Inhibition of Human UGT1A1-Mediated Bilirubin Glucuronidation by

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Inhibition of Human UGT1A1-Mediated Bilirubin Glucuronidation by Polyphenolic Acids Impact Safety of Popular Salvianolic Acid A/B-Containing Drugs and Herbal Products Guo Ma, Ying Zhang, Wenyan Chen, Zhifang Tang, Xiaoming Xin, Ping Yang, Xiaoqin Liu, Weimin Cai, and Ming Hu Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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Molecular Pharmaceutics

Inhibition of Human UGT1A1-Mediated Bilirubin Glucuronidation by Polyphenolic Acids Impact Safety of Popular Salvianolic Acid A/B-Containing Drugs and Herbal Products

Guo Ma†, Ying Zhang†, Wenyan Chen†, Zhifang Tang†, Xiaoming Xin†, Ping Yang†, Xiaoqin Liu†, Weimin Cai*† and Ming Hu*‡

† Department of Clinical Pharmacy, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, P.R. China

‡ Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, 1441 Moursund Street, Houston, Texas 77030, United States

1

ACS Paragon Plus Environment


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Running title: Inhibition of Bilirubin Glucuronidation by Polyphenolic Acids

* Corresponding author Ming Hu, Ph.D., 1441 Moursund Street, Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX 77030, United States. Tel: (713)-795-8320, E-mail: [email protected]

Weimin Cai, Ph.D., 826 Zhangheng Road, Department of Clinical Pharmacy, School of Pharmacy, Fudan University, Shanghai, 201203, P.R. China. Tel.: (86)-21-5198-0023; E-mail: [email protected]

Ming Hu and Weimin Cai are the co-corresponding author.

Number of text pages: 27 Number of tables: 1 Number of figures: 9 Number of references: 49 Number of words in the Abstract: 248 Number of words in the Introduction: 1001 Number of words in the Discussion: 996 Number of tables in the Supporting Information: 14 Number of figures in the Supporting Information: 10

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Abstract Graphic

Blood

Erythrocyte

UCB + Albumin

Sinusoidal

UCB UGT1A1

Hepatocyte

Liver

Smooth ER

SAA,SAB DSI, CDI

BG Canalicular

BileBile BGBG

Bile duct

Intestine

Abstract graphic for inhibition of human UGT1A1-mediated bilirubin glucuronidation by Salvianolic acid A (SAA), Salvianolic acid B (SAB) and polyphenolic acid-rich Salvia miltiorrhiza (Danshen) injections (i.e., DSI and CDI).

"For Abstract graphic Use Only" Title: Inhibition of Human UGT1A1-Mediated Bilirubin Glucuronidation by Polyphenolic Acids Impact Safety of Popular Salvianolic Acid A/B-Containing Drugs and Herbal Products Authors: Guo Ma, Ying Zhang, Wenyan Chen, Zhifang Tang, Xiaoming Xin, Ping Yang, Xiaoqin Liu, Weimin Cai* and Ming Hu*

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ABSTRACT Bilirubin-related adverse reactions (ADR, e.g., jaundice and hyperbilirubinemia) induced by herbs rich in certain polyphenolic acids are widely reported. However, the causes and the mechanisms underlying these ADR are not well understood. The purpose of this paper was to determine the mechanism by which certain polyphenolic acids inhibit UGT1A1-mediated bilirubin glucuronidation, leading to jaundice or hyperbilirubinemia. We investigated in vitro inhibitory effects on bilirubin glucuronidation of salvianolic acid A (SAA), salvianolic acid B (SAB), danshensu (DSS), protocatechuic aldehyde (PA) and rosmarinic acid (RA), as well as two Salvia miltiorrhiza injections (DSI and CDI) rich in polyphenolic acids. The results showed that average formation rates of three bilirubin glucuronides displayed a significant difference (p 98%, Shanghai, China). Danshen injection (DSI, was prepared from the aqueous extract of Danshen; total herb extract 8.530 mg/ml; batch no. 160605) and its negative control injection (DSI control, was prepared by the preparation process of DSI in the absence of the aqueous extract of Danshen) were supplied by Jiangsu Shenlong Pharmaceutical Co., Ltd (Yancheng, Jiangsu, China). Compound danshen injection (CDI, was prepared with the aqueous extracts of Danshen and the steam distillates of Lignum Dalbergia odorifera; total herb extract 11.521 mg/ml; batch no. 160702) and its control injection (CDI control, was prepared by the preparation process of CDI without Danshen) were provided by Guangdong Boluo Pioneer Pharmaceutical Group Co., Ltd. (Huizhou, Guangdong, China). Bilirubin (including three mixed isomers, i.e., bilirubin IX-α 90.11%, XIII-α 3.12% and III-α 5.93%), uridine 5′-diphospho-glucuronic acid trisodium salt (UDPGA) and alamethicin were purchased from Sigma-Aldrich. Corning® Gentest™ pooled human liver microsomes (HLM, ~20-Donor Pool, Mixed Gender) and recombinant human UGT1A1 enzyme (UGT1A1) was purchased from BD Biosciences-Discovery Labware. Inc (Woburn, MA, USA). Formic acid, MgCl2·6H2O, K2HPO4·3H2O, KH2PO4, NaH2PO4·2H2O, Na2HPO4·12H2O, NaCl (all of analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). 12


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Acetonitrile and methanol (Both of HPLC grade) were provided by Fisher Scientific International, Inc. (Fair Lawn, NJ, USA). Argon was purchased from Shanghai Lvmin Gas Co. Ltd. Purified water was prepared in a water purification system (EMD Millipore Corp., Billerica, MA, USA). All other reagents were of analytical grade or better.

Bilirubin Glucuronidation Before the inhibition studies, kinetic parameters of bilirubin glucuronidation, namely, Michaelis–Menten constant (Km), maximum glucuronidation rate (Vmax), and relevant parameters (e.g., substrate inhibition constant, Ksi) were determined by measuring the initial glucuronidation rates of bilirubin over a series of concentrations. The experimental procedures were essentially the same as our recent publication4 with slight modifications. The incubation procedures were as follows: (1) bilirubin (final concentration 0.2–5 µM, which was in the in vivo concentration range of bilirubin24), potassium phosphate buffer (50 mM, pH 7.4), magnesium chloride (0.88 mM), alamethicin (22 µg/mL) and human UGT1A1 SupersomesTM (or pooled human liver microsomes, final concentration 12.5 µg of protein/mL) were mixed in amber glass vials (full of argon) and pre-incubated at 37˚C for 2 min in a shaking water bath; (2) The reaction was initiated by the addition of UDPGA (3.5 mM); (3) The mixture (final volume 200 µL) was incubated at 37˚C for a predetermined period of time (15– 30 min); (4) The reaction was terminated by the addition of 600 µL ice-cold methanol containing 200 mM ascorbic acid. The mixtures were vortexed for 2 min and then 13



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centrifuged at 12,000 rpm for 10 min to precipitate and separate protein. The supernatant was injected into the HPLC system for analysis of bilirubin and its glucuronides (i.e., UCB, BMG1, BMG2 and BDG). Bilirubin was dissolved in DMSO just before adding into the incubation systems. The final DMSO concentration in the incubation mixture was 1%. The samples were stored in amber glass vials, handled and processed under the dim light. Glucuronidation rates were calculated as nanomoles of glucuronide(s) formed per mg SupersomesTM protein amount per reaction time (or nmol/mg protein/min). All experiments were performed in triplicates.

Inhibition of bilirubin glucuronidation Based on the Km values determined in “Bilirubin Glucuronidation”, inhibition of bilirubin glucuronidation was determined at a series of concentrations of the substrates (i.e., bilirubin) surrounding the Km values and the potential inhibitors. Specifically, the final concentrations of bilirubin were 0.2–5 µM in the incubation systems, respectively. The final concentrations of SAA, SAB, DSS, PA, RA, DSI and CDI in the incubation systems were 0–2.96 µg/mL, 0–71.86 µg/mL, 0–39.63 µg/mL, 0–27.62 µg/mL, 0–72.07 µg/mL, 0–128 µg/mL and 0–172.80 µg/mL, respectively. The studied concentrations of these components were related to their in vivo human plasma concentrations.15,26,31-33 The detailed reaction conditions for inhibition of bilirubin glucuronidation were shown in Table S1 (Supporting Information). Concentrations of DSI and CDI were calculated by the concentration of total herbal 14


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extract in the injections, respectively. The experimental procedure was the same as the above-mentioned “Bilirubin Glucuronidation”, except that the substrate and inhibitor were added into the reaction mixtures at the same time. The negative control incubations of DSI and CDI were carried out by using their control injections in the absence of the aqueous extract of Danshen.

Analysis of Bilirubin and Its Glucuronides A Shimadzu LC-2010A HT HPLC system equipped with a quaternary pump, an automatic sampler, a UV–Vis detector, a system controller, a temperature control oven, and a LabSolution workstation (Kyoto, Japan) was used to analyze bilirubin and its glucuronides, as well as the primary constituents in DSI and CDI. The HPLC method for determination of UCB, BMG1, BMG2 and BDG was the same as our recent publication4 with slight modifications. Briefly, UCB, BMG1, BMG2 and BDG were separated on a HPLC column (reverse phase Diamonsil™ C18 column, 200 mm × 4.6 mm, i.d., 5 µm particle size, Dikma) with guard column (Cartridge Guard Column E, Inertsil ODS-SP, 10 mm × 4 mm, GL Sciences Inc.). The mobile phase consisted of 0.1% formic acid in water (A) and 100% acetonitrile (B) was delivered at a flow rate of 1 mL/min. The linear gradient elution program was as follows: 0–9 min, 40–75% B; 9–18 min, 75–95% B; 18–27 min, 95% B; 27–30 min, 95–40% B. The column temperature was 45 ℃. The detection wavelength was 450 nm. The injection volume was 100 µL. Because of lack of commercial products of bilirubin glucuronides, the calibration curves for UCB was used to estimate concentration of 15



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BMG1, BMG2 and BDG as was done previously.4,9,34

Presence of UCB, BMG1, BMG2 and BDG were confirmed by the LC-MS/MS systems consisted of an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to an Applied Biosystems 4000 Q TRAP mass spectrometer (Applied Biosystems Sciex, Ontario, Canada) equipped with a TurboIonSpray™ source for ion production. The chromatographic conditions were essentially the same as the above HPLC method with slight modifications, namely the flow rate was adjusted to 0.8 mL/min and the column temperature was adjusted to 35℃. The injection volume was 10 µL. All detection was performed on electrospray positive ionization mass spectrometry by multiple reaction monitoring (MRM) of the transitions of UCB at m/z 585.3→299.1, both BMG1 and BMG2 at m/z 761.3→585.3, and BDG at m/z 937.3→585.3. The optimal MS parameters were as follows: Nebulizer, heater and curtain gas (all nitrogen) flow rates 30, 30 and 10 units, respectively; ionspray voltage 4500 V; ion source temperature 450 ℃; collision gas 35 units; declustering potentials 50 V. The pause time was set at 10 ms and the dwell time at 100 ms. Data acquisition and integration were controlled by Applied Biosystems Analyst 1.6 Software.

