de-en han identification and characterization of human

1

Identification and Characterization of Human UDP-Glucuronosyltransferases

Responsible for the In Vitro Glucuronidation of Salvianolic acid A

De-en Hana,b, Yi Zhengb, Xijing Chenb, Jiake Heb, Di Zhaob, Shuoye Yangb, Chunfeng

Zhanga*,Zhonglin Yanga,*

aState Key laboratory of Natural Products and Functions, China Pharmaceutical

University, Ministry of Education, Nanjing, P. R. China.

bCenter of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University,

Nanjing, Jiangsu 210009, China.

Copyright 2012 by the Japanese Society for the Study of Xenobiotics (JSSX)C

Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGEReceived; February 25, 2012Accepted; April 19, 2012

Published online; April 27, 2012doi; 10.2133/dmpk.DMPK-12-RG-023

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Running title：Glucuronidation of salvianolic acid A in Vitro

Corresponding Author:

Chunfeng Zhang, PhD

State Key laboratory of Natural Products and Functions, China Pharmaceutical

University 24# Tong Jia Xiang, Nanjing 210009, PR China.

E-mail: [email protected] Phone: +86 25 8337 1694

Zhonglin Yang, PhD

State Key laboratory of Natural Products and Functions, China Pharmaceutical

University 24# Tong Jia Xiang, Nanjing 210009, PR China.

E-mail: [email protected] Phone: +86 25 8337 1694

Fax: +86 25 8337 1694

Number of text pages: 23

Number of tables: 2

Number of figures: 6

Drug Metabolism and Pharmacokinetics (DMPK) Advance Publication by J-STAGE

3

ABSTRACT:

Glucuronidation is an important pathway in the elimination of salvianolic acid A

(Sal A), however the mechanism of UDP-glucuronosyltransferases (UGTs) in this

process remains to be investigated. In this study, the kinetics s of salvianolic acid A

glucuronidation by pooled human liver microsomes (HLMs), pooled human intestinal

microsomes (HIMs) and 12 recombinant UGTs isozymes were investigated. The

glucuronidation of salvianolic acid A can be shown both in HLMs and HIMs with Km

values of 39.84 ± 3.76 and 54.04 ± 4.36µM, respectively. Among the 12 human UGTs

investigated, UGT1A1 and UGT1A9 were the major isoforms that catalyzed the

glucuronidation of salvianolic acid A (Km values of 29.72 ± 2.20 and 24.40 ± 2.60

µM). UGT1A9 showed the highest affinity of salvianolic acid A glucuronidation.

Furthermore, a significant correlation between salvianolic acid A glucuronidation and

propofol glucuronidation (a typical UGT1A9 substrate) was observed. The chemical

inhibition study showed that the IC50 for phenylbutazone inhibition of salvianolic acid

A glucuronidation was 50.3 ± 4.3µM, 39.4 ± 2.9µM by HLMs and UGT1A9,

respectively. Mefenamic acid inhibited salvianolic acid A glucuronidation in UGT1A1

and HLMs with IC50 values of >200 and 12.4 ± 2.2µM, respectively.

Keywords: Salvianolic acid A; UDP-glucuronosyltransferanses; Human Liver

Microsomes; UGT1A1; UGT1A9.


4

Introduction

Glucuronidation reaction catalyzed by the enzyme UDP-Glucuronosyltransferases,

accounts for over thirty-five percent of all phase II drug metabolism.1) Human

UGTs had been identified and classified into two subfamilies (UGT1 and UGT2)

based on gene sequence.2,3) Most of the human UGTs exhibited distinct,

overlapping substrates and selective inhibitors. A few specific reactions of certain

UGT isoforms have been presented, such as the glucuronidation for UGT1A4 of

trifluoperazine,4) UGT1A1 of bilirubin,5) UGT1A9 of propofol,6) UGT1A6 of

serotonin,7) and UGT2B7 of 3´-azido-3´-deoxythy-midine.8) The contribution of

different isoforms to glucuronidation is relied on the relative expression levels in

different tissues. For example, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9,

UGT2B4, UGT2B7 and UGT2B17 have been detected in liver, while UGT1A7,

UGT1A8, UGT1A10, and UGT2A1 are mainly presented in extrahepatic tissues,

although liver is generally considered as the major site of glucuronidation.9)

Glucuronides have more polarity than the parent drugs, and are thus more readily

excreted into bile and urine.

Salvia miltiorrhiza Bunge (Danshen), a well known Chinese herbal medicine, has

been widely used in the treatment of coronary heart disease, cerebrovascular disease,

hepatocirrhosis and hepatitis.10-12) The water-soluble components, extracted from

Salvia miltiorrhiza Bunge by water, are phenolic acids. These components are likely

to undergo phase II conjugation metabolisms,13,14) which result in the formation of a

covalent linkage between a functional group on the parent compound.15) Sal A (Fig. 1)


5

has been identified as one of the major water-soluble active constituents.16) Many

pharmacological studies have demonstrated that Sal A posses obvious biological

activities such as decreasing the infarct area and inhibiting cerebral edema of ischemic

rat by improving regional cerebral blood flow in the ischemic hemisphere, inhibiting

platelet aggregation and scavenging oxygen free radicals in rats.17-19) The

pharmacokinetic of Sal A in rat has been reported after intravenous administration of

Danshen injection;20 Pei et al. have researched the pharmacokinetic of Sal A in rats

after a single intragastric administration.21) Glucuronidation of Sal A has been

identified in rats and the majority of metabolites were appeared in the plasma as

conjugated forms.22) Although the pharmacokinetic and metabolites of Sal A have

been studied, the metabolism characterizes of glucuronidation were still scare.

Identification of isoforms responsible for drug metabolism is important to understand

the possible effect of enzymes on the pharmacokinetics of the administered drugs. In

addition, the identification of drug-metabolizing enzymes will be useful for

characterizing the role of these particular enzymes in potential clinical herbal and

chemical drug interactions and estimating the impact of genetic polymorphisms of

interested enzymes on drug disposition.

This study characterized the enzyme kinetics of Sal A glucuronidation in human

liver (HLMs) and intestine microsomes (HIMs). The recombinant human UGTs and

chemical inhibition study were also employed to identify the human UGT isoforms

responsible for Sal A glucuronidation.


6

Materials and Methods

Chemicals and reagents: Sal A was purchased from Sichuan victory

biotechnology Co., Ltd. (Si Chuan, China). Alamethicin, magnesium chloride,

D-saccharic acid 1,4-lactone, β-D-glucuronoside, UDPGA, propofol, phenylbutazone

and mefenamic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Bilirubin was purchased from the National Institute for the Control of Pharmaceutical

and Biological Products (Beijing, China). Pooled HLMs, HIMs and a panel of

recombinant human UGT SupersomesTM (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9,

1A10, 2B4, 2B7, 2B15, and 2B17) expressed in baculovirus infected insect cells were

purchased from BD Biosciences (Bedford, MA, USA). All other reagents were of

HPLC grade or of the highest grade commercially available.

Identification of Sal A glucuronide by HPLC-MS analysis: The total volumes of

200 µl incubation mixture comprised 50 mM Tris-HCl buffer (pH 6.5), 5 mM MgCl2,

25 µg/ml alamethicin, 0.1 mg/ml human liver microsomes, and 50 µM Sal A. After

preincubation at 37℃ for 10 min, the reaction was initiated by the addition of

UDPGA. At 10min, the reaction was terminated with 200 µl of cold acetonitrile

containing 1% formic acid and 0.3 µg of internal standard (Nateglinide). After

removal of the protein by centrifugation at 16000 g for 10 min, a HPLC-MS-2010EV

mass spectrometer system (Shimadzu, Japan) with electro-spray ionization interface

was used to identify Sal A glucuronide operating in both negative and positive ion

modes from m/z 100 to 800. The detector voltage was put at +1.70 and -1.85 kV for

positive and negative ion mode, respectively. The block heater and the curved


7

desolvation line temperature were both put at 250 °C. Other MS detection conditions

were set as follows: interface voltage, 40 V; voltage, 4 kV; drying gas (N2) pressure,

0.06 MPa; and nebulizing gas (N2) flow, tuned to be 1.5 L/min. Data processing was

performed using HPLC-MS Solution version 3.0 software (Shimadzu, Japan).

Glucuronidation assay of Sal A: The incubation of Sal A with HLMs and HIMs

were performed as depicted above. Detection of Sal A glucuronide was obtained with

injection of 20 µl of the centrifugal supernatant into the HPLC-UV (model 2010C,

Shimadzu, Japan) with the wavelength of 286 nm. Separation was performed on a

Thermo Scientific BDS HYPERSIL C18 column (150 mm × 2.1 mm, i.d., 1.7 µm

particle sizes) maintained at 30 °C and a flow rate of 1mL/min. The gradient, showed

as changes in mobile phase A (acetonitrile) and B (0.1% formic acid water), was as

follows: 0-2 min, hold at 80% B; 2-4 min, a linear decrease from 80% to 20% B; 4-7

min, hold at 80% B.

Quantification of the glucuronide in the incubation mixtures was established using

standard curves of Sal A because of the absence of Sal A glucuronide standards. The

outflows by HPLC from the incubation mixture with HLMs were obtained for the

quantification of Sal A glucuronide. Aliquot of the outflows were hydrolyzed by

β-glucuronidase performed as below. The thoroughly hydrolyzed Sal A glucuronide

was quantified as Sal A by HPLC-UV. The standard curve for Sal A was linear from

0.2 to 200 µM, and the correlation coefficient was > 0.99. The accuracy and precision

of the back-calculated values for each concentration were less than 15%.

Hydrolysis with β-glucuronidase: The eluate corresponding to Sal A glucuronides


8

was dried and redissolved in 1.0 ml of 0.05 M sodium acetate buffer (pH 5.0). Each

sample was incubated in the absence (control) and presence of 100 units of

β-glucuronidase at 37℃ for 24 h. Parts (100 µl) of each incubation were terminated

with 200 µL of ice-cold acetonitrile containing 1% formic acid. The supernatant was

injected to HPLC-UV as described above after removal of the protein by

centrifugation.

Glucuronidation by recombinant UGTs: Incubations in recombinant UGTs were

conducted as described above for liver microsome incubations. Human UGT1A1,

1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 were used to

investigate the glucuronidation of Sal A. Incubation mixtures (total volume 200 µl)

were prepared containing 50 mM Tris-HCl buffer (pH6.5), 5 mM MgCl2, 25 µg/ml

alamethicin, 0.1 mg/ml human liver microsomes, and Sal A. The final concentration

series of Sal A were 50, 200, and 400 µM, yielding a pH of 6.5 for all the UGTs

incubation system.

Kinetic analysis: Sal A concentration series from 1 to 200 µM were performed in

HLMs, HIMs, recombinant UGT1A1 and UGT1A9. Kinetic parameters were

evaluated from the suitable curves using the Graphpad Prism (GraphPad Software Inc.,

San Diego, CA), followed by nonlinear regression analysis. The following equation

was used:

V = Vmax × [S] / (Km + [S])

where V is the rate of reaction, Vmax is the maximum velocity, Km is the Michaelis

constant (substrate concentration at 0.5 Vmax), and [S] is the substrate concentration.


9

Correlation analysis: The glucuronidation of Sal A at a concentration of 50 µM

was conducted in HLMs to assess the individual differences of Sal A glucuronidation

and to correlate with the specific UGTs substrates (the final concentration also 50 µM).

Pearson’s product-moment correlation coefficient (r) was applied to estimate the

relationship between glucuronidation activities for Sal A in HLMs and bilirubin

glucuronidation activity for UGT1A1,4,23) propofol glucuronidation activity for

UGT1A9.24)

Chemical inhibition studies: Sal A glucuronidation activity in HLMs, HIMs,

UGT1A1 and UGT1A9 were determined in the absence or presence of

phenylbutazone or mefenamic acid, known UGTs inhibitors. Phenylbutazone is an

effective inhibitor for UGT1As25) and mefenamic acid is an effective inhibitor for

UGT1A9.26) Sal A (50 µM) was incubated in the absence or presence of either

mefenamic acid (1-50 µM) or phenylbutazone (10-500 µM). All incubations were

incubated for 10 min at a concentration of 0.1 mg/ml protein. The glucuronidation

activities were calculated as a percentage of control concentration, and the IC50,

indicating the concentration that inhibits 50% of control concentration, were

calculated by nonlinear curve fitting with Graphpad Prism (GraphPad Software Inc.,

San Diego, CA).

Results

Identification of Sal A glucuronide formed by HLMs: Sal A glucuronide by

HLMs was first identified by HPLC-ESI-MS (Fig. 2A and 3). Fig. 2A indicated

representative chromatograms of enzymatically Sal A glucuronide incubated with


10

HLMs in the presence of UDPGA. The Sal A glucuronide was eluted at 4.8min in the

reaction mixture. The negative ion mode was adopted for chemical identification

because it is more sensitive than the positive ion mode for Sal A and its metabolites.

The mass spectrum dominated by [M - H]- ion at m/z 669.19, corresponding to Sal A

glucuronides and followed by an ion at m/z 493.38, corresponding to the parent drug

Sal A - H with characteristic m/z 176 loss of the glucuronic acid moiety. The same

metabolite, Sal A glucuronide, was formed in HIMs (figures not shown).

Hydrolysis with β-glucuronidase: Sal A glucuronide was hydrolyzed by

β-glucuronidase and converted to parent Sal A (Fig. 2B). The increase of Sal A was

equivalent to the decrease of Sal A glucuronide after incubation with β-glucuronidase,

while no changes were detected in the control samples without β-glucuronidase.

Kinetics of Sal A glucuronidation in HLMs and HIMs: Kinetic analysis of Sal A

glucuronidation was investigated in HLMs and HIMs. The substrate concentration

glucuronidation velocity curves showed typical Michaelis-Menten kinetics (Fig. 4A

and 4B). Moreover, an Eadie-Hofstee plot was monophasic. The kinetic parameters

fitted the data points to the Michaelis-Menten equation displayed in Table 1.

Incubation of Sal A from 1 to 200 µM with HLMs revealed that the Km, and Vmax

values for Sal A glucuronide were 39.84 ± 3.76 µM, and 23.59 ± 0.52 µmol/(min

mg(protein)), respectively. Simultaneously, incubation of appropriate Sal A

concentrations with HIMs revealed that the Km, and Vmax values for Sal A glucuronide

were 54.04 ± 4.36 µM, 9.28 ± 0.19 µmol/(min mg(protein)), respectively. The in

vitro total intrinsic clearances (calculated as Vmax/Km) of the glucuronidation of Sal A


11

in HLMs and HIMs were 0.59 and 0.17 µl/min/mg protein, respectively.

Sal A glucuronidation in recombinant UGT isoforms: Twelve recombinant

UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15,

and 2B17 were applied to catalyze Sal A glucuronidation and to identify the isozymes

involved in the metabolism. At all of the three Sal A concentrations, UGT1A1 and

UGT1A9 displayed the highest and prominent catalytic activity to Sal A

glucuronidation (Fig. 5). Kinetics analyses of the Sal A glucuronidation in

recombinant UGT1A1 and UGT1A9 were investigated (Fig. 4C and 4D). Sal A

glucuronidation by recombinant UGT1A1 and UGT1A9 also revealed typical

Michaelis-Menten kinetics, similar to that by HLMs and HIMs. Moreover, each

Eadie-Hofstee plot displayed monophasic. The kinetic parameters fitted the data

points to the Michaelis-Menten equation displayed in Table 1. Incubation of Sal A

from 1 to 200 µM with recombinant UGT1A1 and UGT1A9 revealed that the Km,

Vmax, and CLint values for Sal A glucuronide were 29.72 ± 2.20 and 24.40 ± 2.60 µM,

13.65 ± 0.22 and 12.71 ± 0.28 µmol/ (min mg(protein)), 0.46 and 0.52 ml/(min

mg(protein)), respectively. UGT1A3, UGT1A7 and UGT1A8 also showed slight

activity, while other UGT isozymes did not catalyze this reaction. This result

demonstrates that UGT1A1 and UGT1A9 are the most important isoforms involved in

the glucuronidation of Sal A.

Correlation study: Correlation analyses were performed between the Sal A

glucuronosyltransferase activity and UGT1A1 probe substrate bilirubin and UGT1A9

probe substrate propofol glucuronosyltransferase activities in HLMs. The Sal A


12

glucuronosyltransferase activities were significantly related with the propofol

glucuronosyltransferase activities (R2 = 0.961, P < 0.001) (Fig. 6B), but the Sal A

glucuronosyltransferase activities were less extent related with the bilirubin (R2 =

0.637, P < 0.001) glucuronosyltransferase activities (Fig. 6A).

