The enantioselective metabolic mechanism of quizalofop-ethyl and quizalofop-acid enantiomers in animal: Protein binding, intestinal absorption, and: In vitro metabolism in plasma and the microsome

Article abstract of DOI:10.1039/c6ra19391a

To investigate the metabolic mechanism and enantioselectivity of the chiral herbicide quizalofop-ethyl and its primary chiral metabolite quizalofop-acid in animals by oral administration, the effects of the processes involved in digestion, absorption, tra

Full text of DOI:10.1039/c6ra19391a

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The enantioselective metabolic mechanism of quizalofop-ethyl and
quizalofop-acid enantiomers in animal: protein binding, intestinal
absorption, and in vitro metabolism in plasma and the microsome
Yiran Liang, Peng Wang, Donghui Liu, Jing Zhan, Mai Luo, Jiajun Han, Xu Jing,
Guojun Yao, Zhiqiang Zhou*
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College
of Science, China Agricultural University
Abstract
To investigate the metabolic mechanism and enantioselectivity of the chiral
herbicide quizalofopꢀethyl and its primary chiral metabolite quizalofopꢀacid in
animals by oral administration, the effects of the processes involved in digestion,
absorption, transportation and metabolism, such as protein binding (pepsin, trypsin
and serum albumin), intestinal absorption (everted gut sac), and degradation (plasma,
liver microsome and cytosol) were studied in vitro. Protein binding experiments
showed that quizalofopꢀethyl interacted with pepsin, trypsin and serum albumin
without enantioselectivity and the proteins did not stimulate metabolic processes that
would produce quizalofopꢀacid. In the everted gut sac, quizalofopꢀethyl was degraded
rapidly to quizalofopꢀacid without enantioselectivity and the preferential absorption of
(+)ꢀquizalofopꢀacid was observed throughout the gut. In plasma, the metabolic
process of quizalofopꢀethyl was enantioselective with preferential degradation of
(+)ꢀquizalofopꢀethyl and generation of (+)ꢀquizalofopꢀacid. However there was no
enantioselectivity in the degradation of quizalofopꢀethyl or formation of
quizalofopꢀacid in liver microsome. The metabolism of quizalofopꢀacid was
noenantioselective by liver microsome, sulfotransferase and glucuronosyltransferase.
It could be inferred that the enzymes present in the lumen of intestine, blood and liver
might facilitate the rapid transformation of quizalofopꢀethyl to quizalofopꢀacid after
*
Corresponding author at: Department of Applied Chemistry, China Agricultural
University, Yuanmingyuan west road No. 2, Beijing 100193, P.R. China
Tel: +8610 62733547 Email: zqzhou@cau.edu.cn

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oral administration, and the enantioselective absorption of quizalofopꢀacid
enantiomers by intestine was the main reason for the enantioselectivity observed in
rats in vivo.
KEYWORDS: quizalofopꢀethyl; metabolism; enantioselectivity; mechanism
1. Introduction
Pesticides have been widely used in agriculture in the control of pests, weeds and
plant diseases, which have drawn attention as environmental pollutants. Chiral
pesticides made up about 30% of all pesticides used and the proportion is increasing,
with more complex structures coming into use. Enantiomeric pairs of chiral pesticides
share identical physical and chemical properties. However, individual enantiomers
can interact enantioselectively with enzymes or other naturally occurring chiral
1
2
molecules , so they have different physiological and biochemical properties . Some
chiral pesticides are still used as racemates despite the fact that the pesticidal activity
of the individual enantiomers may be different. Sometimes only one enantiomer has
the desired activity and the other may be less effective or even have adverse effects on
nonꢀtarget species. Enantioselective degradation of the enantiomers has been widely
observed in the field and under laboratory conditions 3ꢀ9. Information on
stereoselective behaviors of chiral pesticide contributes to minimize environmental
risk and improve the evaluation of safety conditions for humans and other animals.
The biological behaviors of chiral pesticides can also be stereoselective in
animals. For example, many researchers have reported high enantiomeric ratios for
ɑꢀhexachlorocyclohexane in wildlife 10ꢀ15. Zhang et al. demonstrated that the
degradation of (ꢀ)ꢀfenoxapropꢀethyl was much faster than (+)ꢀfenoxapropꢀethyl in
rabbit plasma 16. Brad et al. proved a greater enantioselective biotransformation rate
of the (+)ꢀenantiomer than (ꢀ)ꢀenantiomer of fipronil in rainbow trout 17. In most cases,
the enantioselectivity of drugs has been mainly attributed to uptake, distribution,
metabolism and excretion. Occasionally, this can also result from selective protein
binding or tissue transport 18ꢀ22. To investigate the enantioselective behaviors of chiral
compounds in animals, in vitro experiments are usually conducted. The liver

