Cytotoxic, Antioxidant and Antimicrobial Properties of Red Sweet Pepper (Capsicum Annuum L. var. Llanerón) Extracts: In Vitro Study

Alcoholic and aqueous extracts were obtained from red sweet pepper (Capsicum annuum L.) by different methodologies to evaluate their cytotoxic, antioxidant and antimicrobial properties. Alcoholic extracts (MFP, MSd, SFP, SDP, SSd) from fresh red sweet pepper (FP) and dry pulp (DP) and seed (Sd) were obtained by maceration (M) and Soxhlet (S) equipment using methanol as extraction solvent; whereas aqueous extracts (LFP, LSd) were obtained by decoction followed by lyophilization (L). Human tumoral cell lines from breast (MCF-7 and SKBr3), prostate (PC3) and cervix (HeLa), and fibroblasts (as control) were used to determine the cytotoxic properties by the MTT assay. Antioxidant and antimicrobial properties were determined by DPPH and disc diffusion method, respectively. The extracts SDP and SFP showed the higher cytotoxic activity. The SDP extract had a significant (P < 0.05) in-vitro effect on HeLa (1.9 ± 1.4 μg/mL) and PC3 (< 1 μg/mL) cells with a moderated impact on fibroblasts (26.1 ± 1.2 μg/mL); whereas, SFP had a significant (p < 0.05) effect on MCF-7 cell line (2.1 ± 1.2 μg/mL) with a moderated impact on fibroblasts (25.9 ± 1.0 μg/mL). The higher antioxidant activity was found for MFP (80.3 ± 0.2%) and SFP extracts (75.5 ± 0.5%). Mild antimicrobial activity was only observed for alcoholic extracts. The results showed the potential of red sweet pepper (C. annuum L.) as a source of antioxidant and cytotoxic compounds, and suggest the need of further studies to isolate and characterize the bioactive compounds that impart those properties.


Introduction
Fruits and vegetables have been recognized as natural sources of various bioactive compounds, the main phytochemical compounds present in risk of developing various chronic diseases due to their antioxidant and radical-scavenging activities (Ayala-Zavala et al., 2011).Peppers are usually consumed as food and used as additives in the food industry.They have a significant role in traditional medicine: in fact, in Indian, Native American, and Chinese traditional medicine, Capsicum species have been used for the treatment of arthritis, rheumatism, stomach aches, skin rashes, dog/snake bites, and flesh wounds.These therapeutic applications are related to the capsaicinoids, phenolic compounds, and carotenoids content of the peppers (Hernandez-Ortega et al., 2012).Different varieties of Capsicum annuum L. have been studied and have shown benefit with respect to some disorders, mainly for those related to mental health.Yazdizadeh Shotorban, Jamei, and Heidari (2012)), in studies performed with rats, established the antioxidant activity of bell pepper, which showed a protective effect on the brain cells.In the same way, Oboh and Rocha (2008) reported that chemical compounds present in unripe hot pepper inhibited lipid peroxidation in rat brain, thus enhancing the protective properties on the brain cells.Sweet pepper (C.annuum L.) is a plant whose fruits have different forms and size, and several colours, ranging from yellow to red, from intense purple to dark green to black, depending on the genotype or the seasonal period of breeding (Nazzaro et al., 2009).This fresh fruit is known as an excellent source of Vitamin C and E as well as provitamin A, carotenoids and polyphenols, and are considered of great importance because of their antioxidant properties (Nazzaro et al., 2009;Shaha, Rahman, & Asrul, 2013;Yazdizadeh Shotorban et al., 2012).The main objective of this research was to evaluate the In vitro cytotoxic properties of aqueous and alcoholic extracts of pulp and seed of red sweet pepper (C.annuum L.) on human tumoral cells of breast, prostate and cervix, in addition to the study of the antioxidant (by DPPH method), and antimicrobial (against Salmonella enterica ser.Enteritidis, Staphylococcus aureus and Listeria monocytogenes) properties of those extracts.

