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Pharmacokinetics and Bioavailability of the Isoflavones Formononetin and Ononin and their In Vitro Absorption in Ussing Chamber and Caco-2 Cell Models Li-Yu Luo, Miao-Xuan Fan, Hai-Yu Zhao, Mingxing Li, Xu Wu, and Wenyuan Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00035 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018
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Journal of Agricultural and Food Chemistry
Pharmacokinetics and Bioavailability of the Isoflavones Formononetin and Ononin and their In Vitro Absorption in Ussing Chamber and Caco-2 Cell Models Li-Yu Luo,† Miao-Xuan Fan,‡ Hai-Yu Zhao,# Ming-Xing Li,§ Xu Wu,§,* and Wen-Yuan Gao†,* †
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin,
China ‡
Beijing Key Laboratory of Analysis and Evaluation on Chinese Medicine, Beijing
Institute of Drug Control, Beijing 102206, China # Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China §
Laboratory of Molecular Pharmacology, Department of Pharmacology, School of
Pharmacy, Southwest Medical University, Luzhou, Sichuan, China
*Corresponding Author: Telephone: +86-13920837932; E-mail:
[email protected] (W.Y. Gao). Telephone: +86-13882770623; E-mail:
[email protected] (X. Wu).
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ABSTRACT
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Formononetin and its glycoside ononin are bioactive isoflavones widely present in
3
legumes. The present study investigated the pharmacokinetics, bioavailability and the
4
in vitro absorption of formononetin and ononin. After an oral administration to rats,
5
formononetin showed a higher systemic exposure over ononin. The oral
6
bioavailability of formononetin and ononin were 21.8% and 7.3%, respectively.
7
Ononin was more bioavailable than perceived, and its bioavailability reached 21.7%
8
when its metabolite formononetin was taken into account. Both formononetin and
9
ononin exhibited better absorption in large intestine segments than that in small
10
intestine segments. Formononetin displayed a better permeability in all intestinal
11
segments over ononin. Transport of formononetin across Caco-2 cell monolayer was
12
mainly through passive diffusion, while ononin was actively pumped out by MRP2
13
but not P-gp. The results provide evidences for better understanding of the
14
pharmacological actions of formononetin and ononin, which advocates more in vivo
15
evaluations or human trials.
16
KEYWORDS: Formononetin; Ononin; Bioavailability; Permeability; Caco-2;
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Ussing chamber
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INTRODUCTION
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Formononetin is a dietary isoflavone and a potent phytoestrogen widely present in
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legumes such as soy, kidney beans and navy beans as free aglycone or the substituted
21
glucoside conjugate ononin.1-3 Formononetin and ononin (Figure 1) are also major
22
types of flavonoes found in various Chinese herbal medicines, such as red clover
23
(Trifolium pratense L.), glycyrrhizae radix (the root of licorice) and astragali radix
24
(the root of Astragalus membranaceus var. mongholicus or A. membranaceus).3-6
25
Dietary intake of isoflavones has been associated with alleviation of osteoporosis,
26
climacteric symptoms and vascular disease,7-9 as well as lower risk of breast cancer
27
and prostate cancer.10-11 Notably, formononetin and ononin have been reported to
28
exhibit remarkable anti-oxidant, anti-inflammatory and hypolipidemic effects in
29
various in vitro and animal models.12-16 Recent reports have demonstrated that
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formononetin had neuroprotective effect in rat models of ischemia/reperfusion injury
31
and traumatic brain injury,17-18 protected C57BL/6 mice from high-fat diet-induced
32
obesity and bone loss through inhibition of adipogenesis,19 and inhibited angiogenesis
33
and tumor growth in breast cancer xenograft models via inhibition of fibroblast
34
growth factor receptor 2 (FGFR2).20
35
In contrast to the widely-explored pharmacological actions, the studies on the in
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vivo fate of formononetin and ononin are limited. Several researchers have
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investigated the pharmacokinetics of formononetin and ononin in rats after oral
38
administration of herbal extracts of Astragali radix and red clover, or Chinese herbal
39
formulas
40
bioavailability of these two individual isoflavones remain to be addressed. On the
41
basis of current knowledge on other isoflavones, low gut permeability and extensive
42
metabolism often lead to poor bioavailability. Previously, it was reported that both
43
hepatic microsomal enzymes and gut microbiota played key roles in extensive
44
metabolism of formononetin and ononin.24-25 However, to date, there is little
45
information available on gut permeability of these two isoflavones.26 Moreover, it is
46
unclear that whether transporters have been involved in their absorption.
containing
these
herbs.21-23
However,
the
pharmacokinetics
and
47
Therefore, the present study aims to investigate the pharmacokinetics and
48
bioavailability of formononetin and ononin in rats, and to monitor their gut
49
permeability properties in Ussing chamber and Caco-2 cell models.
