elucidation of the intestinal absorption mechanism of loganin in...

Research ArticleElucidation of the Intestinal Absorption Mechanism of Loganinin the Human Intestinal Caco-2 Cell Model

Renjie Xu 1 Yichu Yuan2 Jia Qi1 Jia Zhou1 Xiaowen Guo1

Jian Zhang 1 and Ruanjuan Zhan 3

1Department of Pharmacy Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine Shanghai200092 China2Department of Urology Ren Ji Hospital School of Medicine Shanghai Jiao Tong University Shanghai200127 China3Department of Pharmacy The First Affiliated Hospital Wenzhou Medical University Wenzhou325035 China

Correspondence should be addressed to Jian Zhang zhangjianxinhuamedcomcn and Ruanjuan Zhan 335658460qqcom

Received 25 June 2018 Revised 26 August 2018 Accepted 4 December 2018 Published 23 December 2018

Academic Editor Mona Abdel-Tawab

Copyright copy 2018 Renjie Xu et alThis is an open access article distributed under theCreative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Loganin iridoid glycosides is the main bioactive ingredients in the plant Strychnos nux-vomica L and demonstrates variouspharmacological effects though poor oral bioavailability in rats In this study the intestinal absorption mechanism of loganin wasinvestigated using the human intestinal Caco-2 cell monolayer model in both the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) direction additionally transport characteristics were systematically investigated at different concentrations pHstemperatures and potential transporters The absorption permeability (PappAB) of loganin which ranged from 1217 to 1478 times10minus6cms was high at four tested concentrations (5 20 40 and 80120583M) while the major permeation mechanism of loganin wasfound to be passive diffusion with active efflux mediated by multidrug resistance-associated protein (MRP) and breast cancerresistance protein (BCRP) In addition it was found that loganin was not the substrate of efflux transporter P-glycoprotein (P-gp)since the selective inhibitor (verapamil) of the efflux transporter exhibited little effects on the transport of loganin in the humanintestinal Caco-2 cells Meanwhile transport from the apical to the basolateral side increased 209-fold after addition of a MRPinhibitor and 232-fold after addition of a BCRP inhibitor In summary our results clearly demonstrate for the first time a goodpermeability of loganin in the human intestinal Caco-2 cell model and elucidate in detail the intestinal absorption mechanismand the effects of transporters on iridoid glycosides compounds

1 Introduction

Many traditional Chinese medicines (TCM) have beenapplied in modern medicine to promote health and preventdisease TCM have attracted attention due to their distinctivebiological activities without toxicity andor side-effects [1]

Loganin (Figure 1) is an iridoid glycoside extracted fromthe plant Strychnos nux-vomica L additionally it is alsodistributed in the plants Oleander Branch and Columelliaceae[2] In traditional Chinese medicine nux-vomica is used toenhance limb repair after trauma whereas loganin is usedas a central nervous system (CNS) stimulant in modernmedicine Recent studies have shown that loganin inhibitsinflammation [3ndash5] and protects the kidney [6 7] and nerves[8ndash10]

Unfortunately these pharmacological activities ofloganin are curtailed due to its low oral bioavailability [11]

Analogously to other iridoid glycosides it is well knownthat loganin exhibits low water solubility which limits itsabsorption and bioavailability The intestinal absorptionbarrier is a major factor controlling the absorption andoral bioavailability of drugs [12] and it is here that the firststeps of pharmacokinetics occur after oral intake Thereforeexploration of the intestinal absorption mechanism ofloganin is necessary not only for the in vivo pharmacokineticsstudy but also to provide some key information for theireffective delivery systems

The aim of the present study was to further investigatethe intestinal absorption characteristics of loganin by uti-lizing the Caco-2 human intestinal cells model an in vitroabsorption model Human intestinal Caco-2 cell monolayershave been widely used to determine the permeation rateand to examine the permeation mechanisms of bioactivecompounds [13] These cells also express nutrient and drug

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 8340563 8 pageshttpsdoiorg10115520188340563

2 Evidence-Based Complementary and Alternative Medicine

O

O O

OH

H

H

HO

O O

OH

HO OH

Figure 1 The chemical structure of loganin

transporters that allow for the study of carrier mediateduptake and efflux mechanisms [14] Different studies demon-strated that in vivo absorption could be accurately predictedfrom the apparent permeation rate (Papp) across the humanintestinal Caco-2 cell monolayers [15ndash18] Although the Pappvalues obtained from different laboratories differ there is ageneral trend that a high Papp implies effective absorptionA published report using the human intestinal Caco-2 cellsmodel for the study of geniposide [19] a structural analogof loganin showed good intestinal permeability by passivediffusion chiefly The present study is devoted for the firsttime to the investigation of the detailed intestinal absorptionmechanism of loganin and the results will be useful forfurther studies on this type of compounds in vitro and in vivo

2 Materials and Methods

21 Materials The human colon adenocarcinoma cell lineCaco-2 was purchased from the Cell Bank of the Chi-nese Academy of Sciences (Shanghai China) Dulbeccorsquosmodified Eaglersquos medium (DMEM) Hankrsquos buffered saltsolution (HBSS) antibiotic solutions (100000 UL penicillinand 100000mgL streptomycin) fetal bovine serum (FBS)100times nonessential amino acids 100times penicillin and strepto-mycin and 025 trypsin with ethylenediaminetetraaceticacid (EDTA) were from Invitrogen Corp (Carlsbad CA)Transwell permeable polycarbonate inserts (04 120583m) and12-well cell culture plates were obtained from CorningVerapamil MK 571 indomethacin benzbromarone api-genin sodium vanadate and cimetidine were obtained fromAladdin Industrial Inc (Shanghai China) Loganin (purity gt980) and puerarin (internal standard (IS) purity gt 980)were obtained from the National Institute for the Control ofPharmaceutical and Biological Products (Beijing China) Allother reagents were of analytical grade

