research article cytotoxicity and in vivo acute...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 403491 12 pageshttpdxdoiorg1011552013403491

Research ArticleIn Vitro Cytotoxicity and In Vivo Acute and Chronic Toxicity ofXanthii Fructus and Its Processed Product

Jie Yu Mei-Zhen Song Jing Wang Yun-Fei Li Pei Lin Lin Que and Zhaorigetu Bao

Yunnan University of Traditional Chinese Medicine Kunming Yunnan 650500 China

Correspondence should be addressed to Zhaorigetu Bao rigdan126com

Received 27 July 2013 Revised 11 October 2013 Accepted 11 October 2013

Academic Editor Swaran J S Flora

Copyright copy 2013 Jie Yu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Xanthii Fructus (XF) the fruit of Xanthium sibiricum Patr was used in the treatment of rhinitis and related nasal disease Adverseeffects of Xanthii Fructus are frequently reported these years In the paper in vitro renal cytotoxicity and in vivo acute and chronictoxicity researches of Xanthii Fructus (XF) and its processed product (processed Xanthii Fructus (PXF)) were carried out Waterextraction of XF displayed no cell membrane damage effects even in the highest concentration (100 120583gmL) however it mightaffect the function of renal cell mitochondria Acute toxicities were observed only in high and middle dosage groups Fortunatelythe single dose administration of XF or PXF was safe even at the highest daily dosage Twelve-week chronic toxicity assays wereperformed in SD rats with low middle and high dosage Notable changes in body weight and blood cell and BUN and Scr changessporadically occurred in middle and high groups after the 9th week SerumHA and HPCIII values were sustained increasing fromthe 4th week to the 8th week in Group Vmale rats which indicated that the renal fibrosis risks still existed although no fibrosis wasfound in the pathological examination of the liver and kidney

1 Introduction

Concerns about the toxicity of traditional Chinese medicinesare increasing with each passing day after the ldquoaristolochicacid nephropathyrdquo case Aristolochic acids are hypothesizedto be causative agents in Balkan endemic nephropathy and arelated possibly identical condition known as ldquoChinese herbsnephropathyrdquo [1]

Hereafter adverse effects of traditional Chinese medicineother than aristolochic acid related crude drugs such asXanthii Fructus Polygoni Multiflori and Leonuri Herbaare frequently reported [2ndash8] Among them Xanthii Fructusattracts our intensive attention due to its wide range ofapplications in the treatment of rhinitis snuffle nasosinusitisand related nasal disease Xanthii Fructus is the fruit ofXanthium sibiricum Patr [9] Xanthii Fructus is the mostfrequently used crude drug in compound recipes which wereused in the treatment of rhinitis Xanthii Fructus is recordedas nontoxic in the ancient herbal ldquoShen Nong Ben Cao Jingrdquo(Shen Nongrsquos Classic of Materia Medica composed in aboutAD 1082) Nevertheless its toxicity is recognized graduallyas time goes by It is recorded as slight noxious or middle

level noxious in most of the ancient herbals such as ZhengLei Ben Cao (composed in about AD 1082) and Pin Hui JingYao (composed in about AD 1505)

Unfortunately although the toxicity of Xanthii Fructusis clearly alerted in the Pharmacopoeia of China (2010edition) [9] poisoning even death cases are reported con-tinuously after immoderate usage of Xanthii Fructus as foodor medicine The authors have reviewed the clinic toxicityreports of Xanthii Fructus systematically [7] Three deathcases caused by multiple organ failure were reported [3] inTurkey (no clear dosage reported) An outbreak characterizedby vomiting and rapid progression to unconsciousness anddeath was reported in northeastern Bangladesh after theconsumption of Xanthium strumarium seedlings in largequantities due to inaccessibility of other foods [5] Kidneydamage was the most frequently reported adverse effectcaused by Xanthii Fructus [7] Hematuria albuminuriaabnormal renal function and acute renal failure were themost presented renal damage types [2 4] Judging fromall the adverse effects reports we find that most of thesesevere adverse effects are occurred in the high dosage or evenoverdose administration cases

2 BioMed Research International

The toxicity of Xanthii Fructus is usually considered tobe associated with two diterpenoids glycosides atractyloside(AL) and carboxyatractyloside (CAL) The biochemistry andtoxicology of them are well investigated and elucidated bothin in vitro and in vivo [10 11] methods The LD

50of AL is

15mgkg in dog (iv) 434mgkg in mouse (ip) and 143mgkgin rat (ip) According to the latest literature [10] contentof CAL decreased dramatically after processing procedurewhile AL content increased after processing procedure CALwas reported to be about 116mgndash225mgg in XF and 0ndash021mgg in PXF On the other hand AL could not bedetected in XF however the content of AL increased to about017ndash256mgg in PXF However the toxicity mechanismsinvolved are not well investigated

Despite the prevalent reports of their toxicity the toxicityresearch data about XF and PXF still need to be clarifiedespecially their toxicity in normal or middle dosage admin-istration case In this paper we systemically evaluated the invitro and in vivo toxicity of Xanthii Fructus its processedproduct and its major chemical constituent

In vitro renal cytotoxicities were investigated by humankidney HK-2 cell line an immortalized proximal tubularepithelial cell line from normal adult human male kid-ney retaining the phenotype and functional characteristicsindicative of well-differentiated proximal tubular epithelialcell [12] Not only the water extracts of Xanthii Fructusbut also its major chemical constituents atractyloside potas-sium salt (AL) were involved Culture of this cell line haspreviously been employed by several studies to investigatenephrotoxicity of various toxic compounds [13ndash15]

Acute and chronic toxicities were tested in Kunmingmice and SD rats respectively Single dosage of XF or PXFwas given to Kunming mice in order to investigate theiracute toxicity Twelve-week chronic toxicity assay was alsoconducted in rats Systemic toxicity indexes were involved inboth acute and chronic toxicity assays in order to elucidatetheir toxicity comprehensively Some kidney toxicity indexesand early indicators of renal fibrosis were involved in chronictoxicity assays

2 Materials and Methods

21 Chemicals 3-(45)-Dimethylthiahiazo(-z-y1)-35-di-phe-nytetrazoliumromide (MTT) and trypsin (1 250)were acqui-red from Amresco USA Dimethylsulfoxide (DMSO) waspurchased from Sigma USA Other reagents were all aboveanalytical grade Lactate dehydrogenase (LDH) assay kitswere purchased from Changchun Huili Biotech Co LtdChina BUN and Scr assay kits were purchased fromBiosino Bio-technology amp Science Inc China Enzyme-linked immunosorbent assay kits of HA LN and HPC IIIthree early indicators of renal fibrosis were obtained fromCusabio Biotech Co Ltd Wuhan China