Determination of the Primary Constituents in DSI and CDI A Shimadzu LC-2010A HT HPLC system equipped with a quaternary pump, an automatic sampler, a UV-Vis detector, a system controller, a temperature control oven 16


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and a LabSolution workstation (Kyoto, Japan) was used to analyze SAA, SAB, DSS, PA and RA in the DSI and CDI. All these constituents were simultaneously separated on a HPLC column (Diamonsil™ C18 column, 200 mm × 4.6 mm, i.d., 5 µm particle size, Dikma) with guard column (Cartridge Guard Column E, Inertsil ODS-SP, 10 mm × 4 mm, GL Sciences Inc.). The mobile phase consisted of 0.1% formic acid in water (A) and 100% acetonitrile (B) was pumped at a flow rate of 1 mL/min. The linear gradient elution program was as followed: 0–5 min, 11–12% B; 5–10 min, 12– 13% B; 10–15 min, 13-15% B; 15–30 min, 15–20% B; 30–45 min, 20–25% B；45–55 min, 25–30% B；55–65 min, 30–11% B. The column temperature was 35℃. The detection wavelength was 286 nm. The sample injection volume was 10 µL.

Kinetics Analysis Apparent kinetic parameters [e.g., V (reaction velocity), Km (Michaelis–Menten constant), Vmax(maximum reaction velocity), Ksi (substrate inhibition constant) and Ki (inhibition constant)] of bilirubin glucuronidation were obtained by fitting the substrate concentrations and initial reaction rates to the enzyme kinetics equations (e.g., Michaelis–Menten, substrate inhibition or Hill equations) with a weighting of 1/y. Types of inhibition were determined by fitting kinetic data (substrate concentrations, inhibitor concentrations and initial rates) to four reversible inhibition models (i.e., competitive, noncompetitive, uncompetitive, mixed-type inhibition models) using nonlinear regression analysis, respectively.10,35 Goodness of the fitting was evaluated by comparing R2 (square of correlation coefficient) and AIC (Akaike 17



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information criterion) values.36,37 Especially, as an useful graphical tool, the Lineweaver–Burk (double-reciprocals) plots (1/V versus 1/[S]) were used to determine the type and mechanism of enzyme inhibition in the study. Kinetics of the mixed-type inhibition was described by the following Eq. 1

Where S is substrate; I, inhibitor; α, modifying factor; V, Vmax, Km and Ki are the same as above.

In the current analysis, a decrease in the formation of the substrate glucuronide(s) was used to calculate IC50 (i.e., half maximal inhibitory concentration) value in the UGT1A1 inhibition assay. Concentration of the inhibitor that is required to produce 50% inhibition of the enzymatic activity was determined from the curves plotting enzymatic activity versus the logarithm of the inhibitor concentration. The apparent Ki value was determined from the x-intercept of a replot of the mean slopes of the Lineweaver–Burk plots versus concentrations of the inhibitor.37 Finally, mechanism of the inhibition of bilirubin glucuronidation by DSI, CDI and their ingredients were systematically evaluated on the basis of the multiple kinetic parameters (V, Km, Vmax, Ksi, Ki and IC50) and the Lineweaver–Burk plots. Data analysis was performed by GraphPad Prism V5 for Windows (GraphPad Software, San Diego, CA). In addition, in order to evaluate the contribution of SAA and SAB in the DSI or CDI to inhibition of bilirubin glucuronidation, Ki values and IC50 values of SAA and SAB, and their equivalent values in the DSI extract or the CDI extract at different substrate 18


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concentrations were compared, respectively. The equivalent values and contribution ratio (%) of SAA and SAB were estimated based on their Ki and IC50 values, and their contents in the DSI (or CDI) extract, respectively.

Statistical Analysis Statistical analyses were performed by two-way ANOVA or one-way ANOVA using GraphPad Prism V5 software for Windows (GraphPad Software, San Diego, CA). Differences were considered significant when p values were less than 0.05 (p < 0.05).

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RESULTS Bilirubin Glucuronidation Kinetics Inhibition

of

human

UGT1A1-mediated

bilirubin

glucuronidation

was

characterized using IC50 values. IC50 values were dependent on the conditions under which they were generated. For example, concentrations of the substrate and enzyme will have an effect. In order to reliably determine inhibitory effect of glucuronidation and accurately determine the IC50 values, Km values of bilirubin glucuronidation were determined in the present study. The apparent enzyme kinetic parameters and profiles of human UGT1A1 mediated-bilirubin glucuronidation were shown in Table 1 and Figure 2, respectively. The Km values of bilirubin glucuronidation were equal to 0.82 ± 0.15 µM for BMG1, 0.83 ± 0.16 µM for BMG2, 0.44 ± 0.10 µM for BDG, and 0.75 ± 0.14 µM for TBG (TBG = BMG1 + BMG2 + BDG), respectively. Moreover, bilirubin glucuronidation displayed substrate inhibition kinetics in the selected bilirubin concentration range (i.e., 0.2–5 µM).

Bilirubin and its glucuronides (i.e., BMG1, BMG2 and BDG) were identified in the in vitro incubation samples by comparing the retention time and relative elution sequence of various conjugates as reported earlier4,38,39 and matching the corresponding ESI (electrospray ionization)-MS/MS spectra. The representative chromatograms for UCB, BMG1, BMG2 and BDG in the incubation samples were shown in Figure S2 (Supporting Information). UCB was metabolized to BMG1 (glucuronidation at the C8 carbon), BMG2 (glucuronidation at the C12 carbon) and 20


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BDG (glucuronidation at both C8 and C12 carbons) by human UGT1A1. The representative MRM chromatograms and MS/MS spectrum of UCB, BMG1, BMG2, and BDG in the incubation samples were shown in Figure S3 (Supporting Information). With (+) ESI, UCB produced an m/z 585.3 [M+H]+ ion. As the major product ion, the m/z 585.3 ion underwent CID (collision induced dissociation)MS/MS to form m/z 299.1. Since there are two carboxyl groups in the structure, the glucuronidation positions (i.e., C8 and C12) are assigned as the mono-glucuronide isomers BMG1, BMG2 and the di-glucuronide BDG, respectively. With (+) ESI, BMG1 and BMG2 produced m/z 761.3 [M+H]+ ions, which underwent CID-MS/MS to form m/z 585.3. BDG produced m/z 937.3 [M+H]+ ion, which underwent CID-MS/MS to form m/z 585.3. The calibration curves for UCB in the HLM and UGT1A1 standard samples were shown in Figure S4 (Supporting Information).

Contents of the primary active constituents in DSI and CDI Contents of SAA, SAB, DSS, PA, RA and the total crude herbal extract were 0.217, 0.885, 2.264, 0.422, 0.379 and 8.531 mg/mL in the DSI, 0.257, 0.104, 3.116, 0.298, 0.248 and 11.520 mg/mL in the CDI, respectively.

Inhibitory Effect of the Primary Constituents Extracted from Danshen on Bilirubin Glucuronidation In order to explore which constituents in the DSI and CDI inhibited bilirubin glucuronidation, the primary constituents (i.e., SAA, SAB, DSS, PA and RA) 21



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extracted from Danshen were used to co-incubate with bilirubin and human UGT1A1, respectively. The results indicated inhibitory effects (IC50) of these phenolic acid or polyphenol constituents on human UGT1A1-mediated bilirubin glucuronidation demonstrated a significant difference (p < 0.05), and the rank order of their IC50 values was SAA ˂ SAB < RA < DSS < PA at 0.75 µM bilirubin. Their IC50 values against bilirubin glucuronidation for BMG1, BMG2, BDG and TBG displayed the same trend (Figure 3 and Table S2 in the Supporting Information). IC50 values of SAA, SAB, RA, DSS and PA against glucuronidation of bilirubin (0.75 µM) were 0.56 ± 0.04 µg/mL (1.13± 0.08 µM), 7.82 ± 0.11 µg/mL(10.87± 0.16 µM), 53.89 ± 0.91 µg/mL(149.53 ±2.54 µM), 67.72 ± 1.41 µg/mL(340.20 ±6.13 µM) and 101.90 ± 3.82 µg/mL (738.01±20.75 µM) for TBG, respectively. Among the five constituents, SAA and SAB were potent inhibitors of UGT1A1 because of their lower IC50 values. However, DSS, PA and RA exhibited very poor inhibitory effects on bilirubin glucuronidation.

