Accepted Manuscript

Anti-inflammatory effect of Capuli cherry against LPS-induced cytotoxic damage in

RAW 264.7 macrophages

Jos M. Alvarez-Suarez, Estefana Carrillo-Perdomo, Angel Aller, Francesca

Giampieri, Massimiliano Gasparrini, Lien Gonzlez-Prez, Pablo Beltrn-Ayala,
Maurizio Battino
PII: S0278-6915(17)30032-7
DOI: 10.1016/j.fct.2017.01.024
Reference: FCT 8870

To appear in: Food and Chemical Toxicology

Received Date: 16 December 2016

Revised Date: 19 January 2017
Accepted Date: 25 January 2017

Please cite this article as: Alvarez-Suarez, J.M., Carrillo-Perdomo, E., Aller, A., Giampieri, F., Gasparrini,
M., Gonzlez-Prez, L., Beltrn-Ayala, P., Battino, M., Anti-inflammatory effect of Capuli cherry against
LPS-induced cytotoxic damage in RAW 264.7 macrophages, Food and Chemical Toxicology (2017), doi:
10.1016/j.fct.2017.01.024.

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 ACCEPTED MANUSCRIPT

Anti-inflammatory effect of Capuli cherry against LPS-induced cytotoxic damage in RAW 264.7

macrophages

Jos M. Alvarez-Suarez a*, Estefana Carrillo-Perdomo b, Angel Aller b, Francesca Giampieri c,

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Massimiliano Gasparrini c, Lien Gonzlez-Prez d, Pablo Beltrn-Ayala e, Maurizio Battino c,f*

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a
Escuela de Medicina Veterinaria y Zootecnia. Unidad de Investigacin en Biotecnologa y Medio
Ambiente (BIOMA). Universidad de Las Amricas (UDLA), Quito, Ecuador;

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b
Estacin Experimental Santo Domingo-Instituto Nacional de Investigaciones Agropecuarias (EESD-
INIAP). Santo Domingo de los Tschilas, Ecuador;
c

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Dipartimento di Science Cliniche Specialistiche ed Odontostomatologiche (DISCO), Sez. Biochimica,
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Facolt di Medicina, Universit Politecnica delle Marche, Ancona, Italy;
d
Unidad de Investigacin en Biotecnologa y Medio Ambiente (BIOMA). Universidad de Las Amricas
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(UDLA), Quito, Ecuador;

e
Colegio de Administracin y Economa. Universidad San Francisco de Quito (USFQ), Quito, Ecuador;
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f
Centre for Nutrition & Health, Universidad Europea del Atlntico, Santander, Spain;
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*Corresponding author: Prof. Jos M. Alvarez-Suarez, PhD. Unidad de Investigacin en Biotecnologa y

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Medio Ambiente (BIOMA). Universidad de Las Amricas (UDLA) Quito, Ecuador, Tel: (+593 2) 398-
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1000, ext. 7500, E-mail: jose.alvarez@udla.edu.ec and Prof. Maurizio Battino, PhD, DSc, MS, MD (Hon)
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DISCO, Facolt di Medicina, Universit Politecnica delle Marche, Ancona, Italy, Tel.: +39 071 2204646,

E-mail: m.a.battino@univpm.it
 ACCEPTED MANUSCRIPT

Anti-inflammatory effect of Capuli cherry against LPS-induced cytotoxic damage in RAW

264.7 macrophages

Jos M. Alvarez-Suarez a*, Estefana Carrillo-Perdomo b, Angel Aller b, Francesca Giampieri c,

Massimiliano Gasparrini c, Lien Gonzlez-Prez d, Pablo Beltrn-Ayala e, Maurizio Battino c,f*

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a
Escuela de Medicina Veterinaria y Zootecnia. Unidad de Investigacin en Biotecnologa y Medio

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Ambiente (BIOMA). Universidad de Las Amricas (UDLA), Quito, Ecuador;
b
Estacin Experimental Santo Domingo-Instituto Nacional de Investigaciones Agropecuarias

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(EESD-INIAP). Santo Domingo de los Tschilas, Ecuador;
c
Dipartimento di Science Cliniche Specialistiche ed Odontostomatologiche (DISCO), Sez.