22 Processing Procedure of Xanthii Fructus Xanthii Fructuswas purchased from Juhuacun Chinese herbal medicinemarket Kunming Yunnan China in February 2009 (shownin Figure 1) and was identified as fruit of Xanthium sibiricum

Patar by the author Associate Professor Jie Yu Voucher spec-imens were deposited in the Herbarium of PharmacognosyYunnan University of Traditional Chinese Medicine

Processed Xanthii Fructus was stir-fried from XanthiiFructus with mild fire to yellowish brown (Figure 1) Process-ing of Xanthii Fructus was carried out by the authors accord-ing to the procedures documented in the Pharmacopoeia ofChina 2010 edition [9]

23 In Vitro Renal Cytotoxicity Assay

231 Preparation of Exposure Solution of Atractyloside Potas-sium Salt for In Vitro Assay Atractyloside potassium salt(AL) major constituent of XF and PXF was purchased fromSigma USA (structure shown in Figure 2) with purity higherthan 98 The exposure solutions of AL in concentrationfrom 25 to 320 120583M were diluted by RPMI 1640 containing02 fetal bovine serum (FBS)

232 Preparation of Extraction of Xanthii Fructus for In VitroAssay 40 g powder of XF was decocted with water (1000mL800mL and 600mL) for 3 times respectivelyThe extractionswere combined condensed and lyophilized Extraction yieldwas 2315

XF stock solution was achieved by dissolving them inDMSO at a concentration of 10mgmL Then dissolving inDMSO it was further diluted with 02 FBS-RPMI 1640 tograded concentrations from 10 to 100 120583gmL

233 Culture Conditions The whole research was carriedout by HK-2 cells immortalized proximal tubular epithelialcell line which were purchased from Cell Bank KunmingInstitute of Zoology Chinese Academic of Science Cells werecultured in monolayer culture in DMEM medium (GibcoInvitrogenCorp) supplementedwith 10 fetal bovine serum(HyClone)

Cells were seeded at an appropriate cell density in ahumidified incubator (Series II water jacketed CO

2Incubator

Model 3111 Thermo Electron Corporation USA) with 5carbon dioxide and dioxide 95 air at 37∘C for different assayand left to grow to 80-90 confluence

234 MTT Cell Viability Assay This assay procedure wasadopted from the previous report [8] HK-2 cells were seededin 96-well plates (Corning Incorporated) at a cell density of2 times 104 in 200120583Lwell and then incubated for 48 h till 80ndash90 confluenceThen the cell synchronization was routinelyaccomplished by incubating cells in G

0medium for 12 h

before the test Subsequently cells were exposed to fresh G0

medium with various concentrations of test compounds orextractions for 24 h

At the end of the incubation period 10 120583Lwell of5mgmL MTT (Amresco USA) which was dissolved inPBS (pH 74) and sterilized by filtrating through a Milliporefilter (022 120583m) was added in medium for a further 4 hincubation then the medium was removed and 100 120583LDMSO was added to dissolve the resulting intracellularpurple formazan product The 96-well plates were gently

BioMed Research International 3

Xanthii Fructus Processed Xanthii Fructus

Figure 1 Photographs of Xanthii Fructus and its processed product

CH3

OH

O

HOOC

H3C

OHOH2C

OO

OC

OCH2

CH2

CHH3C

minusO3S

minusO3S

Figure 2 Structure of atractyloside potassium salt (AL)

swirled for 5min in room temperature The absorbance wasmeasured at 570 nmThis was conducted by amicrotiter platereader (DNM-9602G Beijing Perlong Medical EquipmentCo Ltd) The wells added with cell and culture medium butwithout stimulus were considered as normal control and thewells added culture medium only were considered as blankcontrol HgCl

2(10 120583M) a typical renal toxicant was used as a

positive control Each concentration of tested compounds orextractions was paralleled for three times and 6 wells wereaveraged in each plate The survival rate of cells in eachconcentration was calculated

235 LDH (Lactate Dehydrogenase) Leakage Assay Thisassay procedure was adopted from the previous report [8]HK-2 cells were seeded in 24-well plates (Corning Incor-porated) at a cell density of 1 times 104 in 500 120583Lwell andthen incubated till the cells reached 80ndash90 confluenceThen the cell synchronization was routinely accomplishedby incubating cells in G

0medium for 12 h before the test

Subsequently cells were exposed to fresh G0medium with

test compounds or extraction for 24 h At the endpoint ofthe incubation period all the culture medium was collectedand the intracellular LDH was ingathered after lysing cellswith 500120583L 01 Triton X-100 (Sigma) The enzyme contentin culture medium and cell lysis solution was analyzed bylactate dehydrogenase kit by an UV detector (725 ShanghaiSpectrum Instruments Co Ltd)The leakage percentage wascalculated by the extracellular LDH activity compared withthe total LDH activity (extracellular + intracellular LDH)HgCl2(10 120583M) was used as a positive control Two wells were

repeated in each 24-well plate and three plates were paralleledfor different concentrations of each compound or extraction

24 In Vivo Acute and Chronic Toxicity Assays

241 Animals Acute toxicity research was carried out onKunming mice of both sexes 70 Kunming mice wereprovided by the Experimental Animal Center of YunnanUniversity of Traditional ChineseMedicineTheywere aged 5weeks andweighed 18sim22 gMice of the same sexwere housedten in a stainless steel cage containing sterile paddy husk asbedding in ventilated animal rooms

Chronic toxicity research was carried out on SD rats ofboth sexes which were also provided by the ExperimentalAnimal Center of Yunnan University of Traditional ChineseMedicine They were aged 6 weeks and weighed 200 plusmn 20 gRats of the same sex were housed four in a stainless steel ratcage containing sterile paddy husk as bedding in ventilatedanimal rooms

All mice and rats were acclimated in the controlledenvironment (temperature 22 plusmn 1∘C 60 plusmn 10 humidityand a 12 h12 h lightdark cycle) with free access to waterand a commercial laboratory complete food All animalexperiments were performed in compliance with the animalexperimental ethics committee of Yunnan University ofTraditional Chinese Medicine All reasonable efforts weremade to minimize the animalsrsquo suffering

242 Preparation of Extraction of Xanthii Fructus for InVivo Toxicity Assay 500 g Xanthii Fructus was immersedin water for an hour Then they were decocted with water(2500mL 1500mL and 1500mL) for 3 times 30mins pertime respectively The extraction was combined and con-densed to 10 gmL (themaximumconcentration that can flowthrough the gavage needle for mice) The same procedurewas adopted in the preparation of processed Xanthii Fructusextraction

The recommended dosages of XF and PXF were both 3ndash10 gper day according to the Pharmacopoeia of the PeoplersquosRepublic of China 2010 edition [9]The lowmiddle and highdosages of XF and PXF in acute toxicity assay for mice were1668 gmL (10 times the highest human dosage) 1668 gmL(100 times the highest human dosage) and 400 gmL (240times the highest human dosage)