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microsome is one of the more widely used in vitro metabolic models because it
contains abundant cytochrome P450 enzymes, which plays an important role in drug
elimination. Everted gut sacs have a relatively large surface area and mucus layer
available for absorption, so they are widely used as an in vitro model to study the
kinetics and mechanisms of drug absorption. In general, in vitro experiments can offer
more information regarding the mechanisms and reduce the massive use of animals,
so they are widely used.
Quizalofopꢀethyl (Fig. 1) is a phenoxy propionate herbicide consisting of a pair
of enantiomers, with the (+)ꢀenantiomer possessing more herbicidal activity than the
(ꢀ)ꢀenantiomer. Quizalofopꢀethyl can undergo deesterification very quickly in many
organisms and its primary metabolite is quizalofopꢀacid. Quizalofopꢀethyl is used for
postemergence control of annual and perennial grass weeds in broadꢀleafed crops
such as rape, sunflower, soybeans, cotton, peanuts, flax, alfalfa, and some vegetables.
Because of the broad scope of its application, humans and many other animals are
exposed to this compound via active or passive processes. The maximum residue limit
(MRL) for quizalofopꢀethyl was 3 mg/kg in sunflower seeds and 1.5 mg/kg in
rapeseed in Canada. In a previous study, quizalofopꢀethyl has been proven to affect
2
the development of rats, leading to a significant decrease in neonatal survival rates .
Results also showed that (+)ꢀquizalofopꢀethyl could induce
a
mixed
cholestatic/hepatocellular liver injury 23. To assess the potential risk of pesticides to
animals, it is important to understand their fate in those animals. For chiral
enantiomers, it is also necessary to determine the mechanism underlying
stereoselective behaviors.
In our previous work performed in rats24, quizalofopꢀethyl was found to be
metabolized very quickly when administered intragastrically, and high concentrations
of the metabolite quizalofopꢀacid was observed in the blood and tissues. In addition,
enantioselectivity of quizalofopꢀethyl and its chiral metabolite was observed.
However, the mechanisms underlying the degradation and enantioselective behaviors
are unclear. In most cases, the fate of drugs in animals after intragastric administration
might be affected by the enzymes in the stomach and intestine, gut absorption, plasma

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and liver metabolism. For this reason, in this work, the enantioselective protein
(pepsin, trypsin and serum albumin) binding, absorption (everted gut sac), and the
metabolism of quizalofopꢀethyl by plasma， rat liver microsome and cytosol were
studied in vitro. The data from in vitro analysis may elucidate the metabolic
mechanism and enantioslectivity in vivo and may improve understanding of the fate
of chiral pesticides in animals.
2 Materials and methods
2.1 Chemicals and Reagents
Racemicꢀquizalofopꢀethyl (racꢀQE, 98%) and racemicꢀquizalofopꢀacid (racꢀQA,
99%) were obtained from Institute for the Control of Agrichemicals, Ministry of
Agriculture of China. Trifluoroacetic acid (TFA), ethyl acetate, ethanol, acetonitrile,
isopropanol, porcine pepsin and trypsin were obtained from Alladin Reagent
(Shanghai, China). βꢀNicotinamide adenine dinucleotide phosphate (NADPH),
uridine 5’ꢀdiphosphate glucuronic acid (UDPGA), 3’ꢀphosꢀphoadenosine
5’ꢀphosphosulfate (PAPS), alamethicin and serum albumin were purchased from
SigmaꢀAldrich (St. Louis, MO). Other solvents or chemicals used in this study were
of analytical grade.
2.2 Animals
SpragueꢀDawley male rats weighing 200ꢀ250 g were obtained from
Experimental Animal Research Institute of China Agriculture University. Prior to the
experiments, the rats were housed in polycarbonate cages placed in wellꢀventilated
house conditions with a 12:12 h light: dark photoperiod. They were allowed free
access to water and standard rat food. The rats were allowed to acclimatize in the
laboratory for seven days before the experiments started. Before the operation, the rats
were fasted overnight. This study and all animal experiments were approved by the
local ethics committee (Beijing Association For Laboratory Animal Science) and
carried out in strict accordance with the Experimental Animal Regulation Ordinances
defined by China National Science and Technology Commission.
2.3 Protein binding of QE with pepsin, trypsin and serum albumin

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Protein binding was studied in vitro by equilibrium dialysis. After 1 mL of
pepsin solution (0.08 mol/L HCl containing 10 g/L pepsin, pH=1.5), trypsin solution
(0.05 mol/L KH2PO4 and Na2HPO4 buffer solution containing 22 g/L trypsin, pH 6.8)
or albumin solution (50 mmol/L TrisꢀHCl buffer solution containing 0.1 mol/L NaCl,
pH 7.4) were placed in a dialysis tube, the dialysis tube was placed in 20 mL
corresponding buffer solution at 37 °C and preincubated for 5 min. RacꢀQE (dissolved
in alcohol, 1% of final volume) was added to the dialysis tube at 10, 30, 50, 70 and
100 mg/L, and the dialysis tube was then incubated at 37 °C. After 24 h, 0.5 mL of the
outer dialysate was withdrawn and added to 0.5 mL 10% HClO4. The samples were
considered invalid if there was white floc precipitation present. Then 1 mL outer and
inner dialysates were collected and 5 mL of iceꢀcold ethyl acetate was immediately
added. The mixture was then stirred for 5 min to terminate the reaction.
2.4 Enantioselective metabolism of QE and QA by rat plasma, liver microsome and
cytosol
Rat blank blood was separated from drugꢀfree heart blood and centrifuged at
4000 rpm for 10 min at 4 °C to obtain plasma. Then the plasma was diluted by
TrisꢀHCl buffer (pH 7.4) to produce the 10% (v/v) plasma solution. RacꢀQE
(dissolved in alcohol, 1% of final volume) was added into the 10% plasma solution
(preincubated at 37 °C for 5 min) at a final concentration of 10 mg/L. Samples (1 mL)
were taken out at 0, 5, 15, 30 and 60 min, and 5 mL of iceꢀcold ethyl acetate was
immediately added. The mixture was then stirred for 5 min to terminate the reaction.
Rat liver microsome and cytosol from male SpragueꢀDawley rats that used to
study the methylated and hydroxylated effects of CYP 450 was prepared according to
a previously described method 25. In brief, after rats were sacrificed, their livers were
quickly removed and immediately placed on ice and thoroughly washed with iceꢀcold
1.15% KCl solution. The pooled livers were minced and immediately homogenized
into three volumes of iceꢀcold SET solution (1 mM ethylenediamine tetraꢀacetic acid
and 50 mM TrisꢀHCl, pH 7.4). Liver microsomes were used to determine CYP 450
and glucuronosyltransferase activity for QA and cytosols were used for the
measurement of sulfotransferase activity for QA. These were prepared using

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differential centrifugation. Protein levels were determined using the Bradford method
26
.
The metabolism of QE in the liver microsome was carried out in a reaction
TrisꢀHCl buffer (50 mM, pH 7.4) containing microsomal protein (1 mg/mL). The
microsomal suspension was preincubated for 5 min at 37 °C and then initiated by
addition of racꢀQE (10 mg/L, dissolved in alcohol, 1% of final volume). The
metabolism of racꢀQA by liver microsome was carried out in a reaction mixture
containing TrisꢀHCl buffer (50 mM, pH 7.4), microsomal protein (1 mg/mL),
NADPH (1.0 mM) and racꢀQA (10 mg/L, dissolved in alcohol, 1% of final volume).
After preincubation for 5 min at 37 °C, the reaction was regenerated by adding
NADPH (1.0 mM).
The metabolism of racꢀQA by sulfotransferase and glucuronosyltransferase was
carried out in this study. The sulfotransferase (SULT) activity for QA was determined
in the reaction mixture consisted of hepatic cytosolic protein (0.1 mg/mL), TrisꢀHCl
buffer (50 mM, pH 7.4), MgCl2 (5 mM) and PAPS (0.2 mol/L). After preincubation
for 5 min at 37 °C, the reaction was regenerated by addition of racꢀQA (10 mg/L,
dissolved in alcohol, 1% of final volume). The glucuronosyltransferase (UGT)
activity for QA was studied in the incubation mixture containing TrisꢀHCl buffer (50
mM, pH 7.4), microsomal protein (0.5 mg/mL), alamethicin (1 mg/10 mg protein) and
racꢀQA (10 mg/L, dissolved in alcohol, 1% of final volume). The reaction was
initiated by the addition of 2 mmol/L UDPGA.
Samples were collected at 0, 10, 20, 40 and 60 min. Then 5 mL of iceꢀcold ethyl
acetate was immediately added. The mixture was then stirred for 5 min to terminate
the reaction.
2.5 In vitro everted gut sac absorption
The aim of this study was to assess the stereoselective absorption of QE and QA
by intestine in vitro. The Sharma method was used for everted rat gut sac experiments
27. After an overnight fast, rats were sacrificed by cervical dislocation, and then the
intestine was rapidly removed and placed in a beaker containing ice cold
KrebsꢀRinger (KꢀR) culture solution (containing 7.8 g NaCl, 0.35 g KCl, 1.37 g

