Research ArticleAntioxidant and Cytotoxicological Effects ofAloe vera Food Supplements

Zaira Loacutepez1 Gabriela Nuacutentildeez-Jinez1 Guadalupe Avalos-Navarro1 Gildardo Rivera2

Joel Salazar-Flores1 Joseacute A Ramiacuterez3 Benjamiacuten A Ayil-Gutieacuterrez2 and Peter Knauth1

1Cell Biology Laboratory Centro Universitario de la Cienega Universidad de Guadalajara Av Universidad 111547810 Ocotlan JAL Mexico2Centro de Biotecnologıa Genomica Instituto Politecnico Nacional Boulevard del Maestro sn 88710 Reynosa TAMPS Mexico3Centro de Excelencia Universidad Autonoma de Tamaulipas Matamoros sn 87000 Cd Victoria TAMPS Mexico

Correspondence should be addressed to Peter Knauth knauthgmxde

Received 25 November 2016 Revised 24 January 2017 Accepted 15 February 2017 Published 7 March 2017

Academic Editor Giuseppe Zeppa

Copyright copy 2017 Zaira Lopez et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Currently food industries use supplements from Aloe vera as highly concentrated powders (starting products) which are addedto the final product at a concentration of 1x meaning 10 gL for decolourized and spray-dried whole leaf powder (WLP) or5 gL for decolourized and spray-dried inner leaf powder (ILG) and also for nondecolourized and belt-dried inner leaf powder(ILF) Flavonoids tannins or saponins could not be detected for any starting product at this concentration and their total phenolconcentration of 68ndash112 120583M gallate-eq was much lower than in fresh extract however their antioxidant capacity of 90ndash123120583Mascorbate-eq for DPPH was similar to the fresh extract Starting products dissolved at 1x had an aloin concentration of 004 to007 ppm a concentration much lower than the industry standard of 10 ppm for foodstuff While decolourized starting products(ie treated with activated carbon) exhibited low cytotoxicity on HeLa cells (CC

50= 15 gL ILG or 50 gL WLP) ILF at CC

50= 1ndash

5 gL exhibited cytotoxic effects that is at concentrations even below the recommended for human consumption Probable causesfor the cytotoxicity of ILF are the exposure to high temperatures (70ndash85∘C) combined with a high fibre content

1 Introduction

The Aloe genus comprises 581 accepted species [1] thatbelong to the family Xanthorrhoeaceae which was previouslycategorized under Liliaceae [2] Aloe vera (L) aka Aloebarbadensis Miller is a perennial leaf-succulent xerophyteTheir leaves consist of an outer waxy cuticle and a singlecell layer of epidermis underneath is an 8ndash10-cell layer ofchlorenchyma cellsThe vascular bundles are located betweenthe thick watery inner parenchyma called Aloe fillet andthe thin chlorenchyma [3] extruded Aloe fillet is called Aloegel

The whole leaf of A vera consists mainly of water andthe residual dry mass of 26 is composed of approximately734 carbohydrate fibres 169 ashes (minerals) 69proteins and 29 lipids [4] Additionally it contains manysecondary metabolites such as complex polyphenols (egtannins and flavonoids) lignins saponins anthraquinones

glycoproteins polysaccharides and enzymes and also smallermetabolites like sterols fatty acids alcohols vitamins aminoacids and saccharides [3 5 6] The Aloe gel consists ofabout 99 water the residual dry mass is composed ofapproximately 355 crude fibres 268 soluble saccharides236 ashes (minerals) 89 proteins and 51 lipids [7]More than 95 of the soluble saccharides are glucose thenonstarch polysaccharides (apart from pectin cellulose andhemicellulose) consist mainly of mannose glucose andgalactose forming 12057314-linked polymers of 30ndash40 kDa [7 8]The thin intermediate layer composed of chlorenchyma andvascular bundles produces a yellow bitter tasting exudatecalled Aloe latex this liquid contains secondary metabolitessuch as glycosylated anthrones (up to 35 aloin A and aloinB) glycosylated chromones (aloesin and aloeresin) glycosy-lated anthraquinones and polyphenols Free anthraquinones(aloe-emodin) are onlyminor components of the exudate andare more abundant in the root [9 10]

HindawiJournal of Food QualityVolume 2017 Article ID 7636237 10 pageshttpsdoiorg10115520177636237

2 Journal of Food Quality

Several positive effects have been attributed to Aloeproducts [11] in fact it has been used in traditional medicinefor centuries therefore the food industry started to useAloe extracts as a supplement for functional food [3 12]Typical health claims for this kind of products are to providerelief from rheumatoid arthritis cancer diabetes digestiveand intestinal disorders or ulcers The related pharmacolog-ical actions include anti-inflammatory antiproliferation andantimicrobial activity as well as laxative and blood glucoselowering effects to prove those claims Aloe gel extractsespecially have been analysed by several research groups[8 13ndash15]

Aloe latex contains even more secondary metabolitesthan Aloe gel and several effects could be attributed tothem for example the induction of apoptosis in several celllines like HL60 HeLa Jurkat and MCF-7 [16ndash19] Anotherdesired effect is that C

8-hydroxy substituted anthranoids act

as laxatives this effect is caused by changes in the colonicmotility and absorption However in vitro studies revealedpotential mutagenic activity of aloe-emodin in the Amestest and carcinogenic activity of 1-hydroxyanthraquinone invivo [20] The safety aspects that have arisen for potentialconsumers are summarized in the report on the safetyassessment of different Aloe products cell culture studies forgel and whole leaf extracts revealed proliferative effects whilethe latex fraction had cytotoxic effects [21] The same reportquoted no acute oral toxicity when mice were fed with 3 gkgof an ethanolic leaf extract even when rats were fed with 1or 10 Aloe extract in drinking water during a period of 55months no negative health effects could be observed whenthe extract was decolourized (ie treated with charcoal toeliminate anthranoids) when left untreated the rats sufferedfrom diarrhoea and a lower growth rate with the 10 extractTheir final conclusion was that Aloe-derived products forcosmetic industries (ie topical use) are considered safeif the anthraquinone concentration is below 50 ppm [21]In 2002 the US Food and Drug Administration (FDA)declared that Aloe extracts as laxative were not generallyrecognized as safe and effective and that more evidencewas needed [22] As a consequence the International AloeScience Council (IASC) established the industry standard fororal consumption to less than 10 ppm aloin [23] Moreoverwithin theNational Toxicology Program (NTP) toxicologicalaspects were reviewed with results pointing to potentiallycarcinogenic effects F344N rats fed during a period of 2years with 1 (nondecolourized) whole leaf extract in drink-ing water developed significantly more intestinal adenomasor carcinomas on the other hand the same treatment didnot significantly increase neoplasms in B6C3F1 mice [24]This report was heavily criticized especially by the IASCbecause the aloin content with approximately 6500 ppm wasmore than 650 times higher than the industry standard [25]Thereafter Sehgal et al [26] reported no negative effects(neither on organs nor on blood values) on F344 rats whenfed during a period of 13 weeks with charcoal decolourizedwhole leaf extract even when the rats consumed 5 to 6times more Aloe extract than the recommended maximumfor humans

Until now most studies have analysed raw extracts fromA vera but the starting material for food industries isprocessed in a very different way thus the safety aspects forthose products have not been fully evaluated meaning that arisk of adverse outcomes or collateral damage to consumersexists In order to address this discrepancy we began toexamine cytotoxic effects of starting products produced andcommercialized by Mexican facilities of a US enterprisepreliminary studies revealed that those powders were notcytotoxic to the cell line HeLa when up to 12 gL (24x)concentrated inner leaf extract or less than 50 gL (5x) wholeleaf extract was used both (concentrated) products had only5-6 ppm of aloin [27] We then continued by analysing thephytochemical components antioxidant activity and theircytotoxic effects in greater detail

2 Materials and Methods

21 Molecular Identification by ITS Sequencing The genomicDNA was extracted with the Wizard Genomic DNA Purifi-cation Kit (Promega Madison USA) Briefly the sample wasfrozen at minus70∘C and ground to a fine powder The nucleiwere then lysed (65∘C) and the RNA was degraded by addingRNase (37∘C) After cooling down to room temperature(RT) the proteins were removed by salt precipitation Aftercentrifugation the supernatant was transferred into a newtube to precipitate the genomic DNA with isopropanol aftercentrifugation the pellet was washed with 70 ethanol air-dried and finally resuspended in buffer

The universal primer pair ITS-1 (51015840-TCC GTAGGT GAACCT GCG G-31015840) and ITS-4 (51015840-TCC TCC GCT TAT TGATAT GC-31015840) were used in the PCR (Polymerase ChainReaction) to amplify the polymorphic Internal TranscribedSpacers (ITS1 and ITS2) and the intervening 58S rRNA generegion as described by White et al [28] The PCR productsof approximately 650 bp were purified with the ExoSAP-ITPCR Product Cleanup (Affymetrix Santa Clara USA) andsequenced by the Sequencing Laboratory of the Center ofGenomic Biotechnology of the Instituto Politecnico Nacional(Cd Reynosa Mexico) The sequence was compared toreported sequences of the GenBank database using theBLAST (Basic Local Alignment Search Tool) algorithm [29]available at the NCBI (National Center for BiotechnologyInformation) website Finally a neighbour-joining phyloge-netic tree was calculated with the program CLC SequenceViewer 76

22 Starting Products of Aloe vera A US enterprise providedthe starting products (powders) from its facilities in Mexicowhich are commercialized as food supplements of A verato food processing industries The whole leaf was mashedfiltered and pasteurized (HTST-juice) decolourized withactivated charcoal filtered through diatomaceous earth anddehydrated by spray-drying resulting in 100x decolourizedwhole leaf powder (WLP) The inner leaf fillet was washedand mashed and the juice was decolourized filtered throughdiatomaceous earth and dehydrated by spray-drying 200xinner leaf gel (ILG) powder or the inner leaf fillet waswashed dehydrated by belt-drying and crushed resulting in

Journal of Food Quality 3

Aloe vera

Whole leaf

Inner leaf llet

Peeled

Pasteurized

HTST-juice

WLP

Belt-dried

Crushed

ILG + ILF = ILB

Decolourized (charcoal)Filtered (diatomite)

Mashed

Press-lteredGround

WashedPasteurized

WashedTrimmed

Cut

Decolourized (charcoal)Filtered (diatomite)

Filtered (4120583m)Filtered (4120583m)Spray-dried (lt60∘C) Spray-dried (lt60∘C)

(70ndash85∘C)

Figure 1 Sample preparation scheme Leaves from Aloe vera arecut washed and trimmed to get the whole leaves For HTST-juicethe whole leaves are ground to a particulate slurry which is press-filtered and the resulting juice is pasteurized Whole leaf powder isproduced by filtration of HTST-juice through diatomaceous earthand the aloin content is then reduced by charcoal treatment whichitself is removed by another filtration the resulting liquid is spray-dried resulting in a 100x concentrated powder (WLP)Thewhole leafcan also be peeled washed and pasteurized to get the inner leaf filletThis fillet can be mashed and then treated like HTST-juice to get a200x concentrated inner leaf gel (ILG) powder or it can be (probablysliced) belt-dried and crushed to get a 200x concentrated innerleaf gel powder rich in fibres (ILF) The main difference betweenWLPILG and ILF is that the latter has no charcoal treatment and isexposed to higher temperatures during the drying process The 1 1mixture of ILG and ILF results in a 200x inner leaf blended (ILB)powder

a powder with a high fibre content 200x dehydrated innerleaf powder with fibre (ILF) (Figure 1) The fourth sampleinner leaf powder blended (ILB) is a mixture of ILG andILF The powders were dissolved in water or culture mediumto different concentrations the enterprise recommended aconcentration of 1x for their products meaning 5 gL for the200x samples (ILG ILB and ILF) or 10 gL for the 100x sample(WLP)When indicated samples were centrifuged for 20minat 15000g or filtered through a nylonmembranewith 020120583mpore size to reduce the fibre contentThe liquid HTST-juice isa preproduct and is not commercialized by this enterprise andwas used by us as an internal control (nearly a nonprocessedextract similar to a raw extract)

23 Phytochemical Screening For the qualitative analysisof flavonoids tannins terpenoids and saponins [30] thesamples were dissolved in distilled water (05 gmL WLP025 gmL ILG 017 gmL ILB and 01 gmL ILF 1 1 vvHTST-juice) and shaken (80 rpm) for 12 h at RT in the darkand subsequently filtered (Whatman No 4) Only positivetested samples were quantified using concentrations of 10 gLfor WLP and 5 gL for ILG ILB or ILF HTST-juice wasdiluted at 1 7 (vv)

Flavonoids 1mL of each sample was mixed with 5mL 1NH4Cl and then 5 drops of conc H

2SO4were added

An unstable yellow colour was formed in the presence offlavonoids

Tannins 1mL of each sample was boiled for 2min and then5 drops of 01 FeCl

3were added forming a brownish green

or blue-black colour in the presence of tannins

Terpenoids 5mL of each sample was mixed with 2mLdichloromethane and then 3mL of conc H