Much lower dosages were adopted in the chronic toxicityassay in order to imitate the human clinicmedication practiceof XF and PXF In the chronic toxicity assay the low middleand high dosages of XF and PXF were 0585 gmL (the sameas the humanmedian dosage) 1755 gmL (3 times the humandosage median dosage) and 5265 gmL (9 times the humandosage median dosage)

243 Acute Toxicity Testing All mice of both sexes wererandomly divided into 7 groups of ten each (Table 1) Themale and female rats were all five in each group All micefasted for 12 h before the administration of each agent GroupA received physiological saline only and served as vehicleGroup B to G received water of XF or PXF in low middleand high dosage respectively (Table 1)

Clinical symptoms of mice after respective treatmentswere monitored closely for 4 hr after gavage Seven-day


Table 1 Mice grouping and treatments in acute toxicity testing

Groups Treatment Dosage (gkgbody weight)

Relative ratio tohuman highestdaily dosage

A Physiological saline 0 mdashB Water extraction of XF 1668 10 timesC Water extraction of XF 1668 100 timesD Water extraction of XF 400 240 timesE Water extraction of PXF 1668 10 timesF Water extraction of PXF 1668 100 timesG Water extraction of PXF 400 240 times

Table 2 Rats grouping and treatments in chronic toxicity testing


Relative ratio tohuman mediandaily dosage

I Physiological saline 0 mdashII Water extraction of XF 0585 1 timeIII Water extraction of XF 1755 3 timesIV Water extraction of XF 5265 9 timesV Water extraction of PXF 0585 1 timeVI Water extraction of PXF 1755 3 timesVII Water extraction of PXF 5265 9 times

Table 3 MTT survival rate and LDH leakage rate of HK-2 cellexposed to atractyloside potassium salt (AL)

Exposure (120583M) MTT survival rate () LDH leakage rate ()Control 100 763 plusmn 000

HgCl2 539 plusmn 148 953 plusmn 193

5 884 plusmn 132 899 plusmn 0311

10 934 plusmn 868 822 plusmn 0571

20 892 plusmn 442 896 plusmn 0198

40 922 plusmn 282 928 plusmn 0421

80 995 plusmn 1533 103 plusmn 125

160 941 plusmn 103 873 plusmn 0132

240 982 plusmn 656 962 plusmn 0744

320 924 plusmn 312 117 plusmn 0981

observationwas carried out on all the survivingmiceMortal-ities clinical signs time of onset duration and reversibilityof toxicity in each group were recorded Gross necropsieswere performed on all animals including those sacrificedmoribund found dead or terminated at 7 days Routineanalysis of blood cells in Groups A B C E and F was carriedout after the sacrifice in the end of the research For lightmicroscopic observations samples from liver kidney lungand heart of each group were fixed in formalin fixative andprocessed routinely for embedding in paraffin Both maleand female mice samples were used in the histopathologicalexamination Tissue sections in 5 120583m thickness were stainedwith hematoxylin and eosin (HampE) and examined under alight microscope

244 Chronic Toxicity Assays A 12-week chronic toxicityassay was executed to investigate the long term toxicity after

normal dosage administration of XF and PXF every day 56rats of both sexes were randomly divided into 7 groups ofeight each (Table 2) The male and female rats were all fourin each group Group I received physiological saline only andserved as vehicle Group II to IV received water extraction ofXF while Group V to VII got PXF water extraction in variousdosages All rats fasted for 2 h before administration of eachagent every day

Body weights were recorded every three days till the endSamples of blood 15ndash2mL in volume were collected from theretro-orbital venous plexus once every week throughout thestudy Blood samples were collected under ether anestheticcondition two hours after the administration of therapeuticagents in the morning Routine analysis of blood cells wasperformed by hematology analyzer (URIT-2900 GuilingChina) Levels of BUNand Scrwere determined by enzymaticcolorimetric method using commercial assay kits (BiosinoBio-technology amp Science Inc) by AB-1020 automatic bio-chemical analyzer (Sunostik Medical Technology Co Ltd)As early indicators of renal fibrosis serum HA LN andHPCIII content values were also tested in our research Theywere analyzed using enzyme-linked immunosorbent assaykits from Cusabio Biotech Co Ltd (Wuhan China) HALN and HPCIII were tested only on weeks 4 8 and 12 Allbioassays were carried out in duplicate

For light microscopic observations samples from liverand kidney of each group were fixed in formalin fixativeand processed routinely for embedding in paraffin Bothmale and female samples were used in the histopathologicalexamination Tissue sections in 5120583m thickness were stainedwith hematoxylin and eosin (HampE) and examined under alight microscope

25 Statistical Analysis All data in this research wereexpressed in the form of mean plusmn SD One-way analysisof variance (ANOVA) was performed when multiple groupcomparisons were carried out Results were classified intothree significance levels using the 119875 value of 005 001 and0001

3 Results

31 In Vitro Renal Cytotoxicity of Atractyloside PotassiumSalt In order to avoid overestimation or underestimationof the toxicity of a substance incubations with variousconcentrations are required to distinguish between effectson specific organelles or general cytotoxicity More than oneassay should be used to determine cell viability in in vitrostudies as this would increase the reliability of the resultsobtained [8 16] In this research both MTT and LDH assaysreflected the function of mitochondria and cell membranewere adopted

As listed in Table 3 AL showed no inhibition on the cellproliferation of HK-2 cells in MTT survival rate assay Thecell survival rate was not affected by the exposure of AL andit almost remained at 100 even at the highest concentrationof 320120583M Meantime the LDH leakage rate was not affectedby the exposure of AL and it almost remained at 10 (similar


Control 10 20 40 60 80 100

0

20

40

60

80

100

120M

TT su

rviv

al ra

te (

)

Concentration of water extraction of Xanthii FructusHgCl2

lowastlowastlowast

(a)

Control 10 20 40 60 80 1000

20

40

60

80

100

120

LDH

leak

age r

ate (

)


lowastlowastlowast

(b)

Figure 3 MTT survival rate (a) and LDH leakage rate (b) of HK-2 cell exposured to water extraction of Xanthii Fructus The lowast indicates asignificant difference compared with control group lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001

to the control group) even at the highest concentration of320 120583M

Judging from these data AL not only showed no inhibi-tion on the cell proferliation of HK-2 cells but also exhibitedno damage on the cell membrane function

32 In Vitro Renal Cytotoxicity of Xanthii Fructus Resultsof renal cytotoxicity assay of water extraction of XF wereshown in Figure 3 MTT survival rates ranged from 70 to90 in the 10 to 80 120583gmL XF water extraction However thesurvival rate decreased to 630 plusmn 27 in the 100 120583gmL XFwater extractionWater extraction of Xanthii Fructus showedmoderate inhibition effects on proliferation of HK-2 cells inhigh dosage (Figure 3(a)) On the other hand we find that theHK-2 cell membrane remains undamaged even in the highest100 120583gmL XF water extraction exposure (Figure 3(b))