Inhibition of Human Liver Microsome-Mediated Bilirubin Glucuronidation by SAA, SAB, DSI and CDI In order to explore the cause and mechanism of salvianolic acid rich products-induced jaundice or hyperbilirubinemia, inhibition of human liver microsomes (HLM)-mediated bilirubin glucuronidation by SAA, SAB, and polyphenolic acid-rich DSI and CDI was investigated. The results indicated that SAA, SAB, DSI and CDI were potent inhibitors of glucuronidation of bilirubin (0.75 µM) 22


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for they displayed small IC50 values (Figure 4). IC50 values of SAA, SAB, DSI and CDI against HLM-mediated bilirubin glucuronidation were 0.59 ± 0.04 µg/mL, 8.09 ± 0.09 µg/mL, 11.28 ± 0.84 µg/mL and 13.88 ± 0.39 µg/mL for TBG, respectively. Their IC50 values against bilirubin glucuronidation for BMG1, BMG2, BDG and TBG displayed the same trend. It meant that DSI, CDI and their components can decrease bilirubin glucuronidation.

Rates of Bilirubin Glucuronidation in the Absence or the Presence of SAA, SAB, DSI and CDI In order to further disclose the cause and mechanism of salvianolic acid rich products-induced jaundice or hyperbilirubinemia, inhibition of human UGT1A1mediated bilirubin glucuronidation by SAA, SAB, DSI and CDI was subsequently investigated. As shown in Figure 5-Figure 8, compared to the control group (in the absence of SAA, SAB, DSI and CDI), average formation rates of BMG1, BMG2, BDG and TBG significantly decreased with increasing concentrations of SAA (from 0 to 2.96 µg/mL), SAB (from 0 to 71.86 µg/mL), DSI (from 0 to 128 µg/mL) or CDI (from 0 to 172.8 µg/mL). Meanwhile, average formation rates of BMG1, BMG2, BDG and TBG firstly increased, and then decreased with increasing bilirubin concentrations regardless if SAA, SAB, DSI and CDI were present. It indicated that human UGT1A1-mediated bilirubin glucuronidation was inhibited by SAA, SAB, DSI and CDI, and the glucuronidation reaction at higher substrate concentration (> 2 µM) was dually inhibited by both the inhibitor (SAA, SAB, DSI or CDI) and bilirubin 23



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itself (substrate inhibition). Regardless of the presence of SAA, SAB, DSI and CDI, the rank order of average formation rates of bilirubin glucuronides remained the same as BMG2 > BMG1 > BDG (p < 0.05, TBG=BMG1+BMG2+BDG). It means that bilirubin glucuronidation by human UGT1A1 displayed positional preference (i.e., regioselectivity).

Inhibitory extents (i.e., % inhibition) of bilirubin glucuronidation increased with increasing the concentrations of the inhibitor SAA, SAB, DSI or CDI, and all decreased with increasing the bilirubin concentrations from 0.2 to 5 µM. Therefore, all the inhibition of bilirubin glucuronidation by SAA, SAB, DSI or CDI displayed concentration dependence (Figure 5–Figure 8 and Figure S5–Figure S8 in the Supporting Information). Compared to that of the control group (in the absence of SAA, SAB, DSI and CDI), formation rates of TBG (0.2 to 5 µM bilirubin) were inhibited 95.87 ± 0.18% to 80.96 ± 1.06% in the presence of 2.96 µg/mL SAA, 90.32 ± 0.42% to 75.71 ± 0.91% in the presence of 71.86 µg/mL SAB, 97.10 ± 0.21% to 88.16 ± 0.39% in the presence of 128 µg/mL DSI; and 93.49 ± 0.56% to 79.40 ± 0.15% in the presence of 172.8 µg/mL CDI, respectively. Compared to that of the control group (in the absence of SAA, SAB, DSI and CDI), the inhibition of formation rates of BMG1, BMG2, BDG displayed the same trend as that of TBG.

IC50 Values of SAA, SAB, DSI and CDI as the Inhibitors of Bilirubin Glucuronidation 24


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Average IC50 values of SAA, SAB, DSI and CDI against BMG1, BMG2, BDG and TBG increased with increasing bilirubin concentrations from 0.2 µM to 5 µM, and demonstrated substrate concentration-dependence. It means that inhibitory effects of SAA, SAB, DSI and CDI on bilirubin glucuronidation decreased with increasing the substrate (bilirubin) concentrations (p < 0.05). Average IC50 values were in the range 0.44 ± 0.02 µg/mL~0.86 ± 0.04 µg/mL, 4.22 ± 0.30 µg/mL~12.50 ± 0.93 µg/mL, 9.29 ± 0.76 µg/mL~18.82 ± 0.63 µg/mL and 9.18 ± 2.00 µg/mL~22.36 ± 1.39 µg/mL for TBG in the presence of SAA, SAB, DSI and CDI, respectively. Average IC50 values of SAA, SAB, DSI and CDI for BMG1, BMG2 and BDG displayed the same trend at different substrate concentrations (Figure 9, Table S3–Table S6 in the Supporting Information).

Inhibitory effect of SAA on bilirubin glucuronidation was more potent than that of SAB (p < 0.05). Effects of SAA and SAB on bilirubin glucuronidation was different with respect to different glucuronidation pathway, with higher inhibition (p < 0.05) on BDG than on BMG1 and BMG2 (IC50 BMG1 = BMG2 > BDG). This means that SAA and SAB both exhibited stronger inhibitory effect on formation of BDG than that of BMG1 and BMG2.

It needs to be pointed out that inhibition of DSI on formation of BMG1, BMG2 and BDG showed no difference in the selected concentration ranges of bilirubin and DSI (p > 0.05). In other words, inhibition of DSI on bilirubin glucuronidation showed no 25



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regioselectivity. However, average IC50 values of CDI on glucuronidation of bilirubin with respect to different concentrations of the substrate demonstrated a significant difference among BMG1, BMG2 and BDG with the lowest IC50 values (or highest inhibition) against BDG (IC50 BMG2 > BMG1 > BDG, p < 0.05). This means that CDI exhibited the strongest inhibitory effect on formation of BDG, and the weakest inhibitory effect on formation of BMG2. Namely, inhibition of bilirubin glucuronidation by CDI displayed regioselectivity.

Type of Inhibition of Bilirubin Glucuronidation by SAA, SAB, DSI and CDI According to the criteria of R2 (better if close to1.0) and AIC (best if the value is the minimal among the models fitted) of selecting the best fit kinetic parameters, kinetics of inhibition of human UGT1A1-mediated bilirubin glucuronidation in the presence of 0–2.96 µg/mL SAA, 0–71.86 µg/mL SAB, 0–128 µg/mL DSI or 0–172.8 µg/mL CDI were all best fitted to the mixed-type inhibition (Table S7-Table S10 in the Supporting Information), respectively. Consistent with kinetic analysis, all the Lineweaver–Burk (double reciprocal) plots shown in Figure 5-Figure 8 also showed that the slopes and the y-intercepts increased along with increasing concentrations of the inhibitor SAA, SAB, DSI and CDI, which were all consistent with characteristics of the mixed-type inhibition.40 Thus, it can be speculated that inhibition kinetics of human UGT1A1-mediated bilirubin glucuronidation by SAA, SAB, DSI and CDI obeyed the mixed-type inhibition, and hence SAA, SAB, DSI and CDI were the mixed-type inhibitors of UGT1A1. 26


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Ki values of inhibition of bilirubin glucuronidation were found to be 0.22 ± 0.03 µg/mL, 4.50 ± 0.84 µg/mL, 6.13 ± 1.05 µg/mL and 7.78 ± 1.94 µg/mL for TBG by SAA, SAB, DSI and CDI, respectively. Ki values of inhibition of bilirubin glucuronidation for BMG1, BMG2 and BDG by SAA, SAB, DSI and CDI were similar to that of TBG, respectively (Table S7-Table S10 in the Supporting Information). These lower Ki values meant that SAA, SAB, DSI and CDI all exhibited potent inhibitory effect on human UGT1A1-mediated bilirubin glucuronidation. Among them, SAA exhibited the most potent inhibitory effect.

Contribution of SAA and SAB to Inhibition of Bilirubin Glucuronidation by DSI and CDI The equivalent values of Ki and IC50 of SAA and SAB in the DSI (or CDI) extract, and contribution ratios (%) of SAA and SAB to inhibition of bilirubin glucuronidation at different substrate concentrations were estimated and shown in Table S11–Table S14 (Supporting Information). The contribution ratios of SAA and SAB to inhibition (Ki) of bilirubin glucuronidation by DSI were 70.77% and 14.13% for TBG, respectively. The contribution ratios of SAA and SAB to inhibition (IC50) of bilirubin glucuronidation by DSI were 48.37%~57.53% and 14.44%~22.83% for TBG at different bilirubin concentrations, respectively. The contribution ratios of SAA and SAB to inhibition (Ki) of bilirubin glucuronidation by CDI were 78.86% and 1.56% for TBG, respectively. The contribution ratios of SAA and SAB to inhibition (IC50) of bilirubin glucuronidation by CDI were 46.53%~60.26% and 1.52%~1.96% for TBG 27



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at different bilirubin concentrations, respectively. All these results indicated that SAA played a dominant role in inhibition of bilirubin glucuronidation by DSI and important role for inhibition by CDI. Compare to SAA, SAB displayed relatively weak inhibitory effect on bilirubin glucuronidation by DSI and CDI. It appeared that inhibitory effect of DSI and CDI on bilirubin glucuronidation is mainly due to inhibitory potential and content (or concentration) of their salvianolic acid constituents (i.e., SAA and SAB). In a word, inhibition of bilirubin glucuronidation by DSI and CDI is strongly associated with the presence of salvianolic acid constituents.

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DISCUSSION Scientific and public interest in the polyphenolic acids SAA, SAB and the herbal injections DSI, CDI containing these phenolic acid constituents have grown enormously in recent years because of their wide spread use in various clinical conditions.27-30,41 DSI and CDI have been widely used with little awareness of the drug interactions with endogenous bilirubin. Case reports have demonstrated that DSI and

CDI

can

lead

to

bilirubin-related

ADR

(e.g.,

jaundice

and

hyperbilirubinemia).16-22 However, the cause and mechanism has not been investigated. This report is the first to disclose that SAA and SAB, as well as two Danshen injections (DSI and CDI) rich in SAA and SAB are the potent inhibitors of UGT1A1. These polyphenolic acids exerted their potent inhibition of human UGT1A1-mediated bilirubin glucuronidation via a mixed-type inhibitory mechanism.

