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Research Article Toxicologic Assessment of a Commercial Decolorized Whole Leaf Aloe Vera Juice Lily of the Desert Filtered Whole Leaf Juice with Aloesorb

lnder Sehgal 12 Wallace D Winters3 Michael Scott3 Andrew David4 Glenn Gillis

Thaya Stoufflet~ Anand Nair2 and Konstantine Kousoulas4

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Aloe vcri a common mgrcd1cn1 m osmcucs 1s mcreasmgl) being onsumcd as a bcCragc upplemcn1 Ahhough conltumcr mlcrcgtl 111 aloe ltkcly ltbullems from 11s altsocial1on w11h several hcahh bcMfm bullconcern has aho been rbullcd by a Nal10nal Toxicology Program Report tlMl a nnndccolcmtcd wlwlc leaf aloe vera cx1rac11akcn Internally by rallt was agtSOtatcd w11h in1cs1111al muosal hyperplasia and uhlmgt1cly mnllgnancy We lctcd a Jccolonzcd whokbull lcaf (l)CWl) tloc vcri lrcucJ 1vilh activated charcoal to remove the latex portion of lhc plant for gcnolox1cuy 1n bacteria ~bullutcubaute toxiot) in BtgtC 311 mice and subchro1Hl 1uxi11y in ~344 rat We found 1lult newt tine wrl JUbullw to~ nongcnotom in h1~lld1nc rcvcrnnn and DNA rrpair alt3ys Fnllmng acute admtnilttrallon n11cc exhibited no advcrlte ltignlt at 3- or 14-da) cvaluJllon periods When fcJ tn male and icmalc F344 rat~ owr 13 weelu DCWl alM I~ tn no 1nx1cuy alt bull-cgt-ed b) hcha~or middotweight gam ftcd conlt11mp11nn1gtrs1n bullighband hema1olog1 or d1n1col thcmtlttry prnlilcs lhcgtlt rdl hJJ cn1lt1mal mucobullal morpholog1c-cxantlncd gru-ly and m1Lrosop1ally-1ha1 were s1nular lo 1ontroh Our stud1elt lthow 1hll oral adm1ni1ra11on of1h1 DC Wl aloe 1uicc halt a d1ffcrc111 loxology pmfilc than 1ha1 of the unlrcalccl aloe JUlt at opcgt-Urltgt UJl IU I weeks

1 Introduction

Aloe vera 1s a common ingredient in cosmclcs skin are products and increasing) beverages and tood products (l) Recent consumer m1eres1 in aloe beverages may stem from 1he association of aloe iuke with a variety of both anec dotal and experimental rcsearch-supporteJ health benefits including the prevention or lrcatmcnl of various tumors [2 ~)arthritis [bullI] diabctcgt [5] enhanced immumty [6) and decreased chokstcrol lccls [7j

Aloe juice is approximate) 99deg0 waler [81 and the remainshyder consists of mmerals vitamins polysachandes lipids

phenolk ompounds and orga111c awls According to the lntcrnallonal Aloe Scientific Council the aloe leaf can he proccsltcd into rwo types of JUiCcgt for ~ommerdal use inner eat gel JUtce and decolonzed whole leaf iwce (9) lnner l~af gel 1u1ce is produced from only thltgt gelatinous fillet of the leaf Dccolorized whole leaf iuictbull b produced by grinding the leaf followed by tre3tmcnl of extracted juice with act1va1cd charcoal 10 remove aloe latex (10] Ch3rcoal treatment is ncce~sary ~ince the latex whkh exists as a separate liquid between the outer nnd and inner fillet gel contains bitter phenoli~ molecules including anthraquinone C and 0 glycos1des anthrone~ and ~ome free anthraquinoncs (II)

The maior C-glrcoside alo1n A is the ma1or anthraqumone 10 3loe l3tex and when oxidi1tgtd yieldlt aloe emodin a lree anthraquinone Ill) Anthraqumonfs are associated with diarrhea i11 vivo 112 13) weight logts gall bladder les1oos renal tubule pigmentation and rcn11 tubule hraline droplets [M] 111 vitro anthraquinones cause mutations in mouse lymphoma (15] and salmonella assars 116) Aloe beverages which follow IASC standards contain lcs than 10 ppm aloin A and tts isomer aloin B [9]

Toxicology ofvarious aloe produtlt halt been examined 111

vivo and these studies collectively suggest that the potential for toxicity depends on the plant semons used m 1u1ce productton Oral consumpllon ofan Clhanol extract ofwhole leaf aloe owr 3 monthgt by mice resulted 10 reproduwve toxicity and increased mortality 117) However subchromc admm1strat1on of aloe taken from the mner leaf gel resulted in no evidence of oral toxicity to rats (18) In 2010 the National Toxicology Program [10 J released a technical report on aloe vera that concluded that there was clear evidence of carcmogcmc activity of a nondccolonzcd whole leaf extract of Aloe vcra in male and female F344N rats based upon increased incidences of adenomas and carcinomas of the large 111test1ne The results of th1~ report have been referred tu on various health-related websites suggcstmg caution in consumpuon of aloe products middot19-22) and have generated substantial interest and concern b) aloe producers btcause the tudy results were based on a nondccolontcd whole leaf extract that 1s not commercially available (23) The -=TP study was designed to delinr nondecolonzcd whole leaf freezeshydncd JUICC powder to test ammal~ through their drinking water at levels up to 30 (11wt) and the ~tudy examined potential toxkologic effects ofaloe 111 both B6C3FI mice and f344 rats over time periods of 14 days 13 weeks and 2 years [lOf

While an increased inciltJence of tumorigenesis was observed 111 the large mtesttnc ol rats 111 the 2 year study no lesions were observed in mice or rats in acute studies hoeer there was decreased ater consumption m both rodmt species at the highest aloe levels During a subchronic pltnod (13 weeks) mice adrrumstered aloe ere similar to controls m most paramdergt ~th the cxccptton of mcreased incidences of mucosa) hyperplasia with goblet ~ell hypershyplasia in the cecum and large mtestme This mucosa) and goblet cell hyperplasia was also observed 111 rats at 13 weeks These rats displayed increased mortaliues and weight losses at the high aloe concentrauons Ratgt exposed for ttvo years ilso were found to have mucosa hyperplasia in th~ colon particularly in the cecum and ascending portions Large inte~tine mucosalgoblct cell hypcrpla1a~ were also observed 111 mke exposed for two rears however these 1mce did not ~howmcrcascd neoplasms and had 11nilar Unibullmiddotal and body weights as control groups

1 he nondecolorized ~hole leaf podcr u~ed b)middot the IP contamcd the b111er latex of the aloe plant and theretorc the addition of thi aloe directly to drmlong water could haw contributed to the studrs outcomes Palatability can tnlluence wa1er consumplion and conceivably could have also contributed to the weight loss and mortality in the two-year study Commercial aloe beverages that contam litt le or no

Journal ofTox1cology

latex anthraqumones mar have yielded different outcomes 111 the subhromlt or chrome exposure test~ 1l1c ~TP abo assessed the acute effects of aloe gel only and decolomed (aellvated charcoal treated) whollt leaf aloe and in these~tud 1cs mice and rat body weights water and feed consumptions organ weights hematology cl1111cal chemistry and urine chemistry were generally similar to control However only the nondccoloriLcd extract was wed in the 13-wcck and 2shyyear studies

Although thc reported incidence of large mte~tmal tumorgt hagt garnered the most attention from the -=TP report the mc1dences ot mtestinal mucosa( hyperplasia were the most consistent observations across species expogted both subchron1cill) ind chronically middot1higt mucoal hyperplasia suggcMs a tox1colog1cal response to one or more whole leaf aloe ltomponcnts In the rat hyperplasia of both the muco~a and goblet cells may indicate cellular damage and could be prognostic oflater tumor development since control rats showed liulc or no hyperplasia at 13 weeks or ademiddot nom1carcinomas of the large intestine al 2 years

To date no evaluation of the de-colorited whole leaf Aloe vera JU1Cc hagt been reported In the present stud) we report the first tox1colog1al evaluation of a commercially available deolomed whole leat (OCL) Aloe vera hevcrage Lil) of the Desert Filtered Whole Leaf Aloe vera Jwce This particushylar product an be considered as being well characterized with regard to high moleculaMbullttght polysacchande content (24 f and 1mmunomodulatmg acthmiddotity 125 26) Testtng mcluJcd assessment ofpotential genotoxictt) 111 v1trigtdnd iltutc tox1c1l) 111 vivo usmg a B6C3FI mouse model Further test mg included a l~middotwcek study utilizmg F344 rats 111 which the DCWl Juke was administered through the rats chow in order to impart a mmimal effect on palatability Examinations included large intesttnil histology to understand 1f aloe bewrages ltleriwd from DCWL aloe vera JUiee produced tox1colog1c signs hsociatcltl with nondecoloritcd whole leaf JUKC