33 In Vivo Acute Toxicity of XF and PXF In the in vivoacute toxicity assay close observations of mice clinic signswere made before dosing and up to four hours immediatelyafter dosing All animals were observed daily for convulsionsexcitement posture piloerection breathing difficulty seda-tion anorexia diarrhea hemorrhage and death during the7-day assay period

Deaths were observed only in high dosage XF and PXFgroup Mortality rate in XF group (80) was significantlyhigher than that in PXF group (40) in both sexes No deathwas found in middle or low dosage group no death wasfound in middle or low dosage group Regular acute toxicitysymptoms and signs such as salivation slow action andconvulsions were observed in middle or high XF and PXFgroups (Table 4)

Postmortem examination did not reveal any gross abnor-mality of the brain and the organs of the chest and abdominalcavities in all treated groups Liver and kidney pathologicalexamination of dead mice showed the congestion and focal

necrosis in the liver and renal tubules (Figure 4) Pathologicaldamage in the heart and lung in high dosage XF and PXFgroups was reported for the first time

Blood samples of high dosage XF and PXF groups werehard to collect due to the close death time Therefore bloodcell analysis was conducted only in themiddle and lowdosageXF and PXF groups (Table 5) Most of the blood cell indexessuch as white blood cells (WBC) red blood cells (RBC)haemoglobin (HGB) and platelet (PLT) in low dosageXF and PXF groups remained at normal level comparedwith control group However the PLT content decreasedsignificantly in high dosage PXF group This indicated thatthrombocytopenia might be an obvious adverse effect of XF

Judging from the mortality rate toxicity symptoms andsigns tissue morphology and blood cell analysis resultsneither XF nor PXF showed acute toxicity in the low dosagegroup That indicated that the single dose administration ofXF or PXF was safe even at 10 times of the allowed humanhighest daily dosage However higher dosages of XF and PXFboth displayed obvious acute toxicity 40 of themice in highdosage PXF group and 80 of the mice in high dosage XFgroup were dead with severe kidney liver heart and lungmorphology changes within the 7-day observation periodFortunately PXF showedmoremoderate acute toxicity effectsthan XF in the same dosage level

34 In Vivo Chronic Toxicity of XF and PXF 12-week admin-istrations of water extraction of XF and PXF in low middleand high dosage were conducted in order to investigate theirchronic toxicity Taking into account the clinic dosage thelowmiddle and high dosages of this chronic toxicity researchwere set as 0585 1755 and 5265 gkg body weight

Nineteen rats were dead during the research procedure(Table 6) Nine of them were dead immediately after ourexperimental operations such as excessive anesthesia andblood sample collection procedure These deaths might be


(a) Normal group (b) Intracellular edema hemorrhage andloosening of cytoplasm were observed indead mice kidney tissue of high dosage XFgroup

(c) Little hemorrhage was observed in deadmice kidney tissue of high dosage PXF group

(d) Normal group (e) Light intracellular edema and hemor-rhage were observed in dead mice liver tissueof high dosage XF group

(f) Light intracellular edema was observedin dead mice liver tissue of high dosage PXFgroup

(g) Normal group (h) Myocardial cell cytoplasm osteoporosisintracellular edema hemorrhage and fattydegeneration were observed in dead miceheart tissue of high dosage XF group

(i) Myocardial cell cytoplasm osteoporosislittle intracellular edema and hemorrhagewere observed in dead mice heart tissue ofhigh dosage PXF group

(j) Normal group (k) Light intracellular edema thickeningalveolar wall and severe bronchial hemor-rhage were observed in dead mice lung tissueof high dosage XF group

(l) Light intracellular edema and littlebronchial hemorrhage were observed indead mice lung tissue of high dosage PXFgroup

Figure 4 Comparison of microscopic morphology in kidney (a b and c) liver (d e and f) heart (g h and i) and lung (j k and l) tissuebetween control group (a and d) high dosage XF group (b and e) and high dosage PXF group (c and f) Original magnification times400


Table 4 Mice grouping treatments and responses in acute toxicity testing

Groups Treatment Dosage (gkg) Male FemaleMortality Symptoms and signs Mortality Symptoms and signs

A Physiological saline 0 0 None 0 NoneB Water extraction of XF 1668 0 None 0 None

C Water extraction of XF 1668 0 Slow respiratory frequency(20) slow action (20) 0

Salivation (20) piloerection(20) fast respiratory frequency

(20) slow action (20)

D Water extraction of XF 400 80Vomiting (20) writhing

reaction (20) fast respiratoryfrequency (20) convulsions

(20)

80 Pupil dilation (20) piloerection(20) convulsions (20)

E Water extraction of PXF 1668 0 None 0 None

F Water extraction of PXF 1668 0 Dark hair (20) salivation (20) 0 Convulsions (20) salivation(20)

G Water extraction of PXF 400 40 Convulsions (20) salivation(20) 40 Convulsions (20)

Table 5 Blood cell analysis of middle and low dosage XF and PXF groups in acute toxicity assay

Groups Treatment WBC (109L) RBC (1012L) HGB (gL) PLT (109L)A Physiological saline 213 plusmn 205 568 plusmn 107 126 plusmn 254 423 plusmn 152

C Water extraction of XF (L) 236 plusmn 093 605 plusmn 0448 162 plusmn 880 343 plusmn 115

D Water extraction of XF (M) 318 plusmn 155 642 plusmn 0697 142 plusmn 223 335 plusmn 128

F Water extraction of PXF (L) 339 plusmn 132 658 plusmn 0649

150 plusmn 208 347 plusmn 130

G Water extraction of PXF (M) 182 plusmn 112

517 plusmn 104

113 plusmn 281

215 plusmn 725lowastlowast

lowastA significant difference compared with control group lowastlowast119875 lt 001A significant difference compared with XF groups in the same dosage 119875 lt 005

Group I Group II

Group VII Group III

(a) Microscopic morphology in kidney Group I only mild edema wasobserved Group II mild-to-moderate tubular edema renal interstitialinflammation tubular exudation and tubular necrosis were observedGroup III mild-to-moderate tubular edema renal interstitial inflamma-tion tubular exudation and tubular necrosis were observed Group VIImild-to-moderate tubular edema renal interstitial inflammation tubularexudation and tubular necrosis were observed