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Biochimica, Facolt di Medicina, Universit Politecnica delle Marche, Ancona, Italy;
d
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Unidad de Investigacin en Biotecnologa y Medio Ambiente (BIOMA). Universidad de Las
Amricas (UDLA), Quito, Ecuador;
e
Colegio de Administracin y Economa. Universidad San Francisco de Quito (USFQ), Quito,
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Ecuador;
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f
Centre for Nutrition & Health, Universidad Europea del Atlntico, Santander, Spain;
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*Corresponding author: Prof. Jos M. Alvarez-Suarez, PhD. Unidad de Investigacin en

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Biotecnologa y Medio Ambiente (BIOMA). Universidad de Las Amricas (UDLA) Quito, Ecuador,

Tel: (+593 2) 398-1000, ext. 7500, E-mail: jose.alvarez@udla.edu.ec and Prof. Maurizio Battino,
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PhD, DSc, MS, MD (Hon) DISCO, Facolt di Medicina, Universit Politecnica delle Marche,
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Ancona, Italy, Tel.: +39 071 2204646, E-mail: m.a.battino@univpm.it

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Abstract

Capuli cherry (Prunus serotina Ehr. subsp. capuli (Cav.) McVaugh) fruits from the inter-Andean

region of Ecuador were analysed to determine their bioactive compounds content, total antioxidant

capacity, radical scavenging activity and their anti-inflammatory and protective effects against the

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cytotoxic damage mediated by lipopolysaccharide (LPS) in RAW 264.7 macrophages. Capuli fruits

proved to be a natural source of bioactive compounds such as anthocyanins, vitamin C and -

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carotene as well as to present an important total antioxidant capacity and radical scavenging

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activities. RAW 264.7 macrophages were incubated with different concentration of Capuli crude

extract and subsequently activated by LPS to determine the markers related to oxidative damage and

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the proinflammatory cytokine production. The markers of oxidative damage, nitrite levels, the
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interleukin 1 messenger RNA levels and the tumor necrosis factor mRNA levels and secretion

were significantly decreased after the pre-incubated with Capuli extract and subsequently stimulated
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with LPS. In summary, Capuli extract attenuated the LPS-induced damage in RAW 264.7
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macrophages due to its antioxidant and anti-inflammatory properties, showing that Capuli fruits may
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represent a relevant source of bioactive compounds with promising benefits for human health.
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Keywords: Prunus serotina, Capuli cherry, Antioxidant capacity, Oxidative stress, Inflammation
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Abbreviations: NO, nitric oxide; TNF-, tumor necrosis factor-; DW, dry weight; TPC, total

phenolic content; GAE, galic acid equivalents; TFC, total flavonoid content; Cateq, catechin

equivalents; ACYs, total anthocyanins content; Pg-3-gluc, pelargonidin 3-glucoside; PgEq, Pg-3-gluc

equivalents; TAC, total antioxidant capacity; TEq, trolox equivalents; TEAC, trolox equivalent

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antioxidant capacity; FRAP, ferric reducing antioxidant power; O2superoxide radical; TE, -

tocopherol equivalents; AscEq, ascorbate equivalents; CCE, capuli crude extract; DMSO, dimethyl

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sulfoxide; LPS, lipopolysaccharide; ROS, reactive oxygen species; DCFH, 2-7-dichlorofluorescin

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diacetate; CAT, catalase; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive

substances; ABTS+, [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation; Nrf2,

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nuclear factor-erythroid 2-related factor 2; AMPK, 5' adenosine monophosphate-activated protein
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kinase; AMPK/Nrf2/ARE, (Kelch ECH associating protein 1/NF-E2-related factor 2/antioxidant

responsive elements) signalling pathway; IL1, interleukin 1 beta.

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1. Introduction

Inflammation constitutes a physiological response of the organisms against the damage to the

tissues. The inflammatory response leads to well-defined processes such as the activation and

recruitment of circulating leukocytes, with the consequent production of high levels of pro-

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inflammatory mediators such as nitric oxide (NO), tumor necrosis factor- (TNF-) and interleukin-

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1 (IL-1) (Kofler et al., 2005). A high production of pro-inflammatory mediators have been closely

related with a deficient activation of macrophages and the pathogenesis of several chronic diseases

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with inflammatory basis such as rheumatoid arthritis, atherosclerosis, diabetes, and cancer (Manzi

and Wasko, 2000; King, 2008; Kundu and Surh, 2008). Therefore, the inhibition of excessive

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inflammatory response has been proposed as a possible way to reduce the damage caused by
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excessive inflammatory response (Kundu & Surh, 2008). In this sense, diet has been considered to

play an important preventive role, since epidemiologic studies have demonstrated that the
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consumption of fruits and vegetables rich in antioxidant compounds has been associated with a lower
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incidence of chronic diseases related with oxidative stress (Forbes-Hernndez et al., 2014; Cassidy et
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al., 2013; Kaliora et al., 2006; Johnsen et al., 2003).

Capuli cherry (Prunus serotina Ehr. subsp. capuli (Cav.) McVaugh) is one of the iconic berries
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used in Ecuador. It is native from North America, but it is distributed throughout the American

continent and considered an invasive species in Europe. Nonetheless, in Central and South America it
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has been domesticated, so the fruits are characterized by a bigger size, fleshiness, juiciness and a
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pleasant sweet taste (McVaugh, 1951). It is a fundamental ingredient of the diet and economy of

native Ecuadorians, who harvest the trees and commercialize the fruits as stewed (Jucho), preserved,

made into jam or liqueur (Dugo et al., 2001). In addition, since prehispanic times different parts of

the plant have been used in traditional medicine to treat various diseases, such as diarrhoea, kidney
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complaints, rheumatism, jaundice, hypertension or respiratory inflammations associated with

coughing and also as blood tonic (INI, 1994; Moerman, 1998). These properties have been associated

with the presence of several bioactive compounds such as flavonoids and tannins (Ordaz-Galindo et

al., 1999), whose antioxidant and antibacterial properties have been extensively studied (Lachman et

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al., 2009; Einbond et al., 2004). Regarding the Capuli cherry, it has been reported to have a high

content of anthocyanins and polyphenols and a high antioxidant activity in accordance with their

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reducing power and radical scavenging activity (Jimenez et al., 2011; Olszewska, 2005a, 2005b).

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Capuli cherry has also been reported to have antimicrobial activity against gram () bacteria

(Salmonella typhimurium, Proteus mirabilis, Escherichia coli and Pseudomona aeruginosa) and the

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gram (+) bacterium, Staphylococcus aureus; and inhibits yeast activity, but no molds (Jimenez et al.,
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2011). On the other hand, the methanol extract prepared from the stem-bark of P. serotina has been

reported to present significant anti-proliferative activity in cancer cells of human colon (Yamaguchi
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et al., 2006) and the crude extract of the plant, as well as some isolates, have showed vasorelaxant

activity dependent on the concentration of compounds such as hyperoside, prunin, and ursolic acid;
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this fact can explain the popular use of the plant for the treatment of hypertension (Ibarra-Alvarado et
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al., 2009). In this regard, although the composition and antioxidant properties of Capuli cherry

support the existence of a potential anti-inflammatory capacity (based on that inflammation is

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mediated by the oxidative burst that occurs in monocytes, neutrophils, eosinophils, and macrophages)
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(Miguel, 2010), to date, no study has addressed it. In this sense, this study sought to determine the
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anti-inflammatory effects of a crude extract of Capuli cherry and the protective effects against

cytotoxic damage mediated by inflammation, through the markers study of oxidative damage and the

expression and secretion of proinflammatory mediators.

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2. Materials & Method

2.1. Capuli fruits analysis

Ripe Capuli cherry (Prunus serotina Ehr. subsp. capuli Cav. McVaugh) fruits (500 g) were

purchased during the berry season (October-December, 2015) on two different occasions from the

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most important popular markets in the cities of Quito (Iaquito and La Ofelia), Salcedo (Mercaldo

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central), Latacunga (Mercado mayorista) and Riobamba (La condamine), provinces of Pichincha,

Cotopaxi, Tungurahua and Chimborazo, respectively, all located in the inter-Andean region of

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Ecuador (Fig. 1). The edible parts were chopped and freeze-dried, after that, samples were ground to

a fine power and stored at - 20 C until analysis.

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Hydroalcoholic extracts was obtained as previously reported by Tulipani et al. (2008). Briefly, 10
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g of fruit were homogenized for 3 min in 100 mL of the extraction solution (methanol and Milli-Q
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water, 80% v/v) and stirring for 2 h in the dark at room temperature. The mixture was then

centrifuged in two sequential times for 15 min at 3500 rpm, and supernatant was filtered through a
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0.45 m Minisart filter (PBI International) before analysis. Hydroalcoholic extracts was used for the
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analysis of total antioxidant capacity, total polyphenols, flavonoids, anthocyanins content as well as

for total antioxidant capacity (TAC), O2 and H2O2 scavenging activities.

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The total phenolic content (TPC) was determined using the Folin-Ciocalteu method (Slinkard &
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Singleton, 1977) and results was expressed as milligrams of galic acid equivalents (GAE) per gram
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of dry weight (DW) of fruits (mg GAE/g DW). Total flavonoid content (TFC) was determined

spectrophotometrically and results were expressed as milligrams of catechin equivalents (Cateq) per

gram of DW of fruits (mg Cateq/g DW) (Dewanto et al., 2002), while total anthocyanins content

(ACYs) content was determined using a modified pH differential method previously described

(Giusti & Wrolstad, 2001) and results were expressed as milligrams of Pg-3-gluc equivalents (PgEq)
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per gram of DW of fruits (mg PgEq/g DW). Vitamin C was determined by reversed-phase HPLC

(Tulipani et al., 2008), while -carotene was extracted and analysed as previously described (Zanatta

& Mercadante, 2007).

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2.2. Quantification of total antioxidant capacity (TAC) and O2 and H2O2 scavenging activities

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The TAC was determined using in parallel the trolox equivalent antioxidant capacity (TEAC)

assay (Re et al., 1999) and the ferric reducing antioxidant power (FRAP) (Benzie & Strain, 1996). In

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both analyses results were expressed as moles of trolox equivalents (TEq) per gram of DW (mol

TEq/g DW). Superoxide radical (O2) scavenging capacities was determined using a colorimetric

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method, based on monitoring the reduction of nitroblue tetrazolium into a purple coloured
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diformazan by reaction with O2 as previously reported (Gomes et al., 2007) The O2 scavenging
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activity was expressed as mol of -tocopherol equivalents (TEq) per g of DW (mol TEq/g DW).

The ability to scavenge hydrogen peroxide (H2O2) was determined according to the method
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previously described (Ruch et al., 1989) and results were expressed as mol of ascorbate equivalents
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(AscEq) per g of DW (mol AscEq/g DW).

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2.3. Evaluation of anti-inflammatory properties

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2.3.1. Cell culture and treatment

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Murine RAW 264.7 macrophages were maintained in RPMI 1640 medium supplemented with

10% fetal bovine serum, 100 U/mL of penicillin, 100 g/mL of streptomycin, in a humidified

atmosphere at 37 C with 5% CO2. Cells were seeded in 96-well plates at 1.5 x 105 cells/well and

allowed do attach to the plate overnight. Hydroalcoholic extract was concentrated under vacuum and

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the capuli crude extract (CCE) was firstly pre-dissolved in dimethyl sulfoxide (DMSO) and then in

PBS until reaching the desired initial concentration. Stock solutions of CCE was prepared in DMSO

(50 mg/ml) and RAW 264.7 macrophages were incubated with 0-50 g/ml of CCE for 24 h,

according to the Trypan blue dye exclusion tests for cytotoxicity studies, and subsequently, they were

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stimulated with 1 g/ml of lipopolysaccharide (LPS) for 24 h. In final dilutions, the DMSO

concentration did not exceeded 1%. Controls were incubated with the same DMSO concentrations as

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were found in the extracts with which the cells were treated.

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2.3.2. Measurement of intracellular ROS level

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Intracellular ROS levels were determined using 2-7-dichlorofluorescin diacetate (DCFH) as
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previously reported (Wang & Joseph, 1999). After treatment cell were incubated with 5 mol/L
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DCFH at 37C for 30 min under light protection. Fluorescence intensity was read at an excitation

wavelength of 485 nm and an emission wavelength of 530 nm. The protein was determined by the
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Bradford method (Bradford, 1976). Data are expressed as arbitrary unit of fluorescent intensity/g
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cell protein.
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2.3.4. Determination of catalase and superoxide dismutase activity and evaluation of lipid
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peroxidation
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Catalase activity (CAT) was determined spectrophotometrically by monitoring H2O2

decomposition at 240 nm (Aebi, 1984). Superoxide dismutase (SOD) activity was determined as

previously reported (Kakkar et al., 1984) whereas lipid peroxidation levels were determined by the

assay of thiobarbituric acid reactive substances (TBARS) (Ohkawa et al., 1979).

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2.3.5. Measurement of nitrite levels

The production of NO was determined using the Griess reagent (Esposito et al., 2013). Cell

culture supernatant (100 L) was combined with 100 L of the Griess reagent and the nitrite

concentration was determined by the absorption of the coloured compound formed by the interaction

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NO2 / Griess reagent at 550 nm using the microplate reader (Thermo Scientific Microplate Reader,

Multiskan EX, USA). NaNO2 (10100 M) was used as standard and results were expressed as M

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of NaNO2/105 cells.

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2.3.6. Enzyme-linked immunosorbent assay for TNF and total RNA isolation and quantitative real-

time polymerase chain reaction

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After LPS-stimulation the culture supernatant was collected to measure TNF levels using an
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enzyme-linked immunosorbent assay (ELISA) kit (Thermo Fisher Scientific) following the

manufacturers protocols.
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Total RNA from cells was extracted using Trizol reagent (Invitrogen Carlsbad, CA, USA), and
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cDNA synthesis and quantitative real-time polymerase chain reaction (qRT-PCR) were following as
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previously described (Park, Rasmussen, Ehlers, Blobaum, Lu, Schlegal, et al., 2008). Primer

sequences for the TNF, IL-1 and GAPDH were as below: TNF, forward primer 5-
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GGCTGCCCCGACTACGT-3, reverse primer 5-ACTTTCTCCTGGTATGAGATAGCAAAT-3;

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il-1, forward primer 5- GTCACAAGAAACCATGGCACAT-3, reverse primer, 5-

GCCCATCAGAGGCAAGGA-3; GAPDH, forward primer 5-

GGTGGTCTCCTCTGACTTCAACA-3, reverse primer, 5-GTTGCTGTAGCCAAATTCGTTGT-

3.

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2.4. Statistical analysis

Statistical analyses were performed using STATISTICA software (Statsoft Inc., Tulsa, OK, USA).

Data are reported as a mean value standard deviation (SD) for three measurements. Data between

different groups were analysed statistically using one-way ANOVA and Turkeys post hoc test; P <

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0.05 was considered as significant and P < 0.01 highly significant.

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3. Results and Discussion

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3.1 Fruits composition, TAC and radical scavenging activity of Capuli fruits

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The content of bioactive compounds in Capuli fruits are presented in Table 1. According to the
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results, Capuli fruits are an important natural source of bioactive compounds such as polyphenols

(flavonoid and anthocyanins), vitamin C and -carotenes. Total phenolic and flavonoid contents
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were within the range of values previously reported in other berries such as strawberry (Tulipani et

al., 2008) and blackberry (Kaume et al., 2012), while it was higher than the total polyphenols content
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reported in raspberries (Rao & Snyder, 2010). The phenolic profile of Capuli fruit has been
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previously studied reporting the presence of several bioactive compounds such as procyanidin dimer
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B, procyanidin trimer B, quercetin glycosides (Quercetin hexoside, Q-3-O-xyloside, Q-3-O-

arabinoside), kaempferol pentoside and hexoside. On the other hand, the total anthocyanin content
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was lower respect with the values reported in strawberry (Giampieri et al., 2012; Tulipani et al.,
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2008) and blackberry (Kaume et al., 2012). In fact, in the studies carried out on the polyphenolic

profile of Capuli only two anthocyanins (cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside) were

identified (Vasco et al., 2009), suggesting the low concentration of these compounds in the fruit.

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Capuli fruit also results as an important source of vitamin C and -carotenes (Table 1). Vitamin C

values were higher than those reported in strawberry (Giampieri et al., 2012; Tulipani et al., 2008),

blackberry (Kaume et al., 2012) and raspberries (Rao & Snyder, 2010), evidencing the high content

of this compound in fresh fruits. On the other hand, since there are few reports of -carotenes content

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in berries, -carotenes content in Capuli cherry was analysed by HPLC. The results showed that

Capuli fruits can be considered a natural source of -carotenes in the diet with contents similar to

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those previously reported in strawberries (Giampieri et al., 2014b).

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The TAC of Capuli hydroalchoholic extracts was determined using in parallel the TEAC and

FRAP assays. The TAC was well evidenced by its capacity to reduce Fe3+ to Fe2+, and to scavenge

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ABTS+, as well as the capacity to scavenge activity against O2 and H2O2 (Table 1), evidencing the
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antioxidant capacity of the extract, in agreement with previous reports indicating the high antioxidant

capacity of Capuli fruits (Vasco et al., 2008) and berries in general (Giampieri et al., 2014b, 2012;
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Kaume et al., 2012; Rao & Snyder, 2010; Tulipani et al., 2008).
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3.2. Effects of CCE on biomarkers of oxidative damage in RAW 264.7 macrophages

Since the antioxidant and anti-inflammatory capacity of berries has been documented (Lee et al.,
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2014), in this study, we studied the anti-inflammatory and the protective effects against cytotoxic
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damage mediated by inflammation effects of CCE obtained from the evaporation of the solvent of the
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hydroalcoholic extract of Andean Capuli cherry. RAW 264.7 macrophages were incubated with

increasing concentrations of the dry extract of CCE (0-50 g/mL) for 24 h, and subsequently, they

were stimulated with 1 g/ml of LPS for 24 h. Preliminary results showed no cytotoxic effect was

found after incubation of cells with t, showing that CCE does not exert negative effect on viability of

RAW 264.7 at the concentrations tested (data not shown). CCE demonstrated its ability to protect
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RAW 264.7 cells against the oxidative damage mediated by inflammation. A burst in the intracellular

ROS levels was found after incubation with LPS compared to control (P < 0.05), while the pre

incubation of RAW 264.7 with CCE was able to protect against oxidative damage in a dose-

dependent manner, with the best results at concentrations between 40-50 g/mL, showing

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intracellular ROS values significantly lower (P < 0.01) compared to LPS-stimulated cells (Fig. 2).

Our results are consistent with those previously reported by Lee et al. (2014) who reported that berry

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anthocyanins fraction was able to protect RAW 264.7 macrophages against the increase of

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intracellular ROS levels after exposure to LPS (Lee et al., 2014). The authors also found that the

nuclear factor-erythroid 2-related factor 2 (Nrf2) plays a critical role in the antioxidant effect exerted

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by polyphenolic extracts of berries through the reduction of intracellular ROS levels, but the
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contribution of the Nrf2 to the inhibition of proinflammatory gene expression by the berry

anthocyanin fractions was not significant in macrophages (Lee et al., 2014). Other studies have also
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reported the protective effect of polyphenolic extracts of berries, mainly anthocyanins, against the

increase of intracellular ROS (Zhang & Tsao, 2016; Giampieri et al., 2014b), showing the protective
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effect of these compounds on the protection against oxidative damage. Moreover, the capacities of
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polyphenols from different natural sources to reduce the intracellular ROS production have been well

documented (Alvarez-Suarez et al., 2016; Zhang & Tsao, 2016; Hooshmand et al., 2015; Del Rio et
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al., 2013; Kang et al., 2011).

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The above mentioned results allows hypothesizing that polyphenols exhibit their protective
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activity through the activation of the signalling pathways related to the antioxidant response and not

directly with the routes related to the inflammatory response. It has been proposed that polyphenols

could exert their antioxidant action in vivo by their ability to up-regulate the activation of the

AMPK/Nrf2/ARE signalling pathway, and the expression of the antioxidant enzymes such as SOD

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and CAT (Alvarez-Suarez et al., 2016; Buendia et al., 2016; Gasparrini et al., 2016). Moreover, it has

been also proposed that Nrf2 is crucial in the Nrf2/ARE signalling pathway and is responsible for the

expression of the above referred enzymes (Lu et al., 2010).

CCE was also able to improve the activity of the antioxidant enzymes CAT and SOD and to

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protect lipids against oxidative damage (Table 2). LPS-stimulation significantly affected the activity

of both enzymes compared to control (P < 0.05), while a higher activity of both enzymes SOD and

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CAT was observed after the pre-incubation of RAW 264.7 cells with CCE at the concentration

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between 30-50 g/mL compared to LPS-stimulated cells. Similar improvements were observed in the

markers of lipid damage. CCE pre-treatment showed no damage on lipids, while the stimulation with

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LPS caused a significant increase in TBARS respect to control (P < 0.05). Pre-incubation with CCE
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at the concentration between 30-50 g/mL significantly protected lipids against oxidative damage,

showing a significant decreasing in the TBARS (P < 0.05) when compared with LPS-stimulated
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cells. Our results are in accordance with those previously reported, which demonstrate the protective
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effect of polyphenols against oxidative damage (Del Rio et al., 2013; Zhang & Tsao, 2016) and
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specifically the polyphenols from berries (Giampieri et al., 2015,2014a; 2012; Lee et al., 2014; Lee et

al., 2013; Nile & Park, 2014).

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3.3 Effect of CCE on nitrite production and TNF secretion and proinflammatory cytokine
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expression in RAW 264.7 macrophages

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The effect of CCE on nitrite production was also determined (Fig. 3A). Nitrite plays an important

role as regulator of inflammatory responses, where the over production of nitrite reacts with

superoxide causing important cytotoxicity and tissue damage (Korhonen et al., 2005). LPS-

stimulation significantly increased the nitrite levels (P < 0.01) compared to control. Moreover, the
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pre-incubation of RAW 264.7 cells with CCE, at the concentration between 30-50 g/mL,

significantly decreased the levels of NO (P < 0.01), compared with LPS-stimulated cells. Our results

are consistent with previously data, demonstrating that polyphenols fraction was able to protect RAW

264.7 macrophages against the increase of NO levels after exposure to LPS (Bu et al., 2008;

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Hooshmand et al., 2015).

TNF secretion into cell culture medium was determined using the ELISA kit (Fig. 3B). TNF

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secretion were significantly (P < 0.01) increased in LPS-stimulated cells compared to control, while

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incubation with CCE decreased significantly (P < 0.01) the levels of TNF compared to LPS-

stimulated cells in a dose-dependent manner, showing the best results at the highest concentration

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(40-50 g/ml). The expression of proinflammatory cytokines TNF and IL-1 was also determined
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after stimulation with LPS in RAW 264.7 cell (Fig. 3C). The stimulation with LPS caused a

significant increase in both TNF and IL-1 messenger RNA levels (mRNA) (P < 0.01) compared
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with control, whereas pre-incubation with CCE repressed TNF and IL-1 mRNA level, in a dose-
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dependent manner, showing the most significant levels of suppression at the highest concentration
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(40-50 g/ml). Our results are consisted with those previously reported by Lee et al. (2014) who

reported that IL-1 mRNA levels were significantly decreased after the pre-incubation of RAW
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264.7cells with berries anthocyanins fraction and then stimulated with LPS. These authors report that

TNF mRNA levels and secretion were also significantly decreased in LPS-treated macrophages
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(Lee et al., 2014). The same authors report also that polyphenols from berries, specially
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anthocyanins, exert their anti-inflammatory effects in macrophages, at least in part, by inhibiting

nuclear translocation of NF-B independent of the Nrf2-mediated pathways (Lee et al., 2014). The

anti-inflammatory properties of flavonoids have been partly attributed to their primary structure, i.e.,

tricyclic aromatic structure of 15 carbon atoms (Hmlinen et al., 2007). Flavonoids are capable to

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inhibit the synthesis and activities of different pro inflammatory mediators (eicosanoids, cytokines,

adhesion molecules and C-reactive protein) as well as has the capacity to inhibit of transcription

factors (NF-kappaB and activating protein-1 (AP-1)) and the activation Nrf2 (Serafini et al., 2010).

Our study constitutes the first report about the anti-inflammatory properties of capuli fruits

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coming from the Andean regions of Ecuador. The findings here exposed allow us to confirm that

fruits of Capuli, like other fruits of the group of berries, prove to be an important source of bioactive

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compounds associated with important biological properties such as anti-inflammatory and

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antioxidant capacities suggesting that Capuli fruits could be used as natural source of antioxidants

compounds and support its possible use, topic or dietary, as a source of compounds with anti-

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inflammatory properties.
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Conflict of interest statement

All the authors declare that there is no conflict of interest for any of them.
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Acknowledgements
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This study was funded by Universidad de Las Amricas (UDLA), Quito, Ecuador. Access to plant

genetic resources has been made on the basis of the FRAMEWORK AGREEMENT FOR ACCESS
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TO PLANT GENETIC RESOURCES: MAE-DNB-CM-2015-0024 celebrated between the Ministry

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of the Environment (MAE), Ecuador, through the Undersecretariat of Natural Heritage; and the

National Institute of Agricultural Research (INIAP) of Ecuador.

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FIGURE CAPTION

Fig. 1. Distribution of the collect areas of Capuli fruits in Ecuador.

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Fig. 2. Intracellular ROS production in RAW 264.7 macrophages. RAW 264.7 macrophages were

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incubated with 0-50 g/ml of CCE for 24 h, and subsequently, they were stimulated with 1 g/ml of

lipopolysaccharide (LPS) for 24 h. Results are reported as mean SD of three experiments. * P <

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# ##
0.05, **P < 0.01, significant differences compared to control; P < 0.05, P < 0.01, significant

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differences between LPS stimulated cells and CCE pre-treated cell.
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Fig. 3. Nitrite production and repression of proinflammatory cytokine expression by CCE in RAW
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264.7 macrophages. RAW 264.7 macrophages were incubated with 0-50 g/ml of CCE for 24 h, and
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subsequently, they were stimulated with 1 g/ml of lipopolysaccharide (LPS) for 24 h. Nitrite levels
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was determine in the cell culture medium (A), Elisa kits was used to measure TNF secretion into

cell culture medium (B) and qRT-PCR analysis was conducted to determined mRNA levels of IL-1
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and TNF (C). Results are reported as mean SD of three experiments. * P < 0.05, **P < 0.01,

significant differences compared to control; # P < 0.05, ##

P < 0.01, significant differences between
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LPS stimulated cells and CCE pre-treated cell.

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Table 1

Composition, total antioxidant capacity and radical scavenging activities of Capul cherry

hydroalchoholic extracts (data are presented as mean SD)

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Parameters Values
Bioactive compounds

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Total phenolic content (TPC) (mg GAE/g DW) 2.81 0.14
Total flavonoid content (TFC) (mg Cateq/g DW) 1.74 0.09
Total anthocyanins content (ACYs) (mg PgEq/g DW) 0.69 0.03

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Vitamin C content (VitC) (mg/100 g DW) 83.52 2.11
-carotene content (CarotC) (g/100 g DW) 2.41 0.23

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Total antioxidant capacity (TAC)
TEAC (mol TEq/g DW) 18.95 1.26
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FRAP (mol TEq/g DW) 12.65 1.18

Radical scavenging activities (RSA)

O2 scavenging activities (mol TpEq/g DW) 7.63 0.25
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H2O2 scavenging activities (mol AscEq/g DW) 3.86 0.31

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Table 2

Protective effect of CCE against the oxidative damage in LPS-stimulated RAW 264.7 in RAW 264.7 macrophages

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CPE (g/mL)

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LPS
Control (1/mL) 5 10 20 30 40 50
a g
CAT (U/mg protein/min) 1.860.09 1.120.06 1.140.02 1.280.02 1.360.01 1.520.02 1.610.03 1.760.02b
g f e d c

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SOD (U/mg protein/min) 78.538.52a 29.424.28g 33.763.89g 38.346.52f 44.684.85e 54.566.23d 66.352.85c 72.8210.25b
TBARS (M) 0.830.01e 1.340.01a 1.310.02a 1.260.01b 0.970.01c 0.920.01d 0.880.01e 0.860.01e

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Data are means standard deviation (SD). Mean values within a row sharing the same letter are not significantly different (P < 0.05).

CCE: capuli crude extract; LPS: lipopolysaccharide; CAT: Catalase; SOD: Superoxide dismutase; TBARS: thiobarbituric acid reactive
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substances. CCE was obtained from the evaporation of the solvent of the hydroalcoholic extract of Capul cherry.
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FIGURE 1

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FIGURE 2

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**

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**,#

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LPS (1g/mL) - + + + + + + +
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CPE (g/mL) - - 5 10 20 30 40 50
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FIGURE 3

A **

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**,#
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LPS (1g/mL) - + + + + + + +
CPE (g/mL) - - 5 10 20 30 40 50

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LPS (1g/mL) - + + + + + + +
CPE (g/mL) - - 5 10 20 30 40 50
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*,##

LPS (1g/mL) - + + + + + + +
CPE (g/mL) - - 5 10 20 30 40 50
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Highlights

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Capuli fruits represent a relevant source of bioactive compounds.

Capuli crude extract shows important antioxidant properties

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Capuli crude extract suppressing the expression and secretion of proinflammatory

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mediators

Capuli crude extract attenuated the LPS-induced cytotoxic damage in RAW 264.7

cells
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