2SO4was added

forming a stable layer A reddish-brown colour in the inter-face indicates positive results

Saponins 5mL of each sample was boiled for 2min dilutedwith water and shaken to form foam then 3 drops of oliveoil were added and shaken vigorously The formation of anemulsion indicated the presence of saponins

Quantification of Total Phenols 75120583L of each centrifugedsample was mixed with 12mL water and 75120583L water-Folin-Ciocalteu reagent (1 1) After 3min 150 120583L of 1 N Na

2CO3

was added and then incubated for 2 h at RT in the dark Alight violet colourwas quantifiedwith a photometer (MecasysOptizen-Pop Daejeon South Korea) at 725 nmThe standardcurve was prepared with up to 1176mM gallate (G7384Sigma-Aldrich St Louis USA) [31]

Quantification of Flavonoids 100 120583L of each sample wasdiluted with 500120583L distilled water and then 30 120583L of 5NaNO

2 after 6min 60 120583L of 10 AlCl

3sdot6 H2O and after fur-

ther 5min 200120583L of 1MNaOHwere addedThismixture wasfilled up to 1mL with distilled water before the absorbanceat 120582 = 510 nm was quantified with a photometer (Optizen-Pop) Up to 034mM (+)-catechin (C1251 Sigma) was usedas standard [32]

24 Chromatographic Analysis The analytical standardsaloin (B6906) and aloe-emodin (93938) were obtainedfrom Sigma-Aldrich and the necessary solvents were pur-chased from J T Baker (Center Valley USA) The stan-dards and samples were analysed by high pressure liquidchromatography (HPLC) using an Agilent 1100 series withdiode array detector (Agilent Santa Clara USA) and aKromasil C-18 column (K08670357 Bohus Sweden) (1)aloin (acetonitrile-water 40 60 vv flow rate 06mLmindetection at 360 nm run time of 25min) and (2) aloe-emodin(methanol-water 7030 vv flow rate 06mLmin detectionat 254 nm run time of 25min) 05 g or 05mL of eachsample was extracted with 5mL 70 ethanol for 30minand filtered (045120583m) before HPLC analysis 20120583L standardor sample was injected with an isocratic elution gradientat RT A calibration curve was done for each standard atconcentrations from 05 to 5 ppm

25 Antioxidant Capacity 10 gL of WLP and 5 gL of ILGILB or ILF were dissolved in distilled water and HTST-juicewas diluted at 1 7 (vv) All standard curves were preparedwith up to 400120583M ascorbate (11140 Sigma)

4 Journal of Food Quality

DPPH For a stock solution 197mg of 22-diphenyl-1-picrylhydrazyl (D9132 Sigma) was dissolved in 100mLmethanol which diluted at 1 455 to a working solution hadan absorbance of sim138 at 120582 = 515 nm 100 120583L of each samplewas mixed with 900120583L DPPH working solution stored for24 h at RT in the dark and centrifuged (1min at 15000g) andthe absorbance was measured at 120582 = 515 nm

ABTS 226mg of 221015840-azinobis(3-ethylbenzothiazoline-6-sulfonate) (11557 Sigma) was dissolved in 10mL acetatebuffer (20mM pH 45) and then mixed with 10mL of144mM (NH

4)2S2O8in acetate buffer This ABTS-working

solution was incubated for 12ndash16 h at RT in the dark and thendiluted at 1 25 with acetate buffer the absorbance was sim124at 120582 = 734 nm 100 120583L of each sample was mixed with 900120583LABTS-working solution and incubated for 4 h at 37∘C in thedark and the absorbance was measured at 120582 = 734 nm

FRAP The ferric reducing antioxidant power-working solu-tion was made by mixing 25mL of TPTZ (10mM 246-tris(2-pyridyl-s-triazine) [93285 Sigma] in 40mM HCl)and 25mL of 20mM FeCl

3sdot6 H2O in distilled water with

25mL of acetate buffer (300mM pH 36) 100 120583L of eachsample was mixed with 900 120583L FRAP-working solution andincubated for 2 h at 37∘C in the dark and the absorbance wasmeasured at 120582 = 593 nm

26 Cell Culture

Cell Line andCulturingConditionsThecell lineHeLa (humancervix-uteri) was purchased from ATCC (Manassas USA)HeLa was grown in DMEM (ATCC) supplemented with10 FBS (Biowest Kansas City USA) at 37∘C 4 CO

2

and 95 RH The samples were dissolved in concentrationsup to 100 gL WLP or 50 gL ILG ILB and ILF (10x) inculture medium pH was adjusted with NaOH (1M) to pH73 and 50120583gmL of gentamycine (Sigma-Aldrich) was addedto avoid bacterial growth HTST-juice was directly added tothe medium without using antibiotics The experiments werepreviously described [27] in short the cells were seeded overcover slides in 12-well microtiter plates (MTP) and incubatedfor 1 d to let them attach the samples were added or themedium was changed for sample-containing medium andthen the cells were grown for one more day prior to analysis

Cell Viability and Necrosis Induction The metabolic activitywas determined by neutral red uptake (NRU) 20 120583LmL of033 neutral red solution (Santa Cruz USA) was addedand incubated for an additional 4 h Afterwards the coverslides werewashed oncewith phosphate buffered saline (PBS)and observed under the microscope (Axioskop 40FL ZeissOberkochen Germany) For quantification after adding30 120583LmL of 033 neutral red solution and incubation thecells were washed once with PBS and the neutral red wasextracted with 800120583L of 1 acetic acid in 50 ethanol duringa period of 15minThe supernatant was centrifuged for 3minat 10000g (Eppendorf 5415D Hamburg Germany) and theabsorbance was measured at 120582 = 690 nm For the WST-quantifyingmethod the cells were exposed to the sample andthe medium was then changed for a fresh culture medium

Table 1 Concentration of aloin and aloe-emodin in differentcommercial starting products at the concentration 1x comparedto the raw extract (HTST-juice) Values are expressed as mean plusmn196lowastSEM (119899 = 3)

Sample (1x) Aloin (120583gmL) Aloe-emodin(120583gmL)

ILG 0036 plusmn 0012 0003 plusmn 0004ILB 0069 plusmn 0007 0050 plusmn 0008ILF 0053 plusmn 0002 0130 plusmn 0010WLP 0046 plusmn 0011 0009 plusmn 0018HTST-juice 1004 plusmn 082 757 plusmn 047

Then 20120583LmL of WST-1 (Clontech Mountain View USA)was added and the cells were incubated for an additional 4 hFinally 800120583Lof themediumwas centrifuged (1min 8000g)to precipitate possible cell debris before measuring theabsorbance at 120582 = 440 nm and 120582 = 690 nm (as background)To detect necrosis 005 (final concentration) trypan blue(Biowest) was added and incubated for 5 to 10min and thecover slides were washed with PBS and then observed underthe microscope

27 Statistical Analysis Values are expressed as means plusmn196 lowast standard error of the mean (196lowastSEM) Differencesbetween groups were determined by one-way analysis ofvariance (ANOVA) and subjected post hoc to Tukeyrsquos HSDmultiple comparison tests using the program SPSS 19 A valueof 119901 lt 005was considered to indicate statistical significance

3 Results

31 Phytochemical Analysis Plant material from the enter-prise which also provided the starting products was iden-tified by ITS sequencing as Aloe verawith 98 identity to thesample KC893746 of the NCBI GenBank database

Most secondary metabolites are found in the Aloe latexrepresentative metabolites were analysed by HPLC to provewhether the decolourization process for the starting productswas efficient and whether those products fulfilled the IASCstandard of lt 10 ppm aloin The aloin content ranged from46 to 138 120583gg dry weight for the 100x or 200x concentratedpowders when those samples were diluted to the recom-mended concentration of 1x (ie 10 gL for WLP or 5 gLfor ILG ILB and ILF) the aloin concentration diminishedaccordingly to only 004ndash007120583gmL Those values wereconsiderably lower than the sim10 120583gmL of the HTST-juice(Table 1) The anthraquinone aloe-emodin was found insimilar concentrations (0003ndash013120583gmL) in 1x solutions(ie 10 gL for WLP or 5 gL for ILG ILB and ILF) and washigh only in the HTST-juice (Table 1)

Tannins or saponins were not detected in those startingproducts and a hint of terpenoids was observable only inHTST-juice Flavonoids were clearly detectable in HTST-juice barely detectable inWLP ILG and ILB and even absentin ILFThe quantitative analysis forAloe gel powders revealedthat the total phenol concentration decreased in conjunction

Journal of Food Quality 5

Table 2 Concentration of total phenols and flavonoids in different commercial starting products at the concentration 1x compared to theraw extract (HTST-juice) Values are expressed as mean plusmn 196lowastSEM (119899 = 9 for total phenols 119899 = 3 for flavonoids) ND not detected

Sample (1x) ILG ILB ILF WLP HTST-juiceTotal phenols[120583M gallate-eq] 1117 plusmn 155 923 plusmn 150 570 plusmn 158 857 plusmn 141 27124 plusmn 877

Flavonoids [120583Mcatechin-eq] ND ND ND ND 382 plusmn 41

ILG ILB ILF WLP HTST-juice0

20406080

100120140

1000

2000

3000

4000

Sample

Asc

orba

te-e

quiv

alen

t (120583

M)

a

d

g

a c

d

f

b

e

h

c

f f

5gL 5gL 5gL 10 gL

Figure 2 Antioxidant capacity of different starting products atthe concentrations of 5 gL for ILG ILB and ILF and 10 gL forWLP (ie 1x) and HTST-juice measured as FRAP (dark grey)ABTS (grey) and DPPH (light grey) Values are expressed as meanplusmn 196lowastSEM (119899 = 7ndash9) Letters indicate statistically significantdifferences

with an increase of the fibre content (Table 2) accordinglyThe HTST-juice had 25 times more total phenols than thestarting products Consistent with the qualitative analysis thequantification of flavonoids resulted to be positive only forthe HTST-juice for all other samples the concentration wasbelow the detection limit (Table 2)

32 Antioxidant Capacity (AOC) AOC was determined bythree independent methods FRAP and ABTS which mea-sure mainly water soluble antioxidants with Single ElectronTransfer (SET) and DPPH which detects methanol solubleantioxidants with SET and H-Atom-Transfer (HAT) mecha-nisms A rather consistent observation for all three methodswas found in the Aloe gel samples the more fibre the producthad the lower the AOC that is AOC was decreasing fromILG via ILB to ILF (Figure 2)WLP had a similar AOC to ILBonly the HTST-juice exhibited 20 to 30 times higher (DPPHandABTS resp) and even 80 times higher (FRAP)AOC thanthe starting products (Figure 2)

33 Cell Viability and Necrosis Results for HeLa viability forthe WLP and ILG samples have been previously reportedby us [27] ILB inhibited metabolic activity of HeLa (NRU)and induced necrosis (trypan blue) at a concentration of15 gL (3x) (Figure 4) and ILF at even 5 gL (1x) (Figure 3)the recommended concentration for human consumption

Moreover at 5 gL ILF only very few cells were still presentand much debris and fibres could be seen (Figure 3) Thiseffect was observed not only after 24 h of incubation butalready after 4 h (data not shown) which means that ILF actsvery fast Only a further reduction of the ILF concentrationto 05 gL abolished the cytotoxic effect (Figure 3) In orderto narrow the cytotoxic principle the sample was centrifugedor filtered The cytotoxicity for ILF decreased slightly nowat 5 gL ILF few cells (and less fibres) could be seen andexhibited metabolic activity visualized as NRU (Figure 3)Centrifugation of ILB hardly reduced its cytotoxicity com-pared to the noncentrifuged sample at 15 gL of centrifugedILB (3x) somemore cells were visible yet many of themwerenecrotic (Figure 4) thus centrifugation to reduce the fibrecontent was less effective for the sample which contained apriori less fibres

Those effects could not be quantified directly using theWST test the sample probably interferedwith the tetrazoliumsalt (and for the same reason LDH release as a necrosisindicator could not be determined) To address this problemthe standard protocol was changed slightly after 4 h ofexposure the ILF-containing medium was changed for freshmedium and then WST was added to determine metabolicactivity The cytotoxicity could be determined like this toCC50asymp 1 gL ILF (02x) Centrifugation as well as filtration

slightly reduced the cytotoxic effect for both techniques toCC50asymp 225 gL ILF (Figure 5) which is still below the

recommended 5 gL for human consumption30 to 80 120583LmL of HTST-juice reduced the metabolic

activity of HeLa to 50 of the control measured quantita-tively as NRUObserving the cells by themicroscope revealedthat at 60 120583LmL HeLa was metabolically still active (redstained with neutral red) at 100 120583LmL it was nearly inactive(faint red) yet normally shaped and at 300120583LmL only fewcells were seen and were not metabolically active (Figure 6)As HTST-juice has sim10 120583gmL aloin the cells had beenexposed to sim1 120583gmL aloin when metabolic activity declined(at 100 120583LmL HTST-juice) but when the cells were exposeddirectly to aloin they did not show any signs of cytotoxiceffects even at concentrations up to 50 120583gmL (Figure 6)