Group I Group II

Group VII Group III

(b) Microscopic morphology in liver Group I mild hepatocytes edemadegeneration was observed Group II mild-to-moderate edema degen-eration mild-to-moderate spotty necrosis focal necrosis and milddegree inflammation were observed Group III Mild-to-moderate edemadegeneration mild-to-moderate spotty necrosis focal necrosis andmild-to-moderate degree inflammation were observed Group VII mild-to-severe edema degeneration mild-to-moderate spotty necrosis and milddegree inflammation were observed

Figure 5 Comparison of microscopic morphology in kidney (a) and liver (b) tissue between Group I (control group) Group II (low dosageof XF) Group III (middle dosage of XF) and Group VII (high dosage of PXF) Original magnification times200


Table 6 Rats mortality in chronic toxicity assays

Groups Treatment MortalityFemale Male

I Physiological saline None None

II Water extraction of XF None 1998771 One dead at the 12W + 3D with 266 g BW(BW reduced gradually)

III Water extraction of XF1 One dead immediately after the blood sample

collection procedure 2W + 1D2 Another one dead due to excessive anesthesia

at the 8W + 1D

1998771 One dead at the 11W + 3D with 340 g BW(BW reduced gradually)


3 Another one dead immediately after the bloodsample collection procedure at the 3W + 1D

IV Water extraction of XF 1 One dead at the 10W + 7D with 170 g BW(BW reduced gradually) None

V Water extraction of PXF1998771 One dead at the 10W + 2D with 248 g BW

(normal BW)2 Another one dead immediately after the blood

sample collection procedure at the 3W + 1D

1 One dead immediately after the blood samplecollection procedure at the 10W + 1D

2ndash4998771 The other three rats were dead at the 7W +6D (283 g BW) 9W + 4D (281 g BW) and 10W+ 4D (242 g BW) respectively Their BWs were

reduced gradually

VI Water extraction of PXF1998771 One dead at the 12W + 1D with 271 g BW




VII Water extraction of PXF

1998771 One dead at the 2W + 6D with 152 g BW (BWreduced dramatically)

2 Another one dead due to excessive anesthesiaat the 8W + 1D


Total 9 11BW body weight2W + 1D the first day of the second week and so forthDeaths of these rats might be most probably related to the experimental operations998771Deaths of these rats might be most probably related to the administrations of XF and PXF

8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

Group I Group II Group III Group IV

Group V Group VI Group VII

lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)

Figure 6 Female and male ratsrsquo body weights at the end of the 8th to 12th weeks The lowast indicates a significant difference compared withcontrol group lowast119875 lt 005


9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks

Group IGroup IVGroup VII

lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)

Figure 7 Female and male ratsrsquo BUN values at the end of the 9th to 12th weeks The lowast indicates a significant difference compared withcontrol group lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 The indicates a significant difference compared with XF groups in the same dosage119875 lt 005 119875 lt 001 and

119875 lt 0001 Only BUN values in high dosage XF and PXF groups were shown in this figure

Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast

Figure 8 Female andmale ratsrsquo Scr values at the 8th and 11th weeksThelowast indicates a significant difference comparedwith control grouplowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 Only Scr values inpartly treatment groups with significant alterations were shown inthis figure

most probably related to the experimental operations Theother 10 rats were dead probably due to the administrationsof XF and PXF Most of the 10 rats were dead during the 10thto 12th weeks Four of them were in XF treatment groups and6 of themwere in PXF groups Four of the 10 rats were femalewhile 6 of the 10 rats were male A total of 1 2 and 1 rats weredead in the lowmiddle and high dosage groups respectivelyA total of 4 1 and 1 rats were dead in the low middle and

high dosage groups respectively No obvious dose-toxicityrelationship was observed

Postmortem examination did not reveal any gross abnor-mality of the brain and the organs of the chest and abdominalcavities Histopathological examinations of kidney liverheart and lung samples were all carried out However noobvious pathological changes were found in the heart andlung samples Liver and kidney pathological examinationof dead mice showed congestion and focal necrosis in theliver and renal tubules (Table 7 and Figure 5) without a dose-dependent relationship

No significant body weight changes were observed inboth male and female rats in the first 8 weeks of this research(data not shown) Obvious body weight changes were foundafter the end of the 8th week (Figure 6) Body weights (BWs)of female rats in all treated groups were reduced althoughonly female rats in Group III (9th 10th and 11th weeks) andGroup VII (11th week) were significantly lighter than thosein Group I (Control I) in statistical level Body weight losseswere more conspicuous in male rats groups The mean bodyweight of Group III was about 80 of the control group

Blood cell analysis was conducted in all treatment groupsfrom the 3rd to the 12th weeks (data not shown) howeverno significant alterations were observed in all groups com-pared to the control group This indicated that the 3-monthadministration would not affect the composition and contentof basic blood cell such as WBC RBC HGB and PLT

Scr and BUN were analyzed from the 3rd to the12th weeks Similar change trends were observed in Scrand BUN results (in Figures 7 and 8 and SupplementaryTables 1ndash4 in Supplementary Material available online athttpdxdoiorg1011552013403491) Increasing BUN com-pared to Group I (control group) was observed only in female


Table 7 Kidney and liver pathological damage occurrence rates ()in chronic toxicity assays

Groups I II III IV V VI VIIKidney

Tubular edema 0 25 50 0 33 0 50Renal interstitialInflammatory infiltration 0 50 75 0 33 0 25

Renal tubular exudation 0 50 75 50 33 0 25Tubular necrosis 0 25 50 0 33 0 25Fibrosis 0 0 0 0 0 0 0

LiverLiver cell edema 0 25 0 25 50 75 25Spotty necrosis 0 75 100 0 50 0 25Focal necrosis 0 25 50 0 25 0 0Inflammation 0 0 50 0 25 0 0Fibrosis 0 0 0 0 0 0 0

Table 8 Serum HA value at the end of the 4th 8th and 12th weeksin chronic toxicity assays (ngmL)

Weeks Female Male4th 8th 12th 4th 8th 12th

I 802 537 553 940 567 573II 607 619 608 615 572 601III 106 568 584 648 602 616IV 530 623 520 559 615 532V 541 579 622 227 509 NoneVI 208 535 613 569 873 533VII 762 590 585 612 528 531None of the results were obtained in Group V male rats due to the fact thatall rats were dead at the end of the 10th week

Table 9 Serum LN value at the end of the 4th 8th and 12th weeksin chronic toxicity assays (ngmL)



rats in Group IV (high dosage XF) and Group VII (highdosage PXF) from the 10th to the 12th weeks (Figure 7)Sporadic and transient Scr increases appeared only in the8th (Group VI) and the 11th (Groups II and VII) weeks forfemale rats and the 8th (Groups III and VI) weeks for malerats (Figure 8) BUN values were even decreased in male ratsin Group VII

Table 10 Serum HPCIII value at the end of the 4th 8th and 12thweeks in chronic toxicity assays (ngmL)