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NaHCO3, 0.02 g MgCl2, 0.32 g NaH2PO4 and 1.48 g glucose in 1000 mL distilled
water). The jejunal segment (10 cm) was flushed with the KꢀR culture solution to
remove the intestinal contents and to remove the underlying mesenterium. The jejunal
segment was then tied at one end, everted with a plastic pipe and removed the mucus
carefully. The serosa compartment was filled with 2 mL of KꢀR culture solution and
the everted intestinal sac was suspended in the KꢀR culture solution (maintained at
37 °C) and continually aerated with 95% O2 and 5% CO2. Then racꢀQE (dissolved in
alcohol, 1% of final volume) was added into the KꢀR culture solution at a
concentration of 10 mg/L. Aliquots of 2 mL serosal solution were taken out from both
the mucosal and serosal side at different points in time (10, 30, 60, 90 and 150 min).
The serosa compartment was immediately washed three times with KꢀR culture
solution and refilled with 2 mL of fresh KꢀR culture solution at each sampling point to
keep the volume of the serosal solution constant.
The integrity of the gut sac incubated with or without QE was verified according
to the previous procedure based on the ratio of the concentration of glucose in the
mucosal side to the serosal side 28. The sacs were incubated in the presence or absence
of racꢀQE (dissolved in alcohol, 1% of final volume). Samples were taken out exactly
as described above and the concentrations of glucose were measured by the glucose
assay kit from Nanjing Jiancheng Bioengineering Institute (China).
2.6 Extraction procedure
One milliliter of the samples (rat plasma, liver microsome, buffer solution,
pepsin and trypsin solution) was extracted by the addition of 5 mL of ethyl acetate and
then acidified with 100 ꢁL of 1 mol/L HCl. The mixtures were vortexed for 5 min and
centrifugation at 3500 rpm for 5 min, the organic phase was transferred into a test
tube. The extraction was repeated. The combined supernatant extract was evaporated
to dryness under nitrogen. Then the residue was redissolved in 0.5 mL of isopropanol,
and filtered through a 0.22 ꢁm syringe filter.
2.7 Analytical procedures
HPLC analysis for the determination of QE and QA was performed using an
Agilent 1200 series HPLC system consisting of Chiralpak IC column (250 × 4.6 mm,

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Daicel Chemical Industries, Tokyo, Japan) with variable wavelength detectorꢀG1314B.
Column temperature was controlled by a column attemperator (Tianjin Automatic
Science Instrument Co. Ltd, China). The mobile phase consisted of nꢀhexane:
isopropanol: TFA (92: 8: 0.1%, v/v/v). The flowꢀrate was maintained at 0.6 mL/min,
and the detection was performed at a wavelength of 230 nm under constant
temperature (15 °C).
2.8 Data analysis
Pesticide protein binding rate was calculated by P = (C1ꢀC2)/C1 × 100%, where
C1 and C2 were the concentrations of pesticide in the inner sideand outer of dialysate.
Enantiomeric fraction (EF) was used to denote stereoselectivity, defined as EF= peak
areas of (+)/ [(+) + (ꢀ)]. The EF of racemate was 0.5, whereas preferential degradation
of one enantiomer made EF deviating 0.5.
All values were analyzed by analysis of variance (ANOVA) and expressed as the
mean ± SD. P values lower than 0.05 were considered to be of statistical significance.
3. Results and discussion
3.1 Method validation
Fig. 1 showed the typical chromatogram of the separation of the enantiomers of
QE and QA. The two pairs of enantiomers could be completely separated on HPLC
with Chiralpak IC column using nꢀhexane/isopropanol/TFA (92: 8: 0.1%, v/v/v) at 0.6
mL/min. As shown in Table 1, for all the enantiomers in the concentration range of
0.3ꢀ6 mg/L, the correlation coefficients (R2) were higher than 0.994, indicating a
functional linear relationship between the concentration and peak area. The extraction
efficiency of (+)ꢀQE, (ꢀ)ꢀQE, (+)ꢀQA and (ꢀ)ꢀQA throughout the linear range ranged
from 88% to 108% with RSD of 3% to 10% (Table S1). The limit of detection (LOD)
and quantitation (LOQ) for all enantiomers were 0.1 and 0.3 mg/L respectively.
3.2 Protein binding of QE with pepsin, trypsin and serum albumin
No QE was detected in the blood, kidneys, lungs, livers, brain, urine or feces
after intragastric administration of QE to rat in the previous study but high levels of
24
(+)ꢀQA were . Dietary food intake is one major route of pesticide into human body.

