research article evaluation of antioxidant capacity of...

Research ArticleEvaluation of Antioxidant Capacity ofSolanum sessiliflorum (Cubiu) Extract An In Vitro Assay

Diego Rocha de Lucena Herrera Mascato1 Janice B Monteiro2

Michele M Passarinho1 Denise Morais Lopes Galeno1 Rubeacuten J Cruz3

Carmen Ortiz4 Luisa Morales5 Emerson Silva Lima6 and Rosany Piccolotto Carvalho1

1Programa Multi-Institucional de Pos-Graduacao em Biotecnologia Universidade Federal do Amazonas69077-000 Manaus AM Brazil2Division of Biochemistry Ponce Health Sciences University School ofMedicine Ponce Research Institute Ponce PR 00732-7004 USA3Biology Department University of Puerto Rico at Ponce Ponce PR 00734 USA4Divisions of Pharmacology amp Toxicology amp Cancer Biology Ponce Health Sciences University School of MedicinePonce Research Institute Ponce PR 00732-7004 USA5Public Health Program Ponce Health Sciences University Ponce PR 00732-7004 USA6Faculdade de Ciencias Farmaceuticas Universidade Federal do Amazonas 69010-300 Manaus AM Brazil

Correspondence should be addressed to Carmen Ortiz caortizstupsmeduand Rosany Piccolotto Carvalho prosanyufamedubr

Received 2 July 2015 Revised 15 October 2015 Accepted 26 October 2015

Academic Editor C S Johnston

Copyright copy 2015 Diego Rocha de Lucena Herrera Mascato et al This is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Cubiu is a vegetable of Solanaceae family native to the Amazon which is widely distributed through Brazil Peru and Colombia Itis used in food medicine and cosmetics by native populations Research has shown that cubiu extracts have antioxidant activitieswith great biological relevance We performed a phytochemical screening to identify the main chemical groups that could conferantioxidant activity to this extract Several tests and qualitative precipitation specific staining for major classes of secondarymetabolites were used Antioxidant capacity in vitro tests (DPPH andABTS) were also used to assess the extractrsquos ability to sequesterfree radicals of 70 hydroethanolic and aqueous extracts of cubiu flour Alkaloids organic acids phenols flavonoid glycosidesand coumarins were found in the hydroethanolic extract while the aqueous extract presented anthocyanins gums tannins andmucilage amino groups and volatile and fixed acids For in vitro tests the IC

50value obtained in the DPPH assay was 6063 plusmn

35 120583gmL while that for the ABTS assay was 2903 plusmn 107120583gmL Although cubiu extracts present chemical compounds directlyrelated to antioxidant activity our results show that it has a low antioxidant activity Additional studies will be needed to isolate andcharacterize specific compounds to further assess antioxidant activity

1 Introduction

Cubiu (Solanum sessiliflorumDunal) also known in Spanish-speaking countries as ldquotupirordquo ldquotopirordquo and ldquococonardquo andknown as ldquoOrinoco applerdquo and ldquopeach tomatordquo in English-speaking countries is a vegetable of the family Solanaceaewhich is native to the Amazon [6] and widely distributedthrough the humid equatorial regions of Brazil Peru and

Colombia Its domestication was made by the Indians ofWestern Amazonia [7] The fruit can weigh between 20 and450 grams and contains between 200 and 500 oval flat seedsThe fruits have the most diverse forms Those cylindrical inshape have generally 4 loci while the heart-shaped roundand flattened ones have between 6 and 8 loci The fruit colorchanges as it matures ranging from green when it is not ripeto yellow-orange when it is mature and finally to red when

Hindawi Publishing CorporationJournal of Nutrition and MetabolismVolume 2015 Article ID 364185 8 pageshttpdxdoiorg1011552015364185

2 Journal of Nutrition and Metabolism

Table 1 Chemical composition of cubiu (Solanum sessiliflorum) in 100 g of whole pulp [1ndash5]

Component Villachica (1996) [4] Pahlen (1977) [3] Andrade et al (1997) [1] Yuyama et al (1997 1998) [2 5]Units (g) 89 91 93 90Energy (kcal) 41 33 31 45Proteins (g) 09 06 mdash 09Lipids (g) mdash 14 mdash 19N-free extract (g) mdash 57 mdash 47Fiber (g) 02 04 mdash 09Ashes (g) 07 09 mdash 09Total sugars () mdash mdash 46 mdashReducing sugars () mdash mdash 39 1Nonreducing sugars () mdash mdash 18 1Soluble solids (∘Brix) mdash 5 8 mdashCitric acid () mdash mdash 08 mdashBrixacidity mdash mdash 59 mdashPhenolics (mg) mdash mdash 144 mdashTannins (mg) mdash mdash 142 mdash

coffee is ready to be consumed The fruit has a pulp colorranging from light yellow to yellowish cream measuringbetween 02 and 25 cm thick [6]

The pulp is the edible part of the fruit As food it isconsumed ldquonaturallyrdquo as strip-like alcoholic beverages jelliesjams juices and sauces [1 8] The macerated leaves of theplant are used by Peru-Brazil indigenous people to avoidthe formation of blisters in the event of skin burns whilethe juice from the fruitrsquos locular cavity is used to alleviatethe itching of spider bites [9] The pure juice is used for thecontrol of cholesterol diabetes excess uric acid and otheraffections caused by kidney and liver failure besides beingused to eliminate lice [9] Previous studies have shown thecomposition of cubiu as presented in Table 1

