Digestion and colonic fermentation of raw and cooked Opuntia ficus-indica

cladodes impacts bioaccessibility and bioactivity

Elsy De Santiagoa, Chris I. R. Gillb, Ilaria Carafac, Kieran M. Touhyc, María-Paz

De Peñaa* and Concepción Cida

a Universidad de Navarra, Facultad de Farmacia y Nutrición, Departamento de Ciencias

de la Alimentación y Fisiología, C/ Irunlarrea 1, E-31008 Pamplona, Spain.

IdiSNA, Navarra Institute for Health Research. Pamplona, Spain.

b Nutrition Innovation Centre for Food and Health, Centre for Molecular Biosciences,

University of Ulster, Cromore Road, Coleraine, Northern Ireland, BT52 1SA, United

Kingdom.

c Nutrition & Nutrigenomics Unit, Department of Food Quality and Nutrition, Research

and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010, San

Michele all’Adige, Trento, Italy.

*Corresponding author: María-Paz de Peña. Tel: +34 948 425600 (806580); Fax: +34

948 425740. E-mail address: mpdepena@unav.es

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ABSTRACT

The bioactivity of (poly)phenols from a food are interplay between the cooking methods

applied and its interaction with the gastrointestinal tract. (Poly)phenolic profile and

biological activity of raw and cooked cactus (Opuntia ficus-indica Mill.) cladodes

following in vitro digestion and colonic fermentation were evaluated. Twenty-seven

(poly)phenols were identified and quantified by HPLC-ESI-MS, with piscidic acid the

most abundant. Throughout the colonic fermentation, flavonoids showed more

degradation than phenolic acids, remained eucomic acid the most relevant after 24 h.

The catabolite 3-(4-hydroxyphenyl)propionic acid was generated after 24 h of

fermentation incubation. Cytotoxicity, genotoxicity and cell cycle analyses were

performed in HT29 cells. Cactus colonic fermentates showed higher cell viability

(80%) compared to the control fermentation with no cactus, and significantly (p<0.05)

reduced H2O2-induced DNA damage in HT29 cells. Results suggest that, although

phenolic compounds were degraded during the colonic fermentation, the biological

activity is retained in colon cells.

KEYWORDS: Opuntia ficus-indica, Polyphenols, Gut microbiota, Antioxidant

activity, DNA damage, cytotoxicity.

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Introduction

Cactus (Opuntia ficus-indica Mill.) is an American native plant, which farming is

slowly extending throughout the World. In Mexico cactus cladodes, known as “nopales”

or “nopalitos”, are highly consumed (6.4 kg per capita per year) 1 after cooking in

different styles (i.e., boiling, griddling, frying and microwaving). It is being recognized

as a great source of (poly)phenols, predominating flavonoids as isorhamnetin,

kaempferol and quercetin derivatives, as well as phenolic acids like piscidic and

eucomic acids 2–4, especially in young cladodes, which are the preferred ones for human

consumption and that have higher amount of (poly)phenolic compounds and

consequently higher antioxidant activity than the old ones 5,6.

The dietary intake of (poly)phenolic compounds has been studied for decades because

of their beneficial and protective effects on different chronic diseases such as cancer,

diabetes and cardiovascular disease due to their antioxidant properties to reduce

inflammation caused by oxidative stress 7. Previously, it has been demonstrated that

after consumption, digestive conditions and enzymes affect (poly)phenols

bioaccessibility of cactus cladodes, degrading or retaining more flavonoids (37-63%

bioaccessibility) than phenolic acids (56-87% bioaccessibility) even both compounds

still remaining after the gastrointestinal digestion process, especially when cactus

cladodes were cooked 8. Then, bioaccessible compounds can be absorbed through the

stomach and small intestine, or most of them could reach the colon and be extensively

catabolised by the human gut microbiota 9.

(Poly)phenols biotransformations can occur in the colon, in which the enzymes of the

gut microbiota act to breakdown complex polyphenolic structures by hydrolysis,

dehydroxylation, deconjugation, demethylation, ring cleavage and decarboxylation

reactions. Some studies have shown the influence of colonic human microbiota on the

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phenolic profile and catabolites produced in some vegetables as pepper 10, and cardoon

11 as well as in coffee 12 and lingonberries 13 .

Otherwise, the remaining polyphenols which reach the colon could modulate metabolic

pathways related to the gut microbiota and cell processes, as inflammation, immunity,

cell proliferation and oxidative stress 14. Recently, studies have reported the biological

and antioxidant activity of (poly)phenols from raspberries after colonic fermentation on

HT29 human colon cells reducing DNA damage 15. Furthermore, extracts from O. ficus-

indica juices of cactus pear fruits have also demonstrated antiproliferative effects on

HT29 cells 16, even intestinal digestion and gut microbiota action were not considered.

In light of the scientific evidence about the influence of gut microbiota on

(poly)phenolic compounds, and subsequently their biological activity, as well as the

increasing interest on cactus cladodes potential bioactivity, the aim of the current study

was to know how the human colonic microbiota affects the dietary (poly)phenolic

compounds of raw and cooked cactus (O. ficus-indica) cladodes, and consequently the

biological activity of nopal on HT29 colorectal cancer cells in a realistic approach (after

gastrointestinal digestion and gut microbiota action).

Material and methods

Chemical and reagents

O. ficus-indica cactus cladodes (“nopales”) for human consumption were purchased

from BioArchen company located in Murcia, Spain (August 2015). Olive and soybean

oils were obtained from local markets. Methanol, acetone, acetonitrile (HPLC grade)

and formic acid (HPLC grade) were purchased from Panreac (Barcelona, Spain).

Potassium chloride and sodium chloride were obtained from Merck (Darmstadt,

Germany). Human saliva α-amylase (852 U/mg protein), pepsin (674 U/mg), pancreatin

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(4xUPS), bile salts (for digestion), sodium hydrogen carbonate, potassium phosphate

monobasic, magnesium sulfate monohydrate as well as the pure standards used for

quantification of phenolic compounds (isorhamnetin, kaempferol and ferulic acid), were

purchased from Sigma-Aldrich (Steinheim, Germany).