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When pesticide enters the digestive system such as stomach or intestine, digestive
proteases (pepsin and trypsin) may be the direct binding targets. After being absorbed,
pesticide may bind to serum albumin which is the most soluble protein in plasma and
can combine with numerous exogenous substances. As the chiral nature and the
variety of functional groups presenting at the surface, pepsin, trypsin and serum
albumin have the ability to discriminate a chiral molecule29. It is therefore important
to consider the influences of the proteinꢀpesticide binding effects of pepsin, trypsin
and serum albumin on the enantioselectivity. Because QE enters the digestive system
mainly in its parent form and is present in the form of QA in the blood, the binding
effects of QE with pepsin and trypsin and QA with serum albumin were studied in this
article 24. The binding properties of pepsin, trypsin with QE are shown in Fig. 2A and
Fig. 2B. After the incubation of QE with pepsin and trypsin, the proteinꢀbinding rate
decreased as the concentration of QE increased and the concentrations of the two
enantiomers of QE were approximately equal at the end of incubation. Similar results
were also found in the incubation of QA with serum albumin (Fig. 2C). No QA was
detected during these experiments. Protein binding effects should not be the reason
for the enantioselectivity or quick metabolism found in vivo.
3.3 Enantioselective degradation kinetic in rat plasma, liver microsome and cytosol
Xenobiotic metabolism is a biphasic process, comprising phase Ι (CYP450 and
esterase) and phase ΙΙ (UGT and SULT) enzyme reactions. Many chiral chemicals
have demonstrated stereoselective metabolism in animals due to the enzymes like
CYP450, esterase, UGT and SULT 30ꢀ34
.
Concentrationꢀtime curves of (+)ꢀ and (ꢀ)ꢀQE in rat plasma at 10 mg/L are shown
in Fig. 3A. The enantioselective metabolic process of QE was very quick in the
plasma with preference of (+)ꢀQE and the t1/2 of (+)ꢀ and (ꢀ)ꢀQE were 2.06 and 2.88
min. Along with the metabolism of QE, QA was rapidly generated and reached 10
mg/L within 20 min with preferential formation of (+)ꢀQA. Fig. 3B showed the
metabolism of QE and QA in rat microsome in vitro. The degradation of QE in liver
microsome could reflect the enantioselective deesterification of QE by esterase in the
liver. The results showed that QE was rapidly metabolized to QA with t1/2 of 3.13 and

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3.29 min for (+)ꢀQE and (ꢀ)ꢀQE, and the degradation was nonenantioselective. The
metabolite QA was quickly generated and the concentrations of the two enantiomers
were approximately equal with the EF value ranging from 0.496 to 0.508, revealing
that the formation of QA in liver microsome was noenantioselective. To further
investigate the effects CYP450 enzymes on the enantioselectivity of QA, the
metabolism of racꢀQA was performed via the NADPHꢀdependent oxidative
metabolism in rat liver microsome. As shown in Fig. 3C, the metabolism of QA by
liver microsome was nonenantioselective with the EF values ranging from 0.51 to
0.50. Fig. 3D shows the influence of sulfotransferase on the enantioselectivity of QA
in vitro. QA could be sulfate conjugated by SULT and the whole process was
nonenantioselective. Similar results were also found in the glucuronidation of QA by
UGT (Fig. 3E).
In this way, the esterase present in plasma and liver could be the reason for the
quick degradation of QE to QA in vivo and the overwhelming amount of (+)ꢀQA in
rat tissues in vivo could not be explained by the preferential metabolism of
(ꢀ)ꢀenantiomers via the effects of liver microsome, sulfotransferase,
glucuronosyltransferase.
3.4 In vitro everted gut sac absorption
Several in vitro methods can be used in the prediction of drug absorption, in
which the everted gut sac model is a relatively simple and very reproducible
technique. In this study, the intestinal absorptive behavior of QE was studied using the
everted gut sac model with KꢀR culture solution in vitro. QE was added to the donor
(mucosal) side of rat jejunum at 10 mg/L. Because glucose is actively transported in
the small intestine, intact sacs concentrate glucose in the acceptor side and maintain
this gradient. In this way, the effects of QE on the viability and integrity of the gut
were examined via the concentration ratio of glucose between the acceptor side and
donor side (Fig. 4A). It was obvious that the ratio increased gradually regardless of
the presence of QE. Thus, in this study, QE did not induce toxicity and had no obvious
influences on the viability or integrity of the gut. Fig. 4B illustrated the distribution of
(+) and (ꢀ)ꢀQA at the donor and acceptor sides. No QE was detected in the donor side