2 Materials and Methods

2 I Prornremwt ofDCfl Aloe era Juice Aloe vcra leaves were harvctcd and mamtamed under cold storage (R C) until processing Time lapse from harvest to process111g wa~ less than six hours The harvested leaves were washed disinshyfected and macerated to yield an mtcrmcdialc raw material rcprcscnlattve of 3 nonltlecolorizeJ whole letf cxtrac as was used 111 the arorcmcn11oncd XTP study The nondecolorized extract was then filtered using carboodiatomaceou~ earth to yield a DCWL extract To this extract a proprietary isolate ofhigh-molecular weight aloe vera polysa((har1dc Alocsorb middotas added at a rate of30 mg solidfluid oune The resulting extractas flash pagtteurited to )leld the commercial product (Lily of the Desert Filtered Whole Leaf Aloe vera juice with Aloesorb) This produbullt was vacuum dned 10 yield a 7 fold concentrate

NMR analy~i~ of the resulting extract established (I) the preence of Aloe vera (b) confirming the presence of aloe vcra polysacchande malic acid and glucose) (2) the

3 Journal ofToXJcology

presence of whole leaf markers (1socuratc and 1~ocitrateshylactonc) and (3) the absence ofadultcrnnt~tah1h7ers in the ltxtmt HPLC analysis confirmed the prcscnctot low levels of anthraquinones (Aloin A al 0868 ppm Alom Bat 1335 ppm and Aloe-emodm at 0200 ppm) 111c~e rc)ult~ qualify the extract a~ a dcolorizcd whole leaf aloe vcra extract

The juice was main tained under constant refrigeration unlll use Pt ior 10 testing the aloe iuice was lyophillzcd and determined to be I065 011 nona4ueous It was also found to be acidic (pH =39) Prior to anal)s1s in genotoxi W) assays the aloe juice was filtered using pos111vc pressure to render 11 sterile and the pH adjusted to i5 to accommodate S11mtme11 bacterial assays that m pH-Mn~111ve In order to max1m1ze the level of aloe admini)kred lo IC)I mice the 7x iu1cc was further concentrated br lroph1hza11on 10 a final concentration of 35x in a Lahconco lyoph1hze1 over a 6-hour period This (Oncentrated JUice wa~ administered in gavage studies A single lot (1021412 4) was used throughout the studies

2 2 Ge1101oxici1y Assays Potential mutagcmioty andor DNA damage were assessed 111 vitro with two bacterial assays An assar for mu1agenes1s was used which is based on the Ames tet utilizing Salmond111ypl111mm11111gttram1AIOO but moJ1ficd for liquid cuJturtgt and a 96 II plate scale The second assay detected potential DA damage utilizmg an E co1 strain comaming a transgene tor beta-galac1os1dase downstream of the SOS-DNA repair promoter $)Stem Both assays were purchased in a commercial format from EBPI B10-Dctcc11on Products (M1ss1ssaugJ 011 Canada) referred to the u~ Muta Chromo Plate and SOSmiddotChromo Test Assays respectively

To test for potential metabolic generation of mutagens DCWI aloe vera juice was also tested 1n the presence of S9 liver extract For testing with Se1111011ce1 the JUJCC was combmed w11h a reaction mix containing growth substances and a pH indictor The 1u1ce was admmis1ered at 2b 1-lx and 7x concentrations As a positive control or d1rec1 actmg mutagenesis (ie independent of metabolic commiddotersion) either sodium az1de (038 microM final concentration) or 2 n11rotluorene (71 4M final concentration) wa~ mcludcd in one plate 2-Aminoamhracene (260 microM final concentration was used as a pos111ve control for mutagenesis requiring metabolism Plates containing no aloe JU1cc were used 10

measure spontaneous mutations over the incubation periods und these blanks were compared to di aloemiddot containing plates The number of positive mutated wells was scored on days 3 bullI and 5 of growth These number were then compared bulltaubullucally to blanks

For the D-A damage assay m E co1 the DCl Juice was tested at levels of 21x 14x 7x 3Sx I 7Sx 088x 044x 022x Ollx and 0055x When d1lut~d the iu1ce was suspcnded m sterile 10 dimethyl sulfoxidc (DMSO) in

Merile 085 saline A positive control for DgtA damage independent of metabolism was mcluded (4 mtroquinoline oxide [4NQO]) and used at lcmiddotcls of 100 bullgmL (526 fM) 5 0 25 125 0625 and 0312511gml J pobullit1vc control for mutagcnesis requiring metabolism was included m another

plate (2 aminoanthracene) and used at 1000 (2611~1) 500 25 0 12 5 625 and 312511gmL 0)A damage walt 4uantishytated ~pectrophotomctrically by color development Data arc expressed as the mean and standard dev1at1on of the SOS inducuon

23 A11ime1h All mice were purchased from Jackson Labo ratonebull [n( and were ordered between 4 and 6 weeks of age After a two-week period of quarantine the mice were numbered for 1dent1fica11on by ear tag Lpon miliation of studies mice were- weighed sorted m10 boxes of S mice each and then randomly as1gned to control or alltgtlt 1uice groups Mice were housed within the Lou1stana State lniwr1t) Dtv1shysion of Laboratory vicdicmes AALAC-approved vivarium in Super Moubulle licro holation 750 boxes that arc racked 111 an E1w1ro Gard R m1crmsolat1on control cage rack (Lab Prodshyucts Seaford DE) Cage changes with fresh l111er and fresh water were provided weekly 10 the mice Mouse boxes were stocked with clcanaurnclaved Bio-Serv (Frenchtown N J) mou)e Igloo and fat-Trac spinning canchmcnt devices and testlctgt pads (Ancare Bellmore NY) All anim11 proctdure~ followed the Nallonal Research Councils GUIDE FOR THE CARE AD USE OF LABORATORY ANIMALS and were first reviewed and approved by the LSU IACUC

F344 rat wmiddotrc purhased from Charles River Laboratoshyricbull and arrived at 7 weeks of age After a two-week period of quarantmc rats were 1dcnttficd by permanent marker on the tail The rat~ were md1v1dually housed Cage boxes were changed ith fresh litter and fresh water weekl) and boxes were stocked lth dean3u1oclaved Crawl Ralls enrichment devices (Bio-Scrv Frenchtown Nj

2A oncrty 111 3- 1111d 14-Day Time Periods m Mrce I)(WL aloe 1uice was adm1111stcred by gavage with a 20 gauge curved s1ainles~ ieel gavage needle (Popper amp Sons Ne H)middotde rark Y) to male and female B6C3Fl mice twice Omiddoter a 24 hour period at lOGo ofhodr weight Seven rruce per test group middotere gavaged either (Oncentrated JWCe or 3ter then observed daily for abnormal behaviors including h~Poactivtty isolation from littcrmatcs ataua dyspnea or prostration In addition mice ere monitored for a weight lo~s of 10deg0 or greater decreased teed comumpuon of 10 or greater and death

Through the oral gavagc female mice consumed an estimated amount ot aloe vcra juice equal to 321 times the amount 1 person would normally drink per 24-hour period Male micr consumed an average of 2i8 times this amount middot1 hcsc csumatcs were determined b) J))llming the recommended daily amount of aloe juice a person drinks to be 80 ouncs (2366 mL) To scale this quantity to a mouse we used body surface area rauo [27] comparing mice groups with a 5 leet 10 inches person weigh mg 70 kg

At the conclusion of the 3- and 14-da) periods gross necrops1es were performed on all animals and final body we1ghh plus weights of the kidney (nght) heart lung liv1r and testes (mules) wen~ recorded Hematologic parameters were assessed on blood samples collec1ed in potassium EDTA container This analysis included RBC Hb HCT RDW MV MCI I MCllC platelets MPV and total WRC~

4 Journal ofToxicology

l1ver sections were screened independently h) 1wo patholomiddot gisis Finding of pathologic anomalies would trigger furiher microscopic cvalua1ion of all organs collected from mice Body weight organ weights and all hematologic and clinical chcmistnmiddot parameters were analyted within each sex hr Students middottest Ith gti~nificmce a P s 005

