mL to prepare a standard stock solution (10 µg/mL). This standard stock solution dmd.aspetjournals.org (10 µg/mL) was diluted with acetonitrile/water (1:1, v/v) to prepare standard working solutions (1, 2, 5, 10, 50, 100, 1000 ng/mL). The standard solutions were stored at -80 ℃

in an airtight container under light-resistant conditions in a freezer. at ASPET Journals on September 23, 2021

Pre-treatment procedure for the test substance. The test substance (0.5 g) was collected and mixed with 25 mL of methanol/water (75:25, v/v). The mixture was sonicated for 15 min and centrifuged at 3,000 rpm for 5 min at 4°C (refrigerated centrifuge 5910

KUBOTA Corporation) to collect the supernatant. The residue was extracted again with 25 mL of methanol/water (50:50, v/v) as described above. The supernatant was combined and diluted with water to prepare the test substance solution. The test substance solution was used for LC-MS/MS analysis.

Pre-treatment procedure for plasma and urine. The solid-phase extraction was adopted as the extraction procedure. Hydrochloric acid was added to 300µL of the plasma, urine sample, or calibration sample to adjust the pH to about 3.0. The resulting mixture (200 µL) was loaded on a SPE plate pre-conditioned with 200 µL of acetonitrile and 200 µL of water. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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This SPE plate was washed with 200 µL of water, and then eluted with 50 µL of acetonitrile.

The eluate was mixed with 50 µL of water, and the resulting solution (sample solution) was used for LC-MS/MS analysis.

Enzyme treatment. Plasma and urine samples were collected, and ammonium acetate buffer (pH 6.0) and β-glucuronidase were added. The mixture was incubated at 37°C for 2 hr. Subsequently, sample for LC-MS/MS analysis was prepared as described in Downloaded from

“Pre-treatment procedure for plasma and urine”.

LC-MS/MS analysis. LC-MS/MS analyses were conducted using the API 5000 system（AB dmd.aspetjournals.org

Sciex Pte. Ltd.）equipped with a 1200 series HPLC system（Agilent Technologies, Inc.）, and Analyst Software, version Ver. 1.4.2 (Applied Biosystems Inc.). The column for

LC-MS/MS analyses was a YMC-Pack ODA-AQ,( 3 μm, 150 × 2.0 mm I.D. ,3 μm, 50 × at ASPET Journals on September 23, 2021

2.0 mm I.D., YMC Co., Ltd). The other conditions were as follows: flow rate; 0.2 mL/min, column temperature; 40 ℃, injection volume; 10 μL, ionization; ESI with positive or negative mode. The mobile phase, Multiple Reaction Monitoring (MRM) scan, full scan, precursor ion scan and product ion scan conditions are summarized in Table 3. Methods 2 and 3 use the same HPLC gradient conditions but different mass spectrometer acquisition parameters as described in Table 4.

Determination of plasma concentration of four active constituents of DKT. Four constituents, hydroxyl-α-sanshool (HAS), hydroxyl-β-sanshool (HBS), [6]-shogaol and

[10]-shogaol were determined in human plasma. The respective standard working solutions were diluted with blank plasma or urine to prepare calibration curves (0.1, 0.2, 0.5, 1, 5, 10 ng/mL). The calibration curve for the analytes was constructed from the peak areas against DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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the concentrations ranged from 0.1 ng/mL to 10 ng/mL. The pre-treatment recovery rate was calculated by comparing the peak area in the standard solution with that in the samples for calibration curve after pre-treatment procedure as described in “Pre-treatment procedure for plasma and urine” paragraph.

Stability of stock solution, extracted sample in autosampler, storage and frees/thaw cycles

were conducted. The quality control samples were prepared by diluting standard working Downloaded from solution with blank plasma.. The performance of the analytical methods for plasma samples was summarized in Table 5. Quality control samples for urine samples have not been dmd.aspetjournals.org prepared in this study.

Pharmacokinetics. Pharmacokinetic parameters were estimated using the WinNonlin version 5.2 (Pharsight Corporation、USA). Maximum concentration and time to maximum at ASPET Journals on September 23, 2021 concentration following drug administration (tmax) were the experimentally observed values.

The area under plasma concentration-time curve from zero to time t (AUC0-t) was calculated from time zero to last detected time. Apparent elimination half-life (t1/2) was calculated divided by loge2/ke where ke means the terminal elimination rate constant. Each calculated parameters were presented mean ± SD. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Results

The structural formulas of the major DKT ingredients and their metabolites are shown in

Figure 1. All of the 44 authentic standards used in the present study are provided in

Supplementary Figure S1.

Analysis of the ingredients derived from Japanese pepper.

Hydroxyl-α-sanshool (HAS) and hydroxyl-β-sanshool (HBS) were detected in the test Downloaded from substances, plasma and urine samples, and γ-sanshool was detected in the test substances and plasma samples (Figure 2). The product ions of those peaks were coincident with those dmd.aspetjournals.org of the authentic standards (Supplementary Figure S2-1). There was an unknown peak

(Rt:16 min) product ions corresponded with those of HAS and HBS. All of the peaks were

detected most prominently at 0.5 hour post-dose, and thereafter decreased throughout the at ASPET Journals on September 23, 2021 sampling period.

Product ion scanning (fixed Q1 to m/z:440 which corresponded to the [M+H]+ of glucuronide conjugates of hydroxyl-sanshool) showed five peaks in both plasma and urine samples. A product ion derived from loss of glucuronic acid (-176) was detected at m/z 264, which corresponded to the [M+H]+ of hydroxy-sanshool (Supplementary Figure S2-2).

Moreover, product ions m/z 107 and 147 corresponding to hydroxy-α-sanshool and hydroxy-β-sanshool, respectively, were detected. In the plasma and urine samples following

β-glucuronidase treatment, there was no peak for hydroxy-sanshool glucuronide, while that of hydroxy-sanshool was detected with increased intensity in urine. Accordingly, the presence of glucuronide conjugates of hydroxy-α-sanshool and hydroxy-β-sanshool were suggested. The product ion spectra of Hydoroxy-a-sanshool an its glucuronide conjugats DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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are shown in Supplementary Figure S2.

