Visual Overview
Abstract
Literature reports that Poria cocos reduces blood lipid levels; however, the underlying mechanism remains unclear. Blood lipid levels are closely related to the enterohepatic circulation of bile acids, where uptake transporters playing a significant role. P. cocos extract is commonly used in traditional prescriptions and food supplements in China. We investigated the effects of P. cocos and its five triterpene acids on bile acid uptake transporters, including intestinal apical sodium-dependent bile acid transporter (ASBT) and hepatic sodium/taurocholate cotransporting polypeptide (NTCP). Triterpene acids were fingerprinted by high-performance liquid chromatography–TripleTOF and quantified by ultraperformance liquid chromatography/tandem mass spectrometry. The inhibitory effect of P. cocos and its five major representative triterpene acids on ASBT and NTCP was investigated by in vitro assays using Xenopus oocytes expressing ASBT and NTCP. P. cocos extract exhibited significant inhibitory effects with half-maximum inhibition constants of 5.89 µg/ml and 14.6 µg/ml for NTCP and ASBT, respectively. Among five triterpene acids, poricoic acid A, poricoic acid B, and polyporenic acid C significantly inhibited NTCP function. Poricoic acid A, poricoic acid B, and dehydrotumulosic acid significantly inhibited ASBT function. The representative triterpene acid, poricoic acid A, was identified as a competitive inhibitor of NTCP with an inhibitory constant of 63.4 ± 18.7 µM. In conclusion, our results indicate that both P. cocos extract and its major triterpenes are competitive inhibitors of ASBT and NTCP. Accordingly, it was suggested that competitive inhibition of these bile acid transporters is one of the underlying mechanisms for the hypolipidemic effect of P. cocos.
SIGNIFICANCE STATEMENT Poria cocos, a commonly used Chinese herbal medicine and food supplement, demonstrates significantly inhibitory effects on the function of apical sodium-dependent bile acid transporter and sodium/taurocholate cotransporting polypeptide. P. cocos has potential to reduce the blood lipid through inhibition of these uptake transporters in enterohepatic circulation of bile acid.
Introduction
Natural medicines and food supplements have been safely and effectively used to treat various metabolic syndromes. Poria cocos is the dried sclerotium of the fungus Poria cocos (Schw.) Wolf (Polyporaceae), known as Fuling in Chinese, is widely used both as a food supplement and traditional Chinese medicine (Wang et al., 2013). As a health-promoting food, P. cocos exerts beneficial effects on food absorption and metabolism. In the Chinese pharmacopoeia, approximately 15% of traditional Chinese prescriptions contain P. cocos (Nie et al., 2020). For example, P. cocos is present in many ancient Chinese prescriptions, including Si-jun-zi decoction and Lin-gui-zhu-gan decoction, used for treating hyperlipidemia (Wu et al., 2009; Liu et al., 2013). P. cocos has also been shown to reduce blood lipid levels, including triglycerides and cholesterol, in both rats and humans (Miao et al., 2016; Mendes, 2018). The major chemical constituents of P. cocos are triterpenes and polysaccharides (Ríos, 2011). Although several studies have investigated the effects of polysaccharides in alleviating hyperlipidemia (Wu et al., 2019), the mechanism of triterpene acids in reducing blood lipids is not yet known.
Bile acids are physiologic detergents that solubilize lipids in the intestinal tract, thereby promoting fat digestion and absorption in the small intestine. The synthesis of bile acids in the liver accounts for a major fraction of daily cholesterol turnover in humans (Chiang, 2013). Hepatic cholesterol is converted to bile acids by oxidation and conjugation reactions in the liver and intestine by several enzymes. Nearly 95% of the bile acid secreted is reabsorbed at the terminal ileum and is recycled via the enterohepatic circulation. Approximately 50% of cholesterol is eliminated from the body by this pathway (Li and Chiang, 2014). Therefore, fecal excretion of bile acids and reduced return to the liver may result in the accumulation of cholesterol in the liver. Both apical sodium-dependent bile acid transporter (ASBT; SLC10A2) and sodium/taurocholate cotransporting polypeptide (NTCP; SLC10A1) located on the luminal surface of ileal enterocytes and the basolateral membrane of the hepatocytes, respectively, are involved in the reuse of bile acids by contributing to the enterohepatic circulation of bile acids and cholesterol homeostasis. Moreover, inhibition of NTCP and ASBT has been reported to alleviate obesity and hypercholesterolemia (Xu et al., 2000; Claro da Silva et al., 2013; Rao et al., 2016; Donkers et al., 2019). Therefore, P. cocos possibly exerts its lipid-lowering effect via inhibition of bile acid uptake transporters in the enterohepatic circulation to decrease the return of bile acids to the liver, thereby increasing the conversion of cholesterol to bile acids.