25 Subcl1ro11ic foxidry i11 F344 Rats Rat~ were d1v1ded into groups of 6 or 7 Con1rol groups received AIM-93G chow (Tdltlab Madison WI) ad libitum while aloc-1rea1ed rats consumed chow forn1ula1ed wi1h concenlratcd aloe JUice 111

place ofwater (Tcklab Madison Wl) for 13 week~ Each week weights of ind1v1dual ral~ and feed consumed were recorded Animals middotere assessed dail) for general behabull1or health and appearance as well as appearance ofstools

At the conclu~1on of the 13 week study period rats were anegtthetizcd wilh 1sollurane for blood colleclion and then humancly eutha111zcd Al 11~cropsy gemral body appearance and external orifice~ were examined prior to examination of internal organs Abdonunal pelvic and 1horacic organs were obserwd 111 5111 then 1he liver kidneys testes (in males) heart and lungs ere removed weighed rinsed w11h phosphate-buffered saline (PBS) and placed into 10~ neutral buffered formalin (NBF) The intelinal tract wa~ examined from th1bull slomach distally and 1hen 1he tract from the cecum distally was labeled us1n) colored thread for cecum ascending transverse and descending colon and reclum The large inlcstines were then removed placed in PBS and secuoned according to anatomic region Intestinal contents were gently flushed with PBS and using a blunl needle 1he ClCUlll was opened along lhc greater curvature and flushed and examined for gross lesions The cecalmiddot colic junction was opened and grossly examined Each large intelttinal region was preserved m 10 NBF The small imestine was opened and grossly examined and preserved if lesions were obbcrved Fixed tissues were embedded in paraffin and sectioned at 7 um onto glass slides and stained with hcmatoxyhn and eosin Histological preparations and n11croscop1c evaluallons were made or the hver cecum and colon tissues (ascending transverse degtccnding) and rectum Each large intestine anatomic region was emiddotaluated and scored using a semiquantitative scale m increments of01 in which 0-09 indicated hypoplastic mucosa 10-19 indishycaled mucosa architecture and cellular popula1ions ~thin expected rangegt 20- 29 indicated moderate hypltrplasia of 1he mucosa cpilhclia and incrca~ed lymphocytic infiltrate andor goblet cell hyperplasia and scrctaon and 30-39 indicated pronounced hyperplasia ofmucosa and goble1 cells lymphoid aggregue~ wilh enlarged germinal centers and infiammatorr 1nfihrate Mucosa ot rats middotas independently scored by three reviewers and the average scores for each large u11estmal region were computed for aloe treaimcnt anltl controls ofeach gtlX

Feed consumption and body we1gh1 changes during the 13-week period were compared using 1wo vay repeated measures AN OVA wnh Bonlerron1 post tests Organ weights were compared u~ing an unpaired 1-1es1 w11h significance set a P S 005 Hematologic tests includtd RBC Hgb

llCT RIJW MCV MCI ~ICI IC platelets MPV plasma protein PCV WBCs nemrnph1ls l)bullmphocytes monocytes and 0~111ophils Clinical chenmtry assays included glucose AST ALT ALP CK T protein albumin globulin cholesterol BU1 ltreallmne calcium phogtphorus sodium po1ass1um chloride bicarbonate and amon gap Samples were compared wilh gender-matched conlrob u~ing an unpa1rtd I lest (P S 005) iargc in1estinal mucosa th1Ckncss scores ere commiddot pared using Mann-Whitney U rank111g to obtain a P value sci as P s 005

26 Verifimtion ofAloe Juice Lc1c 111 Feed To verify DCWL aloe 1uice Jemiddotels in the rnmpounJcd ftgted one JUicc con gttitucnt mahc acid was deiermined in samples or 9 Jx concentrated juice prior to compounding and m samples of thlt final aloe-containing chow A Malate AltSay kit (B1ov1s1on M1lp11as California) was used to measure L(middot) malate The 93x aloe juice contained a mlnsurcd 3620plusmn 180 mM malatc therefore the chow was expec1eltl to contain a malatc of 362 mM bullthe final meagturcd malatc concentrauon exlracteltl from the finished cha was 37 8 t 129 mM or 1044 plusmn 34deg0 of 1he cxpcted

Male rats m this study con~umed an average of 14 3 g aloe chomiddotfda) or 540511mol malateday Female rats con sumcd an average or 913 g of aloe chowday or 344011mol 1mlJtcltlay These malate levels indicate that the male ral~ excnbulltkltl a recommended 80 ounce human consumption by 59-fold and the female rals cxccc1lcll the human consumpmiddot tion of aloe JU ice by 51-fold based on body surface area

3 Results

31 Ge111Jl1mcr1y DCWL aloe middotera JUICC extract when tested at up to a 21x concemra11on wa~ not significantly mutajlenic to te~l Salmo11ela spp bacteria in the absence or presence of S9 hvcr cxlract The blank plaie without $9 showed a similar number of revertant wells in aloe plates of 2lx 14x or 7x (1ablc l(a)) Similarly thl blank plate with $9 diltl not ~how iny significant d1ffcrc1Kcgt from revenant wells in aloe middot S9 plates of Ix I-Ix or 7x (Tble l(b)) The P value was gt005 for all aloe juice conccntrauons without and middot1th lti9 These sta11s11cs applied 10 both the TAIOO and TA9R strains This mutagenirny assay as developed sufficiently to be interpreied between days 3 and 5 A bacteria free pla1c remained w1thouL grow1h 1mlic1ting thal there was no external bacterial contamination m the assay and pogtilive controls for both sodium azid~ and 2 aminoanthraccne plus S9 extra vcrHied as~ay funchon (data not shown)

ltOS 0A repair in E co1 was not activated hy DCWL aloe vera juice extra1 in concentrations ranging from 21-fold oncentrated to approximately 20-fold diluted (hgurlt l(a )) The juic producl also ltlid not ~how ig nificant concentration-related ONA ltlamagelrepa1r in lhe presence of meLabolicaliy active liver cxtracl (Figure l(b)) rhe)c da1a indicate that JUICC does not contain signifi cant level ot active or latent gcnotoxic compounds SOS DNA repair was act1va1ed in the positibulle control 4 ni troquinohne oxide (41-QO) rc~ulung in a concentrationmiddot

5

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Journal ofToxicology

TAKI~ I (a) Hfotgt orafoe IU1e on S 1ypl111111mu111 TAIOO mutagenlt (b) Hfltd uf Jluc JUIltlt un S IHbulll11m1irrum lA98 mu1agcnc1

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Sample No ofpogt1llvc wdh

bull 2 JJ)

No of positive welllt 3 day

Nn nf positive wells Iimiddot 4 dar

Nn nl pn~111vc well~ sJar

llla11k without S9 0 3 3 s Blank+ S9 0 3 II Ill WI Aloc2lx 0 0 0 ()

WL Aloe 2lx + S9 0 l n n Wl Aloe 14x 0 0 0 ()

WI Aloe 14x ~ S9 0 0 II 10 WLAloe7x () 2 I 9

L Aluc 7x + S9 0 0 I fo WL Aloe 7x + 2x S9 u 2 3 s

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Sample No ofpusillw well

ltii 2Jar =-o of pos111vc wells

(ii 3 days gtn of pnsi11vc 1bullells

(e1 4 day~ No of positive wells

re sdar lllnnk without S9 0 0 I 2 Blank~ S9 () 3 17 lU WI Aloc21x 0 0 0 l

WL Aluc 2u +gt9 0 0 15 17 WI Aloel4x 0 0 2 4

WI Alne 14x bull S9 0 0 M In WI Alnc7x 0 0 0 ()

L Aloe -x shy S9 0 0 3 II WL Aloe 7x + 2x S9 0 () 10 15

rlCliRI l HT-1lt of Dl WL aloe Vltf3 JUIlaquo un LgtA Jamaglt rltpair Abgtorbancc rdkca mcJUfltfficnt rsos trangtibullnbull ltJ3middotgd1Jllo1Jdlt) 1nJu11on and subsequcnl ubstrJk unw1011 ~ymholgt represent meanlt of 1nphlt~lc welllt anubatcd m lhc ab~nce or prcltcnltlt of S9 rat liver cxtraltL lrror bars rcprcgtcnl standard dC~JllOnlt

dependent increase m DNA repair gene acll1ty (Figure l(c)) the positive control chemical 2-aminoanthraccne (2-AA) an Bacteria also showed a concentral1on-dcpcnd~nl increase the presence of metabolic extract from rat liver (Figuregt In DNA damage repair gene ac11vlty when cxpos~d 10 l(d))

fou1~c-nl1it deg loc 1u~t 06 o~~---------------~

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6 Journal ofTox1cology

l11 E 2 ~dcctltmiddotd 11rgan wdghts of B6C311 macbull adm1ntgtlcrcd aloe JUlw hy gJlgc

