Detection of unusual carotenoid esters in fresh mango (Mangifera indica L. cv. ‘Kent’)

Phytochemistry 64 (2003) 825–829 www.elsevier.com/locate/phytochem

Detection of unusual carotenoid esters in fresh mango (Mangifera indica L. cv. ‘Kent’) Isabell Potta, Dietmar E. Breithauptb,*, Reinhold Carlec a

Institute for Agricultural Engineering in the Tropics and Subtropics, Hohenheim University, Garbenstraße 9, D-70599 Stuttgart, Germany b Institute of Food Chemistry, Hohenheim University, Garbenstraße 28, D-70599 Stuttgart, Germany c Institute of Food Technology, Hohenheim University, Garbenstraße 25, D-70599 Stuttgart, Germany Received 5 May 2003; received in revised form 26 June 2003

Abstract The carotenoid pattern of mango cv. ‘Kent’ was investigated by LC-(APcI)MS analyses. In solvent extracts from the mesocarp an unusual carotenoid ester was identified as violaxanthin dibutyrate. For unequivocal identification of butyric acid by an independent method, total lipids were isolated by solvent extraction from the fruit flesh and analyzed by GC after saponification and subsequent methylation. Thus, evidence of butyric acid (1.6 area%) was provided. To the best of our knowledge, this is the first report on a xanthophyll dibutyrate in plants. Additionally, further carotenoid peaks were tentatively assigned to 9-cis-violaxanthin and neochrom or luteoxanthin, respectively, by their UV/vis and MS data of the saponified extracts. # 2003 Elsevier Ltd. All rights reserved. Keywords: Mango; Mangifera indica L.; Carotenoids; Carotenoid esters; Violaxanthin esters; LC–(APcI)MS

1. Introduction Beside bananas, mangoes (Mangifera indica L.) of different cultivars are the second most important tropical fruits in terms of production, marketing and consumption. Their economic importance is continuously increasing in the cultivating countries (Yaacob and Subhadrabandhu, 1995). Due to their succulency, exotic flavor, and delicious taste, mango fruits are popular, even in Western countries. Their remarkably high carotenoid content, which is responsible for the yellow to orange color of ripe mango flesh, provides a high provitamin A value and antioxidative capacity. Total carotenoid concentrations are usually in the range of 0.9–9.2 mg/100 g (Litz, 1997) whereas the Indian cultivar ‘Alphonso’ showed exceptionally high values of up to 11 mg/100 g (Padmini and Prabha, 1997; Litz, 1997). Godoy and Rodriguez-Amaya (1989) found that b-carotene generally is the predominant carotenoid, comprising 48– 84% of the total carotenoid concentrations.

* Corresponding author. Tel.: +49-711-459-4094; fax: +49-7114096. E-mail address: [email protected] (D.E. Breithaupt). 0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0031-9422(03)00466-7

From the great number of different cultivars, only few have been analyzed in terms of their carotenoid pattern. Godoy and Rodriguez-Amaya (1989) quantified various carotenoids in Brazilian cultivars, including b-carotene, violaxanthin, mutatoxanthin, and luteoxanthin. After saponification, extracts of fresh and processed mango slices contained numerous carotenoids, mainly consisting of violaxanthin, neochrom, luteoxanthin, and bcarotene (Cano and de Ancos, 1994). Based on their elution order, xanthophyll myristic and palmitic acid esters, respectively, were tentatively identified as genuine compounds of the unprocessed fruit, whereas Wilberg and Rodriguez-Amaya (1995) merely found b-carotene and violaxanthin in saponified fruit extracts. Recently, Mercadante et al. (1997) confirmed b-carotene, transand 9-cis-violaxanthin as major carotenoids of the cultivar ‘Keitt’ by mass spectrometry in the EI mode. The effects of ripening and varietal differences on the carotenoid pattern were investigated in a subsequent study (Mercadante and Rodriguez-Amaya, 1998). Violaxanthin and luteoxanthin isomers in addition to b-carotene proved to be the major carotenoids in ripe fruits of the cultivar ‘Keitt’. Obviously, the carotenoid composition varies, depending on the cultivar, geographic or climatic effects, stage of maturity, fruit processing, and