Among the multiple polyphenolic acid constituents in the DSI and CDI, SAA and SAB, but not DSS, PA and RA, exhibited potent inhibitory effects on bilirubin glucuronidation (Figure 3 and Table S2 in the Supporting Information). Since other compounds in DSI and CDI have not been found to severely inhibit UGT1A1, it is likely that DSI and CDI-induced jaundice or hyperbilirubinemia are mainly attributed to inhibition of UGT1A1 by SAA and SAB, both of which are enriched in these injections. In other words, the salvianolic acid constituents (e.g., SAA and SAB) of DSI and CDI might cause metabolic disorder of bilirubin, which could lead to jaundice or hyperbilirubinemia if not well managed (e.g., long-term, and/or high dose 29



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administration of SAA/SAB rich products).

We believe that bilirubin-related ADRs induced by DSI and CDI were closely related to their polyphenolic acid constituents (i.e., SAA and SAB), even though DSI and CDI are both multi-component mixtures. This is because when we investigated inhibitory effects of DSI control and CDI control, which contained no SAA or SAB, on human UGT1A1-mediated bilirubin glucuronidation, neither showed inhibitory effects on human UGT1A1-mediated bilirubin glucuronidation (Figure S9 and Figure S10 in the Supporting Information). Hence, DSI and CDI-induced jaundice or hyperbilirubinemia were only associated with aqueous extract of Danshen, both of which were enriched with SAA and SAB.

The fact that UGT1A1 has a large binding pocket that may accommodate more than one mode of binding interaction42 is consistent with our results that showed that bilirubin glucuronidation displayed mixed-type inhibition patterns in the presence of SAA and SAB when using the Lineweaver–Burk plots (Figure 5-Figure 8). Lineweaver–Burk plots give a quick, visual impression of type and mechanism of enzyme inhibition, and are often used to distinguish the competitive, noncompetitive, uncompetitive and mixed-type inhibition.40,43,44 It is apparent from examining these plots that the type of inhibition belongs to the mixed-type inhibition within the whole range of inhibitor concentrations tested. The mixed-type inhibition could be the result of a “mixture” of competitive inhibition and noncompetitive inhibition, or 30


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noncompetitive inhibition is sometimes thought of as a special case of mixed-type inhibition.45 Because the inhibitory kinetics of human UGT1A1-mediated bilirubin glucuronidation by SAA, SAB, DSI and CDI were best fitted to the mixed-type inhibition, according to the criteria of R2 (better if close to 1.0) and AIC (best if the value is minimum) 35 (Table S7-Table S10 in the Supporting Information). SAA, SAB, DSI and CDI are all designated as the mixed-type inhibitors of UGT1A1.

A possible impact on UGT1A1-mediated bilirubin glucuronidation is that SAA is also a substrate of UGT1A1 according to a latest publication,46 which showed SAA was the substrate of UGT1A1 and UGT1A9. However, in that paper, UGT1A9 showed the highest affinity for SAA glucuronidation. On the other hand, we did not find glucuronides of SAA and SAB by LC-MS/MS in the present study. It is probably due to the fact that UGT1A1 possess far stronger affinity for bilirubin than that of SAA/SAB, and UGT1A1 only played a minor role in the glucuronidation of SAA/SAB. When comparing the relative potency of SAA vs SAB, we could show that the inhibitory potential of SAA on BMG1, BMG2, BDG and TBG was about 10 fold higher than that of SAB since average Ki and IC50 values of SAA was about 10 times smaller than that of SAB with respect to different substrate concentrations (i.e., 0.2–5 µM bilirubin) (Figure 9, Table S3, Table S4, Table S7, Table S8 in the Supporting Information).

In this study, we clearly showed that SAA and to a lesser extent SAB were mainly 31



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responsible for inhibition of human UGT1A1-mediated bilirubin glucuronidation. However, SAA/SAB rich herbs-induced jaundice or hyperbilirubinemia may also be caused by additional change in dispositional behaviors of bilirubin. For example, in addition to UGT1A1-catalyzed glucuronidation, bilirubin and its metabolites are also transported by several transport systems, e.g., organic anion transporting polypeptides (OATPs), multidrug resistance-associated protein (MRPs) and breast cancer resistance protein (BCRP) at the sinusoidal and/or canalicular membrane of hepatocytes.3,47-49 It is therefore possible, although unlikely, that some of the reported effects of DSI and CDI on bilirubin conjugation and elimination were partially due to their impact on related efflux transporters. In addition, the in vivo study based on gene knock-out mouse and humanized transgenic mouse of UGT1A1 (or the related efflux transporters) should be conducted to further confirm the in vitro experimental results in the future study.

In summary, in the present study we determined that inhibition of human UGT1A1-mediated bilirubin glucuronidation by the polyphenolic acid constituents SAA and SAB in DSI and CDI could be the cause and mechanism of jaundice or hyperbilirubinemia induced by the herbs rich in these polyphenolic acids. Similarly, it can be postulated that SAA, SAB, DSI and CDI will play important roles in inhibition of UGT1A1-mediated detoxification of some drugs (e.g., UGT1A1 substrate drugs). Furthermore, this research will provide an important reference for appreciating the xenobiotics (e.g., herbs)–endobiotics (e.g., bilirubin)–metabolic enzyme (i.e., 32


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UGT1A1) interactions. This appreciation, when fully delineated, will have important clinical significance in disclosing the etiology and pathogenesis of bilirubin-related ADR or malady (e.g., jaundice, hyperbilirubinemia) induced by SAA and/or SAB rich herbs such as DSI, CDI and Dantonic®.

33



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Acknowledgements

The author would like to thank Dr. Qinfeng Xu of Department of Statistics, School of Management, Fudan University, for his help in statistical analyses. This work was supported by the grants from the National Natural Science Funds of China (81374051), the Fundamental Research Funds for the Central Universities from the Ministry of Education of the People's Republic of China (20520133531), the Fudan′s Wangdao Research Program (JMH6285113/014/002) to Guo Ma and the National Institutes of Health (GM070737) to Ming Hu.

34


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Authorship Contributions

Participated in research design: Guo Ma and Ming Hu. Conducted experiments: Guo Ma, Ying Zhang, Wenyan Chen, Zhifang Tang, Xiaoming Xin, and Xiaoqin Liu. Contributed new reagents or analytic tools: Guo Ma, Weimin Cai and Ping Yang, Performed data analysis: Guo Ma and Ying Zhang. Wrote or contributed to the writing of the manuscript: Guo Ma and Ming Hu.

35



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Tables Table 1 Apparent enzyme kinetic parameters of human UGT1A1 mediated-bilirubin glucuronidation (n=3,x ±SD)a BMG1

BMG2

BDG

TBGb

Km(µM)

0.82±0.15

0.83±0.16

0.44±0.10

0.75±0.14

Vmax(nmol/mg/min)

1.35±0.13

1.55±0.16

0.60±0.06

3.51±0.36

Ksi(µM)

7.16±1.96

7.23±2.18

5.64±1.59

6.80±1.92

Kinetic parameters

a

- Eight different concentrations of bilirubin were used to derive the kinetic parameters.

b

- TBG = BMG1 + BMG2 + BDG.

42


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Figure Captions

Figure 1. The chemical structures of the primary constituents of Danshen Injection (DSI) and Compound Danshen Injection (CDI). SAA, salvianolic acid A; SAB, salvianolic acid B; DSS, danshensu, PA, protocatechuic aldehyde; RA, rosmarinic acid.

Figure 2.

Kinetics profiles of human UGT1A1-mediated bilirubin glucuronidation.

BMG1, BMG2, BDG, and TBG represented the profiles of formation rate of BMG1, BMG2, BDG, and TBG at different bilirubin concentrations (i.e., 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM bilirubin), respectively. BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides. TBG = BMG1 + BMG2 + BDG. Black dots (•) and smooth lines (–) denote observed and predicted formation rates of bilirubin glucuronides, respectively. Predicted formation rates of BMG1, BMG2, BDG, and TBG were from substrate inhibition models. Each data point represents the average of three replicates. Experimental details are presented under Materials and Methods. For kinetic parameters, please see Table 1.

Figure 3. Inhibitory effects (IC50) of SAA, SAB, RA, DSS and PA on human UGT1A1-mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition column plots of SAA, SAB, RA, DSS and PA on BMG1, BMG2, BDG and TBG at 0.75 µM bilirubin, respectively. SAA, salvianolic acid A; 43



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SAB, salvianolic acid B; RA, rosmarinic acid; DSS, danshensu, PA, protocatechuic aldehyde; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. IC50 values of SAA, SAB, RA, DSS and PA on BMG1, BMG2, BDG and TBG showed significant difference (p < 0.05), respectively. Concentrations of SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. Concentrations of RA were 0, 0.18, 0.72, 1.80, 3.60, 9.01, 18.02, 36.03, and 72.07 µg/mL. Concentrations of DSS were 0, 0.10, 0.40, 0.99, 1.98, 4.95, 9.91, 19.82, and 39.63 µg/mL. Concentrations of PA were 0, 0.07, 0.28, 0.69, 1.38, 3.45, 6.91, 13.81, and 27.62 µg/mL. Each data point and plot represents the average of three determinations.

Figure 4.

Inhibitory effects (IC50) of SAA, SAB, DSI and CDI on human liver

microsome-mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition (IC50) profiles of SAA, SAB, DSI and CDI on BMG1, BMG2, BDG and TBG at 0.75 µM bilirubin, respectively. SAA, salvianolic acid A; SAB, salvianolic acid B; DSI, Danshen Injection; CDI, Compound Danshen Injection； BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. The IC50 values are expressed as percentages relative to the control, which was determined in the absence of SAA, SAB, DSI and CDI. Concentrations of SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 44


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0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. Concentrations of DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Concentrations of CDI were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL. Each data point and plot represents the average of three determinations.

Figure 5.

Kinetics profiles (A1-D1) and Lineweaver–Burk (double–reciprocal)

plots (1/V versus 1/[S]) (A2–D2) of human UGT1A1-mediated bilirubin glucuronidation in the presence of different concentrations of SAA. SAA, salvianolic acid A; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1+ BMG2 + BDG. A1 and A2 represented BMG1; B1 and B2 represented BMG2; C1 and C2 represented BDG; D1 and D2 represented TBG. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. The embedded figures are the Lineweaver–Burk plots for the same data at the lower SAA concentrations (0, 0.25, 0.49, 0.99, and 1.48 µg/mL). Each data point and plot represents the average of three determinations.

Figure 6.

Kinetics profiles (A1–D1) and Lineweaver–Burk (double–reciprocal)

plots (1/V versus 1/[S]) (A2–D2) of human UGT1A1-mediated bilirubin glucuronidation in the presence of different concentrations of SAB. SAB, salvianolic acid B; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; 45



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BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides. TBG = BMG1 + BMG2 + BDG. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. A1 and A2 represented BMG1; B1 and B2 represented BMG2; C1 and C2 represented BDG; D1 and D2 represented TBG. The embedded figures are the Lineweaver–Burk plots for the same data at the lower SAB concentrations (0, 0.72, 1.80, 3.59, and 7.19 µg/mL). Each data point and plot represents the average of three determinations.

Figure 7.


plots (1/V versus 1/[S]) (A2–D2) of human UGT1A1-mediated bilirubin glucuronidation in the presence of different concentrations of DSI. DSI, Danshen Injection; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. A1 and A2 represented BMG1; B1 and B2 represented BMG2; C1 and C2 represented BDG; D1 and D2 represented TBG. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Concentrations of DSI were denominated in concentrations of total herbal extract in the injection. The embedded figures are the Lineweaver–Burk plots for the same data at the lower DSI concentrations (0, 2, 4, 8, and 16 µg/mL). Each data point and plot represents the average of three determinations. 46


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Figure 8.


plots (1/V versus 1/[S]) (A2–D2) of human UGT1A1-mediated bilirubin glucuronidation in the presence of different concentrations of CDI. CDI, Compound Danshen Injection; BMG1, bilirubin monoglucuronides 1; BMG2, bilirubin monoglucuronides 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. A1 and A2 represented BMG1; B1 and B2 represented BMG2; C1 and C2 represented BDG; D1 and D2 represented TBG. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor CDI were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL. Concentrations of CDI were denominated in concentration of total herbal extract in the injection. The embedded figures are the Lineweaver–Burk plots for the same data at the lower CDI concentrations (0, 2.7, 5.4, 10.8, and 21.6 µg/mL). Each data point and plot represents the average of three determinations.

Figure 9. Inhibitory effects (IC50) of SAA, SAB, DSI and CDI on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrations. SAA, salvianolic acid A; SAB, salvianolic acid B; DSI, Danshen Injection; CDI, Compound Danshen Injection; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 47



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17.97, 35.93, and 71.86 µg/mL. Concentrations of DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Concentrations of CDI were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL. Concentrations of DSI and CDI were denominated in concentrations of total herbal extract in the injection, respectively. The detailed IC50 values see Table S3Table S6 in the Supporting Information.

48


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Figure 1.

OH OH HO

HO

OH OH

HO

COOH

OH

O

O

O

OH

O HO O

OH

O

COOH

O

COOH

HO HO

SAB

SAA HO

O O

HO

COOH

HO

CHO

HO

OH O

OH

OH HO

HO OH

DSS

PA

RA

49



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Figure 2.

50


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Figure 3.

51



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Figure 4.

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Figure 5.

53



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Figure 6.

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Figure 7.

55



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Figure 8.

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Figure 9.

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Supporting Information

Table S1 Test conditions for inhibition of bilirubin glucuronidation by SAA, SAB, DSS, PA, RA, DSI and CDI Substrate

Inhibitor

UGT1A1 Reaction or HLM

Name

Concentration(µM)

Name

time(min)

Concentration(µg/mL) (µg/mL)

bilirubin

0.2, 0.4, 0.75, 1,

SAA

1.5, 2, 3, 5

0, 0.25, 0.49, 0.99,

12.5

15~30

1.48, 1.98, 2.47, 2.96

0.2, 0.4, 0.75, 1,

SAB

1.5, 2, 3, 5

0, 0.72, 1.80, 3.59, 7.19,

17.97,

35.93,

71.86 0.75

DSS

0, 0.10, 0.40, 0.99, 1.98, 4.95, 9.91, 19.82, 39.63

0.75

PA

0, 0.07, 0.28, 0.69, 1.38, 3.45, 6.91, 13.81, 27.62

0.75

RA

0, 0.18, 0.72, 1.80, 3.60,

9.01,

18.02,

36.03, 72.07 0.2, 0.4, 0.75, 1,

a

DSI

1.5, 2, 3, 5

128

0.2, 0.4, 0.75, 1, 1.5, 2, 3, 5 a,b

0, 2, 4, 8, 16, 32, 64,

b

CDI

0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, 172.8

- Concentrations of DSI and CDI were denominated in the concentration of total herbal extract

in the injections, respectively.

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Table S2 Inhibitory effect (IC50, µM) of SAA, SAB, RA, DSS and PA on human UGT1A1-mediated bilirubin glucuronidation (n=3,x ±SD)

a

Inhibitor Bilirubin Glucuronides BMG1

BMG2

BDG

TBG

a

SAA

SAB

RA

DSS

PA

1.21

11.34

142.10

361.67

702.30

± 0.07

± 1.20

±2.94

±5.21

±22.28

1.23

10.38

140.76

333.33

697.27

± 0.03

± 0.45

±3.05

±4.50

±18.68

0.84

5.95

151.03

347.73

828.23

± 0.05

± 0.49

±1.16

±4.90

±21.25

1.13

10.87

149.53

340.20

738.01

± 0.08

± 0.16

±2.54

±6.13

±20.75

- Concentration of biliruin was 0.75 µM.

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Table S3 Inhibitory effect (IC50, µg/mL) of SAA on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrations (n=3,x ±SD) a Bilirubin (µM) 0.2

0.4

0.75

1

1.5

2

3

5

0.43

0.51

0.60

0.68

0.78

0.84

0.91

0.98

±0.03

±0.01

±0.03

±0.05

±0.04

±0.06

±0.08

±0.07

0.43

0.51

0.61

0.67

0.74

0.81

0.87

0.96

±0.03

±0.04

±0.02

±0.02

±0.02

±0.07

±0.05

±0.10

0.36

0.38

0.41

0.46

0.48

0.55

0.58

0.71

±0.03

±0.03

±0.04

±0.02

±0.03

±0.02

±0.03

±0.04

0.44

0.49

0.56

0.62

0.70

0.76

0.80

0.86

±0.02

±0.03

±0.04

±0.02

±0.02

±0.05

±0.01

±0.04

Glucuronides BMG1

BMG2

BDG

TBGb

a

- Concentrations of SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL.

b


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Table S4 Inhibitory effect (IC50, µg/mL) of SAB on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrations (n=3,x ±SD) Bilirubin(µM) Glucuronides BMG1

BMG2

BDG

TBGb

a

0.2

0.4

0.75

1

1.5

2

3

5

5.21

6.87

8.15

9.16

11.04

12.76

13.84

15.36

±0.54

±0.89

±0.41

±0.94

±0.43

±0.77

±1.37

±2.70

5.13

6.34

7.46

8.81

9.97

11.64

13.17

15.73

±0.15

±0.32

±0.94

±1.12

±0.41

±0.66

±0.95

±1.01

2.69

3.38

4.28

4.98

5.66

7.97

8.94

9.75

±0.57

±0.11

±0.29

±0.35

±0.70

±0.56

±0.44

±1.00

4.22

6.64

7.82

8.36

9.41

10.34

11.32

12.50

±0.30

±0.70

±0.11

±0.74

±0.28

±0.65

±0.51

±0.93

a

-Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL.

b


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Table S5 Inhibitory effect (IC50, µg/mL) of DSI on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrations (n=3,x ±SD)

a

Bilirubin (µM) Glucuronides

0.2

0.4

0.75

1

1.5

2

3

5

BMG1

10.59

11.14

11.24

12.15

12.26

16.45

18.36

19.80

±0.27

±0.60

±0.89

±0.88

±0.86

±1.36

±1.25

±0.91

10.27

10.55

11.85

11.97

13.23

15.53

17.38

18.60

±0.70

±1.02

±0.39

±1.51

±1.38

±0.70

±0.31

±1.33

7.88

8.54

9.40

10.93

13.11

16.39

18.03

20.80

±0.68

±1.20

±0.67

±1.69

±1.23

±2.47

±2.11

±2.38

9.29

10.42

10.89

11.85

13.33

15.93

18.12

18.82

±0.76

±0.33

±0.86

±0.43

±0.70

±1.46

±0.71

±0.63

BMG2

BDG

TBG b

a

- Concentrations of the inhibitor DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Concentrations

of DSI were denominated in concentrations of total herbal extract in the injection. b


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Table S6 Inhibitory effect (IC50, µg/mL) of CDI on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrations (n=3,x ±SD )a Bilirubin (µM) Glucuronides

0.2

0.4

0.75

1

1.5

2

3

5

BMG1

9.99

12.54

13.47

14.52

16.73

18.9

20.61

24.86

±1.13

±1.56

±0.83

±1.28

±1.15

±0.98

±1.73

±1.51

10.40

12.86

14.48

16.04

18.58

20.37

21.38

26.78

±1.80

±0.57

±1.07

±1.87

±2.69

±0.85

±2.47

±1.26

7.31

8.94

11.12

12.25

14.17

15.57

18.02

21.04

±1.59

±0.85

±1.09

±1.68

±2.22

±1.33

±2.55

±2.92

9.18

10.96

13.24

14.27

17.01

18.73

19.89

22.36

±2.00

±0.62

±0.63

±0.98

±0.68

±0.64

±1.73

±1.39

BMG2

BDG

TBG b

a

- Concentrations of the inhibitor CDI were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL.

Concentrations of CDI were denominated in concentration of total herbal extract in the injection. b


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Page 64 of 89

Table S7 Apparent enzyme kinetic parameters of glucuronidation of bilirubin in the presence of SAA fitted to different inhibition kinetics model (n=3,x ±SD) Inhibition model

Competitive

Noncompetitive

Uncompetitive

Mixed-type

Inhibition

Inhibition

Inhibition

Inhibition

Km(µM)

0.13±0.03

0.36 ±0.03

0.43 ±0.05

0.24±0.03

Vmax(nmol/mg/min)

0.68±0.02

0.83±0.02

0.85±0.03

0.77±0.02

Ki(µg/mL)

0.05±0.01

0.65±0.03

0.50±0.03

0.23±0.04

Glucuronides

BMG1

α

–

–

–

3.93±0.90

2

0.87

0.94

0.91

0.95

AIC

3.86

-158.38

-65.22

-193.46

Km(µM)

0.17±0.03

0.42±0.03

0.52±0.05

0.29±0.03

Vmax(nmol/mg/min)

0.83±0.03

1.01±0.02

1.04±0.03

0.94±0.02

Ki(µg/mL)

0.06±0.01

0.63±0.02

0.47±0.03

0.26±0.04

R

BMG2

α

–

–

–

3.42±0.74

2

0.88

0.95

0.91

0.96

AIC

50.01

-117.91

-15.97

-149.08

Km(µM)

0.07±0.03

0.19±0.02

0.20 ±0.03

0.13±0.02

Vmax(nmol/mg/min)

0.35±0.01

0.41±0.01

0.41±0.01

0.39±0.01

Ki(µg/mL)

0.02±0.01

0.40±0.02

0.34±0.02

0.14 ±0.03

R

BDG

α

–

–

–

3.54 ±0.88

2

0.85

0.95

0.93

0.96

AIC

-207.49

-436.19

-370.51

-460.50

Km(µM)

0.13±0.03

0.34±0.03

0.41±0.04

0.23±0.02

Vmax(nmol/mg/min)

1.84±0.06

2.23±0.04

2.28±0.07

2.08±0.04

Ki(µg/mL)

0.05±0.01

0.59±0.02

0.45±0.03

0.22±0.03

R

TBG

α

–

–

–

3.80±0.80

2

0.87

0.95

0.92

0.96

AIC

376.24

192.88

293.00

154.95

R

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Table S8 Apparent enzyme kinetic parameters of glucuronidation of bilirubin in the presence of SAB fitted to different inhibition kinetics model (n=3,x ±SD) Inhibition model

Competitive

Noncompetitive

Uncompetitive

Mixed-type

Glucuronides

Inhibition

Inhibition

Inhibition

Inhibition

BMG1

Km(µM)

0.19±0.02

0.40±0.02

0.46±0.03

0.30±0.03

Vmax(nmol/mg/min)

0.66±0.01

0.76±0.01

0.78±0.02

0.72±0.01

Ki(µg/mL)

1.40±0.18

13.27±0.63

10.04±0.59

4.91±0.84

α

–

–

–

4.10±1.07

2

0.92

0.95

0.93

0.95

AIC

-98.83

-182.58

-120.25

-205.95

Km(µM)

0.20±0.03

0.41±0.03

0.49±0.04

0.34±0.03

Vmax(nmol/mg/min)

0.76±0.02

0.87±0.03

0.90±0.02

0.83±0.02

Ki(µg/mL)

1.55±0.21

13.41±0.67

9.99±0.61

5.94±1.16

R

BMG2

α

–

–

–

3.15±0.91

2

0.91

0.94

0.92

0.95

AIC

-22.68

-110.46

-59.62

-122.45

Km(µM)

0.06 ±0.02

0.22±0.02

0.26±0.03

0.15±0.02

Vmax(nmol/mg/min)

0.31±0.01

0.36±0.01

0.37±0.01

0.35±0.01

Ki(µg/mL)

0.29±0.10

6.85±0.40

5.67±0.38

2.05±0.54

R

BDG

α

–

–

–

4.68±1.64

2

0.87

0.92

0.91

0.93

AIC

-258.89

-355.54

-324.18

-369.01

Km(µM)

0.16±0.02

0.36±0.02

0.43±0.03

0.28±0.03

Vmax(nmol/mg/min)

1.72±0.03

1.98±0.03

2.05±0.05

1.89±0.03

Ki(µg/mL)

1.15±0.17

11.91±0.58

9.11±0.54

4.50±0.84

R

TBG

α

–

–

–

3.87±1.07

2

0.91

0.94

0.93

0.95

AIC

293.62

204.39

258.12

186.10

R

65



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Page 66 of 89

Table S9 Apparent enzyme kinetic parameters of glucuronidation of bilirubin in the presence of DSI fitted to different inhibition kinetics model (n=3,x ±SD) Inhibition model

Competitive

Noncompetitive

Uncompetitive

Mixed-type

Glucuronides

Inhibition

Inhibition

Inhibition

Inhibition

BMG1

Km(µM)

0.24±0.03

0.51±0.03

0.62±0.05

0.40±0.03

Vmax(nmol/mg/min)

0.85±0.02

0.99±0.02

1.04±0.03

0.94±0.02

Ki(µg/mL)

1.78 ±0.24

14.53 ±0.60

10.48±0.59

6.89 ± 1.10

α

–

–

–

2.88 ±0.69

R2

0.93

0.96

0.94

0.97

-18.91

-150.85

-68.53

-168.64

Km(µM)

0.25±0.03

0.53±0.03

0.67±0.05

0.43±0.04

Vmax(nmol/mg/min)

0.98±0.02

1.16±0.02

1.22±0.03

1.10±0.02

Ki(µg/mL)

1.95 ±0.26

15.07±0.68

10.70 ±0.64

7.57± 1.34

AIC

BMG2

α

–

–

–

2.68 ±0.71

2

0.92

0.96

0.94

0.96

AIC

53.26

-62.68

7.91

-74.75

Km(µM)

0.05 ±0.02

0.20±0.02

0.22±0.03

0.12±0.02

Vmax(nmol/mg/min)

0.35±0.01

0.41±0.01

0.42±0.01

0.38±0.01

Ki(µg/mL)

0.50 ±0.21

13.75 ±0.83

11.47 ±0.80

2.95±0.80

R

BDG

α

–

–

–

7.33±2.65

2

0.87

0.91

0.90

0.93

AIC

-202.36

-281.36

-246.42

-306.06

Km(µM)

0.19±0.03

0.44±0.03

0.54±0.04

0.33±0.03

Vmax(nmol/mg/min)

2.16±0.05

2.54±0.05

2.64±0.07

2.40±0.05

Ki(µg/mL)

1.56 ±0.23

14.65±0.66

10.84 ±0.64

6.13 ± 1.05

R

TBG

α

–

–

–

3.39±0.86

2

0.92

0.96

0.94

0.96

AIC

367.75

251.26

323.39

231.03

R

66


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Table S10 Apparent enzyme kinetic parameters of glucuronidation of bilirubin in the presence of CDI fitted to different inhibition kinetics model (n=3,x ±SD) Inhibition model

Competitive

Noncompetitive

Uncompetitive

Mixed-type

Inhibition

Inhibition

Inhibition

Inhibition

Km(µM)

0.14±0.03

0.37±0.03

0.45 ±0.04

0.28±0.03

Vmax(nmol/mg/min)

0.64±0.02

0.75±0.02

0.78±0.02

0.71±0.02

Ki(µg/mL)

2.16 ±0.41

25.52 ±1.49

19.43 ±1.33

8.53 ±2.03

Glucuronides

BMG1

α

–

–

–

3.46 ±1.18

2

0.87

0.91

0.90

0.92

AIC

-27.78

-101.04

-67.17

-108.94

Km(µM)

0.16±0.03

0.40±0.03

0.50±0.05

0.33±0.04

Vmax(nmol/mg/min)

0.74±0.02

0.86±0.02

0.90±0.03

0.83±0.02

Ki(µg/mL)

2.81 ±0.53

29.66±1.85

22.16 ±1.60

9.85 ±2.65

R

BMG2

α

–

–

–

2.57 ±0.97

2

0.86

0.90

0.89

0.91

AIC

48.06

-23.43

3.75

-26.30

Km(µM)

0.04 ±0.02

0.20±0.02

0.24±0.03

0.12±0.03

Vmax(nmol/mg/min)

0.31±0.01

0.37±0.01

0.38±0.01

0.35±0.01

Ki(µg/mL)

0.52 ±0.29

18.55 ±1.27

15.50 ±1.20

3.75 ±1.20

R

BDG

α

–

–

–

6.81 ±2.88

2

0.84

0.89

0.87

0.90

AIC

-199.21

-268.18

-243.62

-282.61

Km(µM)

0.12±0.03

0.34±0.03

0.42±0.04

0.26±0.03

Vmax(nmol/mg/min)

1.68 ±0.04

1.98±0.04

2.05±0.06

1.88±0.05

Ki(µg/mL)

1.93 ±0.41

25.51 ±1.54

19.67 ±1.38

7.78 ±1.94

R

TBG

α

–

–

–

3.66 ±1.30

2

0.87

0.91

0.89

0.91

AIC

364.30

291.95

323.54

283.89

R

67



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Page 68 of 89

Table S11 Ki values of SAA, SAB and their equivalent Ki values in the DSI extract on human

UGT1A1-mediated

bilirubin

glucuronidation

at

different

substrate

concentrations a Bilirubin

DSI Ki

Equivalent SAAb@DSI

Equivalent SABb@DSI

glucuronides

(µg/mL)

Ki(µg/mL)c(contribution ratio)d

Ki(µg/mL) c (contribution ratio)d

BMG1

6.89 ±1.10

0.23±[email protected] (76.09%)

4.91±0.84 @1.00 (14.55%)

BMG2

7.57±1.34


5.94±1.16 @1.00 (13.22%)

BDG

2.95±0.80


2.05±0.54 @0.44 (14.92%)

TBG

6.13 ±1.05


4.50±0.84 @ 0.87 (14.13%)

a

- Concentrations of bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of SAA

were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. Concentrations of DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. The contents of SAA and SAB in the DSI extract were 2.54% and 10.37%, respectively. b

-Ki values of SAA (or SAB) .

c

- Equivalent DSI Ki values corresponding to SAA (or SAB) in the DSI extract, namely Ki values

of SAA (or SAB) in the DSI extract is convert to equivalent Ki values of DSI. d

- The contribution ratio of SAA (or SAB) to inhibition of bilirubin glucuronidation by DSI.

68


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Table S12 IC50 values of SAA, SAB and their equivalent IC50 values in the DSI extract on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrations a Bilirubin

Bilirubin

DSI IC50

Equivalent SAAb@DSI IC50

Equivalent SABb@DSI IC50

Conc.

glucuro-

(µg/mL)

(µg/mL)c(contribution ratio)d

(µg/mL) c(contribution ratio)d

(µM)

nides

0.2

BMG1

10.59±0.27

0.43±0.03@ 6.62 (62.55%)

5.21±0.54 @ 2.23 (21.08%)

BMG2

10.27±0.70

0.43±0.03@ 6.23 (60.66%)

5.13±0.15 @ 2.13 (20.76%)

BDG

7.88±0.68

0.36±0.03@ 4.38 (55.60%)

2.69±0.57@ 2.39 (30.38%)

TBG

9.29±0.76

0.44±0.02@ 4.98 (53.63%)

4.22±0.30 @ 2.12 (22.83%)

BMG1

11.14±0.60

0.51±0.01@ 6.18(55.48%)

6.87±0.89 @ 1.87(16.82%)

BMG2

10.55±1.02

0.51±0.04@ 5.54(52.54%)

6.34±0.32 @ 1.82(17.26%)

BDG

8.54±1.20

0.38±0.03@ 4.87 (57.08%)

3.38±0.11 @ 2.24(26.20%)

TBG

10.42±0.33

0.49±0.03@ 5.63(54.01%)

6.64±0.70 @ 1.70(16.27%)

BMG1

11.24±0.89



BMG2

11.85±0.39



BDG

9.40±0.67



TBG

10.89±0.86



BMG1

12.15±0.88

0.68±0.05@ 5.51 (45.38%)

9.16±0.94 @ 1.67 (13.75%)

BMG2

11.97±1.51

0.67±0.02@ 5.43 (45.36%)

8.81±1.12 @ 1.69 (14.09%)

BDG

10.93±1.69

0.46±0.02@ 6.60 (60.35%)

4.98±0.35 @ 2.49 (22.76%)

TBG

11.85±0.43

0.62±0.02@ 5.75 (48.55%)

8.36±0.74 @ 1.74 (14.70%)

BMG1

12.26±0.86

0.78±0.04@ 4.89 (39.92%)


BMG2

13.23±1.38

0.74±0.02@ 6.01 (45.41%)

9.97±0.41 @ 1.82 (13.76%)

BDG

13.11±1.23

0.48±0.03@ 9.09 (69.37%)

5.66±0.70 @ 3.15 (24.02%)

TBG

13.33±0.70

0.70±0.02@ 6.45 (48.37%)

9.41±0.28 @ 1.96 (14.69%)

BMG1

16.45±1.36

0.84±0.06@ 8.18 (49.74%)


BMG2

15.53±0.70

0.81±0.07@ 7.56 (48.70%)


BDG

16.39±2.47

0.55±0.02@ 12.41 (75.69%)

7.97±0.56 @ 3.50 (21.33%)

TBG

15.93±1.46

0.76±0.05@ 8.48 (53.24%)


BMG1

18.36±1.25

0.91±0.08@ 9.41 (51.25%)


BMG2

17.38±0.31

0.87±0.05@ 8.82 (50.74%)


BDG

18.03±2.11

0.58±0.03@ 14.24 (78.96%)


TBG

18.12±0.71

0.80±0.01@ 10.42 (57.53%)


BMG1

19.80±0.91

0.98±0.07@ 10.16 (51.32%)


BMG2

18.60±1.33

0.96±0.10@ 9.15 (49.21%)


BDG

20.80±2.38

0.71±0.04@ 15.48 (74.41%)


TBG

18.82±0.63

0.86±0.04@ 10.46 (55.58%)


0.4

0.75

1

1.5

2

3

5

69



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a

- Concentrations of bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of SAA

were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. Concentrations of DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. The contents of SAA and SAB in the DSI extract were 2.54% and 10.37%, respectively. b

c

- IC50 values of SAA (or SAB).

- Equivalent DSI IC50 values corresponding to SAA (or SAB) in the DSI extract, namely IC50

values of SAA (or SAB) in the DSI extract is convert to equivalent IC50 values of DSI. d

- The contribution ratio of SAA (or SAB) to inhibition of bilirubin glucuronidation by DSI.

70


Page 70 of 89

Page 71 of 89

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Table S13 Ki values of SAA, SAB and their equivalent Ki values in the CDI extract on human

UGT1A1-mediated

bilirubin

glucuronidation

at

different

substrate

concentrations a Bilirubin

CDI Ki

Equivalent SAAb@CDI

Equivalent SABb@CDI

glucuronides

(µg/mL)

Ki(µg/mL)c(contribution ratio)d

Ki(µg/mL) c (contribution ratio)d

BMG1

8.53 ±2.03


4.91±0.84 @0.13 (1.56%)

BMG2

9.85 ±2.65

0.26 ±[email protected] (84.48%)

5.94±1.16 @0.15 (1.49%)

BDG

3.75 ±1.20

0.14 ±[email protected] (59.73%)

2.05±0.54 @0.06 (1.65%)

TBG

7.78 ±1.94

0.22±0.03@ 6.14 (78.86%)

4.50±0.84 @0.12 (1.56%)

a

- Concentrations of bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of CDI

were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL. Concentrations of SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. The contents of SAA and SAB in the CDI extract were 2.23% and 0.90%, respectively. b

- Ki values of SAA (or SAB).

c

- Equivalent CDI Ki values corresponding to SAA (or SAB) in the CDI extract, namely Ki values

of SAA (or SAB) in the CDI extract is convert to equivalent Ki values of CDI. d

- The contribution ratio of SAA (or SAB) to inhibition of bilirubin glucuronidation by CDI.

71



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Page 72 of 89

Table S14 IC50 values of SAA, SAB and their equivalent IC50 values in the CDI extract on human UGT1A1-mediated bilirubin glucuronidation at different substrate concentrationsa Bilirubin

Bilirubin

CDI

Equivalent SAAb@CDI IC50

Equivalent SABb@CDI IC50

Conc.

glucuro-

IC50

(µg/mL)c(contribution ratio)d

(µg/mL) c(contribution ratio)d

(µM)

nides

(µg/mL)

0.2

BMG1

9.99±1.13

0.43±0.03@ 5.18 (51.81%)

5.21±0.54 @ 0.17 (1.73%)

BMG2

10.40±1.80

0.43±0.03@ 5.61 (53.93%)

5.13±0.15 @ 0.19 (1.82%)

BDG

7.31±1.59

0.36±0.03@ 3.31 (45.28%)

2.69±0.57@ 0.18 (2.45%)

TBG

9.18±2.00

0.44±0.02@ 4.27 (46.53%)

4.22±0.30 @ 0.18 (1.96%)

BMG1

12.54±1.56

0.51±0.01@ 7.93(58.90%)

6.87±0.89 @ 0.24 (1.76%)

BMG2

12.86±0.57

0.51±0.04@ 9.17 (63.31%)

6.34±0.32 @ 0.30 (2.06%)

BDG

8.94±0.85

0.38±0.03@ 7.26 (65.26%)

3.38±0.11 @ 0.33 (2.96%)

TBG

10.96±0.62


6.64±0.70 @ 0.24 (1.79%)

BMG1

13.47±0.83

0.60±0.03@ 6.74 (50.06%)


BMG2

14.48±1.07



BDG

11.12±1.09

0.41±0.04@ 6.73 (60.48%)


TBG

13.24±0.63



BMG1

14.52±1.28

0.68±0.05@ 6.91 (47.62%)

9.16±0.94 @0.21 (1.43%)

BMG2

16.04±1.87

0.67±0.02@ 8.56 (53.39%)

8.81±1.12 @ 0.26 (1.64%)

BDG

12.25±1.68

0.46±0.02@ 7.27 (59.39%)

4.98±0.35 @ 0.27 (2.21%)

TBG

14.27±0.98

0.62±0.02@ 7.32 (51.33%)

8.36±0.74 @ 0.22 (1.54%)

BMG1

16.73±1.15

0.78±0.04@ 8.00 (47.83%)

11.04±0.43 @ 0.23 (1.36%)

BMG2

18.58±2.69

0.74±0.02@ 10.40 (55.99%)

9.97±0.41 @ 0.31 (1.68%)

BDG

14.17±2.22

0.48±0.03@ 9.33 (65.83%)

5.66±0.70 @ 0.32 (2.25%)

TBG

17.01±0.68

0.70±0.02@ 9.22 (54.19%)

9.41±0.28 @ 0.28 (1.63%)

BMG1

18.90±0.98

0.84±0.06@ 9.48 (50.18%)

12.76±0.77@ 0.25 (1.33%)

BMG2

20.37±0.85

0.81±0.07@ 11.42 (56.08%)

11.64±0.66 @ 0.32 (1.58%)

BDG

15.57±1.33

0.55±0.02@ 9.83 (63.13%)

7.97±0.56 @ 0.27 (1.76%)