Because NTCP and ASBT regulate the blood lipid levels, it is hypothesized that P. cocos affects their functions in reducing blood lipid levels. Thus, we studied the effect of P. cocos extract and its major triterpene acids (Fig. 1) on bile acid uptake transporters, ASBT and NTCP.
Materials and Methods
Materials and Reagents.
N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethanesulfonate (tauro-nor-THCA-24-DBD) was purchased from Geno Membrane, Inc. (Kanagawa, Japan), and taurocholic acid sodium salt (TCA) was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). [3H] Taurocholic acid (specific activity 20 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Five triterpene acids, including poricoic acid A, poricoic acid B, polyporenic acid C, pachymic acid, and dehydrotumulosic acid, and P. cocos extract (alcohol extract) were purchased from Nanjing Spring and Autumn Biologic Engineering Co., Ltd. (Nanjing, China). Plasmid pcDNA3.1(+)/NTCP and pGEMHE/ASBT were constructed in our laboratory. Chlorzoxazone (used as internal standard) was obtained from Dalian Meilun Biotechnology Co. Ltd., China. Methanol and acetonitrile of chromatographic grade were obtained from Fujifilm-Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Ammonium acetate was procured from Sigma-Aldrich Chemie GmbH Fluka (Netherlands). Female Xenopus laevis frogs were purchased from Kato-S-Science (Chiba, Japan), and all the studies were approved by the Ethics Committee for Animal Experimentation of Kanazawa University (Kanazawa, Japan).
Identification of Triterpenes in P. cocos by High-Performance Liquid Chromatography–TripleTOF.
Triterpene acids were identified by using a high-performance liquid chromatography (HPLC)–TripleTOF system. Five microliter of the extract (100 µg/ml) was injected into the HPLC system (LC-20A; Shimadzu Corporation, Kyoto, Japan) and separated by a reverse phase C18 column (150 × 2.0 mm × 2.1 mm, VP-ODS; Shimadzu) at a flow rate of 0.4 ml/min for 30 minutes. Formic acid (0.1%) (A) and acetonitrile (B) were used as mobile phases. The gradient used was as follows: 0–2 minutes, 20% B; 2–20 minutes, 90% B; 20–24 minutes, 90%–95% B; 27–30 minutes, 95%–5% B; 30 minutes, stop.
The mass spectroscopy data were recorded by a TripleTOF 5600+ system (SCIEX, CA). The mass spectrometer was equipped with an electron spray ionization source in negative ion mode with a declustering potential of –80.0 V and collision energy of –35.0 eV. The TripleTOF 5600+ system was operated in the information-dependent acquisition mode consisting of a time-of-flight (TOF)/mass spectrometry scan type with an accumulation time of 0.05 second. The TOF masses ranged from 50 to 1000 Da. A curtain gas of 35 psi, ion source gas 1 of 55 psi, and ion source gas 2 of 55 psi were optimized. The ion spray voltage floating was set to 4500 V, and the heater interface temperature was 550°C.
Simultaneous Determination of Five Triterpene Acids in P. cocos Extract by Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry.
The content of five triterpene acids in P. cocos extract was quantified by an ultraperformance liquid chromatography (UPLC)–tandem mass spectrometry (MS/MS) system. The UPLC-MS/MS system consisted of a Shimadzu UPLC system (LC-30A; Shimadzu Corporation) and an AB SCIEX mass spectrometer 4000 QTRAP system (AB Sciex Pte. Ltd., Framingham). Analytes were separated using a C18 column (150 × 2.0 mm, 2.1 µm, VP-ODS; Shimadzu) at 40°C with a flow rate of 0.4 ml/min. The mobile phase consisted of 0.1% formic acid and 0.5 mM ammonium acetate aqueous solution (A) and acetonitrile (B). The gradient elution condition was: 0–6 minutes, 40%–90% B; 6–9.2 minutes, 90% B; 9.2–9.5 minutes, 90%–40% B; 9.5–10 minutes, 40% B. The injection volume was 2 µl. The mode of the mass spectrum was multiple reaction monitoring model for the detection, and related mass parameters are shown in Table 1. Five triterpenes were identified and qualified according to the retention time and molecular ion peaks of standard substances.
