- Email: [email protected]
Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts
Food and Chemical Toxicology 48 (2010) 3406–3411
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts C.M. Ajila, L. Jaganmohan Rao, U.J.S. Prasada Rao ⇑ Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 4 May 2010 Accepted 6 September 2010
Keywords: Mango peel Mangifera indica L. HPLC–MS Polyphenolic compounds Carotenoids
a b s t r a c t Mango is one of the important tropical fruits in the world. As it is a seasonal fruit, it is processed for various products. During its processing, peel is one of the major byproducts, which is being wasted. Bioactive conserves were extracted using 80% acetone from peels of raw and ripe mango fruits and subjected to acid hydrolysis. The prominent phenolic compounds identified by HPLC were protocatechuic acid, gentisic acid and gallic acid. The phenolic acid derivatives present in acetone extracts of raw and ripe peels were tentatively identified by LC–MS. Gallic acid, syringic acid, mangiferin, ellagic acid, gentisyl-protocatechuic acid, quercetin were the phenolic compounds identified in both raw and ripe peels, while raw peel showed the presence of glycosylated iriflophenone and maclurin derivatives also. b-Carotene was the major carotenoid followed by violaxanthin and lutein. Thus, both raw and ripe mango peel extracts have different phenolic compounds and carotenoids, which will have various pharmaceutical applications. Ó 2010 Published by Elsevier Ltd.
1. Introduction Epidemiological evidences suggest that diet rich in fruits and vegetables provide health benefits such as prevention of cardiovascular diseases and certain types of cancer (Arouma, 2003; Miller et al., 2000). These health beneficial effects are due to the presence of various bioactive compounds that are present in fruits and vegetables (Ajila et al., 2007a; Hertog et al., 1993; Heim et al., 2002; Parr and Bolwell, 2000). Phenolic compounds and carotenoids are the major bioactive compounds found in fruits and vegetables and these compounds are shown to exhibit antioxidant, antiallergenic, antiarthrogenic, antiinflamatory, antimicrobial and antithrombotic effects (Ajila et al., 2007b; Bravo, 1998; Middleton et al., 2000; Puupponen-Pimia et al., 2001). Phenolic acids like gallic acid and quercitin are claimed to have activity against allergy, hypertension, arthritis and cancer (Borbalan et al., 2003; Fernandez–Pachon et al., 2004). Mangiferin, which is shown to be the predominant constituent of mango stem bark extract is reported to display a variety of pharmacological effects (Guha et al., 1996; Sanchez et al., 2000). Byproducts from different fruit processing industries, which were traditionally treated as environmental pollutants, are being recognized as source for obtaining valuable components. Mango is one of the important tropical fruits in the world and India contributes major part of the world production. India produced 13.5 MMT of mango fruits, while the world production was 33.45 MMT ⇑ Corresponding author. Tel.: +91 0821 2514876; fax: +91 0821 2517233. E-mail address: [email protected] (U.J.S. Prasada Rao). 0278-6915/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.fct.2010.09.012
(FAO, 2007). Mango fruits are processed for various products as it is a seasonal fruit. Peel is one of the major byproducts during mango processing. It constitutes about 15–20% of the fruit weight. At present peel is discarded as waste and becoming a source of pollution. Recently, Ajila et al. (2007a) reported that mango peel extracts from two different mango varieties at different stages of fruit maturity showed good amount of polyphenols and carotenoids. It was also reported that peel extracts exhibited potential antioxidant activities (Ajila et al., 2007b) and protected against H2O2 induced oxidative damage in rat erythrocytes (Ajila and Prasada Rao, 2008). The present study will focus on the identification of bioactive compounds such as phenolic acids and their derivatives, and carotenoids present in peels of ripe and raw fruits of an Indian mango variety. 2. Materials and methods 2.1. Plant materials Badami mango variety grown in CFTRI campus, Mysore, India was used in this study. Mango fruits were harvested at harvest maturity and peel was removed using a sharp knife and the underlying pulp removed by gently scraping with its blunt edge. To obtain the ripe peel, some fruits were kept to ripen at room temperature and the peel was removed as described earlier. The fresh peels thus obtained were used for analysis. 2.2. Chemicals Gallic acid, caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gentisic acid, protocatechuic acid, syringic acid, vanilic acid, b-carotene, violaxanthin and a-tocoferol were obtained from Sigma Fine Chemicals, St. Louis, USA. All other chemicals and solvents were of analytical grade.
3407
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411 2.3. Preparation of acetone extract of mango peel Both raw and ripe mango peels were removed from the fruits, extracted with 80% acetone, centrifuged at 10,000g for 15 min and these extracts were used for the further studies like estimation and identification of phenolic compounds and carotenoids. 2.4. Determination of total phenolics, carotenoids and vitamin E content Polyphenol content in Mango peel extract was determined by using the method described by Hag et al. (2002). The total polyphenols content in the extract was expressed as gallic acid equivalents (GAE). Carotenoid content in peel extract was determined after saponification using the method as described by Litchenthaler (1987). Vitamin E content was determined according to the method described by Rao et al. (2007). a-Tocoferol was used as a standard to estimate total vitamin E content. 2.5. Identification of phenolic compounds in acetone extract by HPLC and LCMS Polyphenols were separated on a reverse phase C18 column (4.6 250 mm) using HPLC system (Shimadzu, Model LC-10A) using a diode array detector (operating at 280 nm and 320 nm). A solvent system consisting of water: methanol: acetic acid (83:15:2) was used as mobile phase (isocratic) at a flow rate of 1 ml/min (Glowniak et al., 1996). Known quantities of phenolic acid standards such as caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid were used for identification and quantification of phenolic acids present in the acetone extracts. HPLC–ESI-MS analyses were done on a Waters platform ZMD 4000 system composed of a micro ZMD mass spectrophotometer, a Waters 2690 HPLC and a Waters 996 photo diode array detector (Waters corporation, MA, USA). Data were collected and processed via a personal computer running Mass Lynx software version 3.1 (Micromass, a diversion of Waters corporation, MA, USA). The samples in 10 ll aliquot were separated on a reversed phase C18 column (4.6 250 mm), using a diode array detector (operating at 280 and 320 nm). A solvent system consisting of water: methanol: acetic acid (83:15:2) was used as mobile phase (isocratic) at a flow rate of 1 ml/min. UV–VIS absorption spectra were recorded on-line during HPLC analysis. Spectral measurements were made over the range of 200–600 nm. The following ion optics was used- capillary voltage 3 kV, cone voltage 100 V and collision voltage 10 V. The source block temperature was 80 °C and the desolvation temperature was 150 °C. ESI-MS was performed using argon as cone gas (50 L/h) and nitrogen as desolvation gas (50 L/h). The electron spray probe flow was adjusted to 70 ml/min. Continuous mass spectra were recorded over the range m/z 100–500 with scan time 1 s and interscan delay 0.1 s. 2.6. Extraction, estimation and identification of phenolic acids liberated from the soluble bound phenolic compounds Phenolic compounds from mango peel (1 g) were extracted with 20 ml of 80% acetone, centrifuged, and the supernatant obtained was subjected to acid hydrolysis to obtain the phenolic acids according to the method of Krygier et al., (1982) with some modifications as described. The pH of the 80% acetone extract was adjusted to 2 with 4 M HCl and refluxed in boiling water bath for 1 h. Phenolic acids were separated by ethyl acetate phase separation (5 50 ml) and the pooled ethyl acetate fractions were treated with anhydrous sodium sulphate for overnight to remove moisture, filtered and evaporated to dryness and re-dissolved in methanol. Phenolic content was determined by the method of Swain and Hillis (1959) and the phenolic acids were identified after separating them on reverse phase C18 column by HPLC. 2.7. Identification of carotenoids by HPLC Carotenoids in the hexane extract were separated on a reverse phase C18 column (4.6 250 mm) using HPLC system (Model LC- 10A, Shimadzu) with a diode array detector (operating at 460 and 425 nm) using a gradient of two solvents, solvent A-100% acetone and solvent B-90% methanol and 10% water (Sarada et al., 2006). The gradient for separation consists of 1% B traversing to 80% in 40 min at a flow rate of 1 ml/min. The detection was carried out using diode array detector at 445 nm and 460 nm. Peak identification was based on the comparison of retention time with authentic standards of carotenoids such as b-carotene, lutein and violaxanthin.
3. Results and discussion 3.1. Polyphenols, carotenoids and vitamin E content in mango peel acetone extract Peel extracts were analyzed for various compounds namely polyphenols, carotenoids and vitamin E. The polyphenol contents
in acetone extracts of raw and ripe fresh peels varied from 55 to 90 mg GAE/g peel (dry weight basis). The carotenoid contents in raw and ripe peel extracts were found to be 81 and 194 lg/g peel, respectively. The vitamin E content in raw peel was 104 lg/g peel while it was 230 lg/g for ripe peel. The polyphenol content was found to be significantly higher in raw peels compared to ripe peel while the carotenoids and vitamin E were found to be significantly higher in ripe peels (Table 1). 3.2. Identification of phenolic acids of peel extract Initially, efforts were made to identify phenolic compounds by separating on C18 column using HPLC. The extracts obtained from each variety were separated into 5–8 peaks. Efforts to identify these peaks by comparing the retention times of authentic standards of phenolic acids, or spiking with known phenolic acids did not help in identifying the peaks, as there were little variations with retention times. This is mainly due to the complex nature of polyphenols. Phenolic acids can be classified as free, soluble and insoluble bound phenolic acids (Renger and Steinhart, 2000). The free forms of phenolic compounds are very rarely present in plants. Majority of phenolic acids exists in the form of ester, acetal, or ethers and some of the phenolic acids are linked either to structural compounds of plants such as protein, dietary fiber or to longer or smaller organic molecules such as glucose, maleic acid or to other natural products (Robbins, 2003; Nardini et al., 2004). Acid hydrolysis and saponification are the commonly used methods for releasing bound phenolics by different workers. Esterases are also used to cleave the ester bond, but not as popular as the other two methods (Robbins, 2003). The phenolic acids in phenolic fractions after acid hydrolysis of raw and ripe mango peels were separated on reverse phase C18 column on HPLC using water: methanol: acetic acid (83:15:2). The compounds eluted were monitored at 280 and 320 nm to detect various compounds. The retention times of these peaks were compared with the authentic standards of phenolic acids. However, wherever there is a little variation in retention times, their identifications were confirmed by spiking with authentic standards. Gallic acid, protocatechuic acid, gentisic acid and syringic acid were the phenolic acids identified in phenolic fractions of raw and ripe mango peel extracts. 3.3. Identification of phenolic compounds in the acetone extract of mango peel As mentioned earlier, phenolic compounds exist as esters or glycosides. In order to identify these phenolic compounds, the acetone extracts of peels from mango fruits were analyzed by LC–ESIMS. The acetone extracts of raw and ripe peels were separated on reverse phase C18 column using conditions described earlier. The LCMS-TIC profiles of polyphenolics in mango peel extracts of raw and ripe peels were shown in Fig. 1. Based on mass spectrometric
Table 1 Total phenolics, carotenoids and vitamin E contents in acetone extracts of mango peel. Parameter
Raw peel
Ripe peel
Total phenolic content* (mg/g GAE) Carotenoid Content* (lg/g) Vitamin E (lg/g)
90.2 ± 0.57b 81.0 ± 0.42a 104 ± 3.2a
54.67 ± 1.5a 194 ± 0.26b 230 ± 12b
Values are expressed on dry weight basis. All data are the mean ± SD of three replicates. Mean value followed by different letters in the same row differ significantly (P 6 0.05). * Ajila et al. 2007b.
3408
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
analysis, phenolic compounds were tentatively identified. The phenolic compounds identified in raw peel extract are listed in Table 2, and shown in Fig. 2a and b. The major peak with a retention time of 2.8 min showed mixture of syringic glycoside and mangiferin pentoside. Mangiferin glycoside is reported to display a variety of health benefits such as hypolipidimic, antidiabetic, antitumor, hepatoprotective, antioxidant and immunomodulative activities (Guha et al., 1996; Percival et al., 2006). Another major peak (9.97 min) was identified as quercetin. Iriflophenone hexoside, maclurin-tri-O-galloyl hexoside, ellagic acid were also identified in the peel extract (Table 2, Fig. 1A). Two minor peaks, eluted very closely (1.68 min, 1.90 min), were identified as gallic acid (sodium adduct) and maclurin hexoside, respectively. Schieber et al. (2000) reported that the peel extracts of Peruvian mango fruits (cv. Kent) revealed that quercitin 3-galactoside and quercitin 3-glucoside are the predominat compounds. Quercitin has vasodialator and antihypertensive effects, and it has been re-
ported to reduce the vascular remodeling associated with elevated blood pressure in spontaneously hypertensive rats (Duarte et al., 2001). A small peak at 6.67 min was identified as gentisyl-protocatechuic acid (Table 2). In case of ripe peel extract most of the polyphenols present were similar to that of raw peel extract (Table 3). Iriflophenone hexoside was not detected in ripe peel extract, while hepta-O-galloyl hexose was identified in ripe peel extract and it was not present in raw peel. The results indicated that there are few differences in the composition of raw and ripe peels of the same variety. Recently, Schieber et al. (2003) and Berardini et al. (2004) reported the identification of polyphenols from Tommy Atkins mango peel. Of the several compounds identified by them, some of them were mangiferin and its derivatives, iriflophenone di-O-galloyl glucoside, maclurin derivatives including maclurin-tri-O-galloyl glucoside, hepta-O-galloyl glucose. They did not report the presence of ellagic acid, syringic or gentisyl or protocatechuic acid derivatives.
A. raw
2.80
100
9.97
%
10.12 10.21 2.33
4.88
1.47
0 5.00
10.00
15.00
20.00
25.00
Time (min)
B. ripe
10.38
100 2.76
%
9.79 1.83
5.03 9.45
6.91
12.42
0 5.00
10.00
15.00
20.00
25.00
Time (min) Fig. 1. LCMS-TIC profiles of phenolic compounds in the mango peel extract.
Table 2 UV spectra and characteristic ions of polyphenols extracted from the raw mango peel. Retention time (min)
Identitya
HPLC-DAD k-max (nm)
MW
HPLC–ESI ( ) MS (m/z)
1.47 1.68 1.90 2.33 2.80
Iriflophenone hexoside Sodium gallate Maclurin hexoside Maclurin-tri-O-galloyl hexoside Syringic acid hexoside + Mangiferin pentoside Ellagic acid(monohydrate) Gentisyl-protocatechuic acid Quercetin (monohydrate)
280; 278; 230; 280; 280; 280; 280; 281 321;
408 170 424 880 360 554 302 290 302
391[M+H H2O] 191[M+Na 2H] 405[M H H2O], 191 879[M H] 361[M+H] 553[M H] 321[M+H+H2O], 169 289[M H], 153 321[M+H+H2O] 301[M H]
4.88 6.67 9.97 a
Tentatively identified.
320 320 280 321 320 320 230 280
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
3409
Fig. 2a. Structure of phenolic compounds from mango peel.
Fig. 2b. Structure of phenolic compounds from mango peel.
3.4. Identification of carotenoids by RP-HPLC The carotenoids present in the raw and ripe mango peels were separated by reverse phase HPLC on C18 column. The carotenoids
were separated into four major peaks with few minor peaks. However, only three peaks were identified using the authentic standard carotenoids (Fig. 3). The carotenoids identified in the mango peel extracts were violaxanthin, lutein and b-carotene. Recently, Chen
3410
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
Table 3 UV spectra and characteristic ions of polyphenols extracted from the ripe mango peel. Retention time (min)
Identitya
HPLC-DAD k-max (nm)
MW
HPLC-ESI ( ) MS (m/z)
1.83 2.42 2.76
Hepta-O-galloyl hexose Gallic acid Syringic acid hexoside + Mangiferin pentoside Ellagic acid (monohydrate) Gentisyl-protocatechuic acid Quercetin (monohydrate)
230; 281; 281; 281; 281 280;
1244 170 360 554 302 290 302
1244[M+] 169[M-H] 361[M+H] 553[M-H] 321[M+H+H2O], 169 289[M H], 153 321[M+H+H2O], 301[M H
5.03 6.91 10.38 a
280 320 324 320 321
Tentatively identified.
et al. (2004) Identified carotenoids such as b-carotene, violaxanthin, neochrome, luteoxanthin, neoxanthin and zeaxanthin in Taiwanese mango pulp. Earlier studies indicted that acetone extract of raw and ripe peels from Badami mango variety exhibited antioxidant property and inhibited hemolysis of erythrocytes (Ajila et al., 2007b; Ajila and Prasada Rao, 2008). The ripe peel extract showed an IC50 value of 3.67 lg of GAE for DPPH radical scavenging activity while it was 1.39 lg of GAE for inhibition of lipid peroxidation (LPO). On the other hand, raw peel extract had an IC50 value of 4.54 lg of GAE for DPPH radical scavenging activity and 2.68 lg of GAE for LPO inhibition. Thus, the extent of DPPH radical scavenging activity and inhibition of LPO were more by the ripe peel extract than that of peel from raw fruit (Ajila et al., 2007b). However, no significant difference was found in the inhibition of erythrocyte hemolysis by raw and ripe peel extracts (Ajila and Prasada Rao, 2008). On the other hand, raw mango fruit peel extract had exhibited lower IC50 value (2 lg of GAE) for lipoxygenase inhibitory activity com-
pared to ripe peel extract which exhibited IC50 value of 4.7 lg of GAE for lipoxygenase inhibitory activity (Ajila et al., 2007b). The differences in their antioxidant potentials of raw and ripe extracts may be due to variations in the composition of polyphenol and carotenoids. In the present study we have seen the differences in the composition of phenolic compounds and carotenoids in the extracts of raw and ripe peels and these differences may be responsible for the differences in their antioxidant potentials on different systems. Aqueous stem bark extract from selected species of mango, which was used in pharmacological formulations and used as a food supplement from Cuba under the brand name of Vimang, has been reported to display a potent in vitro and in vivo antioxidant activity, and anti-inflammatory activity, and also used to prevent age-associated oxidative stress (Martinez et al., 2000; Garrido et al., 2004). Many of these pharmacological properties of mango fruit and stem bark may be attributed to the presence of phytochemicals such as polyphenols, carotenoids and vitamin E, among others (Nunez-Selles et al., 2000).
3
A. raw
0.075
1
Volts
0.050
2 0.025
0.000 0
5
10
15
20
25
30
35
40
Time (min)
2
3
0.10
B. ripe
Volts
1
0.05
0.00 0
5
10
15
20
25
30
35
40
Minutes Fig. 3. HPLC profiles of carotenoids in mango peel; Peak identification: (1) Violaxanthin, (2) Lutein, (3) b-Carotene.
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
4. Conclusions The present study indicates that the peel extracts obtained from raw and ripe mango fruits are rich source of polyphenols and carotenoids. The major phenolic compounds present in mango peel extract were syringic acid, quercitin, mangiferin pentoside, ellagic acid. In addition, the ripe peel contained glycosylated gallic acid. The carotenoids identified in mango peel extracts were b-carotene, lutein and violaxanthine. These phenolic compounds and carotenoids are reported to have antioxidant properties. We have recently reported that mango peel is also rich in soluble and insoluble dietary fibers. As mango is a seasonal fruit, either peel can be used immediately after its removal from the fruit or stored under refrigerated conditions for few days and used for the extraction of bioactive compounds, and the residue obtained can be used as a source of dietary fiber. Therefore, mango peel extract can be used in pharmaceutical applications and the residue remained after the removal of bioactive compounds can be used as a functional food. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements C.M. Ajila thanks Council of Scientific and Industrial Research, New Delhi, for the award of Senior Research Fellowship. References Ajila, C.M., Prasada Rao, U.J.S., 2008. Protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by Mangifera indica L. peel extract. Food Chem. Toxicol. 46, 303–309. 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 Chem. 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 Chem. 105, 982–988. Arouma, O.I., 2003. Methodological considerations for characterizing potential antioxidant actions of bioactive compounds in plant foods. Mutat. Res. 9, 523– 524. Berardini, N., Carle, R., Schieber, A., 2004. Characterization of gallotannins and benzophenone derivatives from mango (Mangifera indica L. cv. ‘Tommy Atkins’) peels, pulp and kernels by high performance liquid chromatography/ electrospray ionization mass spectrometry. Rapid Comm. Mass Spectrom 18, 2208–2216. Borbalan, A.M.A., Zorro, L., Guillen, D.A., Barroso, C.G., 2003. Study of the polyphenol content of red and white grape variety by liquid chromatography–mass spectrometry and its relationship to antioxidant power. J. Chromato A. 1002, 31–38. Bravo, L., 1998. Polyphenols: chemistry, dietary sources. Metabolism and nutraceutical significance. Nutr. Rev. 56, 317–333. Chen, J.P., Tai, C.Y., Chen, B.H., 2004. Improved liquid chromatographic method for determination of carotenoids in Taiwanese mango (Mangifera indica L.). J. Chromatogr. A 1054, 261–268. Duarte, J., Perez-Palencia, R., Vargas, F., Angeles Ocete, M., Perez-Vizcaino, F., Zarzuelo, A., 2001. Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br. J. Pharmacol. 133, 117–124. Fernandez–Pachon, M.S., Villaro, D., Garcia–Parilla, M.C., Troncoso, A.M., 2004. Antioxidant activity of wine and relation with their polyphenolic composition. Anal. Chem. Acta. 513, 113–118. FAO, 2007. Food and Agriculture Organization of the United Nations. Available at: http://faostat.fao.org.
3411
Garrido, G., Gonzalez, D., Lemus, Y., Garcia, D., Lodeiro, L., Quintero, G., 2004. In vivo and in vitro anti-inflammatory activity of Mangifera indica L. exreact (VIMANGÒ). Pharmacol. Res. 50, 143–149. Glowniak, K., Zgorka, G., Kozyra, M., 1996. Solid-phase extraction and reversedphase high-performance liquid chromatography of free phenolic acids in some Echinacea species. J. Chromatogr. 730, 25–29. Guha, S., Ghosal, S., Chattopadhyay, U., 1996. Anti-tumor, immunomodulatory, and anti-HIV effect of mangiferin, a naturally occurring glucosylxanthone. Chemotherapy 42, 443–451. Heim, K.E., Tagliaferro, A.R., Bobilya, D.J., 2002. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationship. The Nutri. Biochem. 13, 572– 584. Hag, M.E.E., Tinay, A.H.E., Yousif, N.E., 2002. Effect of fermentation and dehulling on starch, total polyphenols, phytic acid content and in vitro protein digestibility of pearl millet. Food Chem. 77, 193–196. Hertog, M.G.L., Feskens, E.J.M., Hallman, P.C.H., Katan, M.B., Kromhout, D., 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the zutphan elderly study. Lancet 342, 1007–1014. Krygier, K., Sosulski, F., Hogge, L., 1982. Free, esterified and insoluble bound phenolic acids. 1. Extraction and purification procedures. J. Agric. Food Chem. 30, 330–334. Litchenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–383. Martinez, G., Delgado, R., Perez, G., Garrido, G., Nunez-Selles, A.J., Leon-Fernandez, O.S., 2000. Evaluation of the in vitro antioxidat acivity of Mangifera indica L. Extract (VIMANGO). Phytother. Res. 14, 424–427. Middleton, E., Kandaswami, C., Theoharides, T.C., 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol. Rev. 52, 673–751. Miller, H.E., Rigelhof, F., Marquart, L., Prakash, A., Kanter, M., 2000. Whole grain products and antioxidants. Cereal Food World 45, 59–63. Nardini, M., Natella, F., Scaccini, C., Ghiselli, A., 2004. Phenolic acids from beer are absorbed and extensively metabolized in humans. J. Nutr. Biochem. 17, 14–22. Nunez-Selles, A.J., Velez-Castro, H.T., Aguero-Aguero, J., Gonzalez-Gonzalez, J., Naddeo, F., De Simone, F., Rastrelli, L., 2000. Isolation and quantitative analysis of phenolic constituents, free sugars, fatty acids, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as nutritional supplement. J. Agric. Food Chem. 50, 762–766. Parr, A.J., Bolwell, G.P., 2000. Phenols in the plant and in man. The potential for possible nutritional enhancement of the diet by modifying the phenols content. J. Sci. Food Agric. 80, 985–1012. Percival, S.S., Talcott, S.T., Chin, S.T., Mallak, A.C., Lounds-Singleton, A., Pettit-Moore, J., 2006. Noeplastic transformation of BALB/3T3 cells and cell cycle of HL-60 cells are inhibited by mango (Mangifera indica L.) juice and mango juice extracts. J. Nutr. 136, 1300–1304. Puupponen-Pimia, R., Nohynek, L., Meier, C., Kahkonen, M., Heinonen, M., Hopia, A., Oksman-Caldentey, K.-M., 2001. Antimicrobial properties of phenolic compounds from berries. J. Appl. Microbiol. 90, 494–507. Rao, A.R., Sarada, R., Ravishankar, G.S., 2007. Stabilization of astaxanthin in edible oils and its use as an antioxidant. J. Sci. Food Agric. 87, 957–965. Renger, A., Steinhart, H., 2000. Ferulic acid dehydrodimers as structural elements in cereal dietary fibre. Eur. Food Res. Technol. 211, 422–428. Robbins, R.J., 2003. Phenolic acids in foods: an overview of analytical methodology. J. Agric. Food Chem. 51, 2866–2887. Sanchez, G.M., Re, L., Giuliani, A., Nunez-Selles, A.J., Davison, G.P., Leon-Fernandez, O.S., 2000. Protective effects of Mangifera indica L. Extract, mangiferin and selected antioxidants against TPA-induced bimolecular oxidation and peritoneal macrophage activation in mice. Pharmacol. Res. 42, 565–573. Sarada, R., Vidhyavathi, R., Usha, D., Ravishankar, G.A., 2006. An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis. J. Agric Food Chem. 54, 7585–7588. Schieber, A., Berardini, N., Carle, R., 2003. Identification of flavonol and xanthone glycosides from mango (Mangifera indica L. Cv. ‘‘Tommy Atkins”) peels by highperformance liquid chromatography-electron spray ionization mass spectrometer.. J. Agric. Food Chem. 51, 5006–5011. Schieber, A., Ullrich, W., Carle, R., 2000. Characterization of polyphenols in mango puree concentrate by HPLC with diode array and mass spectrometric detection. Innovative Food Sci. Technol. 1, 161–166. Swain, T., Hillis, W., 1959. The phenolic constituents of Prunus domestica. 1. The quantitative analysis of phenolic constitutents. J. Sci. Food Agric. 10, 63–68.
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts C.M. Ajila, L. Jaganmohan Rao, U.J.S. Prasada Rao ⇑ Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 020, India
a r t i c l e
i n f o
Article history: Received 4 May 2010 Accepted 6 September 2010
Keywords: Mango peel Mangifera indica L. HPLC–MS Polyphenolic compounds Carotenoids
a b s t r a c t Mango is one of the important tropical fruits in the world. As it is a seasonal fruit, it is processed for various products. During its processing, peel is one of the major byproducts, which is being wasted. Bioactive conserves were extracted using 80% acetone from peels of raw and ripe mango fruits and subjected to acid hydrolysis. The prominent phenolic compounds identified by HPLC were protocatechuic acid, gentisic acid and gallic acid. The phenolic acid derivatives present in acetone extracts of raw and ripe peels were tentatively identified by LC–MS. Gallic acid, syringic acid, mangiferin, ellagic acid, gentisyl-protocatechuic acid, quercetin were the phenolic compounds identified in both raw and ripe peels, while raw peel showed the presence of glycosylated iriflophenone and maclurin derivatives also. b-Carotene was the major carotenoid followed by violaxanthin and lutein. Thus, both raw and ripe mango peel extracts have different phenolic compounds and carotenoids, which will have various pharmaceutical applications. Ó 2010 Published by Elsevier Ltd.
1. Introduction Epidemiological evidences suggest that diet rich in fruits and vegetables provide health benefits such as prevention of cardiovascular diseases and certain types of cancer (Arouma, 2003; Miller et al., 2000). These health beneficial effects are due to the presence of various bioactive compounds that are present in fruits and vegetables (Ajila et al., 2007a; Hertog et al., 1993; Heim et al., 2002; Parr and Bolwell, 2000). Phenolic compounds and carotenoids are the major bioactive compounds found in fruits and vegetables and these compounds are shown to exhibit antioxidant, antiallergenic, antiarthrogenic, antiinflamatory, antimicrobial and antithrombotic effects (Ajila et al., 2007b; Bravo, 1998; Middleton et al., 2000; Puupponen-Pimia et al., 2001). Phenolic acids like gallic acid and quercitin are claimed to have activity against allergy, hypertension, arthritis and cancer (Borbalan et al., 2003; Fernandez–Pachon et al., 2004). Mangiferin, which is shown to be the predominant constituent of mango stem bark extract is reported to display a variety of pharmacological effects (Guha et al., 1996; Sanchez et al., 2000). Byproducts from different fruit processing industries, which were traditionally treated as environmental pollutants, are being recognized as source for obtaining valuable components. Mango is one of the important tropical fruits in the world and India contributes major part of the world production. India produced 13.5 MMT of mango fruits, while the world production was 33.45 MMT ⇑ Corresponding author. Tel.: +91 0821 2514876; fax: +91 0821 2517233. E-mail address: [email protected] (U.J.S. Prasada Rao). 0278-6915/$ - see front matter Ó 2010 Published by Elsevier Ltd. doi:10.1016/j.fct.2010.09.012
(FAO, 2007). Mango fruits are processed for various products as it is a seasonal fruit. Peel is one of the major byproducts during mango processing. It constitutes about 15–20% of the fruit weight. At present peel is discarded as waste and becoming a source of pollution. Recently, Ajila et al. (2007a) reported that mango peel extracts from two different mango varieties at different stages of fruit maturity showed good amount of polyphenols and carotenoids. It was also reported that peel extracts exhibited potential antioxidant activities (Ajila et al., 2007b) and protected against H2O2 induced oxidative damage in rat erythrocytes (Ajila and Prasada Rao, 2008). The present study will focus on the identification of bioactive compounds such as phenolic acids and their derivatives, and carotenoids present in peels of ripe and raw fruits of an Indian mango variety. 2. Materials and methods 2.1. Plant materials Badami mango variety grown in CFTRI campus, Mysore, India was used in this study. Mango fruits were harvested at harvest maturity and peel was removed using a sharp knife and the underlying pulp removed by gently scraping with its blunt edge. To obtain the ripe peel, some fruits were kept to ripen at room temperature and the peel was removed as described earlier. The fresh peels thus obtained were used for analysis. 2.2. Chemicals Gallic acid, caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gentisic acid, protocatechuic acid, syringic acid, vanilic acid, b-carotene, violaxanthin and a-tocoferol were obtained from Sigma Fine Chemicals, St. Louis, USA. All other chemicals and solvents were of analytical grade.
3407
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411 2.3. Preparation of acetone extract of mango peel Both raw and ripe mango peels were removed from the fruits, extracted with 80% acetone, centrifuged at 10,000g for 15 min and these extracts were used for the further studies like estimation and identification of phenolic compounds and carotenoids. 2.4. Determination of total phenolics, carotenoids and vitamin E content Polyphenol content in Mango peel extract was determined by using the method described by Hag et al. (2002). The total polyphenols content in the extract was expressed as gallic acid equivalents (GAE). Carotenoid content in peel extract was determined after saponification using the method as described by Litchenthaler (1987). Vitamin E content was determined according to the method described by Rao et al. (2007). a-Tocoferol was used as a standard to estimate total vitamin E content. 2.5. Identification of phenolic compounds in acetone extract by HPLC and LCMS Polyphenols were separated on a reverse phase C18 column (4.6 250 mm) using HPLC system (Shimadzu, Model LC-10A) using a diode array detector (operating at 280 nm and 320 nm). A solvent system consisting of water: methanol: acetic acid (83:15:2) was used as mobile phase (isocratic) at a flow rate of 1 ml/min (Glowniak et al., 1996). Known quantities of phenolic acid standards such as caffeic acid, p-coumaric acid, cinnamic acid, ferulic acid, gallic acid, gentisic acid, protocatechuic acid, syringic acid, vanillic acid were used for identification and quantification of phenolic acids present in the acetone extracts. HPLC–ESI-MS analyses were done on a Waters platform ZMD 4000 system composed of a micro ZMD mass spectrophotometer, a Waters 2690 HPLC and a Waters 996 photo diode array detector (Waters corporation, MA, USA). Data were collected and processed via a personal computer running Mass Lynx software version 3.1 (Micromass, a diversion of Waters corporation, MA, USA). The samples in 10 ll aliquot were separated on a reversed phase C18 column (4.6 250 mm), using a diode array detector (operating at 280 and 320 nm). A solvent system consisting of water: methanol: acetic acid (83:15:2) was used as mobile phase (isocratic) at a flow rate of 1 ml/min. UV–VIS absorption spectra were recorded on-line during HPLC analysis. Spectral measurements were made over the range of 200–600 nm. The following ion optics was used- capillary voltage 3 kV, cone voltage 100 V and collision voltage 10 V. The source block temperature was 80 °C and the desolvation temperature was 150 °C. ESI-MS was performed using argon as cone gas (50 L/h) and nitrogen as desolvation gas (50 L/h). The electron spray probe flow was adjusted to 70 ml/min. Continuous mass spectra were recorded over the range m/z 100–500 with scan time 1 s and interscan delay 0.1 s. 2.6. Extraction, estimation and identification of phenolic acids liberated from the soluble bound phenolic compounds Phenolic compounds from mango peel (1 g) were extracted with 20 ml of 80% acetone, centrifuged, and the supernatant obtained was subjected to acid hydrolysis to obtain the phenolic acids according to the method of Krygier et al., (1982) with some modifications as described. The pH of the 80% acetone extract was adjusted to 2 with 4 M HCl and refluxed in boiling water bath for 1 h. Phenolic acids were separated by ethyl acetate phase separation (5 50 ml) and the pooled ethyl acetate fractions were treated with anhydrous sodium sulphate for overnight to remove moisture, filtered and evaporated to dryness and re-dissolved in methanol. Phenolic content was determined by the method of Swain and Hillis (1959) and the phenolic acids were identified after separating them on reverse phase C18 column by HPLC. 2.7. Identification of carotenoids by HPLC Carotenoids in the hexane extract were separated on a reverse phase C18 column (4.6 250 mm) using HPLC system (Model LC- 10A, Shimadzu) with a diode array detector (operating at 460 and 425 nm) using a gradient of two solvents, solvent A-100% acetone and solvent B-90% methanol and 10% water (Sarada et al., 2006). The gradient for separation consists of 1% B traversing to 80% in 40 min at a flow rate of 1 ml/min. The detection was carried out using diode array detector at 445 nm and 460 nm. Peak identification was based on the comparison of retention time with authentic standards of carotenoids such as b-carotene, lutein and violaxanthin.
3. Results and discussion 3.1. Polyphenols, carotenoids and vitamin E content in mango peel acetone extract Peel extracts were analyzed for various compounds namely polyphenols, carotenoids and vitamin E. The polyphenol contents
in acetone extracts of raw and ripe fresh peels varied from 55 to 90 mg GAE/g peel (dry weight basis). The carotenoid contents in raw and ripe peel extracts were found to be 81 and 194 lg/g peel, respectively. The vitamin E content in raw peel was 104 lg/g peel while it was 230 lg/g for ripe peel. The polyphenol content was found to be significantly higher in raw peels compared to ripe peel while the carotenoids and vitamin E were found to be significantly higher in ripe peels (Table 1). 3.2. Identification of phenolic acids of peel extract Initially, efforts were made to identify phenolic compounds by separating on C18 column using HPLC. The extracts obtained from each variety were separated into 5–8 peaks. Efforts to identify these peaks by comparing the retention times of authentic standards of phenolic acids, or spiking with known phenolic acids did not help in identifying the peaks, as there were little variations with retention times. This is mainly due to the complex nature of polyphenols. Phenolic acids can be classified as free, soluble and insoluble bound phenolic acids (Renger and Steinhart, 2000). The free forms of phenolic compounds are very rarely present in plants. Majority of phenolic acids exists in the form of ester, acetal, or ethers and some of the phenolic acids are linked either to structural compounds of plants such as protein, dietary fiber or to longer or smaller organic molecules such as glucose, maleic acid or to other natural products (Robbins, 2003; Nardini et al., 2004). Acid hydrolysis and saponification are the commonly used methods for releasing bound phenolics by different workers. Esterases are also used to cleave the ester bond, but not as popular as the other two methods (Robbins, 2003). The phenolic acids in phenolic fractions after acid hydrolysis of raw and ripe mango peels were separated on reverse phase C18 column on HPLC using water: methanol: acetic acid (83:15:2). The compounds eluted were monitored at 280 and 320 nm to detect various compounds. The retention times of these peaks were compared with the authentic standards of phenolic acids. However, wherever there is a little variation in retention times, their identifications were confirmed by spiking with authentic standards. Gallic acid, protocatechuic acid, gentisic acid and syringic acid were the phenolic acids identified in phenolic fractions of raw and ripe mango peel extracts. 3.3. Identification of phenolic compounds in the acetone extract of mango peel As mentioned earlier, phenolic compounds exist as esters or glycosides. In order to identify these phenolic compounds, the acetone extracts of peels from mango fruits were analyzed by LC–ESIMS. The acetone extracts of raw and ripe peels were separated on reverse phase C18 column using conditions described earlier. The LCMS-TIC profiles of polyphenolics in mango peel extracts of raw and ripe peels were shown in Fig. 1. Based on mass spectrometric
Table 1 Total phenolics, carotenoids and vitamin E contents in acetone extracts of mango peel. Parameter
Raw peel
Ripe peel
Total phenolic content* (mg/g GAE) Carotenoid Content* (lg/g) Vitamin E (lg/g)
90.2 ± 0.57b 81.0 ± 0.42a 104 ± 3.2a
54.67 ± 1.5a 194 ± 0.26b 230 ± 12b
Values are expressed on dry weight basis. All data are the mean ± SD of three replicates. Mean value followed by different letters in the same row differ significantly (P 6 0.05). * Ajila et al. 2007b.
3408
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
analysis, phenolic compounds were tentatively identified. The phenolic compounds identified in raw peel extract are listed in Table 2, and shown in Fig. 2a and b. The major peak with a retention time of 2.8 min showed mixture of syringic glycoside and mangiferin pentoside. Mangiferin glycoside is reported to display a variety of health benefits such as hypolipidimic, antidiabetic, antitumor, hepatoprotective, antioxidant and immunomodulative activities (Guha et al., 1996; Percival et al., 2006). Another major peak (9.97 min) was identified as quercetin. Iriflophenone hexoside, maclurin-tri-O-galloyl hexoside, ellagic acid were also identified in the peel extract (Table 2, Fig. 1A). Two minor peaks, eluted very closely (1.68 min, 1.90 min), were identified as gallic acid (sodium adduct) and maclurin hexoside, respectively. Schieber et al. (2000) reported that the peel extracts of Peruvian mango fruits (cv. Kent) revealed that quercitin 3-galactoside and quercitin 3-glucoside are the predominat compounds. Quercitin has vasodialator and antihypertensive effects, and it has been re-
ported to reduce the vascular remodeling associated with elevated blood pressure in spontaneously hypertensive rats (Duarte et al., 2001). A small peak at 6.67 min was identified as gentisyl-protocatechuic acid (Table 2). In case of ripe peel extract most of the polyphenols present were similar to that of raw peel extract (Table 3). Iriflophenone hexoside was not detected in ripe peel extract, while hepta-O-galloyl hexose was identified in ripe peel extract and it was not present in raw peel. The results indicated that there are few differences in the composition of raw and ripe peels of the same variety. Recently, Schieber et al. (2003) and Berardini et al. (2004) reported the identification of polyphenols from Tommy Atkins mango peel. Of the several compounds identified by them, some of them were mangiferin and its derivatives, iriflophenone di-O-galloyl glucoside, maclurin derivatives including maclurin-tri-O-galloyl glucoside, hepta-O-galloyl glucose. They did not report the presence of ellagic acid, syringic or gentisyl or protocatechuic acid derivatives.
A. raw
2.80
100
9.97
%
10.12 10.21 2.33
4.88
1.47
0 5.00
10.00
15.00
20.00
25.00
Time (min)
B. ripe
10.38
100 2.76
%
9.79 1.83
5.03 9.45
6.91
12.42
0 5.00
10.00
15.00
20.00
25.00
Time (min) Fig. 1. LCMS-TIC profiles of phenolic compounds in the mango peel extract.
Table 2 UV spectra and characteristic ions of polyphenols extracted from the raw mango peel. Retention time (min)
Identitya
HPLC-DAD k-max (nm)
MW
HPLC–ESI ( ) MS (m/z)
1.47 1.68 1.90 2.33 2.80
Iriflophenone hexoside Sodium gallate Maclurin hexoside Maclurin-tri-O-galloyl hexoside Syringic acid hexoside + Mangiferin pentoside Ellagic acid(monohydrate) Gentisyl-protocatechuic acid Quercetin (monohydrate)
280; 278; 230; 280; 280; 280; 280; 281 321;
408 170 424 880 360 554 302 290 302
391[M+H H2O] 191[M+Na 2H] 405[M H H2O], 191 879[M H] 361[M+H] 553[M H] 321[M+H+H2O], 169 289[M H], 153 321[M+H+H2O] 301[M H]
4.88 6.67 9.97 a
Tentatively identified.
320 320 280 321 320 320 230 280
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
3409
Fig. 2a. Structure of phenolic compounds from mango peel.
Fig. 2b. Structure of phenolic compounds from mango peel.
3.4. Identification of carotenoids by RP-HPLC The carotenoids present in the raw and ripe mango peels were separated by reverse phase HPLC on C18 column. The carotenoids
were separated into four major peaks with few minor peaks. However, only three peaks were identified using the authentic standard carotenoids (Fig. 3). The carotenoids identified in the mango peel extracts were violaxanthin, lutein and b-carotene. Recently, Chen
3410
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
Table 3 UV spectra and characteristic ions of polyphenols extracted from the ripe mango peel. Retention time (min)
Identitya
HPLC-DAD k-max (nm)
MW
HPLC-ESI ( ) MS (m/z)
1.83 2.42 2.76
Hepta-O-galloyl hexose Gallic acid Syringic acid hexoside + Mangiferin pentoside Ellagic acid (monohydrate) Gentisyl-protocatechuic acid Quercetin (monohydrate)
230; 281; 281; 281; 281 280;
1244 170 360 554 302 290 302
1244[M+] 169[M-H] 361[M+H] 553[M-H] 321[M+H+H2O], 169 289[M H], 153 321[M+H+H2O], 301[M H
5.03 6.91 10.38 a
280 320 324 320 321
Tentatively identified.
et al. (2004) Identified carotenoids such as b-carotene, violaxanthin, neochrome, luteoxanthin, neoxanthin and zeaxanthin in Taiwanese mango pulp. Earlier studies indicted that acetone extract of raw and ripe peels from Badami mango variety exhibited antioxidant property and inhibited hemolysis of erythrocytes (Ajila et al., 2007b; Ajila and Prasada Rao, 2008). The ripe peel extract showed an IC50 value of 3.67 lg of GAE for DPPH radical scavenging activity while it was 1.39 lg of GAE for inhibition of lipid peroxidation (LPO). On the other hand, raw peel extract had an IC50 value of 4.54 lg of GAE for DPPH radical scavenging activity and 2.68 lg of GAE for LPO inhibition. Thus, the extent of DPPH radical scavenging activity and inhibition of LPO were more by the ripe peel extract than that of peel from raw fruit (Ajila et al., 2007b). However, no significant difference was found in the inhibition of erythrocyte hemolysis by raw and ripe peel extracts (Ajila and Prasada Rao, 2008). On the other hand, raw mango fruit peel extract had exhibited lower IC50 value (2 lg of GAE) for lipoxygenase inhibitory activity com-
pared to ripe peel extract which exhibited IC50 value of 4.7 lg of GAE for lipoxygenase inhibitory activity (Ajila et al., 2007b). The differences in their antioxidant potentials of raw and ripe extracts may be due to variations in the composition of polyphenol and carotenoids. In the present study we have seen the differences in the composition of phenolic compounds and carotenoids in the extracts of raw and ripe peels and these differences may be responsible for the differences in their antioxidant potentials on different systems. Aqueous stem bark extract from selected species of mango, which was used in pharmacological formulations and used as a food supplement from Cuba under the brand name of Vimang, has been reported to display a potent in vitro and in vivo antioxidant activity, and anti-inflammatory activity, and also used to prevent age-associated oxidative stress (Martinez et al., 2000; Garrido et al., 2004). Many of these pharmacological properties of mango fruit and stem bark may be attributed to the presence of phytochemicals such as polyphenols, carotenoids and vitamin E, among others (Nunez-Selles et al., 2000).
3
A. raw
0.075
1
Volts
0.050
2 0.025
0.000 0
5
10
15
20
25
30
35
40
Time (min)
2
3
0.10
B. ripe
Volts
1
0.05
0.00 0
5
10
15
20
25
30
35
40
Minutes Fig. 3. HPLC profiles of carotenoids in mango peel; Peak identification: (1) Violaxanthin, (2) Lutein, (3) b-Carotene.
C.M. Ajila et al. / Food and Chemical Toxicology 48 (2010) 3406–3411
4. Conclusions The present study indicates that the peel extracts obtained from raw and ripe mango fruits are rich source of polyphenols and carotenoids. The major phenolic compounds present in mango peel extract were syringic acid, quercitin, mangiferin pentoside, ellagic acid. In addition, the ripe peel contained glycosylated gallic acid. The carotenoids identified in mango peel extracts were b-carotene, lutein and violaxanthine. These phenolic compounds and carotenoids are reported to have antioxidant properties. We have recently reported that mango peel is also rich in soluble and insoluble dietary fibers. As mango is a seasonal fruit, either peel can be used immediately after its removal from the fruit or stored under refrigerated conditions for few days and used for the extraction of bioactive compounds, and the residue obtained can be used as a source of dietary fiber. Therefore, mango peel extract can be used in pharmaceutical applications and the residue remained after the removal of bioactive compounds can be used as a functional food. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements C.M. Ajila thanks Council of Scientific and Industrial Research, New Delhi, for the award of Senior Research Fellowship. References Ajila, C.M., Prasada Rao, U.J.S., 2008. Protection against hydrogen peroxide induced oxidative damage in rat erythrocytes by Mangifera indica L. peel extract. Food Chem. Toxicol. 46, 303–309. 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 Chem. 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 Chem. 105, 982–988. Arouma, O.I., 2003. Methodological considerations for characterizing potential antioxidant actions of bioactive compounds in plant foods. Mutat. Res. 9, 523– 524. Berardini, N., Carle, R., Schieber, A., 2004. Characterization of gallotannins and benzophenone derivatives from mango (Mangifera indica L. cv. ‘Tommy Atkins’) peels, pulp and kernels by high performance liquid chromatography/ electrospray ionization mass spectrometry. Rapid Comm. Mass Spectrom 18, 2208–2216. Borbalan, A.M.A., Zorro, L., Guillen, D.A., Barroso, C.G., 2003. Study of the polyphenol content of red and white grape variety by liquid chromatography–mass spectrometry and its relationship to antioxidant power. J. Chromato A. 1002, 31–38. Bravo, L., 1998. Polyphenols: chemistry, dietary sources. Metabolism and nutraceutical significance. Nutr. Rev. 56, 317–333. Chen, J.P., Tai, C.Y., Chen, B.H., 2004. Improved liquid chromatographic method for determination of carotenoids in Taiwanese mango (Mangifera indica L.). J. Chromatogr. A 1054, 261–268. Duarte, J., Perez-Palencia, R., Vargas, F., Angeles Ocete, M., Perez-Vizcaino, F., Zarzuelo, A., 2001. Antihypertensive effects of the flavonoid quercetin in spontaneously hypertensive rats. Br. J. Pharmacol. 133, 117–124. Fernandez–Pachon, M.S., Villaro, D., Garcia–Parilla, M.C., Troncoso, A.M., 2004. Antioxidant activity of wine and relation with their polyphenolic composition. Anal. Chem. Acta. 513, 113–118. FAO, 2007. Food and Agriculture Organization of the United Nations. Available at: http://faostat.fao.org.
3411
Garrido, G., Gonzalez, D., Lemus, Y., Garcia, D., Lodeiro, L., Quintero, G., 2004. In vivo and in vitro anti-inflammatory activity of Mangifera indica L. exreact (VIMANGÒ). Pharmacol. Res. 50, 143–149. Glowniak, K., Zgorka, G., Kozyra, M., 1996. Solid-phase extraction and reversedphase high-performance liquid chromatography of free phenolic acids in some Echinacea species. J. Chromatogr. 730, 25–29. Guha, S., Ghosal, S., Chattopadhyay, U., 1996. Anti-tumor, immunomodulatory, and anti-HIV effect of mangiferin, a naturally occurring glucosylxanthone. Chemotherapy 42, 443–451. Heim, K.E., Tagliaferro, A.R., Bobilya, D.J., 2002. Flavonoid antioxidants: chemistry, metabolism and structure-activity relationship. The Nutri. Biochem. 13, 572– 584. Hag, M.E.E., Tinay, A.H.E., Yousif, N.E., 2002. Effect of fermentation and dehulling on starch, total polyphenols, phytic acid content and in vitro protein digestibility of pearl millet. Food Chem. 77, 193–196. Hertog, M.G.L., Feskens, E.J.M., Hallman, P.C.H., Katan, M.B., Kromhout, D., 1993. Dietary antioxidant flavonoids and risk of coronary heart disease: the zutphan elderly study. Lancet 342, 1007–1014. Krygier, K., Sosulski, F., Hogge, L., 1982. Free, esterified and insoluble bound phenolic acids. 1. Extraction and purification procedures. J. Agric. Food Chem. 30, 330–334. Litchenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148, 350–383. Martinez, G., Delgado, R., Perez, G., Garrido, G., Nunez-Selles, A.J., Leon-Fernandez, O.S., 2000. Evaluation of the in vitro antioxidat acivity of Mangifera indica L. Extract (VIMANGO). Phytother. Res. 14, 424–427. Middleton, E., Kandaswami, C., Theoharides, T.C., 2000. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease and cancer. Pharmacol. Rev. 52, 673–751. Miller, H.E., Rigelhof, F., Marquart, L., Prakash, A., Kanter, M., 2000. Whole grain products and antioxidants. Cereal Food World 45, 59–63. Nardini, M., Natella, F., Scaccini, C., Ghiselli, A., 2004. Phenolic acids from beer are absorbed and extensively metabolized in humans. J. Nutr. Biochem. 17, 14–22. Nunez-Selles, A.J., Velez-Castro, H.T., Aguero-Aguero, J., Gonzalez-Gonzalez, J., Naddeo, F., De Simone, F., Rastrelli, L., 2000. Isolation and quantitative analysis of phenolic constituents, free sugars, fatty acids, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as nutritional supplement. J. Agric. Food Chem. 50, 762–766. Parr, A.J., Bolwell, G.P., 2000. Phenols in the plant and in man. The potential for possible nutritional enhancement of the diet by modifying the phenols content. J. Sci. Food Agric. 80, 985–1012. Percival, S.S., Talcott, S.T., Chin, S.T., Mallak, A.C., Lounds-Singleton, A., Pettit-Moore, J., 2006. Noeplastic transformation of BALB/3T3 cells and cell cycle of HL-60 cells are inhibited by mango (Mangifera indica L.) juice and mango juice extracts. J. Nutr. 136, 1300–1304. Puupponen-Pimia, R., Nohynek, L., Meier, C., Kahkonen, M., Heinonen, M., Hopia, A., Oksman-Caldentey, K.-M., 2001. Antimicrobial properties of phenolic compounds from berries. J. Appl. Microbiol. 90, 494–507. Rao, A.R., Sarada, R., Ravishankar, G.S., 2007. Stabilization of astaxanthin in edible oils and its use as an antioxidant. J. Sci. Food Agric. 87, 957–965. Renger, A., Steinhart, H., 2000. Ferulic acid dehydrodimers as structural elements in cereal dietary fibre. Eur. Food Res. Technol. 211, 422–428. Robbins, R.J., 2003. Phenolic acids in foods: an overview of analytical methodology. J. Agric. Food Chem. 51, 2866–2887. Sanchez, G.M., Re, L., Giuliani, A., Nunez-Selles, A.J., Davison, G.P., Leon-Fernandez, O.S., 2000. Protective effects of Mangifera indica L. Extract, mangiferin and selected antioxidants against TPA-induced bimolecular oxidation and peritoneal macrophage activation in mice. Pharmacol. Res. 42, 565–573. Sarada, R., Vidhyavathi, R., Usha, D., Ravishankar, G.A., 2006. An efficient method for extraction of astaxanthin from green alga Haematococcus pluvialis. J. Agric Food Chem. 54, 7585–7588. Schieber, A., Berardini, N., Carle, R., 2003. Identification of flavonol and xanthone glycosides from mango (Mangifera indica L. Cv. ‘‘Tommy Atkins”) peels by highperformance liquid chromatography-electron spray ionization mass spectrometer.. J. Agric. Food Chem. 51, 5006–5011. Schieber, A., Ullrich, W., Carle, R., 2000. Characterization of polyphenols in mango puree concentrate by HPLC with diode array and mass spectrometric detection. Innovative Food Sci. Technol. 1, 161–166. Swain, T., Hillis, W., 1959. The phenolic constituents of Prunus domestica. 1. The quantitative analysis of phenolic constitutents. J. Sci. Food Agric. 10, 63–68.