The chilean superfruit black-berry Aristotelia chilensis (Elaeocarpaceae), Maqui as mediator in inflammation-associated disorders

Food and Chemical Toxicology xxx (2016) 1e13

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The chilean superfruit black-berry Aristotelia chilensis (Elaeocarpaceae), Maqui as mediator in inflammation-associated disorders Carlos L. Cespedes a, *, Natalia Pavon b, Mariana Dominguez c, Julio Alarcon a, Cristian Balbontin a, Isao Kubo d, Mohammed El-Hafidi e, Jose G. Avila f a

Department of Basic Sciences, Faculty of Sciences, Universidad del Bio Bio. Chillan, Chile
vez”, Juan Badiano 1, Seccio
n XVI, Tlalpan, 14080, M
Departmento de Farmacología, Instituto Nacional de Cardiología “Ignacio Cha exico D.F., Mexico
n, 04510, M
Departamento de Biología Celular y Desarrollo, Laboratorio 305-Sur, Instituto de Fisiología Celular, UNAM. Ciudad Universitaria, Coyoaca exico D.F., Mexico d Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA e
vez”, Juan Badiano 1, Seccio
n XVI, Tlalpan, 14080, M
Departamento de Biomedicina Cardiovascular, Instituto Nacional de Cardiología “Ignacio Cha exico D.F., Mexico f
noma de M
Laboratorio de Fitoquímica, UBIPRO, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Auto exico, Tlalnepantla de Baz, Estado de M
exico, Mexico b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 October 2016 Received in revised form 25 December 2016 Accepted 27 December 2016 Available online xxx

The effects of phytochemicals occurred in fractions and extracts of fruits of “Maqui-berry” (Aristotelia chilensis), on the expression of cyclooxygenase-2 (COX-2), inducible-nitric oxide synthases (iNOS) and the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-activated murine macrophage RAW-264 cells, as well as their antioxidant activities. The MeOH extract (A), acetone/methanol extract (B), fractions F3, F4, subfractions (SF4-SF6, SF7, SF8-SF10, SF11-SF15, SF16SF20), quercetin, gallic acid, luteolin, myricetin, mixtures M1, M2 and M3 exhibited potent antiinflammatory and antioxidant activities. The results indicated that anthocyanins, flavonoids and its mixtures suppressed the LPS induced production of nitric oxide (NO), through the down-regulation of iNOS and COX-2 protein expressions and showed a potent antioxidant activity against SOD, ABTS, TBARS, ORAC, FRAP and DCFH. The inhibition of enzymes and NO production by selected fractions and compounds was dose-dependent with significant effects seen at concentration as low as 1.0e50.0 (ppm) and 5.0e10.0 mM, for samples (extracts, fractions, subfractions and mixtures) and pure compounds, respectively. Thus, the phenolics (anthocyanins, flavonoids, and organic acids) as the fractions and mixtures may provide a potential therapeutic approach for inflammation associated disorders and therefore might be used as antagonizing agents to ameliorate the effects of oxidative stress. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Flavonoids Phenolics Antioxidant activity Anti-inflammatory activity Aristotelia chilensis COX-2 iNOS ORAC FRAP ABTS TBARS

1. Introduction Antioxidants are substances that delay the oxidation process, inhibiting the polymerization chain initiated by free radicals and other subsequent oxidizing reactions (Halliwell and Aruoma, 1991). A growing body of literature points to the importance of natural

* Corresponding author. Biochemistry and Phyto-Chemical Ecology Lab, Basic Sciences Department, Faculty of Sciences, Andres Bello Av. s/n, Chillan, 3780000, Chillan, Chile. E-mail address: [email protected] (C.L. Cespedes).

antioxidants from many plants, which may be used to reduce cellular oxidative damage, provide protection against chronic diseases, including cancer and neurodegenerative diseases, inflammation and cardiovascular diseases (Prior et al., 1998, 2003; Xiao, 2016). Diets rich in saturated fatty acids, together with environmental pollution increase the oxidative damage in body. Given this constant exposure to oxidants, antioxidants may be necessary to counteract chronic oxidative effects, thereby improving the quality of life (Roberts et al., 2003). The increasing interest in the measurement of the antioxidant activity of different plant samples is derived from the overwhelming evidence of the importance of Reactive Oxygen Species

http://dx.doi.org/10.1016/j.fct.2016.12.036 0278-6915/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Cespedes, C.L., et al., The chilean superfruit black-berry Aristotelia chilensis (Elaeocarpaceae), Maqui as mediator in inflammation-associated disorders, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.12.036

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C.L. Cespedes et al. / Food and Chemical Toxicology xxx (2016) 1e13

(ROS), (Taruscio et al., 2004; Prior et al., 2005). On the other hand, the use of traditional medicine is widespread and plants still present a large source of novel bioactive compounds with interesting bioactivities, in which the antioxidants may play a crucial role in health promotion (Schinella et al., 2002; Yan et al., 2002; Xiao, 2015). The numerous benefits attributed to phenolics product € gger, 2015) have risen to a (Balasundram et al., 2006; Xiao and Ho new interest in finding vegetal species with high phenolic content and relevant bioactivity. Berries constitute a rich source of phenolic antioxidants and bioactive properties (Seeram, 2008; Smith et al., 2000). Chilean Maqui ea blackberry- Aristotelia chilensis (Mol) Stuntz (Elaeocarpaceae), an edible black-colored fruit, which reaches its ripeness between December to March, has a popular and very high consume during these months in Central and South Chile and western of Argentina (Rodriguez, 2005). This plant grows in dense populations called “macales”, endemic from Chile together with other two members of this family (Crinodendron patagua Mol. and C. hookerianum Gay). Common names are: Maqui, macqui, clon, maquie, queldron, koelon. Grows on rainforest areas from sea level to 1500 m in template forest, in communities dominated by Nothofagus dombeyi e Austrocedus chilensis from central to southern of Chile and western of Argentina, this is a small tree that dominates the understory of ungrazed N. dombeyi forests together with Alstroemeria aurea, Eucryphia cordifolia, Maytenus boaria, M. chubutensis, M. disticha, Ribes magellanicum, Saxegothaea conspicua, Laurelia sempervirens, L. philippiana, Persea lingue, Cynanchum diemii, Tristerix corymbosus and Chusquea culeou (Vazquez and Simberloff, 2002). Previously, the alkaloids were reported in the leaves of A. chilensis (Bhakuni et al., 1976; Cespedes et al., 1990, 1993; Watson et al., 1989; Silva et al., 1997). On the other hand, in the continuation of the general screening program of Chilean flora with bioactivities, it has been reported a number of bioactivities including antioxidant, cardioprotection, and anti-inflammation among other from fruits of A. chilensis (Cespedes et al., 2008, 2009, 2010a, 2010b, 2010c). Although it has gained popularity as an ethno-medicine for many years, it is used particularly as an anti-inflammatory agent for kidney pain, stomach ulcers, diverse digestive ailments (tumors and ulcers), fever and cicatrization injuries (Bhakuni et al., 1976; Cespedes et al., 2010b, 2010c). Several updated studies report that fruit extracts of A. chilensis possessed anti-inflammatory effect, antioxidant property, antiatherogenic, hypoglycemic, and antihaemolytic activities (Cespedes et al., 2010b; Cespedes et al., 2010c; Romanucci et al., 2016; PoolZobel et al., 1999), inhibit LDL oxidation (Miranda-Rottmann et al., 2002), and the phytochemicals have been reported (Cespedes et al., 2010a; Brauch et al., 2016; Genskowsky et al., 2016; Ruiz et al., 2010, 2016). Furthermore, two important enzymes involved in inflammatory response are inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). iNOS and COX-2 catalyze the synthesis of nitric oxide (NO) and prostaglandin E2 (PGE2), respectively, which in turn cause sepsis, sepsis shock, and systemic inflammatory response syndrome (Liu et al., 2008). Therefore, the evaluation of inhibition of the expression of these enzymes or of their products can give highlights for new knowledge about reducing inflammation and related conditions. Herein, the in vitro antioxidant capacity and anti-inflammatory properties in RAW 264.7 macrophages of extracts, fractions, compounds and mixtures of phytochemicals of fruits of A. chilensis were investigated.

2. Material and methods 2.1. Materials The fruits of A. chilensis were collected from the fields near to
n City, Chile. University Campus of Universidad Del Bio-Bio, Chilla The samples of plants and fruits were identified botanically by Professor Dr. David S. Seigler (Plant Biology Department, curator of Herbarium, University of Illinois, Urbana-Champaign, US) and voucher specimens were deposited at the Herbarium of Departamento de Ciencias Basicas, Facultad de Ciencias, Universidad del Bio Bio, Chillan, Chile. The collected fruits were air-dried and frozen at
80  C until use. 2.2. Sample preparation Fruits were separated in their main morphological parts (seed and pulp), dried and then were milled and two samples of the pulp were extracted, one with methanol (containing 0.1% HCl, extract A) and the other with distilled water (extract D). The methanol extract (A) was dried and re-dissolved in methanol: water (6:4, v/v), then partitioned into acetone/methanol (B) and ethyl acetate (C). The best antioxidant activity was shown by acetone/methanol partition (B), which was further fractionated into four fractions (1e4), elution was carried out with hexane (100%, fraction 1), hexane/ ethyl acetate (1:1, v/v, fraction 2), ethyl acetate/methanol (1:1, v/v, fraction 3), and methanol 100% (fraction 4), by open column chromatography using silica gel (type G, 10e40 mm). Furthermore extracts and fractions were processed as was previously reported (Cespedes et al., 2008, 2009, 2010a) (Scheme 1). 2.3. Chemicals and solvents All reagents used were either analytical grade or chromatographic grade, 2,2’-azobis (2-aminopropane) dihydrochloride (AAPH), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), butylated hydroxy toluene (BHT), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid), quercetin, rutin, ellagic acid, gallic acid, tannic acid, luteolin, myricetin, (þ)-catechin, Folin-Ciocalteu reagent, 2thiobarbituric acid (TBA), 2,4,6-tripyridil-s-triazine (TPTZ), 2,2azino-bis(3-ethylbenzothyazolin-6-sulfonic ammonium)-salt (ABTS), 20 , 7’-dichlorodihydrofluorescin diacetate (DCFH-DA), FeCl3$6H2O, hypoxanthine, xanthine oxidase, dihydroethydium (DHE), fluorescein disodium (FL) (30 ,6’-dihydroxy-spiro[isobenzofuran-1 [3H], 9[9H]-xanthen]-3-one), tetramethoxypropane (TMP), 1,1,3,3-tetraethoxypropane (TEP), tris-hydrochloride buffer, phosphate buffered saline (PBS), phosphatidylcholine, FeSO4, trichloroacetic acid, were purchased from Sigma-Aldrich Química, S.A. de C.V., Toluca, Mexico, or Sigma-Aldrich Química Ltda, Santiago de Chile, Chile. Methanol, CH2Cl2, CHCl3, NaCl, KCl, KH2PO4, NaHPO4, NaOH, KOH, HCl, sodium acetate trihydrate, glacial acetic acid, silica gel GF254 analytical chromatoplates, silica gel grade 60, (70e230, 60A) for column chromatography, methanol, dichloromethane, n-hexane, and ethyl acetate were purchased from MerckMexico, S.A., Mexico D.F., Mexico and Merck-Chile, Santiago de Chile, Chile. 2.4. Reduction of DPPH free radical The extracts and partitions were chromatographed on TLC and examined for antioxidant effects by spraying the TLC plates with DPPH reagent. Specifically, the plates were sprayed with 0.2% DPPH in methanol (Cespedes et al., 2003). Plates were examined 30 min after spraying, and active compounds appeared as yellow spots against a purple background (Marston, 2011). In addition, TLC

Please cite this article in press as: Cespedes, C.L., et al., The chilean superfruit black-berry Aristotelia chilensis (Elaeocarpaceae), Maqui as mediator in inflammation-associated disorders, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.12.036

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Fruits Pulp of A. chilensis Dried Methanol Extract (A) (200 g) EtOH/H2O (6:4, v/v) Acetone/methanol (B) 120 g (60%)

Ethyl acetate (C) 35.0 g (17.5 %)

water (D) 44.5 g (22.25%)

36.0 g for FracƟonaƟon

F-1

F-2

F-3

(Note A)

F-4

Vacuum chromatography on silica-gel

SF1-SF3 SF4-SF6** Triglycerides and fatty acids p-coumaric acid rutin, catechin + epicatechin

SF7**

SF8-SF10**

SF11-SF15**

SF16-SF20**

gentisic acid gallic acid sinapic acid quercetin, myricetin procyanidin B-9 delphinidin-3-glucoside cyanidin-3-glucoside p-coumaric acid p-hydroxybenzoic acid

SF21-SF25**

SF26-SF30**

delphinidin-3-glucoside delphinidin-3,5-diglucoside delphinidin-3-sambubioside cyanidin-3-sambubioside

SF31-SF37** trimers and tetramers of procyanidins

SF38-SF40** free sugars

cyanidin-3-glucoside mixture of procyanidins 4-hydroxybenzoic acid procyanidin B-9 ferulic acid cyanidin-3-sambubioside-5-glucoside mixture of cyanidin + catechin

Scheme 1. Method of obtaining extracts, partitions, fractions. Fraction F-1 (Hexane 100%), fraction F-2 (hexane: ethyl acetate 1: 1), fraction F-3 (ethyl acetate: Methanol 1: 1), fraction F-4 (methanol 100%). Note A: F-3 together with F-4 were collect up and chromatographed on silica-gel by Vacuum chromatography, solvent system starting with n-hexane, ethyl acetate and increasing MeOH-H2O. Furthermore F4 to F30 were chromatographed on Sephadex LH-20 column, solvent system starting with EtOH and going to 100% acetone. (Taken from Cespedes et al., 2010a).

plates were sprayed with 0.05% ?-carotene solution in chloroform, and then detected under UV254 light until the background bleached. Active components appeared as pale yellow spots against a white background. Samples that showed a strong response were selected for fractionations by open column chromatography, using solvents of increasing polarity. Furthermore, each fraction was analyzed with DPPH in microplates of 96 wells as follow: extracts, partitions and fractions (50 mL) were added to 150 mL of DPPH (100 mM, final concentration) in methanol (The microtiter plate was immediately placed in an Biotek™ Model ELx808, Biotek Instruments, Inc., Winooski, VT) and their absorbance at 515 nm was recorded after 30 min (Cuendet et al., 1997). Quercetin and atocopherol were used as standards. 2.5. Oxygen Radical Absorbance Capacity estimation Oxygen Radical Absorbance Capacity measures antioxidant scavenging activity of a sample or standard against peroxyl radicals generated from AAPH at 37  C using FL, and Trolox was used as standard (Ou et al., 2001). The assay was carried out in black-walled 96-well plates (Fischer Scientific, Hanover Park, IL) at 37  C in 75 mM phosphate buffer (pH 7.4). The following reactants were added in the order shown: Sample or Trolox (20 mL; 7 mM final concentration) and fluorescein (120 mL; 70 nM final concentration). The mixture was preincubated for 15 min at 37  C, after which AAPH (60 mL; 12 mM final concentration) was added (final volume 200 mL). The microtiter plate was immediately placed in an Biotek Model FLx800 (Biotek Instruments, Inc., Winooski, VT) fluorescence plate reader set and the fluorescence recorded every minute for 120 min, using an excitation wavelength of 485/20 nm and emission wavelength of 582/20 nm, to reach a 95% loss of fluorescence. Results are expressed as mmol Trolox equivalents (TE) per gram. All tests were conducted in triplicate.

2.6. Ferric reducing antioxidant power estimation The FRAP assay was performed as previously described by Benzie and Strain (1999). Reagents were freshly prepared and mixed in the proportion 10:1:1, for A:B:C, where A ¼ 300 mM sodium acetate trihydrate/glacial acetic acid buffer pH 3.6; B ¼ 10 mM TPTZ in 40 mM HCl and C ¼ 20 mM FeCl3. And catechin was used for a standard curve (5e40 mM final concentration) with all solutions including samples dissolved in sodium acetate trihydrate/glacial acetic acid buffer. The assay was carried out in 96-well plates, at 37  C at pH 3.6, using 10 mL sample or standard plus 95 mL of the mixture of regents shown above. After 10 min incubation at RT, absorbance was read at 593 nm. Results are expressed as mmol catechin equivalents (CatE) per gram of sample. All tests were conducted in triplicate. 2.7. Estimation of total polyphenol content The total phenolic content of extracts was determined using the Folin-Ciocalteu reagent (Singleton et al., 1999): 10 mL sample or standard (10e100 mM catechin) plus 150 mL diluted Folin-Ciocalteu reagent (1:4 reagent:water) was placed in each well of a 96 well plate, and incubated at RT for 3 min. Following addition of 50 mL sodium carbonate (2:3 saturated sodium carbonate: water) and a further incubation of 2 h at RT, absorbance was read at 725 nm. Results are expressed as mmol Cat E per gram. All tests were conducted in triplicate. 2.8. Estimation of lipid peroxidation through rat brain As an index of lipid peroxidation, TBARS levels were measured using rat brain homogenates according to the method described by Ng with some modifications (Ng et al., 2000). Adult male Wistar

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rats (200e250 g) were provided by the Instituto de Fisiología Celular, UNAM, and was approved by the Animal Care and Use Committee (PROJ.eNOM 087-ECOL-SSA 1e2000). Rats were maintained at 25  C on a 12/12 h light-dark cycle with free access to food and water, and sacrificed under mild ether anesthesia. Cerebral tissue was rapidly dissected from the whole brain and homogenized in phosphate-buffered saline (0.2 g KCl, 0.2 g KH2PO4, 8 g NaCl and 2.16 g NaHPO4$7 H2O/L, pH 7.4) to produce a 1 in 10 homogenate, w/v (Rossato et al., 2002). The homogenate was centrifuged for 10 min at 3400 rpm, and the resulting pellet was discarded. Protein content of the supernatant was measured by the method of Lowry (Lowry et al., 1951), and samples adjusted to 2.5 mg protein/mL with PBS. The supernatant (400 mL, 1 mg protein) was pre-incubated with sample (50 mL) at 37  C for 30 min, then peroxidation was initiated by the addition of 50 mL freshly prepared FeSO4 solution (final concentration 10 mM), and incubated at 37  C for an additional 1 h (Ng et al., 2000). The TBARS assay was determined as described by Ohkawa (Ohkawa et al., 1979) with the modification that 0.5 mL TBA reagent (1% thiobarbituric acid in 0.05 N NaOH and 30% trichloroacetic acid, 1:1) was used, and the final solution was cooled on ice for 10 min, centrifuged at 10, 000 rpm for 5 min, and then heated at 95  C in a boiling water bath for 30 min. After cooling on ice, the absorbance was read at 532 nm in a Spectronic Genesys 5 spectrophotometer. Quercetin and BHT were used as positive controls. Concentrations of TBARS were calculated using a TMP standard curve (Esterbauer and Cheeseman, 1990). Results are expressed as nmoles TBARS per mg of protein, with percent inhibition after 30 min calculated as the inhibition ratio (IR), where

IRð%Þ ¼ ðC
EÞ=C  100% Where C is the absorbance of the control and E is the absorbance of the test sample. These values were plotted against the log10 of the concentrations of individual extracts and fractions, and a decrease of 50% in peroxidation was defined as the EC50. 2.9. Determination of malondialdehyde (MDA) an index of lipid peroxidation of liposomes ~ os Liposomes were prepared as described by El-Hafidi and Ban (1997). Two hundred mg of phosphatidylcholine from soybean (sigma) in 2 mL phosphate buffer was sonicated during 30 min. The clear final solution was centrifuged at 12,000 rpm filtered through a column of Sephadex G50 to eliminate all trace of metal resulted from the tip during the sonication. Lipid peroxidation of liposomes was induced by 5 mM of CuSO4 and 1.0 mM of ascorbic acid to generate hydroxyl radicals by Fenton reaction in presence and absence of different plant extracts. The determination of the MDA-equivalent by the formation of thiobarbituric acid reactive substances (TBARS) was used to evaluate the lipid peroxidation in liposome. The fluorescence method was used for TBARS quantification according to the method ~ os (1997), and the procedure was described by El-Hafidi and Ban carried out with 10 mg of phosphatidylcholine liposome. The samples were treated with 0.05 mL of methanol containing 4% of BHT in 1 mL KH2PO4 (0.15 M, pH 7.4), the mixture agitated in vortex 5 s, then incubated for 30 min at 37  C with constant agitation. At the end of incubation, 1.5 mL of 0.8% thiobarbituric acid and 1 mL of acetic acid to 20% pH 3.5 were added. The mixture was heated with boiling water for 1 h, and immediately the samples were placed in ice. The TBARS was extracted by adding 1 mL from KCl (2%) more 5 mL n-butanol. The n-butanol phase separated by centrifugation for 2 min at 755  g and it was carried out to measure the

fluorescence in Fluorimeter (Perkin Elmer Luminescence LS-50B) at the excitation wavelength of 515 nm and the emission wavelength of 553 nm. The concentration of the MDA equivalent (TBARS) was determined by means of a calibration curve, using as standard 1,1,3,3-tetraethoxypropane (Sigma). 2.10. Generation of the superoxide anion radical with the hypoxanthine-xanthine oxidase system For the specific generation of superoxide anion radical, a chemical system was used to involve hypoxanthine and the specific enzyme, xanthine oxidase, whose reaction gives the consequent liberation of the radical anion superoxide. Briefly, in 1 mL of KH2PO4 (10 mM, pH 7.4) containing 5 mM of hypoxanthine, 23 mg of xanthine oxidase and 1 mM of dihydroethidium (DHE), an indicator of superoxide anion radical and it is detected by fluorescence at the excitation wavelength of 488 nm and emission wavelength of 620 nm. The reaction was performed at 37  C in the presence and absence of plant extracts, allowing them to incubate previously for 2 min and then the reaction began with adding the xanthine oxidase (Masuoka et al., 2013). A volume of 100 mL at 100 mg/mL (ppm) of concentration of each sample was used. The inhibition is presented as an average of three measurements in independent experiments. The results are show as percentage of inhibition. 2.11. Generation of the hydroxyl radical by means of the hydrogen peroxide-peroxidase system The reaction of the H2O2 with the peroxidase generates radical hydroxyls (OH) that react with the ABTS. In the presence of hydroxyl radical, ABTS forms a radical cation ABTSþ, a soluble and a green colour end product which is detected by spectrophotometry at 414 nm. Each chemical reaction was carried out in 1 mL of sodium phosphate buffer (30 mM, pH 7.0) containing 20 mM ABTS, 200 mM H2O2 and 25 mg peroxidase. The effect of different plant extracts on the generation of radical hydroxyl was analyzed at 414 nm. The results were expressed as enzymatic activity in percentage of inhibition of ABTS taking in account the extinction coefficient of the ABTS (Llesuy et al., 2001). The inhibition is presented as an average of three measurements in independent experiments. 2.12. Cell culture Raw 264.7 murine macrophage cells were obtained from American Type Culture Collection (ATTC, Rockville, MD, USA). Maintained in Dulbecco's modified Eagles Medium (DMEM) containing 100 units/mL penicillin G sodium, 100 units/mL streptomycin, supplemented with 10% heat inactivated FBS under endotoxin-free conditions at 37  C in a 5% CO2 atmosphere. 2.13. Cell stimulation Raw 264.7 cells were seeded in T-25 tissue culture flasks (3.0  106 cells/flask). Cells were incubated in DMEM for 24 h. The cells were replaced with new media with or without LPS (1.0 mg/ mL), and then the sample (plant extracts, fractions, or pure compounds) was applied and incubated for 12 h. Cells were then washed with PBS and lysed with 250 mM NaCl, 50 mM HEPES (pH 7.9), 5.0 mM EDTA, 0.1% Nonidet p-40, 0.5 mM DTT, 1.0 mM PMSF, 0.5 mM Na-orthovanadate, 3.0 mM NaF and protease inhibitor cocktail 1.0 mL by 1-3 X 107 cells. Protein was determined by the BioRad method.

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2.14. Western blotting analysis

3. Results and discussion

Total protein (40 mg/lane) was on a 7.5% SDS polyacrylamide gel under standard conditions and electro-blotted to a NTT membrane in 15% methanol, 25 mM Tris and 192 mM glycine. The membrane was blocked with nonfat milk in TTBS saline 1 h at 37  C or overnight at 4  C before incubation with primary antibody (1:500 for iNOS, 1:1000 dilutions for COX-2) in 5% milk in TTBS for 1 h at 37  C. After thorough washing, the membrane was incubated with a secondary antibody radish peroxidase (1:25,000) for 1 h at 37  C. The immunoreactive bands were visualized using an enhanced chemoluminescence system Amersham.

3.1. Phytochemical profile

2.15. MTT assay 80,000 cells/well were plated under the same conditions as cell stimulation. After 12 h incubation, MTT (20 mL, 5 mg/mL in PBS) was added to each well and incubated for 1.5 h in a CO2 incubator at 37  C. The medium was removed and DMSO (200 mL) was added. The plate (96 wells) was incubated for another 15 min before measuring the absorbance at 550 nm (Biotek ELx800).

2.16. Measurement of NO formation by iNOS activity in cultured LPS-induced RAW 264.7 cells Macrophage cells were maintained in DMEM supplemented with penicillin/streptomycin and 10% FBS at 37  C, 5% CO2 in humidified air. For evaluating the inhibitory activity of test materials on iNOS, the cells in 10% FBS_/DMEM without were plated in 96well plates (500,000 cells/well), and then incubated for 24 h. The cells were replaced with new media, and then incubated in the medium with 1.0 mg/mL of LPS and test samples. After additional 12 h incubation, the media were removed and analyzed for nitrite accumulation as an indicator of NO production by the Griess reaction. Briefly, 50 mL of Griess reagent (0.1% naphthylethylenediamine and 1% sulfanilamide in 5% H3PO4) were added to 50 mL supernatant from LPS, or sample-treated cells in triplicate. The plates (96 wells) were incubated for 5 min, and then read at 570 nm against a standard curve of sodium nitrite (Biotek ELx800).

5

The phytochemical analysis of extracts and fractions was made according to Scheme 1. The chemical components in different extracts and fractions were identified by hyphenated HPLC-DAD-ESI/ MSn and 1H-13C-NMR. It revealed an ample range of phenolics from hydroxycinnamic derivatives, flavonoids, anthocyanins and polymers as proanthocyanidins, the complete phytochemical profile was published previously (Cespedes et al., 2010a). Briefly, from fractions SF1-SF3 triglycerides and fatty acids were detected; from SF4-SF6 p-coumaric acid, rutin, catechin together with epicatechin were detected; from SF7 p-coumaric and phydroxybenzoic acid were determined as majority compounds; from SF8-SF10 gentisic acid, sinapic acid and procyanidin B9 were determined; from SF11-SF15 gallic acid, quercetin, myricetin, delphinidin-3-glucoside and cyanidin-3-glucoside were determined; from SF16-SF20 cyanidin-3-glucoside, 4-hydroxybenzoic acid, ferulic acid, and cyanidin together with catechin were determined; from SF21-SF25 delphinidin-3-glucoside, delphinidin-3,5diglucoside, delphionidin-3-sambubioside and cyanidin-3sambubioside were determined; from SF26-SF30 procyanidin B9 and cyanidin-3-sambubioside-5-glucoside were determined; from SF31-SF37 several trimers and tetramers of procyanidins were determined; and finally from SF38-SF40 many sugars were detected. The phytochemical profile has many similarities or matches with various investigations reported by relevant authors (Ruiz et al., 2016; Brauch et al., 2016; Nakamura et al., 2014; GironesVilaplana et al., 2014; Ruiz et al., 2010). Additionally, it has been reported the occurrence of an unusual hydroxyindol derivative in the fruits of this Maqui-berry species (Cespedes et al., 2009). Briefly, from acetone partition (B) fractions F-1 to F-4 were obtained throughout flash-column chromatography. The most active fractions F-3 and F-4 were collected and chromatographed using SiO2 (G 60, Merck), which afforded 40 subfractions (SF) SF-1 to SF-40 and finally compounds were purified through Sephadex LH-20 and analyzed through hyphenated techniques, see Scheme 1 (Cespedes et al., 2010a). 3.2. Antioxidant activity

2.17. DCFH-DA assay 80,000 Cells/well were plated under the same condition as cell stimulation at room temperature. After the 12 h incubation, 200 mL plus 100 mM DCFH-DA wer added to the medium per 30 min, the medium was removed and the cells were washed and added with 200 mL PBS pH 7.5 and the plate was placed in the reader and the fluorescence recorded every minute for 120 min, using an excitation l ¼ 485/20 and emission l ¼ 582/20 (Biotek FLx800).

2.18. Statistical analysis Data were analyzed by one-way ANOVA followed by Dunnett's test for comparisons against control. Values of p < 0.05 (*) and p < 0.01 (**) were considered statistically significant and the significant differences between means were identified by GLM Procedures. In addition, differences between treatment means were established with a Student-Newman-Keuls (SNK) test. The I50 values for each analysis were calculated by Probit analysis. Complete statistical analyses were performed using the MicroCal Origin 8.0 statistical and graphs PC program.

Samples were evaluated by ABTS, ORAC, FRAP, DPPH radical scavenging, an estimation of lipid peroxidation in rat brain and in liposomes through the inhibition of formation of thiobarbituric acid reactive species (TBARS), a measure of the superoxide anion radical with the system hypoxanthine-xanthine oxidase and of the radical hydroxyl by means of the system hydrogen peroxideperoxidase was made through the formation of radical ABTSþ. Antioxidant activities were strongly correlated with the content of phenolic in the samples. The most active samples were fractions SF4-SF6, SF7, SF8-SF10, SF11-SF15, M2, M3, quercetin, gallic acid, luteolin, and myricetin in all bioassays used and the samples were compared by activity against butylated hydroxy toluene (BHT), and tocopherol used as positive control. The Fractions SF4-SF6, SF7, SF8-SF10, SF11-SF15, quercetin, gallic acid, luteolin, and myricetin were found to have IC50 values between 4.7 and 2.6 ppm against DPPH and between 5.9 and 2.7 ppm against TBARS formation. Consistent with this finding, methanol extract (A) had the greatest ABTS and FRAP values as percentage of activity of the extracts. Additionally, it was investigated the response to an acute stress such as ischemia/reperfusion applied to the rat heart in vivo treated with a methanol-extract of fruits of A. chilensis. The methanol extract protects against oxidative stress reducing the

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concentration of the MDA a lipid peroxidation index. A. chilensis protected animals from heart damage as observed by the incidence of reperfusion dysrhythmias, and the no-recovery of sinus rhythm. On the other hand, methanol extract was able to prevent these harmful events in the animal's heart by diminishing lipid oxidation (Cespedes et al., 2008). 3.2.1. DPPH and TBARS determination The DPPH radical scavenging assay was first used as a screen for antioxidant components within the primary extracts (Cespedes et al., 2008). As shown in Table 1, the methanol and acetone partitions (A and B, respectively) had the higher inhibitory activity against DPPH radical formation compared to the other partitions and juice (extract E), with I50 values of 1.7 and 7.4 ppm, respectively (Table 1). For extracts, partitions C and D the I50 values were 35.7 and 17.9 ppm, respectively. Almost all these samples exhibited a concentration-dependence in their DPPH radical scavenging activities, particularly A, which showed the highest activity (100% inhibition) at a concentration of 8.6 ppm. This action was greater than that of a-tocopherol (one of the positive controls), which at 31.6 ppm caused only 53.8% quenching and very similar to ferulic and p-coumaric acids with IC50 values of 5.1 and 7.8 ppm, respectively (data were not shown here). Partition B was then loaded onto a silica-gel open chromatography column, from which four fractions were collected (F1-F4). Of these, fractions F-3 and F-4 were the most active, with an IC50 of 7.1 and 4.9 ppm, respectively Table 1 Amounts of samples from fruits of A. chilensis extracts, fractions and compounds needed to inhibit oxidative damage by 50%.a Sample

DPPHb

TBARSc

TBARSd

A B C D E F-1 F-2 F-3 F-4 SF1-SF3 SF4-SF6 SF7 SF8-SF10 SF11-SF15 SF16-SF20 SF21-SF25 SF26-SF30 SF31-SF37 SF38-SF40 Rutin Catechin Quercetin Luteolin Myricetin Gallic acid M1e M2 M3

1.7 ± 0.3b 7.4 ± 1.4b 35.7 ± 3.6a 17.9 ± 9.1a 4.7 ± 0.7b 109.9 ± 9.2c 79.8 ± 5.5d 7.1 ± 1.4b 4.9 ± 0.5b n.d. 3.2 ± 0.4b 17.9 ± 2.1a 20.6 ± 2.5e 8.9 ± 9.2a 6.1 ± 0.9c 11.2 ± 1.8c 6.1 ± 0.9b >250 n.d. 9.7 (15.9 ± 2.8a) 7.6 (26.4 ± 2.9e) 3.01(8.9 ± 1.9a) 19.78 (69.1 ± 5.7d) 4.1 (12.9 ± 2.8a) 0.54 (3.2 ± 0.9b) 19.6 ± 1.91a 8.8 ± 0.08b 5.6 ± 0.06b

2.1 ± 0.1b 3.9 ± 0.2b 20.1 ± 0.8a 11.2 ± 3.2c 5.9 ± 0.9b 131.2 ± 12.5d 90.5 ± 9.9d 9.9 ± 2.7c 6.5 ± 1.3c n.d. 10.9 ± 0.8 40.4 ± 3.2c 35.7 ± 5.9c 4.5 ± 0.2b 9.6 ± 1.1c 31.2 ± 2.9b 9.6 ± 1.1c >250 n.d. 12.09 (19.8 ± 3.8b) 8.68 (29.9 ± 0.9c) 1.89 (5.6 ± 1.2c) 27.25 (95.2 ± 7.1d) 2.51 (7.9 ± 0.9c) 1.9 (11.2 ± 3.2c) 28.9 ± 1.1c 12.7 ± 0.7c 3.8 ± 0.09c

4.9 ± 0.04a 8.5 ± 0.13b 25.3 ± 2.35c 29.6 ± 2.91c 12.0 ± 1.22b 223.2 ± 10.33d 189.8 ± 12.22d 10.4 ± 1.46b 8.9 ± 0.59b n.d. 12.4 ± 1.56b 44.4 ± 4.58e 20.7 ± 2.05c 5.5 ± 0.09a 8.2 ± 0.19b 10.1 ± 1.36b 4.2 ± 0.03a n.d. n.d. 9.3 (15.3 ± 1.55b) 6.6 (22.8 ± 2.14c) 1.2 (3.6 ± 0.01a) 9.7 (33.9 ± 3.78c) 2.8 (8.8 ± 1.25b) 1.7 (10.2 ± 1.88b) 32.3 ± 3.12c 11.5 ± 0.99b 4.3 ± 0.03a

a For extracts, fractions and mixtures values expressed as mg/mL (ppm), for compounds between parenthesis (values expressed as [mM], Mean ± SD, n ¼ 3). Different letters show significant differences at (P < 0.05), using Duncan's multiplerange test. b IC50 for inhibition of diphenyl picryl hydrazyl radical formation. c IC50 for inhibition of peroxidation of lipids, estimated as thiobarbituric acid reactive substances for rat brain procedures. d IC50 for inhibition of peroxidation of lipids, estimated as thiobarbituric acid reactive substances for liposomes procedures. e M1 ¼ quercetin þ rutin, M2 ¼ quercetin þ catechin, M3 ¼ quercetin þ gallic acid. See Methods for details.

(Table 1). In addition to samples A - D, partitions F-1 and F-2 of extract B showed considerable activity to inhibit DPPH free radical almost completely, the IC50 values were 109.9, and 79.8 ppm, respectively (Table 1). So, the lower IC50 value for partition A (1.7 ppm) than that of the partitions from A, might be due to a synergistic effect of the components (mainly hydroxycinnamic acid derivatives, anthocyanidins and flavonoids) within this extract, similar to that reported for components of Vaccinium corymbosum and V. angustifolium fruits (Ehlenfeldt and Prior, 2001; Smith et al., 2000; Lo and Cheung, 2005), where the acetone and MeOH partitions were the most active extracts. 3.2.2. Lipid peroxidation Biomacromolecules (carbohydrates, lipids, proteins, and DNA) in the presence of ROS suffer oxidative damage, the membrane lipids are especially sensitive to this oxidative physiological process (Diplock et al., 1998). From this point, brain and hearth homogenates can be used for the investigation of lipid peroxidation as an assessment of oxidative stress. The capacity for plant extracts to prevent lipid peroxidation was assayed using malondialdehyde formation as an index of oxidative breakdown of membrane lipids, following incubation of rat brain cortical and hearth homogenates with the oxidant chemical species Fe2þ. Ferrous ion both stimulates lipid peroxidation and supports decomposition of lipids peroxides once formed, generating highly reactive intermediates such as hydroxyl radicals, perferryl and ferryl species (Ko et al., 1998). Fraction F4 was the most effective one and fraction F3 was the least effective one, but none were as effective as extracts A or B, quercetin or M3 in inhibiting lipid peroxidation. Table 1 shows the tabulated data that provide IC50 values; extract A clearly showing the greatest activity. Thus extract A reduced lipid peroxidation in a dose-dependent manner, and proved to be an excellent antioxidant, reflected by its low IC50 value when analyzed by both TBARS and DPPH. When the relative contribution of each fraction (F2, F3 and F4) to the total antioxidant activity of partition B was evaluated using DPPH and TBARS, all fractions except fraction F1 showed some protective effect, with IC50 values between 4.9 and 90.5 ppm (Table 1). Fractions F3 and F4 were the most active, with IC50 values of 7.1 and 4.9, and 9.9 and 6.5 ppm, for DPPH and TBARS, respectively (Table 1). Fraction F4 was substantially more active than that of other fractions. It is noteworthy that the value for fraction F4 is very low compared with values for flavonoids and anthocyanins in general, as well as for myricetin or quercetin (Makris and Rossiter, 2001). It has been reported that the antioxidant activity of many compounds of botanical origin is proportional to the phenolic content (Rice-Evans et al., 1997), suggesting a causative relationship between total phenolic content and antioxidant activity (Veglioglu et al., 1998). Halliwell and Gutteridge (1990) has defined antioxidants as substances that, when present at low concentrations compared with an oxidizable compound (e.g. DNA, protein, lipid, or carbohydrate), delay or prevent oxidative damage due to the presence of ROS. These ROS can undergo a redox reaction with phenolics, such that oxidant activity is inhibited in a concentrationdependent manner. At low concentrations of phenolics the main mechanism is the breaking of chain reactions (Rice-Evans, 2000). Thus, total phenolic content is measured in each one of the extracts, partitions and fractions (see Cespedes et al., 2010a,b,c). Extract A, which had the greatest DPPH and TBARS activities reduced MDA generation, had a significantly greater phenolic content than other extracts. The phenolic content of fractions F1 e F4 showed a small but significant increase in phenolic content for fraction F4 over fraction F3, which

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had a similar content similar to that of fraction F2; fraction F1 had significantly lower phenolic content. These findings correlate well with fraction F4 having one of the greatest activities against DPPH and TBARS. 3.2.3. ORAC and FRAP The ORAC and FRAP values for A. chilensis extracts are given in Table 2. The capacity for a compound to scavenge peroxyl radicals generated by spontaneous decomposition of AAPH was estimated in terms of Trolox equivalents, using the ORAC assay (Cao, & Prior, 1999). A wide variety of different phytochemicals from edible plants, purified or as an extract or fraction, have been found active in this assay, including alkaloids, coumarins, flavonoids, phenylpropanoids, terpenoids and phenolic acids (Lopez-Alarcon and Denicola, 2013). Among the extracts assayed here, the values were found to be in the range of 3900e29,600 mmol TE/g extract for ORAC and from 990 to 15,200 mmol Cat E/g extract for the FRAP assay, respectively (Table 2). The same as with the earlier measurements, extract A had the highest activity in both trials, with values of 29,554.5 [mmol TE/g of extract] and 15,210.9 [mmol Cat E/g of extract] for ORAC and FRAP assays, respectively. In similar form extract B show a very good potency with values of 28,100.1 [mmol TE/g] and 9205.4 [mmol Cat E/g] for ORAC and FRAP assays, respectively. The other extracts (C and D) showed values of intermediate potency, 3898.0 and 19,700.9 [mmol TE/g extract] in the ORAC assay, and 6876.5 and 4726.9 [mmol Cat E/g extract] for FRAP assay, respectively (Table 2). Among the fractions, fraction 4 was significantly more than twice as active as any other fraction (Table 2). The FRAP assay showed greater variability (Table 2). Several extracts had very low values and only extracts A, B and fractions F3 and F4 showed substantial activity. Again, A was significantly more active than any other samples (Table 2). Fractions F3 and F4, those

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with substantial phenolic content, were the fractions showing activity in the FRAP assay (Table 2). In agreement with the ORAC assay, it was A, B, and F4 that showed the greatest values (15,210.9, 9205.4, and 11,000.9 [mmol Cat Equiv/g extract], respectively). Those data correlate well with the ORAC values, (Table 2), and with SOD and ABTS values (Table 3). These effects match very well with other studies for flavonoids and phenolics from other sources and plants (Firuzi et al., 2005; Cao et al., 1997; Fukumoto and Mazza, ~ o et al., 2005). 2000; Villan 3.2.4. SOD and ABTS Antioxidant activities bore a direct relationship with the phenolic content of the extracts and fractions. As with DPPH, TBARS, ORAC and FRAP activities, samples A, B, F4, SF11-SF15, SF16SF20, M3, and quercetin were the most active in SOD and ABTS both the assays. Among the fractions, F4 was the most active in both assays. These facts can be correlated correctly between SOD and total polyphenolic composition of all extracts and partitions and between ABTS and total phenolic composition of fractions. The phenolic characterization suggests that the different phytochemical antioxidant components in the active extract and fractions, mainly anthocyanins, cinnamic derivatives and flavonoids, may be involved in the antioxidant mechanism of action and the antioxidant methods gives a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxyl radical of our samples (Cespedes et al., 2010a). Thus, the highest ORAC and SOD numbers of the extracts and fractions show an excellent antioxidant potential (Tables 2 and 3), for instance, the extracts A, B and F4. In addition, the ORAC numbers of fractions showed a very high correlation with polyphenols content (R > 0.95) (data not shown), the same level of correlation was observed between the FRAP numbers and phenolic composition of the extracts and fractions. In the case of the extracts A and B, there is a similar level of correlation

Table 2 Antioxidant Capacity of A. chilensis extracts, fractions and compounds, ameasured with the ORACb and FRAPc assays. Sample

Concentration [mg/mL]

ORAC

Concentration [mg/mL]

FRAP

A B C D E F-1 F-2 F-3 F-4 SF1-SF3 SF4-SF6 SF7 SF8-SF10 SF11-SF15 SF16-SF20 SF21-SF25 SF26-SF30 SF31-SF37 SF38-SF40 Rutin Quercetin Luteolin Myricetin Gallic acid M1 M2 M3

10.0 10.0 10.0 10.0 10.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

29,554.5 ± 95.2a 28,100.1 ± 85.8a 3890.0 ± 9.7d 19,700.9 ± 90.1a 22,500.8 ± 86.2a N.D. 10,223.9 ± 23.2b 15,699.9 ± 98.8b 25,911.5 ± 115.9c n.d. 19,066.9 ± 79.3c 16,001.6 ± 71.5c 20,446.6 ± 91.5c 21,203.3 ± 105.8c 12,536.8 ± 34.8b 8644.3 ± 7.4d 5699.2 ± 6.1d 2678.9 ± 4.6d n.d. 4533.3 ± 8.6d 13,588.8 ± 13.57b 14,885.2 ± 9.2b 14,435.4 ± 8.1b 8992.2 ± 10.3d 4695.2 ± 5.1d 12,388.4 ± 11.8b 16,899.5 ± 19.3b

25.0 25.0 25.0 25.0 25.0 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

15,210.9 ± 24.4d 9205.4 ± 10.5a 6876.5 ± 9.9a 4726.9 ± 8.4b 8771.1 ± 12.9a N.D. 995.5 ± 1.2e 1159.9 ± 9.9e 11,000.9 ± 22.1f n.d. 11,233.4 ± 19.9f 10,987.2 ± 18.7f 12,448.3 ± 15.3f 14,588.2 ± 16.8f 8955.5 ± 5.9a 6894.2 ± 4.6a 3456.1 ± 2.4a 1988.4 ± 1.1e n.d. 5939.2 ± 6.3a 9966.2 ± 7.2a 7589.2 ± 4.9a 8846.9 ± 5.8a 6498.5 ± 4.4a 3211.6 ± 2.2b 6588.2 ± 4.9a 12,899.8 ± 10.7f

a

For detail see Scheme 1. Expressed as mmol TE/g sample, (mmol of Trolox Equivalents/gram sample). Mean ± SD, n ¼ 3. Different letters show significant differences at (P < 0.05), using Duncan's multiple-range test. c Expressed as mmol CatE/g sample, (mmol of Catequin Equivalents/gram sample). Mean ± SD, n ¼ 3. Values with the same letter are not significantly different (P < 0.05). b

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Table 3 Superoxide radical scavenging capacity, and ABTS inhibition of Maqui berry extracts, fractions, mixtures and compounds. sample

SOD inhibition %

ABTSþ inhibition %

A B C D E F-1 F-2 F-3 F-4 SF1-SF3 SF4-SF6 SF7 SF8-SF10 SF11-SF15 SF16-SF20 SF21-SF25 SF26-SF30 SF31-SF37 SF38-SF40 Rutin Catechin Quercetin Luteolin Myricetin Gallic acid M1 M2 M3

95.7 ± 0.9a 93.8 ± 0.5a 12.2 ± 0.2b 5.9 ± 0.1b n.d. 15.9 ± 2.8c 58.3 ± 2.9d 80.2 ± 1.5a 89.7 ± 1.3a 0.5 ± 0.1e 52.8 ± 2.3d 62.4 ± 2.9f 84.2 ± 2.4a 91.8 ± 1.6a 88.6 ± 1.9a 71.2 ± 1.1 g 60.1 ± 0.9 g 35.2 ± 0.4 h 4.6 ± 0.5b 12.0 ± 0.1c 15.9 ± 0.5c 92.1 ± 0.7a 88.1 ± 0.6a 85.0 ± 0.8a 77.5 ± 0.9 g 35.1 ± 0.8 h 39.8 ± 0.3 h 97.9 ± 0.4a

99.8 ± 0.7a 85.9 ± 0.6a 15.8 ± 0.3b 8.1 ± 0.2c n.d. 17.5 ± 0.1d 60.1 ± 0.2e 82.5 ± 0.4a 92.4 ± 0.1a 0.29 ± 0.01e 48.6 ± 0.9f 60.8 ± 1.2e 90.8 ± 0.8a 92.5 ± 0.3a 89.2 ± 0.5a 69.9 ± 1.1 g 66.8 ± 1.2 g 40.6 ± 0.7f 8.0 ± 0.2c 25.8 ± 0.8 h 17.9 ± 1.2 h 90.7 ± 0.8a 94.8 ± 0.4a 92.6 ± 0.6a 80.4 ± 0.8a 44.6 ± 1.2f 50.7 ± 1.7i 95.5 ± 0.2a

For detail see Scheme 1. Mean ± SD, n ¼ 3. Different letters show significant differences at (P < 0.05), using Duncan's multiple-range test.

(R > 0.98) between FRAP numbers and its polyphenolic content (data not shown). 3.3. Cytotoxicity To evaluate whether the inhibition of NO production was possibly caused by the cytotoxicity effect of test extracts or compounds, the viability of test cells was determined by the MTT assay (Kubo et al., 2007). Extracts A e E, subfractions, compounds and mixtures show no significant cytotoxicity to RAW 264.7 cells at test used concentrations (Fig. 1). Thus, the inhibition of NO production in LPS-stimulated RAW 264.7 cells by extracts, and subfractions of fruits from A. chilensis was not due to cytotoxicity. 3.4. NO production Previous studies showed that A. chilensis extract inhibits lipid peroxidation in vitro systems and decreases oxidation of LDL and increases the ratio of HDL-to-LDL, thus decreasing the risk of heart disease (Miranda-Rottmann et al., 2002; Cespedes et al., 2008). Beneficial effects of fruits of A. chilensis extract are most likely due to polyphenols, which are efficient free radical and singlet oxygen scavengers (Yan et al., 2002). Because heart ischemia-reperfusion causes free radical production and A. chilensis polyphenols are effective free radical scavengers, this study was designed to test the hypothesis that Maqui-berry will block free radical formation, thus preventing injury. Indeed, Maqui-berry and their polyphenolic components, significantly reduced NO injury after ischemiareperfusion (Cespedes et al., 2008), which afford protection against oxidative stress and the risk of increasing septic complications. This study shows as Maqui-berry extracts can help in the control of these health risks. i-NOS is expressed in macrophages by stimulation with LPS

Fig. 1. Viability of macrophages RAW 264.7 treated with LPS (1 mg/mL) with and without the assayed samples (Extracts, fractions and compounds). CO ¼ control, LPS ¼ lipopolysaccharide. Data are expressed as the mean ± S.E. at least of three independent experiments. The viability of cells without LPS was 100%. P < 0.05 represent a significant difference compared with values obtained with cells without LPS. A: Ext C, B: Ext D, C: Ext A, D: Ext B, E: Mix F3 þ F4, F: SF4-SF6, G: SF7, H: SF8-SF10. I: SF11-SF15, J: SF16-SF20, K: SF21-SF25, L: SF26-SF30, M: SF31-SF37. For detail see Scheme 1.

among others, increasing NO production. In this study, NO inhibitory activity of extracts and major compounds from ripe fruits of A. chilensis was evaluated by using a LPS-stimulated RAW 264.7 cell assay. In the previous studies, the ethyl acetate extract from the fruits of A. chilensis exhibited excellent inhibitory activity against ROS and a strong anti-inflammatory activity against TPA-induced inflammation in mouse ear edema model (Cespedes et al., 2010a, 2010b). To determine further effects of those extracts and phenolic compounds on NO production, different concentrations of test samples were incubated with LPS-activated RAW 264.7 cell macrophages. As shown in Fig. 2, the nitrite level produced in cultured supernatant of RAW 264.7 cells was markedly elevated for extracts C, D, E, and mix M1 and M2 these samples do not show a significant inhibition at 100 mg/mL, after 24 h of treatment with LPS. However, extract A and B showed an inhibition >50% [4.0 and 3.0 mM of nitrite, respectively] and F4 was the strongest inhibitor with >90% of activity [2.5 mM of nitrite concentration] (Fig. 2). Thus, sample A and B significantly inhibited LPS-induced NO production in a dosedependent manner (Fig. 2). In relation to the fractions, F4 was the strongest inhibitor with an activity greater than 95% at 50.0 mg/mL, agreeing with the inhibition of iNOS enzyme by this fraction, other fraction with a significant activity was F3 with a 60% of inhibition of NO production, but it did not show the same activity against iNOS expression (Fig. 3). The most active compounds inhibiting the NO production were myricetin and quercetin (Fig. 2). Quantifying the production of nitrite is a technique used to determine the indirect production of NO in macrophages, which are known to be capable of reaching produce 4  106 NO molecules per cell from the iNOS enzyme (Dedon and Tannenbaumb, 2004). This determination was commonly made using the Griess test. In conducting the trial with the pure compounds was observed that the compounds quercetin and myricetin were which had a higher inhibition in the production of nitrites used to the maximum concentrations (25 and 50 mM, respectively) and that the other compounds showed no effect

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and 50 mM for both compounds, respectively) as in the expression of the enzyme iNOS, there was a dose kinetics response in which quercetin showed greatest inhibitory activity on the production of nitrite, with respect to myricetin, these data are consistent with inhibition of the expression of the enzyme COX-2, and iNOS enzyme, because it is known that the production of nitrite is closely linked to the expression of COX-2, these effects have been reported previously for flavonoids and anthocyanins (Cheong et al., 2004; Chiang et al., 2005; Nabavi et al., 2015; Hou et al., 2005; Ojeda et al., 2011). 3.5. Effects on iNOS and COX-2 levels in LPS-activated RAW 264.7 macrophages

Fig. 2. Nitric oxide production in macrophage RAW 264.7 cells measured with Griess reagents: Cells stimulated for 24 h (1 mg/mL) only, or LPS, extracts, fractions, compounds and mixtures from fruits of Aristotelia chilensis. A: total ethanol extract (A), B: Acetone partition (B), F3: F-3 fraction from acetone partition, F4:F-4 fraction from Acetone partition, C: SF7, D:SF8-SF10, E:SF11-SF15, F:SF16-SF20, Myr: myricetin, Q:quercetin, L:luteolin, Cat:catechin, M1:quercetin þ rutin, M2:quercetin þ catechin, M3:quercetin þ gallic acid. At the end of incubation, 100 mL of the medium was removed for measuring nitrite production. Control values were obtained in the absence of LPS. Data were derived from three independent experiments and expressed as means ± S.E. P < 0.05. For detail see Scheme 1.

As mentioned in the background, macrophages are cells that are closely related to the inflammatory response, these cells arrive at the site of inflammation, and initiate a series of events and signals triggered in the first place by neutrophils. It is also known that macrophages can secrete protease, eicosanoids, cytokines, ERO and EPM (Nathan, 2002). It is well known that NOS activity is induced by cytokines such as TNF-a and IL-1b that play an essential role in many inflammatory lesions (De Nardin, 2001). NOS catalyzes NO synthesis. There are three isoforms including a neuronal (nNOS), and endothelial (eNOS) and an endotoxin or cytokine-inducible (iNOS) form (Rosen et al., 2002). In spite of iNOS is usually not detectable in healthy tissues, it is expressed after immunological challenge or injury, the expression of iNOS and its enzymatic activity could be observed (Thomsen et al., 1995; Vane et al., 1994;

Fig. 3. Representative western blots. Effects of 1.0 mg/mL of sample. iNOS and COX-2 protein expression in LPS-stimulated RAW 264.7 macrophages. 1: control, 2: LPS, 3: Ext C, 4: Ext D, 5: Ext E, 6: Ext A, 7: Ext B, 8: SF7, 9: SF8-SF10, 10: SF11-SF15, 11: SF16-SF20, 12: myricetin, 13: quercetin, 14:M1, 15: M2, 16: M3. For detail see Scheme 1.

which coincides with the results obtained in the expression of iNOS enzyme (Fig. 3). To check the effect of the compounds quercetin and myricetin an experiment was conducted at different concentrations (10, 25

MacMicking et al., 1997). In this study, the effect of methanol (A), acetone (B), ethyl acetate (C), residue EtOH/H2O (D), water (100%) (E) extracts, subfractions, compounds and mixtures on iNOS and COX-2 expression

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was also investigated. As shown in Fig. 3, unstimulated RAW 264.7 cells (control) showed barely detectable iNOS. In contrast 12 h incubation with LPS resulted in a large increase in iNOS expression. However, at 50 mM quercetin and myricetin completely suppressed the iNOS expression, as well as extracts A and B, the F3F4 fractions, subfractions SF7 to SF16-SF20 and M1 -M3 mixtures at 50 mg/mL (ppm). The proposed mechanism associated with the reduction of NO production are scavenging of NO, suppression of iNOS enzyme activity, inhibition of iNOS gene expression and/or down-regulation of iNOS enzyme by modulation of enzyme activities related to signal transduction, etc. (Park et al., 2000; Sheu et al., 2001; Chiang et al., 2005; Kim et al., 1999; Paul et al., 1995). In the study, the samples inhibited NO production in macrophages via one or more of these mechanisms. Cyclooxygenase (COX) is the rate-limiting enzyme in PG synthesis and exist as two isoforms: constitutive (COX-1) and inducible (COX-2) (Sheu et al., 2001). Thus, like iNOS, COX-2 is an important enzyme that mediates inflammatory processes. Multiple lines of compelling evidence support COX-2 playing a role in the development of tumors (MacMicking et al., 1997). Thus, aberrant or excessive expression of iNOS and COX-2 is implicated in inflammatory disorders and the pathogenesis of cancer. Quercetin and myricetin, acetone extract (B), Subfractions SF7, SF8-SF10, SF11-SF15, SF16-SF20 and Mix M3 not only showed strong inhibitory activity on iNOS expression, but also significantly inhibited the COX-2 expression in LPS-stimulated macrophages. Fig. 3 show that quercetin, myricetin and extract B suppressed the LPS-induced COX-2 expression in a dose dependent manner. Fig. 3 shows that extract B suppressed the LPS-induced COX-2 expression in a dose-dependent manner. Approximately, 40 and 90% reduction was observed at 50 and 100 mg/mL, respectively, as determined by densitometry analysis. At concentrations up to 100 mg/mL extracts A and B can completely inhibit the expression of COX-2 in LPS-stimulated cells (data not show). On the contrary, these extracts inhibit around 40% and completely (100%) the expression of iNOS at 50 and 100 mg/mL, respectively. Thus, one of the mechanisms of extract B inhibition of NO production in LPS-stimulated macrophages is mediated by the down-regulation of iNOS and COX-2 expressions. Further studies on intracellular signaling cascades leading to COX-2 and iNOS reduction by Maqui-berry extracts, fractions and subfractions of this plant are of interest. Additionally, in vivo pharmacological research on the anti-inflammatory and neuroprotective activities of Maqui-Berry extracts should also be addressed. In Fig. 3 is observed that quercetin had an inhibitory activity of the enzyme expression of iNOS, introducing the increased activity to 50 mM and losing almost entirely activity to 10 mM (data not show). In the case of the expression of the enzyme COX-2 was observed a similar effect though minor, this result is consistent with that reported in other articles, showing how the inhibitory activity of this compound (Matsuda et al., 2003; Nabavi et al., 2015), therefore it was considered to the quercetin as a positive control of this trial and in many published articles. The compound myricetin introduced a similar pattern, the expression of iNOS enzyme to the maximum concentration 25 mM, but in the case of COX-2 effect was less than that found the quercetin at the same concentration of 10 mM. The same experiment was carried out with the more polar samples to the same 25 mM without observing significant effect on concentrations (50 and 10 mM) the expression of both enzymes (data not show). To corroborate the effect of quercetin and myricetin an experiment was conducted to dose response (10, 25 and 50 mM respectively). Even so the flavonoid quercetin presented major inhibitory activity of iNOS expression of the enzyme with respect to the myricetin. In the case of the COX-2 enzyme was observed with a similar behavior quercetin although the effect was

smaller than in the expression of iNOS, myricetin did not present a significant effect on the expression of COX-2 and the lowest concentration 10 mM there was a slight stimulation. Moreover, according to a test conducted by Kim et al. (2004) these flavonoids (of flavonols type) comply with the structural characteristics necessary to inhibit the production of nitrites, which are: a ring A, C-5, 7 dihydroxy replaced and a ring B C-20 , 30 dihydroxy replaced in the case of quercetin and myricetin, while a ring2'-hydroxy B C 3'-metoxy substituted for whose replacement metoxylated form provides lower activity as shown in conducted tests, as also it has been reported by Orhan group (Nabavi et al., 2015; Surh et al., 2001). 3.6. Oxidative stress Fig. 4 shows that the compound quercetin presented protective activity against oxidative stress (DCFH), which led to conclude that the inhibitory activity of the expression of iNOS and in the production of nitrites was favored by the antioxidant activity. According to the background oxidative stress is involved at different levels in inflammatory response by stimulating the activity of NF- and production of different mediators such as IL-1, IL-6, TNF-a, IFN-g in the cellular signaling, thus it is possible that quercetin through its antioxidant activity has decreased the studied factors which are involved in the inflammatory response (Nathan, 2002; Nabavi et al., 2015). With regard to the present myricetin despite a slight prooxidant activity also inhibited the expression of iNOS and COX-2 as well as in the production of nitrites, which suggests that the mechanism of action is separated from the production of reactive species. In the case of the other compounds these did not show antioxidant activity at level showed by flavonoids, including phenolics acids mixtures, showed an apparent increase of oxidative stress, like the myricetin. The result obtained with the phenolic acids disagrees with the antioxidant activities reported in the literature (Aldini et al., 2006). Tables 1e3 shows the antioxidant activity of the samples isolated from A. chilensis. It is noted that the flavonoids mixtures showed a larger reduction DPPH radical activity with a 87.25%, followed by phenolic acids

Fig. 4. Inhibition of oxidative stress in macrophage RAW 264.7 cells measured with DCFH cells stimulated for 24 h with LPS (1 mg/mL) only, or LPS and samples from fruits of Maqui berry. 1: control, 2: LPS, 3: Ext C, 4: Ext D, 5: Ext E, 6: Ext A, 7: Ext B, 8: SF7, 9: SF8-SF10, 10: SF11-SF15, 11: SF16-SF20, 12: myricetin, 13: quercetin, 14:M1, 15: M2, 16: M3. For detail see Scheme 1.

Please cite this article in press as: Cespedes, C.L., et al., The chilean superfruit black-berry Aristotelia chilensis (Elaeocarpaceae), Maqui as mediator in inflammation-associated disorders, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.12.036

C.L. Cespedes et al. / Food and Chemical Toxicology xxx (2016) 1e13

activity with a 66.2%. The fractions that contains anthocyanins showed less reduction DPPH radical activity with a 58.13% and 22.48%, respectively. Anthocyanidins did not show activity to this concentration. Similar activity was observed in the trial against TBARS where quercetin and myricetin showed the greatest inhibition on lipid peroxidation (93.28% and 87.53% respectively), followed by phenolic acids (34.29%) and anthocyanins mixtures (28.52%).

4. Concluding remarks In the particular case of flavonoids, it is known that these compounds have a strong antioxidant activity which was reflected in the essays against DPPH, but not in the essay of antioxidant activity in the RAW 264.7 macrophages, this could be due to the hydrophilic properties upon the hydroxyl groups of both the aromatic rings as sugars (in the case of anthocyanins) and do not allow it to cross the lipid membranes and exercise their antioxidant effect for the same reasons it may not have shown effect in inhibiting the expression of COX enzymes
2 and iNOS. With these data highlights the variability of the results of different trials designed to measure the same effect. Besides, the compounds quercetin and myricetin showed antioxidant activity in DPPH test and TBARS. Quercetin is a flavonoid with antioxidant activity reported by its replacement ortho-dihydroxy in the ring B. The inhibiting the expression of COX2 enzymes and iNOS by quercetin and myricetin, as well as the production of nitrite is not new, since it is known that the quercetin interferes with cascades of signals in which, stimulated with LPS is the RAW 264.7 cell line, blocking the main (Xagorari et al., 2002), proinflammatory molecules such as TNF-a. However, it does not take reports of the antioxidant activity of these compounds in RAW 264.7 macrophages using DCFH. According, to these results it is suggested that not all compounds with antioxidant activity in the chemical models may affect activity, and that the antioxidant activity can be a factor that promotes the inhibition of the expression of certain enzymes. The extracts A and B of fruits from A. chilensis and some of their fractions and subfractions exhibited substantial potency in scavenging DPPH-radical and inhibiting lipid peroxidation. Two of the four fractions isolated from B, the F3 and F4 showed potency in scavenging against DPPH-radicals, as well as a strong inhibitory effect against lipid peroxidation, particularly fraction F4. The antioxidant activities, total phenolic content (Table 4) and ORAC and FRAP assays all correlated, suggest but do not prove a causative relationship. It was the acetone partition B that showed that the phenolic compounds present are probably low or medium molecular weight, with relative high polarity. Phytochemical analysis of these extract, partitions and fractions are in progress, and is expected to identify chemical structures of bioactive components that may have a future role in human health maintenance. Many cellular components are sensitive to oxidative damage,

Table 4 Phenolics content of Maqui samples. (Expressed as mmol of Catechin equiv/g of sample). sample

Amount of total phenolics

Extract A Extract B Extract C F-4 F-3 F-2 F-1

17,879.2 ± 335.7 16,554.8 ± 501.0 6399.1 ± 101.4 18,987.6 ± 755.9 13,555.1 ± 310.0 5445.6 ± 198.5 3899.8 ± 95.5

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caused by the presence of nitrogen or oxygen reactive species, including a myriad of different free radicals. Rat brain homogenates, rich in lipids such as polyunsaturated fatty acids can undergo peroxidation. The research showed that the extract A and acetone partition B of fruits from A. chilensis, several fractions and subfractions of that extract, contain antioxidants that can inhibit lipid peroxidation, SOD, and that they have a high phenolic content (Table 4). The relationship between total phenolics with ORAC and FRAP values in all extracts and fractions was similar to those found in other methanol and ethyl acetate plants extracts, and that the values are similar to those of different known fruits and vegetables as prunes, raisins, blueberries, spinach and Broccoli (Aldini et al., 2006; Balasundram et al., 2006; Cao et al., 1997; Cheong et al., 2004; Ehlenfeldt and Prior, 2001; Firuzi et al., 2005; Fukumoto and Mazza, 2000; Hou et al., 2005; Kim et al., 1999; 2004; Ko et al., 1998; Lopez-Alarcon and Denicola, 2013; Matsuda et al., ~o 2003; Nabavi et al., 2015; Seeram, 2008; Smith et al., 2000; Villan et al., 2005). With the aim of to elucidate the sites and mechanism of action, we are on-going studies doing in vivo test of anti-inflammatory activities, those results will be published in a next paper. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported in part by internal grant from Direccion de Investigacion, Universidad del Bio Bio, Chillan, Chile (Grant # 091909-1/R); by grant FONDECYT-2920018, and in part by grants from UC-MEXUS-CONACYT (#2013-02) and UC-CONICYTChile(#2013-02). The authors thanks to Roberto Rodriguez (Facultad de Ciencias Naturales y Oceanograficas, Universidad de Con
n, Concepcio
n, Chile) and Prof David S. Seigler, curator cepcio Herbarium of University of Illinois at Urbana-Champaign, USA, for botanical identification of the plant. We thank Ma. Teresa RamirezApan, and Antonio Nieto for technical assistance: Chemistry Institute, Ana Ma. Garcia-Bores: UBIPRO FES-Iztacala, UNAM, Mexico D.F., Mexico. Anne Murray (ESPM, UC, Berkeley); M.D. thanks CONACyT-Mexico for a doctoral fellowship and to TIES-ENLACES USAID Program for a research fellowship. CLC and IK acknowledge to Seed Funds Program of Conicyt-Chile and UC-Berkeley “2013 UC BerkeleyeChile Seed Grants”, grant (# 2013-02): A New Connection: Potential Cancer Treatment Agents. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.fct.2016.12.036. References Aldini, G., Piccoli, A., Beretta, G., Morazzoni, P., Riva, A., Marinello, C., MaffeiFacino, R., 2006. Antioxidant activity of polyphenols from solid olive residues of c.v. Coratina. Fitoter. 77, 121e128. Balasundram, N., Sundram, K., Samman, S., 2006. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem. 99, 191e203. Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzym. 299, 15e27. Bhakuni, D.S., Bittner, M., Marticorena, C., Silva, M., Weldt, F., Hoeneisen, M., Hartwell, J.L., 1976. Screening of Chilean plant for anticancer activity. J. Nat. Prod. 39, 225e243. Brauch, J.E., Buchweitz, M., Schweiggert, R.M., Carle, R., 2016. Detailed analyses of fresh and dried Maqui (Aristotelia chilensis (Mol.) Stuntz) berries and juice.

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Please cite this article in press as: Cespedes, C.L., et al., The chilean superfruit black-berry Aristotelia chilensis (Elaeocarpaceae), Maqui as mediator in inflammation-associated disorders, Food and Chemical Toxicology (2016), http://dx.doi.org/10.1016/j.fct.2016.12.036