Cubiu is a juicy fruit with humidity between 88 and 93It has a variable soluble solids content (∘Brix) between 5 and8 of which the majority are reducing sugars [1] Since theratio of ∘Brix and acidity of the fruit is low it is consideredacidic This ensures a high dilution factor (great for juiceproduction) and little preference for fresh consumption(due to sourness of the fruit) The composition of phenoliccompounds is also low [1] Taking into consideration thenutritional value of cubiu (Table 2) it presents a high dietarypotential due to its low caloric and high fiber content Thissupports its indication in the diet of hypercholesterolemicand hyperglycemic patients [2]

In a study conducted in Norbert Wiener University inLima (Peru) it was found that administration of cubiuextract (40mLday) for three days decreased the level ofLDL triglycerides serum cholesterol and glucose How-ever it increased serum HDL levels in 100 volunteers ofboth sexes [10] Furthermore it was found that the fruithas a phenolic compound called tannin Phenolic com-pounds are products of secondary plant metabolism whichare essential for their growth and reproduction Tanninsare formed under conditions of stress such as infectioninjury and UV radiation [11] These also act as natural

second class antioxidants [12] According to their modeof action antioxidants can be classified into primary andsecondary Primary antioxidants act by donating hydrogenor electrons to free radicals therefore converting them intostable products andor reacting with free radicals forminglipid-antioxidant complexes that can react with another freeradical Secondary antioxidants act by delaying the initiationstep of autooxidation by different mechanisms includingmetal complexation sequestration of oxygen decompositionof hydroperoxides to form nonradical species absorption ofUV light or deactivation of singlet oxygen species [13]

The phenolic antioxidants interact preferentially with theperoxyl radical due to its prevalence in the autooxidationstep [15] This mechanism of action present in plant extractsplays an important role in reducing lipid oxidation in tissuesplants and animals because it preserves food quality andreduces the risk of developing diseases such as arteriosclerosisand cancer [16 17] Since antioxidants are of great relevancefor the field of medicine today there is a need to investigatethe antioxidant potential of cubiu and to elucidate the role ofthis fruit in the diet of the population

2 Material and Methods

21 Feedstock For this study cubiu fruits used were obtainedfrom the Horticultural Experimental Station Alejo von derPahlen from the National Institute of Amazonian Research(NIAR) located in Manaus AM The fruits were transportedin coolers to the Laboratory of Food and Nutrition at theNIAR where they were processed to obtain cubiu flour asdescribed by da Silva Filho et al (2005) [18]

22 Preparation of Cubiu Flour After the fruits werereceived the ripe intact fruits (free of cuts holes and otherdefects) were selected for processing Fruits were washedbrushed sanitized using a 200 ppm hypochlorite solution for

Journal of Nutrition and Metabolism 3

Table 2 Vitamin and mineral composition of Solanum sessiliflorum in 100 g of cubiu whole pulp according to several studies and percentageof the daily recommendation of the National Research Council (1989) [14]

Component Villachica (1996) [4] Pahlen (1977) [3] Andrade et al (1997) [1] Yuyama et al (1997 1998) [2 5] NRCAscorbic acid (mg) 45 mdash 139 mdash 153Niacin (mg) 23 25 mdash mdash 141Carotene (mg) 02 02 mdash mdash mdashThiamine (mg) 01 03 mdash mdash 154Riboflavin (mg) 01 mdash mdash mdash 66Calcium (mg) 16 12 mdash mdash 12Magnesium (mg) mdash mdash mdash 237 75Phosphorus (mg) 30 14 mdash mdash 18Potassium (mg) mdash mdash mdash 3854 193Sodium (mg) mdash mdash mdash 371 742Copper (mg) mdash mdash mdash 329 146Iron (mg) mdash mdash mdash 324 26Zinc (mg) mdash mdash mdash 157 11Manganese (mg) mdash mdash mdash 97 28

Fruits Selection Washing Sterilization

Shellseed

Washing

Bleaching

Pulping

Milling

Packing

Storing

Drying (65∘C)

Figure 1 Processing of cubiu fruits to obtain the flour as describedby da Silva Filho et al [18]

30minutes and rinsedwith runningwater After this processthe fruits were stripped of their pulp and had the placenta(inner part of the fruit that contains the seeds and the juiceof the locular cavity) and the epicarp being removed with astainless steel knifeThermal bleachingwas then performed at90∘C for 3minutes followed by a heat shock in an ice bath for3 minutes Bleached fruits were packed in 2 kg polyethylenebags and frozen at minus15∘C The fruits were then dried inan oven with forced air circulation at 60∘C for 48 hoursAfter drying the fruits were liquified homogenized packedand frozen at minus15∘C until they were grinded to obtain theflour The flour was packed in plastic bags and stored in afreezer with temperatureminus15∘Cuntil the extract was prepared(Figure 1)

23 Preparations of Cubiu Flour Extracts Cubiu extractswere prepared by dissolving 2 kg of cubiu flour in 2 L ofethanolic solvent The solvent was prepared using 500mLof distilled water and 1500mL of ethyl alcohol (Sigma

MO) Mixtures were then placed on the Ultrasonic Cleaner(UNIQUE Brazil) for 15 minutes to complete homogeniza-tion and filtered using filter paper and a disposable glassfunnel three times Then the extracts were vacuum filteredusing a Buchner funnel and an Erlenmeyer filtration flaskSubsequently the extracts were placed in round bottomflasksfor rotoevaporation with a 25∘Cwater bathThe extracts werereturned to their viscous form and then freeze-dried using alyophilizer After this they were kept in a freezer at minus11∘F forlater use as described by Rogerio (2006) [19]

