Food and Chemical Toxicology 53 (2013) 392–401

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Food and Chemical Toxicology

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Influence of drying and cooking process on the phytochemical content,antioxidant and hypoglycaemic properties of two bell Capsicum annum L. cultivars

Monica Rosa Loizzo a,⇑, Alessandro Pugliese a, Marco Bonesi a, Damiano De Luca b, Nora O’Brien c,Francesco Menichini a, Rosa Tundis a

a Department of Pharmacy, Health Sciences and Nutrition, University of Calabria, 87036 Rende (CS), Italyb CALAB, Laboratorio Chimico Merceologico della Calabria, I-87040 Montalto Uffugo (CS), Italyc Department of Food and Nutritional Sciences, University College Cork, Western Road, Cork, Ireland

a r t i c l e i n f o

Article history:Received 1 November 2012Accepted 7 December 2012Available online 22 December 2012

Keywords:Bell pepperPhytochemical contentApigeninFood processesAntioxidant activityHypoglycemic properties

0278-6915/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2012.12.011

⇑ Corresponding author. Tel.: +39 0984493071; faxE-mail address: mr.loizzo@unical.it (M.R. Loizzo).

a b s t r a c t

The present study evaluates the influence of drying and cooking processes on the health properties of twobell Capsicum annuum L. cultivars Roggiano and Senise compared with fresh peppers. The content of phy-tochemicals decreased in the order fresh > dried > dried frying processes. HPLC analysis was applied toquantify five flavonoids from peppers. Apigenin was identified as main constituent. Its content wasaffected by drying and dried frying processes. The antioxidant activity was evaluated by DPPH, ABTS,b-carotene bleaching test and Fe-chelating activity assay. A comparable radical scavenging activity wasobserved for both cultivars. Interestingly, frying process did not influenced this property. Roggiano pep-pers exhibited the highest antioxidant activity using b-carotene bleaching test with IC50 values of 38.1and 24.9 lg/mL for total extract and n-hexane fraction, respectively. GC–MS analysis of lipophilic fractionrevealed the presence of fatty acids and vitamin E as major components. In the inhibition of the carbo-hydrate-hydrolyzing enzymes fresh Senise peppers exerted the strongest activity against a-amylase withan IC50 value of 55.3 lg/mL. Our results indicate that C. annuum cultivars Roggiano and Senise have aninterestingly potential health benefits not influenced by processes that are used before consumption.

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1. Introduction

Epidemiological and clinical studies have shown that the intakeof vegetables and fruit plays an important role in the protection ofhuman health, reducing the risk of several age-related diseasesincluding cardiovascular diseases, cancers and diabetes type 2(Kritchevsky et al., 1998). Antioxidants have a long history of usein the nutrition/health community and food industry. The tradi-tional understanding has been that antioxidant chemicals promotehealth by removing reactive species that may otherwise exertharmful metabolic effects. By this view, most free radicals andreactive oxygen species (ROS) were considered to be harmful,implying that maximizing antioxidant concentrations could mini-mize the risk for chronic disease (Finley et al., 2011). Many dietarycompounds are capable of negating the danger of ROS: vitamins(particularly A and E), carotenoids, polyphenols and other phyto-chemicals (Mayne, 2003).

Diabetes mellitus (DM) is a group of metabolic disorder charac-terized by elevated blood glucose level resulting from the defects

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in insulin secretion, insulin action, or both, balance of glucosehomeostasis. Between 2010 and 2030, there will be a 69% increasein numbers of adults with diabetes in developing countries and a20% increase in developed countries. However, among the two ma-jor types of diabetes, type 1 and 2, type 2 DM is the commonestform (90–95% of patients) (American Diabetes Association, 2005).Recent studies have shown that the glucose-induced increased lev-els of mitochondrial ROS produced by the mitochondrial electrontransport chain seems to be the causal link between elevated levelsof glucose and the pathways responsible for hyperglycemia-induced vascular complications (Brownlee, 2005). Effective dietarystrategies can contribute to solutions for managing both hypergly-cemia and proper cellular redox status. One therapeutic approachfor treating diabetes in the early stage is to decrease post-prandialhyperglycemia. This is done by retarding the absorption of glucosethrough the inhibition of the carbohydrate-hydrolyzing enzymes,a-amylase and a-glucosidase, in the digestive tract. Inhibitors ofthese enzymes delay carbohydrate digestion and prolong overallcarbohydrate digestion time, causing a reduction in the rate ofglucose absorption and consequently blunting the post-prandialplasma glucose rise (Chiasson,2006; Chen et al., 2006). A maindrawback of currently used a-glucosidase and a-amylase

M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401 393

inhibitors such as acarbose is side effects caused by the excessiveinhibition of pancreatic a-amylase resulting in the abnormalbacterial fermentation of undigested carbohydrates in the colon.Therefore, in recent years, considerable attention has been directedtowards the study on the antioxidant and carbohydrate hydrolyz-ing enzymes inhibitors of fruits, vegetables and spices largelyconsumed.

