Food and Chemical Toxicology 108 (2017) 438e450

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Food and Chemical Toxicology

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The chilean superfruit black- chilensis (), 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, b Departmento de Farmacología, Instituto Nacional de Cardiología “Ignacio Chavez ”, Juan Badiano 1, Seccion XVI, Tlalpan, 14080, Mexico D.F., Mexico c Departamento de Biología Celular y Desarrollo, Laboratorio 305-Sur, Instituto de Fisiología Celular, UNAM. Ciudad Universitaria, Coyoacan, 04510, Mexico D.F., Mexico d Department of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA e Departamento de Biomedicina Cardiovascular, Instituto Nacional de Cardiología “Ignacio Chavez ”, Juan Badiano 1, Seccion XVI, Tlalpan, 14080, Mexico D.F., Mexico f Laboratorio de Fitoquímica, UBIPRO, Facultad de Estudios Superiores-Iztacala, Universidad Nacional Autonoma de Mexico, Tlalnepantla de Baz, Estado de Mexico, Mexico article info abstract

Article history: The effects of phytochemicals occurred in fractions and extracts of of “Maqui-berry” (Aristotelia Received 3 October 2016 chilensis), on the expression of cyclooxygenase-2 (COX-2), inducible-nitric oxide synthases (iNOS) and Received in revised form the production of proinflammatory mediators were investigated in lipopolysaccharide (LPS)-activated 25 December 2016 murine macrophage RAW-264 cells, as well as their antioxidant activities. The MeOH extract (A), Accepted 27 December 2016 acetone/methanol extract (B), fractions F3, F4, subfractions (SF4-SF6, SF7, SF8-SF10, SF11-SF15, SF16- Available online 28 December 2016 SF20), quercetin, gallic acid, luteolin, myricetin, mixtures M1, M2 and M3 exhibited potent anti- inflammatory and antioxidant activities. The results indicated that , flavonoids and its Keywords: Flavonoids mixtures suppressed the LPS induced production of nitric oxide (NO), through the down-regulation of Phenolics iNOS and COX-2 protein expressions and showed a potent antioxidant activity against SOD, ABTS, TBARS, Antioxidant activity ORAC, FRAP and DCFH. The inhibition of enzymes and NO production by selected fractions and com- Anti-inflammatory activity pounds was dose-dependent with significant effects seen at concentration as low as 1.0e50.0 (ppm) and Aristotelia chilensis 5.0e10.0 mM, for samples (extracts, fractions, subfractions and mixtures) and pure compounds, respec- COX-2 tively. Thus, the phenolics (anthocyanins, flavonoids, and organic acids) as the fractions and mixtures iNOS may provide a potential therapeutic approach for inflammation associated disorders and therefore might ORAC be used as antagonizing agents to ameliorate the effects of oxidative stress. FRAP © 2016 Elsevier Ltd. All rights reserved. ABTS TBARS

1. Introduction antioxidants from many , which may be used to reduce cellular oxidative damage, provide protection against chronic dis- Antioxidants are substances that delay the oxidation process, eases, including cancer and neurodegenerative diseases, inflam- inhibiting the polymerization chain initiated by free radicals and mation and cardiovascular diseases (Prior et al., 1998, 2003; Xiao, other subsequent oxidizing reactions (Halliwell and Aruoma, 1991). 2016). Diets rich in saturated fatty acids, together with environ- A growing body of literature points to the importance of natural mental 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). * Corresponding author. Biochemistry and Phyto-Chemical Ecology Lab, Basic The increasing interest in the measurement of the antioxidant Sciences Department, Faculty of Sciences, Andres Bello Av. s/n, Chillan, 3780000, Chillan, Chile. activity of different samples is derived from the over- E-mail address: [email protected] (C.L. Cespedes). whelming 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. C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 439

(ROS), (Taruscio et al., 2004; Prior et al., 2005). On the other hand, 2. Material and methods the use of traditional medicine is widespread and plants still pre- sent a large source of novel bioactive compounds with interesting 2.1. Materials bioactivities, in which the antioxidants may play a crucial role in health promotion (Schinella et al., 2002; Yan et al., 2002; Xiao, The fruits of A. chilensis were collected from the fields near to 2015). University Campus of Universidad Del Bio-Bio, Chillan City, Chile. The numerous benefits attributed to phenolics product The samples of plants and fruits were identified botanically by (Balasundram et al., 2006; Xiao and Hogger,€ 2015) have risen to a Professor Dr. David S. Seigler (Plant Biology Department, curator of new interest in finding vegetal species with high phenolic content Herbarium, University of Illinois, Urbana-Champaign, US) and and relevant bioactivity. constitute a rich source of phenolic voucher specimens were deposited at the Herbarium of Departa- antioxidants and bioactive properties (Seeram, 2008; Smith et al., mento de Ciencias Basicas, Facultad de Ciencias, Universidad del Bio 2000). Chilean Maqui ea blackberry- Aristotelia chilensis (Mol) Bio, Chillan, Chile. The collected fruits were air-dried and frozen Stuntz (Elaeocarpaceae), an edible black-colored , which rea- at 80 C until use. ches its ripeness between December to March, has a popular and very high consume during these months in Central and South Chile 2.2. Sample preparation and western of (Rodriguez, 2005). This plant grows in dense populations called “macales”, Fruits were separated in their main morphological parts (seed endemic from Chile together with other two members of this and pulp), dried and then were milled and two samples of the pulp family (Crinodendron patagua Mol. and C. hookerianum Gay). were extracted, one with methanol (containing 0.1% HCl, extract A) Common names are: Maqui, macqui, clon, maquie, queldron, koe- and the other with distilled water (extract D). The methanol extract lon. Grows on rainforest areas from sea level to 1500 m in template (A) was dried and re-dissolved in methanol: water (6:4, v/v), then forest, in communities dominated by Nothofagus dombeyi e Aus- partitioned into acetone/methanol (B) and ethyl acetate (C). The trocedus chilensis from central to southern of Chile and western of best antioxidant activity was shown by acetone/methanol partition Argentina, this is a small tree that dominates the understory of (B), which was further fractionated into four fractions (1e4), ungrazed N. dombeyi forests together with Alstroemeria aurea, elution was carried out with hexane (100%, fraction 1), hexane/ Eucryphia cordifolia, Maytenus boaria, M. chubutensis, M. disticha, ethyl acetate (1:1, v/v, fraction 2), ethyl acetate/methanol (1:1, v/v, Ribes magellanicum, Saxegothaea conspicua, Laurelia sempervirens, fraction 3), and methanol 100% (fraction 4), by open column L. philippiana, Persea lingue, Cynanchum diemii, Tristerix corymbosus chromatography using silica gel (type G, 10e40 mm). Furthermore and Chusquea culeou (Vazquez and Simberloff, 2002). extracts and fractions were processed as was previously reported Previously, the alkaloids were reported in the of (Cespedes et al., 2008, 2009, 2010a)(Scheme 1). 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 2.3. Chemicals and solvents of the general screening program of Chilean flora with bioactivities, it has been reported a number of bioactivities including antioxi- All reagents used were either analytical grade or chromato- dant, cardioprotection, and anti-inflammation among other from graphic grade, 2,2’-azobis (2-aminopropane) dihydrochloride fruits of A. chilensis (Cespedes et al., 2008, 2009, 2010a, 2010b, (AAPH), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), butylated hydroxy 2010c). Although it has gained popularity as an ethno-medicine toluene (BHT), Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- for many years, it is used particularly as an anti-inflammatory carboxylic acid), quercetin, rutin, ellagic acid, gallic acid, tannic agent for kidney pain, stomach ulcers, diverse digestive ailments acid, luteolin, myricetin, (þ)-catechin, Folin-Ciocalteu reagent, 2- (tumors and ulcers), fever and cicatrization injuries (Bhakuni et al., thiobarbituric acid (TBA), 2,4,6-tripyridil-s-triazine (TPTZ), 2,2- 1976; Cespedes et al., 2010b, 2010c). azino-bis(3-ethylbenzothyazolin-6-sulfonic ammonium)-salt Several updated studies report that fruit extracts of A. chilensis (ABTS), 20,7’-dichlorodihydrofluorescin diacetate (DCFH-DA), possessed anti-inflammatory effect, antioxidant property, anti- FeCl3$6H2O, hypoxanthine, xanthine oxidase, dihydroethydium atherogenic, hypoglycemic, and antihaemolytic activities (Cespedes (DHE), fluorescein disodium (FL) (30,6’-dihydroxy-spiro[iso- et al., 2010b; Cespedes et al., 2010c; Romanucci et al., 2016; Pool- benzofuran-1 [3H], 9[9H]-xanthen]-3-one), tetramethoxypropane Zobel et al., 1999), inhibit LDL oxidation (Miranda-Rottmann (TMP), 1,1,3,3-tetraethoxypropane (TEP), tris-hydrochloride buffer, et al., 2002), and the phytochemicals have been reported phosphate buffered saline (PBS), phosphatidylcholine, FeSO4, tri- (Cespedes et al., 2010a; Brauch et al., 2016; Genskowsky et al., 2016; chloroacetic acid, were purchased from Sigma-Aldrich Química, Ruiz et al., 2010, 2016). S.A. de C.V., Toluca, Mexico, or Sigma-Aldrich Química Ltda, San- Furthermore, two important enzymes involved in inflammatory tiago de Chile, Chile. Methanol, CH2Cl2, CHCl3, NaCl, KCl, KH2PO4, response are inducible nitric oxide synthase (iNOS) and NaHPO4, NaOH, KOH, HCl, sodium acetate trihydrate, glacial acetic cyclooxygenase-2 (COX-2). iNOS and COX-2 catalyze the synthesis acid, silica gel GF254 analytical chromatoplates, silica gel grade 60, of nitric oxide (NO) and prostaglandin E2 (PGE2), respectively, (70e230, 60A) for column chromatography, methanol, dichloro- which in turn cause sepsis, sepsis shock, and systemic inflamma- methane, n-hexane, and ethyl acetate were purchased from Merck- tory response syndrome (Liu et al., 2008). Therefore, the evaluation Mexico, S.A., Mexico D.F., Mexico and Merck-Chile, Santiago de of inhibition of the expression of these enzymes or of their products Chile, Chile. can give highlights for new knowledge about reducing inflamma- tion and related conditions. Herein, the in vitro antioxidant capacity 2.4. Reduction of DPPH free radical and anti-inflammatory properties in RAW 264.7 macrophages of extracts, fractions, compounds and mixtures of phytochemicals of The extracts and partitions were chromatographed on TLC and fruits of A. chilensis were investigated. 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 440 C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450

Fruits Pulp of A. chilensis Dried Methanol Extract (A) (200 g)

EtOH/H2O (6:4, v/v)

Acetone/methanol (B) Ethyl acetate (C) 35.0 g (17.5 %) 120 g (60%) water (D) 44.5 g (22.25%) 36.0 g for FracƟonaƟon

(Note A) F-1 F-2 F-3 F-4 Vacuum chromatography on silica-gel

** ** ** ** ** ** ** ** ** SF1-SF3 SF4-SF6 SF7 SF8-SF10 SF11-SF15 SF16-SF20 SF21-SF25 SF26-SF30 SF31-SF37 SF38-SF40 Triglycerides and fatty acids p-coumaric acid gentisic acid gallic acid delphinidin-3-glucoside trimers and tetramers free sugars rutin, catechin sinapic acid quercetin, myricetin delphinidin-3,5-diglucoside of procyanidins + epicatechin procyanidin B-9 delphinidin-3-glucoside delphinidin-3-sambubioside cyanidin-3-glucoside cyanidin-3-sambubioside p-coumaric acid p-hydroxybenzoic acid 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, 2.6. Ferric reducing antioxidant power estimation and then detected under UV254 light until the background bleached. Active components appeared as pale yellow spots against The FRAP assay was performed as previously described by a white background. Samples that showed a strong response were Benzie and Strain (1999). Reagents were freshly prepared and selected for fractionations by open column chromatography, using mixed in the proportion 10:1:1, for A:B:C, where A ¼ 300 mM so- solvents of increasing polarity. Furthermore, each fraction was dium acetate trihydrate/glacial acetic acid buffer pH 3.6; B ¼ 10 mM analyzed with DPPH in microplates of 96 wells as follow: extracts, TPTZ in 40 mM HCl and C ¼ 20 mM FeCl3. And catechin was used for partitions and fractions (50 mL) were added to 150 mL of DPPH a standard curve (5e40 mM final concentration) with all solutions (100 mM, final concentration) in methanol (The microtiter plate was including samples dissolved in sodium acetate trihydrate/glacial immediately placed in an Biotek™ Model ELx808, Biotek In- acetic acid buffer. The assay was carried out in 96-well plates, at struments, Inc., Winooski, VT) and their absorbance at 515 nm was 37 C at pH 3.6, using 10 mL sample or standard plus 95 mL of the recorded after 30 min (Cuendet et al., 1997). Quercetin and a- mixture of regents shown above. After 10 min incubation at RT, tocopherol were used as standards. absorbance was read at 593 nm. Results are expressed as mmol catechin equivalents (CatE) per gram of sample. All tests were conducted in triplicate. 2.5. Oxygen Radical Absorbance Capacity estimation

Oxygen Radical Absorbance Capacity measures antioxidant 2.7. Estimation of total content scavenging activity of a sample or standard against peroxyl radicals generated from AAPH at 37 C using FL, and Trolox was used as The total phenolic content of extracts was determined using the standard (Ou et al., 2001). The assay was carried out in black-walled Folin-Ciocalteu reagent (Singleton et al., 1999): 10 mL sample or e 96-well plates (Fischer Scientific, Hanover Park, IL) at 37 Cin standard (10 100 mM catechin) plus 150 mL diluted Folin-Ciocalteu 75 mM phosphate buffer (pH 7.4). The following reactants were reagent (1:4 reagent:water) was placed in each well of a 96 well added in the order shown: Sample or Trolox (20 mL; 7 mM final plate, and incubated at RT for 3 min. Following addition of 50 mL concentration) and fluorescein (120 mL; 70 nM final concentration). sodium carbonate (2:3 saturated sodium carbonate: water) and a The mixture was preincubated for 15 min at 37 C, after which further incubation of 2 h at RT, absorbance was read at 725 nm. AAPH (60 mL; 12 mM final concentration) was added (final volume Results are expressed as mmol Cat E per gram. All tests were con- 200 mL). The microtiter plate was immediately placed in an Biotek ducted in triplicate. Model FLx800 (Biotek Instruments, Inc., Winooski, VT) fluorescence plate reader set and the fluorescence recorded every minute for 2.8. Estimation of lipid peroxidation through rat brain 120 min, using an excitation wavelength of 485/20 nm and emis- sion wavelength of 582/20 nm, to reach a 95% loss of fluorescence. As an index of lipid peroxidation, TBARS levels were measured Results are expressed as mmol Trolox equivalents (TE) per gram. All using rat brain homogenates according to the method described by tests were conducted in triplicate. Ng with some modifications (Ng et al., 2000). Adult male Wistar C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 441 rats (200e250 g) were provided by the Instituto de Fisiología fluorescence in Fluorimeter (Perkin Elmer Luminescence LS-50B) at Celular, UNAM, and was approved by the Animal Care and Use the excitation wavelength of 515 nm and the emission wavelength Committee (PROJ.eNOM 087-ECOL-SSA 1e2000). Rats were of 553 nm. The concentration of the MDA equivalent (TBARS) was maintained at 25 C on a 12/12 h light-dark cycle with free access to determined by means of a calibration curve, using as standard food and water, and sacrificed under mild ether anesthesia. Cere- 1,1,3,3-tetraethoxypropane (Sigma). bral tissue was rapidly dissected from the whole brain and ho- mogenized in phosphate-buffered saline (0.2 g KCl, 0.2 g KH PO , 2 4 2.10. Generation of the superoxide anion radical with the 8 g NaCl and 2.16 g NaHPO $7HO/L, pH 7.4) to produce a 1 in 10 4 2 hypoxanthine-xanthine oxidase system homogenate, w/v (Rossato et al., 2002). The homogenate was centrifuged for 10 min at 3400 rpm, and the resulting pellet was For the specific generation of superoxide anion radical, a discarded. Protein content of the supernatant was measured by the chemical system was used to involve hypoxanthine and the specific method of Lowry (Lowry et al., 1951), and samples adjusted to enzyme, xanthine oxidase, whose reaction gives the consequent 2.5 mg protein/mL with PBS. The supernatant (400 mL, 1 mg pro- liberation of the radical anion superoxide. Briefly, in 1 mL of tein) was pre-incubated with sample (50 mL) at 37 C for 30 min, KH PO (10 mM, pH 7.4) containing 5 mM of hypoxanthine, 23 mg then peroxidation was initiated by the addition of 50 mL freshly 2 4 of xanthine oxidase and 1 mM of dihydroethidium (DHE), an in- prepared FeSO solution (final concentration 10 mM), and incubated 4 dicator of superoxide anion radical and it is detected by fluores- at 37 C for an additional 1 h (Ng et al., 2000). cence at the excitation wavelength of 488 nm and emission The TBARS assay was determined as described by Ohkawa wavelength of 620 nm. The reaction was performed at 37 C in the (Ohkawa et al., 1979) with the modification that 0.5 mL TBA reagent presence and absence of plant extracts, allowing them to incubate (1% thiobarbituric acid in 0.05 N NaOH and 30% trichloroacetic acid, previously for 2 min and then the reaction began with adding the 1:1) was used, and the final solution was cooled on ice for 10 min, xanthine oxidase (Masuoka et al., 2013). A volume of 100 mLat centrifuged at 10, 000 rpm for 5 min, and then heated at 95 Cina 100 mg/mL (ppm) of concentration of each sample was used. The boiling water bath for 30 min. After cooling on ice, the absorbance inhibition is presented as an average of three measurements in was read at 532 nm in a Spectronic Genesys 5 spectrophotometer. independent experiments. The results are show as percentage of Quercetin and BHT were used as positive controls. Concentrations inhibition. 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 2.11. Generation of the hydroxyl radical by means of the hydrogen the inhibition ratio (IR), where peroxide-peroxidase system

IRð%Þ¼ðC EÞ=C 100% The reaction of the H2O2 with the peroxidase generates radical hydroxyls (OH) that react with the ABTS. In the presence of hy- þ Where C is the absorbance of the control and E is the absorbance of droxyl radical, ABTS forms a radical cation ABTS , a soluble and a the test sample. These values were plotted against the log10 of the green colour end product which is detected by spectrophotometry concentrations of individual extracts and fractions, and a decrease at 414 nm. Each chemical reaction was carried out in 1 mL of so- fi of 50% in peroxidation was de ned as the EC50. dium phosphate buffer (30 mM, pH 7.0) containing 20 mM ABTS, 200 mM H2O2 and 25 mg peroxidase. The effect of different plant 2.9. Determination of malondialdehyde (MDA) an index of lipid extracts on the generation of radical hydroxyl was analyzed at peroxidation of liposomes 414 nm. The results were expressed as enzymatic activity in per- centage of inhibition of ABTS taking in account the extinction co- Liposomes were prepared as described by El-Hafidi and Banos~ efficient of the ABTS (Llesuy et al., 2001). The inhibition is presented (1997). Two hundred mg of phosphatidylcholine from soybean as an average of three measurements in independent experiments. (sigma) in 2 mL phosphate buffer was sonicated during 30 min. The clear final solution was centrifuged at 12,000 rpm filtered through a 2.12. Cell culture column of Sephadex G50 to eliminate all trace of metal resulted from the tip during the sonication. Lipid peroxidation of liposomes Raw 264.7 murine macrophage cells were obtained from was induced by 5 mM of CuSO and 1.0 mM of ascorbic acid to 4 American Type Culture Collection (ATTC, Rockville, MD, USA). generate hydroxyl radicals by Fenton reaction in presence and Maintained in Dulbecco's modified Eagles Medium (DMEM) con- absence of different plant extracts. taining 100 units/mL penicillin G sodium, 100 units/mL strepto- The determination of the MDA-equivalent by the formation of mycin, supplemented with 10% heat inactivated FBS under thiobarbituric acid reactive substances (TBARS) was used to eval- endotoxin-free conditions at 37 C in a 5% CO atmosphere. uate the lipid peroxidation in liposome. The fluorescence method 2 was used for TBARS quantification according to the method described by El-Hafidi and Banos~ (1997), and the procedure was 2.13. Cell stimulation carried out with 10 mg of phosphatidylcholine liposome. The samples were treated with 0.05 mL of methanol containing 4% of Raw 264.7 cells were seeded in T-25 tissue culture flasks 6 BHT in 1 mL KH2PO4 (0.15 M, pH 7.4), the mixture agitated in vortex (3.0 10 cells/flask). Cells were incubated in DMEM for 24 h. The 5 s, then incubated for 30 min at 37 C with constant agitation. At cells were replaced with new media with or without LPS (1.0 mg/ the end of incubation, 1.5 mL of 0.8% thiobarbituric acid and 1 mL of mL), and then the sample (plant extracts, fractions, or pure com- acetic acid to 20% pH 3.5 were added. The mixture was heated with pounds) was applied and incubated for 12 h. Cells were then boiling water for 1 h, and immediately the samples were placed in washed with PBS and lysed with 250 mM NaCl, 50 mM HEPES (pH ice. 7.9), 5.0 mM EDTA, 0.1% Nonidet p-40, 0.5 mM DTT, 1.0 mM PMSF, The TBARS was extracted by adding 1 mL from KCl (2%) more 0.5 mM Na-orthovanadate, 3.0 mM NaF and protease inhibitor 5 mL n-butanol. The n-butanol phase separated by centrifugation cocktail 1.0 mL by 1-3 X 107 cells. Protein was determined by the Bio- for 2 min at 755 g and it was carried out to measure the Rad method. 442 C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450

2.14. Western blotting analysis 3. Results and discussion

Total protein (40 mg/lane) was on a 7.5% SDS polyacrylamide gel 3.1. Phytochemical profile under standard conditions and electro-blotted to a NTT membrane in 15% methanol, 25 mM Tris and 192 mM glycine. The membrane The phytochemical analysis of extracts and fractions was made was blocked with nonfat milk in TTBS saline 1 h at 37 Corover- according to Scheme 1. The chemical components in different ex- night at 4 C before incubation with primary antibody (1:500 for tracts and fractions were identified by hyphenated HPLC-DAD-ESI/ iNOS, 1:1000 dilutions for COX-2) in 5% milk in TTBS for 1 h at 37 C. MSn and 1H-13C-NMR. It revealed an ample range of phenolics from After thorough washing, the membrane was incubated with a hydroxycinnamic derivatives, flavonoids, anthocyanins and poly- secondary antibody radish peroxidase (1:25,000) for 1 h at 37 C. mers as proanthocyanidins, the complete phytochemical profile The immunoreactive bands were visualized using an enhanced was published previously (Cespedes et al., 2010a). chemoluminescence system Amersham. 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 p- 2.15. MTT assay hydroxybenzoic acid were determined as majority compounds; from SF8-SF10 gentisic acid, sinapic acid and procyanidin B9 were 80,000 cells/well were plated under the same conditions as cell determined; from SF11-SF15 gallic acid, quercetin, myricetin, del- stimulation. After 12 h incubation, MTT (20 mL, 5 mg/mL in PBS) was phinidin-3-glucoside and cyanidin-3-glucoside were determined; added to each well and incubated for 1.5 h in a CO2 incubator at from SF16-SF20 cyanidin-3-glucoside, 4-hydroxybenzoic acid, 37 C. The medium was removed and DMSO (200 mL) was added. ferulic acid, and cyanidin together with catechin were determined; The plate (96 wells) was incubated for another 15 min before from SF21-SF25 delphinidin-3-glucoside, delphinidin-3,5- measuring the absorbance at 550 nm (Biotek ELx800). diglucoside, delphionidin-3-sambubioside and cyanidin-3- sambubioside were determined; from SF26-SF30 procyanidin B9 and cyanidin-3-sambubioside-5-glucoside were determined; from 2.16. Measurement of NO formation by iNOS activity in cultured SF31-SF37 several trimers and tetramers of procyanidins were LPS-induced RAW 264.7 cells determined; and finally from SF38-SF40 many sugars were detected. Macrophage cells were maintained in DMEM supplemented The phytochemical profile has many similarities or matches with penicillin/streptomycin and 10% FBS at 37 C, 5% CO2 in hu- with various investigations reported by relevant authors (Ruiz midified air. For evaluating the inhibitory activity of test materials et al., 2016; Brauch et al., 2016; Nakamura et al., 2014; Girones- on iNOS, the cells in 10% FBS_/DMEM without were plated in 96- Vilaplana et al., 2014; Ruiz et al., 2010). Additionally, it has been well plates (500,000 cells/well), and then incubated for 24 h. The reported the occurrence of an unusual hydroxyindol derivative in cells were replaced with new media, and then incubated in the the fruits of this Maqui-berry species (Cespedes et al., 2009). Briefly, medium with 1.0 mg/mL of LPS and test samples. After additional from acetone partition (B) fractions F-1 to F-4 were obtained 12 h incubation, the media were removed and analyzed for nitrite throughout flash-column chromatography. The most active frac- accumulation as an indicator of NO production by the Griess re- tions F-3 and F-4 were collected and chromatographed using SiO2 action. Briefly, 50 mL of Griess reagent (0.1% naphthylethylenedi- (G 60, Merck), which afforded 40 subfractions (SF) SF-1 to SF-40 amine and 1% sulfanilamide in 5% H3PO4) were added to 50 mL and finally compounds were purified through Sephadex LH-20 and supernatant from LPS, or sample-treated cells in triplicate. The analyzed through hyphenated techniques, see Scheme 1 (Cespedes plates (96 wells) were incubated for 5 min, and then read at 570 nm et al., 2010a). against a standard curve of sodium nitrite (Biotek ELx800). 3.2. Antioxidant activity

2.17. DCFH-DA assay Samples were evaluated by ABTS, ORAC, FRAP, DPPH radical scavenging, an estimation of lipid peroxidation in rat brain and in 80,000 Cells/well were plated under the same condition as cell liposomes through the inhibition of formation of thiobarbituric stimulation at room temperature. After the 12 h incubation, 200 mL acid reactive species (TBARS), a measure of the superoxide anion plus 100 mM DCFH-DA wer added to the medium per 30 min, the radical with the system hypoxanthine-xanthine oxidase and of the medium was removed and the cells were washed and added with radical hydroxyl by means of the system hydrogen peroxide- þ 200 mL PBS pH 7.5 and the plate was placed in the reader and the peroxidase was made through the formation of radical ABTS . fluorescence recorded every minute for 120 min, using an excita- Antioxidant activities were strongly correlated with the content tion l ¼ 485/20 and emission l ¼ 582/20 (Biotek FLx800). 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 2.18. Statistical analysis compared by activity against butylated hydroxy toluene (BHT), and tocopherol used as positive control. The Fractions SF4-SF6, SF7, Data were analyzed by one-way ANOVA followed by Dunnett's SF8-SF10, SF11-SF15, quercetin, gallic acid, luteolin, and myricetin test for comparisons against control. Values of p < 0.05 (*) and were found to have IC50 values between 4.7 and 2.6 ppm against p < 0.01 (**) were considered statistically significant and the sig- DPPH and between 5.9 and 2.7 ppm against TBARS formation. nificant differences between means were identified by GLM Pro- Consistent with this finding, methanol extract (A) had the cedures. In addition, differences between treatment means were greatest ABTS and FRAP values as percentage of activity of the ex- established with a Student-Newman-Keuls (SNK) test. The I50 tracts. Additionally, it was investigated the response to an acute values for each analysis were calculated by Probit analysis. Com- stress such as ischemia/reperfusion applied to the rat heart in vivo plete statistical analyses were performed using the MicroCal Origin treated with a methanol-extract of fruits of A. chilensis. The meth- 8.0 statistical and graphs PC program. anol extract protects against oxidative stress reducing the C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 443 concentration of the MDA a lipid peroxidation index. A. chilensis (Table 1). protected animals from heart damage as observed by the incidence In addition to samples A-D, partitions F-1 and F-2 of extract B of reperfusion dysrhythmias, and the no-recovery of sinus rhythm. showed considerable activity to inhibit DPPH free radical almost On the other hand, methanol extract was able to prevent these completely, the IC50 values were 109.9, and 79.8 ppm, respectively harmful events in the animal's heart by diminishing lipid oxidation (Table 1). So, the lower IC50 value for partition A (1.7 ppm) than that (Cespedes et al., 2008). of the partitions from A, might be due to a synergistic effect of the components (mainly hydroxycinnamic acid derivatives, anthocya- nidins and flavonoids) within this extract, similar to that reported 3.2.1. DPPH and TBARS determination for components of Vaccinium corymbosum and V. angustifolium The DPPH radical scavenging assay was first used as a screen for fruits (Ehlenfeldt and Prior, 2001; Smith et al., 2000; Lo and antioxidant components within the primary extracts (Cespedes Cheung, 2005), where the acetone and MeOH partitions were the et al., 2008). As shown in Table 1, the methanol and acetone par- most active extracts. titions (A and B, respectively) had the higher inhibitory activity against DPPH radical formation compared to the other partitions 3.2.2. Lipid peroxidation and juice (extract E), with I values of 1.7 and 7.4 ppm, respectively 50 Biomacromolecules (carbohydrates, lipids, proteins, and DNA) (Table 1). For extracts, partitions C and D the I values were 35.7 50 in the presence of ROS suffer oxidative damage, the membrane and 17.9 ppm, respectively. Almost all these samples exhibited a lipids are especially sensitive to this oxidative physiological process concentration-dependence in their DPPH radical scavenging ac- (Diplock et al., 1998). From this point, brain and hearth homoge- tivities, particularly A, which showed the highest activity (100% nates can be used for the investigation of lipid peroxidation as an inhibition) at a concentration of 8.6 ppm. This action was greater assessment of oxidative stress. The capacity for plant extracts to than that of a-tocopherol (one of the positive controls), which at prevent lipid peroxidation was assayed using malondialdehyde 31.6 ppm caused only 53.8% quenching and very similar to ferulic formation as an index of oxidative breakdown of membrane lipids, and p-coumaric acids with IC values of 5.1 and 7.8 ppm, respec- 50 following incubation of rat brain cortical and hearth homogenates tively (data were not shown here). Partition B was then loaded onto þ with the oxidant chemical species Fe2 . Ferrous ion both stimulates a silica-gel open chromatography column, from which four frac- lipid peroxidation and supports decomposition of lipids peroxides tions were collected (F1-F4). Of these, fractions F-3 and F-4 were once formed, generating highly reactive intermediates such as the most active, with an IC of 7.1 and 4.9 ppm, respectively 50 hydroxyl radicals, perferryl and ferryl species (Ko et al., 1998). Fraction F4 was the most effective one and fraction F3 was the Table 1 least effective one, but none were as effective as extracts A or B, Amounts of samples from fruits of A. chilensis extracts, fractions and compounds quercetin or M3 in inhibiting lipid peroxidation. Table 1 shows the needed to inhibit oxidative damage by 50%.a tabulated data that provide IC50 values; extract A clearly showing Sample DPPHb TBARSc TBARSd the greatest activity. Thus extract A reduced lipid peroxidation in a ± ± ± dose-dependent manner, and proved to be an excellent antioxi- A 1.7 0.3b 2.1 0.1b 4.9 0.04a fl B 7.4 ± 1.4b 3.9 ± 0.2b 8.5 ± 0.13b dant, re ected by its low IC50 value when analyzed by both TBARS C 35.7 ± 3.6a 20.1 ± 0.8a 25.3 ± 2.35c and DPPH. D 17.9 ± 9.1a 11.2 ± 3.2c 29.6 ± 2.91c When the relative contribution of each fraction (F2, F3 and F4) E 4.7 ± 0.7b 5.9 ± 0.9b 12.0 ± 1.22b to the total antioxidant activity of partition B was evaluated using F-1 109.9 ± 9.2c 131.2 ± 12.5d 223.2 ± 10.33d DPPH and TBARS, all fractions except fraction F1 showed some F-2 79.8 ± 5.5d 90.5 ± 9.9d 189.8 ± 12.22d F-3 7.1 ± 1.4b 9.9 ± 2.7c 10.4 ± 1.46b protective effect, with IC50 values between 4.9 and 90.5 ppm F-4 4.9 ± 0.5b 6.5 ± 1.3c 8.9 ± 0.59b (Table 1). Fractions F3 and F4 were the most active, with IC50 values SF1-SF3 n.d. n.d. n.d. of 7.1 and 4.9, and 9.9 and 6.5 ppm, for DPPH and TBARS, respec- ± ± ± SF4-SF6 3.2 0.4b 10.9 0.8 12.4 1.56b tively (Table 1). Fraction F4 was substantially more active than that SF7 17.9 ± 2.1a 40.4 ± 3.2c 44.4 ± 4.58e SF8-SF10 20.6 ± 2.5e 35.7 ± 5.9c 20.7 ± 2.05c of other fractions. It is noteworthy that the value for fraction F4 is SF11-SF15 8.9 ± 9.2a 4.5 ± 0.2b 5.5 ± 0.09a very low compared with values for flavonoids and anthocyanins in SF16-SF20 6.1 ± 0.9c 9.6 ± 1.1c 8.2 ± 0.19b general, as well as for myricetin or quercetin (Makris and Rossiter, SF21-SF25 11.2 ± 1.8c 31.2 ± 2.9b 10.1 ± 1.36b 2001). SF26-SF30 6.1 ± 0.9b 9.6 ± 1.1c 4.2 ± 0.03a It has been reported that the antioxidant activity of many SF31-SF37 >250 >250 n.d. SF38-SF40 n.d. n.d. n.d. compounds of botanical origin is proportional to the phenolic Rutin 9.7 (15.9 ± 2.8a) 12.09 (19.8 ± 3.8b) 9.3 (15.3 ± 1.55b) content (Rice-Evans et al.,1997), suggesting a causative relationship Catechin 7.6 (26.4 ± 2.9e) 8.68 (29.9 ± 0.9c) 6.6 (22.8 ± 2.14c) between total phenolic content and antioxidant activity (Veglioglu ± ± ± Quercetin 3.01(8.9 1.9a) 1.89 (5.6 1.2c) 1.2 (3.6 0.01a) et al., 1998). Halliwell and Gutteridge (1990) has defined antioxi- Luteolin 19.78 (69.1 ± 5.7d) 27.25 (95.2 ± 7.1d) 9.7 (33.9 ± 3.78c) Myricetin 4.1 (12.9 ± 2.8a) 2.51 (7.9 ± 0.9c) 2.8 (8.8 ± 1.25b) dants as substances that, when present at low concentrations Gallic acid 0.54 (3.2 ± 0.9b) 1.9 (11.2 ± 3.2c) 1.7 (10.2 ± 1.88b) compared with an oxidizable compound (e.g. DNA, protein, lipid, or M1e 19.6 ± 1.91a 28.9 ± 1.1c 32.3 ± 3.12c carbohydrate), delay or prevent oxidative damage due to the M2 8.8 ± 0.08b 12.7 ± 0.7c 11.5 ± 0.99b presence of ROS. These ROS can undergo a redox reaction with M3 5.6 ± 0.06b 3.8 ± 0.09c 4.3 ± 0.03a phenolics, such that oxidant activity is inhibited in a concentration- a For extracts, fractions and mixtures values expressed as mg/mL (ppm), for dependent manner. ± ¼ compounds between parenthesis (values expressed as [mM], Mean SD, n 3). At low concentrations of phenolics the main mechanism is the Different letters show significant differences at (P < 0.05), using Duncan's multiple- range test. breaking of chain reactions (Rice-Evans, 2000). Thus, total phenolic b IC50 for inhibition of diphenyl picryl hydrazyl radical formation. content is measured in each one of the extracts, partitions and c IC50 for inhibition of peroxidation of lipids, estimated as thiobarbituric acid fractions (see Cespedes et al., 2010a,b,c). Extract A, which had the reactive substances for rat brain procedures. greatest DPPH and TBARS activities reduced MDA generation, had a d IC for inhibition of peroxidation of lipids, estimated as thiobarbituric acid 50 significantly greater phenolic content than other extracts. The reactive substances for liposomes procedures. e e fi M1 ¼ quercetin þ rutin, M2 ¼ quercetin þ catechin, M3 ¼ quercetin þ gallic phenolic content of fractions F1 F4 showed a small but signi cant acid. See Methods for details. increase in phenolic content for fraction F4 over fraction F3, which 444 C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 had a similar content similar to that of fraction F2; fraction F1 had with substantial phenolic content, were the fractions showing ac- significantly lower phenolic content. These findings correlate well tivity in the FRAP assay (Table 2). In agreement with the ORAC with fraction F4 having one of the greatest activities against DPPH assay, it was A, B, and F4 that showed the greatest values (15,210.9, and TBARS. 9205.4, and 11,000.9 [mmol Cat Equiv/g extract], respectively). Those data correlate well with the ORAC values, (Table 2), and with 3.2.3. ORAC and FRAP SOD and ABTS values (Table 3). These effects match very well with The ORAC and FRAP values for A. chilensis extracts are given in other studies for flavonoids and phenolics from other sources and Table 2. The capacity for a compound to scavenge peroxyl radicals plants (Firuzi et al., 2005; Cao et al., 1997; Fukumoto and Mazza, generated by spontaneous decomposition of AAPH was estimated 2000; Villano~ et al., 2005). in terms of Trolox equivalents, using the ORAC assay (Cao, & Prior, 1999). A wide variety of different phytochemicals from edible 3.2.4. SOD and ABTS plants, purified or as an extract or fraction, have been found active Antioxidant activities bore a direct relationship with the in this assay, including alkaloids, coumarins, flavonoids, phenyl- phenolic content of the extracts and fractions. As with DPPH, propanoids, terpenoids and phenolic acids (Lopez-Alarcon and TBARS, ORAC and FRAP activities, samples A, B, F4, SF11-SF15, SF16- Denicola, 2013). Among the extracts assayed here, the values SF20, M3, and quercetin were the most active in SOD and ABTS both were found to be in the range of 3900e29,600 mmol TE/g extract for the assays. Among the fractions, F4 was the most active in both ORAC and from 990 to 15,200 mmol Cat E/g extract for the FRAP assays. These facts can be correlated correctly between SOD and assay, respectively (Table 2). total polyphenolic composition of all extracts and partitions and The same as with the earlier measurements, extract A had the between ABTS and total phenolic composition of fractions. highest activity in both trials, with values of 29,554.5 [mmol TE/g of The phenolic characterization suggests that the different extract] and 15,210.9 [mmol Cat E/g of extract] for ORAC and FRAP phytochemical antioxidant components in the active extract and assays, respectively. In similar form extract B show a very good fractions, mainly anthocyanins, cinnamic derivatives and flavo- potency with values of 28,100.1 [mmol TE/g] and 9205.4 [mmol Cat noids, may be involved in the antioxidant mechanism of action and E/g] for ORAC and FRAP assays, respectively. The other extracts (C the antioxidant methods gives a direct measure of hydrophilic and D) showed values of intermediate potency, 3898.0 and 19,700.9 chain-breaking antioxidant capacity against peroxyl radical of our [mmol TE/g extract] in the ORAC assay, and 6876.5 and 4726.9 [mmol samples (Cespedes et al., 2010a). Thus, the highest ORAC and SOD Cat E/g extract] for FRAP assay, respectively (Table 2). Among the numbers of the extracts and fractions show an excellent antioxi- fractions, fraction 4 was significantly more than twice as active as dant potential (Tables 2 and 3), for instance, the extracts A, B and any other fraction (Table 2). F4. In addition, the ORAC numbers of fractions showed a very high The FRAP assay showed greater variability (Table 2). Several correlation with content (R > 0.95) (data not shown), extracts had very low values and only extracts A, B and fractions F3 the same level of correlation was observed between the FRAP and F4 showed substantial activity. Again, A was significantly more numbers and phenolic composition of the extracts and fractions. In active than any other samples (Table 2). Fractions F3 and F4, those 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 ORAC Concentration FRAP [mg/mL] [mg/mL]

A 10.0 29,554.5 ± 95.2a 25.0 15,210.9 ± 24.4d B 10.0 28,100.1 ± 85.8a 25.0 9205.4 ± 10.5a C 10.0 3890.0 ± 9.7d 25.0 6876.5 ± 9.9a D 10.0 19,700.9 ± 90.1a 25.0 4726.9 ± 8.4b E 10.0 22,500.8 ± 86.2a 25.0 8771.1 ± 12.9a F-1 1.0 N.D. 2.5 N.D. F-2 1.0 10,223.9 ± 23.2b 2.5 995.5 ± 1.2e F-3 1.0 15,699.9 ± 98.8b 2.5 1159.9 ± 9.9e F-4 1.0 25,911.5 ± 115.9c 2.5 11,000.9 ± 22.1f SF1-SF3 1.0 n.d. 2.5 n.d. SF4-SF6 1.0 19,066.9 ± 79.3c 2.5 11,233.4 ± 19.9f SF7 1.0 16,001.6 ± 71.5c 2.5 10,987.2 ± 18.7f SF8-SF10 1.0 20,446.6 ± 91.5c 2.5 12,448.3 ± 15.3f SF11-SF15 1.0 21,203.3 ± 105.8c 2.5 14,588.2 ± 16.8f SF16-SF20 1.0 12,536.8 ± 34.8b 2.5 8955.5 ± 5.9a SF21-SF25 1.0 8644.3 ± 7.4d 2.5 6894.2 ± 4.6a SF26-SF30 1.0 5699.2 ± 6.1d 2.5 3456.1 ± 2.4a SF31-SF37 1.0 2678.9 ± 4.6d 2.5 1988.4 ± 1.1e SF38-SF40 1.0 n.d. 2.5 n.d. Rutin 1.0 4533.3 ± 8.6d 2.5 5939.2 ± 6.3a Quercetin 1.0 13,588.8 ± 13.57b 2.5 9966.2 ± 7.2a Luteolin 1.0 14,885.2 ± 9.2b 2.5 7589.2 ± 4.9a Myricetin 1.0 14,435.4 ± 8.1b 2.5 8846.9 ± 5.8a Gallic acid 1.0 8992.2 ± 10.3d 2.5 6498.5 ± 4.4a M1 1.0 4695.2 ± 5.1d 2.5 3211.6 ± 2.2b M2 1.0 12,388.4 ± 11.8b 2.5 6588.2 ± 4.9a M3 1.0 16,899.5 ± 19.3b 2.5 12,899.8 ± 10.7f

a For detail see Scheme 1. b 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). C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 445

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

þ sample SOD inhibition % ABTS inhibition %

A 95.7 ± 0.9a 99.8 ± 0.7a B 93.8 ± 0.5a 85.9 ± 0.6a C 12.2 ± 0.2b 15.8 ± 0.3b D 5.9 ± 0.1b 8.1 ± 0.2c E n.d. n.d. F-1 15.9 ± 2.8c 17.5 ± 0.1d F-2 58.3 ± 2.9d 60.1 ± 0.2e F-3 80.2 ± 1.5a 82.5 ± 0.4a F-4 89.7 ± 1.3a 92.4 ± 0.1a SF1-SF3 0.5 ± 0.1e 0.29 ± 0.01e SF4-SF6 52.8 ± 2.3d 48.6 ± 0.9f SF7 62.4 ± 2.9f 60.8 ± 1.2e SF8-SF10 84.2 ± 2.4a 90.8 ± 0.8a SF11-SF15 91.8 ± 1.6a 92.5 ± 0.3a SF16-SF20 88.6 ± 1.9a 89.2 ± 0.5a SF21-SF25 71.2 ± 1.1 g 69.9 ± 1.1 g SF26-SF30 60.1 ± 0.9 g 66.8 ± 1.2 g SF31-SF37 35.2 ± 0.4 h 40.6 ± 0.7f SF38-SF40 4.6 ± 0.5b 8.0 ± 0.2c Rutin 12.0 ± 0.1c 25.8 ± 0.8 h Fig. 1. Viability of macrophages RAW 264.7 treated with LPS (1 mg/mL) with and Catechin 15.9 ± 0.5c 17.9 ± 1.2 h without the assayed samples (Extracts, fractions and compounds). CO ¼ control, Quercetin 92.1 ± 0.7a 90.7 ± 0.8a LPS ¼ lipopolysaccharide. Data are expressed as the mean ± S.E. at least of three in- Luteolin 88.1 ± 0.6a 94.8 ± 0.4a dependent experiments. The viability of cells without LPS was 100%. P < 0.05 represent Myricetin 85.0 ± 0.8a 92.6 ± 0.6a a significant difference compared with values obtained with cells without LPS. A: Ext C, Gallic acid 77.5 ± 0.9 g 80.4 ± 0.8a 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, M1 35.1 ± 0.8 h 44.6 ± 1.2f J: SF16-SF20, K: SF21-SF25, L: SF26-SF30, M: SF31-SF37. For detail see Scheme 1. M2 39.8 ± 0.3 h 50.7 ± 1.7i M3 97.9 ± 0.4a 95.5 ± 0.2a ± ¼ fi For detail see Scheme 1. Mean SD, n 3. Different letters show signi cant dif- among others, increasing NO production. In this study, NO inhibi- ferences at (P < 0.05), using Duncan's multiple-range test. tory 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 (R > 0.98) between FRAP numbers and its polyphenolic content fruits of A. chilensis exhibited excellent inhibitory activity against (data not shown). ROS and a strong anti-inflammatory activity against TPA-induced inflammation in mouse ear edema model (Cespedes et al., 2010a, 3.3. Cytotoxicity 2010b). To determine further effects of those extracts and phenolic To evaluate whether the inhibition of NO production was compounds on NO production, different concentrations of test possibly caused by the cytotoxicity effect of test extracts or com- samples were incubated with LPS-activated RAW 264.7 cell mac- pounds, the viability of test cells was determined by the MTT assay rophages. As shown in Fig. 2, the nitrite level produced in cultured (Kubo et al., 2007). Extracts A e E, subfractions, compounds and supernatant of RAW 264.7 cells was markedly elevated for extracts mixtures show no significant cytotoxicity to RAW 264.7 cells at test C, D, E, and mix M1 and M2 these samples do not show a significant used concentrations (Fig. 1). Thus, the inhibition of NO production inhibition at 100 mg/mL, after 24 h of treatment with LPS. However, in LPS-stimulated RAW 264.7 cells by extracts, and subfractions of extract A and B showed an inhibition >50% [4.0 and 3.0 mM of ni- fruits from A. chilensis was not due to cytotoxicity. trite, respectively] and F4 was the strongest inhibitor with >90% of activity [2.5 mM of nitrite concentration] (Fig. 2). Thus, sample A 3.4. NO production and B significantly inhibited LPS-induced NO production in a dose- dependent manner (Fig. 2). Previous studies showed that A. chilensis extract inhibits lipid In relation to the fractions, F4 was the strongest inhibitor with peroxidation in vitro systems and decreases oxidation of LDL and an activity greater than 95% at 50.0 mg/mL, agreeing with the in- increases the ratio of HDL-to-LDL, thus decreasing the risk of heart hibition of iNOS enzyme by this fraction, other fraction with a disease (Miranda-Rottmann et al., 2002; Cespedes et al., 2008). significant activity was F3 with a 60% of inhibition of NO produc- Beneficial effects of fruits of A. chilensis extract are most likely due tion, but it did not show the same activity against iNOS expression to polyphenols, which are efficient free radical and singlet oxygen (Fig. 3). scavengers (Yan et al., 2002). Because heart ischemia-reperfusion The most active compounds inhibiting the NO production were causes free radical production and A. chilensis polyphenols are myricetin and quercetin (Fig. 2). Quantifying the production of ni- effective free radical scavengers, this study was designed to test the trite is a technique used to determine the indirect production of NO hypothesis that Maqui-berry will block free radical formation, thus in macrophages, which are known to be capable of reaching pro- preventing injury. Indeed, Maqui-berry and their polyphenolic duce 4 106 NO molecules per cell from the iNOS enzyme (Dedon components, significantly reduced NO injury after ischemia- and Tannenbaumb, 2004). This determination was commonly reperfusion (Cespedes et al., 2008), which afford protection made using the Griess test. In conducting the trial with the pure against oxidative stress and the risk of increasing septic compli- compounds was observed that the compounds quercetin and cations. This study shows as Maqui-berry extracts can help in the myricetin were which had a higher inhibition in the production of control of these health risks. nitrites used to the maximum concentrations (25 and 50 mM, i-NOS is expressed in macrophages by stimulation with LPS respectively) and that the other compounds showed no effect 446 C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450

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

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 Fig. 2. Nitric oxide production in macrophage RAW 264.7 cells measured with Griess by cytokines such as TNF-a and IL-1b that play an essential role in reagents: Cells stimulated for 24 h (1 mg/mL) only, or LPS, extracts, fractions, com- fl pounds and mixtures from fruits of Aristotelia chilensis. A: total ethanol extract (A), B: many in ammatory lesions (De Nardin, 2001). NOS catalyzes NO Acetone partition (B), F3: F-3 fraction from acetone partition, F4:F-4 fraction from synthesis. There are three isoforms including a neuronal (nNOS), Acetone partition, C: SF7, D:SF8-SF10, E:SF11-SF15, F:SF16-SF20, Myr: myricetin, and endothelial (eNOS) and an endotoxin or cytokine-inducible Q:quercetin, L:luteolin, Cat:catechin, M1:quercetin þ rutin, M2:quercetin þ catechin, (iNOS) form (Rosen et al., 2002). In spite of iNOS is usually not M3:quercetin þ gallic acid. At the end of incubation, 100 mL of the medium was detectable in healthy tissues, it is expressed after immunological removed for measuring nitrite production. Control values were obtained in the absence of LPS. Data were derived from three independent experiments and expressed challenge or injury, the expression of iNOS and its enzymatic ac- as means ± S.E. P < 0.05. For detail see Scheme 1. tivity 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 MacMicking et al., 1997). enzyme (Fig. 3). In this study, the effect of methanol (A), acetone (B), ethyl ace- To check the effect of the compounds quercetin and myricetin tate (C), residue EtOH/H2O(D), water (100%) (E) extracts, sub- an experiment was conducted at different concentrations (10, 25 fractions, compounds and mixtures on iNOS and COX-2 expression C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 447 was also investigated. As shown in Fig. 3, unstimulated RAW smaller than in the expression of iNOS, myricetin did not present a 264.7 cells (control) showed barely detectable iNOS. In contrast significant effect on the expression of COX-2 and the lowest con- 12 h incubation with LPS resulted in a large increase in iNOS centration 10 mM there was a slight stimulation. expression. However, at 50 mM quercetin and myricetin completely Moreover, according to a test conducted by Kim et al. (2004) suppressed the iNOS expression, as well as extracts A and B, the F3- these flavonoids (of flavonols type) comply with the structural F4 fractions, subfractions SF7 to SF16-SF20 and M1 -M3 mixtures at characteristics necessary to inhibit the production of nitrites, which 50 mg/mL (ppm). The proposed mechanism associated with the are: a ring A, C-5, 7 dihydroxy replaced and a ring B C-20,30 dihy- reduction of NO production are scavenging of NO, suppression of droxy replaced in the case of quercetin and myricetin, while a ring- iNOS enzyme activity, inhibition of iNOS gene expression and/or 2'-hydroxy B C 3'-metoxy substituted for whose replacement down-regulation of iNOS enzyme by modulation of enzyme activ- metoxylated form provides lower activity as shown in conducted ities related to signal transduction, etc. (Park et al., 2000; Sheu et al., tests, as also it has been reported by Orhan group (Nabavi et al., 2001; Chiang et al., 2005; Kim et al., 1999; Paul et al., 1995). In the 2015; Surh et al., 2001). study, the samples inhibited NO production in macrophages via one or more of these mechanisms. Cyclooxygenase (COX) is the rate-limiting enzyme in PG syn- 3.6. Oxidative stress thesis and exist as two isoforms: constitutive (COX-1) and inducible (COX-2) (Sheu et al., 2001). Thus, like iNOS, COX-2 is an important Fig. 4 shows that the compound quercetin presented protective enzyme that mediates inflammatory processes. activity against oxidative stress (DCFH), which led to conclude that Multiple lines of compelling evidence support COX-2 playing a the inhibitory activity of the expression of iNOS and in the pro- role in the development of tumors (MacMicking et al., 1997). Thus, duction of nitrites was favored by the antioxidant activity. aberrant or excessive expression of iNOS and COX-2 is implicated in According to the background oxidative stress is involved at fl inflammatory disorders and the pathogenesis of cancer. Quercetin different levels in in ammatory response by stimulating the ac- and myricetin, acetone extract (B), Subfractions SF7, SF8-SF10, tivity of NF- and production of different mediators such as IL-1, IL-6, SF11-SF15, SF16-SF20 and Mix M3 not only showed strong inhib- TNF-a, IFN-g in the cellular signaling, thus it is possible that quer- itory activity on iNOS expression, but also significantly inhibited cetin through its antioxidant activity has decreased the studied fl the COX-2 expression in LPS-stimulated macrophages. Fig. 3 show factors which are involved in the in ammatory response (Nathan, that quercetin, myricetin and extract B suppressed the LPS-induced 2002; Nabavi et al., 2015). With regard to the present myricetin COX-2 expression in a dose dependent manner. despite a slight prooxidant activity also inhibited the expression of Fig. 3 shows that extract B suppressed the LPS-induced COX-2 iNOS and COX-2 as well as in the production of nitrites, which expression in a dose-dependent manner. Approximately, 40 and suggests that the mechanism of action is separated from the pro- 90% reduction was observed at 50 and 100 mg/mL, respectively, as duction of reactive species. determined by densitometry analysis. At concentrations up to In the case of the other compounds these did not show anti- fl 100 mg/mL extracts A and B can completely inhibit the expression of oxidant activity at level showed by avonoids, including phenolics COX-2 in LPS-stimulated cells (data not show). acids mixtures, showed an apparent increase of oxidative stress, On the contrary, these extracts inhibit around 40% and like the myricetin. The result obtained with the phenolic acids completely (100%) the expression of iNOS at 50 and 100 mg/mL, disagrees with the antioxidant activities reported in the literature e respectively. Thus, one of the mechanisms of extract B inhibition of (Aldini et al., 2006). Tables 1 3 shows the antioxidant activity of NO production in LPS-stimulated macrophages is mediated by the the samples isolated from A. chilensis. fl down-regulation of iNOS and COX-2 expressions. Further studies It is noted that the avonoids mixtures showed a larger reduc- on intracellular signaling cascades leading to COX-2 and iNOS tion DPPH radical activity with a 87.25%, followed by phenolic acids 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 myr- icetin an experiment was conducted to dose response (10, 25 and 50 mM respectively). Even so the flavonoid quercetin presented 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 major inhibitory activity of iNOS expression of the enzyme with 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: respect to the myricetin. In the case of the COX-2 enzyme was SF8-SF10, 10: SF11-SF15, 11: SF16-SF20, 12: myricetin, 13: quercetin, 14:M1, 15: M2, 16: observed with a similar behavior quercetin although the effect was M3. For detail see Scheme 1. 448 C.L. Cespedes et al. / Food and Chemical Toxicology 108 (2017) 438e450 activity with a 66.2%. The fractions that contains anthocyanins caused by the presence of nitrogen or oxygen reactive species, showed less reduction DPPH radical activity with a 58.13% and including a myriad of different free radicals. Rat brain homoge- 22.48%, respectively. Anthocyanidins did not show activity to this nates, rich in lipids such as polyunsaturated fatty acids can undergo concentration. Similar activity was observed in the trial against peroxidation. The research showed that the extract A and acetone TBARS where quercetin and myricetin showed the greatest inhi- partition B of fruits from A. chilensis, several fractions and sub- bition on lipid peroxidation (93.28% and 87.53% respectively), fol- fractions of that extract, contain antioxidants that can inhibit lipid lowed by phenolic acids (34.29%) and anthocyanins mixtures peroxidation, SOD, and that they have a high phenolic content (28.52%). (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 4. Concluding remarks values are similar to those of different known fruits and vegetables as prunes, raisins, blueberries, spinach and Broccoli (Aldini et al., In the particular case of flavonoids, it is known that these 2006; Balasundram et al., 2006; Cao et al., 1997; Cheong et al., compounds have a strong antioxidant activity which was reflected 2004; Ehlenfeldt and Prior, 2001; Firuzi et al., 2005; Fukumoto in the essays against DPPH, but not in the essay of antioxidant ac- and Mazza, 2000; Hou et al., 2005; Kim et al., 1999; 2004; Ko tivity in the RAW 264.7 macrophages, this could be due to the et al., 1998; Lopez-Alarcon and Denicola, 2013; Matsuda et al., hydrophilic properties upon the hydroxyl groups of both the aro- 2003; Nabavi et al., 2015; Seeram, 2008; Smith et al., 2000; Villano~ matic rings as sugars (in the case of anthocyanins) and do not allow et al., 2005). it to cross the lipid membranes and exercise their antioxidant effect With the aim of to elucidate the sites and mechanism of action, for the same reasons it may not have shown effect in inhibiting the we are on-going studies doing in vivo test of anti-inflammatory expression of COX enzymes 2 and iNOS. With these data high- activities, those results will be published in a next paper. lights the variability of the results of different trials designed to measure the same effect. Conflict of interest Besides, the compounds quercetin and myricetin showed anti- fl oxidant activity in DPPH test and TBARS. Quercetin is a avonoid The authors declare that there are no conflicts of interest. with antioxidant activity reported by its replacement ortho-dihy- droxy in the ring B. The inhibiting the expression of COX2 enzymes Acknowledgements and iNOS by quercetin and myricetin, as well as the production of nitrite is not new, since it is known that the quercetin interferes This work was supported in part by internal grant from Direc- with cascades of signals in which, stimulated with LPS is the RAW cion de Investigacion, Universidad del Bio Bio, Chillan, Chile (Grant 264.7 cell line, blocking the main (Xagorari et al., 2002), pro- # 091909-1/R); by grant FONDECYT-2920018, and in part by grants inflammatory molecules such as TNF-a. However, it does not take from UC-MEXUS-CONACYT (#2013-02) and UC-CONICYT- reports of the antioxidant activity of these compounds in RAW Chile(#2013-02). The authors thanks to Roberto Rodriguez (Facul- 264.7 macrophages using DCFH. tad de Ciencias Naturales y Oceanograficas, Universidad de Con- According, to these results it is suggested that not all com- cepcion, Concepcion, Chile) and Prof David S. Seigler, curator pounds with antioxidant activity in the chemical models may affect Herbarium of University of Illinois at Urbana-Champaign, USA, for activity, and that the antioxidant activity can be a factor that pro- botanical identification of the plant. We thank Ma. Teresa Ramirez- motes the inhibition of the expression of certain enzymes. Apan, and Antonio Nieto for technical assistance: Chemistry Insti- The extracts A and B of fruits from A. chilensis and some of their tute, Ana Ma. Garcia-Bores: UBIPRO FES-Iztacala, UNAM, Mexico fractions and subfractions exhibited substantial potency in scav- D.F., Mexico. Anne Murray (ESPM, UC, Berkeley); M.D. thanks enging DPPH-radical and inhibiting lipid peroxidation. Two of the CONACyT-Mexico for a doctoral fellowship and to TIES-ENLACES four fractions isolated from B, the F3 and F4 showed potency in USAID Program for a research fellowship. CLC and IK acknowl- scavenging against DPPH-radicals, as well as a strong inhibitory edge to Seed Funds Program of Conicyt-Chile and UC-Berkeley effect against lipid peroxidation, particularly fraction F4. The anti- “2013 UC BerkeleyeChile Seed Grants”, grant (# 2013-02): A New oxidant activities, total phenolic content (Table 4) and ORAC and Connection: Potential Cancer Treatment Agents. 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 molec- Transparency document ular weight, with relative high polarity. Phytochemical analysis of these extract, partitions and fractions are in progress, and is ex- Transparency document related to this article can be found pected to identify chemical structures of bioactive components that online at http://dx.doi.org/10.1016/j.fct.2016.12.036. may have a future role in human health maintenance. Many cellular components are sensitive to oxidative damage, References

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