3 days post aloe Male cnnlrnl Alucm01lc Comwl femtic Aloe female lllortahhcs 06 0(1 06 06

Abnormal cllntcal signs 06 016 016 06 Gmltlt ahnnrmalmc 06 06 0h Oftgt

Weight change 1111t h1 0115 103 0868 215 4 12 283 3b7 t 205 Rdauve organ weight

Liver relative weight ISbplusmn 011U 435 t 0113 501 + 017l 497 t 0315 Kidney (n ) 0769plusmn0071 0750 t 0029 072-1 plusmn 00middot17 0679 plusmn 0048 Heart 04(10i()016 0 173 J 00bull1 I 0180 i 0 025 0198 t 0056 Lung 0592 t 0088 0578 plusmn 0062 0667 0031 0663 plusmn Cl Ot9 Testicle (rt) o390 n044 0371 t 0017

14 days post aloe Mortahucs 06 06 Olti 06 Abnormal d1111ltil Mgngt 016 U6 06 06 Gross abnormahllelt 016 06 06 06 Weight Change l 11111 hM 336=191 637 plusmn 0 772 718 t 2 685 t 346 Rela11ve organ weight

L1vlaquor 185 t 0252 465 plusmn 0108 521+0372 j 74 plusmn 0388 Kidney (rt) 076gt plusmn 0072 0750 t 0029 072bull1plusmn00 17 067I plusmn OIM8 Heart 0417 plusmn 0077 0442 0037 0585 0063 0182 i 0061 Lung 0633 0080 0614 t 0650 0715 plusmn 0075 0662 plusmn 0053 ldegl-sllcle (rtl 0390 t OOH 0371 t U017

Vilu~-s preqntN i~ mtin bull J Rdgt1ibullc ltgtllgtn Wlt1gh1bull al tgtCplltbull~d u bull pcrltnl of boJy 1~h1

TAuu 3 Hematology of ~hole blood from B6t3H nucc admmmcrcd aloe Juice b gavage

laramNcrgt-3 Jar ecI cxpourc Control male Aloe m11 lu111rol tmalc Aloe female IIBC (10deg uL) 86 t 021 88plusmn018 87 t 016 88 plusmn 025 Hh(gldL) 13l t033 131bull0211 13-3 plusmn 033 134 t 031 HCT (bl ~1 i +I 2I 141 116 4l2plusmn1l l 121 121 ROW (lo) 12 7 t 045 12iO 30 ID t0-16 II 9 036 ~1CV (fi) 501J018 502plusmn086 116 t OJO 48iplusmnOH MCH (ft) MCHC (gdl)

IUt004 30 i t 0 23

15 3 t 0 18 304 = 032

154 - 0 lR )i0 plusmn 043

middot~ 0 middot~ 312 t 027

Pla1clcts (10 1uL) 1100 0 plusmn 10210 11168 plusmn 129 )I 9522 1 12894 1041 7 plusmn 12875 WllCs (101uL) 22 t 102 33 plusmn 2M 21plusmn012 2 3 t 058 Parameter~l4 dar poltl e~polturlt RBC (106 ul) K7 t 012 87 i 027 84 = 028 87 t 015 Hb(gdL) 132 t 039 Ill +041 131=014 136 t 042 HCT(O) 44 7 t 203 138 121 -1 ~ 087 433 t 148 ROW() 130 bull OH 132-064 14 0 i 353 122 t 0 71 MCV (fl) 51 0 075 501 0middot15 517 plusmn 35bull1 496 t () 211 MtH (ft) 151 t018 1so plusmn oog 158 011 156 t OM MCHC (gdL) 295 1 050 29 8 I 036 306 plusmn 16 315 t 01middot1 Platdcts (10 1 uL) IOM 11 t 1292 11000 i 1045 9552 1 31053 10235 plusmn 162 WBCs (IOJu l ) U tl5 38 t II 31plusmn23 21 t 0 12

tllltl cxpresJ mltbulln 1 J bullp ~ 005 RBC rcJ hlnod di rnunt Hb hemoglobin HtTmiddot htm10ltf11 ~CVmltln corp11gtltulu rnlum bull CHmebullncorpugtltulu hcmoilobin BC total wh1k bkgtod ctll rount RD rtU Mood -II digttnbuuon Jth Mlt HC mbullmiddotbulln oorpuscular htmoglobln oneltntration BCgt while blooJ coll counL

32 liffects of Arnie DCWL Aloe Admi11istm1to11 111 Miu ~ml fem~k mice aft~r both 3 anltl 14 days postadmnusmiddot at 1 and 14 Days Postad111i11istratio11 There were no mor 1rat1on of aloe had body and organ vcights similar to tahties en any mk( f(d high concentrations ot aloe 1mce control mice (Table 2) Necropsy was unremarkable in all Mice wen equivalent to control mic( m behabullior Male nucc

i Journal ofToxicology

TAHLl 4 Chni~al ~hcm1lt1ry ol plaltma from B6C3FI ml(e admm1~tcrcd aloe JUICe by ga-age

Parameicrlt-3 darlt pos1 exposure Conlrol male Aloltmak Con1rol female Alolt lcm1le AIT (U1) 260 I 3S 230 I 54 184 I 20 197 I 22 Al I (UI) fbull12plusmn 4 4 tOSlt t 71 87X t 94 XS Xplusmn 16lt1 CK (UI) 2876 plusmn 2648 1460 plusmn 1147 854 plusmn 292 120 2 t t183

I BIL (mgdL) lt01 SOI SOI so I Tn1al pro1ein 15 plusmn 013 43 plusmn 01middot 41 plusmn 013 40 + 042 llU-1 (mgdi) 18 0 t I 2 180 I I 228 plusmn 19 225 plusmn II Crcaunine (mgdL) 02 so2 so2 so2 lbullarunelltr~-JI Jaygt post expourc ALI (UL) 25 21 74 218 6 5 H2t119 187 bull 2 I

ALI (LiL) 712 180 674 plusmn o 101 3 39 86 166 t K (LIL) 1170 r 143 16J2 ~6J 15Z2 + 10~5 1160+ IH6

TRI (mgdi) so 1 SOI SOI SOI

lolal pro1ein middot8 t 006 10 plusmn 0 8plusmn00 llt02 RUN (mgJL) W8t40 218 I 26 24 3 23 222plusmn 10

Cre~un1ne mlildl) lt02 lt02 so2 lt0 2

Jhlltgt LXprt~~middotd a~ meanplusmn sd bull p oos All alanine amino1ransfltl3Slt activny ALP bulllkbullhnc phogtpha1~ Actmty CK crcunc k111bullmiddot IBil toul b1hrub111 8l1middot biood urcbull nitngtKltn bullLevels for TRU ind (tt3Hnlne Ct~ tt or blow 111Crumcnt quant1tat1on tbrchud

70 17 84 91 98 105 112 119 12ltgt IJJ 140 147 154 l(gti Dbull)gt of bullglt

lemalc al rat eight Molle Jloe r11 Shi ~ Ftmilt -Ontrol nt ~tight ~ 1tlt onttol ru eight

~IGliRt 2 WedJ) bod) wc1ghb ofmilc mJ female ms fod OCWL aloeera d1el over 13 wee~ The ltymhoh rcprcgtcnl mean valult1gt for (Ontrol and aloe-fed groups error birs Jiuw ~tandarJ dcV1auons

1 lematologic values from blood sampled from mice at 3 days and 14 days postcxposurc to the aloe vcra juice were glnlrally gtimilar lo control group mice (Table 3) The few values that appeared to be d1tfcrcnt between groups had vcr) small variances within groups 1hus small d1ffcrcnccs between means were stat1stteally (akulatcd as different llowever in all cases the d1flcrcnces bet 1bullmiddotecn means were 10 or le~ Clinical chemistry values from blood sampled at these 11me periods were also s1m1lar to values for control group mice (Table 4)

Sections ofhver from aloe gavaged and control mice were mtcroscoplcally examined The archnccturc 01 the ussues and cellular morphology wcrc considered l yp1cal of mice at their age (data not sho n) For imlancc male livers from

17 84 9198105il21i9i26133il0117154161 Days of bullglt

_ tcmale nlltgtlt rat 1J _ ~falc il1gtlt rGI ked -amp- 1cmJtc wnlrol ra1 feed -9- ~tile bullontrol rt Iced