50
MATERIALS AND METHODS
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Materials and Reagents. Formononetin, ononin and calycosin-7-O-glucoside
52
(internal standard, IS) were bought from Solarbio (Beijing, China). Sodium carboxyl
53
methyl cellulose (CMC-Na) was purchased from Tianjin Chemical Reagent Company
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(Tianjin, China). Methanol and acetonitrile of HPLC grade were obtained from Merck
55
(Darmstadt, Germany). Deionized water was purified by a Milli-Q purification system
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(Millipore; Bedford, MA). Heparin, dimethyl sulphoxide (DMSO), Hanks’ balanced
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salt solution (HBSS), tween 80, HEPES, verapamil, and pravastatin was purchased
58
from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM),
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fetal bovine serum (FBS) and nonessential amino acids were supplied by Gibco
60
(Grand Island, NY). Caco-2 cells were obtained from the American Type Culture
61
Collection (Rockville, MD).
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Pharmacokinetic Study. The care of animals and all experimental procedures
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were approved by the Committee on Use and Care of Animals of China Academy of
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Chinese Medical Sciences. Male Sprague-Dawley (SD) rats (210-230 g) supplied by
65
the Experimental Animal Centre, China Academy of Chinese Medical Sciences
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(Beijing, China) were maintained under controlled conditions of temperature (22-24
67
o
68
water ad libitum. Right jugular vein cannulation was performed on anesthetized rats
69
on the day before isoflavones administration. The rats were housed individually in
70
metabolic cage, and allowed to recover and fasted overnight with free access to water.
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Right jugular vein was cannulated with polyethylene tube for blood sampling.
C), humidity (55-60 %), and a light/dark cycle of 12/12 h with access to food and
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Rats were randomly grouped into four groups (6 rats per group). Formononetin or
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ononin was dissolved in 0.5% CMC-Na or in 5% tween 80 for oral and intravenous
74
administration, respectively. As tween 80 has been reported to affect transporter
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activity in gut,27 it is not used as a solvent for oral administration. Group 1 and group
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2 rats were orally treated formononetin or ononin (20 mg/kg). Blood (about 0.2 mL)
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were collected in heparinized tube at 0, 5, 15, 30 min, 1, 2, 4, 6, 8 h after drug
78
administration. Group 3 and 4 rats received intravenously formononetin or ononin (4
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mg/kg) via tail vein. Blood (about 0.2 mL) were collected in heparinized tube at 0, 2,
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5, 15, 30 min, 1, 2, 4, 6, 8 h after drug administration. After each sampling, an equal
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volume of heparinized normal saline was given to rats immediately for compensation
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of blood withdrawal. Blood were centrifuged at 1500 g for 10 min to obtain plasma
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and stored at -20 oC until analysis.
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For sample preparation, 80 µL ice-cold methanol containing 100 nM IS was added
85
to 40 µL plasma sample, which was then vortexed for 1 min and centrifuged at 15,000
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g for 10 min at 4 oC. The supernatant was used for LC-MS detection.
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Ussing Chamber. The Ussing chamber system, equipped with intestinal tissues
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from rats, was prepared as previously reported.28-29 In brief, rat gastrointestinal
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segments, including stomach, duodenum, jejunum, ileum, cecum and colon, after
90
removing the muscle layer, were vertically mounted in Ussing chambers. The
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transport buffer of Krebs-Ringer solution (5 mL) was added into both apical and basal
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sides, which was maintained at 37 oC and continuously supplied with 95% O2/5%
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CO2. The available area for drug transportation was 0.5 cm2. The transepithelial
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electrical resistance (TEER) was measured before and after the study. A decrease of
95
TEER (>15%) indicates the leakage of intestinal tissues.30-31 After a 15-min
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equilibration, transport buffer in the apical side was replaced with pre-warmed buffer
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containing 10 µM formononetin or ononin. Samples (100 µL) were collected from the
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basal side at 0, 15, 30, 45, 60 and 120 min. At the end of sampling, 100 µL solution
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was collected from the apical side for analysis to calculate recovery. All samples were
100
stored at -20 oC until analysis.
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For sample preparation, 100 µL samples were mixed with 300 µL ice-cold
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methanol containing 50 nM IS, followed by vortexed for 1 min and centrifuged at
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15,000 g for 10 min at 4 oC. The supernatant was used for LC-MS detection.
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Caco-2 Monolayer Transport Assay. Caco-2 cells were cultured in a DMEM
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medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids
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and 1% penicillin and streptomycin at 37 oC in 5% CO2, and were seeded into 6-well
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transwells (0.4 µM, polyester membrane, 24 mm insert; Corning, NY) at 2 × 105
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cell/mL for 22 days. The integrity of the monolayer was verified by measuring the
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apparent permeability coefficients (Papp) values of the paracellular marker atenolol
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and the transcellular marker propranolol, and the TEER using an epithelial tissue
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voltohmmeter (World Precision Instrments, Sarasota, FL). Monolayers with TEER >
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300 Ω‧cm2 (subtracting the background value of a transwell) before and after
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transport assay and appropriate Papp values for atenolol (around 10-7 cm/s) and
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propranolol (around 10-5 cm/s) were used for transport study.
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To see whether transporters were involved in the absorption of formononetin and
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ononin, the bidirectional transport assays with or without verapamil (a specific P-gp
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inhibitor) or pravastatin (a specific MRP2 inhibitor) were performed. Transport
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studies were conducted at 37 oC, in HBSS containing 25 mM HEPES. Prior to study,
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medium from both sides of the monolayers was removed, and replaced with HBSS
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alone, or HBSS containing verapamil (50 µM) or pravastatin (1 mM). Following a
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15-min incubation, formononetin or ononin (10 µM) was added into the basolateral
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side for the basolateral to apical (B to A) transport study or apical side for the apical
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to basolateral (A to B) transport study. At designated times (0, 15, 30, 60, 90 min),
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100 µL sample was collected from the receiver compartment, and then 100 µL HBSS
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solution was added immediately to maintain a constant volume. At the end of
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sampling, 100 µL solution was collected from the donor compartment for LC-MS
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analysis to calculate recovery. Samples were stored at -20 °C until LC-MS analysis.
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Sample preparation was conducted as described in Ussing chamber study.
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LC-MS Analysis. Quantitative analysis was conducted on an Agilent UHPLC
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1290 system equipped with a vacuum degasser, a binary pump, an autosampler and a
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6460 triple quadruple mass spectrometry.32 A ZORBAX Eclipse Plus C18 column (2.1
132
× 100 mm, 1.7 µm, Agilent Technologies) was used for chromatographic separation.
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The column was maintained at 35 °C. The mobile phases were acetonitrile containing
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0.1% formic acid (A) and water containing 0.1 % formic acid (B). The elution rate
135
was 0.3 mL/min and the gradient program was as follows: 0-1 min 15% B; 1-4 min
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15-60% B; 4-5 min 60-90% B; 5-6 min 90% B. The eluate from column for the first 1
137
min was directed to the waste. The injection volume is 10 µL for both formononetin
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and ononin.
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The mass spectrometer was operated in positive ion mode with an electrospray
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ionization (ESI) interface. Quantitation was performed by multiple reactions
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monitoring (MRM). In the positive mode, the MS/MS parameters were as follows:
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capillary voltage 3500 V; gas temperature 280 oC; sheath gas heater 350 oC; sheath
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gas flow 11 L/min; gas flow 5 L/min. The precursor to product ion pairs for
144
monitoring formononetin, ononin and IS in the MRM mode were m/z 269→197, m/z
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431→269, and m/z 447→285, respectively. The fragmentor was set at 135 V, while
146
collision energies were 45 V for formononetin and 20 V for ononin and IS.
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Data Analysis. Oral bioavailability (F) was calculated as: F = (AUCp.o.*Dosei.v.)/
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(AUCi.v.*Dosep.o.)*100%. Papp value of formononetin and ononin for the bidirectional
149
transport in Caco-2 monolayers and in Ussing chambers was calculated as:33 Papp =
150
(dC/dT*V)/A/C0, where dC/dT is the initial slope of the plot of cumulative
151
concentrations versus time (µM/s); V is the volume of solution in receiver chamber,
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which is 0.5 mL for the apical side and 1.5 mL for the basolateral side in Caco-2
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model, and 5 mL in the Ussing chamber model; A is the surface area of the monolayer,
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which was 1.12 cm2 for the 12-well Transwell plate and 0.5 cm2 for the Ussing
155
chamber; C0 is the initial concentration in donor site (µmol/mL). The Papp values are
156
commonly used for the prediction of absorption of orally administered drugs. It is
157
suggested that drugs with Papp < 1 × 10-6 cm/s, Papp = (1-10) × 10-6 cm/s, and Papp > 10
158
× 10-6 cm/s usually have poor, moderate and good absorptions, respectively.34 An
159
efflux ratio (Papp (B to A)/ Papp (A to B)) larger than 1.5 was used for determining whether
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efflux transporters were involved in the drug transport in Caco-2 model.
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All data are expressed as mean ± standard deviation (SD). Statistical difference
162
was analyzed by GraphPad Prism 7.0 using unpaired student’s t test (for comparison
163
between two groups) or one-way ANOVA with a post hoc Tukey test (for comparison
164
among three or more groups). A p < 0.05 was considered significant.
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RESULTS AND DISCUSSION
166
Pharmacokinetics and Bioavailability of Formononetin and Ononin.
167
Determination of the concentration of formononetin and ononin in rat plasma was
168
performed using LC-MS method. As shown in Table 1, the calibration curves of
169
formononetin and ononin provided good linearity (R2 > 0.99) within the concentration
170
ranges tested. The limits of quantification (LOQs) were 0.5 and 1.0 nM for
171
formononetin and ononin, respectively. The results (Supplementary Table 1) for
172
precision (RSD, from 0.7% to 6.9%), accuracy (standard error, from -8.5% to 9%) and
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extraction recovery (from 89.2 ± 8.2% to 100.7 ± 6.9%) indicated the reliability of
174
methods for determination of formononetin and ononin.
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The plasma concentration versus time curves of formononetin and ononin after
176
oral or intravenous administration are shown in Figure 2, with pharmacokinetic
177
parameters displayed in Table 2. The oral dosages of formononetin and ononin were
178
set at 20 mg/kg based on previous pharmacological studies.35-36 After an oral
179
administration at 20 mg/kg, formononetin was rapidly absorbed and peaked at 30 min,
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which was followed by a quick elimination phase with a half-life of 2.1 h, whereas
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ononin showed a double peak phenomenon in the plasma concentration profile, which
182
were characterized by Tmax1 (or Cmax1) and Tmax2 (or Cmax2) (Table 2). The first peak
183
was observed at around 30 min, indicating a rapid absorption of ononin, then
184
experienced a quick decline and a subsequently small rise that peaked at 4 h after oral
185
administration (Figure 2). The rapid absorption of formononetin and ononin was
186
observed similar to other reported isoflavones such as genistein, daidzein, daidzin and
187
puerarin with Tmax of 0.25-1 h.37-39 Furthermore, in consistent with previous
188
studies,40-41 the double peak phenomenon of ononin has been reported after oral
189
administration of other herbal extracts, which was further discussed in the next
190
section after the gut permeability study.
191
It should be noted that after an oral administration of ononin, formononetin was
192
detected as a metabolite in rat plasma, and displayed a double peak phenomenon. The
193
first peak of formononetin was observed at 1 h post ononin dosing, with a Cmax1 of
194
35.2 ± 10.1 nM, and the second peak was seen at around 6 h, with a larger Cmax2 of
195
76.5 ± 12.5 nM. It has been demonstrated that ononin could be biotranformed into
196
formononetin via colonic microbiota-mediated deglycosylation.25 Moreover, there is
197
evidence that, compared to colonic microbiota, isoflavone glycosides such as
198
quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside can be
199
hydrolyzed, to a lesser extent, by the lactase phlorizin hydrolase (LPH), an enzyme
200
found on the brush border of the mammalian small intestine.42 Although not being
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proved, ononin may be deglycosylated by LPH in small intestine in a way similar to
202
other isoflavone glycosides. Therefore, hydrolysis of ononin at double sites (both
203
small and large intestines) might be responsible for the double peak phenomenon of
204
formononetin after oral administration of ononin.
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Notably, after giving a same oral dose, the systemic exposure of formononetin
206
(AUC0-8 h, 713.4 ± 46.2 nM*h) was about 4.5-fold higher than that of ononin (AUC0-8
207
h,
208
4 mg/kg, formononetin and ononin displayed similar biphasic plasma concentration
209
profiles (Figure 2), with the AUC0-∞ of 652.3 ± 89.1 and 439.5 ± 54.1 nM*h,
210
respectively (Table 2). Based on the results, formononetin was moderately
211
bioavailable in rats with an absolute bioavailability of 21.8%, while ononin had a
212
relatively low bioavailability of 7.3%. Since ononin was rapidly biotransformed in gut
213
to generate formononetin and was further metabolized,43 it is not a surprise that
214
ononin is less bioavailable than formononetin. Besides, the aglycones are generally
215
more liposoluble than their corresponding glycosides, and hence easier to transport
216
across the gut. However, when the formononetin as an active metabolite of ononin
217
was taken into consideration for calculation, the bioavailability of ononin reached
218
21.7%, which was similar to that of formononetin. That is to say, ononin is more
219
bioavailable in vivo than perceived.
160.2 ± 39.1 nM*h). On the other hand, following an intravenous administration at
220
Most human studies of isoflavones and flavonoids have been unsuccessful, which
221
is mainly due to the very poor oral bioavailability.44-45 Isoflavones and flavonoids
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such as chrysin, quercetin and resveratrol usually contain several free hydroxyl groups
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and facilitate rapid intestinal/hepatic phase II metabolism by glucuronidation and/or
224
sulfation for excretion.46-48 It is reported that methylated flavones have increased
225
metabolic stability and improved intestinal transport.49-50 Thomas Walle et al. showed
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that 5,7-dimethoxyflavone and 5,7,4’-trimethoxyflavone had higher bioavailability
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and tissue distribution in rat in contrast to the unmethylated chrysin and apigenin.51
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Formononetin is a naturally occurring methylated isoflavone. Compared with other
229
hydroxylated ones such as biochanin A, formononetin has been demonstrated to
230
display slower glucuronidation in intestinal and hepatic microsomes and higher gut
231
permeability.52 The moderately bioavailable formononetin might have benefited from
232
its methylation status in its structure.
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Absorption of Formononetin and Ononin at Different Gastrointestinal
234
Segments. As one of the key limiting steps for entering circulation system, how gut
235
permeability contribute to the bioavailability differences of the parent formononetin
236
and ononin is further evaluated using a Ussing chamber model. Samples were
237
determined using LC-MS method, and the calibration curves and quantification limits
238
are shown in Table 1. The Papp values for absorption of formononetin and ononin at
239
different intestinal segments are displayed in supplementary Table 2 and Figure 3. As
240
shown in supplementary Table 2, the recoveries of the two compounds in the assay
241
were generally above 80% (except formononetin using duodenum segment and
242
ononin using jejunum segment with mean recovery of 79%), indicating that the Papp
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values were within acceptable accuracy.53-54 We chose 10 µM of formononetin and
244
ononin for the Ussing chamber study, because initial results showed su‧cient
245
isoflavones at the donor and receiver sides to allow accurate measurement.
246
The results (Figure 3 and supplementary Table 2) showed that both formononetin
247
and ononin were better absorbed in the large intestine segments (cecum and colon),
248
with Papp values of (5.98-6.10) × 10-6 cm/s (formononetin) and (1.16-1.91) × 10-6 cm/s
249
(ononin), in comparison to that in the small intestine segments (duodenum, jejunum
250
and ileum), with Papp values of (1.52-2.45) × 10-6 cm/s (formononetin) and (0.10-0.25)
251
× 10-6 cm/s (ononin). Similar result has also been reported on biochanin A.52 This
252
might be explained by the site-specific distribution of these isoflavones in the gut tract.
253
It was found that genistein had a high tissue distribution in gut, with a highest
254
residence in cecum at specific time point after administration in rats.39 In this study,
255
the site-specific absorption behavior was more obvious for ononin, which supported
256
the finding that ononin showed a double peak phenomenon in its plasma
257
concentration profile. It is anticipated that, after oral administration, ononin had a
258
small absorption in small intestine, and most parts were absorbed in the large intestine.
259
The fact that double peak phenomenon did not occur after intravenous administration
260
of ononin indicated that it was not due to enterohepatic cycling. Thus, it is highly
261
possible that double-site absorption leads to the double peak phenomenon of oral
262
ononin. On the other hand, although formononetin showed a better permeability in the
263
large intestine segments, it had a moderate absorption (Papp > 1 × 10-6 cm/s) in the
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small intestines segments. Due to the much longer length of small intestine than large
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intestine, formononetin should have a longer residence in the small intestine. In this
266
regard, small intestine may be the main site responsible for formononetin absorption,
267
and this might be the main reason for why formononetin did not display double peak
268
phenomenon in its plasma concentration profile.
269
Interestingly, ononin could not transport across stomach tissue, whereas
270
formononetin showed a poor absorption with a Papp value of (0.20 ± 0.04) × 10-6 cm/s.
271
This is in consistent with previous report that certain isoflavonoid aglycones such as
272
daidzein and genistein but not their glycosides can be absorbed in stomach.55 It was
273
found that after only 3 minutes of administration, daidzein and genistein and their
274
metabolites were detected in the rat plasma. Compared to the isoflavone aglycones,
275
their glucosides, daidzin and genistin, were found in the plasma with only a few
276
minutes’ delay. The delay was due to the time required by the administered glucosides
277
to pass across stomach before reaching the small intestine where they were absorbed.
278
Therefore, both isoflavones and the glucosides are rapidly absorbed in rats regardless
279
of their permeability difference in stomach. This supported that both formononetin
280
and ononin had a similar Tmax at around 30 min.
281
It should be noted that, in all segments of intestines, the Papp values of
282
formononetin were significantly higher than that of ononin (Figure 3), indicating a
283
better permeability of the more liposoluble formononetin. Importantly, the relatively
284
low permeability (Papp < 2 × 10-6 cm/s) of ononin in gut is proposed as an alternative
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contributor for its poor bioavailability.
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Transport of Formononetin and Ononin across Caco-2 Cell Monolayer.
287
Caco-2 model is widely applied to predict drug transport via different ways, including
288
the passive diffusion and paracellular routes, the transporters-mediated route and
289
transcytosis, across the human intestinal epithelium.56 In vitro permeability of
290
formononetin and ononin was carried out in this model. In the present permeability
291
assay, recoveries of formononetin and ononin were above 77% (Table 3), suggesting
292
an acceptable accuracy for the Papp values.57-58 At 10 µM, both formononetin and
293
ononin did not cause cytotoxicity on Caco-2 cells for 24 h incubation, suggesting that
294
formononetin and ononin at the selected concentration would not affect the integrity
295
of Caco-2 cell monolayer.
296
The Papp (A to B) values for formononetin and ononin across Caco-2 cell monolayers
297
are summarized in Table 3. The results showed a moderate absorption of
298
formononetin with a Papp
299
absorption of ononin with a Papp (A to B) value of (3.58 ± 0.25) ×10-7 cm/s, which was 1
300
order of magnitude lower than that of formononetin. These data were in consistent
301
with the Ussing chamber results, which again indicated that the better permeability of
302
formononetin might contribute to its higher bioavailability over ononin. High
303
permeability (> 5 ×10-6 cm/s) of isoflavones in Caco-2 cell model has been reported
304
previously regardless of the methylated status in structures.26, 59 Based on previous
(A to B)
value of (5.14 ± 0.22) ×10-6 cm/s, and a poor
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reports59 and the present study, the more liposoluble isoflavones generally have better
306
permeability over others, especially the unmethylated ones or the glycosides.
307
However, higher gut permeability is not always correlated with higher bioavailability.
308
Genistein was reported to less permeable than the more liposoluble daidzein in gut,26
309
whereas it was found that genistein was more bioavailable than daidzein.60
310
To investigate whether efflux transporters were involved in the absorption,
311
bi-directional transport assay was performed. The bilateral Papp values are shown in
312
Table 3. The efflux ratio (Papp (B to A)/Papp (A to B)) of formononetin was within the range
313
of 1.0-1.5, indicating that there was no significant difference between permeability in
314
A to B direction and that in B to A direction, and transportation of formononetin
315
across intestinal epithelial cells was mainly through passive diffusion.
316
On the other hand, the transportation of ononin from A to B direction was
317
significantly lower than that from B to A direction, with an efflux ration of 1.656,
318
suggesting that ononin might be substrate of efflux transporters. The addition of
319
verapamil (a specific P-gp inhibitor) did not affect the bilateral transport of ononin,
320
whereas pravastatin (a specific MRP2 inhibitor) significantly inhibited the preferential
321
transportation of ononin from B to A direction. The results indicated that ononin was a
322
substrate of MRP2 but not P-gp, and its low bioavailability might be partially
323
attributed to MRP2-mediated efflux during absorption. This was in consistent with
324
other report where they found that transport of the flavonoid glycoside
325
quercetin-4’-β-glucoside was specifically through MRP2 but not P-gp.61 MRP2 ACS Paragon Plus Environment
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governs the transportation of its substrates across the intestinal wall. Previous studies
327
have demonstrated that MRP2 mostly expresses at the apical side of epithelia in small
328
intestine including duodenum, jejunum and ileum, with almost no expression in the
329
entire colon segment.62 The lower permeability of ononin in the small intestine
330
compared to the large intestine might be due to the differential efflux activity of
331
MRP2 in these intestinal segments.
332
In summary, the present study revealed the pharmacokinetic properties of
333
formononetin and ononin after oral and intravenous administration in rats.
334
Formononetin had a bioavailability of 21.8%, and was absorbed in all gastrointestinal
335
segments with varied permeability. In Caco-2 model, formononetin showed moderate
336
absorption via passive diffusion. Meanwhile, ononin displayed a bioavailability of
337
7.3%. When formononetin as an active metabolite of ononin was taken into
338
consideration, the bioavailability of ononin reached 21.7%. Ononin was poorly
339
absorbed in all intestine segments of rats, and had a poor permeability in Caco-2
340
monolayer, which was identified as a substrate of MRP2 but not P-gp. These findings
341
facilitated the understanding of the in vivo pharmacological actions of formononetin
342
and ononin from a pharmacokinetic view, and also implicated that both formononetin
343
and ononin, due to their moderate bioavailability, might have a potential for more in
344
vivo evaluations or even human trials.
345
ABBREVIATIONS
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DMEM, Dulbecco’s modified Eagle’s medium; FGFR2, Fibroblast growth factor
347
receptor 2; LPH, lactase phlorizin hydrolase; MRM, multiple reaction monitoring;
348
MRP2, multidrug resistance protein 2; Papp, apparent permeability coefficient; TEER,
349
transepithelial electrical resistance.
350
SUPPORTING INFORMATION
351
Table 1 about the validation of LC-MS method for determining formononetin and
352
ononin in rat plasma in terms of precision, accuracy and recovery, and table 2 about
353
the Papp values and recoveries of formononetin and ononin at different gastrointestinal
354
segments in Ussing chambers.
355
ACKNOWLEDGEMENT OF FINANCIAL SUPPORT
356
This work was supported by the Funding of Hong Kong, Macao & Tianwan Science
357
and Technology Cooperation Project (2015DFM30030), and the Fundamental
358
Research Funds for the Central Public Welfare Research Institutes (ZZ10-007).
359 360
REFERENCES
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Figure Captions: Figure 1. Structures of the isoflavone formononetin and its glycoside ononin. Figure 2. Plasma concentration versus time curves of formononetin (A and B) and/or ononin (C and D) after oral or intravenous administration in rats. Rats (n = 6 in each group) were treated with formononetin or ononin via p.o. (20 mg/kg) or i.v. (4 mg/kg). Blood were obtained through cannulated right jugular at the designated time points. Plasma concentrations of formononetin or ononin were analyzed by LC-MS method. Data are represented as mean ± SD (n = 6). Figure 3. In vitro absorption of formononetin (A) and ononin (B) at different gastrointestinal segments in Ussing chamber model. Gastrointestinal segments, including stomach, duodenum, jejunum, ileum, cecum and colon, were mounted in the Ussing chambers after removal of the muscle layer. Transfer buffer containing 10 µM formononetin or ononin was added in the apical side. Apparent permeability coefficients (Papp) values for formononetin (A) and ononin (B) after 120-min transport are shown. Statistical analysis was performed using Graphpad Prism 7.0. Data are obtained from three independent experiments and are displayed as mean ± SD (n = 3).
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Table 1. Calibration and Limits of Quantification (LOQ) for Formononetin and Ononin in Different Matrices. Compounds
Matrices
Calibration curvesa
Ranges (nM)
R2
LOQ (nM)
Formononetin
Plasma HBSS Krebs-Ringer buffer Plasma HBSS Krebs-Ringer buffer
Y = 9.501*10-3X + 4.301*10-4 Y = 0.0338X - 3.088*10-4 Y = 0.0549X + 0.00101
5-2000 1-500 1-500
0.991 0.989 0.994
0.5 0.2 0.2
Y = 0.0169X + 8.09857*10-4 Y = 0.0750X - 0.00107 Y = 0.0969X + 8.090*10-4
5-2000 1-400 1-400
0.990 0.990 0.997
1.0 0.5 1.0
Ononin
a
Y represents peak area ration of analyte and IS while X is the concentration of analyte. The injection volume is 10 µL for formononetin and ononin. The calibration curve of each analyte in different matrices was constructed by plotting peak area ratio of analyte and IS versus spiked concentration. The LOQs were determined using the signal to noise (S/N) ratio of 10.