24 Assessment of Antioxidant Capacity

241 DPPH Method The DPPH assay is based on thereduction of 22-diphenyl-1-picrylhydrazyl radical (DPPH)through the donation of a hydrogen atom of the testcompound to the radical of the molecule [20] Measure-ment of DPPH radical scavenging activity was performedaccording to themethodology described byMolyneux (2004)[21] with some modifications Initially 2mg of DPPH wasdissolved in 12mL of absolute ethanol In a flat bottom96-well plate 270120583L of DPPH solution was mixed with30 120583L ethanol Thereafter the test consisted of 270120583L of thesolution of DPPH mixed with 30 120583L of 70 hydroethanoliccubiu extract The reaction was incubated for 30 minutes atroom temperature in the absence of light After incubationthe absorbance was read at 517 nm To express antioxidantactivity IC

50was calculated that is the minimum concen-

tration required to reduce the initial DPPH reaction by 50Ascorbic acid was used as standard The antioxidant activitywas calculated using the following formula

Inhibition = 100 minus (Abs sampleAbs control

) times 100 (1)

242 ABTS Method This method is based on the reductionof ABTS (221015840azinobis-3-ethylbenzothiazoline-6-sulfonicacid) and was performed as described by Re et al (1999) [22]


with modifications ABTS was dissolved in MiliQ water toa 7mM concentration ABTS∙+ was produced by reactingABTS stock solution with 5mM potassium persulfate andkept in the dark at room temperature for 12ndash16 h OnceABTS∙+ is formed MiliQ water was added to the solution(dilution 1 7) A 96-well plate flat-bottomed was added by270120583L of ABTS solution with 30 120583L of water This solutionwas monitored by reading at 714 nm in a microplate reader(DTX 800 Beckman CA USA) to obtain absorbance ofapproximately 100 (control) Then 30 120583L of cubiu extractfrom different concentrations was added to 270120583L of ABTS∙+and the reaction was incubated for 15min in the dark atroom temperature After incubation the absorbance at714 nm was measured Gallic acid in the same cubiu extractconcentrations was measured following the same proceduresdescribed above and was used as positive controls Theantioxidant activity was calculated using the followingequation


) times 100 (2)

25 Systematic Analysis in Phytochemistry This assay ana-lyzes all the qualitative features of the principal chemicalgroups active in natural products by means of staining orprecipitation reactions The systematic testing for phyto-chemical analysis was performed according toMoreira (1979)using maceration aqueous extraction and hydroethanolicextraction [23]

26 Preparation of Aqueous and Hydroethanolic CubiuExtract The extracts were performed by dissolving 40 g ofthe powder of dried cubiu fruits in 200mL of 70 ethylalcohol or water The mixture was placed in an ultrasoundat 50∘C for 10minThemash was filtered through filter paperand supplemented with the same solvent volume to 200mLThe extract was kept refrigerated until completion of thephytochemical assays

27 Assessment for the Presence of Alkaloids Organic Acidsand Phenols Experiments were conducted using generalalkaloid reagents (Mayer Dragendorff Bouchardat andBertrand as described in Table 4) as follows 50mL of thehydroethanolic extract was evaporated to dryness in a waterbath at 70∘C followed by dissolution of the residue in 1mLof ethanol and 20mL of 1 HCl Extracts were transferred totest tubes and the reagent was added on each oneThe appear-ance of precipitate indicated a positive reaction To retest15mL of the aqueous extract was transferred to a separatingfunnel and alkalized with ammonium hydroxide (pH 10)An etherchloroform (3 1) extraction was performed and theextract obtained was also tested for alkaloids The remainingsolution prepared for the study of alkaloids was dried andredissolved in 5mL of distilled water The pH of the solutionwas measured where an acidic pH indicated the presence oforganic acids Two drops of 1 FeCl

2aqueous solution were

added to the solution obtained in the study of organic acidsto confirm the presence of phenols

28 Assessment for the Presence of Flavonoid GlycosidesOxalic-Boric Complex (R Wilson-Taubock) Pacheco andShinoda Tests 10mL of each of the extractions was driedusing a water bath Five drops of acetone and 30mg of amixture of boric acid and oxalic acid at a ratio of 1 1 wereadded to the dried extracts After stirring and drying themixture 5mL of ethyl ether was added and the solution wastransferred to test tubes Solutions were seen under UV lightThe reaction is considered positive when the result has agreenish yellow fluorescent appearance For the Pacheco testa few crystals of NaOAc and 100 120583L of acetic anhydride wereadded to the dried extractThemixture was heated in a waterbath and 100 120583L of concentrated HCl was added The resultof the reaction is considered positive when there was purplecoloring For the Shinoda test 5mL of the hydroethanolicextract was mixed with 200mg of magnesium turnings and1mL of HCl The formation of orange color indicates thepresence of flavonoids

29 Assessment for the Presence of Coumarins Thirtymilliliters of hydroethanolic extract was acidified to pH1 and concentrated to 10mL in a water bath at 60∘C Theresidual extract was mixed with 5mL of deionized waterand transferred into a separation funnel with ethyl etherin 3 portions of 10mL The volume of the organic extractwas reduced to 5mL in a water bath at 60∘C Three dropsof hydroethanolic extract were placed in two points of apremarked filter paper and left to dry and 1 drop of 1 NNaOHwas added on each spot The stains were covered with a coinand observed under UV light A blue or yellow-greenfluorescence indicated a positive reaction

210 Assessment for the Presence of Anthraquinones 20mLof hydroethanolic extract was brought to a boil under refluxfor 15 minutes by adding 3mL of 10 H

2SO4 After cooling

the mixture was transferred to a separatory funnel alongwith 30mL of distilled water to perform an extraction with10mL of toluene The extract was concentrated to 10mL andmixed with 10mL of NaOH The appearance of pink or redcolor indicated the presence of hydroxy-anthraquinones andnaphthoquinones

211 Assessment for the Presence of Sterols and Triterpenes20mL of hydroethanolic extract was evaporated to performan extraction with dichloromethane Extracts were concen-trated (3mL) and mixed with 2mL of acetic anhydride and3 drops of H

2SO4 The development of blue-green color

demonstrated the presence of steroids andor triterpenes

212 Assessment for the Presence of Anthocyanin and SaponinGlycosides Three portions (5mL each) of aqueous extractwere neutralized with 5 KOH until a pH of 55 was reachedChanges in color indicated the presence of the neutralizedportions of anthocyanin glycosides To test for the presenceof saponin glycosides the remaining solution was stirredvigorously for 5minThe appearance of persistent foam in thesample indicated the presence of saponins This was furtherconfirmed by adding 1 HCl to the sample


213 Assessment for the Presence of Cyanogenic Glycosides15mL of the aqueous extract was transferred to a test tubeAfter adding 1mL of 1NH

2SO4 a strip of picrosodium paper

was secured inside the tube while it was in a water bath at60∘C for 30minutesThe appearance of red color on the paperindicated the presence of cyanogenic glycosides

214 Assessment for the Presence of Gums Tannins andMucilage Five drops of 10 basic acetate and neutral leadacetate were added to 2 portions of 5mL of aqueous extractThe precipitate formation is indicative of the presence ofgums mucilages and tannins To assess the presence oftannins 5 drops of 1 FeCl

2were added to 5mL of the

aqueous extract Upon dark precipitate formation 5mL ofthe aqueous extract was transferred to a flat bottom flask toadd 5 drops of 37 formaldehyde and 4mLHClThemixturewas left to reflux for 1 hour and when it cooled it was filteredand washed with distilled water and alcohol A few drops of5 KOH were added to the material retained on the filterThe appearance of color indicated the presence of condensedtannins The filtrate was then mixed with 10 drops of 1FeCl3 the formation of a dark blue precipitate confirmed the

presence of hydrolyzable tannins

215 Assessment for the Presence of Amino Groups Afterconcentrating 10mL of the aqueous extract at a temperatureof 50∘C 5 drops of concentrated extract were dropped on afilter paper After drying the filter paper was sprayed witha solution of ninhydrin in butanol and heated at 90ndash100∘Cfor 15min The appearance of blue-violet color indicated thepresence of amino groups

216 Assessment for the Presence of Volatile and Fixed Acids10mL of aqueous extract was acidified with 1NH

2SO4and

brought to a boil in a water bath A pH strip was used tomeasure the acidity of the vapors produced during boilingThe acidic color indicates the presence of volatile acids20mL of aqueous extract was transferred to a distillationflask along with 2mL of 1NNaOH The mixture was left toreflux for 30 minutes and after cooling and acidifying with1NH2SO4an extraction with ethyl ether was performedThe

extracts obtained were filtered and evaporated to drynessAfter heating the residue for 10min at 100∘C and adding5mL of 1NNH

4OH it was filtered again Three drops were

transferred to a filter paper to obtain a spot of 1 cm indiameter The paper was dried at 100∘C for 10 minutesand treated with Nesslerrsquos reagent The color developmentindicated the presence of fixed acids

3 Results and Discussion

The results for the systematic phytochemistry analysis can beseen in Tables 3 and 5

In this first set of assays we obtained positive results forthe presence of alkaloids and most of them precipitated inneutral or slightly acidic medium It should be emphasizedthat these precipitates can also be caused by proteins purinesalpha pyrones certain coumarins lignans and phenolic

Table 3 Tests for the 70 hydroethanolic extract of the fruitSolanum sessiliflorum Dunal

Compound classes assessed Test resultsAlkaloidsMayer reagent minus

Dragendorff reagent ++Bouchardat reagent minus

Bertrand reagent ++Reaction of confirmation Dragendorff and Bertrand

Organic acids ++ pH = 55ndash6Phenols +++Heterogeneous flavonoidsTaubock or Oxalo-boric reaction +++Pacheco reaction minus

Shinoda reaction +Coumarins +Anthraquinones minus

Sterols and Triterpenes minus

+++ strongly positive++ positive+ traitsminus negative

Table 4 Reagent composition and color of precipitate for thedifferent tests used to determine the presence of alkaloids on the 70hydroethanolic extract of the Solanum sessiliflorum Dunal fruit

RGA Composition Color ofprecipitate

(1) Mayer Potassium-mercuric iodide Orange(2) Dragendorff Bismuth nitrate White(3) Bertrand Silicotungstic acid White(4) BouchardWagner Potassium triiodide Brown

Table 5 Tests for the aqueous fruit extract of Solanum sessiliflorumDunal

Presence of chemical groups Test resultsAnthocyanin heterosides ++Saponin glycosides minus

Cyanogenic glycosides minus

Gums tannins and mucilage +++Tannins +Amine groups +++Volatile acids ++Fixed acids ++++ strongly positive++ positive+ traitsminus negative

hydroxyl compounds Negative results with these reagents areindicative of the absence of alkaloids however the formationof precipitates can only be considered as probable presencethereof [24]

The acidity of the fruit was confirmed through themeasure of pH of 55ndash6 This correlated with the resultsshown by Andrade et al 1997 where it was calculated usingthe ratio of Brix (soluble solids content) and the acidity ofthe fruit yielding a low ratio therefore showing its acidicpotential


It also presented a strong positive result for the presenceof phenols in contrast with the findings reported in a studyby Andrade et al (1997) in which a relatively low amountof phenolic compounds (144mg) in 100 g of cubiu wholepulp was found [1] According to Moreira and Mancini-Filho (2004) phenolic compounds are natural second classor secondary antioxidants [12] This class of antioxidantsacts by delaying the initiation of autooxidation by differentmechanisms for example hydroperoxide decomposition toform nonradical species [13] This finding confirms thepotential antioxidant activity of cubiu

In our study we also found evidence of the presenceof flavonic heterosides Previous studies have shown thatflavonoids exert several activities over different biological sys-tems showing antimicrobial antiviral antiulcerogenic cyto-toxic antineoplastic antioxidant antihepatotoxic antihyper-tensive hypolipidemic anti-inflammatory and antiplateletactivities Flavonoids have also been found to increase capil-lary permeability and inhibit protein exudation and leukocytemigration [25] These effects may be related to the inhibitoryeffect that flavonoids exert over different enzyme systemsincluding hydrolases isomerases oxygenases oxidoreduc-tases polymerases phosphatases proteins and amino acidoxidases phosphokinases [26] The presence of coumarinscan be related to antioxidant activities along with other pos-siblemechanisms such as anti-inflammatory antispasmodicantitumor properties and interaction with several enzymes

In the second set of assays (Table 5) the presence ofanthocyanin heterosides acids (volatile and fixed) and ingreater quantity amino groups and gums mucilages andtannins was noted

The anthocyanin or anthocyanin heterosides are sub-stances belonging to the flavonoid familyThese are pigmentsthat give color to flowers fruits leaves stems and roots ofplants [27] In previous studies anthocyanins were found tohave an inhibitory action on lipid peroxidation [28] and aprotective action over vascular epithelial cells against reactiveoxygen species principally to H

2O2-induced loss of cell

viability [29]Gums are generally considered a pathological result due

to physical injury suffered by plant tissues by the actionof microorganisms or due to unfavorable conditions suchas drought Mucilage gums were detected by precipitationin tube assays However neither of both has antioxidantcharacteristics [30] The amino groups were detected by thepresence of purple coloring They are compounds bound toan NH

2radical structurally similar to the alkaloids but with

no oxidative activity due to the gradual reduction of theradicals present in their structures [24]

The antioxidant activity of the 70 hydroethanolicextract of cubiu was evaluated using in vitro assays DPPHand ABTS In this study the sequestering capacity of thehydroethanolic extract of cubiu was expressed as the finalconcentration of extract required to inhibit oxidation ofDPPHby 50 (Figure 2)The antioxidants in the extract reactwith DPPH which is a stable free radical and convert it into22-diphenyl-1-picryl hydrazine The degree of discolorationindicates the antioxidant potential of the extract An extractthat has a high antioxidant potential has a low IC

50[31]

01020304050607080

0 200 400 600 800 1000

Inhi

bitio

n (

)

R2 = 099IC50 = 6063 plusmn 35

Concentration (120583gmL)

Figure 2 Colorimetric assessment of antioxidant capacity using inthe DPPH assay Values represent mean plusmn standard deviation of themean of replicate readings (119899 = 3) The IC

50values denote the

concentration of the sample which is required to scavenge 50 offree radicals

Our results show that IC50of cubiu was 6063 plusmn 35 120583gmL a

high value when compared with ascorbic acid (IC50of 274 plusmn

03 120583gmL) which used as a reference substanceIn a study comparing the antioxidant activity of native

fruits the lower IC50

values were obtained by gallic acid(138mgmL) ethanolic and aqueous pequi bark extracts(944 and 1798 gmL resp) ethanolic cagaita seed extract(1415mgmL) and ethanolic extract of araticum seed andpeel (3097 and 4918mgmL resp) [32] In a study thatevaluated the antioxidant activity of several brands of orangejuice using the DPPH assay it was found that the oneswith activity were the orange file (6624) and the orangebahia (6032) [33] However it is difficult to comparethe antioxidant activity of different samples because theauthors used different dilutions of the samples to conduct theanalysis since each sample has a different antioxidant powerFurthermore the ways to analyze and display the results aredifferent Stratil et al (2007) reported that the comparisonof results regarding the antioxidant capacity published inindividual methods and among groups that use the samemethod is often problematic [34]

Regarding the results obtained by the ABTS method(Figure 3) the values obtained were 2903 plusmn 107 120583gmL In astudy performed in the Singapore market the ABTS methodwas used to measure the Equivalent Antioxidant CapacityL-Ascorbic Acid (EACA) and the results were expressedin mg of ascorbic acid (AA) per 100 g of the homogenatecontaining the fruit extract In this study the fruit with thehighest antioxidant capacity was sapodilla (3396mg100 g)followed by strawberry (472mg100 g) plum (312mg100 g)and carambola (278mg100 g) [35] Dos Santos et al (2008)evaluated the antioxidant capacity of commercial acai pulpusing the ABTS method and expressing the results as TEAC(Trolox Equivalent Antioxidant Capacity) The TEAC of acaipulp ranged from 1021 to 5247mM of Troloxg sample Thisconfirms the difficulty of comparing the antioxidant activityreported in different studies since each one uses a differentmethod of analysis [36]

The results obtained in this study were conflicting whencompared to those presented in other studies Nascimento


01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107

Figure 3 Results from the ABTS test Values represent mean plusmnstandard deviation of the mean of replicate readings (119899 = 3)The IC

50values denote the concentration of the sample which is

required to scavenge 50 of free radicals

and Pereira (2011) used the DPPH method and stated thatcubiu extracts obtained from methanol fraction containedthe highest antioxidant activity (between 100 and 200mEBHTg of freeze-dried material) [37] which explains thefact that in this study the antioxidant activity was found tobe low since the hydroethanolic extract was used Anotherinteresting finding of the study is that of all the extractsobtained from different parts of the fruit the bark extractstood out with higher antioxidant activity which is usuallydiscarded in food preparations Ledur et al (2012) used theDPPHmethod to evaluate the antioxidant capacity of the 70hydroethanolic extract of cubiuThe bark extract showed thelowest IC

50when compared to the seed and pulp extracts

(2787mgmL and 30998mgmL resp) [38] These valuesare lower than the results obtained in our study however theyconfirm the fact that cubiu has little antioxidant capacity

4 Conclusion

To achieve the objective of the study flour of cubiu (Solanumsessiliflorum Dunal) fruit was prepared and used for thepreparation of the 70 hydroethanolic extract that wasfurther analyzed using different phytochemical assays andantioxidant capacity tests From the phytochemicals assaysthe presence of phenolic compounds and flavonoids wasnoted Both of these substances are recognized in the liter-ature for their great antioxidant potential However despiteconfirmation of the presence of these compounds cubiuextract showed a low antioxidant activity when compared toother fruits such as sapodilla strawberry orange cherry [35]and many others known for their great antioxidant effect

Conflict of Interests

The authors declare no conflict of interests

Acknowledgments

This research was supported by the RCMI programG12MD007579 (Janice B Monteiro) and the MBRS-RISE

program funded by RISE Grant R25GM082406 from theNational Institutes of Health (Carmen Ortiz) at PonceSchool of Medicine and Health Sciences and Puerto RicoClinical and Translational Research Consortium GrantU54MD007587 The authors would like to acknowledgethe support of the Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq) and the Fundacao deAmparo a Pesquisa do Estado do Amazonas (FAPEAM)

References

[1] J S Andrade I M A Rocha and D F Silva Filho ldquoCarac-terısticas fısicas e composicao quımica de frutos de populacoesnaturais de cocona (Solanum sessiliflorum Dunal) encontradasna Amazonia Centralrdquo Revista Brasileira de Fruticultura 1997

[2] L KO Yuyama J P Aguiar S HMMacedo T Gioia andD FSilva Filho ldquoComposicao centesimal de diversas populacoes decubiu (Solanum sessiliflorum Dunal) da Estacao Experimentaldo Instituto Nacional de Pesquisas da Amazonia INPArdquo inProceedings of the Anais do II Simposio Latino Americano deCiencias de Alimentos Campinas Brazil 1997

[3] A V D Pahlen ldquoCubiu (Solanum topiro (Humb amp Bonpl))uma fruteira da Amazoniardquo Acta Amazonica vol 7 no 3 pp301ndash307 1977

[4] H Villachica ldquoCocona (Solanum sessiliflorum Dunal)rdquo in Fru-tales y Hortalizas Promisorios de la Amazonıa pp 98ndash102Secretaria Pro-Tempore Lima Peru 1996

[5] L K O Yuyama ldquoConteudos minerais em algumas populacoesde cocona (Solanum sessiliflorum Dunal) dados preliminaresrdquoin Anuais do Congresso Brasileiro de Ciecia e Tecnologia Rio deJaneiro Brazil 1998

[6] D F da Silva Filho J S de Andrade C R Clement F MMachado and H Noda ldquoCorrelacoes fenotıpicas geneticas eambientais entre descritores morfologicos e quimicos em frutosde cubiu (Solanum sessiliflorum Dunal) da amazoniardquo ActaAmazonica vol 29 no 4 pp 503ndash511 1999

[7] R E Schultes ldquoAmazonian cultigens and their northwardand westward migrations in pre-columbian timesrdquo in Pre-Columbian Plant Migration D Stone Ed pp 19ndash38 HarvardUniversity Press Cambridge Mass USA 1984

[8] D F de Silva Filho H Noda and C R Clement ldquoGeneticvariability of economic characters in 30 accessions of cubiuSolanum sessiliflorum Dunal Solanaceae evaluated in CentralAmazoniardquoRevista Brasileira deGenetica vol 16 no 2 pp 409ndash417 1993

[9] J Salick ldquoCocona (Solanum sessiliflorum Dunal) an overviewof productions and breeding potentialsrdquo in Proceedings of theInternational Symposium on New Crops for Food IndustryUniversity Southampton Southampton UK 1989

[10] M A Pardo S ldquoEfecto de Solanum sessiliflorum Dunal sobre elmetabolismo lipıdico y de la glucosardquo Ciencia e Investigacionvol 7 no 2 pp 44ndash48 2004

[11] M Naczk and F Shahidi ldquoExtraction and analysis of phenolicsin foodrdquo Journal of Chromatography A vol 1054 no 1-2 pp 95ndash111 2004

[12] A V B Moreira and J Mancini-Filho ldquoInfluencia dos compos-tos fenolicos de especiarias sobre a lipoperoxidacao e o perfillipıdico de tecidos de ratosrdquo Revista de Nutricao vol 17 no 4pp 411ndash424 2004

[13] G O Adegoke M Vijay Kumar A G Gopala Krishna M CVaradaraj K Sambaiah and B R Lokesh ldquoAntioxidants and


lipid oxidation in foodsmdasha critical appraisalrdquo Journal of FoodScience and Technology vol 35 no 4 pp 283ndash298 1998

[14] National Research Council Recomended Dietary AllowancesMA Press 1989

[15] E A Decker ldquoStrategies for manipulating the prooxida-tiveantioxidative balance of foods to maximize oxidative sta-bilityrdquo Trends in Food Science amp Technology vol 9 no 6 pp241ndash248 1998

[16] N Ramarathnam T Osawa H Ochi and S Kawakishi ldquoThecontribution of plant food antioxidants to human healthrdquoTrends in Food Science amp Technology vol 6 no 3 pp 75ndash821995

[17] M Namiki ldquoAntioxidantsantimutagens in foodrdquo CriticalReviews in Food Science and Nutrition vol 29 no 4 pp 273ndash300 1990

[18] D F da Silva Filho L K Yuyama J P Aguiar M C Oliveiraand L H Martins ldquoCaracterizacao e avaliacao do potencialagronomico e nutricional de etnovariedades de cubiu (SolanumsessiliflorumDunal) da AmazoniardquoActa Amazonica vol 35 no4 pp 399ndash405 2005

[19] A P Rogerio Estudo da atividade anti-inflamatoria analgesicaanti-edematogenica e antipiretica do extrato de Lafoensia pacarie do acido elagico [PhD thesis] Faculdade de Ciencias Far-maceuticas de Ribeirao Preto Universidade de Sao Paulo SaoPaulo Brazil 2006

[20] O I Aruoma J P E Spencer DWarren P Jenner J Butler andB Halliwell ldquoCharacterization of food antioxidants illustratedusing commercial garlic and ginger preparationsrdquo Food Chem-istry vol 60 no 2 pp 149ndash156 1997

[21] P Molyneux ldquoThe use of the stable free radical diphenylpicryl-hydrazyl (DPPH) for estimating antioxidant activityrdquo Songk-lanakarin Journal of Science and Technology vol 26 no 2 pp211ndash219 2004

[22] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[23] E A Moreira ldquoMarcha sistematica de analise em fitoquımicardquoTribuna Farmaceutica vol 47 no 1 pp 1ndash19 1979

[24] C M O Simoes E P Schenkel G Gosmann J C P MelloL A Mentz and P R Petrovick Farmacognosia Da Planta aoMedicamento Florianopolis Porto Alegre Brazil 2001

[25] L E Pelzer T Guardia A O Juarez and E Guerreiro ldquoAcuteand chronic antiinflammatory effects of plant flavonoidsrdquo Far-maco vol 53 no 6 pp 421ndash424 1998

[26] L R Ferguson ldquoRole of plant polyphenols in genomic stabilityrdquoMutation ResearchFundamental and Molecular Mechanisms ofMutagenesis vol 475 no 1-2 pp 89ndash111 2001

[27] P Markakis ldquoStability of anthocyanins in foodsrdquo in Antho-cyanins As Food Colors P Markakis Ed chapter 6 pp 163ndash180Academic Press 1982

[28] M S Narayan K A Naidu G A Ravishankar L Srini-vas and L V Venkataraman ldquoAntioxidant effect of antho-cyanin on enzymatic and non-enzymatic lipid peroxidationrdquoProstaglandins Leukotrienes and Essential Fatty Acids vol 60no 1 pp 1ndash4 1999

[29] K A Youdim A Martin and J A Joseph ldquoIncorporationof the elderberry anthocyanins by endothelial cells increasesprotection against oxidative stressrdquo Free Radical Biology andMedicine vol 29 no 1 pp 51ndash60 2000

[30] B Nair ldquoSustainable utilization of gum and resin by improvedtapping technique in some speciesrdquo in Harvesting of Non-WoodForest Products International Agro-Hydrology Research andTraining Center Menemen Turkey 2000

[31] G K Jayaprakasha and B S Patil ldquoIn vitro evaluation of theantioxidant activities in fruit extracts from citron and bloodorangerdquo Food Chemistry vol 101 no 1 pp 410ndash418 2006

[32] R Roesler L G Malta L C Carrasco R B Holanda C A SSousa and G M Pastore ldquoAtividade antioxidante de frutas docerradordquo Food Science and Technology (Campinas) vol 27 no1 pp 53ndash60 2007

[33] M A L Couto and S G Canniatti-Brazaca ldquoQuantification ofvitamin C and antioxidant capacity of citrus varietiesrdquo Cienciae Tecnologia de Alimentos vol 30 supplement 1 pp 15ndash19 2010

[34] P Stratil B Klejdus and V Kuban ldquoDetermination of phenoliccompounds and their antioxidant activity in fruits and cerealsrdquoTalanta vol 71 no 4 pp 1741ndash1751 2007

[35] L P Leong and G Shui ldquoAn investigation of antioxidantcapacity of fruits in Singapore marketsrdquo Food Chemistry vol76 no 1 pp 69ndash75 2002

[36] G M Dos Santos G A Maia P H M de Sousa J MC da Costa R W de Figueiredo and G M do PradoldquoCorrelacao entre atividade antioxidante e compostos bioativosde polpas comerciais de acaı (Euterpe oleraceaMart)rdquo ArchivosLatinoamericanos de Nutricion vol 58 no 2 pp 187ndash192 2008

[37] G S Nascimento and I R O Pereira ldquoAvaliacao da atividadeantioxidante das partes da fruta Cubiu (Solanum sessiliflorum)rdquoin Jornada de Iniciacao Cientıfica Universidade PresbiterianaMackenzie Sao Paulo Brazil 2011

[38] P C Ledur F Rogalski RMOliveira C PMostardeiro G F FS Montagner and I B M Cruz Determinacao da CapacidadeAntioxidante do Extrato Bruto de Solanum sessiliflorum (Cubiu)UNIFRA 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Chemical composition of cubiu (Solanum sessiliflorum) in 100 g of whole pulp [1ndash5]

Component Villachica (1996) [4] Pahlen (1977) [3] Andrade et al (1997) [1] Yuyama et al (1997 1998) [2 5]Units (g) 89 91 93 90Energy (kcal) 41 33 31 45Proteins (g) 09 06 mdash 09Lipids (g) mdash 14 mdash 19N-free extract (g) mdash 57 mdash 47Fiber (g) 02 04 mdash 09Ashes (g) 07 09 mdash 09Total sugars () mdash mdash 46 mdashReducing sugars () mdash mdash 39 1Nonreducing sugars () mdash mdash 18 1Soluble solids (∘Brix) mdash 5 8 mdashCitric acid () mdash mdash 08 mdashBrixacidity mdash mdash 59 mdashPhenolics (mg) mdash mdash 144 mdashTannins (mg) mdash mdash 142 mdash

coffee is ready to be consumed The fruit has a pulp colorranging from light yellow to yellowish cream measuringbetween 02 and 25 cm thick [6]

The pulp is the edible part of the fruit As food it isconsumed ldquonaturallyrdquo as strip-like alcoholic beverages jelliesjams juices and sauces [1 8] The macerated leaves of theplant are used by Peru-Brazil indigenous people to avoidthe formation of blisters in the event of skin burns whilethe juice from the fruitrsquos locular cavity is used to alleviatethe itching of spider bites [9] The pure juice is used for thecontrol of cholesterol diabetes excess uric acid and otheraffections caused by kidney and liver failure besides beingused to eliminate lice [9] Previous studies have shown thecomposition of cubiu as presented in Table 1

Cubiu is a juicy fruit with humidity between 88 and 93It has a variable soluble solids content (∘Brix) between 5 and8 of which the majority are reducing sugars [1] Since theratio of ∘Brix and acidity of the fruit is low it is consideredacidic This ensures a high dilution factor (great for juiceproduction) and little preference for fresh consumption(due to sourness of the fruit) The composition of phenoliccompounds is also low [1] Taking into consideration thenutritional value of cubiu (Table 2) it presents a high dietarypotential due to its low caloric and high fiber content Thissupports its indication in the diet of hypercholesterolemicand hyperglycemic patients [2]

In a study conducted in Norbert Wiener University inLima (Peru) it was found that administration of cubiuextract (40mLday) for three days decreased the level ofLDL triglycerides serum cholesterol and glucose How-ever it increased serum HDL levels in 100 volunteers ofboth sexes [10] Furthermore it was found that the fruithas a phenolic compound called tannin Phenolic com-pounds are products of secondary plant metabolism whichare essential for their growth and reproduction Tanninsare formed under conditions of stress such as infectioninjury and UV radiation [11] These also act as natural

second class antioxidants [12] According to their modeof action antioxidants can be classified into primary andsecondary Primary antioxidants act by donating hydrogenor electrons to free radicals therefore converting them intostable products andor reacting with free radicals forminglipid-antioxidant complexes that can react with another freeradical Secondary antioxidants act by delaying the initiationstep of autooxidation by different mechanisms includingmetal complexation sequestration of oxygen decompositionof hydroperoxides to form nonradical species absorption ofUV light or deactivation of singlet oxygen species [13]

The phenolic antioxidants interact preferentially with theperoxyl radical due to its prevalence in the autooxidationstep [15] This mechanism of action present in plant extractsplays an important role in reducing lipid oxidation in tissuesplants and animals because it preserves food quality andreduces the risk of developing diseases such as arteriosclerosisand cancer [16 17] Since antioxidants are of great relevancefor the field of medicine today there is a need to investigatethe antioxidant potential of cubiu and to elucidate the role ofthis fruit in the diet of the population

2 Material and Methods

21 Feedstock For this study cubiu fruits used were obtainedfrom the Horticultural Experimental Station Alejo von derPahlen from the National Institute of Amazonian Research(NIAR) located in Manaus AM The fruits were transportedin coolers to the Laboratory of Food and Nutrition at theNIAR where they were processed to obtain cubiu flour asdescribed by da Silva Filho et al (2005) [18]

22 Preparation of Cubiu Flour After the fruits werereceived the ripe intact fruits (free of cuts holes and otherdefects) were selected for processing Fruits were washedbrushed sanitized using a 200 ppm hypochlorite solution for





Shellseed

Washing

Bleaching

Pulping

Milling

Packing

Storing

Drying (65∘C)










) times 100 (1)





) times 100 (2)









2SO4 After cooling
















2SO4and




































50[31]

01020304050607080

0 200 400 600 800 1000

Inhi

bitio

n (

)

R2 = 099IC50 = 6063 plusmn 35



50values denote the










01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107







4 Conclusion




Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Shellseed

Washing

Bleaching

Pulping

Milling

Packing

Storing

Drying (65∘C)










) times 100 (1)





) times 100 (2)









2SO4 After cooling
















2SO4and




































50[31]

01020304050607080

0 200 400 600 800 1000

Inhi

bitio

n (

)

R2 = 099IC50 = 6063 plusmn 35



50values denote the










01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107







4 Conclusion




Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















) times 100 (2)









2SO4 After cooling
















2SO4and




































50[31]

01020304050607080

0 200 400 600 800 1000

Inhi

bitio

n (

)

R2 = 099IC50 = 6063 plusmn 35



50values denote the










01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107







4 Conclusion




Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























2SO4and




































50[31]

01020304050607080

0 200 400 600 800 1000

Inhi

bitio

n (

)

R2 = 099IC50 = 6063 plusmn 35



50values denote the










01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107







4 Conclusion




Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























50[31]

01020304050607080

0 200 400 600 800 1000

Inhi

bitio

n (

)

R2 = 099IC50 = 6063 plusmn 35



50values denote the










01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107







4 Conclusion




Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















01020304050607080

0 200100 400300 500 600

Inhi

bitio

n (

)


R2 = 09814IC50 = 2903 plusmn 107







4 Conclusion




Acknowledgments



References














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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