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since the first time point (10 min). However, a large amount of QA was observed,
indicating that QE was metabolized quickly in mucosal side of the jejunum. The rapid
metabolism of QE might be caused by the esterases present in the lumen of the
intestine, which from enterocytes and pancreatic secretions and could rapidly
hydrolyze many kinds of esterꢀtype drugs 35ꢀ37. The two enantiomers of QA were
formed in equal amounts in the donor side without enantioselectivity, and great
enrichment of (+)ꢀQA was found in the acceptor side with the EF value increasing to
0.88 after 150 min. Stereoselectivity may take place during the processes of
absorption and transport because the membrane phospholipids are chiral. Many
studies have reported the enantioselective absorption and disposition of chiral drugs
38ꢀ40. In this study, enantioselective absorption by intestine should be the main reason
for the observed enrichment of (+)ꢀQA after oral administration in rat in vivo.
In this way, when the oral administrated QE reached the intestine, QE was
hydrolyzed rapidly in the lumen of the intestine to QA which was absorbed
enantioselectively by intestine reaching into the blood or tissues mainly in the form of
(+)ꢀQA.
4. Conclusions
In this study, protein binding, plasma, liver microsome and cytosol degradation,
and everted gut sac absorption were performed to investigate the enantioselective
metabolism behavior of orally ingested QE in rats. The protein binding process of QE
with pepsin, trypsin, and serum albumin was noenantioselective and no degradation to
QA was found in this process. The elimination of QE in plasma was quick with slight
enantioselectivity, while there was no difference in the formation of the two QA
enantiomers. The formation and degradation of QA was noenantioselective in the liver
microsome and cytosol, and the metabolism of QE to QA was rapidly. Results showed
the metabolism of QE to QA was also very quick in the intestine and the absorption of
QA was enantioselective with preference of (+)ꢀQA. The results suggested that the
esterase present in the tissues might lead to the quick metabolism of QE to QA and
that the absorption of QA by intestine might contribute to the enantioselectivity in

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vivo.
Acknowledgement
This work was supported by NewꢀStar of Science and Technology and by
Beijing Nova program YETP0323, Supported by the National Natural Science
Foundation of China (Contract Grants 21277171，21337005 ) .
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Table. 1. Validation of QE and QA enantiomer analysis.
Linear range
Enantiomers
(+)ꢀQE
Linear equation
R2
(mg/L)
0.3ꢀ6
0.3ꢀ6
0.3ꢀ6
0.3ꢀ6
LOD (mg/L) LOQ (mg/L)
y=69.48x+15.62 0.9994
y=69.82x+21.83 0.9995
y=130.96x+5.91 0.9999
y=131.12x+4.25 0.9997
0.1
0.1
0.1
0.1
0.3
0.3
0.3
0.3
(ꢀ)ꢀQE
(+)ꢀQA
(ꢀ)ꢀQA

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Fig. 1. Chemical structures and typical chromatogram of the separation of the enantiomers of
QE and QA.

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Fig. 2. The protein binding of QE with pepsin (A), trypsin (A) and albumin (C).

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Fig. 3. Degradation of QE by 10% rat plasma (A), microsome (B), and QA by microsome (C),
sulfotransferase (D) and glucuronosyltransferase (E).

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Fig. 4. Glucose transport in everted gut sac (A) and the concentration-time curves of QA in
mucosal (B) and serosal side (C).

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