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storage conditions during shipment (e.g. Mercadante and Rodriguez-Amaya, 1998). The composition of mango lipids has been studied by several groups. Padmini and Prabha (1997) investigated biochemical changes during acetylene-induced ripening in the cultivar Alphonso and found C16:0, C16:1, and C18:1 to be the main fatty acids of neutral lipids. Shibahara et al. (1986) described an unconventional octadecenoic acid [cis-vaccenic acid, C18:1($-7)] as a typical constituent of mango lipids. However, C16:0, C16:1, and C18:1 dominated the fatty acid spectrum. The USDA Nutrient Database for Standard Reference (1999) and Souci and Bosch (1994) listed C18:3 ($-3) as a characteristic fatty acid. However, no evidence for the presence of fatty acids with an acyl chain shorter than C12 was obtained in those reports. In this paper unusual carotenoid esters of ripe mango flesh, cv. ‘Kent’, tentatively identified as violaxanthin esters in a previous investigation (Pott et al., 2003) are characterized by LC–(APcI)MS analysis. The occur-

rence of violaxanthin dibutyrate is reported for the first time. To support the presence of butyric acid, the fatty acid pattern of mango flesh was studied.

2. Results and discussion For the characterization of free and acylated carotenoids in the ripe mesocarp of mango cv. ‘Kent’, unsaponified as well as saponified extracts were analyzed by HPLC and LC–(APcI)MS. The native carotenoid pattern of the ripe flesh is dominated by three intense peaks (Fig. 1A). Co-chromatography with an authentic b-carotene standard allowed unequivocally assignment of 6 to all-trans-b-carotene. Furthermore, the genuine extract showed a large number of minor peaks with retention times typical of free ( < 15 min), mono- (15–25 min) and diacylated carotenoids (> 30 min). Analytical proof of the presence of carotenoid esters was provided by analysis of saponified samples (Fig. 1B). Three peaks

Fig. 1. HPLC chromatograms (extended section) of a normal (A) and a saponified (B) extract of ripe mango mesocarp (cv. ‘Kent’). Peak assignment: 1: violaxanthin; 2: neochrom or luteoxanthin (tentatively identified); 3: unidentified free xanthophyll (m/z 601); 4: violaxanthin dibutyrate; 5: unidentified esterified xanthophyll; 6: b-carotene. Table 1 LC–(APcI)MS (positive mode) and UV/vis data of carotenoids from mango mesocarp (cv. ‘Kent’) extracts Peak

Carotenoid

UV/vis data (nm)

(APcI)MS data (m/z)

1 2 3 4 5 6

Violaxanthin Neochrom/luteoxanthina cis-Violaxanthina Violaxanthin dibutyrate cis-Violaxanthin dibutyratea b-Carotene

416/440/470 400/422/450 414/436/466 416/440/470 414/436/466 426/452/478

601 (100%)/583 (15%) 601 (100%)/583 (29%) 601 (100%)/583 (15%) 741 (100%)/653 (4%), 723 (28%)/635 (6%) 741 (100%)/653 (3%), 723 (28%)/635 (7%) 537 (100%)

Peak numbering is according to Fig. 1. UV/vis data (DAD) are indicated in the solvents during LC–MS analyses. a Tentatively identified, based on the molecular mass and the UV/vis spectrum.