No obvious differences between samples from the 2 male and the 2 female individuals were observed.

Analysis of the ingredients derived from processed ginger.

The full scan, precursor ion scan, and product ion scan revealed ginger ingredients in the

standard solution. However, peaks corresponding to the authentic standards were not Downloaded from detected in the plasma or urine by any of the above analyses.

The Multiple Reaction Monitoring (MRM) chromatograms of plasma detected trace dmd.aspetjournals.org amounts of the peaks which were coincident with the peaks of [6]-shogaol, [8]-shogaol,

[10]-shogaol, [6]-gingerol, [8]-gingerol, [10]-gingerol, [10]-gingerdione,

[10]-dehydrogingerdione, and [6]-paradol (Figure 3). In the same way, the MRM scan of at ASPET Journals on September 23, 2021 urine detected peaks of [6]-shogaol, [8]-shogaol, [8]-gingerol, [10]-gingerol,

[10]-gingerdion, [10]-dehydrogingerdione, and [6]-paradol (Figure 3). The MRM chromatograms also detected the peaks that coincided with the 4’-O-glucuronide conjugates of [6]-shogaol, [8]-shogaol, [10]-shogaol, [6]-gingerol, [8]-gingerol and

[10]-gingerol in plasma and urine (Figure 3). All of the peaks were detected most prominently at 0.5 hour post-dose, and thereafter decreased throughout the sampling period.

The plasma and urine samples treated with β-glucuronidase were analyzed by the product ion scan method. [6]-shogaol, [6]-gingerol, [8]-gingerol, [10]-gingerol, [6]-paradol,

[6]-gingerdiol, and (E)-9-(4-hydroxy-3-methoxyphenyl)-7-oxonon-4-enoic acid were detected, and the product ion spectra of these ingredients coincided with those of the DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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authentic standards. Accordingly, the presence of glucuronide conjugates of [6]-shogaol,

[8]-shogaol, [10]-shogaol, [6]-gingerol, [8]-gingerol, [10]-gingerol, [6]-paradol,

[6]-gingerdiol, and (E)-9-(4-hydroxy-3-methoxyphenyl)-7-oxonon-4-enoic acid, were suggested. Subsequently, plasma and urine samples were analyzed by the product ion scan method to identify [M−H]－ corresponding to [6]-shogaol sulfate (m/z:355), [8]-shogaol sulfate (m/z:383), [10]-shogaol sulfate (m/z:411), [6]-gingerol sulfate (m/z:373), Downloaded from

[8]-gingerol sulfate (m/z:401), and [10]-gingerol sulfate (m/z:429). The analysis included the identification of product ions ([M−H]－ of the above ingredients) derived from the loss dmd.aspetjournals.org of sulfates and main product ions of [6]-shogaol, [8]-shogaol, [10]-shogaol, [6]-gingerol,

[8]-gingerol, and [10]-gingerol. That is, peaks corresponding to [6]-shogaol and

[6]-gingerol sulfates were detected along with m/z 275 and 293, which corresponded to the at ASPET Journals on September 23, 2021 loss of sulfates. m/z 139 and 99 are the main product ions of [6]-shogaol and [6]-gingerol,

- and m/z 80 corresponded to sulfate moiety (SO3 ). Accordingly, the presence of a sulfate conjugate of [6]-shogaol and [6]-gingerol was suggested.

No obvious differences between samples from the 2 male and the 2 female individuals were observed.

Analysis of the ingredients derived from ginseng.

The full scan, precursor ion scan, and product ion scan revealed ginseng ingredients in the standard solution. However, peaks corresponding to the authentic standards were not detected in the plasma or urine by any of the above analyses.

The MRM chromatograms of plasma detected several peaks which were coincident with the peaks of ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rb2, ginsenoside Rc, DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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ginsenoside Rf and (20R)-ginsenoside Rg3, and the MRM chromatograms of urine detected the peaks of ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rf, ginsenoside Rg2, and ginsenoside Rh1 (Figure 4). Unlike the peaks derived from Japanese pepper and ginger, the peaks derived from ginseng increased gradually during the sampling period.

No obvious differences between samples from the 2 male and the 2 female individuals were

observed. Downloaded from

A summary of detected ingredients and their glucronide conjugates are listed in

Supplementary Table S1. dmd.aspetjournals.org Plasma concentrations of four active constituents of DKT

Plasma concentration of hydroxyl-α-sanshool (HAS), hydroxyl-β-sanshool (HBS),

[6]-shogaol and [10]-shogaol which have known pharmacological activities and detected at ASPET Journals on September 23, 2021 enough strength for determination were determined in human plasma (Fig. 5).

HAS reached a maximum concentration (221 ± 33 ng/mL) at the time of 0.625 ± 0.217 hr and eliminated with elimination half life (t1/2) of 1.27 ± 0.61 hr. The area under curve from tome zero to last detection time (AUC0-8) was 606 ± 280 hr*ng/mL.HBS reached a maximum concentration (64.1 ± 12.4 ng/mL) at the time of 0.625 ± 0.217 hr and eliminated with t1/2 of 1.49 ± 0.33 hr. The AUC0-8 was 167 ± 70.3 hr*ng/mL.

[6]-shogaol reached a maximum concentration (0.098 ± 0.030 ng/mL) at the time of 0.5 hr.

The AUC0-4 was 0.353 ± 0.174 hr*ng/mL. Elimination half life was not calculated because the time points were insufficient. [10]-shogaol reached a maximum concentration (0.255 ±

0.116 ng/mL) at the time of 1.13 ± 0.54 hr. AUC0-2 was 0.505 ± 0.245 hr*ng/mL.

