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Food Chemistry 121 (2010) 429–436

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Antioxidant and antiproliferative activities of mango (Mangifera indica L.) flesh and peel Hana Kim a,1, Jeong Yong Moon a,1, Hyeonji Kim a, Dong-Sun Lee a, Moonjae Cho b, Hyung-Kyoon Choi c, Young Suk Kim d, Ashik Mosaddik a,f, Somi Kim Cho a,e,f,* a

Faculty of Biotechnology, College of Applied Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea Department of Medicine, Medical School, Jeju National University, Jeju 690-756, Republic of Korea College of Pharmacy, Chung-Ang University, Seoul 156-756, Republic of Korea d Department of Food Science and Technology, Ewha Womans University, Seoul 120-750, Republic of Korea e The Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University, Jeju 690-756, Republic of Korea f Subtropical Horticulture Research Institute, Jeju National University, Jeju 690-756, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 25 August 2009 Received in revised form 25 October 2009 Accepted 15 December 2009

Keywords: Antioxidant activity Antiproliferative activity Electron spin resonance (ESR) Free radical scavenging activity Mango flesh Mango peel

a b s t r a c t The antioxidant and antiproliferative properties of flesh and peel of mango (Mangifera indica L.) were investigated. The cytoprotective effect of mango flesh and peel extracts on oxidative damage induced by H2O2 in a human hepatoma cell line, HepG2, were determined, and the underlying mechanism was examined by a single-cell electrophoresis assay (comet assay). Treatment of HepG2 cell with mango peel extract prior to oxidative stress was found to inhibit DNA damage. The free radical scavenging activities of mango flesh and peel extracts were evaluated by electron spin resonance (ESR). The mango peel extract exhibited stronger free radical scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and alkyl radicals than mango flesh extract, regardless of ripeness. Similarly, peel extract exhibited significant antiproliferative effect against all tested cancer cell lines, compared to that of flesh extract, in a dose-dependent manner. The result also showed that the antiproliferative activity of mango flesh and peel extracts correlated with their phenolic and flavonoid contents. Thus, mango peel, a major by-product obtained during the processing of mango product, exhibited good antioxidant activity and may serve as a potential source of phenolics with anticancer activity. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction The importance of dietary polyphenols has been underlined because of their antioxidative activities (Fresco, Borges, Diniz, & Marques, 2006) and anticarcinogenic effects; they inhibit cancer cell proliferation in vitro (Scalbert, Johnson, & Saltmarsh, 2005). Fruits and vegetables contain many antioxidant compounds, including phenolic compounds, carotenoids, anthocyanins, and tocopherols (Naczk & Shahidi, 2006). Specially, fruit peels are rich in polyphenolic compounds, flavonoids, ascorbic acid, and many other biologically active components having positive influences on health (Leontowicz et al., 2003). Mango (Mangifera indica L.), which belongs to the family Anacardiaceae, is one of the most popular tropical fruits, followed by banana, pineapple, papaya, and avocado. The mango plant has been the focus of attention of many researchers searching for potent * Corresponding author. Mailing address: 66 Jejudaehakno, Jeju 690-756, Republic of Korea. Tel.: +82 64 754 3348; fax: +82 64 756 3351. E-mail address: [email protected] (S.K. Cho). 1 These authors equally contributed to this work. 0308-8146/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.12.060

antioxidants. Parts of the mango, such as stem bark, leaves, and pulp are known for various biomedical applications, including antioxidative and free radical scavenging (Ajila, Bhat, & Prasada Rao, 2007a; Rocha Ribeiro et al., 2007), anti-inflammatory (Hernandez, Rodriguez, Delgado, & Walczak, 2007), and anticancer (Percival et al., 2006) activities. In a recent study, aqueous and ethanolic extracts of mango leaves were reported to be ideal antioxidants (Ling et al., 2009). Despite these reports, few scientific investigations have examined the importance of mango peels in terms of antioxidant and anticancer activities. Because mango is a seasonal fruit, approximately 20% of fruits are processed for products, such as puree, nectar, pickles, and canned slices that are popular worldwide (Loelillet, 1994). Peel is a major by-product of such processing; mango peels are not currently used commercially, but are discarded as waste and are becoming a source of pollution (Ajila, Naidu, Bhat, & Prasada Rao, 2007b). Peel has been found to be a good source of phytochemicals, such as polyphenols, carotenoids, vitamin E and vitamin C (Ajila et al., 2007a) and it exhibited good antioxidant properties (Ajila et al., 2007b). Recently polyphenol profiles of mango by-product including peel have been reported using HPLC-MSn analysis

