Accepted Manuscript

May the superfruit red guava and its processing waste be a

potential ingredient in functional foods?

Renan da Silva Lima, Sandra Regina Salvador Ferreira, Luciano

Vitali, Jane Mara Block

PII: S0963-9969(18)30845-7
DOI: doi:10.1016/j.foodres.2018.10.053
Reference: FRIN 8023
To appear in: Food Research International
Received date: 30 May 2018
Revised date: 14 October 2018
Accepted date: 19 October 2018

Please cite this article as: Renan da Silva Lima, Sandra Regina Salvador Ferreira, Luciano
Vitali, Jane Mara Block , May the superfruit red guava and its processing waste be a
potential ingredient in functional foods?. Frin (2018), doi:10.1016/j.foodres.2018.10.053

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 ACCEPTED MANUSCRIPT

May the superfruit red guava and its processing waste be a potential ingredient
in functional foods?

Renan da Silva Limaa, Sandra Regina Salvador Ferreirab, Luciano Vitalic, Jane Mara Blocka*

*
Corresponding author at: Federal University of Santa Catarina, Agricultural Sciences Center,
Food Science and Technology Department, Rodovia Admar Gonzaga, 1346, Itacorubi, CEP:
88034-001, Florianópolis, SC, Brazil.
Corresponding author email: [email protected]

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a
Department of Food Science and Technology, Federal University of Santa Catarina,

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Florianópolois, SC, Brazil
b
Department of Food Engineering, Federal University of Santa Catarina, Florianópolis, SC,

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Brazil
c

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Department of Chemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil

Email addresses: [email protected] (R.S. Lima); [email protected] (S.R.S. Ferreira);

[email protected] (L. Vitali); [email protected] (J.M. Block).
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ABSTRACT
Guava (Psidium guajava L.), a popular fruit in tropical countries, can be consumed in natura
or transformed into many products. Guava’s processing waste (seed, peel, and pulp leftovers)
can represent up to 30% of the fruit’s volume. Evidence indicates that this fraction still holds
a significant amount of antioxidant substances, such as phenolic compounds. Therefore, the
objective of this study was to investigate the phytochemical composition (total phenolics,
flavonoids, flavonols, and condensed tannins), phenolic profile, and antioxidant activity
(DPPH, modified DPPH, ABTS, and FRAP) of guava’s pulp and waste extracts (GPE and
GWE) obtained from guava’s pulp and waste powders (GPP and GWP). The extraction was
performed using a probe-type ultrasound for 2 min at 25 ºC, using ethanol:water (30:70, v/v)

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as the solvent (15 mL for 1 g of sample). The phenolic profile of guava’s powders and

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extracts was performed by LC-ESI-MS/MS analysis. GPE showed the highest concentration
of total phenolics (2348.3 mg GAE 100 g-1 by Folin-Ciocalteu and 518.00 mg GAE 100 g-1

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by Prussian Blue), and the greater in vitro antioxidant activity among all the assays tested. On
the other hand, GWE was found to hold a higher amount of flavonoids (1006.08 mg QE 100

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g-1 ), flavonols (352.59 mg QE 100 g-1 ), and condensed tannins (1466.9 mg CE 100 g-1 ).
Overall, 37 phenolic compounds were identified among all the samples, with 14 compounds
being reported for the first time for red guava. Ellagic acid was the major phenolic present in
all samples. The results showed that guava’s pulp and waste are a rich source of phenolics
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compounds and may be used as an ingredient for the development of functional foods.

Keywords: guava by-products; ultrasound-assisted extraction; ellagic acid; antioxidants;

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polyphenols; flavonoids.

* Abbreviations:
GPP: Guava’s pulp powder; GPE: Guava’s pulp extract; GWP: Guava’s waste powder; GWE: Guava’s
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waste extract
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1. Introduction

Guava (Psidium guajava L.) is one of the most popular exotic fruits in tropical
countries because of its slightly sweet taste and bland aroma. It has drawn the attention of
researchers due to its high phenolic content and other antioxidant substances, which could
potentially classify guava as a superfruit. The color of guava’s pulp varies according to its
type, with red guava being superior in flavor and antioxidant compounds, such as
carotenoids, when compared to the white variety (Yadava, 1996). Guava is a versatile fruit,
therefore, it may be consumed in natura or processed into juices, jams, pie fillings, and other
products. This fruit and its by-products have a potential for being incorporated into healthier

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processed foods because of their sensory characteristics and the presence of bioactive

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compounds.
Brazil, with a guava production of 424,305 tons, is the third largest guava producer in
the world (Triechel, 2016). The red varieties ‘Pedro Sato’, along with ‘Paluma’, are the main

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ones used both for in natura consumption and industry processing (Souza, 2014). Moreover,

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it is estimated that 55% of the total guava volume produced in Brazil is processed, which
corresponds to 233,368 tons. Triechel (2016) reported that 30% of the total volume of
processed guava is lost in the form of waste (seeds, peel, and pulp leftovers). Therefore, a
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total of 70,010 tons of residue is produced and, in many cases, discarded, despite its high
amount of bioactive compounds.
Red guava has been previously analyzed for its phytochemical composition by some
authors, and it has shown high contents of total phenolics (Vasco et al., 2008) and flavonoids
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(Kong et al., 2010). Phenolic compounds are a group of secondary metabolites found in
plants, characterized by the presence of phenol units in its structure. The main phenolic group
is the flavonoids, consisting of a 15-carbon skeleton, with two phenyl rings linked to a
heterocyclic ring. Other phenolic classes include phenolic acids, flavonols (a subgroup of
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flavonoids), and tannins, which are flavanol's oligomers and polymers, having three or more
phenol units, being classified into condensed and hydrolyzed tannins (gallic acid esters).
These substances are responsible for the astringency and bitterness of the fruits. Besides, the
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multiple hydroxyl groups present in the phenolic’s chemical structures enable them to act as
antioxidants by donating H+ ions to free radicals, stabilizing them, and interrupting the
oxidation chain reactions (Shi et al., 2005; Gülçin, 2012).
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Oxidation reactions represent a problem for the food industry, especially in products
rich in unsaturated fatty acids, promoting quality losses and reduced shelf life (Gülçin, 2012).
Besides, the excessive production of free radicals in human cells is related to the
development of chronic diseases, such as cancer and Alzheimer (Poprac et al., 2017).
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Antioxidant substances may be used to retard oxidation in foods as well as to develop food
products with functional properties. These antioxidants can be chemically synthesized or
extracted from natural sources, such as plants, fruits, and vegetables (Gülçin, 2012).
Studies have shown that phenolic-rich extracts obtained from natural sources present
pharmacological activity. Terminalia Muelleri, which contains high amounts of gallic acid,
ellagic acid, among other phenolics, exhibits anti-inflammatory and analgesic activities, as
well as hepatoprotective action (Fahmy et al., 2017). Furthermore, Euphorbia hirta linn.
ethanolic extract, rich in flavonoids and tannins, has demonstrated wound healing effect
(Tuhin et al., 2017). Polyphenols present in honey also have shown health benefits. Hossen et
al. (2017) reviewed the existing literature on the pharmacological effects of honey’s
phenolics, such as gallic acid, ellagic acid, vanillic acid, quercetin, myricetin, and others.
Evidence of several health effects, such as oxidative stress reduction, hepatoprotective effect,
anti-carcinogenic activity, induction of apoptosis, anti-microbial activity has been reported.
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This fact suggests that the natural-extracted polyphenols may be a source for the production
of supplements and nutraceuticals.
Fruits are also extensively researched as a source for the extraction of phenolics.
Some of them are being considered “superfruits”, which is a new term recently addressed to
exotic fruits containing high levels of antioxidant compounds, such as phenolics, carotenoids,
ascorbic acid, among others (Chang et al., 2018). Also, as already stated by many authors, the
by-products of superfruits are a potential source for the production of powdered ingredients
to be used for the formulation of value-added functional foods (Chang et al., 2018; Peixoto et
al., 2016; Clerici & Carvalho-Silva, 2011; Schreckinger et al., 2010).
However, the majority of studies addressing the extraction of bioactive compounds

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from guava use large amounts of toxic organic solvents, such as methanol, hydrochloric acid

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(Ademiluyi et al., 2016; Contreras-Calderón et al., 2011; Rojas-Garbanzo et al., 2017),
formic acid (Flores et al., 2015), and ethyl acetate (Ongphimai et al., 2013), which can harm

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the environment and the health of professionals involved in it. In addition, many of these
studies use conventional extraction techniques, such as maceration (Musa et al., 2011; El

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Anany et al., 2013; Ademiluyi et al., 2016) and Soxhlet extraction (Ongphimai et al., 2013).
These techniques are laborious and time-consuming, preventing them from being used on a
commercial scale.
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In contrast, newer green extraction methods such as ultrasound-assisted extraction,
pressurized liquid extraction, supercritical fluid extraction, may reduce and/or eliminate the
use of toxic organic solvents, decrease extraction time, allow the use of mild extraction
conditions to prevent the degradation of target compounds, and scale up for commercial
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implementation (Selvamuthukumaran & Shi, 2017).

Ultrasound-assisted extraction (UAE) has been considered as an alternative to the
classical methods since it shows potential for scaling up, as it requires less solvent and
shorter extraction times. The probe-type UAE is the most promising method once the
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ultrasonic waves are delivered directly into the sample, requiring a reduced extraction time
when compared with the ultrasound bath (Chemat et al., 2017). The probe-type UAE of
bioactive compounds from red guava hasn’t been reported in the literature so far.
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Depending on the extraction technique employed, as well as the methods of

preparation and analysis of the samples, the results reported on the literature for the bioactive
compounds in red guava can be very discrepant. In this study, phenolic-rich extracts from
dehydrated guava’s pulp and processing waste using probe-type ultrasound-assisted
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extraction were obtained and their phenolic profile, phytochemical composition, and
antioxidant activity were determined. In order to evaluate how the extraction process
interferes with the content of the compounds analyzed, the phenolic profiles of guava’s pulp
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and waste powders were also analyzed.

2. Material and Methods

2.1. Samples
Fresh red guavas (Psidium guajava L. cv. Pedro Sato) were purchased at a local
market in October 2017 at maturation stage III (Total Soluble Solids = 8.23%), according to
the classification proposed by Azzolini et al. (2004). The guava fruits were sanitized in a 4%
sodium hydrochloride solution for 30 min. Then, the samples were pulped in a pulper
(Brameitar sieve 2 mm, Campinas, Brazil), and the pulp and processing waste (seeds, skin,
and pulp leftovers) were collected separately. The guava pulp and waste were dried,
following the protocol suggested by Nunes et al. (2016), with modifications, at a temperature
of 55 ºC for 10 h in an oven with air circulation (TE – 394/2, Tecnal, Piracicaba-SP, Brazil).
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The final moisture of the dried samples was 17% for the pulp and 10% for the waste. The
dried samples were milled with a hammermill (TE - 090, Tecnal, Piracicaba-SP, Brazil) and
the particle size of the guava pulp powder (GPP) and waste powder (GWP) was adjusted to
0.500-0.550 mm. GPP and GWP were kept in amber flasks, flushed with nitrogen, and stored
at -24 ºC until further analysis.

2.2. Chemicals
Folin-Ciocalteau phenol reagent, gallic acid, vanillin, (+)-catechin hydrated, ABTS
[2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)], DPPH (2,2-diphenyl-1-
picrylhydrazyl), TPTZ (2,2′- 2,4,6-tripyridyltriazine), Trolox, and the ultra-pure phenolic

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standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). The standards were

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prepared as a stock solution (1000 mg L-1 in 100% chromatographic grade methanol), stored
at -24 ºC, and used to prepare the calibration curves by appropriate dilution from the mixture.

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All other chemical reagents and solvents used in the experiment were of analytical grade
(P.A.) obtained from Neon and Sigma–Aldrich.

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2.3. Ultrasound-assisted extraction (UAE)
Samples of GPP and GWP (1 g) were mixed with 15 mL of the extracting solvent,
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composed of ethanol:water (30:70, v/v). Then, the mixture was taken to a probe-type
ultrasonic device (QR500, Eco-Sonics, Indaiatuba-SP, Brazil) at a frequency of 20 kHz and
power of 500 W, where the extraction took place for 2 minutes at 25 ºC. The experimental
conditions for the extraction were determined in preliminary tests (results not shown). Next,
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the extracts were centrifuged for 10 min at 4000 rpm and vacuum-filtered in a sintered glass
funnel. The guava pulp extract (GPE) and waste extract (GWE) were concentrated in a rotary
evaporator (model 558, Fisatom, São Paulo-SP, Brazil) at 40 ºC to evaporate the solvent,
flushed with nitrogen, and stored at -24 ºC until further analysis.
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2.4. Characterization of the extracts

2.4.1. Determination of total phenolic content (TPC): Folin-Ciocalteu assay
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The total phenolic content by Folin-Ciocalteu assay was determined following the
protocol by Singleton & Rossi (1965), with modifications. Briefly, 25 μL of GPE and GWE
samples diluted with ultrapure water (1:50, v/v) were mixed with 25 μL of Folin-Ciocalteau
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phenol reagent, previously diluted with ultrapure water (1:3, v/v), and placed in a 96-well
microplate. Next, 200 μL of ultrapure water was added and this mixture rested for 5 min.
After that, 25 μL of a 10% sodium carbonate solution was added, and the plate was kept in
the dark for 60 min at 25 ºC. The blank was composed of ultrapure water instead of the
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extracts. Then, the absorbance was measured at  725 nm with the use of a microplate
reader (Spectramax Paradigm, Molecular Devices, San Jose-CA, USA). In order to construct
a calibration curve, gallic acid (98% purity) diluted in ethanol (final concentration of 0.2 mg
mL-1 ) was used at concentrations ranging from 0 to 136 mg L-1 . The results were expressed
as mg of gallic acid equivalents per 100 g of sample (mg GAE 100 g-1 of GPE or GWE).

2.4.2. Determination of total phenolic content (TPC): prussian blue assay

The total phenolic content by the Prussian blue assay was determined following the
protocol by Price & Butler (1977) modified by Margraf et al. (2015). 100 μL of diluted GPE
and GWE samples (1:50, v/v), along with 100 μL of a 0.5 mM iron (III) chloride hexahydrate
solution, was added to a microplate and allowed to react for 2 min. Then, 100 μL of a 0.5
mM potassium ferricyanide solution was added to the microplate. The blank was composed
of ultrapure water instead of the extracts. The absorbance was read at = 725 nm. A
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calibration curve was built with a 100 mg L-1 gallic acid solution, using concentrations
ranging from 0 to 40 mg L-1 . The results were expressed as mg of gallic acid equivalents per
100 g of sample (mg GAE 100 g-1 of GPE or GWE).

2.4.3. Determination of Flavonoids

Flavonoids were analyzed according to Lees & Francis (1972). Briefly, the samples
were diluted with ultrapure water (1:20, v/v for GPE and 1:30, v/v for GWE) and their
absorbance was measured at 374 nm in a spectrophotometer (SP 2000, Belphotonics,
Piracicaba-SP, Brazil). Eq. 1 and 2 were used to calculate the concentration of total
flavonoids in the samples.

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𝑣𝑜𝑙𝑢𝑚𝑒𝑛𝑜𝑛−𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑒𝑥𝑡𝑟𝑎 𝑐𝑡 × 𝑣𝑜𝑙𝑢𝑚𝑒𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 = (Eq. 1)
𝐴𝑙𝑖𝑞𝑢𝑜𝑡 𝑢𝑠𝑒𝑑 𝑡𝑜 𝑑𝑖𝑙𝑢𝑡𝑒 𝑡ℎ𝑒 𝑠𝑎𝑚𝑝𝑙𝑒

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𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑎𝑡 374 𝑛𝑚 × 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑇𝑜𝑡𝑎𝑙 𝐹𝑙𝑎𝑣𝑜𝑛𝑜𝑖𝑑𝑠 = (Eq. 2)
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The results were expressed as mg of quercetin equivalent per 100 g of sample (mg QE 100 g-
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of GPE or GWE).
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2.4.4. Determination of Flavonols
Flavonols were determined by the method described by Yermakov et al. (1987), with
modifications. An aliquot of 80 μL of diluted GPE and GWE samples (1:50, v/v) was placed
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in a microplate and mixed with 80 μL of a 2% hexahydrate aluminum chloride (III) ethanolic

solution along with 120 μL of a 50 g L-1 sodium acetate solution. The microplate was
incubated in the dark for 2.5 h at 25 ºC. The blank was composed of ultrapure water instead
of the extracts. The absorbance was measured at 440 nm. A calibration curve was
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prepared with a quercetin stock solution, with concentrations varying from 0 to 80 mg L-1 .
The results were expressed as mg of quercetin equivalents per 100 g of sample (mg QE 100
g-1 of GPE or GWE).
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2.4.5. Determination of Condensed Tannins

Condensed tannins were determined according to the protocol described by
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Horszwald & Andlauer (2011). First, samples of GPE and GWE were diluted with methanol
(1:50, v/v). Then, an aliquot of 25 μL of the diluted samples was placed in a microplate along
with a mixture of 150 μL of a 4% vanillin methanolic solution and 25 μL of a 32% sulphuric
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acid methanolic solution. The microplate was allowed to stand in the dark for 15 min at room
temperature. The blank was composed of methanol instead of the extracts. The absorbance
was measured at  = 500 nm. A catechin methanolic solution (final concentration of 300 mg
L-1 ) was used at concentrations ranging from 0 to 240 mg L-1 for calibration curve. The
results were expressed as mg of catechin equivalents per 100 g of sample (mg CE 100 g-1 of
GPE or GWE).

2.4.6. DPPH Assay

The DPPH assay followed the methodology developed by Brand-Williams et al.
(1995), with modifications. Briefly, 40 μL of diluted GPE and GWE samples (1:50, v/v) were
placed in a microplate. Then, 260 μL of a 0.10 mM DPPH methanolic solution was added to
the wells containing the samples. The microplate was kept in the absence of light for 30 min
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at 25 ºC and the absorbance was measured at  = 517 nm. The blank was composed of
ultrapure water instead of the extracts. The antioxidant power was calculated by Eq. 3.

𝐴517 𝑠𝑎𝑚𝑝𝑙𝑒
% 𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = [1 − ( )] 𝑥 100 (Eq. 3)
𝐴517 𝑏𝑙𝑎𝑛𝑘

2.4.7. Modified DPPH Assay

The methodology used was developed by Brand-Williams et al. (1995) and modified
by Staško et al. (2007). 40 μL of diluted GPE and GWE samples (1:50, v/v) were transferred
to a microplate, where 260 μL of a 0.10 mM DPPH ethanolic solution was added, along with

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260 μL of a 50 mM monobasic sodium phosphate buffer solution pH 6.0. The microplate was

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kept in the dark for 30 min at 25 ºC. The decrease in absorbance was recorded at  = 525 nm.
The blank was composed of ultrapure water instead of the extracts. The antioxidant activity

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was calculated using Eq. 4.

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𝐴525 𝑠𝑎𝑚𝑝𝑙𝑒
% 𝑆𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = [1 − ( )] 𝑥 100 (Eq. 4)
𝐴525 𝑏𝑙𝑎𝑛𝑘 NU
2.4.8. ABTS Assay
The protocol used for this determination is reported by Rufino et al. (2007). First,
GPE and GWE samples were diluted in ethanol (1:20, v/v). An aliquot of 10 μL of the diluted
samples was placed in a microplate, then, 290 μL of ABTS + radical solution was added and
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the microplate was kept in the dark for 6 min. The blank was composed of ethanol instead of
the extracts. The decrease in absorbance was recorded at  = 734 nm. A calibration curve
was constructed with the use of a 300 mg L-1 ascorbic acid stock solution. The concentrations
ranged between 30 and 300 mg L-1 . Results were expressed as mmol of ascorbic acid
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equivalents per 100 g of sample (mmol AAE 100 g-1 of GPE or GWE).

2.4.9. FRAP Assay

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The method for this assay was described by Pulido et al. (2000). First, FRAP reagent
was prepared by mixing 2.5 mL of a 10 mmol/L TPTZ solution (3.12 g of TPTZ diluted with
50 mL of a 40 mmol/L solution of hydrochloric acid) with 25 mL of a buffer solution of
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sodium acetate pH 3.6, and 2.5 mL of a 20 mmol/L solution of Iron (III) chloride
hexahydrate. Then, 20 μL of GPE and GWE diluted samples (1:50, v/v) were mixed with 280
μL of FRAP reagent in a microplate. The samples were incubated at 37 ºC for 30 min. The
blank was composed of ultrapure water instead of the extracts. The decrease in absorbance
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was recorded at nm. A calibration curve was built with a 150 mg L-1 ascorbic acid
stock solution, with concentrations ranging from 15 to 150 mg L-1 . The results were
expressed as mmol of ascorbic acid equivalent per 100 g of sample (mmol AAE 100 g-1 of
GPE or GWE).

2.5. Identification and quantification of phenolic compounds by LC-ESI-MS/MS

2.5.1. Sample preparation

The preparation of samples followed the protocol suggested by Schulz et al. (2015),
with modifications. Briefly, samples of guava’s pulp and waste powders and extracts (GPP,
GWP, GPE, and GWE, respectively) were subjected to acid hydrolysis by adding 5 mL of
methanol and 5 mL of a 6 mol L-1 hydrochloric acid solution to either 1 g of the powder
sample or 1 mL of the extract sample. The mixture was taken to an oven (400/D200 °C, New
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Ethics, São Paulo-SP, Brazil) at 85 ºC for 30 min. Then, the pH was adjusted to 2,0 using a 6
mol L-1 sodium hydroxide solution. Next, acidified samples were partitioned with 10 mL of
diethyl ether using a centrifuge (Z 200 A, Hermle Labortechnik, Wehingen, Germany) at
4000 rpm for 10 min. This process was repeated two more times for each sample. The
supernatants were combined in a bottom-round flask and the solvent was removed in a rotary
evaporator at 40 ºC until dryness. Then, the dried sample was resuspended in 1 mL of
chromatographic grade methanol and diluted 10 times with methanol:water (70:30. v/v) for
the injection in the LC-ESI-MS/MS system.

2.5.2. LC-ESI-MS/MS Analysis

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The identification and quantification of phenolic compounds were performed in a

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high-performance liquid chromatography (HPLC) system (1200 Series, Agilent
Technologies, Waldbronn-BW, Germany), following the methodology described by Schulz et
al. (2015). A Synergi column (4.0 μm, 2.0 x 150 mm d.i.; Phenomenex, Torrance-CA, USA)

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was used for liquid chromatographic separation, under gradient elution condition. Mobile

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phases were composed of mixtures of methanol 95% and water 5% (v/v), channel A, and
water and formic acid 0.1% (v/v), channel B. The separation was carried out at 30 °C using
segmented elution gradient as follows: 0–5 min, 10% A; 5–7 min, 90% A; 7–10 min, 90% A;
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10–17 min, 10% A. Between the analyses, the column was conditioned for 5 min with the
proportion of the initial mobile phase of the separation. The running flow rate was 150
μL.min-1 . Sample sizes of 10 μL were injected into the equipment.
The HPLC system was coupled to a mass spectrometry system composed by a hybrid
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triple quadrupole/linear ion trap mass spectrometer (Q Trap 3200 Applied Biosystems/MDS
Sciex, Concord-ON, Canada). The mass spectrometer was operated in negative electrospray
(TurboIonSpray Applied Biosystems/MDS Sciex, Concord-ON, Canada) ionization mode.
The MS/MS parameters were: capillary needle maintained at −4500 V; curtain gas at 10 psi;
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the temperature at 400 °C; gas 1 and gas 2 at 45 psi; and CAD gas, medium. The software
Analyst version 1.5.1 was used for the HPLC–ESI-MS/MS system control and data analysis.
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2.6. Statistical Analysis

Statistical data analysis was carried out with using the R-project 3.4.3 (2017)
software. The results were subjected to one-way analysis of variance (ANOVA), and the
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means were compared with the use of Tukey posthoc test (p < 0.05). All the analyses were
performed in triplicate and the results were expressed as means  standard deviation (SD).
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3. Results and Discussion

3.1. Characterization of the extracts

Figure 1 shows the results for TPC, flavonoids, flavonols, condensed tannins, and
antioxidant activity (DPPH, modified DPPH, ABTS, and FRAP) of the guava’s pulp and
waste extracts.
The results for all the parameters determined were significantly different between
GPE and GWE (t-test, p < 0.05). GPE presented a higher phenolic content by both the Folin-
Ciocalteu and the Prussian Blue assay. The discrepancy between the results can be explained
by the limitations of each technique. The Folin-Ciocalteu method relies on the reduction of
molybdenum and tungsten by substances such as polyphenols, ascorbic acid, reducing sugars
and amino acids, which is recorded by the decrease in absorbance. Therefore, this assay can
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overestimate the total phenolic content by the presence of these other reducing agents in the
sample (Everette et al., 2010). On the other hand, the Prussian Blue assay is less affected by
substances other than phenolics. Thus, this method returns a total phenol estimation much
closer to the actual amount (Berker et al., 2012).
Other studies have investigated the total phenolic content (TPC) of Psidium guajava,
using the Folin-Ciocalteu assay, obtaining significantly lower amounts of phenolic
compounds. Isabelle et al. (2010) found a TPC of 1.75 mg GAE g-1 FW (or 175 mg GAE 100
g-1 FW) for freeze-dried guava pulp. Vasco et al. (2008) reported a TPC of 462 mg GAE 100
g-1 FW from freeze-dried guava, using methanol followed by acetone as solvents. Ademiluyi
et al. (2016) obtained a TPC of 66.1 mg GAE 100 g-1 for red guava using maceration with a

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methanol:HCl solution (20:1, v/v) as a solvent. Souza et al. (2011) reported a TPC of 24.63

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mg GAE 100 g-1 for guava’s industrial waste, using maceration with water as a solvent. El
Anany et al. (2013) extracted the phenolic compounds from raw guava seeds using a shaker

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incubator with a 70% ethanol solution as a solvent, and found a TPC of 973.80 mg GAE 100
g-1 . The use of different extraction techniques with distinct parameters could have led to such

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variation of results, including those reported in the present article.
Flavonoids were observed at high concentrations in both studied samples. GWE
presented 50% more flavonoids and three times more flavonols when compared to GPE.
Kong et al. (2010) found a total flavonoid content of 427.35 mg QE 100 g-1 for guava
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industrial waste, which may be considered a fairly lower content when compared to the
amount found in the present work. The authors conducted their phenolic extraction in an
orbital shaker for 5 h under a temperature of 60 ºC and pH 2 in their optimized condition. The
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exposure of the sample to a high temperature for such a long time could explain the
decreased flavonoid yield compared to the results determined in this study, which used room
temperature for only 2 min in the extraction procedure.
Condensed tannins, as well as flavonoids and flavonols, also showed to be more
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concentrated in the processing waste fraction of guava (1081.5 mg 100 g-1 for GPE and
1466.9 mg 100 g-1 for GWE). Moreno et al. (2014) analyzed the polyphenols of freeze-dried
guava (Psidium guajava) flour as well as the fresh fruit. The authors found a total condensed
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tannins concentration of 8.51 mg 100 g-1 in fresh guava, and 208.02 mg 100 g-1 in guava
flour. The yield in both samples is fairly lower than the values obtained in the present study.
Regarding the antioxidant activity determinations, results for the modified DPPH
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were lower than those obtained using the classic assay. This difference was expected since
the traditional DPPH assay measures the decrease in absorbance when an antioxidant diluted
in ethanol or methanol reduces the DPPH* radical to DPPHH. However, this assay can be
affected by the deprotonation of DPPHH. Therefore, the modified DPPH assay aims at the
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reduction of this effect by increasing the solubility of the antioxidants present in the sample,
by conducting the experiment in an ethanol:water (1:1, v/v) solution (Staško et al., 2007).
GPE showed a higher antioxidant activity than GWE in all the four determinations.
This could be related to the fact that GPE presents a higher TPC when compared to GWE,
indicating that the antioxidant capacity has a direct relationship to the total phenol content
rather than to other specific phenolic groups. On the other hand, guava pulp is also rich in
compounds such as carotenoids, which may have interfered with this result (Verma et al.,
2015; Santos et al., 2018).
Other authors have reported a lower antioxidant capacity determined by the same
methods. Ongphimai et al. (2013) found a scavenging activity of 22% for insoluble phenolic
extract and 20% for soluble phenolic extract from Psidium guajava by the classic DPPH
method. Contreras-Calderón et al. (2011) analyzed whole Brazilian guava and found 44.8
mol TE g-1 FW (or 4.48 mmol TE 100 g-1 FW) in the ABTS assay, and 39.9 mol TE 100 g-
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1
FW (or 3.39 mmol TE 100 g-1 FW) in the FRAP assay. Nunes et al. (2016) analyzed whole
Psidium guajava cv. Pedro Sato and found 4.2 mmol Fe2+ 100 g-1 in the FRAP assay.
Thaipong et al. (2006) studied four guava genotypes. The ABTS results ranged from 29.6 to
37.9 mol TE g-1 (or 2.96 to 3.79 mmol TE 100 g-1 ) while the FRAP results ranged from 15.5
to 33.3 mol TE g-1 (or 1.55 to 3.33 mmol TE 100 g-1 ).

3.2. Phenolic Profile

Table 1 shows the phenolic profile of GPP, GPE, GWP, and GWE. The parameters

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for the identification and quantification of phenolic compounds, such as retention time,

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parent ion, quantitative ion, and limits of identification and quantification can be found in the
Supplementary Material section (Table 3).
Among the 47 phenolics tested, a total of 37 were detected throughout the four

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samples (Table 1), which are predominantly composed of phenolic acids and flavonoids,

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although other phenolic classes were also detected. GWE was the richest one, yielding a total
of 34 identified compounds, closely followed by GPP with 33 compounds, GPE with 32, and
GWP with 31 phenolics.
Ellagic acid (13) was the major phenolic found in all samples (7.6179 mg g-1 in GPE,
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7.5466 mg g-1 in GPP, 2.7725 mg g-1 in GWP, and 1.6000 mg g-1 in GWE). Analyzing the
concentration of this compound, it is possible to notice that the majority of ellagic acid is
retained in the pulp fraction of guava. Ellagic acid is extensively investigated throughout
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clinical studies, both in vitro and in vivo, since it is linked to potential anti-cancer (Núñez-
Sánches et al., 2016; Ceci et al., 2016) and anti-diabetes properties (Mehta et al., 2017;
Nankar & Doble, 2017). Vanillic acid (8), gallic acid (9), and quercetin (28) are also among
the major compounds encountered. The chemical structures of the main phenolic compounds
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identified in the samples are shown in Figure 2.

The majority of studies analyzing the phenolic profile of Psidium guajava generally
reported gallic acid (Zulkifli et al., 2012) and quercetin (Flores et al., 2015; Lin & Yin, 2012)
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as the major compound. Although these compounds were found in high amounts, the major
phenolic found in our study was ellagic acid (Lin & Yin, 2012; Flores et al., 2013). This
could be caused mainly by differences in extraction technique and solvent, as well as, the
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methodology used for the identification and quantification of the phenolic compounds.
The antioxidant activity of phenolic compounds is related to their chemical structure
and their capacity to act as a radical scavenging agent (Chen et al., 2017). The phenolic
profile of GPE and GWE may explain the fact that the samples showed a high radical
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scavenging activity observed by the antioxidant activity assays. Both GPE and GWE showed
a great variety of flavonoids, which relies on the capacity of donating hydrogen atoms to free
radicals in order to cease chain reactions. Quercetin, the main flavonoid found in GPE and
GWE, presents in its structure an ortho-dihydroxy substitution in the B-ring, which stabilizes
the radical form and participates in electron delocalization; a 2,3-unsaturation, and a 4-
carbonyl group in the C-ring. These structural characteristics are required for maximum
radical scavenging potential in flavonoids (Chen et al., 2017; Rice-Evans et al., 1996).
The antioxidant activity of phenolic acids is also dependent on the number of
hydroxyl groups available in the molecule with no steric hindrance. Thus, the compound is
able to donate a hydrogen atom to stabilize the free radical. Gallic acid, present in GPE and
GWE, contains three available hydroxyl groups in its structure, which contributes to an
increased antioxidant activity (Chen et al., 2017; Rice-Evans et al., 1996). Similarly, the
chemical reactivity of ellagic acid, the main phenolic in GPE and GWE, is due to the
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presence of multiple hydroxyl groups. Besides, the electrophilic aromatic substitution of

ellagic acid’s electron-rich aromatic rings plays an important role in stabilizing the free
radicals (Ahmed et al., 2016).
In addition, ellagic acid and gallic acid have demonstrated pharmacological action.
Fahmy et al. (2015) studied the effects of phenolic-rich extracts, containing ellagic acid and
gallic acid, from Terminalia muelleri against liver injuries. The extract (400 mg kg-1 ) was
capable of reducing the elevation of carbon tetrachloride (CCl4 ), a hepatoxin and nephrotoxin
present in humans and animals that generates free radicals due to its metabolic conversion
pathways. The same effect was also observed by Al-Sayed et al. (2015) for a phenolic-rich
extract from Bauhinia hookeri. The protective action of Bauhinia hookeri was attributed to

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the presence of polyphenols, which enhanced the antioxidant activity, decreasing the lipid

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peroxidation.
Our extraction technique, coupled with the LC-ESI-MS/MS analysis, allowed the

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identification of 14 phenolics that are being reported for the first time for red guava, which
are 4-hydroxymethylzenzoic acid (4), umbelliferone (36), mandelic acid (3), syringaldehyde

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(34), galangin (19), eriodictyol (22), aromadendrin (23), hispidulin (27), taxifolin (29), and
carnosol (37), which were detected in all samples. Also, methoxyphenylacetic acid (7) was
detected in GPE, GWE, and GPP. Salicylic acid (1) and fustin (24) were only identified in
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GWE, and coniferaldehyde (33) was present just in GWE. It has been reported for these
compounds a potential action in the prevention and treatment of degenerative diseases.
Umbelliferone has been linked to anti-carcinogenic (Muthu et al., 2013), cardioprotective
(Lou et al., 2017), and anti-diabetic (Noawaboot et al., 2015) effects. Salicylic acid and
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coniferaldehyde have also been related to anti-carcinogenic potential (Ai et al., 2015; Kim et
al., 2016).
Once the guava pulp and processing waste were analyzed separately, it was possible
to identify the fraction of the fruit in which each phenolic is predominantly located after the
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fruit has been processed. Also, for the majority of the detected phenolics, their amount varied
significantly between the powder and the extract samples. This could be related to the
extraction process prior to the LC-ESI-MS/MS analysis, once the extract samples were first
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submitted to ultrasonic extraction, which did not take place for the powders, as stated in item
2.5.1.
The results from Table 1 show that the powder samples presented a TPC greater than
the extracts, both for guava’s pulp (GPP = 14.71 mg g-1 , GPE = 10.63 mg g-1 ) and guava’s
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waste (GWP = 7.559 mg g-1 , GWE = 4.327 mg g-1 ). As for the extracts, it is interesting to
observe that the TPC results given by the LC-ESI-MS/MS analysis are closer to the TPC
results estimated by the Prussian Blue Assay (GPE = 5.180 mg g-1 , GWE = 3.990 mg g-1 )
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than the ones obtained by the Folin-Ciocalteu method (GPE = 23.48 mg g-1 , GWE = 15.78
mg g-1 ). Therefore, the present study reaffirms the potential of the Prussian Blue assay to be
used as a tool for TPC estimation. The phenolic content ratio for the guava extracts and
powders were calculated, and Figure 3 shows that the compounds salicylic acid (1), 4-
hydroxymethylbenzoic acid (5), and umbelliferone (7) were only quantified in guava’s
extract samples. These results indicate that UAE was able to release specific phenolics that
could not be isolated in a significant amount during the sample preparation of powders prior
to the LC-ESI-MS/MS analysis. The losses of some phenolics from the powders to the
extracts were, in most cases, similar in the pulp and the waste fractions. However, the
differences in yield between pulp and waste were substantial for p-coumaric acid (8), syringic
acid (15), kaempferol (23), epicatechin (28), and hispidulin (29).
Table 2 shows that studies on the phenolic profile of guava or its fractions and by-
products are scarce. The highest number of phenolic compounds in red guava was found in
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the present work when compared to other studies. The results may be explained by the
differences in the solvent system, the preparation of the samples prior to the phenolic
extraction, the variable extraction conditions (technique, temperature, time), and distinct
methodology of analysis.
Overall, the phenolic profile of guava powders (GPP and GWP) and extracts (GPE
and GWE) showed a great variety of compounds in all samples. The concentration and the
presence of different compounds may explain the high total amount of phytochemicals found,
as well as the antioxidant activity demonstrated by guava’s extracts. In addition, based on the
number of phytochemical compounds identified in large amount in this study, we suggest that
guava may fit the criteria to be named a superfruit.

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4. Conclusion

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The phytochemical composition of guava’s pulp and waste extracts showed that

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guava is a rich source of substances with antioxidant properties, such as flavonoids, flavonols
and condensed tannins. The guava processing waste presents a higher concentration in such
compounds when compared to the pulp. This fact highlights the importance of giving a
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proper destination for the guava waste, which should be considered a valuable processing co-
product rather than a residue. Therefore, we suggest that GWE may be used as an ingredient
in different food formulations.
The Prussian Blue method for GPE and GWE exhibited values much closer to those
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obtained by the LC-ESI-MS/MS analysis when compared to the Folin-Ciocalteu method.

This assay, due to the overestimation of results, is not recommended to be used for total
phenol estimation. Therefore, the Prussian Blue assay should be adopted for this purpose,
followed by the LC-ESI-MS/MS analysis.
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The increased content of total phenolics, flavonoids, flavonols, condensed tannins,

and antioxidant activity compared to other studies in the literature could be mainly attributed
to the use of a probe-type UAE. Unlike classical methods, the ultrasonic extraction carries
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two important physical phenomena, which are the breaking of the material’s cell walls and
the diffusion of the solvent through the matrix, washing out the target compounds. Therefore,
the probe-type UAE promotes a release of bioactive substances in a much more efficient way
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when compared with those methods.

In addition, solvent choice, sample preparation, and different analysis protocols could
also have contributed to the discrepancy with results from other studies. Therefore, the
comparison of results between different studies from the literature is only possible when the
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parameters used were the same.

Lastly, guava may be considered a superfruit with a rich variety of phenolic
compounds. Our further work includes the investigation of other non-conventional extraction
techniques to obtain extracts rich in bioactive compounds; the identification and
quantification of guava’s carotenoids; and the application of guava’s extracts and powders for
the development of functional and healthy foods.
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Acknowledgements
The authors are grateful to the National Council of Technological and Scientific
Development (CNPq) for granting a scholarship to Renan da Silva Lima. We also thank
Guilherme Corrêa Danielski for proof-reading this article and helping with some of the
editing, and Professor Gianluca Li Puma (Loughborough University) for proof-reading the
revised manuscript.

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Table 1. Phenolic profile of guava’s pulp powder (GPP, mg g-1 of sample) and extract (GPE, mg g-1 of
sample), and guava’s waste powder (GWP, mg g-1 of sample) and extract (GWE, mg g-1 of sample).

Phenolic Compound GPP GPE GWP GWE

Phenolic Acids

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nd nd nd 0.0108 ±

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Salicylic acid* 0.0018

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0.4884 0.2613 0.5457 ± 0.1790 ±
2 Cinnamic acid ± 0.1222a,b ± 0.0205b,c 0.0837a 0.0166c

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0.0443 <LOQ <LOQ <LOQ
Mandelic acid* ±
3
0.0193
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0.0354 <LOQ 0.0092 ± <LOQ
4 a b
4-Hydroxymethylbenzoic acid* ± 0.0052 0.0029
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0.1196 0.0924 ±
5
Protocatechuic acid <LOQ ± <LOQ 0.0710a
0.0423a
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0.0556 0.0176 0.0274 ± 0.0150 ±

6 p-Coumaric acid ± ± 0.0013b 0.004b,c
0.0104a 0.0017c
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Methoxyphenylacetic acid* 0.1091± <LOQ nd 0.1021 ±

7
0.0098a 0.0144a
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0.7730 0.9732 0.1899 ± 1.2346 ±

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Vanillic acid ± ± 0.0169c 0.2363a

8 b a,b
0.1682 0.1532

4.1576 0.2720 1.5014 ± 0.3504 ±

b c
Gallic acid ± ± 0.6181 0.0160
9 a c
0.2223 0.0433

0.1509 0.0543 0.0771 ± 0.0254 ±

10 Ferulic acid ± ± 0.0397a 0.0078a
0.0937a 0.0123a

0.4308 0.0870 0.2441 ± 0.0320 ±

11 Syringic acid ± ± 0.0079b 0.0329d
0.011a 0.0118c
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0.0459 0.0452 0.1067 ± 0.0406 ±

12 Sinapic acid ± ± 0.0109a 0.0044b
0.0069b 0.0049b

7.547 7.618 2.773 1.600

13 Ellagic acid ± 0.2183a ± ± 0.3642b ±
a c
1.227 0.0667

0.0149 0.0115 0.0114 0.0124 ±

14 Chlorogenic acid ± ± ± 4.122-4 b

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6.873-4 a 0b 1.376-4 b

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0.0031 0.0034 <LOQ 0.0074 ±

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15 Rosmarinic acid ± ± 0.0053a
b -4 b
0.0012 2.315

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Flavonoids
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16 Chrysin <LOQ nd nd nd
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0.1062 0.0065
17 Pinocembrin ± ± nd nd
0.0132a 5.998-4 b
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0.0051 0.0056 0.0050 ± 0.0053 ±

18 Apigenin ± ± 3.357-5 b 4.031-4 a,b
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2.688-4 a,b 2.350-4 a

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0.0152 0.0151 0.0292 ± 0.0224 ±

19 Galangin* ± ± 0.0057a 0.0012 a,b
4.370-4 c 3.567-4 b,c
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0.0194 0.0151 0.0124 ± 0.0164 ±

20 Naringenin ± ± 1.744-4 c 0b
a b
0 0.0019

nd <LOQ nd <LOQ
21 Kaempferol
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0.0031 0.0031 0.0030 ± 0.0035 ±

22
Eriodictyol* ± ± 5.172-8 a 9.766-4 a
4.352-4 a 6.523-4 a

0.0686 0.0265 0.0193 ± 0.0283 ±

a b b b
23 Aromadendrin* ± 0.0062 ± 0.0050 0.0035 0.0015

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24 Fustin* nd nd nd <LOQ

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0.0304 0.0239 0.1241 ± 0.0256 ±
25 Catechin ± ± 0.0094
a
7.228
-4 b

3.932-4 b 2.409-4 b
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0.0312 nd 0.1759 ± <LOQ
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Epicatechin ± 0.0181a
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b
0.0044

Hispidulin* <LOQ <LOQ <LOQ <LOQ

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0.1920 0.7801 0.7813 ± 0.2363 ±

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28 Quercetin ± ± 0.0434a 0.0279b

b a
0.1046 0.1798
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0.0693 0.0382 0.0256 ± 0.0482 ±

29 Taxifolin* ± ± 0.0059c 0.0112a,b
0.0112a 0.0015b,c
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0.1088 0.1815 0.1139 ± 0.1575 ±

30 Myricetin ± ± 0.0158b 0.0091a
b a
0.0048 0.0386

0.0353 0.0227 ±
31 Isoquercetin ± <LOQ 0.0025b <LOQ
a
0.0036

Phenolic Aldehydes
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32 Vanillin 0.0716 0.0410 0.6015 ± 0.0441 ±

± 0.0469b ± 0.5258a 0.0019c
6.531-4 c

Coniferaldehyde* nd nd 0.0503 ± nd
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0.0135

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0.0380 <LOQ 0.0710 ± <LOQ

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Syringaldehyde* ± 0.0053b 0.0413a
34

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0.0111 0.0100 0.0171 ± 0.0141 ±

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35 Sinapaldehyde ± ± 3.820-4 a 0.0031a
3.819-4 b
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Coumarin

0.0066 0.0078 0.0065 ± 0.0084 ±

36 Umbelliferone* b a
± ± 0 0.0020
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-4 b -4 a
1.324 1.334

Phenolic Diterpene
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0.0148 0.0146 ± 0.0146 ±

37 a b -8 b
Carnosol* 0.0436 ± 0 ± 0 2.131
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3.197-8 b

Total Phenolic Content (mg g-1 )

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14.71 10.63 7.559 4.327

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Not detected; * Reported for the first time; <LOQ – not quantifiable. Results followed by the same letter in
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the row do not differ significantly (t-test, p < 0.05).

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Table 2. Phenolic profile of guava reported in different studies.

Study Sample Extraction Process Identification Number of

Method identified
phenolic
Ademiluyi at P. guajava L. (giant Maceration with Gas
al., 2016 white, small white, methanol:HCl chromatography
stripped, and pink) (20:1, v/v) for 24 h 14
oven dried at 35 ºC at room temperature

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Zulkifli et al., Fresh peels of P. Samples boiled in High-

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2012 guajava L. water with a 1:20 performance 8
peel to water ratio liquid

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chromatography
(HPLC)

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Flores et al., Freeze-dried pulp of Blender extraction
2015 P. guajava L. with
(Homestead, Barbie CH3 OH/H2 O/formic HPLC-PDA 21
Pink, Thai Maroon, acid (70:25:5) for 5
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Sardina 1, Sardina 2, min at room
Yen 2, and Sayla) temperature

Rojas-Garbano Freeze-dried peel and Vortex-extracted (1

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et al., 2017 pulp of P. guajava L. min) and bath UHPLC-DAD- 29 phenolics and
cv. ‘Criolla’ ultrasound- MS/MS 34 polar
extracted (20 min) compounds
using methanol +
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water (9 + 1, v + v)

Nunes et al., Whole P. guajava L. Extracted with

2016 cv. Pedro Sato oven- ethanol:water HPLC-DAD 13
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dried at 55 ºC (80:20, v/v) for 10

min
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Ongphimai et Whole freeze-dried Soxhlet-extracted 8 soluble

al., 2013 P. guajava L. with ethyl acetate HPLC phenolic acids
for 10 h and 6 insoluble
phenolic acids
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Lin & Yin, Whole fresh P. Blender-extracted 9 in the aqueous

2012 guajava L. cv. Pearl white 100% water HPLC extract and 10 in
or water:ethanol the ethanolic
(50:50, v/v) for 24 h extract
at 25 ºC

Flores et al., Whole freeze-dried Blender-extracted

2013 P. with HPLC-PDA 9
friedrichsthalianum methanol:water
(70:30, v/v) for 5
min at room
temperature
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Figure 1. Total phenolic content, flavonoids, flavonols, condensed tannins, and antioxidant activity
(DPPH, modified DPPH, ABTS, and FRAP) of guava’s pulp and waste extracts (GPE and GWE,
respectively).

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Abbreviations: TPC (FC) = total phenolic content by the Folin-Ciocalteu assay, TPC (PB) = total phenolic
content by the Prussian Blue assay, CT = Condensed Tannins
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Figure 2. Chemical structures of the major phenolic compounds identified in samples of guava’s pulp
powder and extract (GPP and GPE) and guava’s waste powder and extract (GWP and GWE).
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Figure 3. Ratio between guava’s pulp (GP) extract and powder, and guava’s waste (GW) extract and
powder**.

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** The number of the corresponding phenolic compound can be seen in Table 1. * Ratio = ∞. * Values
greater than 1.0 mean that the extract sample exhibited a higher yield than the powder sample for that
specific phenolic.
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HIGHLIGHTS
 Guava and its by-products are rich in flavonoids, flavonols, and condensed tannins.
 Prussian Blue assay allows a more accurate estimate of the total phenol content.
 37 compounds were identified in guava and its by-products by LC-ESI-MS/MS.
 14 phenolic compounds are being reported for the first time in red guava.
 Guava can be called a superfruit due to its concentration of antioxidants.

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