I 339 0974 316 914 227 115II 138 148 371 248 0528 NDIII 132 0758 144 805 245 396IV 0986 310 110 116 108 846V 705 171 396 943 557 NoneVI 663 838 174 746 460 721VII 391 133 0757 157 650 868None of the results were obtained in Group V male rats due to the fact thatall rats were dead at the end of the 10th weekND not detected

In conclusion low and middle dosage of XF and PXFwould not induce serum BUN alteration even in a three-month chronic researchHigh dosage PXF even decreased theBUN compared to high dosage XF in male rats in the 9th10th and 11th weeks Scr alterations were not sustained butirreversible in both female andmale rats in all dosage XF andPXF groups

As early indicators of renal fibrosis serum HA LN andHPCIII content valueswere also tested in our research (Tables8 9 and 10) Some transient increasing of them were foundAlthough no HA LN and HPCIII increase was found atthe end of the research we still noticed that serum HAand HPCIII values were sustained increasing from the 4thweek to the 8th week in Group V male rats Whether thisincrease is related to the death of these rats still need to beinvestigated That indicated that the renal fibrosis risks stillexisted although no fibrosis was found in the pathologicalexamination of liver and kidney

4 Discussion

Adverse effects of Xanthii Fructus were frequently reportedin recent years although the toxicity of Xanthii Fructus isclearly alerted in the Pharmacopoeia of China (2010 edition)Moreover its basic toxicity data and related mechanism werenot well investigated In this manuscript we systemicallyevaluated the in vitro and in vivo toxicity of Xanthii Fructusits processed product and its major chemical constituent

In the HK-2 renal cells water extraction of XF dis-played no cell membrane damage effects even in the highest100 120583gmL concentration however it might affect the func-tion of renal cell mitochondria

Atractyloside was frequently reported to be a toxic sub-stance of Xanthii Fructus The toxic mechanisms of atracty-loside were also well explained Atractyloside competitivelyinhibits the adenine nucleoside carrier in isolated mito-chondria and thus blocks oxidative phosphorylation [10]Atractyloside inhibits gluconeogenesis and fatty acid oxida-tion but accelerates anaerobic glycolysis and glycogenolysisThe metabolic alterations in humans and animals includean acute hyperglycaemic phase due to rapid depletion ofskeletal muscle and hepatic glycogen inhibition of glycogen


synthesis and a subsequent marked hypoglycaemic phaseThe rapidly declining plasma glucose resulted in insufficientglucose delivery to the brain to sustain normal functions Asthe severe hypoglycaemia persisted depression of respirationhypoxaemia tissue hypoxia (diminished oxygen consump-tion) acidosis convulsion coma and in some cases deathfollowed However in this experiment atractyloside didnot show such obviously toxic effects as other publishedresearches We consulted this to the differences between invitro and in vivo research results Limited metabolic enzymeand limited exposure time might restrict the exposure ofatractyloside toxicity Moreover toxic effects induced byatractyloside might be mediated by its in vivo metabolitewhich could not happen in our in vitro experiment system

In vivo toxicity assays provided more information andbasic data for the clinical usage of Xanthii Fructus BothXF and PXF displayed severe acute toxicity in 240 timesof human highest dosage however this dosage was tooextreme in the clinical usage Fortunately the single doseadministration of XF and PXF was safe even at 10 times ofallowed human highest daily dosage No obvious behavioralchanges blood cell analysis results changes and generalorgan lesions were observed in this dosage

Chronic toxicity assays focused on the more commondosage with high probability of occurrence in clinical prac-tice In order to evaluate their toxicity to kidney BUN andScr were tested as the function index of kidneyThe BUN testalong with the creatinine test is primarily used to evaluatekidney function in a wide range of circumstances to helpdiagnose kidney disease and to monitor people with acuteor chronic kidney dysfunction or failure Increased BUNlevels suggest impaired kidney function This may be due toacute or chronic kidney disease damage or failure It mayalso be due to a condition that results in decreased bloodflow to the kidneys such as congestive heart failure shockstress recent heart attack or severe burns due to conditionsthat cause obstruction of urine flow or due to dehydrationMoreover HA LN and PCIII the major ingredients ofextracellular matrix (ECM) were also evaluated Synthesisand accumulation of large quantities ECM were foundationsof kidney fibrosis

Twelve-week administrations of low middle and highdosage of XF and PXF were performed in SD rats Notablechanges in body weight and blood cell and BUN and Scrchanges occurred sporadically in middle and high dosagegroups after the 9th week Although some liver and kidneypathological damage was found in the high dosage groupsno early stage changes of renal fibrosis were found

All the above results indicated that the single use of even10 times the permitted highest dosage of XF and PXF is safeHowever long-term usage of overdose XF and PXF was notentirely safe Adverse effects or even death casesoccurred aftercontinuous administration of 3 times or 9 times the permitteddosage of XF and PXF for 2 months

Abbreviations

AL Atractyloside potassium saltBUN Blood urea nitrogen

BW Body weightDMSO DimethylsulfoxideECM Extracellular matrixFBS Fetal bovine serumLDH Lactate dehydrogenaseHA Hyaluronic acidHGB HaemoglobinHPCIII Human procollagen type IIILD50 Median lethal dose

LN LamininMTT 3-(45)-Dimethylthiahiazo(-z-y1)-35-

di-phenytetrazoliumromideNRU Neutral red uptake assayPLT PlateletPXF Processed Xanthii FructusRBC Red blood cellsRPMI 1640 Roswell Park Memorial Institute

medium 1640Scr Serum creatinineWBC White blood cellsXF Xanthii Fructus

Conflict of Interests

The authors declare that there is no conflict of interests Theydeclare that they have no financial and personal relationshipswith other people or organizations that can inappropriatelyinfluence their work there is no professional or other per-sonal interest of any nature or kind in any product serviceandor company that could be construed as influencing theposition presented in this paper

Authorsrsquo Contribution

Jie Yu and Mei Zhen Song contribute equally to the work

References

[1] M E De Broe ldquoChinese herbs nephropathy and balkan ende-mic nephropathy toward a single entity aristolochic acidnephropathyrdquo Kidney International vol 81 no 6 pp 513ndash5152012

[2] J S Li ldquo6 poisoning cases induced by xanthii fructusrdquo PeoplersquosMilitary Surgeon vol 10 pp 31ndash32 1981

[3] M Turgut C C Alhan M Gurgoze et al ldquoCarboxyatractylo-side poisoning in humansrdquo Annals of Tropical Paediatrics vol25 no 2 pp 125ndash134 2005

[4] W Q Su ldquoSiberian cocklour fruit-induced adverse drug reac-tions literature analysis of 95 Casesrdquo Pharmacy Today vol 18no 5 pp 44ndash46 2008