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(Barreto et al., 2008). Polyphenol content of peel was reported to be more than that of flesh (Lakshminarayana, Subhadra, & Subramanyam, 1979). Though considerable work has been done with regard to antioxidants and polyphenols of mango, very few reports are available with regard to mango peel. Thus, we evaluated the ability of extract of mango peel to function as an antioxidant and anticarcinogenic agent, using electron spin resonance (ESR) and cell-based assay. The ESR trapping technique is based on the measurement of transitions of unpaired electrons in a magnetic field and provides a sensitive, direct, and accurate means of monitoring reactive species at room temperature (Antolovich, Prenzier, Patsalides, McDonald, & Robards, 2002). As it has been reported that different antioxidant compounds exhibit differential scavenging activity on various reactive oxygen species (Wang & Jiao, 2000) and that reaction with hydroxyl radicals is non-specific, while reaction with other radicals is more specific (Singh, Kunwar, Srinjivasan, Nanjan, & Priyadarsini, 2009), the abilities of mango extracts to scavenge various free radicals were determined by ESR spectrometer. Therefore, one of the aims of this study was to evaluate the antioxidant property of mango peel and flesh extracts prepared at two different ripening stages to scavenge DPPH radicals, hydroxyl radicals, and alkyl radicals using the ESR technique. Additionally, the ability of the mango peel extract to inhibit proliferation of various cancer cells was investigated. Because the elimination of cancer in the early stages is an integral part of chemoprevention, measuring antiproliferative properties against cancer cells using common assays, such as the MTT assay, provide useful insight on the chemoprotective potential of natural extracts. Our results showed the ability of mango peel extract for inhibiting the growth of the tested cancer cell lines, to varying degrees. The HeLa cell (human cervical cancer cell) line was highly susceptible to unripe mango peel (UMP) extract-induced toxicity, whereas CCD-25Lu (human normal lung fibroblast) cells were not. Thus, the objective of this study was to examine the efficacy of mango peel as an antioxidant with an inhibitory effect on human cancer cell proliferation in comparison to the part and the ripeness of the mango. 2. Materials and methods 2.1. Materials Mangos (Mangifera indica L. cv. Irwin) used in this study were obtained from a local farm in Jeju, Korea. Mango fruits were continuously observed on the tree during ripening and selected fruits, firm-green (unripe) and soft-yellowish (ripe) mangos, were harvested, shipped on the same day, and processed upon receipt. 2.2. Chemicals Folin–Ciocalteu’s phenol reagent, rutin, gallic acid, ferrous sulfate heptahydrate (FeSO47H2O), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2,20 -azobis (2-amidinopropane) hydrochloride (AAPH), a-(4-pyridyl-1-oxide)N-t-butylnitrone (4-POBN), and 2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma Chemical Co. (St. Louis, MO). Dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Amresco Inc. (Solon, OH). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), trypsin/EDTA, foetal bovine serum (FBS), penicillin, and streptomycin were purchased from Invitrogen Life Technology Inc. (Grand Island, NY). All other chemicals and reagents used were of analytical grade.

2.3. Preparation of the fruit extracts Unripe mango flesh (UMF), unripe mango peel (UMP), ripe mango flesh (RMF), and ripe mango peel (RMP) were obtained by manually removing peel and edible flesh from the seed kernel. A kitchen peeler and spoon were used to separate flesh from peel. Samples were lyophilized for 2 days, then pulverized, and extracted with 80% ethanol by sonicating for 3 days at room temperature. The extracts were filtered, concentrated with a vacuum rotary evaporator under reduced pressure at 40 °C and lyophilized. The extracts were dissolved in dimethyl sulfoxide (DMSO) as 200 mg/ml and diluted with phosphate-buffered saline (PBS, pH 7.4) to give final concentrations.

