Abstract
Daikenchuto (DKT), 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 multiherbal medicine consisting of Japanese pepper (zanthoxylum fruit), processed ginger, and ginseng with maltose as an additive. Despite substantial research on the pharmacological activities of DKT and its ingredients, the lack of studies on absorption, distribution, metabolism, and excretion of DKT 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 seven conditions of liquid chromatography and mass spectrometric analysis, which enabled the identification of 44 ingredients of DKT component herbs. We investigated the plasma and urine profiles of these ingredients 0.5 to 8 h after oral administration of 15.0 g of DKT in four healthy volunteers. The results indicated that 1) hydroxy-α-sanshool and [6]-shogaol, the prominent peaks in plasma derived from Japanese pepper and ginger, respectively, were detected at 0.5 h 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 hydroxy-sanshools, shogaols, and gingerols were detected in plasma and urine; and 4) no obvious differences between samples from the two male and the two female individuals were observed. These results provide a strong basis for future studies on pharmacokinetics and pharmacology of DKT.
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 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 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.
Daikenchuto (DKT) is widely prescribed for patients with gastrointestinal obstruction such as postoperative ileus, 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 United States 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 patients with Parkinson's disease (Sakakibara et al., 2005) have also been investigated.
DKT consists of processed ginger rhizome, ginseng, Japanese pepper (zanthoxylum fruit), 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 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 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). Furthermore, several pharmacological effects of DKT ingredients have 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). Hydroxy-α-sanshool (HAS) has recently been reported to be an inhibitory ligand for two-pore domain potassium channels (KCNK3 and others), which may regulate the excitability of enteric neurons (Bautista et al., 2008). Despite 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 the efficacy and pharmacology of DKT.
ADME studies of Kampo medicines are extraordinarily challenging. In general, like DKT, multiple constituents are contained in a single Kampo formulation, and the amount of each constituent is minute. Furthermore, most Kampo medicines are administered orally and 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 and Kitajima, 2009). For these reasons, there have been only a few reports on ADME of Kampo medicines.
However, recent advancements in mass spectrometry technology have opened the way to profiling and elucidating trace amounts of ingredients contained in these complex 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, has been reported to our knowledge. Administration of polyherbal compounds may result in an ADME profile different from that for 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 hydroxy-β-sanshool (HBS), by use of liquid chromatography-tandem mass spectrometry (LC-MS/MS). These 44 constituents were subsequently investigated in human plasma and urine from four healthy volunteers after oral administration of 15.0 g of DKT. Furthermore, the conjugated forms of DKT metabolites were analyzed by digesting plasma samples with enzymatic hydrolysis.
Materials and Methods
Chemicals and Reagents.
Tsumura Daikenchuto Extract Granules was manufactured by Tsumura & Co. (Ami, Ibaraki, Japan). 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. 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, MN). Water was purified using a pure water supply system (Milli-Q; Nihon Millipore Ltd., Tokyo, Japan). An Oasis HLB μElution Plate (Nihon Waters K.K., Tokyo, Japan) was used as the solid-phase extraction (SPE) plate. β-Glucuronidase (EC 3.2.1.31) was obtained from 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-glucuronide, [6]-shogaol 4′-O-glucuronide, [8]-gingerol 4′-O-glucuronide, [8]-shogaol 4′-O-glucuronide, [10]-gingerol 4′-O-glucuronide, [10]-shogaol 4′-O-glucuronide, 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, 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, and (20R)-protopanaxadiol. All of these authentic standards were reliably identified with spectral methods such as NMR, mass, and IR spectrometry. The structural formulas of the authentic standards are provided in Supplemental Fig. S1.
Clinical Trial Design.
This study was conducted in accordance with the ethical principles of the Declaration of Helsinki, and consistent with Good Clinical Practice guidelines. The study was approved by an independent ethics committee at the Kochi Medical School before recruitment commenced. Before initiation of study procedures, all volunteers gave their written informed consent for participation in the study.
Four healthy volunteers (two males and two females) aged 21 to 32 (25.0 ± 4.2) years, with a body mass index of 18.5 to 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 Granules and fasted for 12 h before and 4 h after administration. Each participant then consumed a standardized meal. In addition, the participants refrained from Japanese pepper-, ginseng-, and ginger-containing foods for 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 h after administration and were immediately centrifuged (3000 rpm, 15 min). Urine samples (50 ml each) were collected the day before the administration and at 0 to 4 h and 4 to 8 h 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 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 (10 μg/ml) was diluted with acetonitrile-water (1:1, v/v) to prepare standard working solutions (1, 2, 5, 10, 50, 100, and 1000 ng/ml). The standard solutions were stored at −80°C in an airtight container under light-resistant conditions in a freezer.