22 Cell Culture Cells were maintained in DMEM with10 FBS 1times nonessential amino acids and 1times penicillinand streptomycin at 37∘C with 5 CO2 Cells of passage

35-40 were used in this study to maintain relatively constantcellular phenotypes The medium was replaced every 2-3days during incubation After reaching approximately 80confluence the cells were detached using 002 ethylenedi-aminetetraacetic acid (EDTA) and 005 trypsin at a densityof 53times104 cellscm2 on a 60-mm plastic culture dish for theuptake experiment or a 12-mm polyester membrane insert ina 12-well plate for the permeation experiment The media inthe culture plates were changed every two days for the firstweek after seeding and replaced daily afterwardThe integrityof the cell monolayers was examined by measuring thetransepithelial electrical resistance (TEER) with a Millicell-ERS-electrode (Millipore Corp Billerica MA USA) After21-23 days monolayers with a TEER value (TEER=[TEERtest-TEERbackground]timesarea) above 500Ωcm

2 were used during thetransport experiments

23 Cytotoxicity Assay Cell viability was assessed using anMTT assay Briefly 01mL of human intestinal Caco-2 cellswas seeded onto 96-well plates at a density of 1times105 cellsper well in a 96-well plate The cells were grown in anatmosphere with 5 CO2 and 95 relative humidity overapproximately 48 h Subsequently the culture medium wasreplaced with HBSS containing different concentrations ofloganin (01-200 120583M) the final concentration of dimethylsulfoxide (DMSO) in HBSS remained at lt1 The negativecontrol was HBSS containing 1 DMSO The cells wereexposed to the drug for 2 h Then 02mL of 05mgmLMTT solution was added to each well and incubated for4 h in dark The medium was then removed the MTT-formazan crystals were solubilized by incubating with 150 120583Lof DMSOwith gentle shaking for 10min and absorbance wasdetermined at 490 nm in a Multiskan Spectrum microplatereader (Thermo Labsystems MA USA) In each MTT assayevery sample was tested in five replicates and the viabilityof the nontreated control cells was arbitrarily defined as100

24 Transport Experiments Before the experiments thehuman intestinal Caco-2 cell monolayers were washed twicewith HBSS medium (pH 74) The transport experimentswere conducted by adding the drug solutions (containing1 DMSO) to either the apical (AP 05mL) or basolateralside (BL 10mL) while the receiving chamber containedthe corresponding volume of blank HBSS medium Themonolayers were incubated at 37∘C and 50120583L sampleswere taken at 15 30 45 60 90 and 120min from theacceptor compartment and the volume was then immedi-ately replaced with50 120583L of fresh prewarmed blank HBSSTEER measurements for assessing the membrane integritytook place before and after the experiment The sampleswere frozen immediately and stored below minus80∘C beforeanalysis

The loganin transport (20 120583M) at 4∘C and 37∘C wasevaluated in the AP to BL direction to investigate the effectof temperature The effect of pH on the loganin transport (20120583M) in theAP toBL directionwas studied using the followingpH combinations for the HBSS in the acceptordonor com-partments 6074 and 7474

Evidence-Based Complementary and Alternative Medicine 3

Several efflux and influx transporters were investigatedfor their effects on the transport flux of loganin One hundred120583M verapamil was added to evaluate the selectivity of P-glycoprotein (P-gp) [20] inhibiting the efflux by multidrugresistance-associated proteins (MRPs) was undertaken byadding 100120583M MK571 50120583M benzbromarone and 200120583Mindomethacin [21ndash23] 25 120583M apigenin was used to inves-tigate breast cancer resistance protein (BCRP) [24] 50 120583Msodium vanadate and cimetidine were used as Na+K+pumpand organic anion transporters (OATs) inhibitors [25]

25 Sample Processing In a 15mL centrifuge tube an aliquotof 5 120583L of the internal standard working solution (5 ngmL)was added to 50 120583L of collected sample followed by the sup-plementary addition of 145 120583L of acetonitrile (41 vv) After1min of vortexing and 10min centrifugation at 14000 rpmthe supernatant (2120583L) was injected into the UPLC-MSMSsystem for direct analysis

26 UPLC-MSMS Analytical Methods Chromatographicand mass spectrometry detection were performed accordingto a previous method [11] Briefly loganin and puerarin(internal standard IS) were isolated using a ZORBAX EclipsePlus C18 column (50mm times 21mm 18120583m) with an API5500triple-quadrupole mass spectrometer (Applied Biosystems-SCIEX Concord Canada) The mobile phase consisted ofwater-formic acid (10005 vv) (solvent A) and 100 ace-tonitrile (solvent B) A gradient program was used for theUPLC separation with a flow rate of 04mlmin The initialcomposition of the mobile phase was 5 solvent B adjustedto 0-31min (5-63 solvent B) 31-32min (63-95 solventB) 32-42 (95 solvent B) and 42-43min (5 solventB) and followed by a 10min re-equilibration to the initialcondition

