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Bioactive Constituents, Metabolites, and Functions

Brazilian biodiversity fruits: discovering bioactive compounds from underexplored sources Katia Regina Biazotto, Leonardo Mendes de Souza Mesquita, Bruna Vitória Neves, Anna Rafaela Cavalcante Braga, Marcelo Tangerina, Wagner Vilegas, Adriana Zerlotti Mercadante, and Veridiana V. De Rosso J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05815 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 2, 2019

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Journal of Agricultural and Food Chemistry

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Brazilian biodiversity fruits: discovering bioactive compounds

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from underexplored sources

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Katia Regina Biazotto†, Leonardo Mendes de Souza Mesquita†, Bruna Vitória Neves†,

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Anna Rafaela Cavalcante Braga†, Marcelo Marucci Pereira Tangerina‡, Wagner Vilegas§,

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Adriana Zerlotti Mercadante∥ and Veridiana Vera de Rosso†*

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†* Department of Biosciences, Federal University of São Paulo (UNIFESP), Rua Silva

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Jardim 136, Santos, Brazil, CEP 11015-020.

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‡Departament of Botany, Institute of Biosciences, University of São Paulo, CEP 05508-

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090, São Paulo – SP, Brazil

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§UNESP - São Paulo State University/Coastal Campus of São Vicente, Laboratory of

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Bioprospection of Natural Products (LBPN), São Vicente, São Paulo, Brazil.

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∥Department of Food Science, Faculty of Food Engineering, University of Campinas

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(UNICAMP), Campinas, Brazil, CEP 13083-862.

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* Corresponding Author:

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†* Department of Biosciences, Federal University of São Paulo (UNIFESP), Rua Silva

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Jardim 136, Santos, Brazil, CEP 11015-020. E-mail: [email protected]

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ACS Paragon Plus Environment

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ABSTRACT

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Large segments of the Brazilian population still suffer of malnutrition and diet-related

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illnesses. In contrast, many fruits provided of native biodiversity is an underexploited

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source of bioactive compounds and unknown to consumers. The phytochemical

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composition of nine underexplored Brazilian fruits was determined. Carotenoids and

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anthocyanins were identified and quantified by HPLC-PDA-MS/MS; and phenolic

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compounds and iridoids were identified by FIA-ESI-IT-MS/MS, totaling the identification

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of 84 compounds. In addition, the chemical structure and pathway mass fragmentation

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of new iridoids from jenipapo (Genipa americana) and jatoba (Hymenae coubaril) are

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proposed. The highest level of carotenoids was registered in pequi (Caryocar brasiliense)

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(10,156.21μg/100g edible fraction), while the major total phenolic content was found in

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Cambuci (Campomanesia coubaril), 221.70mg GAE/100g. Anthocyanins were quantified

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in jabuticaba (Plinia cauliflora) 45.5mg/100 g and pitanga (Eugenia uniflora) 81.0mg/100

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g. Our study illustrates the chemical biodiversity of underexplored fruits from Brazil,

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supporting the identification of new compounds and encouraging the study of more

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food matrices not yet investigated.

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Keywords: Chemical biodiversity, Brazilian fruits, carotenoids, anthocyanins, phenolic

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compounds, iridoids

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INTRODUCTION

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Nations (FAO/ONU), about 50,000 species of edible plants are available worldwide;

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however, 90% of the energy demands of humans are supplied by only 15 species,

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especially rice, corn, and wheat.1 Brazil is a megadiverse country, home to 18% of all

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plant biodiversity on the planet. Nevertheless, it still suffers from the effects of

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malnutrition, micronutrient deficiencies such as Fe and vitamin A, and, more recently,

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obesity and chronic diseases. Data from the Brazilian National Survey on Demography

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and Health of Children and Women2 pointed out that 17.4% of children under five years

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old have vitamin A deficiency, and the highest prevalence rates were found in the

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Southeast (21.6%) and Northeast (19%) regions of Brazil.2 Overweight (BMI  25 kg/m2)

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and obesity (BMI  30 kg/m2) affected 56.7% and 18.9% of the Brazilian adult population

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in 2016.3

According to estimates of the Food and Agriculture Organization of the United

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In recent years, growing evidence has shown that consuming fruits and

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vegetables reduces the risk of mortality from various diseases, especially cardiovascular

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diseases and cancers. Several fruits from different Brazilian biomes are rich in bioactive

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compounds, such as carotenoids and phenolic compounds.4,5 However, the Brazilian

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Household Budget Survey (2008-2009) verified that the daily consumption of fruits and

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vegetables is only 150 g/day, corresponding to the 177 g of Retinol Equivalent (RE)/day

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because of the contribution of -carotene, α-carotene, and -cryptoxanthin6, and

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representing only 22.1% of the recommended vitamin A intake,7 expressed in RE (26.4%

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women, 18.9% men), or 11.1% if expressed in retinol activity equivalents. A similar result

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was observed for the consumption of phenolic compounds (460 mg/day) derived mainly


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from beverages (48.9%), especially coffee, since the average consumption of fruits is

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only 43.2 mg/day.8

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Besides the low consumption of fruits and vegetables compared to the

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recommendation (400 g fruit + vegetable/day – excluding potatoes and starchy tubers),9

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it is worth mentioning that the most consumed fruits in Brazil are banana, orange, and

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apple. Fruits from the Brazilian biodiversity are poorly consumed, and the only exception

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is açai (3.0 g/day). One of the reasons to explain this low consumption is the lack of

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knowledge about the nutritional composition of native species and poor scientific

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evidence of their health benefits. In addition, one must implement public policies that

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can support the farming/forestry of these species, besides ensuring their acquisition and

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consumption by specific population groups.10

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One success case of the use of Brazilian native biodiversity as a social and

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environmental agent of change occurs with jussara (Euterpe edulis Mart.). Several

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studies identified and quantified its nutritional composition and bioactive

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compounds4,11 and suggested it has high antioxidant and anti-inflammatory

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activities.12,13 Functional products were also developed using jussara pulp,14 and other

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studies involving in vitro fermentation and the bioaccessibility of its bioactive

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compounds were recently published.15–17 This case is an example of how create an

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enabling environment for biodiversity to improve nutrition.

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Moreover, other initiatives, such as Projeto Jussara (Jussara Project),18 are

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needed to expand the species of Brazilian biodiversity that must be studied and receive

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help from governments by public policies to stimulate productive chains. The

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importance of biodiversity for food systems, biome preservation, rural development,

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healthy agriculture, and promotion of access to adequate feeding for all the population


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involves a holistic approach for sustainable development, and the first step to it is

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improving the nutritional knowledge about unexplored specimens.19

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In this context, this study aimed to characterize the phytochemical composition

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of some underexplored tropical fruits from the Brazilian biodiversity, identifying and

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quantifying their major bioactive compounds.

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MATERIAL AND METHODS

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Samples. Each fruit was harvested from three different geographic locations of

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two Brazilian biomes: Atlantic Forest and Cerrado. These fruits were obtained in

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Brazilian protected areas or produced in small family farms. The project was registered

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(no. 50313) and the authorization for collection of the biological material was obtained

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from the Biodiversity Authorization and Information System (SISBIO – Sistema de

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Autorização e Informação em Biodiversidade) from the Chico Mendes Institute for

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Biodiversity Conservation (ICMBIO – Instituto Chico Mendes de Conservação da

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Biodiversidade). The collection, transport, and processing of the biological samples for

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botanical identification of the species was performed according to Fidalgo & Bononi.20

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The material was identified, and the vouchers were deposited and registered at the

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Herbarium of Santa Cecília University (HUSC), in Santos, state of São Paulo (Brazil), which

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has an international registration at the Index Herbariorum of the New York Botanical

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Garden. Access to the photographic record of the collected material can be obtained at

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www.splink.org.br using the voucher number shown in Table 1.

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Each sample consisted of 3 to 10 kg of fruits, according to the processing yield to

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obtain the edible fraction. All the fruits were sanitized with water to remove sediment

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and possible contaminants. The extraction procedures were carried out only with the


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edible parts of each fruit (Table 1), following the same pattern of ingestion from local

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communities. The fruits were collected between July 2015 and July 2016. After the

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removal of the inedible fraction, the samples were immediately lyophilized in a freeze-

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drier and stored at -40 °C until analysis.

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Analysis of carotenoids, anthocyanins, and other bioactive compounds.

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Carotenoids were extracted with acetone from all lyophilized fruits (1 g), transferred to

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petroleum ether/diethyl ether (2:1), and saponified with 10% methanolic KOH overnight

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at room temperature in the dark. The solution was then washed until being alkali-free

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and concentrated to dryness.5 The experimental conditions for separation,

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identification, and quantification by HPLC-DAD-APCI-MS/MS (San Jose, CA, USA) were

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the same as previously described.4,5

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Carotenoids were individually quantified by HPLC-DAD using five-point analytical

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curves of all-trans-lutein (1.0-50.0 μg/mL), all-trans-β-cryptoxanthin (1.0-60 μg/mL), all-

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trans-β-carotene (1.0-50 μg/mL), and all-trans-lycopene (1.0-50 μg/mL). The limit of

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detection (LOD) was calculated using the parameters of each standard curve: LOD = 3.3

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× SD/S, where SD is the standard deviation of the response and S is the slope of the

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curve. For all analytical curves of carotenoids, r2 = 0.99, the limit of detection was 0.1

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μg/mL and the limit of quantification was 0.5 μg/mL. The carotenoid concentration was

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expressed in μg/100 g edible fraction.

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Anthocyanins were extracted from lyophilized pulp and peel (2 g) of pitanga and

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jabuticaba fruits using 100 mL of 0.5% HCl in methanol. The slurry was filtered and

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concentrated in a rotary evaporator (T < 38 oC) to yield the crude extract.4,21 The crude

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extract was diluted in water containing 5% formic acid/methanol (85:15, v/v)


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immediately before analysis by HPLC-DAD-ESI-MS/MS. The anthocyanin separation and

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identification were conducted as previously described.4,22

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The anthocyanins were quantified by HPLC-DAD using a five-point analytical

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curve of cyanidin 3-glucoside (0.5-10.0 mg/mL), r2 = 0.998; the limit of detection was

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0.05 mg/mL and the limit of quantification was 0.1 mg/mL. The concentration was

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expressed in mg of cyanidin 3-glucoside/100 g of edible fraction.

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Phenolic compounds and iridoids were extracted from all fruits using 2 g of

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lyophilized pulp with 50 mL of methanol/water (8:2 v/v) by agitation with a magnetic

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homogenizer (Tecnal, Piracicaba, Brazil) for 20 min. The slurry was filtered, the solids

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were extracted with an additional 50 mL of methanol/water (8:2) two times more, and

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the extract was concentrated in a rotary evaporator (T < 38 °C) until methanol

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evaporation. The extract was used to determine the total phenolic compound content

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by Folin-Ciocalteu reagent, as previously reported.23 The same extract was characterized

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using FIA-ESI-IT-MSn (Flow Injection Analysis - Electrospray - Ion Trap - Tandem Mass

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Spectrometry) and dereplication strategies. Each extract was dissolved in

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methanol/water (8:2 v/v) and infused in the electrospray ionization (ESI) source (direct

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infusion). MS analyses were carried out in a Thermo Fischer Scientific (San Jose, CA, USA)

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LTQ XL mass spectrometer equipped with an ESI source, linear ion trap analyzer, and

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Xcalibur software for data processing. The capillary voltage was set at -35 V, the spray

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voltage at 5 kV, and the tube lens at −200 V. The capillary temperature was 280 °C. Data

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were acquired in MS1 and MSn scanning modes using a syringe pump (flow rate 20

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μL/min). For the IT-MSn experiments, the m/z range of 100-2000 Da was monitored

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using the collision-induced dissociation technique, helium gas, isolation width of m/z 1.5


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and activation time of 30 ms. For each ion it was applied a normalized collision energy

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of 20% up to 50% depending on the fragmentation pattern observed.

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All the compounds were proposed based on their fragmentation patterns compared to the available literature data.

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Statistical analysis. All assays were performed in triplicate for each sample and

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the results expressed as mean ± standard deviation (SD); the differences were significant

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at p < 0.05. The statistical analysis was performed using Microcal Origin 8.0.

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Comparisons by similarity index. Carotenoids and methanolic extracts (phenolic

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compounds and iridoids) were subjected to similarity analysis. For carotenoid extracts,

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retention time and mass/charge ratio (m/z) were the criteria for distinction. For

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methanolic extracts, the m/z was the criterion for distinguishing each molecule detected

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in the analyses. A matrix of presence (1) and absence (0) of chemical markers was

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constructed (for each extract) for clustering data and is shown in the supplementary

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material. The data in this matrix were subjected to similarity analysis conducted by

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Jaccard similarity coefﬁcient, which describes how similar the fruits are in terms of

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shared chemical compounds.24

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RESULTS AND DISCUSSION

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Nine Brazilian tropical fruits from five different botanical families were selected

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to be evaluated in this study. Table 1 shows all fruits analyzed and their respective

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botanical identifications and geographical locations.

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Carotenoid composition. Table 2 shows the characteristics of the carotenoids

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separated from all the fruits investigated. They were identified by applying the following

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mutual data: elution order on C30 column, chromatography with authentic standards,


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UV-visible spectrum (λmax, spectral fine structure, cis peak intensity), and mass

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spectrum, compared with data obtained in the literature. A comprehensive description

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of carotenoid identification was previously described by our research group.4,5 Table 2

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shows the characteristics of 23 peaks found in the studied fruits; from them, only one

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was not identified. Table 3 shows the total and major carotenoid content in each fruit.

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The minor carotenoids identified and quantified and all carotenoid chromatograms are

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presented in the Supporting Information (Table 1S and 2S; Figure 1S and 2S).

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Most of the evaluated fruits presented (all-E)-lutein and (all-E)-β-carotene as

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major carotenoids, except pequi and pitanga fruits. Pequi showed an unidentified peak

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(peak 3) and all-trans-zeaxanthin as main carotenoid components; and in pitanga (all-E)-

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β-cryptoxanthin and (all-E)-lycopene were quantified as the major carotenoids.

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According to Table 3, pequi show the highest levels of carotenoids (10156.21 ± 453

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µg/100 g edible portion) considering all the studied fruits. Carotenoids were not

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detected in jenipapo.

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Samples collected from different locations showed statistical differences in the

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carotenoid composition for all fruits studied in this research. These differences could be

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associated to several parameters, such as the differences between biomes (Atlantic

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Forest and Cerrado), volume of rainfall, seasoning conditions, edaphic characteristics,

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among others. In fact, our results showed that differences in carotenoid contents mainly

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occur because of the geographic location of the plantation. Pitanga from EUB presented

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between 3.7 and 4.5 more (all-E)-β-cryptoxanthin content than those from samples from

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EUC and EUA, respectively. Similar results for pitanga fruits collected in different regions

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of Brazil were already reported. Regarding (all-E)-lycopene, 7300 µg/100 g were found

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in fruits collected in the Northeast region, 7110 µg/100 g in those from the Southeast


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and 1400 µg/100 g in those from the South; for (all-E)-β-cryptoxanthin, 4700 µg/100 g

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were found in fruits from the Northeast, 1180 µg/100 g in those from the Southeast and

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1280 µg/100 g in fruits from the South.25,26

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Although cagaita also belongs to the genus Eugenia, the major carotenoids of E.

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dysenterica fruits are different from those of E. uniflora (pitanga). The major carotenoids

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of cagaita fruits are (all-E)-lutein and (all-E)-β-carotene, which showed different

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amounts in the three evaluated locations (EDA, EDB, and EDC). Ribeiro et al.27 evaluated

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cagaita fruits from the Midwest region of Brazil and found that the mean value of total

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carotenoids was 822 µg/100 g, around 2.6 times greater than the highest result in our

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study, with (all-E)-lutein as major carotenoid (181 µg/100 g). Cardoso et al.28 reported

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770 µg/100 g of total carotenoids for cagaita fruits (Southeast region), a value around

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2.4 times greater than the highest result in our study (319.30 µg/100 g – EDB fruits). In

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addition, the fruits evaluated by Cardoso et al.28 had α-carotene (310 µg/100 g), β-

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carotene (390 µg/100 g), and lycopene (60 µg/100 g) as major carotenoids, showing that

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fruits from different localities can produce different types of carotenoids.

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Araçá fruits from Southeast Brazil (São Paulo state) were already evaluated by

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our research group,4 with 77.7 µg/100 g of total carotenoids, with (all-E)-β-

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cryptoxanthin (26.4 µg/100 g) as the major carotenoid, followed by (all-E)-β-carotene

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(20 µg/100 g). In the present study, (all-E)-β-carotene was detected as major carotenoid,

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followed by (all-E)-lutein, which was detected as the third major carotenoid by da Silva

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et al.4

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Pereira et al.29 reported 41.22 µg/100 g of total carotenoids in araçá fruits grown

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in Southern Brazil (Brazilian region with lower temperatures), with (all-E)-lutein being

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the major carotenoid (26.38 µg/100 g). Although our fruits (from the three localities)


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had (all-E)-lutein as major carotenoid, our lower result is around 2.5 times greater than

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that previously reported. We cannot claim this with certainty, but fruits from warmer

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locations may show higher levels of carotenoids than fruits from colder sites.26

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Cambuci fruits are known for being rich in phenolic compounds, and that is why

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few studies approach their carotenoid composition. The total carotenoid content of the

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cambuci was the lowest among all the fruits evaluated (43.87 – 83.23 μg/100g), except

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for jenipapo, where the presence of carotenoids was not detected. Pereira et al.29

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reported a total of carotenoids of 305.53 µg/100 g in C. xanthocarpa, and cryptoxanthin

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(121.08 µg/100 g) and lutein (81.91 µg/100 g) were the major ones.

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Data on carotenoid content in berries are scarce in the literature. Probably, their

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reddish-purplish color can lead to the carotenoid color to be hidden. Although Inada et

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al.30 reported that the only carotenoid that could be identified and quantified in

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jabuticaba fruits (Myrciaria jaboticaba) was (all-E)-β-carotene (873 µg/100 g), our study

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presented different results, with (all-E)-lutein as the major carotenoid. Zanatta &

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Mercadante31 evaluated the fruit Myrciaria duvia (a berry called camu-camu) from two

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different localities: one from a Cerrado region (Southeast Brazil – 429 m at sea level)

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with a total carotenoid content of 1,095.3 µg/100 g, which represents around three

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times more than the values found for the same fruit from the coastal region (Atlantic

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Forest). Nevertheless, our results for jabuticaba (PCA, PCB, and PCC) showed a higher

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carotenoid content compared to some world-marketed berries, such as strawberries

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(Fragaria vesca) and red currant (Ribes rubrum), which are not considered rich sources

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of carotenoids.32

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Mangaba is a typical fruit from the Brazilian Northeast region (region with the

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highest temperatures) and inhabits the ecosystems of Cerrado, Caatinga, and Savanna


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(ecosystems with low rainfall).33 Rufino et al.34 reported a total carotenoid content of

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300 µg/100 g for mangaba fruits from Caatinga ecosystems, representing about three

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times more than that reported by Cardoso et al.35 (110 µg/100 g), who evaluated fruits

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from Cerrado. To our knowledge, our study is the first to report the profile of

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carotenoids of mangaba fruits, which have (all-E)-lutein and (all-E)-β-carotene as major

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carotenoids. Our results show that mangaba fruits from Atlantic Forest (HSC – Southeast

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Brazil) have higher total carotenoid content compared to mangaba fruits from Cerrado

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(HSA and HSB). This fact suggests that although carotenoids are known for showing

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higher levels in regions of high temperatures and luminosity, other factors may

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positively contribute to the increase. Yuan et al.36 reported that horticultural crops could

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generate accumulate diverse levels and varieties of carotenoids even within the same

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species.

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Jatobá, pitanga and pequi are the fruits with the highest total carotenoid

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contents in this study. To the best of our knowledge, this is the first research to

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accurately report the chromatographic profile of jatobá, which has (all-E)-lutein and (all-

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E)-β-carotene as major carotenoids. Cardoso et al.37 evaluated H. stigonocarpa fruits

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from Cerrado, also popularly known as jatobá, and reported a total of 400 µg/100 g,

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similar to the value found in our study (HCC: 345.21 µg/100 g). Pequi CBB fruits have

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about two times more carotenoids than EUB (second best result of our study). Ribeiro

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et al.27evaluated pequi fruits from five different Brazilian regions, with a range from

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3,700.08 to 18,700.00 µg/100 g of total carotenoids, suggesting that carotenoid content

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is affected by the native region of the pequi fruit.

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The similarity index comparison from carotenoid identified of all fruits evaluated

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shared 24% similarity (Figure 1). The cluster was divided into two main groups. The first


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group shares 38% of the carotenoids and is composed by mangaba (HSA, HSB, and HSC)

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and cagaita (EDA and EDB). These fruits share the carotenoids (15Z)-lutein, (9Z)-

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violaxanthin, and 5,6-epoxy-β-carotene, which were not detected in the other fruits. The

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second group shares 27.5% similarity and is composed by pitanga (EUA, EUB, and EUC),

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jatobá (HCA, HCB, and HCC), araçá (AAU, AAB, and AAC), pequi (CBA, CBB, and CBC),

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jabuticaba (PCA, PCB, and PCC), cambuci (CPA, CPB, and CPC), and cagaita (EDC). Into

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this group, all fruits presented carotene derivatives, but with some subdivisions (Figure

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1). The first one is composed by pitanga, because this was the only fruit that showed

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rubixanthin and lycopene derivatives, and therefore it distances itself from the other

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fruits. There is a subgroup with 60% similarity between jatobá and araçá, because they

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are the only ones that share the presence of (9Z)-lutein and absence of (all-E)-

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zeaxanthin. Pequi presented one unidentified carotenoid, (all-E)-neoxanthin, and (9Z)-

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violaxanthin, which causes a subgrouping of this fruit into the second group. Cagaita

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(EDC) shares more carotenoids (presence and absence) with cambuci than with same-

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species fruits from other localities (EDA/EDB). All fruits presented (all-E)-lutein and (all-

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E)-β-carotene (except mangaba).

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Anthocyanin composition. Anthocyanins were only detected in jabuticaba and

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pitanga fruits, both from the Myrtaceae family. The anthocyanins found in both species

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were cyanidin 3-rutinoside and cyanidin 3-glucoside. The chromatographic profile of

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both fruits was very similar (Supporting information – Figure 3S); the anthocyanin

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contents were expressed in terms of cyanidin 3-glucoside and are presented in the

313

supplementary material (Table 3S). The anthocyanin quantities ranged from 23.5 ± 1.7

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to 45.5 ± 2.6 mg/100 g for jabuticaba and from 22.9 ± 0.5 to 81.0 ± 5.3 mg/100 g for


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pitanga. As also occurred for the carotenoid composition data, the anthocyanin content

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was statistically different considering the place of origin of the fruit.

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Pitanga and jabuticaba are considered sources of anthocyanins, as well as

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Dovyalis abyssinica (41.96 mg/100 g), Cyphomandra betacea (8.48 mg/100 g),5

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Malpighia emarginata (6.5-8.4 mg/100 g),38 and Euterpe edulis (91.52-236.19 mg C3G

320

equivalent/100 g dry matter).39 Recently, the importance of anthocyanins has been

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threatened, since they show low bioavailability. Despite the beneficial properties of

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anthocyanins, their effectiveness at preventing or treating a range of diseases depends

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on their bioavailability.17 However, it is known that these pigments are bioconverted

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into phenolic acids because of the action of intestinal microorganisms, and therefore

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some authors attribute prebiotic action to them.17

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Phenolic compounds. The identification of these compounds was based on the

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main molecular ions and on some of the useful observed fragmentations. The

328

compounds listed in Table 4 were restricted to those in which [M-H]− ions were clearly

329

detected. Among the observed ions, some were too small to allow structural analyses.

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Among the secondary metabolites detected in extracts, we identified ellagitannins,

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flavonoids (quercetin, myricetin, apigenin, and luteolin derivatives), gallic and quinic

332

acids. All analyses were conducted in negative ion ionization mode, which provided a

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very sensitive and selective method, being an excellent way to identify the compounds

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present in the extracts. The compounds were organized from highest to lowest m/z

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(Table 4).

336

Ellagitannin derivatives. Ellagitannins are the compounds with the highest m/z

337

detected in this study (compounds 1-7, 9, 10, and 19). They were detected only in fruits

338

from the Myrtaceae family (cambuci, pitanga, mangaba, jabuticaba, and araçá).


14

Page 15 of 55


339

Ellagitannins are a hydrolyzable tannin group, which has a typical fragmentation of 152

340

Da [M-H-152]−, corresponding to the loss of a galloyl moiety and/or a typical

341

fragmentation

342

hexahydroxydiphenoyl (HHDP) groups. The ions at m/z 935 and m/z 785 were chosen to

343

represent the fragmentation model of the ellagitannin class. The MS2 fragmentation of

344

the deprotonated molecule [M-H]- at m/z 935 produced fragments at m/z 917 [M-H-

345

18]−, which represents a neutral loss of water, along with the product ions at m/z 783

346

[M-H-152]− and at m/z 633 [M-H-302]−. The MS³, MS4, MS5, and MS6 fragmentations

347

were performed in order to confirm the identification of this compound (Table 4),

348

proposed as di-HHDP-galloyl hexoside (Figure 2A). The MS² spectrum of the precursor

349

ion at m/z 785 also presented ellagitannin fragmentation. The MS2 spectrum generated

350

the product ions at m/z 767 [M-H-18]−, m/z 633 [M-H-152]−, m/z 419 [M-H-152-152-18-

351

44]−, and m/z 301 [M-H-484]−. The schematic representation of this fragmentation is

352

shown in Figure 2B.

of

302

Da

[M-H-302]−,

which

indicates

the

presence

of

353

The occurrence of ellagitannin derivatives is common in fruits from the

354

Combretaceae, Lythraceae, Melastomataceae, Punicaceae, and Trapaceae families.40 In

355

the Myrtaceae family, the target of our study, we could detect 12 ellagitannin

356

derivatives by mass spectrometry analysis. The isolation and identification of

357

compounds with high molecular weight require the use of complex steps of separation

358

and purification. Moreover, ellagitannins are very unstable, with complex molecular

359

structures, thermolabile, and photosensitive. Furthermore, because of their structural

360

complexity, these compounds are poorly absorbed by the organism (low bioavailability)

361

and are bioconverted into lower molecular weight compounds.37 The consumption of

362

ellagitannins has a prebiotic-like effect and has been associated with different health


15


Page 16 of 55

363

benefits.41 Urolithins are intestinal microbial metabolites produced from ellagitannin

364

and ellagic acid rich foods that are much better absorbed, with numerous benefits, such

365

as anti-inflammatory, anticarcinogenic, antiglycative, antioxidant, and antimicrobial

366

effects.42 Recently, Selma et al.43 reported the importance of different types of urolithins

367

(urolithins A, B, and isourolithin A) and discussed their potential of novel probiotics with

368

applications in the development of functional foods and nutraceuticals.

369

Flavonoids and diarylketone derivatives. Several known flavonoids, their

370

glycosyl derivatives and diarylketone derivatives were identified in all fruits evaluated.

371

Nine different aglycones were detected: maclurin, quercetin, cirsiliol, myricetin,

372

isorhamnetin, phlorizin, kaempferol, diosmetin, and apigenin, and their derivatives.

373

Diarylketone derivatives were detected in jabuticaba (compounds 11 and 25). The MS²

374

spectrum of ion m/z 727 produced two major fragments: the ion at m/z 531 [M-H-152-

375

44]− arose from the loss of a galloyl moiety (Figure 2C), whereas the ion at m/z 261 [M-

376

H-466]− arose from the loss of a di-O-galloyl-glucoside, which led us to propose the

377

presence of a maclurin derivative, named maclurin-di-O-galloyl-hexoside. In the same

378

pattern of fragmentation, the m/z 575 was identified, and it represents maclurin mono-

379

O-galloyl-hexoside [m/z 727 – m/z 575 = 152 Da, because of a galloyl moiety].

380

Quercetin derivatives were detected in five fruits: cambuci, pitanga, mangaba,

381

jabuticaba, and araçá. Several substituents were detected in the quercetin aglycone. The

382

MSn fragmentation of the precursor ion at m/z 751 (Figure 2D) produced some

383

diagnostic fragments: the ion at m/z 599 [M-H-152]− is characteristic of a galloyl moiety

384

and the ion at m/z 301 [M-H-450]− arose from the loss of a di-O-galloyl-rhamnoside [M-

385

H-152-152-146]− (compound 8). Therefore, we suggest the identification of a quercetin-


16

Page 17 of 55


386

di-O-galloyl-rhamnoside. The ion at m/z 585 showed the same pathway of

387

fragmentation and represents quercetin-O-galloyl-pentoside (compound 24).

388

The compound at m/z 685 was assigned as quercetin-O-malonyl-galloyl-

389

rhamnoside (compound 12); the MSn fragmentation of this precursor ion led to the

390

product ions at m/z 667 [M-H-18]−, due to a neutral loss of water; m/z 523 [M-H-162]−,

391

typical fragmentation of a hexose moiety; and m/z 345 [M-H-340]−, due to a loss of

392

rhamnoside + galloyl moiety + half of a malonyl group. The ion at m/z 301 [M-H-384]−

393

confirms the presence of a quercetin aglycone (Figure 2E).

394

A cirsiliol derivative (m/z 329) was detected in a jatobá fruit extract (HCC locality).

395

The MS² spectrum of the ion at m/z 653 showed major fragments at m/z 635 [M-H-18]−

396

(neutral loss of water), 491 [M-H-162]− (loss of a hexoside), 447, and 329 (cirsiliol

397

aglycone). The MS³ [653 → 491] spectrum showed fragments at m/z 431, 401, 343, 329

398

[M-H-162]−, due to a sequential loss of a hexoside moiety. We proposed the

399

identification of a cirsiliol-di-hexoside (compound 14).

400

Myricetin derivatives (m/z 317) were detected in pitanga, cagaita, jabuticaba,

401

and araçá fruits. The MSn spectrum of the ion at m/z 631 generated the product ions at

402

m/z 613 [M-H-18]− (neutral loss of water), 479 [M-H-152]− (loss of a galloyl moiety), and

403

317 [M-H-152-162]− (loss of galloyl + hexose moiety). The ion at m/z 317 confirms the

404

presence of a myricetin aglycone. The MS² spectrum of the ion at m/z 449(a) led to the

405

product ions at m/z 431 [M-H-18]− (neutral loss of water), m/z 345, and m/z 317 [M-H-

406

132]− (loss of a pentoside). The MS³ [449 → 317] generated the product ions at m/z 299,

407

271, 245, 195, 167, and 138 (myricetin confirmation ions).

408

Isorhamnetin derivatives were detected in jatobá (HCC), pitanga, jabuticaba, and

409

mangaba samples. Isorhamnetin is an O-methylated quercetin with m/z 315. The MS2


17


Page 18 of 55

410

spectrum of the ion at m/z 639 generated the major fragment at m/z 477 [M-H-162]−

411

(loss of a hexoside) and the MS³ [639 → 477] generated the major fragment ion at m/z

412

315 [M-H-162-162]− (sequential loss of hexosides). This fragmentation pattern allowed

413

us to infer the presence of a isorhamnetin-dihexoside (compound 15). The MSn spectrum

414

of the ion at m/z 623 led to the product ions at m/z 461 [M-H-162]− (loss of a hexoside)

415

and 315 [M-H-162-146]− (loss of a rutinoside group), tentatively identified as

416

isorhamnetin-O-rutinoside.

417

Only one phloretin derivative was detected in this study, in jabuticaba samples.

418

The MSn spectrum at m/z 597 showed the major fragment ion at m/z 434 [M-H-162]−

419

(loss of a hexoside group); the confirmation ion at m/z 273 occurred because of a

420

concomitant loss of a second hexoside unit. We identified the ion at m/z 597 as

421

phloretin-di-hexoside (compound 21). Kaempferol aglycone (m/z 285) was detected in

422

pitanga, jabuticaba, and mangaba fruits. The ion at m/z 593 was identified as

423

kaempferol-rutinoside (compound 23). The MS² spectrum generated the major product

424

ion at m/z 285 [M-H-308]−, due to the loss of a rutinoside group. The MS² spectrum of

425

the precursor ion at m/z 447(c) generated the major fragment ion at m/z 285 [M-H-

426

162]−, attributed to the loss of a hexoside group, characteristic of a kaempferol-hexoside

427

(compound 38).

428

Diosmetin aglycone (m/z 299) was detected in jatobá (HCC) and mangaba fruits.

429

The MS² spectrum of the ion at m/z 445 generated the major fragment ions at m/z 427

430

[M-H-18]− (neutral loss of water), 415, 383, 329, and 299 [M-H-146]− (loss of a

431

deoxyhexose moiety), tentatively identified as diosmetin-deoxyhexoside (compound

432

39). Apigenin aglycone (m/z 269) was detected in jatobá (HCC), mangaba, and pequi

433

fruits. The MS² spectrum of the ions at m/z 431 and 401 showed the same base peak at


18

Page 19 of 55


434

m/z 269, referent to the loss of a hexoside and a rhamnoside, respectively. A self-

435

proposed flavanone identification was made to araçá for the ion at m/z 297. The

436

pathway fragmentation is proposed in Figure 2F. The MS² spectrum of the ion at m/z 297

437

generated the major fragments: m/z 295 [M-H-2]−, because of double bond formation

438

in ring C (2-3); m/z 269 [M-H-28]−, due to the loss of a dimethyl group; and m/z 251 [M-

439

H-46]−, because of methoxy and methyl groups. The ion at m/z 595 was identified as

440

[2M-H]− adduct of the ion at m/z 297.

441

A wide variety of aglycones and substituents of flavonoids were detected in our

442

results. All biological functions depend on events that occur at the molecular level. Thus,

443

one must know the molecular structure to infer a function relationship.44 Flavonoid

444

derivatives, especially their glycosides, are the most common group of polyphenolic

445

plant secondary metabolites. They are the most vital phytochemicals in diets and are of

446

great general interest because of their diverse bioactivity.45 Most flavonoids appear in

447

the form of glycosides under natural conditions. However, some changes may occur

448

because of abiotic/biotic stress conditions of the plant, and these modifications often

449

change their solubility, reactivity, and stability.46 There are two ways in which flavonoids

450

can be absorbed by the body: through the small intestine or through the colon before

451

absorption.47 This depends on the physicochemical properties of the food, such as

452

molecular size, configuration, lipophilicity, and solubility.48 The sugar and substituent

453

moieties of flavonoids are important to determine their bioavailability.44 In our results,

454

we found galloyl, malonyl, and glycosyl flavonoids.

455

Galloyl-hexoside flavonoids were detected in cambuci, pitanga, cagaita, and

456

jabuticaba, all from the Myrtaceae family. Some studies have already detected galloyl

457

substituents in flavonoid aglycones in the Myrtaceae family.49–51 These compounds have


19


Page 20 of 55

458

many biological functions, such as antioxidant, anti-inflammatory, neuronal cellular

459

protecting, anti-diabetic, and anti-tumor effects.52,53

460

Although there are numerous studies related to the identification, isolation, and

461

biological activity of flavonoids, very little is known about how the flavonoid

462

glycosylation affects bioactivity in vivo.54 One flavanone aglycone was detected in araçá

463

fruits (m/z 297: 5-dihydroxy-7-methoxy-6,8-dimethylflavanone). Oikawa et al.55 isolated

464

one flavanone from Myrceugenia euosma (Myrtaceae) called 5,7-dihydroxy-6,8-

465

dimethylflavanone (m/z 283), which has −14 Da than m/z 297, suggesting that the

466

flavanone proposed in our study has an additional methyl radical. Araçá fruits are the

467

only ones of the Myrtaceae family evaluated in this study that have this compound,

468

which reflects the smaller similarity between this species and the others of the

469

Myrtaceae family. Therefore, the fruits analyzed in this study (except for jenipapo) have

470

a wide variety of types of flavonoid derivatives.

471

Total phenolic content was different in each locality of cambuci, pitanga, cagaita,

472

araçá, mangaba, and pequi fruits (Table 5). The Myrtaceae family showed higher

473

phenolic content compared to the other families evaluated in this study (p=0.0320).

474

Pequi has the lowest phenolic content compared to the other fruits.

475

Iridoid Composition. Iridoids were detected only in jatobá and jenipapo fruits

476

(table 6). The deprotonated molecule at m/z 565 was tentatively identified as gardoside

477

derivative (HCA and HCB). The MS2 spectrum of ion 565 generated the major fragments

478

at 355 [M-H-210]− and 337 [M-H-228]− relative to the loss of one dehydrated and one

479

hydrated aglycone respective. MS³ [565 → 355] generated the major fragment ion at

480

m/z 337 [M-H-18]− (loss of water) and m/z 225 [M-H-130]−. The m/z 555 was tentatively

481

identified as genipin derivative (HCA and HCB). The MS/MS fragments at 392 [M-H-162]−


20

Page 21 of 55


482

(Retro-Diels Alder fragmentation) and the confirmation at m/z 225 [M-H-330]− relative

483

to the aglycone and were considered to propose the identification. Jenipapo (GAA and GAC) has three different ions with the main aglycone at m/z

484 485

349,

486

tetrahydro-1Hcyclopenta[c]pyran-4-carboxylic acid (Figure 3A). The MS² fragmentation

487

of ion 349 led to m/z 243 [M-H-106]− (structural rearrangement) (Figure 3A) and m/z 225

488

[M-H-106-18]− (loss of water). The compound at m/z 515 [M-H]− showed MSn fragments

489

at m/z 349 [M-H-166]− (due to a phenolic acid substituent loss) and m/z 331 [M-H-184]−

490

(further loss of water – Figure 3B), and was identified as 1-hydroxy-5-(4’-

491

methoxybenzoyl)-7-(5’’-methoxygalloyl)-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-

492

4-carboxylic acid. Similarly, the MSn fragmentation of the ion at m/z 531 [M-H]− led to

493

m/z 349 [M-H-182]− (due to the same phenolic acid substituent loss with one more

494

hydroxyl group). This compound was named 1-hydroxy-5-(4’-methoxybenzoyl)-7-(3’’-

495

hydhoxy-5’’-methoxygalloyl)-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylic

496

acid (Figure 3C).

497

tentatively

identified

as

1,7-dihydroxy-5-(4’methoxybenzoate)4a,5,6,7a-

Jenipapo (GAB) showed another three iridoids with m/z 273, 241, and 213. The

498

difference

between

these

compounds

is

the

substituent

499

cyclopentanopyran. The m/z 273 was tentatively identified as 1-hydroxy-6-hydroxy-5-

500

methoxy-7-methoxymethyl—4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylic

501

acid (Figure 3D), with MS/MS fragment ions at m/z 255 [M-H-18]− (loss of water), m/z

502

241 [M-H-32]− (loss of MeOH), m/z 229 [M-H-44]− (loss of CO2), and m/z 197 [M-H-76]−

503

(loss of methanol and CO2). The ion at m/z 241 [M-H]− was tentatively identified as 1-

504

hydroxy-5,7-dimethoxy-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylic acid

505

(Figure 3E), with MS/MS fragments at m/z 209 [M-H-32]− (loss of MeOH) and m/z 165


linked

to

the

21


Page 22 of 55

506

[M-H-76]− (loss of MeOH and CO2). The ion at m/z 273 was tentatively identified as 1-

507

hydroxy-7-methoxy-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylic

508

(Figure 3F), with MS/MS fragments at m/z 181 [M-H-32]− (loss of methanol), m/z 169

509

[M-H-44]− (loss of CO2), and m/z 137 [M-H-76]− (loss of methanol and CO2).

acid

510

Iridoid derivatives are monoterpenes with a cyclopentanopyran ring and occur

511

naturally in plants, but, in contrast to polyphenols, are rarely found in edible fruits.56

512

Apocynaceae, Scrophulariaceae, Diervillaceae, Lamiaceae, Loganiaceae, Fabaceae, and

513

Rubiaceae are the main families that show iridoids in their constitution.57 There are

514

many benefits in an iridoid-rich ingestion, such as antiaging,58 anti-inflammatory,58 and

515

antidiabetic effects.59

516

Although jenipapo and jatobá fruits are rich in iridoids,60–62 no iridoids detected

517

in the fruit extracts were identified before. However, based on the main fragmentations,

518

we could propose their molecular structures, but it is recommended that work be

519

dedicated to the isolation of these compounds for accurate identification by Nuclear

520

Magnetic Resonance (NMR). Our proposals were based on the main iridoid aglycones

521

found in the genera Genipa and Hymenaea: genipin and gardoside derivatives. Popov &

522

Handijeva63 proposed the main pathway fragmentations of iridoids by electron impact

523

ionization mass spectrometry, such as: [M-H-228]− due to aglycone loss, [M-H-162]−

524

because of a Retro-Dies-Alder loss or sugar moieties, and the classical fragments [M-H-

525

76]−, [M-H-44]−, [M-H-32]−, and [M-H-18]−. These fragmentation pathways were also

526

observed by Ren et al.64 with the electrospray ionization mass spectrometry (ESI-MSn)

527

technique, the same used in this study. Furthermore, based on the structural

528

rearrangements of the iridoid fragmentation pathways, we propose for the ion at m/z

529

349 the structure called 1,7-dihydroxy-5-(4’ methoxybenzoate) 4a,5,6,7a-tetrahydro-


22

Page 23 of 55


530

1H-cyclopenta[c]pyran-4-carboxylic acid (detected in Genipa americana fruits, locality A

531

and C). Moreover, jenipapo and jatobá are the only fruits studied in this research that

532

show different chemical composition of the methanolic extract between the three

533

localities. GAA and GCC have the same compounds: m/z 531, 515, and 349 (100%

534

similarity), while GAB presents the ions at m/z 276, 241, and 213 (0% similarity

535

compared to the other fruits).

536

The results obtained from Cluster Analysis of methanolic extract divided the

537

fruits into four distinct groups, as can be seen in Figure 4. One can also observe a high

538

diversity of secondary metabolites in these fruits, since many compounds are found in

539

only one species. The first group was formed by the samples of Myrtaceae fruits, which

540

are the only ones with ellagitannin derivatives, and they share some flavonoids, such as

541

compound 8 (m/z 751), detected in cambuci, cagaita, and jabuticaba, and compounds

542

16, 36, and 53, detected in pitanga, mangaba, and jabuticaba. Besides that, fruits from

543

the Myrtaceae family had the highest values of phenolic content (Table 5). Into this

544

group, we can highlight a strong similarity between pitanga and jabuticaba (~50%). Only

545

araçá presented flavanones (m/z 297 and 595). Despite a low similarity (~15%), the

546

second group is constituted by pequi and mangaba (Figure 1B), both having low phenolic

547

content and the compound 46 in common. The third group is represented by samples

548

of murici. These fruits have only 5% similarity compared to the other fruits analyzed.

549

Besides, HCA and HCB have a different composition compared to the HCC fruits (~15%),

550

which have flavonoids and iridoids. Jenipapo fruits form the fourth group and do not

551

have any compounds in common with the other fruits analyzed (0% similarity). In

552

addition, fruits from GAA and GAC also do not have any compounds in common with


23


Page 24 of 55

553

those detected in the locality GAC. All compounds from jenipapo detected in this study

554

belong to the class of iridoids.

555

The same pattern of differentiation occurred in total phenolic content, since GAA

556

and GAC have statistically equivalent values compared to GAB. Jatobá fruits from locality

557

A and B have 100% similarity between each other (HCA and HCB). When compared to

558

HCC, they show 11.1% similarity. The ion at m/z 555 (shanzhiside aglycone derivative)

559

was the only ion detected in the three localities. We highlight that the content and

560

proportion of secondary metabolites may differ depending on ecological factors in areas

561

where the plants are grown.65 However, our data do not allow us to infer with precision

562

the real reason for these differences.

563

Our results show that the Brazilian fruit biodiversity is positively correlated with

564

the number of secondary metabolites. We detected 51 phenolic compounds (between

565

flavonoids and phenolic acids), 08 iridoids, 23 carotenoids, and 2 anthocyanins in nine

566

fruits from the Brazilian biodiversity. This shows an excellent resource with

567

technological and economic potential still unexplored, mainly for the nutritional and

568

pharmaceutical sectors. Some experimental studies in the field of nutrition, published

569

in high-quality editorial journals, often do not evaluate the chemical characterization of

570

the diet offered to patients and/or animals, and are based only on the literature to

571

justify the nutraceutical effects found in some foods. This study proves that the same

572

fruit can have a varied content of bioactive compounds when subjected to different

573

types of cultivation and environmental conditions. Therefore, analyses of chemical

574

composition are always of extreme importance. In addition, it enters as a database with

575

several proposals for identification of bioactive compounds, which will help several

576

future studies.


24

Page 25 of 55


577

Hence, one must know the composition of the bioactive compounds present in

578

the natural resources from this country. Despite the inherent evidence that the fruits of

579

the Brazilian biodiversity have an immense nutritional and nutraceutical potential, it is

580

important to ensure the human right of adequate food to people that have difficult

581

access to food. This knowledge promotes food and nutritional security, achieving social

582

inclusion and citizenship. There are still few studies that detail the composition of these

583

fruits.

584 585

ASSOCIATED CONTENT

586

Supporting Information Available: Carotenoid content from nine fruits (Table 1S and

587

Table 2S) and carotenoid chromatograms (Figure 1S and Figure 2S). Anthocyanins

588

composition from pitanga and jabuticaba fruits (Table 3S) and anthocyanins

589

chromatogram (Figure 3S). This material is available free of charge via the Internet at

590

http:// pubs.acs.org.

591 592

FUNDING

593

We thank the Biodiversity for Food and Nutrition Project, Brazilian Environmental

594

Ministry, FUNBIO – Biodiversity Fund, Bioversity International, FAO, ONU –

595

Environmental and GEF – Global Environmental Facility for financial support. AZM

596

thanks FAPESP (proc. 2013/07914-8). VVR thanks CNPq (303956/2015-1).

597 598

References

599

(1)

600

FAO - Food and Agriculture Organization of the United Nations. Biodiversity and Nutrition: A Common Path. Nutrition and Consumer Protection Division. 2010.


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Brazil; Ministry of Health. Brazilian National Survey on Demography and Health of Children and Women (2006): Dimensions of Reproduction and Child Health. 2009.

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Brazil; Ministry of Health. Secretary of Health Surveillance. Vigitel Brazil 2016:

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Surveillance of Risk Factors and Protection for Chronic Diseases by Telephone Survey:

605

Estimates of Frequency and Sociodemographic Distribution of Risk Factors and Protection

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for Chronic Diseases in the Capitals of the 26 Brazilian States and in the Federal District

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in 2016. 2017.

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(4)

Silva, N. A. da; Rodrigues, E.; Mercadante, A. Z.; De Rosso, V. V. Phenolic Compounds and

609

Carotenoids from Four Fruits Native from the Brazilian Atlantic Forest. J. Agric. Food

610

Chem. 2014, 62 (22), 5072–5084.

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(5)

de Rosso, V. V.; Mercadante, A. Z. Identification and Quantification of Carotenoids, by

612

HPLC-PDA-MS/MS, from Amazonian Fruits. Journal of agricultural and food chemistry

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2007, 55 (13), 5062–5072.

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(6)

Vargas-Murga, L.; de Rosso, V. V.; Mercadante, A. Z.; Olmedilla-Alonso, B. Fruits and

615

Vegetables in the Brazilian Household Budget Survey (2008–2009): Carotenoid Content

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and Assessment of Individual Carotenoid Intake. Journal of Food Composition and

617

Analysis 2016, 50, 88–96.

618

(7)

Institute of Medicine (IOM). Food and Nutrition Board, 2001. Dietary References Intakes

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for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese,

620

Molybdenum, Nickel, Silicon, Vanadium, and Zinc. 2001.

621

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Corrêa, V. G.; Tureck, C.; Locateli, G.; Peralta, R. M.; Koehnlein, E. A. Estimate of

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Consumption of Phenolic Compounds by Brazilian Population. Revista de Nutrição 2015,

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FAO/WHO. Diet, Nutrition and the Prevention of Chronic Diseases. Report of a Joint FAO/WHO Expert Consultation. 2003.


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Hunter, D.; Özkan, I.; Moura de Oliveira Beltrame, D.; Samarasinghe, W. L. G.; Wasike, V.

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W.; Charrondière, U. R.; Borelli, T.; Sokolow, J. Enabled or Disabled: Is the Environment

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Schulz, M.; Borges, G. da S. C.; Gonzaga, L. V.; Seraglio, S. K. T.; Olivo, I. S.; Azevedo, M.

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S.; Nehring, P.; de Gois, J. S.; de Almeida, T. S.; Vitali, L. Chemical Composition, Bioactive

631

Compounds and Antioxidant Capacity of Juçara Fruit (Euterpe Edulis Martius) during

632

Ripening. Food Research International 2015, 77, 125–131.

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Table 1. Sample characterization of all fruits studied. Common name Botanical name Family (Portuguese)

Edible fraction

Campomanesia coubaril

Myrtaceae

Cambuci

Mesocarp and endocarp

Eugenia uniflora

Myrtaceae

Pitanga

Epicarp and mesocarp

Eugenia dysenterica

Myrtaceae

Cagaita


Plinia cauliflora

Myrtaceae

Jabuticaba


Psidium cattleianum

Myrtaceae

Araçá

All fruit

Hymenae coubaril

Fabaceae

Jatobá

Mesocarp

Hancornia speciose

Apocynaceae

Mangaba

Epicarp and Mesocarp

Caryocar brasiliense

Caryocaraceae

Pequi

Mesocarp

Genipa Americana

Rubiaceae

Jenipapo

Mesocarp

n.r.: not registered.


Code (locality) - Geographical coordinate CPA (23°29'21.4"S 46°42'52.9"W) CPB (23°22'35.2"S 45°26'51.9"W) CPC (23°42'28.6"S 46°24'18.4"W) EUA (15o55'31"S 46o10'57"W) EUB (23°53'14.6"S 48°00'16.4"W) EUC (23°29'21.4"S 46°42'52.9"W) EDA (16°40'47,4''S 43°51’29,7''W) EDB (16°48’46,1''S 44°18’07,0''W) EDC (15°55'30,6''S 46°10’57,1''W) PCA (23°29'21.4"S 46°42'52.9"W) PCB (21°46'20.6"S 47°05'03.1"W) PCC (15°55'06.3"S 46°06'23.4"W) AAU (15°59'39,2''S 46°18’49,4''W) AAB (15°59'39,2''S 16°18’32,3''W) AAC (16°36'17,9'S 44°05’56,1''W) HCA (21°50'36,1''S 47°25’24,5''W) HCB (46°48'46,1''S 44°18’07,0’’W) HCC (21°47'46,6''S 47°22’44,9''W) HSA (15°55'06.3"S 46°06'23.4"W) HSB (16°40'47,4''S 43°51'29,7''W) HSC (17°56'65" S 46°43.33"W) CBA (16°36'17,9''S 44°05'56,1''W) CBB (16°21'49,0''S 44°06’57,5''W) CBC (15°55'31,8''S 45°10’59,0''W) GAA (16°36'17,9''S 44°05'56,0''W) GAB (23°25'11,8''S 45°38'31,6''W) GAC (16°38'46,9''S 44°11'23,2''W)

Voucher specimens n.r. n.r. n.r. N° HUSC 12101 N° HUSC 11449 N° HUSC 11445 N° HUSC 11444 N° HUSC 11443 N° HUSC 11453 N° HUSC 11446 N° HUSC 11448 N° HUSC 11447 N° HUSC 11454 N° HUSC 11455 N° HUSC 11456 N° HUSC 11451 N° HUSC 11459 N° HUSC 11441 N° HUSC 12102 N° HUSC 12103 N° HUSC 12104 N° HUSC 11459 N° HUSC 11458 N° HUSC 11441 N° HUSC 11448 N° HUSC 11449 N° HUSC 11454

37


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Table. 2 Chromatographic, UV−Vis and mass spectroscopy characteristics of carotenoids from the nine fruits obtained from HPLC-DAD-MS/MS. Peak

Carotenoid

tRa (min)

λmax (nm)b

% III/II

1

(all-E)-neoxanthin

6.1

416, 440, 469

86

0

601

2

(9Z)-neoxanthin

8.7

327, 415, 438, 468

80

14

601

3

not identified

8.9

420, 445, 472

50

0

601

4

(all-E)-neochrome

10.3

398, 420, 447

80

0

601

5

(13Z)-lutein

12.3

328, 410, 435,467

17

32

569

6

(9Z)-violaxanthin

12.8

326, 410, 435, 463

63

14

601

7

(13'Z)-lutein

13.7

336, 410, 435, 465

18

40

569

8

(all-E)-lutein

15.8

418, 443, 471

60

0

569

9

(9Z)-lutein

18.8

326, 417, 439, 466

57

18

569

10

(all-E)-zeaxanthin

18.9

418, 447, 470

33

0

569

11

5,8-epoxy-β-cryptoxanthin

20.2

418, 426, 452

50

0

569

% AB/II


[M + H]+ (m/z)

MS/MS (m/z) 583[M+H-18], 565[M+H-18-18], 547[M+H-18-18-18], 509[M+H-92], 491[M+H-18-92], 221 583[M+H-18], 565[M+H-18-18], 547[M+H-18-18-18], 509[M+H-92], 491[M+H-18-92], 221 583[M+H-18], 565[M+H-18-18], 547[M+H-18-18-18], 509[M+H-92], 491[M+H-18-92], 221 583[M+H-18], 565[M+H-18-18], 547[M+H-18-18-18], 509[M+H-92], 491[M+H-18-92], 221 551 [M+H-18], 533[M+H-18-18], 477[M+H-92] 583[M+H-18], 565 [M+H-18-18], 491[M+H-18-92], 221 551[M+H-18], 533[M+H-18-18], 477[M+H-92], 463[M+H-106], 459[M+H-18-92] 551[M+H-18], 533[M+H-18-18], 477[M+H-92], 463[M+H-106], 459[M+H-18-92] 551[M+H-18], 533[M+H-18-18], 477[M+H-92], 463[M+H-106], 459[M+H-18-92] 551[M+H-18], 533[M+H-18-18], 463[M+H-106] 551[M+H-18], 459[M+H-18-92], 221

38

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12 13 14 15 16 17 18 19

(all-E)-α-cryptoxanthin (all-E)-β-cryptoxanthin 5,6-epoxy-β-carotene (13Z)-β-carotene (all-E)-α-carotene (9Z)-α-carotene (all-E)-β-carotene (9Z)-β-carotene

24.3 28.4 31.5 32.4 35.1 36.4 39.5 41.8

418, 445, 471 420, 450, 476 418, 446, 470 336, 418, 444, 469 420, 445, 472 330, 420, 444, 472 421, 451, 476 331, 418, 446, 471

50 30 20 17 50 60 25 25

0 0 0 46 0 09 0 19

553 553 553 537 537 537 537 537

20

(all-E)-rubixanthin

46.9

439, 463, 492

36

0

553

21

(9Z)-rubixanthin

47.9

350, 437, 460, 488

30

30

553

535[M+H-18], 461[M+H-92] 535[M+H-18], 461[M+H-92] 535[M+H-18], 461[M+H-92], 205 444[M-92] 481[M+H-56], 444[M-92] 481[M+H-56], 444[M-92] 444[M-92] 444[M-92] 535[M+H-18], 497[M+H-56], 461[M+H-92] 535[M+H-18], 497[M+H-56], 461[M+H-92]

290, 360, 440, 466, 75 12 537 467[M+H-69], 444[M+H-92] 497 23 (all-E)-lycopene 83.9 447, 474, 505 75 0 537 467[M+H-69], 444[M+H-92] a Retention time on C column; b Linear gradient of methanol/MTBE; λ 30 max: maximum absorption wavelength (nm); % III/II: spectral fine structure; % AB/II: 22

(9Z)-lycopene

55.7

intensity of cis peak.


39


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Table. 3 Total and major carotenoids from nine Brazilian fruits from three different locations. Total carotenoids* Major carotenoids* Fruit Location (μg/100g edible fraction) (μg/100g edible fraction) (all-E)-lutein (all-E)-β-carotene a b Cagaita EDA 305.15 ± 23.8 89.0 ± 5.38 74.6 ± 7.99b a a EDB 319.30 ± 7.89 109.3± 3.12 84.13± 2.24b EDC 269.96 ± 15.3a 63.4 ± 8.80c 155.8 ± 7.99a (all-E)-lutein (all-E)-β-carotene Jatobá HCA 2675.90 ± 56.2b 1270.48 ± 26.1a 804.58 ± 49.5b HCB 4074.74 ± 92.5 a 1595.51 ± 162.1a 1326.15 ± 47.4a c b HCC 1171.19 ± 172.0 539.93 ± 88.7 345.21 ± 42.5c (all-E)-lutein (all-E)-β-carotene b b Jabuticaba PCA 152.40 ± 6.61 91.00 ± 4.99 32.60 ± 4.80b b b PCB 154.74 ± 11.99 82.23 ± 8.35 39.94 ± 3.74b PCC 326.70 ± 63.0a 172.54 ± 38.3a 99.04 ± 19.7a (all-E)-lutein (all-E)-β-carotene Cambuci CPA 83.23 ± 1.98 a 22.61 ± 0.37b 29.62 ± 1.29a CPB 43.87 ± 6.15 b 13.45 ± 0.16c 9.52 ± 1.72b a a CPC 81.69 ± 4.63 34.14 ± 1.06 27.00± 3.17a (all-E)-lutein (all-E)-β-carotene b b Araçá AAU 61.44 ± 2.66 13.10 ± 0.47 21.27 ± 0.95b b b AAB 49.37 ± 3.61 8.25 ± 0.91 16.42 ± 1.01b a a AAC 110.06 ± 17.79 38.30 ± 5.24 47.55 ± 4.65a not identified (all-E)-zeaxanthin Pequi CBA 8600.87 ± 147.2b 3943.22 ± 78.5a 1564.77 ± 58.3ab a a CBB 10156.21 ± 453.2 4675.13 ± 93.9 2074.19 ± 274.5a CBC 6090.66 ± 267.4c 2675.53 ± 400.9b 1092.11 ± 117.1b (all-E)-β-cryptoxanthin (all-E)-lycopene Pitanga EUA 1748.06 ± 69.8b 548.41 ± 17.0b 288.71 ± 62.3b b b EUC 1902.32 ± 176.7 666.22 ± 50.8 184.40 ± 13.6b a a EUB 5880.98 ± 434.5 2467.71 ± 189.1 1047.26 ± 11.3a


40

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(all-E)-lutein Mangaba HAS 101.12 ± 43.96± 5.4a b HSB 80.76 ± 6.4 20.17 ±1.32b HSC 160.11 ± 14.3a 48.82 ± 4.5a * mean ± standard deviation; Jenipapo (GAA, GAB, GAC): carotenoid not detected 0.2b

(all-E)-β-carotene 29.44 ± 1.5b 25.34 ± 3.08b 37.42 ± 3.8a


41


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Table 4. Mass spectroscopy characteristics of phenolic compounds from nine Brazilian fruits from three different locations. ID

Compound

[M-H]m/z 951

1

trisgalloyl-HHDP-hexoside isomer

2

trigalloyl-HHDP-hexoside

937

3

di-HHDP-galloylhexoside

935

4

ellagitannin derivative

801

5


799

6

digalloyl-HHDP-hexoside

785

MS/MS

Fruit

MS²: 907 [M-H-44]-, 836, 783 MS³ [951 → 907]: 889, 782 [M-H-125]-, 763, 605, 301 MS4 [951 → 907 →782]: 764, 708, 481 [M-H-302]-, 301 MS5 [951 → 907 → 782 → 481]: 301 [M-H-180]-

CPA/CPB/CPC

66

MS²: 919 [M-H-18]-, 845, 812, 785[M-H-152]-, 767, 451, 301, 275 MS³ [937 → 919]: 767, 750 [M-H-169]-, 727 MS²: 917 [M-H-18]-, 783 [M-H-152]-, 765 [M-H-170]-, 633, 451 [M-H-152-170-162]-, 301 [M-H-152-170-162-150]MS³ [935 → 917]: 749 [M-H-168]MS4 [935 → 917 → 749]: 597 [M-H-152]-, 491, 405, 338 MS5 [935 → 917 → 749 → 597]: 445 [M-H-152]MS6 [935 → 917 → 749 → 597 → 445]: 275 [M-H-170]MS²: 757 [M-H-44]-, 631 MS³ [801 → 757]: 633 [M-H-124]-, 613, 605 MS4 [801 → 757 → 633]: 463 [M-H-170]MS5 [801 → 757 → 633 → 463]: 301 MS²: 755 [M-H-44]-, 727, 647 [M-H152]-, 601, 526 MS³ [799 → 755]: 727, 603 [M-H152]MS4 [799 → 755 → 603]: 433 [M-H-170]-, 391, 361, 337, 288 MS5[799 → 755 → 603 → 433]: 288 MS²: 767 [M-H-18]-,633 [M-H152]-, 419, 301 MS³ [785 → 633]: 615 [M-H-18]-, 301 [M-H-302]MS4 [785 → 633 → 301]: 257, 185

EDA/EDB/EDC

67

CPA/CPB/CPC EDA/EDB/EDC

66

CPA/CPB/CPC

68

PCA/PCB/PCC

-

CPA/CPB/CPC EDA/EDB/EDC EUA/EUB/EUC

69


Ref.

42

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7

pedunculagin I

783

8

quercetin-di-O-galloyl-rhamnoside

751

9


737

10

galloyl proanthocyanidin dimer

729

11

maclurin-di-O-galloyl-hexoside

727

12

quercetin-O-malonyl-galloylrhamnoside

685

13

methyl-digalloyl-dihexoside

659

14

cirsiliol-dihexoside

653

MS²: 765 [M-H-18]-, 631 [M-H-152]-, 613, 481, 451, 275, 229 MS³ [783 → 765]: 613 [M-H-152]-, 597 [M-H-168]MS4 [783 → 765 → 597]: 553 [M-H-44]-, 453, 445, 427, 301 MS4: [783 → 765 → 613]: 461 [M-H-152]-,425, 403, 301 MS5 [783 → 765 → 597 → 553]: 295 MS²: 733 [M-H-18]-, 707 [M-H-44]-, 599 [M-H-152]-, 583 [M-H-168]-, 393, 449, 393, 367, 301 [M-H-450]MS³ [751 → 599]: 581 [M-H-18]-, 555 [M-H-44]-,431 [M-H168]-, 393 [M-H-206]-, 367 [M-H-232]MS4 [751 → 599 → 431]: 413, 387 MS2: 705, 585 [M-H-152]-, 435 [M-H-302]MS³ [737 → 705]: 661 [M-H-44]MS³ [737 → 435]: 377, 305 MS4 [737 → 705 → 661]: 529 [M-H-132]MS²: 669, 625, 619, 579, 577, 572 [M-H-152]MS³ [729 → 572]: 559 MS²: 575 [M-H-152]-, 557 [M-H-170]MS³ [727 → 575]: 531, 423 [M-H-152]-, 405, 362, 261, 235, 191 MS4 [727 → 575 → 423]: 379 [M-H-152]-, 363 MS²: 667 [M-H-18]-, 625, 595 [M-H-90]-, 523 [M-H-162]-, 301 MS³ [685 → 667]: 521 [M-H-146]-, 538, 380, 320 MS4 [685 → 667 → 521]: 301, 155 MS²: 641 [M-H-18]-, 627, 615, 524, 492, 478, 395, 317 MS³ [659 →641]: 609, 565, 479, 313 MS4 [659 → 641 →609]: 591, 565 [M-H-44]-, 487, 400 MS²: 635, 491 [M-H-162]-, 447, 329 MS³: 431, 401, 343, 329 [M-H-162]-


CPA/CPB/CPC EDA/EDB/EDCPCA/PCB /PCC/EUA/EUB/EUC

70

CPA/CPB/CPC/PCA/PCB /PCC/EDA/EDB/EDC

71

EDA/EDB/EDC

-

AAU/AAB/AAC

72

PCA/PCB/PCC

73

EDA/EDB/EDC

66

EDA/EDB/EDC

74

HCC

Self proposed

43


15

Isorhamnetin-dihexoside

639

16

myrecitin-galloyl-hexoside

631

17

isorhamnetin-rutinoside

623

18

ellagic acid-galloyl-hexoside

615

19

galloyltannin derivative

613

20 21

rutin phloretin-di-hexoside

609 597

22

5-dihydroxy-7-methoxy-6,8dimethylflavanone [2M-H]-

595

23 24

kaempferol-rutinoside quercetin-O-galloyl-pentoside

593 585

25

maclurin mono-O-galloyl-hexoside

575

MS²: 607 [M-H-32]-, 477 [M-H-162]-, 417, 377, 329 MS³ [639 → 477]: 315[M-H-162]MS²: 587, 571, 499 [M-H-132]-, 479 [M-H-152]-, 451 [M-H180]-, 525, 317 MS³ [631→ 451]: 436 [M-H-15]-, 433 [M-H-18]-, 407, 379, 351, 317 MS³ [631 → 479]: 332, 317[M-H-162]MS²: 604 [M-H-18]-, 551, 477 [M-H-146]-, 461 [M-H-162]MS³ [623 → 461]: 315 [M-H-146]-, 161, 135 MS4[623 → 461 → 315]: 135 [M-H-180]MS² 463, 301 MS³ [615 → 463]: 301 [M-H-162] MS4 [615 → 463 → 301]: 273, 257, 239, 193, 179, 151, 107 MS²: 595 [M-H-18], 565, 461 [M-H-152]-, 401, 301 MS³ [613 → 461]: 402, 373, 301 [M-H-160]MS4[613 → 461 → 301]: 257 [M-H-44]MS²: 343, 301 [M-H-308]MS²: 507 [M-H-90]-,477 [M-H-120]-, 435 [M-H-162]-, 387 MS³ [597 → 477]: 459 [M-H-18]-, 387 [M-H-90]-, 357 [M-H120]MS³ [597 → 435]: 406, 308 MS4 [597 → 477 → 387]: 357, 326, 315, 239, 209 MS²: 297 MS³ [595 → 297]: 295, 251, 203, 181, 160, 131 MS4 [595 → 297 → 295]: 251, 223, 203, 181 MS²: 458, 429, 315, 285 [M-H-308]MS²: 433 [M-H-152]-, 357, 301 MS³ [585 → 433]: 301 [M-H-162]MS4[585 → 433 → 301]: 273, 240, 179, 151 MS²: 556 [M-H-18]-, 531 [M-H-44]-, 423 [M-H-152]MS³ [575 → 423]: 363 [M-H-60]-, 269 [M-H-154]-


Page 44 of 55

HCC PCA/PCB/PCC/EUA/EU B/EUC/EDA/EDB/EDC

Self proposed 75

HSA/HSB/HSC

-

EDA/EDB/EDC

50

CPA/CPB/CPC

68

HSA/HSB/HSC PCA/PCB/PCC

76

AAU/AAB/AAC

55

HSA/HSB/HSC EDA/EDB/EDC

75

PCA/PCB/PCC

73

44

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26

caffeoyl-O-hexagalloyl

493

27

digalloyl-hexoside

483

28

ellagic acid-hexoside

481

29

isorhamnetin-hexoside

479

30

gallic acid derivative

467

31

HHDP-hexoside

465

32

quercetin-O-hexoside or isorhamnetin-O-rhamnoside

463

33

methyl-ellagic acid-rhamnoside

461

34

myricetin-pentoside

449A

35

HHDP-rhamnoside

449B

MS4 [575 → 423 → 363]: 162 [M-H-100]MS²: 477 [M-H-16]-, 331 [M-H-162]-, 313 [M-H-180]MS³ [493 → 331]: 315, 303, 275, 241, 169 MS²: 439, 331 MS³ [483 → 331]: 313, 271, 193, 169 [M-H-162]-, 125 MS²: 465 [M-H-16]-, 391 [M-H-90]-, 361 [M-H-120]-, 319 [M-H-162]-, 301, 275 MS³ [481 → 319]: 304 [M-H-15]-, 301, 275, 258, 194 MS³ [481 → 301]: 300, 285, 257 MS4 [481 → 319 → 304]: 283, 258, 173, 127 MS²: 315 [M-H162]-, 299 [M-H-180]-, 285 [M-H-195]-, 255, 213, 161 MS³ [479 → 315]: 301, 241, 179, 193, 151, 137 MS³ [479 → 299]: 285 [M-H-15]-, 255, 213, 161 MS4 [479 → 299 → 285]: 269 [M-H-15]-, 242, 210, 200 MS²: 449 [M-H-18]-, 377 [M-H-90]-, 351[M-H-116]-, 305 [M-H-162]-, 231, 169 MS³ [467 → 351]: 333 [M-H-18]-, 307, 261 [M-H-90]-, 231 [M-H-120]-, 189 [M-H-162]-, 131 MS4 [467 → 351 → 231]: 215, 185, 172, 144, 132 MS²: 403, 363, 321 [M-H-144]-, 282 MS³ [465 → 321]: 303 [M-H-18]MS²: 419, 331, 315[M-H-146]-, 301 [M-H-162]MS³ [463 → 315]: 288, 245, 191, 178, 151, 137 MS²: 417 [M-H-44]-, 355, 315 [M-H-146]MS³ [461 → 315]: 301 [M-H-15]MS²: 431, 345, 317 [M-H-132]MS³ [449 → 317]: 299, 271, 245, 195, 167, 138 MS²: 431, 303[M-H-146]-, 285, 258, 151 MS³ [449 → 303]: 285, 177, 125


EDA/EDB/EDC

75

CPA/CPB/CPC EDA/EDB/EDC AAU/AAB/AAC

4

PCA/PCB/PCC EUA/EUB/EUCHSA/HSB /HSC

-

AAU/AAB/AAC/CBA/CB B/CBC

77

CBA/CBB/CBC

78

EDA/EDB/EDCPCA/PCB /PCCHSA/HSB/HSCEUA /EUB/EUC CPA/CPB/CPCHSA/HSB/ HSC EDA/EDB/EDC/AAU/AA B/AAC HCA/HCB

75

-

68

79

-

45


36 37 38

isorhamnetin-O-pentoside or quercetin-O-rhamnoside ellagic acid-rhamnoside

447a

MS²: 315 [M-H-132]-, 301 [M-H-146]-

447b

MS²: 429, 403, 323, 315 [M-H-132]-, 301 [M-H-146] MS³ [447 → 315]: 301 MS²: 301 [M-H-146]-, 285 [M-H-162]MS³ [447 → 301]: 273, 239, 192, 179, 151, 107 MS²: 427, 415, 383, 329, 299 [M-H-146]-, 265 MS²: 301 [M-H-132]MS³ [433 → 301]: 299, 273, 239, 193, 179, 151, 121

447c

39 40

quercetin-rhamnoside or kaempferol-hexoside diosmetin-rhamnoside quercetin-pentoside

41

apigenin-hexoside

431

42

apigenin-pentoside

401

43

ethyl gallate-hexoside

359

44

chlorogenic acid

353

MS²: 197 MS³ [359 → 197]: 179 [M-H-18]-, 153 [M-H-18]MS²: 191 [M-H-162]-, 183, 177, 123

45

caffeoyl-hexoside

341

MS²: 326, 297 [M-H-44]-, 185 [M-H-156]-, 179, 161

46 47 48

gallic acid-hexoside ellagic acid Quercetin

331 301a 301b

MS²: 313, 271, 169 [M-H-162]MS²: 283, 257, 229, 185 MS²: 257, 229,185, 179, 151

49

5-dihydroxy-7-methoxy-6,8dimethylflavanone quinic acid

297

MS²: 295, 269, 251, 223, 203, 181, 159 MS³ [297 → 295]: 251, 205, 203, 181 MS²: 172, 163, 110

50

445 433

191

MS² 415, 387, 305, 283, 269 [M-H-162]MS³ [431 → 269]: 233, 209 MS²: 269 [M-H-132]-


Page 46 of 55

EDA/EDB/EDC/PCA/PC B/PCC CPA/CPB/CPC

72

PCA/PCB/PCC EUA/EUB/EUC HSA/HSB/HSCHCC PCA/PSCB/PCCEDA/ED B/EDCEUA/EUB/EUC AAU/AAB/AACHCA/HC B HSA/HSB/HSCHCC/CBA /CBB/CBC HSA/HSBHSC/CBA/CBB /CBC EDA/EDB/EDC

-

80

81

79

EUA/EUB/EUCHSA/HSB HSC/CBA/CBB/CB/PCA/ PCB/PCC EUA/EUB/EUCCBA/CBB /CBC PCA/PCB/PCC CPA/CPB/CPC EDA/EDB/EDCPCA/PCB /PCC/EUA/EUB/EUC AAU/AAB/AAC

82

83

55

CPA/CPB/CPC/HSAHSB/ HSC/ AAU/AAB/AAC

-

46

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51

gallic acid

169

MS²: 140, 125

CPA/CPB/CPC/EDA/DB/ EDC Campomanesia Coubaril: CPA, CPB, CPC; Eugenia uniflora: EUA, EUB, EUC; Eugenia dysenterica: EDA, EDB, EDC; Plinia cauliflora: PCA, PCB, PCC; Psidium cattleianum: AAU, AAB, AAC; Hancornia speciosa: HSA, HSB, HSC; Caryocar brasiliense: CBA, CBB, CBC;.


-

47


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Table 5. Total phenolic content from nine Brazilian fruits from three different locations. Fruits

Cambuci

Pitanga

Cagaita

Jabuticaba

Araçá

Jatobá

Mangaba

Pequi

Jenipapo

CPA

Total phenolics* [mg GAE]/100g EF 221.70 ± 24.69a

CPB

142.60 ± 8.14b

CPC

111.97 ± 5.91b

EUA

110.93 ± 22.69b

EUB

185.71 ± 14.94a

EUC

140.26 ± 9.46b

EDA

101.16 ± 9.43b

EDB

91.74 ± 10.89a

EDC

45.21 ± 6.13a

PCA

129.51 ± 12.66a

PCB

107.11 ± 8.13a

PCC

109.65 ± 10.84a

AAU

67.72 ± 3.18a

AAB

85.30 ± 6.77b

AAC

68.16 ± 6,15a

HCA

115.25 ± 23.41a

HCB

96.39 ± 6.55a

HCC

104.59 ± 5.55a

HAS

45.46 ± 1.86a

HSB

38.22 ± 2.41b

HSC

38.22 ± 1,60b

CBA

26.91 ± 11.49a

CBB

25.34 ± 2.05a

CBC

9.16 ± 0.74b

GAA

2.29 ± 0.18b

GAB

3.39 ± 0.02a

GAC

2.23 ± 0.08b

Locality

Comparisons F = 40.5861 P = 0.0007 F = 15.4439 P = 0.005 F = 32.9619 P = 0.001 F = 3.1948 P = 0.1133 F = 9.6419 P= 0.0139 F = 1.2941 P = 0.3416 F = 24.6935 P = 0.0019 F = 63.4851 P = 0.0003 H* = 5.6 P = 0.0608

* mean ± standard deviation; EF: Edible Fraction; Campomanesia Coubaril: CPA, CPB, CPC; Eugenia uniflora: EUA, EUB, EUC; Eugenia dysenterica: EDA, EDB, EDC; Plinia cauliflora: PCA, PCB, PCC; Psidium cattleianum: AAU, AAB, AAC; Hymenae coubaril: HCA, HCB, HCC; Hancornia speciosa: HSA, HSB, HSC; Caryocar brasiliense: CBA, CBB, CBC; Genipa americana: GAA, GAB, GAC.


48

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Table 6. Mass spectroscopy characteristics of iridoids from Hymenae coubaril and Genipa americana from three different locations. ID Compound [M-H]MS/MS Fruit m/z 1 gardoside derivative 565 MS²: 575, 423, 355 [M-H-210]-, 337, 225 HCA/HCB MS³ [565 → 355]: 337 [M-H-18]-, 225, 207 MS4 [565 → 355 → 337]: 225, 207, 139 MS5 [565 → 355 → 337 → 207]: 178, 165 [M-H-44]MS6 [565 → 355 → 337 → 207 → 165]: 149, 135, 121 2 genipin derivative 555 MS²: 299, 225 HCA/HCB/HCC MS³ [555 → 299]: 225, 207, 179, 165 MS4[555 → 299 → 225]: 207, 165,124 3 1-hydroxy-5-(4’-methoxybenzoyl)-7- 531 MS²: 513 [M-H-18]-, 443, 365, 349 [M-H-182]-, 331 GAA/GAC (3’’-hydhoxy-5’’-methoxygalloyl)MS³ [531 → 349]: 287, 242, 209, 183, 121 4a,5,6,7a-tetrahydro-1Hcyclopenta[c]pyran-4-carboxylic acid 4 1-hydroxy-5-(4’-methoxybenzoyl)-7- 515 MS²: 471 [M-H-44]-, 349[M-H-166]-, 353 [M-H-162]-, GAA/GAC (5’’-methoxygalloyl)-4a,5,6,7a331 tetrahydro-1H-cyclopenta[c]pyranMS³ [515 → 349]: 287, 261, 165, 159 4-carboxylic acid 5 1,7-Dihydroxy-5-(4’ 349 MS²: 331 [M-H-18]-, 305 [M-H-44]-, 287, 243, 209, 183, GAA/GAC methoxybenzoate) 4a,5,6,7a165, 139 tetrahydro-1H cyclopenta[c]pyran-4carboxylic acid 6 1-dydroxy-6-hydroxy-5-methoxy-7273 MS²: 255 [M-H-18]-, 241[M-H-32]-, 219 [M-H-54]GAB methoxymethyl-4a,5,6,7aMS³ [273 → 241]: 211, 209, 167, 139, 119 tetrahydro-1H-cyclopenta[c]pyran4-carboxylic acid 7 1-dydroxy-5,7-dimethoxy-4a,5,6,7a- 241 MS²: 225 [M-H-16]-, 209 [M-H-32]GAB tetrahydro-1H-cyclopenta[c]pyranMS³ [241 → 209]: 179, 165, 121 4-carboxylic acid


Ref. Self proposed

Self proposed Self proposed

Self proposed

Self proposed

Self proposed

Self proposed

49


8

1-dydroxy-7-methoxy-4a,5,6,7a213 MS²: 195 [M-H-18]-, 181 [M-H-32]-, 169 [M-H-44]-, 137, tetrahydro-1H-cyclopenta[c]pyran119 4-carboxylic acid Hymenae coubaril: HCA, HCB, HCC; Genipa americana: GAA, GAB, GAC.


Page 50 of 55

GAB

Self proposed

50

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Figure 1. Cluster of similarity index comparison from carotenoid identified in all fruits evaluated. HCA, HCB, HCC: Hymenae coubaril; HSA, HSB, HSC: Hancornia speciose; CBA, CBB, CBC: Caryocar brasiliense; AAU, AAB, AAC: Psidium cattleianu; CPA, CPB, CPC: Campomanesia coubaril; EDA, EDB, EDC: Eugenia dysenterica; EUA, EUB, EUC: Eugenia uniflora; PCA, PCB, PCC: Plinia cauliflora; GAA, GAB, GAC: Genipa Americana.


51


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Figure 2. Phenolic compounds identification from fruit methanolic extracts, using mass spectra. (2A) MS² of the di-HHDP-galloyl-hexoside; (2B) MS² of the digalloyl-HHDP-hexoside; (2C) MS² of the maclurin-di-O-galloyl-hexoside; (2D) MS² of the quercetin-di-O-galloyl-rhamnoside; (2E) MS² of the quercetin-O-malonyl-galloyl-rhamnoside;

(2F)

MS²

of

the

5-dihydroxy-7-methoxy-6,8-

dimethylflavanone.


52

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Figure 3. Iridoid tentative identification from fruit methanolic extracts, using mass spectra. (3A) MS² of the 1,7-dihydroxy-5-(4’methoxybenzoate)4a,5,6,7a-tetrahydro-1Hcyclopenta[c]pyran-4-carboxylic acid; (3B) MS² of the 1-hydroxy-5-(4’-methoxybenzoyl)-7-(5’’-methoxygalloyl)-4a,5,6,7a-tetrahydro1H-cyclopenta[c]pyran-4-carboxylic acid; (3C) MS² of the 1-hydroxy-5-(4’-methoxybenzoyl)-7-(3’’hydhoxy-5’’-methoxygalloyl)-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylic acid; (3D) MS² of

the

1-hydroxy-6-hydroxy-5-methoxy-7-methoxymethyl—4a,5,6,7a-tetrahydro-1H-

cyclopenta[c]pyran-4-carboxylic acid; (3E) MS² of the 1-hydroxy-5,7-dimethoxy-4a,5,6,7a-tetrahydro1H-cyclopenta[c]pyran-4-carboxylic acid; (3F) MS² of the 1-hydroxy-7-methoxy-4a,5,6,7a-tetrahydro1H-cyclopenta[c]pyran-4-carboxylic acid.


53


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Figure 4. Cluster of similarity index comparison from methanolic extracts (phenolic compounds and iridoids identified of all fruits evaluated). EDA, EDB, EDC: Eugenia dysenterica; HSA, HSB, HSC: Hancornia speciose; EUA, EUB, EUC: Eugenia uniflora; HCA, HCB, HCC: Hymenae coubaril; AAU, AAB, AAC: Psidium cattleianu; CBA, CBB, CBC: Caryocar brasiliense; PCA, PCB, PCC: Plinia cauliflora; CPA, CPB, CPC: Campomanesia coubaril.


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Journal of Agricultural and Food Chemistry Brazilian biodiversity fruits

Araçá (Psidium cattleianum) Pitanga (Eugenia uniflora)

Cagaita (Eugenia dysenterica) Pequi (Cariocar brasiliense)

Jenipapo (Genipa Americana)

Cambuci (Campomanesia coubaril) Mangaba (Hancornia speciose)

Jabuticaba (Plinia cauliflora)

Jatobá (Hymenae coubaril)


Brazilian biodiversity fruits: discovering bioactive ... - ACS Publications

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