Metabolite Profiling, Pharmacokinetics, and In Vitro...
Transcript of Metabolite Profiling, Pharmacokinetics, and In Vitro...
Research ArticleMetabolite Profiling Pharmacokinetics and In VitroGlucuronidation of Icaritin in Rats by Ultra-Performance LiquidChromatography Coupled with Mass Spectrometry
Beibei Zhang Xiaoli Chen Rui Zhang Fangfang Zheng Shuzhang Du and Xiaojian Zhang
Department of Pharmacy The First Affiliated Hospital of Zhengzhou University Zhengzhou Henan 450052 China
Correspondence should be addressed to ShuzhangDu dushuzhang911163com andXiaojian Zhang zhangxiaojian_yxb163com
Received 5 April 2017 Accepted 23 May 2017 Published 10 July 2017
Academic Editor Filomena Conforti
Copyright copy 2017 Beibei Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Icaritin is a naturally bioactive flavonoid with several significant effects This study aimed to clarify the metabolite profilingpharmacokinetics and glucuronidation of icaritin in rats An ultra-performance liquid chromatography coupled with massspectrometry (UPLC-MS) assay was developed and validated for qualitative and quantitative analysis of icaritin Glucuronidationrates were determined by incubating icaritin with uridine diphosphate glucuronic acid- (UDPGA-) supplemented microsomesKinetic parameters were derived by appropriate model fitting A total of 30 metabolites were identified or tentatively characterizedin rat biosamples based on retention times and characteristic fragmentations following proposed metabolic pathway which wassummarized Additionally the pharmacokinetics parameters were investigated after oral administration of icaritin Moreovericaritin glucuronidation in rat liver microsomes was efficient with CLint (the intrinsic clearance) values of 112 and 156mLminmgfor icaritin-3-O-glucuronide and icaritin-7-O-glucuronide respectively Similarly the CLint values of icaritin-3-O-glucuronideand icaritin-7-O-glucuronide in rat intestine microsomes (RIM) were 145 and 086mLminmg respectively Taken altogetherdehydrogenation at isopentenyl group and glycosylation and glucuronidation at the aglycone were main biotransformation processin vivo The general tendency was that icaritin was transformed to glucuronide conjugates to be excreted from rat organism Inconclusion these results would improve our understanding of metabolic fate of icaritin in vivo
1 Introduction
Herba Epimedii the dried aerial parts of Epimedium L(Berberidaceae) are a widely used Chinese medicine forimpotence bone loss and cardiovascular diseases [1ndash3]Prenylflavonoids are reported to be a group of major activeconstituents present in Epimedium for the antioxidativestress anti-inflammatory antitumor and antiosteoporosisactivities [4ndash8] Icaritin is the common aglycone with manybiological effects especially antiosteoporosis activities [5 7]Besides icaritin could induce cell death in activated hepaticstellate cells through mitochondrial activated apoptosis andameliorate the development of liver fibrosis in rats [9] Mean-while icaritin is able to target androgen receptor and andro-gen receptor COOH-terminal truncated splice variants toinhibit androgen receptor signaling and tumor growth withno apparent toxicity [10] Additionally icaritin has neuro-protective effects against MPP+-induced toxicity in MES235
cells IGF-I receptor mediated activation of PI3KAkt andMEKERK12 pathways are involved in the neuroprotectiveeffects of icaritin against MPP+-induced neuronal damage[11] Recently icaritin had been shown as a potential agentfor the treatment of systemic lupus erythematosus [12]
These biological activities above had stimulated increas-ing interests in the in vivo metabolism of icaritin or itsrelated prenylflavonoids Poor bioavailability of prenylatedflavonoids results from their poor intrinsic permeation andtransporter-mediated efflux by the human intestinal Caco-2model and the perfused rat intestinal model [13] Meanwhileit is shown that Epimedium flavonoids could be hydrolyzedinto secondary glycosides or aglycone by intestinal flora orenzymes thereby enhancing their absorption and antiosteo-porosis activity [14] So far numerous researches of totalprenylflavonoids or individual flavonoid had been conductedin the fields of in vivo metabolites profiling biliary excre-tion and pharmacokinetics [15ndash19] Generally the in vivo
HindawiJournal of Analytical Methods in ChemistryVolume 2017 Article ID 1073607 13 pageshttpsdoiorg10115520171073607
2 Journal of Analytical Methods in Chemistry
metabolismofHerba Epimedii extracts or its prenylflavonoidscould easily bemetabolized in gastrointestinal tract followingdeglycosylation reaction Additionally icaritin was easilymetabolized into glucuronidation conjugates to be prefer-entially eliminated and excreted from rat organism [16 1820] Though the data on metabolic researches of icaritinabounds its metabolic profile is not so clear It is essential tosystematically characterize the in vivo metabolites in orderto better understand its mechanism of action Hence thepresent study aimed to conduct the metabolites screeningquantitative determination and in vitro glucuronidation oficaritin
Recently liquid chromatography coupledwithmass spec-trometry (LC-MS) had been widely introduced to rapidlyscreen trace components in biological samples [21 22] Inthis study icaritin-related metabolites were analyzed basedon characteristic fragmentation by UPLC-MS after oraladministration Meanwhile possible disposing pathway oficaritinwas proposed Furthermore aUPLC-MSmethodwasdeveloped and applied to perform the pharmacokinetics oficaritin Moreover glucuronidation rates were determinedby incubating icaritin with uridine diphosphate glucuronicacid- (UDPGA-) supplemented rat liver microsomes (RLM)and rat intestinemicrosomes (RIM) Kinetic parameters werederived by appropriatemodel fitting Icaritin was subjected tosignificant hepatic and gastrointestinal glucuronidation
2 Materials and Methods
21 Materials Icaritin epimedin C icariside I icarisideII and desmethylicaritin (purity gt 98) were purchasedfrom Nanjing Jingzhu Medical Technology Co Ltd Uridinediphosphate glucuronic acid (UDPGA) magnesium chloride(MgCl2) alamethicin D-saccharic-1 and 4-lactone wereprovided from Sigma-Aldrich (St Louis MO) Rat livermicrosomes (RLM) and rat intestinemicrosomes (RIM)wereprepared in our laboratory based on the protocol [21] HPLCgrademethanol and acetonitrile were purchased fromDikmaScientific and Technology Co Ltd All other chemicals wereof analytical grade
22 Animals Male Sprague-Dawley rats (180sim220) g wereprovided by Guangdong Medical Laboratory Animal CenterThe rats were kept in an animal room at constant temperature(24 plusmn 2)∘C and humidity (60 plusmn 5) with 12 h of lightdarkper day and free access to water and food The animalprotocols were approved and conducted in accordance withthe guidelines of Laboratory Animal Ethics Committee ofZhengzhou University
23 Samples Collection and Preparation for Qualitative Anal-ysis After the rats were fasted for 12 h with free access towater before experiments icaritin dissolved in 03 sodiumcarboxymethyl cellulose solution was orally administrated torats at a dose of 100mgkg Blood samples were collectedfrom external jugular vein into heparinized tubes and wereseparated by centrifuging at 13800119892 for 10min at 4∘Crespectively Bile samples were collected and recorded during
0ndash24 h period after an abdominal incision anesthetized with10 aqueous chloral hydrate The urine and feces sampleswere collected separately during 0ndash24 h period after oraladministration Small intestinal samples were obtained afteroral administration for 24 h All blank samples were obtainedin the same way
Before experiments all biosamples were stored at minus20∘CIn this work solid phase extraction method was applied topretreat all samples Before use C18 columns (3 cm3 60mg)were first preconditioned and equilibrated with 3mL ofmethanol and 3mL of water respectively Urine samples wereevaporated and concentrated at 40∘Cunder reduced pressureFeces samples and small intestinal samples were dried in airand stirred into powder And then they were treated withan ultrasonic bath for 30min The filtrate was combined andevaporated to dryness at 40∘C in vacuum The residue wasreconstituted with water Plasma urine bile feces and smallintestinal samples were loaded on pretreated columns Theresidue was reconstituted in 200120583L of 60 methanol andfiltered through a 022 120583mmembrane until injection
24 Samples Preparation for Quantitative Analysis Plasmasample (200120583L) was treated with methanol (12mL) afterwhich the mixture was vortex-mixed for 30 s and centrifugedat 13800119892 for 10min at 4∘C The supernatant was thentransferred and evaporated to dryness using N2 at roomtemperature The residue was dissolved in 200 120583L of 60methanol and was then injected into the UPLC-MS system
25 Preparation of Standard Solutions Blank rat plasmawas spiked with standard working solutions to achieve finalconcentration of icaritin of 20 40 160 640 1280 2560and 5120 ngmL All reference standard solutions were storedat 4∘C until use
26 Glucuronidation Assay Icaritin was incubatedwith RLMand RIM to determine the rates of glucuronidation aspublished references previously [23] Briefly the incubationmixture mainly contained 50mM Tris-hydrochloric acidbuffer (pH = 74) 088mM MgCl2 22 120583gmL alamethicin44mM saccharolactone and 35mM UDPGA The reactionwas terminated by adding ice-cold acetonitrile The sampleswere vortexed and centrifuged at 13800119892 for 10min Thesupernatant was subjected to UPLC-MS analysis All experi-ments were performed in triplicate
27 UPLC-MS Conditions UPLC was performed using anACQUITY UPLC system (Waters Milford MA USA)Separation was achieved on a Waters BEH C18 column(17 120583m 21 times 50mm) maintained at 35∘C The mobile phaseconsisted of water (A) and acetonitrile (B) (both containing01 formic acid) and the flow rate was 05mLmin Thegradient elution program was as follows 0min 15 B 3min35 B 7min 60 B 8min 100 B An aliquot of 4 120583L samplewas then injected into the UPLC-MS system