The MS analysis was carried out in multiple reactionmonitoring (MRM) mode by monitoring the ion transi-tions from [M+H]+ mz 4082997888rarr2290 for loganin andmz [M+H]+ 4172997888rarr2969 for puerarin (IS) The MSMSconditions were as follows ion spray source temperatureat 550∘C ionspray voltage (IS) at 5500V Gas 1 and Gas 2(nitrogen) at 40 psi and collision energy (CE) at 12 eV forloganin and 32 eV for IS

27 Statistical Analysis UPLC-MSMS data acquisition wasperformed using Analyst 152 and MultiQuant 211 software(Applied Biosystems) The data were analyzed by SPSSSoftware The apparent permeability coefficient (Papp) wascalculated according to Papp = (dQdt)(AsdotC0)

where dQdt is the steady-state flux (nmols) A is thesurface area of the insert (cm2 ) C0 is the initial concentrationThe efflux ratio (P) was determined by calculating the ratio ofPappBA versus PappAB as in

P =PappBAPappAB

(1)

Statistical analysis was performed with analysis of variance(ANOVA) after confirmation of normal distributions

Table 1 Survival Rates of human intestinalCaco-2 cells treatedwithloganin (n = 5)

Concentration (120583M) Survival Rates ()Loganin

01 10766plusmn1091 10873plusmn2545 10657plusmn8310 10622plusmn10950 10345plusmn33100 10145plusmn65200 9533plusmn53aData represent the mean plusmn SD from five replicates

3 Results and Discussion

31 Human Intestinal Caco-2 Cell Viability Assay The viabil-ity of human intestinal Caco-2 cells was directly measuredby the MTT test to evaluate the cytotoxicity of loganin andthe results were shown in Table 1 As shown in Table 1the concentrations of loganin ranging from 01 to 100 120583Mwere nontoxic to the human intestinal Caco-2 cells after 4 hexposure When 200 120583M of loganin was added the humanintestinal Caco-2 cellswere inhibited by 467Generally cellsurvival rates of less than 50 of the controls were consideredas the reduction ofmitochondrial activity while an inhibitionrate of less than 10 indicated that the compounds at theconcentrations were nontoxic to the cells [26] The resultsindicated that in our experimental design concentrationsof approximately 5 20 40 and 80120583M could be chosen forloganin in the following studies

32 Development of LCMSMSMethod to Quantify LoganinThe retention time for loganin and IS was approximately139 and 132min respectively The chromatogram showedbaseline separation of loganin and IS without any interfer-ence from the endogenous components The representativechromatograms are presented in Figure 2 A typical cali-bration curve equation for loganin was y = 00496x+02201(R2=09981) where y represents the ratio of the loganinpeak area to the puerarin peak area (x is the concentrationof loganin in rat plasma and R is about the correlationcoefficient) The lower limit of quantification (LLOQ) inthe collected samples was 2 ngmL and linear ranges from2ngmL to 1750ngmL

33 Effects of Concentration and Time on Transcellular Trans-port of Loganin The transport of different concentrations (520 40 and 80120583M) of loganin was investigated in both direc-tions and both the absorptive (PappAB) the and secretory(PappBA) permeability of loganin were estimated (Figure 3)As shown in Figure 3 PappAB of loganin ranged from 1217 to1478times10minus6cms (AP to BL) and from 1113 to 1296times10minus6cms(BL to AP) A close correlation between the permeabilityacross human intestinal Caco-2 cell monolayers and theabsorption after oral administration in vivohas been obtainedfor several compounds It is well known that compounds with


00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 300

50100150200250300350400450500550600650700750800

0200400600800

100012001400160018002000220024002600280030003200138

198283

019114

110103 185091 217 256 271080

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 3005

101520253035404550556065707580 190 302

194 263

286139 273175080 246131 232

209 224114 187 256161 288029

Loganin

(a1) (a2)

(b1) (b2)

IS

0102030405060708090

100110120130140150160

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30Time (min)Time (min)

Time (min)00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30

Time (min)

014 023

034 038 049

061

070

087

093 105 113

137

141 154

165

170

182

197

147 190

230

218

236

249

255

262

271 280

291 304

201

Figure 2 Typical chromatogram of a separation of loganin (1) and IS (2) in fresh blank HBSS (A) and a sample (B) 5 minutes after transportexperiments by LCMSMS (4597ngmL for loganin and 008ngmL for IS)

A-B

80molL40molL

20molL5molL

B-A

80molL40molL

20molL5molL

0

200

400

600

800

1000

1200

1400

1600

Wei

ght (

ng)

20 40 60 80 100 120 1400Time (min)

20 40 60 80 100 120 1400Time (min)

0200400600800

10001200140016001800

Wei

ght (

ng)

Figure 3 Time Course of loganin transport across human intestinal Caco-2 cell monolayers at different concentrations (n = 3)


pH 60 pH 74 4∘C 37∘C

lowast lowast

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)

Figure 4 Apparent permeability coefficient (PappAB 60min) of loganin (20 120583M) in the AP-to-BL direction in human intestinal Caco-2 cellspreviously treated at different PH values and temperatures (n = 3 p lt 005)