4 Discussion

Starting products for foodstuff from Aloe vera are mostlycommercialized as concentrated powders but the qualityamong them can vary considerably The products can beold (lt10 of polysaccharide acemannan) and suffer frombacterial fermentation (gt10 lactate) or other adulterations(typically with maltodextrin or citrate) Previous analysis

6 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 3 Effect of ILF on HeLa cells measured as NRU all images at 100x magnification and 400x magnification at the upper leftcorner Growth control wo ILF (a) at 05 gL (b) at 5 gL (c) and at 5 gL centrifugation (d) The recommended concentration for humanconsumption is 5 gL (1x) Red stained cells indicate metabolic activity and functional lysosomes

of those products revealed large differences among themalthough all have been certified by IASC For instance theacemannan content of different 200x inner leaf gel powdersranged between 4 and 9 and the aloin concentrationvaried between ldquonot detectablerdquo and up to 15 ppm [33] Thealoin concentrations for our samples ranged from 46 to138 120583gg dry weight but as the samples are 100 or 200 timesconcentrated the aloin concentration in the final productfor the consumer (meaning 10 gL for WLP or 5 gL for ILGILB and ILF ie at 1x) ranged between 004 and 007 120583gmLA comparison to the aloin content of the HTST-juice (sim10 120583gmL) demonstrates that not only the decolourization butalso the filleting and dehydration process is very effective inreducing the aloin content It is known that heat treatment(as of 80∘C) halves the aloin content of fresh Aloe geljuice within 4 h [34] The biological activity for the highlyconcentrated powders cannot be estimated according to theirchemical constitution as can readily be done for raw extracts[35] Therefore the principal constituents of our samplesregarding potentially bioactive molecules were determinedtannins saponins and terpenoids were not detected in any ofthe highly concentrated powders For fresh Aloe gel Luciniet al [5] determined 1276mmolkg gallate-eq total phenolsand 016mmolkg rutin-eq flavonoids For the HTST-juicewe found with 27mM gallate-eq total phenols and 004mMcatechin-eq flavonoids similar values but in our powdersamples in 10 gL WLP and 5 gL ILG ILB or ILF (1x)

respectively we found only 007 to 011mM gallate-eq totalphenols and flavonoids could not be detected This indicatesthat the treatment for reducing the aloin content also reducesthe content of other biologically active compounds Manystudies on the effects ofA verawere done with freshmaterialbut as the constitution of those starting products differsconsiderably from the fresh material their properties shouldbe analysed in order to address (or not) effects to endproducts

For all four powder samples when dissolved at 10 gLWLP or 5 gL ILG ILB and ILF (1x) we found AOCvalues ranging from 894 to 1228120583M ascorbate-eq for theDPPH test Lucini et al [5] using the same test measured108 120583Mtrolox-eq for fresh whole leaf extract and 54 120583Mtrolox-eq for Aloe gel extract those values are comparablebecause ascorbate and trolox have nearly the same redoxpotential [36]The decolourization process used to lower thealoin concentration also reduced the total phenol and theflavonoid content and alongwith this theAOCOnly the ILFpowder is not decolourized but dried by heat compared to thedecolourized sample (ILG) ILF has a higher aloin and aloe-emodin content but less total phenols and thus the lowestAOC of all the samples

To evaluate the consequences of these constitutional dif-ferences among the samples we performed cytotoxicologicalstudies we differentiated between a reduction of metabolicactivity (WST and NRU) and necrosis induction (trypan

Journal of Food Quality 7

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 4 Effect of ILB on HeLa cells measured as trypan blue all images at 400x magnification Growth control wo ILB (a) at 5 gL (b) at15 gL (c) and at 15 gL centrifugation (d) The recommended concentration for human consumption is 5 gL (1x) Blue stained cells indicatenecrotic cell death

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

001 01 1 1000000Concentration (gL)

Figure 5 Effects of ILF (square) centrifuged ILF (circle) andfiltered ILF (triangle) on the metabolic activity of HeLa quantifiedas relative WST activity Growth control activity was set to 100for each experiment The recommended concentration for humanconsumption is 5 gL (1x) Values are expressed asmean plusmn 196lowastSEM(119899 = 3 for ILF filtered 119899 = 4)

blue) In our case the lowest cytotoxicity on HeLa wasexhibited by both decolourized samples with 50 gL WLP(5x) or 25 gL ILG (5x) followed by approximately 15 gL ILB(3x) ILF showed cytotoxic effects in a range of 1ndash5 gL (02ndash1x) But as HeLa was not inhibited by up to 50 ppm aloin we

conclude that aloin itself is not responsible for cytotoxicityof the samples Moreover flavonoids tannins and saponinswere not detected in the sample powders therefore thesecompounds should not contribute to cytotoxicity either Thedifference between the gel fillet samples is that while ILF is awashed heat-dried and powdered fillet that still contains allof the Aloe gel fibres ILG is a decolourized and spray-driedfillet extract whose Aloe gel fibres are mostly removed whenmaking an extract (Figure 1) samples comparable to ILGhave a fibre content ranging from 30 to 64 while samplessimilar to ILF have up to 184 fibre content [35] Thesefibres contain among other polysaccharides acemannans apolymer of C

2 C3 or C6O-acetylated mannose interspaced

by a few glucose residues and branched by some 120572-16-bond galactose residues [7 8] To prove a possible cytotoxiceffect by the fibres alone the ILF sample was centrifugedor filtered and the cytotoxicity reduced slightly from CC

50

= 1 gL for the nontreated to about CC50

= 225 gL forthe centrifuged or filtered sample Thus by centrifugationor filtration the cytotoxic component could not be readilyremoved Another major difference between the samples isthe heat treatment which causes changes in the acemannancomposition compared to fresh Aloe gel When Aloe gelis dehydrated debranching reactions occur at temperaturesstarting at 60∘C especially for terminal galactose at tem-peratures starting at 70∘C deacetylation reactions increaseand the polymers gain molecular weight this is followed bybrowning reactions which start at 80∘C [37]The changes for

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

Journal of Food Quality 3

Aloe vera

Whole leaf

Inner leaf llet

Peeled

Pasteurized

HTST-juice

WLP

Belt-dried

Crushed

ILG + ILF = ILB

Decolourized (charcoal)Filtered (diatomite)

Mashed

Press-lteredGround

WashedPasteurized

WashedTrimmed

Cut

Decolourized (charcoal)Filtered (diatomite)

Filtered (4120583m)Filtered (4120583m)Spray-dried (lt60∘C) Spray-dried (lt60∘C)

(70ndash85∘C)

Figure 1 Sample preparation scheme Leaves from Aloe vera arecut washed and trimmed to get the whole leaves For HTST-juicethe whole leaves are ground to a particulate slurry which is press-filtered and the resulting juice is pasteurized Whole leaf powder isproduced by filtration of HTST-juice through diatomaceous earthand the aloin content is then reduced by charcoal treatment whichitself is removed by another filtration the resulting liquid is spray-dried resulting in a 100x concentrated powder (WLP)Thewhole leafcan also be peeled washed and pasteurized to get the inner leaf filletThis fillet can be mashed and then treated like HTST-juice to get a200x concentrated inner leaf gel (ILG) powder or it can be (probablysliced) belt-dried and crushed to get a 200x concentrated innerleaf gel powder rich in fibres (ILF) The main difference betweenWLPILG and ILF is that the latter has no charcoal treatment and isexposed to higher temperatures during the drying process The 1 1mixture of ILG and ILF results in a 200x inner leaf blended (ILB)powder

a powder with a high fibre content 200x dehydrated innerleaf powder with fibre (ILF) (Figure 1) The fourth sampleinner leaf powder blended (ILB) is a mixture of ILG andILF The powders were dissolved in water or culture mediumto different concentrations the enterprise recommended aconcentration of 1x for their products meaning 5 gL for the200x samples (ILG ILB and ILF) or 10 gL for the 100x sample(WLP)When indicated samples were centrifuged for 20minat 15000g or filtered through a nylonmembranewith 020120583mpore size to reduce the fibre contentThe liquid HTST-juice isa preproduct and is not commercialized by this enterprise andwas used by us as an internal control (nearly a nonprocessedextract similar to a raw extract)

23 Phytochemical Screening For the qualitative analysisof flavonoids tannins terpenoids and saponins [30] thesamples were dissolved in distilled water (05 gmL WLP025 gmL ILG 017 gmL ILB and 01 gmL ILF 1 1 vvHTST-juice) and shaken (80 rpm) for 12 h at RT in the darkand subsequently filtered (Whatman No 4) Only positivetested samples were quantified using concentrations of 10 gLfor WLP and 5 gL for ILG ILB or ILF HTST-juice wasdiluted at 1 7 (vv)

Flavonoids 1mL of each sample was mixed with 5mL 1NH4Cl and then 5 drops of conc H

2SO4were added

An unstable yellow colour was formed in the presence offlavonoids

Tannins 1mL of each sample was boiled for 2min and then5 drops of 01 FeCl

3were added forming a brownish green

or blue-black colour in the presence of tannins

Terpenoids 5mL of each sample was mixed with 2mLdichloromethane and then 3mL of conc H

2SO4was added

forming a stable layer A reddish-brown colour in the inter-face indicates positive results

Saponins 5mL of each sample was boiled for 2min dilutedwith water and shaken to form foam then 3 drops of oliveoil were added and shaken vigorously The formation of anemulsion indicated the presence of saponins

Quantification of Total Phenols 75120583L of each centrifugedsample was mixed with 12mL water and 75120583L water-Folin-Ciocalteu reagent (1 1) After 3min 150 120583L of 1 N Na

2CO3

was added and then incubated for 2 h at RT in the dark Alight violet colourwas quantifiedwith a photometer (MecasysOptizen-Pop Daejeon South Korea) at 725 nmThe standardcurve was prepared with up to 1176mM gallate (G7384Sigma-Aldrich St Louis USA) [31]

Quantification of Flavonoids 100 120583L of each sample wasdiluted with 500120583L distilled water and then 30 120583L of 5NaNO

2 after 6min 60 120583L of 10 AlCl

3sdot6 H2O and after fur-

ther 5min 200120583L of 1MNaOHwere addedThismixture wasfilled up to 1mL with distilled water before the absorbanceat 120582 = 510 nm was quantified with a photometer (Optizen-Pop) Up to 034mM (+)-catechin (C1251 Sigma) was usedas standard [32]

24 Chromatographic Analysis The analytical standardsaloin (B6906) and aloe-emodin (93938) were obtainedfrom Sigma-Aldrich and the necessary solvents were pur-chased from J T Baker (Center Valley USA) The stan-dards and samples were analysed by high pressure liquidchromatography (HPLC) using an Agilent 1100 series withdiode array detector (Agilent Santa Clara USA) and aKromasil C-18 column (K08670357 Bohus Sweden) (1)aloin (acetonitrile-water 40 60 vv flow rate 06mLmindetection at 360 nm run time of 25min) and (2) aloe-emodin(methanol-water 7030 vv flow rate 06mLmin detectionat 254 nm run time of 25min) 05 g or 05mL of eachsample was extracted with 5mL 70 ethanol for 30minand filtered (045120583m) before HPLC analysis 20120583L standardor sample was injected with an isocratic elution gradientat RT A calibration curve was done for each standard atconcentrations from 05 to 5 ppm

25 Antioxidant Capacity 10 gL of WLP and 5 gL of ILGILB or ILF were dissolved in distilled water and HTST-juicewas diluted at 1 7 (vv) All standard curves were preparedwith up to 400120583M ascorbate (11140 Sigma)

4 Journal of Food Quality

DPPH For a stock solution 197mg of 22-diphenyl-1-picrylhydrazyl (D9132 Sigma) was dissolved in 100mLmethanol which diluted at 1 455 to a working solution hadan absorbance of sim138 at 120582 = 515 nm 100 120583L of each samplewas mixed with 900120583L DPPH working solution stored for24 h at RT in the dark and centrifuged (1min at 15000g) andthe absorbance was measured at 120582 = 515 nm

ABTS 226mg of 221015840-azinobis(3-ethylbenzothiazoline-6-sulfonate) (11557 Sigma) was dissolved in 10mL acetatebuffer (20mM pH 45) and then mixed with 10mL of144mM (NH

4)2S2O8in acetate buffer This ABTS-working

solution was incubated for 12ndash16 h at RT in the dark and thendiluted at 1 25 with acetate buffer the absorbance was sim124at 120582 = 734 nm 100 120583L of each sample was mixed with 900120583LABTS-working solution and incubated for 4 h at 37∘C in thedark and the absorbance was measured at 120582 = 734 nm

FRAP The ferric reducing antioxidant power-working solu-tion was made by mixing 25mL of TPTZ (10mM 246-tris(2-pyridyl-s-triazine) [93285 Sigma] in 40mM HCl)and 25mL of 20mM FeCl

3sdot6 H2O in distilled water with

25mL of acetate buffer (300mM pH 36) 100 120583L of eachsample was mixed with 900 120583L FRAP-working solution andincubated for 2 h at 37∘C in the dark and the absorbance wasmeasured at 120582 = 593 nm

26 Cell Culture

Cell Line andCulturingConditionsThecell lineHeLa (humancervix-uteri) was purchased from ATCC (Manassas USA)HeLa was grown in DMEM (ATCC) supplemented with10 FBS (Biowest Kansas City USA) at 37∘C 4 CO

2

and 95 RH The samples were dissolved in concentrationsup to 100 gL WLP or 50 gL ILG ILB and ILF (10x) inculture medium pH was adjusted with NaOH (1M) to pH73 and 50120583gmL of gentamycine (Sigma-Aldrich) was addedto avoid bacterial growth HTST-juice was directly added tothe medium without using antibiotics The experiments werepreviously described [27] in short the cells were seeded overcover slides in 12-well microtiter plates (MTP) and incubatedfor 1 d to let them attach the samples were added or themedium was changed for sample-containing medium andthen the cells were grown for one more day prior to analysis

Cell Viability and Necrosis Induction The metabolic activitywas determined by neutral red uptake (NRU) 20 120583LmL of033 neutral red solution (Santa Cruz USA) was addedand incubated for an additional 4 h Afterwards the coverslides werewashed oncewith phosphate buffered saline (PBS)and observed under the microscope (Axioskop 40FL ZeissOberkochen Germany) For quantification after adding30 120583LmL of 033 neutral red solution and incubation thecells were washed once with PBS and the neutral red wasextracted with 800120583L of 1 acetic acid in 50 ethanol duringa period of 15minThe supernatant was centrifuged for 3minat 10000g (Eppendorf 5415D Hamburg Germany) and theabsorbance was measured at 120582 = 690 nm For the WST-quantifyingmethod the cells were exposed to the sample andthe medium was then changed for a fresh culture medium

Table 1 Concentration of aloin and aloe-emodin in differentcommercial starting products at the concentration 1x comparedto the raw extract (HTST-juice) Values are expressed as mean plusmn196lowastSEM (119899 = 3)

Sample (1x) Aloin (120583gmL) Aloe-emodin(120583gmL)

ILG 0036 plusmn 0012 0003 plusmn 0004ILB 0069 plusmn 0007 0050 plusmn 0008ILF 0053 plusmn 0002 0130 plusmn 0010WLP 0046 plusmn 0011 0009 plusmn 0018HTST-juice 1004 plusmn 082 757 plusmn 047

Then 20120583LmL of WST-1 (Clontech Mountain View USA)was added and the cells were incubated for an additional 4 hFinally 800120583Lof themediumwas centrifuged (1min 8000g)to precipitate possible cell debris before measuring theabsorbance at 120582 = 440 nm and 120582 = 690 nm (as background)To detect necrosis 005 (final concentration) trypan blue(Biowest) was added and incubated for 5 to 10min and thecover slides were washed with PBS and then observed underthe microscope

27 Statistical Analysis Values are expressed as means plusmn196 lowast standard error of the mean (196lowastSEM) Differencesbetween groups were determined by one-way analysis ofvariance (ANOVA) and subjected post hoc to Tukeyrsquos HSDmultiple comparison tests using the program SPSS 19 A valueof 119901 lt 005was considered to indicate statistical significance

3 Results

31 Phytochemical Analysis Plant material from the enter-prise which also provided the starting products was iden-tified by ITS sequencing as Aloe verawith 98 identity to thesample KC893746 of the NCBI GenBank database

Most secondary metabolites are found in the Aloe latexrepresentative metabolites were analysed by HPLC to provewhether the decolourization process for the starting productswas efficient and whether those products fulfilled the IASCstandard of lt 10 ppm aloin The aloin content ranged from46 to 138 120583gg dry weight for the 100x or 200x concentratedpowders when those samples were diluted to the recom-mended concentration of 1x (ie 10 gL for WLP or 5 gLfor ILG ILB and ILF) the aloin concentration diminishedaccordingly to only 004ndash007120583gmL Those values wereconsiderably lower than the sim10 120583gmL of the HTST-juice(Table 1) The anthraquinone aloe-emodin was found insimilar concentrations (0003ndash013120583gmL) in 1x solutions(ie 10 gL for WLP or 5 gL for ILG ILB and ILF) and washigh only in the HTST-juice (Table 1)

Tannins or saponins were not detected in those startingproducts and a hint of terpenoids was observable only inHTST-juice Flavonoids were clearly detectable in HTST-juice barely detectable inWLP ILG and ILB and even absentin ILFThe quantitative analysis forAloe gel powders revealedthat the total phenol concentration decreased in conjunction

Journal of Food Quality 5

Table 2 Concentration of total phenols and flavonoids in different commercial starting products at the concentration 1x compared to theraw extract (HTST-juice) Values are expressed as mean plusmn 196lowastSEM (119899 = 9 for total phenols 119899 = 3 for flavonoids) ND not detected

Sample (1x) ILG ILB ILF WLP HTST-juiceTotal phenols[120583M gallate-eq] 1117 plusmn 155 923 plusmn 150 570 plusmn 158 857 plusmn 141 27124 plusmn 877

Flavonoids [120583Mcatechin-eq] ND ND ND ND 382 plusmn 41

ILG ILB ILF WLP HTST-juice0

20406080

100120140

1000

2000

3000

4000

Sample

Asc

orba

te-e

quiv

alen

t (120583

M)

a

d

g

a c

d

f

b

e

h

c

f f

5gL 5gL 5gL 10 gL

Figure 2 Antioxidant capacity of different starting products atthe concentrations of 5 gL for ILG ILB and ILF and 10 gL forWLP (ie 1x) and HTST-juice measured as FRAP (dark grey)ABTS (grey) and DPPH (light grey) Values are expressed as meanplusmn 196lowastSEM (119899 = 7ndash9) Letters indicate statistically significantdifferences

with an increase of the fibre content (Table 2) accordinglyThe HTST-juice had 25 times more total phenols than thestarting products Consistent with the qualitative analysis thequantification of flavonoids resulted to be positive only forthe HTST-juice for all other samples the concentration wasbelow the detection limit (Table 2)

32 Antioxidant Capacity (AOC) AOC was determined bythree independent methods FRAP and ABTS which mea-sure mainly water soluble antioxidants with Single ElectronTransfer (SET) and DPPH which detects methanol solubleantioxidants with SET and H-Atom-Transfer (HAT) mecha-nisms A rather consistent observation for all three methodswas found in the Aloe gel samples the more fibre the producthad the lower the AOC that is AOC was decreasing fromILG via ILB to ILF (Figure 2)WLP had a similar AOC to ILBonly the HTST-juice exhibited 20 to 30 times higher (DPPHandABTS resp) and even 80 times higher (FRAP)AOC thanthe starting products (Figure 2)

33 Cell Viability and Necrosis Results for HeLa viability forthe WLP and ILG samples have been previously reportedby us [27] ILB inhibited metabolic activity of HeLa (NRU)and induced necrosis (trypan blue) at a concentration of15 gL (3x) (Figure 4) and ILF at even 5 gL (1x) (Figure 3)the recommended concentration for human consumption

Moreover at 5 gL ILF only very few cells were still presentand much debris and fibres could be seen (Figure 3) Thiseffect was observed not only after 24 h of incubation butalready after 4 h (data not shown) which means that ILF actsvery fast Only a further reduction of the ILF concentrationto 05 gL abolished the cytotoxic effect (Figure 3) In orderto narrow the cytotoxic principle the sample was centrifugedor filtered The cytotoxicity for ILF decreased slightly nowat 5 gL ILF few cells (and less fibres) could be seen andexhibited metabolic activity visualized as NRU (Figure 3)Centrifugation of ILB hardly reduced its cytotoxicity com-pared to the noncentrifuged sample at 15 gL of centrifugedILB (3x) somemore cells were visible yet many of themwerenecrotic (Figure 4) thus centrifugation to reduce the fibrecontent was less effective for the sample which contained apriori less fibres

Those effects could not be quantified directly using theWST test the sample probably interferedwith the tetrazoliumsalt (and for the same reason LDH release as a necrosisindicator could not be determined) To address this problemthe standard protocol was changed slightly after 4 h ofexposure the ILF-containing medium was changed for freshmedium and then WST was added to determine metabolicactivity The cytotoxicity could be determined like this toCC50asymp 1 gL ILF (02x) Centrifugation as well as filtration

slightly reduced the cytotoxic effect for both techniques toCC50asymp 225 gL ILF (Figure 5) which is still below the

recommended 5 gL for human consumption30 to 80 120583LmL of HTST-juice reduced the metabolic

activity of HeLa to 50 of the control measured quantita-tively as NRUObserving the cells by themicroscope revealedthat at 60 120583LmL HeLa was metabolically still active (redstained with neutral red) at 100 120583LmL it was nearly inactive(faint red) yet normally shaped and at 300120583LmL only fewcells were seen and were not metabolically active (Figure 6)As HTST-juice has sim10 120583gmL aloin the cells had beenexposed to sim1 120583gmL aloin when metabolic activity declined(at 100 120583LmL HTST-juice) but when the cells were exposeddirectly to aloin they did not show any signs of cytotoxiceffects even at concentrations up to 50 120583gmL (Figure 6)

4 Discussion

Starting products for foodstuff from Aloe vera are mostlycommercialized as concentrated powders but the qualityamong them can vary considerably The products can beold (lt10 of polysaccharide acemannan) and suffer frombacterial fermentation (gt10 lactate) or other adulterations(typically with maltodextrin or citrate) Previous analysis

6 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 3 Effect of ILF on HeLa cells measured as NRU all images at 100x magnification and 400x magnification at the upper leftcorner Growth control wo ILF (a) at 05 gL (b) at 5 gL (c) and at 5 gL centrifugation (d) The recommended concentration for humanconsumption is 5 gL (1x) Red stained cells indicate metabolic activity and functional lysosomes

of those products revealed large differences among themalthough all have been certified by IASC For instance theacemannan content of different 200x inner leaf gel powdersranged between 4 and 9 and the aloin concentrationvaried between ldquonot detectablerdquo and up to 15 ppm [33] Thealoin concentrations for our samples ranged from 46 to138 120583gg dry weight but as the samples are 100 or 200 timesconcentrated the aloin concentration in the final productfor the consumer (meaning 10 gL for WLP or 5 gL for ILGILB and ILF ie at 1x) ranged between 004 and 007 120583gmLA comparison to the aloin content of the HTST-juice (sim10 120583gmL) demonstrates that not only the decolourization butalso the filleting and dehydration process is very effective inreducing the aloin content It is known that heat treatment(as of 80∘C) halves the aloin content of fresh Aloe geljuice within 4 h [34] The biological activity for the highlyconcentrated powders cannot be estimated according to theirchemical constitution as can readily be done for raw extracts[35] Therefore the principal constituents of our samplesregarding potentially bioactive molecules were determinedtannins saponins and terpenoids were not detected in any ofthe highly concentrated powders For fresh Aloe gel Luciniet al [5] determined 1276mmolkg gallate-eq total phenolsand 016mmolkg rutin-eq flavonoids For the HTST-juicewe found with 27mM gallate-eq total phenols and 004mMcatechin-eq flavonoids similar values but in our powdersamples in 10 gL WLP and 5 gL ILG ILB or ILF (1x)

respectively we found only 007 to 011mM gallate-eq totalphenols and flavonoids could not be detected This indicatesthat the treatment for reducing the aloin content also reducesthe content of other biologically active compounds Manystudies on the effects ofA verawere done with freshmaterialbut as the constitution of those starting products differsconsiderably from the fresh material their properties shouldbe analysed in order to address (or not) effects to endproducts

For all four powder samples when dissolved at 10 gLWLP or 5 gL ILG ILB and ILF (1x) we found AOCvalues ranging from 894 to 1228120583M ascorbate-eq for theDPPH test Lucini et al [5] using the same test measured108 120583Mtrolox-eq for fresh whole leaf extract and 54 120583Mtrolox-eq for Aloe gel extract those values are comparablebecause ascorbate and trolox have nearly the same redoxpotential [36]The decolourization process used to lower thealoin concentration also reduced the total phenol and theflavonoid content and alongwith this theAOCOnly the ILFpowder is not decolourized but dried by heat compared to thedecolourized sample (ILG) ILF has a higher aloin and aloe-emodin content but less total phenols and thus the lowestAOC of all the samples

To evaluate the consequences of these constitutional dif-ferences among the samples we performed cytotoxicologicalstudies we differentiated between a reduction of metabolicactivity (WST and NRU) and necrosis induction (trypan

Journal of Food Quality 7

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 4 Effect of ILB on HeLa cells measured as trypan blue all images at 400x magnification Growth control wo ILB (a) at 5 gL (b) at15 gL (c) and at 15 gL centrifugation (d) The recommended concentration for human consumption is 5 gL (1x) Blue stained cells indicatenecrotic cell death

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

001 01 1 1000000Concentration (gL)