During sample preparation, original stock solutions (5 mg/ml) of poricoic acid A, poricoic acid B, polyporenic acid C, pachymic acid, and dehydrotumulosic acid were dissolved in DMSO. The working standard solutions of five analytes at concentrations of 1000, 500, 200, 100, 50, 20, and 10 ng/ml were prepared by diluting the stock solutions with acetonitrile. The chlorzoxazone working solution, used as the internal standard (IS), was prepared using acetonitrile at a final concentration of 5 µg/ml. Working standard solution (50 µl) and 50 µl of IS working solution were added to an eppendorf tube and mixed to get calibration curve samples. The stock solution (5 mg/ml) of P. cocos extract was prepared and diluted to 5 µg/ml by acetonitrile. Next, 50 µl of extract samples were mixed with 50 µl IS working solution. After the mixtures were vortexed for 5 minutes, four parallel samples were withdrawn for UPLC-MS/MS analysis.
Uptake Assays in X. laevis Oocytes.
Human NTCP and ASBT plasmid DNAs cloned in pcDNA3.1(+) and pGEMHE vectors were linearized by SmaI (New England Biolabs Inc, Ipswich, UK) and NheI (Takara Bio Inc., Ostu, Japan), respectively. Then, complementary RNAs of NTCP and ASBT were synthesized for injecting into oocytes by in vitro transcription method using T7 mMESSAGE-mMACHINE kit (Ambion, Austin, TX).
Female X. laevis frogs were anesthetized in a mixture of ice and water for 30 minutes. The oocytes were dissected from ovaries and extracted from X. laevis (15–20 minutes in the collagenase solution, 1 mg/ml). After digestion by collagenase solution, oocytes were washed in the oocyte Ringer 2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2·6H2O and 5 mM HEPES, pH 7.4) for 10 times and in modified Barth’s saline (MBS; 88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4, 2.4 mM NaHCO3, and 10 mM HEPES, pH 7.4) for five times, respectively. Oocytes were defolliculated one by one under the microscope. After 24-hour culture in the MBS solution with 50 mg/l gentamicin at 18°C, defolliculated oocytes between stage IV and VI were microinjected with 50 nl complementary RNA solution (500 ng/μl) or pure water as control. After 48-hour incubation in the MBS solution, oocytes were used in the uptake assays.
Tauro-nor-THCA-24-DBD (10 µM), a fluorescent derivative of bile acid, was used as the probe substrate of NTCP and ASBT (Yamaguchi et al., 2010; De Bruyn et al., 2014; Zhu et al., 2021) in the uptake studies. All oocytes were preincubated in the uptake buffer (MBS) for 5 minutes at 25°C. Oocytes injected with pure water were used as a reference. Uptake assays were performed at 25°C for 45 minutes (ASBT) and 30 minutes (NTCP) in the presence and absence of test compounds, including P. cocos extract and five triterpenes. TCA (25 µM) was used as a positive control. All experiments were terminated by removing the uptake buffer. The oocytes were washed thrice with ice-cold uptake buffer. The whole process is shown in Fig. 2. In the uptake assays of [3H] taurocholic acid (0.125 μCi/ml), the concentration of substrate was 5 µM, and the uptake time was 10 minutes.
Detection of Substrate Tauro-nor-THCA-24-DBD.
For sample preparation, all oocytes were disrupted in ice-cold methanol (500 µl). A 450-µl aliquot of the supernatant of each sample was withdrawn, vortexed for 5 minutes, and centrifuged at 15,000 rpm for 15 minutes. The resultant samples were volatilized for 3 hours to dryness, and each sample was dissolved in 100 µl of mobile phase for measurement. Samples in the uptake assays were quantified by an HPLC system (Jasco 2057 Plus, Japan) with a fluorescence detector (Jasco FP2020 Plus, Japan). Tauro-nor-THCA-24-DBD analysis was performed using a reverse phase Mightysil RP-18 GP column (4.6 mm × 250 mm, 5 µm). The flow rate was 1 ml/min, and the eluent mobile phase consisted of 45% 10 mM ammonium acetate and 55% acetonitrile. The injection volume of samples was 50 µl. The excitation and emission wavelengths for detection were set at 454 nm and 570 nm, respectively.