[5] E S Gurley M Rahman M J Hossain et al ldquoFatal outbreakfrom consuming xanthium strumarium seedlings during timeof food scarcity in northeastern Bangladeshrdquo PloS one vol 5no 3 article e9756 2010

[6] J Yu J Wang R Zhao Z Bao and S Cai ldquoAdvances in studiesof renal adverse reactions of leonurus heterophyllusrdquo ChinaJournal of Chinese Materia Medica vol 35 no 9 pp 1213ndash12162010


[7] J Wang C L Mo L Que G Zhaori and J Yu ldquoAdvances instudies on adverse effects of xanthii fructusrdquoChinese Traditionaland Herbal Drugs vol 42 no 3 pp 613ndash616 2011

[8] J Yu J Xie X J Mao et al ldquoHepatoxicity of major constituentsand extractions of radix polygoni multiflori and radix polygonimultiflori praeparatardquo Journal of Ethnopharmacology vol 137no 3 pp 1291ndash1299 2011

[9] Chinese Pharmacopeia Commission Pharmacopoeia of thePeoplersquos Republic of China vol 1 ChinaMedico-PharmaceuticalScience amp Technology Beijing China 2010

[10] RDuo Y Chen YH Liu et al ldquoInfluence of processing on con-tents of carboxyatractyloside and atractyloside in Xanthii Fruc-tusrdquo Chinese Traditional Patent Medicine vol 35 no 2 pp 353ndash356 2013

[11] D K Obatomi N T K Thanh S Brant and P H Bach ldquoThetoxic mechanism and metabolic effects of atractyloside inprecision-cut pig kidney and liver slicesrdquoArchives of Toxicologyvol 72 no 8 pp 524ndash530 1998

[12] M J Ryan G Johnson J Kirk S M Fuerstenberg R A Zagerand B Torok-Storb ldquoHK-2 an immortalized proximal tubuleepithelial cell line from normal adult human kidneyrdquo KidneyInternational vol 45 no 1 pp 48ndash57 1994

[13] S Koul S Fu and H Koul ldquoOxalate exposure promotes rein-itiation of the DNA synthesis and apoptosis of HK-2 cells a lineof human renal epithelial cellsrdquoAnnals of theNewYorkAcademyof Sciences vol 1010 pp 292ndash295 2003

[14] AOrtiz P Justo A Sanz et al ldquoTubular cell apoptosis and cido-fovir-induced acute renal failurerdquo Antiviral Therapy vol 10 no1 pp 185ndash190 2005

[15] R A Zager ldquoPolyene antibiotics relative degrees of in vitrocytotoxicity and potential effects on tubule phospholipid andceramide contentrdquoAmerican Journal of Kidney Diseases vol 36no 2 pp 238ndash249 2000

[16] E M Scheers B Ekwall and P J Dierickx ldquoIn vitro long-termcytotoxicity testing of 27 MEIC chemicals on Hep G2 cellsand comparison with acute human toxicity datardquo Toxicology inVitro vol 15 no 2 pp 153ndash161 2001

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

ToxicologyJournal of


StrokeResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


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AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


The toxicity of Xanthii Fructus is usually considered tobe associated with two diterpenoids glycosides atractyloside(AL) and carboxyatractyloside (CAL) The biochemistry andtoxicology of them are well investigated and elucidated bothin in vitro and in vivo [10 11] methods The LD

50of AL is

15mgkg in dog (iv) 434mgkg in mouse (ip) and 143mgkgin rat (ip) According to the latest literature [10] contentof CAL decreased dramatically after processing procedurewhile AL content increased after processing procedure CALwas reported to be about 116mgndash225mgg in XF and 0ndash021mgg in PXF On the other hand AL could not bedetected in XF however the content of AL increased to about017ndash256mgg in PXF However the toxicity mechanismsinvolved are not well investigated

Despite the prevalent reports of their toxicity the toxicityresearch data about XF and PXF still need to be clarifiedespecially their toxicity in normal or middle dosage admin-istration case In this paper we systemically evaluated the invitro and in vivo toxicity of Xanthii Fructus its processedproduct and its major chemical constituent

In vitro renal cytotoxicities were investigated by humankidney HK-2 cell line an immortalized proximal tubularepithelial cell line from normal adult human male kid-ney retaining the phenotype and functional characteristicsindicative of well-differentiated proximal tubular epithelialcell [12] Not only the water extracts of Xanthii Fructusbut also its major chemical constituents atractyloside potas-sium salt (AL) were involved Culture of this cell line haspreviously been employed by several studies to investigatenephrotoxicity of various toxic compounds [13ndash15]

Acute and chronic toxicities were tested in Kunmingmice and SD rats respectively Single dosage of XF or PXFwas given to Kunming mice in order to investigate theiracute toxicity Twelve-week chronic toxicity assay was alsoconducted in rats Systemic toxicity indexes were involved inboth acute and chronic toxicity assays in order to elucidatetheir toxicity comprehensively Some kidney toxicity indexesand early indicators of renal fibrosis were involved in chronictoxicity assays

2 Materials and Methods

21 Chemicals 3-(45)-Dimethylthiahiazo(-z-y1)-35-di-phe-nytetrazoliumromide (MTT) and trypsin (1 250)were acqui-red from Amresco USA Dimethylsulfoxide (DMSO) waspurchased from Sigma USA Other reagents were all aboveanalytical grade Lactate dehydrogenase (LDH) assay kitswere purchased from Changchun Huili Biotech Co LtdChina BUN and Scr assay kits were purchased fromBiosino Bio-technology amp Science Inc China Enzyme-linked immunosorbent assay kits of HA LN and HPC IIIthree early indicators of renal fibrosis were obtained fromCusabio Biotech Co Ltd Wuhan China

22 Processing Procedure of Xanthii Fructus Xanthii Fructuswas purchased from Juhuacun Chinese herbal medicinemarket Kunming Yunnan China in February 2009 (shownin Figure 1) and was identified as fruit of Xanthium sibiricum

Patar by the author Associate Professor Jie Yu Voucher spec-imens were deposited in the Herbarium of PharmacognosyYunnan University of Traditional Chinese Medicine

Processed Xanthii Fructus was stir-fried from XanthiiFructus with mild fire to yellowish brown (Figure 1) Process-ing of Xanthii Fructus was carried out by the authors accord-ing to the procedures documented in the Pharmacopoeia ofChina 2010 edition [9]

23 In Vitro Renal Cytotoxicity Assay

231 Preparation of Exposure Solution of Atractyloside Potas-sium Salt for In Vitro Assay Atractyloside potassium salt(AL) major constituent of XF and PXF was purchased fromSigma USA (structure shown in Figure 2) with purity higherthan 98 The exposure solutions of AL in concentrationfrom 25 to 320 120583M were diluted by RPMI 1640 containing02 fetal bovine serum (FBS)