FtGVRF 3 Weekly feed consumpllon of male Jud female rat~ fod UCWL aloe era d1lt1uwr13 ks- The wmbollt rcprcltcnl mean valuelt for control and aloemiddotfbullJ grouplt error barlt how tanJard Je~a11onlt

both control and mice treated with the test extract showed moderate cytoplasmic vacuolation u1stanccs of binudcatc hcpatoq1es and extramedullary hematopoiesis (FMI I) 1he female m1~c treated w11h the test extract as wel l as controls showed muhiloal EM I some cytoplasm1c vawolntion and bmudcatl hcpatoq te- but tliJ not display the occasional mild hepuocdlular necrogtb seen in malegt No pathologic features were determmcd to be exclus1middotely or predominantly as~oc1ated llth test group~

JJ Subd1m111r Fud Ad111111istrario11 13-Veek Rar Sridies During the course ol study there were no mortahltes m any group DltWL aloe vera-fed rats were equivalent to control rats 111 behavior Both body weight gams anti feed


TAHLf 5 The effect of aloe vera administration for 13 weeks on rat organ weights

Parameter Mak CODI rol WLalo~ male Control remak WL aloe female 13 week

Mortalities 07 on 016 07

Abnormal cJlmcal signs 07 017 16 017

Gross abnormalltics at necropsy 017 017 06 017

body wdght change 1903 plusmn 663 1775 plusmn 23IO 1436 plusmn582 1444 plusmn 1074 Relative organ weight

Liver 376 plusmn 02gt3 382 plusmn 0178 330 plusmn 0103 328 plusmn 016bull1

Kidney (rt) 0338 plusmn 0059 0374 plusmn 0070 0334 0045 0328 plusmn 0072 Kidney (If) 0334 plusmn 0077 0412 plusmn 0098 0352 plusmn 0049 0336 plusmn 0061

Heart 0325 plusmn 0034 029 I plusmn 0050 0119 plusmn0051 0376 plusmn 0044

Lung 0490 =0081 0550 plusmn 0080 05-10 plusmn 0059 0197 plusmn 0082

All values expr~ued as meanplusmn nandard dcvmlon middotPS llOS Clt1111pamJ wilh iltndcr control Relalivc organ weigb1s arc expressed isbull pe-cn1 ltgtflgtody weight

TAnLn 6 The effccl of aloe vera admini$tra11on for 13 weeks on rat hematologies

Paran1eter Conlrol male WI aloe male Control female WL aloe female RBC (lobull1u L 795 plusmn 028 828 plusmn 03lt1 726 plusmn 059 6911 plusmn 042

Hgb (gldL) 1254 plusmn 032 1294 plusmn 050 1232 plusmn 020 1180 plusmn 071 HCT() 38i6 =113 4009 plusmn 133 3768plusmn121 3583 plusmn 197 RDW( l99 plusmn 030 ll89 plusmn OIJ 810 plusmn 508 1129plusmn 03~ MCV (fl 4873 plusmn 054 4866 plusmn 059 5105 plusmn 067 5160 plusmn 097 MCH (fl) 1579 plusmn 022 1573 plusmn 014 1667 t 045 1700 t 077

MCHC(gdLl 321010n 3299 plusmn 037 3267 +058 3294 t 0S2 Platelets (JO uL) 61343 plusmn 484 1 58657 plusmn 66 12 56917 plusmn 8441 56443 plusmn 6143 MPV (it) 767 plusmn 061 740 =013 772 plusmn 030 Z99 t 051 Plasma protem (gdL) 756 + 061 1203 t 1322 680 plusmn 0A6 647 plusmn 069 PCV () 3843 plusmn 113 3929 =160 3617 plusmn 098 3471plusmn160 WBCs ( 101u I) 473 plusmn 056 517 plusmn 119 3lt12 plusmn 053 307 plusmn 109 Neutrophils () ll89 plusmn 027 013 plusmn 024 052 plusmn 010 061plusmn023 Lymphocytes() 351plusmn 061 396 plusmn 095 267 plusmn 049 231 plusmn090 Monocytes () 014 plusmn 005 021 plusmn 011 015 plusmn 005 013 plusmn OIO Eosinophih ( 007 t 005 U06 t 005 003 plusmn 005 004 I 005

All values cxpre$$Cltl u mcbulln plusmn bulltandard delatlon bullP S 005 ltiu11pare1i wllh gender control

consumption of rats fed lht test extract were equivalent to the same parameters m the control males and females (Figures 2 and 3)

One female rat died during transport from the vendor and thus the control group of females contained 6 rats while all olh~r groups contained 7 One control female rat developed a facial swelling during the study This swelling was diagnosed to be an abscessed tooth The ral was treated with an antibiotic (enrofloxacin) and nonsteroidal anti-innammatory (mdoxicam) for a one-week period This female responded well to therapy and was included in final data analyses

Al the studys conclusion weights of organs were not significantly different from control in either sex (Table 5) and gross necropsy was otherwise unremarkable I lematologic analyses showed that no parameter assessed m raL~ fed with the DCWL aloe vera differed significantly from the respective comrol group (Table 6 Clinical chemistry values from blood were al~o generally not different from control group mice (Table 7) There were no differences in males however in females values for albu1mn and cholesterol were lower in rats fed the test extract versus control rats Cholesterol was reduced in males fed the Lest extract but this cWference wa~ not statistically significant


iABU 7 middot1h~ dfc11 ofaloe a admtn1gttralon for 13 weeklt on rat d1mcal lthem1ltt1rc

lJrltlllldltr (luu (mfdl)

Control mal~ 221 0 t 6 13

WL alw male 2007 t 5992

ltomrul female 1903 t 826

WLaloe female 1689 + 3428

A~ I (UL) 6111 1 J1SO 6233 845 7117 t 1898 68M i 1227 t11 (UL) J529 I 522 4167plusmn 1395 2933 I 1029 25116 I 445 ALI (UL) 1470 1577 1528 I 591 16717 =1258 150H 2881 lt - CUL) 190 H 1 113 IS 35300 plusmn 38035 10 150 z 7202 23521 r 13217 Tmal protein (gdL) 581 047 602 I 043 567 r045 5 25 I 052 Alhumin (gdl) I 06 t 0 2middot1 307 011gt 323 plusmn 027 2111 023 tlobuhn (gdL) 276 t 026 295 =u 28 242 =023 2J7 plusmn 031 Cholcltlerol (mgdL) 1037 1721 9483 + 61i5 1028 995 M oo 1 12 sr OLIN mgJL) 21N206 10li t 160 1733 791 1886 +l16 ( rcallmnc (mgdL) 0 31t0035 032 =0032 o ~4 =ooi 030 t 003 ltaloum (mg JL) 899 1169 937 0 67 8 25 J 116 716 I 094 Pholtphorult (mgdl) 319 0 11 100plusmn 0H l57 plusmn 038 03 I 055 SoJ1um (mmolL) 1411 t I 22 1407 I 151 M06 I 197 1421I157 lomum (mmolL) 32bull1 bull 030 350 I 018 275 I 014 259 I 020 Chloride (mmolL) 1033 I 315 1018 t 232 1062 I 214 1106 t bull161 lhcarhonale (rnmol1) 2500 200 2657 t 270 232 plusmn 363 20lt19 I 268 Anton_s1p (mmolL) 16ll I 150 IS77 t 077 1373 I 167 1367 1 ISI All v1luu oxpremd u me1n + st~nd~rJ drv1111on middotI 005 compbullrtJ with ibullnder control

1hc ccum (including the ceci1-ohc 1u1Khon) lSccndmiddot mg lransvcrse and dcsccndmg colon and rectum were opened and exam111ed for gross abnormah11e~ (Ie masses or ulcerative lesions) No abnormali11es were found 111 aloe or comrol rats of either gender

4 Discussion

In lhts study we sough1 lo de1erm1ne 1t oral adm1111strallon ot onenlraled levels of a commcrciall available DCWI aloe JU ice Lily of the Desert Filtered Whole Leaf Aloe vera Juice with Alocsorb produced geno1ox1C11y 111 lltro acutesubacute tox1ci1y in mice or subchronic 1ox1c1l) Ill ratgt ln agreement wilh lhc rc~ults of others who have lCgthd alot Ta from the mner gel fillet IJSJ we found no evidene to gtUpport geno1ox1c effects in baclerial a~ays