TBG

18.73±0.64

0.76±0.05@ 10.29 (54.96%)

10.34±0.65 @ 0.31 (1.63%)

BMG1

20.61±1.73

0.91±0.08@ 10.41 (50.51%)

13.84±1.37 @ 0.28 (1.34%)

BMG2

21.38±2.47

0.87±0.05@ 11.72 (54.80%)

13.17±0.95 @ 0.31 (1.46%)

BDG

18.02±2.55

0.58±0.03@ 12.48 (69.28%)

8.94±0.44 @ 0.33 (1.81%)

TBG

19.89±1.73

0.80±0.01@ 11.03 (55.44%)

11.32±0.51@ 0.31 (1.58%)

BMG1

24.86±1.51

0.98±0.07@ 14.06 (56.57%)

15.36±2.70 @ 0.36 (1.46%)

BMG2

26.78±1.26


15.73±1.01 @ 0.41 (1.53%)

BDG

21.04±2.92

0.71±0.04@ 13.90 (66.08%)

9.75±1.00@ 0.41 (1.94%)

TBG

22.36±1.39

0.86±0.04@ 12.96 (57.98%)

12.50±0.93 @ 0.36 (1.61%)

0.4

0.75

1

1.5

2

3

5

72


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a

- Concentrations of bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of CDI

were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL. Concentrations of SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Concentrations of SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. The contents of SAA and SAB in the CDI extract were 2.23% and 0.90%, respectively. b

- IC50 values of SAA (or SAB).

c

- Equivalent CDI IC50 values corresponding to SAA (or SAB) in the CDI extract, namely IC50

values of SAA (or SAB) in the CDI extract is convert to equivalent IC50 values of CDI. d

- The contribution ratio of SAA (or SAB) to inhibition of bilirubin glucuronidation by CDI.

73



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Supporting Information Figure Captions Figure S1. The chemical structures of bilirubin and its glucuronides. UCB was metabolized to BMG1, BMG2 and BDG by UGT1A1. UCB, unconjugated bilirubin； BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; UGT1A1, recombinant human UGT1A1 enzyme.

Figure S2. Representative chromatograms for bilirubin glucuronidation in human UGT1A1 incubation system. A, blank sample (the incubation sample in the absence of bilirubin); B, bilirubin standard sample (the incubation sample in the absence of UDPGA); C, bilirubin glucuronidation sample (the incubation sample in the presence of bilirubin and UDPGA) in the human UGT1A1 incubation system. UCB, unconjugated bilirubin; BMG1, bilirubin monoglucuronide1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide. Incubations were conducted with 1 µM bilirubin at 12.5 µg/mL UGT1A1 protein concentration for 15 min.

Figure S3. Representative MRM chromatograms (A, B, C) and MS/MS spectrum (D) of UCB, BMG1, BMG2, and BDG in the incubation samples for human UGT1A1 medicated-bilirubin glucuronidation. A, MRM chromatogram of UCB (retention time 26.03 min) from bilirubin standard sample (the incubation sample in the absence of UDPGA); B and C, MRM chromatograms of BMGs [including BMG1 (retention time 11.37 min) and BMG2 (retention time 10.50 min)] and BDG (retention time 7.04 min) 74


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from bilirubin glucuronidation sample (the incubation sample in the presence of bilirubin and UDPGA) in the human UGT1A1 incubation system. D, MS/MS spectrum of UCB (Q1/Q3 masses is 585.3/299.1), BMGs (BMG1 and BMG2, Q1/Q3 masses are both 761.3/585.3), and BDG (Q1/Q3 masses is 937.3/585.3). In the “D”, the molecular ion 937.3 [M+H]+, 761.3 [M+H]+ and their MS2 fragment at 585.3 [M+H-Glu]+ denoted BDG, BMGs (including the isomers BMG1 and BMG2) and BMGs, bilirubin monoglucuronides; UCB, respectively. UCB, unconjugated bilirubin； BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide. Incubations were conducted with 1 µM bilirubin at 12.5 µg/mL UGT1A1protein concentration for 30 min.

Figure S4 The calibration curves for UCB in the HLM and UGT1A1 standard samples. UCB, unconjugated bilirubin; HLM, human liver microsome; UGT1A1, UDP-glucuronosyltransferases1A1.The final concentrations of UCB in the standard samples were 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 8 µM, respectively. The limit of detection (LOD), the lower limit of quantification (LLOQ) and linear range for UCB in the both HLM and UGT1A1 standard samples were 0.005, 0.01, and 0.01–8 µM, respectively. The LOD was calculated as the final concentration of bilirubin producing a signal-to-noise ratio of 3. The LLOQ was considered as the lowest concentration of the calibration curve.

Figure S5.

Concentration-dependent inhibitory effects (IC50) of SAA on human 75



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UGT1A1-mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition (IC50) profiles of SAA on BMG1, BMG2, BDG and TBG at different concentrations of bilirubin, respectively. SAA, salvianolic acid A; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. The IC50 values are expressed as percentages relative to the control, which was determined in the absence of SAA. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor SAA were 0, 0.25, 0.49, 0.99, 1.48, 1.98, 2.47, and 2.96 µg/mL. Each data point represents the average of three determinations.

Figure S6.

Concentration-dependent inhibitory effects (IC50) of SAB on human

UGT1A1- mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition (IC50) profiles of SAB on BMG1, BMG2, BDG and TBG at different concentrations of bilirubin, respectively. SAB, salvianolic acid B; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. The IC50 values are expressed as percentages relative to the control, which was determined in the absence of SAB. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor SAB were 0, 0.72, 1.80, 3.59, 7.19, 17.97, 35.93, and 71.86 µg/mL. Each data point represents the average of three determinations. 76


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Figure S7. Concentration-dependent inhibitory effects (IC50) of DSI on human UGT1A1- mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition (IC50) profiles of DSI on BMG1, BMG2, BDG and TBG at different concentrations of bilirubin, respectively. DSI, Danshen Injection; BMG1, bilirubin monoglucuronides 1; BMG2, bilirubin monoglucuronides 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. The IC50 values are expressed as percentages relative to the control, which was determined in the absence of DSI. Concentrations of the substrate bilirubin were 0.2, 0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor DSI were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Concentrations of DSI were denominated in concentrations of total herbal extract in the injection. Each data point represents the average of three determinations.

Figure S8.

Concentration-dependent inhibitory effects (IC50) of CDI on human

UGT1A1-mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition (IC50) profiles of CDI on BMG1, BMG2, BDG and TBG at different concentrations of bilirubin, respectively. CDI, Compound Danshen Injection; BMG1, bilirubin monoglucuronides 1; BMG2, bilirubin monoglucuronides 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. The IC50 values are expressed as percentages relative to the control, which was determined in the absence of CDI. Concentrations of the substrate bilirubin were 0.2, 77



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0.4, 0.75, 1, 1.5, 2, 3, and 5 µM. Concentrations of the inhibitor CDI were 0, 2.7, 5.4, 10.8, 21.6, 43.2, 86.4, and 172.8 µg/mL. Concentrations of CDI were denominated in concentration of total herbal extract in the injection. Each data point represents the average of three determinations.

Figure S9. Inhibitory effects of DSI control on human UGT1A1-mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition column plots of DSI control on BMG1, BMG2, BDG and TBG at 0.75 µM bilirubin, respectively. DSI, Danshen Injection; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. Compared to that in absence of DSI control, the remaining percentage (i.e., % control activity) of BMG1, BMG2, BDG and TBG showed no significant difference (p > 0.05) at different DSI control concentrations. Concentrations of the DSI control (corresponds to the total herbal extract of Danshen, in fact in the absence of the extract) were 0, 2, 4, 8, 16, 32, 64, and 128 µg/mL. Each data point represents the average of three determinations.

Figure S10.

Inhibitory effects of CDI control (i.e., Jiangxiang Injection) on Human

UGT1A1-mediated bilirubin glucuronidation. BMG1, BMG2, BDG and TBG in the figure are the inhibition column plots of Jiangxiang (i.e., Lignum Dalbergia odorifera) Injection on BMG1, BMG2, BDG and TBG at 0.75 µM bilirubin, respectively. CDI, Compound Danshen Injection; BMG1, bilirubin monoglucuronide 1; BMG2, bilirubin 78


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monoglucuronide 2; BDG, bilirubin diglucuronide; TBG, total bilirubin glucuronides; TBG = BMG1 + BMG2 + BDG. Compared to that in the absence of Lignum Jiangxiang (Dalbergia odorifera) Injection, the remaining percentage (i.e., % control activity) of BMG1, BMG2, BDG and TBG showed no significant difference (p > 0.05) at different Jiangxiang Injection concentrations. Concentrations of Jiangxiang Injection (corresponds to the concentrations of total herbal extract of Lignum

Dalbergia odorifera) were 0, 1.37, 2.74, 5.48, 10.95, 21.90, 43.80, and 87.60 µg/mL. Each data point represents the average of three determinations.

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Figure S1. H2C O

CH3

O

H 3C

UGT1A1

NH

HN

N

HN

CH2

UGT1A1

H3C

H 2C O

O

CH3

H2 C

HOOC

CH3

O

CO2H

O

H3C NH

HN

N

HN

BMG2

CH2

O

CH3

H3C

OH

HO

O

O

OH

NH

HN

N

HN

CH2

H3C HO2C

H 3C

CH3

HOOC

HO

COOH

H2C O

UCB

O

HO

CH3

O O

OH

UGT1A1

HN

N

HN

H3C HO2C HO HO

CH2

UGT1A1

CH3 COOH

O O OH

O

O O

O

BDG

H 3C NH

CH3

O

BMG1

80


HO

CO2H OH OH

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Figure S2.

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Figure S3.

82


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Figure S4.

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Figure S5.

84


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Figure S6.

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Figure S7.

86


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Figure S8.

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Figure S9.

88


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Figure S10.

89


Inhibition of Human UGT1A1-Mediated Bilirubin Glucuronidation by

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