Pretreatment 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 3000 rpm for 5 min at 4°C (refrigerated centrifuge 5910; Kubota Corporation, Osaka, Japan) 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.
Pretreatment Procedure for Plasma and Urine.
Solid-phase extraction was adopted as the extraction procedure. Hydrochloric acid was added to 300 μl of the plasma, urine sample, or calibration sample to adjust the pH to approximately 3.0. The resulting mixture (200 μl) was loaded on an SPE plate preconditioned with 200 μl of acetonitrile and 200 μl of water. 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 h. Subsequently, a sample for LC-MS/MS analysis was prepared as described under Pretreatment Procedure for Plasma and Urine.
LC-MS/MS Analysis.
LC-MS/MS analyses were conducted using the API 5000 system (AB SCIEX, Tokyo, Japan) equipped with a 1200 series HPLC system (Agilent Technologies, Inc., Santa Clara, CA), and Analyst Software (version 1.4.2; AB SCIEX). The column for LC-MS/MS analyses was a YMC-Pack ODA-AQ (3 μm, 150 × 2.0 mm i.d.; 3 μm, 50 × 2.0 mm i.d.; YMC Co., Ltd., Kyoto, Japan). The other conditions were as follows: flow rate, 0.2 ml/min; column temperature, 40°C; injection volume, 10 μl; and ionization, electrospray ionization 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 Concentrations of Four Active Constituents of DKT.
Four constituents, HAS, 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, and 10 ng/ml). The calibration curve for the analytes was constructed from the peak areas against the concentrations ranging from 0.1 to 10 ng/ml. The pretreatment recovery rate was calculated by comparing the peak area in the standard solution with that in the samples for the calibration curve after the pretreatment procedure as described under Pretreatment Procedure for Plasma and Urine.
Stability of the stock solution, extracted sample in the autosampler, and storage and freeze/thaw cycles was determined. The quality control samples were prepared by diluting the standard working solution with blank plasma. The performance of the analytical methods for plasma samples is summarized in Table 5. Quality control samples for urine samples were not prepared in this study.
Pharmacokinetics.
Pharmacokinetic parameters were estimated using WinNonlin (version 5.2; Pharsight Corporation, Mountain View, CA). Maximum concentration and time to maximum concentration after drug administration (tmax) were the experimentally observed values. The area under the plasma concentration-time curve from zero to time t (AUC0–t) was calculated from time 0 to last detected time. Apparent elimination half-life (t1/2) was calculated divided by loge2/ke where ke is the terminal elimination rate constant. Calculated parameters are presented as means ± S.D.
Results
The structural formulas of the major DKT ingredients and their metabolites are shown in Fig. 1. All of the 44 authentic standards used in the present study are provided in Supplemental Fig. S1.
Analysis of the Ingredients Derived from Japanese Pepper.
HAS and HBS were detected in the test substances and in plasma and urine samples, and γ-sanshool was detected in the test substances and plasma samples (Fig. 2). The product ions of those peaks were coincident with those of the authentic standards (Supplemental Fig. S2-1). There was an unknown peak (retention time 16 min) whose product ions corresponded with those of HAS and HBS. All of the peaks were detected most prominently at 0.5 h postdose and thereafter decreased throughout the sampling period.
Product ion scanning (fixed Q1 to m/z 440, which corresponded to the [M + H]+ of glucuronide conjugates of hydroxy-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 (Supplemental Fig. 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 after β-glucuronidase treatment, there was no peak for hydroxy-sanshool glucuronide, whereas that for 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 hydroxy-α-sanshool and its glucuronide conjugates are shown in Supplemental Figure S2. No obvious differences between samples from the two male and the two 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 detected in the plasma or urine by any of the above analyses.
The MRM chromatograms of plasma detected trace 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 (Fig. 3). Likewise, the MRM scan of urine detected peaks of [6]-shogaol, [8]-shogaol, [8]-gingerol, [10]-gingerol, [10]-gingerdione, [10]-dehydrogingerdione, and [6]-paradol (Fig. 3). The MRM chromatograms also detected 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 (Fig. 3). All of the peaks were detected most prominently at 0.5 h postdose, 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 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 was 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), [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 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 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 the 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 that were coincident with the peaks of ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rb2, ginsenoside Rc, 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 (Fig. 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 two male and the two female individuals were observed. A summary of detected ingredients and their glucuronide conjugates is found in Supplemental Table S1.
