Phenolic content and antioxidant and antimutagenic activities in tomato peel, seeds, and byproducts.

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Phenolic content, and antioxidant and antimutagenic activities in tomato peel and seeds, and tomato by-products Maribel Valdez-Morales, Laura Gabriela Espinosa-Alonso, Libia C Espinoza-Torres, Francisco Delgado-Vargas, and Sergio Medina-Godoy J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5012374 • Publication Date (Web): 03 May 2014 Downloaded from http://pubs.acs.org on May 25, 2014

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Journal of Agricultural and Food Chemistry

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Phenolic content, and antioxidant and antimutagenic activities in tomato peel and seeds, and tomato by-products

Maribel Valdez-Moralesa, Laura Gabriela Espinosa-Alonsoa, Libia Citlali Espinoza-Torresb, Francisco Delgado-Vargasc, and Sergio Medina-Godoya*.

a

Instituto Politécnico Nacional, Centro Interdisciplinario de Investigación para el

Desarrollo Integral Regional CIIDIR, Unidad Sinaloa. Laboratorio de Alimentos Funcionales, Departamento de Biotecnología Agrícola, Guasave, Sinaloa, México C.P. 81101. b

Instituto Tecnológico de Estudios Superiores de Guasave. Carretera Internacional

y entronque a la Brecha, Ej. El Burrioncito, Guasave, Sinaloa. México c

Universidad Autónoma de Sinaloa. Facultad de Ciencias Químico-Biológicas,

Natural Products Laboratory. Av. de las Américas y Josefa Ortiz de Domínguez s/n. Ciudad Universitaria, Culiacán, Sinaloa. México.

* Corresponding author: Sergio Medina-Godoy. Tel. +52-(687)87-29626; fax: +52-(687)87-29625. E-mail address: [email protected]

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Abstract

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The phenolic content, antioxidant and antimutagenic activity from peel and seeds

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of different tomato types (Grape, Cherry, Bola and Saladette type), and simulated

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tomato industrial by-products, were studied. Methanolic extracts were used to

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quantify total phenolic content, groups of phenolic compounds, antioxidant

6

activities, and, the profile of phenolic compounds (by HPLC). Antimutagenic activity

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was determined by Salmonella typhimurium assay. The total phenolic content and

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antioxidant activity of tomato and tomato by-products were comparable or superior

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than previously reported for whole fruit, and tomato pomace. Phenolic compounds

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with important biological activities, such as caffeic acid, ferulic acid, chlorogenic

11

acids, quercetin-3-β-O-glycoside, and quercetin, were quantified. Differences in all

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phenolic determinations due tomato type and part of the fruit analyzed were

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observed, peel from Grape type showed the best results. Positive antimutagenic

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results were observed in all samples. All evaluated materials could be used as a

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source of potential nutraceutical compounds.

16 17 18 19 20 21 22

Keywords: tomato by-products, phenolic compounds, in vitro bioactivity,

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nutraceutical profiting.


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Introduction

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By considering the cultivated area, tomato is the second most important crop in the

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world. Mexico has the 9th place in production (∼2 million metric tons/year)1 and

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Sinaloa Stated is the main producer with 37% of the national production. The main

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cultivated varieties in Sinaloa are “Saladette” or “Roma”, “Bola”, “Cherry type”, and

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“Grape type”. Tomatoes are used as food in fresh or processed (e.g. paste, juice

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and sauce) forms and worldwide consumption has increased in the last two

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decades.2 In our country, approximately 120,000 tons of tomato are industrialized

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annually, generating 6,000 ton of by-products;3 these residues are mainly

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composed by peel and seeds, which are usually used as feed4 and not for human

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consumption.

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On the other hand, the whole tomato fruit has an important content of C and E

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vitamins and some minerals such as K, P, and Mg;5,6 carotenoids, mainly

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lycopene; and important phenolic compounds such as caffeic acid, ferulic acid,

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kaempherol, quercetin, and others, have been identified in the fruit.7,8,9 In this way,

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tomato seed and peel generated as by-products, contain important quantities of the

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above mentioned compounds too; and even fiber, fatty acids, sitosterol,

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isorhamnetin, myricetin, and others.5,8,10,11 Although it has been found that the

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application of heating or storing processes decreases the content of these

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compounds, in tomato studies have shown that these compounds remain at

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acceptable levels, and they show nutraceutical activity.2,11 Furthermore, the

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consumption of fresh tomatoes, processed tomatoes, and dietary supplements

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based on tomato fruit, have been associated with prevention of some diseases

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such as: cancer, cardiovascular diseases and Alzheimer.2,7,12 Most important 3 ACS Paragon Plus Environment


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biological activities demonstrated in tomato fruit, tomato food products and by-

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products

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inflammatory.2,13,14, Traditionally, bioactivity of tomato and their products has been

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attributed to carotenoids;7 although, phenolic compounds have shown antioxidant

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and cytotoxic activities in vitro, in both processed products and fresh tomato.10,15

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Phenolic compounds are a wide group of chemicals that contains one or more

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phenyl rings in its structure, with at least one hydroxyl bonded to the phenyl

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structure.16 The phenolic compounds mostly distributed in foods are phenolic acids

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and flavonoids (60% and 30% of the total phenolic consumed in the diet,

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respectively); several reports describe their attributes as natural and potent

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antioxidant, anti-carcinogenic, chemopreventive agents, and other bioactivities.15,16

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An important issue is that bioactivity of the phenolic compounds from any natural

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source depends on the quality and quantity of compounds that are present, and the

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phenolic concentration could be affected by both cultivation system and genotype

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of the source.15,16,17 By that reason, it is necessary to strengthen the search of

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nutraceutical compounds as phenolics from several sources, including tomato by-

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products from different varieties.

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The purpose of this work was to determinate the phenolic content, and antioxidant

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and antimutagenic activities of peel and seed of different tomato cultivars from

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Sinaloa Mexico. In order to take advantage of these by-products to enrich animal

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or human food, and have ecological and economic benefits reducing the tomato

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agricultural and industrial wastes.

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Materials and methods

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Chemicals and reagents

are:

antimicrobial,

antioxidant,

anti-mutagenic,


and

anti-

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HPLC-grade acetonitrile and methanol were purchased from Karal (Leon, Gto.,

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Mexico). Minimal glucose agar and Oxoid nutrient broth were bought from Oxoid

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Microbiology Products (Thermo Fisher Scientific, New York, NY, USA). Solvents

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and other reagents, including the phenolic standards (gallic acid, caffeic acid,

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ferulic acid, p-coumaric acid, o-coumaric acid, (+)-catechin hydrate, quercetin

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anhydrous, rutin hydrate, kaempherol, isorhamnetin and myricetin) were ACS

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grade solvents or reactives, and pure grade standards (Sigma Chemical Co., St.

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Louis MO, US)

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Biological material

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Organic growth tomatoes, cultivated in Sinaloa (a state from the Northwest of

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Mexico) during the autumn/winter 2010-2011 season, of four varieties (Saladette,

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Bola, Grape and Cherry) were collected (20 kg/ cultivar) in the breaker stage

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(USDA, 1991) during the morning and within the period December 17th, 2010 and

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January 23th, 2011. Collected tomato materials were transported to the laboratory

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and stored at 20 °C until they reached the red stage (last stage in the color scale).

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Commercial Saladette tomato (20 kg) was bought in a local market to get

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simulated industrial by-products, because is the main material used for regional

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tomato processing industry to produce tomato paste.

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Color analysis

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The color of ripe tomatoes was measured with a chroma meter CR-400 (Konica

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Minolta Sensing, Inc, Japan), ten whole fruits of each type were evaluated in five

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points, three equidistant equatorial points and two axial. Chroma and a*/b* data

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were calculated from L*, a*, and b*CIELab parameters, using a white standard CR-

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Sample processing

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For the chemical analysis, the peel and seeds of all tomato types were manually

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separated from the pulp, frozen (-20 °C) and freeze-dried (Labconco CO., Kansas-

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City, MO, US). The simulated commercial tomato by-products were prepared as

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suggested by an Engineer of a local processor tomato industry (personal

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communication); ripe Saladette tomato (20 kg) was cut into cubes of 2 cm3, ground

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in a conventional blender until juice appearance and heated at 80 °C during 1 h in

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an aluminum pot with constant stirring to get the higher yield of juice. Mixture was

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filtered with a gauze swab, juice was discarded, and the remnants of peel and

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seeds were manually separated, frozen at -20 °C and freeze-dried. Dried by-

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products were ground in a laboratory mill (Thomas Wiley, Thomas Scientific,

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Swedesboro, NJ, USA) until we had a fine flour (80 mesh size), and stored (4 °C/

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protected from light) until analyses. Three biological replicates of each variety of

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tomato were processed.

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Proximal composition analysis

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Proximal composition was determined by the following methods described in the

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AOAC (2005) standard methodologies:18 930.15 for humidity; weight difference

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method 942.05 for ashes; 976.05 for total nitrogen; 976.05 for total lipids; and

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978.10 for total crude fiber. All quantifications were conducted by triplicate.

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Preparation of the methanolic extract

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Peel and seed flour were extracted as described by Ferreres et al.10 with some

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modifications. Samples (0.5 g) were suspended in 5 mL of methanol in 15 mL

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plastic tubes, sonicated during 1 h (mixed with a vortex every 10 min), transfered

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into 125 mL Erlenmeyer flasks, and made up to 20 mL with methanol. Then, 6 ACS Paragon Plus Environment

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samples were incubated for 22 h (25 °C/darkness/220 rpm), centrifuged at 1409 g/

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3 min, in a rotor J600R2004 (Solbat S.A. de C.V., Puebla de Zaragoza, Mexico),

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the supernatant was decanted and stored at -20 °C, and the pellet was re-

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extracted adding 20 mL of methanol and following the previously described steps.

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After re-extraction, both supernatants were combined and concentrated to dryness

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at 37 °C in a rotary evaporator (Yamato, Santa Clara, CA, USA). The extract were

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reconstituted with 3 mL of methanol, filtered through 0.22 µm Durapore syringe

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filters (Millipore, Carrigtwohill, Co. Cork, Ireland) and kept stored at -20 °C until use

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(this was called methanolic crude extract). Each tomato sample was made by

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triplicate.

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Total polyphenol content

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The total polyphenol content of crude methanolic extract was quantified using the

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Folin-Ciocalteu spectrophotometric method, according to Nurmi et al.19 with

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modifications made by Ramírez-Mares et al.20 Briefly, methanolic crude extract was

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dissolved in methanol (1:30 p/v), 100 µL of the solution was mixed with 1 mL of 0.5

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N Folin-Ciocalteu's phenol reagent and 2 mL of a 20% (w/v) Na2CO3 solution;

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mixture was allowed to stand for 15 min at darkness and absorbance was

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measured at 734 nm. Gallic acid (GA) and (+) catechin hydrate (CAT) were used to

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prepare calibration curves and results were reported as mg of gallic acid

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equivalents per g of flour (mg GAE/g) or catechin equivalents (mg CE/g). Groups of

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phenolic compounds were quantitated by spectrophotometry21 at: 320 nm for

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tartaric esters of phenolic acids and 404 nm for flavonoids; in a Thermo Spectronic

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Genesys 2 spectrophotometer (Thermo Fisher Scientific, New York, NY, USA). 7 ACS Paragon Plus Environment


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Results of groups of phenolic compounds were expressed as caffeic acid or

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quercetin equivalents per g of peel or seed flour, for phenolic tartaric esters or

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flavonoids, respectively.

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Antioxidant activity

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The antioxidant activity of crude methanolic extracts was measured by the DPPH,

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22

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radicals, and we measured spectrophotometrically their decrease in absorbance

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(as the effect of the phenolic compounds presents in the samples). In the ABTS

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method, the ABTS•+ radical was chemically prepared (7 mM in methanol), the

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ABTS reagent was mixed with potassium persulfate (2.45 mM), incubated during

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16 h at room temperature, and diluted with methanol to get an absorbance value of

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0.70 ± 0.02 at 734 nm. To measure the antioxidant activity, 10 µL of the

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concentrated methanol crude extract (controls or standard) was mixed with 1000

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µL of ABTS•+, vortex gently during 20 s, and absorbance at 734 nm was registered.

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For DPPH assay, the radical (125 µM in methanol) was daily prepared and kept at

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4 °C/darkness. Assay was done in a 96-well microplate, 20 µL of the concentrated

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methanol crude extract (controls or standard) was mixed with 200 µL of DPPH

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solution and absorbance (515 nm) was read at 0, 10, 20, 30, 40, 50, 60, 70, 80,

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and 90 min with a DTX 880 Multimode Detector, Beckman Coulter (Brea, CA,

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USA). Trolox standard curve was constructed at 30 min.

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For the ORAC (Oxygen radical absorbance capacity, performed fluorometrically)

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assay,24 analysis was done at 37 °C in pH 7.4, 75 mM phosphate buffer, peroxyl

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radical was generated using AAPH (70 mM), and fluorescein (87µM) was the

ABTS23 and ORAC24 methods. DPPH and ABTS are free stable oxidative


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substrate. 25 µL of diluted (1:100) sample (control or standard) was mixed with 200

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µL of fluorescein and 50 µL of AAPH, and the decrease of fluorescence was

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calculated in a DTX 880 Multimode Detector, Beckman Coulter (Brea, CA, USA),

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using the following conditions: excitation wavelength of 485 nm, and emission

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wavelength of 530 nm; 76 cycles were read, every 2 min. In all cases we report

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antioxidant activity as Trolox equivalents, a standard curve was prepared with a

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correlation coefficient up to R=0.99, and the equivalents was calculated as µmol

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eq. Trolox/100 g of flour. Three independent experiments were conducted in each

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assay.

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Profile of phenolics by HPLC-DAD

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The HPLC profile of phenolics in the methanolic crude extract was established with

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a Dionex UltiMate 3000 liquid chromatograph, equipped with a titanium quaternary

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pump (LPG-3400AB), an autosampler (WPS-3000TBPL), column oven and a

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photodiode array detector DAD-3000(RS) (scan range 190–800

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speed 2 nm/s) from Thermo Scientific (Thermo Fisher Scientific, New York, NY,

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USA). Separation was carried out using an analytical column Acclaim® 120 A

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(C18, 5 µm, 120 Å, 4.6 X 250 mm), from Dionex (Thermo Fisher Scientific, New

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York, NY, USA), at ambient temperature. Elution was done with a gradient of two

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solvents: water acidified with acetic acid (pH 2.8) (A) and acetonitrile (B). The

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gradient for phenolic acids25 was 100% phase A, 6% phase B from 0-8 min, 6% to

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12% B from 8-14 min, 12% to 20% B from 14-18 min; 20% to 35% B from 18-24

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min, 35% to 95% B from 24-27 min, 95% to 100% B from 27-30 min, and 100% B

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to 100% A in 10 min. The fixed wavelengths were 260, 270, 275, 280, 285, 290, 9 ACS Paragon Plus Environment

nm and scan


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295 and 300 nm. Gradient for the elution of flavonoids was 100% A, 10% B from 0-

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2.5 min, 10% to 12% B from 2.5-6 min, 12% to 23% B from 6-18 min, 23% to 35%

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B from 18-24 min, 35% to 95% B from 24-27 min, 95% to 100% B from 27-30 min,

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and 100% B to 100% A in 10 min; 260 and 342 nm were the fixed wavelengths for

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flavonoids. The flow rate was 0.5 mL/min. The injection volume was 10 µL.

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Chromatographic peaks were identified by comparing the retention times and UV-

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Visible absorption of pure standards (i.e. gallic, caffeic, ferulic, p-coumaric and o-

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coumaric acids; and flavonoids: (+) catechin hydrate, quercetin, rutin hydrate,

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kaempherol, isorhamnetin and myricetin). A standard curve was constructed with

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the mix of all phenolic acids and another with the mix of all flavonoids. Each

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sample was injected three times. The data were processed using Chromeleon 7.0

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software (Dionex, Thermo Fisher Scientific, New York, NY, USA).

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Antimutagenic activity test

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Antimutagenicity of the methanolic crude extracts was determined, the extracts

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were concentrated to dryness under a nitrogen flow, and the solid extract was

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resuspended in sterilized DMSO. The antimutagenic activity was done by the

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microsuspension assay using YG1024 Salmonella typhimurium as tester strain,

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and 1-nitropyrene as mutagen, according to Santos-Cervantes et al. (2007).26 A

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dose-response curve of mutagen, and different sample concentrations was

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constructed. An aliquot (10 µL) of resuspended extract was homogenized with the

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components of the incubation mix, including 1-nitropyrene. A mix of bacteria plus

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mutagen was used as a positive control of mutation reversion. The capacity of 10 ACS Paragon Plus Environment

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tomato seed or peel extracts to prevent the action of 1-nitropyrene against the cells

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was measure with the follow equation:

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% Antimutagenic activity= ((His+ revertants of positive control plate – His+

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revertants of sample test plate) / His+ revertants of positive control plate) *100

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Statistical analysis

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A totally random experimental design was used to evaluate the color parameters

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and, a factorial design 5 × 2 for the chemical and antimutagenic analysis, the first

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factor was tomato type, and the second one was the anatomic part of the tomato

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fruit. ANOVA analysis and a multiple ranges test (Tukey, p≤0.05) were done with

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the SAS 7.0 statistic program (SAS Institute Inc., Cary, North Carolina). Correlation

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test between total phenolics/ individual phenolic content and antioxidant or

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antimutagenic activity were done through SAS 7.0 using the Pearson´s correlation

223

coefficient option. Three biological replicates and at least two independent

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experiments were performed.

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Results/Discussion

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Color and proximal composition

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Maturity stage was not significantly different among tomato varieties considered in

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this study. Tomato color was uniform inter and intra-tomato varieties, as indicated

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by the low variability of the color parameters (L*, a*, b*, chroma and a*/b* values)

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and their statistical similarity (Table 1). The ripeness stage for these tomatoes

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corresponded with that accepted for marketing.27

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The proximal composition of peel and seed of all tomato types can be observed in

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Figure 1. All measured parameters were higher than the average values

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previously reported for whole tomato fruit,28 except total lipids in peel;6 and were 11 ACS Paragon Plus Environment


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closer than those reported for seed and peel from tomato pomace.11,29 Notable

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protein content was found in all seed samples, above of 24 g/100g, closer than the

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reported by Fuentes et al. (2013) for tomato seed from pomace.29 Saladette type

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had the highest protein percentage (28.7 g/100g of flour); and, a high ash content

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was quantified in the majority of tomato types (6.7 g/100g of flour, on average), the

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mentioned parameter was superior to that reported by different authors in flour of

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tomato by-products (5 g/100 g of flour).5,6,29 Crude fiber of seed plus peel of each

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tomato type, was superior than the previously reported for tomato pomace.28 The

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by-products contained the highest fiber content (38.5 g/100 g of flour) (p ≤ 0.05).

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This effect could be the result of the heat process, as it has been previously

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reported in other vegetables where insoluble dietary fiber increased as result of a

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cooking process.30 Total lipids in peel correspond to 3% of the proximal

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composition, and are represented by waxes, mainly.15

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represented up to 15 g/100 g of flour, and in the case of Grape seeds, these

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compounds reached up to 20 g/100 g of flour, it could be important analyze the

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fatty acids profile and other hydrophobic compounds as phytosterols, in order to

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determinate a potential commercial use. Analysis of variance (ANOVA) showed

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that all proximal composition parameters were affected by tomato type, the part of

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the fruit analyzed (peel or seed), and the interaction of both factors.

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High protein and total fiber content found in our samples, suggest that the peel or

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seed of tomato dry flour can be used as source of those substances to enrich

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animal or human food.

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Total phenolic content, groups of phenolic compounds, and antioxidant

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capacity 12 ACS Paragon Plus Environment

In seeds, total lipids

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Crude methanolic extracts showed levels of phenolic content (Table 2) comparable

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with other fruits recognized for their phenolic content, such as kiwi and grapes.31

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The ranges were from 71.6 to 351.6 mg eq of gallic acid (GA)/100 g of peel flour

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samples (208.2 mg eq of GA/100 g of flour, on average), and from 67.3 to 121.8

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mg eq of GA/100 g of seed flour (95.2 mg eq of GA/100 g of flour, on average);

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these values were superior to the reported for peel and seed from several tomato

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varieties, and tomato by-products.17,28 In the case of the total phenolic content

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expressed as mg eq of (+) catechin (CAT), data was ranged between 464.3 and

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1824.1 mg eq of CAT/100 g of peel flour samples (1136.8 mg eq of CAT/100 g of

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flour, on average); and between 438.3 and 682.8 mg eq of CAT/100 g of seed flour

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(569.4 mg eq of CAT/100 g, on average) in seed samples, and was superior to the

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previously reported for George et al. (2004), in tomato peel.32 There were found

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statistical significant differences (p≤ 0.05) due to the interaction of tomato type and

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part of the fruit, and is notable the presence of higher amount of phenolic

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compounds in peel flour than seed flour, as has been reported by Chandra et al.

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(2013).17 Grape peel flour was the material with the best total phenolic content in

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both ways to express phenolic concentration (351.6 mg eq GA/100 g of flour, and

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1824.1 mg eq CAT/100 g of flour); while, by-products seed flour showed the lowest

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phenolic content even (67.3 eq GA/100 g of flour and 464.3 mg eq CAT/100 g of

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flour) than Saladette (by-products were produced from a Saladette tomato type);

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perhaps, this decrease in phenolic content could be due to the heat processing.33

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In addition, phenolics oxidation could have occurred during homogenization of the

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fresh fruit due to polyphenol oxidase;33 but the total phenolic concentration in by-

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products remains attractive to use them as a source of phenolic compounds. 13 ACS Paragon Plus Environment


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Sagdic et al.34 in 2011, reported the content of total phenolic compounds in by-

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products from grape fruit, our data obtained in Cherry and Grape peel flours were

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comparable to the reported for these authors. Differences in the phenolic content

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for effect of tomato genotype have been reported,17,32 the highest amount of

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phenolic compounds found in Cherry and Grape tomato types, can be due to that

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these genotypes are less genetically manipulated than Saladette and Bola, and it is

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known that the crops that have been largely genetically improved, show reduced

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concentration of some metabolites, even phenolic compounds.35 The amount of the

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different phenolic groups can be observed in Table 1. The major group was

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flavonoids, ranged from 83.7 mg/100 g of flour in by-products, to 572.2 mg/100 g of

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Grape peel flour; and in the case of seed samples, flavonoids were quantified from

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50.0 to 69.2 mg/100 g, in by-products and Grape flour, respectively. On the other

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hand, phenolic tartaric esters ranged from 3.1 to 16.9 mg/100 g, for by-products

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and Cherry peel flours, respectively; meanwhile in seeds decrease the content until

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2.6 times, ranged from 3.2 to 6.3 mg/100 g, for by-products and Grape seed,

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respectively. In the same way as happened with total phenolic content, flavonoids

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and esters phenolics concentration were higher in the peel flour than seed flour, in

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the most of cases. Statistical significant differences (p ≤0.05) were observed

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between treatments. We observed that approximately 60% of total phenolic content

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corresponding to flavonoids and 30% to phenolic acids, in the same way that has

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been found in several samples.36 Oomah et al.21 reported levels of flavonols similar

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to those that we found in tomato peel, in hull fractions and residues from red and

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green lentis; but this previous study was done with acetone and hot water extracts,

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and the proportion of phenolic compounds extracted, vary widely according to the 14 ACS Paragon Plus Environment

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extraction solvent, so our results and those reported previously, are not completely

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comparable.

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Antioxidant activity was determined with three methodologies: ABTS, DPPH and

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ORAC, and results were expressed as µmol eq. Trolox/100 g of flour (peel or seed)

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in all cases. Peel samples showed the best results, in all cases. ABTS and DPPH

312

methodologies have a similar chemical fundament, but the data obtained with

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these two methods were different between the same samples. ABTS measured a

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major antioxidant activity than DPPH, in the peel samples of all evaluated

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genotypes; except in by-products, in which case there was no significant difference

316

(p ≤ 0.05) in the antioxidant activity due to the radical used (ABTS or DPPH). In

317

contrast, in seed samples, DPPH methodology showed the best results in all

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genotypes. Due to the previous described behavior, it can be inferred that the

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quality of the extracted compounds are different between peel and seeds samples;

320

because both extracts reacted differently on the same free radical, as observed

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before.37 The best results were obtained in Grape genotype, again. This sample

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showed 405.7 µmol eq. Trolox/100 g of flour by ABTS, and 181.4 µmol eq.

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Trolox/100 g of flour by DPPH, in peel; and, 139.6 µmol eq. Trolox/100 g of flour by

324

ABTS, and 156.2 µmol eq. Trolox/100 g of flour with DPPH methodology, in seed

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sample. Antioxidant activity determined by ABTS was superior that the previously

326

reported in aqueous extracts from tomato plants, and yellow Cherry whole tomato

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fruits.2,32

328

Antioxidant capacity calculated with ORAC methodology was significantly higher

329

than those quantified with ABTS and DPPH methodologies (p ≤ 0.05); values were 15 ACS Paragon Plus Environment


.

330

in the range between 705.3 and 4131.7 µmol eq. Trolox/100 g of flour in peel

331

samples, and from 1028.3 to 1825.0 µmol eq. Trolox/100 g of flour, in the case of

332

seed samples. In both cases the lowest activity corresponded to by-products. The

333

highest activity was measured in Grape tomato type (p ≤ 0.05). Despite this,

334

antioxidant activity of the by-products remained in high levels.21,37 Several studies

335

have shown that ORAC methodology is more adequate to quantify antioxidant

336

activity, due to its resemblance to a biological process in vivo, regarding the

337

peroxyl free radicals. It is however desirable that our results were validated through

338

an in vivo antioxidant activity methodology, reducing confounding factors that affect

339

the action of phenolic compounds over free radicals. ORAC methodology does not

340

take into account those factors, thus further studies are required.38 There was

341

observed an influence of the tomato type, as has been reported before by Chandra

342

et al. (2012)17, the part of the fruit analyzed, and the interaction of this two factors

343

on the antioxidant activity measured by the three methodologies used.

344

Correlations analysis (Pearson, SAS 7.0) between total phenolic and

345

antioxidant activity, and between groups of phenolic compounds and antioxidant

346

activity, were done (data no shown). We calculated R values greater than 0.8, in all

347

cases, except when we correlate flavonoids with antioxidant activity determined by

348

DPPH method. R values superiors than 0.8 are considering higher. It has been

349

reported that not all extractable compounds, that can be measured as total phenols

350

or belonging to a phenolic group, may have the capacity to act against a free

351

radical used in a particular methodology to determine antioxidant activity 16 ACS Paragon Plus Environment

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Page 17 of 36


.

352

(scavenging activity, mainly), this may be due to structural aspects or steric

353

impediments of the different compounds that are presents in the extract.34,39

354

Phenolic compounds profile by HPLC-DAD

355

Results of phenolic compounds profile by HPLC-DAD of each tomato

356

sample (peel and seed flour) are summarized in Table 3, and Figure 2 show the

357

chromatograms obtained in peel of Grape tomato type. We have identified and

358

quantified sixteen compounds in total, eight phenolic acids and eight flavonoids. Is

359

interesting state that by-products not presented the lowest concentration of

360

individual phenolic compounds, always; but the trend was to diminish the content

361

of the evaluated compounds in by-products, may be by the heat processing

362

effect,29 as has been mentioned before in this report. Flavonoids were found in

363

higher concentration than phenolic acids in all tomato types (as happened in

364

phenolic groups evaluation). The most abundant phenolic acids were: caffeic acid,

365

in concordance with the reported before in whole red tomato fruit,40 followed by

366

vanillic, ferulic, sinapic, and chlorogenic acids; in the case of flavonoides, the most

367

concentrated

368

kaempherol. Rutin (and its diverse chemical derivates) are localized in the last

369

steps of the phenolic biosynthesis route, and are related with a commercial mature

370

stage of fruits.41 The content of all phenolic acids and flavonoids determined by

371

HPLC-DAD, in our samples, were three to five times greater than the evaluated

372

with the total phenolic methodology (Folin-Ciocalteu) used. There are diverse

373

reports about the individual content of phenolic compounds in tomato fruits, and

374

seeds generated as by-product, including an effort to characterize tomato

were:

quercetin-3-β-O-glycoside,

rutin,


isorhamnetin,

and


.

375

metabolome, from a mix of 96 cultivars from Iberic peninsula; these works were

376

done by HPLC, and HPLC coupled to mass spectrometry.2,11,12,29,42 The main

377

phenolic compounds found in tomato previous reports (caffeic, ferulic, p-cumaric,

378

and sinapic acids, rutin, quercetin, and kaempherol) are almost the same that we

379

found in our work, and were presents in comparable levels, than those reported by

380

other authors. There have been reported attractive phenolic compounds as caffeic,

381

ferulic and chlorogenic acids, and rutin, in tomato industrialized products (tomato

382

juices and ketchup) in important levels, and we observed the presence of these

383

phenolics in our by-products samples. Therefore we can to mention that these

384

compounds remain after a heat or cooking process.11

385

Attractive biological activities have been reported in the phenolic compounds found

386

in the higher concentrations, in this work; i.e, ferulic and sinapic acids have been

387

correlated with antioxidant and anti-inflammatory activities, and acts as

388

chemiprotectors agents, if they are frequently consumed in the diet;43 and rutin and

389

quercetin are able to suppress mutagenesis and have antitumoral capacity.43

390

Mathematical correlation analysis (data no shown) revealed that the compounds

391

that showed the highest level of correlations (R=0.8) with antioxidant activity were

392

those that were detected in high levels; but only quercetin-3-β-O-glycoside,

393

quercetin, naringenin showed correlation with antioxidant activity evaluated with at

394

least two methodologies. The high correlations found between individual phenolic

395

concentrations and antioxidant activity, in many cases, suggest that the antioxidant

396

activity determined in this work could be given by the action of the quantified 18 ACS Paragon Plus Environment

Page 18 of 36

Page 19 of 36


. 397

phenolic compounds or by the interaction between the different compounds

398

present in the extract; although further studies are necessary to prove this.

399

Antimutagenic activity test

400

Antimutagenic activity, expressed as percentage of mutagenic inhibition, as a

401

response to methanolic tomato peel and seed extracts was determinated (Figure

402

3). A dose-response curve using 0.0, 5.0, 10.0, 20.0, 30.0, and 40.0 mg/mL of solid

403

extract (using seed Saladette tomato type extract), was constructed, no differences

404

in the percentage of mutagenic inhibition were obtained at concentrations equal or

405

greater than 20 mg/mL; by that reason, the following experiments were done at 20

406

mg/mL extract concentration. There were no statistical significant differences (p ≤

407

0.05) between peel and seeds flours, except for by-products flours, where peel

408

samples had a higher antimutagenic activity than seed samples. Percentage of

409

mutagenic inhibition were between 40% and 60%; peel extracts from by-products

410

showed an antimutagenic activity greater than Bola and Saladette tomato types

411

and very similar to the activities determined in Cherry and Grape genotypes. We

412

suggested that the heat process did not affect compounds, which conferred the

413

antimutagenic activity. Bunkova et al.,44 reported in antimutagenic studies made

414

with green tea (using TA98 S. typhimurium strain,) that percentage of mutagenic

415

inhibition between 40% and 60% represents an antimutagenic activity from

416

moderate to strong.

417

Correlations analysis between total phenolic content and antimutagenic activity;

418

groups of phenolic compounds and antioxidant activity; and also, profile of

419

individual phenolic compounds (by HPLC-DAD) and antimutagenicity, were done.

420

We were not able to obtain a strong linear, second or third grade correlations, 19 ACS Paragon Plus Environment


.

421

between all evaluated parameters and the percentage of antimutagenicity. In

422

relation with this negative correlation, it could be happening that the antimutagenic

423

activity observed results from the action of several compounds (or their

424

interactions), some of them not identified with the used methodology. More work is

425

needed in order to determine which compounds impart the antimutagenic activity

426

observed in our extracts, and their mechanisms of action.

427

Finally, we can affirm that methanolic extracts from peel and seed of several

428

tomato types, cultivated in the Northwest of Mexico, including industrial by-

429

products, can be a potential source of compounds with an attractive antioxidant

430

and antimutagenic activities. These extracts could be utilized as nutraceutical

431

ingredients in animal or human food, or as nutritional supplements. For this reason,

432

further studies are needed to complete the metabolite characterization of these

433

materials, and to realize in vivo assays.

434

Acknowledgments

435

Special acknowledgment to farmers from Guasave and Culiacán, Sinaloa, to

436

provide us the biological material. To Basilio Heredia and Rosalva Carolina Valdez

437

Agramón, for technical facilities.

438

References

439

(1) SIAP. Servicio de información agroalimentaria y pesquera, Secretaría de

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agricultura, ganadería desarrollo rural, pesca y alimentación de México. 2010.

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Lamuela- Raventós, R. M. Changes in phenolic content of tomato products during

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(44) Bunkova, R.; Marova, I.; Nemec, M. Antimutagenic properties of green tea. Plant Foods Hum. Nutr. 2005, 60, 25-29(5).

586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607

The first author is grateful to the National Council of Science and Technology (CONACyT) of México for the scholarship granted to achieve her postdoctoral studies. To the Biotechnology Network of Instituto Politécnico Nacional through Secretaría de Investigación y Posgrado (SIP-IPN) for part of the financial founding to realize this work ACS Paragon Plus Environment


608

Figure captions

609

Figure 1.

610

Proximal composition of flour from peel (pane A) and seeds (pane B) of

611

different tomato types, and tomato by-products.

612

Data are the average of three replicates ± standard deviation. Statistical analysis of

613

proximal composition was no shown in this figure; but significant differences

614

between treatments were observed.

615 616

Figure 2.

617 618 619

HPLC-DAD chromatograms of phenolic compounds standards and phenolic and flavonoids determined in a methanolic extract from peel of tomato Grape.

620 621 622 623 624 625 626

Pane A, 1: phenolic acids standard mix, absorbance at 260 nm, labelled peaks in order from left to right are: gallic, protochatecuic, chlorogenic, caffeic, vanillic, elagic, p-coumaric, ferulic, sinapic, and trans-cinnamic. Pane A, 2: phenolic acids detected in Grape peel´s tomato methanolic extract at 260 nm (retention times): 23.7 min=chlorogenic acid, 27.1 min=caffeic acid, 27.9 min=vanillic acid, 31.3 min=p-coumaric acid, 31.8 min=ferulic acid, 32.3 min=sinapic acid and 38.9 min=trans-cinnamic acid; other signals: unknown.

627 628 629 630 631 632 633 634

Pane B, 1: flavonoids standard mix, absorbance at 360 nm, identities of the labelled peaks from left to right are: catechin, epicatechin, rutin, quercetin 3-βglucoside, epicatechin gallate, myricetin, quercetin, apigenin, naringenin, kaempherol, and isorhamnetin. Pane B, 2: flavonoids found in grape´s peel tomato methanolic extract, main signals detected at 360 nm, retention times of the identified compounds are: 27.7 min=rutin, 29.1 min=quercetin 3-β-glucoside, 33.3 min=myricetin, 35.1 min=quercetin, 37.9 min=apigenin, 38.4 min=naringenin, 39.0 min=kaempherol and 40.1 min=isorhamnetin; other signals=unknown.

635 636

Figure 3.

637

Antimutagenic activity of methanolic extracts (20 mg solid extract/mL) from

638

peel and seed´s flour from different tomato types.

ACS Paragon Plus Environment

Page 28 of 36

Page 29 of 36


.

639

Values are the mean of three replicates of two independent experiments. Different

640

letters means statistically significant difference between treatments (Multiple range

641

test; Tukey, p≤0.05).

642 643 644 645 646 647 648 649 650 651

652

653

654

655

656

657

658

659



Page 30 of 36

Table 1. Parameters of Color of Whole Tomato Fruits from Several Varieties Cultivated in the Northwest of Mexico (X ± SD, n = 10)a. Tomato type Cherry Grape Bola Saladette By-products a

L* 31.4 ± 1.45 b 34.2 ± 1.58 a 36.7 ± 1.30 a 35.6 ± 1.36 z 34.8 ± 1.80 a

a* 13.8 ± 1.64 ab 17.2 ± 1.50 a 15.9 ± 1.74 a 15.9 ± 1.35 a 17.5 ± 2.60 a

b* 11.2 ± 1.29 b 13.0 ± 0.96 ab 15.1 ± 0.96 a 15.0 ± 0.92 a 12.9 ± 2.10 ab

Chroma 17.5 ± 3.72 a 21.6 ± 1.36 a 20.1 ± 3.14 a 19.4 ± 4.03 a 18.2 ± 4.40 a

a*/b* 1.24 ± 0.17 a 1.32 ± 0.15 a 1.05 ± 0.13 ab 1.06 ± 0.09 ab 1.37 ± 0.11 a

Values marked by the same letter within the same column mean are not statistically

significant differences (Multiple range test; Tukey, p ≤ 0.05).


Page 31 of 36


.

Table 2. Total Phenolic Content, Groups of Phenolics, and Antioxidant Capacity of Methanolic Extracts from Peel or Seed´s Flour of Different Tomato Varieties (X ± SD/100 g, n = 3).a Groups of phenolics Sample type

Total phenols d

Peel Cherry Grape Bola Saladette By-products Seed Cherry Grape Bola Saladette By-products

Tartaric esters Flavonoids f g mg eq CA mg eq QUER 11.2 ± 0.3 b 218.3 ± 37.7 b 16.9 ± 0.1 a 572.2 ± 174.4 a 8.9 ± 0.8 d 225.8 ± 37.2 b 10.2 ± 0.1 c 216.5 ± 55.0 b 3.1 ± 0.5 g 83.7 ± 1.0 c

e

mg eq GA mg eq CAT 266.3 ± 13.6 b 1481.8 ± 36.6 b 351.6 ± 12.7 a 1824.1 ± 68.7 a 157.8 ± 19.5 d 922.6 ± 34.6 c 192.8 ± 0.2 c 991.3 ± 34.5 c 71.6 ± 2.7 g 464.3 ± 6.7 fg 121.8 ± 3.5 e 113.3 ± 4.9 e 99.6 ± 2.1 f 73.8 ± 9.8 g 67.3 ± 0.3 h

c

h

Antioxidant capacity (mM eq Trolox )

b

682.8 ± 16.7 d 648.8 ± 24.2 de 589.9 ±10.1 e 486.7 ± 11.6 f 438.8 ± 6.7 g

6.3 ± 0.5 e 6.3 ± 0.3 e 4.8 ± 0.8 f 3.3 ± 0.9 g 3.2 ± 0.4 g

60.1 ± 10.4 c 69.2 ± 8.3 c 53.1 ± 5.0 c 57.0 ± 8.5 c 50.0 ± 23.4 c

ABTS 209.2 ± 31.6 b 405.7 ± 29.7 a 201.4 ± 29.0 b 196.3 ± 16.2 b 47.9 ± 5.0 e

DPPH

ORAC

171.0 ± 10.1 ab 2812.6 ± 235.9 b 181.4 ± 20.2 a 4131.7 ± 86.8 a 155.5 ± 20.2 ab 2188.6 ± 110.7 bcd 168.9 ± 19.5 ab 2188.6 ± 110.7 bc 51.4 ± 10.2 d 705.3 ± 20.3 f

85.0 ± 6.8 cd 92.4 ± 17.7 cd 139.6 ± 1.1 c 156.2 ± 17.4 ab 49.4 ± 1.2 e 118.8 ± 5.2 bc 67.4 ± 4.2 de 92.4 ± 17.6 cd 35.1 ± 8.1 e 83.6 ± 6.8 cd

1404.7 ± 238.7 cdef 1825.0 ± 375.4 cde 1213.7 ± 20.0 cdef 1146.5 ± 218.5 def 1028.3 ± 78.9 ef

a

Values marked by the same letter within the same column mean are not statistically significant differences (Multiple range test;

Tukey, p≤0.05). b

Total phenols were quantified with the Folin-Ciocalteu method; cGroup of phenolics were determined by direct measurement

according to Oomah et al. (2011); d µg equivalents of gallic acid (GA) per 100 g of sample; e µg equivalents of (+) catechin (CAT) per 100 g of sample; f µg equivalents of caffeic acid (CA) per 100 g of sample; g µg equivalents of quercetin (QUER) per 100 g of sample; h

µmol equivalents of Trolox per 100 g of sample.



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.

Table 3. Profile of phenolic compounds, phenolics acids and flavonoids, in peel and seed of different tomato types cultivated in the Northwest of Mexico (X ± SD, n = 3)a.

Phenolic acid

Peel

Seeds

Flavonoids Peel

Seeds

mg / 100 g DWb Cherry Grape Bola Saladette By-products

Caffeic acid

Vanilic acid

Ferulic acid

5.11 ± 1.24 cd 22.06 ± 3.24 a 7.85 ± 0.00 bc 10.51 ± 0.38 b 1.05 ± 0.17 e

1.57 ± 0.11 d 7.02 ± 0.74 b 11.27 ± 0.50 a 3.07 ± 0.18 c 0.00 ± 0.00 e

1.36 ± 0.05 e 3.32 ± 0.27 b 1.60 ± 0.02 e 4.56 ± 0.22 a 3.67 ± 0.24 b

2.66 ± 0.80 cde 4.22 ± 0.17 a 3.45 ± 0.32 abc 2.77 ± 0.06 bcd 0.00 ± 0.00 f

Cherry Grape Bola Saladette By-products

2.19 ± 0.17 de 1.67 ± 0.16 e 2.13 ± 0.03 de 0.95 ± 0.04 e 1.95 ± 0.04 de

0.00 ± 0.00 e 0.00 ± 0.00 e 0.00 ± 0.00 e 2.01 ± 0.27 d 0.00 ±0.00 e

1.67 ± 0.00 e 4.31 ± 0.17 a 2.75 ± 0.11 c 2.19 ± 0.09 d 2.20 ± 0.03 d

2.11 ± 0.04 de 1.82 ± 0.15 e 3.56 ± 0.15 ab 2.87 ± 0.18 bcd 3.08 ± 0.28 cd

1.41 ± 0.08 b 0.61 ± 0.04 c 0.29 ± 0.07 c 0.10 ± 0.02 c 0.05 ± 0.01 c

0.79 ± 0.00 bc 3.96 ± 0.15 a 1.31 ± 0.27 bc 0.90 ± 0.21 bc 1.58 ± 0.44 b

Kaempherol

Naringenin

Quercetin

Chlorogenic acid ρ-cumaric acid Trans-cinnamic acid 3.24 ± 0.72 a 1.52 ± 0.29 b 0.23 ± 0.05 de 2.01 ± 0.15 b 1.54 ± 0.56 b 2.42 ± 0.28 b 1.52 ± 0.15 b 1.57 ± 0.15 b 0.87 ± 0.03 c 0.41 ± 0.10 c 0.64 ± 0.04 c 0.22 ± 0.06 de 0.10 ± 0.01 c 1.36 ± 0.06 bc 0.56 ± 0.01 cd 0.72 ± 0.04 c 0.16 ± 0.01 e 0.83 ± 0.03 c 0.10 ± 0.00 e 3.01 ± 0.31 a

Total

0.00 ± 0.00 d 0.72 ± 0.18 b 0.95 ± 0.13 ab 0.00 ± 0.00 d 0.00 ± 0.00 d

15.7 43.3 29.1 22.2 6.7

0.00 ± 0.00 d 0.11 ± 0.01 d 1.17 ± 0.22 a 0.40 ± 0.00 c 0.00 ± 0.00 d

8.9 12.6 12.0 9.5 11.9

Myricetin

Total

2.65 ± 0.11 b 3.17 ± 0.12 a 3.16 ± 0.05 a 1.03 ± 0.03 d 0.89 ± 0.01 d

1.06 ± 0.20 cd 3.74 ± 0.58 a 3.70 ± 0.49 a 2.04 ± 0.07 b 0.33 ± 0.01 ed

0.00± 0.00 d 4.86 ± 0.39 a 2.52 ± 0.36 b 1.66 ± 0.09 c 0.00 ± 0.00 d

2.58 ± 0.18 a 2.08 ± 0.22 b 0.19 ± 0.01 e 1.48 ± 0.30 c 0.04 ± 0.00 e

1.34 ± 0.03 a 0.82 ± 0.04 b 0.95 ± 0.04 b 1.55 ± 0.23 a 0.23 ± 0.03 c

0.16 ± 0.00 c 0.50 ± 0.16 abc 0.54 ± 0.03 abc 0.66 ± 0.05 ab 0.33 ± 0.02 bc

27.5 77.4 40.9 53.8 12.2

Cherry Grape Bola Saladette By-products

0.56 ± 0.00 e 0.00 ± 0.00 g 0.34 ± 0.02 f 0.00 ± 0.00 g 1.36 ± 0.06 c

1.10 ± 0.01 c 2.01 ± 0.22 b 0.00 ± 0.00 e 0.46 ± 0.01 cde 0.00 ± 0.00 e

0.35 ± 0.06 d 0.00 ± 0.00 d 0.34 ± 0.03 d 0.00 ± 0.00 d 0.16 ± 0.01 d

0.28 ± 0.01 e 0.90 ± 0.20 d 0.17 ± 0.04 e 0.31 ± 0.02 c 0.08 ± 0.03 e

0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 d

0.29 ± 0.04 bc 0.88 ± 0.39 a 0.34 ± 0.03 bc 0.34 ±0.03 bc 0.34 ± 0.04 bc

9.3 9.9 7.1 7.4 6.7

2.76 ± 0.58 fg 3.53 ± 1.10 ef 2.68 ± 0.00 fg 2.94 ± 0.09 fg 2.06 ± 0.15 g

a

Apigenin

Gallic acid

mg / 100 g Quercetin-3-O-βRutin DWb glucoside Cherry 15.12 ± 2.04 d 4.56 ± 0.49 e Grape 47.99 ± 2.71 a 14.19 ± 0.17 b Bola 18.30 ±0.05 c 11.85 ± 0.41 c Saladette 25.24 ± 0.23 b 20.09 ± 0.13 a By-products 4.47 ± 0.10 e 5.95 ± 0.12 d 3.94 ± 0.07 e 2.61 ± 0.14 e 3.23 ± 0.01 e 3.37 ± 0.03 e 2.68 ± 0.16 e

Isorhamnetin

Sinapic acid

Values marked by the same letter within the same column mean statistically significant difference between treatments (Multiple

range test; Tukey, p≤0.05). b

Dry weight


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Figure 1.



Figure 2.


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Page 35 of 36


Figure 3.



Table of contents


Page 36 of 36

Tomato waste: Carotenoids content, antioxidant and cell growth activities.

Evaluation of total phenolic compounds and insecticidal and antioxidant activities of tomato hairy root extract.

Comparative Assessment of Phenolic Content and in Vitro Antioxidant Capacity in the Pulp and Peel of Mango Cultivars.

Investigation of total phenolic content and antioxidant activities of Azadirachta indica roots.

Total Phenolic, Flavonoid, Tomatine, and Tomatidine Contents and Antioxidant and Antimicrobial Activities of Extracts of Tomato Plant.

Phenolic content and antioxidant capacity in algal food products.

A correlation between tocopherol content and antioxidant activity in seeds and germinating seeds of soybean cultivars.

Lipids, proteins, phenolic composition, antioxidant and antibacterial activities of seeds of peanuts (Arachis hypogaea l) cultivated in Tunisia.

Antioxidant and cytotoxic activities investigation of tomato seed extracts.

Total phenolic content and antioxidant activities of pomegranate juice and whey based novel beverage fermented by kefir grains.

Phenolic acid composition, antioxidant activity and phenolic content of tarhana supplemented with oat flour.

Phenolic Compounds in the Potato and Its Byproducts: An Overview.

Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars.

Variation in phenolic compounds and antioxidant activity in apple seeds of seven cultivars.

Evaluation of the antimutagenic, antigenotoxic, and antioxidant activities of Eriobotrya japonica leaves.

Effect of pineapple waste powder on probiotic growth, antioxidant and antimutagenic activities of yogurt.

A study on antimicrobial, antioxidant and antimutagenic activities of Elaeagnus angustifolia L. leaves.

Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid.

Biological activities of commercial bee pollens: antimicrobial, antimutagenic, antioxidant and anti-inflammatory.

Olive Mill Waste Extracts: Polyphenols Content, Antioxidant, and Antimicrobial Activities.

Phenolic Proﬁle and Antioxidant Activity of Melon (Cucumis Melo L.) Seeds from Pakistan.

Effect of nitrogen fertilisation and irrigation on phenolic content, phenolic acid composition, and antioxidant activity of winter wheat grain.

In vitro and in vivo antioxidant and antimutagenic activities of polyphenols extracted from hops (Humulus lupulus L.).

Phytochemical Contents and Antioxidant and Antiproliferative Activities of Selected Black and White Sesame Seeds.