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in the polar region of the chromatogram (1–3 in Fig. 1B) showed identical quasimolecular ions (m/z 601, [M+H]+). The loss of water from [M+H]+ led to m/z 583 [M+H–H2O]+ (Table 1). Since all ions were protonated in the APcI+ mode, 1–3 were assigned to xanthophylls with molecular masses of 600 Da. Based on its molecular mass, retention time, and characteristic UV/vis spectrum, 1 was identified as violaxanthin (Fig. 2a). On the basis of the UV/vis data reported for the main carotenoids present in genuine mango extracts (Cano and de Ancos, 1994), 2 was tentatively assigned to neochrom and luteoxanthin (398/422/444 nm or 398/418/442 nm), respectively (Figs. 2b,c). Due to the lack o>f reference material, comparison of the UV/vis spectra and the MS data of 3 with literature data (e.g. Ko¨st, 1988) did not allow the unequivocal identification of this carotenoid. Because of missing a cis-peak in its UV/vis

Fig. 2. Structures of carotenoids identified in mango fruits (Mangifera indica L.) cv. ‘Kent’. Assignment: (1) violaxanthin; (2) neochrom; (3) luteoxanthin; (4) b-carotene.

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spectrum, 3 may be assigned to a cis-isomer of violaxanthin possessing a peripheral double bond. This assumption is in agreement with data given by Mercadante et al. (1997), proposing a 3 nm hypsochromic shift for 9-cis-violaxanthin (412/437/465 nm) compared to the all-trans isomer. Unexpectedly, 4 and 5 disappeared after saponification, suggesting that both carotenoids were acylated, in spite of their short retention times. The mass spectrum of 4 is depicted in Fig. 3. The fragmentation pattern is characterized by two paths, affording different daughter ions. The loss of water from the quasimolecular ion (m/z 741) yields m/z 723. Subsequent elimination of butyric acid (88 Da) as neutral molecule results in m/z 635. Alternatively, removal of a fatty acid residue (88 Da) with subsequent elimination of water from the resulting daughter ion (m/z 653) occurs. Identical fragmentation was observed for 5 which is in accordance with mass spectroscopic data earlier reported for violaxanthin diesters of potatoes (Breithaupt and Bamedi, 2002). The loss of 88 Da unambiguously proved 4 and 5 to be diacylated with butyric acid. Since the UV/vis spectrum of 4 was in accordance with data reported for violaxanthin, 4 is attributed to be violaxanthin dibutyrate. The UV/vis data of 5 are identical to 3 (formed after saponification), suggesting 3 to be the parent carotenoid of 5 (Table 1). Based on its fragmentation pattern and respective UV/vis data 5 was tentatively identified as cis-violaxanthin dibutyrate. The short retention times of both, 4 and 5, indicate ‘‘polar’’ xanthophyll fatty acid esters with very short acyl chains, thus supporting their structural assignment. The origin of 2 may be ascribed to the saponification of minor carotenoid esters, eluting between 5 and 6 (all-trans -b-carotene).

Fig. 3. Mass spectrum (APcI+ mode) of violaxanthin dibutyrate (corresponding to 4, Fig. 1).

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Table 2 Fatty acid distribution (C4:0 to C18:3) of total lipids obtained from mango mesocarp, calculated as area% FAME/total FAME

described by Pott et al. (2003). Aliquots of 20 ml were used for analysis.

Fatty acid methyl ester (FAME)

3.2. Saponification

Butyric (C4:0)

Relative area (area%) 1.60.5

Shibahara et al. (1986) (wt.%) –

Myristic (C14:0)

2.40.4

5.7

Palmitic (C16:0)

24.01.5

22.2

Palmitoleic (C16:1, $-7)

20.31.7

17.3

Stearic (C18:0)

1.20.3

0.5

Oleic (C18:1, $-9)

8.91.3

15.8

cis-Vaccenic acid (C18:1, $-7)

7.10.9

14.5

Linoleic (C18:2, $-6)

6.21.2

0.6

Linolenic (C18:3, $-3)

31.73.1

9.7

Minor compounds below 0.5 area% were not listed. Values are presented as arithmetic meansstandard deviations of five independent determinations. For comparison, the fatty acid composition of mangoes of Philippine origin (Shibahara et al., 1986) is presented.

Total lipids were isolated by solvent extraction from mango mesocarp and analyzed by GC after saponification and subsequent methylation. For calculation of the fatty acid composition, unusual cis-vaccenic acid [C18:1($-7)] formerly described by Shibahara et al. (1986) was considered in comparison (Table 2). The identity of cis-vaccenic acid was verified by standard addition experiments. GC analyses provided evidence of butyric acid—albeit in low concentrations of approximately 1.6%—in total mango lipids. Thus, the presence of butyric acid in the lipid fraction of mango mesocarp was ascertained by two independent methods. Although butyric acid has been described as volatile compound being responsible, together with numerous other constituents, for the inherent aroma profile of mango (Bautista et al., 1998; Adedeji et al., 1992), this is the first report on plant carotenoids being acylated with butyric acid. As reported by Mercadante and Rodriguez-Amaya (1998) xanthophyll esters are prone to degrade during fruit processing. Consequently, upon conventional and solar drying violaxanthin dibutyrate was completely degraded (Pott et al., 2003). Therefore, the occurrence of xanthophyll esters may be taken as a marker of unprocessed mango products.

For identification of parent carotenoids, samples were saponified in diethyl ether by addition of methanolic KOH (30%, w/v). The procedure was carried out as described by Breithaupt (2000). 3.3. HPLC and LC–(APcI)MS analyses LC–(APcI)MS (positive mode) was run on an HP1100 HPLC system, coupled to a Micromass (Manchester, UK) VG platform II quadrupole mass spectrometer. For carotenoid separation, a YMC (Schermbeck, Germany) C30 analytical column (5.0 mm, 2504.6 mm I.D.) including a C30 guard column (5.0 mm, 104.0 mm I.D.) was used and kept at 35  C. For gradient elution two mobile phases A and B consisting of MTBE, methanol, and water, each, were applied: A=90:6:4 (v/v/v); B=15:81:4 (v/v/v). The following gradient was used (min/% A): 0/99; 39/44; 45/0; 50/99; 55/99 (flow rate: 1 ml/min). Further MS and HPLC parameters have been detailed by Breithaupt et al. (2002). 3.4. Isolation and transesterification of total lipids from mango mesocarp Mango mesocarp was dried in vacuo over night (70  C). For fat isolation, 25 g of dried flesh were extracted with n-hexane using a Soxhlet device (4 h). The organic phase was concentrated to 5 ml. An aliquot (1 ml) was evaporated to dryness (N2), the resulting residue suspended in methanolic KOH solution (0.5 N, 1 ml) and treated as described by Breithaupt and Schwack (2000). After transesterification with boron trifluoride-methanol reagent, an aliquot of the supernatant organic phase (1 ml) was used for analysis of the fatty acid methyl esters (FAME) by GC. FAME were identified by comparison of their retention times with a standard mixture in n-hexane (1 mg/ml each). Since accurate quantification was not intended, the fatty acid distribution was calculated as area% FAME/total FAME, irrespective of their relative response factors. 3.5. GC analyses

3. Experimental 3.1. Preparation of samples Fresh mango fruits, cv. ‘Kent’ (Brazil), were purchased from a retail shop in Stuttgart. The ripe flesh was prepared and extracted for HPLC and LC–(APcI)MS as

Gas chromatography was performed using a Carlo Erba (Hofheim, Germany) Vega Series 2 gas chromatograph GC 6000 equipped with a flame ionization detector (FID) and a J&W Scientific (Folsom, CA, USA) fused-silica capillary column (30 m0.53 mm) wallcoated with DB-23 (0.5 mm film thickness). Further

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parameters were previously reported by Breithaupt and Schwack (2000).

Acknowledgements The authors are grateful to the ‘Ministerium fu¨r Wissenschaft, Forschung und Kunst, Baden-Wu¨rttemberg, Germany’ for financial support.

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