Figure 5 Effects of ILF (square) centrifuged ILF (circle) andfiltered ILF (triangle) on the metabolic activity of HeLa quantifiedas relative WST activity Growth control activity was set to 100for each experiment The recommended concentration for humanconsumption is 5 gL (1x) Values are expressed asmean plusmn 196lowastSEM(119899 = 3 for ILF filtered 119899 = 4)

blue) In our case the lowest cytotoxicity on HeLa wasexhibited by both decolourized samples with 50 gL WLP(5x) or 25 gL ILG (5x) followed by approximately 15 gL ILB(3x) ILF showed cytotoxic effects in a range of 1ndash5 gL (02ndash1x) But as HeLa was not inhibited by up to 50 ppm aloin we

conclude that aloin itself is not responsible for cytotoxicityof the samples Moreover flavonoids tannins and saponinswere not detected in the sample powders therefore thesecompounds should not contribute to cytotoxicity either Thedifference between the gel fillet samples is that while ILF is awashed heat-dried and powdered fillet that still contains allof the Aloe gel fibres ILG is a decolourized and spray-driedfillet extract whose Aloe gel fibres are mostly removed whenmaking an extract (Figure 1) samples comparable to ILGhave a fibre content ranging from 30 to 64 while samplessimilar to ILF have up to 184 fibre content [35] Thesefibres contain among other polysaccharides acemannans apolymer of C

2 C3 or C6O-acetylated mannose interspaced

by a few glucose residues and branched by some 120572-16-bond galactose residues [7 8] To prove a possible cytotoxiceffect by the fibres alone the ILF sample was centrifugedor filtered and the cytotoxicity reduced slightly from CC

50

= 1 gL for the nontreated to about CC50

= 225 gL forthe centrifuged or filtered sample Thus by centrifugationor filtration the cytotoxic component could not be readilyremoved Another major difference between the samples isthe heat treatment which causes changes in the acemannancomposition compared to fresh Aloe gel When Aloe gelis dehydrated debranching reactions occur at temperaturesstarting at 60∘C especially for terminal galactose at tem-peratures starting at 70∘C deacetylation reactions increaseand the polymers gain molecular weight this is followed bybrowning reactions which start at 80∘C [37]The changes for

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

4 Journal of Food Quality

DPPH For a stock solution 197mg of 22-diphenyl-1-picrylhydrazyl (D9132 Sigma) was dissolved in 100mLmethanol which diluted at 1 455 to a working solution hadan absorbance of sim138 at 120582 = 515 nm 100 120583L of each samplewas mixed with 900120583L DPPH working solution stored for24 h at RT in the dark and centrifuged (1min at 15000g) andthe absorbance was measured at 120582 = 515 nm

ABTS 226mg of 221015840-azinobis(3-ethylbenzothiazoline-6-sulfonate) (11557 Sigma) was dissolved in 10mL acetatebuffer (20mM pH 45) and then mixed with 10mL of144mM (NH

4)2S2O8in acetate buffer This ABTS-working

solution was incubated for 12ndash16 h at RT in the dark and thendiluted at 1 25 with acetate buffer the absorbance was sim124at 120582 = 734 nm 100 120583L of each sample was mixed with 900120583LABTS-working solution and incubated for 4 h at 37∘C in thedark and the absorbance was measured at 120582 = 734 nm

FRAP The ferric reducing antioxidant power-working solu-tion was made by mixing 25mL of TPTZ (10mM 246-tris(2-pyridyl-s-triazine) [93285 Sigma] in 40mM HCl)and 25mL of 20mM FeCl

3sdot6 H2O in distilled water with

25mL of acetate buffer (300mM pH 36) 100 120583L of eachsample was mixed with 900 120583L FRAP-working solution andincubated for 2 h at 37∘C in the dark and the absorbance wasmeasured at 120582 = 593 nm

26 Cell Culture

Cell Line andCulturingConditionsThecell lineHeLa (humancervix-uteri) was purchased from ATCC (Manassas USA)HeLa was grown in DMEM (ATCC) supplemented with10 FBS (Biowest Kansas City USA) at 37∘C 4 CO

2

and 95 RH The samples were dissolved in concentrationsup to 100 gL WLP or 50 gL ILG ILB and ILF (10x) inculture medium pH was adjusted with NaOH (1M) to pH73 and 50120583gmL of gentamycine (Sigma-Aldrich) was addedto avoid bacterial growth HTST-juice was directly added tothe medium without using antibiotics The experiments werepreviously described [27] in short the cells were seeded overcover slides in 12-well microtiter plates (MTP) and incubatedfor 1 d to let them attach the samples were added or themedium was changed for sample-containing medium andthen the cells were grown for one more day prior to analysis

Cell Viability and Necrosis Induction The metabolic activitywas determined by neutral red uptake (NRU) 20 120583LmL of033 neutral red solution (Santa Cruz USA) was addedand incubated for an additional 4 h Afterwards the coverslides werewashed oncewith phosphate buffered saline (PBS)and observed under the microscope (Axioskop 40FL ZeissOberkochen Germany) For quantification after adding30 120583LmL of 033 neutral red solution and incubation thecells were washed once with PBS and the neutral red wasextracted with 800120583L of 1 acetic acid in 50 ethanol duringa period of 15minThe supernatant was centrifuged for 3minat 10000g (Eppendorf 5415D Hamburg Germany) and theabsorbance was measured at 120582 = 690 nm For the WST-quantifyingmethod the cells were exposed to the sample andthe medium was then changed for a fresh culture medium

Table 1 Concentration of aloin and aloe-emodin in differentcommercial starting products at the concentration 1x comparedto the raw extract (HTST-juice) Values are expressed as mean plusmn196lowastSEM (119899 = 3)

Sample (1x) Aloin (120583gmL) Aloe-emodin(120583gmL)

ILG 0036 plusmn 0012 0003 plusmn 0004ILB 0069 plusmn 0007 0050 plusmn 0008ILF 0053 plusmn 0002 0130 plusmn 0010WLP 0046 plusmn 0011 0009 plusmn 0018HTST-juice 1004 plusmn 082 757 plusmn 047

Then 20120583LmL of WST-1 (Clontech Mountain View USA)was added and the cells were incubated for an additional 4 hFinally 800120583Lof themediumwas centrifuged (1min 8000g)to precipitate possible cell debris before measuring theabsorbance at 120582 = 440 nm and 120582 = 690 nm (as background)To detect necrosis 005 (final concentration) trypan blue(Biowest) was added and incubated for 5 to 10min and thecover slides were washed with PBS and then observed underthe microscope

27 Statistical Analysis Values are expressed as means plusmn196 lowast standard error of the mean (196lowastSEM) Differencesbetween groups were determined by one-way analysis ofvariance (ANOVA) and subjected post hoc to Tukeyrsquos HSDmultiple comparison tests using the program SPSS 19 A valueof 119901 lt 005was considered to indicate statistical significance

3 Results

31 Phytochemical Analysis Plant material from the enter-prise which also provided the starting products was iden-tified by ITS sequencing as Aloe verawith 98 identity to thesample KC893746 of the NCBI GenBank database

Most secondary metabolites are found in the Aloe latexrepresentative metabolites were analysed by HPLC to provewhether the decolourization process for the starting productswas efficient and whether those products fulfilled the IASCstandard of lt 10 ppm aloin The aloin content ranged from46 to 138 120583gg dry weight for the 100x or 200x concentratedpowders when those samples were diluted to the recom-mended concentration of 1x (ie 10 gL for WLP or 5 gLfor ILG ILB and ILF) the aloin concentration diminishedaccordingly to only 004ndash007120583gmL Those values wereconsiderably lower than the sim10 120583gmL of the HTST-juice(Table 1) The anthraquinone aloe-emodin was found insimilar concentrations (0003ndash013120583gmL) in 1x solutions(ie 10 gL for WLP or 5 gL for ILG ILB and ILF) and washigh only in the HTST-juice (Table 1)

Tannins or saponins were not detected in those startingproducts and a hint of terpenoids was observable only inHTST-juice Flavonoids were clearly detectable in HTST-juice barely detectable inWLP ILG and ILB and even absentin ILFThe quantitative analysis forAloe gel powders revealedthat the total phenol concentration decreased in conjunction

Journal of Food Quality 5

Table 2 Concentration of total phenols and flavonoids in different commercial starting products at the concentration 1x compared to theraw extract (HTST-juice) Values are expressed as mean plusmn 196lowastSEM (119899 = 9 for total phenols 119899 = 3 for flavonoids) ND not detected

Sample (1x) ILG ILB ILF WLP HTST-juiceTotal phenols[120583M gallate-eq] 1117 plusmn 155 923 plusmn 150 570 plusmn 158 857 plusmn 141 27124 plusmn 877

Flavonoids [120583Mcatechin-eq] ND ND ND ND 382 plusmn 41

ILG ILB ILF WLP HTST-juice0

20406080

100120140

1000

2000

3000

4000

Sample

Asc

orba

te-e

quiv

alen

t (120583

M)

a

d

g

a c

d

f

b

e

h

c

f f

5gL 5gL 5gL 10 gL

Figure 2 Antioxidant capacity of different starting products atthe concentrations of 5 gL for ILG ILB and ILF and 10 gL forWLP (ie 1x) and HTST-juice measured as FRAP (dark grey)ABTS (grey) and DPPH (light grey) Values are expressed as meanplusmn 196lowastSEM (119899 = 7ndash9) Letters indicate statistically significantdifferences

with an increase of the fibre content (Table 2) accordinglyThe HTST-juice had 25 times more total phenols than thestarting products Consistent with the qualitative analysis thequantification of flavonoids resulted to be positive only forthe HTST-juice for all other samples the concentration wasbelow the detection limit (Table 2)

32 Antioxidant Capacity (AOC) AOC was determined bythree independent methods FRAP and ABTS which mea-sure mainly water soluble antioxidants with Single ElectronTransfer (SET) and DPPH which detects methanol solubleantioxidants with SET and H-Atom-Transfer (HAT) mecha-nisms A rather consistent observation for all three methodswas found in the Aloe gel samples the more fibre the producthad the lower the AOC that is AOC was decreasing fromILG via ILB to ILF (Figure 2)WLP had a similar AOC to ILBonly the HTST-juice exhibited 20 to 30 times higher (DPPHandABTS resp) and even 80 times higher (FRAP)AOC thanthe starting products (Figure 2)

33 Cell Viability and Necrosis Results for HeLa viability forthe WLP and ILG samples have been previously reportedby us [27] ILB inhibited metabolic activity of HeLa (NRU)and induced necrosis (trypan blue) at a concentration of15 gL (3x) (Figure 4) and ILF at even 5 gL (1x) (Figure 3)the recommended concentration for human consumption

Moreover at 5 gL ILF only very few cells were still presentand much debris and fibres could be seen (Figure 3) Thiseffect was observed not only after 24 h of incubation butalready after 4 h (data not shown) which means that ILF actsvery fast Only a further reduction of the ILF concentrationto 05 gL abolished the cytotoxic effect (Figure 3) In orderto narrow the cytotoxic principle the sample was centrifugedor filtered The cytotoxicity for ILF decreased slightly nowat 5 gL ILF few cells (and less fibres) could be seen andexhibited metabolic activity visualized as NRU (Figure 3)Centrifugation of ILB hardly reduced its cytotoxicity com-pared to the noncentrifuged sample at 15 gL of centrifugedILB (3x) somemore cells were visible yet many of themwerenecrotic (Figure 4) thus centrifugation to reduce the fibrecontent was less effective for the sample which contained apriori less fibres

Those effects could not be quantified directly using theWST test the sample probably interferedwith the tetrazoliumsalt (and for the same reason LDH release as a necrosisindicator could not be determined) To address this problemthe standard protocol was changed slightly after 4 h ofexposure the ILF-containing medium was changed for freshmedium and then WST was added to determine metabolicactivity The cytotoxicity could be determined like this toCC50asymp 1 gL ILF (02x) Centrifugation as well as filtration

slightly reduced the cytotoxic effect for both techniques toCC50asymp 225 gL ILF (Figure 5) which is still below the

recommended 5 gL for human consumption30 to 80 120583LmL of HTST-juice reduced the metabolic

activity of HeLa to 50 of the control measured quantita-tively as NRUObserving the cells by themicroscope revealedthat at 60 120583LmL HeLa was metabolically still active (redstained with neutral red) at 100 120583LmL it was nearly inactive(faint red) yet normally shaped and at 300120583LmL only fewcells were seen and were not metabolically active (Figure 6)As HTST-juice has sim10 120583gmL aloin the cells had beenexposed to sim1 120583gmL aloin when metabolic activity declined(at 100 120583LmL HTST-juice) but when the cells were exposeddirectly to aloin they did not show any signs of cytotoxiceffects even at concentrations up to 50 120583gmL (Figure 6)

4 Discussion

Starting products for foodstuff from Aloe vera are mostlycommercialized as concentrated powders but the qualityamong them can vary considerably The products can beold (lt10 of polysaccharide acemannan) and suffer frombacterial fermentation (gt10 lactate) or other adulterations(typically with maltodextrin or citrate) Previous analysis