Bell pepper (Capsicum annuum L.) is a non-pungent variety ofpepper which is the world’s second most important Solanaceousvegetable after tomato (Vengaiah and Pandey, 2007). It is eatenas raw, dried and cooked and also used commonly in makingpaste, pickle and sauce. Drying of peppers all over the world iscarried out by either sunlight or dryers using solar collectors(Doymaz and Pala, 2002). Most of the Italian pepper varietieshave an extremely limited geographic distribution and showadaptation to local pedo-climatic conditions (Nervo et al.,2004). Capsicum annuum cultivar Roggiano owes its peculiaritiesto the means of production remained unchanged over time. Thisvegetable growth in the middle valley of the Crati river (Calabria,Italy) and has a unique climate and soil conditions that conferorganoleptic qualities superior to those of other types of peppersproduced in the region. Roggiano pepper being awarded bothprotected geographical identification (IGP) and protected desti-nation of origin (DOP) denomination in 1996. Roggiano pepperis consumed fresh salad, fried with potatoes in the other and/or eggplant, and in others it is sun-dried and eaten during thewinter, and fried with turnip or broccoli or after dried processcut to pieces in the omelette or pasta with beans or chickpeas.Capsicum annuum cultivar Senise are among the most represen-tative products of the Lucanian region (Italy), typical of the cul-ture and gastronomical tradition of the valleys between the Sinniand Agri rivers. Senise pepper being awarded the IGP (ProtectedGeographical Indication) denomination in 1996. Senise peppersare sold both fresh and dried, threaded onto strings called‘‘serte’’, or ground into powder. The ‘‘serte’’ are made by thread-ing the peppers one by one from their stalk with a needle andarranging them spirally around the string until they between150 and 200 cm in length. Senise pepper may be eaten freshor dried with uses similar to Roggiano cultivar, while the powderis used in the preparation of the sausage. Both bell peppers areoften consumed dried after frying in olive oil. Dehydration is oneof the most widely used methods for vegetables preservation. Itsmain objective is the removal of water to the level at whichmicrobial spoilage and deterioration reactions are minimized.However, it is well known that during hot-air drying, vegetablesundergo physical, structural, chemical and nutritional changesthat can affect quality attributes like texture, color, flavour andnutritional value (Di Scala and Crapiste, 2008). Reports on the ef-fects of cooking on the antioxidant compounds in vegetableshave been inconclusive. There are reports demonstrating anenhancement or no change in antioxidant activity of vegetables(Gahler et al.,2003; Turkmen et al., 2005) while others have indi-cated a deterioration of activity after thermal treatment (Ismailet al., 2004; Zhang and Hamauzu, 2004).

To our knowledge, there are no published reports on the influ-ence of drying and cocking process on chemical composition andhypoglycemic properties of these bell peppers largely consumedin Italy. Therefore we focused our interest on the determinationof the total phenols, flavonoids and carotenoids content, HPLCflavonoids profile, GC–MS analysis of lipophilic fractions and theantioxidant capacity of bell peppers using different in vitro assays:2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging as-say, antioxidant capacity determined by radical cation (ABTS�+),b-carotene bleaching test and the Fe2+-metal chelating assay. Inorder to evaluate the hypoglycaemic potential, a-amylase anda-glucosidase inhibition assays were also performed.

2. Experimental

2.1. Chemicals and reagents

The following chemicals were obtained from Sigma–AldrichS.p.a. (Milan, Italy): potato starch, sodium phosphate, sodium chlo-ride, a-amylase from porcine pancreas (EC 3.2.1.1), a-glucosidasefrom Saccharomyces cerevisiae (EC 3.2.1.20), maltose, sodium ace-tate, sodium potassium tartrate, potassium hydrogen carbonate,3,5-dinitrosalicylic acid, o-Dianisidine Color Reagent (DIAN), PGOEnzymes Solution (PGO), Acetic acid, perchloric acid, basic bismuthnitrate, potassium iodide, phosphoric acid, sodium hydroxyde,HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid),sodium phosphate monobasic, chlorogenic acid, quercetin,kaempferol, luteolin, rutin, apigenin, anhydrous sodium sulfate,ascorbic acid, propyl gallate, 2,2-diphenyl-1-picrylhydrazyl (DPPH),Folin–Ciocalteu reagent, Tween 20, sodium phosphate buffer,sodium potassium tartrate tetrahydrate, tripyridyltriazine (TPTZ),2,20-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)solution, Trolox, potassium persulphate, b-carotene, Tween 20,linoleic acid, FeCl2, ferrozine. Acarbose from Actinoplanes sp. wasobtained from Serva (Heidelberg, Germany). Methanol, ethanol,n-hexane, chloroform, sodium sulfate, dimethylsulphoxide (DMSO),H2SO4, chloroform, sodium carbonate, sodium nitrite, aluminumtrichloride, NaOH, perchloric acid, HCl, NaOH, were obtained fromVWR International s.r.l. (Milan, Italy).

2.2. Pepper

The fruits of C. annuum cultivars Roggiano and Senise used inthis study were purchased in the local market in Calabria and Basil-icata (Italy) during August–September 2009. These cultivars at thefully maturity stage are characterized by a length of about 11–14and 12–17 cm for Senise and Roggiano peppers, respectively. Pep-pers were examined for integrity and absence of dust and insectcontamination. Samples were divided into three different groups:a) fresh, b) sun-dried, c) sun-dried and cooked by frying in extravirgin olive oil following the traditional cooking process.

2.3. Drying and frying process

Bell peppers selected for group b) and c) were sun-dried at 30–35 �C for two weeks. For frying process extra virgin olive oil(300 mL) was placed into a non-stick frying pan (diameter 20 cm)and heated. Temperature was 170 ± 5 �C to achieve uniform cookingwithout external burning. Frying time ranged from 5 to 8 min sincepeppers were browned. Fried peppers were then placed in a cleandry grill for 5 min, allowing for the excess oil. After each frying oper-ation, the frying pan was thoroughly cleaned and the used oil was re-placed with fresh. Samples group c) were taken for analysis.

2.4. Extraction procedure

Fresh, dried and fried bell peppers (200 g) were extracted byethanol (350 mL) at room temperature (48 h). Extraction proce-dure was repeated for 3 times. The ethanol solutions were com-bined and dried to obtain the total extract. In order to operate aseparation of non polar compounds, ethanol extract was solubi-lized with MeOH/H2O (8:2) and extracted with n-hexane to obtaina lipophilic fraction. Yields are reported in Table 1.

2.5. Determination of total phenol content

The amount of total phenol in pepper samples was determined bythe Folin–Ciocalteu method (Gao et al., 2000). Briefly, the extract

Table 1Extraction yield and phytochemicals content of C. annuum Roggiano and Senise cultivars.

Ethanol extracta n-Hexane fractiona Phenolsb Flavonoids Carotenoidse

Total contentc Apigenind

Roggiano cultivarFresh pepper 4.5 ± 0.9 0.05 ± 0.006 127.5 ± 3.1 46.0 ± 2.1 26.1 ± 0.6 2.5 ± 0.8Dried pepper 0.6 ± 0.05 0.3 ± 0.04 116.7 ± 3.7 44.5 ± 2.3 17.4 ± 0.5 2.0 ± 0.6Dried frying pepper 20.4 ± 1.8 13.6 ± 1.2 15.0 ± 1.1 15.0 ± 0.4 4.7 ± 0.1 1.5 ± 0.5

Senise cultivarFresh pepper 8.4 ± 0.7 0.1 ± 0.03 224.5 ± 4.2 48.5 ± 3.0 13.4 ± 0.3 6.5 ± 0.7Dried pepper 9.8 ± 0.8 0.5 ± 0.06 195.5 ± 3.7 37.0 ± 2.1 11.2 ± 0.3 6.0 ± 0.8Dried frying pepper 21.5 ± 1.6 10.3 ± 1.1 24.5 ± 1.1 11.5 ± 0.9 5.4 ± 0.1 4.0 ± 0.8

Data are expressed as mean ± S.D. (n = 3).a Extraction yield (%).b mg chlorogenic acid equivalents/g extract.c mg quercetin equivalents/g extract.d mg/g extract.e mg b-carotene equivalents/g extract.

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was mixed with 0.2 mL Folin–Ciocalteu reagent, 2 mL of distilledwater and 1 mL of 15% Na2CO3. The absorbance was measured at765 nm using a UV–Vis Jenway 6003 spectrophotometer after 2 hincubation at room temperature. The levels of total phenols contentwere determined in triplicate. Chlorogenic acid was used as a stan-dard and the total phenols content was expressed as chlorogenicacid equivalents in mg per g extract (Table 1).

2.6. Determination of total flavonoid content

The flavonoids content was determined spectrophotometricallyusing a method based on the formation of a flavonoid-aluminumcomplex (Yoo et al., 2008). One milliliter of the extract was addedto a 10 mL volumetric flask. Distilled water was added to make avolume of 5 mL. At zero time, 0.3 mL of 5% (w/v) sodium nitritewas added to the flask. After 5 min, 0.6 mL of 10% (w/v) AlCl3

was added and then at 6 min 2 mL of 1 M NaOH were also addedto the mixture, followed by the addition of 2.1 mL distilled water.Absorbance at 510 nm was read immediately. Quercetin was cho-sen as a standard and the levels of total flavonoid content weredetermined in triplicate and expressed as quercetin equivalentsin mg per g extract (Table 1).

2.7. Determination of total carotenoid content

The total carotenoid content was determined by measuring theabsorption of lipophilic fractions at k = 460 nm (Gao et al., 2000).Triplicate aliquots of fractions were analyzed spectrophotometri-cally in a UV–vis Jenway 6003 spectrophotometer. One ml of thelipophilic layer (0.1 g/mL) was added to 0.5 mL of 5% NaCl, vor-texed for 30 s and centrifuged for 10 min at 4500 rpm. The super-natant (100 lL) was diluted with 0.9 mL of n-hexane andmeasured. b-Carotene was used as a standard. The total carote-noids contents were determined in triplicate and expressed asb-carotene equivalents in mg per g extract (Table 1).

2.8. Flavonoids HPLC analysis

Flavonoid analysis was performed using the slightly modifiedmethod previously described (Tundis et al., 2011). Briefly, bell pep-per extract was hydrolyzed (2.8 M HCl in 60% MeOH (v/v) at 90 �Cfor 10 min). The residue was treated with ethyl acetate three times,dried by N2 and dissolved again in 40% MeOH (v/v) for HPLC anal-ysis. HPLC analyses were realized using an HPLC system HP 1100equipped with a pump, UV–vis detector (280 nm), column oven,injector and a C18 RP column (Phenomenex Luna 5 lm C18,

250 � 4.60 mm). The mobile phase was H2O/formic acid (0.1%)(A) and methanol (B) with a flow rate of 1 mL min�1 (2 min 100%A; 8 min 80% A; 55 min 100% B; 65 min 100% A). Quercetin, luteo-lin, rutin, apigenin and kaempferol were selected as markers.

2.9. GC–MS analysis

Pepper n-hexane fractions were analyzed by Gas Chromatogra-phy-Mass Spectrometry (GC–MS). GC–MS analyses were carriedout using a Hewlett–Packard 6890 gas chromatograph equippedwith an HP-5 non polar capillary column (30 m length, 0.25 mmi.d., 0.25 lm film thickness) and interfaced with a Hewlett Packard5973 Mass Selective. Ionization of the sample components wasperformed in electron impact mode (EI, 70 eV). The carrier gaswas helium (1 mL/min) and the analytical conditions were as fol-low: oven temperature was 5 min isothermal at 50 �C, then 50–250 �C at a rate of 5 �C/min; then held isothermal for 10 min. Theinjector and detector temperatures were 250 and 280 �C, respec-tively. For analysis, lipophilic fractions were dissolved in acetone(ca. 1 mg/mL) and aliquots (1 lL) were directly injected. Constitu-ents were tentatively identified by gas chromatography by com-parison of their retention indices with those of the literature orwith those of authentic compounds available in our laboratory.Further tentative identification was made by comparison of theirmass spectra with those stored in Wiley 138, Wiley 275 and NIST98 libraries.

2.10. DPPH assay

This experimental procedure was described by Loizzo et al.(2012). In an ethanol solution of DPPH radical (final concentrationwas 1.0 � 10�4 M), extracts at different concentrations wereadded. The reaction mixtures were shaken and kept in the darkfor 30 min. The absorbance of the resulting solutions was mea-sured in 1 cm cuvettes using a Perkin Elmer Lambda 40 UV/VISspectrophotometer at k = 517 nm against blank without DPPH. Adecrease in the absorbance of the DPPH solution indicates an in-crease of DPPH radical scavenging activity.

2.11. ABTS test

ABTS assay was based on the method previously described byLoizzo et al. (2012) with slight modifications. ABTS radical cation(ABTS+) was produced by the reaction of a 7 mM ABTS solutionwith 2.45 mM potassium persulphate. The mixture was stored inthe dark at room temperature for 12 h before use. The ABTS+

M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401 395

solution was diluted with ethanol to an absorbance of 0.70 ± 0.05at k = 734 nm. After addition of 25 lL of pepper extract or Troloxstandard to 2 mL of diluted ABTS+ solution, absorbance was mea-sured at exactly 6 min after mixing. Appropriate solvent blankswere run in each assay. The scavenging ability of sample was cal-culated according to the following equation:

ABTS scavenging activity ð%Þ ¼ ½ðA0 � AÞ=A0� � 100

where A0 is the absorbance of the control reaction and A is theabsorbance in the presence of samples.

2.12. b-carotene bleaching test

Antioxidant activity was determined using the b-carotenebleaching test with some modifications (Menichini et al., 2009).Briefly, 1 mL of b-carotene solution (0.2 mg mL�1 in chloroform)was added to 0.02 mL of linoleic acid and 0.2 mL of 100% Tween20. After evaporation of chloroform and dilution with water(100 mL), 5 mL of the emulsion were transferred into different testtubes containing 0.2 mL of samples in 70% ethanol at different con-centrations. Propyl gallate at the same concentration of sampleswas used as standard. The tubes were shaken and placed at 45 �Cin a water bath for 60 min. The absorbance of the samples, stan-dard and control was measured at k = 470 nm against a blank con-sisting of an emulsion without b-carotene. The measurement wascarried out at t = 0 and successively at 30 and 60 min. All sampleswere assayed in triplicate and the mean value calculated.

2.13. Fe2+ chelating activity assay

The chelating activity of total extracts and lipophilic fractionsfor ferrous ions Fe2+ was measured according to the method of Di-nis et al. (1994). Briefly, to 0.5 mL of sample, 1.6 mL of deionizedwater and 0.05 mL of FeCl2 (2 mM) was added. After 30 s, 0.1 mLferrozine (5 mM) was added. Ferrozine reacted with the divalentiron to form stable magenta complex species that were very solu-ble in water. After 10 min at room temperature, the absorbance ofthe Fe2+-ferrozine complex was measured at k = 562 nm. The che-lating activity of the extract for Fe2+ was calculated using equation:

Chelating rate ¼ ðA0 � A1Þ=A0 � 100

where A0 was the absorbance of the control (blank, without sample)and A1 was the absorbance in the presence of the sample.

2.14. a-Amylase inhibitory activity

The a-amylase inhibition assay was performed using the meth-od previously described (Tundis et al., 2007). Briefly, a starch solu-tion (0.5% w/v) was obtained by stirring 0.125 g of potato starch in25 mL of 20 mM sodium phosphate buffer with 6.7 mM sodiumchloride, pH 6.9 at 65 �C for 15 min. The a-amylase solution wasprepared by mixing 0.0253 g of a-amylase in 100 mL of cold dis-tilled water. Samples were dissolved in buffer to give final concen-tration from 12.50 to 1 lg/mL. The colorimetric reagent wasprepared mixing a sodium potassium tartrate solution (12.0 g ofsodium potassium tartrate, tetrahydrate in 8.0 ml of 2 M NaOH)and 96 mM 3,5-dinitrosalicylic acid solution. Control, total extractsand lipophilic fractions were added to starch solution and left toreact with a-amylase solution at 25 �C for 5 min. The reactionwas measured over 3 min. The generation of maltose was quanti-fied by the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, the product being detectable at k = 540 nm. Inthe presence of an a-amylase inhibitor less maltose will be pro-duced and the absorbance value would decrease.

2.15. a-Glucosidase inhibitory activity

The a-glucosidase inhibition was measured through a modifiedSigma–Aldrich bioassay method (Loizzo et al., 2008). A maltosesolution (4% w/v) was prepared by dissolving 12 g of maltose in300 mL of 50 mM sodium acetate buffer. The enzyme solutionwas prepared by mixing 1 mg of a-glucosidase (10 units/mg) in10 mL of ice-cold distilled water. Samples were dissolved in thebuffer to give a final concentration ranging from 5 to 1 lg/mL.The DIAN solution was prepared by dissolving 1 tablet in 25 mLof distilled water, while PGO system-color reagent solution wasprepared fresh by dissolving 1 capsule in 100 mL of ice-cold dis-tilled water. In the first step both control and samples were addedto maltose solution and left to equilibrate at 37 �C. The reactionwas started by adding a-glucosidase solution and tubes were leftto incubate at 37 �C for 30 min. After this time perchloric acid solu-tion (4.2% w/v) was added to stop reaction. In the second step thegeneration of glucose was quantified by the reduction of DIAN. Thesupernatant of tube of step one was mixed with DIAN and PGO andwas left to incubate at 37 �C for 30 min. The absorbance of DIANwas measured at k = 500 nm.

2.16. Statistical analysis

All experiments were carried out in triplicate. Data were ex-pressed as means ± standard deviation (S.D.). The concentrationgiving 50% inhibition (IC50) was calculated by nonlinear regressionwith the use of Prism GraphPad Prism version 4.0 for Windows(GraphPad Software, San Diego, CA, USA). The dose–response curvewas obtained by plotting the percentage inhibition versus concen-tration. Differences within and between groups were evaluated byone-way analysis of variance test (ANOVA) followed by a multi-comparison Dunnett’s test compared with the positive controls.

3. Results and discussion

3.1. Phytochemical analysis

As reported in Table 1, the extraction yields of Roggiano and Se-nise bell peppers were in the range of 0.6–21.5% for total extractand 0.05–13.6% for n-hexane fractions. Peppers are a good sourceof several health-promoting compounds including phenols, flavo-noids, carotenoids, vitamins and capsaicinoids. The content ofextractable phenol compounds was analyzed by Folin–Ciocalteumethod and expressed as mg/g extract. Both fresh and dried Rog-giano peppers exhibited a comparable phenol content of 127.5and 116.7 mg/g extract, respectively. A drastically reduction wasobserved after frying process with a value of 15 mg/g extract. Asimilar decrease was observed with Senise cultivar where the esti-mated total phenol content is 224.5 and 24.5 mg/g extract in freshand fried peppers, respectively. Processes influenced also the totalflavonoids content (Table 1).

On the basis of our experience and a perusal analysis of litera-ture we decided to quantified by HPLC the most common agly-cones recognized in pepper fruits (Howard et al., 2000; Tundiset al., 2011; Kim et al., 2011; Bae et al., 2012). Among selectedflavonoids as markers (quercetin, luteolin, rutin, apigenin andkaempferol) apigenin content was found in the range 26.1–4.7 mg/g extract for Roggiano peppers and 13.4–5.4 mg/g extractfor Senise peppers. Quercetin and luteolin are present in trace inboth fresh bell peppers. A twofold content of apigenin was foundin fresh Roggiano peppers respect to Senise peppers. Interestingly,this flavone is not identified in hot peppers (Tundis et al., 2011; Baeet al., 2012; Tundis et al., 2012). On the contrary apigenin wasidentified in Malaysian bell peppers (272 mg/kg) (Miean and

Table 2Fatty acids composition (abundance %) of bell peppers.

C.annuum

PA PAM PAE SA LA LAM LAE LN LNM LNE

RoggianoFresh 12.6 9.6 1.2 1.2 1.4 10.0 10.8 0.3 10.2 10.0Dried 11.1 10.5 0.6 0.8 0.8 10.3 12.5 0.4 12.8 11.6Dried

frying13.9 14.8 1.9 1.6 3.0 12.8 15.8 2.3 16.7 13.2

SeniseFresh 1.9 1.3 5.4 0.8 1.9 0.2 12.3 1.4 0.3 4.9Dried 1.6 1.9 6.1 1.9 2.4 0.3 15.3 1.9 0.2 4.4Dried

frying2.9 3.4 4.7 1.4 3.5 1.9 19.9 4.5 1.7 8.7

PA, palmitic acid; PAM, palmitic acid methyl ester; PAE, palmitic acid ethyl ester;SA, stearic acid; LA, linoleic acid; LAM, linoleic acid methyl ester; LAE, linoleic acidethyl ester; LN, linolenic acid; LNM, linolenic acid methyl ester; LNE, linolenic acidethyl ester.

396 M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401

Mohamed, 2001). It was demonstrated that drying processes maya) enhance the enzymatic degradation, b) determine the decompo-sition/combination of phenols with other plant components, thuslower the amount of phenolic compounds (Miean and Mohamed,2001). This evidence is clearly visible also in our results. Cookingmethods alter positively the sensory attributes of pepper but cangreatly affect their contents of bio-functional compounds. Effectof cooking process evaluated by our research group is in disagree-ment with Turkmen et al. (2005) that reported how several cook-ing method including boiling, steaming and microwaving couldincrease the phenols content in peppers. Increments of phenoliccontent of pepper by cooking have been attributed to dehydrationof food matrix and an improved extractability of phenols from thefood (Schweiggert et al., 2006). Cooking is able to inactivate thepolyphenol oxidase enzyme during heating, leading to the inhibi-tion of phenols degradation. However, significant losses of phenolcompounds by household cooking or industrial heat processinghave been reported in peppers (green, yellow, and red) and othervegetables by Chuah et al. (2008).

Fried foods remain very popular world-wide due to their uniqueand delicious sensory characteristics (Gertz, 2000). Despite thecommon belief, frying process is considered to have almost thesame or even less effect on nutrient losses compared to other cook-ing methods (Bognar, 1998). Furthermore, the nutritive value offood increases due to the absorption of frying oils, which are richin unsaturated fatty acids and vitamin E (Andrikopoulos et al.,1989; Fillion and Henry, 1998). Extra virgin olive oil is uniqueamong cooking oils because of its high monounsaturated fatty acidcontent and the presence of health-promoting constituents,namely polyphenols, terpenoids, squalene and tocopherols (Owenet al., 2000). During frying the condensation caused by water losstogether with the absorption of extra virgin olive oil are expectedto increase the polyphenol concentrations in fried vegetables,while on the other hand the loss of polyphenols due to oxidationis expected to have the opposite result. In our samples, frying pro-cess drastically loss polyphenols originally present in peppers. Theabove mentioned results are in disagreement with those reportedby Kalogeropoulos et al. (2007) that reported an overall polyphe-nols retention of 38.5% for green fried pepper. However in Roggi-ano cultivar these phytochemicals are reduced by frying processto 0.35 mg/g extract. This evidence is in disagreement with Ewaldet al. (1999) that reported how boiling, microwaving, frying, didnot affect the levels of polyphenols. Both cultivars were tradition-ally air-dried before consumption. Dried process did not signifi-cantly influenced the bell pepper phenol content.

Peppers are also a good source of carotenoids, which can vary incomposition and concentration owing to differences in geneticsand maturation (Markus et al., 1999). The pigments capsanthin,capsanthin 5,6-epoxide, and capsorubin were exclusive to the Cap-sicum genus. In particular, Ha et al. (2007) analyzed the accumula-tion of carotenoids in Capsicum fruits at fully ripen maturity stageconcluding that capsanthin was found to be the major component(80%) of the total carotenoids in the ripe fruits of red peppers. Thelevels of other b-carotenoids including b-carotene, b-cryptoxan-thin, and zeaxanthin varied from 1.5% to 16%, and that of luteinwas never detected above 0.3% of the total carotenoid content inmost of the red peppers. The total carotenoid content was deter-mined by measuring the absorption of lipophilic fractions at460 nm. b-Carotene was used as a standard. In fact, Suzuki et al.(2007) reported that normally significant positive correlationswere observed between b-carotene and capsanthin contents. Basedon these observations, irrespective of the total quantity of carote-noids and their relative proportion of capsanthin in C. annuumfruit, it is assumed that the ratio of b-carotene to capsanthin is con-stant and the ratio of 1:10 could thus potentially be used as an in-dex to analyze the content of these carotenoids. Senise cultivar

showed the highest total carotenoid content with values of 6.5,6.0 and 4.0 mg/g extract for fresh, dried and fried peppers, respec-tively. Both technological process (drying and frying) did not sig-nificantly reduced the amount of these phytochemicals. Perusalanalysis of literature revealed that significant differences inb-carotene content among bell pepper cultivar are evident (Deepaet al., 2006). The high or low carotenoid content for a given cultivardepends upon on various factors: the level of expression of thegenes governing carotenogenesis, physiological and morphologicalcharacteristics intrinsic to the cultivar, and pedo-climatic growthconditions. All these factors taken together may influence theperformance of a cultivar with respect to phytochemical content.Moreover, carotenoids have been shown to vary with cultivars,maturity and processing conditions (Howard et al., 2000; Daoodet al., 2006). In particular, Chuah et al. (2008) reported that cookingdid not affect the carotenoid content since it is stable for up 30 minin the cooked tissue also if boiling process is applied. Recently,Ornelas-Paz et al. (2011) reported that carotenoid losses were sig-nificantly higher in non pungent (31–53%) than in pungent peppers(3–24%). This evidence was supported by a research of Daood et al.(2006) that demonstrated how the capsaicinoids in pungent varie-ties of peppers can affect the chemical interactions of carotenoidsin the tissues of pungent peppers and thereby provide substantialprotection against thermal destruction. In agreement with Orn-elas-Paz et al. (2011) capsaicinoids were not detected in Seniseand Roggiano cultivars (method of analysis not reported). So theycould not influence the losses of carotenoids during cooking.

The n-hexane fraction of both bell pepper cultivars was ana-lyzed by GC–MS. Results are reported in Table 2. C. annuum cultivarRoggiano was characterized by palmitic acid, methyl and ethyllinoleate and methyl and ethyl linolenate as major constituents.Ethyl linoleate, ethyl linolenate and ethyl palmitate were identifiedas most abundant fatty acids in Senise n-hexane fraction. Compar-ing fresh bell peppers, Roggiano cultivar was characterized by amajor content of palmitic acid and its methyl ester, methyl linole-ate, methyl linolenate, ethyl linolenate. Interestingly, both driedfrying samples are characterized by the highest linoleic acid withpercentage of 3.0% and 3.5% for Roggiano and Senise cultivars,respectively. Linoleic acid is an unsaturated x-6 fatty acid and isthe most abundant polyunsaturated fatty acid in the diet. A num-ber of studies have shown that diabetics require higher than nor-mal intakes of C18:2. Because diabetics have consistently beenshown to have above normal levels of C18:2 while having lowerthan normal levels of c-linolenic acid, it is believed that diabeticshave impaired D-6-desaturase activity. Moreover, several investi-gation demonstrated that linoleic acid from food could attenuatediabetic complications since it improve insulin sensitivity (Davìet al., 2010).

Table 3Antioxidant and metal chelating activity of Roggiano and Senise bell peppers.

DPPH Assay (IC50 lg/mL) b-Carotene bleaching test (IC50 lg/mL) ABTS Assay (TEAC value) Fe2+ chelating activity (IC50 lg/mL)

30 min 60 min

Fresh pepperRoggianoEtOH 58.0 ± 1.8** 196.2 ± 2.8** >1000 1.8 ± 0.7** 65.2 ± 2.8**

n-Hexane 75.3 ± 2.8** 105.7 ± 2.2** 308.9 ± 2.4** 8.9 ± 0.9** 189.1 ± 3.7**

SeniseEtOH 55.0 ± 1.8** 143.9 ± 1.6** 258.9 ± 4.1** 2.4 ± 0.8** 36.7 ± 1.8**

n-Hexane 75.8 ± 2.8** 105.7 ± 2.5** 668.9 ± 2.7** 11.5 ± 0.9** 88.7 ± 4.0**

Dried pepperRoggianoEtOH 55.1 ± 1.9** 38.1 ± 1.5** 155.6 ± 2.8** 5.7 ± 0.9** 131.4 ± 2.8**

n-Hexane 186.4 ± 3.4** 24.9 ± 1.1** 178.0 ± 3.1** 12.5 ± 1.1** 189.1 ± 3.7**

SeniseEtOH 52.1 ± 1.9** 43.7 ± 1.9** 298.6 ± 3.9** 12.6 ± 1.1** 90.4 ± 3.8**

n-Hexane 181.4 ± 3.4** 26.94 ± 1.9** 63.6 ± 1.8** 21.4 ± 1.5** 153.7 ± 4.4**

Dried frying pepperRoggianoEtOH 61.1 ± 2.7** 493.1 ± 3.3** 497.8 ± 3.8** 10.4 ± 1.7** 142.9 ± 3.1**

n-Hexane 248.9 ± 1.8** 177.0 ± 2.8** 797.4 ± 3.5** 24.2 ± 3.9** 270.8 ± 6.1**

SeniseEtOH 60.1 ± 2.7** 97.6 ± 1.6** 102.1 ± 2.1** 27.4 ± 2.7** 211.8 ± 3.5**

n-Hexane 251.6 ± 1.8** 177.0 ± 1.8** 248.6 ± 1.6** 48.9 ± 2.9** 270.4 ± 5.1**

Positive controlAscorbic acid 5.0 ± 0.8 – – 0.96 ± 0.03 –Propyl gallate – 1.0 ± 0.01 1.0 ± 0.01 – –EDTA – – – 1.27 ± 0.05

Data are expressed as mean ± S.D. (n = 3). Differences within and between groups were evaluated by one-way analysis of variance test ⁄⁄⁄p < 0.0001 followed by a multi-comparison Dunnett’s test.** p < 0.01 compared with the positive controls: ascorbic acid, BHT and EDTA.

M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401 397

Generally, in our bell pepper samples frying process resulted ina higher concentration of fatty acids and their methyl and ethyl es-ters. In fact, as evidenced in literature, the increase in the lipid con-tent through frying process may reflect the uptake of vegetable oilcomponents. Cooking oil mixed with pepper tissues and added ex-tra to the total lipid value. The higher fatty acids content could berelated to the more and longer time associated with cooking oil(Xiao and Babb, 2007).

Both fresh and dried Roggiano peppers are also characterized bya high vitamin E content with a percentage of 4.3% and 4.8%,respectively while a content of 0.9% and 1.0% was found for freshand dried pepper from Senise cultivar. Vitamin E could act as anti-oxidant and scavenger of hydroxyl radicals. The controversialquestion of research community, of course, is whether this chem-ical ‘antioxidant’ potential of tocopherols is relevant to their phys-iological action (Brigelius-Flohé and Davies, 2007).

3.2. Antioxidant activity

Reactive oxygen species (ROS), including superoxide anion rad-ical (O2��), hydroxyl radical (�OH), Organic oxygen radical (RO� andROO�), singlet oxygen (1O2), hydrogen peroxide (H2O2) and organicperoxide (ROOH), possess strong bioactivity. It has been found thatfree radicals are closely related to many pathological and physio-logical phenomenon, such as aging, tumor, inflammation, muta-tion, atherosclerosis, cardiovascular, cerebral ischemia anddiabetes. DPPH� and ABTS� are stable free radicals, which have beenwidely accepted as a tool for estimating free radical scavengingactivities of antioxidants (Krishnaiah et al., 2010). All samples werecapable of scavenging both DPPH� and ABTS�� radicals in a concen-tration dependent-manner (Table 3, Fig. 1). Pepper total extractsexhibited the highest activity. In particular, IC50 values of 58.0and 55.0 lg/mL were estimated for fresh Roggiano and Senise cul-tivar while IC50 values of 55.1 and 52.1 lg/mL were found for driedRoggiano and Senise peppers. Frying dried pepper did not signifi-

cantly reduce the ability of samples to scavenge the DPPH� radical.A lower activity was observed with n-hexane fractions (valuesranging from 75.3 to 251.6 lg/mL for fresh Roggiano peppers andfried Senise peppers). A radical scavenging activity against ABTS�

radical comparable with positive control was obtained with the to-tal extract of both fresh peppers (TEAC value 1.8 and 2.4 for Roggi-ano and Senise cultivars, respectively).

Drying and frying process drastically reduced the ability of sam-ples to scavenge the ABTS� radical. Our results are in disagreementwith Arslan and Ozcan (2011) that reported how the antioxidantactivity of red bell pepper from Turkey was significantly affectedby the drying conditions. In particular, the TEAC value was5564.60 and 1733.33 lmol TEAC 100/g for dried and raw pepper.The DPPH radical scavenging capacity of the dried and raw pepperwas 76.14% and 48%. Previously Deepa et al. (2006) reportedthe DPPH radical scavenging activity of some red bell peppercultivar founding a percentage of inhibition from 25% to 72% forParker and Flamingo pepper cultivars, respectively. A lowerpercentage of inhibition against DPPH radical was calculated forbell pepper cultivar Arian, Marona, Zorro, Y-43-09 and Y-43-07(Ghasemnezhad et al., 2011). Matsufuji et al. (2007) revealed thatamong analyzed five different bell pepper ‘‘Signal Red’’ bell pepperthat possessed higher levels of carotenoids and a-tocopherolexhibited the strongest radical scavenging activity. It has beenreported that a-tocopherol, ascorbic acid and carotenoids reactwith DPPH, and the order of the activity according to the reportswas: carotenoids � ascorbic acid >a-tocopherol, although thebioactivity of carotenoids was affected by the chemical structure(Jiménez-Escrig et al., 2000).

The potential of bell peppers to inhibit lipid peroxidation wasevaluated using the b-carotene/linoleic acid bleaching test, whichmeasures the capacity for inhibiting the conjugated diene hydro-peroxides formation upon linoleic acid oxidation. The n-hexanefraction exhibited the highest antioxidant activity with IC50

values of 24.9 and 26.9 lg/mL for Roggiano and Senise cultivar,

Fig. 1. Radical scavenging activity by DPPH test of fresh and processed C. annuum Roggiano (A) EtOH extract and (B) n-hexane fraction and Senise (C) EtOH extract and (D) n-hexane fraction. Data are mean ± S.D. (n = 3).

Fig. 2. Effect of fresh and processed C. annuum Roggiano (A) EtOH extract and (B) n-hexane fraction and Senise (C) EtOH extract and (D) n-hexane fraction on Fe2+-ferrozineformation. Data are means of percentage of inhibition of chromogen formation ± SD (n = 3).

398 M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401

M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401 399

respectively, at 30 min of incubation. Frying process drastically re-duced this ability since the measured IC50 value for Roggiano pep-per was 797.4 lg/mL at 60 min of incubation. An interestinginhibition of the discoloration of b-carotene was observed alsowith total extract of both dried Roggiano and Senise cultivar withIC50 values of 38.1 and 43.7 lg/mL, respectively.

Previously, we have investigated dried C. annuum var. acumina-tum and C. annuum var. cerasiferum founding a remarkable inhibi-tion of linoleic acid oxidation with IC50 values of 17.2 and 10 lg/mLat 60 min, respectively (Tundis et al., 2011). The ability of driedpeppers to inhibit linoleic acid oxidation could be attributed tocarotenoids and capsaicinoids content. This pungent componentscan inhibit iron mediated lipid peroxidation and copper-dependentoxidation of low-density lipoprotein, an effect ascribed to theircapacity to form complexes with reduced metals and act as hydro-gen donors (Rosa et al., 2002). The lower bioactivity exhibited byour samples in b-carotene bleaching test respect to the previouslyinvestigated pepper could be explained by the absence of capsaici-noids in our samples.

Transition metals Cu2+ and Fe2+ are involved in oxidative reac-tions. The Fe2+-chelating activity was determined by measuringthe formation of the Fe2+-ferrozine complex, and these activitieswere compared with the chelating activity of the synthetic metalchelator ethylenediamine tetra-acetic acid (EDTA). Generally totalextracts, rich in phenols, exhibited a remarkable metal chelatoractivity (Fig. 2). Fresh Senise total extract exhibited the highestactivity with an IC50 value of 36.7 lg/mL. A lower activity was ob-served after drying (IC50 value of 90.4 lg/mL) and frying (IC50 valueof 211.8 lg/mL) process. It is well known accepted that phenolcompounds are effective metal chelators. Because iron-mediateddamage to biomolecules such as lipids and DNA is implicated indisease development, the iron-chelating mechanism of polyphenolantioxidant activity must be fully explored in addition to radicalscavenging to understand polyphenol antioxidant behavior (Perronand Brumaghim, 2009).

Previously Sim and Sil (2008) analyzed the chelating activitiesof both red pepper pericarp and seed extracts on ferrous ionsfounding a chelating activities of 34% and 6% for pericarp and seed,respectively at 2.5 lg/mL. It was reported that chelating agents,which form r-bonds with metals, are effective as secondary anti-oxidants because they reduce the redox potential, thereby stabiliz-ing the oxidized form of metal ions.

3.3. Carbohydrate-hydrolyzing enzymes inhibition

In our continuous search of hypoglycaemic properties of spicesincluding peppers, in this work we analyzed for the first time theability of bell peppers to inhibit carbohydrate-hydrolyzingenzymes a-amylase and a-glucosidase. All samples exhibited

Table 4Carbohydrate hydrolyzing enzymes activity (IC50 lg/mL) of C. annuum Roggiano and Senis

Fresh pepper Dried pepp

a-Amylase a-Glucosidase a-Amylase

RoggianoEtOH 68.9 ± 1.8** 65.9 ± 1.9** 223.5 ± 3.7n-Hexane 171.9 ± 2.8** 326.1 ± 2.4** 352.8 ± 2.9

SeniseEtOH 55.3 ± 1.8** 83.1 ± 1.9** 174.5 ± 3.7n-Hexane 142.7 ± 2.8** 310.9 ± 2.4** 352.8 ± 2.9

Data are given as the mean ± S.D (n = 3). Differences within and between groups werecomparison Dunnett’s test.** p < 0.01 compared with the positive control acarbose (a-amylase: IC50 50.0 ± 0.9 lg/m

concentration–response relationship (Table 4, Fig. 3). Total extractof fresh pepper exhibited significant a-amylase and a-glucosidaseinhibitory activity with IC50 values of 55.3 and 83.1 lg/mL for Se-nise cultivar, respectively, and 68.9 and 65.9 lg/mL for Roggianocultivar, respectively. The ability of Senise and Roggiano pepperto inhibit the a-amylase enzyme is comparable to the positivecontrol acarbose (IC50 value of 50.0 lg/mL). Both drying and fryingprocess reduced the ability of pepper extracts to exert theinhibition of carbohydrate-hydrolyzing enzymes. In particular,total extract exhibited IC50 values of 259.7 and 187.9 lg/mLagainst a-amylase after frying process (3.8–3.4 times lowerthat fresh peppers, respectively). A same trend was observedagainst a-glucosidase with IC50 values of 846.4 and 740.7 lg/mL(12.8–8.9 times lower than fresh peppers, respectively).

The ability of pepper to inhibit both enzymes maybe is relatedwith phenols content since total extract showed the highest IC50

values. Generally, the lipophilic fraction exhibited a lower bioactiv-ity than the total extract, but a similar trend of bioactivity reduc-tion was observed after technological processes of drying andfrying.

The obtained results are better than those obtained in our pre-vious investigations regard the ability of several pungent pepper toinhibit carbohydrate-hydrolyzing enzymes (Menichini et al., 2009;Tundis et al., 2011; Tundis et al., 2012). In fact, Habanero pepperstotal extract inhibited a-amylase and a-glucosidase with IC50 val-ues of 131 and 265 lg/mL, respectively, while dried C. annuum var.cerasiferum showed IC50 values of 256.8 and 356.8 lg/mL againsta-amylase and a-glucosidase, respectively. Kwon et al. (2007)demonstrated the ability of yellow sweet pepper to inhibit the a-glucosidase from different source (rat intestine and yeast) whilea lower bioactivity was observed with a-amylase.

The most abundant flavonoid identified in both pepper culti-vars namely apigenin demonstrated to inhibit the a-glucosidasewith a percentage of 43% at 200 lM (Tadera et al., 2006). At thesame time it is of interest that administration of this flavone for10 consecutive days in alloxan-treated diabetic animals increasedthe levels of serum insulin and hardly inhibited the glycation ofplasma proteins (Panda and Kar, 2007; Liu et al., 2012). Unableto conclude that apigenin is responsible of the a-amylase inhibi-tory activity, we could speculate that other phytocomplex constit-uents are able to inhibit the enzyme. Moreover, recently Chaiyasitet al. (2009) evidenced by in vivo study that pepper consumptionmight have clinical implications in the management of type 2diabetes. Twelve healthy volunteers were treated with 5 g of C.frutescens each day. After measurement of Oral Glucose ToleranceTest (OGTT) and insulin level in plasma, authors concludedthat pepper consumption was associated with a decrease inplasma glucose levels and the maintenance of insulin levelsin vivo.

e cultivars.

er Dried frying pepper

a-Glucosidase a-Amylase a-Glucosidase

** 392.7 ± 3.6** 259.7 ± 2.9** 846.4 ± 3.5**

** 344.9 ± 2.9** 448.5 ± 3.5** 736.9 ± 3.9**

** 325.9 ± 3.6** 187.9 ± 2.9** 740.7 ± 3.5**

** 338.5 ± 2.9** 448.5 ± 3.5** 442.2 ± 3.9**

evaluated by one-way analysis of variance (ANOVA) test completed with a multi-

l; a-glucosidase: IC50 35.5 ± 1.2 lg/ml).

Fig. 3. Effect of EtOH extract of fresh and processed C. annuum Roggiano against a-amylase (A) and a-glucosidase (B) and Senise against a-amylase (C) and a-glucosidase (D).Data are mean ± S.D. (n = 3).

400 M.R. Loizzo et al. / Food and Chemical Toxicology 53 (2013) 392–401

4. Conclusions

Bell pepper are largely consumed worldwide especially dried.Roggiano and Senise varieties after receiving quality mark IGPand DOP are largely consumed in Italy and exported in other coun-tries as typical south Italian food. In this work we have evaluatedthe phenols, flavonoids and carotenoids content and the bioactivityof these two bell pepper cultivars. Samples were analyzed freshand after drying and frying process. Although reports on the effectsof drying and cooking processes on different vegetables have beeninconclusive, our results clearly evidenced that drying process al-low to permit the retention of phytochemicals in pepper that areable to exert their bioactivity. On the contrary frying process dras-tically reduced the phytochemical content especially of phenolsand consequently reduced both antioxidant activity and inhibitionof carbohydrate-hydrolyzing enzymes. At the same time both pro-cesses did not affect the value of carotenoids content in these pep-per. Collectively our results would contribute to the controversialliterature about the impact of technological process on healthycompounds in food and to promote the consumption of thesetwo traditional bell pepper.

Conflict of Interest

None declared.

Acknowledgements

This work was supported by European Community POR CalabriaFSE 2007/2013 and ‘‘Carmencita Verre’’ fellowship. The authorsthank Dr. R. De Maio for technical assistance.

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