The UPLC system was coupled to a Waters Xevo TQD(Waters Milford MA USA) with electrospray ionization
Journal of Analytical Methods in Chemistry 3
The operating parameters were as follows capillary volt-age 25 kV (ESI+) sample cone voltage 300V extractioncone voltage 40 V source temperature 100∘C desolvationtemperature 300∘C and desolvation gas flow 800 Lh Themethod employed lock spray with leucine enkephalin (mz5562771 in positive ion mode and mz 5542615 in negativeion mode) to ensure mass accuracy
28 Pharmacokinetic Application After fasting with freeaccess to water for 12 h icaritin was given to rats as a dosage of100mgkg Plasma samples were then obtained at 0083 02505 1 2 3 4 6 8 12 24 36 and 48 h after administration Forpharmacokinetic application DAS 20 was used to calculatethe pivotal pharmacokinetic parameters
29 Enzymes Kinetic Evaluation Serial concentrations oficaritin (04sim20120583M) were incubated with RLM and RIM todetermine icaritin glucuronidation rates The kinetic modelsMichaelis-Menten equation and substrate inhibition equa-tion were fitted to the data of metabolic rates versus substrateconcentrations and displayed in (1) and (2) respectivelyAppropriate models were selected by visual inspection ofthe Eadie-Hofstee plot [24] Model fitting and parameterestimation were performed by Graphpad Prism V5 software(San Diego CA)
The parameters were as follows 119881 is the formation rateof product 119881max is the maximal velocity 119870m is the Michaelisconstant and [119878] is the substrate concentration 119870si is thesubstrate inhibition constant The intrinsic clearance (CLint)was derived by 119881max119870m for Michaelis-Menten and substrateinhibition models
119881 =119881max times [119878]
119870m + [119878] (1)
119881 =119881max times [119878]
119870m + [119878] (1 + [119878] 119870si) (2)
3 Results
31 Fragment Pattern of Icaritin As had already beenreported in the previous study [16] besides the typicaladduct ion [M+Na]+ at mz 3911151 (C21H20O6Na) and[M+H]+ at mz 3691336 (C21H21O6 minus05 ppm) the ionat mz 3130714 (C17H13O6) in positive ion mode wasconsidered as the characteristic fragment ion (see Fig-ure S1a in the Supplementary Material available online athttpsdoiorg10115520171073607)
32 Screening of Metabolites On the basis of MSMS frag-mentation pattern the metabolites were deduced clarifyingthe general metabolism in vivo The extracted ion chro-matograms (EICs) of prototype (M0) and metabolites (M1simM30) were shown in Figure 1 while the individual EICsof M1simM30 were exhibited in Figure S2 The UV MS andMSMS data of M0simM30 were all exhibited in Table 1
33 Structure Elucidation of Metabolites
M0 (Parent Drug) M0 (720min C21H20O6 minus05 ppm) inbiological samples was unambiguously identified by compar-ing with references
M15 and M23 (Hydration of Isopentene Group) Based onthe [M+H]+ ion at mz 3871445 (C21H23O7 03 ppm) and[MminusH]minus ion at mz 3851286 (C21H21O7) the molecular for-mula of M15 (434min) and M23 (497min) was determinedas C21H22O7 with one H2O more than M0 The MSMSspectrum of [M+H]+ ion (C21H23O7) showed predominant[M+HminusH2O]
+ ion at mz 3691343 and 3130715 (FigureS1b) which indicated that M15 and M23 were the hydrationproducts at isopentene group of icaritin and agreed withprevious study of icariin [18]
M25 (Demethylation of Flavonoid Aglycone) According tothe [M+H]+ ion at mz 3551180 (C20H19O6 minus06 ppm) and[MminusH]minus ion atmz 3531036 (C20H17O6) the formula ofM25(508min) was supposed as C20H18O6 with a methyl groupless than M0 The MSMS experiments (Figure S1c) showeda significant loss of neutral loss of C4H8 (560626Da) fromthe ion at mz 3551180 to 2990582 in positive ion mode orfrom the ion atmz 3531036 to 2970373 in negative ionmodeMeanwhile the demethylation position was purposed at 41015840position of B ring of flavonoid aglycone Moreover M25 wasidentified as desmethylicaritin by comparison of referencestandard
M30 (Dehydrogenation of Isopentene Group) The formulaof M30 (636min) was C21H18O6 with two hydrogensfewer than M0 based on the [M+H]+ ion at mz 3671180(C21H19O6 minus05 ppm) and [MminusH]minus ion at mz 3651047(C21H17O6) In MSMS spectrum (Figure S1d) the ions atmz 3520948 and 3130721 were attributed to obvious loss ofCH3 (150235Da) and C4H6 (540470Da) group respectivelywhich indicated that the dehydrogenation position was atisopentene group [18]
M27simM29 (Hydroxylation of Isopentene Group) From the[M+H]+ ion at mz 3851288 (C21H21O7 03 ppm) and[MminusH]minus ion at mz 3831175 (C21H19O7) the formulae ofM27 (537min 120582max 268 nm) M28 (554min) and M29(566min) were speculated as C21H20O7 which was oneoxygen more than M0 In (+) ESI-MSMS spectrum (FigureS1e) the ion atmz 3851288 could lose aH2OandC4H6 groupto produce the daughter ions at 3671186 ([M+HminusH2O]
+) and3130715 ([M+HminusC4H6]
+) respectively This illustrated thatM27simM29 were tentatively characterized as the hydroxylatedproducts of M0 at the isopentene group
M14 M16 M17 M19 M20 and M21 (Glycosylation ofFlavonoid Aglycone) M21 (488min) was given a [M+H]+ ionatmz 5151927 (C27H31O10 19 ppm) and [MminusH]minus ion atmz5131766 (C27H29O10) in full scan mass spectrum The ion atmz 5151927 could easily yield the characteristic fragmentions at mz 3691340 and 3130726 by subsequent loss ofC6H10O4 and C4H8 (Figure S1f) So M21 could be the gly-cosylation product of M0 by adduct of rhamnose (C6H10O41460579Da) Similarly M17 (453min C27H30O11 15 ppm)
4 Journal of Analytical Methods in Chemistry
100 200 300 400 500 600 700 800 900
0
100
()
M0
M1
M13M18
Time
1 TOF MS ES+369134 + 721198 + 545166 00200 Da839e3
(a)
100 200 300 400 500 600 700 800 900
0
100
() M0M1
M2M3
M4
M5M6
M8
M10
M13M18 M23
M25M27
M28M29
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561161 + 545166 + 387144 + 355118 + 385129386e4
(b)
100 200 300 400 500 600 700 800 900
0
100
()
M0M1 M2 M3
M4
M5M6
M7M8
M9M10M11
M13M18
M22M24
M26
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561162 + 545166 00200 Da491e4
(c)
100 200 300 400 500 600 700 800 900
0
100
() M0
M13M17
M18M20
M21M23M25
M27M28
M29 M30
1 TOF MS ES+369134 + 545166 + 531187 + 66125 + 515193 + 387144 + 355118 + 385129 + 367118177e5
Time
(d)
100 200 300 400 500 600 700 800 900
M0
M1 M2
M3M4
M5 M6M7
M8M9
M10M11
M12
M13M14
M15M16M17M18M19M20
M21M22M23M24
M25M26
M27M28 M29
M300
100
()
Time
1 TOF MS ES+Sum 00200 Da548e5
(e)
Figure 1 EICs of all metabolites in rat biosamples after oral administration of icaritin (a) Plasma (b) urine (c) bile (d) feces (e) intestine
Journal of Analytical Methods in Chemistry 5
Table1UPL
C-MSanalysisof
icaritinandits
observed
metabolitesinbiosam
ples
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M0
720
272
3691336
ndash05
C 21H20O6
3911152
369
1336
3130714
2150748
3671
1803520941
3090
4172970
399
2810
44325304
85PU
BFI
Icaritin
M1
248
270
7211978
ndash03
C 33H36O18
7431882
7211978
5451666
369
1345
3130717
7191
8255431521
3671
194
PUBI
Icaritin-di-O
-gluA
M2
262
269
5631768
05
C 27H30O13
5631768
3871
442
3691
3323130716
561160
838
512
88UBI
Hydrated
icaritin-O-gluA
M3
289
345
5311511
15C 26H26O12
53115113
5511
662990
576
5291
348
353
1027
1190504
UBI
Desmethylicaritin-
O-gluA
M4
315
341
5611617
16C 27H28O13
5611617
385
1301
3671186
5591456
383
1175
36711351750263
UBI
Hydroxylated
icaritin-O-gluA
M5
322
341
5611613
09
C 27H28O13
5611613
385
1296
5591458
383
1172
1750262
UBI
Hydroxylated
icaritin-O-gluA
M6
336
345
5311503
00
C 26H26O12
5311503
355
1175
3290
7413
140455
5291
345
353
1025
1190506
UBI
Desmethylicaritin-
O-gluA
M7
341
341
5611605
ndash05
C 27H28O13
5611605
385
1295
3130715
559146
038
311
71BI
Hydroxylated
icaritin-O-gluA
M8
346
270
7211982
03
C 33H36O18
72119825451662
369
1340
3130717
7191
8235431519
3671
196
UBI
Icaritin-di-O
-gluA
M9
352
341
5611608
0C 27H28O13
561160
838
512
835591454
383
1170
1750260
BIHydroxylated
icaritin-O-gluA
M10
358
269
5631761
ndash07
C 27H30O13
5631761
3871
452
3691
3503130735
5611605
385
1289
UBI
Hydrated
icaritin-O-gluA
6 Journal of Analytical Methods in Chemistry
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M11
374
341
5611615
12C 27H28O13
5611615
385
1292
3671186
5591462
383
1175
BIHydroxylated
icaritin-O-gluA
M12
429
na
5431501
ndash04
C 27H26O12
5431501
3671
189
5411343
365
1034
3510
7651750221
1130253
IDehydrogenated
icaritin-O-gluA
M13
434
271
5451663
07
C 27H28O12
56714865451663
369
1335
3130716
5431501
3671
188
3520948
3090
408
2970
3982810
498
PUBF
IIcaritin-O-gluA
M14
440
270
8233024
ndash01
C 39H50O19
8233024
369
1324
3130721
8212
8636592
331
3671
1683510
867
IEp
imedin
C
M15
445
273
387144
0ndash10
C 21H22O7
3871
4403691
338
3130722
385
1288
IHydratedicaritin
M16
449
270
8233021
ndash05
C 39H50O19
8233021
369
1336
3130725
8212
8606592
333
3671
1653510
869
IEp
imedin
Ciso
mer
M17
453
270
5311866
15C 27H30O11
5311866
369
1350
3130718
5291
705
3671
182
3520948
2970
395
2530496
FIIcariside
I
M18
457
271
5451661
04
C 27H28O12
5451661
369
1346
3130723
5431505
3671
183
3520945
2970
394
PUBF
IIcaritin-O-gluA
M19
459
271
6472
349
14C 32H38O14
6692
2156472
349
5151930
369
1346
3130724
6452168
3671
162
3510
8633230945
2950595
IIcaritin-rha-xyl
M20
461
270
6612
503
11C 33H40O14
6832326
6612
503
369
1339
3130719
6592
343
3671
165
3510
86529506
022170
499
FIIcaritin-rha-rha
M21
488
269
5151927
19C 27H30O10
53717575151927
369
1340
3130726
5131766
3671
157
3520900
3230910
29506
042170
498
FIIcariside
II
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal ofInternational Journal ofPhotoenergy
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Analytical Methods in Chemistry
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Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Analytical ChemistryInternational Journal of
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Quantum Chemistry
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Organic Chemistry International
ElectrochemistryInternational Journal of
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CatalystsJournal of
2 Journal of Analytical Methods in Chemistry
metabolismofHerba Epimedii extracts or its prenylflavonoidscould easily bemetabolized in gastrointestinal tract followingdeglycosylation reaction Additionally icaritin was easilymetabolized into glucuronidation conjugates to be prefer-entially eliminated and excreted from rat organism [16 1820] Though the data on metabolic researches of icaritinabounds its metabolic profile is not so clear It is essential tosystematically characterize the in vivo metabolites in orderto better understand its mechanism of action Hence thepresent study aimed to conduct the metabolites screeningquantitative determination and in vitro glucuronidation oficaritin
Recently liquid chromatography coupledwithmass spec-trometry (LC-MS) had been widely introduced to rapidlyscreen trace components in biological samples [21 22] Inthis study icaritin-related metabolites were analyzed basedon characteristic fragmentation by UPLC-MS after oraladministration Meanwhile possible disposing pathway oficaritinwas proposed Furthermore aUPLC-MSmethodwasdeveloped and applied to perform the pharmacokinetics oficaritin Moreover glucuronidation rates were determinedby incubating icaritin with uridine diphosphate glucuronicacid- (UDPGA-) supplemented rat liver microsomes (RLM)and rat intestinemicrosomes (RIM) Kinetic parameters werederived by appropriatemodel fitting Icaritin was subjected tosignificant hepatic and gastrointestinal glucuronidation
2 Materials and Methods
21 Materials Icaritin epimedin C icariside I icarisideII and desmethylicaritin (purity gt 98) were purchasedfrom Nanjing Jingzhu Medical Technology Co Ltd Uridinediphosphate glucuronic acid (UDPGA) magnesium chloride(MgCl2) alamethicin D-saccharic-1 and 4-lactone wereprovided from Sigma-Aldrich (St Louis MO) Rat livermicrosomes (RLM) and rat intestinemicrosomes (RIM)wereprepared in our laboratory based on the protocol [21] HPLCgrademethanol and acetonitrile were purchased fromDikmaScientific and Technology Co Ltd All other chemicals wereof analytical grade
22 Animals Male Sprague-Dawley rats (180sim220) g wereprovided by Guangdong Medical Laboratory Animal CenterThe rats were kept in an animal room at constant temperature(24 plusmn 2)∘C and humidity (60 plusmn 5) with 12 h of lightdarkper day and free access to water and food The animalprotocols were approved and conducted in accordance withthe guidelines of Laboratory Animal Ethics Committee ofZhengzhou University
23 Samples Collection and Preparation for Qualitative Anal-ysis After the rats were fasted for 12 h with free access towater before experiments icaritin dissolved in 03 sodiumcarboxymethyl cellulose solution was orally administrated torats at a dose of 100mgkg Blood samples were collectedfrom external jugular vein into heparinized tubes and wereseparated by centrifuging at 13800119892 for 10min at 4∘Crespectively Bile samples were collected and recorded during
0ndash24 h period after an abdominal incision anesthetized with10 aqueous chloral hydrate The urine and feces sampleswere collected separately during 0ndash24 h period after oraladministration Small intestinal samples were obtained afteroral administration for 24 h All blank samples were obtainedin the same way
Before experiments all biosamples were stored at minus20∘CIn this work solid phase extraction method was applied topretreat all samples Before use C18 columns (3 cm3 60mg)were first preconditioned and equilibrated with 3mL ofmethanol and 3mL of water respectively Urine samples wereevaporated and concentrated at 40∘Cunder reduced pressureFeces samples and small intestinal samples were dried in airand stirred into powder And then they were treated withan ultrasonic bath for 30min The filtrate was combined andevaporated to dryness at 40∘C in vacuum The residue wasreconstituted with water Plasma urine bile feces and smallintestinal samples were loaded on pretreated columns Theresidue was reconstituted in 200120583L of 60 methanol andfiltered through a 022 120583mmembrane until injection
24 Samples Preparation for Quantitative Analysis Plasmasample (200120583L) was treated with methanol (12mL) afterwhich the mixture was vortex-mixed for 30 s and centrifugedat 13800119892 for 10min at 4∘C The supernatant was thentransferred and evaporated to dryness using N2 at roomtemperature The residue was dissolved in 200 120583L of 60methanol and was then injected into the UPLC-MS system
25 Preparation of Standard Solutions Blank rat plasmawas spiked with standard working solutions to achieve finalconcentration of icaritin of 20 40 160 640 1280 2560and 5120 ngmL All reference standard solutions were storedat 4∘C until use
26 Glucuronidation Assay Icaritin was incubatedwith RLMand RIM to determine the rates of glucuronidation aspublished references previously [23] Briefly the incubationmixture mainly contained 50mM Tris-hydrochloric acidbuffer (pH = 74) 088mM MgCl2 22 120583gmL alamethicin44mM saccharolactone and 35mM UDPGA The reactionwas terminated by adding ice-cold acetonitrile The sampleswere vortexed and centrifuged at 13800119892 for 10min Thesupernatant was subjected to UPLC-MS analysis All experi-ments were performed in triplicate
27 UPLC-MS Conditions UPLC was performed using anACQUITY UPLC system (Waters Milford MA USA)Separation was achieved on a Waters BEH C18 column(17 120583m 21 times 50mm) maintained at 35∘C The mobile phaseconsisted of water (A) and acetonitrile (B) (both containing01 formic acid) and the flow rate was 05mLmin Thegradient elution program was as follows 0min 15 B 3min35 B 7min 60 B 8min 100 B An aliquot of 4 120583L samplewas then injected into the UPLC-MS system
The UPLC system was coupled to a Waters Xevo TQD(Waters Milford MA USA) with electrospray ionization
Journal of Analytical Methods in Chemistry 3
The operating parameters were as follows capillary volt-age 25 kV (ESI+) sample cone voltage 300V extractioncone voltage 40 V source temperature 100∘C desolvationtemperature 300∘C and desolvation gas flow 800 Lh Themethod employed lock spray with leucine enkephalin (mz5562771 in positive ion mode and mz 5542615 in negativeion mode) to ensure mass accuracy
28 Pharmacokinetic Application After fasting with freeaccess to water for 12 h icaritin was given to rats as a dosage of100mgkg Plasma samples were then obtained at 0083 02505 1 2 3 4 6 8 12 24 36 and 48 h after administration Forpharmacokinetic application DAS 20 was used to calculatethe pivotal pharmacokinetic parameters
29 Enzymes Kinetic Evaluation Serial concentrations oficaritin (04sim20120583M) were incubated with RLM and RIM todetermine icaritin glucuronidation rates The kinetic modelsMichaelis-Menten equation and substrate inhibition equa-tion were fitted to the data of metabolic rates versus substrateconcentrations and displayed in (1) and (2) respectivelyAppropriate models were selected by visual inspection ofthe Eadie-Hofstee plot [24] Model fitting and parameterestimation were performed by Graphpad Prism V5 software(San Diego CA)
The parameters were as follows 119881 is the formation rateof product 119881max is the maximal velocity 119870m is the Michaelisconstant and [119878] is the substrate concentration 119870si is thesubstrate inhibition constant The intrinsic clearance (CLint)was derived by 119881max119870m for Michaelis-Menten and substrateinhibition models
119881 =119881max times [119878]
119870m + [119878] (1)
119881 =119881max times [119878]
119870m + [119878] (1 + [119878] 119870si) (2)
3 Results
31 Fragment Pattern of Icaritin As had already beenreported in the previous study [16] besides the typicaladduct ion [M+Na]+ at mz 3911151 (C21H20O6Na) and[M+H]+ at mz 3691336 (C21H21O6 minus05 ppm) the ionat mz 3130714 (C17H13O6) in positive ion mode wasconsidered as the characteristic fragment ion (see Fig-ure S1a in the Supplementary Material available online athttpsdoiorg10115520171073607)
32 Screening of Metabolites On the basis of MSMS frag-mentation pattern the metabolites were deduced clarifyingthe general metabolism in vivo The extracted ion chro-matograms (EICs) of prototype (M0) and metabolites (M1simM30) were shown in Figure 1 while the individual EICsof M1simM30 were exhibited in Figure S2 The UV MS andMSMS data of M0simM30 were all exhibited in Table 1
33 Structure Elucidation of Metabolites
M0 (Parent Drug) M0 (720min C21H20O6 minus05 ppm) inbiological samples was unambiguously identified by compar-ing with references
M15 and M23 (Hydration of Isopentene Group) Based onthe [M+H]+ ion at mz 3871445 (C21H23O7 03 ppm) and[MminusH]minus ion at mz 3851286 (C21H21O7) the molecular for-mula of M15 (434min) and M23 (497min) was determinedas C21H22O7 with one H2O more than M0 The MSMSspectrum of [M+H]+ ion (C21H23O7) showed predominant[M+HminusH2O]
+ ion at mz 3691343 and 3130715 (FigureS1b) which indicated that M15 and M23 were the hydrationproducts at isopentene group of icaritin and agreed withprevious study of icariin [18]
M25 (Demethylation of Flavonoid Aglycone) According tothe [M+H]+ ion at mz 3551180 (C20H19O6 minus06 ppm) and[MminusH]minus ion atmz 3531036 (C20H17O6) the formula ofM25(508min) was supposed as C20H18O6 with a methyl groupless than M0 The MSMS experiments (Figure S1c) showeda significant loss of neutral loss of C4H8 (560626Da) fromthe ion at mz 3551180 to 2990582 in positive ion mode orfrom the ion atmz 3531036 to 2970373 in negative ionmodeMeanwhile the demethylation position was purposed at 41015840position of B ring of flavonoid aglycone Moreover M25 wasidentified as desmethylicaritin by comparison of referencestandard
M30 (Dehydrogenation of Isopentene Group) The formulaof M30 (636min) was C21H18O6 with two hydrogensfewer than M0 based on the [M+H]+ ion at mz 3671180(C21H19O6 minus05 ppm) and [MminusH]minus ion at mz 3651047(C21H17O6) In MSMS spectrum (Figure S1d) the ions atmz 3520948 and 3130721 were attributed to obvious loss ofCH3 (150235Da) and C4H6 (540470Da) group respectivelywhich indicated that the dehydrogenation position was atisopentene group [18]
M27simM29 (Hydroxylation of Isopentene Group) From the[M+H]+ ion at mz 3851288 (C21H21O7 03 ppm) and[MminusH]minus ion at mz 3831175 (C21H19O7) the formulae ofM27 (537min 120582max 268 nm) M28 (554min) and M29(566min) were speculated as C21H20O7 which was oneoxygen more than M0 In (+) ESI-MSMS spectrum (FigureS1e) the ion atmz 3851288 could lose aH2OandC4H6 groupto produce the daughter ions at 3671186 ([M+HminusH2O]
+) and3130715 ([M+HminusC4H6]
+) respectively This illustrated thatM27simM29 were tentatively characterized as the hydroxylatedproducts of M0 at the isopentene group
M14 M16 M17 M19 M20 and M21 (Glycosylation ofFlavonoid Aglycone) M21 (488min) was given a [M+H]+ ionatmz 5151927 (C27H31O10 19 ppm) and [MminusH]minus ion atmz5131766 (C27H29O10) in full scan mass spectrum The ion atmz 5151927 could easily yield the characteristic fragmentions at mz 3691340 and 3130726 by subsequent loss ofC6H10O4 and C4H8 (Figure S1f) So M21 could be the gly-cosylation product of M0 by adduct of rhamnose (C6H10O41460579Da) Similarly M17 (453min C27H30O11 15 ppm)
4 Journal of Analytical Methods in Chemistry
100 200 300 400 500 600 700 800 900
0
100
()
M0
M1
M13M18
Time
1 TOF MS ES+369134 + 721198 + 545166 00200 Da839e3
(a)
100 200 300 400 500 600 700 800 900
0
100
() M0M1
M2M3
M4
M5M6
M8
M10
M13M18 M23
M25M27
M28M29
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561161 + 545166 + 387144 + 355118 + 385129386e4
(b)
100 200 300 400 500 600 700 800 900
0
100
()
M0M1 M2 M3
M4
M5M6
M7M8
M9M10M11
M13M18
M22M24
M26
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561162 + 545166 00200 Da491e4
(c)
100 200 300 400 500 600 700 800 900
0
100
() M0
M13M17
M18M20
M21M23M25
M27M28
M29 M30
1 TOF MS ES+369134 + 545166 + 531187 + 66125 + 515193 + 387144 + 355118 + 385129 + 367118177e5
Time
(d)
100 200 300 400 500 600 700 800 900
M0
M1 M2
M3M4
M5 M6M7
M8M9
M10M11
M12
M13M14
M15M16M17M18M19M20
M21M22M23M24
M25M26
M27M28 M29
M300
100
()
Time
1 TOF MS ES+Sum 00200 Da548e5
(e)
Figure 1 EICs of all metabolites in rat biosamples after oral administration of icaritin (a) Plasma (b) urine (c) bile (d) feces (e) intestine
Journal of Analytical Methods in Chemistry 5
Table1UPL
C-MSanalysisof
icaritinandits
observed
metabolitesinbiosam
ples
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M0
720
272
3691336
ndash05
C 21H20O6
3911152
369
1336
3130714
2150748
3671
1803520941
3090
4172970
399
2810
44325304
85PU
BFI
Icaritin
M1
248
270
7211978
ndash03
C 33H36O18
7431882
7211978
5451666
369
1345
3130717
7191
8255431521
3671
194
PUBI
Icaritin-di-O
-gluA
M2
262
269
5631768
05
C 27H30O13
5631768
3871
442
3691
3323130716
561160
838
512
88UBI
Hydrated
icaritin-O-gluA
M3
289
345
5311511
15C 26H26O12
53115113
5511
662990
576
5291
348
353
1027
1190504
UBI
Desmethylicaritin-
O-gluA
M4
315
341
5611617
16C 27H28O13
5611617
385
1301
3671186
5591456
383
1175
36711351750263
UBI
Hydroxylated
icaritin-O-gluA
M5
322
341
5611613
09
C 27H28O13
5611613
385
1296
5591458
383
1172
1750262
UBI
Hydroxylated
icaritin-O-gluA
M6
336
345
5311503
00
C 26H26O12
5311503
355
1175
3290
7413
140455
5291
345
353
1025
1190506
UBI
Desmethylicaritin-
O-gluA
M7
341
341
5611605
ndash05
C 27H28O13
5611605
385
1295
3130715
559146
038
311
71BI
Hydroxylated
icaritin-O-gluA
M8
346
270
7211982
03
C 33H36O18
72119825451662
369
1340
3130717
7191
8235431519
3671
196
UBI
Icaritin-di-O
-gluA
M9
352
341
5611608
0C 27H28O13
561160
838
512
835591454
383
1170
1750260
BIHydroxylated
icaritin-O-gluA
M10
358
269
5631761
ndash07
C 27H30O13
5631761
3871
452
3691
3503130735
5611605
385
1289
UBI
Hydrated
icaritin-O-gluA
6 Journal of Analytical Methods in Chemistry
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M11
374
341
5611615
12C 27H28O13
5611615
385
1292
3671186
5591462
383
1175
BIHydroxylated
icaritin-O-gluA
M12
429
na
5431501
ndash04
C 27H26O12
5431501
3671
189
5411343
365
1034
3510
7651750221
1130253
IDehydrogenated
icaritin-O-gluA
M13
434
271
5451663
07
C 27H28O12
56714865451663
369
1335
3130716
5431501
3671
188
3520948
3090
408
2970
3982810
498
PUBF
IIcaritin-O-gluA
M14
440
270
8233024
ndash01
C 39H50O19
8233024
369
1324
3130721
8212
8636592
331
3671
1683510
867
IEp
imedin
C
M15
445
273
387144
0ndash10
C 21H22O7
3871
4403691
338
3130722
385
1288
IHydratedicaritin
M16
449
270
8233021
ndash05
C 39H50O19
8233021
369
1336
3130725
8212
8606592
333
3671
1653510
869
IEp
imedin
Ciso
mer
M17
453
270
5311866
15C 27H30O11
5311866
369
1350
3130718
5291
705
3671
182
3520948
2970
395
2530496
FIIcariside
I
M18
457
271
5451661
04
C 27H28O12
5451661
369
1346
3130723
5431505
3671
183
3520945
2970
394
PUBF
IIcaritin-O-gluA
M19
459
271
6472
349
14C 32H38O14
6692
2156472
349
5151930
369
1346
3130724
6452168
3671
162
3510
8633230945
2950595
IIcaritin-rha-xyl
M20
461
270
6612
503
11C 33H40O14
6832326
6612
503
369
1339
3130719
6592
343
3671
165
3510
86529506
022170
499
FIIcaritin-rha-rha
M21
488
269
5151927
19C 27H30O10
53717575151927
369
1340
3130726
5131766
3671
157
3520900
3230910
29506
042170
498
FIIcariside
II
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 3
The operating parameters were as follows capillary volt-age 25 kV (ESI+) sample cone voltage 300V extractioncone voltage 40 V source temperature 100∘C desolvationtemperature 300∘C and desolvation gas flow 800 Lh Themethod employed lock spray with leucine enkephalin (mz5562771 in positive ion mode and mz 5542615 in negativeion mode) to ensure mass accuracy
28 Pharmacokinetic Application After fasting with freeaccess to water for 12 h icaritin was given to rats as a dosage of100mgkg Plasma samples were then obtained at 0083 02505 1 2 3 4 6 8 12 24 36 and 48 h after administration Forpharmacokinetic application DAS 20 was used to calculatethe pivotal pharmacokinetic parameters
29 Enzymes Kinetic Evaluation Serial concentrations oficaritin (04sim20120583M) were incubated with RLM and RIM todetermine icaritin glucuronidation rates The kinetic modelsMichaelis-Menten equation and substrate inhibition equa-tion were fitted to the data of metabolic rates versus substrateconcentrations and displayed in (1) and (2) respectivelyAppropriate models were selected by visual inspection ofthe Eadie-Hofstee plot [24] Model fitting and parameterestimation were performed by Graphpad Prism V5 software(San Diego CA)
The parameters were as follows 119881 is the formation rateof product 119881max is the maximal velocity 119870m is the Michaelisconstant and [119878] is the substrate concentration 119870si is thesubstrate inhibition constant The intrinsic clearance (CLint)was derived by 119881max119870m for Michaelis-Menten and substrateinhibition models
119881 =119881max times [119878]
119870m + [119878] (1)
119881 =119881max times [119878]
119870m + [119878] (1 + [119878] 119870si) (2)
3 Results
31 Fragment Pattern of Icaritin As had already beenreported in the previous study [16] besides the typicaladduct ion [M+Na]+ at mz 3911151 (C21H20O6Na) and[M+H]+ at mz 3691336 (C21H21O6 minus05 ppm) the ionat mz 3130714 (C17H13O6) in positive ion mode wasconsidered as the characteristic fragment ion (see Fig-ure S1a in the Supplementary Material available online athttpsdoiorg10115520171073607)
32 Screening of Metabolites On the basis of MSMS frag-mentation pattern the metabolites were deduced clarifyingthe general metabolism in vivo The extracted ion chro-matograms (EICs) of prototype (M0) and metabolites (M1simM30) were shown in Figure 1 while the individual EICsof M1simM30 were exhibited in Figure S2 The UV MS andMSMS data of M0simM30 were all exhibited in Table 1
33 Structure Elucidation of Metabolites
M0 (Parent Drug) M0 (720min C21H20O6 minus05 ppm) inbiological samples was unambiguously identified by compar-ing with references
M15 and M23 (Hydration of Isopentene Group) Based onthe [M+H]+ ion at mz 3871445 (C21H23O7 03 ppm) and[MminusH]minus ion at mz 3851286 (C21H21O7) the molecular for-mula of M15 (434min) and M23 (497min) was determinedas C21H22O7 with one H2O more than M0 The MSMSspectrum of [M+H]+ ion (C21H23O7) showed predominant[M+HminusH2O]
+ ion at mz 3691343 and 3130715 (FigureS1b) which indicated that M15 and M23 were the hydrationproducts at isopentene group of icaritin and agreed withprevious study of icariin [18]
M25 (Demethylation of Flavonoid Aglycone) According tothe [M+H]+ ion at mz 3551180 (C20H19O6 minus06 ppm) and[MminusH]minus ion atmz 3531036 (C20H17O6) the formula ofM25(508min) was supposed as C20H18O6 with a methyl groupless than M0 The MSMS experiments (Figure S1c) showeda significant loss of neutral loss of C4H8 (560626Da) fromthe ion at mz 3551180 to 2990582 in positive ion mode orfrom the ion atmz 3531036 to 2970373 in negative ionmodeMeanwhile the demethylation position was purposed at 41015840position of B ring of flavonoid aglycone Moreover M25 wasidentified as desmethylicaritin by comparison of referencestandard
M30 (Dehydrogenation of Isopentene Group) The formulaof M30 (636min) was C21H18O6 with two hydrogensfewer than M0 based on the [M+H]+ ion at mz 3671180(C21H19O6 minus05 ppm) and [MminusH]minus ion at mz 3651047(C21H17O6) In MSMS spectrum (Figure S1d) the ions atmz 3520948 and 3130721 were attributed to obvious loss ofCH3 (150235Da) and C4H6 (540470Da) group respectivelywhich indicated that the dehydrogenation position was atisopentene group [18]
M27simM29 (Hydroxylation of Isopentene Group) From the[M+H]+ ion at mz 3851288 (C21H21O7 03 ppm) and[MminusH]minus ion at mz 3831175 (C21H19O7) the formulae ofM27 (537min 120582max 268 nm) M28 (554min) and M29(566min) were speculated as C21H20O7 which was oneoxygen more than M0 In (+) ESI-MSMS spectrum (FigureS1e) the ion atmz 3851288 could lose aH2OandC4H6 groupto produce the daughter ions at 3671186 ([M+HminusH2O]
+) and3130715 ([M+HminusC4H6]
+) respectively This illustrated thatM27simM29 were tentatively characterized as the hydroxylatedproducts of M0 at the isopentene group
M14 M16 M17 M19 M20 and M21 (Glycosylation ofFlavonoid Aglycone) M21 (488min) was given a [M+H]+ ionatmz 5151927 (C27H31O10 19 ppm) and [MminusH]minus ion atmz5131766 (C27H29O10) in full scan mass spectrum The ion atmz 5151927 could easily yield the characteristic fragmentions at mz 3691340 and 3130726 by subsequent loss ofC6H10O4 and C4H8 (Figure S1f) So M21 could be the gly-cosylation product of M0 by adduct of rhamnose (C6H10O41460579Da) Similarly M17 (453min C27H30O11 15 ppm)
4 Journal of Analytical Methods in Chemistry
100 200 300 400 500 600 700 800 900
0
100
()
M0
M1
M13M18
Time
1 TOF MS ES+369134 + 721198 + 545166 00200 Da839e3
(a)
100 200 300 400 500 600 700 800 900
0
100
() M0M1
M2M3
M4
M5M6
M8
M10
M13M18 M23
M25M27
M28M29
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561161 + 545166 + 387144 + 355118 + 385129386e4
(b)
100 200 300 400 500 600 700 800 900
0
100
()
M0M1 M2 M3
M4
M5M6
M7M8
M9M10M11
M13M18
M22M24
M26
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561162 + 545166 00200 Da491e4
(c)
100 200 300 400 500 600 700 800 900
0
100
() M0
M13M17
M18M20
M21M23M25
M27M28
M29 M30
1 TOF MS ES+369134 + 545166 + 531187 + 66125 + 515193 + 387144 + 355118 + 385129 + 367118177e5
Time
(d)
100 200 300 400 500 600 700 800 900
M0
M1 M2
M3M4
M5 M6M7
M8M9
M10M11
M12
M13M14
M15M16M17M18M19M20
M21M22M23M24
M25M26
M27M28 M29
M300
100
()
Time
1 TOF MS ES+Sum 00200 Da548e5
(e)
Figure 1 EICs of all metabolites in rat biosamples after oral administration of icaritin (a) Plasma (b) urine (c) bile (d) feces (e) intestine
Journal of Analytical Methods in Chemistry 5
Table1UPL
C-MSanalysisof
icaritinandits
observed
metabolitesinbiosam
ples
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M0
720
272
3691336
ndash05
C 21H20O6
3911152
369
1336
3130714
2150748
3671
1803520941
3090
4172970
399
2810
44325304
85PU
BFI
Icaritin
M1
248
270
7211978
ndash03
C 33H36O18
7431882
7211978
5451666
369
1345
3130717
7191
8255431521
3671
194
PUBI
Icaritin-di-O
-gluA
M2
262
269
5631768
05
C 27H30O13
5631768
3871
442
3691
3323130716
561160
838
512
88UBI
Hydrated
icaritin-O-gluA
M3
289
345
5311511
15C 26H26O12
53115113
5511
662990
576
5291
348
353
1027
1190504
UBI
Desmethylicaritin-
O-gluA
M4
315
341
5611617
16C 27H28O13
5611617
385
1301
3671186
5591456
383
1175
36711351750263
UBI
Hydroxylated
icaritin-O-gluA
M5
322
341
5611613
09
C 27H28O13
5611613
385
1296
5591458
383
1172
1750262
UBI
Hydroxylated
icaritin-O-gluA
M6
336
345
5311503
00
C 26H26O12
5311503
355
1175
3290
7413
140455
5291
345
353
1025
1190506
UBI
Desmethylicaritin-
O-gluA
M7
341
341
5611605
ndash05
C 27H28O13
5611605
385
1295
3130715
559146
038
311
71BI
Hydroxylated
icaritin-O-gluA
M8
346
270
7211982
03
C 33H36O18
72119825451662
369
1340
3130717
7191
8235431519
3671
196
UBI
Icaritin-di-O
-gluA
M9
352
341
5611608
0C 27H28O13
561160
838
512
835591454
383
1170
1750260
BIHydroxylated
icaritin-O-gluA
M10
358
269
5631761
ndash07
C 27H30O13
5631761
3871
452
3691
3503130735
5611605
385
1289
UBI
Hydrated
icaritin-O-gluA
6 Journal of Analytical Methods in Chemistry
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M11
374
341
5611615
12C 27H28O13
5611615
385
1292
3671186
5591462
383
1175
BIHydroxylated
icaritin-O-gluA
M12
429
na
5431501
ndash04
C 27H26O12
5431501
3671
189
5411343
365
1034
3510
7651750221
1130253
IDehydrogenated
icaritin-O-gluA
M13
434
271
5451663
07
C 27H28O12
56714865451663
369
1335
3130716
5431501
3671
188
3520948
3090
408
2970
3982810
498
PUBF
IIcaritin-O-gluA
M14
440
270
8233024
ndash01
C 39H50O19
8233024
369
1324
3130721
8212
8636592
331
3671
1683510
867
IEp
imedin
C
M15
445
273
387144
0ndash10
C 21H22O7
3871
4403691
338
3130722
385
1288
IHydratedicaritin
M16
449
270
8233021
ndash05
C 39H50O19
8233021
369
1336
3130725
8212
8606592
333
3671
1653510
869
IEp
imedin
Ciso
mer
M17
453
270
5311866
15C 27H30O11
5311866
369
1350
3130718
5291
705
3671
182
3520948
2970
395
2530496
FIIcariside
I
M18
457
271
5451661
04
C 27H28O12
5451661
369
1346
3130723
5431505
3671
183
3520945
2970
394
PUBF
IIcaritin-O-gluA
M19
459
271
6472
349
14C 32H38O14
6692
2156472
349
5151930
369
1346
3130724
6452168
3671
162
3510
8633230945
2950595
IIcaritin-rha-xyl
M20
461
270
6612
503
11C 33H40O14
6832326
6612
503
369
1339
3130719
6592
343
3671
165
3510
86529506
022170
499
FIIcaritin-rha-rha
M21
488
269
5151927
19C 27H30O10
53717575151927
369
1340
3130726
5131766
3671
157
3520900
3230910
29506
042170
498
FIIcariside
II
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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CatalystsJournal of
4 Journal of Analytical Methods in Chemistry
100 200 300 400 500 600 700 800 900
0
100
()
M0
M1
M13M18
Time
1 TOF MS ES+369134 + 721198 + 545166 00200 Da839e3
(a)
100 200 300 400 500 600 700 800 900
0
100
() M0M1
M2M3
M4
M5M6
M8
M10
M13M18 M23
M25M27
M28M29
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561161 + 545166 + 387144 + 355118 + 385129386e4
(b)
100 200 300 400 500 600 700 800 900
0
100
()
M0M1 M2 M3
M4
M5M6
M7M8
M9M10M11
M13M18
M22M24
M26
Time
1 TOF MS ES+369134 + 721198 + 563177 + 531151 + 561162 + 545166 00200 Da491e4
(c)
100 200 300 400 500 600 700 800 900
0
100
() M0
M13M17
M18M20
M21M23M25
M27M28
M29 M30
1 TOF MS ES+369134 + 545166 + 531187 + 66125 + 515193 + 387144 + 355118 + 385129 + 367118177e5
Time
(d)
100 200 300 400 500 600 700 800 900
M0
M1 M2
M3M4
M5 M6M7
M8M9
M10M11
M12
M13M14
M15M16M17M18M19M20
M21M22M23M24
M25M26
M27M28 M29
M300
100
()
Time
1 TOF MS ES+Sum 00200 Da548e5
(e)
Figure 1 EICs of all metabolites in rat biosamples after oral administration of icaritin (a) Plasma (b) urine (c) bile (d) feces (e) intestine
Journal of Analytical Methods in Chemistry 5
Table1UPL
C-MSanalysisof
icaritinandits
observed
metabolitesinbiosam
ples
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M0
720
272
3691336
ndash05
C 21H20O6
3911152
369
1336
3130714
2150748
3671
1803520941
3090
4172970
399
2810
44325304
85PU
BFI
Icaritin
M1
248
270
7211978
ndash03
C 33H36O18
7431882
7211978
5451666
369
1345
3130717
7191
8255431521
3671
194
PUBI
Icaritin-di-O
-gluA
M2
262
269
5631768
05
C 27H30O13
5631768
3871
442
3691
3323130716
561160
838
512
88UBI
Hydrated
icaritin-O-gluA
M3
289
345
5311511
15C 26H26O12
53115113
5511
662990
576
5291
348
353
1027
1190504
UBI
Desmethylicaritin-
O-gluA
M4
315
341
5611617
16C 27H28O13
5611617
385
1301
3671186
5591456
383
1175
36711351750263
UBI
Hydroxylated
icaritin-O-gluA
M5
322
341
5611613
09
C 27H28O13
5611613
385
1296
5591458
383
1172
1750262
UBI
Hydroxylated
icaritin-O-gluA
M6
336
345
5311503
00
C 26H26O12
5311503
355
1175
3290
7413
140455
5291
345
353
1025
1190506
UBI
Desmethylicaritin-
O-gluA
M7
341
341
5611605
ndash05
C 27H28O13
5611605
385
1295
3130715
559146
038
311
71BI
Hydroxylated
icaritin-O-gluA
M8
346
270
7211982
03
C 33H36O18
72119825451662
369
1340
3130717
7191
8235431519
3671
196
UBI
Icaritin-di-O
-gluA
M9
352
341
5611608
0C 27H28O13
561160
838
512
835591454
383
1170
1750260
BIHydroxylated
icaritin-O-gluA
M10
358
269
5631761
ndash07
C 27H30O13
5631761
3871
452
3691
3503130735
5611605
385
1289
UBI
Hydrated
icaritin-O-gluA
6 Journal of Analytical Methods in Chemistry
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M11
374
341
5611615
12C 27H28O13
5611615
385
1292
3671186
5591462
383
1175
BIHydroxylated
icaritin-O-gluA
M12
429
na
5431501
ndash04
C 27H26O12
5431501
3671
189
5411343
365
1034
3510
7651750221
1130253
IDehydrogenated
icaritin-O-gluA
M13
434
271
5451663
07
C 27H28O12
56714865451663
369
1335
3130716
5431501
3671
188
3520948
3090
408
2970
3982810
498
PUBF
IIcaritin-O-gluA
M14
440
270
8233024
ndash01
C 39H50O19
8233024
369
1324
3130721
8212
8636592
331
3671
1683510
867
IEp
imedin
C
M15
445
273
387144
0ndash10
C 21H22O7
3871
4403691
338
3130722
385
1288
IHydratedicaritin
M16
449
270
8233021
ndash05
C 39H50O19
8233021
369
1336
3130725
8212
8606592
333
3671
1653510
869
IEp
imedin
Ciso
mer
M17
453
270
5311866
15C 27H30O11
5311866
369
1350
3130718
5291
705
3671
182
3520948
2970
395
2530496
FIIcariside
I
M18
457
271
5451661
04
C 27H28O12
5451661
369
1346
3130723
5431505
3671
183
3520945
2970
394
PUBF
IIcaritin-O-gluA
M19
459
271
6472
349
14C 32H38O14
6692
2156472
349
5151930
369
1346
3130724
6452168
3671
162
3510
8633230945
2950595
IIcaritin-rha-xyl
M20
461
270
6612
503
11C 33H40O14
6832326
6612
503
369
1339
3130719
6592
343
3671
165
3510
86529506
022170
499
FIIcaritin-rha-rha
M21
488
269
5151927
19C 27H30O10
53717575151927
369
1340
3130726
5131766
3671
157
3520900
3230910
29506
042170
498
FIIcariside
II
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
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Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
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CatalystsJournal of
Journal of Analytical Methods in Chemistry 5
Table1UPL
C-MSanalysisof
icaritinandits
observed
metabolitesinbiosam
ples
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M0
720
272
3691336
ndash05
C 21H20O6
3911152
369
1336
3130714
2150748
3671
1803520941
3090
4172970
399
2810
44325304
85PU
BFI
Icaritin
M1
248
270
7211978
ndash03
C 33H36O18
7431882
7211978
5451666
369
1345
3130717
7191
8255431521
3671
194
PUBI
Icaritin-di-O
-gluA
M2
262
269
5631768
05
C 27H30O13
5631768
3871
442
3691
3323130716
561160
838
512
88UBI
Hydrated
icaritin-O-gluA
M3
289
345
5311511
15C 26H26O12
53115113
5511
662990
576
5291
348
353
1027
1190504
UBI
Desmethylicaritin-
O-gluA
M4
315
341
5611617
16C 27H28O13
5611617
385
1301
3671186
5591456
383
1175
36711351750263
UBI
Hydroxylated
icaritin-O-gluA
M5
322
341
5611613
09
C 27H28O13
5611613
385
1296
5591458
383
1172
1750262
UBI
Hydroxylated
icaritin-O-gluA
M6
336
345
5311503
00
C 26H26O12
5311503
355
1175
3290
7413
140455
5291
345
353
1025
1190506
UBI
Desmethylicaritin-
O-gluA
M7
341
341
5611605
ndash05
C 27H28O13
5611605
385
1295
3130715
559146
038
311
71BI
Hydroxylated
icaritin-O-gluA
M8
346
270
7211982
03
C 33H36O18
72119825451662
369
1340
3130717
7191
8235431519
3671
196
UBI
Icaritin-di-O
-gluA
M9
352
341
5611608
0C 27H28O13
561160
838
512
835591454
383
1170
1750260
BIHydroxylated
icaritin-O-gluA
M10
358
269
5631761
ndash07
C 27H30O13
5631761
3871
452
3691
3503130735
5611605
385
1289
UBI
Hydrated
icaritin-O-gluA
6 Journal of Analytical Methods in Chemistry
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M11
374
341
5611615
12C 27H28O13
5611615
385
1292
3671186
5591462
383
1175
BIHydroxylated
icaritin-O-gluA
M12
429
na
5431501
ndash04
C 27H26O12
5431501
3671
189
5411343
365
1034
3510
7651750221
1130253
IDehydrogenated
icaritin-O-gluA
M13
434
271
5451663
07
C 27H28O12
56714865451663
369
1335
3130716
5431501
3671
188
3520948
3090
408
2970
3982810
498
PUBF
IIcaritin-O-gluA
M14
440
270
8233024
ndash01
C 39H50O19
8233024
369
1324
3130721
8212
8636592
331
3671
1683510
867
IEp
imedin
C
M15
445
273
387144
0ndash10
C 21H22O7
3871
4403691
338
3130722
385
1288
IHydratedicaritin
M16
449
270
8233021
ndash05
C 39H50O19
8233021
369
1336
3130725
8212
8606592
333
3671
1653510
869
IEp
imedin
Ciso
mer
M17
453
270
5311866
15C 27H30O11
5311866
369
1350
3130718
5291
705
3671
182
3520948
2970
395
2530496
FIIcariside
I
M18
457
271
5451661
04
C 27H28O12
5451661
369
1346
3130723
5431505
3671
183
3520945
2970
394
PUBF
IIcaritin-O-gluA
M19
459
271
6472
349
14C 32H38O14
6692
2156472
349
5151930
369
1346
3130724
6452168
3671
162
3510
8633230945
2950595
IIcaritin-rha-xyl
M20
461
270
6612
503
11C 33H40O14
6832326
6612
503
369
1339
3130719
6592
343
3671
165
3510
86529506
022170
499
FIIcaritin-rha-rha
M21
488
269
5151927
19C 27H30O10
53717575151927
369
1340
3130726
5131766
3671
157
3520900
3230910
29506
042170
498
FIIcariside
II
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
6 Journal of Analytical Methods in Chemistry
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M11
374
341
5611615
12C 27H28O13
5611615
385
1292
3671186
5591462
383
1175
BIHydroxylated
icaritin-O-gluA
M12
429
na
5431501
ndash04
C 27H26O12
5431501
3671
189
5411343
365
1034
3510
7651750221
1130253
IDehydrogenated
icaritin-O-gluA
M13
434
271
5451663
07
C 27H28O12
56714865451663
369
1335
3130716
5431501
3671
188
3520948
3090
408
2970
3982810
498
PUBF
IIcaritin-O-gluA
M14
440
270
8233024
ndash01
C 39H50O19
8233024
369
1324
3130721
8212
8636592
331
3671
1683510
867
IEp
imedin
C
M15
445
273
387144
0ndash10
C 21H22O7
3871
4403691
338
3130722
385
1288
IHydratedicaritin
M16
449
270
8233021
ndash05
C 39H50O19
8233021
369
1336
3130725
8212
8606592
333
3671
1653510
869
IEp
imedin
Ciso
mer
M17
453
270
5311866
15C 27H30O11
5311866
369
1350
3130718
5291
705
3671
182
3520948
2970
395
2530496
FIIcariside
I
M18
457
271
5451661
04
C 27H28O12
5451661
369
1346
3130723
5431505
3671
183
3520945
2970
394
PUBF
IIcaritin-O-gluA
M19
459
271
6472
349
14C 32H38O14
6692
2156472
349
5151930
369
1346
3130724
6452168
3671
162
3510
8633230945
2950595
IIcaritin-rha-xyl
M20
461
270
6612
503
11C 33H40O14
6832326
6612
503
369
1339
3130719
6592
343
3671
165
3510
86529506
022170
499
FIIcaritin-rha-rha
M21
488
269
5151927
19C 27H30O10
53717575151927
369
1340
3130726
5131766
3671
157
3520900
3230910
29506
042170
498
FIIcariside
II
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 7
Table1Con
tinued
Num
ber
119905 119877UV
[M+H]+
Error
Form
ula
(+)E
SI-M
SMS
(ndash)E
SI-M
SMS
Sources
Characteriz
ation
min
nmpp
m
M22
493
300
5431500
ndash06
C 27H26O12
5651453
5431500
3671
172
5411345
365
1033
1750222
1130254
BIDehydrogenated
icaritin-O-gluA
M23
497
273
387144
503
C 21H22O7
3871
4453691
343
3130715
3851286
UFI
Hydratedicaritin
M24
502
300
5431505
04
C 27H26O12
5431505
3671
134
3130720
5411346
365
1032
1750223
1130255
BIDehydrogenated
icaritin-O-gluA
M25
508
270
3551180
ndash06
C 20H18O6
355
1180
2990
582
353
1036
3090
409
2970
3732810
481
UFI
Desmethylicaritin
M26
513
271
5451664
09
C 27H28O12
56715405451664
369
1343
3130720
2990
585
5431504
3671
180
3520942
2970
391
BIIcaritin-O-gluA
M27
537
268
3851288
03
C 21H20O7
4071136
385
1288
36711863130715
383
1175
3651035
1750226
UFI
Hydroxylated
icaritin
M28
554
268
3851285
ndash05
C 21H20O7
385
1285
3671181
3130719
383
1170
3651046
UFI
Hydroxylated
icaritin
M29
566
na
3851290
09
C 21H20O7
385
1290
3671176
3130712
383
1172
3671043
1750262
UFI
Hydroxylated
icaritin
M30
636
272
3671180
ndash05
C 21H18O6
3671
1803520948
3130721
2970
395
2530496
365
1047
3510
765
1750223
1130250
FIDehydrogenated
icaritin
M0parent
drugM
1simM34m
etabolitesna
notavailablePplasmaU
urineB
bileFfecesIintestin
egluA
glucuronide
conjugatesglcglucoserharhamno
sexylxylose
means
thatthem
etabolitesa
reexactly
identifi
edwith
references
tand
ards
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
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Carbohydrate Chemistry
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Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
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Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
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Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
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Analytical ChemistryInternational Journal of
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Quantum Chemistry
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Organic Chemistry International
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CatalystsJournal of
8 Journal of Analytical Methods in Chemistry
was the glucose conjugate of M0 while M19 (459minC32H39O14 14 ppm) andM20 (461min C33H41O14 11 ppm)were the xylose and rhamnose glycosylation derivates ofM21respectively M14 M17 and M21 were identified as epimedinC icariside I and icariside II respectively
M14 (440min 120582max 270 nm C39H51O19 minus01 ppm) andM16 (449min 120582max 270 nm C39H51O19 minus05 ppm) bothwith the formula of C39H50O19 (Figure S1g) were tenta-tively characterized as the glucose glycosylation conjugateof M20 These glycosylation reactions were the same as themetabolism of epimedin C in rats reported in reference (Liuet al 2011) By comparingwith referencesM14M17 andM21were identified as epimedin C icariside I and icariside IIrespectively And the MSMS spectra of M17 M19 and M20were shown in Figures S1hndashS1j respectively
M1 M8 M13 M18 and M26 (Glucuronidation of FlavonoidAglycone) In full scan mass spectrum M13 (434min) M18(457min) and M26 (510min) all exhibited the [M+H]+ ionat mz 5451663 (C27H29O12 07 ppm) and [MminusH]minus ion atmz 5431501 (C27H27O12) with a formula of C27H28O12 of1760325Da larger than M0 The MSMS spectrum (FigureS1k) displayed an obvious loss of C6H8O6 group fromparent ion at mz 5451663 to the daughter ion at mz3691338 which suggested an existing glucuronic acid ofthese three metabolites Just like reported studies [16]monoglucuronide conjugate and diglucuronide conjugatewere widely distributed in biological samples after oraladministration of Epimedium-related total flavonoids orindividual flavonoid Therefore M13 M18 and M26 weretentatively identified as monoglucuronidation conjugate ofM0 while M1 (248min C33H36O18 minus03 ppm) and M8(345min C33H36O18 03 ppm) (Figure S1l) were character-ized as diglucuronidation derivates based on two moleculesof C6H8O6 fragment larger than M0
Similarly M2 (262min 120582max 269 nm C27H30O1305 ppm) and M10 (357min 120582max 269 nm C27H30O13minus07 ppm) with the MSMS spectrum shown in Figure S1mwere tentatively considered as the monoglucuronidationproducts of M15 and M23 M3 (289min 120582max 345 nmC26H26O12 15 ppm) and M6 (336min 120582max 345 nmC26H26O12 0 ppm) were characterized as monoglucuronideconjugate of M25 The MSMS spectrum of M3 and M6was exhibited in Figure S1n Meanwhile M4 (316min120582max 341 nm C27H28O13 16 ppm) M5 (321min 120582max341 nm C27H28O13 09 ppm) M7 (340min 120582max 341 nmC27H28O13 minus05 ppm) M9 (350min 120582max 341 nmC27H28O13 0 ppm) and M11 (374min 120582max 341 nmC27H28O13 12 ppm) were regarded as monoglucuronidationderivates of M27simM29 and their MSMS spectrum wasdisplayed in Figure S1o M12 (429min not available120582max C27H26O12 minus04 ppm) M22 (494min 120582max300 nm C27H26O12 minus06 ppm) and M24 (502min 120582max300 nm C27H26O12 04 ppm) were tentatively identifiedas glucuronidation conjugates of M30 And their MSMSspectrum was shown in Figure S1p
34 Method Validation Themethod was validated for speci-ficity linearity extraction recovery matrix effects precision
accuracy and stability according to the US Food DrugAdministration guidelines for bioanalytical method valida-tion [25]
Specificity was determined by comparing the chro-matograms obtained for six blank plasma samples blankplasma samples spiked with standard solutions at LLOQconcentrations and drug plasma samples obtained 4 h afteroral administration As shown in Figure S3 no interferencepeaks were detected at the retention times of icaritin
The LOD and LOQ were calculated as 3-fold and 10-foldof the ratio of signal-to-noise respectively The LLOQ wasdefined as the lowest concentration in the calibration curvewith accuracy of 80sim120 and precision of 20 Calibra-tion curves were acquired by plotting peak area (119910) versusrespective plasma concentrations (119909) using a 11199092 weightingfactor and linear least-squares regression analysis A series ofstandard solutions were used to generate calibration curveThe correlation coefficients (1199032) of calibration curves weregreater than 09926 within 20sim5120 ngmL and LLOQ was20 ngmLThe regression equations correlation coefficientsand LLOQ were shown in Table S1
The experiments to evaluate matrix effect and recov-ery were conducted by the protocol [26] According tothe protocol the peak areas from QC samples at threeconcentrations were defined as A1 those from extractedcontrol plasma reconstituted with standard solutions at 40640 and 2560 ngmL were A2 The responses of icaritinfound by direct injection of the corresponding pure referencestandards at three QC levels were A3 The matrix effectand recovery were calculated as follows matrix effect () =A2A3times100 Recovery () = A1A2 times 100The results (asshown in Table S2) illustrated that matrix effect was between891 and 1135 and the recovery was from 963 to 1027
The accuracy and interintraday precision of the methodwere evaluated by determining six replicates of QC sampleson three consecutive days The measured concentrationsof QC samples were determined with a calibration curveobtained on the same day Relative error and relative standarddeviation were used to describe accuracy and interintradayprecision respectively They both should not exceed 15 Asexhibited in Table S3 the intraday and interday precisionwere less than 132 and 102 respectively while theintraday and interday precision of LLOQ were no more than174 and 156 respectively
Stability of icaritin in rat plasma was assessed underdifferent conditions at three concentration levels includingextracted samples for 12 h at room temperature kept atminus20∘Cfor 60 h three cycles of freezing atminus20∘Cand thawing at 25∘Cand plasma sample at room temperature for 8 h Each wascompared by three QC replicates of the same concentrationwith a calibration curve in the same day The RE was within138 and RSD was less than 113 Stability results (TableS4) indicated that icaritin were stable under different storageconditions
35 Pharmacokinetics Application Themean concentration-time profiles of these bioactive components were shown inFigure 2 The main pharmacokinetic parameters were illus-trated in Table 2 In this study119862max was (2945plusmn227) ngmL
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 9
0 12 24 36 48 60
Time (hr)
0
80
160
240
320
400
Mea
n (n
gm
L)
Figure 2 Concentration-time curve of icaritin in rat plasma after oral administration
Table 2 Pharmacokinetic parameters of icaritin in rat plasma afteroral administration
Parameters 100mgkg119879max (h) 53 plusmn 11
119862max (ngmL) 2945 plusmn 227
11990512 (h) 83 plusmn 10
AUC0minus119905 (ngsdothmL) 30485 plusmn 2890
AUC0minusinfin (ngsdothmL) 31450 plusmn 3023
MRT0minus119905 (h) 96 plusmn 11
MRT0minusinfin (h) 109 plusmn 13
when119879max was (53plusmn11) h after oral administrationThe areaunder the concentration-time curve (AUC0minusinfin) and meanresidence time (MRT0minusinfin) were (31450plusmn3023) ngsdothmL and(109 plusmn 13) h respectively The results illustrated that icaritinhad a poor absorption after oral administration The reasonmay be that icaritin stepped into small intestine to undergomass phase I and phase II metabolism by intestinal floraespecially the glycosylation and glucuronidation conjugates
36 Glucuronidation of Icaritin in RLM and RIM Due to lackof reference standard quantification of icaritin glucuronidewas based on the standard curve of the parent compound(icaritin) according to the assumption that parent compoundand its glucuronide have closely similar UV absorbancemaxima [27ndash29] The detection wavelength of icaritin andicaritin glucuronides was 270 nm The linear range of icar-itin was 002sim20120583M with LOD (119878119873 = 3sim5) and LOQ(119878119873 = 8sim10) of 001 and 002 120583M respectively And theacceptable linear correlation (119884 = 12149119883) was confirmedby correlation coefficients (1199032) of 09994 The accuracy andprecision of the intraday and interday error were both lessthan 34 There were no matrix effects observed and no
other sample preparation performed except those mentionedin the manuscript
Kinetic profiling revealed that formation of icaritin-3-O-glucuronide (M13) and icaritin-7-O-glucuronide (M18) inRLM was well modeled by the substrate inhibition equation(Figure 3(a)) whereas they followed the classical Michaelis-Menten kinetics in RIM (Figure 3(b)) In contrast theglucuronide formation of M13 (406 nmolminmg) and M18(239 nmolminmg) in RLM was similar as well as M13(1188 nmolminmg) and M18 (823 nmolminmg) in RIMIcaritin glucuronidation in RLM was efficient (CLint = 112and 156mLminmg for M13 and M18 resp) following thesubstrate inhibition kinetics with 119870m values of 362 and153 120583M respectively Similarly the CLint values of M13 andM18 in RIM were 1446 and 0861mLminmg respectivelywhereas the119870m values of M13 and M18 in RIM in Michaelis-Menten model were 822 and 956 120583M respectively Inaddition 119870i values of M13 and M18 in RLM were 1131 and1707 120583M respectively The detailed parameters of M13 andM18 were listed in Table 3
4 Discussion
Normally only the prototypes or metabolites in blood witha high enough exposure in target organs for a finite periodof time are considered as potential effective components fortherapeutic benefits [30] In this study M0 M1 and M13were themain xenobiotics in plasma (Figure 1(a)) whichmaybe the potential in vivo effective components directly Aftercirculation M2 M5 M13 M23 and M28 were passed outwith the urine (Figure 1(b))
Due to poor oral bioavailability several components werelimited to be absorbed in blood But they could influenceintestinal dysfunction to exert efficacy by their prototypessecondary metabolites or finally the aglycone in intestinal
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
10 Journal of Analytical Methods in Chemistry
Table 3 Kinetic parameters of icaritin glucuronidation by RLM and RIM (mean plusmn SD)
Protein source Metabolite 119881max(nmolminmg)
119870m(120583M)
119870i(120583M)
CLint(mLminmg) Model
RLM M13 406 plusmn 070 362 plusmn 099 1131 plusmn 351 112 plusmn 036 SIM18 239 plusmn 026 153 plusmn 034 1707 plusmn 438 156 plusmn 038 SI
RIM M13 1188 plusmn 060 822 plusmn 092 NA 145 plusmn 018 MMM18 823 plusmn 063 956 plusmn 151 NA 086 plusmn 015 MM
Note SI substrate inhibition model MM Michaelis-Menten model NA not available
G1
G2
00
05
10
15
20
25
V(n
mol
min
mg)
50 15 20 2510
Icaritin (120583M)
V(G
1)
V(G
2)
VC VC
(a)
G1
G2
50 15 20 2510
Icaritin (120583M)
0
2
4
6
8
10
V(n
mol
min
mg)
V(G
1)
V(G
2)
VC VC
(b)
Figure 3 Kinetic profiles for glucuronidation of icaritin by various types of microsomes (a) Pooled rat liver microsomes (RLM) (b) pooledrat intestine microsomes (RIM) In each panel the insert figure showed the corresponding Eadie-Hofstee plot
tract [31]Massivemetabolites containingM6 M8 M13 M17M25 M28 and M30 were detected in rat feces and smallintestinal samples (Figure 1(d)) Moreover icaritin under-went phase II metabolism by main conjugating enzymesincluding UDP-glucuronosyltransferases (UGTs) to produceextensive mono- or diglucuronic acid conjugates In rat bileM3 M6 M13 M18 and M24 mainly were biotransformed inrat liver and excreted into bile (Figure 1(c))
Characterization of icaritin glucuronidation assumed agreat role in the understanding of its pharmacokinetics andbioavailability Oral bioavailability is a major factor in deter-mining the biological actions of icaritin in vivo following oraladministration of the compound [32] This study suggestedthat the oral bioavailability of icaritin would be influenced byfirst-pass glucuronidation in the liver The glucuronidationactivity was obtained by kinetic profiling and modelingKinetic profiling required the determination of the rates oficaritin glucuronidation at a series of icaritin concentrationsThe relative activities of RLM and RIM toward icaritinglucuronidation were evaluated by the derived CLint values(Table 3) Use of CLint (=119881max119870m) as an indicator of enzymesactivity was advantageous because (1) CLint represents thecatalytic efficiency of the enzyme and is independent of thesubstrate concentration (2) compared with other kineticparameters such as119870m and119881max CLint is more relevant in anattempt to predict hepatic clearance in vivo [33] ThereforeCLint values were used to determine icaritin glucuronidationactivity in this study
Based on the metabolite profiles the metabolic pathwaysof icaritin were proposed and shown in Figure 4(a) andthe metabolic sites were shown in Figure 4(b) In summaryicaritin was hard to be absorbed into the rat blood Insmall intestine icaritin could form flavonoid glycoside bythe sequential glycosylation metabolism Meanwhile icaritincould easily conjugate with a glucuronic acid to form phaseII metabolites in liver which indicated that the biliaryclearance was one of the major routes of excretion PhaseI metabolism of icaritin mainly included demethylationdehydrogenation and hydration The general tendency wasthat the saponins were metabolized and transformed into thehigh polarmetabolites to be eliminated and excreted from therat organism
5 Conclusion
As a result a total of 30 metabolites were identified or ten-tatively characterized based on the retention time behaviorsand fragmentation patterns Dehydrogenation at isopentenylgroup and glycosylation and glucuronidation at the flavonoidaglycone were the main biotransformation process of icaritinin vivo Meanwhile a validated method was successfullyapplied to a pharmacokinetic study Moreover icaritin glu-curonidation in RLM was efficient with CLint values of112 and 156mLminmg for M13 and M18 respectivelySimilarly the CLint values of M13 and M18 in RIM were145 and 086mLminmg respectively Taken altogether this
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 11
HOHO HO HO
HOHOHOHO
HO
HO
HO HOHO
OH
OH
OH OH OH
OHOHOH
OH
OH
OH
OH
OH
OH
OH
GlcO HO GlcO
OH OH
OH
OH OHOH
OHOH
OH OHOH
OH OH
OHO
OH OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O-Rha-RhaO-Rha-Rha
O-Rha-XylO-Rha
O
O
O
O
O
O
gluA
gluA
2gluA
gluA
gluA
M2 M10
M15 M23
M13 M18 M26
M1 M8
M25
M0
M21
M17
M3 M6
M30
M19
M20
M4 M5 M7 M9 M11
M27 M28 M29
M12 M22 M24
M14 M16
H2O
H2O
+ gluA
Glucuronidation
Gluc
uron
idati
on
Glucuronidation
GlucuronidationDemethylation
Hydration HydroxylationDehydrogenation
Gly
cosy
latio
n
GlycosylationGlycosylation
Glycosylation
Glycosylation
Glucuronidati
on Glucuronidation
gluA glucuronide conjugates Glc glucose Rha rhamnose Xyl xyloseRed parent drug blue phase I metabolites
(a)
Icaritin(M0)
ig
Excretionthrough the feces
Intestine
M0 M0Dehydrogenation
HydroxylationHydroxylation
Demethylation
Dehydrogenation
HydroxylationHydroxylation
Demethylation
Glycosylation
M30M27ndashM29
M30M27ndashM29
M15 M23
M25
M15 M23
M25
M14ndashM17M19ndashM21
Portal vein
Bile duct M1ndashM13 M18M22 M24 M26 Glucuronidation
Liver
Systematiccirculation
Kidney Metabolitesdetectedin urine
(b)
Figure 4 The proposed metabolic pathway (a) and metabolic sites (b) of icaritin in rats
study could provide an experimental basis to understand themetabolic fate of icaritin in rat
Conflicts of Interest
The authors have declared no conflicts of interest
Authorsrsquo Contributions
Beibei Zhang Shuzhang Du and Xiaojian Zhang conceivedand designed the experiments Beibei Zhang and Xiaoli Chenperformed the experiments Beibei Zhang and Rui Zhangcontributed analytic tools Beibei Zhang and Fangfang Zheng
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
12 Journal of Analytical Methods in Chemistry
performed data analysis Beibei Zhang and Shuzhang Duwrote the paper
References
[1] Editorial Committee of Pharmacopoeia of Ministry of HealthPR China The Pharmacopeoia of People
1015840
s Republic of China(Part 1) Chemical Industry Press Beijing China 2015
[2] H Ma X He Y Yang M Li D Hao and Z Jia ldquoThegenus Epimedium an ethnopharmacological and phytochem-ical reviewrdquo Journal of Ethnopharmacology vol 134 no 3 pp519ndash541 2011
[3] F Xu Y Ding Y Guo et al ldquoAnti-osteoporosis effect ofEpimedium via an estrogen-like mechanism based on a system-level approachrdquo Journal of Ethnopharmacology vol 177 pp 148ndash160 2016
[4] J Zhou J Wu X Chen et al ldquoIcariin and its derivativeICT exert anti-inflammatory anti-tumor effects and modulatemyeloid derived suppressive cells (MDSCs) functionsrdquo Interna-tional Immunopharmacology vol 11 no 7 pp 890ndash898 2011
[5] S-H Chen M Lei X-H Xie et al ldquoPLGATCP compositescaffold incorporating bioactive phytomolecule icaritin forenhancement of bone defect repair in rabbitsrdquo Acta Biomate-rialia vol 9 no 5 pp 6711ndash6722 2013
[6] X Lai Y Ye C Sun et al ldquoIcaritin exhibits anti-inflammatoryeffects in the mouse peritoneal macrophages and peritonitismodelrdquo International Immunopharmacology vol 16 no 1 pp41ndash49 2013
[7] S Peng G Zhang B-T Zhang et al ldquoThe beneficial effect ofIcaritin on osteoporotic bone is dependent on the treatmentinitiation timing in adult ovariectomized ratsrdquo Bone vol 55 no1 pp 230ndash240 2013
[8] S W Lei G Cui G P H Leung et al ldquoIcaritin protectsagainst oxidative stress-induced injury in cardiac H9c2 cellsvia AktNrf2HO-1 and calcium signalling pathwaysrdquo Journalof Functional Foods vol 18 pp 213ndash223 2015
[9] J Li P Liu R Zhang et al ldquoIcaritin induces cell death inactivated hepatic stellate cells through mitochondrial activatedapoptosis and ameliorates the development of liver fibrosis inratsrdquo Journal of Ethnopharmacology vol 137 no 1 pp 714ndash7232011
[10] F Sun I R Indran Z W Zhang et al ldquoA novel prostate cancertherapeutic strategy using icaritin-activated arylhydrocarbon-receptor to co-target androgen receptor and its splice variantsrdquoCarcinogenesis vol 36 no 7 pp 757ndash768 2015
[11] M-C Jiang X-H Chen X Zhao X-J Zhang and W-FChen ldquoInvolvement of IGF-1 receptor signaling pathway inthe neuroprotective effects of Icaritin against MPP+-inducedtoxicity in MES235 cellsrdquo European Journal of Pharmacologyvol 786 pp 53ndash59 2016
[12] J Liao Y Liu H Wu et al ldquoThe role of icaritin in regulatingFoxp3IL17a balance in systemic lupus erythematosus and itseffects on the treatment ofMRLlprmicerdquoClinical Immunologyvol 162 pp 74ndash83 2016
[13] Y Chen Y H Zhao X B Jia and M Hu ldquoIntestinalabsorption mechanisms of prenylated flavonoids present in theheat-processed Epimedium koreanum Nakai (Yin Yanghuo)rdquoPharmaceutical Research vol 25 no 9 pp 2190ndash2199 2008
[14] J Zhou Y H Ma Z Zhou Y Chen Y Wang and X GaoldquoSpecial section on drug metabolism and the microbiomeintestinal absorption and metabolism of epimedium flavonoids
in osteoporosis ratsrdquo Drug Metabolism and Disposition vol 43no 10 pp 1590ndash1600 2015
[15] Y-T Wu C-W Lin L-C Lin A W Chiu K-K Chen and T-H Tsai ldquoAnalysis of biliary excretion of icariin in ratsrdquo Journal ofAgricultural and Food Chemistry vol 58 no 18 pp 9905ndash99112010
[16] H Zhao M Fan L Fan J Sun and D Guo ldquoLiq-uid chromatography-tandem mass spectrometry analysis ofmetabolites in rats after administration of prenylflavonoidsfrom Epimediumsrdquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 878 no 15-16 pp 1113ndash1124 2010
[17] Q Chang G-N Wang Y Li L Zhang C You and Y ZhengldquoOral absorption and excretion of icaritin an aglycone andalso active metabolite of prenylflavonoids from the Chinesemedicine Herba Epimedii in ratsrdquo Phytomedicine vol 19 no 11pp 1024ndash1028 2012
[18] Q Qian S-L Li E Sun et al ldquoMetabolite profiles of icariinin rat plasma by ultra-fast liquid chromatography coupled totriple-quadrupoletime-of-flight mass spectrometryrdquo Journal ofPharmaceutical and Biomedical Analysis vol 66 pp 392ndash3982012
[19] Y Jin C-SWu J-L Zhang and Y-F Li ldquoA new strategy for thediscovery of epimedium metabolites using high-performanceliquid chromatography with high resolution mass spectrome-tryrdquo Analytica Chimica Acta vol 768 no 1 pp 111ndash117 2013
[20] L Cui F Xu J Jiang et al ldquoIdentification of metabolites ofepimedinA in rats usingUPLCQ-TOF-MSrdquoChromatographiavol 77 no 17-18 pp 1223ndash1234 2014
[21] H Liu H Sun D Lu et al ldquoIdentification of glucuronidationand biliary excretion as the main mechanisms for gossypolclearance in vivo and in vitro evidencerdquo Xenobiotica vol 44no 8 pp 696ndash707 2014
[22] F Li Y Tan H Chen et al ldquoIdentification of schisandrin asa vascular endothelium protective component in YiQiFuMaipowder injection using HUVECs binding and HPLC-DAD-Q-TOF-MSMS analysisrdquo Journal of Pharmacological Sciences vol129 no 1 pp 1ndash8 2015
[23] D Lu ZMa T Zhang X Zhang andBWu ldquoMetabolismof theanthelmintic drug niclosamide by cytochrome P450 enzymesand UDP-glucuronosyltransferases metabolite elucidation andmain contributions from CYP1A2 and UGT1A1rdquo Xenobioticavol 46 no 1 pp 1ndash13 2016
[24] J M Hutzler and T S Tracy ldquoAtypical kinetic profiles in drugmetabolism reactionsrdquo Drug Metabolism and Disposition vol30 no 4 pp 355ndash362 2002
[25] US FDA Guidance for Industry Bioanalytical Method Valida-tion USDepartment of Health andHuman Services Center forDrug Evaluation and Research (CDER) httpwwwfdagovcderguidance 2001
[26] B K Matuszewski M L Constanzer and C M Chavez-EngldquoMatrix effect in quantitative LCMSMS analyses of biologicalfluids a method for determination of finasteride in humanplasma at picogram per milliliter concentrationsrdquo AnalyticalChemistry vol 70 no 5 pp 882ndash889 1998
[27] J Troberg E Jarvinen G Ge L Yang andM Finel ldquoUGT1A10Is a high activity and important extrahepatic enzyme why hasits role in intestinal glucuronidation been frequently underesti-matedrdquoMolecular Pharmaceutics Ahead of Print 2016
[28] D Lu H Liu W Ye Y Wang and B Wu ldquoStructure- andisoform-specific glucuronidation of six curcumin analogsrdquoXenobiotica pp 1ndash10 2016
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Journal of Analytical Methods in Chemistry 13
[29] H Sun H Wang H Liu X Zhang and B Wu ldquoGlucuronida-tion of capsaicin by liver microsomes and expressed UGTenzymes reaction kinetics contribution of individual enzymesand marked species differencesrdquo Expert Opinion on DrugMetabolism and Toxicology vol 10 no 10 pp 1325ndash1336 2014
[30] X Wang ldquoStudies on serum pharmacochemistry of traditionalChiese medicinerdquo World Science And TechnologyModerniza-tion of Traditional Chinese Medicine vol 4 no 2 pp 1ndash4 2002
[31] R Zhang S Gilbert X Yao et al ldquoNatural compound methylprotodioscin protects against intestinal inflammation throughmodulation of intestinal immune responsesrdquo PharmacologyResearch Perspectives vol 3 article e00118 no 2 2015
[32] B Wu K Kulkarni S Basu S Zhang and M Hu ldquoFirst-passmetabolism via UDP-glucuronosyltransferase a barrier to oralbioavailability of phenolicsrdquo Journal of Pharmaceutical Sciencesvol 100 no 9 pp 3655ndash3681 2011
[33] B Wu D Dong M Hu and S Zhang ldquoQuantitative predictionof glucuronidation in humans using the in vitro-in vivo extrap-olation approachrdquo Current Topics in Medicinal Chemistry vol13 no 11 pp 1343ndash1352 2013
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of
Submit your manuscripts athttpswwwhindawicom
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Inorganic ChemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 201
International Journal ofInternational Journal ofPhotoenergy
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Carbohydrate Chemistry
International Journal ofInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Advances in
Physical Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom
Analytical Methods in Chemistry
Journal of
Volume 2014
Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
SpectroscopyInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Medicinal ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Chromatography Research International
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Applied ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Theoretical ChemistryJournal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Spectroscopy
Analytical ChemistryInternational Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Journal of
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Quantum Chemistry
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
Organic Chemistry International
ElectrochemistryInternational Journal of
Hindawi Publishing Corporation httpwwwhindawicom Volume 2014
Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014
CatalystsJournal of