Elimination half life was not calculated because the time points were insufficient. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Discussion

DKT is a mixture of herbs which are familiar to most Japanese people. Ginseng is a frequently used herbal medication and/or supplement, and ginger and Japanese pepper are commonly-used spices in the standard Japanese diet. Therefore, detailed instructions to avoid foods and medications containing these herbs from 3 days before DKT administration

were given to the volunteers, and the herbs were carefully excluded from the controlled Downloaded from meal during blood and urine sampling.

In order to detect as many compounds as possible, 15 g of DKT was administered as a dmd.aspetjournals.org single dose instead of the usual thrice-daily regimen. Off-label use of single dose of 15g of

DKT has a long track record of safety, and in the present study, no toxic or adverse effects were reported except for a mild irritation at the time of taking the medication. at ASPET Journals on September 23, 2021

Because of DKT’s characteristics containing a large number but small amount of chemical compounds, a combination of full scan precursor ion scan, product ion scan and MRM method for identification of the ingredients was used. The MRM method is the most sensitive method for LC-MS/MS detection, however this method requires authentic standards for identification. We used 44 authentic standards which were selected based on the extensive literature review of bioactive components of DKT. Three chromatographic conditions were needed to cover all of the authentic standards.

While many pharmacokinetic studies have been conducted for ginseng and ginger, no such study on Japanese pepper has been reported. However, the pepper and its major ingredients,

HAS and HBS, have been reported to have unique biological activities and play a critical role in the beneficial effects of DKT. An earlier study has shown that the administration of DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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the pepper at a dose equivalent to that contained in DKT ameliorated intestinal manipulation-induced delay of gastrointestinal transit, as previously demonstrated by administration of DKT alone (Tokita et al., 2007b). DKT has been reported to decrease intestinal adhesion induced by dusting talc over intestines, and this anti-adhesion effect has been attributed to the pepper (Hayakawa et al., 1999; Tokita et al., 2007a). HAS has been

known to stimulate contractility in intestinal muscle strips in an organ bath which was Downloaded from antagonized by capsazepine, a TRPV1 inhibitor (Satoh et al., 2001a). HAS has been reported to have a receptor agonist activity to TRPV1- and TRPA1- transfected HEK293 dmd.aspetjournals.org cells (Koo et al., 2007; Riera et al., 2009). In addition, at lower concentrations, HAS has been purported to inhibit two pore domain potassium channels KCNK3, KCNK9, and

KCNK18 (Bautista et al., 2008), which are thought to regulate the background current of at ASPET Journals on September 23, 2021

KCNKs-expressing enteric neuron by altering the balance between intracellular and extracellular K+ concentrations (Matsuyama et al., 2008). Although a more complete investigation of pharmacokinetics and tissue/blood distribution of HAS is warranted in the future, the present study indicates for the first time that this important compound is absorbed into the blood in measurable amounts.

In this study, shogaols and gingerols were detected in plasma within 30 min after dosing and decreased thereafter. These findings corroborate the results of a previous study where maximal concentration of [6]-gingerol in rat plasma was reached within 10 min after oral administration (Jiang et al., 2008). On the other hand, Zick SM et al. reported that no free

[6]-gingerol, [8]-gingerol, [10]-gingerol nor [6]-shogaol were found but instead, their conjugates were detected in human plasma after oral dosing of ginger extract capsules DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #33589, 18

(Zick et al., 2008). The maximum dosage given to a human in Zick’s study contained 21.5 mg of [6]-gingerol, 7.20 mg of [8]-gingerol, 16.8 mg of [10]-gingerol and 3.68 mg of

[6]-shogaol. Based on the HPLC analysis, 15 g of DKT used in the present study contained

3.69 mg of [6]-gingerol, 0.53 mg of [8]-gingerol, 0.89 mg of [10]-gingerol and 2.22 mg of

[6]-shogaol. It is therefore clear that the difference in the contents of gingerols and shogaols

cannot explain the discrepancy between the results of the former study and our study. Downloaded from

Differences in race or diet may affect the bioavailability of these compounds, and the varying methods for extraction, separation or detection may yield different results. It is also dmd.aspetjournals.org possible that the co-existence of other herbal ingredients with ginger alters the absorption of gingerols and shogaols. Self-formulation of micelles of ginseng saponins such as ginsenosides has been reported to prevent permeation or absorption through the cell at ASPET Journals on September 23, 2021 membrane of mucosal cells of the gastrointestinal tract. Incorporation of lipidophilic compound into the micelle may enhance the bioavailability of ingredients of ginger and ginseng (Xiong et al., 2008). Ginseng saponin’s effect on enhancement of the blood concentrations of salvianolic acids, a medication for treatment of myocardial ischemia in

China, has also been reported (Yang et al., 2008).

In various in vitro experimental systems, DKT ingredients such as HAS, shogaols, gingerols, ginsenosides and their metabolites exhibited pharmacological effects at the range of 0.1 ~ 100uM (see the references in Table 1). Though the quantitative method of the present study has not been validated and therefore gives only an approximation, the peak concentration of HAS (215ng/ml = 0.81μM) may be enough to exert some of its pharmacological activities. Although the blood concentrations of shogaols, gingerols and DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #33589, 19

ginsenoside Rb1 found in the present study were extremely low, it must be noted that their peak concentrations were missed with the sampling protocol employed in the study. Further, previous studies on DKT have addressed the possibility that DKT ingredients can affect gastrointestinal functions directly from the gut lumen, presumably without absorption into the blood stream (Kawasaki et al, 2007; Jin et al, 2001). Extensive ADME studies including

analysis of tissue distribution of DKT ingredients are necessary to clarify this point. Downloaded from

In summary, the major representative ingredients of DKT, which have been reported to have various pharmacological effects relevant to DKT’s clinical efficacy, have been dmd.aspetjournals.org detected in human plasma and urine. The data presented in this paper provide the basis for pharmacokinetic and pharmacodynamic research on DKT for assessing and clarifying its diverse bioactivities. Subsequent pharmacokinetic studies which focus on validating the at ASPET Journals on September 23, 2021 quantity of selected ingredients are currently in progress.

DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #33589, 20

Acknowledgment

We would like to thank Dr. Ken-ichiro Hayashi (Medi-Chem Business Segment, Mitsubishi

Chemical Medience Corporation, Kumamoto, Japan) for the analysis of DKT constituents. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #33589, 21

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Figure Legends

Figure 1. The structures of main DKT ingredients and their conjugated metabolites. Filled arrow indicates glucuronide conjugation. Open arrow indicates sulfate conjugation. The structural formulas of sevaral glucuronide conjugates of shogaols and gingerols were

determined using the authentic standards synthesized in our laboratory. The complete list of Downloaded from investigated compounds and in the present study is given as Supplementary Tables S1.

dmd.aspetjournals.org Figure 2. MRM chromatograms of authentic standards, plasma, and urine samples for ingredients derived from Japanese pepper. 1. hydroxy-α-sanshool, 2. hydroxy-β-sanshool, 3.

γ-sanshool. A calibration sample contains 10ng/ml of each authentic standard. at ASPET Journals on September 23, 2021

Figure 3. MRM chromatograms of authentic standards, plasma, and urine samples for ingredients derived from processed ginger. 4. [6]-gingerol, 5.[6]-shogaol, 6. [10]-gingerol,

7. [10]-gingerdione, 8. [10]-shogaol, 9. [8]-gingerol, 10. [6]-paradol , 11. [8]-shogaol, 12.

[10]-dehydrogingerdione, 13. [6]-gingerol 4’-O-glucronide, 14. [6]-shogaol

4’-O-glucronide, 15. [8]-gingerol 4’-O-glucronide, 16. [8]-shogaol 4’-O-glucronide, 17.

[10]-gingerol 4’-O-glucronide, 18. [10]-shogaol 4’-O-glucronide. A calibration sample contains 10ng/ml of each authentic standard.

Figure 4. MRM chromatograms of authentic standards, plasma, and urine samples for glucuronide derived from ginseng. 19. ginsenoside Rg1, 20. ginsenoside Rb1, 21. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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ginsenoside F2, 22. (20S)-ginsenoside Rh2, 23. ginsenoside Re, 24. ginsenoside Rb2, 25. ginsenoside Rc, 26. ginsenoside Rd, 27, ginsenoside Rg3, 28. ginsenoside Rf, 29, ginsenoside Rg2 30. ginsenoside Rh1, 31. (20R)-ginsenoside Rg3. A calibration sample contains 10ng/ml of each authentic standard.

Figure 5. A semi-logarithmic plot of plasma concentrations of main DKT ingredients in Downloaded from human healthy volunteers. The results of quantitation of ingredients in Japanese pepper (A) and processed ginger (B) are shown. A: hydroxy-α-sanshool (closed circle), dmd.aspetjournals.org hydroxy-β-sanshool (closed triangle). B: [10]-shogaol (closed circle), [6]-shogaol (closed square). Values are means ± standard deviations (n=4). at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 1. Major ingredients and their biological activities relevant to DKT's efficacy

Source Major active Known biological Known molecular ingredients activities interactions Zanthoxylum fruit hydroxyl-α-sanshool Prokinetic effect 1) TRPV1/TRPA1 stimulation 2) KCNK3 inhibition 3) Ginger shogaols, gingerols Anti-emetic effect 4) TRPV1 stimulation Downloaded from Prokinetic effect 5) 9) Relax muscle 6) Inhibition of Increase intestinal Ca(++) channels 10) blood flow 7) 5-HT3 receptor Anti-inflammatory blocking 11) dmd.aspetjournals.org effect 8) Ginseng ginsenosides Alleviation of TRPV1 modulation noxious pain 12) 15) Anti-inflammatory Modulation of 13) 16)

effect Ca(++) channels at ASPET Journals on September 23, 2021 Amelioration of Inhibition of accelerated various ion-gated intestinal transit 14) channels 17) 1) Tokita Y et al, Inflammopharmacology. 2007;15:65-6; 2) Koo JY et al, Eur J Neurosci. 2007;26:1139-47; 3) Bautista DM et al, Nat Neurosci. 2008;11:772-9; 4) Kawai T et al, Planta Med. 1994;60:17-20; 5) Ghayur MN et al, Int J Food Sci Nutr. 2006;57:65-73; 6) Hashimoto K et al, Planta Med. 2002;68:936-9; 7) Murata P et al, Life Sci. 2002;70:2061-70; 8) Pan MF et al, Mol Nutr Food Res. 2008;52:1467-77, 9) Iwasaki Y et al, Nutr Neurosci. 2006;9:169-78, 10) Ghayur MN et al, J Pharm Pharmacol. 2008;60:1375-83; 11) Abdel-Aziz H et al, Planta Med. 2005 Jul7:609-1; 12) Kim-JH, Biol. Pharm. Bull. 28; 2120—2124; 13) Wang J et al, Exp Mol Med. 2008;40:686-98; 14) Hahimoto K et al, J Ethnopharmacol. 2003, 84:115-9; 15) Jung SY et al, Mol Cells. 2001;12:342-6; 16) Rhim H et al, Eur J Pharmacol. 2002;436:151-8. 17) CNS Drug Rev. 2007, Nah SY et al, 13:381-404. DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 2. Clinical and Demographic Characteristics of Study Participants Code age Height(cm) Weight(kg) BMI(kg/m2) Sex AW01-01 32 165.4 66.0 24.1 Male AW01-02 23 174.1 66.0 21.8 Male AW01-03 24 166.0 52.1 18.9 Female AW01-04 21 156.0 45.1 18.5 Female