Chemical inhibition analysis: The inhibitory effects of mefenamic acid and

phenylbutazone on Sal A glucuronidation in HLMs, HIMs, UGT1A1, and UGT1A9

were investigated. As shown in Table 2, the UGT activity for the formation of the

metabolites in HLM, HIM, UGT1A1, and UGT1A9 was prominently inhibited by

phenylbutazone. Similar IC50 values for phenylbutazone inhibition of HLMs, HIMs,

UGT1A1, and UGT1A9 were displayed as follow: 50.3 ± 4.3 µM, 53.2 ± 4.6 µM,

43.1 ± 5.2 µM, and 39.4 ± 2.9 µM, respectively. Mefenamic acid seemed generally to

be more effective in inhibition than phenylbutazone besides UGT1A1, Moreover,

mefenamic acid displayed differential inhibitory profile on the enzyme activity,

however, the inhibition of UGT1A1 (IC50 over 200 µM) was more weaken than that

investigated for other enzyme preparations. The IC50 values for mefenamic acid

inhibition of HLMs, HIMs, and UGT1A9 were calculated to be 12.4 ± 2.2 µM, 9.8 ±

1.1 µM, and 9.1 ± 1.6 µM, respectively.


13

Discussion

In this study, we examined Sal A glucuronidation in HLMs, HIMs, human

recombinant UGTs and compared the enzyme kinetic parameters. Shen et al. had

found that glucuronidation was an elimination pathway of Sal A in rats. 23) In this

study, the same metabolite was detected in human microsomal incubations. Based on

the screening experiment conducted with baculosome of 12 recombinant UGTs,

UGT1A1, UGT 1A3, UGT 1A7, UGT1A8 and UGT1A9 were isoforms responsible

for the glucuronidation of Sal A. Due to the relatively pronounced catalytic activity,

kinetic experiments were carried out in recombinant UGT1A1 and UGT1A9.

It is noticeable that UGT1A9 has been involved in the metabolic elimination of

many types of endogenous compounds (fatty acids) 27-29) and xenobiotic compounds

(phenols, pimary amines, and flavones)30). The kinetic studies of Sal A

glucuronidation by UGT1A1 and UGT1A9 revealed that these two isoforms had

similar apparent intrinsic clearance (Vmax/Km) values, although the apparent intrinsic

clearance value of UGT1A9 was higher than that of UGT1A1, which could be

contributed to the relative low Km value of UGT1A9 (Table 1). Moreover, the Km

values in recombinant UGT1A9 and UGT1A1 were lower than those in HLMs and

HIMs. It has been reported that long-chain unsaturated fatty acids could be considered

as potent competitive inhibitors of UGT1A1, UGT1A6, and UGT1A9.27) In addition,

correlation analysis and chemical inhibition study were applied to distinguish the

relative contributions of the UGT isoforms. The high correlation between Sal A

glucuronide formation and propofol O-glucuronidation was showed in Fig. 6B, and


14

the inhibitory effects of phenylbutazone and mefenamic acid on HLMs and HIMs

with similar IC50 values were listed in Table 2. Mefenamic acid is a known potent

inhibitor of UGT1A9 and UGT2B7,26,31) and was considered as a specific inhibitor of

UGT1A9 in this study，because UGT2B7 was not involved in Sal A glucuronidation.

The intrinsic clearance value in HIMs was about one-third of that in HLMs, which

suggested that the intestine might not be the main glucuronidation metabolic site but

intestine could still attribute to presystemic elimination after oral administration of Sal

A. Therefore, the liver might play important role in Sal A metabolic clearance via

glucuronidation. However, because no HLMs and HIMs were obtained from the same

donor, we could not detect their exact contribution. Therefore, considering the

intrinsic clearance of Sal A by glucuronidation, how to maintain the therapeutic

plasma concentration of Sal A remains to be studied. In addition, the role of Sal A

glucuronidation activity in the intestine needs to be further investigated.

In conclusion, Sal A is glucuronidated in the presence of UDPGA in vitro

incubation systems. UGT1A1 and UGT1A9 are identified as the major isoforms

responsible for Sal A glucuronidation.


15

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efficiently glucuronidated by microsomes of the human gastrointestinal tract.

Biochim. Biophys. Acta., 1675:120-129 (2004).

30) Tukey, R.H., and Strassburg, C.P.: Human UDP-glucuronosyltransferases:

metabolism, expression, and disease. Annu. Rev. Pharmacol. Toxicol.,

40:581-616 (2000).

31) Gaganis, P., Miners, J.O., and Knights, K.M.: Glucuronidation of fenamates:

kinetic studies using human kidney cortical microsomes and recombinant

UDP-glucuronosyltransferase (UGT) 1A9 and 2B7. Biochem. Pharmacol.,

73:1683-1691 (2007).


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Table 1. Kinetic parameters of salvianolic acid A glucuronidation in HLMs, HIMs,

and recombinant UGT1A1 and UGT1A9.

Source of

UGT

Km (µM) Vmax (µmol/(min

mg(protein)))

Vmax/Km (ml/(min mg(protein)))

HLMs 39.84 ± 3.76 23.59 ± 0.52 0.59

HIMs 54.04 ± 4.36 9.28 ± 0.19 0.17

UGT1A1 29.72 ± 2.20 13.65 ± 0.22 0.46

UGT1A9 24.40 ± 2.60 12.71 ± 0.28 0.52

salvianolic acid A was incubated with liver microsomes, recombinant UGT1A1 and

UGT1A9, and UDP-glucuronic acid for 10 min. The kinetic parameters were

calculated with GraphPad Prism software. Each value represents mean ± S.D. of

triplicate determinations.


21

Table 2. Inhibition of salvianolic acid A glucuronidation in HLMs, HIMs, and

recombinant UGT1A1 and UGT1A9 with mefenamic acid and phenylbutazone.

IC50 (µM) UGT Source

Mefenamic Acid Phenylbutazone

HLMs 12.4 ± 2.2 50.3 ± 4.3

HIMs 9.8 ± 1.1 53.2 ± 4.6

UGT1A1 IC50 > 200 43.1 ± 5.2

UGT1A9 9.1 ± 1.6 39.4 ± 2.9

Salvianolic acid A (50 µM) was incubated with microsomes (0.1 mg/ml) and UDPGA

(5 mM) for 10 min in the presence of mefenamic acid (1-50 µM) or phenylbutazone

(10-500 µM). The IC50 values are given as mean ± computer-calculated S.D. of the

triplicate determinations.


22

Figure legends

Fig. 1 - Chemical structure of salvianolic acid A.

Fig. 2 - Representative HPLC-ESI-MS chromatograms of the formation of salvianolic

acid A glucuronide in human liver microsomes and hydrolyzing of salvianolic acid A

glucuronide by β-D-glucuronidase. (A) human liver microsomes (0.1 mg/ml protein)

was incubated at 37℃ for 10 min with 50 µM salvianolic acid A in the presence of

5mM UDPGA; (B) salvianolic acid A glucuronide was hydrolyzed by

β-D-glucuronidase at 37℃ for 24h. Metabolite, salvianolic acid A, and Nateglinide

were eluted at 4.8, 5.5, and 8.5 min, respectively.

Fig. 3 - Representative mass spectrum of salvianolic acid A glucuronide formed from

salvianolic acid A by human liver microsomes. The spectrum was taken at the

retention time of 4.8min in Fig. 1A.

Fig. 4 - Kinetics of salvianolic acid A glucuronidation in human liver microsomes (A),

human intestinal microsomes (B), recombinant UGT1A1 (C), and recombinant

UGT1A9 (D). The concentration of salvianolic acid A ranged from 1 to 200 µM. The

formation of salvianolic acid A glucuronide was determined as described above. Each

inset shows the Eadie-Hofstee plot of the experimental data. Each incubation was

performed by triplicate determinations.


23

Fig. 5 - Salvianolic acid A glucuronidation by recombinant human UGTs. salvianolic

acid A was incubated with recombinant human UGT1A1, UGT1A3, UGT1A4,

UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15,

and UGT2B17 at 37°C for 10 min. A substrate concentration of (50, 200, and 400 µM)

and a protein concentration of 0.1 mg of protein/ml were used. Data represent the

mean of triplicate determinations.

Fig. 6 - Correlation between overall salvianolic acid A glucuronidation and other

glucuronosyl-transferase activities in human liver microsomes. salvianolic acid A (50

µM) was incubated with individual HLMs (0.1 mg of protein/ml) at 37°C for 10 min.

bilirubin glucuronidation (A) and propofol glucuronidation (B), both typical reactions

for UGT1A1 and UGT1A9, respectively. Data represent the mean of triplicate

determinations.


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Fig.1


25

Fig.2


26

Fig. 3


27

Fig.4


28

Fig.5


29

Fig.6


de-en han identification and characterization of human

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