Inhibition Kinetics Assays.
Poricoic acid A was used as the representative inhibitor of NTCP in assays for assessing the inhibition kinetics. The experiments were conducted using a series of substrate concentrations (5, 10, 20, 40, and 80 µM) in the presence and absence of poricoic acid A (5, 25, and 75 µM). Samples were prepared by the method described above.
Data Analysis.
Inhibition kinetic parameters were estimated using nonlinear least-squares analysis using GraphPad Prism 7.0 (GraphPad Software Inc.). The IC50 and the inhibition ratio (the percent inhibition of the ASBT and NTCP uptake activity) was calculated using the following equations:where [I] is inhibitor concentration.where (accumulation)Transporter,I and (accumulation)Control,I represent tauro-nor-THCA-24-DBD accumulation (pmol/oocyte) in ASBT/NTCP-expressing or control oocytes, respectively at a given inhibitor concentration (I). The terms (accumulation)Transporter,0 and (accumulation)Control,0 represent tauro-nor-THCA-24-DBD accumulation (pmol/oocyte) in ASBT/NTCP-expressing or control oocytes in the absence of inhibitors, respectively.
The type of inhibition and inhibition constant (Ki) for NTCP-mediated tauro-nor-THCA-24-DBD transport was determined by fitting competitive, noncompetitive, and uncompetitive models to the untransformed data by nonlinear regression analysis using GraphPad Prism 7.0. The best-fit model was assessed by R square of goodness of fit. Equation used for each inhibition model were as follows:where S represents the concentration of tauro-nor-THCA-24-DBD, I represents the concentration of poricoic acid A, v represents the rate of tauro-nor-THCA-24-DBD transport, Vmax represents the maximal rate of tauro-nor-THCA-24-DBD transport, Km represents the tauro-nor-THCA-24-DBD concentration that yields one-half maximal velocity of tauro-nor-THCA-24-DBD transport, and Ki represents the inhibition constant. All experiments were conducted at least three times, and results are expressed as means ± S.E.M.
Results
Fingerprints of Triterpene Acids in P. cocos by HPLC-TripleTOF.
According to the precise molecular mass and fragmentation information from previous reports (Zhao et al., 2013; Wu et al., 2016; Qian et al., 2018; Zou et al., 2019), eight triterpene acids in the P. cocos extract were identified (Fig. 3). These were divided into lanostane and 3, 4-secolanostane skeletons (Ríos, 2011). The closed structure of lanostane imparts stability to the parent nucleus tetracyclic triterpenes in triterpene acids. Therefore, most of the quasimolecular ion peaks were selected both as precursor ions and fragment ions. 3,4-Secolanostane skeletons can be identified more precisely because the opening ring in them produces fragment ions. The possible chemical structures of the identified triterpene acids are shown in Fig. 3, and the chromatographic and mass spectral data of eight analyzed compounds are listed in Table 2. Five representative commercially available triterpenes included poricoic acid A, poricoic acid B, polyporenic acid C, pachymic acid, and dehydrotumulosic acid, with retention times of 15.93, 15.11, 16.71, 19.50, and 15.57 minutes, respectively.
Simultaneous Determination of Five Triterpene Acids in P. cocos Extract by UPLC-MS/MS.
Five triterpene acids in P. cocos extract were determined simultaneously. A series of standard mixture solutions of these five triterpene acids was used to determine the linearity between the standard mixture concentration and peak areas. The concentration ranges of five triterpene acids in the calibration curves are all from 10 to 1000 ng/ml. The regression equations of the standard calibration curve for five triterpene acids were y = 0.00148x + 0.000201 (poricoic acid A, R2 = 0.995), y = 0.00173x + 0.00246 (poricoic acid B, R2 = 0.996), y = 0.00267x − 0.00195 (dehydrotumulosic acid, R2 = 0.994), y = 0.00246x + 0.00315 (polyporenic acid C, R2 = 0.995), and y = 0.00361x + 0.00522 (pachymic acid, R2 = 0.994), respectively. In the content analysis, five triterpene acids occupied 26.08% of the P. cocos extract. Among the five triterpene acids in the P. cocos extract, poricoic acid A had the highest content, accounting for 18.03% of P. cocos extract (Table 3).
In Vitro Transporter Inhibition Study Using X. laevis Oocytes.
