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 (wileyonlinelibrary.com) DOI 10.1002/jsfa.9224 Camu-camu (Myrciaria dubia) from commercial cultivation has higher levels of bioactive compounds than native cultivation (Amazon Forest) and presents antimutagenic effects in vivo Luciana Azevedo,a Paula F de Araujo Ribeiro,b Jéssica A de Carvalho Oliveira,a Maryana G Correia,a Flávia M Ramos,b Eduardo B de Oliveira,b Frederico Barrosb* and Paulo C Stringhetab Abstract BACKGROUND: Camu-camu (Myrciaria dubia) is a typical Amazonian fruit and has high antioxidant capacity due to its high levels of vitamin C and phenolic compounds. This study aimed to determine the phytochemicals, antioxidant capacity and antimutagenic effects of camu-camu fruits with different maturity stages grown in dry (commercial cultivation) or flooded environments (native cultivation, Amazon). RESULTS: Total polyphenols, ascorbic acid and in vitro antioxidant capacity levels were higher in ripe fruits grown in a commercial cultivation. The extracts from ripe camu-camu grown in a commercial cultivation exerted antioxidant effects and high percentage of protection against doxorubicin and 1,2-dimethylhydrazine in all tested systems (liver, bone marrow and gut), for three camu-camu extract concentrations (17, 85 and 170 mg kg−1 body weight), as follows: bone marrow minocronucleus (37.91%, 41.75%, 43.95%); micronucleus gut test (61.01%, 64.40%, 50.28%); apoptosis index (60.26%, 62.44%, 58.22%); comet assay through the tail moment (71.64%, 72.31%, 70.70%), percent DNA in the tail (64.54%, 68.75%, 76.79%) and tail intensity (76.43%, 81.02%, 68.33%). CONCLUSION: The results of this study contribute to increasing the production of camu-camu fruits grown in dry environments and their use as a health-promoting food. © 2018 Society of Chemical Industry Keywords: Myrciaria dubia; camu-camu; antimutagenic effects; phytochemicals INTRODUCTION Camu-camu has been under the spotlight because of its high The Amazon region is distinguished by its great biodiversity of antioxidant capacity, due to the chemical profile containing signif- fruits containing remarkable nutritional composition, therapeutic icant levels of vitamin C and phenolic compounds such as ellagic 6,7 value, and thus agribusiness potential, such as guaraná (Paullinia acid and anthocyanins. As is well known, regular intake of foods cupana Kunth), castanha-do-brasil (Bertholletia excelsa Humb. & containing antioxidants may prevent chronic diseases, such as 8,9 Bonpl.), açaí (Euterpe oleracea Mart.), cupuaçu (Theobroma gran- inflammations, mutations, cancer and atherosclerosis. All these diflorum), araçá-pera (Psidium acutangulum) and pupunha (Bactris associated factors have created great interest in camu-camu pro- gasipaes Kunth).1–3Among them, camu-camu (Myrciaria dubia)is duction and, as consequence, commercial cultivation in dry envi- a typical Amazonian fruit4 with natural occurrence during periods ronments has been increasing. However, the concentrations of of flooding, near courses of rivers and lakes in the Amazon For- est. Camu-camu stands out above other plant species by its eco- nomic potential, being mainly exported by Peru in the form of flour ∗ Correspondence to: F Barros, Departamento de Tecnologia de Alimen- (76%), extract (13.4%) and dehydrated (6.2%).5 New ways of pro- tos, Universidade Federal de Viçosa, Viçosa, Minas Gerais 36570-000, Brazil. cessing this fruit are being studied in order to serve an increased E-mail: [email protected] demand of its consumption as a functional ingredient for com- a Faculdade de Nutrição, Universidade Federal de Alfenas, Minas Gerais, Brazil mercial use, with wide application such as in pharmaceutical man- ufacture, or in food processing, such as meat, bakery and dairy 624 b Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, products.4 Minas Gerais, Brazil J Sci Food Agric 2019; 99: 624–631 www.soci.org © 2018 Society of Chemical Industry Commercial camu-camu as functional food www.soci.org these chemical compounds can vary within the fruit, depending The total polyphenol content was estimated using on the environment (dry or flooded), where the fruit is grown, and Folin–Ciocalteu reagent, according to spectrophotometric also on its ripeness stage.10,11 In spite of the increase in applications methodology.16 For accurate determination of the amount of of camu-camu from commercial cultivation as a functional food, polyphenols, samples were primarily eluted by Waters C18 car- there is still a lack of knowledge regarding its biological effects, tridge separation17 and the results were expressed as mg gallic such as antioxidant capacity and mutagenic/antimutagenic activ- acid equivalents (GAE) 100 g−1 dry weight. The content of total ity, when compared to fruits collected from flooded natural envi- anthocyanins was determined by spectrophotometry, accord- ronments in the Amazon region. ing to Lees and Francis.18 Readings were taken at 535 nm and In this study, we have chosen the micronucleus assay to detect the results were expressed as mg cyanidin 3-glucoside 100 g−1 mutagenic/antimutagenic activity, which is widely applicable to pitted fruit or skin. Antioxidant capacity (μmol Trolox equiv- different cell types with potential for detection of both aneugen alents g−1 sample) of the extracts was measured in vitro by and clastogen damage.12 Although the micronucleus test is most 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and frequently used to evaluate bone marrow, the gut micronucleus 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays as described by Phyu assay considers the gastrointestinal tract and its contact with food. and Tangpong19 and Re et al.20 Another important test applied for this purpose is the comet assay. This assay considers damage prior to the repair system when cells Extraction and quantification of vitamin C (ascorbic acid), with increased DNA damage display increased DNA migration, -carotene, and free and total ellagic acid resembling the image of a comet, indicating the amount of DNA The extraction and quantification of vitamin C in the skin and breakage in the cell.13 All these assays are complementary and pitted fruits of camu-camu were performed according to Cam- consist of an evaluation trial of the multi-endpoint assays in vivo.14 pos et al.21 The extraction of -carotene from the skin and pitted The objectives of this study were therefore to determine fruits of camu-camu was performed according to Rodriguez et al.22 the chemical profile (e.g. levels of major phytochemicals and The identification and quantification of -carotene in these sam- antioxidant capacity) of camu-camu fruits grown in differ- ples were performed by high-performance liquid chromatogra- ent environments (dry and flooded), with different stages of phy (HPLC) according to Pinheiro Sant’Ana et al.23 and results were ripeness, and to perform mutagenic/antimutagenic and antiox- expressed as μg 100 g−1 dry weight. idant multi-endpoint in vivo assays in these fruits, in order to The extraction and quantification of free and total ellagic acid in obtain more complete information about the health benefits of the skin and pitted fruits of camu-camu were performed accord- camu-camu. ing Pinto et al.,24 with modifications. Quantification of free ellagic acid (mg 100 g−1 dry weight) was performed by HPLC using a 4.0 × 250 mm Dionex Acclaim 120 C18 column (5 μm) in reverse MATERIALS AND METHODS phase and UV–visible detector (Shimadzu SPD-10 AV), with detec- Plant material and moisture content determination tion at 254 nm. Fruits were collected in the Amazonas and Roraima states in the Brazilian Amazon Forest. Camu-camu from the Amazon, cultivated Experimental design for the in vivo study in dry conditions, was collected in the Yuricam Farm, located at km Crude extracts from camu-camu fruits at optimum ripeness stage, 100 of highway AM-010, in Rio Preto da Eva (latitude 2∘ 41′ 56′′ from the Amazon region, were obtained according to the proce- S, longitude 59∘ 42′ 00′′ W). Camu-camu from Roraima, cultivated dure described above and used in the in vivo study. in a flooded environment, was collected from the margins of the The animals used in this study were handled in accordance middle Rio Branco, located 30 km north of Boa Vista (latitude 2∘ with the Ethical Principles for Animal Research adopted by the 49′ 12′′ N, longitude 60∘ 40′ 19′′ W) in April 2011. Both unripe Brazilian College of Animal Experimentation (COBEA) with pro- and ripe fruits were collected from Amazonas (dry environment), tocol approved by the University’s Ethical Committee for animal characterized by their skin color: green in unripe fruit and red in research (23087.006783/2012-70). Male Swiss mice, 4–5 weeks old, ripe fruit. Only ripe fruits were collected from Roraima (flooded weighing 19.1 ± 1.5 g, were divided into eight groups. The groups environment), because access was restricted by the flooding of the received the crude extracts of camu-camu in three concentra- Amazon River, covering part of the camu-camu bushes for almost tions based on their total polyphenol concentrations (17, 85 and −1 −1 the entire period of fruit growth. Each experimental unit consisted 170 mg kg body weight (b.w.)) or water (10 mL kg b.w.) by oral of 10 randomly chosen fruits. For analyses performed with pitted gavage twice a day, for 15 days. This dosage used in our study 25 fruits, the seeds were manually separated and the remainder (pulp was based on studies carried out by Scalbert et al. and Scal- 26 and skin) was ground in a mixer (model Gourmet DMX433, Dellar, bert and Williamson. They reported a total polyphenol intake of −1 China). In another experiment, the skins were manually separated approximately 1 g d for a person weighing about 60 kg, corre- −1 from the fruits and ground, using the same equipment.