Plasma Concentrations of Four Active Constituents of DKT.
Plasma concentrations of HAS, HBS, [6]-shogaol, and [10]-shogaol, which have known pharmacological activities and were detected with 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 h and was eliminated with t1/2 of 1.27 ± 0.61 h. The area under the curve from time zero to the last detection time (AUC0–8) was 606 ± 280 h · ng/ml. HBS reached a maximum concentration (64.1 ± 12.4 ng/ml) at the time of 0.625 ± 0.217 h and was eliminated with t1/2 of 1.49 ± 0.33 h. The AUC0–8 was 167 ± 70.3 h · ng/ml.
[6]-Shogaol reached a maximum concentration (0.098 ± 0.030 ng/ml) at the time of 0.5 h. The AUC0–4 was 0.353 ± 0.174 h · ng/ml. The 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 h. AUC0–2 was 0.505 ± 0.245 h · ng/ml. The elimination half-life was not calculated because the time points were insufficient.
Discussion
DKT is a mixture of herbs that 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 meal during blood and urine sampling.
To detect as many compounds as possible, 15 g of DKT was administered as a single dose instead of the usual three times daily regimen. Off-label use of a single dose of 15 g 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.
Because DKT contains a large number but small amount of chemical compounds, a combination of a 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 that were selected on the basis of an extensive literature review of bioactive components of DKT. Three chromatographic conditions were needed to cover all of the authentic standards.
Although 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 showed that the administration of 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 antiadhesion 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 that was antagonized by capsazepine, a TRPV1 inhibitor (Satoh et al., 2001a). HAS has been reported to have receptor agonist activity to TRPV1- and TRPA1-transfected HEK293 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 KCNKs expressing enteric neurons 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 in which a 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 et al. (2008) reported that no free [6]-gingerol, [8]-gingerol, [10]-gingerol, or [6]-shogaol was found, but instead their conjugates were detected in human plasma after oral dosing of ginger extract capsules. The maximum dosage given to a human in the study of Zick et al. (2008) 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. Differences in race or diet may affect the bioavailability of these compounds, and the various methods for extraction, separation, or detection may yield different results. It is also possible that the coexistence 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 membrane of mucosal cells of the gastrointestinal tract. Incorporation of lipidophilic compounds into the micelle may enhance the bioavailability of ingredients of ginger and ginseng (Xiong et al., 2008). The saponin effect of ginseng 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 in the range of 0.1 to 100 μM (see references in Table 1). Although the quantitative method of the present study has not been validated and therefore gives only an approximation, the peak concentration of HAS (215 ng/ml = 0.81 μM) may be enough to exert some of its pharmacological activities. Although the blood concentrations of shogaols, gingerols, and ginsenoside Rb1 found in the present study were extremely low, it must be noted that their peak concentrations were missed with the sampling protocol used in the study. Furthermore, 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 bloodstream (Jin et al., 2001; Kawasaki et al., 2007). Extensive ADME studies including analysis of tissue distribution of DKT ingredients are necessary to clarify this point.
In summary, the major representative ingredients of DKT, which have been reported to have various pharmacological effects relevant to its clinical efficacy, have been detected in human plasma and urine. The data presented in this article provide the basis for pharmacokinetic and pharmacodynamic research on DKT for assessing and clarifying its diverse bioactivities. Subsequent pharmacokinetic studies that focus on validating the quantity of selected ingredients are currently in progress.
Acknowledgments.
We thank Dr. Ken-ichiro Hayashi (Drug Development Service Segment, Mitsubishi Chemical Medience Corporation, Kumamoto, Japan) for the analysis of DKT constituents.
Footnotes
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033589.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- DKT
- daikenchuto
- TRP
- transient receptor potential
- HAS
- hydroxy-α-sanshool
- ADME
- absorption, distribution, metabolism, and excretion
- HBS
- hydroxy-β-sanshool
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- HPLC
- high-performance liquid chromatography
- SPE
- solid-phase extraction
- MRM
- multiple reaction monitoring.
- Received April 6, 2010.
- Accepted August 5, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics