Journal of Ethnopharmacology 155 (2014) 1382–1387

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Research Paper

Biodistribution evaluation of icaritin in rats by ultra-performance

liquid chromatography–tandem mass spectrometry
Shuang-Qing Zhang n
Department of Nutrition and Metabolism, National Institute for Nutrition and Food Safety, China Center for Disease Control and Prevention, Beijing 100050,
China

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Icaritin (ICT) is a major bioactive prenylflavonoid derivative contained
Received 7 May 2014 in the Epimedium which is a widely used herbal medicine for the treatment of infertility, impotence,
Received in revised form cardiovascular and skeletal diseases listed in the Chinese Pharmacopoeia. The aim of this study is to
10 July 2014
investigate the tissue distribution of ICT in rats by ultra-performance liquid chromatography–tandem
Accepted 19 July 2014
mass spectrometry (UPLC–MS/MS)
Available online 30 July 2014
Materials and methods: ICT was intraperitoneally administrated to rats for 7 consecutive days at dose
Keywords: levels of 20, 40 and 60 mg/kg/day, respectively. Various tissue homogenates were pretreated by protein
Icaitin precipitation with acetonitrile. ICT and internal standard coumestrol were separated on a BEH C18 column
UPLC–MS/MS
with a gradient mobile phase and detected using precursor-product ion transitions of m/z 367.1-297.1
Biodistribution
for ICT and 267.0-211.1 for coumestrol at the negative ionization mode, respectively.
Results: ICT was widely distributed in rat's various tissues and its concentrations in tissues increased
with elevated doses. A sensitive and reliable UPLC–MS/MS method was firstly established to quantify ICT
in rat tissues. The lower limit of quantification was 0.5 ng/mL based on 100 μL of tissue homogenates.
The intra- and inter-day accuracy at all levels fell in the ranges of 90.8–103.4% and 91.6–100.3%, and the
intra- and inter-day precision (RSD) were in the ranges of 2.9–10.5% and 2.6–9.1%, respectively.
Conclusions: The UPLC–MS/MS showed good accuracy, precision and recovery and was suitable for the
quantification of ICT in rat tissues. Wide distribution of ICT could helpfully elucidate systemic effects and
various functions of ICT.
& 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
The Epimedium has been used as a tonic, aphrodisiac and
antirheumatic for the treatment of infertility, impotence, cardio-
vascular and skeletal diseases in China, Japan and Korea for more
than 2000 years (Ma et al., 2011). Icaritin (ICT, Fig. 1A) is
recognized as a major bioactive prenylflavonoid derivatives con-
tained in plants of the Epimedium (Ma et al., 2011). ICT has been
demonstrated to possess several pharmacological activities. These
include the osteoprotective effect as seen by its ability to increase
the formation of new osteoblast and reducing osteoclastogenesis
(Huang et al., 2007a), increasing the differentiation of mesenchy-
mal stem cells (Sheng et al., 2013), the reduction of incidence in
steroid-associated osteonecrosis in rabbits (Zhang et al., 2009), as
well as the prevention of osteoporosis in ovariectomised rats (Peng
et al., 2013). Its anti-tumorigenesis function in regards to breast
n
Tel./fax: þ 86 10 87708717.
E-mail address: zhangshq@hotmail.com
(Tiong et al., 2012), prostate (Chen et al., 2010, Huang et al.,
2007b), endometrial (Tong et al., 2011), chronic myeloid leukemia
(Li et al., 2013a) and renal cancer cells (Li et al., 2013b) has been
demonstrated. Its cardiovascular improvements, via initiating
cardiomyocyte differentiation from rat embryonic stem cells have
also been exhibited (Zhu and Lou, 2005;Wo et al., 2008). As well
as, neurological protection from beta-amyloid induced neurotoxi-
city in an Alzheimer model has been shown (Wang et al., 2007).
A phase I clinical trial of ICT for the treatment of advanced breast
cancer showed that ICT exhibited good safety and tolerance, and a
phase II clinical trial of ICT for the treatment of advanced
hepatocellular carcinoma is ongoing (Clinicaltrials.Gov, 2014). As
such, accurate and simple methods to measure ICT concentration
in tissues and plasma are critical for the development of ICT as a
pharmaceutical-quality health intervention.
There have been three publications for the quantification of ICT
from the consumption of crude extracts from the Epimedium
species or as a pure compound in plasma, urine and bile (Shen
et al., 2007; Shen et al., 2009; Chang et al., 2012). However, there
have been various shortcomings for those methods. Shen et al.

http://dx.doi.org/10.1016/j.jep.2014.07.045
0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
 S.-Q. Zhang / Journal of Ethnopharmacology 155 (2014) 1382–1387
Fig. 1. Chemical structures of (A) ICT and (B) coumestrol.
reported gas chromatography-mass spectrometry (GC–MS) (Shen
et al., 2007) and liquid chromatography–tandem mass spectro-
metry (Shen et al., 2009) (LC–MS/MS) methods for assay of ICT in
human and rat serum, respectively, in which the former involved
laborious precolumn derivatization and the latter employed a
time-consuming multiple-step liquid–liquid extraction and large
amount of rat plasma (0.5 mL). In addition, ICT was detected by
GC–MS and LC–MS/MS after administration of Epimedium crude
extract containing icariin, icariside and epimedin, which were
finally metabolized to ICT resulting in increasing actual ICT
concentration in plasma, compared to pure ICT (Shen et al.,
2009, 2007). Therefore, elevation of actual ICT concentration due
to the biotransformation with administration of Epimedium extract
led to faulty calculation of pharmacokinetic parameters compared
to pure ICT, unfortunately, no attention was paid to that. Chang
et al. described high performance liquid chromatography with a
ultraviolet detection method for analysis of ICT in rat plasma, bile
and urine, which suffered from low quantification limit of 20 ng/
mL resulting in failure detection of parent ICT in biological matrix
(Chang et al., 2012).
To our knowledge, no analytical method has been published to
quantify ICT in rat tissues (liver, spleen, kidney, heart, lung,
muscle, thymus and brain). Therefore, we have developed a robust,
fast and sensitive ultra-performance liquid chromatography–tan-
dem mass spectrometry (UPLC–MS/MS) method employing a
simple one-step protein precipitation for measurement of ICT in
rat tissues. Moreover, this method has also been successfully
applied to the quantification of pure ICT in rat tissues after
intraperitoneal administration of ICT at various dose levels.
2. Materials and methods
2.1. Materials
ICT was provided by Ctech Scientific Pte Ltd. (Singapore).
Coumestrol (Fig. 1B) as an internal standard (IS), formic acid,
1383
ammonium formate and Cremophor EL were purchased from
Sigma (St. Louis, MO, USA). HPLC grade acetonitrile was obtained
from Merck (Darmstadt, Germany). HPLC grade water was pro-
duced with Sartorius Arium 611VF water purification system
(Milan, Italy). All other reagents were commercially available and
of analytical grade.
2.2. LC–MS/MS instrumentation
A Waters Acquity TQD UPLC–MS/MS system consisted of a
binary solvent manager, a sample manager, a column oven and a
TQD triple quadrupole mass spectrometer with an orthogonal
electrospray ionization source Z-spray. All data were acquired and
processed using MassLynx version 4.1 software.
2.3. Chromatographic conditions
The chromatographic analysis was carried out utilizing a
Waters UPLC BEH C18 analytical column (2.1 mm  50 mm,
1.7 mm particle size) protected by a Waters VanGuard guard
column (2.1 mm  5 mm, 1.7 mm particle size) of the same packing
material. Column temperature was maintained at 40 1C. The
mobile phase consisted of two eluents, namely, elution A (95:
5 acetonitrile/water containing 2 mM ammonium formate and
0.05% formic acid) and elution B (water with 2 mM ammonium
formate and 0.05% formic acid, pH3.0), delivered at a constant flow
rate of 0.3 mL/min. The gradient elution was: 0–1.5 min, 10% A;
1.5–2.2 min linear from 10% to 90% A; 2.2–5.0 min, 90% A; 5.0–
5.5 min linear from 90% to 10% A, returned to initial conditions;
5.5–6.5 min 10% A, equilibration of the column. Total run time per
sample was 6.5 min and all injection volumes were 4 μL at a mode
of partial loop with needle overfill. Between injections, the
sampling needle was washed for 2 min each with a weak wash
of 1200 μL (90% water, 10% acetonitrile) and a strong wash of
600 μL (acetonitrile).
2.4. Mass spectrometric conditions
The Acquity UPLC was coupled to a TQD mass spectrometer
operated in negative electrospray ionization mode. Quantification
was performed using multiple reaction monitoring (MRM) mode
to monitor precursor-product ion transitions of m/z 367.1-297.1
for ICT, 267.0-211.1 for the IS at negative ionization mode. The
optimal instrument operating conditions were as follows: capil-
lary, 3.0 kV; cone, 60 V; source temperature, 150 1C; and desolva-
tion temperature, 300 1C. Nitrogen gas flow was 600 L/h and 50
L/h for desolvation and cone, respectively. Argon was employed as
a collision gas with a flow rate of 0.11 mL/min. The collision
voltage values of ICT and the IS were 27 and 29 V, respectively.
2.5. Preparations of stock and standard working solutions
Stock solutions of ICT (500 μg/mL) and coumestrol (100 μg/mL)
were prepared in acetonitrile and stored at  20 1C. Working
standard solutions of ICT were prepared in acetonitrile at concen-
trations from 5 to 200 ng/mL. Working IS was 1 μg/mL by diluting
the stock solution of coumestrol.
2.6. Sample preparation
Rat tissue homogenates were homogenized using a high-speed
homogenizer by adding purified water to liver, spleen, kidney, heart,
lung, muscle, thymus and brain tissues at a ratio of 5: 1. The calibration
standards samples for ICT were prepared by spiking 90 μL of blank
tissue homogenates with 10 μL of ICT working solution. The ultimate
concentrations of ICT in rat tissue homogenates were 0.5–20 ng/mL.
1384 S.-Q. Zhang / Journal of Ethnopharmacology 155 (2014) 1382–1387
Quality control (QC) samples in rat tissue homogenates were prepared
in the similar way with the three ICT concentrations of 1, 8 and 15
ng/mL.
To 100 mL of tissue homogenate sample was added 10 mL of
coumestrol solution. Following the addition of 200 μL of acetoni-
trile, the sample was vortex-mixed for 5 min and centrifuged at
10,000 g for 10 min. The supernatant was transferred to an
autosampler vial, and a 4-μL aliquot of the sample was injected
into UPLC–MS/MS. Samples with concentrations above the upper
limit of quantification (ULOQ) were diluted with blank tissue
homogenate and reanalyzed.
2.7. Method validation
2.7.1. Selectivity and matrix effect
Assay selectivity was evaluated in rat tissue homogenates.
Blank rat tissue homogenates were obtained from six animals
and the results were compared with those obtained from ICT
solution at the lower limit of quantification (LLOQ). At levels of 1,
8 and 15 ng/mL, the matrix effect was tested by comparing the
response peaks of the extracted blank tissue homogenates spiked
with the drug to the samples which were injected directly with
the drugs.
2.7.2. Extraction recovery
The extraction recovery of ICT from tissue homogenates was
tested at low, middle and high concentrations of 1, 8 and 15 ng/mL
in the range of calibration curve. The ICT peak areas in tissue
homogenates were compared to those obtained from injecting the
equivalent amount of the drug directly.
2.7.3. Linearity
The linearity of the assay was assessed by calibration curves
ranging from 0.5 to 20 ng/mL for ICT in various tissue homoge-
nates. The calibration curve was established by plotting peak area
ratios of ICT to coumestrol versus nominal concentration.
2.7.4. Accuracy and precision
The intra- and inter-day accuracy and precision of the assay
were evaluated by analysis of low, middle and high QC samples in
six replicates against calibration standards on the same day over
three days. The accuracy and precision were calculated and
expressed as the percentage value of observed concentration to
theoretical concentration and the relative standard deviation
(RSD), respectively. For acceptable intra- and inter-day values,
the accuracy should be within 85–115% with an exception of 80–
120% for the LLOQ level. The precision at each concentration level
from the nominal concentration was expected to be within 715%
except LLOQ, for which it should be within 720%.
2.7.5. Carryover and dilution integrity
Carryover studies were performed in order to evaluate any
interference on the samples that followed the high level standard.
Carryover in the blank sample following ULOQ should not be
greater than 20% of LLOQ and 5% for the IS. Furthermore, the
evaluation of carryover was performed by comparing the average
results of the blank tissue samples assayed three times before and
three times after running ULOQ (20 ng/mL). The dilution integrity
experiment was performed with a purpose to validate the dilution
test to be carried out on higher analyte concentrations (above
ULOQ), which may be encountered during real tissue samples
analysis. Dilution integrity experiment was performed at 20 μg/mL
(1000-fold of ULOQ). Six replicate samples were diluted to 1/2000
(10 ng/mL) by spiking blank tissue homogenate and their concen-
trations were calculated by applying the dilution factor of 2000
against calibration curve. Accuracy and precision should be
within 715%.
2.7.6. Stability
The test included short-term temperature, post-preparative
and long-term temperature stabilities which covered the actual
experimental conditions that the samples may experience. The
stability of ICT in tissue homogenates was evaluated using 1 and
15 ng/mL QC samples. Short-term temperature stability samples
containing ICT were left to stand at ambient temperature for 6 h
until pretreatment. The post-preparative stability of the prepared
tissue samples was determined after keeping the samples at
ambient temperature for 8 h. The long-term stability was evalu-
ated after keeping the tissue samples frozen at 80 1C for
7 consecutive days. Thereafter, samples were analyzed and the
resulted values for these samples were then compared to nominal
concentration. The analytes were considered stable under different
conditions when a deviation of less than 715% from the nominal
value was obtained.
2.8. Pharmacokinetics and biodistribution of ICT
The analytical method was applied to tissue distribution of rats
after intraperitoneal administration of ICT. Adult female Sprague-
Dawley rats, weighing 2427 12 g, were housed in a 12 h light-dark
cycle and temperature-controlled facility for at least 7 days prior
to the study. The animals were deprived of food overnight for 12 h,
although they were allowed free access to drinking water before
and during the course of experiments. ICT was dissolved in a
mixture solvent containing Cremophor EL: ethanol: PEG 400:
saline (13:7:40:40, v/v). Multiple doses of ICT were intraperitone-
ally injected to rats daily for 7 consecutive days at dose levels of
20, 40 and 60 mg/kg/day, respectively. At designated time-points
after the last dosing on the 7th day, blood was collected in a
heparin-coated tube and plasma was separated by centrifugation
for 10 min. Immediately after the last time-point blood collection,
rats were sacrificed and tissues (liver, spleen, kidney, heart, lung,
muscle, thymus and brain) were immediately excised. These
tissues were rinsed with ice-old saline and lightly blotted dry to
remove any excess blood. All the samples were stored at  80 1C
until analysis within a week.
3. Results and discussion
3.1. Liquid chromatography–mass spectrometry
As the signals of ICT and coumestrol were stronger at ESI 
mode as compared to the ESI þ mode, the ESI  mode was selected
for the determination of analytes in the experiment. In Q1 MS full
scan spectra, the analytes gave predominant singly charged pre-
cursor ions at m/z of 367.1 for ICT and precursor ion at m/z of 267.0
for the IS, respectively. Different volume ratios of methanol-water
and acetonitrile-water combinations were explored as a mobile
phase, along with formic acid, ammonium acetate and ammonium
formate buffers. It was observed that gradient conditions of
methanol and ammonium formate buffer as the mobile phase
was most appropriate for a faster elution, better efficiency and
peak shape.
3.2. Selectivity and matrix effect
Method selectivity was evaluated in rat tissue homogenates and
no significant endogenous interfering response peaks were observed
at or near the retention times of ICT (3.12 min) and coumestrol
(2.66 min) in the chromatograms of tissue homogenates (Fig. 2).
 S.-Q. Zhang / Journal of Ethnopharmacology 155 (2014) 1382–1387
Fig. 2. Representative MRM chromatograms of ICT in heart tissue homogenate (I: m/z
367.1-297.1 for ICT; II: m/z 267.0-211.1 for coumestrol) for (A) a blank heart
homogenate; (B) a heart homogenate spiked with ICT at LLOQ level; (C) a heart sample
obtained 8 h on the 7th day after intraperitoneal administration of ICT at a dose of
20 mg/kg/day.
Chromatographic conditions may cause co-elution of endogenous
compounds that are undetected by the MS–MS but may affect the
ionization efficiency. Therefore, the effect of matrix on the response
1385
of the analytes was also evaluated. As shown in Table 1, the RSD
value of matrix effect at each concentration is not greater than 10.5%.
3.3. Extraction recovery
For the extraction of ICT from rat tissues, a higher recovery was
obtained using acetonitrile compared to ethyl acetate, therefore,
acetonitrile was used an extraction solvent. The extraction recov-
eries of ICT from tissue homogenates at levels of 1, 8 and 15 ng/mL
are shown in Table 1. The mean extraction recoveries of ICT in
tissues were in the range of 85.5–92.1% with a RSD value of less
than 12.5%. These results indicated that there was no relevant
difference in extraction recovery at different concentrations of ICT.
3.4. Linearity and lower limit of quantification
Calibration curve was generated by least-squares linear regres-
sion weighting by the reciprocal of the concentration in tissue
homogenates. All linear equations and correlation coefficients of
ICT in tissue homogenates are shown in Table 2. The linear range
was 0.5–20 ng/mL in tissue homogenates and correlation coeffi-
cients of all calibration curves were greater than 0.98. The non-
zero standards showed less than 15% deviation at all concentration
points, and showed less than 20% deviation at the concentration of
0.5 ng/mL. The LLOQ for ICT was 0.5 ng/mL in tissue homogenates.
3.5. Accuracy and precision
The results of accuracy and precision are presented in Table 1.
The intra- and inter-day accuracy for ICT at 1, 8 and 15 ng/mL
levels in rat tissue homogenates fell in the ranges of 90.8–103.4%
and 91.6–100.3%, and the intra- and inter-day precision (RSD) were
in the ranges of 2.9–10.5% and 2.6–9.1%, respectively. These results
demonstrated that the values were within the acceptable range,
and that the method was sufficiently accurate and precise.
3.6. Carryover and dilution integrity
There was no carryover observed in the presence of ULOQ
(20 ng/mL of ICT and 100 ng/mL of IS), which was due to the
specificity of the LC–MS/MS method. The dilution integrity test
demonstrated an accuracy range of 94.3–107.9% with a RSD range
of 4.9–12.7% for all diluted tissue samples.
3.7. Stability
The stability results showed that tissue homogenate samples
could be prepared at ambient temperature during the period of a
minimum of 6 h without any indication of degradation and
processed tissue samples of ICT were stable for 8 h. These studies
also suggested that rat tissue samples containing ICT could be
stored at  80 1C for at least 7 days without significant loss of
compound, as shown in Table 3.
3.8. Pharmacokinetics and biodistribution of ICT
The method was successfully applied to the quantification of
ICT in plasma and tissues after multiple intraperitoneal adminis-
tration of ICT to rats at dose levels of 20, 40 and 60 mg/kg/day. The
concentration-time profiles of ICT in plasma are shown in Fig. 3.
The ICT levels in rat tissues at 8 h post-dosing are shown in Fig. 4.
The maximum concentration was reached at 1 h for the dose levels
of 20 and 60 mg/kg/day, while the value for 40 mg/kg was 0.5 h.
Area under curve from 0 to 8 h (AUC0–8 h) values for 20, 40 and
60 mg/kg/day were 1963, 7450 and 15885 h ng/mL, respectively,
and the values linearly correlated with the doses (r¼ 0.993). The
1386 S.-Q. Zhang / Journal of Ethnopharmacology 155 (2014) 1382–1387

Table 1
Accuracy, precision and recovery of the method for the determination of ICT in rat tissues. Data are expressed in % (n¼6).

Tissues Concentration (ng/mL) Intra-day Inter-day Extraction recovery Matrix effect

Accuracy RSD Accuracy RSD Accuracy RSD Accuracy RSD

Liver 1 90.8 5.6 91.6 6.3 91.7 7.2 93.6 9.2

8 95.3 3.3 97.1 3.5 90.7 4.8 97.6 5.1
15 94.8 2.9 95.8 2.6 88.6 2.6 98.3 3.4
Spleen 1 95.2 6.1 93.4 4.6 87.1 8.1 92.7 8.8
8 95.9 5.3 95.7 5.2 89.5 7.9 94.2 6.3
15 97.3 4.8 95.3 4.6 91.4 4.5 96.4 4.7
Kidney 1 96.2 5.6 93.5 6.4 89.4 7.3 91.0 7.1
8 97.8 6.1 96.0 5.7 88.2 6.5 96.2 7.3
15 101.9 5.2 98.1 5.1 87.0 4.4 95.7 6.2
Heart 1 97.7 6.6 100.3 7.5 85.5 5.3 95.2 5.9
8 95.6 4.8 96.6 8.4 89.7 7.8 94.9 7.6
15 98.4 3.2 105.1 5.7 88.2 8.7 98.3 8.5
Lung 1 94.6 4.4 95.6 9.1 87.6 7.5 89.7 9.2
8 97.2 7.5 94.0 6.4 88.2 5.8 93.6 4.8
15 97.9 6.1 92.3 4.5 87.7 6.8 96.1 7.3
Muscle 1 94.7 8.7 93.2 8.6 87.0 7.9 90.2 8.3
8 103.4 7.1 99.9 6.3 92.1 7.3 95.5 6.2
15 98.3 4.7 95.1 7.1 89.4 8.1 93.4 6.9
Thymus 1 92.1 7.5 95.4 9.1 90.6 9.3 95.4 6.3
8 96.6 8.7 97.1 7.6 89.0 5.8 94.3 5.7
15 98.9 6.8 97.6 5.2 90.2 6.1 96.7 3.8
Brain 1 98.2 10.5 93.4 8.1 87.6 12.5 95.1 10.5
8 97.1 8.7 95.4 6.9 85.8 8.4 93.4 7.7
15 101.9 6.8 98.8 7.8 89.3 6.9 92.3 5.8

Table 2
Calibration curves of ICT in rat tissue homogenates.

Tissues Calibration curve Correlation coefficient (r)

Liver Y ¼ 0.070176X þ 0.053509 0.9920

Spleen Y ¼ 0.150110X  0.158606 0.9845
Kidney Y ¼ 0.062727X þ0.053908 0.9964
Heart Y ¼ 0.054312X þ 0.126048 0.9928
Lung Y ¼ 0.055540X þ0.004854 0.9966
Muscle Y ¼ 0.050380X  0.033120 0.9947
Thymus Y ¼ 0.156779X  0.023125 0.9966
Brain Y ¼ 0.117516X  0.010976 0.9917

Table 3
Stability results for ICT in rat tissue homogenate. Data are expressed in % (n ¼3).

Tissues Concentration Short-term Post-preparative Long-term

(ng/mL)
Accuracy RSD Accuracy RSD Accuracy RSD
Fig. 3. Concentration-time profiles of ICT in rat plasma on the 7th day after rats
Liver 1 93.2 7.3 97.4 5.2 96.4 6.9 were intraperitoneally injected with ICT at dose levels of 20, 40 and 60 mg/kg/day
15 97.4 3.1 98.5 3.4 98.6 5.0 for 7 consecutive days (n¼ 4).
Spleen 1 94.3 9.4 96.1 6.6 97.1 6.5
15 97.6 2.9 98.2 3.7 98.2 3.1
Kidney 1 93.4 6.4 99.4 7.9 99.1 5.3 Icaritin-7-glucuronide, (6R)-6-(3,5-dihydroxy-2-(4-methoxyphe-
15 98.3 2.8 97.8 6.3 97.0 4.1 nyl)-8-(3-methylbut-2-enyl)-4-oxo-4H-chromen-7-yloxy)-3,4,5-
Heart 1 101.2 7.9 98.5 7.7 95.6 8.2 trihydroxytetrahydro-2H-pyran-2-carboxylic acid, was found to be
15 98.4 3.2 100.7 8.2 98.6 4.6
the major metabolite in rat plasma and tissues in the preliminary
Lung 1 97.8 5.7 96.6 6.4 96.4 7.8
15 98.6 4.9 99.3 5.9 96.3 4.7
investigation.
Muscle 1 96.8 8.3 98.4 8.0 93.2 4.1
15 98.4 2.7 99.6 5.8 95.3 4.4
Thymus 1 98.3 5.5 95.2 6.4 96.7 7.6 4. Conclusion
15 98.5 3.8 103.9 5.5 98.5 5.7
Brain 1 93.2 7.1 104.3 6.8 97.5 11.6
15 103.2 6.4 98.4 5.7 95.4 8.6 A specific, sensitive and rapid UPLC–MS/MS method for the
quantification of ICT in rat tissues has been firstly developed and
validated, and found to be accurate and precise for the analysis of
ICT in tissues. The pretreatment of biological specimens combines
biodistribution results demonstrated that ICT was widely distrib- only a rapid one-step protein precipitation method with MRM
uted in rat's various tissues and its concentrations in tissues detection to achieve the selective and specific quantification of ICT
increased with elevated doses. At 8 h after the last dosing, the with low sample volumes. The method has been applied success-
content of ICT remained relatively high in liver and muscle. fully, for the first time, to quantitatively analyze ICT in rat plasma
 S.-Q. Zhang / Journal of Ethnopharmacology 155 (2014) 1382–1387
Fig. 4. Distribution profiles of ICT in rat tissues at 8 h on the 7th day after rats were
intraperitoneally injected with ICT at dose levels of 20, 40 and 60 mg/kg/day for
7 consecutive days (n¼ 4).
and tissues after intraperitoneal administration. Wide distribution
of ICT could helpfully elucidate systemic effects and various
functions of ICT.
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