The inhibitory effect of five triterpene acids and P. cocos extract on ASBT- and NTCP-mediated tauro-nor-THCA-24-DBD uptake was assessed using X. laevis oocytes expressing ASBT and NTCP, respectively. Tauro-nor-THCA-24-DBD is a substrate of ASBT and NTCP (Yamaguchi et al., 2010; De Bruyn et al., 2014; Zhu et al., 2021). Oocytes expressing ASBT and NTCP exhibited a higher uptake of tauro-nor-THCA-24-DBD compared with those injected with water as a reference. Oocytes expressing ASBT were treated with five triterpene acids with different concentrations of 1, 5, 10, 50, and 100 µM and P. cocos extract with concentrations of 1, 5, 10, 50, and 100 µg/ml. For NTCP, 1, 5, 10, 50, and 100 µM of five triterpene acids and 0.1, 5, 10, 50, and 100 µg/ml P. cocos extract were used as inhibitors.
To verify the reliability of the uptake experimental model, 25 µM TCA was used as a positive control inhibitor. The inhibition ratio for TCA was 34.3% in the ASBT-mediated uptake assay and 49.3% in the NTCP-mediated uptake assay. The results of inhibition study by P. cocos are shown in Figs. 4 and 5, and the IC50 values are summarized in Table 4 (the noncorrected data are shown in Supplemental Figs. 1 and 2). P. cocos extract, poricoic acid A, poricoic acid B, and dehydrotumulosic acid inhibited ASBT with IC50 values of 14.6 ± 1.42 µg/ml, 39.7 ± 1.63, 67.3 ± 1.28, and 38.7 ± 1.32 µM concentrations. Pachymic acid did not inhibit ASBT up to 100 µM. Polyporenic acid C inhibited ASBT; however, its IC50 value was higher than 100 µM (Fig. 4D). Regarding NTCP, IC50 values of P. cocos extract, poricoic acid A, poricoic acid B, and polyporenic acid C were 5.89 ± 1.48 µg/ml, 14.3 ± 1.47, 31.1 ± 1.16, and 38.6 ± 1.47, whereas IC50 values of dehydrotumulosic acid and pachymic acid were higher than 100 µM (Fig. 5). Poricoic acid A strongly inhibited both ASBT and NTCP.
Inhibition Kinetics Ki of Poricoic Acid A on NTCP.
Poricoic acid A, as a representative inhibitor, was used at varying concentrations of 0, 5, 25, and 75 µM to study inhibition kinetics because of its strongest inhibitory effect on NTCP-mediated tauro-nor-THCA-24-DBD. The uptake of tauro-nor-THCA-24-DBD in the presence of poricoic acid A was analyzed by competitive, noncompetitive, and uncompetitive models in the nonlinear regression. The best-fit model was assessed by R square of goodness of fit. The R square in the competitive, noncompetitive, and uncompetitive model is 94.6, 93.5 and 92.3, respectively. Therefore, the best fitting is the competitive model. The uptake of tauro-nor-THCA-24-DBD in the presence of poricoic acid A by fitting the competitive model is shown in Fig. 6. The result demonstrates that poricoic acid A competitively inhibits NTCP. Estimated inhibition constant Ki value of poricoic acid A on NTCP was 63.4 ± 18.7 µM.
Discussion
Hyperlipidemia is an emerging health problem worldwide that increases the risk of more serious diseases such as atherosclerosis and coronary heart disease (Sorokin et al., 2007). Therefore, regulating the levels of cholesterol and triglycerides by controlling the synthesis and transport of bile acids is crucial. ASBT and NTCP are uptake transporters responsible for enterohepatic circulation of bile acid. Accordingly, we investigated the mechanism underlying the lipid-lowering effects of P. cocos extract and its five major triterpene acids on ASBT and NTCP.
The extract of P. cocos has complex components. The raw material and extraction methods could result in variations in their composition. Although our results are specific to one alcohol extract of P. cocos obtained commercially, five triterpene acids with the range of concentrations tested in the uptake assays encompass the possible variable concentration levels in the different ethanol-extractable P. cocos, including this specific extract and other similarly prepared extracts.
In the concentration-dependent uptake of the probe substrate, tauro-nor-THCA-24-DBD, the evaluated Km for NTCP was 22.1 ± 2.4 μM (Supplemental Fig. 3). It is close to the Km (10.5 ± 2.9 μM) of utilizing TCA as the substrate (Masuda et al., 2014). For NTCP, the affinity of tauro-nor-THCA-24-DBD is considerably close to the affinity of taurocholate. Km of TCA and tauro-nor-THCA-24-DBD for ASBT is 66.0 ± 10.6 μM and 40.6 ± 14.0 μM, respectively (Zhu et al., 2021). The Michaelis constants of two substrates for both NTCP and ASBT are close. The inhibitory effect on poricoic acid A was compared by using TCA and tauro-nor-THCA-24-DBD as the substrates in vitro. The IC50 values are 10.85 ± 1.04 and 14.3 ± 1.47 µM, respectively. It had low propensity to generate the different values (Supplemental Fig. 4). Therefore, uptake assays using tauro-nor-THCA-24-DBD as an in vitro probe substrate to determine IC50 values are feasible.
Poricoic acid A exhibited the strongest inhibitory effect on both NTCP- and ASBT-mediated uptake among five triterpene acids. The inhibitory effect could be attributed to the structural similarity between triterpene acids and bile acids. Results of kinetic assays confirmed poricoic acid A as a competitive inhibitor of bile acids during NTCP-mediated uptake. The observed IC50 for P. cocos extract in this study converted to the concentration of single compound according to their contents in P. cocos extract are all lower than 10 μM. These concentrations are all lower than IC50 values observed when assessing a single compound. Thus, other compounds in the P. cocos extract could contribute to the inhibition of ASBT and NTCP, or only a mixture including different triterpene acids such as the P. cocos extract mentioned above could inhibit ASBT and NTCP.
The IC50 values of uptake transporters of ASBT and NTCP in the enterohepatic circulation indicated a possibility that increased bile acid excretion regulated in vivo bile acid synthesis. The recommended dose of P. cocos extract ranges from 10 to 30 mg as a food supplement (data provided by the manufacturer). The concentration of extract in the intestine can be up to 120 µg/ml (30 mg/250 ml water), which is eight times higher than the IC50 in ASBT. However, concentrations of these components in the blood are still unknown because of the complexity of traditional Chinese medicine and lack of corresponding pharmacokinetic data. Therefore, more pharmacokinetic studies are required to obtain clinical relevance.
The currently available lipid-lowering drugs in the market, such as bile acid sequestrants as the combined medication of statins, reduce the reabsorption of bile salts. However, their therapeutic effects are associated with adverse reactions (McCrindle, 2003). Moreover, difficulties in their preparation and administration prevent their use as first-line drugs. Compared with these clinically available drugs, P. cocos, as a food supplement, can be used in daily life and has been proved to treat obesity (Mendes, 2018). Therefore, P. cocos extract and its triterpene acids represent potential therapeutics for hyperlipidemia. The mechanism should be further proved in the animal assessment level in the future.
Conclusion
In the present study, five representative triterpene acids in the alcohol extract of P. cocos were identified and quantified. Both P. cocos extract and its main triterpene acids displayed an inhibitory effect on the functions of ASBT and NTCP in the enterohepatic circulation of bile acids, thus increasing the conversion of blood cholesterol to bile acids. This could be one of the mechanisms responsible for lowered blood lipid levels observed in vivo after administration of P. cocos.
Authorship Contributions
Participated in research design: Chen, Tamai, Lu.
Conducted experiments: Cai, Zhu.
Performed data analysis: Cai, Cheng.
Wrote or contributed to the writing of the manuscript: Cai, Kong, Tamai, Lu.
Footnotes
- Received November 10, 2020.
- Accepted February 18, 2021.
This work was supported by National New Drug Innovation Program of China [No. 2017ZX09301004], China Pharmaceutical University Exploration World Program Student Scholarship, Grant-in-Aid for Scientific Research (B) [16H05111], and Grant-in-Aid for Challenging Exploratory Research [20K21474] from the Japan Society for the Promotion of Science (JSPS).
↵This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- ASBT
- apical sodium-dependent bile acid transporter
- HPLC
- high-performance liquid chromatography
- IS
- internal standard
- MBS
- modified Barth’s saline
- MS/MS
- tandem mass spectrometry
- NTCP
- sodium/taurocholate cotransporting polypeptide
- tauro-nor-THCA-24-DBD
- N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethanesulfonate
- TCA
- taurocholic acid sodium salt
- TOF
- time of flight
- UPLC
- ultraperformance liquid chromatography
- Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics