Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN: 0717-7917 [email protected] Universidad de Santiago de Chile

CÉSPEDES, Carlos L.; ALARCON, Julio; AVILA, Jose G.; NIETO, Antonio Anti-inflammatory Activity of chilensis Mol. (Stuntz) () Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 9, núm. 2, marzo, 2010, pp. 127-135 Universidad de Santiago de Chile Santiago, Chile

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Artículo Original | Original Article Anti-inflammatory Activity of Mol. (Stuntz) (Elaeocarpaceae). [Actividad anti-inflamatoria de Aristotelia chilensis Mol. (Stuntz) (Elaeocarpaceae).]

Carlos L CÉSPEDES*1, Julio ALARCON 1, Jose G AVILA2 , Antonio NIETO3 1Plant Biochemistry and Phytochemical Ecology Laboratory, Department of Basic Sciences, University of Bío-Bío, Chillán, Chile. 2Phytochemical Laboratory, UBIPRO, FES-Iztacala, UNAM, 3Laboratorio de Pruebas Biológicas, Instituto de Química, UNAM.

Abstract

Context: “Chilean Black-” Aristotelia chilensis is a wild that growth in southern Chile. This fruit possess a strong antioxidant activity and is commonly used in foods and beverages in Chile. Objective: The anti-inflammatory activity of the extracts, fractions and subfractions of this fruit are investigated here for the first time. Materials and methods: Extracts, fractions and subfractions were analyzed for their content in total phenolics and the effects in the TPA-induced inflammation in ear of the mouse ear edema induced by single doses of TPA were investigated. In addition, the antioxidant activity was investigated against DPPH, Crocin and TBARS. Results: The results showed that extract B, fraction F-4, and ovatifolin, quercetin, myricetin, luteolin and diosmetin used as pattern compounds were the most active samples together with those subfractions rich in phenolic compounds. Thus, SF11- SF15, SF16-SF20, and SF21-SF25 showed are the best subfractions inhibitors in similar form to indomethacin a known selective COX inhibitor that have an EI50 of 0.11 mg/ear. Results demonstrated that these samples strongly inhibited the induced inflammation in ear of the mouse edema in TPA inflammation model, with EC50 values ranging from 0.3 to 11.8 μg/mL. Discussion and conclusion: These findings demonstrate that the and their constituents of A. chilensis have excellent anti-inflammatory activities and thus have great potential as a source for natural health products. Additionally, these findings showed that the flavonoids, phenolic acids and present in this fruit may be responsible of the antioxidant activity observed.

Keywords: Aristotelia chilensis, anti-inflammatory activity, antioxidants, DPPH, crocin, TBARS.

Resumen

Contexto: “Chilean Blackberry” Aristotelia chilensis es un fruto silvestre que crece en el sur de Chile. Este fruto posee una fuerte actividad antioxidante y comúnmente es usado en alimentos y bebidas en Chile. Objetivo: Se investigo la actividad anti-inflamatoria de los extractos, fracciones y subfracciones de este fruto y son informados aquí por primera vez. Materiales y métodos: Los extractos, fracciones y subfracciones fueron analizados por su contenido total de fenoles y se investigo el efecto sobre la inflamación en oreja de rata a través de la inducción con TPA en dosis sencillas. Además se investigo la actividad antioxidante frente a DPPH, Crocina y TBARS. Resultados: Los resultados muestran que el extracto B, la fracción F-4, y ovatifolina, quercetina, myricetina, luteolina y diosmetina, que se usaron como muestras patrones, fueron las mas activas junto con aquellas subfracciones ricas en compuestos fenólicos. Asi,

SF11-SF15, SF16-SF20, y SF21-SF25 mostraron ser las mejores subfracciones inhibitorias en una forma similar a indometacina un conocido inhibidor selectivo de COX que tiene un EI50 de 0.11 mg/oreja. Los resultados demuestran que estas muestras inhiben fuertemente la inflamación inducida en el modelo del edema en oreja de rata, con valores de EC50 entre 0.3 a 11.8 μg/mL. Discusión y conclusión: Estos hallazgos demuestran que los frutos y sus constituyentes de A. chilensis poseen una excelente actividad anti-inflamatoria, y así tienen un gran potencial como una fuente de productos naturales saludables. Adicionalmente, estos hallazgos muestran que los flavonoides, ácidos fenólicos y antocianinas presentes en este fruto podrían ser los responsables de la actividad antioxidante observada.

Palabras Clave: Aristotelia chilensis, anti-inflammatory activity, antioxidants, DPPH, crocin, TBARS.

Recibido | Received: November 1, 2009 Aceptado en Versión Corregida | Accepted in Corrected Version: January 4, 2010 Publicado en Línea | Published Online March 25, 2010 Declaración de intereses | Declaration of interests: authors have no competing interests. Financiación | Funding: This work was supported in part by internal grant from Department of Basic Sciences, University of Bio-Bio, Chillan, Chile. This article must be cited as: Carlos L. Céspedes, Julio Alarcon, Jose G. Avila Acevedo, A. Nieto. 2010. Anti-inflammatory Activity of Aristotelia chilensis Mol. (Stuntz) (Elaeocarpaceae). Bol Latinoam Caribe Med Aromat 9(2):127 – 135. {EPub 25, March 2010}. *Contactos | Contacts: E-mail address: [email protected], [email protected]; Phone: +56-42-253049, Fax: +-56-42-253046

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INTRODUCTION high consume during these months in Central and South Chile and western of . A growing body of literature points to the Previously, we have reported the alkaloid importance of natural antioxidants from many composition of the of A. chilensis , which may be used to reduce cellular (Cespedes et al., 1990; Cespedes et al., 1993; oxidative damage, not only in foods, but also in Cespedes, 1996). The botanical characteristics the human body (Prior et al., 2003; Haliwell and were reported previously (Cespedes et al., 1996; Aruoma, 1991). This may provide protection 2008; 2010; Silva et al., 1997). against chronic diseases, including cancer and This plant has enjoyed popularity as an neurodegenerative diseases, inflammation and ethno-medicine for many years, used cardiovascular disease (Prior et al., 2005). particularly as an anti-inflammatory agent, Adverse conditions within the environment, kidneys pains, stomach ulcers; diverse digestive such as smog and U.V.-radiation, in addition to ailments (tumors and ulcers), fever and diets rich in saturated fatty acids, increase cicatrization injuries (Bhakuni et al., 1976), and oxidative damage in the body. Given this the have traditionally been consumed as constant exposure to oxidants, antioxidants may treatment for diarrhea and dysentery and the be necessary to counteract chronic oxidative Araucanian people prepare a liquor with an effects, thereby improving the quality of life ethanolic macerated solution that is used in (Roberts et al., 2003; Cespedes et al., 2010). religious ritual know as “machitun” or The increasing interest in the measurement of “nguillatun” and as daily beverages (Muñoz- the antioxidant activity of different plant Pizarro, 1966). samples is derived from the overwhelming Up-to-date some studies reports that the juice evidence of the importance of Reactive Oxygen (an aqueous extract) from fruits of A. chilensis Species (ROS), including superoxide (O ˙ˉ), 2 has a good antioxidant activity against FRAP peroxyl (ROO), alkoxyl (RO), hydroxyl analysis but not reduce endogenous oxidative (OH˙ˉ), and nitric oxide (NO) radicals in aging DNA damage in human colon cells (Pool-Zobel and chronic disease (Fernandes et al., 2004). et al., 1999), an effective capacity to inhibit the Several methods have been developed to cooper-induced LDL oxidation in vitro and the measure the antioxidant activity in biological induction of intracellular oxidative stress samples, including the oxygen radical absorption induced by hydrogen peroxide in human capacity (ORAC), ferric reducing antioxidant endothelial cells culture (Miranda-Rottmann et power (FRAP), 2,2-diphenyl-1-picryl-hydrazil al., 2002), other study report only the partial (DPPH) radical scavenging and inhibition of composition of anthocyanidins constituents of formation of thiobarbituric acid reactive species the juice (Escribano-Bailon et al., 2006), and (TBARS) (Taruscio et al., 2004; Schinella et al., recently was reported the inhibitory activity 2002; Prior et al., 2003; Prior et al., 2005). against aldose reductase by an extract rich in The use of traditional medicine is widespread anthocyanins of this fruit (Kraft et al., 2007). and plants still present a large source of novel Subsequently, we have some recent reports active biological compounds with different about the effects of MeOH extract from ripe activities, including anti-inflammatory, anti- fruits of A. chilensis on isquemic/reperfusion cancer, anti-viral, anti-bacterial and system, several antioxidant activities of that cardioprotective activities (Seigler 1998; extract and its relationship between total Schinella et al., 2002; Yan et al., 2002). phenolic levels and the cardioprotective effect Berries constitute a rich dietary source of (Cespedes et al., 2008; 2010). In other recent phenolic antioxidant and bioactive properties works, in addition to a phytochemical profile (Pool-Zobel et al., 1999; Smith et al., 2000). composition by NMR, HPLC, and GC/MS Chilean wild black-berry Aristotelia chilensis analyses, the ethanolic extract, fractions and (Mol) Stuntz (Elaeocarpaceae), an edible black- some phytochemicals that occurs in the fruit colored fruit, which reach its ripeness between were assayed against ORAC, FRAP, DPPH and December to March, have a popular and very www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.9 (2) 2010 | 128

Céspedes et al. Anti-inflammatory activity of Aristotelia chilensis

TBARS as an index of lipid peroxidation in of University of Bio-Bio, Campus Chillan. The liposomes from brain homogenate (Cespedes et collected fruits were air-dried and prepared for al., 2010) and the presence of 3-hydroxyindole extraction. in this fruit was reported together with its antioxidant activity (Cespedes et al., 2009). Chemicals and solvents In the continuation of our general screening All reagents used were either analytical grade program of Chilean flora with biological or chromatographic grade, 2,2’-azobis (2- activities (Cespedes et al., 2001), a re- aminopropane) dihydrochloride (AAPH), 2,2- examination of the EtOH extract of fruits of A. diphenyl-1-picryl-hydrazyl (2,2-Diphenyl-1- chilensis (Elaeocarpaceae) has been initiated. (2,4,6-Trinitrophenyl), DPPH), Butylated Thus, in the present work, we designed to Hydroxy Toluene (BHT), 2[3]-t-Butyl-4- investigate the anti-inflammatory activity in the hydroxytoluene (THQ), 2[3]-tert-butyl-4- TPA-induced inflammation in ear of mice model hydroxyanisole (BHA), 2[3]-tert- of the EtOH, acetone extracts, fractions, and butylhydroquinone monomethyl ether (TBH), subfractions, the occurrence of phenolic ethylenediaminetetraacetic acid (EDTA), bovine compounds, and its relationship with its serum albumin, Percoll, Trolox (6-hydroxy- phytochemical contents (Cespedes et al., 2010; 2,5,7,8-tetramethylchroman-2-carboxylic acid), 2009) of these extracts, fractions and sub- quercetin, Folin-Ciocalteu reagent, 2- fractions from ripe fruits of A. chilensis. thiobarbituric acid (TBA), FeSO4, trichloroacetic The aim of this work was to evaluate the anti- acid, gentisic acid (2,5-dihydroxybenzoic acid), inflammatory activity of EtOH, acetone, ethyl gallic acid, p-coumaric acid, o-coumaric acid, acetate and MeOH/H2O extracts from ripe fruits propil-gallate, quercetin (3,3;,4’,5,7- and subfractions from SF4 to SF37 isolated from pentahydroxyflavone), myricetin (3,3’,4’,5,5’,7- F-3 and F-4 fractions (see Table 1 and scheme hexahydroxyflavone), kaempferol (3,4’,5,7- 1). The anti-inflammatory effect on the 12-O- tetrahydroxyflavone), (±)-catechin hydrate, (-)- tetradecanoyl phorbol acetate (TPA)-induced catechin gallate, (-)-gallocatechin, gallocatechin- mouse ear edema test was used (Tubaro et al., gallate, -carotene, saffron, crocin, sorbitol, 1985; De Young et al., 1989; Paya et al., 1993). tricine, and trizma-hydrochloride were Additionally, we are reporting the purchased from Sigma-Aldrich Química, S.A. de phytochemical analysis of the bioactive fraction C.V., Toluca, Mexico, or Sigma, St. Louis, MO. F-4 and the antioxidant activity of the Glycosides of anthocyanidins (cyanidin 3,5- subfractions. diglucoside, delphinidin 3,5-diglucoside, Continuously, we are working in a more cyanidin, delphinidin) were purchased from complete metabolomic profile of the fruits and Fluka, (Fluka-Sigma-Aldrich Química, S. A. de leaves of this plant and in the evaluation of C. V., Toluca, Mexico), samples of luteolin, additional biological activities of leaves. diosmetin and proanthocyanidins were a gift from Prof. Dr. David Seigler University of MATERIAL AND METHODS Illinois at Urbana-Champaign. Methanol, CH2Cl2, CHCl3, NaCl, KCl, Plant material KH2PO4, NaHPO4, NaOH, KOH, HCl, sodium Fruits of Aristotelia chilensis (Mol) Stuntz acetate trihydrate, glacial acetic acid silica gel (Elaeocarpaceae) were collected from fields at GF254 analytical chromatoplates, Sephadex LH- foothills of Los at the Araucanian 20, silica gel grade 60, (70-230, 60A) for Region, near to Temuco City, Chile, in January, column chromatography, n-hexane, and ethyl 2006. Voucher specimens are deposited at the acetate were purchased from Merck-Mexico, Herbarium (CONC) of Departamento de S.A., Mexico. Indomethacin, quercetin, Botánica, Facultad de Ciencias Naturales y myricetin, luteolin, diosmetin and ovatifolin Oceanograficas, Universidad de Concepción, were used as pattern samples. Concepción, Chile and in the botany Collection

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Céspedes et al. Anti-inflammatory activity of Aristotelia chilensis

Apparatus (edema A – edema B/edema A) x 100, where A UV Spectronic model Genesys 5 edema A = edema induced by TPA alone and spectrophotometer was used for biological and edema B = edema induced by TPA plus sample. spectrophotometric analyses. Fluorimetric measurements were determined with TURNER Estimation of lipid peroxidation Barnstead-Thermolyne, model Quantech S5 As an index of lipid peroxidation, TBARS Fluorometer, with 420, 440, 470, 550, and 650 levels were measured using rat brain Turner filters. HPLC Hewlett-Packard, Series homogenates according to the method described 1050, with diode array detector, and UV by Ng with some modifications (Ng et al., detector at 254, 280, 365 and 520 nm, 2000), and as is described in Dominguez et al., column YMC C18-Pack ODS-AM-303, 2005. Results are expressed as nanomoles of AM12S05-2546 WT, 250 x 4.6 mm, ID S- TBARS per milligram of protein, with percent inhibition after 30 min calculated as the 5um, 12nm; movil phase inhibition ratio (IR), where C) absorbance of the water/methanol/acetonitrile (50:35:15), control and E) absorbance of the test sample. isocratic, pressure 212 bar; it was prepared These values were plotted against the log of the 300 µL of each sample in amber vials and concentrations of individual extracts and injected 20 µL of each sample. fractions, and a decrease of 50% in peroxidation was defined as the EC50 (Dominguez, et al., Obtention of extracts, fractions, subfractions 2005). and sample preparation All extracts, fractions and subfractions were Reduction of the 2,2-diphenyl-1- obtained as described in scheme 1. The picrylhydrazyl radical (DPPH) composition of each subfraction was reported in Extracts and partitions were Cespedes et al. 2010. chromatographed on TLC and examined for antioxidant effects by spraying the TLC plates Anti-inflammatory activity with DPPH reagent. Specifically, the plates were The assay of TPA-induced ear edema in mice sprayed with 0.2% DPPH in methanol. was based on the described method (Tubaro et Quercetin and α-tocopherol were used as al., 1985; Merlos et al., 1991; Della Loggia et standards (Dominguez, et al., 2005). al., 1996). Groups of 5 male CD-1 mice (25- 30g) were anaesthetized with Imalgen. A Bleaching of crocin. solution of 12-O-tetradecanoylphorbol-13- The solutions were placed under UV254 light. acetate (TPA, 2.5 g) in acetone (10L) was Following the decrease of absorbance, bleaching topically applied to both faces (5L each face) of crocin and fluorescence emission at 440 and of the right ear of the mice. The left are received 470 nm were monitored with time each 5 min only acetone. Solutions of 0.05, 0.1 and 0.5 mg (Cespedes et al, 2008; Dominguez et al., 2005). in 20 L of acetone of the extracts, sub-fractions and quercetin, ovatifolin, diosmetin, luteolin, Statistical analysis myricetin and indomethacine as references, these Data shown in table 1 is the mean results solutions were applied to both faces of the right obtained with means of five animals and are ear (10L each face) 10 min after TPA presented as mean  standard errors of the mean treatment. Control animals received only (SEM). Data were subjected to analysis of acetone. Fours hours later the animals were variance (ANOVA) with significant differences killed by cervical dislocation. A 9 mm diameter between means identified by GLM Procedures. plug was removed from each ear. The swelling The results are given in the text as probability was assessed as the difference in weight between values, with p < 0.05 adopted as the criterion of the right and left ear plugs. The % inhibition of significance, differences between treatments edema was calculated by the equation: % = means were established with a Dunnett’s test. www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.9 (2) 2010 | 130

Céspedes et al. Anti-inflammatory activity of Aristotelia chilensis

Table 1. Amounts of phenolic content (mg/L ± Standard error) from extracts, fractions and some compounds from Table 2. Amounts of Phenolic content (mg/L ± Standard error) from subfractions§ of A. chilensis needed to inhibit acetone partition B of A. chilensis and indomethacin needed a for inhibitory effect on the TPA-induced inflammation in oxidative damage by 50% . mice modela. EI=Edema Inhibition (%). e Sampleb DPPHc TBARSd Crocin f b Samples / Dose EI50 (mg/ear) c 13.4 Indo-methacin 0.11 SF4-SF6 23.8 7.9 Af 3.6 c 18.9 f c SF 37.4 11.8 B 1.8 7 f 5.9 d 12.0 C SF8-SF10 47.6 9.9 Df 6.7 d d f 0.7 F-1 10.9 SF11-SF15 1.3 (2.2) 1.9 d F-2 11.8 f 0.8 c SF -SF 2.3 (3.8) 2.1 F-3 1.1 16 20 c F-4 1.9 5.2 (7.8)f 2.3 1.1 d SF21-SF25 SF4-SF6 6.7 d 0.9 SF7 10.1 SF26-SF30 17.1 3.9 d SF8-SF10 11.0 2.4 c SF31-SF31 19.7 10.1 SF11-SF15 0.3 c SF16-SF20 0.9 22.9 c Myricetin 21.0 9.7 SF21-SF25 1.0 c 21.0 SF26-SF30 3.8 Quercetin 19.98 2.90 c SF31-SF37 4.1 10.1 Quercetin 0.16 c α-Tocopherol 11.9 3.92 0.068 c Ovatifolin a Diosmetin 0.45 c Values expressed as g/mL (ppm), Mean Confidence Luteolin 0.25 c Interval 95%, n = 3. Different letters show significant 0.17 c differences at (P < 0.05), using Duncan’s multiple-range Myricetin b a Effects on ear edema of female mice CD-1. Means of five test. See scheme 1 for an explanation of extracts and c d animals in independent experiments. Data expressed as % partitions. IC50 for inhibition of DPPH radical formation. of the mean ± SD of weigh of ear. All data analyzed with t- IC50 for inhibition of peroxidation of lipids, estimated as student test. b Each value correspond to concentration that thiobarbituric acid reactive substances. Values are inhibits 50% of edema development during bioassay stage. expressed as g/mL (ppm), See Methods for details. Mean c P < 0.05 d P < 0.01 e Not determined. f A: ± SD, n = 3. Different letters show significant differences e Methanol/water (6:4) extract. B: Acetone extract. C: Ethyl at (P < 0.05), using Duncan’s multiple-range test. IC50 for f acetate extract. D: MeOH/H2O Residue. (Cespedes et al., bleaching of crocin. The values between parenthesis 2010) correspond to the assay made with 50 μM of final concentration of DPPH. For methodology used see Dominguez et al., 2005. §Values of extracts A, B, C, D, E, and fractions F-1, F-2, F-3 and F-4, were reported in Cespedes et al., 2010, here are show only SF from which were isolated different compounds, see scheme 1.

Figure 2. HPLC-DAD of F-4, all peaks were identified comparing with data bases, patterns and authentic samples. For GC/MS analyses of peak at 24.9 min, see Cespedes et al., 2009.

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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%). Extract E correspond water 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. (The phytochemical composition of each subfraction in Cespedes et al., 2010).

The EC50 values for each activity were calculated showed in Table 1. All samples used in this study by Probit analysis on the basis of the percentage of showed a dose-dependent anti-inflammatory activity. inhibition obtained at each concentration of the These effects were compared with those produced samples. EC50 is the concentration producing 50% by the commercially available anti-inflammatory drug inhibition. Completely statistical analysis was indomethacin and ovatifolin (Cespedes et al., 2000), performed by means of the MicroCal Origin 8.0 together with quercetin, myricetin, luteolin and statistical and graphs PC program. diosmetin as natural compound (Cespedes et al., 2001), (Table 1). All of compounds assayed inhibited RESULTS AND DISCUSSION the TPA-induced inflammation (data not show). On the other hand, extract B, fraction F-4, and Anti-inflammatory activity. subfractions SF11-SF15, SF16-SF20 were as well as The results of anti-inflammatory activities of active as indomethacin at 0.11 mg/ear a selective extracts A, B, C, D, fractions F-1 to F-4, and cyclo-oxygenase (COX) inhibitor (Table 1). Is subfractions SF4 to SF37 are outlined in Table 1. These important mention that F-4 showed a very good anti- findings shows that the TPA-induced inflammation in inflammatory activity. This action could be attributed mouse method was well inhibited mainly by extracts to a synergic effect proportionated by the phenolic rich A, B, F-4, SF11-SF15, SF16-SF20, SF21-SF25 and SF26– composition observed in this fraction Fig. 2. SF30 with EI50 of 3.6, 1.8, 1.9, 0.3, 0.9, 1.0 and 3.8 On the other hand, a decrease in the anti- mg/ear, respectively. Additionally, quercetin, inflammatory activity was observed when F-1, F-2, ovatifolin, diosmetin, luteolin, myricetin and SF7, SF8-SF10, SF26-SF30, and SF31-SF37, which have indomethacin showed EI50 0.16, 0.068, 0.45, 0.25, 0.17 sugared components. A similar effect, but in minor and 0.11 mg/ear, respectively used as pattern samples. percentage was observed when was used diosmetin The bioassay was carried out between 0.01 and 15.0 instead of luteolin. Surprisingly, myricetin showed an mg/ear with all samples being extract B, fraction F-4, intermediate activity, since at 0.17 mg/ear showed a subfractions SF11-SF15, SF16-SF20, and SF21-SF25, 50.0% of inhibition. ovatifolin, quercetin, myricetin, luteolin and diosmetin the most active samples, therefore with these samples was made a curve of dose-response, obtaining the EI50 www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.9 (2) 2010 | 132

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Antioxidant activities. DPPH and TBARS investigation of lipid peroxidation as an assessment of evaluation oxidative stress. The capacity for plant extracts to The DPPH radical scavenging assay was used first prevent lipid peroxidation was assayed using as a screen for antioxidant components within the malondialdehyde formation as an index of oxidative primary extracts (Dominguez et al., 2005; Cespedes, breakdown of membrane lipids, following incubation El-Hafidi, Pavon & Alarcon, 2008). The results of of rat brain cortical and hearth homogenates with the 2+ antioxidant activity of extracts A, B, C, D, E, and oxidant chemical species Fe . Ferrous ion both fractions F-1 to F-4, were published previously at stimulates lipid peroxidation and supports Cespedes et al., 2010. As shown in Table 2, the decomposition of lipids peroxides once formed, generating highly reactive intermediates such as subfractions SF11-SF15, SF16-SF20, and SF21-SF25 had the highest inhibitory activity against DPPH radical hydroxyl radicals, perferryl and ferryl species (Ko et formation compared to the other partitions with IC50 al., 1998). Against TBARS SF11-SF15, SF16-SF20 and values of 1.3, 2.3 and 5.2 ppm, respectively. Almost SF21-SF25 were most effective in similar form to all these samples exhibited a concentration- quercetin or BHT in inhibiting lipid peroxidation. dependence manner in their DPPH radical scavenging SF11-SF15 had the greatest activity and reduced lipid peroxidation in a dose-dependent manner, and proved activities, particularly SF11-SF15, and SF16-SF20 which showed the highest activity (100% inhibition) at a to be an excellent antioxidant, reflected by its low IC50 concentration of 4.9 and 15.5 ppm, respectively (data value (1.9 ppm) when analyzed by both TBARS and not show). This action was greater than that of - DPPH (Table 2), at the same level than quercetin and tocopherol, which at 31.6 ppm caused only 53.8% α-tocopherol whom shows IC50 of 2.9 and 3.92 ppm, quenching and very similar to ferulic and p-coumaric against TBARS formation, respectively (Table 2). When the relative contribution of each subfraction acids with IC50 values of 5.1 and 7.8 ppm, respectively (data not shown), similar performance was observed to the total antioxidant activity was evaluated using against crocin inhibitory activity (Table 2). TBARS, all samples showed some protective effect, In addition to pattern samples the subfractions all the IC50 values of all subfractions are shown in Table 2. SF11-SF15 and SF16-SF20 were the most active, SF11-SF15, SF16-SF20, and SF21-SF25 showed considerable activity, quenching DPPH radical with IC50 values of 1.9 and 2.1 ppm, respectively. It is reduction completely (100 % of inhibition, data not noteworthy that the value for SF11-SF15 is very low show). Nevertheless, SF -SF , SF -SF and SF - compared with values for flavonoids and anthocyanins 4 6 26 30 31 in general, as well as for myricetin or quercetin (data SF37 showed a moderate activity their IC50 values were 23.8, 17.1 and 19.7 ppm, respectively (Table 2), these not show) (Makris & Rossiter, 2001; Lo & Cheung, subfractions showed to have the highest concentration 2005). of anthocyanins, and reached the 100% of inhibition at CONCLUSIONS similar concentrations than SF16-SF20, and SF21-SF25. The lowest I50 value for SF11-SF15, SF16-SF20, (1.3 and In general these compounds that occur in these 2.3 ppm, respectively) than for any of the other Aristotelia species have been considered as the active subfractions, might be due to a synergistic effect of the principles of many anti-inflammatory plants. Thus, components due to extraction procedures (mainly many phenolic acids, anthocyanins and flavonoids gallic acid, quercetin, myricetin, delphinidin-3- type have shown inhibitory activities on nitric oxide glucoside and cyaniding-3-glucoside, (scheme 1)) implicated in physiological and pathological process inside this subfraction, similar to that reported for as chronic inflammation (Matsuda et al., 2000; components of Vaccinium corymbosum and V. Odontuya et al., 2005). angustifolium fruits (Ehlenfeldt and Prior, 2001, Smith These finding shows that the anthocyanins, et al., 2000, Lo & Cheung, 2005), where the acetone flavonoids and phenolic acids may be responsible of and MeOH partitions were the most active extracts. the anti-inflammatory activity of this fruit. We are Of the many biological macromolecules, including working in the kinetic of inhibition of these plant carbohydrates, lipids, proteins, and DNA, that can extracts and compounds as anti-inflammatory and undergo oxidative damage in the presence of ROS, additionally we are dissecting the sites and mechanism membrane lipids are especially sensitive to oxidation of action as iNOS, COX, and TNF, among others. from this physiological process (Diplock et al., 1998). For this reason, brain homogenates were used for the www.blacpma.org Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas Vol.9 (2) 2010 | 133

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