In previously reported 111 vivo tox1c1ty tests aloe denvashylives w11hout the anthraquinone conta111111g latex are not associated w11h adverse efft-cts keno ct al [28] tested dried powder from lhc mner leaf fillet 111 a feed s1udy wi1h lischer 344 ralgt and found no loxicily More rcc~nll) Tanaka el al (291 reported no mortalities no abnormalities al nccrops) and no differences in body weight ga111 alter 14 days u1 a rat ~1udy 1his study tested an aloe vern gel cx1rac1ed wilh supercnucal carbon d1ox1de adm1ms1ered as a single oral dose of 150 mgkg ln our m vnmiddoto assl)S C also found no cVJdencc of aloe tox1a1y In mice our dCUle sludies Jcmons1ra1ed no mor1alttitgt no changltmiddots m bcha~or body we1gh1 or organ eights I Iema1ologmiddot and d1111Cal chemis1ry values were generally not significantly d1flere11t compared lo con1roh ahhough 111 some instances a measured value for one parameter was d1poundferen1 from lhe ~on1rol al P 005 If the mouse showed no other subJCCt1vcob1cc11vc change

~uggcgttmg 1oxici1y considered lhese values lo be lhc oasional oulliergt which middotould be an11c1pa1cd A1vcn the large number of mdl1dual parameters quanlimed and ~e did 1101 feel lhat lhee wcre b1ologically relevant differences between aloc-trea1ed and un1rea1ed groups

Ra1s fed DCWL aloe vera juice over 13 weeks also disshyplayed no adverse signs Necropsy evalualions hcmalology and clinical chc1ms1ricgt were gtnerally similar to lhe con1 rol groupraquo he ltlilfcrcnce in plasma album111 was not foh 10 be a toxk effccl s111cc we found no differences in tolal protein globulln or scrum pro1em m females and bcausc albumin in males led wi1h the les1 extrac1 wa~ not d1tTeren1 (P ~ 094) One notable effect wa~ lhe significandy reduced cholesterol concen1ra11on m females Cholesterol has been reported to be dtereagted m ra1s tha1 were admm1stered aloe wra prcv1ousl) (30 31) thus lh1s resul1 m our analsis igt not ~urprising I luseini e1 al [7] have reported that aloe ict rcducd 1holcsmiddot tcrol tn human tn a clinical trial however lhee clinical results have not been verified mother studies to date Thi~ 3~ a rcla11vcly short lcrmed study with young healthy animals and this effect may have been more pronounced andor ~ccn In malegt over a longer term or in aged animals S1ud1e cmploymg mnatdy hypercholesterol rat models have shown a grea1cr cholcs1erol reducing effec1 of aloe [32] Natural produclgt that lower cholesterol arc sought alter by consumer ot supplements Therefore although this s1udy was nol one of eflicacr de1ermma1ion u is a notable observalon

Of particular 1mercs1 m our 13-week ra1 s1udy lhere was an anal)middots1s of lhc large inlestinc for ~igns of multosal pathology fhcsc pathologies were repor1ed in 1144 rats with water adm1111s1ercd nondccolonzcd whole-leaf aloe Cra cx1ract (10] and feamred a signlficandy increased incidence and severity of mucosa hyperplasia with goble1 cell h)bullpcrpld~ia lhal wa~ more pronounced m male rals Our

10 Journal ofToiucology

TABLI $ Conumpllon ofAloe Cra by rats In this lttudy lttSUbull N ngt drinking wata bullludr

Target maloc JL1d Maho aud molanty in Conu111plion ol Weight ol rats at umoVday malic acidDuc ofaloe conccntrnt10n dosed form (mM) docd form ( bull da bull) I weeks (g) consumed100 g body wc1gh1

880 male 200 0 male 2128 m NTP 1 m water 19RO (ppm) 14 R

2246 fomalc M26 female 2328 female 362 mmolkg 1130 male 23Mmalc nso malelrcscni study 100gkg 378feed 913 female 1500 female 2293 female

bull token from middotrbk H4 Targltl vbulllucgt for malo J bullJmln1lt1erod to rts m thlt lmiddotbullcbullr iuJy 31 bullpprox1m3lltlv 4 wks pg 260 Nl P study ~lta-surrd 1n Aloe feed extract bull - 11 Ja1iubiincJ lrom Tbullbkgt 11 bullnd J2 pgs 2~J bullnd llmiddotl XTP lt1udi Aumlts I ~nm flaquod - I ml sohd

data obtained using a DCL aloe vcra jutCe contrast w11h these premiddotiou~ results No gross oi nuaocopi~ Cidcncc of intestinal pathologies was observed Ral mtestmal sections from both control and rats fed with the test extract displayed typical mucosal phenotypes including sections wuh variable levds of focal lrmphoid cdlularity in lhl limina propria and occasional variations in the dgrt of fibrou) li~ue between ~rypts however crypts were um form m lcng1h and depth and goblet cells were evenly distributed and not OCr productive with mucous secretions

The differences in findings between our present study and that of the NTP are most likely due to the different aloe middotera 1uices tltsted We hae not identified spcCtfic component~ 111 nondecolorizcd whole-leaf aloe extract that are reduced or absent in purified whole leaf beverage~ and which may have led to the mucosal response observed b) the NTP Howshyever allractive candidates are the anthraqulnones associated wtth the latex in whole leaf juice hut largely absent from activated charcoal-treated aloe Most aloe an1hraqumones exist as glycosides which arc rarely mutagcmc (33] Free 1nthraqu111ones released m animals after intestinal microbial oxidation of the glycos1dic bond) haw been associated with in vitro mutagenesis although aloe-emodin the predomimiddot nant anthraqumone released by microbial actton on aloin glycosides has been repeatedly found to be negative m a variet) of m vo assays (reviewed by (31)) Longer termed animal ~tudies particularly w11h a ~~muiw pecie) could rebullcal toxtcologic or carcinogenic tgtffoct~ not )Cen _th acute assays and thlt rat model seem) well suited to potentiate toxic and carcinogenic effects of aloes anthi aqumones Rat~ (a~ well as mice) have substan11al quanti1ie~ of microbial nora throughout their upper as well as lower gastrom1estishynal tracts (34] mcludmg those such 1 f11bacrcriim1 spp which arc capable of oxidizing the barbalom C-glycosidic bond (35 36J ln contrast human~ have a relatively sterile upper c1 tract [32] and anthroid glycosidcs pass mLacL to the colon before limited C glycosidase activity occurs [31] Other aloe vera components such as pol)sac~handes that serve as growth substrate~ for pound11badrri11111 (Y] could alter mt~tmal flora 111 favor of glycosidasc-capable bacteria The rat may there1ore be an ideal sentmel for mtcstmal toxicities associated with lhe phenoltcs 111 iloe however extrapolation of carcinogenic ouicom should account for the degree of relative human exposure to the tree anthraqmnones and the levels of anthraqumone glycMide 1n the aloe iu1ce extract~ tested

middot1 he levels of aloe wra consumed by rats m the prt1gtent study are similar to a concentration consumed by rats in the 2middotrear ral NTP study (see Table 8) The NTP study used dried nondecolorized whole leaf aloe JUiee powder at levels of 0 05 10 and 15 in dnnkmg water At the 10 level male rats in the NTP study drunk an average of 21UO mlsdJ) at ipproximately the 4-week timl point 1hl drinkmg water contained a target of 1980 ppm of t11dlic acid This equates lo 2128 bullmol malateday 100 g rat body weight Female~ consumed a similar amount In our stud- male and female~ consumed approXllllatcl) 232 micromol malic acidday 100 g weight These data indicate that the rat~ in our study consumed an amount of aloe JUice equivalent to tho 10 aloe Jnnking water m the TP Mudy by mahc acid equiviknq

In gtUmmary after assessmg a DCWL aloe beverage for both genetic and 111 1bull1vo tox1c1ty we found no adverse effects asodated with high intake of aloe at acute or subchromc penod Importantly we did not find hypcrplastic mucosal changes that were so notable in a recenl report using nondeshycoloncd wholemiddotleaf aloe Therefore it is reasonable to believe that compoMnts 111 the untreated leafextract arc responsible [or the large mtestmal hyperplas1ic reactions at I~ we4ks

Abbreviations

RSC Red blood cell rnunt Hb Hemoglobm HCT Hematouil MCV Mean corpuscular volume MCH Mean corpuscular hemoglobin WUlt Total white blood cell count ROW Red blood cell distribution width MCHC Mean corpuscular hemoglobin concentration PCV Packed cell volume ALT Alanine ammotransferase activity ALP Alkalme phosphatase ac11v11y CK Creatmc kinase TBIL Total bthrubm BUN Blood urea nnrogen PPM Parts per mtlhon DCWL Decolomed whole leaf 2-AA- 2-ammoanthracene 4-IQO 11 N11rolt1uinoline 1 oxide