Mean 25.0 165.4 57.3 20.8 Downloaded from

S.D 4.2 6.4 9.0 2.3 Max 32.0 174.1 66.0 24.1

Min 21.0 156.0 45.1 18.5 dmd.aspetjournals.org

at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

DMD #33589, 31

Table 3 Analytical conditions used for the sample analysis HPLC MS/MS condition Method Target Compounds condition mode Q1(m/z) Q3(m/z) gradient of 264.3 107.1 hydroxyl-α-sanshool 1 30-90% (B) positive 264.3 107.1 hydroxyl-β-sanshool over 30 min 274.3 133.1 γ-sanshool 277.3 137.1 [6]-shogaol (positive) 333.4 137.1 [10]-shogaol (positive) positive/ 293.1 557.0 [6]-gingerol (negative) negative 349.2 57.0 [10]-gingerol (negative) Downloaded from 347.2 211.1 [10]-gingerdion (negative) 303.2 167.0 [8]-shogaol gradient of 321.2 57.0 [8]-gingerol negative 2 15-95% (B) 345.2 149.0 [10]-dehydrogingerdion

over 15 min 277.1 141.0 [6]-paradol dmd.aspetjournals.org 453.3 137.3 [6]-shogaol 4’-O-glucronide 481.3 137.3 [8]-shogaol 4’-O-glucronide 509.3 137.3 [10]-shogaol 4’-O-glucronide positive 471.3 177.2 [6]-shogaol 4’-O-glucronide 499.3 177.2 [8]-shogaol 4’-O-glucronide 527.3 177.2 [10]-shogaol 4’-O-glucronide at ASPET Journals on September 23, 2021

1109.6 325.1 ginsenoside Rb1 801.5 423.4 ginsenoside Rf 785.5 407.4 ginsenoside F2 623.4 407.2 (20S)-ginsenoside Rh2 ginsenoside Rb2, ginsenoside gradient of 1079.6 457.1 Rc 3 15-95% (B) positive 947.5 407.3 ginsenoside Rd, ginsenoside over 15 min 785.5 407.3 Re ginsenoside Rg3 801.5 423.3 ginsenoside Rg1 785.4 423.3 ginsenoside Rg2 785.5 407.3 (20R)-ginsenoside Rg3 639.3 423.3 ginsenoside Rh1 Mobile phase： (A) 0.2vol% acetic acid in water (B) acetonitrile

DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 4. Optimized mass spectrometer acquisition parameters used for analyte quantitation.

Method CUR GS1 GS2 TEM CAD (psi) CE (V) (psi) (psi) (psi) (°C)

1 40 40 50 600 4 25 2 10 30 60 600 4 25 3 30 30 30 200 5 25

CUR, Curtain Gas; GS, Ion source gas; TEM, Temperature; CAD, Collision Gas; CE, Downloaded from

Collision energy. dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 5 Summary of validation results of analytical methods for plasma samples

Hydroxy-α-sanshool Hydroxy-β-sanshool [6]-shogaol [10]-shogaol

Linearity 0.9998 0.9997 0.9999 0.9994 Correlation coefficient ( r ) Accuracy 96.7 - 118.8 96.3 - 128.7 96.2 - 105.5 83.9 - 116.8 (%)

Precision 0.40 - 3.93 0.40 - 4.92 0.15 - 3.03 1.44 - 10.46 (%) Lower Limit of Quantification

0.1 0.1 0.1 0.1 Downloaded from (ng/mL） † Pre-treatment recovery 100.3 102.3 120.1 59.5 (%) Stability in Plasma* 98.7 99 95.3 108.6 8 ng/mL, room temperature, 4 hour dmd.aspetjournals.org

Stability in autosampler* 92.9 98.5 110.7 106.4 8 ng/mL, 7 °C, 24 hour

Stability in Plasma* 91.1 104.6 106.5 108.3 8 ng/mL, -20 °C, one month

Freeze and Thaw Stability* at ASPET Journals on September 23, 2021 86.8 105.5 95.3 108.6 8 ng/mL, three cycles †; Peak area of calibration curve /Peak area of standard solution *; Observed concentration after storage or treatment / Observed concentration at the initial ×100

DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 DMD Fast Forward. Published on August 5, 2010 as DOI: 10.1124/dmd.110.033589 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from dmd.aspetjournals.org at ASPET Journals on September 23, 2021 Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition

Supplementary Data for DMD-2010-033589

Title: Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) .

Authors: Jun Iwabu, et al.

Manuscript for Drug Metabolism and Disposition

Manuscript ID: DMD-2010-033589 Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition

Supplementary Figure S1. Structural formulas of DKT ingredients and possible DKT metabolites

H OH N

hydroxy-ααα-sanshool O No. Compound 1 hydroxy-α-sanshool

H OH N

hydroxy-βββ-sanshool O No. Compound 2 hydroxy-β-sanshool

N H γγγ−γ−−−sanshool

No. Compound 3 γ-sanshool Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition Supplementary Figure S1. continued

O R2 R3

O (CH2)nCH3

No. Compound R1 R2 R3 n R1O 4 [6]-gingerol H OH H 4 6 [10]-gingerol H OH GlcA 8 7 [10]-gingerdione H O 8 9 [8]-gingerol H OH H 6

10 [6]-paradol H R7,R 8=H 4 13 [6]-gingerol 4'-O-glucronide GlcA OH H 4 15 [8]-gingerol 4'-O-glucronide GlcA OH GlcA 6 17 [10]-gingerol 4'-O-glucronide GlcA OH GlcA 8

GlcA,D-glucuronic acid

O No. Compound R n O 4 5 [6]-shogaol H 4 (CH2)nCH3 8 [10]-shogaol H 8 11 [8]-shogaol H 6 14 [6]-shogaol 4'-O-glucronide GlcA 4 R4O 16 [8]-shogaol 4'-O-glucronide GlcA 6 18 [10]-shogaol 4'-O-glucronide GlcA 8

GlcA,D-glucuronic acid Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition Supplementary Figure S1. continued

O R5

O No. Compound R5 n (CH2)nCH3 12 [10]-dehydrogingerdion O 8

38 [6]-dehydroparadol H2 4 HO OH R6 R7 O R8

No. Compound R R R HO 6 7 8 32 [6]-gingerdiol OH H (CH 2)4CH 3

35 5-hydroxy-9-(4-hydroxy-3-methoxyphenyl)-7-oxonanoic acidR10 ,R 11 =O (CH 2)3COOH