6 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 3 Effect of ILF on HeLa cells measured as NRU all images at 100x magnification and 400x magnification at the upper leftcorner Growth control wo ILF (a) at 05 gL (b) at 5 gL (c) and at 5 gL centrifugation (d) The recommended concentration for humanconsumption is 5 gL (1x) Red stained cells indicate metabolic activity and functional lysosomes

of those products revealed large differences among themalthough all have been certified by IASC For instance theacemannan content of different 200x inner leaf gel powdersranged between 4 and 9 and the aloin concentrationvaried between ldquonot detectablerdquo and up to 15 ppm [33] Thealoin concentrations for our samples ranged from 46 to138 120583gg dry weight but as the samples are 100 or 200 timesconcentrated the aloin concentration in the final productfor the consumer (meaning 10 gL for WLP or 5 gL for ILGILB and ILF ie at 1x) ranged between 004 and 007 120583gmLA comparison to the aloin content of the HTST-juice (sim10 120583gmL) demonstrates that not only the decolourization butalso the filleting and dehydration process is very effective inreducing the aloin content It is known that heat treatment(as of 80∘C) halves the aloin content of fresh Aloe geljuice within 4 h [34] The biological activity for the highlyconcentrated powders cannot be estimated according to theirchemical constitution as can readily be done for raw extracts[35] Therefore the principal constituents of our samplesregarding potentially bioactive molecules were determinedtannins saponins and terpenoids were not detected in any ofthe highly concentrated powders For fresh Aloe gel Luciniet al [5] determined 1276mmolkg gallate-eq total phenolsand 016mmolkg rutin-eq flavonoids For the HTST-juicewe found with 27mM gallate-eq total phenols and 004mMcatechin-eq flavonoids similar values but in our powdersamples in 10 gL WLP and 5 gL ILG ILB or ILF (1x)

respectively we found only 007 to 011mM gallate-eq totalphenols and flavonoids could not be detected This indicatesthat the treatment for reducing the aloin content also reducesthe content of other biologically active compounds Manystudies on the effects ofA verawere done with freshmaterialbut as the constitution of those starting products differsconsiderably from the fresh material their properties shouldbe analysed in order to address (or not) effects to endproducts

For all four powder samples when dissolved at 10 gLWLP or 5 gL ILG ILB and ILF (1x) we found AOCvalues ranging from 894 to 1228120583M ascorbate-eq for theDPPH test Lucini et al [5] using the same test measured108 120583Mtrolox-eq for fresh whole leaf extract and 54 120583Mtrolox-eq for Aloe gel extract those values are comparablebecause ascorbate and trolox have nearly the same redoxpotential [36]The decolourization process used to lower thealoin concentration also reduced the total phenol and theflavonoid content and alongwith this theAOCOnly the ILFpowder is not decolourized but dried by heat compared to thedecolourized sample (ILG) ILF has a higher aloin and aloe-emodin content but less total phenols and thus the lowestAOC of all the samples

To evaluate the consequences of these constitutional dif-ferences among the samples we performed cytotoxicologicalstudies we differentiated between a reduction of metabolicactivity (WST and NRU) and necrosis induction (trypan

Journal of Food Quality 7

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 4 Effect of ILB on HeLa cells measured as trypan blue all images at 400x magnification Growth control wo ILB (a) at 5 gL (b) at15 gL (c) and at 15 gL centrifugation (d) The recommended concentration for human consumption is 5 gL (1x) Blue stained cells indicatenecrotic cell death

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

001 01 1 1000000Concentration (gL)

Figure 5 Effects of ILF (square) centrifuged ILF (circle) andfiltered ILF (triangle) on the metabolic activity of HeLa quantifiedas relative WST activity Growth control activity was set to 100for each experiment The recommended concentration for humanconsumption is 5 gL (1x) Values are expressed asmean plusmn 196lowastSEM(119899 = 3 for ILF filtered 119899 = 4)

blue) In our case the lowest cytotoxicity on HeLa wasexhibited by both decolourized samples with 50 gL WLP(5x) or 25 gL ILG (5x) followed by approximately 15 gL ILB(3x) ILF showed cytotoxic effects in a range of 1ndash5 gL (02ndash1x) But as HeLa was not inhibited by up to 50 ppm aloin we

conclude that aloin itself is not responsible for cytotoxicityof the samples Moreover flavonoids tannins and saponinswere not detected in the sample powders therefore thesecompounds should not contribute to cytotoxicity either Thedifference between the gel fillet samples is that while ILF is awashed heat-dried and powdered fillet that still contains allof the Aloe gel fibres ILG is a decolourized and spray-driedfillet extract whose Aloe gel fibres are mostly removed whenmaking an extract (Figure 1) samples comparable to ILGhave a fibre content ranging from 30 to 64 while samplessimilar to ILF have up to 184 fibre content [35] Thesefibres contain among other polysaccharides acemannans apolymer of C

2 C3 or C6O-acetylated mannose interspaced

by a few glucose residues and branched by some 120572-16-bond galactose residues [7 8] To prove a possible cytotoxiceffect by the fibres alone the ILF sample was centrifugedor filtered and the cytotoxicity reduced slightly from CC

50

= 1 gL for the nontreated to about CC50

= 225 gL forthe centrifuged or filtered sample Thus by centrifugationor filtration the cytotoxic component could not be readilyremoved Another major difference between the samples isthe heat treatment which causes changes in the acemannancomposition compared to fresh Aloe gel When Aloe gelis dehydrated debranching reactions occur at temperaturesstarting at 60∘C especially for terminal galactose at tem-peratures starting at 70∘C deacetylation reactions increaseand the polymers gain molecular weight this is followed bybrowning reactions which start at 80∘C [37]The changes for

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

Journal of Food Quality 5

Table 2 Concentration of total phenols and flavonoids in different commercial starting products at the concentration 1x compared to theraw extract (HTST-juice) Values are expressed as mean plusmn 196lowastSEM (119899 = 9 for total phenols 119899 = 3 for flavonoids) ND not detected

Sample (1x) ILG ILB ILF WLP HTST-juiceTotal phenols[120583M gallate-eq] 1117 plusmn 155 923 plusmn 150 570 plusmn 158 857 plusmn 141 27124 plusmn 877

Flavonoids [120583Mcatechin-eq] ND ND ND ND 382 plusmn 41

ILG ILB ILF WLP HTST-juice0

20406080

100120140

1000

2000

3000

4000

Sample

Asc

orba

te-e

quiv

alen

t (120583

M)

a

d

g

a c

d

f

b

e

h

c

f f

5gL 5gL 5gL 10 gL

Figure 2 Antioxidant capacity of different starting products atthe concentrations of 5 gL for ILG ILB and ILF and 10 gL forWLP (ie 1x) and HTST-juice measured as FRAP (dark grey)ABTS (grey) and DPPH (light grey) Values are expressed as meanplusmn 196lowastSEM (119899 = 7ndash9) Letters indicate statistically significantdifferences

with an increase of the fibre content (Table 2) accordinglyThe HTST-juice had 25 times more total phenols than thestarting products Consistent with the qualitative analysis thequantification of flavonoids resulted to be positive only forthe HTST-juice for all other samples the concentration wasbelow the detection limit (Table 2)

32 Antioxidant Capacity (AOC) AOC was determined bythree independent methods FRAP and ABTS which mea-sure mainly water soluble antioxidants with Single ElectronTransfer (SET) and DPPH which detects methanol solubleantioxidants with SET and H-Atom-Transfer (HAT) mecha-nisms A rather consistent observation for all three methodswas found in the Aloe gel samples the more fibre the producthad the lower the AOC that is AOC was decreasing fromILG via ILB to ILF (Figure 2)WLP had a similar AOC to ILBonly the HTST-juice exhibited 20 to 30 times higher (DPPHandABTS resp) and even 80 times higher (FRAP)AOC thanthe starting products (Figure 2)

33 Cell Viability and Necrosis Results for HeLa viability forthe WLP and ILG samples have been previously reportedby us [27] ILB inhibited metabolic activity of HeLa (NRU)and induced necrosis (trypan blue) at a concentration of15 gL (3x) (Figure 4) and ILF at even 5 gL (1x) (Figure 3)the recommended concentration for human consumption

Moreover at 5 gL ILF only very few cells were still presentand much debris and fibres could be seen (Figure 3) Thiseffect was observed not only after 24 h of incubation butalready after 4 h (data not shown) which means that ILF actsvery fast Only a further reduction of the ILF concentrationto 05 gL abolished the cytotoxic effect (Figure 3) In orderto narrow the cytotoxic principle the sample was centrifugedor filtered The cytotoxicity for ILF decreased slightly nowat 5 gL ILF few cells (and less fibres) could be seen andexhibited metabolic activity visualized as NRU (Figure 3)Centrifugation of ILB hardly reduced its cytotoxicity com-pared to the noncentrifuged sample at 15 gL of centrifugedILB (3x) somemore cells were visible yet many of themwerenecrotic (Figure 4) thus centrifugation to reduce the fibrecontent was less effective for the sample which contained apriori less fibres

Those effects could not be quantified directly using theWST test the sample probably interferedwith the tetrazoliumsalt (and for the same reason LDH release as a necrosisindicator could not be determined) To address this problemthe standard protocol was changed slightly after 4 h ofexposure the ILF-containing medium was changed for freshmedium and then WST was added to determine metabolicactivity The cytotoxicity could be determined like this toCC50asymp 1 gL ILF (02x) Centrifugation as well as filtration

slightly reduced the cytotoxic effect for both techniques toCC50asymp 225 gL ILF (Figure 5) which is still below the

recommended 5 gL for human consumption30 to 80 120583LmL of HTST-juice reduced the metabolic

activity of HeLa to 50 of the control measured quantita-tively as NRUObserving the cells by themicroscope revealedthat at 60 120583LmL HeLa was metabolically still active (redstained with neutral red) at 100 120583LmL it was nearly inactive(faint red) yet normally shaped and at 300120583LmL only fewcells were seen and were not metabolically active (Figure 6)As HTST-juice has sim10 120583gmL aloin the cells had beenexposed to sim1 120583gmL aloin when metabolic activity declined(at 100 120583LmL HTST-juice) but when the cells were exposeddirectly to aloin they did not show any signs of cytotoxiceffects even at concentrations up to 50 120583gmL (Figure 6)

4 Discussion

Starting products for foodstuff from Aloe vera are mostlycommercialized as concentrated powders but the qualityamong them can vary considerably The products can beold (lt10 of polysaccharide acemannan) and suffer frombacterial fermentation (gt10 lactate) or other adulterations(typically with maltodextrin or citrate) Previous analysis

6 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 3 Effect of ILF on HeLa cells measured as NRU all images at 100x magnification and 400x magnification at the upper leftcorner Growth control wo ILF (a) at 05 gL (b) at 5 gL (c) and at 5 gL centrifugation (d) The recommended concentration for humanconsumption is 5 gL (1x) Red stained cells indicate metabolic activity and functional lysosomes

of those products revealed large differences among themalthough all have been certified by IASC For instance theacemannan content of different 200x inner leaf gel powdersranged between 4 and 9 and the aloin concentrationvaried between ldquonot detectablerdquo and up to 15 ppm [33] Thealoin concentrations for our samples ranged from 46 to138 120583gg dry weight but as the samples are 100 or 200 timesconcentrated the aloin concentration in the final productfor the consumer (meaning 10 gL for WLP or 5 gL for ILGILB and ILF ie at 1x) ranged between 004 and 007 120583gmLA comparison to the aloin content of the HTST-juice (sim10 120583gmL) demonstrates that not only the decolourization butalso the filleting and dehydration process is very effective inreducing the aloin content It is known that heat treatment(as of 80∘C) halves the aloin content of fresh Aloe geljuice within 4 h [34] The biological activity for the highlyconcentrated powders cannot be estimated according to theirchemical constitution as can readily be done for raw extracts[35] Therefore the principal constituents of our samplesregarding potentially bioactive molecules were determinedtannins saponins and terpenoids were not detected in any ofthe highly concentrated powders For fresh Aloe gel Luciniet al [5] determined 1276mmolkg gallate-eq total phenolsand 016mmolkg rutin-eq flavonoids For the HTST-juicewe found with 27mM gallate-eq total phenols and 004mMcatechin-eq flavonoids similar values but in our powdersamples in 10 gL WLP and 5 gL ILG ILB or ILF (1x)

respectively we found only 007 to 011mM gallate-eq totalphenols and flavonoids could not be detected This indicatesthat the treatment for reducing the aloin content also reducesthe content of other biologically active compounds Manystudies on the effects ofA verawere done with freshmaterialbut as the constitution of those starting products differsconsiderably from the fresh material their properties shouldbe analysed in order to address (or not) effects to endproducts