Chemicals for the batch culture nutrient medium for the in vitro fermentation as bile

salts, yeast extract, buffered peptone water, sodium chloride, Tween 80, hemin, vitamin

K, L-cystein hydrochloride monohydrate, resazurin redox indicator, sodium hydrogen

carbonate, potassium phosphate monobasic, magnesium sulfate monohydrate, potassium

hydrogen phosphate, calcium chloride hexahydrate were purchased from Sigma-Aldrich

(St. Louis, MO, USA) and Applichem (Darmstadt, Germany).

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS) and penicillin

streptomycin (Pen Strep) were obtained from Gibco Life Technologies Ltd (Paisley,

Scotland, UK). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

was acquired from Promega (Madison, USA). All other chemicals for tissue culture and

biological activity assays were purchased from Sigma-Aldrich Company Ltd (Dorset,

England, UK) unless otherwise specified.

Samples preparation

Cactus cladodes were washed and the thorns were manually removed. Then, they were

cut into small pieces and divided into six portions (300 g for each one). One portion was

used as raw sample and the other five were processed by the different cooking methods

(boiling, microwaving, griddling and frying in olive or in soybean oil) as shown in

Table 1. Afterwards, each raw and cooked sample was lyophilized in a freeze dryer

Cryodos-80 (Telstar, Terrasa, Spain) and stored at -18 C.

In vitro gastrointestinal digestion and colonic fermentation

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A simulated digestion model was performed according to Minekus et al. (2014) 17 and

Monente et al. (2015) 18 adapted to our laboratory. Three steps were carried out to

simulate the oral (with -amylase), gastric (with pepsin at pH 3) and small intestine

(with pancreatin and bile salts at pH 7) conditions. Details of the in vitro gastrointestinal

process are described in De Santiago et al. (2018) 8. Each cactus sample was digested in

duplicate and then the two repetitions were mixed and homogenized. After intestinal

digestion, samples were lyophilized in a freeze dryer Cryodos-80 (Telstar), and stored at

-18 C until in vitro fermentation process.

The digesta raw and cooked cactus cladodes were subjected to an in vitro fermentation

with human faecal samples to simulate the condition present in the colon following the

method described by Koutsos et al. (2017) 19 and briefly described below.

The composition for 1 L of growth medium was 2 g of peptone, 2 g of yeast extract, 0.1

g of NaCl, 0.04 g of K2HPO4, 0.04 g of KH2PO4, 0.01 g of MgSO4 x 7H2O, 0.04 g of

CaCl2 x 6H2O, 2 g of NaHCO3, 2 mL of Tween 80, 0.05 g of hemin dissolved in 1 mL

of 4 M NaOH, 10 mL of vitamin K, 0.5 g of L-cysteine HCl, 0.5 g of bile salts and 4

mL of resazurin solution (0.025%, w/v) as an anaerobic indicator. The growth medium

was sterilized at 121 °C for 15 min in glass vessels (280 mL) before sample preparation.

The fermentation process of digesta raw and cooked cactus as well as the control (with

no cactus cladodes) was developed using anaerobic, stirred, pH (5.3-5.7) and

temperature controlled faecal batch cultures. Glass water-jacketed vessels were

sterilized and filled aseptically with 67.5 mL of pre-sterilized basal nutrient medium.

The medium was then gassed overnight with nitrogen (>99% purity) to maintain

anaerobic conditions. The following day and before the inoculation, each vessel was

dosed with 0.75 g of digesta cactus, for a final concentration of 1% (w/v). Three

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independent experiments were performed with fresh human faecal samples collected in

an anaerobic jar from three healthy faecal donors who were free of any known

metabolic and gastrointestinal diseases, were not taking probiotic or prebiotic

supplements, followed a polyphenol-free diet for 2 days and had not taken antibiotics 3

months before faecal collection. Faecal slurry was prepared by homogenizing the faeces

in pre-reduced phosphate buffered saline (PBS). The temperature was set to 37 °C using

a circulating water-bath and the vessels were inoculated with 7.5 mL faecal slurry (10%

w/v of fresh human faeces) for a period of 24 h, during which samples were collected at

4 time points (0, 5, 10 and 24 h). Samples were centrifuged at 13,500 rpm at 4 °C for 5

min and stored at −80 °C for further analysis.

Identification and quantification of (poly)phenolic compounds by HPLC-ESI-MS

(Poly)phenolic compounds from fermented, raw and cooked, cactus cladodes were

extracted following the method described in Juániz et al. (2017) 11. Briefly, 0.1 mL of

methanol/acidified water (0.1% formic acid) (80:20 v/v) was added to 0.1 mL of

fermented samples, vortexed and centrifuged at 14,000 rpm for 10 min. Then,

supernatants were transferred to vials to carry out HPLC-ESI-MS analysis, which was

performed using an HPLC unit model 1200 (Agilent Technologies, Palo Alto, CA,

USA) equipped with a Triple Quadrupole Linear Ion Trap Mass Spectrometer 3200 Q-

TRAP (AB Sciex, Framingham, MA, USA). The column used was a CORTECS® C18

(3x75 mm, 2.7 µm) from Waters (Milford, MA, USA).

For HPLC separation, mobile phase A was 0.1% (v/v) formic acid in water and mobile

phase B was acetonitrile. Separations were carried out with an injection volume of 4 μL,

column oven temperature of 30 C and elution flow rate of 0.35 mL/min. The mobile

phases comprised a program of 0–1.20 min, 5% B; 1.20–8.80 min, 5–11.4% B; 8.80-10

min, 11.4–11% B; 10–30 min, 11-30% B; 30-32 min, 30-100% B and then return to 5%

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B in 2 min and maintained isocratic until the end of the analysis (38 min) to re-

equilibrate the column. Mass analyses were performed in negative ionization mode,

with the turbo heater maintained at 500 °C and Ion Spray voltage set at -3500. Nitrogen

was used as nebulizing, turbo heater and curtain gas and was set at the pressure of 40,

50 and 35 psi, respectively. Chromatograms were acquired using Analyst software 1.6.3

(AB Sciex, Framingham, MA, USA).

For the identification of the phenolic compounds, a preliminary analysis was carried out

in a full scan from 100 to 1000 m/z, and a consecutively selective product ion mode

analysis with specific m/z. Identification was achieved by multiple reaction monitoring

(MRM). Two transitions were studied for each phenolic compound. The first transition,

corresponding to the most abundant fragment, was used as quantifier ion, and the

second as qualifier ion. Details of the (poly)phenols identification are shown in the

Supporting Information Table S1 (Supplementary Material).

Phenolic acids were expressed as ferulic acid equivalents, whereas isorhamnetin and

kaempferol derivatives were quantified with their respective aglycones using calibration

curves. Results were expressed as milligrams of each compound per millilitre of batch

culture (µg /mL).

Biological activity assays

Tissue culture

Biological activity of fermented cactus cladodes was performed using HT29 human

colorectal adenocarcinoma cells acquired from the European Collection of Cell Cultures

(ECACC). Cells were grown in tissue culture flasks and maintained in DMEM

supplemented with FBS (10%) and Pen Strep (1%). They were incubated at 37 °C with

5% CO2 and sub-cultured every 2 days by the addition of trypsin (0.25% trypsin-EDTA)

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for 10 min at 37 °C and centrifuged at 1200 rpm for 3 min. After that, the supernatant

was decanted and cells were re-suspended in the appropriate medium.

Colonic fermented samples were centrifuged at 13,500 rpm at 4 °C for 5 min and the

supernatant was first filtered by 0.45 m and then by 0.22 m filters, before incubation

with HT29 cells. Twenty-four hours was selected as the exposure time for all in vitro

studies with fermented samples, as it is generally considered to reflect the average

colonic transit time.

Cytotoxicity assay

Raw and cooked cactus cladodes colonic fermentates were diluted in DMEM at 10%

and 20% (v/v) to evaluate cytotoxicity by the MTT colorimetric assay, following the

method described by McDougall et al. (2017) 15. First, HT29 cells were seeded in 96

multi-well plates (Costar, Cambridge, MA, USA) at a concentration of 1.5 x 104 cells

per well. After 2 days of incubation at 37 °C, media was replaced with fermented raw

and cooked cactus samples at 10% and 20% (v/v) and then incubated for 24 h. The

wells were washed and refreshed with media. Thereafter, 15 L of MTT (5 mg/ mL)

were added to each well. After 4 h at 37 °C, lysis was carried out with 100 L of

solubilizing solution (2-isopropanol with triton X-100 (10% v/v)) and measured using a

plate reader (Alpha, SLT Rainbow Thermo, Antrim, UK) at a wavelength of 570 nm,

reference 650nm. Each sample was measured in octuple and results are presented as

percentage cell viability compared to untreated cells. The experiment was repeated

independently three times.

Genotoxicity assay (Comet assay)

The effect of colonically fermented raw and cooked cactus on colonocyte DNA damage

was determined using the Comet assay according to the method described by

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McDougall et al. (2017) 15. HT29 cells were pre-incubated with the samples for 24 h.

The anti-genotoxic effect was assessed by treating pre-incubated HT29 cells with

hydrogen peroxide (75 M H2O2), or with PBS as their respective control. Briefly, the

cells were reconstituted in 85 μL of 0.85% low melting point agarose in PBS and

maintained in a water bath at 37 °C. This suspension was mixed with 1% normal

agarose to previously prepared gels on frosted slides and coverslips were added. The

slides were subjected to lysis buffer (2.5 M NaCl, 100 mM Na2EDTA, 10 mM TRIS)

for 1 h at 4 °C and placed in electrophoresis buffer for 20 min at 25 V 300 mA.

Subsequently, slides were washed (3 × 5 min) in neutralisation buffer (0.4 M TRIS HCl,

pH = 7.5) at 4 °C. All slides were stained with ethidium bromide (20 μL of 20 μg/mL)

prior to scoring. Images were analysed at 400 × magnification using a Nikon eclipse

600 epifluorescence microscope. The percentage (%) tail DNA was recorded using

Komet 5.0 image analysis software (Kinetic Imaging Ltd, Liverpool, UK). For each

slide, 50 cells were scored and the mean was calculated. Results are presented as mean

percentage (%) tail DNA. The experiment was repeated independently 3 times and

positive (H2O2) and negative controls (PBS) were included in all experiments, as well as

cells without fermented treatment.

Cell cycle analysis

Cell cycle analysis of fermented raw and cooked cactus was determined using

propidium iodide staining and measured using flow cytometry (Ormerod, 2000 20) in

order to analyse the DNA content to provide an indication of the number of cells in the

phases of the cell cycle and if the concentration selected of fermented cactus cladodes

have an effect on them. Briefly, HT29 cells were seeded at a density of 1 x 106 cells per

mL for 24 h before treatment with each fermented extract at 10%. After fermentate

treatment exposure, the suspension was centrifuged at 1,200 rpm for 5 min, the

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supernatant discarded and the pellet re-suspended in 1 mL of ice cold PBS. Then, the

suspension was centrifuged at 4,000 rpm for 5 min, the supernatant was discarded and

the cells were re-suspended in 1 mL of ice cold ethanol/PBS (90:10) and incubated at -

20 C overnight. After incubation, cells were washed twice with ice cold PBS and the

staining buffer (100 L/mL of RNase A, 10 L/mL of propidium iodide, 0.1% NP40

and 50 L/mL tri sodium citrate in PBS) was added. The cells were then incubated at 37

°C, 5% CO2 for 30 min and the fluorescence emission spectra of propidium iodide was

collected at 585 nm, using Beckman-Coulter Gallios flow cytometer (Brea, CA).

Subsequently these emission spectra were analysed for DNA content using Gallios

Acquisition Software. The experiment was performed as triplicate independent

experiments and results are presented as a percentage (%) of cell count.

Statistical analysis

The mean of each data set was used for statistical analysis. The Shapiro-Wilk test was

used to test for normality. Analysis of variance was applied to test for significant

differences between means using one-way analysis of variance (ANOVA, Dunnett T

and T3 tests). A Bonferroni test was applied as a posteriori test for significant

differences among fermentation time points. Significance was accepted at p < 0.05.

Analysis was carried out using SPSS (version 20 for Windows).

Results and discussion

Colonic catabolism of (poly)phenolic compounds

The (poly)phenolic composition of fermentates over time is reported in Table 2.

Twenty-seven (poly)phenolic compounds were identified and quantified in baseline (0

h) of fermented cactus cladodes, being phenolic acids the most abundant in all samples,

accounted for 78-88% of the total content. Piscidic acid (Fig. 1a) was the main

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compound (52-65% total (poly)phenolic compounds), followed by eucomic acid (Fig.

1b) (15-36% total (poly)phenolic compounds). Griddled cactus cladodes presented the

highest amount of piscidic acid, and microwaved samples had higher amount of

eucomic acid than the rest of cooking treatments. Flavonoids were also present in cactus

cladodes colonic fermentates, being kaempferol derivatives (6-13% total) and

isorhamnetin derivatives (4-7% total) mainly in their glycoside forms. Griddled cactus

cladodes presented the highest amount of kaempferol derivatives whilst microwaved

samples contributed with the highest isorhamnetin derivatives content at the beginning

of colonic fermentation (0 h). These (poly)phenolic profiles are consistent with those of

previous studies in O. ficus-indica cactus cladodes without gastrointestinal digestion,

even quercetin derivatives were also quantified 2-4,8. Enzymes and digestive conditions

higher degraded or retained flavonoids than phenolic acids, but 4-7%, 22-30% and 15-

23% of the total (poly)phenolic content for kaempferol, isorhamnetin and quercetin

derivatives remained bioaccessible after gastrointestinal digestion, respectively 8.

Nevertheless, in the present study quercetin derivatives were not detected at any time

point of the fermentation, may be due to the rapid metabolism of quercetin glycosides at

the starting of colonic fermentation. Actually, it has been previously demonstrated a fast

degradation within the first 15 min after colonic fermentation in quercetin derivatives

from raw and cooked pepper 10 as well as in apples 19. Similarly, isorhamnetin

derivatives were faster metabolized by gut microbiota since the beginning of colonic

fermentation than kaempferol ones, accounting for 5-8% and 6-14% of the total

(poly)phenolic compounds, respectively.

Changes in the amount of (poly)phenols throughout the faecal fermentations were

observed. Even though a degradation of the compounds was shown, some (poly)phenols

were still present after 24 h of faecal fermentation. Both kaempferol and isorhamnetin

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derivatives were rapidly catabolised by human microbiota, showing low amounts, traces

or even no detected at the end of the 24 h of colonic fermentation (Fig. 2a). In

particular, minor flavonoids were completely degraded in most of the samples after 24 h

of colonic fermentation, except in griddled and microwaved, in which remained

available or in traces.

Since the first step of the faecal incubation, the hydrolysis of flavonoid glycosides and

the consequent formation of aglycones took place, especially from the isorhamnetin

derivatives. The highest amount of isorhamnetin was detected in microwaved samples

after 10 h of faecal incubation but then it decreased. Kaempferol derivatives were also

degraded after 24 h fermentation, but their respective aglycone was not detected due to

the low amount of its native (poly)phenols. Some increases in flavonoids during the

fermentation were also observed, may be due to the release from the food matrix.

Particularly, methoxy kaempferol hexoside I increased after 5 and 10 h of incubation in

all samples, except in both fried samples. Moreover, although at the end of the 24 h

fermentation it decreased, the final amount was higher than in the beginning in raw,

microwaved and griddled cactus cladodes.

Regarding phenolic acids, eucomic acid was the most stable during the 24 h of faecal

incubation (Fig. 2b). Although it was degraded, it still remained in high amounts in all

samples, especially in microwaved and griddled ones (Table 2). Conversely, piscidic

acid increased after 5 h of incubation in all samples; but was almost completely

degraded after 24 h. However, the degradation was lower in griddled samples. Likewise,

feruloylglucose I and II were rapidly degraded after 24 h of faecal fermentation, but

remained in traces in griddled and microwaved cactus cladodes.

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Eucomic acid were more stable under colonic conditions probably due to the resistance

to gastrointestinal and colonic microflora enzymes, as well as the release from the cell

walls by the microbiota action 21. However, ferulic acid derivatives were rapidly

degraded in all samples may be due to the catabolism related to microbial feruloyl

esterases which are present in the colonic inoculum and degraded the compounds 22.

After 24 h colonic fermentation, the catabolite 3-(4-hydroxyphenyl)propionic acid was

found in trace amounts maybe produced via demethoxylation from ferulic acid

derivatives 12, as well as via dehydroxylation of 3-(3,4-dihydroxyphenyl)propionic acid

derived from ferulic acid and the ring fission of quercetin, isorhamnetin and kaempferol

derivatives 10,11,23.

Metabolic products depend on (poly)phenolic compounds present and the

interindividual variation in the gut microbial composition and enzymatic capacities

leading to different catabolite profile 12. However, in the present study 3-(3,4-

dihydroxyphenyl)propionic acid was present in all the volunteers, even in traces.

Further investigation is needed to evaluate the absorption of bioactive compounds from

cactus cladodes taking into account the gastrointestinal digestion and microbiota

conditions.

Biological activity in colon cells

The biological activity of cactus cladodes submitted to the most common culinary

techniques (boiling, griddling and frying) in human colorectal cells was evaluated after

gastrointestinal digestion and the action of gut microbiota in HT29 cells to simulate

physiological conditions. Raw samples and control (with no cactus) fermentates were

used as positive and negative controls.

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No cytotoxic activity was observed in HT29 cells treated with fermented raw and

cooked cactus cladodes at 10% and 20% (v/v) after 24 h of incubation, compared to

untreated cells (Fig. 3). Likewise, no significant differences (p<0.05) were observed

among cooking treatments. Nevertheless, cell viability was significant lower (p<0.05) in

control fermentation (with no cactus cladodes) at 20% (v/v) than in untreated cells and

did not present a cell viability higher than 80%, like most of the other treated cells with

cactus at 20% (v/v), suggesting a potential cytotoxicity due to a high concentration.

Therefore, a 10% (v/v) concentration was used for the comet and cell cycle assays. Up

to our knowledge, cytoxicity of cactus cladodes after colonic fermentation has not been

previously evaluated. Similarly, Serra et al. (2013) 16 reported no cytotoxic activity of

O. ficus-indica fruit juices in a Caco-2 human colon cell model.

The effect of cactus cladodes colonic fermentates against DNA damage induced by

oxidation in colonocytes was assessed by the comet assay (Fig. 4). The percentage of

DNA in the tail induced by H2O2 was significantly (p<0.05) reduced by raw and cooked

cactus cladodes colonic fermentates (34-38%) in comparison with the control

fermentate (with no cactus cladodes) (45%). The reduction in the H2O2-induced DNA

damage of fermented cactus cladodes may be attributed to the (poly)phenolic

compounds which still remained after colonic fermentation, including eucomic acids.

Similarly, studies in digested blackcurrant 24 and elderberry 25 have observed a reduction

in H2O2-induced DNA damage in NCM460 (non-tumorigenic colon cell line) and in

Caco-2 cells. Other studies have evaluated the bioactivity of Opuntia ficus-indica

cladodes, showing a protection against oxidative stress biomarkers and apoptosis in cell

cultures associated with flavonoids such as isorhmanetin and kaempferol 26 as well as

phenolic acids including eucomic and piscidic acids 27. However, the role of

fermentation on the bioactivity of the extracts was not considered. A similar

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consideration to this study was those reported with colonic fermented raspberry,

strawberry and blackcurrant which exerted a significant (p<0.05) reduction of H2O2-

induced DNA damage in HT29 cell line 28,29, demonstrating that antioxidant activity,

and specifically potential antigenotoxic activity of (poly)phenols is retained after

colonic fermentation.

Finally, the effect of cactus cladodes colonic fermentates on the HT29 cell cycle was

evaluated (Fig. 5) in order to know whether they affect the DNA content in every phase

of the cell cycle and consequently HT29 cells proliferation. No significant effect

(p<0.05) was observed in each stage of the HT29 cell cycle after 24 h of incubation with

colonic fermented cactus cladodes. No significant differences (p<0.05) were also

observed among fermented samples. Results are consistent with Coates et al. (2007)

who reported not significant changes in cell cycle proliferation of HT29 cells pre-treated

with colonic fermented berries. In contrast, extracts from O. ficus-indica fruit juice

induced a cell cycle arrested in the G1 phase in HT29 cells 16, attributing this effect to

the betalains.

In summary, results of the in vitro colonic fermentation of raw and cooked cactus

cladodes showed that flavonoids tended to be degraded more than phenolic acids, but

they are still available after 24 h of colonic incubation, including eucomic acid. Results

also suggest that remained (poly)phenols after the action of colonic microbiota still have

antioxidant and genoprotective properties which can exert into the intestinal cells.

Therefore, the current study provides a basis for further investigations about the cactus

cladodes bioactive components potentially available to be absorbed, and to exert their

beneficial effects in colon and other target organs after ingestion and to understand their

mechanism of action in vivo.

Acknowledgment

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We thank Keith Thomas for his kind help.

Supporting Information

Mass spectrometric characteristics of (poly)phenolic compounds identified in this study

(Table S1).

Mass chromatograms of phenolic profile after colonic fermentation at 0 h in full scan

mode of a) control fermentation (sample with no cactus cladodes), b) griddled cactus

cladodes (Figure S1).

Mass chromatograms of phenolic profile after colonic fermentation at 0 h in Multiple

Reaction Monitoring (MRM) mode of a) control fermentation (sample with no cactus

cladodes), b) griddled cactus cladodes (Figure S2).

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Funding

This research was funded by the Spanish Ministry of Economy and Competitiveness

(AGL2014-52636-P). E.D.S. expresses their gratitude to the Association of Friends of

the University of Navarra and La Caixa Banking Foundation for the grants received.

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Figure captions

Figure 1. Structures of phenolic acids, a) Piscidic acid. b) Eucomic acid.

Figure 2. Degradation of (poly)phenolic compounds of griddled cactus cladodes during

24 h colonic fermentation. a) Total kaempferol derivatives, total isorhamnetin

derivatives and isorhamnetin aglycone. b) Total phenolic acids, piscidic acid and

eucomic acid. Different letters indicate significant differences (p<0.05) during each

time point of the colonic fermentation.

Figure 3. The cytotoxic effect of 20% (v/v) and 10% (v/v) fermented cactus cladodes on

HT29 cells. Data are presented as means of 3 independent experiments +/- SD

compared to the untreated cells as a control. One-way ANOVA and Post Hoc test

Dunnett’s T *p<0.05.

Figure 4. Antigenotoxic activity of fermented cactus cladodes 10% (v/v) on DNA

damage in HT29 cells challenged with 75 μM H2O2. Data are presented as means of 3

independent experiments +/- SD compared to the control fermentation. One-way

ANOVA and Post Hoc test Dunnett’s T *p<0.05.

Figure 5. Effects of fermented cactus cladodes 10% (v/v) on HT29 cell cycle. Data are

presented as means of 3 independent experiments +/- SD compared to the untreated

cells as a control.

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Tables

Table 1. Cooking Conditions Applied to Cactus Cladodes.

Cooking technique

Time Temperature Amount of water or oil

Equipment

Boiling 10 min 100 °C 600 mL Stainless steel pot

Microwaving 5 min - - Silicone case (Lékué, Barcelona, Spain) and microwave oven (Whirlpool, Michigan, USA) at 900W

Griddling 5 min & 5 min 150 °C & 110 °C - Non-stick griddle (Jata Electro, Vizcaya, Spain)

Frying in olive/ soybean oil

10 min & 5 min 100 °C & 90 °C 30 mL Non-stick frying pan

24

540541

542543

544545

Table 2. Native (Poly)phenolic Compounds and Catabolites Produced During Colonic Fermentation of Raw and Cooked Cactus Cladodes. Results are Expressed as Mean ± Standard Deviation (µg (Poly)phenolic Compound/mL) (n=3).

CompoundsRaw Boiled Microwaved Griddled Fried in olive oil Fried in soybean oil

µg /mL µg /mL µg /mL µg /mL µg /mL µg /mL

Phenolic acidsPiscidic acid

0 h5 h10 h24 h

250.59 ± 76.17265.01 ± 33.27249.05 ± 86.67

0.46 ± 0.15

140.53 ± 24.75159.95 ± 23.06140.77 ± 73.26

0.12 ± 0.05

333.09 ± 32.90422.39 ± 21.15431.91 ± 11.54

1.47 ± 0.56

355.75 ± 11.86384.77 ± 17.70302.27 ± 93.99

32.31 ± 6.42

182.60 ± 8.31205.54 ± 12.26137.83 ± 85.08

0.16 ± 0.09

177.79 ± 46.77212.97 ± 96.91147.98 ± 98.80

0.45 ± 0.12

Feruloylglucose I

0 h5 h10 h24 h

0.95 ± 0.510.15 ± 0.200.01 ± 0.00

nd

0.54 ± 0.300.07 ± 0.03

ndnd

1.17 ± 0.290.76 ± 0.030.03 ± 0.01

tr

0.84 ± 0.130.28 ± 0.200.60 ± 0.23

tr

0.41 ± 0.180.06 ±0.09

trtr

0.54 ± 0.29trtrtr

Feruloylglucose II0 h5 h10 h24 h

1.14 ± 0.430.48 ± 0.570.01 ± 0.00

nd

0.70 ± 0.270.28 ± 0.36

trnd

1.12 ±0.191.06 ± 0.410.24 ± 0.29

tr

1.33 ± 0.250.77 ± 0.430.60 ± 0.23

tr

0.77± 0.17trndnd

0.81 ± 0.240.16 ± 0.21

ndnd

Eucomic acid0 h5 h10 h24 h

178.06 ± 51.20163.38 ± 43.33105.72 ± 16.6671.50 ± 30.68

33.01 ± 17.8456.99 ± 27.8958.21 ± 25.3942.98 ± 2.31

161.75 ± 12.80184.41 ± 19.23208.89 ± 11.54154.08 ± 2.88

100.91 ± 15.31105.63 ± 30.81118.23 ± 25.6081.09 ± 18.66

54.86 ± 3.9657.13 ± 13.2052.14 ± 16.0735.29 ± 12.68

64.91 ± 10.1655.89 ± 15.6353.66 ± 10.4936.31 ± 19.81

TOTAL PHENOLIC ACIDS0 h5 h10 h24 h

430.75 ± 126.65429.01 ± 130.27354.92 ± 117.91

71.96 ± 50.23

174.78 ± 66.34217.29 ± 75.34198.98 ± 58.3743.10 ± 30.31

497.12 ± 158.46608.63 ± 199.85641.07 ± 206.10155.55 ± 107.92

458.83 ± 167.45491.44 ± 181.50421.18 ± 142.59113.40 ± 34.50

238.64 ± 85.86259.72 ± 104.41189.97 ± 60.5935.45 ± 24.84

244.05 ± 83.53269.02 ± 110.35201.64 ± 66.6936.77 ± 25.36

Isorhamnetin derivatesIsorhamnetin

0 h5 h10 h24 h

0.11 ± 0.141.37 ± 0.860.84 ± 0.690.73 ± 0.93

0.18 ± 0.211.30 ± 0.861.38 ± 0.580.68 ± 0.55

0.28 ± 0.420.78 ± 0.804.74 ± 2.012.89 ± 2.49

0.22 ± 0.351.17 ± 0.641.85 ± 1.501.63 ± 0.26

0.20 ± 0.280.75 ± 0.351.58 ± 1.681.77 ± 1.24

0.15 ± 0.260.75 ± 0.561.08 ± 0.340.49 ± 0.46

Isorhamnetin hexose rhamnose hexoside I

0 h5 h10 h24 h

0.02 ± 0.00trtrnd

trtrndnd

0.02 ± 0.010.03 ± 0.000.01 ± 0.00

tr

0.02 ± 0.000.01 ± 0.00

trtr

0.01 ± 0.00trtrtr

0.01 ± 0.00trtrnd

25

546547548

Isorhamnetin hexose rhamnose hexoside II0 h5 h10 h24 h

trtrtrnd

trtrndnd

trtrtrnd

trtrtrnd

trtrtrnd

trtrtrnd

Isorhamnetin hexose rhamnose hexoside III0 h5 h10 h24 h

trtrtrnd

trtrtrnd

0.01 ± 0.00trtrnd

0.01 ± 0.00trtrnd

trtrtrnd

trtrtrnd

Isorhamnetin di-hexoside I0 h5 h10 h24 h

0.15 ± 0.090.14 ± 0.120.09 ± 0.110.06 ± 0.08

0.12 ± 0.060.13 ± 0.040.07 ± 0.070.01 ± 0.02

0.24 ± 0.0250.35 ± 0.020.27 ± 0.070.15 ± 0.12

0.22 ± 0.020.22 ± 0.070.14 ± 0.100.01 ± 0.01

0.12 ± 0.010.10 ± 0.060.06 ± 0.050.04 ± 0.05

0.13 ± 0.050.15 ± 0.010.14 ± 0.01

ndIsorhamnetin di-hexoside II

0 h5 h10 h24 h

trtrtrnd

trtrtrnd

0.01 ± 0.00trtrnd

0.01 ± 0.00trtrnd

trtrtrnd

trtrtrnd

Isorhamnetin rutinoside rhamnoside0 h5 h10 h24 h

0.17 ± 0.100.09 ± 0.110.08 ± 0.07

tr

0.10 ± 0.03trtrnd

0.25 ± 0.110.35 ± 0.000.25 ± 0.070.07 ± 0.05

0.24 ± 0.080.20 ± 0.080.10 ± 0.07

tr

0.14 ± 0.050.10 ± 0.03

trtr

0.13 ± 0.070.08 ± 0.070.06 ± 0.05

ndIsorhamnetin rutinoside rhamnoside II

0 h5 h10 h24

0.18 ± 0.110.14 ± 0.120.09 ± 0.110.02 ± 0.03

0.12 ± 0.040.12 ± 0.09

trtr

0.23 ± 0.100.38 ± 0.010.34 ± 0.010.20 ± 0.11

0.22 ± 0.070.23 ± 0.090.19 ± 0.090.04 ± 0.06

0.12 ± 0.030.08 ± 0.070.03 ± 0.03

tr

0.13 ± 0.090.15 ± 0.000.15 ± 0.00

ndIsorhamnetin hexose pentoside

0 h5 h10 h24 h

2.36 ± 1.831.73 ± 1.571.25 ± 1.480.30 ± 0.48

2.29 ± 1.401.63 ± 1.850.85 ± 1.16

tr

4.63 ± 2.128.08 ± 1.226.54 ± 1.712.71 ± 0.22

4.39 ± 1.883.92 ± 1.761.69 ± 0.270.94 ± 0.77

2.57 ± 1.191.95 ± 1.600.49 ± 0.63

tr

2.77 ± 2.063.40 ± 1.262.84 ± 1.13

trIsorhamnetin rutinoside I

0 h5 h10 h24 h

0.71 ± 0.520.82 ± 0.760.86 ± 1.180.30 ± 0.48

0.83 ± 0.650.72 ± 0.200.53 ± 0.01

tr

0.58 ± 0.131.39 ± 0.562.02 ± 1.122.11 ± 0.01

0.60 ± 0.171.06 ± 0.521.17 ±1.011.03 ± 0.83

0.42 ± 0.140.80 ± 0.360.59 ± 0.530.22 ± 0.23

0.42 ± 0.150.66 ± 0.670.91 ± 0.780.02 ± 0.03

Isorhamnetin rutinoside II0 h5 h10 h24 h

19.12 ± 9.096.15 ± 5.721.76 ± 1.050.07 ± 0.01

12.00 ± 6.721.97 ± 0.950.20 ± 0.07

tr

34.58 ± 7.5630.61 ± 8.67

12.80 ± 10.270.51 ± 0.51

30.53 ± 9.7524.51 ± 11.1610.65 ± 4.062.02 ± 1.41

13.26 ± 3.408.36 ± 2.980.79 ± 0.28

tr

17.02 ± 5.317.57 ± 3.533.42 ± 3.13

trUnknown isorhamnetin derivative

0 h5 h10 h24 h

0.75 ± 0.530.33 ± 0.420.22 ± 0.13

nd

0.41 ± 0.260.20 ± 0.27

trnd

1.00 ± 0.581.37 ± 0.151.07 ± 0.220.24 ± 0.17

0.89 ± 0.490.71 ± 0.360.31 ± 0.150.16 ± 0.08

0.54 ± 0.320.40 ± 0.030.05 ± 0.03

tr

0.56 ± 0.440.49 ± 0.300.35 ± 0.20

ndTOTAL ISORHAMNETIN DERIVATES

0 h 23.57 ± 6.23 16.04 ± 3.90 41.81 ± 10.29 37.33 ± 9.09 17.36 ± 4.32 21.32 ± 5.56

26

5 h10 h24 h

10.78 ± 2.045.19 ± 0.631.49 ± 0.26

6.06 ± 0.773.03 ± 0.530.70 ± 0.48

43.34 ± 9.9928.03 ± 4.298.87 ± 1.24

32.02 ± 7.9516.11 ± 3.405.84 ± 0.80

12.53 ± 2.683.61 ± 0.552.03 ± 0.95

13.25 ± 2.628.97 ± 1.310.52 ± 0.33

Kaempferol derivatesKaempferol hexoside dirhamnoside I

0 h5 h10 h24 h

0.24 ± 0.160.16 ± 0.210.17 ± 0.18

nd

0.18 ± 0.090.16 ± 0.21

trnd

0.40 ± 0.210.65 ± 0.060.58 ± 0.080.32 ± 0.18

0.32 ± 0.140.30 ± 0.150.17 ± 0.130.12 ± 0.16

0.20 ± 0.070.23 ± 0.050.05 ± 0.04

tr

0.20 ± 0.120.23 ± 0.000.05 ± 0.04

nd

Kaempferol hexoside dirhamnoside II

0 h5 h10 h24 h

0.14 ± 0.100.07 ± 0.070.05 ± 0.07

tr

0.09 ± 0.05trtrnd

0.17 ± 0.110.29 ± 0.020.20 ± 0.010.04 ± 0.05

0.15 ± 0.070.13 ± 0.080.06 ± 0.050.04 ± 0.05

0.10 ± 0.050.06 ± 0.040.13 ± 0.22

nd

0.10 ± 0.060.09 ± 0.010.05 ± 0.04

ndKaempferol hexose pentoside

0 h5 h10 h24 h

1.28 ± 0.880.69 ± 0.710.35 ± 0.410.08 ± 0.14

0.83 ± 0.490.51 ± 0.670.26 ± 0.360.01 ± 0.02

1.91 ± 1.082.63 ± 0.122.06 ± 0.050.93 ± 0.18

1.38 ± 0.661.09 ± 0.500.55 ± 0.360.12 ± 0.16

0.77 ± 0.360.50 ± 0.440.20 ± 0.20

tr

0.88 ± 0.671.02 ± 0.340.68 ± 0.26

trKaempferol rutinoside I

0 h5 h10 h24 h

0.10 ± 0.070.18 ± 0.070.21 ± 0.21

tr

0.07 ± 0.060.19 ± 0.010.16 ± 0.14

nd

0.11 ± 0.060.20 ± 0.110.33 ± 0.240.58 ± 0.01

0.09 ± 0.040.14 ± 0.080.15 ± 0.140.07 ± 0.10

0.07 ± 0.050.10 ± 0.060.06 ± 0.070.08 ± 0.11

0.08 ± 0.040.11 ± 0.090.16 ± 0.09

trKaempferol rutinoside II

0 h5 h10 h24 h

0.06 ± 0.030.13 ± 0.10

0.11 ± 0.166tr

0.07 ± 0.020.16 ± 0.080.02 ± 0.00

nd

0.05 ± 0.000.23 ± 0.130.23 ± 0.180.08 ± 0.08

0.06 ± 0.030.13 ± 0.080.15 ± 0.160.08 ± 0.11

0.10 ± 0.050.06 ± 0.040.13 ± 0.22

tr

0.06 ± 0.040.16 ± 0.080.28 ± 0.08

trKaempferol rutinoside III

0 h5 h10 h24 h

0.17 ± 0.050.28 ± 0.300.02 ± 0.02

nd

0.20 ± 0.140.04 ± 0.03

ndnd

2.58 ± 3.692.51 ± 2.820.19 ± 0.240.01 ± 0.02

1.53 ± 1.801.91 ± 2.510.35 ± 0.140.08 ± 0.07

0.64 ± 0.910.14 ± 0.120.04 ± 0.05

nd

1.28 ± 1.931.43 ± 1.990.63 ± 0.90

nd

Kaempferol rutinoside IV

0 h5 h10 h24 h

4.20 ± 4.021.10 ± 1.360.53 ± 0.240.01 ± 0.01

2.58 ± 2.67trtrnd

8.18 ± 6.6214.04 ± 2.492.44 ± 3.270.11 ± 0.14

6.34 ± 5.215.55 ± 4.311.45 ± 0.850.34 ± 0.57

1.78 ± 1.461.23 ± 0.260.17 ± 0.08

nd

3.25 ± 2.902.08 ± 1.130.96 ± 1.13

trKaempferol glucoside I

0 h5 h10 h24 h

0.22 ± 0.230.14 ± 0.200.08 ± 0.130.06 ± 0.09

0.21 ± 0.170.19 ± 0.270.14 ± 0.20

nd

0.27 ± 0.240.46 ± 0.430.54 ± 0.500.38 ± 0.21

0.26 ± 0.140.32 ± 0.180.32 ± 0.180.19 ± 0.06

0.11 ± 0.080.15 ± 0.100.07 ± 0.090.05 ± 0.03

0.10 ± 0.080.19 ± 0.15

0.11 ± 0.012nd

Kaempferol glucoside II0 h 0.10 ± 0.10 0.10 ± 0.09 0.22 ± 0.19 0.23 ± 0.02 0.06 ± 0.04 0.07 ± 0.09

27

5 h10 h24 h

0.05 ± 0.060.04 ± 0.06

nd

trtrnd

0.15 ± 0.200.01 ± 0.01

tr

0.22 ± 0.090.12 ± 0.070.02 ± 0.03

0.05 ± 0.06trnd

0.02 ± 0.010.04 ± 0.05

nd

Methoxy kaempferol hexoside I

0 h5 h10 h24 h

11.71 ± 7.3614.89 ± 21.4718.08 ± 27.2913.84 ± 21.34

15.45 ± 12.4018.79 ± 25.6215.47 ± 19.71

0.51 ± 0.08

22.13 ± 16.0735.27 ± 25.5443.48 ± 35.1636.44 ± 15.75

24.01 ± 17.0832.53 ± 16.6531.11 ± 27.4830.42 ± 15.00

10.04 ± 7.484.03 ± 2.032.09 ± 0.531.37 ± 0.58

4.99 ± 1.363.61 ± 2.402.35 ± 0.821.51 ± 0.94

Methoxy kaempferol hexoside II

0 h5 h10 h24 h

12.41 ± 10.604.08 ± 5.520.29 ± 0.260.02 ± 0.03

12.30 ± 9.941.71 ± 2.390.50 ± 0.70

tr

25.96 ± 26.4421.44 ± 28.51

0.60 ± 0.070.26 ± 0.37

28.14 ± 23.5225.31 ± 19.4810.93 ± 12.62

0.16 ± 0.12

8.81 ± 6.116.29 ± 6.701.20 ± 0.96

tr

7.56 ± 10.301.97 ± 2.340.33 ± 0.290.03 ± 0.04

TOTAL KAEMPFEROL DERIVATES0 h5 h10 h24 h

30.63 ± 4.7521.77 ± 4.4419.92 ± 5.4014.01 ± 5.63

32.08 ± 5.5121.75 ± 6.5216.55 ± 6.230.53 ± 0.35

61.98 ± 9.4477.89 ± 11.6850.66 ± 12.9239.16 ± 10.91

62.51 ± 10.2867.64 ± 11.4945.35 ± 9.5031.63 ± 9.14

22.67 ± 3.6912.87 ± 2.064.04 ± 0.691.51 ± 0.66

18.56 ± 2.5310.92 ± 1.175.83 ± 0.671.55 ± 0.87

3-(4-hydroxyphenyl)propionic acid0 h5 h10 h24 h

ndndndtr

ndndndtr

ndndndtr

ndndndtr

ndndndtr

ndndndtr

TOTAL (POLY)PHENOLIC COMPOUNDS0 h5 h10 h24 h

484.95 ± 60.93461.56 ± 63.22380.03 ± 55.2987.46 ± 18.51

222.90 ± 28.98245.10 ± 38.03218.55 ± 40.5944.32 ± 17.44

600.91 ± 70.90729.85 ± 91.64719.75 ± 95.72203.58 ± 34.00

558.67 ± 71.30591.10 ± 79.95482.64 ± 64.89150.86 ± 19.76

278.67 ± 38.15285.13 ± 43.11197.61 ± 32.2638.99 ± 11.63

283.94 ± 37.80293.19 ± 46.12216.44 ± 33.6038.83 ± 13.58

nd = not detectedtr = traces

28

549550

551

Figure 1

a) Piscidic acid

b) Eucomic acid

29

552553

554555

556557558559

560561

562

Figure 2

a)

b)

30

563564

565

566567568

569

Figure 3

Untreate

d

Control fe

rmentati

on 20%

Raw ca

ctus 2

0%

Boiled ca

ctus 2

0%

Griddled ca

ctus 2

0%

Fried Oliv

e cactu

s 20%

Untreate

d

Control fe

rmentati

on 10%

Raw ca

ctus 1

0%

Boiled ca

ctus 1

0%

Griddled ca

ctus 1

0%

Fried Oliv

e cactu

s 10%

0102030405060708090

100

*

ns nsns

ns ns ns ns ns ns

Cell

viab

ility

(%)

31

570571

572573574575

Figure 4

0

10

20

30

40

50

60

*

Tail

DN

A (%

)

32

576577

578579580581582583584

Figure 5

Sub G0 G0/G1 S-Phase G2/M0

10

20

30

40

50

60 Untreated

Control fermentation 10%

Raw cactus 10%

Boiled cactus 10%

Griddled cactus 10%

Fried olive oil cactus 10%

Cell

coun

t (%

)

ns

ns

ns

ns

ns = not significant

33

585586

587588589590591

Table of Contents (TOC) Graphic

34

592593

594595596597