Vegetable
Red sweet pepper (Capsicum annuum L. var Llanerón) commonly called in Venezuela "Ají dulce Llanerón" is a cultivated variety in the Venezuelan plains, specifically in the Guárico state, and is characterized by having an elongated shape, orange or red colour when ripening and with intense flavour.Red sweet pepper was harvested in summer (March to August) and supplied commercially to Caracas city.They were selected in a local supermarket (Caracas, Venezuela) at commercial ripeness and maintained at 7 °C until processing.

Preparation of Red sweet pepper seeds and pulp methanolic extracts by Soxhlet apparatus
Samples of the seed and pulp, from red sweet pepper (300 g) were dried at room temperature (25-27 °C) and then finely ground with a ceramic mortar, and submitted to an extraction process (individually) with methanol, analytic grade (99.8%) (J.T. Baker, Phillispsburg, NJ) in Soxhlet apparatus for 12 hours at a ratio of 1:2 (vegetable sample: methanol) (at solvent boiling temperature of approx.50 °C) as was suggested by Leong, Alviarez, Compagnone, and Suarez (2009).Then, the mixtures were vacuum filtered (paper Whatman N°1).The solvent was removed in vacuo using a rotator evaporator at 45-50 °C for 60 min.approximately or until dryness in order to obtain the sweet pepper raw extracts from fresh pulp (FP), dried pulp (DP) and seed (Sd).

Preparation of Red sweet pepper seeds and pulp methanolic extracts by maceration
Samples of the seed and pulp, from red sweet pepper (200 g) were dried at room temperature (25-27 °C) and then finely ground with a ceramic mortar and submitted to maceration using

Preparation of Red sweet pepper aqueous extract by decoction
The aqueous extracts of red sweet pepper seed and pulp were obtained by decoction according to methodology used by Suarez et al. (2006).For these, 500 g of the seed or pulp were boiled in 500 mL of distilled water for 30 minutes.The decoction was then vacuum filtered, and the aqueous extracts were stored in glass vials, and frozen for subsequent lyophilization to produce powdered forms of sweet pepper fresh pulp extract (LFP) and sweet pepper seed extract (LSd) lyophilized.

Cytotoxicity assay
Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide) assay, which is based on the ability of viable cells to metabolically reduce a yellow tetrazolium salt (MTT; Sigma) to purple crystals of formazan (Mosmann, 1983).This reaction takes place when mitochondrial reductases are active.Cells were grown in 96-well plates (5Ö10 5 cells/well) and incubated at 37°C for 72 hours with the extracts of the red sweet pepper seed and pulp obtained by different extraction methods at concentrations of 0, 0.001, 0.01, 0.1, 1, 5, 10, 15, 25 and 100 µg/ml, respectively, in a humidified atmosphere with 5% CO 2 .The lyophilized extracts were diluted in water and extracts obtained by maceration and Soxhlet were diluted in DMSO.The final concentration of the DMSO in culture medium was always lower than 1%, a concentration that has neither cytotoxic effect nor causes any interference with the colorimetric detection methods).After incubation, the medium was removed and the cells were treated with 100 µL MTT for 3 hours at 37°C.Subsequently, 100 µL DMSO was added to the mixture.The solubilized formazan product was quantified with the help of a microplate reader TECAN-Sunriseat 570 nm (Tecan Group LTD, Männedorf, Switzerland).Taxol (Bristol-Myers Squibb, USA) was used as a positive control in the assay.

Selectivity index for cytotoxicity assay in tumoral cells
The selectivity index (SI) was calculated as the IC 50 (control cells)/IC 50 (tumor cells line) ratio.A selectivity index > 1 indicated that the cytotoxicity on tumor cells surpassed that on healthy non-tumor cells (Callacondo-Riva, Quispe-Mauricio, Lindo-Gamarra, & Vaisberg, 2008).