For all four powder samples when dissolved at 10 gLWLP or 5 gL ILG ILB and ILF (1x) we found AOCvalues ranging from 894 to 1228120583M ascorbate-eq for theDPPH test Lucini et al [5] using the same test measured108 120583Mtrolox-eq for fresh whole leaf extract and 54 120583Mtrolox-eq for Aloe gel extract those values are comparablebecause ascorbate and trolox have nearly the same redoxpotential [36]The decolourization process used to lower thealoin concentration also reduced the total phenol and theflavonoid content and alongwith this theAOCOnly the ILFpowder is not decolourized but dried by heat compared to thedecolourized sample (ILG) ILF has a higher aloin and aloe-emodin content but less total phenols and thus the lowestAOC of all the samples

To evaluate the consequences of these constitutional dif-ferences among the samples we performed cytotoxicologicalstudies we differentiated between a reduction of metabolicactivity (WST and NRU) and necrosis induction (trypan

Journal of Food Quality 7

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 4 Effect of ILB on HeLa cells measured as trypan blue all images at 400x magnification Growth control wo ILB (a) at 5 gL (b) at15 gL (c) and at 15 gL centrifugation (d) The recommended concentration for human consumption is 5 gL (1x) Blue stained cells indicatenecrotic cell death

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

001 01 1 1000000Concentration (gL)

Figure 5 Effects of ILF (square) centrifuged ILF (circle) andfiltered ILF (triangle) on the metabolic activity of HeLa quantifiedas relative WST activity Growth control activity was set to 100for each experiment The recommended concentration for humanconsumption is 5 gL (1x) Values are expressed asmean plusmn 196lowastSEM(119899 = 3 for ILF filtered 119899 = 4)

blue) In our case the lowest cytotoxicity on HeLa wasexhibited by both decolourized samples with 50 gL WLP(5x) or 25 gL ILG (5x) followed by approximately 15 gL ILB(3x) ILF showed cytotoxic effects in a range of 1ndash5 gL (02ndash1x) But as HeLa was not inhibited by up to 50 ppm aloin we

conclude that aloin itself is not responsible for cytotoxicityof the samples Moreover flavonoids tannins and saponinswere not detected in the sample powders therefore thesecompounds should not contribute to cytotoxicity either Thedifference between the gel fillet samples is that while ILF is awashed heat-dried and powdered fillet that still contains allof the Aloe gel fibres ILG is a decolourized and spray-driedfillet extract whose Aloe gel fibres are mostly removed whenmaking an extract (Figure 1) samples comparable to ILGhave a fibre content ranging from 30 to 64 while samplessimilar to ILF have up to 184 fibre content [35] Thesefibres contain among other polysaccharides acemannans apolymer of C

2 C3 or C6O-acetylated mannose interspaced

by a few glucose residues and branched by some 120572-16-bond galactose residues [7 8] To prove a possible cytotoxiceffect by the fibres alone the ILF sample was centrifugedor filtered and the cytotoxicity reduced slightly from CC

50

= 1 gL for the nontreated to about CC50

= 225 gL forthe centrifuged or filtered sample Thus by centrifugationor filtration the cytotoxic component could not be readilyremoved Another major difference between the samples isthe heat treatment which causes changes in the acemannancomposition compared to fresh Aloe gel When Aloe gelis dehydrated debranching reactions occur at temperaturesstarting at 60∘C especially for terminal galactose at tem-peratures starting at 70∘C deacetylation reactions increaseand the polymers gain molecular weight this is followed bybrowning reactions which start at 80∘C [37]The changes for

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

6 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 3 Effect of ILF on HeLa cells measured as NRU all images at 100x magnification and 400x magnification at the upper leftcorner Growth control wo ILF (a) at 05 gL (b) at 5 gL (c) and at 5 gL centrifugation (d) The recommended concentration for humanconsumption is 5 gL (1x) Red stained cells indicate metabolic activity and functional lysosomes

of those products revealed large differences among themalthough all have been certified by IASC For instance theacemannan content of different 200x inner leaf gel powdersranged between 4 and 9 and the aloin concentrationvaried between ldquonot detectablerdquo and up to 15 ppm [33] Thealoin concentrations for our samples ranged from 46 to138 120583gg dry weight but as the samples are 100 or 200 timesconcentrated the aloin concentration in the final productfor the consumer (meaning 10 gL for WLP or 5 gL for ILGILB and ILF ie at 1x) ranged between 004 and 007 120583gmLA comparison to the aloin content of the HTST-juice (sim10 120583gmL) demonstrates that not only the decolourization butalso the filleting and dehydration process is very effective inreducing the aloin content It is known that heat treatment(as of 80∘C) halves the aloin content of fresh Aloe geljuice within 4 h [34] The biological activity for the highlyconcentrated powders cannot be estimated according to theirchemical constitution as can readily be done for raw extracts[35] Therefore the principal constituents of our samplesregarding potentially bioactive molecules were determinedtannins saponins and terpenoids were not detected in any ofthe highly concentrated powders For fresh Aloe gel Luciniet al [5] determined 1276mmolkg gallate-eq total phenolsand 016mmolkg rutin-eq flavonoids For the HTST-juicewe found with 27mM gallate-eq total phenols and 004mMcatechin-eq flavonoids similar values but in our powdersamples in 10 gL WLP and 5 gL ILG ILB or ILF (1x)

respectively we found only 007 to 011mM gallate-eq totalphenols and flavonoids could not be detected This indicatesthat the treatment for reducing the aloin content also reducesthe content of other biologically active compounds Manystudies on the effects ofA verawere done with freshmaterialbut as the constitution of those starting products differsconsiderably from the fresh material their properties shouldbe analysed in order to address (or not) effects to endproducts

For all four powder samples when dissolved at 10 gLWLP or 5 gL ILG ILB and ILF (1x) we found AOCvalues ranging from 894 to 1228120583M ascorbate-eq for theDPPH test Lucini et al [5] using the same test measured108 120583Mtrolox-eq for fresh whole leaf extract and 54 120583Mtrolox-eq for Aloe gel extract those values are comparablebecause ascorbate and trolox have nearly the same redoxpotential [36]The decolourization process used to lower thealoin concentration also reduced the total phenol and theflavonoid content and alongwith this theAOCOnly the ILFpowder is not decolourized but dried by heat compared to thedecolourized sample (ILG) ILF has a higher aloin and aloe-emodin content but less total phenols and thus the lowestAOC of all the samples

To evaluate the consequences of these constitutional dif-ferences among the samples we performed cytotoxicologicalstudies we differentiated between a reduction of metabolicactivity (WST and NRU) and necrosis induction (trypan

Journal of Food Quality 7

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 4 Effect of ILB on HeLa cells measured as trypan blue all images at 400x magnification Growth control wo ILB (a) at 5 gL (b) at15 gL (c) and at 15 gL centrifugation (d) The recommended concentration for human consumption is 5 gL (1x) Blue stained cells indicatenecrotic cell death

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

001 01 1 1000000Concentration (gL)

Figure 5 Effects of ILF (square) centrifuged ILF (circle) andfiltered ILF (triangle) on the metabolic activity of HeLa quantifiedas relative WST activity Growth control activity was set to 100for each experiment The recommended concentration for humanconsumption is 5 gL (1x) Values are expressed asmean plusmn 196lowastSEM(119899 = 3 for ILF filtered 119899 = 4)

blue) In our case the lowest cytotoxicity on HeLa wasexhibited by both decolourized samples with 50 gL WLP(5x) or 25 gL ILG (5x) followed by approximately 15 gL ILB(3x) ILF showed cytotoxic effects in a range of 1ndash5 gL (02ndash1x) But as HeLa was not inhibited by up to 50 ppm aloin we

conclude that aloin itself is not responsible for cytotoxicityof the samples Moreover flavonoids tannins and saponinswere not detected in the sample powders therefore thesecompounds should not contribute to cytotoxicity either Thedifference between the gel fillet samples is that while ILF is awashed heat-dried and powdered fillet that still contains allof the Aloe gel fibres ILG is a decolourized and spray-driedfillet extract whose Aloe gel fibres are mostly removed whenmaking an extract (Figure 1) samples comparable to ILGhave a fibre content ranging from 30 to 64 while samplessimilar to ILF have up to 184 fibre content [35] Thesefibres contain among other polysaccharides acemannans apolymer of C

2 C3 or C6O-acetylated mannose interspaced

by a few glucose residues and branched by some 120572-16-bond galactose residues [7 8] To prove a possible cytotoxiceffect by the fibres alone the ILF sample was centrifugedor filtered and the cytotoxicity reduced slightly from CC

50

= 1 gL for the nontreated to about CC50

= 225 gL forthe centrifuged or filtered sample Thus by centrifugationor filtration the cytotoxic component could not be readilyremoved Another major difference between the samples isthe heat treatment which causes changes in the acemannancomposition compared to fresh Aloe gel When Aloe gelis dehydrated debranching reactions occur at temperaturesstarting at 60∘C especially for terminal galactose at tem-peratures starting at 70∘C deacetylation reactions increaseand the polymers gain molecular weight this is followed bybrowning reactions which start at 80∘C [37]The changes for

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

Journal of Food Quality 7

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 4 Effect of ILB on HeLa cells measured as trypan blue all images at 400x magnification Growth control wo ILB (a) at 5 gL (b) at15 gL (c) and at 15 gL centrifugation (d) The recommended concentration for human consumption is 5 gL (1x) Blue stained cells indicatenecrotic cell death

0

20

40

60

80

100

120

Rela

tive a

ctiv

ity (

)

001 01 1 1000000Concentration (gL)

Figure 5 Effects of ILF (square) centrifuged ILF (circle) andfiltered ILF (triangle) on the metabolic activity of HeLa quantifiedas relative WST activity Growth control activity was set to 100for each experiment The recommended concentration for humanconsumption is 5 gL (1x) Values are expressed asmean plusmn 196lowastSEM(119899 = 3 for ILF filtered 119899 = 4)

blue) In our case the lowest cytotoxicity on HeLa wasexhibited by both decolourized samples with 50 gL WLP(5x) or 25 gL ILG (5x) followed by approximately 15 gL ILB(3x) ILF showed cytotoxic effects in a range of 1ndash5 gL (02ndash1x) But as HeLa was not inhibited by up to 50 ppm aloin we

conclude that aloin itself is not responsible for cytotoxicityof the samples Moreover flavonoids tannins and saponinswere not detected in the sample powders therefore thesecompounds should not contribute to cytotoxicity either Thedifference between the gel fillet samples is that while ILF is awashed heat-dried and powdered fillet that still contains allof the Aloe gel fibres ILG is a decolourized and spray-driedfillet extract whose Aloe gel fibres are mostly removed whenmaking an extract (Figure 1) samples comparable to ILGhave a fibre content ranging from 30 to 64 while samplessimilar to ILF have up to 184 fibre content [35] Thesefibres contain among other polysaccharides acemannans apolymer of C

2 C3 or C6O-acetylated mannose interspaced

by a few glucose residues and branched by some 120572-16-bond galactose residues [7 8] To prove a possible cytotoxiceffect by the fibres alone the ILF sample was centrifugedor filtered and the cytotoxicity reduced slightly from CC

50

= 1 gL for the nontreated to about CC50

= 225 gL forthe centrifuged or filtered sample Thus by centrifugationor filtration the cytotoxic component could not be readilyremoved Another major difference between the samples isthe heat treatment which causes changes in the acemannancomposition compared to fresh Aloe gel When Aloe gelis dehydrated debranching reactions occur at temperaturesstarting at 60∘C especially for terminal galactose at tem-peratures starting at 70∘C deacetylation reactions increaseand the polymers gain molecular weight this is followed bybrowning reactions which start at 80∘C [37]The changes for

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

8 Journal of Food Quality

50120583m

(a)

50120583m

(b)

50120583m

(c)

50120583m

(d)

Figure 6 Effects of HTST-juice on HeLa measured as NRU red stained cells indicate metabolic activity (magnification 100x) 60120583LmL (a)100 120583LmL (b) 300 120583LmL (c) and HTST-juice and 50 120583gmL aloin (d)

pasteurized Aloe gel are similar deacetylation and debranch-ing reactions are observed as well as molecular weight gains[38] Deacetylation reactions cause major structural changeswithin acemannans which affect their biological activityperhaps because of an altered affinity to members of themannose receptor family however cytotoxic effects were notobserved [39] Thus it is likely that the fibre itself and evenafter being altered by heat treatment did not induce thecytotoxic effect either On the other hand the browning ofAloe samples at higher temperatures indicates that complexMaillard reactions occurred although the precise changeswhich introduce those reactions still remain unclear TheILF sample received a heat treatment (belt-drying) at highertemperatures (70ndash85∘C) than all other samples and thusit is reasonable that the heat treatment possibly combinedwith a high fibre content introduces a toxic principle theexact nature and mode of action remain to be elucidated Wefound only one study on starting products which reported acytotoxic concentration of CC

50= 027 gL1 for a heat-dried

gel powder on HeLa [40] which is in a similar range to whatwe have found for ILF On the other hand the same studyreported a cytotoxic concentration of CC

50= 041 gL1 for a

whole leaf powder which would be considerably lower thanour result of about 50 gL WLP (5x)