II Journal of Toxicology

Conflict of Interests

Financial sponsorship was provided through collaborative agreement wi1h STampT consultants STampT consultants have received aloe juice from the manufacturer of DCWL aloe vcra gel in order lo conduct safely studies including those presented here G Gillis is employed by LOD and supplied juice characterization data Coauthors (M Scott and W D Winters) who consult for STampT consultants participated in the studys design All data collect1on analym and interpreshytation were done exclusively by coauthorgt at Louisiana State Universitys SVM

Acknowledgments

lh authors gratefully acknowledge the Division of Laborashytory Animal Medicine staff and allcndmg vctcnnanans at the LSU SVM The) also thank Dr Nobuko Wakamatsu for her suggestions ~nd helpful reading of the paper

References

(1) Almendarez Aloe Vera from Standards to Scacncc 2012 hup fwww 1ascorg

(21 I lt De ~telo A G Santos f I C Ile Amorim S C 0 asamento and U P De Albuquerque Med1nal plints 11gtd as antuumor agents m Rra11I tn cthnohotanKal approach Fi1dmcemiddot Btised Comple111e111nr1 11ml Al1er11111lr fedicine vol 2011 Arudc ID 36535914 pages 2011

(1] I I Kuo T C l in and C c l m lhe a1111prolifrrat1w acl1V11) of aloc-cmodin 1s through p53middot dependent and pl-dcpendcnl a11op1011i pathway m human hrpllonrn icll lonelt lfe Srie11rcs vol 71 no 16 pp 1879-1892 2002

[middot11 l Cowan Oral Aloe vcra a~ a treatment for ostcoarthnusmiddot a ummMymiddot Brili$1 fuurmtl ofCon111111111) NtminJI vol 15 no I pp 280-2822010

SI M Tanaka r ~hsawa Y ho ci al IJcn11fica11on of fiw phytostcrol~ trom Aloe era gel i inti d1abet1bull compounds 8iolvgical and Pharmaceullcal Bullttm vol l no 7 pp l 118shylmiddotl22 W06

(6) I gtawa111 Aloe wra 1u1cc the magtC put1un htmiddot lrnrgt ufl1111i11 012 h1tpart1cleL1mcolind1amd1a11megtiom20121un26

(7) H L Hugtcinigt K1anbakh1 R Haj1ighacc and F H Uabagluan An11-hypcrglyccm1c nnd an11-hypercholcMerolcm1c effects of Aloe vcra leaf gel Jn hypcrhpilkmil type 2 dlabctilt patk1m a randomized double-blind plnccbomiddotgtntrollcd clm1cal trial 1111111111 Meilim vol 78 nu 4 pp 311-316 2012

(RI J H Hamman Composition and apphca1Uln of Aloe veri leaf gel Mnlwllts vol 13 no 8 pp 1599 1616 2008

[) lnkrnallunal Aloe Science Cuu1ml Plthllllll Papltr of thlt lnshytcrnallonal Aloe Sc1cnt1tic Counul on lhc Z-a11onal Togtshy1cology Program Study of OrallymiddotlngcgttCd Aloe Vera 2011 httpwww1asorgpdfbull 110middot127_roMllOn-italcmenLIP ltONiUlfRSpdf

[10) at1onal foxicology Program Report lox1cologr ind Car cmugltnltSIS Studll~ uf a Nondwlurttltd hullt Leaf Extract of Aloe BarbadcnbullbullS Villcr(Uoc vcral m 1middot34-1N Rats and B6C3FI Mice (Jnnk1ng waler gttudy) NTI 1d1111lJI lkpurt gtencgt 577 NIH Publication 2011

(II I K lark Analyi1f n phciohc compounJgt m Aloe Pccicgt by high pcrformincc liquid chromatography Pl1gtmiddot1orl1tm1cal 111tl)Sbull~ bullll 9 no 4 pp 186-191 1998

[12) L r ~cnddbach A rcicw of the toxmiy and laquouunogcn1c1t) ofanthraqumnne denvat1ws Toxicology vol 57 no 3 pp 227shy240 19119

(13) ll K P11d K Patel and V Tahilyani lfarbaloln a concise rcporl of 11~ pharmacological and inalytical apcctgt Asum lltulfibull 01m111I of lrop1rnl B1omedmne vol 2 no O pp 835shy838 2012

(14) lumnal To~1wlogy Program NTP togt1wlogy mJ ronoshygcnlt1 lud1c oi F100JX (CAS 0 511-li2-I focd ~tudte~ m B44 N UIS and B6C3Fl mice 111101wl ToungtlllK) Pmgram 1i-1h111((1 Rt-port mes no 493 pp 1-278 001

(15) O luller I Fckert I K lulz and H Stopper Grn(ll1lxic1t) uf 1he laxo111vc drug componentgt emoJm aloc-cmodm and dan1hrun 111 mammalian cells topo1somerJ~e II mtJiateIgt M11tatio11 Htsfarc1 wl 371 no 3middot4 pp 165 173 1996

(161 J Weltltndorl 11 larquardl B Pog111ky M l1n111111iak J Schmidt and 11 Marquardt Genotoxidty nf na1urally ourmiddot ring hydrox)middotanthraquinnnlaquo Mu111riigt11 Resenrcl1 vol 2bull10 no I pp 1-12 19JO

[17) A 11 Shah ~ Qureshi M Tanq ind A M Aged foxmty stuJ1c on six plant uJ in the 1n1uitlonal Arab sygttcm of mcdwnc Plbull) tothuap) Roearcl1 vol 3 no pp 2$-29 19119

IS I n Williamlt c A Burdock L Shin ct al Safety tudics ondudrd on a propnetM lugh-punty Aloe vcra inner leaf hllet prcparauon Qma1nxmiddot Rexularory 1ox1rnltgtJtV a111I PharmashymiddotoOg) OI 57 no 1 pp 90-98 2010

[19) ht tpww dnndlt01111ta1111nbullmiddotgtupplc111cntgtl ngrcJJc111shymonomiddot607-A ll llmiddotapxact1vcI ngrcJ1cnt ld-607ampactivclngrc dicntN1mc-A IOb

(20] htlpw wwcancr orgtrcat menttreatmc111andMJccffccts w111pk111cntHya1Hlahernativc111cdidnchcrbgtvita111i11sandshy111111crllt1loe

( 1] htt)lmiddot w1t ntmiddotbull1tc1tn11t (omartHmiddot1edn2UllcaltH_middotVlnmiddot elttrJd middotg1vc rah tumours html

[22) httpff ww nlmn1hgobullmedltneplus1drugmlonaturil1gt07 html

(231 0 Po1middotcll lTP and the -=cN for Cnlt1lt ltanagcmcnt 2010 http (ww IJlt nrglln1dcAoe I0_021SJAOpltlf

[2middot1) S A Rolt 1 A rlltnhl) ind S P 1lkiru Quanmammiddotc inalmiddot )degI nf Aloe Vera muUagmous polyltachariJc In commercial Aloe vrra productmiddot fournnl oj AOAC l111err1111iu1111I vol RO no 2 pp 4~~-4gt7 l)J7

[25] N Pugh~ A Ros ~1 J EISohly and 0 S PagtLO Charaten zauon of alocndc a new l11gh-111olecular-we1ght polysacchandc from Aloe vcra wllh potent lmmunoltlutllllatory dlllV1ty 11urshy11al ot~rirnlt11r11I and Food Chenustry vol 49 no 2 pp 1030shy1034 2001

26] G ltJ1Jh middotrhc 1mgin1I supcquRc-1ns1dcout studies of the heJhh hcntli1 of Alot vera Colkgt of Scit11rt 1111J Ttltl11111logy vol 9 pp 60 63 009

127 R [) hltdler Simplified cakulalJon ol boJ) urfalt era 7l1c Xtw fr111lm1d oumal o Med1cu1t vol 317 no 17 3ntdc 1098 19R7

18) Y lkltno ( K Hubbard ~ Lmiddot B I Yu and J I Hcrhhy The influence of long-term Aloe era mgctaon on agemiddot related d1gteagtc m male hgtehcr 344 rats Phy1u1l1ert1py Rccarch vol 16 no II pp 712-7111 2002

Journal ofToxicology12

(211 M fanaka M Yamada T Toida and K lwa1suki Safe1y eval shyuahnn ofsupercritical carbon dioxide extract ofAloe vcra gd oumal ofFond Science vol 77 no 1 pp T2-T9 2012

(30] It Maharian P Nagar and L Narnpoo1hiri Effec1 of Aloe harhndensis Mill Formulation ltln lelro~ole induced polycylttic ovarian syndrome rat model nurnal of iy11rveda mid 111egrashytive iWetlici11e vol I no 4 pp 273-279 2010

[31] B 0 Lim N S Seong R W lthouc ct al bffica~y of dietary Aloe wra suppl1nwnta1ion on hepatic cholesterol and ox1Ja1 lve stalUs tn agcltI ratsmiddot wmnl ufNutritrmwl Science and Vitaminolo[y vol 49 no 4 pp 292-296 2003

(321 S Rajasekaran K Ravi K Sivagnanarn and S Subramanim Bencfic1al effects of Aloe vcra leaf gel extract on lipid profile status m ra1s with strepwzowcin diabetes Cimcal and Experishymental P1nr111ncology and Physiology vol 33 no 3 pp 232-237 2006

[33] n llrusack and U Menggt Ase~gtlllclll of the geno1ox1c risk from laxalivc senna products Envrronmenta and Molecular M1lllt1[e11esis vol 29 no I pp 1-9 1997

[341 T T Kararli Comparison of the ga~lrointcsli nal analom)bull physiology and b1ochemls1ry of human~ and comm1gtnly ueltI laboratory animalsmiddot Biop111rmc1Ce11tics anti Dni~ Dispositrm1 vol 16 no S pp 351-380 1995

135] R I Wang WW Cao and C E Cerniglia PCR delcclion and quanutation of predominant anacrltgtbic bacteria m human and animal fecal samplegt llpplietl 1111d tnv1ronmtbullnltll Micr11bl(Jfugy vol 62 no 1 pp 1242-1247 1996

1361 M llauori T Kanda Y Z Shu T Akao K Kobashl and T Narnba Metabolism of harbaloin by 111tcstJnal bacteria Chemrca amt N111r1111ccr111irnl 8ulldi11 vol 36 no 11 pp 4462shy4466 1988

(37] A A Salyers S E H West J R Vcrccllom and I D W1lktns Fcrmcnlalmn uf mucins and plant polysaccharidcgt by anaerobic balttcria from the human colonmiddot Applied and Ellvironmentlt1 Microbiolngy vol 34 no 5 pp 5211-533 1977

The maior C-glrcoside alo1n A is the ma1or anthraqumone 10 3loe l3tex and when oxidi1tgtd yieldlt aloe emodin a lree anthraquinone Ill) Anthraqumonfs are associated with diarrhea i11 vivo 112 13) weight logts gall bladder les1oos renal tubule pigmentation and rcn11 tubule hraline droplets [M] 111 vitro anthraquinones cause mutations in mouse lymphoma (15] and salmonella assars 116) Aloe beverages which follow IASC standards contain lcs than 10 ppm aloin A and tts isomer aloin B [9]

Toxicology ofvarious aloe produtlt halt been examined 111

vivo and these studies collectively suggest that the potential for toxicity depends on the plant semons used m 1u1ce productton Oral consumpllon ofan Clhanol extract ofwhole leaf aloe owr 3 monthgt by mice resulted 10 reproduwve toxicity and increased mortality 117) However subchromc admm1strat1on of aloe taken from the mner leaf gel resulted in no evidence of oral toxicity to rats (18) In 2010 the National Toxicology Program [10 J released a technical report on aloe vera that concluded that there was clear evidence of carcmogcmc activity of a nondccolonzcd whole leaf extract of Aloe vcra in male and female F344N rats based upon increased incidences of adenomas and carcinomas of the large 111test1ne The results of th1~ report have been referred tu on various health-related websites suggcstmg caution in consumpuon of aloe products middot19-22) and have generated substantial interest and concern b) aloe producers btcause the tudy results were based on a nondccolontcd whole leaf extract that 1s not commercially available (23) The -=TP study was designed to delinr nondecolonzcd whole leaf freezeshydncd JUICC powder to test ammal~ through their drinking water at levels up to 30 (11wt) and the ~tudy examined potential toxkologic effects ofaloe 111 both B6C3FI mice and f344 rats over time periods of 14 days 13 weeks and 2 years [lOf

While an increased inciltJence of tumorigenesis was observed 111 the large mtesttnc ol rats 111 the 2 year study no lesions were observed in mice or rats in acute studies hoeer there was decreased ater consumption m both rodmt species at the highest aloe levels During a subchronic pltnod (13 weeks) mice adrrumstered aloe ere similar to controls m most paramdergt ~th the cxccptton of mcreased incidences of mucosa) hyperplasia with goblet ~ell hypershyplasia in the cecum and large mtestme This mucosa) and goblet cell hyperplasia was also observed 111 rats at 13 weeks These rats displayed increased mortaliues and weight losses at the high aloe concentrauons Ratgt exposed for ttvo years ilso were found to have mucosa hyperplasia in th~ colon particularly in the cecum and ascending portions Large inte~tine mucosalgoblct cell hypcrpla1a~ were also observed 111 mke exposed for two rears however these 1mce did not ~howmcrcascd neoplasms and had 11nilar Unibullmiddotal and body weights as control groups

1 he nondecolorized ~hole leaf podcr u~ed b)middot the IP contamcd the b111er latex of the aloe plant and theretorc the addition of thi aloe directly to drmlong water could haw contributed to the studrs outcomes Palatability can tnlluence wa1er consumplion and conceivably could have also contributed to the weight loss and mortality in the two-year study Commercial aloe beverages that contam litt le or no

Journal ofTox1cology

latex anthraqumones mar have yielded different outcomes 111 the subhromlt or chrome exposure test~ 1l1c ~TP abo assessed the acute effects of aloe gel only and decolomed (aellvated charcoal treated) whollt leaf aloe and in these~tud 1cs mice and rat body weights water and feed consumptions organ weights hematology cl1111cal chemistry and urine chemistry were generally similar to control However only the nondccoloriLcd extract was wed in the 13-wcck and 2shyyear studies

Although thc reported incidence of large mte~tmal tumorgt hagt garnered the most attention from the -=TP report the mc1dences ot mtestinal mucosa( hyperplasia were the most consistent observations across species expogted both subchron1cill) ind chronically middot1higt mucoal hyperplasia suggcMs a tox1colog1cal response to one or more whole leaf aloe ltomponcnts In the rat hyperplasia of both the muco~a and goblet cells may indicate cellular damage and could be prognostic oflater tumor development since control rats showed liulc or no hyperplasia at 13 weeks or ademiddot nom1carcinomas of the large intestine al 2 years

To date no evaluation of the de-colorited whole leaf Aloe vera JU1Cc hagt been reported In the present stud) we report the first tox1colog1al evaluation of a commercially available deolomed whole leat (OCL) Aloe vera hevcrage Lil) of the Desert Filtered Whole Leaf Aloe vera Jwce This particushylar product an be considered as being well characterized with regard to high moleculaMbullttght polysacchande content (24 f and 1mmunomodulatmg acthmiddotity 125 26) Testtng mcluJcd assessment ofpotential genotoxictt) 111 v1trigtdnd iltutc tox1c1l) 111 vivo usmg a B6C3FI mouse model Further test mg included a l~middotwcek study utilizmg F344 rats 111 which the DCWl Juke was administered through the rats chow in order to impart a mmimal effect on palatability Examinations included large intesttnil histology to understand 1f aloe bewrages ltleriwd from DCWL aloe vera JUiee produced tox1colog1c signs hsociatcltl with nondecoloritcd whole leaf JUKC

2 Materials and Methods

2 I Prornremwt ofDCfl Aloe era Juice Aloe vcra leaves were harvctcd and mamtamed under cold storage (R C) until processing Time lapse from harvest to process111g wa~ less than six hours The harvested leaves were washed disinshyfected and macerated to yield an mtcrmcdialc raw material rcprcscnlattve of 3 nonltlecolorizeJ whole letf cxtrac as was used 111 the arorcmcn11oncd XTP study The nondecolorized extract was then filtered using carboodiatomaceou~ earth to yield a DCWL extract To this extract a proprietary isolate ofhigh-molecular weight aloe vera polysa((har1dc Alocsorb middotas added at a rate of30 mg solidfluid oune The resulting extractas flash pagtteurited to )leld the commercial product (Lily of the Desert Filtered Whole Leaf Aloe vera juice with Aloesorb) This produbullt was vacuum dned 10 yield a 7 fold concentrate

NMR analy~i~ of the resulting extract established (I) the preence of Aloe vera (b) confirming the presence of aloe vcra polysacchande malic acid and glucose) (2) the
























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