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Table 2 Pharmacokinetic Parameters of Formononetin and Ononin after an Oral or Intravenous Administration. Formononetin Ononin Parameters 20 mg/kg, 20 mg/kg, p.o. 4 mg/kg, 4 mg/kg, i.v. p.o. i.v. Ononin Formononetin 1302.8 ± Cmax (nM) 302.1 ± (1) 74.6 ± (1) 35.2 ± 10.1 1142.2 ± 129.1 35.9 26.1*** (2) 76.5 ± 12.5 103.2 (2) 34.0 ± 8.4 AUC0-8 h 317.2 ± 49.1 439.5 ± 713.4 ± 652.3 ± 89.1 160.2 ± 39.1### 54.1† (nM*h) 46.2 AUC0-∞ 757.7 ± 689.5 ± 92.1 173.3 ± 43.1§§§ 463.8 ± (nM*h) 48.2 60.1‡ Tmax (h) 0.5 ± 0.0 (1) 0.5 ± 0.0 (1) 1.0 ± 0.0 (2) 4.0 ± 0.0 (2) 5.7 ± 0.0 t1/2 (h)
2.10 ± 0.28
2.23 ± 0.6
1.82 ± 0.56
-
1.92 ± 0.8
Vz (L/kg) CL (L/h/kg) F%
-a -
13.9 ± 1.0 4.3 ± 0.8
-
-
18.4 ± 1.5 6.6 ± 0.9
21.8%
-
7.3% (21.7%b)
-
-
a
“-”, not applicable.
b
The calculation has included both AUC of formononetin and ononin after oral ononin administration.
Data are expressed as mean ± SD (n=6). ***p < 0.001, ###p < 0.001, or §§§p < 0.001, compared with the Cmax, AUC0-8 h, and AUC0-∞ of p.o. formononetin, respectively;
†
p < 0.05, or
‡
p < 0.05, compared
with the AUC0-8 h, and AUC0-∞ of i.v. formononetin, respectively. Cmax, peak plasma concentration after drug administration; AUC, area under the curve; Tmax, time at peak concentration; t1/2, half life; Vz, volume of distribution; CL, clearance; F, bioavailability.
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Table 3 Apparent Permeability Coefficients (Papp) Values and Efflux Ratio of Formononetin and Ononin Determined in Caco-2 Model. Recoveryb
Papp (×10-6 cm/s) Efflux ratioa
Isoflavones
Formononetin Ononin
A to B
B to A
A to B
B to A
5.143 ± 0.220
5.515 ± 0.139
1.174
77.8 ± 4.7 95.1 ± 5.3
0.358 ± 0.025*** 0.593 ± 0.127#
1.656
87.4 ± 5.7 87.9 ± 10.8
Ononin+Verapamil
0.349 ± 0.151
0.661 ± 0.102†
1.893
81.8 ± 9.1 86.2 ± 1.3
Ononin+Pravastatin
0.443 ± 0.117
0.417 ± 0.390
0.941
78.9 ± 4.2 90.0 ± 13.3
a
Efflux Ratio, Papp (B to A)/Papp (A to B), where Papp (B to A) is the average of the permeability coefficient
from basolateral (B) to apical (A); Papp (A to B) is the average of the permeability coefficient from A to B. b
Recovery was calculated as the ratio of total amount of drugs detected in A and B sides after
90-min transport study and the nominated amounts initially added. Verapamil (a specific P-gp inhibitor) and pravastatin (a specific MRP2 inhibitor) were used to investigate whether transporters were involved in the bidirectional transportation of isoflavones. Data are expressed as mean ± SD (n = 3). ***p < 0.001, compared with Papp (A to B) of formononetin; #p < 0.05, compared with Papp (A to B) of ononin; †p < 0.05, compared with Papp (A to B) of ononin in the presence of verapamil. Papp, apparent permeability coefficients.
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