O OH No. Compound O 36 4-(4-hydroxy-3-methoxyphenyl)butanoic acid HO O O

O OH

No. Compound HO 37 (E )-9-(4-hydroxy-3-methoxyphenyl)-7-oxonon-4-enoic acid Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition

RRR11101000 RRR999 Supplementary Figure S1. continued OOOHOHHH

No. Compound R9 R10 R11 R12

19 ginsenoside Rg1 OGlc CH 3 Oglc H

20 ginsenosise Rb1 OGlc(6-1)Glc CH 3 H Glc(2-1)Glc

21 ginsenoside F2 OGlc CH 3 H Glc RRR11121222OOO 22 ginsenoside Rh2 OH CH 3 H Glc RRR 111111 23 ginsenoside Re OGlc CH 3 OGlc(2-1)Rha H

24 ginsenoside Rb2 OGlc(6-1)Ara p CH 3 H Glc(2-1)Glc

25 ginsenoside Rc OGlc(6-1)Ara f CH 3 H Glc(2-1)Glc

26 ginsenoside Rd OGlc CH 3 H Glc(2-1)Glc

27 ginsenoside Rg3 OH CH 3 H Glc(2-1)Glc

28 ginsenoside Rf OH CH 3 OGlc(2-1)Glc H

29 ginsenoside Rg2 OH CH 3 OGlc(2-1)Rha H

30 Ginsenoside Rh1 OH CH 3 OGlc H

31 (20 R )-ginsenoside Rg3 CH 3 OH H Glc(2-1)Glc

33 ginsenoside F1 OGlc CH 3 OHH

34 (20 R )-ginsenoside Rh1 CH 3 OH OGlc H

39 (20 R )-ginsenoside Rh2 CH 3 OH H Glc

40 (20 S )-protopanaxatriol OH CH 3 OHH

41 (20 R )-protopanaxatriol CH 3 OHOHH

42 Compound K OGlc CH 3 HH

43 (20 S )-protopanaxadiol OH CH 3 HH

44 (20 R )-protopanaxadiol CH 3 OHHH Glc, β-D-glucopyranosyl; Ara p, α-L-arabinopyranosyl; Ara f, α-L-arabinofuranosyl; Rha, α-L-rhamnopyranosyl. Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition

Supplementary Figure S2-1. Product ion scan chromatograms and Product ion scan spectra (hydroxy- ααα-sanshool) of the Calibration samples, plasma (0.5 hr) and Urine (0 - 4 hr)

Product ion scan chromatogram Product ion scan spectra (1) Calibration sample

TIC: from Sample 3 (Plasma 0.5 h (Pro-1)) of 091104 04.wiff (Turbo Spray) Max. 2.2e8 cps. +MS2 (264.30) CE (25): Period 1, 16.277 to 16.731 min from Sample 2 (STD (S-7) product scan-1) of 091 02802.wiff (Turbo Spray) Max. 1.8e6 cps. 16.51 2.2e8 1.8e6 107.1 2.1e8 hydroxy-α-sanshool 1.7e6 2.0e8 1.6e6 1.9e8 1.5e6 1.8e8 79.1 1.7e8 1.4e6 m/z 107 1.6e8 1.3e6 1.5e8 1.2e6 1.4e8 1.3e8 1.1e6 1.0e6 1.2e8 147.2 + 1.1e8 hydroxy-β-sanshool 9.0e5 m/z 147 [M+H] 1.0e8 8.0e5 Intensity, cps Intensity, Intensity, cps Intensity, 105.1 9.0e7 7.0e5 72.2 8.0e7 hydroxy-sanshool isomer 91.1 6.0e5 7.0e7 17.15 81.1 6.0e7 γ-sanshool 5.0e5 133.1 246.2 139.1 246 5.0e7 4.0e5 93.0 119.2 4.0e7 3.0e5 67.1 3.0e7 16.02 95.0 2.0e5 2.0e7 131.1 149.3 55.1 69.2 175.0 1.0e5 97.9 121.1 129.0 152.1 157.2 1.0e7 26.87 77.3 83.0 109.0 191.2 0.0 12.07 57.2 59.2 65.1 166.3178.0 204.2 218.0 223.3 241.2 264.3 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 23 0 240 250 260 270 280 290 300 Time, min m/z, amu (2) Plasma TIC: from Sample 2 (STD (S-7) product scan-1) of 09 102802.wiff (Turbo Spray) Max. 2.7e8 cps. +MS2 (264.30) CE (25): Period 1, 16.335 to 16.756 min from Sample 3 (Plasma 0.5 h (Pro-1)) of 091104 04.wiff (Turbo Spray) Max. 2.4e6 cps. 2.7e8 26.87 107.2 hydroxy-α-sanshool γ-sanshool 2.4e6 2.6e8 2.3e6 2.2e6 2.4e8 2.1e6 79.1 2.2e8 2.0e6 hydroxy-β-sanshool 1.9e6 m/z 107 2.0e8 1.8e6 1.7e6 + 1.8e8 1.6e6 16.49 1.5e6 [M+H] 17.13 1.6e8 1.4e6 m/z 147 1.3e6 147.2 1.4e8 1.2e6 1.1e6 Intensity, cps Intensity, Intensity, cps Intensity, 1.2e8 1.0e6 72.2 105.1 91.0 246 1.0e8 9.0e5 8.0e5 81.1 8.0e7 7.0e5 133.1 139.1 246.2 6.0e5 93.0 119.2 6.0e7 5.0e5 67.0 27.07 4.0e5 95.1 4.0e7 3.0e5 131.2 149.3 2.0e5 55.1 69.1 175.0 2.0e7 71.2 98.1 121.1 128.9 157.2 1.0e5 83.0 142.0 15.97 53.2 58.0 65.2 86.1 111.1 166.2 178.1 191.2 204.1 218.3 228.3 235.5 244.2 264.3 0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 23 0 240 250 260 270 280 290 300 Time, min m/z, amu