Finally we could show that the standard procedures toreduce the aloin content also reduced other biologically activecompounds for instance tannins saponins and terpenoidsNevertheless the antioxidant capacity was still comparable tofresh extracts On the other hand we detected that the fibre

content correlated with a very fast (less than 4 h) cytotoxiceffect and could exclude the aloin content being responsiblefor that The strong cytotoxic effect is probably not causedexclusively by the high fibre content itself but by chemicalreactions that occur when those fibres are exposed to heattreatment as was done for ILF however the nature of thecytotoxic substance has yet to be elucidated Therefore more(cyto)toxicological studies with starting products should bedone especially when those products receive a heat treatmentand contain a high fibre content

Abbreviations

ABTS 221015840-Azinobis(3-ethylbenzothiazoline-6-sulfonate)

AOC Antioxidant capacityATCC American Type Culture CollectionDMEM Dulbeccorsquos modified Eaglersquos mediumDPPH 22-Diphenyl-1-picrylhydrazylFBS Fetal bovine serumFDA Food and Drug AdministrationFRAP Ferric reducing antioxidant powerHPLC High pressure liquid chromatographyHTST High temperature short timeILB Aloe inner leaf blended powderILF Aloe inner leaf fibre powderILG Aloe inner leaf gel powderISAC International Aloe Science CouncilNRU Neutral red uptake

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

Journal of Food Quality 9

NTP National Toxicology ProgramPBS Phosphate buffered salineRH Relative humidityRT Room temperatureWLP Aloe whole leaf powderWST Water soluble tetrazolium

Disclosure

Gildardo Rivera holds a scholarship from the Comision deOperacion y Fomento de Actividades Academicas (COFAA-Instituto Politecnico Nacional) and from the Programa deEstımulos al Desempeno de los Investigadores (EDI-InstitutoPolitecnico Nacional)

Competing Interests

The authors report no competing interests regarding thepublication of this paper

Acknowledgments

The authors are grateful to the Aloe vera producers inTamaulipas Mexico for providing the samples used in thisstudyThis research was funded by a public project (FOMIX-Tamaulipas 195867) and therefore there was no influence bythe industries that provided the samples

Endnotes

1 We assume these concentrations The authors stated inthe Materials and Methods that they dissolved 10 gL =10000mgL powder in culture medium (resulting for atypical 100x concentrated starting product powder in afinal concentration of 1x) and from this solution theymade serial dilutions which (as we suppose) range from001 to 1000mgL The authors claim that the dilutionsrange from 001 to 1000mgmL = gL but then thehighest concentration (ie 1000mgmL = 1000 gL =1 kgL)wouldmean a 100-fold higher concentration thanthe initial solution (and it is hard to imagine dissolving1 kg 100x Aloe powder in 1 L of water or medium)therefore we suppose a typo (mL versus L)

References

[1] The Plant List ldquoA working list of all plant speciesrdquo 2013 httpwwwtheplantlistorgtpl11searchq=aloe

[2] The Angiosperm Phylogeny Group (APG) ldquoAn ordinal clas-sification for the families of flowering plantsrdquo Annals of theMissouri Botanical Garden vol 85 no 4 pp 531ndash553 1998

[3] R Upton and P Axentiev Eds Aloe Vera Leaf Aloe Vera LeafJuice Aloe Vera Inner Leaf Juice Standards of Identity Analysisand Quality Control AHP 2012

[4] M Ahmed and F Hussain ldquoChemical composition and bio-chemical activity of Aloe vera (Aloe barbadensisMiller) leavesrdquoInternational Journal of Chemical and Biochemical Sciences vol3 pp 29ndash33 2013

[5] L Lucini M Pellizzoni R Pellegrino G P Molinari andG Colla ldquoPhytochemical constituents and in vitro radicalscavenging activity of different Aloespeciesrdquo Food Chemistryvol 170 pp 501ndash507 2015

[6] F Nejatzadeh-Barandozi ldquoAntibacterial activities and antioxi-dant capacity of Aloe verardquo Organic and Medicinal ChemistryLetters vol 3 no 1 p 5 2013

[7] A Femenia E S Sanchez S Simal and C Rossello ldquoCom-positional features of polysaccharides from Aloe vera (Aloebarbadensis Miller) plant tissuesrdquo Carbohydrate Polymers vol39 no 2 pp 109ndash117 1999

[8] T Reynolds and A C Dweck ldquoAloe vera leaf gel a reviewupdaterdquo Journal of Ethnopharmacology vol 68 no 1-3 pp 3ndash37 1999

[9] T Reynolds ldquoThe compounds in Aloeleaf exudates a reviewrdquoBotanical Journal of the Linnean Society vol 90 no 3 pp 157ndash177 1985

[10] T Reynolds ldquoAloe chemistryrdquo in Aloes The Genus Aloe TReynolds Ed vol 38 pp 54ndash89 CRC Press Boca Raton FlaUSA 2004

[11] C Domınguez-Barradas G E Cruz-Morales and C Gonzalez-Gandara ldquoPlantas de uso medicinal de la reserva ecologicalsquoSierra deOtontepecrsquo municipio de Chontla VeracruzMexicordquoCienciaUAT vol 9 pp 41ndash52 2015

[12] L E Newton ldquoAloes in habitatrdquo in Aloes The genus Aloe TReynolds Ed vol 38 pp 19ndash30 CRC Press Boca Raton FlaUSA 2004

[13] M Foster D Hunter and S Samman ldquoEvaluation of the nutri-tional and metabolic effects of Aloe verardquo in Herbal MedicineS Wachtel-Galor Ed pp 37ndash54 CRC Press Boca Raton FlaUSA 2nd edition 2011

[14] V Steenkamp and M J Stewart ldquoMedicinal applications andtoxicological activities of Aloeproductsrdquo Pharmaceutical Biol-ogy vol 45 no 5 pp 411ndash420 2007

[15] B K Vogler and E Ernst ldquoAloe vera a systematic review of itsclinical effectivenessrdquoBritish Journal of General Practice vol 49no 447 pp 823ndash828 1999

[16] E J Buenz ldquoAloin induces apoptosis in Jurkat cellsrdquo Toxicologyin Vitro vol 22 no 2 pp 422ndash429 2008

[17] H C Chen W T Hsieh W C Chang and J G Chung ldquoAloe-emodin induced in vitro G2M arrest of cell cycle in humanpromyelocytic leukemia HL-60 cellsrdquo Food and Chemical Toxi-cology vol 42 no 8 pp 1251ndash1257 2004

[18] J-M Guo B-X Xiao Q Liu S Zhang D-H Liu and Z-HGong ldquoAnticancer effect of aloe-emodin on cervical cancer cellsinvolves G2M arrest and induction of differentiationrdquo ActaPharmacologica Sinica vol 28 no 12 pp 1991ndash1995 2007

[19] J Jose S Sudhakaran T M Sumesh Kumar S Jayaraman andE Jayadevi Variyar ldquoA comparative evaluation of anticanceractivities of flavonoids isolated from Mimosa pudica Aloe veraand Phyllanthus niruri against human breast carcinoma cell line(MCF-7) using MTT assayrdquo International Journal of Pharmacyand Pharmaceutical Sciences vol 6 no 2 pp 319ndash322 2014

[20] B A P Van Gorkom E G E De Vries A Karrenbeld and JH Kleibeuker ldquoReview article anthranoid laxatives and theirpotential carcinogenic effectsrdquo Alimentary Pharmacology andTherapeutics vol 13 no 4 pp 443ndash452 1999

[21] C I R E Panel ldquoFinal report on the safety assessment ofAloe andongensis extract Aloe andongensis leaf juice Aloearborescens leaf extract Aloe arborescens leaf juice Aloearborescens leaf protoplasts Aloe barbadensis flower extract

10 Journal of Food Quality

Aloe barbadensis leaf Aloe barbadensis leaf extract Aloe bar-badensis leaf juice Aloe barbadensis leaf polysaccharides Aloebarbadensis leaf water Aloe ferox leaf extract Aloe ferox leafjuice and Aloe ferox leaf juice extractrdquo International Journal ofToxicology vol 26 no 2 pp 1ndash50 2007

[22] Food and Drug Administration and HHS ldquoStatus of certainadditional over-the-counter drug category II and III activeingredients Final rulerdquo Federal Register vol 67 no 90 pp31123ndash31125 2002

[23] IASC Certification Program Policies and Operational Proce-dures SOP Final SOP International Aloe Science Council 2012

[24] M D Boudreau P W Mellick G R Olson R P Felton B TThorn andFA Beland ldquoClear evidence of carcinogenic activityby a whole-leaf extract of Aloe barbadensis Miller (Aloe vera)in F344N ratsrdquo Toxicological Sciences vol 131 no 1 pp 26ndash392013

[25] S Dentali ldquorsquoNondecolorizedrsquo essential qualifier for NTP Aloevera studymaterialrdquo Toxicological Sciences vol 133 no 2 article342 2013

[26] I SehgalW DWintersM Scott et al ldquoToxicologic assessmentof a commercial decolorized whole leaf Aloe vera juice lily ofthe desert filtered whole leaf juice with Aloesorbrdquo Journal ofToxicology vol 2013 Article ID 802453 12 pages 2013

[27] Z Lopez P Knauth G Avalos-Navarro et al ldquoCytotoxic effectsof Sabila (Aloe vera) commercial products as food supplementrdquoJournal of Chemical Biological and Physical Sciences vol 4 pp47ndash54 2014

[28] T J White T Burns S Lee and JW Taylor ldquoAmplification anddirect sequencing of fungal ribosomal rna genes for phyloge-neticsrdquo in PCR Protocols A Guide to Methods and ApplicationsM A Innis D H Gelfand J J Sninsky and T J White Edspp 315ndash322 American Press Cambridge Mass USA 1990

[29] S F Altschul T L Madden A A Schaffer et al ldquoGappedBLAST and PSI-BLAST a new generation of protein databasesearch programsrdquo Nucleic Acids Research vol 25 no 17 pp3389ndash3402 1997

[30] H O Edeoga D E Okwu and B O Mbaebie ldquoPhytochemicalconstituents of some Nigerian medicinal plantsrdquo African Jour-nal of Biotechnology vol 4 no 7 pp 685ndash688 2005

[31] K Thaipong U Boonprakob K Crosby L Cisneros-Zevallosand D Hawkins Byrne ldquoComparison of ABTS DPPH FRAPandORACassays for estimating antioxidant activity fromguavafruit extractsrdquo Journal of Food Composition and Analysis vol 19no 6-7 pp 669ndash675 2006

[32] M Liu X Q Li C Weber C Y Lee J Brown and R HLiu ldquoAntioxidant and antiproliferative activities of raspberriesrdquoJournal of Agricultural and Food Chemistry vol 50 no 10 pp2926ndash2930 2002

[33] A Bozzi C Perrin S Austin and F Arce Vera ldquoQuality andauthenticity of commercial Aloe vera gel powdersrdquo Food Chem-istry vol 103 pp 22ndash30 2007

[34] X L Chang CWang Y Feng and Z Liu ldquoEffects of heat treat-ments on the stabilities of polysaccharides substances andbarbaloin in gel juice from Aloe vera Millerrdquo Journal of FoodEngineering vol 75 no 2 pp 245ndash251 2006

[35] C E Turner D A Williamson P A Stroud and D JTalley ldquoEvaluation and comparison of commercially availableAloe vera L products using size exclusion chromatographywith refractive index and multi-angle laser light scatteringdetectionrdquo International Immunopharmacology vol 4 no 14pp 1727ndash1737 2004

[36] S S Marques L M Magalhaes I V Toth and M A SegundoldquoInsights on antioxidant assays for biological samples based onthe reduction of copper complexesmdashthe importance of analyti-cal conditionsrdquo International Journal of Molecular Sciences vol15 no 7 pp 11387ndash11402 2014

[37] A Femenia PGarcıa-Pascual S Simal andC Rossello ldquoEffectsof heat treatment and dehydration on bioactive polysaccharideacemannan and cell wall polymers from Aloe barbadensisMillerrdquoCarbohydrate Polymers vol 51 no 4 pp 397ndash405 2003

[38] V M Rodrıguez-Gonzalez A Femenia R F Gonzalez-Laredoet al ldquoEffects of pasteurization on bioactive polysaccharideacemannan and cell wall polymers from Aloe barbadensisMillerrdquo Carbohydrate Polymers vol 86 no 4 pp 1675ndash16832011

[39] J Chokboribal W Tachaboonyakiat P Sangvanich V Ruang-pornvisuti S Jettanacheawchankit and P ThunyakitpisalldquoDeacetylation affects the physical properties and bioactivityof acemannan an extracted polysaccharide from Aloe verardquoCarbohydrate Polymers vol 133 pp 556ndash566 2015

[40] L H Du Plessis and J H Hamman ldquoIn vitro evaluation of thecytotoxic and apoptogenic properties of Aloewhole leaf and gelmaterialsrdquoDrug and Chemical Toxicology vol 37 no 2 pp 169ndash177 2014

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology