All these b 8,9 in As is well known, regular intake of foods 6,7 Eduardo B de Oliveira, b Jéssica A de Carvalho ) from commercial b b body weight), as follows: bone marrow minocronucleus 1 Correspondence to:tos, Universidade F Federal Barros, deE-mail: Viçosa, [email protected] Departamento Viçosa, Minas de Gerais Tecnologia 36570-000, . de Alimen- Faculdade de Nutrição, Universidade Federal de Alfenas, Minas Gerais, Brazil Departamento de Tecnologia deMinas Alimentos, Gerais, Universidade Brazil Federal de Viçosa, − Camu-camu has been under the spotlight because of its high a ∗ b antioxidant capacity, due to the chemical profile containingicant signif- levels of vitamin Cacid and and phenolic . compounds such as ellagic associated factors have created greatduction interest in and, as camu-camu consequence, pro- commercial cultivationronments in has dry envi- been increasing. However, the concentrations of containing antioxidants mayinflammations, prevent mutations, chronic cancer diseases, and atherosclerosis. such as )is antioxidant capacity levels were higher in ripe grown in a Bactris Paullinia Humb. & Flávia M Ramos, in vitro a Theobroma gran- ) is a typical Amazonian and has high antioxidant capacity due to its high New ways of pro- dubia 5 ) and pupunha ( Myrciaria dubia Bertholletia excelsa and Paulo C Stringheta Paula F de Araujo Ribeiro, Myrciaria dubia a Mart.), cupuaçu ( b* ; camu-camu; antimutagenic effects; phytochemicals with natural occurrence during periods 4 : 624–631 www.soci.org © 2018 Society of Chemical Industry Psidium acutangulum 99 Among them, camu-camu ( Maryana G Correia, 1–3 a 2019; Euterpe oleracea Myrciaria dubia 4 Kunth).

Kunth), castanha-do-brasil ( ), araçá-pera (

levels of vitamin Cantimutagenic and effects phenolic of compounds. camu-camuenvironments This (native fruits cultivation, study Amazon). with aimed different to maturity determine stages the grown phytochemicals,RESULTS: in antioxidant Total dry capacity polyphenols, (commercial and ascorbic cultivation) or acid flooded and commercial cultivation. The extracts fromhigh ripe percentage of camu-camu protection against grown doxorubicin and in 1,2-dimethylhydrazine infor a all three tested commercial systems camu-camu (liver, cultivation bone marrow exerted extract and antioxidant gut), concentrations effects (17, and 85 and 170 mg kg diflorum gasipaes J Sci Food Agric INTRODUCTION The Amazon region isfruits distinguished containing remarkable by nutritional its composition, therapeutic value, great and biodiversity thus agribusiness of potential, such as guaraná ( cupana

Forest) and presents antimutagenic effects compounds than native cultivation (Amazon Abstract BACKGROUND: Camu-camu ( cultivation has higher levels of bioactive vivo Frederico Barros Bonpl.), açaí ( Luciana Azevedo, Camu-camu ( (wileyonlinelibrary.com) DOI 10.1002/jsfa.9224 Research Article Received: 10 April 2018 Revised: 22 June 2018 Accepted article published: 27 June 2018 Published online in Wiley Online Library: 3 August 2018 cessing this fruit are beingdemand studied in of order its to consumption servemercial an as use, with increased a wide application functional such as ingredientufacture, in pharmaceutical for or man- com- in foodproducts. processing, such as meat, bakery and dairy of flooding, near coursesest. of Camu-camu rivers stands and out lakes abovenomic other in potential, being the mainly exported species by Amazon by in For- its the(76%), form eco- of extract flour (13.4%) and dehydrated (6.2%). a typical Amazonian fruit Keywords: (37.91%, 41.75%, 43.95%); micronucleus gut testassay (61.01%, 64.40%, through 50.28%); the apoptosis tail index moment (60.26%,(76.43%, (71.64%, 62.44%, 81.02%, 58.22%); 72.31%, comet 68.33%). 70.70%), percent DNA in the tail (64.54%, 68.75%,CONCLUSION: The 76.79%) results and of tail this intensity study contributeand to their increasing use the as production a of health-promoting© camu-camu food. 2018 fruits Society grown of in dry Chemical Industry environments Oliveira,

624 625 1 22 − by car- 8ani- et al. 18 = and Scal- n in vitro 8 animals in b.w.) by oral 25 1 m) in reverse = − μ ), both of them n and results were 1 et al. − 23 mol Trolox equiv- 8 animals) received μ study. = et al. -carotene in these sam- n 𝛽 column (5 , Bergamo Laboratory, Italy; in vivo study wileyonlinelibrary.com/jsfa 18 ® 8) were used for the micronu- = dry weight. The content of total n -carotene from the skin and pitted 1 in vivo − 𝛽 20 . Groups 5, 6, 7 and 8 ( Readings were taken at 535 nm and 1 − 18 dry weight. et al. b.w. We therefore provided crude extracts They reported a total polyphenol intake of 1 for a person weighing about 60 kg, corre- 1 − − 1 26 and the results were expressed as mg gallic − dry weight) was performed by HPLC using a 17 with modifications. Quantification of free ellagic For accurate determination of the amount of and Re 1.5 g, were divided into eight groups. The groups 1 − 24 ± 19 g 100 g , 16 μ body weight (b.w.)) or water (10 mL kg sample) of the extracts was measured 1 The extraction of ), as a single i.p. dose, and the other half ( − 1 1 et al. − 21 − 250 mm Dionex Acclaim 120 C et al. -azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and × ′ The animals used in this study were handled in accordance The total polyphenol content was estimated using The extraction and quantification of free and total ellagic acid in -carotene, and free and total ellagic acid with the Ethical PrinciplesBrazilian for College Animal of Research Animaltocol adopted approved Experimentation by by (COBEA) the the with University’sresearch (23087.006783/2012-70). Ethical Male pro- Swiss mice, Committee 4–5 for weeks old, animal weighing 19.1 Crude extracts from camu-camu fruits atfrom optimum ripeness the stage, Amazon region, weredure obtained described according above and to used the in proce- the received the crudetions extracts based of on their camu-camu total170 in polyphenol mg kg concentrations three (17, concentra- 85 and Folin–Ciocalteu reagent,methodology. according to spectrophotometric alents g phase and UV–visible detector (Shimadzu SPD-10tion AV), with at detec- 254 nm. Experimental design for the fruits of camu-camu was performed according to Rodriguez mals) received oral gavage of 1,2-dimethylhydrazineride dihydrochlo- (DMH; Sigma, St Louis, MO, USA) (30 mg kg approximately 1 g d pitted fruit or skin. Antioxidant capacity ( Extraction and quantification of vitamin C𝜷 (ascorbic acid), The extraction andpitted quantification fruits of of vitamin camu-camu Cpos were in performed the according skin to and Cam- The identification and quantification of ples were performed byphy (HPLC) high-performance according to liquid Pinheiro Sant’Ana chromatogra- anthocyanins was determineding by to Lees spectrophotometry, and accord- Francis. tridge separation sponding to 17 mg kg acid (mg 100 g expressed as polyphenols, samples were primarily eluted by Waters C each group) received a physiological solutionThe (PS; animals NaCl 0.9% that w/v). received DXR ( cleus (MN), comet and oxidative stress assays, and the other half 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays as described byand Phyu Tangpong bert and Williamson. 2,2 the skin and pitted fruitsing of Pinto camu-camu were performed accord- 4.0 the results were expressed as mg cyanidin 3-glucoside 100 g with about 100%, 500%body and weight. 1000% Before the totalthe end polyphenol of animals intake the from by experiment groupsdoxorubicin (day chloridate 1, (DXR; 14), Rubidox 2, half 3 of and 4 ( 30 mg kg acid equivalents (GAE) 100 g with a volume of 10 mL kg gavage twice a day,was for based 15 days. on This studies dosage carried used out in by our Scalbert study ∘ ′′ 14 . 56 ′ 41 in vivo ∘ C). The final extract pH was ∘ 1 15 W) in April 2011. Both unripe ± ′′ assays in these fruits, in order to 19 ′ In spite of the increase in applications 40 Although the micronucleus test is most 10,11 ∘ W). Camu-camu from Roraima, cultivated All these assays are complementary and 12 in vivo ′′ : 624–631 © 2018 Society of Chemical Industry 13 ). The suspension was allowed to stand in the 00 99 1 ′ − 42 ∘ C to constant weight. 2019; ∘ N, longitude 60 ′′ 12 ′ The objectives of this study were therefore to determine The moisture level of the samples was determined by gravimetric In this study, we have chosen the micronucleus assay to detect dark for 24 h under refrigeration (7 corrected to about 2.0. obtain more complete informationcamu-camu. about the health benefits of the chemical profileantioxidant (e.g. levels capacity) ofent of major environments camu-camu phytochemicals (dryripeness, and fruits and and to flooded), grown performidant with mutagenic/antimutagenic in multi-endpoint and different antiox- differ- stages of frequently used to evaluate boneassay considers marrow, the gastrointestinal the tract gut and its micronucleus contactAnother with important food. test applied for this purpose isThis the assay comet considers assay. damage prior towith the repair increased system when DNA cells damageresembling the display image increased of a DNAbreakage comet, migration, indicating in the the amount cell. of DNA and ripe fruits were collectedcharacterized from by Amazonas their (dry skin environment), color:ripe green fruit. in Only unripe ripe fruitenvironment), and because fruits access red was were restricted in by collected the flooding fromAmazon of the River, Roraima covering part (flooded of thethe camu-camu entire bushes period for of fruit almost growth. Eachof experimental unit 10 consisted randomly chosen fruits.fruits, For the analyses seeds performed were manually with separated and pitted theand remainder skin) (pulp was ground in aChina). mixer In (model another Gourmet experiment, DMX433, the Dellar, skinsfrom were the manually fruits separated and ground, using the same equipment. drying at 105 J Sci Food Agric Extraction and quantification of total polyphenols, anthocyanins and antioxidant capacity Crude extracts fromwere obtained the by macerating skin 10 g and ofsolution sample (700 mL with pitted L 100 mL fruits ethanol of camu-camu S, longitude 59 MATERIALS AND METHODS Plant material and moisture content determination Fruits were collected in theBrazilian Amazon Amazonas Forest. and Camu-camu from Roraima the Amazon, states cultivated in in dry conditions, the was collected in the100 Yuricam Farm, located of at km highway AM-010, in Rio Preto da Eva (latitude 2 Commercial camu-camu as functional foodthese chemical compounds can varyon the within environment the (dry or fruit, flooded), depending wherealso on the its fruit ripeness is stage. grown, and www.soci.org of camu-camu from commercial cultivationthere as is a still functional a food, such lack as of antioxidant capacity knowledge and regarding mutagenic/antimutagenicity, its activ- when biological compared to effects, fruitsronments collected in from the flooded Amazon natural region. envi- mutagenic/antimutagenic activity, which isdifferent widely cell applicable types to with potentialand for clastogen detection damage. of both aneugen consist of an evaluation trial of the multi-endpoint assays 49 in a flooded environment, wasmiddle collected Rio from the Branco, margins located of 30 km the north of Boa Vista (latitude 2 2 𝜒 et al. of skin 1 : 624–631 , from São − 1 dry weight, 99 − -values were 1 P − ) and 2019; andexpressedas g 100 g 31 μ et al. tests (growth performance of pulp, 210 mg g (73–142 1 J Sci Food Agric 11 Euterpe oleracea − 35 in vivo dry weight) for fruits grown in a dry 1 The lower concentration of polyphe- − 33 ) (3268 and 3584 mg GAE 100 g , Table 1) were slightly higher than the values 1 of seed. The levels of total polyphenols were − 1 − 34 protein, using Bradford’s method. 1 -carotene was higher in the skins (Table 1), which shows g 100 g − 𝛽 μ ) than in those from flooded) environment (12 (Table 217 1). mg GAE The highest polyphenol content observed in (17 970 mg AGE 100 g 1 1 − − 32 -carotenefoundinthecamu-camufruitsfromdryenvironment 𝛽 Moreover, besides anthocyanins, it was also observed that the There were no differences in the total content The fruits (ripe or unripe) grown in both environments (dry Thermal stress is one of the factors responsible for the increase of Myrciaria jaboticaba (197.22 microtiter plate, as proposed by Rahman Statistical analysis The experiment was conducteddomized design according (CRD) to with three a replications. Analysis(ANOVA) completely of and variance ran- the Tukey average comparisonthe test chemical were analysis applied data, to and and nutrient utilization, oxidative stress and comet assay). The higher in the ripe100 g fruits from dry environment (14 750 mg GAE level of between camu-camu fruits grown in dry(Table or 1), flooded environments despite their different climaticlight conditions (temperature, and water)pounds during formation. On the fruit other hand, there development werein no the anthocyanins and unripe pitted fruits phenolic or skins com- (Tableby 1), which the can be explained physiological changesis during associated with the an maturation increasedleading activity process. to chlorophyll of degradation It chlorophyllase and enhanced enzyme accumulation of anthocyanins, turning the fruits red inpurple the at intermediate the stage end and of maturation. the importance of consuming theof whole camu-camu fruit. Levels 0.05 or less. test was used for comparison oftical micronucleus number. program The used statis- was SAS9.2, licensed (Statistical for Analysis UFV System), (Federal version The University results were of considered statistically Viçosa, significant Brazil) if in 2008. RESULTS AND DISCUSSION Chemical analyses A comprehensive evaluation of camu-camu from its native flooded environment and its dry cultivation,profile, including mutagenic the and phytochemical genotoxic activities,study. were provided in this and flooded) exhibited the sameThey content had, of pulp, on skin average, and seed. 520 mg g nmol mg Paulo state). and 260 mg g environment in the southwest of Brazil. polyphenol levels in fruits. found by Zanatta and Mercadante 100 g this study waset slightly al. lower than that observed by Genovese respectively). nols in the fruits grownpart in flooded because cultivation of (Table the 1) exposurenian may to rivers be is water high, in causing when accumulation of the water around levelwhich the decreases plant, of its temperature. Amazo- The levels of totalcamu-camu, polyphenols regardless of of the growing region, arethe far content greater than reported for someof fruits these compounds, considered such as classical açai ( sources ( )to 1 − www.soci.org L Azevedo L blood magnifi- μ × © 2018 Society of Chemical Industry and with a green filter. ) for 30 s and rinsed in 1 − × magnification. For the gut × Two thousand polychromatic ery- 27 ). 1 C. The colon was removed, opened lon- − ∘ et al. v. 1.5, TriTek freeware). The parameters for 80 TM L low-melting-point agarose (5.0 mL L − with some adjustments. For the staining pro- μ 12 , with some modifications. For this test, 7 13.0). The fluorescent DNA marker (stained with The apoptosis index (AI %) was estimated as the per- 30 , et al. ≥ 29 et al. et al. The apoptotic cells were identified as previously described 8) that received DMH were used for the gut micronucleus 28 ) for 15 min at room temperature, rinsed with distilled water = 1 − n Comet (single-cell gel electrophoresis assay) andstress oxidative assays The peripheral blood cometAzevedo assay was executed as described in centage of apoptotic cells in relation to the total number of cells. cedure, the slides were firstL subjected to hydrolysis in HCl (5 mol wileyonlinelibrary.com/jsfa ( (MN) test and apoptosis assays. At thewere end of anesthetized the with study, all ketamine animals nized and by exsanguination. xylazine At necropsy, and the bone thenand marrow colon cells, eutha- were liver removed from the animals.with The PS liver and stored was at perfused gitudinally, fixed flat in buffered formalin for 24 h,ethanol then solution placed in (700 an mL L was mixed with 73 absolute ethanol. For each animal, 1000 coloncounted epithelial manually cells using were a whole numbergle of continuous row crypts of epithelium in cells which could be a discernedproximal sin- from end the adjacent to the musclemucosal layer surface to analyzed the in distal a end lightcation. at microscope For the at analysis 1000 of apoptosis, the ‘Swisshematoxylin–eosin rolls’ were (HE). stained with For thea identification total of of 20 apoptotic perpendicular cells, well-orientedeach crypts animal, were counting examined the in total numberone. of epithelial cells in each form a microgel. The slidesand were then after immersed in 24 h lysisDNA solution, the gel blades electrophoresis were andsolution left placed (pH to in rest aRed (20 min) horizontal Gel, in 1:10 tank 000 anmicroscope of dilution), alkaline with was a magnification visualized ofFifty using 400 cells a were fluorescence randomlyof counted 100 from cells each werewere blade counted captured and in on a each asoftware total animal; computer (CometScore and the processed resulting by images image analysis by Risio three times, soaked in Schiff reagentin (Merck, Germany) the for dark, 90 min, then washedwashed, for the 5 min slides in wereFine running counterstained Chemicals water. After LTDA, with Brazil) being Fast (5.0 mg Green mL (Vetec Bone marrow micronucleus, gut micronucleus andassays apoptosis These analyses were carriedand out antimutagenicity for properties assayingThe of bone the camu-camu marrow mutagenicity micronucleus crude testdescribed extracts. was by conducted MacGregor as originally throcytes (PCE) were analyzed perusing animal in a slides blindly light scored micronucleus microscope and at apoptosis 1000 assays,the colons euthanized animals, were washed removed withwaste from 0.9% and cut NaCl longitudinally to in removethe the fecal anus. direction The gut from micronucleus the assay was caecumVanhauwaert performed to according to the analysis of DNA damage included:of tail moment the (TM), tail intensity (TI) andof DNA DNA content in in theused. tail). tail For Levels of the the of cometof oxidative (percent the stress glutathione reduced assay were (GSH) mouse determined and liver using oxidized was a (GSSG) method forms for a 96-well

626 627 b Taking 130.36e 80.2b 118.29b 0.08b 1.21b 10.93c 5.37a 79.4ab 16.44c 39 ± ± ± ± ± ± ± ± ± 85.02c 10 611.08 40.22b 357.29 65.66b 997.07 3.54b 28.71 1.20b 171.87 48.9c 569.23 0.15a 86.70 29.12c 978.64 23.3b 1003.88 ± ± ± ± ± ± ± ± ± wileyonlinelibrary.com/jsfa Ripe pitted fruit Ripe skin Cultivation in flooded environment Moreover, the reduction of the apop- 40 80a 210.52 70a,b 945.70 101.19f 12 216.84 50.73c 1124.37 41.26a 348.31 2.88a 27.50 36.77d 1209.08 0.51b 91.79 ± ± ± ± ± ± ± ± standard deviation. ± ), as follows: bone marrow micronucleus (37.91%, 1 − 56.5a 1049.49 a 1.06c 50.82 6.89ab ND 158.86 0.50b 86.20 14.40b 961.21 68.01b 981.15 30.96b 768.25 99.94b 9889.79 30.36a 857.97 ± ± ± ± ± ± ± ± ± These results indicate that camu-camu extracts have a protec- performed assays whenIn compared opposition, to for theirsystems all – negative endpoints gut, tested controls. bonesome in marrow level these and of three(Table blood kinds protective 3). – of effect camu-camu These against exerted camu-camu results DXR extract (Table highlight as 2) observeddamage the or by or DMH the protective increase significant ofwith potential reduction the camu-camu of extract of repair and system DXRrespective in or positive DMH, the control when groups groups compared (G1: treated with protective DXR effects varied or among assays DMH and only). biological systems,it These but was clear that theof camu-camu them extract at exerted some protection level,and in in 170 the all mg kg three extract concentrations (17, 85 41.75%, 43.95%; Table 2); micronucleus gut50.28%; test (61.01%, Table 64.40%, 3);Table 3); apoptosis comet assay index (Fig.72.31%, 1) 70.70%), (60.26%, percent through DNA in the the 62.44%, tail tailand (64.54%, tail 68.75%, moment 76.79%) intensity 58.22%; (76.43%, (71.64%, 81.02%, 68.33%). tive effect inprimary the DNA first lesions steps (genotoxicity) suchalkali of as labile mutation, strand sites. breaks protecting and/or Thisage cells was observed from concluded in by the thecells comet in microgels, assay, reduction electrophoresis which and in fluorescent is staining dam- ofThis based DNA. on technique the detects lysis primarysystem, of DNA nuclear division damage, and before conversion the into repair mutations. tosis level (Table 3) intreated with the camu-camu epithelial extract colon may cellsthe have decrease occurred from of as the mutated a cells. groups result of into account thatmosome the breakage micronuclei and/or chromosome are loss indicativeonto (aneuploidy) progeny passed of cells after fixed complete nuclear chro-of division, gut the or reduction bone marrowthe micronucleus positive frequencies may effects be of dueing upon to the the camu-camu DNA repair extractwith system. many Our compounds authors present concerning act- observation the ability agrees ofto phenolic stimulate compounds DNA repair pathways,tion or through mRNA stabilization. transcription regula- ) ) 1 1 − − and 34 38.20c 1328.50 21.53c 1370.64 63.4c 619.98 11.13a 472.80 47.9ab 1109.62 2.12b 18.13 0.24a 86.20 180d 13 348.97 ± ± ± ± ± ± ± ± ) (Table 1). 32 et al. 1 Unripe − and could be pitted fruit Ripe skin Unripe skin Cultivation in dry environment 36 ) (6–26 and 170–470 mg 18.7a 1022.65 5.51a 11 252.75 1.57c 25.75 0.06a 92.3 112.79a 1066.50 5.02ab ND 223.14 23.47b 764.69 49.5c 282.48 17.65a 1058.54 ± ± ± ± ± ± ± ± ± This ratio was similar to that Ripe 209 24 pitted fruit 1520.45 1418.25 14 749.93 ) 1 fresh weight), and pulp of açaí (68.5 mg − ) 425.44 ) 17.75 1 1 ) ellagic acid in the ripe pitted camu-camu 1 − − 1 − : 624–631 © 2018 Society of Chemical Industry − Fragaria ananassa highlighting their nutritional importance. ) 197.22 99 ) 1100.54 1 1 − using camu-camu from the Brazilian states of − 37,38 32 2019; g 100 g Chemical analyses of pitted fruits and skins of camu-camu grown in different environments (amounts in 100 g of dry sample) μ et al. juice), ) 1 4.78 mg 100 g 1 dry weight, respectively). mol Trolox equivalents − (dry weight), while the free compound corresponds to only mol Trolox equivalents study μ − μ ± 1 1 ) ) 1 1 − − − − 100 g g cyanidin-3-glucoside 100 g g Fruit produced in Roraima (ripe); GAE: gallic acid equivalent; Values represent the average of three replicates Fruit produced in Amazonas (ripe). -Carotene ( The high antioxidant capacity (DPPH and ABTS, Table 1) found The camu-camu fruit presented a high level of ascorbic acid, The contents of free and total ellagic acid are presented in Values followed by the samea letter, in the same line,b do not differ among them by Tukey test at 5% significance level. Determinations Moisture (%) 92.21 Table 1. Vitamin C (mg 100 g ABTS ( Total polyphenols (mg GAE Total ellagic acid (mg 100 g 𝛽 Total anthocyanins (mg Free ellagic acid (mg 100 g DPPH ( in camu-camu fruits fromthe both dry environments environment was and also ripe higheris stage. in an The unripe important camu-camu source fruit high of enough to ellagic increase the acid; fruit antioxidant however, capacity its significantly, perhaps level influenced was by not itspolyphenols. lower Similar levels of results anthocyanin were and found total by Rufino which was higher in the dry environment (1100.54 mg 100 g Table 1. Ripe fruits grown inproportion of a free flooded ellagic environment acid had and,of in a consequence, its higher lower bonded content form (e.g. ellagitanninsthe or total glycosylated). and In free addition, ellagic acid contents(pitted were fruit greater and in green skin). fruits The levels of either free (17.75 mg 100 g J Sci Food Agric Commercial camu-camu as functional food www.soci.org 3.3% of the total determined (16 mg 100 g dry weight). fruit were higher than in the classical sourcessuch of these as compounds, strawberry ( 100 mL The camu-camu extractseffects of were the testedgroups drugs against with DXR the three andplay mutagenic DMH concentrations any in of genotoxic, animals. mutagenic camu-camu The or did treatment oxidative not stress dis- effects in all In vivo Pará and São Paulo, respectively, whichretains confirms its that antioxidant camu-camu potential even outside of its native region. Only 10 g camu-camu fruit would provideamount the daily of recommended ascorbic acid for an adult (65–90 mg) or total (425 mg 100 g than in the flooded environment (945.70 mg 100 g Genovese in camu-camu cultivated inBrazil), a which dry presented environment a (São content100 Paulo g of state, total ellagic acid of 480 mg comparable and even higher than other fruits(42.45 such as strawberries 100 g a b 1 ), − et al. in vitro %AI : 624–631 Myrciaria reduction 99 and This results in b.w., 85 mg kg Myrciaria dúbia 2019; 44 1 − in vivo ). 2 𝜒 -glutamyl cysteinyl glycine) ), 17 mg kg 𝛾 cells AI% J Sci Food Agric Total 0.05 ( G1 (positive control); for apoptosis: < apoptotic < P G4; = Myrciaria dubia cells Total G3 = a G1; G2, G3 and G4 0.2 0.16 0.240.220.13 0.120.21 0.11 0.04 0.07 37.91 41.75 0.130.14 0.15 0.05 43.95 0.14 0.05 Our results showed that the levels of GSH of groups ) compared with positive control (G1). < ± ± ± ± ± ± ± ± 31 2 𝜒 O. %MN 2 reduction 0.05 ( in the frequencies of micronucleated polychromatic erythrocytes to H < 2 P O These antioxidant effects are confirmed by the parameter 2 the DXR inhibitory activityfinally of the (iv) topoisomerase preventing IIleads DNA enzyme; to lesions and inter- such and as intra-strand crosslinking, DNA which damage. systems. glutathione (GSH: tripeptide thiol, (Fig. 1), considered an indicator ofreduced oxidative sulfhydryl stress, which form exists (GSH) in anddisulfide its oxidized (GSSG). form, glutathione Thesepathway structures of take GSH peroxidase,H part which in catalyzes the the conversion enzymatic of the accumulation ofare repaired DNA by strand the cell, breaks, cause which, mutations in unless they treated with DXRlevels plus as the camu-camu negativephenolic extract control. extracts reached This means the decreasedproduction that of same oxidative the free radicals camu-camu stress andpeptide consequently by saving (GSH). the reducing The reduced higher the formation (Fig. of 1e), GSSG indicatingand in oxidized. that Conversely, the the high higher DXR dosethe levels of group DXR extract of antagonized was drug, GSH leading were towards used the same level of GSSG as the positive control (G1); G2 < • 41 50.86 61.00 90.97 61.07 31.07 50.88 30.96 61.06 ...... (Myrciaria dubia) www.soci.org L Azevedo negative control. (ii) acting = b.w.); PS, physiological solution (NaCl, 0.9% w/v); extract camu-camu ( cells %MN Total 42 1 PS − © 2018 Society of Chemical Industry (iii) decreasing + micronucleated 43 b.w.; AI, apoptosis index. 1 Conclusions: G2, G3 and G4 − Frequency of micronucleated gut epithelial cells Frequency of apoptotic cells (2003) Conclusions: for gut micronuclei assay, G5 30 et al. et al. b.w.); PS, physiological solution (NaCl, 0.9%, w/v); extract, camu-camu ( 1 b.w., 170 mg kg gut epithelial cells micronuclei test and apoptotic cells for different concentrations of extract of camu-camu ( − PS 16 000 8 PS 46.78 42 0.53 906.38 0.88 0.10 DMH 45.45 129 1.61 50.28 985.50 4.00 0.41 58.22 DXR 12 000 17 1 PSPS 46.24 52.63 35 46 0.44 0.58 944.75 885.13 1.25 1.50 0.13 0.17 DMHDMH 45.45 42.94 110 104 1.57 1.49 61.01 64.40 863.00 851.29 3.86 3.71 0.45 0.44 60.26 62.44 DXRDXRPS 16 000PS 16 000 16 18 000 14 17 000 5 9 − + + + + + + + + + + + + 1 1 1 1 − 1 1 1 1 1 1 1 1 − − − − − − − − − − − in vivo DMH (positive control) and extracts b.w.; PCE/NCE, polychromatic erythrocytes/normochromatic erythrocytes ratio; MNPCE, polychromatic erythrocytes micronucleus; 1 < − 8000 per group. b.w., 85 mg kg = Results of Theeffectsofdifferentconcentrationsofcamu-camu DMH 1 − + ) 2000 analyzed cells/animal. = Calculated as described in Azevedo Total cells Calculated as described in Azevedo The results demonstrate that the phytochemical compounds G2 / extract 17 mg kg G7 / extract 85 mg kg G8 / extract 170 mg kg G3 / extract 85 mg kg G4 / extract 170 mg kg G3 / extract 85 mg kg G4 / extract 170 mg kg G7 / extract 85 mg kg G8 / extract 170 mg kg 17 mg kg G5 / negative control PSG6 / extract 17 mg kg 48.78 41 0.51 990.13 1.13 0.11 G5 / negative control PSG6 / extract 17 mg kg 16 000 7 N a b DMH, 1,2-dimethylhydrazine dihydrochloride(30 mg kg (MNPCE) in the bone marrow of mice Group / treatment Cells MNPCEdubia PCE/NCE %MN %MN reduction G1 / positive control DMHG2 / extract 17 mg kg 49.69 218 2.73 1007.57 8.00 0.79 Group / treatments Cell/ crypt Table 3. a DXR, doxorubicin (30 mg kg b.w., 170 mg kg G1 / positive control DXR 16 000 25 Table 2. extracts from camu-camu extract play a roleacting in DMH as and DXR a metabolism, protectivetheir barrier carcinogenic against intermediates. DNA Thesecrude damage extract results without induced are purification by that relatedmixture corresponds to to of a a complex camu-camugistic polyphenols, effects which in might theseof exert antimutagenic a effects. syner- dose-dependent Moreover,complexity the effect of lack the may Camu-camu beits crude bioavailability addressed extract and, and by consequently, colon biological the protective potential. effects matrix Thus, againstpounds DMH regarding may damage, act the the by extract reducingdiazonium the com- availability ions of the generatedincreasing active methyl by the its repairferase protein metabolism (MGMT) activity under in the formation of of the O6-MeG adducts. methylguanine liver, or methyltrans- wileyonlinelibrary.com/jsfa free radicals and the lipid peroxidation produced; Additionally, the camu-camu phytochemicalsagainst could DXR have mutagenicity acted viasibilities: (i) the its following activation four different and pos- consequent decrease in the OH through stabilizingthe these performance free of endogenous radicals antioxidants; generated, increasing

628 629 b.w.); PS, 1 − oxidative stress in vivo b.w.) given to three animal 1 − wileyonlinelibrary.com/jsfa ): (a–c) comet assay parameters; Myrciaria dubia 48 activities, measured by botha single-electron hydrogen transfer atom assaysacid. and transfer assay, than gallic acid and ascorbic We gratefully acknowledge FundaçãoEstado de de Amparo Minas à Gerais Pesquisa (FAPEMIG),volvimento do Conselho Científico Nacional e de Tecnológico Desen- (CNPq)Coordenação (483138/2013-4) and de Aperfeiçoamento de(CAPES) Pessoal for financial de support. Nível Superior CONFLICT OF INTEREST The authors have no conflict of interest to declare. CONCLUSION In this study, we havean demonstrated important that source the of camu-camulevels fruit bioactive in is compounds, fruits present from atcompared higher a to dry those environment fromronment. (commercial a This cultivation) flooded suggests (Amazon that Forest,a their Brazil) feasible commercial envi- nutritional, cultivation functional can andover, be economic camu-camu alternative. More- extracts decreased the ACKNOWLEDGEMENTS and exerted protection against the mutagenic effectson of bone the marrow drugs and gut micronuclei, apoptosiswhich are and able comet to assay, predict the risk of cancer.vide These new findings may insights pro- into the functional potentialbeneficial of effects camu-camu on with general health. More 47 In Fig. 1(f) experiment, 31 standard deviation. Different letters within same parameter indicate a significant difference ± in vivo attributed to camu-camu 46 protein). GSH, glutathione reduced form; GSSG, glutathione disulfide form; DXR, doxorubicin (30 mg kg et al. 1 − comet assay and oxidative stress for different concentrations of camu-camu ( : 624–631 © 2018 Society of Chemical Industry 99 in vivo 2019; Results of 45 0.05, Tukey test). < Overall, the results of this study indicated that reactive oxygen (d) (e) (f) (a) (b) (c) P physiological solution (NaCl, 0.9%w/v); extracts 0, 17, 85 and 170 mean the water and dosages of camu-camu extract (mg kg groups by oral gavage.( Quantitative data are the mean species (ROS) scavenging plays a centraland role in antimutagenic the antigenotoxic effects observed in the antioxidants a fundamental roleage in assayed the by reduction thethe of comet content DNA test, of dam- and saponins,vitamin associated flavonoids this C, and effect tannins, bytion with together of with the metal followingwhich and/or antioxidant can the mechanisms: decrease eliminationtion of oxygen chela- of free toxicityflavonoids radicals, antioxidant to present both in enzymes, the this of fruit.that triggered cells Moreover, there radical and by is clear scavengingand induc- evidence the substituents activity anthocyanin of dependsROS-quenching the on efficiency heterocyclic among the rings, different which polyphenol compounds. explains the J Sci Food Agric (d–f) glutathione levels (nmol mg negative control (Fig. 1e).two In cellular addition, thiols theleading ratio (GSH/GSSG) to its between is reduction these in affected many organs by of the oxidative body. stress, Figure 1. Commercial camu-camu as functional food www.soci.org it is possible tothe observe lowest that level, this in ratiothe the gradually highest increased positive concentration from control, of tocan camu-camu be the extracts. explained highest based These on level, results antioxidant the in capacity positive and relationship total between phenolic the content,activity whose biological is based onnumber of its hydroxyl extent, groups, nature,capacity. complexation position and of their substituents, absorption specifically, ellagitannins, found(total ellagic at acid), were high reported to levels exhibit stronger in antioxidant this study which can be attributedpounds, carotenoids to and the vitamins from combinationcamu-camu the crude of complex extract. mixtures phenolic Silva of com- , . 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