The ripe pulp of Mangifera indica L.: A rich source of phytosterols and other lipophilic phytochemicals

Thechemicalcompositionofthelipophilicextractsoftheripepulpofmangoesfromtwelvecultivarsof Mangifera indica L.from MadeiraIsland wasinvestigatedbygas chromatography – mass spectrometry (GC – MS) for the ﬁ rst time. The ripe pulp of these mango cultivars showed analogous amounts of lipophilic extractives, aswellas sim- ilarqualitativechemicalcompositions.Thepredominantcompoundswerefreeandglycosylatedsterolsandfatty acids,representing44.8 – 70.7%and22.6 – 41.9%,respectively,ofthetotalamountoflipophiliccomponents.Small-er amounts of long chain aliphatic alcohols and α -tocopherol were also identi ﬁ ed. These data indicate that the investigated mango cultivars are a rich source of valuable phytochemicals, contributing to the intake of at least 9.5 – 38.2 mg of phytosterols (free and glycosylated) and 0.7 – 3.9 mg of fatty acids ( ω − 3 and ω − 6) per 100 g of fresh mango, with recognizable bene ﬁ cial effects on human nutrition and health.


Introduction
The mango fruit, one of the most important tropical fruits in the world, enjoys the status of "the king of fruits" as a result of its unique flavor, fragrance and appearance (Singh, Singh, Sane, & Nath, 2013). The Mangifera indica L. species, belonging to the Mangifera genus, Anacardiaceae family and Sapindales order, is the most important edible species and its fruit shows a pronounced diversity in size, shape, color, flavor, seed size, and chemical composition (Stafford, 1983), depending on the cultivar (Othman & Mbogo, 2009), edaphoclimatic conditions (Léchaudel & Joas, 2006) and postharvest storage (Nunes, Emond, Brecht, Dea, & Proulx, 2007). 'Kent', 'Tommy Atkins', 'Haden', and 'Keitt' are the most popular export mango cultivars (Sauco, 2004).
M. indica L. species, native from Southeast Asia, are widely cultivated at both tropical and subtropical latitudes (Kaira, Tandon, & Singh, 1995;Ueda, Sasaki, Utsunomiya, Inaba, & Shimabayashi, 2000), over a harvested area of approximately 5 million ha in 94 countries (FAOSTAT, 2011). Over the last decade, mango cultivated area increased by 41.8%, and is expected to increase even more due to the growing consumption of fresh fruit and processed products. The annual world production accounted in 2011 for ca. 38 million tonnes, with India as the major producer (15 million tonnes), Mexico and India as the major exporters (275 and 260 thousand tonnes in 2010, respectively), while the European Union and United States of America were the main importers (369 and 320 thousand tonnes in 2010, respectively) of mango fruits (FAOSTAT, 2011). In Madeira Island, a Portuguese Mediterranean subtropical region with adequate climate to the growth cycle of tropical and subtropical fruits (e.g. banana (Musa acuminata Colla), avocado (Persea americana Mill.) and annona (Annona cherimola Mill.)), mango was introduced after the second half of the eighteenth century. Nevertheless, it was only in the twentieth century that the plantations of this fruit attained a commercial dimension and became an important crop for the island's economy.
The mango fruit, with an average annual worldwide per capita consumption of 3.42 kg (CBI Report, 2011), is one of the nutritionally richest fruits, providing about 64-86 cal per 100 g (Rathore, Tariq, Shehla, & Soomro, 2007), with 32-200 mg per 100 g of vitamin C (Akinyele & Keshinro, 1980). When regularly consumed, this fruit can be a valuable dietary source of many phytochemicals (Haard & Chism, 1996) that provide several human health benefits (Singh et al., 2013). Several studies have addressed the phytochemical composition of diverse mango plant tissues, namely leaves, stem bark, peel, pulp and kernel, given their medicinal applications (Masibo & He, 2009). For example, Garido et al. (2001) found different polyphenols, steroids, flavonoids and tannins with antinociceptive and anti-inflammatory actions in M. indica L. stem bark extracts that could be used to improve the life quality in patients suffering from high stress levels. The mango seeds of M. indica L., with a broad antimicrobial spectrum (Kabuki et al., 2000) and a significant anti-diarrheal activity (Sairam et al., 2003), showed potential as food additives for extending the shelf-life of a variety of food products. Nevertheless, there is still a lack of detailed studies on the phytochemical composition of mango pulp, particularly on the lipophilic components, with only a study reporting the sterol composition of mango from China (Han, Yang, & Feng, 2008).
The present study is part of a global project concerning sub-tropical fruits' nutritional and functional values, aiming to add value to the fruits and by-products, by promoting an increase in consumption and market competitiveness of this sector. To the best of our knowledge, no studies about the chemical composition of lipophilic extracts of ripe mango pulp of 'Tommy Atkins', 'Rosa', 'OTT', 'Anderson', 'Rubro Brasil', 'Osteen', 'Tolbert', 'Irwin', 'Gleen', 'Gomera I', 'Gomera II' and 'Gomera III' cultivars, growing in Madeira Island, have been published until now. In this context, this work aims at establishing the lipophilic extractives profile (fatty acids, sterols, long chain aliphatic alcohols and other compounds) of mango pulp by gas chromatography-mass spectrometry (GC-MS) analysis and to link them with the potential health benefits of these mango pulp cultivars growing under the Mediterranean subtropical climate conditions of that island. From a commercial point of view, the evaluation of the selected mango cultivars could provide information to farmers about the cultivars with a higher commercial added-value, in order to compete favorably for local and export markets.

Sample preparation and physicochemical parameters
Mango fruits (M. indica L.) without evidence of physical or pathological injuries were selected from Centro de Fruticultura Subtropical do Funchal, Madeira Island, Portugal (32°38′ 52″ N, 16°57′ 44″ W). The mature green fruits from 'Tommy Atkins', 'Rosa', 'OTT', 'Anderson', 'Rubro Brasil', 'Osteen', 'Tolbert', 'Irwin', 'Gleen', 'Gomera I', 'Gomera II' and 'Gomera III' cultivars were hand harvested and then left to reach full ripeness at room temperature (20-23°C). Fruit firmness was determined after removing the skin on two opposite sides in the middle of each fruit using a pressure-testing instrument (Model FT 327) fitted with an 11.3 mm cylindrical plunger. The force required to penetrate into the flesh was expressed in Newtons (N). The fruits were immediately peeled (peel was fully discarded), sliced, quick-frozen in liquid nitrogen and lyophilized. Fresh slices of each sample were used to measure fruit water content through a Gibertini-Eurotherm balance, at 105°C and Brix using a digital Brix refractometer from ATAGO. The frozen samples were lyophilized and milled to pass through a 40-60 mesh sieve and stored (humidity c.a. 5%) in dark at −18°C for further analyses.

Extraction
Three powdered samples (20 g) of each cultivar were Soxhlet extracted with dichloromethane for 6 h. The solvent was evaporated to dryness, the lipophilic extracts were weighted and the results were expressed in percent of dry weight (% dw). Dichloromethane was selected as a fairly specific solvent for lipophilic extractives isolation for analytical purposes.
Compounds were identified as TMS derivatives by comparing their mass spectra with the GC-MS spectral library (Wiley-NIST Mass Spectral Library 1999) and their retention times with published data obtained under the described experimental conditions (Oliveira et al., 2006(Oliveira et al., , 2008, and also by comparing their fragmentation profiles with published data or by injection of standards. For semi-quantitative analysis, GC-MS was calibrated with pure reference compounds, representative of the major lipophilic extractive families (stigmasterol, octadecanoic acid, ferulic acid and nonadecan-1-ol) relative to tetracosane. The respective response factors were calculated as an average of six GC-MS runs. For tocopherol the response factor of stigmasterol was used. Each aliquot was injected in triplicate. The presented results are the average of the concordant values obtained for the six aliquots (less than 5% variation between injections of the same aliquot and between aliquots of the same mango cultivar extracts).

Results and discussion
The physicochemical characteristics, namely weight, length, pulp/ seed ratio, water content, pulp firmness, total soluble solids (TSS) and pH, of the twelve mango cultivars investigated in this study, are given in Table 1. All the values obtained are comparable to values previously reported for other mango varieties/cultivars (Charoensiri, Kongkachuichai, Suknicom, & Sungpuag, 2009;Liu et al., 2013;Pleguezuelo, Zuazo, Fernández, & Tarifa, 2012). Mangoes have high water content, with the cultivar 'Tolbert' and 'Gomera II' presenting the highest (86.4%) and the lowest (75.1%) values, respectively. Firmness was evaluated when the mangoes reached the mature stage, and 'Rosa' presented the highest pulp firmness with 1.52 N. Total soluble solids (TSS) determination expressed as°Brix, is usually used as an estimation of the sugar content of fruit. Generally, the TSS in mangoes range from 7.0 to 17.4°Brix, depending on the variety, the production place and maturity stage (Lucena, Assis, Alves, Silva, & Enéas, 2007), and good quality mango for fresh consumption should have a TSS between 13 and 15°Brix (Rovira & Alvarez, 1990). In the present study, the lowest°Brix was observed for 'OTT' (11.0°Brix), the highest one for 'Gomera III' (19.3°Brix), and except for 'OTT', all cultivar showed TSS above 12°Brix. Finally, the pH of the studied samples varied between 5.02 for 'Anderson' and 3.41 for 'OTT'. The differences among them can be attributed to the different cultivars, edaphoclimatic conditions and fruit maturity. In fact, it is known that during mango ripening process the acidity decreased and pH increased, due to the cell metabolization of volatile organic acids and non-volatile constituents (Tucker, 1993).
The lipophilic extractives yields from the ripe pulp of mango cultivars were quite similar, with values ranging from 0.56 to 1.34% of dry material for 'Gomera I' and 'Tommy Atkins', respectively, as shown in Fig. 1. These lipophilic extractives contents are similar to those found in other tropical fruits e.g. in the unripe pulp of banana (Oliveira et al., 2008).
The composition of the lipophilic extracts of the ripe mango pulp was analyzed in detail by GC-MS, and the identities and abundances of the identified compounds are summarized in Table 2. The predominant lipophilic compounds were a series of free sterols that accounted for nearly 32.8-54.2% of all identified compounds. Free fatty acids (C12-C25) were also very abundant accounting for 22.6-41.9% of all lipophilic compounds. Additionally, minor amounts of long chain aliphatic alcohols (C14-C30) were also identified in the extracts. The relative abundance of the identified compounds and their families differs somewhat between cultivars, as illustrated in Table 2 and Fig. 2. The presence of these classes of compounds was already reported in other tissues (e.g. stem bark, peel, kernel) of M. indica L. (Gaydou, 1984;Masibo & He, 2009;Muchiri, Mahungu, & Gituanja, 2012).
Free sterols are the most abundant class of lipophilic compounds present in the ripe mango pulps, accounting for 343 and 1030 mg kg −1 of dry material for 'Gomera I' and 'OTT', respectively (Table 2). β-Sitosterol is definitely the major component of this family in all pulp samples, representing between 51.0 ('Irwin') and 69.1% ('Gomera I') of total sterol contents and between 20.1 ('Osteen') and 36.4% ('Rubro Brasil') of the total lipophilic extractives ( Table 2). Other identified free sterols include campesterol (52-174 mg kg −1 of dry material), fucosterol (23-146mg kg −1 of dry material), stigmasterol (24-82 mg kg −1 of dry material), 24-methylenecholesterol (1-50 mg kg −1 of dry material) and 24-methylenecycloartanol (7-108 mg kg −1 of dry material). Generally, the human intake of phytosterols varies from about 145 to 405 mg per day (Sánchez-Moreno, De Pascual-Teresa, De Ancos, & Cano, 2012), and, although fruits in general are not considered good sources of sterols, 'Gomera II' (e.g.) can contribute to the intake of ca. 23.4 mg of free phytosterols per 100 g of fresh mango. This value is in agreement with the average value of 24.4 mg per 100 g of edible portion of mango reported by Han et al. (2008). Hence, the ripe mango pulps from these twelve cultivars can contribute to the intake of natural phytosterols in the human diets, which appear to be a practical and safe option for reducing cholesterol levels in the population (Piironen, Lindsay, Miettinen, Toivo, & Lampi, 2000;Quílez, García-Lorda, & Salas-Salvadó, 2003).
Long chain fatty acids represent about 22.6-41.9% of the lipophilic components of ripe mango pulps. Whereas the cultivars 'Tommy Atkins' and 'Osteen' presented the higher amounts of fatty acids (940 and 1108 mg kg −1 of dry material, respectively), 'Gomera I' presented the lowest one (353 mg kg −1 of dry material). The identified fatty acids ranged from dodecanoic to pentacosanoic acids, including five unsaturated structures (C16 and C18), one diacid (nonadioic acid) and one ω-hydroxy fatty acid (Table 2). Hexadecanoic acid is the most abundant saturated fatty acid, with the highest content observed in the cultivar 'Osteen' (311 mg kg −1 of dry material) and the lowest in the 'Gomera I' (107 mg kg −1 of dry material). Unsaturated fatty acids were also present in high amounts (158-612 mg kg −1 of dry material), with octadec-9-enoic acid as the major compound of this group, with the highest content observed in the cultivar 'Tommy Atkins' (327 mg kg −1 of dry material) and the lower in the 'Gomera I' (53 mg kg −1 of dry material), followed by octadeca-9,12,15-trienoic acid (an ω−3 fatty acid) with 29-198 mg kg −1 of dry pulp, hexadec-9-enoic acid with 19-110 mg kg −1 of dry pulp and octadeca-9,12-dienoic acid (an ω−6 fatty acid) with 10-57 mg kg −1 of dry pulp. Minor amounts of 22-hydroxydocosanoic (1-7 mg kg −1 of dry pulp) and nonadioic acids (1-2 mg kg −1 of dry pulp) were also found in all twelve extracts of ripe mango pulps. Contrary to saturated and monounsaturated fatty acids that are nonessential dietary lipids, polyunsaturated fatty acids, like octadeca-9,12dienoic (ω−6) and octadeca-9,12,15-trienoic (ω−3) acids, are essential nutrients that must be obtained from the diet because they are not synthesized in the human body (Sánchez-Moreno et al., 2012). Hence, these mango pulps can also contribute to the intake of the above ω−3 and ω−6 fatty acids, with 'Osteen' contributing to the higher intake of octadeca-9,12,15-trienoic acid with ca. 3.4 mg per 100 g of fresh mango, and 'Tommy Atkins' and 'Osteen' to the higher intake of octadeca-9,12-dienoic acid with ca. 0.9 mg per 100 g of fresh mango. The role of fatty acids in the human health, especially ω−3 and ω−6 fatty acids, is mainly associated with the prevention, delay, or treatment of chronic and acute diseases, such as cancer, cardiovascular diseases, osteoporosis, and immune disorders (Chen, McClements, & Decker, 2013;Sánchez-Moreno et al., 2012;Simopoulos, 1999Simopoulos, , 2008. Long-chain aliphatic alcohols (LCAA) were also detected in the ripe mango pulps (49-107 mg kg −1 of dry material), representing only a small fraction (2.5-5.5%) of the total amount of lipophilic extractives ( Table 2). The most abundant LCAA found are triacontan-1-ol, octacosan-1-ol and hexadecan-1-ol, with 9-47, 6-44 and 14-41 mg kg −1 of dry material, respectively. Reports on the role of LCAA in human health suggest a decrease of the low-density lipoprotein (LDL) cholesterol and an increase of the high-density lipoprotein (HDL) cholesterol (Hargrove, Greenspan, & Hartle, 2004), with their regular consumption.
Finally, other compounds like monoglycerides, α-tocopherol, transferulic acid and tricosane were also detected in smaller amounts (Table 2). Only three monoglycerides were identified (25-131 mg kg −1 Table 2 Compounds identified in the lipophilic extracts of ripe pulp from mango cultivars expressed in mg kg −1 of dry material. a

Conclusions
The present work represents the first study on the lipophilic components from the ripe pulp of twelve mango cultivars of the M. indica L. species, namely 'Tommy Atkins', 'Rosa', 'OTT', 'Anderson', 'Rubro Brasil', 'Osteen', 'Tolbert', 'Irwin', 'Gleen', 'Gomera I', 'Gomera II' and 'Gomera III', cultivated in Madeira Island. The major groups of compounds identified in the lipophilic fraction of the extractives consisted mainly of sterols and fatty acids, followed by long-chain aliphatic alcohols. Considerable amounts of steryl glucosides and steryl esters were also detected. Among all the identified compounds, the presence of phytosterols (and derivatives) and ω−3 and ω−6 fatty acids with well-established beneficial nutritional and health effects, contributes to the valorization of these mango cultivars as sources of valuable phytochemicals.