(3) Urine

TIC: from Sample 7 (Urine 0-4 h (Pro-1)) of 091104 04.wiff (Turbo Spray), Smoothed Max. 2.6e6 cps. +MS2 (264.30) CE (25): Period 1, 16.369 to 16.815 min from Sample 7 (Urine 0-4 h (Pro-1)) of 0911040 4.wiff (Turbo Spray) Max. 5.6e4 cps. 2.6e6 16.55 137.1 hydroxy-α-sanshool 5.5e4 2.4e6 12.09 5.0e4 2.2e6 4.5e4 2.0e6 1.8e6 hydroxy-sanshool isomer 4.0e4 1.6e6 3.5e4 29.87 1.4e6 3.0e4 hydroxy-β-sanshool 28.46 29.57 [M+H]+ 1.2e6 12.48 15.95 28.59 Intensity, cps Intensity, Intensity, cps Intensity, 2.5e4 12.42 16.77 1.0e6 14.98 26.29 27.11 2.0e4 m/z 107 8.0e5 14.82 17.16 m/z 147 1.5e4 246 17.81 25.28 6.0e5 18.99 20.68 21.05 22.72 24.38 107.2 264.1 1.0e4 79.1 4.0e5 147.3 246.2 5000.0 72.3 81.1 91.1 105.1 133.3 59.2 119.2 139.1 2.0e5 93.0 95.1 217.3 70.1 82.9 109.1 122.0 149.3 174.9 205.2 221.1 57.7 116.9 141.1166.0 190.5 177.9 236.1 247.4 0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 23 0 240 250 260 270 280 290 300 Time, min m/z, amu Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition

Supplementary Figure S2-2. Product ion scan chromatograms and product ion spectra of the Urine (0 -4 hr) before and after enzyme treatment (Japanese pepper).;Precursor ion: m/z 440 (hydroxy-ααα-sanshool glucuronide). ,:loss of glucuronic acid (-176)

Urine before enzyme treatment Urine after enzyme treatment

XIC of +MS2 (440.30) CE (25): 263.6 to 264.8 amu fr om Sample 3 (Urine 0-4h (Pro-1-2)) of 09121002.wiff (Turbo Spray), Smoothed Max. 2.0e7 cps. XIC of +MS2 (440.30) CE (25): 264.0 to 265.0 amu fr om Sample 3 (Urine 0-4h-enzyme (Pro-1-2)) of 100108 01.wiff (Turbo Spray) Max. 5.9e4 cps. 11.87 2.0e7 1.9e6 1.9e7 1.8e6 1.8e7 c 1.7e6 1.7e7 b 1.6e6 1.6e7 1.5e6 1.5e7 1.4e6 1.4e7 1.3e6 1.3e7 a 1.2e6 1.2e7 d 1.1e6 1.1e7 1.0e6 1.0e7 9.0e5 Intensity, cps Intensity, Intensity, cps Intensity, 9.0e6 5.81 8.0e5 8.0e6 11.45 7.0e6 7.0e5 6.0e6 6.0e5 5.0e6 5.0e5 4.0e6 4.0e5 3.0e6 e 3.0e5 2.0e5 2.0e6 13.39 1.0e6 1.0e5 6.08 6.59 8.06 5.41 9.25 10.8712.16 14.1413.32 15.41 16.93 17.92 19.47 21.29 22.73 0.0 0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time, min Time, min

a. hydroxy-α-sanshool glucuronide-1 b. hydroxy-α-sanshool glucuronide-2 c. hydroxy-α-sanshool glucuronide-3

+MS2 (440.30) CE (25): 5.680 to 6.048 min from Sam ple 3 (Urine 0-4h (Pro-1-2)) of 09121002.wiff (Turb o Spray) Max. 8.0e5 cps. +MS2 (440.30) CE (25): 11.242 to 11.611 min from S ample 3 (Urine 0-4h (Pro-1-2)) of 09121002.wiff (Tu rbo Spray) Max. 1.2e6 cps. +MS2 (440.30) CE (25): 11.711 to 12.130 min from S ample 3 (Urine 0-4h (Pro-1-2)) of 09121002.wiff (Tu rbo Spray) Max. 2.5e6 cps.

264.2 246.2 246.2 8.0e5 1.20e6 2.5e6 1.15e6 2.4e6 7.5e5 1.10e6 2.3e6 m/z 246 7.0e5 1.05e6 2.2e6 1.00e6 2.1e6 6.5e5 9.50e5 2.0e6 1.9e6 6.0e5 9.00e5 1.8e6 8.50e5 m/z 246 [M+H]+ 5.5e5 1.7e6 8.00e5 1.6e6 5.0e5 7.50e5 264.3 1.5e6 7.00e5 4.5e5 1.4e6 6.50e5 1.3e6 6.00e5 264.3 4.0e5 1.2e6

440 cps Intensity, 5.50e5 Intensity, cps Intensity, [M+H]+ Intensity, cps Intensity, [M+H]+ 1.1e6 3.5e5 m/z 246 5.00e5 1.0e6 3.0e5 4.50e5 9.0e5 4.00e5 8.0e5 2.5e5 3.50e5 7.0e5 147.2 2.0e5 3.00e5 6.0e5 440 440 2.50e5 5.0e5 1.5e5 246.1 2.00e5 4.0e5 440.2 147.2 72.2 1.0e5 1.50e5 3.0e5 1.00e5 72.3 2.0e5 149.2 5.0e4 218.1 107.2 133.1 175.0 151.1 422.2 5.00e4 107.1 133.1 149.3 175.0 1.0e5 81.1 138.1 204.2 360.2 81.0 93.1 422.4 440.3 67.1 93.1 139.0 422.3 440.3 60.4 72.4 85.0 97.2 113.2 159.1 201.3 228.0 247.0 263.2 271.2 306.2 322.1 348.2 386.4 404.2 54.9 67.1 204.3 218.4 255.0 259.3274.1 288.2 306.1 324.1 351.3 360.3 399.1 404.4 55.2 191.2 204.2 230.0 248.1 266.0 288.2306.0 311.2 350.3 386.4368.3 404.2 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 40 0 420 440 460 480 500 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 40 0 420 440 460 480 500 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 40 0 420 440 460 480 500 m/z, amu m/z, amu m/z, amu d. hydroxy-α-sanshool glucuronide-4 e. hydroxy-α-sanshool glucuronide-5