Papp values less than 1times10minus6cms are considered to demon-strate low absorption (lt30) while compounds with Pappvalues between 1times10minus6 and 1times10minus5cms are considered to havea moderate absorption (30-70) and those compoundswith Papp values of more than 1times10minus5cms are consideredto have a high absorption (gt70) [27] The Papp valuesdetermined in the present study indicated good intestinalabsorption of loganin At first instance this high permeabilityobserved in the Caco-2 model seems to be inconsistent withthe previously reported loworal bioavailability of loganin [11]Besides its low water solubility a strong metabolism may beregarded as the major cause of the low oral bioavailabilityof loganin in vivo In fact other iridoid glycosides suchas geniposide [28 29] catalpol [30] and gentiopicroside[31 32] were effectively metabolized by liver or intestinalmicroflora All in all the in vivo processing of drug is complexand additional research is needed to explain the low oralbioavailability

The transport of loganin in the Caco2 cell monolayer wasplotted versus time at 15 30 45 60 90 and 120min for everyconcentration range The concentration of loganin increasedalmost linearly with time within the first 60min After60min the concentration gradient between the two sides hadgreatly decreased resulting in reduced transport and curvesplateau (Figure 3)This indicated that the transport of loganinwas driven by a concentration gradient and passive diffusionrepresents themain transportmechanism for loganin on bothsites

34 Effect of pH and Temperature on the Uptake of Loganinby Human Intestinal Caco-2 Cells The effects of differentpH conditions and temperature on the uptake of loganin byhuman intestinal Caco-2 cells were investigated as shownin Figure 4 The PappAB values of loganin at pH 74 weresignificantly higher than those at pH6 (plt005) indicating aneasier transport of loganin at a higher pH (74) than at lowera pH (60)

A decrease in temperature reduces cellular metabolismand acts as an inhibitor of energy-dependent transport[33] As shown in Figure 4 incubation at low temperature(4∘C) significantly reduced the uptake of loganin as PappABdecreased from 1131 times 10minus6cms to 712times10minus6cms

These data show that loganin transport is both pH-dependent and temperature-dependent indicating that sometransporters may be involved in the efflux of loganin Infact a pH dependent transport has been reported before forBCRP being more efficient at lower pH irrespective of thedissociation status of the substrate [34 35] It can therefore beassumed that loganin is also a substrate of BCRP This couldbe indeed verified in the next experiments

35 Effects of Transporters on Transcellular LoganinTransportVarious inhibitors were investigated for their effects on thetransport flux of loganin across the human intestinal Caco-2cells (Table 2) As shown in Table 2 no significant effects wereobserved in the Papp values after pretreatment with 50120583Mof sodium vanadate or cimetidine suggesting that the influxtransporters like OATs or Na+K+ pump contributed little totheir transport

P-glycoprotein (P-gp) has been confirmed as one of themain transporters that influencing drug transport in theintestine [36]The P-gp inhibitor verapamil may significantlyincrease the transport of some compounds from the APto BL side However it seemed that loganin was not asubstrate of P-gp as the PappAB difference between theverapamil and the control groups was insignificant In addi-tion to the P-gp transporter MRP and BCRP transportersare also members of the adenosine triphosphate-bindingcassette (ABC) superfamily [37] which is related to theprocesses of drug absorption and distribution As shownin Table 2 in the presence of 100120583M MK 571 a MRPsinhibitor the PappAB value of 20120583M loganin was both almost2-fold that of the control group whereas its PappBA valuewas reduced by 667 resulting in a reduction in effluxratio from 09 to 014 (p lt 001) This implied that theMRPs transporters governed the loganin secretion Earlierobservations indicated that Caco-2 cells express more MRP2and MRP3 than MRP1 and MRP5 moreover MRP2 andMRP3 were the main apically and basolaterally localizedMRPs of the Caco-2 cells respectively [21 38] Thereforethe inhibitory effect of MK-571 implied that MRP2 may beprimarily responsible for the loganin efflux in the AP to BLdirectionThis result was validated by the PappABvalueswhenloganin efflux was inhibited by two additional inhibitors


Table 2 Inhibitory effects on bidirectional loganin transport in human intestinal Caco-2 cell monolayers (n = 3)

Inhibitors Transporter concentration (120583M) PappAB (times10minus6cms) PappBA(times10minus6cms) P Modulatory effectControl 130plusmn07 117plusmn09 09 Efflux transportersVerapamil P-gp 100 111plusmn17 93plusmn17 084 -MK 571 MRPs 100 272plusmn41lowastlowast 39plusmn02lowast 014 +Indomethacin MRP2 200 247plusmn48lowastlowast 44plusmn11lowast 018 +Benzbromarone MRP2 MRP3 50 295plusmn59lowastlowast 67plusmn09lowast 023 +Apigenin BCRP 25 301plusmn62lowastlowast 123plusmn25 041 +Infflux transportersSodium vanadate Na+K+pump 50 123plusmn07 90plusmn11 073 -Cimetidine OATs 50 110plusmn15 99plusmn20 09 -lowastDenotes results significantly different from those of the control experiments (p lt 005)lowastlowastDenote results significantly different from those of the control experiments (p lt 0001)+ Indicates that the inhibitor has significant effect on the loganin transport

benzbromarone and indomethacin Indomethacin is aMRP2inhibitor thatmarkedly increased the PappAB value of loganin(20-fold p lt 001) Benzbromarone an inhibitor of MRP2and MRP3 [21 23] enhanced loganin transport just likeindomethacin (p lt 001) Seemingly MRP3 has little effecton the transport of loganin The result turned out to bethat apically localized MRPs especially MRP2 were the mainefflux protein in intestinal absorption mechanism of loganinThe observed reduced PappBA values in the presence of MK571 indomethacin and benzbromarone compared to thecontrol may be attributed to the inhibition of the basolaterallylocalized MRPs [39 40]

Moreover loganin was found to be a substrate of BCRPas in the presence of apigenin a BCRP inhibitor the PappABvalue was about 23-fold that of the control group

4 Conclusions

In the present study it was shown that the transport ofloganin is complex and involves dual processes (a) passivediffusion as the main absorption mechanism and (b) a majorrole of transporter mediated active efflux Efflux transportersBCRP and MRP are vital for loganin transport in the intes-tine The elucidated loganin absorption mechanism providesuseful information for the study of pharmacokinetics Fur-ther studies are needed to explain the low oral bioavailabilityabout loganin

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that they have no conflicts of interest

Authorsrsquo Contributions

Renjie Xu and Yichu Yuan contributed equally to this work asfirst authors

Acknowledgments

This work was partially supported by the Science andTechnology Commission of Shanghai Municipality (Projectnos 18ZR1424900 16401900500 and 154119639900) by theCross-Fund of Biomedical Engineering of Shanghai JiaotongUniversity (No YG2016MS77) and by Shanghai Health andFamily Planning Commission (201840121)

References

[1] W-H Tsai C-H Wu C-H Cheng and C-T Chien ldquoBa-Wei-Di-Huang-Wan through its active ingredient loganin coun-teracts substance P-enhanced NF-120581BICAM-1 signaling in ratswith bladder hyperactivityrdquo Neurourology and Urodynamicsvol 35 no 7 pp 771ndash779 2016

[2] G Chao X Tian W Zhang X Ou F Cong and T SongldquoBlocking Smad2 signalling with loganin attenuates SW10 cellcycle arrest induced by TNF-120572rdquo PLoS ONE vol 12 no 5 2017

[3] W Ma K J Wang C-S Cheng et al ldquoBioactive compoundsfrom Cornus officinalis fruits and their effects on diabeticnephropathyrdquo Journal of Ethnopharmacology vol 153 no 3 pp840ndash845 2014

[4] M-J Kim G-S Bae and I-J Jo ldquoLoganin protects againstpancreatitis by inhibiting NF-120581B activationrdquo European Journalof Pharmacology vol 765 pp 541ndash550 2015

[5] Y Li Z Li L Shi et al ldquoLoganin inhibits the inflammatoryresponse in mouse 3T3L1 adipocytes and mouse modelrdquo Inter-national Immunopharmacology vol 36 pp 173ndash179 2016

[6] K Liu H Xu G Lv et al ldquoLoganin attenuates diabeticnephropathy in C57BL6J mice with diabetes induced by strep-tozotocin and fed with diets containing high level of advancedglycation end productsrdquo Life Sciences vol 123 pp 78ndash85 2015

[7] M Zhao J Tao D Qian et al ldquoSimultaneous determinationof loganin morroniside catalpol and acteoside in normal andchronic kidney disease rat plasma by UPLC-MS for investigat-ing the pharmacokinetics of Rehmannia glutinosa and Cornusofficinalis Sieb drug pair extractrdquo Journal of Chromatography Bvol 1009-1010 pp 122ndash129 2016

[8] H Kim K Youn and M R Ahn ldquoNeuroprotective effect ofloganin against A12057325-35-induced injury via NF-120581B-dependent


signaling pathway in PC12 cellsrdquo Food Function pp 1108ndash11162015

[9] Y-T Tseng C-S Chen Y-J Jong F-R Chang and Y-CLo ldquoLoganin possesses neuroprotective properties restoresSMN protein and activates protein synthesis positive regulatorAktmTOR in experimentalmodels of spinalmuscular atrophyrdquoPharmacological Research vol 111 pp 58ndash75 2016

[10] L Yao S-X Peng Y-D Xu et al ldquoUnexpected Neuro-protective Effects of Loganin on 1-Methyl-4-Phenyl-1236-Tetrahydropyridine-Induced Neurotoxicity and Cell Death inZebrafishrdquo Journal of Cellular Biochemistry vol 118 no 3 pp615ndash628 2017

[11] R Xu J Qi R-J Zhan et al ldquoComparative pharmacokinetics offour active components on normal and diabetic rats after oraladministration of Gandi capsulesrdquo RSC Advances vol 8 no 12pp 6620ndash6628 2018

[12] X Liu S Zheng Y Qin et al ldquoExperimental Evaluation of theTransport Mechanisms of PoIFN-120572 in Caco-2 Cellsrdquo Frontiersin Pharmacology vol 8 2017

[13] Y Peng M Fan C Peng M Wang and X Li ldquoAlleviatingthe Intestinal Absorption of Rhein in Rhubarb through HerbCompatibility in Tiaowei Chengqi Tang in Caco-2 CellsrdquoEvidence-Based Complementary and Alternative Medicine vol2018 2018

[14] F Zhou W Huang M Li Y Zhong M Wang and B LuldquoBioaccessibility and Absorption Mechanism of Phenyletha-noid Glycosides Using Simulated DigestionCaco-2 IntestinalCell Modelsrdquo Journal of Agricultural and Food Chemistry vol66 no 18 pp 4630ndash4637 2018

[15] XWang G Liu Y Yang XWuW Xu and X Yang ldquoIntestinalAbsorption of Triterpenoids and Flavonoids fromGlycyrrhizaeradix et rhizoma in the Human Caco-2Monolayer Cell ModelrdquoMolecules vol 22 no 10 p 1627 2017

[16] Y-F Yang Q-L Zhou and X-W Yang ldquoElucidation of Com-patibility Interactions of Traditional Chinese Medicines InVitro Absorptions Across Caco-2 Monolayer of Coptidis Rhi-zoma and Euodiae Fructus in Zuojin and Fanzuojin Formulasas A Caserdquo Phytotherapy Research vol 31 no 8 pp 1220ndash12292017

[17] Q Zhao Y Zou C Huang P Lan J Zheng and S OuldquoFormation of a Hydroxymethylfurfural-Cysteine Adduct andIts Absorption and Cytotoxicity in Caco-2 Cellsrdquo Journal ofAgricultural and Food Chemistry vol 65 no 45 pp 9902ndash99082017

[18] L-Y Luo M-X Fan H-Y Zhao M-X Li X Wu and W-YGao ldquoPharmacokinetics and Bioavailability of the IsoflavonesFormononetin and Ononin and Their in Vitro Absorption inUssing Chamber and Caco-2 Cell Modelsrdquo Journal of Agricul-tural and Food Chemistry vol 66 no 11 pp 2917ndash2924 2018

[19] D Yu Y Zhang L Guo Q Zhang and H Zhu ldquoStudy on theAbsorption Mechanism of Geniposide in the Chinese FormulaHuang-Lian-Jie-Du-Tang in Ratsrdquo AAPS PharmSciTech vol 18no 4 pp 1382ndash1392 2017

[20] B Zhang X-M Zhu J-N Hu et al ldquoAbsorptionmechanism ofginsenoside compound K and its butyl and octyl ester prodrugsin caco-2 cellsrdquo Journal of Agricultural and Food Chemistry vol60 no 41 pp 10278ndash10284 2012

[21] H M Prime-Chapman R A Fearn A E Cooper V Mooreand B H Hirst ldquoDifferential multidrug resistance-associatedprotein 1 through 6 isoform expression and function in humanintestinal epithelial Caco-2 cellsrdquo The Journal of Pharmacology

and Experimental Therapeutics vol 311 no 2 pp 476ndash4842004

[22] L Zhang G Lin B Kovacs M Jani P Krajcsi and Z ZuoldquoMechanistic study on the intestinal absorption and dispositionof baicaleinrdquo European Journal of Pharmaceutical Sciences vol31 no 3-4 pp 221ndash231 2007

[23] D Ursic K Berginc S Zakelj and A Kristl ldquoInfluence ofluminal monosaccharides on secretion of glutathione conju-gates from rat small intestine in vitrordquo International Journal ofPharmaceutics vol 381 no 2 pp 199ndash204 2009

[24] S Zhang X Yang andM E Morris ldquoCombined effects of mul-tiple flavonoids on breast cancer resistance protein (ABCG2)-mediated transportrdquo Pharmaceutical Research vol 21 no 7 pp1263ndash1273 2004

[25] M N Chabane A Al Ahmad J Peluso C D Muller and GUbeaud ldquoQuercetin and naringenin transport across humanintestinal Caco-2 cellsrdquo Journal of Pharmacy and Pharmacologyvol 61 no 11 pp 1473ndash1483 2009

[26] B Wahlang Y B Pawar and A K Bansal ldquoIdentification ofpermeability-related hurdles in oral delivery of curcumin usingthe Caco-2 cell modelrdquo European Journal of Pharmaceutics andBiopharmaceutics vol 77 no 2 pp 275ndash282 2011

[27] S Yee ldquoIn vitro permeability across Caco-2 cells (colonic) canpredict in vivo (small intestinal) absorption in manmdashfact ormythrdquo Pharmaceutical Research vol 14 no 6 pp 763ndash766 1997

[28] Y C Hou S Y Tsai P Y Lai Y S Chen and P D L ChaoldquoMetabolism and pharmacokinetics of genipin and geniposidein ratsrdquo Food and Chemical Toxicology vol 46 no 8 pp 2764ndash2769 2008

[29] J-S Yim Y-S Kim S-K Moon et al ldquoMetabolic activitiesof ginsenoside Rb1 baicalin glycyrrhizin and geniposide totheir bioactive compounds by human intestinal microflorardquoBiological amp Pharmaceutical Bulletin vol 27 no 10 pp 1580ndash1583 2004

[30] J Tao M Zhao D Wang et al ldquoBiotransformation andmetabolic profile of catalpol with human intestinal microfloraby ultra-performance liquid chromatography coupled withquadrupole time-of-flight mass spectrometryrdquo Journal of Chro-matography B vol 1009-1010 pp 163ndash169 2016

[31] L J Wang and Q Xu ldquoMechanism of anti-inflammatoryaction of Huanglian Jiedu decoction a traditional Chineseprescriptionrdquo Zhongguo Zhong Yao Za Zhi vol 25 no 8 pp493ndash496 2000

[32] A I El-Sedawy M Hattori T Namra and K K KobashiildquoMetabolism of Gentiopicroside (Gentiopicrin) by HumanIntestinal BacteriardquoChemicalampPharmaceutical Bulletin vol 37no 9 pp 2435ndash2437 1989

[33] R A Rocha VDevesa andDVelez ldquoIn vitro study of intestinaltransport of fluoride using the Caco-2 cell linerdquo Food andChemical Toxicology vol 55 pp 156ndash163 2013

[34] P Breedveld D Pluim G Cipriani et al ldquoThe effect of low pHon breast cancer resistance protein (ABCG2)-mediated trans-port of methotrexate 7-hydroxymethotrexate methotrexatediglutamate folic acid mitoxantrone topotecan and resvera-trol in in vitro drug transportmodelsrdquoMolecular Pharmacologyvol 71 no 1 pp 240ndash249 2007

[35] M Jani C Ambrus R Magnan et al ldquoStructure and functionof BCRP a broad specificity transporter of xenobiotics andendobioticsrdquoArchives of Toxicology vol 88 no 6 pp 1205ndash12482014


[36] H Li J Li L Liu et al ldquoElucidation of the Intestinal AbsorptionMechanism of Celastrol Using the Caco-2 Cell TranswellModelrdquo Planta Medica vol 82 no 13 pp 1202ndash1207 2016

[37] L Bromberg and V Alakhov ldquoEffects of polyether-modifiedpoly(acrylic acid) microgels on doxorubicin transport inhuman intestinal epithelial Caco-2 cell layersrdquo Journal of Con-trolled Release vol 88 no 1 pp 11ndash22 2003

[38] J Duan Y Xie H Luo G Li T Wu and T Zhang ldquoTransportcharacteristics of isorhamnetin across intestinal Caco-2 cellmonolayers and the effects of transporters on itrdquo Food andChemical Toxicology vol 66 pp 313ndash320 2014

[39] D Keppler ldquoMultidrug resistance proteins (MRPs ABCCs)Importance for pathophysiology and drug therapyrdquo Handbookof Experimental Pharmacology vol 201 pp 299ndash323 2011

[40] Z Yuan S T Zhang B Jin T Li and C Ma Zhongguo ZhongYao Za Zhi Transportmechanismof isorhapontigeninbased onhuman intestinal Caco-2 cells Zhongguo Zhong Yao Za Zhi 4587-592 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018


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EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


O

O O

OH

H

H

HO

O O

OH

HO OH

Figure 1 The chemical structure of loganin

transporters that allow for the study of carrier mediateduptake and efflux mechanisms [14] Different studies demon-strated that in vivo absorption could be accurately predictedfrom the apparent permeation rate (Papp) across the humanintestinal Caco-2 cell monolayers [15ndash18] Although the Pappvalues obtained from different laboratories differ there is ageneral trend that a high Papp implies effective absorptionA published report using the human intestinal Caco-2 cellsmodel for the study of geniposide [19] a structural analogof loganin showed good intestinal permeability by passivediffusion chiefly The present study is devoted for the firsttime to the investigation of the detailed intestinal absorptionmechanism of loganin and the results will be useful forfurther studies on this type of compounds in vitro and in vivo

2 Materials and Methods

21 Materials The human colon adenocarcinoma cell lineCaco-2 was purchased from the Cell Bank of the Chi-nese Academy of Sciences (Shanghai China) Dulbeccorsquosmodified Eaglersquos medium (DMEM) Hankrsquos buffered saltsolution (HBSS) antibiotic solutions (100000 UL penicillinand 100000mgL streptomycin) fetal bovine serum (FBS)100times nonessential amino acids 100times penicillin and strepto-mycin and 025 trypsin with ethylenediaminetetraaceticacid (EDTA) were from Invitrogen Corp (Carlsbad CA)Transwell permeable polycarbonate inserts (04 120583m) and12-well cell culture plates were obtained from CorningVerapamil MK 571 indomethacin benzbromarone api-genin sodium vanadate and cimetidine were obtained fromAladdin Industrial Inc (Shanghai China) Loganin (purity gt980) and puerarin (internal standard (IS) purity gt 980)were obtained from the National Institute for the Control ofPharmaceutical and Biological Products (Beijing China) Allother reagents were of analytical grade

22 Cell Culture Cells were maintained in DMEM with10 FBS 1times nonessential amino acids and 1times penicillinand streptomycin at 37∘C with 5 CO2 Cells of passage

35-40 were used in this study to maintain relatively constantcellular phenotypes The medium was replaced every 2-3days during incubation After reaching approximately 80confluence the cells were detached using 002 ethylenedi-aminetetraacetic acid (EDTA) and 005 trypsin at a densityof 53times104 cellscm2 on a 60-mm plastic culture dish for theuptake experiment or a 12-mm polyester membrane insert ina 12-well plate for the permeation experiment The media inthe culture plates were changed every two days for the firstweek after seeding and replaced daily afterwardThe integrityof the cell monolayers was examined by measuring thetransepithelial electrical resistance (TEER) with a Millicell-ERS-electrode (Millipore Corp Billerica MA USA) After21-23 days monolayers with a TEER value (TEER=[TEERtest-TEERbackground]timesarea) above 500Ωcm

2 were used during thetransport experiments

23 Cytotoxicity Assay Cell viability was assessed using anMTT assay Briefly 01mL of human intestinal Caco-2 cellswas seeded onto 96-well plates at a density of 1times105 cellsper well in a 96-well plate The cells were grown in anatmosphere with 5 CO2 and 95 relative humidity overapproximately 48 h Subsequently the culture medium wasreplaced with HBSS containing different concentrations ofloganin (01-200 120583M) the final concentration of dimethylsulfoxide (DMSO) in HBSS remained at lt1 The negativecontrol was HBSS containing 1 DMSO The cells wereexposed to the drug for 2 h Then 02mL of 05mgmLMTT solution was added to each well and incubated for4 h in dark The medium was then removed the MTT-formazan crystals were solubilized by incubating with 150 120583Lof DMSOwith gentle shaking for 10min and absorbance wasdetermined at 490 nm in a Multiskan Spectrum microplatereader (Thermo Labsystems MA USA) In each MTT assayevery sample was tested in five replicates and the viabilityof the nontreated control cells was arbitrarily defined as100

24 Transport Experiments Before the experiments thehuman intestinal Caco-2 cell monolayers were washed twicewith HBSS medium (pH 74) The transport experimentswere conducted by adding the drug solutions (containing1 DMSO) to either the apical (AP 05mL) or basolateralside (BL 10mL) while the receiving chamber containedthe corresponding volume of blank HBSS medium Themonolayers were incubated at 37∘C and 50120583L sampleswere taken at 15 30 45 60 90 and 120min from theacceptor compartment and the volume was then immedi-ately replaced with50 120583L of fresh prewarmed blank HBSSTEER measurements for assessing the membrane integritytook place before and after the experiment The sampleswere frozen immediately and stored below minus80∘C beforeanalysis

The loganin transport (20 120583M) at 4∘C and 37∘C wasevaluated in the AP to BL direction to investigate the effectof temperature The effect of pH on the loganin transport (20120583M) in theAP toBL directionwas studied using the followingpH combinations for the HBSS in the acceptordonor com-partments 6074 and 7474








P =PappBAPappAB

(1)










00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 300

50100150200250300350400450500550600650700750800

0200400600800

100012001400160018002000220024002600280030003200138

198283

019114

110103 185091 217 256 271080

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 3005

101520253035404550556065707580 190 302

194 263

286139 273175080 246131 232

209 224114 187 256161 288029

Loganin

(a1) (a2)

(b1) (b2)

IS

0102030405060708090

100110120130140150160

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30Time (min)Time (min)

Time (min)00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30

Time (min)

014 023

034 038 049

061

070

087

093 105 113

137

141 154

165

170

182

197

147 190

230

218

236

249

255

262

271 280

291 304

201


A-B

80molL40molL

20molL5molL

B-A

80molL40molL

20molL5molL

0

200

400

600

800

1000

1200

1400

1600

Wei

ght (

ng)

20 40 60 80 100 120 1400Time (min)

20 40 60 80 100 120 1400Time (min)

0200400600800

10001200140016001800

Wei

ght (

ng)



pH 60 pH 74 4∘C 37∘C

lowast lowast

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)














4 Conclusions


Data Availability






Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























P =PappBAPappAB

(1)










00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 300

50100150200250300350400450500550600650700750800

0200400600800

100012001400160018002000220024002600280030003200138

198283

019114

110103 185091 217 256 271080

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 3005

101520253035404550556065707580 190 302

194 263

286139 273175080 246131 232

209 224114 187 256161 288029

Loganin

(a1) (a2)

(b1) (b2)

IS

0102030405060708090

100110120130140150160

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30Time (min)Time (min)

Time (min)00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30

Time (min)

014 023

034 038 049

061

070

087

093 105 113

137

141 154

165

170

182

197

147 190

230

218

236

249

255

262

271 280

291 304

201


A-B

80molL40molL

20molL5molL

B-A

80molL40molL

20molL5molL

0

200

400

600

800

1000

1200

1400

1600

Wei

ght (

ng)

20 40 60 80 100 120 1400Time (min)

20 40 60 80 100 120 1400Time (min)

0200400600800

10001200140016001800

Wei

ght (

ng)



pH 60 pH 74 4∘C 37∘C

lowast lowast

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)














4 Conclusions


Data Availability






Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 300

50100150200250300350400450500550600650700750800

0200400600800

100012001400160018002000220024002600280030003200138

198283

019114

110103 185091 217 256 271080

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 3005

101520253035404550556065707580 190 302

194 263

286139 273175080 246131 232

209 224114 187 256161 288029

Loganin

(a1) (a2)

(b1) (b2)

IS

0102030405060708090

100110120130140150160

00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30Time (min)Time (min)

Time (min)00 02 04 06 08 10 12 14 16 18 20 22 24 26 28 30

Time (min)

014 023

034 038 049

061

070

087

093 105 113

137

141 154

165

170

182

197

147 190

230

218

236

249

255

262

271 280

291 304

201


A-B

80molL40molL

20molL5molL

B-A

80molL40molL

20molL5molL

0

200

400

600

800

1000

1200

1400

1600

Wei

ght (

ng)

20 40 60 80 100 120 1400Time (min)

20 40 60 80 100 120 1400Time (min)

0200400600800

10001200140016001800

Wei

ght (

ng)



pH 60 pH 74 4∘C 37∘C

lowast lowast

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)














4 Conclusions


Data Availability






Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















pH 60 pH 74 4∘C 37∘C

lowast lowast

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)

0

2

4

6

8

10

12

14

P app

A-B

(

-cm

s)














4 Conclusions


Data Availability






Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of
























4 Conclusions


Data Availability






Acknowledgments


References

















































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















elucidation of the intestinal absorption mechanism of loganin in...

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