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Hipólito Cortez-Madrigal2, María Valentina Angoa-Pérez2, Estefanía Elizabeth Cázares-. Álvarez3. 1CONACYT ..... C.T. Cuevas-Arias, O. Vargas, A. Rodríguez.

JCBPS; Section B; August 2016 – October 2016, Vol. 6, No. 4; 1135-1144.

E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online at www.jcbsc.org Section B: Biological Sciences

CODEN (USA): JCBPAT

Research Article

Total phenolic content, total flavonoids and antioxidant capacity of methanolic extracts from Solanum ferrugineum Jacq. (Solanaceae) José Roberto Medina-Medrano1 *, Monserrat Vázquez-Sánche z1 , Edgar Villar-Luna1 , Hipólito Cortez-Madrigal2 , María Valentina Angoa-Pérez2 , Estefanía Elizabeth CázaresÁlvarez3 1

CONACYT - Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, Unidad Michoacán. Justo Sierra No. 28 Jiquilpan, Michoacán, México. C. P. 59510 Tel: 01 (353) 53-30218 and (353) 53-30083 ext. 82951 2

Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el Desarrollo Integral Regional, Unidad Michoacán. Justo Sierra No. 28 Jiquilpan, Michoacán, México. C.P. 59510 Tel: 01 (353) 53-30218 and (353) 53-30083 3

Universidad de La Ciénega del Estado de Michoacán de Ocampo. Avenida Universidad 3000, Col. Lomas de la Universidad, Sahuayo, Michoacán. C.P. 59103 Tel: 01 (353) 532-0762. Received: 04 August 2016; Revised: 18 August 2016; Accepted: 24 August 2016

Abstract: In vitro total phenolic content (TPC), total flavonoid content (TFC) and antioxidant capacity of methanol extracts of Solanum ferrugineum were analyzed to determine its potential as a source of natural antioxidants using 2,2-Diphenyl-lpicrylhydrazyl (DPPH), 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and total antioxidant capacity (TAC). TPC in fruits, reported in mg gallic acid equivalents (GAE)/g dry tissue (DT) showed similar values to those reported for cranberry. TFC content in fruits, reported in mg catechin equivalents (CE)/g DT, showed higher values than those reported for black mulberry. A relevant scavenging activity of ABTS radical 1135

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was exhibited by extracts of leaves and fruits of S. ferrugineum (86.97±0.79% and 57.80±1.19% inhibition respectively) and EC50 values similar to those reported for grape seed. Leaf and fruit extracts showed a TAC in mg ascorbic acid equivalents (AAE)/g DT similar to those in tomato. Analyzed S. ferrugineum leaves and fruits synthesize significant concentrations of flavonoids with relevant antioxidant capacity. Keywords: scavenging activity, Torva section, wild plant, flavonoid, Cherry eggplant INTRODUCTION The Solanaceae family is an economically important and cosmopolitan family, comprising approximately 3,000 species and 90 genera. The family includes globally important food crops such as potato (Solanum tuberosum L.), tomato (Solanum lycopersicum L.), eggplant (Solanum melongena L.),and pepper (Capsicum spp.) as well as a wide number of plants used as a drug source, such as tobacco (Nicotiana tabacum L.), Datura, and Atropa belladonna L., a source of atropine 1 . The Solanum L. genera is the most diverse genera within the family, encompassing 1,500 species2 . This genera also includes a large number of wild plants3 . There is a recent growing interest in the ethnobotanical uses of wild edible plants and their uses as sources of natural antioxidants4 (Ahmad et al. 2016). Solanum ferrugineum Jacq. (Cherry eggplant) is a wild species which can be found from Mexico to Costa Rica 5,6 . In Mexico this species distributes in the Western part of the country, from Sonora to Chiapas states7 and it frequently grows in conifer and oak forests, semi-deciduous and deciduous tropical forests8 . S. ferrugineum belongs to the Torva section and S. chrysotrichum Schltdl., S. dasyanthum Brandegee, S. donianum Walp, S. hispidum Pers., S. rudepannum Dunal and S. torvum Sw. are also found in Mexico9 . Some of them have been reported to have potential medicinal uses, as is the case of S. chrysotrichum, S. hispidum, S. rudepannum and S. torvum10-12 , and to serve as food, such as S. torvum fruits13 . It is necessary, thus, to keep exploring the potential of other species of the Torva section distributed in Mexico, which could be used, for instance, as a source of phenolic compounds. Phenolic compounds are a large class of plant secondary metabolites, varying in complexity from simple organic acids to complex polyphenols, such as tannins14 . Phenolic compounds are found in all plants, which collectively synthesize tens of thousands of different chemical structures characterized by hydroxylated aromatic ring(s). The largest family of phenolic compounds include simple phenols, such as benzoic and cinnamic acids, coumarins, tannins, lignins, lignans, stilbenes and flavonoids 15 . Flavonoids in particular—but also some other phenolic compounds—have been reported to have a wide range of pharmacological activities, including antibacterial, antiviral, anti-inflammatory, antilipidemic, antidiabetic, neuroprotective, hepatoprotective and cardioprotective properties16-20 . These properties have been related to the strong antioxidant nature of many phenolic compounds and their capacity to scavenge 'reactive oxygen species' (ROS). The objective of this study was to determine total phenolic content, total flavonoids and antioxidative capacity of the methanolic extracts of Solanum ferrugineum in order to assess its potential as a source of natural antioxidants. MATERIAL AND METHODS Chemical substances and reagents : The Folin-Ciocalteu reagent, gallic acid standards, catechin, DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid]), potassium persulfate, ammonium molybdate, and ascorbic acid were adquired at Sigma-Aldrich (St. Louis, MO, USA). The solvents methanol and ethanol were adquired at JT Baker (Phillipsburg, NJ, USA). 1136

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Plant material: Solanum ferrugineum plants were collected at the Fray Dominguez town, in Pajuacarán, located Northwest of the Mexican state of Michoacán. Leaves and fruits were separated from the plants and dried in a stove at 40°C during 96 h. Each sample of tissue was stored at room temperature in the dark until they were used. Preparation of extracts: The extraction of phenolic compounds was performed following MedinaMedrano et al.21 — 1 g of dry tissue, ground by maceration in 20 mL of 80% methanol (v/v), for 24h, agitated at 100 rpm, in the dark, at room temperature. Extracts were centrifuged at 4,800x g for 5 min at room temperature and the supernatants formed the total extract. Different aliquots were taken for determining phenolic content, total flavonoids, and antioxidant capacity. Determination of total phenolic content: The concentration of the total phenolic content (TPC) of each sample was determined using the Folin-Ciocalteu reagent, following Nurmi et al.22 . The phenolic content was calculated using a concentration curve of gallic acid (A 760 = 0.0097[gallic acid] − 0.0494, r = 0.9949), built with six concentrations of the compound (0–92 μg/mL). The phenolic content was expressed as milligrams of gallic acid equivalents per gram of dry tissue (mg GAE/g DT). Determination of total flavonoids content: The total flavonoids content (TFC) of each sample was determined following Heimler et al.23 —using a catechin standard curve (A 425 = 0.0878[catechin] + 0.0004, r = 0.9945), built with five concentrations of catechin (0-0.7 mg/mL). The total flavonoids content was expressed as milligrams of catechin equivalents per gram of dry tissue (mg CE/g DT). Determination of the scavenging capacity of the DPPH free radical: The determination of the scavenging capacity of free radicals was performed through the DPPH • (2,2-diphenyl-1-picrylhydrazyl) method described by Barriada-Bernal et al.24 with some modifications. A DPPH • solution (62 µg/mL metanol) was prepared. To determine the scavenging capacity, 900 µL of DPPH • reagent were mixed with 100 µL of extract, in different concentrations (1, 2.5 and 3.75 mg/mL) and they were incubated at room temperature for 10 min. After incubation, the absorbance was measured at 523 nm. Absolut methanol was used as control. The scavenging effect of DPPH • was measured using the next formula: DPPH scavenging effect (%) = [(A) control − (A) sample / (A) control ] × 100 Effective concentration at 50%, defined as the antioxidant concentration needed to decrease the initial DPPH• concentration by 50% (EC50 ) was determined by linear regression. The following curve, made with DPPH• (2-60 µg/mL), was used to estimate DPPH • concentration (µg/mL) in the reaction medium: A 523 = 0.0309 [DPPH•] + 0.0319, r = 0.9996. Antiradical activity was expressed in terms of EC 50 in milligrams per milliliter (mg/ml). Determination of the scavenging capacity of the ABTS free radical: The ABTS assay (2,2'-azino-bis[3ethylbenzothiazoline-6-sulphonic acid]) was performed following Lee et al.25 . The cationic radical ABTS was generated from the oxidation reaction with 1 mL of ABTS (7 mmol/L) and 17.6 µL of potassium persulfate solution (140 mmol/L), mixed in the dark at room temperature for 12 h. The ABTS•+ solution was diluted with absolute ethanol to obtain an absorbance of 0.700 ± 0.01 at 734 nm. When the radical was formed, an aliquot of the extract (500 µL) was mixed with 500 µL of ABTS•+solution—the absorbance was registered at 734 nm after 6 min. The assay was performed with three concentrations of the extract (1, 2.5

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and 3.75 mg/mL). The blank was prepared under the conditions described previously, substituting the extract for an equal volume of methanol 80% (v/v). The activity percentage was calculated with the formula: ABTS scavenging effect (%) = [(A) control − (A) sample / (A) control ] × 100 The extract concentration that provided a 50% inhibition of the ABTS• + radical (EC50 ) was calculated from a graphic built with the inhibition percentage versus the extract concentration. Total antioxidant capacity: The total antioxidant capacity (TAC) was evaluated following Prieto et al.26 . In this method, the reduction of Mo (VI) to Mo (V) by an antioxidant forms a green phosphate/Mo complex (V) measured at an acid pH. TAC values were expressed as milligrams of ascorbic acid equivalents/ mL (AAE/mL), which were calculated using the following curve A 695 = −0.2365+4.2133 [ascorbic acid], correlation coefficient r = 0.9987, generated with six concentrations of ascorbic acid (0.1-1mg/mL). Data analysis: The determination of phenolic and flavonoids contents, and the evaluations of the antioxidant capacity were performed by triplicate, and the corresponding results were subjected to a variance analysis (ANOVA). Differences between values with a p