2.4. Colour measurement The skin colour of mango samples was measured with a tristimulus colorimeter (Chroma Meter CR-300, Minolta Co. Ltd., Japan) in the L, R, and Y colour space. Two evenly distributed places along the equator were selected and a mean value was used. Three values were obtained: L measures lightness and varies from 100 for perfectly reflective white to zero for perfectly absorptive black, R measures redness when positive, gray when zero, and greenness when negative, and Y measures yellowness when positive, gray when zero, and blueness when negative.

2.5. Determination of firmness The firmness of the mangos was measured using a texture analyzer (TA-XT 2/25, Stable Micro Systems Co. Ltd., Godalming, England) fitted with parallel plates using a 5-mm plunger. The conditions were as follows: pre-test speed: 5.0 mm/s, test speed: 2.0 mm/s, return speed: 5.0 mm/s, and the maximum force during compression was recorded in newtons.

2.6. Determination of total phenolics and flavonoids Total phenolics were determined according to the method of Cheung, Cheung, and Ooi (2003), with slight modifications. A 1.5 ml aliquot of sample was mixed with 0.5 ml of Folin-Ciocalteu’s phenol reagent. After 3 min, 1 ml Na2CO3 (10% v/v) was added to the mixture. The reaction was kept in the dark for 30 min, after which its absorbance was read at 725 nm using a UV 1800 spectrophotometer (Shimadzu, Tokyo, Japan). The results were expressed as gallic acid equivalents (GAE) in mg/g of dried sample. The flavonoid content was measured using a colorimetric assay developed previously (Zhishen, Mengcheng, & Jianming, 1999). Absorbance was read at 510 nm against the blank (PBS) and flavonoid content was expressed as rutin equivalents (RE) in mg/g of dried sample. All analyses were done at least in triplicate.

2.7. Cell culture Cancer cell lines, including AGS, a human gastric adenocarcinoma cell line, HeLa, a human cervix adenocarcinoma cell line, HepG2, a human hepatocarcinoma cell line, and CCD-25Lu, a human lung fibroblast cell line, were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). The cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or RPMI 1640 containing 10% (v/v) heat-inactivated foetal bovine serum (FBS), 100 units/ml penicillin, and 100 lg/ml streptomycin. Cells were maintained in a humidified incubator at 37 °C in a 5% CO2 atmosphere.


2.8. Cell viability assay The effect of the extracts on the viability of various cancer cell lines was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) colorimetric assay (Hansen, Nielsen, & Berg, 1989). Briefly, exponential-phase cells were collected and transferred to a 96-well plate (2 103–2 104 cells per well). The cells were then incubated for 72 h in the presence of various concentrations of the extracts. After incubation, 0.1 mg of MTT were added to each well and the cells were incubated at 37 °C for 4 h. The medium was then carefully removed and DMSO (150 ll) were added to each well to dissolve the formazan crystals. The plates were read immediately at 570 nm on a microplate reader (Tecan, Salzburg, Austria). 2.9. Cytotoxicity assay To evaluate the ability of the extracts to protect cultured cells from oxidant-induced cell death, the MTT colorimetric assay was performed to determine the cell viability (Hansen et al., 1989). HepG2 cells were cultured at a density of 1 104 cells/well in 96-well plates for 16 h, washed twice using PBS, and pretreated with 80% ethanol extracts. After 1 h incubation, 200 lM of H2O2 solution were added to the wells, and the cells were re-incubated for 2 h. MTT reagent (5 mg/ml) was added to each well, and the plate was incubated at 37 °C for an additional 4 h. Then the medium was removed and the intracellular formazan product was dissolved in DMSO. Absorbance at 570 nm of the mixture was detected using microplate reader (Tecan, Salzburg, Austria). Cell viability was expressed as a percentage of counts relative to the untreated control cells. 2.10. Determination of DNA damage (comet assay) The comet assay was conducted according to Tice et al. (2000), with slight modification. Cells in a 24-well plate were incubated with the extracts for 1 h, after which 200 lM of H2O2 were added to the cells for 15 min. Harvested cells were mixed with 100 ll of 0.7% low-melting point agarose (LMPA) and added to slides precoated with 1.0% normal melting agarose (NMA). After solidification of the agarose, the slides were covered with another 100 ll of 0.5% LMPA and then immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% sodium lauroylsarcosine and 1% Triton X-100) at 4 °C for 1.5 h. The slides were then placed into an electrophoresis tank containing unwinding buffer (300 mM NaOH and 10 mM Na2EDTA, pH 13.0) for 40 min. For electrophoresis of the DNA, an electric current of 25 V/300 mA was applied at 4 °C for 20 min. The slides were washed three times with a neutralising buffer (0.4 M Tris–HCl, pH 7.5) at 4 °C for 10 min, then treated with ethanol for 5 min before staining with 50 ll ethidium bromide (20 lg/ml). Measurements were made by image analysis (Kinetic Imaging Komet 5.0, UK) and fluorescence microscopy (Leica DMLB, Germany), determining the percentage of fluorescence in the tail (tail intensity, TI; 50 cells from each of two replicate slides). 2.11. Determination of free radical scavenging activity using ESR spectrometry 2.11.1. DPPH radical scavenging 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was measured using the method described by Nanjo et al. (1996). A 30 ll aliquot of each sample in PBS (or PBS itself as a control) was added to 30 ll of DPPH (60 lM) in ethanol solution. After mixing vigorously for 10 s, the solutions were transferred into a 50-ll Teflon capillary tube and fitted into the cavity of the ESR spectrometer. The spin adduct was measured on an ESR spectrometer ex-

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actly 2 min later. Measurement conditions were as follows: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 5 mW, gain 6.3 105, and temperature 297 K. The radical scavenging activity of the extracts of mango was calculated by scavenging rate = [(h0 hx)/h0] 100%, where h0 and hx are the ESR signal intensities of samples in the absence and presence of the extracts, respectively. 2.11.2. Hydroxyl radical scavenging Hydroxyl radicals were generated by the Fenton reaction, and reacted rapidly with nitrone spin trap DMPO: the resultant DMPO-OH adduct was detectable with an ESR spectrometer (Rosen & Rauckman, 1984). The ESR spectrum was recorded 2.5 min after mixing in a phosphate buffer solution (pH 7.4) with 20 ll of 0.3 M DMPO, 20 ll of 10 mM FeSO4, 20 ll of 10 mM H2O2 20 using a JESFA electron spin resonance spectrometer (JEOL, Tokyo, Japan) set at the following conditions: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 1 mW, gain 6.3 105, and temperature 298 K. The radical scavenging activity of the extracts of mango was calculated by scavenging rate = [(h0 hx)/h0] 100%, where h0 and hx are the ESR signal intensities of samples in the absence and presence of the extracts, respectively. 2.11.3. Alkyl radical scavenging Alkyl radicals were generated by AAPH. The PBS (pH 7.4) reaction mixtures containing 40 mM AAPH, 40 mM 4-POBN, and the indicated concentrations of tested samples, were incubated at 37 °C in a water bath for 30 min (Hiramoto, Johkoh, Sako, & Kikugawa, 1993), and then transferred to a 50-ll Teflon capillary tube. The spin adduct was recorded on a JES-FA ESR spectrometer. Measurement conditions were as follows: central field 3475 G, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 10 mW, gain 6.3 105, and temperature 298 K. The radical scavenging activity of the extracts of mango was calculated by scavenging rate = [(h0 hx)/h0] 100%, where h0 and hx are the ESR signal intensities of samples in the absence and presence of the extracts, respectively. 2.12. ABTS radical cation-scavenging Determination of the antioxidant capacity was as detailed previously by Giao et al., 2007. ABTS (2,20 -azino-bis(3-ethylbenzthiazoline-6-sulfonic) acid) was dissolved in water, to a 7 mM concentration. ABTS radical cation (ABTS+) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 16 h before use. To obtain an absorbance of 0.700 ± 0.005 at 734 nm, measured with a UV 1800 spectrophotometer (Shimadzu), the stock solution was diluted with as much ultra-pure water as necessary. A 100-ll of sample in PBS was added to 900 ll of this diluted solution, and the absorbance at 734 nm was determined after 2 min initial mixing. The antioxidant solution reduced the radical cation to ABTS, which reduced the colour. The extent of decolorization was calculated as the percentage reduction in absorbance. 2.13. Statistical analyses All experiments were conducted in triplicate. The results were analyzed using the Statistical Package of Social Science Software (SPSS 12.0 for Windows, 2003, SPSS Inc, Chicago, IL). Data were analyzed using one-way ANOVA, followed by LSD. The data are expressed as means ± SD. A value of p < 0.05 was deemed to be statistically significant. Pearson’s correlations were used to

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Table 1 Characteristics of mango samples. Samples

Colour a

Unripe mango Ripe mango a b c

Table 2 Total phenolic and total flavonoid content of mango extracts. Firmness b

c

L

R

Y

(N)

41.01 ± 0.74 39.95 ± 4.10

11.15 ± 1.35 12.99 ± 6.06

15.50 ± 0.50 15.29 ± 3.10

52.73 ± 10.44 32.01 ± 15.17

L: Lightness. R: Redness. Y: Yellowness.

Samples

Total phenolic content (mg GAE/g)

Total flavonoid content (mg RE/g)

UMF UMP RMF RMP

27.8 ± 2.21 92.6 ± 3.40 26.9 ± 3.76 70.1 ± 4.61

8.15 ± 1.54 22.2 ± 3.32 3.30 ± 0.79 21.2 ± 2.47

All data are presented as mean ± SD of the three replicates. Samples are unripe mango flesh (UMF), unripe mango peel (UMP), ripe mango flesh (RMF), and ripe mango peel (RMP). GAE: gallic acid equivalents, RE: rutin equivalents.

determine the relationship between antioxidant content and cell viability. 3. Results and discussion

mango. The subjectively judged firmness of fruit, based on our criteria, corresponded to the instrumentally determined firmness values.

3.1. Colour and firmness measurements of mangos The mango samples harvested at the firm and soft stages were assessed by the colour of their skins. The subjective colours for unripe and ripe mangos were yellowish-green and yellowish-red, respectively. Colour readings recorded with a Hunter colour meter are shown in Table 1. A significant R colour difference existed between unripe mango and ripe mango, whereas there were no significant L or Y colour differences. The firmness reading of unripe mango was 52.7 N, which was 1.6-fold greater than that of ripe

3.2. Total phenolic and flavonoids content of ethanolic extract of mango fruit Phenolics, commonly found in fruits, have been reported to exhibit antioxidant activity, due to the reactivity of the phenol moiety, and have the ability to scavenge free radicals, via hydrogen donation or electron donation (Shahidi & Wanasundara, 1992). A causative relationship has been demonstrated between total phenolic content and antioxidant activity (Jayaprakasha & Patil,

Fig. 1. Protective effect of ethanolic extracts of unripe mango flesh (UMF), unripe mango peel (UMP), ripe mango flesh (RMF), and ripe mango peel (RMP) on H2O2-induced cytotoxicity in HepG2 cells. (A) Cultures were treated with the noted concentrations of mango extracts for 1 h and then incubated with 200 lM H2O2 for 2 h. Cell viability was determined by the MTT assay. Cell viability without treatment was taken as 100%. (B) Cultures treated with 12.5–100 lM of the positive controls, quercetin and catechin, for 1 h and then incubated with 200 lM H2O2 for 2 h. (C) Comet assay of H2O2-induced DNA damage in HepG2 cells. Cells were treated with 200 lg/ml of extracts for 1 h prior to the incubation with 200 lM of H2O2 for 15 min. Data are expressed as mean ± SD.


2007). Additionally, many individual phenolic compounds that have antioxidant activity in fruits cannot be identified and measured by HPLC methods (Ferreira et al., 2002). Thus, quantification of polyphenols was conducted using the Folin–Ciocalteu’s phenol (FC) reagent. As shown in Table 2, the total polyphenolic content in 80% ethanol extract of unripe mango peel (UMP) was 92.62 mg GAE/g. This was approximately three-fold higher than both unripe mango flesh (UMF) and ripe mango flesh (RMF). These results are consistent with previously reported results, in which the total polyphenolic content in 80% acetone extract of raw mango peels ranged from 90 to 110 mg/g peel, and 55 to 100 mg/g in ripe peel, depending on the variety (Ajila et al., 2007b). The content of flavonoids in UMP and RMP was 22.16 and 21.16 mg RE/g, respectively, 3- and 6-fold higher than that of UMF and RMF, respectively. Our results showed that mango peel contained more phenolics and flavonoids than mango flesh, regardless of ripeness. 3.3. Protective effect of mango extract on H2O2-induced cytotoxicity in HepG2 cells The role of ethanolic extracts of mango samples, UMP, UMF, RMP and RMF, in the protection of the HepG2 cells from H2O2-induced oxidative stress was evaluated using the MTT assay. The HepG2 cell line is a reliable model that is well characterized and is widely used for biochemical and nutritional studies of many antioxidants and conditions (Alia, Ramos, Mateos, Bravo, & Goya, 2005). Our results showed that exposure of cells to 200 lM H2O2 for 2 h without pretreatment with mango extracts caused cell

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death to increase by 50% (Fig. 1A). Pre-incubation of cells with UMP or RMP, but not UMF or RMF extracts, for 1 h prior to inducing the oxidative stress, greatly inhibited cell death. Cell viability was restored, back to nearly 80% by pretreatment with mango extract at a concentration of 50 lg/ml and 200 lg/ml of UMP and RMP, respectively. Similar effect was also observed for the positive control, quercetin and catechin at concentrations of 12.5–100 lM (Fig. 1B). This result indicated that UMP and RMP extracts had a protective effect against H2O2-induced oxidative stress in HepG2 cells. 3.4. Effect of extracts of mango on H2O2-induced DNA damage We used the comet assay to investigate the effect of mango extracts on H2O2-induced DNA damage (Fig. 1C). The comet assay, a relatively fast, simple, and sensitive technique for the analysis of DNA damage in all cell types, has been successfully used to assess interactions of antioxidants with genotoxicants. It has also proven to be a valid technique to evaluate whether antioxidants/micronutrients are able to protect the integrity of genetic material (Heaton et al., 2002). Our results indicated that the tail DNA percentage of the cells exposed to 200 lM H2O2 without treatment with mango extracts increased up to 60% compared with non-treated cells (control). When HepG2 cells were pretreated with 200 lg/ml UMP or RMP extracts, DNA damage induced by 200 lM H2O2 decreased dramatically. These results suggest that UMP and RMP extracts protected cells from DNA damage induced by H2O2. Cells pretreated with UMF or RMF were not protected from DNA damage.

Fig. 2. Effects of ethanolic extracts of unripe mango flesh (UMF), unripe mango peel (UMP), ripe mango flesh (RMF), ripe mango peel (RMP), and catechin (CAT) on several radicals. Data showed the percent of DPPH radical (A), hydroxyl radical (B), alkyl radical (C), and ABTS radical cation (D), scavenging activity of extracts. An appropriate amount of catechin was used as a positive control in each experiment. Data are expressed as mean ± SD.

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3.5. Free radical scavenging activity of mango extracts

radical scavenging activity of natural antioxidants (Singh & Rajini, 2004). The radical scavenging activity of UMP increased with increasing concentrations, with 1.72%, 48.00%, and 92.57% scavenging activity (calculated from the ESR signal intensity) for 12.5 lg/ml, 25 lg/ml, and 50 lg/ml extracts, respectively, whereas that of RMP sharply increased, with 3.99%, 6.67%, and 81.86% scavenging activity for 12.5 lg/ml, 25 lg/ml, and 50 lg/ml extracts, respectively (Fig. 2A). The UMF and RMF exhibited some DPPH radical scavenging activity at high concentrations (1000 lg/ml), but the activities of UMF and RMF were far lower than those of the unripe mango extracts, and were undetectable at 50 lg/ml. The scavenging activity of catechin, a positive control was 91.37% at

Since ESR spin trapping provides a sensitive, direct and accurate means of monitoring reactive species, this study used ESR to compare the DPPH, hydroxyl and alkyl radical scavenging abilities of the 80% methanol extracts of mango flesh and peel. 3.5.1. DPPH radical scavenging activity DPPH, a stable free radical, decreases significantly on exposure to proton radical scavengers. This assay was used to evaluate the free radical scavenging activity in foods and biological systems (Sánchez-Moreno, 2002). It has also been used to evaluate the free

(B)

(A) 120

100

HeLa cell viability (%)

AGS cell viability (%)

100 80 60 40

80 60 40 20

20 0

120

0

125

250

500

0

1000

0

125

(C)

(D)

120

CCD-25Lu cell viability (%)

HepG2 cell viability (%)

100 80 60 40 20 0

0

125

250

250

500

1000

Concentration (µg/mL)

Concentration (µ µg/mL)

500

140 120 100 80 60 40 20 0

1000

0

125

250

500

1000



(E) 120

AGS HeLa HepG2

Cell viability (%)

100 80 60 40 20 0

0

12.5

25

50

100

Concentration (µM) Fig. 3. Inhibition of cancer cell growth by mango extracts measured by the MTT assay. Cells (2 103–2 104 cells per well) were incubated with extracts or positive control (quercetin) for 72 h. Error bars represent ± SD from three separate experiments. Key: unripe mango flesh, s unripe mango peel, . ripe mango flesh, 4 ripe mango peel. (A) Human gastric cancer cells (AGS), (B) human cervical cancer cells (HeLa), (C) human hepatocarcinoma cells (HepG2) and (D) human normal lung fibroblasts (CCD-25 Lu). Three different cancer cells were treated with quercetin as a positive (E). ( AGs; s HeLa; . HepG2).

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6.25 lg/ml. Thus, UMP and RMP possessed hydrogen-donating abilities, suggesting the potential to react with the DPPH radical. 3.5.2. Hydroxyl radical scavenging activity Hydroxy radicals are the major active oxygen species causing lipid peroxidation and enormous biological damage. (Huang et al., 2002). In this study, the hydroxyl radicals generated in the Fe2+/ H2O2 system were trapped by a DMPO-forming spin adduct that could be detected using an ESR spectrometer (Bindoli, Rigobello, & Deeble, 1992). As shown in Fig. 2B, the scavenging activities of UMF, UMP, RMF, and RMP extracts at 500 lg/ml were 91.05%, 88.86%, 91.61%, and 89.61%, respectively. There was no significant difference among the extracts and the activities were higher than that of catechin, which was used as a positive control, and exhibited 89.53% scavenging activity at 1000 lg/ml. 3.5.3. Alkyl radical scavenging activity Alkyl radicals are a primary intermediate in many hydrocarbon reactions, and can be easily detected with ESR, a technique that has been found to be very useful in the characterization of solid surfaces and in the elucidation of active surface sites, as well as surface reactions (Adebajo & Gesser, 2001). Similar to the results of DPPH radical scavenging activities, UMF and RMF extracts exhibited no activity up to 200 lg/ml, while UMP and RMP extracts showed 90.39% and 86.95% radical scavenging activities, respectively, at 200 lg/ml. In the presence of the UMP and RMP extracts, the activities increased with increasing concentration. Catechin, a positive control, showed 87.34% radical scavenging activity at 50 lg/ml (Fig. 2C). 3.5.4. ABTS radical cation-scavenging activities The ABTS radical cation-scavenging activities of mango extracts obtained from different fruit parts of different ripeness are shown in Fig. 2D. UMF and RMF extracts did not show ABTS radical cation-scavenging activity up to 200 lg/ml, whereas UMP extracts exhibited 67.49% radical scavenging activity at 200 lg/ml. The radical scavenging activities of UMP and RMP extracts increased, in a dose-dependent manner. Catechin, used as a positive control, showed higher activity than those of the tested extracts. 3.6. Effect of mango extracts on cell viability Deregulation of cell proliferation, together with suppressed apoptosis, is a minimal, common platform for all cancer evolution and progression (Evan & Vousden, 2001). Uncontrolled cell division is the primary key in the progression of a cancer tumour. We found proliferation was inhibited in human gastric cancer AGS cells, human cervical cancer HeLa cells, and human hepatocarcinoma HepG2 cells in a dose-dependent manner after exposure to mango peel extracts (125-1000 lg/ml; Fig. 3). UMP and RMP extracts showed greater antiproliferative activities than UMF and RMF extracts, demonstrating that mango peel extracts significantly enhanced the inhibition of proliferation of the tested human cancer cell lines, regardless of ripeness. Among the mango extracts, UMP showed relatively potent antiproliferative activities on both AGS and HeLa cells, whereas no significant antiproliferative activity was seen in normal human lung fibroblasts (CCD-25Lu cells). A similar effect was also observed for the positive control, quercetin, at a concentration of 12.5–100 lM (Fig. 3E). Previous studies have shown that natural antioxidants from fruits or vegetables can inhibit cancer cell growth (Eberhardt, Lee, & Liu, 2000). Thus, correlations were tested between the polyphenolic and flavonoid contents of the extracts with the antiproliferative capacities measured by MTT assays. Table 3 summarizes the Pearson’s correlation coefficients between all analyses carried out on the extracts. Negative correlations were found

Table 3 Pearson’s correlation coefficients (r) between the analysis parameters. Samples

Contents

Cell proliferation

UMF

Polyphenol Flavonoid

0.075 0.075

0.256 0.256

0.661** 0.661**

UMP


0.867** 0.867**

0.835** 0.835**

0.797** 0.797**

RMF


0.598** 0.598**

0.034 0.034

0.645** 0.645**

RMP


0.936** 0.791**

0.950** 0.816**

0.661** 0.533*

AGS

* **

HeLa

HepG2

The value was significant at p < 0.01. The value was significant at p < 0.01.

between cell proliferation of HeLa, AGS, and HepG2 cells and total polyphenol content of RMP or UMP, ranging from r = 0.661 to 0.950. Negative correlation coefficients of 0.867, 0.835, and 0.797 were found between the flavonoid content of peel extracts and the proliferation activity of AGS, HeLa, and HepG2 cells for UMP extract (p < 0.01), respectively. These results indicate that antiproliferative effects of mango extracts may be obtained due to a combination effect of polyphenols and flavonoids present in the extracts. 4. Conclusion The present study indicated that the mango peel contained more polyphenols and flavonoids than flesh and exhibited good antioxidant activity by effectively scavenging various free radicals, such as DPPH radicals, hydroxyl radicals and alkyl radicals. In addition, it has been demonstrated that the mango peel is a potential antiproliferative agent. The antioxidant and antiproliferative activities of mango peel might be due to the synergistic actions of bioactive compounds present in them. Thus, mango peel, a by-product of mango processing industry, shows potential as a functional food or value added ingredient. However, these findings warrant extensive studies on the chemical profiles and mechanistic action of antiproliferative and antioxidant activities of the by-product of mango and such studies are currently underway in our lab. Acknowledgments This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (No. R01-2007-000-20492-0), Republic of Korea. References Adebajo, M. O., & Gesser, H. D. (2001). ESR study of alkyl radicals adsorbed on porous Vycor glass: I. Build up of mehyl and ethyl radicals. Applied Surface Science, 171, 120–124. Ajila, C. M., Bhat, S. G., & Prasada Rao, U. J. S. (2007a). Valuable components of raw and ripe peels from two Indian mango varieties. Food Chemistry, 102, 1006–1011. Ajila, C. M., Naidu, K. A., Bhat, S. G., & Prasada Rao, U. J. S. (2007b). Bioactive compounds and antioxidant potential of mango peel extract. Food Chemistry, 105, 982–988. Alia, M., Ramos, S., Mateos, R., Bravo, L., & Goya, L. (2005). Response of the antioxidant defense system to t-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). Journal of Biochemical and Molecular Toxicology, 19, 119–128. Antolovich, M., Prenzier, P. D., Patsalides, E., McDonald, S., & Robards, K. (2002). Methods for testing antioxidant activity. The Analyst, 127, 183–198. Barreto, J. C., Trevisan, M. T. S., Hull, W. E., Erben, G., Brito, E. S., Pfundstein, B., et al. (2008). Characterization and quantitation of polyphenolic compounds in bark, kerne, leaves and peel of mango (Mangifera indica L.). Journal of Agricultural and Food Chemistry, 56, 5599–5610.

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