+MS2 (440.30) CE (25): 12.432 to 12.700 min from S ample 3 (Urine 0-4h (Pro-1-2)) of 09121002.wiff (Tu rbo Spray) Max. 2.1e5 cps. +MS2 (440.30) CE (25): 13.269 to 13.604 min from S ample 3 (Urine 0-4h (Pro-1-2)) of 09121002.wiff (Tu rbo Spray) Max. 1.3e5 cps. 246.2 2.1e5 1.29e5 264.2 2.0e5 1.25e5 1.20e5 1.9e5 1.15e5 1.8e5 1.10e5 1.7e5 1.05e5 1.6e5 m/z 246 1.00e5 9.50e4 1.5e5 9.00e4 1.4e5 8.50e4 [M+H]+ 1.3e5 8.00e4 1.2e5 [M+H]+ 7.50e4 m/z 246 1.1e5 7.00e4 6.50e4 1.0e5 6.00e4 Intensity, cps Intensity,

264.2 cps Intensity, 9.0e4 5.50e4 440 8.0e4 5.00e4 7.0e4 440 4.50e4 6.0e4 4.00e4 3.50e4 5.0e4 3.00e4 4.0e4 2.50e4 3.0e4 2.00e4 90.2 147.2 1.50e4 246.1 2.0e4 147.2 107.0 1.00e4 72.1 133.1 175.0 133.2 175.0 386.6 440.2 1.0e4 440.3 72.2 107.0 149.1 247.1 67.3 81.1 119.3 131.2 229.2 360.4 386.4 399.1 422.3 5000.00 81.0 404.3 422.2 98.3 157.2 199.3 225.0 239.0 259.2 285.4 305.9 311.3 340.3 67.2 114.2 170.1 183.2 221.1 230.2 265.2 283.3 306.0 324.3 350.3 360.3 368.3 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 40 0 420 440 460 480 500 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 40 0 420 440 460 480 500 m/z, amu m/z, amu Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition Supplementary Table S1 ．List of DKT ingredients in human plasma and urine detected by LC/MS/MS

Test Plasma Urine Name substance Plasma Gluc. Sulf. Urine Gluc. Sulf. 1 Hydroxy-α-sanshool ＋＋＋－＋＋－ 2 Hydroxy-β-sanshool ＋＋＋－＋＋－ 3 γ-Sanshool ＋＋－－＋－－ 4 [6]-gingerol ＋＋ + － + 5 [6]-shogaol ＋＋ + ＋ + 6 [10]-gingerol ＋＋ nd nd ＋ nd 7 [10]-gingerdion ＋＋ nd nd ＋ nd 8 [10]-shogaol ＋＋ nd － nd 9 [8]-gingerol ＋＋ nd ＋ nd 10 paradol ＋＋＋ 11 [8]-shogaol ＋＋ nd ＋ nd 12 [10]-dehydrogingerdion ＋＋ nd nd ＋ nd nd 13 [6]-gingerol 4’-O-glucronide nd ＋＋ 14 [6]-shogaol 4’-O-glucronide nd ＋＋ 15 [8]-gingerol 4’-O-glucronide nd ＋＋ 16 [8]-shogaol 4’-O-glucronide nd ＋＋

+: detected, nd: not detected, -: not measured, Gluc.: glucuronide, Sulf.:sulfate Profiling of the compounds absorbed in human plasma and urine after oral administration of a traditional Japanese (Kampo) medicine daikenchuto (DKT) . Jun Iwabu et al., Drug Metagolism and Disposition

Supplementary Table S1 ． continued

Test Plasma Urine Name substance Plasma Gluc. Sulf. Urine Gluc. Sulf. 17 [10]-gingerol 4’-O-glucronide nd ＋＋ 18 [10]-shogaol 4’-O-glucronide nd ＋＋ 19 ginsenoside Rg1 ＋ + + 20 ginsenoside Rb1 ＋ + + 21 ginsenoside F2 nd nd nd 22 (20S)-ginsenoside Rh2 ＋ nd nd 23 ginsenoside Re ＋ nd nd 24 ginsenoside Rb2 ＋ + nd 25 ginsenoside Rc ＋ + nd 26 ginsenoside Rd ＋ nd nd 27 ginsenoside Rg3 ＋ nd nd 28 ginsenoside Rf ＋ + + 29 ginsenoside Rg2 ＋ nd + 30 ginsenoside Rh1 ＋ nd + 31 (20R)-ginsenoside Rg3 ＋ + 32 gingerdiol ＋ nd nd nd nd nd nd

Supplementary Table S1 ． continued

Test Plasma Urine Name substance Plasma Gluc. Sulf. Urine Gluc. Sulf. 33 ginsenoside F1 ＋ nd nd nd 34 (20R)-ginsenoside Rh1 ＋ nd nd nd 4-Hydroxy-6-oxo-8-(4-hydroxy-3- 35 nd nd nd nd methoxyphenyl)octanoic acid 36 4-(4-hydroxy-3-methoxyphenyl)butanoic acid nd nd nd nd (E)-8-(4-hydroxy-3-methoxyphenyl)-6-oxooxt- 37 nd nd nd nd 4-enoic acid, 38 dehydroparadol nd nd nd nd 39 (20R)-ginsenoside Rh2 ＋ nd nd nd 40 (20S)-protopanaxatriol nd nd nd nd 41 (20R)-protopanaxatriol nd nd nd nd 42 Compound K nd nd nd nd 43 (20S)-protopanaxadiol nd nd nd nd 44 (20R)-protopanaxadiol nd nd nd nd

+: detected, nd: not detected, -: not measured, Gluc.: glucuronide, Sulf.:sulfate