232 Preparation of Extraction of Xanthii Fructus for In VitroAssay 40 g powder of XF was decocted with water (1000mL800mL and 600mL) for 3 times respectivelyThe extractionswere combined condensed and lyophilized Extraction yieldwas 2315

XF stock solution was achieved by dissolving them inDMSO at a concentration of 10mgmL Then dissolving inDMSO it was further diluted with 02 FBS-RPMI 1640 tograded concentrations from 10 to 100 120583gmL

233 Culture Conditions The whole research was carriedout by HK-2 cells immortalized proximal tubular epithelialcell line which were purchased from Cell Bank KunmingInstitute of Zoology Chinese Academic of Science Cells werecultured in monolayer culture in DMEM medium (GibcoInvitrogenCorp) supplementedwith 10 fetal bovine serum(HyClone)

Cells were seeded at an appropriate cell density in ahumidified incubator (Series II water jacketed CO

2Incubator

Model 3111 Thermo Electron Corporation USA) with 5carbon dioxide and dioxide 95 air at 37∘C for different assayand left to grow to 80-90 confluence

234 MTT Cell Viability Assay This assay procedure wasadopted from the previous report [8] HK-2 cells were seededin 96-well plates (Corning Incorporated) at a cell density of2 times 104 in 200120583Lwell and then incubated for 48 h till 80ndash90 confluenceThen the cell synchronization was routinelyaccomplished by incubating cells in G

0medium for 12 h

before the test Subsequently cells were exposed to fresh G0

medium with various concentrations of test compounds orextractions for 24 h

At the end of the incubation period 10 120583Lwell of5mgmL MTT (Amresco USA) which was dissolved inPBS (pH 74) and sterilized by filtrating through a Milliporefilter (022 120583m) was added in medium for a further 4 hincubation then the medium was removed and 100 120583LDMSO was added to dissolve the resulting intracellularpurple formazan product The 96-well plates were gently




CH3

OH

O

HOOC

H3C

OHOH2C

OO

OC

OCH2

CH2

CHH3C

minusO3S

minusO3S































5 884 plusmn 132 899 plusmn 0311

10 934 plusmn 868 822 plusmn 0571

20 892 plusmn 442 896 plusmn 0198

40 922 plusmn 282 928 plusmn 0421

80 995 plusmn 1533 103 plusmn 125

160 941 plusmn 103 873 plusmn 0132

240 982 plusmn 656 962 plusmn 0744

320 924 plusmn 312 117 plusmn 0981







3 Results




Control 10 20 40 60 80 100

0

20

40

60

80

100

120M

TT su

rviv

al ra

te (

)


lowastlowastlowast

(a)

Control 10 20 40 60 80 1000

20

40

60

80

100

120

LDH

leak

age r

ate (

)


lowastlowastlowast

(b)
































(20)












517 plusmn 104

113 plusmn 281



Group I Group II

Group VII Group III


Group I Group II

Group VII Group III










at the 8W + 1D










reduced gradually










8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks



lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)



9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




CH3

OH

O

HOOC

H3C

OHOH2C

OO

OC

OCH2

CH2

CHH3C

minusO3S

minusO3S































5 884 plusmn 132 899 plusmn 0311

10 934 plusmn 868 822 plusmn 0571

20 892 plusmn 442 896 plusmn 0198

40 922 plusmn 282 928 plusmn 0421

80 995 plusmn 1533 103 plusmn 125

160 941 plusmn 103 873 plusmn 0132

240 982 plusmn 656 962 plusmn 0744

320 924 plusmn 312 117 plusmn 0981







3 Results




Control 10 20 40 60 80 100

0

20

40

60

80

100

120M

TT su

rviv

al ra

te (

)


lowastlowastlowast

(a)

Control 10 20 40 60 80 1000

20

40

60

80

100

120

LDH

leak

age r

ate (

)


lowastlowastlowast

(b)
































(20)












517 plusmn 104

113 plusmn 281



Group I Group II

Group VII Group III


Group I Group II

Group VII Group III










at the 8W + 1D










reduced gradually










8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks



lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)



9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













5 884 plusmn 132 899 plusmn 0311

10 934 plusmn 868 822 plusmn 0571

20 892 plusmn 442 896 plusmn 0198

40 922 plusmn 282 928 plusmn 0421

80 995 plusmn 1533 103 plusmn 125

160 941 plusmn 103 873 plusmn 0132

240 982 plusmn 656 962 plusmn 0744

320 924 plusmn 312 117 plusmn 0981







3 Results




Control 10 20 40 60 80 100

0

20

40

60

80

100

120M

TT su

rviv

al ra

te (

)


lowastlowastlowast

(a)

Control 10 20 40 60 80 1000

20

40

60

80

100

120

LDH

leak

age r

ate (

)


lowastlowastlowast

(b)
































(20)












517 plusmn 104

113 plusmn 281



Group I Group II

Group VII Group III


Group I Group II

Group VII Group III










at the 8W + 1D










reduced gradually










8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks



lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)



9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control 10 20 40 60 80 100

0

20

40

60

80

100

120M

TT su

rviv

al ra

te (

)


lowastlowastlowast

(a)

Control 10 20 40 60 80 1000

20

40

60

80

100

120

LDH

leak

age r

ate (

)


lowastlowastlowast

(b)
































(20)












517 plusmn 104

113 plusmn 281



Group I Group II

Group VII Group III


Group I Group II

Group VII Group III










at the 8W + 1D










reduced gradually










8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks



lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)



9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




















(20)












517 plusmn 104

113 plusmn 281



Group I Group II

Group VII Group III


Group I Group II

Group VII Group III










at the 8W + 1D










reduced gradually










8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks



lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)



9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








at the 8W + 1D










reduced gradually










8 9 10 11 12200

250

300Female

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks



lowastlowast

lowastlowast

(a)



8 9 10 11 12

300

350

400

450 Male

Body

wei

ght a

t the

end

of th

e wee

k (g

)

Weeks

lowastlowast

(b)



9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


9 10 11 123456789

101112131415161718

FemaleBU

N v

alue

at th

e end

of t

he w

eek

(mm

olL

)

Weeks


lowast

lowastlowast

lowastlowast

(a)


9 10 11 123456789

101112131415161718

Male

BUN

val

ue at

the e

nd o

f the

wee

k (m

mol

L)

Weeks

(b)



Gro

up I

Gro

up V

I

Gro

up I

Gro

up II

Gro

up V

II

Gro

up I

Gro

up II

I

Gro

up V

I

0

10

20

30

40

50

60

70

80

90

Male8th week

Female11th week

Female8th week

Scr v

alue

in th

e end

of t

he w

eek

(um

olL

)

Treatments

lowastlowast

lowast lowastlowast

lowastlowast


























4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



















4 Discussion










Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of







Abbreviations










References






















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of
















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

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