Determination of antioxidant properties
The radical scavenging activity of red sweet pepper extracts was performed by the method described by Celep, Aydin, and Yesilada (2012).
Each extract (0.2 ml) was mixed with 2.8 ml of freshly prepared 0.06 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrish, St. Louis, USA) solution in methanol.The mixture was shaken with a vortex mixer and incubated in the dark for 30 min at room temperature.The absorbance was measured at 517 nm (just after added the extract and after 30 min. of dark incubation).Ascorbic acid (176µg/ml) was used as reference substance (control).The absorbance was measured at 517 nm.DPPH radical-scavenging activity was calculated as follows: DPPH radical -scavenging activity (%) = Where, Abscontrol, is the absorbance value of the Ascorbic acid, and Abssample, is the absorbance of the extract solution.

Antimicrobial activity by disc diffusion method
The method used in this experiment was the suggested by Kirby-Bauer (Davidson & Parish, 1989).Sterile filter-paper discs (Whatmann N°1) measuring 9 mm in diameter were dipped in solutions containing aqueous or alcoholic extracts from red sweet pepper seed and pulp individually prepared at different concentrations (0.0; 0.1; 0.3; 0.5 % v/v).Agar disc diffusion method was used for screening of antimicrobial activity of each extract against Salmonella enterica ser.Enteritidis, Staphylococcus aureus and Listeria monocytogenes.The discs impregnated with extracts were put on Müller-Hinton agar (Himedia, Mumbai, India) plates previously inoculated with each pathogenic microorganism at 10 8 CFU/ml, and them incubated at 37 °C for 24-48 h.The size of inhibition zone diameters surrounding filter paper disc was measured using a vernier caliper (Hauptner, Solingen, Germany).Each extract was assayed on two occasions and in duplicate (n = 4).Sterile distilled water was used as negative control.

Statistical analysis
All experiments of cytotoxicity were performed at least three times.The values of IC 50 of natural products were determined by a non-linear regression of individual experiments using the program GraphPad Prism v.5.02 (GraphPad Software, San Diego, CA, USA).Analysis of variance (ANOVA) in the antimicrobial and antioxidant activity of aqueous and alcoholic extracts of Red sweet pepper seed and pulp were carried out to detected differences statistically significant (p< 0.05) among their concentrations, using statistic package Statgraphics Centurion XVI (StatPoint Technologies).Multiple range tests, using the Fisher's LSD method, were then applied to determine which extracts and concentrations of each extract were significantly (p< 0.05) different.
3 Results and Discussion
The results showed that SDP and SFP extract had a highly selective cytotoxic action on HeLa and PC3, and MCF-7, respectively, because their Selectivity Index (SI) values were higher than 1.0 (Table 2).

Antioxidant properties
The free radical scavenging potential of both seed and pulp extracts from red sweet pepper (C.annuum L.) was determined In vitro through the DPPH method just after adding the sample and after 30 min.Both extracts (pulp and seed) showed a higher antioxidant activity at 30 min than at 0 min ( Sweet peppers (C.annuum L.) are known as an excellent source of vitamins A and C as well as flavonoids and phenolic compounds, which are important antioxidant components that may reduce the risk of diseases (Nazzaro et al., 2009;Yazdizadeh Shotorban et al., 2012).Antioxidants are powerful substances naturally present in vegetables and fruits that can neutralize free radicals before they damage the body's cells (Oboh & Rocha, 2007;Watjen, Chovolou, Kampkötter, & Kahl, 2007).The antiradical activity of flavonoids and phenolics is principally based on the redox properties of their hydroxy groups and the structural relationships between different parts of their chemical structure (Materska & Perucka, 2005).

Antimicrobial properties
Lyophilized extracts of red sweet pepper (C.annuum L.) pulp and seed did not show antimicrobial properties against L. monocytogenes, S. enterica ser.Enteritidis and S. aureus.In contrast, both pulp and seed alcoholic extracts obtained by maceration and Soxhlet extraction showed slight antimicrobial properties (Inhibi-     annuum peppers.Some of these compounds such as polyphenols and other antioxidants can affect the growth and metabolism of fungi, insects and bacteria (Alberto, Canavosio, & de Nadra, 2006;Padilha et al., 2015).Similarly, Davidson and Taylor (2007) reported that polyphenols act on cytoplasm membranes causing leakage cell as well as on the membrane proteins such as ATPase.In the same way, there are other compounds presents in Capsicum peppers that have antimicrobial activity such as capsaicinoids (Omolo et al., 2014;Akhtar et al., 2017), which are compounds whose concentrations vary significantly among varieties of pepper.Capsaicinoids are compounds naturally present in pungent peppers, being the capsaicin the most widely studied.Sweet pepper does not contain capsaicin; however, it has capsinoids such as nordihydrocapsiate (Kobata et al., 1999), which is a compound with a similar structure to capsaicinoids, which present an ester group instead of amide group.The slight antimicrobial properties detected in the present research for the methanolic extracts of red sweet pepper could be due to polyphenols, other antioxidants and nordihydrocapsiate.However it is necessary to carry out future studies to confirm this fact because there is little research work cited in the literature with respect to sweet pepper.

Conclusions
The use of phytochemicals present in fruits and vegetable has gained worldwide acceptance as a novel source of chemopreventive agents against cancer cells, because the non-nutrient phytochemicals have showed that can either block or reverse multistage carcinogenesis.The results of this research showed the strong cytotoxic and antioxidant effects that some red sweet pepper (C.annuum L.) extracts could have.Tumoral cell lines from HeLa, MCF-7 and PC3 were more sensitive to red sweet pepper (C.annuum L.) extracts than SKBr3.Alcoholic extracts of fresh pulp obtained by maceration and Soxhlet equipment showed the higher antioxidant activity in comparison with the rest of extracts.The alcoholic extracts showed only slight antimicrobial properties.These findings demonstrated the potential of red sweet pepper as a source of antioxidant and cytotoxic compounds, and suggest the need for further studies to determine which compounds impart these properties to this fruit and which molecular mechanisms are associated with its antitumoral activity.

Table 1 :
Cytotoxic properties of Red sweet pepper (Capsicum annuum L.) pulp and seed extracts on various human tumor cell lines a Values represent the mean of three (3) determinations ± standard deviation.IC 50 : inhibitory concentration 50%; MFP: Sweet pepper fresh pulp extract obtained by maceration.LFP: Sweet pepper fresh pulp extract obtained by lyophilization.SFP: Sweet pepper fresh pulp extract obtained by Soxleth equipment.Sweet pepper dried pulp extract obtained by Soxleth equipment.MSd: Sweet pepper seed extract obtained by maceration.LSd: Sweet pepper seed extract obtained by lyophilization.SSd Sweet pepper seed extract obtained by Soxleth equipment.Taxol: chemical compound used as control.WA: without activity.HeLa (human cervix carcinoma); PC3 (human prostate carcinoma); MCF-7 (breast carcinoma, without over-expression of the HER2/c-erb-2 gene); SKBr3 (breast carcinoma, in which the HER2/c-erb-2 gene is over-expressed); Fibroblasts (healthy cells of human connective tissue, used as control)

Table 2 :
Values of selectivity index (SI) of the Red sweet pepper (Capsicum annuum L.) seed and pulp extracts Values represent the mean of three (3) determinations.MFP: Sweet pepper fresh pulp extract obtained by maceration.LFP: Sweet pepper fresh pulp extract obtained by lyophilization.SFP: Sweet pepper fresh pulp extract obtained by Soxleth equipment.SDP: Sweet pepper dried pulp extract obtained by Soxleth equipment.MSd: Sweet pepper seed extract obtained by maceration.LSd: Sweet pepper seed extract obtained by lyophilization.SSd Sweet pepper seed extract obtained by Soxleth equipment.Taxol: chemical compound used as control.WA: a

Table 3 :
Antioxidant properties of Red sweet pepper (Capsicum annuum L.) pulp and seed extracts

Table 4 :
Antimicrobial properties of Red sweet pepper (Capsicum annuum L.) pulp and seed alcoholic extracts against L. monocytogenes.S. Enteritidis.and S. aureus